Lippincotts illustrated reviews microbiology pdf năm 2024
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Lir microbiology South Asian Edition is the updated version of one of the favourite tools for students to learn microbiology. Part of the popular Lippincott illustrated Reviews series, this proven approach uses clear, concise writing and hundreds of dynamic illustrations to take students into the realms of the Microbial world. The contents of the book have been extensively revised and updated in order to make them relevant for the countries in South Asia. In keeping with the revised competency-based medical curriculum for undergraduates, this book lays adequate stress on clinical applications of diagnostic. Simply head over to the account section in settings and click on “Cancel Subscription” - it’s as simple as that. After you cancel, your membership will stay active for the remainder of the time you’ve paid for. Learn more here. Can/how do I download books? 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Learn more here. Is Lippincott® Illustrated Reviews: Microbiology an online PDF/ePUB? Yes, you can access Lippincott® Illustrated Reviews: Microbiology by Cynthia N. Cornelissen, Marcia Metzgar Hobbs in PDF and/or ePUB format, as well as other popular books in Medicine & Medical Microbiology & Parasitology. We have over one million books available in our catalogue for you to explore. Cynthia Nau Cornelissen, Ph.D. Department of Microbiology and Immunology School of Medicine Virginia Commonwealth University Richmond, Virginia Bruce D. Fisher, M.D. Department of Medicine Jersey Shore University Medical Center Neptune, New Jersey Department of Medicine University of Medicine and Dentistry of New Jersey– Robert Wood Johnson Medical School Piscataway, New Jersey Richard A. Harvey, Ph.D. Department of Biochemistry University of Medicine and Dentistry of New Jersey– Robert Wood Johnson Medical School Piscataway, New Jersey iv v Acknowledgments Cynthia Nau Cornelissen is grateful to her husband, Christopher and to her children, Jeremy and Emily, for their support and encouragement during this endeavor. Bruce D. Fisher is grateful to his wife Doris, for constant support and encouragement; to Elliot Frank, MD, FACP, Chair of the Department of Medicine at Jersey Shore University Medical Center, esteemed colleague and consummate clinician-educator; and to Donald Armstrong, MD, MACP, for mentorship, guidance, and inspiration for over 35 years. Richard Harvey is grateful to the many friends and colleagues who generously contributed their time and effort to help us make this book as accurate and useful as possible. Without talented artists, an Illustrated Review would be impossible, and we have been particularly fortunate in working with Michael Cooper throughout this project. His artistic sense and computer graphics expertise have greatly added to our ability to bring microbiology “stories” alive for our readers. We are also highly appreciative of Dr. Hae Sook Kim and Linda Duckenfield, SM/MT (ASCP), for assistance in preparing photomicrographs. The editors and production staff of Lippincott Williams & Wilkins were a constant source of encouragement and discipline. We particularly want to acknowledge the tremendously supportive and creative contributions of our editor, Susan Rhyner, whose imagination and positive attitude helped us bring this complex project to completion. The design, content, editing, and assembly of the book have been greatly enhanced through the efforts of Kelly Horvath. vi Acquisitions Editor: Susan Rhyner Product Manager: Angela Collins Marketing Manager: Joy Fisher-Williams Vendor Manager: Alicia Jackson Cover Design: Holly McLaughlin Development Editor: Kelly Horvath Third Edition Copyright © 2013 Lippincott Williams & Wilkins, a Wolters Kluwer business 351 West Camden Street Baltimore, MD 21201 Two Commerce Square; 2001 Market Street Philadelphia, PA 19103 Printed in China All rights reserved. This book is protected by copyright. No part of this book may be reproduced or transmitted in any form or by any means, including as photocopies or scanned-in or other electronic copies, or utilized by any information storage and retrieval system without written permission from the copyright owner, except for brief quotations embodied in critical articles and reviews. Materials appearing in this book prepared by individuals as part of their official duties as U.S. government employees are not covered by the above-mentioned copyright. To request permission, please contact Lippincott Williams & Wilkins at Two Commerce Square, 2001 Market Street, Philadelphia, PA 19103, via email at [email protected], or via website at lww.com (products and services). 9 8 7 6 5 4 3 2 1 Library of Congress Cataloging-in-Publication Data Cornelissen, Cynthia Nau. Microbiology / Cynthia Nau Cornelissen, Bruce D. Fisher, Richard A. Harvey. 3rd ed. p. ; cm. (Lippincott's illustrated reviews) Includes index. Rev. ed. of: Microbiology / Richard A. Harvey, Pamela C. Champe, Bruce D. Fisher. c2007. Summary: "Lippincott's Illustrated Reviews: Microbiology, Third Edition enables rapid review and assimilation of large amounts of complex information about medical microbiology. The book has the hallmark features for which Lippincott's Illustrated Reviews volumes are so popular: an outline format, 450 full-color illustrations, end-of-chapter summaries, review questions, plus an entire section of clinical case studies with full-color illustrations. NEW TO THIS EDITION: an online testbank of 100 review questions"--Provided by publisher. ISBN 978-1-60831-733-2 (pbk.) I. Fisher, Bruce D., M.D. II. Harvey, Richard A., Ph. D. III. Title. IV. Series: Lippincott's illustrated reviews. [DNLM: 1. Microbiological Phenomena--Outlines. QW 18.2] 579--dc23 DISCLAIMER Care has been taken to confirm the accuracy of the information present and to describe generally accepted practices. However, the authors, editors, and publisher are not responsible for errors or omissions or for any consequences from application of the information in this book and make no warranty, expressed or implied, with respect to the currency, completeness, or accuracy of the contents of the publication. 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Lippincott Williams & Wilkins customer service representatives are available from 8:30 am to 6:00 pm, EST. vii viii Unique Clinical Features Summaries of bacteria and their diseases Gram (+) cocci • Catalase (+) Colonies are yellow • Nonmotile • Do not form spores cocci tending to occur • Round in bunches like grapes anaerobic • Facultative organisms on enriched media • Cultured containing broth and/or Staphylococcus species Summary of common diseases Staphylococcus aureus • Skin and soft URINARY TRACT INFECTIONS A Common causes of UTI1 • Septicemia tissue infections • Necrotizing pneumonia • Toxic shock syndrome • Food poisoning (antibiotic therapy not used) blood • Osteomyelitis • Septic arthritis • Endocarditis B Classification of pathogens Staphylococcus aureus on blood agar surrounded by zone of β hemolysis. Staphylococcus aureus cultured from a wound infection Methicillin susceptible 1 Oxacillin 1 Nafcillin Escherichia coli Gram (+) cocci Staphylococcus saprophyticus 10 20 30 40 50 Ciprofloxacin Methicillin resistant (community-acquired; mild-moderate infection) 80 90 1 Doxycycline Approximate prevalence (%) Common Methicillin resistant sis C complaintss Other enterobacteria (community-acquired; uir Other genera of Enterobacteriaceae, such severeasinfection) Escherichia coli • • 2 Vancomycin2 1Most isolates resistant to penicillin G 2Used in methicillin-resistant isolates 1 Trimthoprim/ sulfmethoxazole 60 70 • Cystitis in women 1 Oxacillin 1 Nafcillin Pseudomonas aeruginosa 0 Staphylococcus saprophyticus and heart valves 1 Vancomycin Escherichia coli Klebsiella Proteus Pseudomonas aeruginosa Proteus • Infections of catheters (health-care associated) Gram (–) rods Klebsiella Staphylococcus epidermidis Methicillin resistant Staphylococcus saprophyticus •
DYSURIA LUMBAR PAIN • 108 1 Daptomycin Klebsiella, Enterobacter, Proteus, and Serratia, which can be found as normal inhabitants of the large intestine, include organisms that are primarily opportunistic and often nosocomial pathogens. They all frequently colonize hospitalized patients, especially in association with antibiotic treatment, indwelling catheters, or invasive procedures, causing extra-intestinal infections such as those of the urinary tract. 1 Linezolid 1 Vancomycin 2 Quinupristindalfopristin 2 Teicoplanin Carbuncle caused by Staphylococcus aureus Note: Treatment of MRSA may vary by the type and on location of infection. Furuncle caused by Staphylococcus aureus These organisms produce exotoxins. Wide-spread antibiotic resistance among these organisms necessitates sensitivity testing to determine the appropriate antibiotic treatment. 104 100 Staphylococcus saprophyticus 98 Normal 94 • •
Pseudomonas P d aeruginosa i • FEVER •
CHILLS
Staphylococcal scalded skin syndrome Folliculitis caused by Fo Staphylococcus aureus Sta St
sta staphylococcal disease. 1 Indicates first-line drugs; 2 Superficial impetigo indicates alternative drugs. Figure 33.4 (continued on the next page) Disease summary of urinary tract infections. 1Uncomplicated cystitis. Illustrated Case Studies Case 1: Man with necrosis of the great toe This 63-year-old man with a long history of diabetes mellitus was seen in consultation because of an abrupt deterioration in his clinical status. He was admitted to the hospital for treatment of an ulcer, which had been present on his left great toe for several months. Figure 34.1 shows a typical example of perforating ulcer in a diabetic man. Because of the inability of medical therapy (multiple courses of oral antibiotics) to resolve the ulcer, he underwent amputation of his left leg below the knee. On the first postoperative day he developed a temperature of 101oF, and on the second postoperative day he became disoriented and his temperature reached 105.2oF. His amputation stump was mottled with many areas of purplish discoloration, and the most distal areas were quite obviously necrotic (dead). Crepitus (the sensation of displacing gas when an area is pressed with the fingers) was palpable up to his patella. An X-ray of the left lower extremity showed gas in the soft tissues, extending beyond the knee to the area of the distal femur. A Gram stain of a swab from the necrotic tissue is shown in Figure 34.2. Double stranded Enveloped Herpesviridae Polymorphonuclear leukocyte Epstein-Barr virus virus, Type 1 Herpes simplex virus, Type 2 Human cytomegalovirus Human herpesvirus, Type 8 Varicella-zoster virus Gram positive Herpes simplex bacillus Quick Review Common characteristics • • • • • Figure 34.1 Perforating ulcer of the great toe. Figure 34.2 Gram stain of material swabbed from deep within a crepitant area. There are numerous polymorphonuclear leukocytes, and many large gram-positive bacilli, as well as a few gram-negative bacilli and cocci. Linear, double-stranded DNA genome Replicate in the nucleus Envelope contains antigenic, species-specific glycoproteins In the tegument between the envelope and capsid are a number of virus-coded enzymes and transcription factors essential for initiation of the infectious cycle All herpesviruses can enter a latent state following primary infection, to be reactivated at a later time ix Contents UNIT I: The Microbial World Chapter Chapter Chapter Chapter Chapter 1: 2: 3: 4 5: Introduction to Microbiology 1 Normal Flora 7 Pathogenicity of Microorganisms 11 Diagnostic Microbiology 19 Vaccines and Antimicrobial Agents 33 UNIT II: Bacteria Chapter Chapter Chapter Chapter Chapter Chapter Chapter Chapter Chapter Chapter Chapter Chapter Chapter Chapter 6: 7: 8: 9: 10: 11: 12: 13: 14: 15: 16: 17: 18: 19: Bacterial Structure, Growth, and Metabolism 49 Bacterial Genetics 59 Staphylococci 69 Streptococci 79 Gram-positive Rods 91 Neisseriae 101 Gastrointestinal Gram-negative Rods 111 Other Gram-negative Rods 129 Clostridia and Other Anaerobic Rods 149 Spirochetes 161 Mycoplasma 171 Chlamydiae 177 Mycobacteria and Actinomycetes 185 Rickettsia, Erhlichia, Anaplasma and Coxiella 197 UNIT III: Fungi and Parasites Chapter 20: Fungi 203 Chapter 21: Protozoa 217 Chapter 22: Helminths 227 UNIT IV: Viruses Chapter Chapter Chapter Chapter Chapter Chapter Chapter Chapter Chapter 23: 24: 25: 26: 27: 28: 29: 30: 31: Introduction to the Viruses 233 Nonenveloped DNA Viruses 245 Enveloped DNA Viruses 255 Hepatitis B and Hepatitis D (Delta) Viruses Positive-strand RNA Viruses 283 Retroviruses and AIDS 293 Negative-strand RNA Viruses 309 Double-stranded RNA Viruses: Reoviridae Unconventional Infectious Agents 327 273 323 UNIT V: Clinical Microbiology Review Chapter Chapter Chapter Index 32: Quick Review of Clinically Important Microorganisms 33: Disease Summaries 367 34: Illustrated Case Studies 393 419 331 x UNIT I: The Microbial World 1 Introduction to Microbiology I. OVERVIEW Microorganisms can be found in every ecosystem and in close association with every type of multicellular organism. They populate the healthy human body by the billions as benign passengers (normal flora, see p. 7) and even as participants in bodily functions. For example, bacteria play a role in the degradation of intestinal contents. In this volume, we primarily consider the role of microorganisms (that is, bacteria, fungi, protozoa, helminths, and viruses) in the initiation and spread of human diseases. Those relatively few species of microorganisms that are harmful to humans, either by production of toxic compounds or by direct infection, are characterized as pathogens. Most infectious disease is initiated by colonization (the establishment of proliferating microorganisms on the skin or mucous membranes) as shown in Figure 1.1. The major exceptions are diseases caused by introduction of organisms directly into the bloodstream or internal organs. Microbial colonization may result in: 1) elimination of the microorganism without affecting the host; 2) infection in which the organisms multiply and cause the host to react by making an immune or other type of response or 3) a transient or prolonged carrier state. Infectious disease occurs when the organism causes tissue damage and impairment of body function. COLONIZATION Organisms become part of resident flora Organisms eliminated by host defenses INFECTION Carrier state (no apparent symptoms, but organisms may shed) II. PROKARYOTIC PATHOGENS Organisms eliminated by host defenses Pathogenicity All prokaryotic organisms are classified as bacteria, whereas eukaryotic organisms include fungi, protozoa, and helminths as well as humans. Prokaryotic organisms are divided into two major groups: the eubacteria, which include all bacteria of medical importance, and the archaebacteria, a collection of evolutionarily distinct organisms. Cells of prokaryotic and eukaryotic organisms differ in several significant structural features as illustrated in Figure 1.2. INFECTIOUS DISEASE Figure 1.1 Some possible outcomes following exposure to microorganisms. 1 2 1. Introduction To Microbiology CHARACTERISTIC PROKARYOTIC CELLS EUKARYOTIC CELLS Chromosome Usually single, circular1 Multiple Nucleus No nuclear envelope or nucleoli Membrane bound, nucleoli present Membrane-bound organelles Not present Present (examples include mitochondria and endoplasmic reticulum) Cell wall Usually present, many contain peptidoglycan Present in plant cells, no peptidoglycan Plasma membrane No carbohydrates, most lack sterols Sterols and carbohydrates present Ribosome 70S 80S (70S in organelles) Average size 0.2–2 mm in diameter 10–100 mm in diameter Figure 1.2 Comparison of prokaryotic and eukaryotic cells. 1Some bacteria have more than one circular molecule as their genome. Vibrios, for example, have two circular chromosomes. Borrellia have linear chromosomes and a wide array of different sized plasmids.
III. FUNGI Fungi are nonphotosynthetic, generally saprophytic, eukaryotic organisms. Some fungi are filamentous and are commonly called molds, whereas others (that is, the yeasts) are unicellular (see p. 203). Fungal reproduction may be asexual, sexual, or both, and all fungi produce spores. Pathogenic fungi can cause diseases, ranging from skin infections (superficial mycoses) to serious, systemic infections (deep mycoses). IV. PROTOZOA Protozoa are single-celled, nonphotosynthetic, eukaryotic organisms that come in various shapes and sizes. Many protozoa are free living, but oth- VII. Organizing The Microorganisms
1,000 mm =1m UNAIDED EYE ers are among the most clinically important parasites of humans. Members of this group infect all major tissues and organs of the body. They can be intracellular parasites, or extracellular parasites in the blood, urogenital region, or intestine. Transmission is generally by ingestion of an infective stage of the parasite or by insect bite. Protozoa cause a variety of diseases that are discussed in Chapter 21, p. 217. 3 Worms 10 mm = 10-2 m 1 mm = 10-3 m LIGHT MICROSCOPE VI. VIRUSES Viruses are obligate intracellular parasites that do not have a cellular structure. Rather, a virus consists of molecule(s) of DNA (DNA virus) or RNA (RNA virus), but not both, surrounded by a protein coat. A virus may also have an envelope derived from the plasma membrane of the host cell from which the virus is released. Viruses contain the genetic information necessary for directing their own replication but require the host’s cellular structures and enzymatic machinery to complete the process of their own reproduction. The fate of the host cell following viral infection ranges from rapid lysis and release of many progeny virions to gradual, prolonged release of viral particles. 100 mm = 10-1 m 100 μm = 10-4 m Large protozoa Eukaryotic cells 10 μm = 10-5 m Most bacteria VII. ORGANIZING THE MICROORGANISMS 1 μm = 10-6 m The authors have adopted two color-coded graphic formats: 1) an expanded hierarchical organization and 2) lists of clinically important bacteria and viruses. Rickettsiae Chlamydiae
100 nm = 10-7 m ELECTRON MICROSCOPE A hierarchical organization resembles a family tree. These graphs in Figures 1.4 and 1.5 divide bacteria and viruses into groups based on the characteristics of the microorganisms. Mitochondria Mycoplasma Viruses 10 nm = 10-8 m Proteins Prions 1 nm = 10-9 m Small molecules Figure 1.3 Relative size of organisms and molecules. 4 1. Introduction To Microbiology Classified according to: Medically Important Bacteria Physical properties of cell wall Lacking cell wall Rigid cell wall Flexible cell wall Morphology Simple unicellular Filamentous Growth inside or outside host cell Obligate intracellular parasite Free living Gram-staining property Gram-positive Gram-negative Shape Cocci Rods Cocci Rods Oxygen tolerance Aerobes Anaerobes Figure 1.4 Hierarchical classification of clinically important bacteria according to six distinguishing characteristics. Medically Important Viruses RNA Double stranded Icosahedral Nonenveloped DNA Single stranded Single stranded Nonenveloped Nonenveloped Negative strand Helical Enveloped Double stranded Enveloped Positive strand Nonenveloped Icosahedral Enveloped Icosahedral Figure 1.5 Classification of medically important virus families. Helical VII. Organizing The Microorganisms 5 CHAPTER 10 CHAPTER 8 Staphylococcus aureus Staphylococcus epidermidis Staphylococcus saprophyticus CHAPTER 19 Coxiella burnetii Ehrlichia species Rickettsia species Bacillus anthracis Bacillus cereus Corynebacterium diphtheriae Erysipelothrix rhusiopathiae Lactobacillus species Listeria monocytogenes Propionibacterium acnes CHAPTER 12 Campylobacter jejuni Campylobacter fetus Enterobacter species Escherichia coli Helicobacter pylori Klebsiella oxytoca Klebsiella pneumoniae Proteus species Providencia species CHAPTER 11 CHAPTER 9 CHAPTER 18 Actinomyces israelii Arachnia propionica Mycobacterium avium intracellulare Mycobacterium bovis Mycobacterium kansasii Mycobacterium leprae Mycobacterium tuberculosis Nocardia asteroides Norcardia brasiliensis Salmonella typhimurium Enterococcus faecium Enterococcus faecalis Peptostreptococcus faecalis Streptococcus agalactiae Streptococcus bovis Streptococcus mutans Streptococcus pneumoniae Streptococcus pyogenes CHAPTER 17 Acinetobacter species Moraxella catarrhalis Neisseria gonorrhoeae Neisseria meningitidis Gram (+) cocci Gram (+) bacilli Gram (–) cocci Gram (–) rods Anaerobic organisms Spirochetes Mycoplasma Chlamydia Other CHAPTER 15 Borrelia burgdorferi Borrelia recurrentis Leptospira interrogans Treponema pallidum Serratia marcescens Shigella sonnei Vibrio cholerae Vibrio parahaemolyticus Yersinia enterocolitica Yersinia pseudotuberculosis CHAPTER 13 Bartonella species CHAPTER 14 Bordetella parapertussis Bacteroides fragilis Bordetella pertussis Clostridium botulinum Brucella species Clostridium difficile Chlamydia pneumoniae Chlamydia psittaci Chlamydia trachomatis Salmonella typhi Clostridium perfringens Clostridium tetani Fusobacterium Prevotella melaninogenica Burkholderia mallei Francisella tularensis Haemophilus influenzae Legionella pneumophila Pasteurella multocida Pseudomonas aeruginosa Pseudomonas pseudomallei Yersinia pestis CHAPTER 16 Mycoplasma hominis Mycoplasma incognitus Mycoplasma pneumoniae Ureaplasma urealyticum Figure 1.6 Medically important bacteria discussed in this book, organized into similar groups based on morphology, biochemistry, and/or staining properties. 6 1. Introduction To Microbiology CHAPTER 24 CHAPTER 24 Parvovirus B19 Single stranded Nonenveloped Double stranded Nonenveloped Adenoviruses BK virus Human papilloma viruses JC virus Double stranded Enveloped CHAPTERS 25, 26 Single stranded Positive strand Icosadedral Nonenveloped CHAPTER 27 Coxsackievirus Echovirus Enterovirus Hepatitis A virus Hepatitis E virus Norwalk virus Poliovirus Rhinovirus Single stranded Positive strand Icosadedral or helical Enveloped Single stranded Negative strand Helical Enveloped CHAPTER 29 California encephalitis virus Ebola virus Hantaan viruses Human parainfluenza virus types 1, 2, 3, and 4 Influenza virus types A and B Junin virus La Crosse encephalitis virus Lassavirus Lyssavirus (rabies virus) Lymphocytic choriomeningitis virus Machupo virus Marburg virus Measles virus Mumps virus Respiratory syncytial virus TYPE OF GENOME DNA viruses Double stranded, Icosahedral Nonenveloped CHAPTERS 27, 28 CHAPTER 30 Rotaviruses Chikungunya virus Coronavirus Dengue fever virus Eastern and Western equine encephalitis viruses Hepatitis C virus Human immunodeficiency viruses 1 and 2 Human T-cell leukemia viruses 1 and 2 Japanese encephalitis Rubella virus St. Louis encephalitis virus Tick-borne encephalitis virus Venezuelan equine encephalitis virus West Nile virus Yellow fever virus PRESENCE OF ENVELOPE Enveloped STRANDEDNESS OF GENOME Single-stranded nucleic acid Enveloped DNA RNA viruses Nonenveloped Epstein-Barr virus Hepatitis B virus Herpes simplex virus types 1 and 2 Human cytomegalovirus Human herpesvirus types 6, 7, and 8 Molluscum contagiosum virus Vaccinia virus Varicella-zoster virus Variola virus Nonenveloped RNA Double-stranded nucleic acid Figure 1.7 Medically important viruses discussed in this book, organized into similar groups based on the nature of the genome and the presence or absence of a lipid envelope. 2 Normal Flora
II. THE HUMAN MICROBIOME The human microbiome is the total number and diversity of microbes found in and on the human body. In the past, the ability to cultivate organisms from tissues and clinical samples was the gold standard for identification of normal flora and bacterial pathogens. However, the recent application of culture-independent molecular detection methods based on DNA sequencing (see p. 28) indicates that the human body contains a far greater bacterial diversity than previously recognized. Unlike classic microbiologic culture methods, molecular detection requires neither prior knowledge of an organism nor the ability to culture it. Thus, molecular methods are capable of detecting fastidious and nonculturable species. Even using advanced molecular techniques, it is difficult to define the human microbiome because microbial species present vary from individual to individual as a result of physiologic differences, diet, age, and geographic habitat. Despite these limitations, it is useful to be aware of the dominant types and distribution of resident flora, because such knowledge provides an understanding of the possible infections that result from injury to a particular body site. A Gram (+) cocci Staphylococcus aureus Staphylococcus epidermidis Streptococcus species Gram (+) bacilli Corynebacterium species Propionibacterium acnes Skin “Skin popping” (injection of drugs below the skin rather than directly into a vein), breaks the skin barrier and may result in soft tissue infections caused by introduction of normal skin flora into subcutaneous tissue. B Figure 2.1 A. Examples of bacteria that inhabit the skin. B. Arm of individual who injects drugs by “skin popping.” 7 8 2. Normal Flora III. DISTRIBUTION OF NORMAL FLORA IN THE BODY Gram (+) cocci Staphylococcus aureus Staphylococcus epidermidis Streptococcus species Gram (+) rods Corynebacterium species Propionibacterium acnes Gram (–) cocci Moraxella Neisseria species Figure 2.2 Examples of bacteria that inhabit the conjunctival sac. [Note: Tears, which contain the antimicrobial enzyme lysozyme, help limit the bacterial population of the conjunctiva.] Gram (+) cocci Staphylococcus species Streptococcus sanguinis Streptococcus mutans Gram (+) rods Corynebacterium species Propionibacterium acnes Gram (–) cocci Neisseria species Gram (–) rods Haemophilus species Anaerobic organisms Bacteroides species Fusobacterium Prevotella species Spirochetes Borrelia Treponema Other Actinomyces The organism most often implicated in plaque formation is Streptococcus mutans, which produces the gelatinous glucans that constitute plaque material. S. mutans, accompanied by other acidproducing species, then adhere to the plaque and demineralize the tooth surface. The most common sites of the body inhabited by normal flora are, as might be expected, those in contact or communication with the outside world, namely, the skin, eye, and mouth as well as the upper respiratory, gastrointestinal, and urogenital tracts. A. Skin Skin can acquire any bacteria that happen to be in the immediate environment, but this transient flora either dies or is removable by washing. Nevertheless, the skin supports a permanent bacterial population (resident flora), residing in multiple layers of the skin (Figure 2.1). The resident flora regenerate even after vigorous scrubbing. 1. Estimate of the skin microbiome using classical culture techniques: Staphylococcus epidermidis and other coagulase-negative staphylococci (see p. 76) that reside in the outer layers of the skin appear to account for some 90 percent of the skin aerobes. Anaerobic organisms, such as Propionibacterium acnes, reside in deeper skin layers, hair follicles, and sweat and sebaceous glands. Skin inhabitants are generally harmless, although S. epidermidis can attach to and colonize plastic catheters and medical devices that penetrate the skin, sometimes resulting in serious bloodstream infections. 2. Estimate of the skin microbiome using molecular sequencing techniques: The estimate of the number of species present on skin bacteria has been radically changed by the use of the 16S ribosomal RNA gene sequence (see p. 28) to identify bacterial species present on skin samples directly from their genetic material. Previously, such identification had depended upon microbiological culture, upon which many varieties of bacteria did not grow and so were not detected. Staphylococcus epidermidis and Staphylococcus aureus were thought from culture-based research to be dominant. However DNA analysis research finds that, while common, these species make up only 5 percent of skin bacteria. The skin apparently provides a rich and diverse habitat for bacteria. B. Eye The conjunctiva of the eye is colonized primarily by S. epidermidis, followed by S. aureus, aerobic corynebacteria (diphtheroids), and Streptococcus pneumoniae. Other organisms that normally inhabit the skin are also present but at a lower frequency (Figure 2.2). Tears, which contain the antimicrobial enzyme lysozyme, help limit the bacterial population of the conjunctiva. C. Mouth and nose Figure 2.3 Examples of bacteria that inhabit the mouth. The mouth and nose harbor many microorganisms, both aerobic and anaerobic (Figure 2.3). Among the most common are diphtheroids (aerobic Corynebacterium species), S. aureus, and S. epidermidis. In IV. Beneficial Functions Of Normal Flora addition, the teeth and surrounding gingival tissue are colonized by their own particular species, such as Streptococcus mutans. [Note: S. mutans can enter the bloodstream following dental surgery and colonize damaged or prosthetic heart valves, leading to potentially fatal infective endocarditis.] Some normal residents of the nasopharynx can also cause disease. For example, S. pneumoniae , found in the nasopharynx of many healthy individuals, can cause acute bacterial pneumonia, especially in older adults and those whose resistance is impaired. [Note: Pneumonia is frequently preceded by an upper or middle respiratory viral infection, which predisposes the individual to S. pneumoniae infection of the pulmonary parenchyma.] D. Intestinal tract In an adult, the density of microorganisms in the stomach is relatively low (103 to 105 per gram of contents) due to gastric enzymes and acidic pH. The density of organisms increases along the alimentary canal, reaching 108 to 1010 bacteria per gram of contents in the ileum and 1011 per gram of contents in the large intestine. Some 20 percent of the fecal mass consists of many different species of bacteria, more than 99 percent of which are anaerobes (Figure 2.4). Bacteroides species constitute a significant percentage of bacteria in the large intestine. Escherichia coli, a facultatively anaerobic organism, constitutes less than 0.1 percent of the total population of bacteria in the intestinal tract. However, this endogenous E. coli is a major cause of urinary tract infections. E. Urogenital tract The low pH of the adult vagina is maintained by the presence of Lactobacillus species, which are the primary components of normal flora. If the Lactobacillus population in the vagina is decreased (for example, by antibiotic therapy), the pH rises, and potential pathogens can overgrow. The most common example of such overgrowth is the yeast-like fungus, Candida albicans (see p. 213), which itself is a minor member of the normal flora of the vagina, mouth, and small intestine. The urine in the kidney and bladder is sterile but can become contaminated in the lower urethra by the same organisms that inhabit the outer layer of the skin and perineum (Figure 2.5). IV. BENEFICIAL FUNCTIONS OF NORMAL FLORA Normal flora can provide some definite benefits to the host. First, the sheer number of harmless bacteria in the lower bowel and mouth make it unlikely that, in a healthy person, an invading pathogen could compete for nutrients and receptor sites. Second, some bacteria of the bowel produce antimicrobial substances to which the producers themselves are not susceptible. Third, bacterial colonization of a newborn infant acts as a powerful stimulus for the development of the immune system. Fourth, bacteria of the gut provide important nutrients, such as vitamin K, and aid in digestion and absorption of nutrients. [Note: Although humans can obtain vitamin K from food sources, bacteria can be an important supplemental source if nutrition is impaired.] 9 Gram (+) cocci Enterococcus species Peptostreptococcus species Staphylococcus species Streptococcus viridans Gram (–) rods Enterobacter species Escherichia coli Klebsiella species Proteus species Pseudomonas species Anaerobic organisms Bacteroides species Clostridium species Lactobacillus species Bacteroides fragilis causes intraabdominal abscesses, and additional intestinal bacteria commonly cause peritonitis following appendicitis, diverticulitis, or other trauma resulting in perforation of the intestine. Other Actinomyces species Figure 2.4 Examples of bacteria that inhabit the gastrointestinal tract. 10 2. Normal Flora Gram (+) cocci Staphylococcus species Gram (+) bacilli Corynebacterium species Lactobacillus species Gram G ram (–) rods Klebsiella species Proteus species Pseudomonas s species Anaerobic organisms Bacteroides species Mycoplasma Mycoplasma species Other Actinomyces species The most common example of an organism that overgrows in the absence of normal vaginal flora, such as Lactobacillus, is the yeast-like fungus, Candida albicans.
Figure 2.5 Examples of bacteria that inhabit the vagina. Study Questions Choose the ONE correct answer 2.1 The primary effect of lactobacilli in the adult vagina is to A. B. C. D. E. maintain an alkaline environment. maintain an acidic environment. produce a protective mucus layer. increase fertility. keep the menstrual cycle regular. 2.2 A patient presents with severe colitis associated with an overgrowth of Clostridium difficile in the lower bowel. The most likely cause of this condition is A. B. C. D. E. botulinum food poisoning. a stomach ulcer. a compromised immune system. antibiotic therapy. mechanical blockage of the large intestine. Correct answer = B. Lactobacilli produce acid that, in turn, inhibits the growth of potential pathogenic bacteria and fungi. None of the other answers are known to be attributed to lactobacilli. Correct answer = D. Antibiotic therapy can reduce normal flora in the bowel, allowing pathogenic organisms normally present in low numbers to overgrow. None of the other answers explains the overgrowth of Clostridium difficile. 2.3 The predominant bacterial species that colonizes the human skin is A. B. C. D. E. Lactobacillus. Candida albicans. Streptococcus pneumoniae. Staphylococcus epidermidis. Bacterioides fragilis. Correct answer = D. Human skin normally contains up to 10,000 Staphylococcus epidermidis per cm2. Other colonizing bacteria may be present but in much lower numbers. Candida albicans is a yeast-like fungus, not a bacterium. 3 Pathogenicity of Microorganisms I. OVERVIEW Subclinical A pathogenic microorganism is defined as one that is capable of causing disease. Some microorganisms are unequivocally pathogenic, whereas others (the majority) are generally harmless. An organism may invade an individual without causing infectious disease when the host’s defense mechanisms are successful. The occurrence of such asymptomatic infections can be recognized by the presence of antibody against the organism in the patient’s serum. Some infections result in a latent state, meaning that the organism is dormant but may be reactivated with the recurrence of symptoms. Moreover, some pathogens cause disease only under certain conditions (for example, being introduced into a normally sterile body site or infection of an immunocompromised host). Figure 3.1 summarizes some of the terms used to describe the diversity of infections. II. BACTERIAL PATHOGENESIS Although the mechanism of infectious process may vary among bacteria, the methods by which bacteria cause disease can, in general, be divided into several stages (Figure 3.2). Pathogenicity of a microorganism depends on its success in completing some or all of these stages. The terms “virulence” and “pathogenicity” are often used interchangeably. However, virulence can be quantified by how many organisms are required to cause disease in 50 percent of those exposed to the pathogen (ID50, where I = Infectious and D = Dose), or to kill 50 percent of test animals (LD50, where L = Lethal). The number of organisms required to cause disease varies greatly among pathogenic bacteria. For example, less than 100 Shigella cause diarrhea by infecting the gastrointestinal (GI) tract, whereas the infectious dose of Salmonella is approximately 100,000 organisms. The infectious dose of a bacterium depends primarily on its virulence factors. The probability that an infectious disease occurs is influenced by both the number and virulence of the infecting organisms and the strength of the host immune response opposing infection. A. Virulence factors Virulence factors are those characteristics of a bacterium that enhance its pathogenicity, that is, properties that enable a microorganism to establish itself and replicate on or within a specific host. • An infection with no detectable symptoms • Example: asymptomatic gonorrhea Latent • An infection with the potential to become active at some time • Examples: Treponema pallidum (syphilis) and Mycobacterium tuberculosis (tuberculosis) Opportunistic • An infection due to an organism that generally does not cause disease unless normal host defenses are compromised • Example: Pneumocystis pneumonia in patients with HIV Primary • Infection by an organism that may become latent and later cause other disease manifestations • Example:Treponema pallidum (syphilis) Secondary • a) Reactivation of a latent infection, or b) the second stage of an infection • Examples: a) Mycobacterium tuberculosis (tuberculosis) b) Treponema pallidum (syphilis) Mixed • Two or more bacteria infecting the same tissue • Example: Pelvic inflammatory disease may be initiated by infection with N. gonorrhoeae or C. trachomatis but other organisms including anaerobes play important roles in progression of the disease. Pyogenic • Pus forming • Example: staphylococcal and streptococcal infections Fulminant • Infections that occur suddenly and intensely • Example: Necrotizing fasciitis from Streptococcus pyogenes, also called “flesheating bacteria” [Note: fulminant is derived from the Latin word for lightning (fulmen).] Figure 3.1 Terms used to describe infections. 11 12 3. Pathogenicity Of Microorganisms Some of the more important steps in the infectious process are reviewed below. Entry into the host, with evasion of host primary defenses 1 Inhalation Oral 1. Entry into the host: The first step of the infectious process is the entry of the microorganism into the host by one of several ports: via the respiratory, GI, or urogenital tract or through skin that has been cut, punctured, or burned. Once entry is achieved, the pathogen must overcome diverse host defenses before it can establish itself. These include phagocytosis; the acidic environments of the stomach and urogenital tract; and various hydrolytic and proteolytic enzymes found in the saliva, stomach, and small intestine. Bacteria that have an outer polysaccharide capsule (for example, Streptococcus pneumoniae and Neisseria meningitidis) have a better chance of surviving these primary host defenses. Skin (direct contact, cuts, vectorborne transmission) Urogenital 2. Adherence to host cells: Some bacteria (for example, Rectal 2 Escherichia coli, see p. 111) use pili to adhere to the surface of host cells. Group A streptococci have similar structures (fimbriae, see p. 80). Other bacteria have cell surface adhesion molecules or particularly hydrophobic cell walls that allow them to adhere to the host cell membrane. In each case, adherence enhances virulence by preventing the bacteria from being carried away by mucus or washed from organs with significant fluid flow, such as the urinary and the GI tracts. Adherence also allows each attached bacterial cell to form a microcolony. A striking example of the importance of adhesion is that of Neisseria gonorrhoeae in which strains that lack pili are not pathogenic (see p. 101). Adhesion of the microorganism to host cells Pili (or other adhesion molecules) Glycolipid 3. Invasiveness: Invasive bacteria are those that can enter host cells Host cell membrane or penetrate mucosal surfaces, spreading from the initial site of infection. Invasiveness is facilitated by several bacterial enzymes, the most notable of which are collagenase and hyaluronidase. These enzymes degrade components of the extracellular matrix, providing the bacteria with easier access to host cell surfaces. Many bacterial pathogens express membrane proteins known as "invasins" that interact with host cell receptors, thereby eliciting signaling cascades that result in bacterial uptake by induced phagocytosis. Invasion is followed by inflammation, which can be either pyogenic (involving pus formation) or granulomatous Glycoprotein 3 Invasion of the host Bacteria 4 Propagation of the organism 5 Damage to host cell by bacterial toxins or immune response of the host. 6 Progression or resolution of the disease. Toxin Damage mediated by host immune response Figure 3.2 Mechanism of infectious process. Bacteria eliminated or contained (immune response, antimicrobial therapy) II. Bacterial Pathogenesis 13 (having nodular inflammatory lesions), depending on the organism. The pus of pyogenic inflammations contains mostly neutrophils, whereas granulomatous lesions contain fibroblasts, lymphocytes, and macrophages. A 4. Iron sequestering: Iron is an essential nutrient for most bacteria. B To obtain the iron required for growth, bacteria produce iron-binding compounds, called siderophores. These compounds capture iron from the host by chelation, and then the ferrated siderophore binds to specific receptors on the bacterial surface. Iron is actively transported into the bacterium, where it is incorporated into essential compounds such as cytochromes. The pathogenic Neisseria species are exceptions in that they do not produce siderophores but instead utilize host iron-binding proteins, such as transferrin and lactoferrin, as iron sources. They do so by expressing dedicated receptors that bind to these host proteins and remove the iron for internalization. 1 The membrane recognition portion of the toxin (B) binds to the cell membrane. Diphtheria toxin A B Cell membrane CELL Receptor for toxin 2 A B After entering the cell, the A subunit dissociates and cleaves or modifies a target molecule within the cell. 5. Virulence factors that inhibit phagocytosis: The most important e subunit Active tox of toxin antiphagocytic structure is the capsule external to the cell wall, such as in S. pneumoniae and N. meningitidis. A second group of antiphagocytic factors are the cell wall proteins of gram-positive cocci, such as protein A of staphylococcus and M protein of group A streptococci (see pp. 70, 80). A B 6. Bacterial toxins: Some bacteria cause disease by producing toxic substances, of which there are two general types: exotoxins and endotoxin. Exotoxins, which are proteins, are secreted by both gram-positive and gram-negative bacteria. In contrast, endotoxin, which is synonymous with lipopolysaccharide (LPS), is not secreted but instead is an integral component of the cell walls of gram-negative bacteria. a. Exotoxins: These include some of the most poisonous sub- stances known. It is estimated that as little as 1 μg of tetanus exotoxin can kill an adult human. Exotoxin proteins generally have two polypeptide components (Figure 3.3). One is responsible for binding the protein to the host cell, and one is responsible for the toxic effect. In several cases, the precise target for the toxin has been identified. For example, diphtheria toxin is an enzyme that blocks protein synthesis. It does so by attaching an adenosine diphosphate–ribosyl group to human protein elongation factor EF-2, thereby inactivating it (see p. 92). Most exotoxins are rapidly inactivated by moderate heating (60o C), notable exceptions being staphylococcal enterotoxin and E. coli heatstable toxin (ST). In addition, treatment with dilute formaldehyde destroys the toxic activity of most exotoxins but does not affect their antigenicity. Formaldehyde-inactivated toxins, called toxoids, are useful in preparing vaccines (see p. 36). Exotoxin proteins are, in many cases, encoded by genes carried on plasmids or temperate bacteriophages. An example is the diphtheria exotoxin that is encoded by the tox gene of a temperate bacteriophage that can lysogenize Corynebacterium diphtheriae. Strains of C. diphtheriae that carry this phage are pathogenic, whereas those that lack the phage are nonpathogenic. EF-2 Nicotinamide N A NAD+ EF-2 EF F-2 Diphtheria toxin ADPR Toxin ADP-ribosylates host protein elongation factor, stopping protein synthesis. NAD+ G Protein Nicotinamide A ADPR G Pro Protein ro ot ot Cholera toxin Toxin ADP-ribosylates a G protein of intestinal mucosal cells, resulting in ionic imbalance and loss of water. A Pro ro ot Protein Amino acids Shiga toxin Toxin cleaves host ribosomal RNA, resulting in inhibition of protein synthesis. Toxin cleaves a protein involved in release of neurotransmitters. Neurotransmitters A Tetanus toxin Figure 3.3 Action of exotoxins. ADP = adenosine diphosphate; ADPR = adenosine diphosphate ribose; NAD+ = nicotinamide adenine dinucleotide. 14 3. Pathogenicity Of Microorganisms b. Endotoxins: These are heat-stable, LPS components of the Sick 1 2 The microorganism must always be found in similarly diseased animals but not in healthy ones. The microorganism must be isolated from a diseased animal and grown in pure culture. outer membranes of gram-negative (but not gram-positive) bacteria. They are released into the host’s circulation following bacterial cell lysis. LPS consists of polysaccharide composed of repeating sugar subunits (O antigen), which protrudes from the exterior cell surface; a core polysaccharide; and a lipid component called lipid A that is integrated into the outer leaflet of the outer membrane. The lipid A moiety is responsible for the toxicity of this molecule. The main physiologic effects of LPS endotoxin are fever, shock, hypotension, and thrombosis, collectively referred to as septic shock. These effects are produced indirectly by macrophage activation, with the release of cytokines, activation of complement, and activation of the coagulation cascade. Death can result from multiple organ failure. Elimination of the causative bacteria with antibiotics can initially exacerbate the symptoms by causing sudden massive release of endotoxin into the circulation. Although gram-positive bacteria do not contain LPS, their cell wall peptidoglycan and teichoic acids can elicit a shock syndrome similar to that caused by LPS but usually not as severe. B. Host-mediated pathogenesis The pathogenesis of many bacterial infections is caused by the host response rather than by bacterial factors. Classic examples of host response–mediated pathogenesis are seen in diseases such as gram-negative bacterial sepsis, tuberculosis, and tuberculoid leprosy. The tissue damage in these infections is caused by various cytokines released from the lymphocytes, macrophages, and polymorphonuclear leukocytes at the site of infection or in the bloodstream. Often the host response is so intense that host tissues are destroyed, allowing remaining bacteria to proliferate. Sick 3 4 The isolated microorganism must cause the original disease when inoculated into a susceptible animal. The microorganism can be reisolated from the experimentally infected animal. Figure 3.4 Koch’s postulates.
III. Viral Pathogenesis that is unable to cause disease in healthy, immunocompetent individuals but can infect people whose defenses have been impaired.] Robert Koch, a 19th century German microbiologist, recognized this dilemma and defined a series of criteria (Koch’s postulates) to confirm the causative microbial agent of a disease (Figure 3.4). [Note: Although these criteria has been successful in establishing the etiology of most infections, it fails if the causative organism cannot be cultured in vitro.] E. Infections in human populations Bacterial diseases may be communicable from person to person or noncommunicable. For example, cholera is highly communicable (the disease-causing organism, Vibrio cholerae, is easily spread), whereas botulism is noncommunicable because only those people who ingest the botulinum exotoxin are affected. Highly communicable diseases, such as cholera, are said to be contagious and tend to occur as localized epidemics in which the disease frequency is higher than normal. When an epidemic becomes worldwide, it is called a pandemic. Pandemics, such as the 1918 influenza pandemic, arise because the human population has never been exposed to and, therefore, has no immunity against the specific strain of influenza virus. III. VIRAL PATHOGENESIS 15 infections that result A Viral in host cell death B Viral infections that result in transformation of the host cell Some viral infections result in the persistence of the viral genome inside a host cell with no production of progeny virus. Host genome C Viral infections that result in host cell fusion Viral proteins Host cell Viruses can replicate only inside living cells. Consequently, the first pathogenic manifestations of viral infection are seen at the cellular level. The course of events following initial exposure to some viruses may include rapid onset of observable symptoms, which is referred to as an acute infection. Alternatively, the initial infection by other viruses may be mild or asymptomatic. Following the initial infection, the most common outcome is that the virus is cleared completely from the body by the immune system. For some viruses, the initial infection is followed by either a persistent infection or a latent infection. A. Viral pathogenesis at the cellular level Cells show a variety of different responses to viral infection, depending on the cell type and virus. Many viral infections cause no apparent morphologic or functional changes in the cell. When changes do occur, several (potentially overlapping) responses can be recognized (Figure 3.5). 1. Cell death: A cell can be directly killed by the virus. In most cases, this is due to the inhibition of synthesis of cellular DNA, RNA, and protein. Some viruses have specific genes responsible for this inhibition. Dead or dying cells release a brood of progeny viruses that repeat the replication process. Examples of viruses that kill their host cells are adenovirus (see p. 250) and poliovirus (see p. 283). 2. Transformation: Some viruses transform normal cells into malig- nant cells. In many ways, this is the opposite of cell death, because malignant cells have less fastidious growth requirements Host cell nucleus Virion Fu Fusion Giant multinucleate cell D Cytopathic effects Cell rounding Stainable viral proteins Cell disintegration Figure 3.5 Types of viral pathogenesis at the cellular level. 16 3. Pathogenicity Of Microorganisms than do normal cells, and they have an indefinitely extended lifetime. Transformation is an irreversible genetic process caused by the integration of viral DNA into the host’s DNA (see p. 243). Infection 3. Cell fusion: Infection of cells with certain viruses causes the cells to fuse, producing giant, multinucleate cells. Viruses with this property include herpesviruses (see p. 257) and paramyxoviruses (see p. 312). The ability of infected cells to fuse is apparently due to virus-induced changes in the structure of the cell membrane. BODY SURFACE MPH N LYMPH NODE BLOOD (primary viremia) BONE MARROW LIVER SPLEEN BLOOD VESSEL (endothelium) BLOOD (secondary viremia) NASAL AND ORAL MUCOUS MEMBRANE SKIN easle Measles Rubella ubell BRAIN LUNG CNS SALIVARY GLAND KIDNEY Cytomegalovirus egalo Poliovirus oviru Figure 3.6 Examples of dissemination of virus to secondary sites in the body. 4. Cytopathic effect: Cytopathic effect (CPE) is a catch-all term that refers to any visible change in appearance of an infected cell, for example, cell rounding, patches of stainable viral proteins inside the cell, and cell disintegration. Some viruses can be roughly identified by the time of onset and pattern of CPE in cell culture as well as by the types of cells in which these viruses cause CPE. B. Initial infections Following initial multiplication at the primary site of entry, the viral infection may remain localized or become disseminated. The infection may be asymptomatic (unapparent). Alternatively, typical symptoms of disease may occur, often in two temporally distinct forms: 1) early symptoms at the primary site of infection and 2) delayed symptoms due to dissemination from the primary site, causing infection of secondary sites. Virus transmission can occur before symptoms of the generalized illness are apparent, making it difficult to control the spread of viral diseases. 1. Routes of entry and dissemination to secondary sites: Common routes by which viruses enter the body are essentially the same as for bacterial infections (that is, through the skin or respiratory, GI, or urogenital tracts). In each case, some viruses remain localized and cause disease that is largely restricted to the primary site of infection. Other viruses undergo multiplication in cells at the primary site, which may be accompanied by symptoms, followed by invasion of the lymphatic system and the blood. [Note: The presence of virus in the blood is termed viremia.] Virus is disseminated throughout the body via the bloodstream and can infect cells at secondary sites characteristic for each specific virus type, thus causing the disease typically associated with that species (Figure 3.6). 2. Typical secondary sites of localization: Secondary sites of infec- tion determine the nature of the delayed symptoms and, usually, the major characteristics associated with the resulting disease. Viruses frequently exhibit tropism for specific cell types and tissues. This specificity is usually caused by the presence of specific host cell surface receptors recognized by particular viruses. Although any tissue or organ system is a potential target for virus infection, the fetus represents an especially important site for secondary localization of virus infections. Virus from the maternal circulation infects cells of the placenta, thereby gaining access to the fetal circulation and, ultimately, to all tissues of the developing III. Viral Pathogenesis fetus (Figure 3.7). Fetal death or developmental abnormalities are often the result. Neonatal infection can also occur during birth when the fetus comes into contact with infected genital secretions of the mother or after birth when the infant ingests infected breast milk. 3. Virus shedding and mode of transmission: The mode of trans- mission of a viral disease is largely determined by the tissues that produce progeny virus and/or the fluids into which they are released. These are not necessarily the secondary sites of infection but, in fact, are often the site of primary infection at a time before symptoms are apparent. The skin, respiratory and GI tracts, and bodily fluids are commonly sites of viral shedding. 17 A Infected mothers can transmit viral infections to their offspring by three routes: 1 4. Factors involved in termination of acute infection: In a typical, uncomplicated, acute infection, virus is totally eliminated from the host in 2 to 3 weeks. This outcome is primarily a function of the host’s immune system, with involvement of both cell-mediated and humoral responses. The relative importance of these two responses depends on the virus and the nature of the disease. Modes of vertical virus transmission In utero by transplacental spread Virus s 2 During delivery through an infected birth canal 3 After birth by ingestion of breast milk
response to virus infection is a generalized inflammatory response, accompanied by nonspecific killing of infected cells by natural killer cells. This latter activity, enhanced by interferon and other cytokines, begins well before the virus-specific immune response. Later, cytolysis by virus-specific cytotoxic T lymphocytes that recognize virus peptides displayed on the cell surface also eliminates infected cells. These cellular responses are especially significant in that they help limit the spread of the infection by killing infected cells before they have released progeny virus. Cell surface immunodeterminants recognized by T cells are often derived from nonstructural or internal proteins of the virus. Thus, this response complements the inactivation of free virus by humoral antibody, which is directed against capsid or envelope proteins. b. Humoral response: Although circulating antibodies may be directed against any virus protein, those that are of greatest significance in controlling an infection react specifically with epitopes on the surface of the virion and result in inactivation of the virus’s infectivity. The process is called neutralization. This response is of primary importance in suppressing diseases that involve a viremic stage, but secretory antibodies (for example, immunoglobulin A) also play an important protective role in primary infections of the respiratory and GI tracts. Humoral antibodies also take part in killing infected cells by two mechanisms. The first is antibody-dependent, cell-mediated cytotoxicity, in which natural killer cells and other leukocytes bearing Fc receptors bind to the Fc por tions of antibodies that are complexed to virus antigens on the surface of the infected cell and kill it. The second mechanism is complement-mediated lysis of infected cells to which virus-specific antibody has bound. Virus in milk viruses transmitted B Some from mother to infant Herpes simplex virus types 1 and 2 Human cytomegalovirus Human immunodeficiency virus Rubella virus Figure 3.7 Mother-to-infant (vertical) transmission of viral infections. 18 3. Pathogenicity Of Microorganisms Study Questions Choose the ONE correct answer. 3.1 Endotoxin belongs to a class of biological molecules called A. B. C. D. E. mucopolysaccharides. lipopolysaccharides. nucleic acids. proteins. peptidoglycans. Correct answer = B. Endotoxin is an integral lipopolysaccharide constituent of the outer membrane of gram-negative bacteria. The peptidoglycans, present in large amounts in gram-positive bacteria can be toxic but are not considered endotoxin. 3.2 Exotoxins belong to a class of biologic molecules called A. B. C. D. E. mucopolysaccharides. lipopolysaccharides. nucleic acids. proteins. peptidoglycans. 3.3 The mechanism of action of diphtheria toxin is to A. B. C. D. E. disrupt the cell membrane. block nucleic acid synthesis. block protein synthesis. interfere with neurotransmission. destroy the cell nucleus. 3.4 A 48-year-old woman presented at the emergency room complaining of urinary urgency and flank pain. Microscopic examination of a urine sample revealed gram-negative rods. Prior to initiation of antibiotic therapy, she abruptly developed fever, chills, and delirium. Hypotension and hyperventilation rapidly followed. These observations suggest that the patient is responding to the release of bacterial A. B. C. D. E. collagenase. exotoxin. hyaluronidase. lipopolysaccharide. peptidoglycan. Correct answer = D. Exotoxins are secreted toxic proteins that, in many cases, have a well-defined cellular site of action. Correct answer = C. Diphtheria toxin inactivates the polypeptide elongation factor EF-2, blocking protein synthesis. Tetanus toxin interferes with neurotransmission. Correct answer = D. The patient is most likely suffering from septic shock. In two thirds of patients, septic shock results from infection with gram-negative bacteria, such as Escherichia coli, Klebsiella, Enterobacter, Proteus, Pseudomonas, and Bacteroides. Septicemia is more common in persons whose resistance is already compromised by an existing condition. The gram-negative bacteria release endotoxin or heat-stable, lipopolysaccharide (LPS) components of the outer membranes. The main physiologic effects of LPS endotoxin are fever, hypotension, and thrombosis, collectively referred to as septic shock. Death can result from multiple organ failure. Gram-positive bacteria release exotoxins that can elicit a shock syndrome, but response is usually not as severe as that of gram-negative septic shock. 4 Diagnostic Microbiology I. OVERVIEW Identifying the organism causing an infectious process is usually essential for effective antimicrobial and supportive therapy. Initial treatment may be empiric, based on the microbiologic epidemiology of the infection and the patient’s symptoms. However, definitive microbiologic diagnosis of an infectious disease usually involves one or more of the following five basic laboratory techniques, which guide the physician along a narrowing path of possible causative organisms: 1) direct microscopic visualization of the organism, 2) cultivation and identification of the organism, 3) detection of microbial antigens, 4) detection of microbial DNA or RNA, and 5) detection of an inflammatory or host immune response to the microorganism (Figure 4.1). Direct microscopic visualization 1 Cultivation and identification 2 II. PATIENT HISTORY AND PHYSICAL EXAMINATION A clinical history is the most important part of patient evaluation. For example, a history of cough points to the possibility of respiratory tract infection, whereas dysuria (painful or difficult urination) suggests urinary tract infection. A history of travel to developing countries may implicate exotic organisms. For example, a patient who recently swam in the Nile has an increased risk of schistosomiasis. Patient occupations may suggest exposure to certain pathogens, such as brucellosis in a butcher or anthrax in farmers. Even the age of the patient can sometimes guide the clinician in predicting the identity of pathogens. For example, a grampositive coccus in the spinal fluid of a newborn infant is unlikely to be Streptococcus pneumoniae (pneumococcus) but most likely to be Streptococcus agalactiae (group B). This organism is sensitive to penicillin G. By contrast, a gram-positive coccus in the spinal fluid of a 40year-old patient is most likely to be S. pneumoniae. This organism is frequently resistant to penicillin G and requires treatment with a thirdgeneration cephalosporin (such as cefotaxime or ceftriaxone) or vancomycin. The etiology implied by the patient’s age may thus guide initial therapy. A physical examination often provides confirmatory clues to the presence and extent (localized or disseminated) of disease. For example, erythema migrans (a large skin lesion with a bright red outer border and partially clear central area; see p. 165) indicates early localized Lyme disease. Clues to the presence of bacteremia (a disseminated 3 Detection of microbial antigens Detection of microbial RNA or DNA 5 4 Detection of host immune response Figure 4.1 Laboratory techniques that are useful in diagnosis of microbial diseases. 19 20 4. Diagnostic Microbiology 1 infection) may include chills, fever (or sometimes hypothermia), or cardiovascular instability heralding septic shock. Physical signs of pulmonary consolidation suggest pneumonia. If stupor and stiff neck are included in this constellation of findings, the organism causing the pneumonia may have spread to the meninges, warranting a further search for it in the cerebrospinal fluid (CSF). All laboratory studies must be directed by the patient’s history and physical examination and then evaluated, taking into consideration the sensitivity and specificity of the test. Heat-fix specimen to slide. Flood slide with crystal violet solution; allow to act for 1 minute. Crystal violet solution III. DIRECT VISUALIZATION OF THE ORGANISM 2 In many infectious diseases, pathogenic organisms (excluding viruses) can often be directly visualized by microscopic examination of patient specimens, such as sputum, urine, and CSF. The organism’s microscopic morphology and staining characteristics can provide the first screening step in arriving at a specific identification. The organisms to be examined do not need to be alive or able to multiply. Microscopy yields rapid and inexpensive results and may allow the clinician to initiate treatment without waiting for the results of a culture, as noted in the spinal fluid example in the previous paragraph. Rinse the slide, then flood with iodine solution; allow iodine to act for 1 minute. Before acetone decolorization (next step), all organisms appear purple, that is, gram-positive. Iodine solution
3 Because unstained bacteria are difficult to detect with the light microscope, most patient material is stained prior to microscopic evaluation. The most common and useful staining procedure is the Gram stain, which separates bacteria into two classifications according to their cell wall composition. If a clinical specimen on a microscope slide is treated with a solution of crystal violet and then iodine, the bacterial cells will stain purple. If the stained cells are then treated with a solvent, such as alcohol or acetone, grampositive organisms retain the stain, whereas gram-negative species lose the stain, becoming colorless (Figure 4.2). Addition of the counterstain safranin stains the clear, gram-negative bacteria pink or red. Most, but not all, bacteria are stainable and fall into one of these two groups. [Note: Microorganisms that lack cell walls, such as mycoplasma, cannot be identified using the Gram stain.] Rinse off excess iodine. Decolorize with acetone for approximately 5 seconds (time depends on density of specimen). Acetone 4 Wash slide immediately in water. After acetone decolorization, those organisms that are gramnegative are no longer visible. 5 Apply safranin counterstain for 30 seconds. 6 Safranin Figure 4.2 Steps in Gram stain method. Key: = Gram-positive violet. \= Gram-negative red. \= Colorless. Wash in water, blot, and dry in air. Gram-negative organisms are visualized after application of the counterstain. III. Direct Visualization Of The Organism 21 1. Gram stain applications: The Gram stain is important therapeuti- cally because gram-positive and gram-negative bacteria differ in their susceptibility to various antibiotics, and the Gram stain may, therefore, be used to guide initial therapy until the microorganism can be definitively identified. In addition, the morphology of the stained bacteria can sometimes be diagnostic. For example, gram-negative intracellular diplococci in urethral pus provide a presumptive diagnosis of gonorrhea. Gram stains of specimens submitted for culture are often invaluable aids in the interpretation of culture results. For example, a specimen may show organisms under the microscope but appear sterile in culture media. This discrepancy may suggest the presence of either fastidious organisms (bacteria with complex nutrient requirements) that are unable to grow on the culture media employed or fragile organisms, such as gonococcus or anaerobic organisms, that may not survive transport. In these cases, direct visualization with the Gram stain may provide the only clue to the nature, variety, and relative number of infecting organisms. Figure 4.3 Mycobacterium tuberculosis stained with acid-fast stain. 2. Gram stain limitations: The number of microorganisms required is relatively high. Visualization with the Gram stain requires greater than 10 4 organisms/mL. Liquid samples with low numbers of microorganisms (for example, in CSF), require centrifugation to concentrate the pathogens. The pellet is then examined after staining. B. Acid-fast stain Stains such as Ziehl-Neelsen (the classic acid-fast stain) are used to identify organisms that have waxy material (mycolic acids) in their cell walls. Most bacteria that have been stained with carbolfuchsin can be decolorized by washing with acidic alcohol. However, certain acid-fast bacteria retain the carbolfuchsin stain after being washed with an acidic solution. The most clinically important acid-fast bacterium is Mycobacterium tuberculosis, which appears pink, often beaded, and slightly curved (Figure 4.3). Acidfast staining is reserved for clinical samples from patients suspected of having mycobacterial infection. Figure 4.4 India ink preparation of Cryptococcus neoformans in cerebrospinal fluid. These yeast cells are identified by large transparent capsules that exclude the India ink particles.
Figure 4.5 Fungi in unstained nasal sinus exudate, made distinct from other materials (such as cells) with potassium hydroxide. 22 4. Diagnostic Microbiology IV. GROWING BACTERIA IN CULTURE Culturing is routine for most bacterial and fungal infections but is rarely used to identify helminths or protozoa. Culturing of many pathogens is straightforward, for example, streaking a throat swab onto a blood agar plate in search of group A β-hemolytic streptococcus. However, certain pathogens are very slow growing (for example, M. tuberculosis) or are cultured only with difficulty (for example, Bartonella henselae ). Microorganisms isolated in culture are identified using such characteristics as colony size, shape, color, Gram stain, hemolytic reactions on solid media, odor, and metabolic properties. In addition, pure cultures provide samples for antimicrobial susceptibility testing (see p. 30). The success of culturing depends on appropriate collection and transport techniques and on selection of appropriate culture media, because some organisms may require special nutrients. Also, some media are used to suppress the growth of certain organisms in the process of identifying others (see p. 23) A. Specimen collection Figure 4.6 With anaerobic transport media containing a nonnutritive medium that retards diffusion of oxygen after addition of the specimen, microorganisms may remain viable for up to 72 hours. It is important to use an airless syringe for liquid specimens, such as pus, rather than a swab in a tube— especially when transport medium is not available. Also, if promptly transported in a syringe, the relative proportions of each morphology in mixed infections is visible. Many organisms are fragile and must be transported to the laboratory with minimal delay. For example, gonococci and pneumococci are very sensitive to heating and drying. Samples must be cultured promptly, or, if this is not possible, transport media must be used to extend the viability of the organism to be cultured. When anaerobic organisms are suspected, the patient’s specimen must be protected from the toxic effect of oxygen (Figure 4.6). B. Growth requirements All clinically important bacteria are heterotrophs (that is, they require organic carbon for growth). Heterotrophs may have complex or simple requirements for organic molecules. [Note: Organisms that can reduce carbon dioxide and, therefore, do not require organic compounds for cell growth, are called autotrophs.] Most bacteria require varying numbers of growth factors, which are organic compounds required by the cell to grow, but which the organism cannot itself synthesize (for example, vitamins). Organisms that require either a large number of growth factors or must be supplied with very specific ones are referred to as fastidious. C. Oxygen requirements Bacteria can be categorized according to their growth responses in the presence and absence of oxygen. Strict aerobes cannot survive in the absence of oxygen and produce energy only by oxidative phosphorylation. Strict anaerobes generate energy by fermentation or by anaerobic respiration and are killed in the presence of oxygen. Facultative anaerobes can grow in the absence of oxygen but grow better in its presence. Aerotolerant anaerobes have mechanisms to protect themselves from oxygen (therefore, being able to grow in its presence or absence) but do not use oxygen in their metabolism. Finally, microaerophiles require oxygen for their metabolism but cannot survive at atmospheric levels of oxygen. Microaerophiles are found in lakes and wet soil where the oxygen concentration is within an acceptable range. IV. Growing Bacteria In Culture 23
or heart infusions are useful in growing fastidious organisms. For example, sheep blood agar contains protein sources, sodium chloride, and 5 percent sheep blood and supports the growth of most gram-positive and gram-negative bacteria isolated from human sources (see p. 89). However, Haemophilus influenzae and Neisseria gonorrhoeae, among others, are highly fastidious organisms. They require chocolate agar, which contains red blood cells (RBCs) that have been lysed (see p. 130). This releases intracellular nutrients, such as hemoglobin, hemin (“X” factor), and nicotinamide adenine dinucleotide (“V” factor), required by these organisms. Enriched media are useful for culturing normally sterile body fluids, such as blood and CSF, in which the finding of any organisms provides reasonable evidence for infection by that organism. Failure to culture an organism may indicate that the culture medium is inadequate or that the incubation conditions do not support bacterial growth. 2. Selective media: The most commonly used selective medium is MacConkey agar (see p. 115), which supports the growth of most gram-negative rods, especially the Enterobacteriaceae, but inhibits growth of gram-positive organisms and some fastidious gram-negative bacteria, such as Haemophilus and Neisseria species. Growth on blood agar and chocolate agar but not MacConkey agar suggests a gram-positive isolate or a fastidious gram-negative species. In contrast, most gram-negative rods often form distinctive colonies on MacConkey agar. This agar is also used to detect organisms able to metabolize lactose (Figure 4.7). Clinical samples are routinely plated on blood agar, chocolate agar, and MacConkey agar. Hektoen enteric agar is also a selective medium that differentiates lactose/sucrose fermenters and nonfermenters as well as H2S producers and nonproducers. It is often used to culture Salmonella and Shigella species. ThayerMartin agar is another selective medium composed of chocolate agar supplemented with several antibiotics that suppress the growth of nonpathogenic Neisseria and other normal and abnormal flora. This medium is normally used to isolate gonococci. When submitting samples for culture, the physician must alert the laboratory to likely pathogens whenever possible, especially when unusual organisms are suspected. This allows inclusion of selective media that might not be used routinely. It also alerts the labo- Bacteria that ferment lactose produce acids that decrease the pH of agar. The acidity causes a pH indicator in the agar to turn pink. Figure 4.7 Lactose-fermenting, gram-negative rods produce pink colonies on MacConkey agar. 24 4. Diagnostic Microbiology ratory to hold specimens longer if a slow-growing organism, such as Nocardia, is suspected. Catalase test – + The most widely used identification scheme involves determining the morphologic and metabolic properties of the unknown bacterium and comparing these with properties of known microorganisms. Alternate identification schemes using nucleic acid–based methods are discussed on p. 29. Immunologic methods used in diagnosis are described on p. 25. It is essential to start identification tests with pure bacterial isolates grown from a single colony. Bubbles indicate production of oxygen gas by catalase. Oxidase test – +
Dark, oxidized product results from cytochrome c oxidase activity. Different bacteria produce varying spectra of enzymes. For example, some enzymes are necessary for the bacterium’s individual metabolism, and some facilitate the bacterium’s ability to compete with other bacteria or establish an infection. Tests that measure single bacterial enzymes are simple, rapid, and generally easy to interpret. They can be performed on organisms already grown in culture and often provide presumptive identification. 1. Catalase test: The enzyme catalase catalyzes the degradation of Urease test pH indicator in agar turns dark due to production of NH3 by urease. hydrogen peroxide to water and molecular oxygen (H2O2 → H2O + O 2). Catalase-positive organisms rapidly produce bubbles when exposed to a solution containing hydrogen peroxide (Figure 4.8). The catalase test is key in differentiating between many gram-positive organisms. For example, staphylococci are catalase positive, whereas streptococci and enterococci are catalase negative. The production of catalase is an important virulence factor because H2O2 is antimicrobial, and its degradation decreases the ability of neutrophils to kill invading bacteria. 2. Oxidase test: The enzyme cytochrome c oxidase is part of elec- + tron transport and nitrate metabolism in some bacteria. The enzyme can accept electrons from artificial substrates (such as a phenylenediamine derivative), producing a dark, oxidized product (see Figure 4.8). This test assists in differentiating between groups of gram-negative bacteria. Pseudomonas aeruginosa, for example, is oxidase positive. – – + Coagulasepositive bacteria promote clotting of serum. Coagulase test Figure 4.8 Tests commonly used in identifying bacteria. 3. Urease: The enzyme urease hydrolyzes urea to ammonia and car- bon dioxide (NH2CONH2 + H2O → 2NH3 + CO2). The ammonia produced can be detected with pH indicators that change color in response to the increased alkalinity (see Figure 4.8). The test helps to identify cer tain species of Enterobacteriaceae, Corynebacterium urealyticum, and Helicobacter pylori. 4. Coagulase test: Coagulase is an enzyme that causes a clot to form when bacteria are incubated with plasma (see Figure 4.8). The test is used to differentiate Staphylococcus aureus (coagulase positive) from coagulase-negative staphylococci. VI. Identification Of Bacteria 25
A Microbiology laboratories are increasingly using automated methods to identify bacterial pathogens. For example, in the Vitek System, small plastic reagent cards containing microliter quantities of various biochemical test media in 30 wells provide a biochemical profile that allows for organism identification (Figure 4.9). An inoculum derived from cultured samples is automatically transferred into the card, and a photometer intermittently measures color changes in the card that result from the metabolic activity of the organism. The data are analyzed, stored, and printed in a computerized database. There are many commercial variants of these automated systems and several can be used for simultaneous identification and antimicrobial susceptibility determination. Reagent wells
1 These tests measure the presence of metabolic pathways in a bacterial isolate, rather than a single enzyme. Commonly used assays include those for oxidation and fermentation of different carbohydrates, the ability to degrade amino acids, and use of specific substrates. A widely used manual system for rapid identification of members of the family Enterobacteriaceae and other gram-negative bacteria makes use of twenty microtubes containing substrates for various biochemical pathways. The test substrates in the microtubes are inoculated with the bacterial isolate to be identified, and, after 5 hours incubation, the metabolic profile of the organism is constructed from color changes in the microtubes. These color changes indicate the presence or absence of the bacteria’s ability to metabolize a particular substrate. The results are compared with a data bank containing test results from known bacteria (Figure 4.10). The probability of a match between the test organism and known pathogens is then calculated. Test inoculum enters the port and is distributed among 30 wells, each containing a different biochemical test medium. B Time 2 VI. IMMUNOLOGIC DETECTION OF MICROORGANISMS Time Color changes in wells result from metabolic activity of the organism. Figure 4.9 A. Vitek test card containing test wells. B. Color of well changes with time. In the diagnosis of infectious diseases, immunologic methods take advantage of the specificity of antigen–antibody binding. For example, All positive results ONPG ADH LDC ODC CH H2S URE TDA IND VP GEL GLU MAN INO SOR RHA SAC MEL AMY ARA ONPG ADH LDC ODC CH H2S URE TDA IND VP GEL GLU MAN INO SOR RHA SAC MEL AMY ARA All negative results Figure 4.10 Rapid manual biochemical system for bacterial identification. Different appearances of the upper and lower pairs of wells indicate the positive or negative ability of a bacterium to utilize each substrate. 26 4. Diagnostic Microbiology A fixed amount of antigen is added to test serum. If antibody is present in the serum, immune complexes will form. Antibody Immune complex
Antigen Serum without ut antibody known antigens and antibodies are used as diagnostic tools in identifying microorganisms. In addition, serologic detection of a patient’s immune response to infection, or antigenic or nucleic acid evidence of a pathogen in a patient’s body fluids, is frequently useful. Immunologic methods are useful when the infecting microorganism is difficult or impossible to isolate or when a previous infection needs to be documented. Most methods for determining whether antibodies or antigens are present in patients’ sera or other body fluids require some type of immunoassay procedure such as those described in this section. Serum with antibody Complement is then added to the mixture. If complexes are present, they will fix the complement and sequester it. Complement Immune complex These methods of identification are often rapid and show favorable sensitivity and specificity. However, unlike microbial culturing techniques, these immunologic methods do not permit further characterization of the microorganism, such as determining its antibiotic sensitivity or characteristic metabolic patterns. 1. Quellung reaction: Some bacteria having capsules can be identi- fied directly in clinical specimens by a reaction that occurs when the organisms are treated with serum containing specific antibodies (see Figure 9.10). The Quellung reaction makes the capsule more refractile and thus more visible, but the capsule does not actually swell. This method can be used for all serotypes of S. pneumoniae, H. influenzae type b, and Neisseria meningitidis groups A and C. 2. Slide agglutination test: Some microorganisms, such as RBCs and subagglutinating amounts of anti-erythrocyte antibody are added. Salmonella and Shigella species, can be identified by agglutination (clumping) of a suspension of bacterial cells on a microscopic slide. Agglutination occurs when a specific antibody directed against the microbial antigen is added to the suspension, causing cross-linking of the bacteria. B. Identification of serum antibodies Antibody against red bllod cell (RBC) Complement Co omple ement If complement has s not been bound, anti-erythrocyte antibody plus complement lyses s the RBCs. If complement was bound by immune complexes, there will be insufficient complement to lyse RBCs. Detection in a patient’s serum of antibodies that are directed against microbial antigens provides evidence for a current or past infection with a specific pathogen. A discussion of the general interpretation of antibody responses includes the following rules: 1) antibody may not be detectable early in an infection, 2) the presence of antibodies in a patient’s serum cannot differentiate between a present and a prior infection, and 3) a significant rise in antibody titer over a 10 to14-day period does distinguish between a present or prior infection. Techniques such as complement fixation and agglutination can be used to quantitate antimicrobial antibodies. 1. Complement fixation: One older but still useful method for detect- Figure 4.11 Complement fixation. ing serum antibody directed against a specific pathogen employs the ability of antibody to bind complement (Figure 4.11). A patient’s serum is first incubated with antigen specific for the suspected infectious agent, followed by the addition of complement. If the patient’s serum does contain immunoglobulin (Ig) G or IgM antibodies that target the specific antigen (indicating past or current infection), then the added complement will be sequestered in VII. Immunologic Detection Of Microorganisms an antigen–antibody–complement complex (“complement fixation”). Next, sensitized (antibody-coated) indicator sheep RBCs are added to the solution. If complement has been fixed (because the patient’s serum contained antibodies against the added antigen), then little complement will be available to bind to the antibody–RBC complexes, and the cells will not lyse. If complement has not been depleted by initial antigen–antibody complexes (because the patient’s serum does not contain antibodies to the specific antigen), the complement will bind to the antibody–RBC complexes, causing the cells to lyse. As hemolyzed RBCs release hemoglobin, the reaction can be monitored with a spectrophotometer. 27 A Antigen Bound antibody Latex bead 2. Direct agglutination: Direct bacterial agglutination testing is sometimes ordered when a suspected pathogen is difficult or dangerous to culture in the laboratory. This test measures the ability of a patient's serum antibody to directly agglutinate specific killed (yet intact) microorganisms. This test is used to evaluate patients suspected of being infected by Brucella abortus or Francisella tularensis, among others. 3. Direct hemagglutination: Antibodies directed against RBCs can arise during the course of various infections. For example, such antibodies are typically found during infectious mononucleosis caused by Epstein-Barr virus (see p. 267). When uncoated (native) animal or human RBCs are used in agglutination reactions with serum from a patient infected with such an organism, antibodies to RBC antigens can be detected. The patient’s antibodies cause the RBCs to clump. This test is, therefore, a direct hemagglutination reaction. In the case of some diseases, including pneumonia caused by Mycoplasma pneumoniae, IgM autoantibodies may develop that agglutinate human RBCs at 4oC but not at 37oC. This is termed the “cold agglutinins” test. C. Other tests used to identify serum antigens or antibodies 1. Latex agglutination test: Latex and other particles can be readily coated with either antibody (for antigen detection) or antigen (for antibody detection). Addition of antigen to antibody-coated latex beads causes agglutination that can be visually observed (Figure 4.12). For example, such methods are used to rapidly test CSF for antigens associated with common forms of bacterial or fungal meningitis. When antigen is coated onto the latex bead, antibody from a patient’s serum can be detected. Latex agglutination tests are widely used for the identification of β-hemolytic streptococci group A. 2. Enzyme-linked immunosorbent assay: Enzyme-linked immunosorbent assay (ELISA) is a diagnostic technique in which antibody specific for an antigen of interest is bound to the walls of a plastic microtiter well (Figure 4.13). Patient serum is then incubated in the wells, and any antigen in the serum is bound by the antibody on the well walls. The wells are then washed, and a second antibody is added. This one is also specific for the antigen but Particle agglutination B + – Figure 4.12 A. Schematic representation of antigens agglutinating latex beads with bound antibody. B. Photograph of agglutination reaction. 28 4. Diagnostic Microbiology 1 Antibody specific for an antigen of interest is bound to the walls of a plastic microtiter well. Antigen 2 Patient serum is incubated in the well. Any antigen in the serum is bound by the antibody on the well walls. recognizes epitopes different from those bound by the first antibody. After incubation, the wells are again washed, removing any unattached antibody. Attached to the second antibody is an enzyme, which, when presented with its substrate, produces a colored product, the intensity of the color produced being proportional to the amount of bound antigen. ELISAs can also be used to detect or quantitate antibody in a patient’s serum. In this instance, the wells are coated with antigen specific for the antibody in question. The patient’s serum is allowed to react with the bound antigen, the wells are washed, and a secondary antibody (that recognizes the initial antibody) conjugated to a color product–producing enzyme is added to the well. After a final washing, substrate for the bound enzyme is added to the well, and the intensity of the colored product can be measured. 3. Fluorescent-antibody tests: Organisms in clinical samples can be Wash wells to remove unbound antigen. Enzyme Antibody Add second, enzyme-labeled antibody specific for a different epitope on the antigen. 3 Enzyme-labeled antibody is added to the well and binds to the antigen. W h wells Wash ll tto remove unbound antibody. Add substrate for attached enzyme. Substrate 4 Product Enzyme makes colored product from added substrate. Intensity of color produced is proportional to the amount of bound antigen. Figure 4.13 Principle of enzyme-linked immunosorbent assay (ELISA). detected directly by specific antibodies coupled to a fluorescent compound such as fluorescein. In the direct immunofluorescence antibody technique, a sample of concentrated body fluid (for example, CSF or serum), tissue scraping (for example, skin), or cells in tissue culture is incubated with a fluorescein-labeled antibody directed against a specific pathogen. The labeled antibody bound to the microorganism absorbs ultraviolet light and emits visible fluorescence that can be detected using a fluorescence microscope. A variation of the technique, the indirect immunofluorescence antibody technique, involves the use of two antibodies. The first is unlabeled antibody (the target antibody), which binds a specific microbial antigen in a sample such as those described above. This clinical sample is subsequently stained with a fluorescent antibody that recognizes the target antibody. Because a number of labeled antibodies can bind to each target antibody, the fluorescence from the stained microorganism is intensified. VII. NUCLEIC ACID–BASED TESTS The most widely used methods for detecting microbial DNA fall into three categories: 1) direct hybridization (nonamplified assay), 2) amplification methods using the polymerase chain reaction (PCR)1 or one its variations, and 3) DNA microarrays. Although not likely to completely replace culture techniques in the near future, nucleic acid–based tests for the diagnosis of infectious diseases are gaining wider acceptance as more products approved by the Food and Drug Administration become commercially available. A. Direct detection of pathogens without target amplification This highly specific method of pathogen detection involves identification of the DNA of the pathogen in a patient sample or, more commonly, organisms isolated in culture. The basic strategy is to detect a relatively short sequence of nucleotide bases of DNA (target 1See INFO LINK Chapter 33 Lippincott’s Illustrated Reviews: Biochemistry for a more detailed presentation of the techniques used in molecular biology. VII. Nucleic Acid–Based Tests 29 sequence) that is unique to the pathogen. This is done by hybridization with a probe, a single-stranded piece of DNA (usually labeled with a fluorescent molecule) containing a complementary sequence of bases. [Note: In bacteria, DNA sequences coding for 16S ribosomal RNA sequences (rRNA) are commonly used targets because each microorganism contains multiple copies of its specific 16S rRNA gene, thereby increasing the sensitivity of the assay.] When the probe is bound to the target, the label will give off a signal after the free probe is washed away. A limitation of standard direct probe hybridization is the requirement for a 104 or greater number of copies of target nucleic acid for detection. B. Nucleic acid amplification for diagnosis Nucleic acid amplification overcomes the principal limitation of direct detection with nucleic acid probes by selectively amplifying specific DNA targets present in low concentrations. The bacterial 16S rRNA gene has emerged as the most useful marker for microbial detection and identification. Ribosomal DNA genes contain highly conserved areas (that are used as targets for primers) separated by internal transcribed sequences containing variable, species-specific regions. These sequences are like fingerprints. Comparing certain locations on a 16s rRNA gene with a database of known organisms allows the identification of organisms. For virus detection, primers are constructed to target highly conserved DNA or RNA sequences unique to the pathogen. Amplification and detection of the viral genomes are highly sensitive and are especially valuable when the viral load is too low to be detected by culture or when results are needed rapidly. 1. Conventional polymerase chain reaction: In this method, DNA polymerase repetitively amplifies targeted portions of DNA (ideally sequences that are highly conser ved and unique to the pathogen). Each cycle of amplification doubles the amount of DNA in the sample, leading to an exponential increase in DNA with repeated cycles of amplification. The amplified DNA sequence can then be analyzed by gel electrophoresis, Southern blotting, or direct sequence determination. 2. Real-time polymerase chain reaction: This variant of PCR com- bines nucleic acid amplification and fluorescent detection of the amplified product in the same closed automated system. Realtime PCR limits the risk of contamination and provides a rapid (30–40 minutes) diagnosis. Real-time PCR is a quantitative method and allows the determination of the concentrtion of pathogens in various samples. 3. Advantages of polymerase chain reaction: Methods employing nucleic acid–amplification techniques have a major advantage over direct detection with nucleic acid probes because amplification methods allow specific DNA or RNA target sequences of the pathogen to be amplified millions of times without having to culture the microorganism itself for extended periods. PCR also permits identification of noncultivatable or slow-growing microorganisms, such as mycobacteria, anaerobic bacteria, and viruses. Nucleic acid–amplification methods are sensitive, specific ORGANISM Bacillus anthracis Bordetella pertussis Chlamydia trachomatis Cytomegalovirus Enterovirus Epstein-Barr virus Hepatitis B virus Hepatitis C virus Herpes simplex virus Human immunodeficiency virus Human papilloma virus Methicillin-resistant Staphylococcus aureus Mycobacterium tuberculosis Neisseria gonorrhoeae SARS coronavirus Vancomycin-resistant enterococci Varicella zoster virus Variola virus West Nile virus Figure 4.14 Commercial nucleic acid– amplification systems for diagnosis of infectious diseases. The table is not intended to be all inclusive. 30 4. Diagnostic Microbiology for the target organism, and are unaffected by the prior administration of antibiotics. A 4. Applications: Nucleic acid–amplification techniques are gener- Organism to be cultured Sterile nutrient agar ally quick, easy, and accurate. A major use of these techniques is for the detection of organisms that cannot be grown in vitro or for which current culture techniques are insensitive. Moreover, they are useful in the detection of organisms that require complex media or cell cultures and/or prolonged incubation times (Figure 4.14). 5. Limitations: PCR amplification is limited by the occurrence of spu- rious false-positives due to cross-contamination with other microorganisms’ nucleic acid. PCR tests are often costly and require skilled personnel. Disks containing different antibiotics Incubate 24–48 hours (antibiotics diffuse into the agar away from the disk)
Zone of inhibition of growth Although microarrays are now routinely used to measure gene expression, the technique is an emerging technology in the diagnostic microbiology laboratory. Microarrays have the unprecedented potential to simultaneously detect and identify many pathogens from the same specimen. For example, an oligo nucleotide microarray targeting the 16S rRNA gene has been developed for the detection of a panel of forty predominant human intestinal bacterial pathogens in human fecal samples. 1. Diagnostic use of microarrays: A DNA microarray consists of No zone of inhibition nism is Organism tant to resistant microbial agent antimicrobial Large zone of inhibition Organism is sensitive to ntimicrobia agent antimicrobial B microscopic spots of immobilized DNA oligonucleotides, each containing specific DNA sequences, known as probes. The probes are constructed to be complementary to specific gene sequences of interest in suspected pathogens. DNA of the microorganism obtained from a clinical specimen, known as the target, is extracted and amplified using PCR and fluorescent labeling techniques. The target DNA is exposed to the probe microarray. If the labeled DNA from the microorganism and the immobilized probe have a complementary base sequence, they will hybridize, thereby increasing fluorescence intensity. After washing off of nonspecific bonding sequences, only strongly paired strands will remain hybridized and fluoresce. The intensity of fluorescence at each spot is a measure of the amount of that particular microbial DNA in the sample. Correlating fluorescence with the identity of the probe allows for the detection and quantitation of specific pathogens. VIII. SUSCEPTIBILITY TESTING Figure 4.15 A. Outline of disk-diffusion method for determining the sensitivity of bacteria to antimicrobial agents. B. Photograph of culture plate with antibiotic-impregnated disks. After a pathogen is cultured, its sensitivity to specific antibiotics serves as a guide in choosing antimicrobial therapy. Some pathogens, such as Streptococcus pyogenes and N. meningitidis, usually have predictable sensitivity patterns to certain antibiotics. In contrast, most gram-negative bacilli, enterococci, and staphylococcal species show unpredictable sensitivity patterns to various antibiotics and require susceptibility testing to determine appropriate antimicrobial therapy. VIII. Susceptibility Testing 31
1 Tubes containing varying concentrations of antibiotic are inoculated with test organism. 64 32 16 8 4 2 1 0.5 Relative antibiotic concentration
Lowest antibiotic concentration Highest antibiotic concentration Growth of microorganism is measured after 24 hours of incubation. 2 64 32 16 8 4 2 1 0.5 Bacterial growth No bacterial growth Minimal inhibitory concentration (MIC) is the lowest concentration of antibiotic that inhibits bacterial growth (equals 2 in this example).
10 6 Number of viable bacteria As noted above, antimicrobial drugs may be bacteriostatic or bactericidal. Bacteriostatic drugs arrest the growth and replication of bacteria at serum levels achievable in the patient, thereby limiting the spread of infection while the body’s immune system attacks, immobilizes, and eliminates the pathogens. If the drug is removed before the immune system has scavenged the organisms, enough viable organisms may remain to begin a second cycle of infection. For example, Figure 4.17 shows a laboratory experiment in which the growth of bacteria is arrested by the addition of a bacteriostatic agent. Note that viable organisms remain even in the presence of the bacteriostatic drug. By contrast, addition of a bactericidal agent kills bacteria, and the total number of viable organisms decreases. Although practical, this classification may be too simplistic because it is possible for an antibiotic to be bacteriostatic for one organism and bactericidal for another (for example, chloramphenicol is bacteriostatic against gramnegative rods and bactericidal against pneumococci). Control (no drug) 10 5 10 4 Bacteriostatic agent (such as chloramphenicol) added 10 3 10 2 Bactericidal agent (such as penicillin) added Time Addition of drug Figure 4.17 Effects of bactericidal and bacteriostatic drugs on the growth of bacteria in vitro. 32 4. Diagnostic Microbiology Study Questions Choose the ONE correct answer. 4.1 Choose the item that correctly matches the microorganism with an appropriate stain or preparation. A. Mycobacterium tuberculosis with India ink B. Fungi with KOH C. Cryptococcus neoformans in cerebrospinal fluid with Ziehl-Neelsen (classic acid-fast stain) D. Chlamydia with Gram stain E. Escherichia coli (gram-negative bacterium) with crystal violet followed by treatment with acetone 4.2 Which one of the following media is most suitable for identifying Neisseria gonorrhoeae in a cervical swab? A. B. C. D. E. Sheep blood agar Chocolate agar MacConkey agar Thayer-Martin medium Hektoen enteric agar 4.3 A 57-year-old man complains of fever, headache, confusion, aversion to light, and neck rigidity. A presumptive diagnosis of bacterial meningitis is made. Antimicrobial therapy should be initiated after which one of the following occurrences? A. Fever is reduced with antipyretic drugs. B. Samples of blood and cerebrospinal fluid have been taken. C. A Gram stain has been performed. D. The results of antibacterial drug susceptibility tests are available. E. Infecting organism(s) have been identified by the microbiology laboratory. Correct answer = B. Treatment with KOH dissolves host cells and bacteria, allowing fungi to be visualized. Mycobacterium tuberculosis is stained by the Ziehl-Neelsen stain (the classic acid-fast stain). Cryptococcus neoformans in cerebrospinal fluid is visualized with India ink. Organisms that are intracellular, such as Chlamydia, or that lack a cell wall, such as Mycoplasma or Ureaplasma, are not readily detected by Gram stain. Most bacteria stain purple with crystal violet and iodine. If the stained cells are then treated with acetone, gram-positive organisms retain the stain, whereas gram-negative species, such as Escherichia coli, lose the stain, becoming colorless. Visualization of E. coli requires the addition of the counterstain safranin, which stains gram-negative bacteria pink or red. Correct answer = D. Thayer-Martin medium, which is composed of chocolate agar supplemented with several antibiotics, suppresses the growth of nonpathogenic Neisseria and other normal and abnormal flora, but permits the growth of gonococcus. Sheep blood agar supports the growth of most bacteria, both grampositive and gram-negative. Chocolate agar provides the growth requirements for fastidious organisms such as Haemophilus influenzae or Neisseria gonorrhoeae , as well as for most other less-fastidious bacteria. MacConkey agar supports most gram-negative rods, especially the Enterobacteriaeceae, but inhibits growth of gram-positive organisms and some fastidious gram-negative bacteria, such as Haemophilus and Neisseria species. Hektoen enteric agar is also a selective medium often used to culture Salmonella and Shigella species. Correct answer = B. Bacterial meningitis is a medical emergency that requires immediate diagnosis and treatment. Specimens for possible microbial identification must be obtained before drugs are administered whenever possible. Therapy should not be delayed until laboratory results are available. Vaccines and Antimicrobial Agents 5
Yearly incidence 1,000,000 900,000 100,000 III. ACTIVE IMMUNIZATION Active immunization is achieved by injection of viable or nonviable pathogens, or purified pathogen product, prompting the immune system to respond as if the body were being attacked by an intact infectious Pertussis 10,000 1,000 Mumps 100 II. PASSIVE IMMUNIZATION Passive immunization is achieved by injecting a recipient with preformed immunoglobulins (Igs) obtained from human (or, occasionally, equine) serum. Passive immunization provides immediate protection to individuals who have been exposed to an infectious organism and who lack active immunity to that pathogen. Because passive immunization does not activate the immune system, it generates no memory response. Passive immunity dissipates after a few weeks to months as the Igs are cleared from the recipient’s serum. Two basic formulations of prepared Igs have been developed: one from the serum of pooled human donors and one from serum obtained from hyperimmune donors (Figure 5.2). Circles ( ) at upper ends of arrows indicate incidence of the disease before vaccine was available. For example, rubeola (measles) affected about 900,000 individuals during peak years. 67 10 0 Tetanus Rubella Rubeola 1 Poliomyelitis Diphtheria Arrow heads ( ) show incidence of the disease in 2009 after vaccines were in wide use. For example, rubeola (measles) affected 67 individuals. Figure 5.1 Incidence of vaccine-preventable diseases in the United States at their highest incidence and in 2009. [Note: Y axis is a logarithmic scale.] 33 34 5. Vaccines And Antimicrobial Agents STANDARD HUMAN IMMUNOGLOBULINS Hepatitis A or measles Used in individuals who may have been exposed to these viruses. HYPERIMMUNE HUMAN IMMUNOGLOBULINS Botulism Depending on the time lapse between exposure to the toxin and administration of the antitoxin, therapy can reduce the time course and severity of symptoms of the disease. Diphtheria The antibodies only neutralize toxin before its entry into cells, so it is important that the antitoxin be administered as soon as a clinical diagnosis has been made, before laboratory confirmation. Hepatitis B Used to prevent infection after exposure to hepatitis virus, for example, through contaminated blood. Rabies Used in combination with rabies vaccine to prevent rabies after a bite from a rabid animal. Tetanus Used in combination with tetanus booster vaccine to prevent tetanus after a deep puncture wound. Varicella zoster Used to prevent disseminated disease in those who are immunosuppressed and may have been exposed to the virus. Figure 5.2 Immunoglobulins used for passive immunization. microorganism. Whereas passive immunization provides immediate protection, active immunization may require several days to months to become effective. Active immunization leads to prolonged immunity and is generally preferred over the short-term immunity provided by passive immunization with preformed Igs. Simultaneous administration of active and passive immunizations may be required after exposure to certain infections such as hepatitis B. A. Formulations for active immunization Vaccines are 1) live, attenuated microorganisms; 2) killed microorganisms; 3) microbial extracts; 4) vaccine conjugates; or 5) inactivated toxins (toxoids). Both bacterial and viral pathogens are targeted by these diverse means. 1. Live pathogens: When live pathogens are used, they are attenu- ated (weakened) to preclude clinical consequences of infection. Attenuated microbes reproduce in the recipient, typically leading to a more robust and long-lasting immune response than can be obtained through vaccination with killed organisms. However, with live, attenuated vaccines, there is a possibility that the attenuated vaccine strain will revert to an active pathogen after administration to the patient. For example, vaccine-associated poliomyelitis occurs following administration of approximately 1 of every 2.4 million doses of live polio vaccine. All recent cases of polio in the United States are vaccine associated. Also, live, attenuated vaccines should not be given to immunocompromised individuals because there is the potential for a disseminated infection. 2. Killed microorganisms: Killed vaccines have the advantage over attenuated microorganisms in that they pose no risk of vaccineassociated infection. As noted above, killed organisms often provide a weak or short-lived immune response. Some vaccines, such as polio and typhoid vaccines, are available both in live and killed versions. 3. Microbial extracts: Instead of using whole organisms, vaccines can be composed of antigen molecules (often those located on the surface of the microorganism) extracted from the pathogen or prepared by recombinant DNA techniques. The efficacy of these vaccines varies. In some instances, the vaccine antigen is present on all strains of the organism, and the vaccine, thus, protects against infection by all strains. With other pathogens, such as pneumococcus, protective antibody is produced against only a specific capsular polysaccharide, one among more than 80 distinct types. Immunity to one polysaccharide type does not confer immunity to any other type. For this reason, the pneumococcal vaccine is composed of 23 different polysaccharides, comprising the antigens produced by the most common types of diseasecausing pneumococci. Some pathogens, such as influenza virus, frequently change their antigenic determinants. Therefore, influenza virus vaccines must also change regularly to counter the different antigens of influenza A and B virus strains in circulation. In the case of rhinovirus infections (the leading cause of the common cold) at least 100 types of the virus are known. It is not III. Active Immunization 35 feasible to develop a vaccine that confers protection to this large number of antigenic types. 200 0 100 0 by chemically altering the natural toxin or by engineering bacteria to produce harmless variants of the toxin. Vaccines containing toxoid are used when the pathogenicity of the organism is a result of the secreted toxin. Depending on the specific vaccine, administration is generally via intramuscular or subcutaneous routes. Figure 5.5 shows the formulation of some of the vaccines currently licensed in the United States. Details of the various vaccines are presented in the chapters in which the target microorganisms are discussed. B. Types of immune response to vaccines Vaccines containing killed pathogens (such as hepatitis A or the Salk polio vaccine) or antigenic components of pathogens (such as hepatitis B subunit vaccine) do not enter host cells, thereby eliciting a primary B cell–mediated humoral response. These antibodies are ineffective in attacking intracellular organisms. By contrast, attenuated live vaccines (usually viruses) do penetrate cells. This results in the production of intracellular antigens that are displayed on the surface of the infected cell, prompting a cytotoxic T-cell response, which is effective in eliminating intracellular pathogens. 10 0 In 2004, the incidence had declined to 1.4 per 100,000 children. 5 0 87 91 93 95 97 Year 99 01 03 Figure 5.3 Incidence of infection in children due to Haemophilus influenzae type b (Hib) following introduction of conjugate vaccine in 1987. Vaccine made from polysaccharide conjugated with protein produces higher antibody titers than vaccine containing unconjugated polysaccharide. 5,000 Antibody titer (against meningococcus) 5. Toxoids: These are derivatives of bacterial exotoxins produced Reported cases per 100,000 population 4. Vaccine conjugates: Vaccines can produce humoral immunity through B cell proliferation leading to antibody production, which may or may not involve helper T cells. For example, pneumococcal polysaccharide and the polysaccharide of Hib induce B-cell typespecific protective antibody without involvement of helper T cells. These T cell–independent responses are characterized by low antibody titers, particularly in children younger than age 18 months. Thus, conventional H. influenzae polysaccharide vaccine does not provide protection for children ages 3 to 18 months. Consequently, this organism has, in the past, produced severe infections in this age group. However, by covalently conjugating the Haemophilus polysaccharide to a protein antigen, such as diphtheria toxoid, H. influenzae vaccines produce a robust T cell–dependent antibody response even in 3-month-old infants. Figure 5.3 shows the decreased incidence of H. influenzae disease following introduction of conjugated vaccine. Conjugate vaccines are also currently available for Streptococcus pneumoniae and Neisseria meningitidis. Figure 5.4 shows the favorable antibody response to conjugated polysaccharide obtained from N. meningitidis. Before the introduction of Hib vaccine in 1987, the incidence of Hib invasive disease among children younger than age 5 years was approximately 100 per 100,000 children. 1,000 Meningococcal C conjugate Meningococcal polysaccharide Hepatitis B (control) 100 10 120°C). During storage at room temperature in the anaerobic environment provided by the foil, spores germinate, and toxin forms. Heating at 100°C would kill most C. botulinum because the bacterium is in its vulnerable, vegetative state. Heat would also inactivate toxin produced during room-temperature storage. However, any remaining spores would not be killed. 7 Bacterial Genetics I. OVERVIEW Because a single type of molecule, DNA, is the genetic material of all cellular organisms from bacteria to humans, basic genetic phenomena (that is, gene mutation, gene replication, and gene recombination) are much the same for all life forms. The prototypic organism used in microbial genetic studies for the past 50 years is the enteric, gram-negative Escherichia coli (see p. 111). An aspect of microbial genetics of great clinical importance is the ability of bacteria to transfer genes, especially genes for antibiotic resistance, to other bacteria both within and between species. Such transfer allows the flow of antibiotic resistance genes from nonpathogenic bacterial populations to pathogenic populations, as well as between pathogens, with potentially dire consequences for public health. CHROMOSOME Circular, double-stranded DNA 3,000 genes (3,000 kilobases) Single copy per cell Highly folded in cell • • • • II. THE BACTERIAL GENOME Cell wall The genome of an organism is defined as the totality of its genetic material. For bacteria, the genome often consists of a single chromosome that carries all of the essential genes and one or more varieties of plasmid that generally carry nonessential genes (Figure 7.1). A. The chromosome All of the essential genes and many nonessential genes of the bacterium are generally carried on a single, long piece of circular, double-stranded DNA. This molecular structure is called the “chromosome” by analogy with the heredity-carriers of eukaryotic cells. Most bacteria have chromosomes that contain 2,000 to 4,000 genes. B. Pathogenicity islands Pathogenicity islands are discrete genetic elements that encode virulence factors, such as toxins, adhesins, secretion systems, and iron transport proteins. These islands, which range in size from 10 to BACTERIUM PLASMID double-stranded DNA • Circular, genes (5–100 kilobases) • 5–100 • 1–20 copies per cell Figure 7.1 The bacterial genome. [Note: Helical double-stranded DNA is shown as two concentric circles.] 59 60 7. Bacterial Genetics Attachment of phage to cell surface receptor Bacteriophage 0 Cell wall of bacterium 2 MI S 1 NUTE Injection of phage DNA into cell 200 kB, can be horizontally transferred between bacteria, resulting in enhanced virulence and fitness in the recipient. [Note: Horizontal gene transfer is any process (such as transformation, transduction, or bacterial conjugation) in which an organism incorporates genetic material from another organism without being the progeny of that organism. By contrast, vertical transfer occurs when an organism receives genetic material from its ancestor, for example, a species from which it has evolved.] Pathogenicity islands differ from the rest of the chromosome in G+C content and are usually flanked in the recipient's chromosome by repeated sequence elements or genes that encode tRNAs. C. Plasmids DNA 1 4 2 5 III. BACTERIOPHAGE Assembly of new phage particles 5 Complete phage particle MIN U TE S Phage proteins S 1 0 MIN U TE Lysis of cell and release of progeny phage 3 0 S 3 MIN U TE Synthesis of new phage DNA and phage proteins Typically, bacteria contain small DNA circles (plasmids), which range in size from 1.5 kilobase (kb) pairs to 120 kb pairs (less than one tenth the size of the bacterial chromosome). Plasmids replicate independently of the chromosome and can exist in the cell as one copy or as many copies. Plasmids can carry genes that encode toxins or proteins that promote the transfer of the plasmid to other cells but usually do not include genes that are essential for cell growth or replication. Many plasmids contain mobile DNA sequences (transposons) that can move between plasmids and between plasmids and the chromosome (see pp. 63–64). Transposons, the repository for many antibiotic resistance genes, are responsible for the ability of some plasmids to integrate into the chromosome. MIN U TE Figure 7.2 Bacteriophage replication. Clock indicates total elapsed time starting with attachment at t = 0. [Note: Bacterial chromosome and plasmid are not shown.] A bacteriophage (phage) is a virus that replicates inside a bacterial cell. It consists of nothing more than a piece of nucleic acid encapsulated in a protective protein coat. Depending on the phage, the nucleic acid can be DNA or RNA, double stranded or single stranded, and range in size from about 3,000 bases (3 genes) to about 200,000 bases (200 genes). The typical replicative cycle (Figure 7.2) begins with attachment of the phage to receptors on the cell surface, followed by injection of the nucleic acid into the bacterial cell, leaving all or most of the protein outside the cell. [Note: This is in contrast to viral infection of vertebrate cells, in which the entire virus is taken up by the cell, and its nucleic acid released intracellularly (see pp. 236–237).] The phage nucleic acid encodes proteins that take over the cell’s biosynthetic machinery to replicate the phage's own genetic material and to synthesize phagespecific proteins. When sufficient coat proteins and new phage DNA have accumulated, these components self-assemble into mature phage particles, with the DNA encapsulated by the phage coat. Release of new phage particles is accomplished by a phage-specific enzyme (lysozyme) that dissolves the bacterial cell wall. The number of phage particles in a sample can be determined by a simple and rapid plaque assay. If a single phage particle is immobilized in a confluent bacterial lawn growing on a nutrient agar surface, this phage, within a few hours, will produce millions of progeny at the expense of neighboring bacterial cells, leaving a visible “hole” or plaque in the otherwise opaque lawn (Figure 7.3). Phage are classified as virulent or temperate, depending on the nature of their relationship to the host bacterium. IV. Gene Transfer 61
Mixture of 108 uninfected bacterial cells and a single phage-infected cell in melted agar. Solidification of top agar layer immobilizes all cells. A bacterium infected with a temperate phage can have the same fate as a bacterium infected with a virulent phage (lysis rapidly following infection). However, an alternative outcome is also possible: Namely, after entering the cell, the phage DNA, rather than replicating autonomously, can integrate into the chromosome of the host cell. In this state (prophage), the expression of phage genes is repressed indefinitely by a regulatory protein encoded within the phage genome. No new phage particles are produced, the host cell survives, and the phage DNA replicates as part of the host's chromosome. C. Lysogenic bacteria Lysogenic bacteria carry a prophage. This phenomenon is termed “lysogeny,” and the bacterial cell is said to be “lysogenized.” Nonlysogenic bacteria can be made lysogenic by infection with a temperate phage. The association of prophage and bacterial cell is highly stable but can be destabilized by various treatments, such as exposure to ultraviolet light, that damage the host DNA. When DNA damage occurs, repression of phage genes is lifted, and the prophage excises from the host chromosome, replicates autonomously, and produces progeny phage particles. The host cell is lysed just as with a virulent phage. The emergence of the virus from its latent prophage state is called induction. The acquisition by bacteria of properties due to the presence of a prophage is called lysogenic conversion. 12 hours Phage replicate in the infected cell. The cell lyses, releasing progeny that infect adjacent cells. These cells lyse, and the cycle repeats. IV. GENE TRANSFER Genes can be transferred from one bacterial cell to another by three distinct mechanisms: conjugation, transduction, and transformation. Because some types of transferred DNA do not contain an origin of replication, these genes will only be passed on to succeeding generations if the transferred DNA becomes incorporated into the recipient chromosome, which has an origin of replication. Plasmids contain their own origin of replication and can, therefore, be maintained in a host through subsequent generations without being integrated into the chromosome. A. Conjugation Conjugation is the process by which bacteria transfer genes from one cell to another by cell-to-cell contact. The donor (male) and recipient (female) cells must have the proper genetic constitution to adhere to each other, and they form a cytoplasmic bridge between Top view of agar plate Growth of uninfected bacteria creates an opaque lawn except at the location of the original infected cell, where there is a plaque (hole) containing millions of phage. Figure 7.3 Visual detection of bacteriophage by the plaque method. 62 7. Bacterial Genetics 1 An F+ and an F– cell make contact and form a cytoplasmic bridge. F Plasmid F+ CELL F – CELL 2 One strand of the plasmid is nicked. 5' the cells through which DNA can pass. Specifically, the process requires the presence on the donor cell of a hairlike projection called a sex pilus that makes contact with a specific receptor site on the surface of the recipient cell. This contact results in the formation of a relatively stable cell pair and the initiation of DNA transfer (Figure 7.4). B. Transduction Transduction refers to transfer of genes from one cell to another via a phage vector without cell-to-cell contact. There are two ways in which this can occur: generalized transduction and specialized transduction. In each case, the transducing phage is a temperate phage, so that the recipient cell survives the phage infection. 3' 1. Generalized transduction: In generalized transduction, a random 3 Strand elongation at the 3' end displaces the 5' end into the bridge. 5' 2. Specialized transduction: In specialized transduction, only certain 3' 4 fragment of bacterial DNA is accidentally encapsulated in a phage protein coat in place of the phage DNA (Figure 7.5A). When this rare phage particle infects a recipient cell, it injects the bacterial DNA fragment into the cell. If this fragment becomes integrated into the recipient chromosome by recombination, the recipient cell will be stably transduced. The single strand is cut after one complete circle is transferred. At the same time, the complementary plasmid strand is synthesized in the F – cell. bacterial genes, located on the bacterial chromosome in close proximity to the prophage insertion site of the transducing phage, are transduced (Figure 7.5B). The phage acquires the bacterial genes by a rare, abnormal excision from the bacterial chromosome. A specialized transducing phage particle contains both phage and bacterial DNA joined together as a single molecule. After infecting another cell, this joint molecule integrates into the recipient chromosome just as phage DNA normally does in the process of becoming a prophage. C. Transformation 3' 5 F+ CELL 5' The ends are ligated, restoring the doublestranded circular configuration. F+ CELL Figure 7.4 Cell-to-cell transfer of a conjugative plasmid (chromosomal DNA is not shown). Transformation is the transfer of genes from one cell to another by means of naked DNA. The discovery of transformation in 1928, one of the most important in all of biology, led eventually to the identification of DNA as the genetic material. Studies of the transformation phenomenon itself revealed that the ability of a cell to be transformed (called competence) depends on a physiologic state of the cell that allows DNA to cross the cell membrane. As free, doublestranded DNA enters the recipient cell, one of the two strands is destroyed by nucleases. The remaining single strand invades the resident chromosome, seeking a region of sequence homology. If such a sequence is found, the invading strand replaces one of the two resident strands by a complex cut-and-paste process.
A 63 Generalized Transduction Infecting phage B Specialized Transduction UV induction Bacterial chromosome a+ Integrated prophage Phage DNA Phage replication and fragmentation of bacterial DNA (a+ = any bacterial gene) s+ Normal excision of prophage Rare abnormal excision of prophage picks up the adjacent s+ gene (s+ = special bacterial gene) Phage DNA a+ s+ a+ s+ Lysis s+ Lysis a+ These are normal, nontransducing phage. s+ This rare phage, which has accidentally packaged the a+ gene, can transduce an a– cell to a+. Lysis s+ These are normal, nontransducing phage. s+ s+ s+ These phage, which carry the s+ gene, can transduce an s– cell to s+. Figure 7.5 Certain phage can package bacterial genes and transfer them to other bacteria (transduction). By one mechanism (A) any bacterial gene can be transferred. By a second mechanism (B) only certain genes can be transferred, namely those in close proximity to a prophage. A. Mutations Strictly speaking, any change in the structure of genetic material or, more specifically, any change in the base sequence of DNA, is called a mutation. Some mutations are unstable (that is, they frequently revert back to their original state), and others do not noticeably affect the organism. Mutations that come under study are usually those that are stable and that cause some change in the characteristics of the organism. Mutations can be classified according to the kind of chemical change that occurs in the DNA or, when the mutation affects a protein-coding gene, by the effect the mutation has on the translation of the message. B. Mobile genetic elements In recent years, it has been recognized that the arrangement of genes in the genome of bacteria (and probably all organisms) is not entirely static. Certain DNA segments, called transposons, have the ability to move from place to place on the chromosome and into and out of plasmids. Transposons do not exist as segments free of the genome but only as segments within the genome. There are two 64 7. Bacterial Genetics PLASMID CHROMOSOME 2 1 Transposon can jump into and out of a plasmid. Transposon at this original site can jump to other sites, leaving behind a copy of itself. TRANSPOSON abc Left inverted repeat Transposase Resolvase Antibiotic resistance These two genes control the movement of the transposon. cba Right inverted repeat Figure 7.6 A replicative transposon can move from place to place in the chromosome, leaving a copy of itself behind at the previous site. general types of transposons: replicative and nonreplicative. A replicative transposon leaves a copy of itself at the original location. Thus, the transposition process doubles the number of copies of the transposon. A nonreplicative transposon does not leave a copy of itself at the original location. If transposition inserts a transposon into a functional gene, the function of the gene is generally destroyed (this was the original basis by which transposons were discovered). Transposons can, thus, be viewed as internal mutagenic agents. The transposition process and the structure of a typical replicative transposon are shown in Figure 7.6. This transposon has three genes and a length of about five kilobases. The transposase and resolvase genes code for enzymes involved in the transposition process, whereas the antibiotic resistance gene is a “passenger.” The transposon is bounded by short (about 50 bases), inverted repeats. These inverted repeats are the elements recognized by the transposase as it initiates the transposition. Mobile genetic elements are probably responsible for most of the genetic variability in natural bacterial populations and for the spread of antibiotic resistance genes. C. Mechanisms of acquired antibiotic resistance Acquired antibiotic resistance requires a temporary or permanent gain or alteration of bacterial genetic information. Although most resistance genes are plasmid mediated, plasmid-mediated traits can interchange with chromosomal elements. Transfer of genetic material from plasmid to chromosome can occur by simple recombinational events, but the process is greatly facilitated by transposons. Many resistance genes, such as plasmid-mediated β-lactamases, tetracycline-resistance genes, and aminoglycoside-modifying enzymes, are organized on transposons. Resistance to antibiotics is accomplished by five principal mechanisms, three of which are shown in Figure 7.7. VI. Gene Regulation 65 1. Decreased uptake of antibiotic: Gram-negative organisms can 2. Antibiotic efflux: Some gram-negative organisms encode multi- component membrane-imbedded efflux systems that recognize and pump out diverse, toxic substances, including detergents and antibiotics. Expression of these systems is generally tightly regulated and often induced by the presence of substrates recognized by the pump. Antibiotic target Antibiotic-sensitive cell limit the penetration of certain agents, including β-lactam antibiotics, tetracyclines, and chloramphenicol, as a result of alteration in the number and structure of porins (proteins that form channels) in the outer membrane. Ab Ab 3. Alteration of the target site for antibiotic: Streptococcus pneumo- DECREASED PERMEABILITY niae resistance to β-lactam antibiotics, for example, involves alterations in one or more of the major bacterial penicillin-binding proteins (see p. 75), which results in decreased binding of the antibiotic to its target. 4. Acquisition of the ability to destroy or modify the antibiotic: 5. Acquisition of a new target: Some Staphylococcus aureus iso- lates, for example, are vancomycin resistant due to expression of newly acquired genes that modify the D-ala-D-ala (the target of vancomycin) residues on the stem peptide, converting them instead to D-ala-D-lac. Although this new target is effectively polymerized to form a peptidoglycan network with sufficient stability, D-ala-D-lac is not bound by vancomycin, and, therefore, the antimicrobial agent is no longer effective. Ab Antibiotic-resistant cells Examples of antibiotic inactivating enzymes include: 1) β-lactamases that hydrolytically inactivate the β-lactam ring of penicillins, cephalosporins, and related drugs; 2) acetyltransferases that transfer an acetyl group to the antibiotic, inactivating chloramphenicol or aminoglycosides; and 3) esterases that hydrolyze the lactone ring of macrolides. Ab ALTERATION OF TARGET Ab Ab INACTIVATION OF ANTIBIOTIC Ab VI. GENE REGULATION Many bacteria can manufacture most of the organic compounds (such as amino acids, nucleotides, carbohydrates, lipids) that they need, and, in this regard, they are more versatile than higher organisms. This metabolic resourcefulness is a distinct advantage when the organism is in a nutritionally poor environment but is extremely wasteful in a nutritious environment if the bacterium must keep all of the unneeded biosynthetic enzymes ready. Therefore, bacteria have evolved various mechanisms for producing certain metabolic enzymes only when they are needed. Most regulation in bacteria involves control of transcription, rather than control of translation of the mRNA into protein. The following classical example describes the mechanisms that regulate expression of the lac operon in E. coli. A. Negative control (repression) Lactose is a disaccharide composed of glucose and galactose. The first step in lactose metabolism is cleavage into monosaccharide units, a job performed by the enzyme β-galactosidase. To avoid being Ab Figure 7.7 Three common mechanisms of antibiotic resistance. 66 7. Bacterial Genetics A Negative control Repressor gene Promoter Operator Lac operon genes i P O z y a i P O z y a Binding of repressor to the operator prevents expression of lac operon genes. Repressor i P O z y a Transcription of lac mRNA When the inducer binds to the repressor, it inactivates it, allowing lac operon gene expression. Inducer (lactose) B Positive control Operon OFF (because repressor is active) i P O z y a Operon OFF (because repressor is active; CAP is irrelevant) i P O z y a P O z y a CAP–cAMP complex Operon ON (because repressor is inactive and CAP is active) i Transcription of lac mRNA CAP–cAMP complex serves as an activator of lac operon gene expression in the absence of repressor. Operon OFF (because CAP is inactive) i P O z y a Figure 7.8 Bacterial genes can be controlled negatively by repressors or positively by activators. CAP = catabolite activator protein; cAMP = cyclic adenosine monophosphate.
67 68 7. Bacterial Genetics Study Questions Choose the ONE correct answer 7.1 A lysogenic bacterium A. B. C. D. E. carries a prophage. causes lysis of other bacteria on contact. cannot support the replication of a virulent phage. is often a human pathogen. is usually not capable of conjugal genetic transfer. 7.2 Which one of the following statements concerning plasmids is true? A. All plasmids can be transfered between bacteria by conjugation. B. Much of the information coded in the plasmid is essential to the survival of the bacteria cell. C. Resistance plasmids carry genes for antibiotic resistance. D. Resistance plasmids cannot be transferred to other bacterial cells. E. Plasmids lack an origin of replication. 7.3 What occurs when a temperate bacteriophage enters a state called “lysogeny”? A. B. C. D. E. Most viral genes are expressed. The bacterial cell is lysed. Many new viruses are produced. Most normal bacterial functions are turned off. The virus may become integrated into the host genome. 7.4 A virulence factor can be transferred from one strain of bacteria to another in a genetic process that is independent of cell-to-cell contact between the donor and the recipient. Addition of DNase does not interfere with the transfer of the virulence factor either. From these characteristics, which of the following processes is involved in this genetic transfer? A. B. C. D. E. Conjugation Transformation Transduction Transposition Transversion Correct answer = A. A lysogenic bacterium can generate phage because it carries phage genes in a latent state (prophage). Lysogeny does not impart any special lytic properties to the bacterium nor, in general, does it affect conjugal transfer or the ability to support the replication of other unrelated phage. The presence of a prophage can convert certain bacteria to human pathogens, but such cases are rare. Correct answer = C. Plasmids are small, circular, supercoiled DNA molecules found in some bacteria. They usually do not carry essential genes, but some plasmids, such as R (resistance) plasmids, carry genes coding for antibiotic resistance. All plasmids have their own origin of replication, so that they are replicated along with the host chromosome and passed along to progeny cells. Only some plasmids possess genes that allow for transmittal to other bacteria by the process of conjugation. Correct answer = E. There are two types of bacteriophages: lytic and temperate. The distinction is made according to the life cycle of the bacteriophage. On entering a bacterium, lytic phages produce phage nucleic acids and proteins, assemble many new phage particles, lyse the cell, and release the progeny phage. Temperate phages, however, can penetrate the bacterium and enter a dormant state called lysogeny, in which most viral genes are repressed. Bacterial functions remain active and the bacterium is not harmed. Some dormant phages replicate as plasmids; others, such as phage λ (lambda), become integrated into the host genome as prophages. The prophage DNA is replicated along with the host DNA as the bacterium grows and divides. Correct answer = C. Transduction is the process by which genetic material is transferred from donor to recipient within a bacteriophage. This process does not require cell-to-cell contact and is resistant to DNase. Conjugation requires cellto-cell contact between the donor and receipient cells. Transformation involves the exchange of naked DNA between donor and recipient in the absence of cell-to cell contact. However, DNA transformation is sensitive to DNase treatment. Transposition is the process in which a transposon excises from one location and integrates in another location within the same bacterial cell. Thus this process would not explain the transfer of a genetic marker between different bacterial cells. Transversion is not a means of genetic exchange. 8 Staphylococci I. OVERVIEW Staphylococci and streptococci (see Chapter 9) constitute the main groups of medically important gram-positive cocci. Staphylococcal infections range from the trivial to the rapidly fatal. They can be very difficult to treat, especially those contracted in hospitals, because of the remarkable ability of staphylococci to become resistant to antibiotics. Staphylococci are ubiquitous in nature, with about a dozen species occurring as part of human flora. The most virulent of the genus, Staphylococcus aureus, is one of the most common causes of bacterial infections, and is also an important cause of food poisoning and toxic shock syndrome. Among less virulent staphylococcal species, Staphylococcus epidermidis is an important cause of prosthetic implant infections, whereas Staphylo coccus saprophyticus causes urinary tract infections, especially cystitis in women. Figure 8.1 summarizes the staphylococci described in this chapter. Free-living Bacteria Gram-positive Cocci Gram-negative Rods Staphylococcus Staphylococcus aureus S Staphylococcus epidermidis S Staphylococcus saprophyticus S Enterococcus Peptostreptococcus II. GENERAL FEATURES Staphylococci generally stain darkly gram positive (Figure 8.2). They are round rather than oval and tend to occur in bunches like grapes. Because growth of staphylococci requires supplementation with various amino acids and other growth factors, they are routinely cultured on enriched media containing nutrient broth and/or blood (see p. 23). Staphylococci are facultatively anaerobic organisms. They produce catalase, which is one feature that distinguishes them from the catalase-negative streptococci. The most virulent species of staphylococcus is S. aureus, almost all isolates of which secrete coagulase, an enzyme that causes citrated plasma to clot. Other species that occasionally cause disease and lack coagulase are often referred to as coagulasenegative staphylococci. Staphylococci are hardy, being resistant to heat and drying, and thus can persist for long periods on fomites (inanimate objects), which can then serve as sources of infection. Frequent handwashing before and after contact with food or potentially infected individuals decreases the transmission of staphylococcal disease. Streptococcus Figure 8.1 Classification of Staphylococci. S See pp. 349–350 for summaries of these organisms. Figure 8.2 Gram stain of Staphylococcus aureus. 70 8. Staphylococci III. STAPHYLOCOCCUS AUREUS Infection S. aureus disease may be largely or wholly the result of actual invasive infection. Colonization Infection
Intoxication S. aureus disease may be largely or wholly the result of toxins in the absence of infection (“pure” toxicoses, such as food poisoning). Toxin S. aureus Infection and intoxication Generally, significant host compromise is required for S. aureus infection, such as a break in the skin or insertion of a foreign body (for example, wounds, surgical infections, or central venous catheters), an obstructed hair follicle (folliculitis), or a compromised immune system. S. aureus disease may be: 1) largely or wholly the result of actual invasive infection, overcoming host defense mechanisms, and the production of extracellular substances which facilitate invasion; 2) a result of toxins in the absence of invasive infection (“pure” toxinoses); or 3) a combination of invasive infection and intoxication (Figure 8.3). A. Epidemiology
Toxin Virulence factors are the genetic, biochemical, or structural features that enable an organism to produce disease. The clinical outcome of an infection depends on the virulence of the pathogen and the opposing effectiveness of the host defense mechanisms. S. aureus expresses many potential virulence factors (Figure 8.4). [Note: Coagulase activity results in localized clotting, which restricts access by polymorphonuclear neutrophils (PMNs) and other immune defenses. This would make coagulase a virulence factor, even though mutants lacking the ability to make this factor remain virulent in animal models]. For the majority of diseases caused by S. aureus, pathogenesis depends on the combined actions of several virulence factors, so it is difficult to determine precisely the role of any given factor. 1. Cell wall virulence factors: a. Capsule: Most clinical isolates express a polysaccharide Figure 8.3 Causes of disease resulting from infection with Staphylococcus aureus. “microcapsule” of Types 5 or 8. The capsule layer is very thin but has been associated with increased resistance to phagocytosis. Clinical isolates produce capsule but expression is rapidly lost upon in vitro cultivation. b. Protein A: Protein A is a major component of the S. aureus cell wall. It binds to the Fc region of IgG, exerting an anti-opsonin (and therefore strongly antiphagocytic) effect. c. Fibronectin-binding protein: Fibrinectin-binding protein (FnBP) and other staphylococcal surface proteins promote binding to mucosal cells and tissue matrices. d. Clumping factor: This FnBP enhances clumping of the organ- isms in the presence of plasma. 71 III. Staphylococcus aureus 2. Cytolytic exotoxins: α, β, γ, and δ Toxins attack mammalian cell (including red blood cell) membranes, and are often referred to as hemolysins. α Toxin is the best studied, and is chromosomally encoded. It polymerizes into tubes that pierce membranes, resulting in the loss of important molecules and, eventually, in osmotic lysis. 3. Panton-Valentine leukocidin: This pore-forming toxin lyses PMNs. Production of this toxin makes strains more virulent. This toxin is produced predominantly by community-acquired methicillin-resistant S. aureus (MRSA) strains (see p. 74). 4. Superantigen exotoxins: These toxins have an affinity for the T- cell receptor–major histocompatibility complex Class II antigen complex. They stimulate enhanced T-lymphocyte response (as many as 20 percent of T cells respond, compared with 0.01 percent responding to the usual processed antigens). This difference is a result of their ability to recognize a relatively conserved region of the T-cell receptor. This major T-cell activation can cause toxic shock syndrome, primarily by release into the circulation of inordinately large amounts of T-cell cytokines, such as interleukin-2 (IL2), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α). SUPERANTIGEN EXOTOXINS have an affinity for the T cell • Toxins receptor–MHC Class II antigen complex. stimulate an enhanced T• Toxins lymphocyte response. activation can cause toxic • T-cell shock by release of large amounts of T-cell cytokines. Antigenpresenting cell T cell Excess cytokines IL-2 IFN-γ TNF-α T Superantigen Protein A binds to the Fc moiety of IgG, exerting an antiphagocytic effect. IgG
D, E, and G) are produced by approximately half of all S. aureus isolates. When these bacteria contaminate food and are allowed to grow, they secrete enterotoxin, ingestion of which can cause food poisoning. [Note: The toxin stimulates the vomiting center in the brain by binding to neural receptors in the upper gastrointestinal (GI) tract.] Enterotoxins are superantigens that are even more heat-stable than S. aureus . Therefore, organisms are not always recovered from incriminated food but the toxin may be recovered. b. Toxic shock syndrome toxin (TSST –1): This is the classic cause of toxic shock syndrome (TSS). Because of similarities in molecular structure, it is sometimes referred to as staphylococcal enterotoxin F, although it does not cause food poisoning when ingested. Staphylococcus Fibronectin Fibronectin-binding proteins promote binding to mucosal cells and tissue matrices. ENZYMES • Coagulase • Catalase • Hyaluronidase • Fibrinolysin SLIME PRODUCTION (particularly S. epidermidis)
causes scalded skin syndrome in children. The toxin cleaves desmoglein 1, which is a component of desmosomes (cell structures specialized for cell-to-cell adhesion). Cleavage results in loss of the superficial skin layer. C. Clinical significance
CYTOLYTIC EXOTOXINS Exotoxins attack mammalian cell (including red blood cell) membranes and are often referred to as hemolysins. Figure 8.4 Virulence factors that may play a role in the pathogenesis of staphylococcal infections. MHC = major histocompatibility complex; IL = interleukin; IFN = interferon; TNF = tumor necrosis factor; IgG = immunoglobulin G. 72 8. Staphylococci SKIN AND SOFT TISSUE INFECTIONS • Furuncles, carbuncles • Wound infection (traumatic, surgical) • Cellulitis • Impetigo RESPIRATORY • Pneumonia complication of influenza. The localized host response to staphylococcal infection is inflammation, characterized by swelling, accumulation of pus, and necrosis of tissue. Fibroblasts and their products may form a wall around the inflamed area, which contains bacteria and leukocytes. This creates a characteristic pus-filled boil or abscess. Serious consequences of staphylococcal infections occur when the bacteria invade the bloodstream. The resulting septicemia (the presence and persistence of pathogenic microorganisms or their toxins in the blood) may be rapidly fatal. Bacteremia (the presence of viable bacteria circulating in the bloodstream) may result in seeding internal abscesses, skin lesions, or infections in the lung, kidney, heart, skeletal muscle, or meninges. 1. Localized skin infections: The most common S. aureus infections MUSCULOSKELETAL • Osteomyelitis • Arthritis CARDIOVASCULAR • Endocarditis • Septicemia GENITOURINARY TRACT • Renal carbuncle • Lower urinary tract infection DISEASES CAUSED BY TOXINS • Toxic shock syndrome (TSS) • Scalded skin syndrome • Food poisoning (gastroenteritis) Figure 8.5 Diseases caused by Staphylococcus aureus. are small, superficial abscesses involving hair follicles (folliculitis) or sweat or sebaceous glands (see Figure 8.12). For example, the common sty (external hordeolum) is created by infection of an eyelash follicle. Subcutaneous abscesses called furuncles (boils) often form around foreign bodies such as splinters. These generally respond to local therapy, that is, removal of the foreign body, soaking, and drainage as indicated. Carbuncles are larger, deeper, multiloculated skin infections that can lead to bacteremia and require antibiotic therapy and debridement. Impetigo is usually a localized, superficial, spreading crusty skin lesion generally seen in children. It can be caused by S. aureus, although more commonly by Streptococcus pyogenes (see p. 80), or both organisms together. Human staphylococcal infections usually remain localized at the portal of entry by normal host defenses. 2. Deep, localized infections: These may be metastatic from superfi- cial infections or skin carriage or may result from trauma. S. aureus is the most common cause of acute and chronic infection of bone marrow. S. aureus is also the most common cause of acute infection of joint space in children (septic joint). [Note: Septic joints are medical emergencies because pus can rapidly cause irreparable cartilage damage. They must be treated promptly with drainage and an antibiotic.] 3. Acute endocarditis: Generally associated with intravenous drug abuse, acute endocarditis is caused by injection of contaminated preparations or by needles contaminated with S. aureus . S. aureus also colonizes the skin around the injection site, and if the skin is not sterilized before injection, the bacteria can be introduced into soft tissues and the bloodstream, even when a sterilized needle is used. An abscess in any organ or tissue is cause to suspect S. aureus, although many other bacteria can cause abscesses. 4. Septicemia is a generalized infection with sepsis or bacteremia that may be associated with a known focus (for example, a septic joint) or not (an occult focus). 5. Pneumonia: S. aureus is a cause of severe, necrotizing pneumonia. III. Staphylococcus aureus 73 6. Nosocomial infections: S. aureus is one of the most common causes of hospital-associated infections, often of wounds (surgical, decubital) or bacteremia associated with catheters (see Figure 8.10). Progression to septicemia is often a terminal event. 7. Toxinoses: These are diseases caused by the action of a toxin, frequently when the organism that secreted the toxin is undetectable. Toxinoses caused by S. aureus include: a. Toxic shock syndrome: TSS results in high fever, rash (resem- bling a sunburn, with diffuse erythema followed by desquamation), vomiting, diarrhea, hypotension, and multiorgan involvement (especially GI, renal, and/or hepatic damage). An outbreak of TSS occurred in the late 1970s among menstruating women. It was shown to be related to the use of hyperabsorbant tampons by women who happened to be vaginally colonized by toxic shock syndrome toxin–(TSST)–positive strains of S. aureus. [Note: These tampons stimulated TSST expression, resulting in entry of the toxin into the circulation in the absence of true infection.] The incidence has decreased markedly since such tampons were removed from the market. Of the few cases of TSS that occur currently, approximately half are associated with ordinary S. aureus infections. Of the remainder, many result from a circulating enterotoxin rather than TSST. Figure 8.6 shows the desquamation (peeling or scaling of the skin) seen in TSS. Toxic shock syndrome is characterized by fever, hypotension, multisystem organ dysfunction, and an erythematous rash with desquamation occurring during convalescence.
food contaminated with enterotoxin-producing S. aureus. Often contaminated by a food handler, these foods tend to be protein rich (for example, egg salad or cream pastry) or salty, like ham (S. aureus is salt tolerant), and improperly refrigerated. These heat-resistant toxins are able to withstand subsequent reheating. Symptoms, such as nausea, vomiting, and diarrhea, are acute following a short incubation period (less than 6 hours) and are triggered by local actions of the toxin on the GI tract rather than from infection. See p. 372 for a summary of foodborne illness. The short incubation period of staphylococcal food poisoning occurs because the toxin in the food has already been formed by the staphylococci before the food is ingested. c. Scalded skin syndrome: This involves the appearance of superficial bullae resulting from the action of an exfoliative toxin that attacks the intercellular adhesive of the stratum granulosum, causing marked epithelial desquamation (see Figure 8.12). The bullae may be infected or may result from toxin produced by organisms infecting a different site. D. Laboratory identification Identification of an isolate as a staphylococcus relies largely on microscopic and colony morphology and catalase positivity (Figure Figure 8.6 Desquamation of skin in toxic shock syndrome. 74 8. Staphylococci The test for catalase is performed by removing a colony to a slide with a drop of 3% hydrogen peroxide. Catalase-positive cultures produce O2 bubbles. Here it is demonstrated more dramatically directly on a plate. 8.7). Bacteria stain strongly gram-positive, and are frequently seen in grapelike clusters (see Figure 8.2). S. aureus is distinguished from the coagulase-negative staphylococci primarily by coagulase positivity. In addition, S. aureus colonies tend to be yellow (hence “aureus,” meaning golden) and hemolytic (see Figure 8.12), rather than gray and nonhemolytic like the coagulase-negative staphylococci. S. aureus is also distinguished from most coagulase-negative staphylococci by being mannitol-positive. E. Immunity
Percent resistant 1. Hospital-acquired methicillin-resistant S. aureus (MRSA): In 70 0 1997 1999 2001 2003 2005 Year Figure 8.8 Trends in the prevalence of methicillin resistant strains of Staphylococcus aureus. recent decades, a high percentage (often in the range of 50 percent) of hospital S. aureus isolates has been found to be also resistant to methicillin or oxacillin. Antibiotic resistance is caused by chromosomal acquisition of the gene for a distinct penicillinbinding protein (PBP, see p. 57), PBP-2a. This protein codes for a new peptidoglycan transpeptidase with a low affinity for all currently available β-lactam antibiotics, and thus renders infections with MRSA unresponsive to β-lactam therapy. Compared with methicilllin-sensitive S. aureus, MRSA infections are associated with worse outcomes, including longer hospital and intensive care unit stays, longer durations of mechanical ventilation, and higher mortality rates. MRSA strains are also frequently resistant to many other antibiotics, some being sensitive only to glycopeptides such as vancomycin. 2. Community-acquired MRSA (CA-MRSA): Community acquired MRSA infections were documented in the mid-1990s, occurring in individuals who had no previous risk factors for MRSA infections, such as exposure to hospital. The most common clinical manifestations of CA-MRSA are skin and soft tissue infections such as abscesses or cellulitis (Figure 8.9). Less commonly, CA-MRSA can also cause severe diseases such as necrotizing pneumonia, osteomyelitis, and septicemia. Community-acquired MRSA has a III. Staphylococcus aureus DRUG 75 HA-MRSA (Hospital strain) CA-MRSA (Community strain) Characteristics of patients Patients are typically elderly, debilitated, and/or chronically ill. Patients are typically young and healthy. Children students, athletes, and military service personnel are at risk. Infection site Bacteremia commonly occurs with no obvious infection site. Infection of surgical wounds, open ulcer, intravenous line, and urinary catheters often occur. Infections often occur in skin and soft tissues, producing cellulitis and abscesses. Infections include necrotizing community pneumonia, septic shock, and bone and joint infections. Transmission Transmission occurs within health care settings. Only rarely is transmission among household contacts. Medical history Infections more likely in patients with a history of MRSA infections, recent surgery, admission to a hospital or nursing home. Antibiotic use, dialysis and permanent indwelling catheters are risk factors. Virulence of infecting strain Spread of infection in the community is limited. PVL genes are usually absent. Antibiotic susceptibility Multidrug antibiotic resistance often occurs, resulting in a limited choice of effective therapeutic agents. Transmission occurs in the community. May spread in families, sport teams, and other risk groups. Patients show no significant medical history or health care contact. Spread of infection in the community readily occurs. PVL genes are often present, predisposing to necrotising soft tissue or lung infections. CA-MRSA strains are often more virulent than HA-MRSA, but they tend to be susceptible to a broader array of antibiotics. Figure 8.9 Comparison of hospital-acquired methicillin-resistant Staphylococcus aureus (HA-MRSA) with community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). PVL = Panton-Valentine leukocidin. number of characteristics that help distinguish it from hospitalassociated MRSA. For example, CA-MRSA has a characteristic pattern of DNA fragments obtained upon enzymic cleavage and electrophoresis, and it produces specific toxins. CA-MRSA also exhibits a unique antibiotic resistance pattern, that is, CA-MRSA is sensitive to many antibiotics that do not show much activity against hospital-associated MRSA. These antibiotics include ciprofloxacin and clindamycin, with some CA-MRSA even sensitive to erythromycin, gentamicin, rifampin, tetracycline, and/or trimethoprim -sulfamethoxazole. Emerging antibiotic-resistant strains of S. aureus that infect otherwise healthy individuals (community-acquired infections) are often more virulent than the more common strains that originate in hospitals. 3. Vancomycin resistance: Vancomycin has been the agent of choice for empiric treatment of life-threatening MRSA S. aureus infections. Unfortunately, in 1997, several MRSAs were isolated that had also acquired low-level vancomycin resistance. The incidence of vancomycin resistance has increased steadily, prompting the use of alternative drugs such as quinupristin-dalfopristin, linezolid, and daptomycin. These agents have good in vitro activity against MRSA and most other clinically important gram-positive bacterial pathogens. 76 8. Staphylococci G. Prevention There is no effective vaccine against S. aureus. Infection control procedures, such as barrier precautions and disinfection of hands and fomites, are important in the control of nosocomial S. aureus epidemics. IV. COAGULASE-NEGATIVE STAPHYLOCOCCI Of 12 coagulase-negative staphylococcal species that have been recovered as normal commensals of human skin and anterior nares, the most abundant and important is S. epidermidis. For this reason some clinical laboratories designate all coagulase-negative staphylococci as S. epidermidis, a practice that is not encouraged. The second most important coagulase-negative staphylococcus is S. saprophyticus , which has a special medical niche. Coagulase-negative staphylococcal species are important agents of hospital-acquired infections associated with the use of implanted prosthetic devices and catheters. A. Staphylococcus epidermidis Figure 8.10 Staphylococcus epidermidis attached by its biofilm and growing on the surface of a cathether.
Species Frequency of disease Coagulase Color of colonies Mannitol fermentation Novobiocin resistance
Common + Golden yellow + –
Common – White – –
Occasional – Variable – + Figure 8.11 Summary of various species of staphylococci. IV. Coagulase-Negative Staphylococci 77 Gram (+) cocci • Catalase (+) Nonmotile Colonies are yellow • • Do not form spores cocci tending to occur • Round in bunches like grapes anaerobic • Facultative organisms on enriched media • Cultured containing broth and/or Staphylococcus species Staphylococcus aureus • Skin and soft • Septicemia tissue infections • Necrotizing pneumonia • Osteomyelitis • Toxic shock syndrome • Septic arthritis • Food poisoning • Endocarditis (antibiotic therapy not used) blood Staphylococcus aureus on blood agar surrounded by zone of β hemolysis. Staphylococcus aureus cultured from a wound infection Methicillin susceptible 1 Oxacillin 1 Nafcillin Staphylococcus epidermidis • Infections of catheters Methicillin resistant Staphylococcus saprophyticus • Cystitis in women and heart valves (health-care associated) Ciprofloxacin 1 Oxacillin 1 Vancomycin 1 Nafcillin Methicillin resistant (community-acquired; mild-moderate infection) 2 Vancomycin2 1Most isolates resistant to penicillin G 2Used in methicillin-resistant isolates 1 Trimthoprim/ sulfmethoxazole 1 Doxycycline Methicillin resistant (community-acquired; severe infection) 1 Daptomycin 1 Linezolid 1 Vancomycin 2 Quinupristindalfopristin 2 Teicoplanin Carbuncle caused by Staphylococcus aureus Note: Treatment of MRSA may vary by the type and location of infection. Staphylococcal scalded skin syndrome Folliculitis caused by Staphylococcus aureus Figure 8.12 Summary of staphylococcal disease. 1 Indicates first-line drugs; 2 Furuncle caused by Staphylococcus aureus Superficial impetigo indicates alternative drugs. 78 8. Staphylococci Study Questions Choose the ONE best answer 8.1 A 32-year-old woman became ill 4 days after the onset of her menstrual period. She presented in the emergency room with fever (104°F; normal = 98.6°F), elevated white blood cell count (16,000/mm3; normal = 4,000 to 10,000/mm3), and an erythematous, sunburnlike rash on her trunk and extremities. She complained of fatigue, vomiting, and diarrhea. She had recently eaten at a fast-food restaurant, but otherwise had prepared all her meals at home. The patient described most likely has: A. B. C. D. E. 8.2 staphylococcal food poisoning. scalded skin syndrome. infection with a Staphylococcus saprophyticus. chickenpox. toxic shock syndrome. A 57-year-old man arrives at the emergency room complaining of weakness, fatigue, and intermittent fever that has recurred for several weeks. The patient had a cardiac valvular prosthesis implanted 5 years earlier. Physical examination reveals petechiae (pinpoint, nonraised, purplish red spots caused by intradermal hemorrhage) on the chest and stomach. Blood cultures grew catalase-positive, coagulasenegative, cocci. The gram-positive organisms failed to ferment mannitol, and their growth was inhibited by novobiocin. What is the most likely infectious agent? A. B. C. D. E. Correct answer = E. The patient shows signs of toxic shock syndrome. Toxic shock syndrome as defined in the outbreak of the late ‘70s and early ‘80s included an erythematous/peeling rash (not purpuric) and was caused by overproduction of toxic schock syndrome toxin (TSST)-1 by colonizing S. aureus triggered by something in hyperabsorbent tampons. Many signs and symptoms are the results of the superantigen activity of TSST, which activates a whole subclass of T cells, causing overproduction of cytokines. S. saprophyticus is a frequent cause of cystitis in women, but is not associated with toxic shock syndrome. Staphylococcus aureus Staphylococcus epidermidis Staphylococcus saprophyticus Streptococcus pneumoniae Streptococcus agalactiae 8.3. An 18-month-old child was brought the pediatrician's office with what appeared to be a sunburn, although the parents denied that the child had been over exposed to the sun. The parents did recall seeing an area of redness and small blisters on the child's arm the night before. Which of the following virulence factors is critical to this disease manifestation? A. Toxic shock syndrome toxin. B. Panton-Valentine Leukocidin C. Protein A D. Capsule E. Exfoliatin Correct answer = B. The patient is probably suffering from bacterial endocarditis caused by S. epidermidis infection of the prosthetic heart valve. S. epidermidis is a coagulase-negative organism that is unable to ferment mannitol and is sensitive to novobiocin but usually resistant to penicillin. Patients with congenital heart malformations, acquired valvular defects (for example, rheumatic heart disease), prosthetic valves, and previous bacterial endocarditis show an increased incidence of bacterial endocarditis. Intravenous drug users also have a high risk for infection. S. pneumoniae and S. agalactiae can be ruled out, because streptococci are catalase negative, which is a feature that distinguishes them from catalase-positive staphyococci. Correct answer = E. Exfoliatin is a virulence factor, produced by some Staphylococcus aureus strains, cleaves desmosomes, resulting in loss of the outer layers of skin. This manifestation is also known as scalded skin syndrome. The toxic shock syndrome toxin is a superantigen produced by some S. aureus strains. This toxin causes systemic effects and has been associated with tampon use. PantonValentine Leukocidin is a hemolysin that lyses white blood cells and is produced by many communityacquired MRSA strains. Protein A is a virulence factor that allows S. aureus to evade an immune response by binding the Fc region of IgG, resulting in the inverse orientation of the antibody. Thus, the antibody cannot effectively opsonize the bacterium. The thin microcapsule of S. aureus is also associated with immune evasion. 9 Streptococci I. OVERVIEW Streptococci and staphylococci (see Chapter 8) constitute the main groups of medically important gram-positive cocci. Streptococci are grampositive, nonmotile, and catalase negative. Clinically important genera include Streptococcus and Enterococcus (Figure 9.1). They are ovoid to spherical in shape and occur as pairs or chains (see Figure 9.16). Most are aerotolerant anaerobes because they grow fermentatively even in the presence of oxygen. Because of their complex nutritional requirements, blood enriched medium is generally used for their isolation. Diseases caused by this group of organisms include acute infections of the throat and skin caused by group A streptococci (Streptococcus pyogenes); female genital tract colonization, resulting in neonatal sepsis caused by group B streptococci (Streptococcus agalactiae); pneumonia, otitis media, and meningitis caused by Streptococcus pneumoniae; and endocarditis caused by the viridans group of streptococci. II. CLASSIFICATION OF STREPTOCOCCI Streptococci can be classified by several schemes, for example, by the hemolytic properties of the organisms, and according to the presence of specific surface antigens determined by immunologic assays. A. Hemolytic properties on blood agar α-Hemolytic streptococci cause a chemical change in the hemoglobin of red cells in blood agar, resulting in the appearance of a green pigment that forms a ring around the colony (see Figure 9.16). β-Hemolytic streptococci cause gross lysis of red blood cells, resulting in a clear ring around the colony (see Figure 9.16). γ-Hemolytic is a term applied to streptococci that cause no color change or lysis of red blood cells. The traditional division of streptococci based on the ability of the bacterial colony to hemolyze erythrocytes in the blood agar medium is still considered the first step in the classification of streptococci. Free-living Bacteria Gram-positive Gram-negative Cocci Rods Staphylococcus Streptococcus Streptococcus agalactiae S Streptococcus bovis Streptococcus mutans Streptococcus pneumoniae Streptococcus pyogenes S S Enterococcus Enterococcus faecium S Enterococcus fecalis S Figure 9.1 Classification of streptococci. S See pp. 350–351 for summaries of these organisms.
79 80 9. Streptococci stance. The clinically most important groups of β-hemolytic streptococci are Types A and B (Figure 9.2). Commercial kits in which groupspecific antisera are coupled to latex beads are now widely used for identification of β-hemolytic streptococci. Streptococci Lancefield groups Others A B C D... T U α Streptococcus bovis or γ β Streptococcus agalactiae Streptococcus pyogenes β Streptococcus pneumoniae α α Streptococcus mutans or γ III. GROUP A β-HEMOLYTIC STREPTOCOCCI S. pyogenes, the most clinically important member of this group of grampositive cocci, is one of the most frequently encountered bacterial pathogens of humans worldwide. It can invade apparently intact skin or mucous membranes, causing some of the most rapidly progressive infections known. A low inoculum suffices for infection. Some strains of S. pyogenes cause postinfectious sequelae, including rheumatic fever and acute glomerulonephritis. Nasopharyngeal carriage is common especially in colder months and particularly among children. Unlike staphylococcal species, S. pyogenes does not survive well in the environment. Instead, its habitat is infected patients and also normal human carriers in whom the organism resides on skin and mucous membranes. S. pyogenes is usually spread person to person by skin contact and via the respiratory tract. Key: α = α-Hemolytic β = β-Hemolytic γ = γ-Hemolytic α or = α or γ hemolytic γ Figure 9.2 Classification schemes for streptococci.
N-Terminus tive tissue, forms the outermost layer of the cell. This capsule is not recognized as foreign by the body and, therefore, is nonimmunogenic. The capsule is also antiphagocytic. 2. Cell wall: The cell wall contains a number of clinically important M Protein components. Beginning with the outer layer of the cell wall, these components include the following (Figure 9.3): a. M protein: S. pyogenes is not infectious in the absence of M Fimbria Cell wall C-Terminus Cell membrane Figure 9.3 Schematic representation of the streptococcal M protein. protein. M proteins extend from an anchor in the cell membrane, through the cell wall and then the capsule, with the Nterminal end of the protein exposed on the surface of the bacterium. M proteins are highly variable, especially the N-terminal regions, resulting in over 80 different antigenic types. Thus, individuals may have many S. pyogenes infections throughout their lives as they encounter new M protein types for which they have no antibodies. M proteins are antiphagocytic and they form a coat that interferes with complement binding. b. Group A-specific C-substance: This component is composed of rhamnose and N-acetylglucosamine. [Note: All group A streptococci, by definition, contain this antigen.] c. Protein F (fibronectin-binding protein) mediates attachment to fibronectin in the pharyngeal epithelium. M proteins and lipoteichoic acids also bind to fibronectin. III. Group A, β-Hemolytic Streptococci 3. Extracellular products: Like Staphylococcus aureus (see p. 70),
81 Streptococcus pyogenes Cytokines Pyrogenic exotoxins
Cause various effects, including the rashes seen in scarlet fever and streptococcal toxic shock disease.
Streptolysin O Streptolysin S Damage mammalian cells, resulting in cell lysis and release of lysosomal enzymes. Fibrin clot Plasmin Streptokinase Catalyzes conversion of plasminogen to plasmin, causing lysis of clots, facilitating the rapid spread of organisms. C5a
C5 C5a C5a peptidase
Inactivates complement component C5a. 1. Acute pharyngitis or pharyngotonsilitis: Pharyngitis is the most common type of S. pyogenes infection. S. pyogenes pharyngitis (“strep throat”) is associated with severe, purulent inflammation of the posterior oropharynx and tonsillar areas (see Figure 9.16). [Note: If a sunburnlike rash develops on the neck, trunk, and extremities in response to the release of pyrogenic exotoxin to which the patient does not have antibodies, the syndrome is designated scarlet fever.] Many strep throats are mild, and many sore throats caused by viruses are severe. Hence, laboratory confirmation is important for accurate diagnosis and treatment of streptococcal pharyngitis, particularly for the prevention of subsequent acute rheumatic fever and rheumatic heart disease. 2. Impetigo: Although S. aureus is recovered from most contemporary cases of impetigo (see p. 72), S. pyogenes is the classic cause of this syndrome. The disease begins on any exposed surface (most commonly, the legs). Typically affecting children, it can cause severe and extensive lesions on the face and limbs (see Figure 9.16). Impetigo is treated with a topical agent such as mupirocin, or systemically with penicillin or a first-generation cephalosporin such DNA Streptodornases DNAses that degrade the viscous DNA in necrotizing tissue or exudates, aiding the spread of infection. Hyaluronic acid Hyaluronidase Disrupts the organization of ground substance, facilitating the spread of infection. Figure 9.4 Cytolytic toxins and other exoenzymes produced by Streptococcus pyogenes. 82 9. Streptococci 1 Day 0: Right lower leg was edematous with an erythematous area below the knee. as cephalexin, which are effective against both S. aureus and S. pyogenes. 3. Erysipelas: Affecting all age groups, patients with erysipelas experience a fiery red, advancing erythema, especially on the face or lower limbs (see Figure 9.16). 4. Puerperal sepsis: This infection is initiated during, or following soon after, the delivery of a newborn. It is caused by exogenous transmission (for example, by nasal droplets from an infected carrier or from contaminated instruments) or endogenously, from the mother’s vaginal flora. This is a disease of the uterine endometrium in which patients experience a purulent vaginal discharge and are systemically ill. 2 3 Day 2: Initial debridement revealed necrotic tissue with many layers of thrombosed blood vessels. Day 6: Radical débridement was performed because the infectious process was progressing toward the knee. Subsequent skin grafts (not shown) took well, and the wound healed without complications. Figure 9.5 Necrotizing fasciitis in a 59-year-old woman. 5. Invasive group A streptococcal disease: Common during the first half of the century, invasive group A streptococcal (GAS) disease became rare until its resurgence during the past decade. Patients may have a deep local invasion either without necrosis (cellulitis) or with it (necrotizing fasciitis/myositis) as shown in Figure 9.5. [Note: The latter disease led to the term “flesh-eating bacteria.”] Invasive GAS disease often spreads rapidly, even in otherwise healthy individuals, leading to bacteremia and sepsis. Symptoms may include a toxic shock–like syndrome, fever, hypotension, multiorgan involvement, a sunburnlike rash, or a combination of these symptoms. 6. Streptococcal toxic shock syndrome: This syndrome is defined as isolation of group A β-hemolytic streptococci from blood or another normally sterile body site in the presence of shock and multiorgan failure. The syndrome is mediated by the production of streptococcal pyrogenic exotoxins that function as superantigens causing massive, nonspecific T-cell activation and cytokine release. Patients may initially present with flulike symptoms, followed shortly by necrotizing soft tissue infection, shock, acute respiratory distress syndrome, and renal failure. Treatment must be prompt and includes antistreptococcal antibiotics, usually consisting of high-dose penicillin G plus clindamycin. 7. Post-streptococcal sequelae a. Acute rheumatic fever: This autoimmune disease occurs 2 to 3 weeks after the initiation of pharyngitis. It is caused by crossreactions between antigens of the heart and joint tissues, and the streptococcal antigen (especially the M protein epitopes). It is characterized by fever, rash, carditis, and arthritis. Central nervous system manifestions are also common including Sydenham's chorea, symptoms of which are uncontrolled movement and loss of fine motor control. Rheumatic fever is preventable if the patient is treated within the first 10 days following onset of acute pharyngitis. b. Acute glomerulonephritis: This rare, postinfectious sequela occurs as soon as 1 week after impetigo or pharyngitis III. Group A, β-Hemolytic Streptococci 83 ensues, due to a few nephritogenic strains of group A streptococci. Antigen–antibody complexes on the basement membrane of the glomerulus initiate the disease. There is no evidence that penicillin treatment of the streptococcal skin disease or pharyngitis (to eradicate the infection) can prevent acute glomerulonephritis. E. Laboratory identification Rapid latex antigen kits for direct detection of group A streptococci in patient samples are widely used. In a positive test, the latex particles clump together, whereas in a negative test, they stay separate, giving the suspension a milky appearance (Figure 9.6). These tests have high specificity but variable sensitivity compared with culture techniques. Specimens from patients with clinical signs of pharyngitis and a negative antigen detection test should undergo routine culturing for streptococcal identification. Depending on the form of the disease, specimens for laboratory analysis can be obtained from throat swabs, pus and lesion samples, sputum, blood, or spinal fluid. S. pyogenes forms characteristic small, opalescent colonies surrounded by a large zone of β hemolysis on sheep blood agar (see Figure 9.16). [Note: Hemolysis of the blood cells is caused by steptolysin S, which damages mammalian cells resulting in cell lysis.] This organism is highly sensitive to bacitracin, and diagnostic disks with a very low concentration of the antibiotic inhibit growth in culture. S. pyogenes is also catalase negative and optochin resistant. Group A C-substance can be identified by the precipitin reaction. Serologic tests detect a patient’s antibody titer to streptolysin O (ASO test) after group A streptococcal infection. Anti-DNase B titers (ADB test) are particularly elevated following streptococcal infections of the skin. Positive Negative
IV. GROUP B β-HEMOLYTIC STREPTOCOCCI Group B streptococci, represented by the pathogen S. agalactiae, are gram-positive, catalase-negative organisms. S. agalactiae is found in Figure 9.6 Latex agglutination for identification of group A β-hemolytic streptococci. 84 9. Streptococci Figure 9.7 Streptococcus pneumoniae are gram-positive, nonmotile, encapsulated, lancet-shaped cocci. Severity of disease Prevalence of disease (Estimated number of cases per year) Most Meningitis 1,400 17,000 Bacteremia Pneumonia 71,000 5–7 Million Otitis media Least Figure 9.8 Comparison of severity and prevalence of some pneumococcal infections in children in the United States. the vaginocervical tract of female carriers, and the urethral mucous membranes of male carriers as well as in the gastrointestinal (GI) tract. S. agalactiae can be transmitted sexually among adults and from an infected mother to her infant at birth. Group B streptococci are a leading cause of meningitis and septicemia in neonates, with a high mortality rate. They are also an occasional cause of infections in postpartum women (endometritis) and individuals with impaired immune systems, in whom the organism may cause septicemia or pneumonia. Samples of blood, cervical swabs, sputum, or spinal fluid can be obtained for culture on blood agar. Latex agglutination tests can also demonstrate the presence of group B antigen in these samples. Group B streptococci are β hemolytic, with larger colonies and less hemolysis than group A. Most isolates remain sensitive to penicillin G and ampicillin, which are still the antibiotics of choice (see Figure 9.16). In life-threatening infections, an aminoglycoside can be added to the regimen. [Note: Pregnant carriers should be treated with ampicillin during labor if risk factors such as premature rupture of membranes or prolonged labor are present.] Intrapartum prophylaxis of group B streptococcal carriers and administration of antibiotics to their newborns reduce neonatal group B streptococcal sepsis by as much as 90 percent.
85
Streptococcus pneumoniae Autolysin Aut 1. Capsule: The S. pneumoniae polysaccharide capsule is both antiphagocytic and antigenic. Antiphagocytic properties of the capsule protect the bacteria from polymorphonuclear leukocyte attack, facilitating growth of the bacteria prior to the appearance of anticapsular antibodies. There are approximately 85 distinct capsular serotypes, some of which endow strains with greater virulence than others, as reflected by the fact that about 20 serotypes account for the vast majority of pneumococcal infections. occus pneumoniae pne Streptococcus (lysed) neumolysi Pneumolysin 2. Pili: Pili enable the attachment of encapsulated pneumococci to the epithelial cells of the upper respiratory tract. Not all pneumococci are piliated, but those clinical isolates that express pili are more virulent. The genes required for regulation and assembly of the pilus are not present in all pneumococcal strains, but they can be horizontally transferred between strains on a pathogenicity “islet”, which is small pathogenicity island. The chromosomal region responsible for production of the pneumococcal pilus is called the rlrA islet, named for the regulatory gene (rlrA) that is required for expression. 3. Choline-binding protein A: Choline binding protein A is a major Damages mammalian cell membranes Figure 9.9 Cytolytic toxins produced by Streptococcus pneumoniae. adhesin allowing the pneumococcus to attach to carbohydrates on epithelial cells of the human nasopharynx. 4. Autolysins: Autolysins are enzymes that hydrolyze the components 5. Pneumolysin: Although retained within the cytosol of intact pneu- mococci, pneumolysin is thought to be an important virulence factor by virtue of its ability to attack mammalian cell membranes, causing lysis once it is released by autolysin from the interior of the bacterium. Pneumolysin binds to cholesterol and therefore interacts indiscriminately with all cell types. This toxin stimulates production of proinflammatory cytokines, inhibits the activity of polymorphonuclear leukocytes and activates complement. C. Clinical significance Streptococcus pneumoniae is a major cause of community-acquired pneumonia, particularly in older adults. 120 Cases per 100,000 population of a biological cell in which it is produced. LytA, B and C are peptidoglycan-hydrolyzing enzymes that are present in the bacterial cell wall and are normally inactive. However, these enzymes are readily activated (for example, by surface-active agents, β-lactam antibiotics, or stationary phase), resulting in cell lysis. Autolysin is thus responsible for the release of intracellular virulence factors (notably, pneumolysin). Streptococcus pneumoniae 100 Mycoplamsma pneumoniae 80 60 Legionella species 40 Chlamydia pneumoniae 20 0 18–34 35–49 50–64 65–79 \>80 Age group (yr) 1. Acute bacterial pneumonia: A leading cause of death, especially in older adults and those whose resistance is impaired, this disease is caused most frequently by S. pneumoniae (Figure 9.10). Pneumonia is frequently preceded by an upper or middle respiratory viral infection, which predisposes to S. pneumoniae infection Figure 9.10 Age-specific rates of communityacquired pneumonia caused by specific pathogens. 86 9. Streptococci Inhibition by optochin Growth of colonies of Streptococcus pneumoniae is inhibited by optochin contained in the disk applied to the blood agar plate. of pulmonary parenchyma. Mechanisms by which virus infection predisposes an individual to streptococcal pneumonia include increased volume and viscosity of secretions that are more difficult to clear and secondary inhibition of the action of bronchial cilia by viral infection. 2. Otitis media: The most common bacterial infection of children, this disease (which is characterized by earache) is most frequently caused by pneumococcus, followed by Haemophilus influenzae and Moraxella catarrhalis (see p. 391). The traditional empiric treatment of pneumococcal otitis media with a β-lactam antibiotic (with or without a penicillinase-inhibitor) has been threatened by the spread of penicillin-resistant pneumococci. A 3. Bacteremia/sepsis: In the absence of a focus of infection bac- Optochin disk Quellung reaction Capsules of Streptococcus pneumoniae swell in the presence of specific pneumococcal antiserum. B teremia/sepsis is commonly caused by pneumococcus, especially in individuals who are functionally or anatomically asplenic. This includes people with sickle cell disease who infarct their spleen and are functionally asplenic, although they still have a remnant of anatomical spleen. 4. Meningitis: H. influenzae was formerly the leading cause of bacte- rial meningitis in the United States. After a vaccine was developed against this organism, S. pneumoniae became the most common cause of adult bacterial meningitis (see p. 376). This disease has a high mortality rate, even when treated appropriately. D. Laboratory identification Negative Positive C Specimens for laboratory evaluation can be obtained from a nasopharyngeal swab, blood, pus, sputum, or spinal fluid. α-Hemolytic colonies appear when S. pneumoniae is grown on blood agar overnight under aerobic conditions at 37oC. Lancet-shaped, grampositive diplococci are observed on a Gram stain of the sample. Growth of these bacteria is inhibited by low concentrations of the surfactant optochin, and the cells are lysed by bile acids (Figure 9.11). Capsular swelling is observed when the pneumococci are treated with type-specific antisera (the Quellung reaction). E. Treatment Negative Positive Lysis by bile acids Bile acids, such as sodium deoxycholate, dissolve Streptococcus pneumoniae and clear the turbidity of a heavy inoculum of organisms. Figure 9.11 Laboratory tests useful in the identification of Streptococcus pneumoniae.
VI. Enterococci 87
There are two types of pneumococcal vaccine: pneumococcal polysaccharide vaccine (PPV) and pneumococcal conjugate vaccine (PCV13). 1. Pneumococcal polysaccharide vaccine: Introduced in the United 2. Pneumococcal conjugate vaccine 13: The polyvalent PCV 13, licensed in the United States in 2010, is effective in infants and toddlers (ages 6 weeks to 5 years). It is made up of 13 pneumococcal antigens conjugated to CRM197, a mutant nontoxic diphtheria toxin. Significant declines in the incidence of invasive pneumococcal disease occurred as a result of introduction of this and an earlier generation heptavalent conjugated vaccine (PCV 7). (Figure 9.12). In addition, the vaccines prevented greater numbers of invasive pneumococcal cases through indirect effects on pneumococcal transmission (that is, herd immunity) than through its direct effect of protecting vaccinated children. Young children do not elicit an immune response to oligosaccharide-only vaccines. However, if the oligosaccharide is conjugated to a protein, a protective immune response develops. 100 Cases s per 100,000 population States in 1983, PPV immunizes against 23 serotypes of S. pneumoniae and is indicated for the protection of high-risk individuals older than age 2 years. This vaccine protects against the pneumococcal strains responsible for 85 to 90 percent of infections, including prominent penicillin-resistant strains. PCV7 target g population 80 1998–1999) 2000 2001 2002 2003 60 40 20 0 65 Age group (yr) Figure 9.12 Incidence of vaccine-type invasive pneumococcal disease before and after the introduction of pneumococcal conjugate vaccine (PCV7), by age and year. VI. ENTEROCOCCI Enterococci contain a C-substance that reacts with group D antisera. Therefore, in the past, they were considered group D streptococci. Today, DNA analysis and other properties have placed them in their own genus, Enterococcus. The clinically most important species are E. faecalis and E. faecium. Enterococci can be α-, β-, or nonhemolytic. As a rule, enterococci are not very virulent, but they have become prominent as a cause of nosocomial infections as a result of their multiple antibiotic resistance. Figure 9.13 shows the microscopic appearance of E. faecalis. A. Epidemiology Enterococci are part of the normal fecal flora. However, they can also colonize oral mucous membranes and skin, especially in hospital settings. These organisms are highly resistant to environmental and chemical agents and can persist on fomites. B. Diseases Enterococci seldom cause disease in normal, healthy individuals. However, under conditions in which host resistance is lowered or the integrity of the gastrointestinal or genitourinary tract has been Figure 9.13 Enterococcus fecalis showing chain formation characteristic of Streptococcus. 88 9. Streptococci 1 Ampicillin1 1 Gentamicin1 2 Vancomycin2 Quinupristin + 2 dalfopristin3 2 Linezolid3 1 Many isolates show resistance to combination therapy with β-lactam plus an aminoglycoside. 2 Many isolates show resistance to vancomycin. 3 Used to treat infection with vancomycinresistant infections with Enterococcus faecium. Figure 9.14 Antimicrobial agents useful in treating infections caused by enterococci. disrupted (for example, by instrumentation), enterococci can spread to normally sterile sites, causing urinary tract infections, bacteremia/sepsis, endocarditis, biliary tract infection, or intra-abdominal abscesses. C. Laboratory identification Enterococci are distinguished from the non–group D streptococci by their ability to survive in the presence of bile, and to hydrolyze the polysaccharide esculin, producing black colonies on esculin-containing plates. Unlike nonenterococcal group D streptococci, enterococci grow in 6.5 percent NaCl, and yield a positive pyrazin amidase (PYR) test. E. faecalis can be distinguished from E. faecium by their fermentation patterns, which are commonly evaluated in clinical laboratories D. Treatment Enterococci are naturally resistant to β-lactam antibiotics and aminoglycosides, but are sensitive to the synergistic action of a combination of these classes. In the past, the initial regimens of choice were penicillin +/– streptomycin or ampicillin +/– gentamicin (Figure 9.14). However, acquired resistance determinants in many current strains negate this synergy. In addition, isolates frequently have natural or acquired resistances to many other antibiotic classes, including glycopeptides such as vancomycin. Newer antibiotics, such as the combination of quinupristin and dalfopristin, are used to treat vancomycin-resistant infections. However, some enterococcal strains are resistant to all commercially available antibiotics. [Note: E. faecium is more likely to be vancomycin or multiply resistant than E. faecalis.] E. Prevention The rise of nosocomial infections by multiple drug–resistant enterococci is largely the result of selection due to high antibiotic usage in hospitals. Judicious use of antibiotics is an important factor in controlling the emergence of these infections. VII. NONENTEROCOCCAL GROUP D STREPTOCOCCI Figure 9.15 Streptococcal endocarditis showing vegetation of the mitral valve leaflet. [Note: Vegetation is a tissue outgrowth composed of fibrin, bacteria, and aggregated blood platelets adherent to a diseased heart valve.] Streptococcus bovis is the most clinically important of the nonenterococcus group D streptococci. Part of normal fecal flora, they are either α− or nonhemolytic. S. bovis occasionally causes urinary tract infections and endocarditis, the latter especially in association with colon cancer. The organism is bile and esculin positive, but is PYR-negative, and does not grow in 6.5 percent salt (unlike the enterococci). It tends to be sensitive to penicillin and other antibiotics. VIII. VIRIDANS STREPTOCOCCI The viridans group of streptococci includes many gram-positive, catalase-negative, α− or γ−hemolytic species that constitute the main facultative oral flora. The viridans streptococci are relatively avirulent, but VIII. Viridans Streptococci 89 Gram (+) cocci Ovoid to spherical in • shape, occurring as pairs Streptococcus species or chains Nonmotile, catalase • negative Most are aerotolerant • anaerobes because the grow fermentatively even in the presence of oxygen • Culture on blood agar α-Hemolytic streptococci on blood agar β-Hemolytic streptococci on blood agar Streptococcus pyogenes (Gram stain) Streptococcus pyogenes (group A, β-hemolytic) Streptococcus agalactiae (group B, β-hemolytic) and septicemia in neonates • Meningitis Endometritis • Septicemia or pneumonia • in individuals with impaired pharyngitis or • Acute pharyngotonsillitis rheumatic fever • Acute • Erysipelas sepsis • Puerperal group A streptococcal • Invasive disease • immune systems Diabetic foot infections • Acute bacterial pneumonia • Otitis media • Meningitis 1 Penicillin G6 1 Cefotaxime 1 Penicillin G1,2 1 Penicillin G4 2 Clarithromycin3 2 Azithromycin3 2 An aminoglycoside5 1 Ceftriaxone 2 Vancomycin7 1S. pyogenes has not acquired resistance to penicillin G. 2Clindamycin may be added to pencillin G for soft tissue infection such as necrotizing fasciitis. 3For penicillin-allergic patient. 4All isolates remain sensitive to penicillin G and ampicillin. 5In life-threatening infections, an aminoglycoside can be added to the regimen. Facial erysipelas Figure 9.16 Summary of streptococcal disease. Streptococcus pneumoniae (α-hemolytic) Impetigo 1 6Penicillin G has been the drug of choice, but resistant strains are regularly seen. 7Most resistant strains remain sensitive to vancomycin. Streptococcal pharyngitis Indicates first-line drugs; 2 indicates alternative drugs. 90 9. Streptococci Streptococcus mutans and other members of the viridans group cause dental caries. In patients with abnormal or damaged heart valves, they can also infect these valves during a bacteremia, causing endocarditis (Figure 9.15). Therefore, at-risk patients with rheumatic, congenital, or sclerotic valvular disease should receive prophylactic penicillin before undergoing dental procedures. Figure 9.16 summarizes streptococcal disease. Study Questions Choose the ONE correct answer 9.1 Which of the following statements is correct? A. Streptococci are catalase positive. B. Growth of Streptococcus pneumoniae is not sensitive to optochin. C. Streptococcus pyogenes is highly sensitive to bacitracin. D. Streptococci are obligate anaerobes. E. Enterococcus faecalis is β-hemolytic. Correct answer = C. Streptococcus pyogenes is highly sensitive to bacitracin, and diagnostic disks with a very low concentration of the antibiotic inhibit growth in culture. All streptococci are catalase negative. The growth of Streptococcus pneumoniae is inhibited by optochin. Most streptococci are aerotolerant anaerobes, and grow fermentatively even in the presence of oxygen. Enterococcus faecalis is γ hemolytic (no hemolysis). 9.2 A 55-year-old man was admitted to a local hospital with fever and chills. The patient was human immunodeficiency virus positive and had received multiple courses of antibiotics. Blood cultures grew gram-positive cocci, which tested positive with group D streptococcal antisera. The isolate was resistant to penicillin and vancomycin. Which one of the following is the most likely pathogen? A. B. C. D. E. Streptococcus pneumoniae Enterococcus faecium Streptococcus pyogenes Streptococcus agalactiae Streptococcus mutans 9.3 A 65-year-old male presents to his family physician with a rapid onset fever, chest pain and cough productive of rusty-yellow sputum. Chest X-ray shows focal lobar infiltrates. A Gram stain of a sputum sample contained many polymorphonuclear leukocytes and extracellular gram-positive diplococci. Capsule-specific antibodies bound to the diplococci resulted in a positive Quellung reaction. Which of the following is the most likely pathogen? A. Streptococcus pneumoniae B. Enterococcus faecium C. Streptococcus pyogenes D. Streptococcus agalactiae E. Enterococcus faecalis Correct answer = B. Enterococcus faecium is most likely to be vancomycin or multiply drug-resistant. The other organisms are sensitive to vancomycin. Correct answer = A. The most common cause of community acquired pneumonia in this age group is Streptococcus pneumoniae . The X-ray and microbiological findings are most consistent with a diagnosis of pneumococcal pneumonia. Following treatment, this patient should be advised to be vaccinated with the 23-valent pneumococcal vaccine. Streptococcus pyogenes does not typically present as pneumonia. Streptococcus agalactiae generally afflicts neonates. The enterococci (Enterococcus faecium and Enterococcus faecalis) do not exhibit the Quellung reaction and do not present as community acquired pneumonia. 10 Gram-positive Rods I. OVERVIEW The gram-positive rods discussed in this chapter (Figure 10.1) are not closely related, and they do not cause similar clinical conditions. The genus Corynebacterium includes Corynebacterium diphtheriae, the cause of the prototypic toxin-mediated disease diphtheria, as well as several usually harmless human commensals. Bacillus is a large genus of spore-forming bacteria, principally of soil origin. Anthrax is caused by Bacillus anthracis. Listeria monocytogenes causes various types of infection in populations such as newborns, pregnant women, and the immunocompromised. II. CORYNEBACTERIA Corynebacteria are small, slender, pleomorphic, gram-positive rods of distinctive morphology that tend to stain unevenly. They are nonmotile and unencapsulated, and they do not form spores. Corynebacterium is a large genus of diverse habitat. Most species are facultative anaerobes, and those associated with humans, including the pathogen C. diphtheriae, grow aerobically on standard laboratory media such as blood agar. Free-living Bacteria Gram-positive Cocci Gram-negative Rods Corynebacteriun Corynebacterium diphtheriae S Bacillus Bacillus anthracis S Bacillus cereus Listeria Listeria monocytogenes S Propionibacterium
pharynx of carriers and in patients with diphtheria. This disease is a local infection, usually of the throat, and the organism is primar- Propionibacterium acnes Lactobacillus Erysipelothrix Erysipelothrix rhusiopathiae Figure 10.1 Classification of gram-positive rods. S See pp. 338, 332, and 343 for summaries of these organisms. 91 92 10. Gram-positive Rods 1 ily spread by respiratory droplets, usually by convalescent or asymptomatic carriers. It is less frequently spread by direct contact with an infected individual or a contaminated fomite. A membrane receptor recognizes and binds a portion of the toxin (fragment B). A 2. Pathogenesis: Diphtheria is caused by the local and systemic effects of a single exotoxin that inhibits eukaryotic protein synthesis. The toxin molecule is a heat-labile polypeptide that is composed of two fragments, A and B. Fragment B binds to susceptible cell membranes and mediates the delivery of fragment A to its target. Inside the cell, fragment A separates from fragment B and catalyzes a reaction between nicotine adenine dinucleotide (NAD+) and the eukaryotic polypeptide chain elongation factor, EF-21 (Figure 10.2). The toxin is encoded on a β-corynephage and only those strains in which the phage is integrated into the C. diphtheriae chromosome produce toxin. Toxin gene expression is also regulated by environmental conditions. Low iron conditions induce toxin expression, whereas high iron condtions repress toxin production. Diphtheria toxin B A B Cell membrane CELL Receptor for toxin A B 2 The toxin enters the cell by receptor-mediated endocytosis and dissociates into fragments A and B. 3. Clinical significance: Infection may result in one of two forms of clinical disease, respiratory or cutaneous, or in an asymptomatic carrier state. Active fragment of toxin
ized infection, usually of the throat. The infection produces a distinctive thick, grayish, adherent exudate (pseudomembrane) that is composed of cell debris from the mucosa and inflammatory products (see Figure 10.5). It coats the throat and may extend into the nasal passages or downward in the respiratory tract, where the exudate sometimes obstructs the airways, even leading to suffocation. As the disease progresses, generalized symptoms occur caused by production and absorption of toxin (Figure 10.3). Although all human cells are sensitive to diphtheria toxin, the major clinical effects involve the heart and peripheral nerves. Cardiac conduction defects and myocarditis may lead to congestive heart failure and permanent heart damage. Neuritis of cranial nerves and paralysis of muscle groups, such as those that control movement of the palate or the eye, are seen late in the disease. A B The A fragment is translocated to the cytosol, where it catalyzes the transfer of adenosine diphosphate ribose (ADPR) from NAD+ to EF-2. 3 NAD+ EF-2 4 Nicotinamide A EF-2 EF F-2
ADPR The ADPR-elongation factor complex is inactivated, and peptide synthesis stops. Figure 10.2 Action of diphtheria toxin. NAD+ = nicotine adenine dinucleotide; EF-2 = eukaryotic polypeptide chain elongation factor. result in introduction of C. diphtheriae into the subcutaneous tissue, leading to a chronic, nonhealing ulcer with a gray membrane. Rarely, exotoxin production leads to tissue degeneration and death. 4. Immunity: Diphtheria toxin is antigenic and stimulates the produc- tion of antibodies that neutralize the toxin’s activity. [Note: Formalin treatment of the toxin produces a toxoid that retains the antigenic- INFO LINK 1See Chapter 31 in Lippincott’s Illustrated Reviews: Biochemistry for a discussion of polypeptide chain elongation. II. Corynebacteria 93 ity but not the toxicity of the molecule. This is the material used for immunization against the disease (see p. 36).] 5. Laboratory identification: The presumptive diagnosis and deci- sion to treat for diphtheria must be based on initial clinical observation. Diphtheria should be considered in patients who have resided in or traveled to an area in which diphtheria is prevalent, when they have pharyngitis, low-grade fever, and cervical adenopathy (swelling of the neck). Erythema of the pharynx progressing to adherent gray pseudomembranes increases suspicion of diphtheria. However, a definitive diagnosis requires isolation of the organism, which must then be tested for virulence using an immunologic precipitin reaction to demonstrate toxin production. C. diphtheriae can be isolated most easily from a selective medium, such as Tinsdale agar (see Figure 10.5), which contains potassium tellurite, an inhibitor of other respiratory flora, and on which the organism produces several distinctive black colonies with halos (see Figure 10.5). C. diphtheriae from clinical material or culture has a distinctive morphology when stained, for example, with methylene blue. This morphology includes characteristic bands and reddish (polychromatic) granules that are often seen in thin, sometimes club-shaped rods that appear in clumps, suggestive of Chinese characters or picket fences (see Figure 10.4). This presentation is often referred to as a “palisade arrangement” of cells. Initial decision to treat for diphtheria must be based on clinical observation. Culture and assay for toxin production are required for confirmation of the diagnosis. Figure 10.3 Diphtheria with marked swelling of the lymph nodes in the neck. 6. Treatment: Treatment of diphtheria requires prompt neutralization of toxin, followed by eradication of the organism. A single dose of horse serum antitoxin inactivates any circulating toxin, although it does not affect toxin already bound to a cell-surface receptor. [Note: Serum sickness caused by a reaction to the horse protein may cause complications in approximately 10 percent of patients.] C. diphtheriae is sensitive to several antibiotics, and passive immunization with preformed diphtheria toxin antibodies is a mandatory part of treatment of diphtheria. Because diphtheria is highly contagious, suspected diphtheria patients must be isolated. Antibiotic treatment, such as erythromycin or penicillin (Figure 10.5), slows the spread of infection and, by killing the organism, prevents further toxin production. Supportive care directed especially at respiratory and cardiac complications is an essential part of the management of patients with diphtheria. 7. Prevention: The cornerstone of diphtheria prevention is immu- nization with toxoid, usually administered in the DTaP triple vaccine, together with tetanus toxoid and pertussis antigens (see p. 38). The initial series of injections should be started in infancy. Booster injections of diphtheria toxoid (with tetanus toxoid) should be given at approximately 10-year intervals throughout life. The control of an epidemic outbreak of diphtheria involves rigorous immunization and a search for healthy carriers among patient contacts. Figure 10.4 Corynebacterium diphtheriae. 94 10. Gram-positive Rods Gram (+) rods Small, slender, pleomorphic rods • form characteristic clumps that look like Chinese characters or a picket fence. They are nonmotile and • unencapsulated. Most species are facultative • anaerobes. Culture aerobically on selective • medium, such as Tinsdale agar Corynebacteria species Corynebacterium diphtheriae Gram stain Corynebacterium diphtheriae grown on tellurite blood medium containing tellurite (an inhibitor of the other respiratory flora). Corynebacterium diphtheriae • Diphtheria1 1 Erythromycin 2 Penicillin G Corynebacterium diphtheriae infection of the throat. Gross swelling and congestion of the whole pharyngeal and tonsillar area, with a gray exudate covering the tonsil. 1Treatment of diphtheria requires prompt neutralization of toxin, followed by eradication of the organism. A single dose of horse serum antitoxin inactivates any circulating toxin, although it does not affect toxin already bound to a cell-surface receptor. Figure 10.5 Summary of Corynebacterium diphtheriae disease. 1 Indicates first-line drugs; 2 indicates alternative drugs.
III. BACILLUS SPECIES Species of the genus Bacillus are gram-positive, form endospores, and are either strict aerobes or aerotolerant anaerobes (that is, they can grow in the presence of oxygen, but do not require it). Most of the 70 or so species of Bacillus are found in soil and water and are usually encountered in the medical laboratory as airborne contaminants. B. anthracis, the cause of the disease anthrax, is clinically the most important member of this genus. A. Bacillus anthracis Anthrax is a rare disease in the United States. For example, from 1984 to 1997, only three case of cutaneous anthrax were reported. III. Bacillus Species 95 However, in 2001, 20 new cases occurred—11 cutaneous and 11 inhalation anthrax. These infections resulted from probable exposure to B. anthracis powder sent through the mail. 1. Epidemiology: Anthrax is an enzootic disease of worldwide occur- rence. [Note: The term enzootic disease applies to a population of animals (equivalent to endemic disease in a human population, that is, its occurrence changes little over time). This is as compared to an epizootic disease, which attacks a large number of animals at the same time (similar to a human epidemic).] Anthrax affects principally domestic herbivores (for example, sheep, goats, and horses) and is transmitted to humans by contact with infected animal products or contaminated dust (Figure 10.6). Infection is usually initiated by the subcutaneous inoculation of spores through incidental skin abrasions. Less frequently, the inhalation of spore-laden dust causes a pulmonary form of anthrax. [Note: Sometimes an occupational hazard, this form of pneumonia is known as “woolsorter’s disease.”] B. anthracis spores may remain viable for many years in contaminated pastures and in bones, wool, hair, hides, and other animal materials. These spores, like those of clostridia (see p. 53), are highly resistant to physical and chemical agents. In the United States, a veterinary vaccine in widespread use makes domestic animal sources of the disease quite rare. Contaminated agricultural imports may account for the few cases seen and lead occasionally to the quarantine of goods from endemic areas. B. anthracis is a potential bioterrorism agent because it can be easily grown in large quantities. Moreover, the spores are resistant to destruction and can be formulated into an aerosol for wide dissemination. Physicians must be prepared to recognized anthrax even though it is rarely seen in the United States. Bioterro Bioterrorism Inha Inhalation In nha ha of contaminated con c co cont ont dust (Woolsorter's (Wo (Woo (W Woo oo disease) Contact with contaminated animal products (hides, wool, hair) hair).. 2. Pathogenesis: B. anthracis produces a unique capsule that is comprised of poly-D-glutamic acid and is anti phagocytic. Elaboration of this capsule is essential for full virulence. The organism also produces two plasmid-coded exotoxins: edema toxin and lethal toxin. Both toxins are AB type toxins with activity and binding domains. The binding subunit shared by both toxins is called protective antigen (so named because of its use in producing protective anthrax vaccines). This domain mediates cell entry of both toxins. The activity subunits are called edema factor and lethal factor. Edema factor is a calmodulin-dependent adenylyl cyclase, which causes elevation of intracellular cAMP, resulting in the severe edema usually seen in B. anthracis infections. Lethal factor is responsible for tissue necrosis. Lethal factor complexed with protective antigen is known as lethal toxin, whereas edema factor complexed with protective antigen is known as edema toxin. 3. Clinical significance a. Cutaneous anthrax: About 95 percent of human cases of anthrax are cutaneous. Upon introduction of organisms or spores that germinate, a papule develops. It rapidly evolves into a painless, black, severely swollen “malignant pustule,” Spores contaminate nta aminate hide of herbivores biivores (cattle, sheep, ep p, goats) Figure 10.6 Anthrax in animal and human hosts. 96 10. Gram-positive Rods which eventually crusts over. The organisms may invade regional lymph nodes and then the general circulation, leading to fatal septicemia. Although some cases remain localized and heal, the overall mortality in untreated cutaneous anthrax is about 20 percent. b. Pulmonary anthrax (woolsorter’s disease): Caused by inhala- tion of spores, the pulmonary form is characterized by progressive hemorrhagic lymphadenitis (inflammation of the lymph nodes), hemorrhagic mediastinitis (inflammation of the mediastinum) and has a mortality rate approaching 100 percent if left untreated. 4. Laboratory identification: B. anthracis is easily recovered from clinical materials, where it is often present in massive numbers. Microscopically, the organisms appear as blunt-ended bacilli that occur singly; in pairs; or frequently in long chains (Figure 10.7). They do not sporulate often in clinical samples but do so in culture. The spores are oval and centrally located. On blood agar, the colonies are large, grayish, and nonhemolytic, with an irregular border. Unlike many bacillus species, B. anthracis is nonmotile Gram (+) rods bacilli that occur singly; • inBlunt-ended pairs; or, frequently, in long Bacillus species chains. Usual nonhemolytic culture on blood agar Form endospores––oval and • centrally located. Nonmotile with a capsule that is • antiphagocytic. Facultative or strictly aerobic • organisms. • Culture on blood agar. Gram stain of Bacillus anthracis culture smear showing typical bacilli with highly refractile unstained spores Bacillus anthracis • Cutaneous anthrax 1 Ciprofloxacin anthrax (woolsorter’s disease) • Pulmonary Multidrug therapy is recommended . 1 Ciprofloxacin 1 Rifampin 1 Clindamycin 1 or Ciprofloxacin 1 Rifampin 1 Vancomycin Cutaneous anthrax Figure 10.7 Summary of anthrax disease. 1 Indicates first-line drugs. Chest radiograph of a patient with pulmonary anthrax, showing widening of the mediastinum. IV. Listeria and is encapsulated in vivo. A direct immunofluorescence assay aids in identification of the organism. 5. Treatment: B. anthracis is sensitive to a variety of antibiotics. Cutaneous anthrax responds to ciprofloxacin (see Figure 10.7). Penicillin is not recommended because of inducible β-lactamase in B. anthracis. Multidrug therapy (for example, ciprofloxacin plus rifampin plus vancomycin) is recommended for inhalation anthrax. Aggressive therapy is indicated for inhalation anthrax both because of the severity of the disease and the fact that the disease is often not diagnosed until late in the course of the illness. 6. Prevention: A cell-free vaccine is available for workers in high-risk occupations (see p. 37). Postexposure prophylaxis with ciprofloxacin or doxycycline is recommended. [Note: Because of the resistance of endospores to chemical disinfectants, autoclaving is the most reliable means of decontamination.] B. Other bacillus species Uncommonly, other species of bacillus are implicated in opportunistic lesions, particularly following trauma or the placement of artificial devices and catheters. A commonly identified species is Bacillus cereus. Strains of this species produce a tissue-destructive exotoxin. B. cereus also causes food poisoning by means of enterotoxins with either emetic or diarrheal effects. IV. LISTERIA Listeria species are slender, short, gram-positive rods (see Figure 10.9). They do not form spores. Sometimes they occur as diplobacilli or in short chains, and they are avid intracellular parasites that may be seen within the cytoplasm of host cells in tissue samples. Listeria species are catalase-positive, and display a distinctive tumbling motility by light microscopy in liquid medium, which is most active after growth at 25oC. These characteristics distinguish it from Streptococcus (catalase negative) or Corynebacterium (nonmotile) species, both of which may be confused morphologically with Listeria. Listeria species grow on a variety of enriched media. A. Epidemiology Listeria monocytogenes is the only species that infects humans, although the Listeria species are widespread among animals in nature. Listeria infections, which may occur as sporadic cases or in small epidemics, are usually foodborne. For example, studies have shown that 2 to 3 percent of processed dairy products (including ice cream and cheese), 20 to 30 percent of ground meats, and a majority of retail poultry samples are contaminated with L. monocytogenes. [Note: Because L. monocytogenes is capable of growth at 4°C, refrigeration does not reliably suppress its growth in food.] One to 15 percent of healthy humans are asymptomatic intestinal carri- 97 98 10. Gram-positive Rods 1 Listeria is phagocytosed by a macrophage and incorporated into a phagolysosome. ers of the organism. Listeria infections are most common in pregnant women, fetuses and newborns, and in immunocompromised individuals, such as older adults and patients receiving corticosteroids. In the United States, some 2,000 cases are reported each year, with 450 deaths and 100 stillbirths. Blood cultures are indicated in pregnant febrile women when no alternate pathology (for example, urinary tract infection) is readily detected. B. Pathogenesis Listeria 2 The bacterial product listeriolysin O lyses the phagolysosome, allowing the escape of the listeria. 3 Listeria multiplies and assembles an actin filament tail that pushes the bacterium to the surface of the macrophage.
MACROPHAGE 4 A pseudopod extension forms, facilitating transfer of the listeria into another phagocyte. Septicemia and meningitis are the most commonly reported forms of L. monocytogenes infection (listeriosis). A variety of focal lesions are less frequently seen such as granulomatous skin lesions. Pregnant women, usually in the third trimester, may have a milder “flulike” illness. In this as well as in asymptomatic vaginal colonization, the organism can be transmitted to the fetus and result in spontaneous abor tion. Alter natively, the organism can also be transmitted to a newborn following birth, resulting in neonatal meningitis. (L. monocytogenes is a relatively common cause of newborn meningitis). Immunocompromised individuals, especially those with defects in cellular immunity, are susceptible to serious generalized infections. D. Laboratory identification The organism can be isolated from blood, cerebrospinal fluid, and other clinical specimens by standard bacteriologic procedures. On blood agar, L. monocytogenes produces a small colony surrounded by a narrow zone of β hemolysis (Figure 10.9). Listeria species can be distinguished from various streptococci by morphology, motility, and the production of catalase. Figure 10.8 Life cycle of Listeria monocytogenes in host macrophages.
99 Gram (+) rods Slender, short rods, sometimes • occurring as diplobacilli or in short Listeria species chains Listeria monocytogenes on blood agar Listeria monocytogenes in cerebrospinal fluid (Gram stain) • Intracellular parasites • Catalase positive Distinctive tumbling motility in • liquid medium Grow facultatively • enriched media on various Listeria monocytogenes • Listeriosis 1 2 Ampicillin Trimethoprim/ sulfamethoxazole Figure 10.9 Summary of Listeria species. 1 Indicates first-line drugs; 2 indicates alternative drugs. prim/sulfamethoxazole (see Figure 10.9). Prevention of L. monocytogenes infections can be accomplished by proper food preparation and handling, as well as removal of contaminated products from the food supply.
100 10. Gram-positive Rods Study questions Choose the ONE correct answer 10.1 A diagnosis of diphtheria is confirmed by: A. microscopic appearance of organisms stained with methylene blue. B. isolation of a typical colony on Tinsdale agar. C. isolation of typical organisms from materials such as blood, showing invasiveness. D. detection of β phage plaques in cultures of suspicious isolates. E. demonstration of toxin production by a suspicious isolate. 10.2 Listeria monocytogenes shows which of the following characteristics? A. B. C. D. E. It can grow at refrigerator temperatures (4°C). It is an extracellular pathogen. It is catalase negative. It is a gram-negative coccus. It is strictly a human pathogen. 10.3 A 26-year-old woman, 8 months pregnant, visits her obstetrician complaining of fever, myalagia and backache of recent onset. Three weeks earlier, the patient had been a weekend guest at a rural farmhouse, where all the food was reported to be “unprocessed” and “natural.” A culture of the patient’s blood shows gram-positive rods that are catalase positive and display a distinctive tumbling motility in liquid medium. What is the most likely source of the woman’s infection? A. B. C. D. E. Correct answer = E. Observation of diphtheria toxin production is required to prove the diagnosis. Items A and B are presumptive indicators. β Phage is a temperate phage, and lytic activity is not observed. Cornyebacterium diphtheriae is noninvasive, and the organism (but not the toxin) is recovered only from surface infections such as those of the oropharynx and skin lesions. Correct answer = A. Listeria. monocytogenes grows optimally at 30 to 37°C, but is capable of growth at 4°C. Thus, refrigeration does not reliably suppress its growth in food. L. monocytogenes is a catalase-positive, gram-positive, obligate intracellular pathogen. These organisms are found in cattle, other warmblooded animals, and fish, where they can cause disease. Correct answer = B. The woman is most likely experiencing listerosis. Listeriae are common in the gastrointestinal tract and milk of cattle, but are normally killed by pasteurization. Unpasteurized milk was presumably consumed at the farm. Well-done roast beef Fresh, raw cow’s milk Home-baked bread Homemade applesauce Baked apple pie 10.4 A 45-year-old cattle rancher presents to his physician with a wound on his forearm that resembles a large scab. Samples collected from the wound were cultured and examined. The bacteria recovered were Gram positive, nonmotile rods with square ends. The cultured bacteria formed irregularly shaped, nonhemolytic colonies on blood agar plates and individual cells from the plates had a centrally located spore. What is the most likely cause of this infection? A. Listeria monocytogenes B. Staphylococcus aureus C. Legionella pneumophila D. Corynebacterium diphtheriae E. Bacillus anthracis Correct answer = E. This cattle rancher is suffering from cutaneous anthrax, which is an occupational hazard. The scab like wound is called an eschar and results from localized edema and tissue destruction caused by the two toxins produced by Bacillus anthracis. The microbiological characteristics of the organism are consistent with a diagnosis of B. anthracis infection. The other microorganisms do not have the chacteristics described. 11 Neisseriae I. OVERVIEW The genus Neisseria consists of gram-negative, aerobic cocci. Two Neisseria species are pathogenic for humans—Neisseria gonorrhoeae (commonly called gonococcus), the causal agent of gonorrhea and Neisseria meningitidis (commonly called meningococcus), a frequent cause of meningitis. Gonococci and meningococci are nonmotile diplococci that cannot be distinguished from each other under the microscope. However, they can be differentiated in the laboratory by sugar-use patterns, and the sites of their primary infections. Both bacteria are classified as pyogenic cocci because infections by these organisms are also characterized by the production of purulent (puslike) material comprised largely of white blood cells. Neisseriae and organisms that are easily confused with them are discussed in this chapter (Figure 11.1.) Free-living Bacteria Gram positive Cocci Gram negative Rods Cocci II. NEISSERIA GONORRHOEAE Enteric rods Nonenteric rods Neisseria Gonorrhea is one of the most frequently reported infectious diseases in the United States. The causal agent, N. gonorrhoeae, a gram-negative diplococcus, is frequently observed within the polymorphonuclear leukocytes of clinical samples obtained from infected patients (Figure 11.2). N. gonorrhoeae is usually transmitted during sexual contact or, more rarely, during the passage of a baby through an infected birth canal. It does not survive long outside the human body because it is highly sensitive to dehydration. Neisseria gonorrhoeae Neisseria meningitidis S S Moraxella Moraxella catarrhalis Acinetobacter Acinetobacter
Figure 11.1 Classification of Neisseria and related organisms. S See pp. 345– 346 for summaries of these organisms. 1. Pili: These hairlike surface appendages are made of helical aggregates of repeating peptide subunits called pilin. Pili enhance attachment of the organism to host epithelial and mucosal cell surfaces. They are, therefore, important virulence factors. Pili are also antigenic. At least twenty gonococcal genes code for pilin, most of which are not expressed at any given time because they lack promoters (that is, they are “silent”). By shuffling and recombining chromosomal regions of these genes, a single strain of N. gonorrhoeae can, at different times, synthesize (“express”) multiple pilins that have different amino acid sequences. This process, known as antigenic variation by gene conversion, allows the 101 102 11. Neisseriae organism to produce antigenically different pilin molecules at high frequency (Figure 11.3). 2. Lipooligosaccharide: Gonococcal lipooligosaccharides (LOS) have Gonococcus Figure 11.2 Presence of Neisseria gonorrhoeae in polymorphonuclear leukocytes in urethral discharge. shorter, more highly branched, nonrepeat O-antigenic side chains than do lipopolysaccharides found in other gram-negative bacteria (see p. 13). The bactericidal antibodies in normal human serum are IgM molecules directed against LOS antigens. The gonococcus is also capable of high-frequency variation of the LOS antigens presented on the cell surface. Variation occurs as a consequence of phase variation of one of several genes involved in the biosynthesis of LOS. If the biosynthetic gene is in the off phase, terminal saccharide moieties cannot be added, resulting in presentation of an antigenically distinct LOS molecule. 3. Porin proteins: The gonococcus expresses a single porin type, known as PorB. Different strains express either PorB1A or PorB1B; however, the porin proteins are not subject to a high frequency phase or antigenic variation like other outer membrane antigens. 4. Opacity proteins: Opacity (Opa) proteins (formerly called PII pro- Promoter Silent genes A B C Gonococcal pilin genes of different antigenic types A B C Expression locus A Type A gene is copied and inserted into the expression locus next to the promoter, leading to production of pilin protein that is antigenically type A. A B C B Insertion of other genes into the expression locus leads to production of antigenically different pilin molecules. Figure 11.3 Antigenic variation in the gonococcus. teins) are so named due to their tendency to impart an opaque quality to gonococcal colonies. The gonococcus has the capacity to express up to 11 different Opa proteins, but generally only one or a few are expressed simultaneously. Opa proteins are subject to phase variation by virtue of the presence of numerous polymeric repeats (CTCTT) in the coding regions. If an Opa protein is expressed, an increase or decrease in the number of repeats shifts the protein out of the reading frame, resulting in phase variation to the off phase. Different Opa proteins bind to distinct receptors on host cells. Therefore shifting expression from one Opa protein to another results in changes in host cell tropism. B. Pathogenesis Pili and Opa proteins facilitate adhesion of the gonococcus to epithelial cells of the urethra, rectum, cervix, pharynx, and conjunctiva, thereby making colonization possible. In addition, both gonococci and meningococci produce an IgA protease that cleaves IgA1, helping the pathogen to evade immunoglobulins of this subclass. The gonococcus requires iron for growth and survival in vivo. The pathogen acquires this necessary nutrient by expression of specific transport systems that remove and internalize the iron from human iron binding proteins including transferrin, lactoferrin and hemoglobin. To establish infection in human males, the gonococcus must express proteins that facilitate iron acquisition from either transferrin or lactoferrin. C. Clinical significance Gonococci most often colonize the mucous membrane of the genitourinary tract or rectum. There, the organisms may cause a localized infection with the production of pus or may lead to tissue invasion, chronic inflammation, and fibrosis. A higher proportion of females than males are generally asymptomatic, and these individuals act as the reservoir for maintaining and transmitting gonococcal infections. [Note: More than one sexually transmitted disease (STD) may be II. Neisseria gonorrhoeae 103 acquired at the same time, such as, gonorrhea in combination with syphilis (Treponema pallidum infection), Chlamydia, human immunodeficiency virus, or hepatitis B virus. Patients with gonorrhea may, therefore, need treatment for more than one pathogen.] 1. Genitourinary tract infections: Symptoms of gonococcal infection are more acute and easier to diagnose in males. The patient typically presents with a yellow, purulent urethral discharge and painful urination (Figure 11.4). In females, infection occurs in the endocervix and extends to the urethra and vagina. A greenish-yellow cervical discharge is most common, often accompanied by intermenstrual bleeding. The disease may progress to the uterus, causing salpingitis (inflammation of the fallopian tubes), pelvic inflammatory disease (PID), and fibrosis. Infertility occurs in approximately 20 percent of women with gonococcal salpingitis, as a result of tubal scarring. N. gonorrhoeae is a common cause of pelvic inflammatory disease in females. 2. Rectal infections: Prevalent in men who have sex with men, rectal infections are characterized by constipation, painful defecation, and purulent discharge. 3. Pharyngitis: Pharyngitis is contracted by oral-genital contact. Infected individuals may show a purulent pharyngeal exudate, and the condition may mimic a mild viral or a streptococcal sore throat (see p. 81). Figure 11.4 Urethral discarge of gonorrhea. Inflammation and edema appear 2 to 5 days after birth. These symptoms are more severe than those observed with chlamydia infection. 4. Ophthalmia neonatorum: This infection of the conjunctival sac is acquired by newborns during passage through the birth canals of infected mothers (Figure 11.5). If untreated, acute conjunctivitis may lead to blindness. Treatment is systemic ceftriaxone IM or IV in a single dose. Infants born to mothers who are known to have a birth canal infected with gonococcus or are at high risk of having this are also given a systemic dose of ceftriaxone prophylactically, even in the absence of clinically evident ophthalmia. Topical erythromycin ointment is only used for routine prophylaxis in circumstances of relatively low risk. Figure 11.5 Gonococcal ophthalmia neonatorum. 5. Disseminated infection: Most strains of gonococci have a limited ability to multiply in the bloodstream. Therefore, bacteremia with gonococci is rare. In contrast, meningococci multiply rapidly in blood (see p. 105). However, some strains of N. gonorrhoeae do invade the bloodstream and may result in a disseminated infection in which the organism can cause fever; a painful, purulent arthritis; and small, single, scattered pustules on the skin, whose base becomes erythematous (red) due to dilation or congestion of capillaries. Necrosis may develop. [Note: Gonococcal infection is the most common cause of septic arthritis in sexually active adults.] Disseminated infections are seen in both men and women but are more common in women, particularly during pregnancy and menses. All patients treated for disseminated infection should also receive a 7-day course of doxycycline to eliminate potentially concurrent infection with Chlamydia. Gonorrhea is most common in adolescents and young adults (Figure 11.6). (A summary of organisms causing the most common STDs is presented in Figure 33.2.) Rate per 100,000 population Men 600 300 0 Age 0 Women 300 600 (years) 10–14 15–19 20–44 25–29 30–34 35–39 40–44 45–54 55–64 65+ Total Figure 11.6 Incidence of gonorrhea in the United States (2009) according to age and sex. 104 11. Neisseriae D. Laboratory identification Culture of cervical sample on plain chocolate agar allows growth of contaminating organisms. In the male, the finding of numerous neutrophils containing gramnegative diplococci in a smear of urethral exudate permits a provisional diagnosis of gonococcal infection and indicates that the individual should be treated. In contrast, a positive culture is needed to diagnose gonococcal infection in the female as well as at sites other than the urethra in the male. If disseminated gonococcal infection is suspected, appropriate cultures should be set up as indicated, for example, of skin lesions, joint fluid, and blood. 1. Growth conditions for culture: N. gonorrhoeae grows best under Culture on Thayer-Martin chocolate agar medium (containing vancomycin, colistin, trimethoprim, and nystatin) allows selective growth of Neisseria. Figure 11.7 Left: Mixed growth on plain chocolate agar. Right: Pure culture on ThayerMartin chocolate agar medium. aerobic conditions, and most strains require enhanced CO 2. N. gonorrhoeae utilizes glucose as a carbon and energy source but not maltose, lactose, or sucrose. [Note: N. meningitidis utilizes both glucose and maltose (see p. 107).] All members of the genus are oxidase-positive. [Note: The oxidase test (see p. 24) is used to identify Neisseriae, but does not distinguish between gonococci, meningococci, and nonpathogenic Neisseriae.] 2. Selective media: Gonococci, like pneumococci, are very sensitive to heating or drying. Cultures must be plated promptly or, if this is not possible, transport media must be used to extend the viability of the organism to be cultured. Thayer-Martin medium (chocolate agar supplemented with several antibiotics that suppress the growth of nonpathogenic Neisseriae and other normal and abnormal flora) is typically used to isolate gonococci (Figure 11.7). The use of this medium is important for cultures that are typically obtained from sites such as the genitourinary tract or rectum, where there is normally an abundance of flora. On nonselective media, the normal flora overgrows the gonococci. Culture of N. gonorrhoeae on Thayer-Martin agar remains the “gold standard” for diagnosis. E. Treatment and prevention More than 20 percent of current isolates of N. gonorrhoeae are resistant to penicillin, tetracycline, cefoxitin, and/or spectinomycin. Penicillin-resistant organisms are called PPNG—penicillinaseproducing N. gonorrhoeae. These strains contain plasmids that carry the gene for β-lactamase of the TEM type (encoded in a transposable element), such as is seen in Escherichia coli and Haemophilus influenzae. The frequency of PPNG in the United States is now sufficiently high that penicillin is no longer recommended for treatment of gonorrhea. However, most organisms still respond to treatment with third-generation cephalosporins; for example, a single intramuscular dose of ceftriaxone is the recommended therapy for uncomplicated gonococcal infections of the urethra, endocervix, or rectum. Intramuscular spectinomycin is indicated in patients who are allergic to cephalosporins. Many patients with gonorrhea have coexisting chlamydial infections. Therefore, doxycycline, a tetracycline effective against Chlamydia, is often included as part of the treatment regimen for gonorrhea. Prevention of gonorrhea involves evaluation and management of sexual contacts of the patient, generally using a dose of an effective antibiotic prophylactically in an exposed individual even in the absence of symptoms. The use of barrier methods is also a III. Neisseria meningitidis 105 preventive measure against gonorrhea as is the case for all sexually transmitted infections. No vaccine is available for gonorrhea. III. NEISSERIA MENINGITIDIS N. meningitidis is one of the most frequent causes of meningitis. Infection with N. meningitidis can also take the form of a fulminant meningococcemia, with intravascular coagulation, circulatory collapse, and potentially fatal shock, but without meningitis. In each case, symptoms can occur with extremely rapid onset and great intensity. Outbreaks of meningitis, most common in winter and early spring, are favored by close contact between individuals, such as occurs in schools, institutions, and military barracks. Severe epidemics also occur periodically in developing nations, such as in sub-Saharan Africa and Latin America. N. meningitidis tends to strike young, previously well individuals and can progress over a matter of hours to death. A. Structure Figure 11.8 Smear of purulent cerebrospinal fluid showing Neisseria meningitidis. Like N. gonorrhoeae, N. meningitidis is a nonmotile, gram-negative diplococcus, shaped like a kidney bean, which always appears in pairs (Figure 11.8). It is also piliated and the pili allow attachment of the organism to the nasopharyngeal mucosa where it is harbored both in carriers and in those with meningococcal disease. When meningococcus is isolated from blood or spinal fluid, it is invariably encapsulated. The meningococcal polysaccharide capsule is antiphagocytic and, therefore, the most important virulence factor. [Note: Antibodies to the capsule carbohydrate are bactericidal.] EPIDEMIOLOGIC CLASSIFICATION 1. Serogroups: The polysaccharide capsule is antigenically diverse, Serogroups (>13) Polysaccharide capsule Serotypes (>20) Outer membrane proteins which allows the identification of at least 13 capsular polysaccharide types, called serogroups (Figure 11.9). Most infections are caused by serogroups A, B, C, W-135, and Y, although approximately 90 percent of cases of meningococcal disease are caused by serogroups A, B, and C. Serogroup A is usually responsible for massive epidemics in developing countries. In the United States, N. meningitidis serogroup B is the predominant cause of disease and mortality, followed by group C. Organisms that do not have a capsule are called unencapsulated. 2. Serotypes: A second classification system called serotyping (serotypes 1, 2,...20) is also a serologic classification (see Figure 11.9) that is based on the properties of the outer membrane proteins (see p. 102). The meningococcus expresses PorA- and PorB-type porins. There is no predicable relationship between serogroups and serotypes. B. Epidemiology Transmission occurs through inhalation of respiratory droplets from a carrier or a patient in the early stages of the disease. In addition to contact with a carrier, risk factors for disease include recent viral or mycoplasma upper respiratory tract infection, active or passive smoking, and complement deficiency. In susceptible persons, pathogenic strains may invade the bloodstream and cause systemic ANTIGENIC DETERMINANT Figure 11.9 Antigenic determinants of Neisseria meningitidis. 106 11. Neisseriae illness after an incubation period of 2 to 10 days. The incidence of meningococcal disease in the United States is highest among infants younger than age 1 year (Figure 11.10). An incidence peak among adolescents and young adults led the Centers for Disease Control to recently recommend vaccination of this at-risk group. Humans are the only natural host. Rate/100,000 60
0 >1 3 6 9 1 Months 4 7 10 13 16 19 Years Antiphagocytic properties of the meningococcal capsule aid in the maintenance of infection. LOS, released during autolysis and in outer membrane vesicles, is responsible for the toxic effects found in disseminated meningococcal disease. As noted on p. 102, gonococci and meningococci make an IgA protease that cleaves IgA1 and, thus, helps the pathogens to evade immunoglobulins of this subclass. [Note: Nonpathogenic Neisseriae do not make this protease.] D. Clinical significance Figure 11.10 Incidence of meningococcal infection according to age.
Figure 11.11 Petechial and/or purpuric rash and neck extension characteristic of meningococcal meningitis. serves as a barrier to bacteria. Therefore, most persons colonized by N. meningitidis remain well. As a rare event, meningococci penetrate this barrier and enter the bloodstream where they rapidly multiply (meningococcemia). In patients with fulminant septicemia, meningococci can be detected in peripheral blood smears—an unusual occurrence. If the disease is not severe, the patient may have only a fever and other nonspecific symptoms. However, the organism can seed from the blood to other sites, for example, by crossing the blood-brain barrier and infecting the meninges. There they multiply and induce an acute inflammatory response, accompanied by an influx of polymorphonuclear leukocytes, resulting in a purulent meningitis. Joint symptoms and a petechial and/or purpuric rash are also commonly observed in meningococcal infections (Figure 11.11). Within several hours the initial fever and malaise can evolve into severe headache, a rigid neck, vomiting, and sensitivity to bright lights—symptoms characteristic of meningitis. Coma can occur within a few hours. A summary of the major organisms causing meningitis is shown in Figure 33.5, p. 376. The gold standard for diagnosis of systemic meningococcal infection is the isolation of N. meningitidis from a usually sterile body fluid, such as blood or cerebrospinal fluid (CSF). In performing a Gram stain on CSF, the clinical sample is centrifuged to concentrate the organisms, because 105 to 106 bacteria per ml are required for this test. 2. Septicemia: Meningococci can cause a life-threatening septicemia in an apparently healthy individual in less than 12 hours. Up to 30 percent of patients with meningitis progress to fulminant sep- III. Neisseriae meningitidis 107 ticemia. In this condition, the clinical presentation is one of severe septicemia and shock, for which the bacterial endotoxin (LOS) is largely responsible. Acute, fulminant meningococcal septicemia is seen mainly in very young children (the Waterhouse-Friderichsen syndrome). It is characterized by large, purple, blotchy skin hemorrhages, vomiting and diarrhea, circulatory collapse, adrenal necrosis, and death within 10 to 12 hours. Neisseria meningitidis
Under the light microscope, N. meningitidis obtained from CSF and skin lesion aspirates appear as gram-negative diplococci, often in association with polymorphonuclear leukocytes (see Figure 11.8). Carriers can be detected by culturing swabs from the nasopharyngeal region. (utilized) (utilized) (not utilized) 1. Culture conditions: Meningococci are cultured on chocolate agar with increased CO2. The sample must be plated promptly or, if this is not possible, transport medium must be used to extend the viability of the organism to be cultured. Unlike gonococci, meningococci are usually cultured from CSF or blood, which are normally sterile; therefore a selective medium is not required, and plain chocolate agar is sufficient. [Note: Thayer-Martin medium (see p. 104) is required for samples obtained from a skin lesion or nasopharyngeal swab, to eliminate contaminating organisms.] Neisseria gonorrhoeae 2. Additional tests: All Neisseria species are oxidase-positive. To differentiate between species, sugar utilization tests are used (Figure 11.12). N. meningitidis utilizes both glucose and maltose, whereas N. gonorrhoeae uses only glucose (see Figure 11.12). In bacterial meningitis, the CSF shows increased pressure, elevated protein, decreased glucose (partly resulting from its consumption as a bacterial nutrient), and many neutrophils. The presence of an infecting organism or of antigenic capsular substance confirms the diagnosis. Figure 11.13 compares the characteristics of N. gonorrhoeae and N. meningitidis. F. Treatment and prevention Bacterial meningitis is a medical emergency. Accordingly, antibiotic treatment cannot await a definitive bacteriologic diagnosis. High fever, headache, and a rash typical of meningococcal infection are treated immediately in an effort to prevent progression to fulminant septicemia which has a high mortality rate. Blood cultures should be drawn and antibiotic therapy should not be delayed while waiting for Glucose Maltose Sucrose (utilized) (not utilized) (not utilized) Figure 11.12 Neisseria meningitidis produces acid from oxidation of glucose and maltose, but not from sucrose. The acid turns the pH indicator phenol red from red to yellow. PLASMIDS VACCINE AVAILABLE POLYSACCHARIDE CAPSULE β-LACTAMASE PRODUCTION OXIDASE – Common – – Common + + Rare Serogroups A, C, W-135, Y + None + GLUCOSE UTILIZATION MALTOSE UTILIZATION Neisseria gonorrhoeae + Neisseria meningitidis + Figure 11.13 Differential bacteriologic features of Neisseria gonorrhoeae and Neisseria meningitidis. 108 11. Neisseriae lumbar puncture to be performed. Pretreatment with antibiotics can substantially diminish the probability of a positive CSF culture but the diagnosis can often still be established from the pretreatment blood cultures; and organisms may continue to be visible on Gram stain of the CSF. Meningitis can be effectively treated with penicillin G or ampicillin (both of which can pass the inflamed blood-brain barrier) in large intravenous doses. When the etiology of the infection is unclear, cefotaxime or ceftriaxone is recommended. Prompt treatment reduces mortality to about 10 percent. Because of the intense inflammatory reaction that accompanies bacterial meningitis, many authorities recommend a dose of the corticosteroid dexamethasone shortly prior to, or together with, the first dose of antibiotic. 1. Diagnosis: Gram stains on CSF can be performed immediately, and latex agglutination tests with serogroup-specific anticapsular antibody can be used to obtain rapid presumptive identification of serogroup-specific meningococci in CSF. SEROGROUP CLASSIFICATION COMMENT A Usually responsible for massive epidemics in developing countries. B Does not elicit an effective immune response. B, C Responsible for most endemic meningitis in the United States. A, C, W-135,Y Effective capsular vaccine is available. Figure 11.14 Characteristics of the common serogroups of Neisseria meningitidis. 2. Vaccines: A conjugate meningococcal vaccine (MCV4) was approved in the United States in 2005 for use in adolescents and adults ages 11 to 55 years, and has replaced the unconjugated polysaccharide vaccine. MCV4 is a tetravalent vaccine that contains capsular polysaccharides from serogroups A, C, W-135, and Y conjugated to diphtheria toxoid. The conjugated vaccines elicit a T celldependent memory response that increases the effectiveness of the vaccine, resulting in an improved primary response to the vaccine and a strong response on subsequent exposure to the pathogen. The serogroup B polysaccharide capsule is a self-antigen and therefore does not elicit an effective immune response. Figure 11.14 summarizes vaccines and serogroups. 3. Prophylaxis: Prophylactic rifampin is given to family members because of the inevitability of their close contact and thus exposure. Other drugs used for prophylaxis include oral ciprofloxacin and intramuscular ceftriaxone. Figure 11.15 summarizes the diseases caused by Neisseria species. IV. MORAXELLA Members of the genus Moraxella are nonmotile, gram-negative coccobacilli that are generally found in pairs. Moraxella are aerobic, oxidasepositive, fastidious organisms that do not ferment carbohydrates. The most impor tant pathogen in the genus is Moraxella (for mer ly, Branhamella) catarrhalis. This organism can cause infections of the respiratory system, middle ear, eye, CNS, and joints.
109 Neisseria meningitidis in cerebrospinal fluid of patient with meningitis. Neisseria gonorrhoea in clumps within polymorphonuclear leukocytes in urethral exudate. Gram (–) cocci bean”– • “Kidney shaped diplococci important in • Pili attachment to Neisseria species mucosa • Oxidase positive • Aerobic • Nonmotile • Pyogenic Neisseria gonorrhoeae not utilized • Maltose for energy on • Grown Thayer-Martin • medium No polysaccharide capsule Neisseria gonorrhoeae infection of human epithelial cells. A bacteria microcolony is seen attaching to the host cell via surface appendages called Type IV pili (cobwed-like structures), which induce the formation of host cell microvilli. (Photos courtesy of Dustin L. Higashi, Al Agellon, and Magdalene So). • Ophthalmia neonatorum • Uncomplicated gonorrhea 1 Ceftriaxone1 1Systemic 1 Erythromycin2 1 Silver nitrate2 1 Ceftriaxone1 1 Doxycycline2 1A single 1-gram dose of azithromycin is used in persons with cephalosporin allergy. 2Topical for routine prophylaxis in circumstances of relatively low risk 2A tetracycline is added when Chlamydia is a suspected co-pathogen. Neisseria meningitidis • • • utilized for • Maltose energy on chocolate • Grown agar common cause • Most of meningitidis in Meningitis Meningococcemia Waterhouse-Friderichsen syndrome 1 Penicillin G1 2 Cefotaxime 2 Ceftriaxone 1Resistant strains have emerged, and sensitivity testing should be performed. Note: Rifampin can be used prophylactically to treat family members or other close contacts of an infected individual Figure 11.15 Summary of Neisseria diseases. 1 Purpuric rash characteristic of meningococcemia. • • persons between ages 2 and 18 years. Polysaccharide capsule Vaccine for serogroups Y, W-135, C, and A Indicates first-line drugs; 2 indicates alternative drugs. 110 11. Neisseriae Study Questions Choose the ONE best answer 11.1 A 20-year-old, sexually-active female presents at her family physician's office with fever, painful arthritis of the right knee, and several small pustules on her extremities. Material from the pustules and joint fluid were collected for culture on modified Thayer-Martin medium. Which of the following results are consistent with a diagnosis of gonococcal infection? A. Growth of small colonies consisting of gram-negative diplococci. Bacteria grown on plates are catalase and oxidase positive. B. Growth of small colonies consisting of gram-positive cocci. Bacteria growth on plates are catalase and oxidase positive. C. Growth of small colonies consisting of gram-negative diplococci. Bacteria growth on plates are catalase and oxidase negative. D. Growth of large mucoid colonies consisting of gram-negative bacilli. Bacteria growth on plates are catalase and oxidase negative. E. Growth of gram-negative diplococci within polymorphonuclear leukocytes. Bacteria can utilize glucose and maltose as a carbon sources. 11.2 Which of the following neisserial virulence factors is subject to high-frequency antigenic variation by a mechanism involving recombination between silent and expressed chromosomal loci? A. B. C. D. E. Lipooligosaccharide Capsule Porin Pilin Opacity proteins 11.3 Which of the following neisserial virulence factors is part of the tetravalent vaccine that protects against some but not all serogroups of Neisseria meningitidis? A. Lipooligosaccharide B. Capsule C. Porin D. Pilin E. Opacity proteins Correct answer = A. Gonococcal infection is the most common cause of septic arthritis in sexually active adults. Neisseria gonorrhoeae can be cultured from the joint fluid and pustular material, following dissemination from the genital tract to the skin and joints. Gonococci grow on modified Thayer-Martin medium to form small colonies that are oxidase positive and catalase positive. N. gonorrhoeae following Gram stain appears as a gram-negative diplococcus. Although N. gonorrhoeae is often found within polymorphonuclear leukocytes when clinical specimens are stained directly, these human cells would not be present after culturing on Modified Thayer-Martin medium. Correct answer = D. Althoough the synthesis of lipooligosaccharide (LOS) is phase variable, the mechanism does not involve recombination between silent and expressed genes. LOS varies by a mechanism known as slipped-strand mispairing, which results in changes in the number of single nucleotide repeats within the LOS biosynthetic genes. If the biosynthetic gene has the appropriate number of repeats, the gene is in frame, which results in enzymatic modification of the LOS structure. Capsule and porin are not subject to high frequency variation. Opacity proteins (Opa) vary by a mechanism similar to that described for LOS. Slipped-strand mispairing results in changes in the number of CTCTT repeats within the opa structural gene. Some Opa proteins are synthesized because the number of repeats results in the proteins being in frame. Other Opa proteins are not expressed because the number of repeats within the gene results in the protein being out of frame. Correct answer = B. N. meningitidis has a polysaccharide capsule that is an important surface-exposed virulence factor. The chemical composition of the capsule defines the serogroup of the meningococcal strain. There are 13 known serogroups. The meningococcal vaccine contains 4 of the 13 different capsule types, making it a tetravalent vaccine. Note that serogroup B capsule is a self-antigen and is therefore not part of the vaccine. Serogroup B N. meningitidis is endemic in industrialized countries but the vaccine is not protective against this serogroup. 12 Gastrointestinal Gram-negative Rods I. OVERVIEW All of the organisms covered in this chapter are routinely found in the gastrointestinal (GI) tract of humans or other animals. Many also have alternative habitats in soil or water. All are relatively hardy but are sensitive to drying, and all grow in the presence or absence of oxygen, being facultative anaerobes. They contain lipopolysaccharide (LPS), which is both antigenic and an important virulence factor (endotoxin). These gram-negative rods belong to diverse taxonomic groups. These facultative organisms constitute only a fraction of the total microbial flora of the GI tract as most bowel organisms are either gram-positive or gram-negative anaerobes. Different enteric gram-negative rods cause diseases within the GI tract, outside the GI tract, or in both locations. For example, diseases caused by members of the genera Escherichia, Salmonella, Yersinia, and Campylobacter can be both GI and extraintestinal; those caused by members of the genera Shigella, Helicobacter, and Vibrio are primarily GI; and those caused by members of the genera Enterobacter, Klebsiella, Serratia, and Proteus are primarily extraintestinal. Fecal contamination is commonly important in the transmission of those organisms that cause GI diseases. Gramnegative rods discussed in this chapter are listed in Figure 12.1. Free-living Bacteria Gram-negative Cocci Nonenteric Rods Enteric Rods Campylobacter Campylobacter fetus Campylobacter jejuni S Enterobacter Escherichia Escherichia coli S Helicobacter II. ESCHERICHIA COLI Escherichia coli is part of the normal flora of the colon in humans and other animals but can be pathogenic both within and outside of the GI tract. [Note: The differences in the degree of virulence of various E. coli strains is correlated with the acquisition of plasmids, integrated prophages, and pathogenicity islands.] E. coli has fimbriae or pili that are important for adherence to host mucosal surfaces, and different strains of the organism may be motile or nonmotile. Most strains can ferment lactose (that is, they are Lac+) in contrast to the major intestinal pathogens, Salmonella (see p. 115) and Shigella (see p. 119), which cannot ferment lactose (that is, they are Lac–). E. coli produces both acid and gas during fermentation of carbohydrates. Helicobacter pylori S Klebsiella Klebsiella oxytoca Klebsiella pneumoniae Figure 12.1 Classification of enteric gramnegative rods (figure continues on the next page). S See pp. 335, 339, and 341 for summaries of these organisms. 111 112 12. Gastrointestinal Gram-negative Rods A. Structure and physiology
Enteric Rods (continued) Proteus Providencia Salmonella Salmonella enteritidis Salmonella typhi S Salmonella typhimurium S Serratia Serratia marcescens Shigella Shigella sonnei S Vibrio Vibrio cholerae S Vibrio parahaemolyticus
Yersinia Yersinia enterocolitica Yersinia pseudotuberculosis Figure 12.1 (continued) Classification of enteric gramnegative rods. S See pp. 347, 348, and 353 for summaries of these organisms. Transmission of intestinal disease is commonly by the fecal–oral route, with contaminated food and water serving as vehicles for transmission. At least five types of intestinal infections that differ in pathogenic mechanisms have been identified (Figure 12.3): enterotoxigenic (ETEC), enteropathogenic (EPEC), enterohemorrhagic (EHEC), enteroinvasive (EIEC), and enteroaggregative (EAEC). E. coli all are basically the same organism, differing only by the acquisition of specific pathogenic traits. EHEC E. coli infection should be suspected in all patients with acute bloody diarrhea, particularly if associated with abdominal tenderness and absence of fever. 1. Enterotoxigenic E. coli: ETEC are a common cause of traveler’s FLAGELLUM LIPOPOLYSACCHARIDE • O antigen • H antigen CAPSULE • K antigen Figure 12.2 Electron micrograph of Escherichia coli showing virulence factors. diarrhea. Transmission occurs through food and water contaminated with human waste or by person-to-person contact. ETEC colonize the small intestine (pili facilitate the binding of the organism to the intestinal mucosa). In a process mediated by enterotoxins (see p. 51), ETEC cause prolonged hypersecretion of chloride ions and water by the intestinal mucosal cells, while inhibiting the reabsorption of sodium. The gut becomes full of fluid, resulting in significant watery diarrhea that continues over a period of several days. Enterotoxins include a heat-stable toxin (ST) that works by causing an elevation in cellular cyclic guanosine monophosphate (cGMP) levels, whereas a heat-labile toxin (LT) causes elevated cyclic adenosine monophosphate (cAMP) (Figure 12.4). [Note: LT is essentially identical to cholera toxin (see p. 121).] 2. Enteropathogenic E. coli: EPEC are an important cause of diar- rhea in infants, especially in locations with poor sanitation. The newborn becomes infected perinatally. The EPEC attach to mucosal cells in the small intestine by use of bundle-forming pili II. Escherichia coli 113 THERAPY1 STRAIN Escherichia coli ABBREVIATION SYNDROME Enterotoxigenic E. coli ETEC Watery diarrhea Antibiotics may be useful.2 Enteropathogenic E. coli EPEC Watery diarrhea of long duration, mostly in infants, often in developing countries Antibiotics may be useful.2 Enterohemorrhagic E. coli EHEC Bloody diarrhea; Hemorrhagic colitis and hemolytic uremic syndrome (HUS) Avoid antibiotics because of the possible risk of potentiating HUS. Enteroinvasive E. coli EIEC Bloody diarrhea Rehydrate and correct electrolyte abnormalities. Enteroaggregative E. coli EAEC Persistent watery diarrhea in children and patients infected with HIV Rehydrate and correct electrolyte abnormalities. Figure 12.3 Characteristics of intestinal infections caused by Eschericia coli. Fluoroquinolones are commonly used in adults for traveler’s diarrhea but are not recommended for children. 1Rehydration and correction of electrolyte abnormalities are essential for all diarrheal illnesses. 2Rifaximin is approved for the treatment of diarrhea caused by noninvasive strains of E. coli in patients age12 years and older. Rifaximin is a nonabsorable, gastrointestinal-selective, oral antibiotic. (BfpA). Characteristic lesions in the small intestine called attaching and effacing lesions (A/E), in addition to destruction of the microvilli, are caused by injection of effector proteins into the host cell by way of a type III secretion system (T3SS). EPEC cells are presented at the apex of pedestals elicited by dramatic cytoskeletal rearrangements, induced by the T3SS effectors. EPEC are not invasive and, thus, do not cause bloody diarrhea. Toxins are not elaborated by EPEC strains. Watery diarrhea results, which, on rare occasions, may become chronic. 1 LT binds to a receptor and activates adenylate cyclase. 3 IINTESTINAL LUMEN LT LT Elevated levels of cAMP cause active secretion of ions and water. HCO3– K+ Na+ Cl– H2O 3. Enterohemorrhagic E. coli: EHEC bind to cells in the large intes- tine via BfpA and, similar to EPEC, produce A/E lesions. However, in addition, EHEC produce one of two exotoxins (Shiga-like toxins 1 or 2), resulting in a severe form of copious, bloody diarrhea (hemorrhagic colitis) in the absence of mucosal invasion or inflammation. Serotype O157:H7 is the most common strain of E. coli that produce Shiga-like toxins. This strain is also associated with outbreaks of a potentially life-threatening, acute renal failure (hemolytic uremic syndrome, or HUS) characterized by fever, acute renal failure, microangiopathic hemolytic anemia and thrombocytopenia in children younger than ages 5 to 10 years. The primary reservoir of EHEC is cattle. Therefore, the possibility of infection can be greatly decreased by thoroughly cooking ground beef and pasteurizing milk. 4. Enteroinvasive E. coli: EIEC cause a dysentery-like syndrome with fever and bloody stools. Plasmid-encoded virulence factors are nearly identical to those of Shigella species. These virulence factors allow the invasion of epithelial cells (Ipa) and intercellular spread by use of actin-based motility. In addition, EIEC strains produce a hemolysin (HlyA). INTESTINAL MUCOSA + + Adenylate ATP cyclase cAMP ATP cAMP ATP cAMP 2 Adenylate cyclase produces elevated levels of cAMP. Figure 12.4 The action of Escherichia coli LT (heat-labile toxin). [Note: ST (heat-stable toxin) activates guanylate cyclase, causing production of cyclic guanosine monophosphate (cGMP) that also causes secretion.] 114 12. Gastrointestinal Gram-negative Rods 5. Enteroaggregative E. coli: EAEC also cause traveler’s diarrhea and persistent diarrhea in young children. Adherence to the small intestine is mediated by aggregative adherence fimbriae. The adherent rods resemble stacked bricks and result in shortening of microvilli. EAEC strains produce a heat-stable toxin that is plasmid encoded. An outbreak of E. coli infections in Germany in 2011, resulting in many cases of HUS and several deaths, was caused by a hybrid strain. The causative agent was an EAEC strain that had acquired the phage-encoded gene to produce Shiga-like toxin 2. The resulting strain was capable of tight adherence to the small intestine in addition to toxin production, which resulted in HUS. C. Clinical significance: extraintestinal disease The source of infection for extraintestinal disease is frequently the patient's own flora, in which the individual’s own E. coli is nonpathogenic in the intestine. However, it causes disease in that individual when the organism is found, for example, in the bladder or bloodstream (normally sterile sites). 1. Urinary tract infection: E. coli is the most common cause of uri- nary tract infection (UTI), including cystitis and pyelonephritis. Women are particularly at risk for infection. Uncomplicated cystitis (the most commonly encountered UTI) is caused by uropathogenic strains of E. coli, characterized by P fimbriae (an adherence factor) and, commonly, hemolysin, colicin V, and resistance to the bactericidal activity of serum complement. Complicated UTI (pyelonephritis) may occur in settings of obstructed urinary flow, which may be caused by nonuro pathogenic strains. 2. Neonatal meningitis: E. coli is a major cause of this disease occurring within the first month of life. The K1 capsular antigen, which is chemically identical to the polysaccharide capsule of group B Neisseria meningitidis, is particularly associated with such infections. 3. Nosocomial (hospital-acquired) infections: These include sep- sis/bacteremia, endotoxic shock, and pneumonia. D. Laboratory identification 1. Intestinal disease: Because E. coli is normally part of the intestinal flora, detection in stool cultures of disease-causing strains is generally difficult. EIEC strains often do not ferment lactose and may be detected on media such as MacConkey agar (see p. 23). EHEC, unlike most other strains of E. coli, ferment sorbitol slowly, if at all, and may be detected on MacConkey sorbitol agar. Current molecular techniques, such as polymerase chain reaction, may be employed to identify E. coli strains producing Shiga-like toxins. 2. Extraintestinal disease: Isolation of E. coli from normally sterile body sites (for example, the bladder or cerebrospinal fluid) is diagnostically significant. Specimens may be cultured on MacConkey III. Salmonella 115 Gram (–) rods Escherichia species MacConkey agar • Short rods • Facultative anaerobe • Ferments glucose • Most strains ferment lactose • Catalase positive • Oxidase negative • Culture on MacConkey agar
Escherichia coli • Urinary tract infection (UTI) 1 Ciprofloxacin 1 Trimethoprim/ sulfamethoxazole Figure 12.5 Summary of Escherichia species. • Local or systemic disease Test for sensitivity Empiric therapy may include: 1 1 Ampicillin 1 Cefotaxime 1 An aminoglycoside 1 Ciprofloxacin 1 Trimethoprim/sulfamethoxazole Indicates first-line drugs. agar. Strains of E. coli can be further characterized on the basis of serologic tests. E. Prevention and treatment Intestinal disease can best be prevented by care in selection, preparation, and consumption of food and water. Maintenance of fluid and electrolyte balance is of primary importance in treatment. Antibiotics may shorten duration of symptoms, but resistance is nevertheless widespread. Extraintestinal diseases require antibiotic treatment (Figure 12.5). Antibiotic sensitivity testing of isolates is necessary to determine the appropriate choice of drugs. III. SALMONELLA Members of the genus Salmonella can cause a variety of diseases, including gastroenteritis and enteric (typhoid) fever. Although Salmonella classification has undergone numerous revisions, currently, all strains affecting humans are grouped in a single species, Salmonella enteritidis , which has approximately 2,500 different serotypes, or serovars, including the clinically significant serotypes Typhimurium and Typhi. Most strains of Salmonella are Lac– and produce acid and gas during fermentation of glucose. They also produce H2S from sulfur-containing amino acids. • Meningitis in infants 1 Cefotaxime 116 12. Gastrointestinal Gram-negative Rods VILLI 1 LUMEN OF GUT Ingested Salmonella enter small intestinal cells by endocytosis. EPITHELIAL CELLS
Salmonella are widely distributed in nature. Serovar Typhi is an exclusively human pathogen, whereas other strains are associated with animals and foods (for example, eggs and poultry). Fecal–oral transmission occurs and Salmonella serovar Typhi may involve chronic carriers. Pet turtles have also been implicated as sources of infection. Young children and older adults are particularly susceptible to Salmonella infection. Individuals in crowded institutions may also be involved in Salmonella epidemics. M MACROPHAGES
SUBMUCO OSA SUBMUCOSA Salmonella invade epithelial cells of the small intestine. Disease may remain localized or become systemic, sometimes with disseminated foci. The organisms are facultative, intracellular parasites that survive in phagocytic cells (Figure 12.6). C. Clinical significance 2 Salmonella pass through endothelial cells to the submucosa, where they are taken up by macrophages. Salmonella infection can cause both intestinal and extraintestinal diseases. 1. Gastroenteritis: This localized disease (also called salmonellosis) is caused primarily by serovars Enteriditis and Typhimurium. Salmonellosis is characterized by nausea, vomiting, and diarrhea (usually nonbloody), which develop generally within 48 hours of ingesting contaminated food or water. Fever and abdominal cramping are common. In uncompromised patients, disease is generally self-limiting (48 to 72 hours), although convalescent carriage of organisms may persist for a month or more. More than 95 percent of cases of Salmonella infection are foodborne, and salmonellosis accounts for approximately 30 percent of deaths resulting from foodborne illnesses in the United States. 3 Macrophages carry Salmonella to the reticuloendothelial system where bacteria multiply intracellularly, causing lymphoid hyperplasia and hypertrophy. BOWEL 4 Salmonella reenter the bowel via the liver and gallbladder. Figure 12.6 Mechanism of Salmonella infection causing enteric fever. 2. Enteric or typhoid fever: This is a severe, life-threatening sys- temic illness, characterized by fever and, frequently, abdominal symptoms. It is caused primarily by serovar Typhi. Nonspecific symptoms may include chills, sweats, headache, anorexia, weakness, sore throat, cough, myalgia, and either diarrhea or constipation. About 30 percent of patients have a faint and evanescent (transient) maculopapular rash on the trunk (rose spots). The incubation period varies from 5 to 21 days. Untreated, mortality is approximately 15 percent. Among survivors, the symptoms generally resolve in 3 to 4 weeks. Timely and appropriate antibiotic therapy reduces mor tality to less than 1 percent and speeds resolution of fever. Complications can include intestinal hemorrhage and/or perforation and, rarely, focal infections and endocarditis. A small percentage of patients become chronic carriers. [Note: Infected gallbladders are the main source of chronic carriage.] Typhoid fever remains a global health problem. In the United States, however, typhoid fever has become less prevalent and is now primarily a disease of travelers and immigrants. III. Salmonella 117 Gram (–) rods Salmonella species Salmonella typhi Serovars Enteritidis and Typhimurium • Enterocolitis (gastroenteritis, food poisoning) Antibiotics are not normally used except in immunocompromised individuals to prevent systemic spread of the infection.They may be used in individuals older than age 50 years to prevent seeding of atherosolerotic plaques. Salmonella species on MacConkey agar • Short, flagellated rods • Facultative anaerobes • Ferment glucose • Do not ferment lactose • Catalase positive • Oxidase negative • Culture on MacConkey agar Serovar Typhi • Enteric (typhoid) fever and paratyphoid fever 1 Ceftriaxone 1 Ciprofloxacin Figure 12.7 Summary of Salmonella disease. 1 Indicates first-line drugs. 3. Other sites of Salmonella infection: Sustained bacteremia is often associated with vascular Salmonella infections that occur when bacteria seed atherosclerotic plaques. Salmonella can also cause abdominal infections (often of the hepatobiliary tract and spleen); osteomyelitis; septic arthritis; and, rarely, infections of other tissues or organs. Chronic carriage of non-typhoidal serovars may develop, although this is rare. D. Laboratory identification In patients with diarrhea, Salmonella can typically be isolated from stools on MacConkey agar or selective media (Figure 12.7). For patients with enteric fever, appropriate specimens include blood, bone marrow, urine, stool, and tissue from typical rose spots (if they are present). E. Treatment and prevention For gastroenteritis in uncompromised hosts, antibiotic therapy is often not needed and may prolong the convalescent carrier state. For enteric fever, appropriate antibiotics include β-lactams and fluoro quinolones (see Figure 12.7). Prevention of Salmonella infection is accomplished by proper sewage disposal, correct handling of food, and good personal hygiene. Two different vaccines are available to prevent typhoid fever: One vaccine is delivered orally and consists of live attenuated Salmonella serovar Typhi. The other vaccine consists of the Vi capsular polysaccharide and is delivered parenterally. Vaccination is recommended for people who travel from developed countries to endemic areas including Asia, Africa, and Latin America. 118 12. Gastrointestinal Gram-negative Rods IV. CAMPYLOBACTER Members of the genus Campylobacter are curved, spiral, or S-shaped organisms that microscopically resemble vibrios (Figure 12.8). A single, polar flagellum provides the organism with its characteristic darting motility. Somatic, flagellar, and capsular antigens all contribute to the numerous serotypes. Most Campylobacter are microaerophilic (that is, they require oxygen but at lower concentrations than that found in air). Members of this genus use a respiratory pathway and do not ferment carbohydrates. Campylobacter infect the intestine and can cause ulcerative, inflammatory lesions in the jejunum, ileum, or colon. Bacteremia may occur. Figure 12.8 Micrograph showing the S-shaped cells of Campylobacter jejuni. Bacteria that may cause food poisoning due to preformed toxins Bacillus cereus Clostridium botulinum Clostridium perfringens Staphylococcus aureus Because the toxins are ingested preformed and no microbial growth within the host is required, symptoms occur rapidly, usually within 2–12 hour Bacteria that may cause foodborne illness after food is ingested Campylobacter jejuni Escherichia coli Salmonella species Listeria monocytogenes Shigella species Vibrio cholerae Because microbial growth within the host is required, symptoms occur more slowly, usually after at least 24 hours. Figure 12.9 Characteristics of common forms of bacterial foodborne illness.
Campylobacter are widely distributed in nature as commensals of many different vertebrate species, including mammals and fowl, both wild and domestic. These serve as reservoirs of infection. Campylobacter are transmitted to humans primarily via the fecal–oral route through direct contact, exposure to contaminated meat (especially poultry), or contaminated water supplies. B. Pathogenesis and clinical significance Campylobacter may cause both intestinal and extraintestinal disease. The characteristics of some common forms of bacterial foodborne illness are shown in Figure 12.9. [Note: Food infection should be distinguished from food poisoning. Food infections (like Campylobacter) have longer incubation periods and require colonization by the bacterium. Food poisonings have shorter incubation periods and only require ingestion of the toxin.] C. jejuni typically causes an acute enteritis in otherwise healthy individuals following a 1- to 7-day incubation. The disease lasts days to several weeks and, generally, is self-limiting. Symptoms may be both systemic (fever, headache, myalgia) and intestinal (abdominal cramping and diarrhea, which may or may not be bloody). Campylobacter jejuni is a cause of both traveler’s diarrhea and pseudoappendicitis (symptoms simulating appendicitis without inflammation of the appendix). Bacteremia (often transient) may occur, most often in infants and older adults. Sustained bacteremia usually reflects host compromise. Complications include septic abortion, reactive arthritis, and Guillain-Barré syndrome. Important virulence factors include a cytotoxin that may be involved in inflammatory colitis and an enterotoxin (related to cholera toxin) that results in increased adenylyl cyclase activity and, therefore, electrolyte and fluid imbalance. Campylobacter is currently one of the leading causes of foodborne disease in the United States. C. Laboratory identification Campylobacter can be isolated from feces using special selective media and microaerophilic conditions. Because of their small size, these organisms are not retained by bacteriologic filters that hold back most other bacteria. Thus, filtration of the fecal suspension may enhance recovery rate. Presumptive diagnosis can be made on
Gram (–) rods 119 Campylobacter species • Curved, spiral, or S-shaped rods Single, polar flagellum, resulting in • characteristic darting motion • Microaerophilic • Do not ferment carbohydrates Culture on selective medium (blood • agar containing antibiotics to inhibit Campylobacter jejuni • Acute enteritis • Traveler's diarrhea • Pseudoappendicitis 1 Erythromycin growth of other fecal flora) 1 Ciprofloxacin Campylobacter jejuni Campylobacter jejuni (Preston selective medium) Figure 12.10 Summary of Campylobacter disease. 1 Indicates first-line drugs. the basis of finding curved organisms with rapid, darting motility in a wet mount of feces. D. Treatment and prevention Diarrhea should be treated symptomatically with fluid and electrolyte replacement. For patients with more severe symptoms (for example, high fever, bloody diarrhea, worsening illness, or illness of more than 1 week’s duration), antibiotics should be administered. For C. jejuni, ciprofloxacin is the drug of choice, but other antibiotics are also effective (Figure 12.10). For Campylobacter fetus, ampicillin or third-generation cephalosporins are effective. Thorough cooking of potentially contaminated foods (for example, poultry) and pasteurization of milk and milk products are essential to prevention of campylobacteriosis. Also, surfaces used to prepare raw meat or poultry should be disinfected before using them for uncooked food such as salads.
Shigella are typically spread from person to person, with contaminated stools serving as a major source of organisms. Humans are the only natural host for Shigella species. Flies and contaminated food or water can also transmit the disease. Shigellosis has a low infectious dose: Approximately 10–100 viable organisms are sufficient to cause disease. Therefore, secondary cases within a household are common, particularly under conditions of crowding or poor sanitation. The 40 serotypes of Shigella are organized into four groups (A, B, C, and D) based on the serologic relatedness of their polysaccharide O antigens. Group D ( Shigella sonnei ) is the 120 1 12. Gastrointestinal Gram-negative Rods Ingested Shigellae enter large intestinal and rectal cells by endocytosis. 2 LUMEN OF GUT VILLI Shigellae escape from the endocytic vesicles and multiply inside the cell, protected from macrophages. EPITHELIAL CELLS 4 A mucosal abscess forms as the cells die, causing diarrhea with blood, mucus, and painful abdominal cramping. MACROPHAGES 3 Shigellae invade neighboring cells. Figure 12.11 Mechanism of Shigella infection causing diarrhea. serogroup found most commonly in the United States. Shigella flexneri is the second most common species isolated in the United States and has been associated with outbreaks among sexually active men who have sex with men. Shigella dysenteriae causes the most serious infections, including HUS similar to that caused by EHEC. S. dysenteriae type 1 produces Shiga toxin, which is structurally and genetically very similar to Shiga-like toxins 1 and 2 produced by E. coli virotypes. All Shiga and Shiga-like toxins are capable of resulting in HUS in susceptible individuals. B. Pathogenesis and clinical significance Shigellae invade and destroy the mucosa of the large intestine. Infection rarely penetrates to deeper layers of the intestine and does not lead to bacteremia (Figure 12.11). The Shigellae invade the colonic epithelium by expression of plasmid-encoded virulence genes that encode a type III secretion system. Injection of effector proteins results in bacterial engulfment. The same plasmid encodes proteins that allow the Shigellae to polymerize actin at one pole, thereby propelling the bacterium though the cytoplasm and into adjacent cells. This virulence plasmid is also possessed by EIEC. An exotoxin (Shiga toxin) with enterotoxic and cytotoxic properties has been isolated from S. dysenteriae type 1, and its toxicity results in the development of hemorrhagic colitis and HUS. Shigellae cause classic bacillary dysentery, characterized by diarrhea with blood, mucus (“currant jelly” stools), and painful abdominal cramping. The disease is generally most severe in the very young; older adults; and among malnourished individuals, in whom shigellosis may lead to VI. Vibrio 121 Shigella species Gram (–) rods Shigella sonnei Bacillary dysentery (shigellosis) Nonmotile and • nonencapsulated • Cannot ferment lactose Most strains do not produce • gas in a mixed-acid • 1 Azithromycin 1 Ciprofloxacin fermentation of glucose Hektoen agar Gram stain Figure 12.12 Summary of Shigella disease. 1 Indicates first-line drugs. severe dehydration and, sometimes, death. Among uncompromised populations, untreated dysentery commonly resolves in a week but may persist longer. C. Laboratory identification During acute illness, organisms can be cultured from stools using differential, selective Hektoen agar or other media specific for intestinal pathogens. D. Treatment and prevention Antibiotics (for example, ciprofloxacin or azithromycin) can reduce the duration of illness and the period of shedding organisms but usage is controversial because of widespread antibiotic resistance (Figure 12.12). Protection of the water and food supply and personal hygiene are crucial for preventing Shigella infections. Candidate vaccines in advanced development stages include a conjugate vaccine composed of O-antigen polysaccharides from Shigellae and a live attenuated vaccine. VI. VIBRIO Members of the genus Vibrio are short, curved, rod-shaped organisms. Vibrios are closely related to the family Enterobacteriaceae. They are rapidly motile by means of a single polar flagellum. [Note: This contrasts with the peritrichous flagella (distributed all over the surface) of the motile Enterobacteriaceae.] O and H antigens are both present, but only O antigens are useful in distinguishing strains of vibrios that cause epidemics. Vibrios are facultative anaerobes. The growth of many Vibrio strains either requires or is stimulated by NaCl. Pathogenic vibrios include: 1) Vibrio cholerae, serogroup O1 strains that are associated with epidemic cholera; 2) non-O1 V. cholerae and related strains that cause sporadic cases of choleralike and other illnesses; and 3) Vibrio parahaemolyticus and other halophilic vibrios, which cause gastroenteritis and extraintestinal infections. Culture on selective medium • such as Hektoen agar 122 12. Gastrointestinal Gram-negative Rods A. Epidemiology Cholera toxin binds to receptor. 1 Cholera toxin C GM1 ganglioside receptor A A B B B B B Gs-protein Inactive adenylate cyclase "A" subunit enters the cell membrane, activating Gs, which, in turn, activates adenylate cyclase. 2 H2O HCO 3 K+ Na+ ClB B B A B B ATP Active adenylate cyclase 3 Adenylate cyclase produces elevated cAMP. 4 cAMP + PPi + cAMP causes active secretion of ions and water. Figure 12.13 Action of cholera toxin. cAMP = cyclic adenosine monophosphate, PPi = pyrophosphate.
1See Chapter 8 in Lippincott’s Illustrated Reviews: Biochemistry for a discussion of the mechanism of action of Gs proteins. VII. Yersinia 123 Gram (–) rods Vibrio species • Short, curved, rod shaped Rapidly motile as a result of single • polar flagellum • Facultative anaerobes Growth of many Vibrio strains • requires or is stimulated by NaCl Culture on blood or MacConkey agar • Vibrio cholerae • Cholera 1 Doxycycline 1 Ciprofloxacin Vibrio cholerae (colorized scanning electron micrograph) Figure 12.14 Summary of Vibrio disease. 1 Indicates first-line drugs. chemical testing is necessary for specific identification of V. cholerae. E. Treatment and prevention Replacement of fluids and electrolytes is crucial in preventing shock and does not require bacteriologic diagnosis. Antibiotics (doxycycline is the drug of choice) can shorten the duration of diarrhea and excretion of the organism (Figure 12.14). Prevention relies primarily on public health measures that reduce fecal contamination of water supplies and food. Adequate cooking of foods can minimize transmission. Vaccines that are only modestly protective are available in many other countries but not in the United States. F. Vibrio parahaemolyticus and other halophilic, noncholera vibrios These organisms are characterized by a requirement for higherthan-usual concentrations of NaCl and their ability to grow in 10 percent NaCl. They are common in coastal seawaters. Vibrio parahaemolyticus is associated with outbreaks of GI illness that result from ingestion of contaminated and inadequately cooked seafood, especially shellfish and crustaceans. The disease is selflimiting, and antibiotics do not alter the course of infection. Neither human carriers nor other mammalian reservoirs have been identified. Other halophilic, noncholera vibrios are associated with soft tissue infections and septicemia resulting either from contact of wounds with contaminated seawater or from ingestion of contaminated seafood. For soft tissue infections, prompt administration of antibiotics, such as tetracycline, fluoroquinolones or cefotaxime, is important, and surgical drainage/debridement may be required. Bacteremia is associated with high mortality, especially when caused by Vibrio vulnificus. VII. YERSINIA The genus Yersinia includes three species of medical importance: Yersinia enterocolitica and Yersinia pseudotuberculosis, both potential pathogens of the GI tract that are discussed in this chapter, and Yersinia pestis, the etiologic agent of bubonic plague, which is dis- 124 12. Gastrointestinal Gram-negative Rods cussed in Chapter 13 (see p. 143). Y. enterocolitica and Y. pseudotuberculosis are both motile when grown at 25oC but not at 37oC. Multiple serotypes of both species exist, and, as with Y. pestis, the type III secretion system and Yop proteins are virulence factors for avoidance of phagocytosis. In contrast to most pathogenic Enterobacteriaceae, these strains of Yersinia grow well at room temperature as well as at 37oC. Most strains are Lac–. A. Pathogenesis and clinical significance Infection occurs via ingestion of food that has become contaminated through contact with colonized domestic animals, abattoirs, or raw meat (especially pork). Y. enterocolitica is a relatively uncommon cause of enterocolitis in the United States, and Y. pseudotuberculosis is even rarer. Infection results in ulcerative lesions in the terminal ileum, necrotic lesions in Peyer patches, and enlargement of mesenteric lymph nodes. Enterocolitis caused by Yersinia is characterized by fever, abdominal pain, and diarrhea. When accompanied by right lower quadrant tenderness and leukocytosis, the symptoms are clinically indistinguishable from appendicitis. Symptoms commonly resolve in 1 to 3 weeks. Sequelae may include reactive polyarthritis and erythema nodosum. Other, less common clinical presentations include exudative pharyngitis and, in compromised patients, septicemia. B. Laboratory identification Yersinia can be cultured from appropriate specimens on MacConkey or cefsulodin-irgasan-novobiocin (CIN, a medium selective for Yersinia) agars. Identification is based on biochemical screening. In the absence of a positive culture, serologic tests for anti-Yersinia antibodies may assist in diagnosis. C. Treatment and prevention Reducing infections and outbreaks rests on measures to limit potential contamination of meat, ensuring its proper handling and preparation. Antibiotic therapy (for example, with ciprofloxacin or trimethoprim-sulfamethoxazole) is essential for systemic disease Gram (–) rods Yersinia species Yersinia enterocolitica (gastroenteritis) • Yersiniosis • Septicemia 1 Ciprofloxacin1 1 Trimethoprim/ sulfamethoxazole Gram stain of Yersinia enterocolitica 1Antibiotic therapy is essential for systemic disease (sepsis). Figure 12.15 Summary of Yersinia enterocolitica disease. 1 Indicates first-line drugs. • Motile • No capsule Found in contaminated water • supplies, unpasteurized milk, contaminated food. VIII. Helicobacter 125 (sepsis), but is of questionable value for self-limited diseases such as enterocolitis (Figure 12.15). VIII. HELICOBACTER Members of the genus Helicobacter are curved or spiral organisms (Figure 12.16). They have a rapid, corkscrew motility resulting from multiple polar flagella. Helicobacter pylori, the species of human significance, is microaerophilic, and produces urease. It causes acute gastritis and duodenal and gastric ulcers. H. pylori (and several other Helicobacter species) are unusual in their ability to colonize the stomach, where low pH normally protects against bacterial infection. H. pylori infections are relatively common and worldwide in distribution. A. Pathogenesis Transmission of H. pylori is thought to be from person to person, because the organism has not been isolated from food or water. Untreated, infections tend to be chronic, even lifelong. H. pylori colonizes gastric mucosal (epithelial) cells in the stomach and metaplastic gastric epithelium in the duodenum or esophagus but does not colonize the rest of the intestinal epithelium. The organism survives in the mucus layer that coats the epithelium and causes chronic inflammation of the mucosa (Figure 12.17). Although the organism is noninvasive, it recruits and activates inflammatory cells. Urease released by H. pylori produces ammonia ions that neutralize stomach acid in the vicinity of the organism, favoring bacterial multiplication. Ammonia may also both cause injury and potentiate the effects of a cytotoxin produced by H. pylori. Helicobacter pylori MUCOUS LAYER Figure 12.16 Helicobacter pylori in a gastric pit. 1 Urea NH3 CO2
NH3 NH3 NH3 Hemin Urea 2
Cytotoxin NH3 3 CONNECTIVE TISSUE Figure 12.17 Helicobacter pylori infection, resulting in ulceration of the stomach.
126 15. Gastrointestinal Gram-negative Rods Helicobacter species Gram (–) rods • Curved or spiral rods Multiple polar flagella, • which give organism rapid, Helicobacter pylori • Acute gastritis 1 1 Amoxicillin 1 Clarithromycin 1 Proton pump inhibitor corkscrew motility • Urease positive Culture on selective • medium containing antibiotics 1A number of alternate multi-drug regimens have have been shown to be effective in eradicating H. pylori. Helicobacter pylori (Note: Young cultures grown in vitro frequently stain gram-positive) Figure 12.18 Summary of Helicobacter disease. 1 Helicobacter pylori colonies on an agar plate Indicates first-line drugs.
IX. Other Enterobacteriaceae 127 IX. OTHER ENTEROBACTERIACEAE Other genera of Enterobacteriaceae, such as Klebsiella, Enterobacter, Proteus, and Serratia, which can be found as normal inhabitants of the large intestine, include organisms that are primarily opportunistic and often nosocomial pathogens. Widespread antibiotic resistance among these organisms necessitates sensitivity testing to determine the appropriate antibiotic treatment. A. Enterobacter Enterobacter species are motile and Lac+. They rarely cause primary disease in humans but frequently colonize hospitalized patients, especially in association with antibiotic treatment, indwelling catheters, and invasive procedures. These organisms may infect burns, wounds, and the respiratory (causing pneumonia) and urinary tracts. B. Klebsiella Klebsiellae are large, nonmotile bacilli that possess a luxurious capsule (Figure 12.19). They are Lac +. Klebsiella pneumoniae and Klebsiella oxytoca cause necrotizing lobar pneumonia in individuals compromised by alcoholism, diabetes, or chronic obstructive pulmonary disease. K. pneumoniae also causes UTI and bacteremia, particularly in hospitalized patients. The organism formerly known as Calymmatobacterium granulomatis has been reclassified as Klebsiella granulomatis, based upon genome sequence analysis. K. granulomatis causes donovanosis, or granuloma inguinale, which is a sexually transmitted infection that is rare in the United States but endemic in Africa, India, South America, and Australia. The disease presents, after a prolonged incubation period, as subcutaneous nodules that break down, revealing one or more painless granulomatous lesions. The gram-negative rods can be identified within mononuclear cells by staining the material collected from the border of lesions. C. Serratia Serratia are motile and ferment lactose slowly, if at all. The species of Serratia that most frequently causes human infection is Serratia marcescens. Serratia can cause extraintestinal infections such as those of the lower respiratory and urinary tracts, especially among hospitalized patients. D. Proteus, Providencia, and Morganella Members of these genera are agents of urinary tract and other extraintestinal infections. Proteus species are relatively common causes of uncomplicated as well as nosocomial UTI. Other extraintestinal infections, such as wound infections, pneumonias, and septicemias, are associated with compromised patients. Proteus organisms produce urease, which catalyzes the hydrolysis of urea to ammonia. The resulting alkaline environment promotes the precipitation of struvite stones containing insoluble phosphates of magnesium and phosphate. Figure 12.19 Micrograph showing rod-shaped Klebsiella pneumoniae cells. 128 12. Gastrointestinal Gram-negative Rods Study Questions Choose the ONE correct answer 12.1 A Lac+, glucose-fermenting, gram-negative rod isolated from a previously healthy child with bloody diarrhea is most likely to be: A. Shigella sonnei. B. Pseudomonas aeruginosa. C. Escherichia coli. D. Salmonella enterica. E. Helicobacter pylori. 12.2 A male older adult, hospitalized and recovering from cardiac bypass surgery, develops pneumonia. Sputum culture reveals a gram-negative rod that produces a green pigment but does not ferment carbohydrates. The most likely organism is: A. Klebsiella pneumoniae. B. Serratia species. C. Proteus species. D. Enterobacter species. E. Pseudomonas aeruginosa. 12.3 An older, alcoholic male develops severe, necrotizing lobar pneumonia. The organism is Lac+ and produces a luxuriant capsule. The most likely agent is: A. Klebsiella pneumoniae. B. Serratia species. C. Yersinia pseudotuberculosis. D. Pseudomonas aeruginosa. E. Campylobacter fetus. 12.4 A young man returned from a backpacking trip in Mexico suffering from a high fever, pain in the abdomen, and watery diarrhea. The emergency room doctor noted a faint rash on the patient's abdomen and chest. A blood specimen was collected and plated on MacConkey agar, incubated at 37°C in ambient air. Lac– colonies grew on the plates. The cultured organism was a gram-negative rod that did not produce Shiga or Shiga-like toxins. The most likely etiological agent for this man's disease is: A. Enterohemorrhagic Escherichia coli B. Shigella dysenteriae C. Salmonella typhi D. Helicobacter pylori E. Campylobacter jejuni Correct answer = C. Escherichia coli is Lac+, and enteroinvasive strains characteristically cause a dysentery-like syndrome. Shigella characteristically causes bloody diarrhea (dysentery) but is Lac–. Pseudomonas aeruginosa characteristically causes infections in compromised hosts and is Lac–. Salmonella is also Lac–. Helicobacter pylori causes gastritis. Correct answer = E. All five organisms are opportunists capable of causing pneumonia in compromised patients. However, the first four are members of the family Enterobacteriaceae and, by definition, can ferment carbohydrates. In addition, none of these organisms are known to produce a green pigment, although Serratia may produce a red pigment. Pseudomonas aeruginosa is an obligate aerobe that uses respiratory pathways exclusively. Production of green pyocyanin pigment regularly occurs. Correct answer = A. The combination of necrotizing pneumonia and an alcoholic patient suggests Klebsiella pneumoniae, and the laboratory data (Lac + and a luxuriant capsule) are consistent. Although Serratia can cause pneumonia in compromised patients, necrosis is not a characteristic feature. Moreover, the organism ferments lactose slowly, if at all, and does not have a luxuriant capsule. Yersinia pseudotuberculosis is Lac – and rarely causes pneumonia. Pseudomonas aeruginosa can cause pneumonia in compromised patients but does not ferment lactose. Campylobacter fetus typically causes bacteremia and disseminated infections. Correct answer = C. This person is suffering from typhoid fever, caused by Salmonella enterica serovar typhi . Both enterohemorrhagic Escherichia coli and Shigella dysenteriae produce Shiga or Shiga-like toxins, which were not detected in this case. Both Helicobacter pylori and Campylobacter jejuni are curved organisms, which is not consistent with the cell morphology of the organism causing this infection. Moreover, neither H. pylori nor C. jejuni can be cultured on typical primary plating media such as MacConkey agar. Other Gramnegative Rods 13
II. HAEMOPHILUS Cells of Haemophilus influenzae—the major human pathogen of this genus—are pleomorphic, ranging from coccobacilli to long, slender filaments. H. influenzae may produce a capsule (six capsular types have been distinguished) or may be unencapsulated (Figure 13.2). The capsule is an important virulence factor. Serious, invasive H. influenzae disease is associated particularly with capsular type b (Hib), which is composed of polyribose phosphate. Hib is especially important as a pathogen of young children, although it can cause disease in individuals of all age groups. Nontypeable (unencapsulated) strains may also cause serious disease and are a significant cause of pneumonia among older adults and individuals with chronic lung disease. Free-living Bacteria Gram-negative Cocci Enteric Rods Nonenteric Rods Bartonella species Bordetella parapertussis Bordetella pertussis S Brucella species S Francisella tularensis S Haemophilus influenzae S Legionella pneumophila S Pasteurella multocida Pseudomonas aeruginosa Yersinia pestis S S Figure 13.1 Classification of other gramnegative rods. S See pp. 333, 334, 340, 342, 346, and 353 for summaries of these organisms. 129 130 13. Other Gram-negative Rods A. Epidemiology
Capsule
Figure 13.2 Haemophilus influenzae (electron micrograph) showing thick capsules.
Contiguous spread (often involving unencapsulated strains) from site of colonization in the respiratory tract Otitis media Sinusitis Disseminated spread (often involving capsular type b strains) via blood stream Meningitis Epiglottitis Bronchopneumonia
Septic arthritis Figure 13.3 Infections caused by Haemophilus influenzae. A definitive diagnosis generally requires identification of the organism (for example, by culture on chocolate agar). H. influenzae is fastidious and requires supplementation with hemin, factor X, and nicotinamide adenine dinucleotide (NAD+), factor V. H. influenzae can be cultured on chocolate agar (lysed blood cells provide these growth factors) but cannot be grown on blood or MacConkey agar. Isolation from normally sterile sites and fluids, such as blood, cerebrospinal fluid (CSF), and synovial fluid, is significant, whereas isolation from pharyngeal cultures is inconclusive. Rapid diagnosis is crucial because of the potentially fulminant course of type b infections. In cases of meningitis, Gram staining of CSF commonly reveals pleomorphic, gram-negative coccobacilli (Figure 13.5). Type b capsule may be identified directly in CSF, either by the capsular swelling (quellung) reaction (see p. 26) or by immunofluorescent staining (see p. 28). Capsular antigen may be detected in CSF or other body fluids using immunologic tests, such as latex agglutination, countercurrent immunoelectrophoresis, ELISA, and radio immunoassay. 131
Incidence (cases per 100,000 persons) II. Haemophilus Haemophilus influenzae, type b (Hib) 25 PRP VACCINES INTRODUCED 20 15 10 PRP-CONJUGATE 5 VACCINES INTRODUCED 1980 1990 2000 Figure 13.4 Incidence of Haemophilus influenzae type b meningitis in a pediatric population in the United States. PRP = polyribose phosphate. Gram (–) rods Pleomorphic in shape, • ranging from small Haemophilus species coccobacilli to long slender filaments Obligate parasites, • requiring hemin and NAD + for growth Culture on chocolate agar • containing hemin and Haemophilus influenzae on chocolate agar Gram stain of Haemophilus influenzae NAD+ Haemophilus influenzae media • Otitis • Sinusitis • Pneumonia meningitis • Bacterial Epiglottitis • Septic arthritis • 1 Ampicillin/clavulanate1 1 Cefotaxime2 1 Ceftriaxone2 2 Trimethoprimsulfamethoxazole3 1Non-life-threatening illness 2Meningitis, epiglottitis, and other life-threatening illness 3Respiratory infections and otitis; not meningitis Figure 13.5 Summary of Haemophilus disease. adenine dinucleotide. Gross specimen from patient with bacterial meningitis, showing copious purulent exudate covering base of brain 1 Indicates first-line drugs; 2 indicates alternative drugs. NAD = nicotinamide 132 13. Other Gram-negative Rods III. BORDETELLA Bordetella pertussis and Bordetella parapertussis are the human pathogens of this genus. The former causes the disease pertussis (also known as whooping cough), and the latter causes a mild pertussis-like illness. Whooping cough is a highly contagious disease and a significant cause of morbidity and mortality worldwide (51 million cases and 600,000 deaths each year). Members of the genus Bordetella are aerobic. They are small, encapsulated coccobacilli that grow singly or in pairs. They can be serotyped on the basis of cell-surface molecules including adhesins and fimbriae. A. Epidemiology The major mode of transmission of Bordetella is via droplets spread by coughing, but the organism survives only briefly outside the human respiratory tract. The incidence of whooping cough among different age groups can vary substantially, depending on whether active immunization of young children is widespread in the community. In the absence of an immunization program, disease is most common among young children (ages 1 to 5 years). Adolescent and adult household members, whose pertussis immunity has disappeared, are an important reservoir of pertussis for young children. B. Pathogenesis
Bordetella pertussis Pertussis toxin Dermonecrotic toxin Lymphocytosis, sensitization to histamine, activation of insulin production resulting in hypoglycemia Causes vasoconstriction and ischemic necrosis Filamentous hemagglutinin Tracheal cytotoxin Facilitates attachment of bacteria to ciliated epithelial cells Inhibits cilia movement and regeneration of damaged cells Adenylyl cyclase toxin Fimbriae Decreased chemotaxis and phagocytosis of bacteria Promote attachment of bacteria to host cells Figure 13.6 Toxins and virulence factors produced by Bordetella pertussis. III. Bordetella 133 and other virulence factors that interfere with ciliary activity, eventually causing death of these cells (Figure 13.6). C. Clinical significance The incubation period for pertussis generally ranges from 1 to 3 weeks (Figure 13.7). The disease can be divided into two phases: catarrhal and paroxysmal. 1. Catarrhal phase: This phase begins with relatively nonspecific symptoms, such as rhinorrhea, mild conjunctival infection (hyperemia, or bloodshot conjunctivae), malaise, and/or mild fever, and then progresses to include a dry, nonproductive cough. Patients in this phase of disease are highly contagious. 2. Paroxysmal phase: With worsening of the cough, the paroxysmal phase begins. The term “whooping cough” derives from the paroxysms of coughing followed by a “whoop” as the patient inspires rapidly. Large amounts of mucus may be produced. Paroxysms may cause cyanosis and/or end with vomiting. [Note: Whooping may not occur in all patients.] Pertussis typically causes leukocytosis that can be quite striking as the total white blood cell count sometimes exceeds 50,000 cells/μ L (nor mal range = 4,500–11,000 white blood cells/μL), with a striking predominance of lymphocytes. Following the paroxysmal phase, convalescence requires at least an additional 3 to 4 weeks. During this period, secondary complications, such as infections (for example, otitis media and pneumonia) and central nervous system (CNS) dysfunction (for example, encephalopathy or seizures), may occur. Disease is generally most severe in infants. D. Laboratory identification Presumptive diagnosis may be made on clinical grounds once the paroxysmal phase of classic pertussis begins. Pertussis may be suspected in an individual who has onset of catarrhal symptoms within 1 to 3 weeks of exposure to a diagnosed case of pertussis. Culture of B. pertussis on Bordet-Gengou or Regan-Lowe media (selective and enrichment media) from the nasopharynx of a symptomatic patient supports the diagnosis. The organism produces pin- Intensity of symptoms Incubation 0 1 Catarrhal phase Paroxysmal phase Runny nose Malaise Fever Cough Vomiting Leukocytosis 2 3 4 Weeks Figure 13.7 Clinical presentation of Bordetella pertussis disease. 5 Convalescence 6 7 8 9 10 134 13. Other Gram-negative Rods point colonies in 3 to 6 days on selective agar medium (for example, one that contains blood and charcoal), which serves to absorb and/or neutralize inhibitory substances and is supplemented with antibiotics to inhibit growth of normal flora. More rapid diagnosis may be accomplished using a direct fluorescent antibody test to detect B. pertussis in smears of nasopharyngeal specimens. Serologic tests for antibodies to B. pertussis are primarily useful for epidemiologic surveys. E. Treatment Erythromycin is the drug of choice for infections with B. pertussis, both as chemotherapy (where it reduces both the duration and severity of disease) and as chemoprophylaxis for household contacts (see Figure 13.9). For erythromycin treatment failures, trimethoprim-sulfamethoxazole is an alternative choice. Patients are most contagious during the catarrhal stage and during the first 2 weeks after onset of coughing. Treatment of the infected individuals during this period limits the spread of infection among household contacts. Incidence (per 100,000) 100 5 mm is interpreted as positive in the following populations: who have had contact • Persons with infectious individuals with an abnormal • Persons chest radiograph HIV-infected and other immuno• suppressed persons An induration of >10 mm is interpreted as positive in the following populations: • Foreign-born persons from highprevalence countries • Residents of prisons, nursing • • homes, and other institutions Healthcare workers Persons with other medical risk factors An induration of >15 mm is interpreted as positive in the following populations: • Persons with no risk factors Figure 18.8 Interpretations of the Mantoux skin test for tuberculosis. 190 18. Mycobacteria And Actinomycetes a. Identification in clinical specimens: A microscopic search for Figure 18.9 Mycobacterium tuberculosis colonies grown on LowensteinJensen medium. acid-fast bacilli using techniques such as the Ziehl-Neelsen stain is the most rapid test for mycobacteria. However, M. tuberculosis cannot be reliably distinguished on morphologic grounds from other pathogens in the genus, from some saprophytic mycobacterial species that may contaminate glassware and reagents in the laboratory, or from those mycobacteria that may be part of the normal flora. Therefore, a definitive identification of M. tuberculosis can only be obtained by culturing the organism or by using one of the newer molecular methods described below. Although 2 to 8 weeks are required to culture the tubercle bacillus because of its slow growth on laboratory media, such cultures can detect small numbers of organisms in the original sample. Figure 18.9 shows a culture of M. tuberculosis. Isolation of the organism is essential for determining its antibiotic sensitivity, in addition to confirming the specific identity of the bacillus by growth and biochemical characteristics. b. Nucleic acid amplification: Molecular techniques are increas- ingly important in the diagnosis of tuberculosis because they have the potential to shorten the time required to detect and identify M. tuberculosis in clinical specimens. For example, the amplified M. tuberculosis direct test uses enzymes that rapidly make copies of M. tuberculosis 16S ribosomal RNA, which can be detected using genetic probes. The sensitivity of the test ranges from 75 to 100 percent, with a specificity of 95 to 100 percent, and it is used for patients whose clinical smears are positive for acid-fast bacilli and whose cultures are in progress. A second technique, the polymerase chain reaction (PCR), amplifies a small portion of a predetermined target region of the M. tuberculosis DNA. Using human sputum, commercial PCR kits can confirm the diagnosis of tuberculosis within 8 hours, with a sensitivity and specificity that rivals culture techniques. In addition, PCR analysis facilitates DNA fingerprinting of specific strains, allowing studies of the progress of epidemics. 7. Treatment: Several chemotherapeutic agents are effective against
have been isolated even prior to drug treatment. Therefore, the standard procedure is to begin treatment with two or more drugs to prevent outgrowth of resistant strains. Sensitivity tests, administered as soon as sufficient cultured organisms are available, are an important guide to modifying treatment. In most parts of the United States, 8 to 14 percent of M. tuberculosis strains are resistant to one or more of the primary drugs II. Mycobacteria 191 when initially isolated from new cases of tuberculosis. The higher incidence of multiple drug–resistant strains (MDR-TB) in some locations and patient populations (for example, prisons) is a cause for great concern. b. Course of treatment: Clinical tuberculosis requires a long course of treatment because of the characteristics of the organisms and the lesions they produce. For example, as intracellular pathogens, the bacilli are shielded from drugs that do not penetrate host cells, and large cavities with avascular centers are penetrated by drugs with difficulty. Further, in chronic or arrested tubercles, the organisms are nonproliferating and, therefore, not susceptible to many antimicrobial agents. Until recently, 12 to 18 months of drug administration was thought to be required for a clinical cure. In recent years, short courses of 6 months, beginning with a daily dose of a combination of drugs and later by twice-weekly doses, have been successful in curing uncomplicated tuberculosis (Figure 18.10). If the drugs are effective in the pulmonary form of tuberculosis, sputum acid-fast bacteria smears become negative, and the patient becomes noninfectious in 2 to 3 weeks. c. Directly observed therapy: Patient compliance is often low when Short therapy (6 months) effective in some patients with uncomplicated tuberculosis multiple drug schedules last for 6 months or longer. One successful strategy for achieving better treatment completion rates is “directly observed therapy,” in which patients take their medication while being supervised and observed. Some healthcare providers have embraced the concept of directly observed therapy, whereas others regard the strategy as expensive and intrusive, suitable only for individuals who have a history of noncompliance. 8. Prevention: Public health measures, such as tuberculin tests, chest radiographs, case registries, and contact tracing have done much to control tuberculosis at the population level. Standard therapy (12 to 18 months)
culin-positive but asymptomatic, chemotherapy is indicated in several situations, usually with the single antibiotic isoniazid. For example, people in whom a recent skin test conversion is documented or tuberculin-positive patients who need immunosuppressive therapy for another illness can be protected from active tuberculosis by this treatment. b. Vaccines: A vaccine against tuberculosis has been available since early in the 20th century. It is produced from Bacille Calmette-Guérin (BCG), an attenuated strain of Mycobacterium bovis. When injected intradermally, it can confer tuberculin hypersensitivity and an enhanced ability to activate macrophages that kill the pathogen. This vaccine is about 80 percent protective against serious forms of tuberculosis, such as meningitis in children, and has been used in mass immunization campaigns by the World Health Organization Figure 18.10 Duration of treatment for tuberculosis. 192 18. Mycobacteria And Actinomycetes In the United States, BCG vaccine is recommended only for tuberculinnegative individuals under sustained heavy risk of infection. and in several European countries. However, public health officials in the United States recommend that vaccination be considered only for tuberculin-negative individuals under sustained heavy risk of infection, such as special groups of healthcare workers and those at high risk in areas where MDR-TB is common (Figure 18.11). Vaccination results in conversion from PPD negative to PPD positive, thus obviating the utility of the only available surveillance method. B. Mycobacterium leprae Figure 18.11 Bacille Calmette-Guérin (BCG) vaccine is used throughout the world, but seldom in the United States. Leprosy, called Hansen’s disease in publications of the United States Public Health Service, is rare in this country, but a small number of cases, both imported and domestically acquired, are reported each year. Worldwide, it is a much larger problem, with an estimated 10 to 12 million cases. Dozens of cases in the United States have been linked to contact with or ingestion of armadillos, a known reservoir of the pathogen. 1. Pathogenicity: Mycobacterium leprae is transmitted from human Tuberculoid leprosy occur as large maculae • Lesions in cooler body tissues, such as • • skin (especially nose and outer ears), testicles, and superficial nerve endings Cell-mediated immune response Low infectivity Progression of disease Lepromatous leprosy • • • Extensive erythematous macules, papules, or nodules; extensive destruction of skin Immunity severely depressed High infectivity Figure 18.12 Classification of leprosy. to human through prolonged contact, for example, between exudates of a leprosy patient’s skin lesions and the abraded skin of another individual. The infectivity of M. leprae is low, and the incubation period protracted, so that clinical disease may develop years or even decades after initial contact with the organism. 2. Clinical significance: Leprosy is a chronic granulomatous condi- tion of peripheral nerves and mucocutaneous tissues, particularly the nasal mucosa. It occurs as a continuum between two clinical extremes: tuberculoid and lepromatous leprosy (Figure 18.12). In tuberculoid leprosy, the lesions occur as large maculae (spots) in cooler body tissues, such as skin (especially the nose, outer ears, and testicles), and in superficial nerve endings. Neuritis leads to patches of anesthesia in the skin. The lesions are heavily infiltrated by lymphocytes and giant and epithelioid cells, but caseation does not occur. The patient mounts a strong cell-mediated immune response and develops delayed hypersensitivity, which can be shown by a skin test with lepromin, a tuberculin-like extract of lepromatous tissue. There are few bacteria in the lesions (paucibacillary). The course of lepromatous leprosy is slow but progressive (Figure 18.13). Large numbers of organisms are present in the lesions and reticuloendothelial system (multibacillary), the results of a severely depressed immune system. No well-formed granulomas emerge. 3. Laboratory identification: M. leprae is an acid-fast bacillus. It has not been successfully maintained in artificial culture but can be grown in the footpads of mice and in the armadillo, which is a natural host and reservoir of the pathogen. Laboratory diagnosis of lepromatous leprosy, in which organisms are numerous, involves acid-fast stains of specimens from nasal mucosa or other infected areas. In tuberculoid leprosy, organisms are extremely rare, and diagnosis depends on clinical findings and the histology of biopsy material. III. Actinomycetes 193 4. Treatment and prevention: Several drugs are effective in the treat- ment of leprosy, including sulfones such as dapsone, rifampin, and clofazamine (see Figure 18.14). Treatment is prolonged, and combined therapy is necessary to ensure the suppression of resistant mutants. The fact that vaccination with BCG (see p. 192) has shown some protective effect in leprosy has encouraged further interest in vaccine development. Thalidomide, an inhibitor of tumor necrosis factor-α, is being distributed under tight restrictions for use as a treatment for erythema nodosum leprosum, a serious and severe skin complication of leprosy. III. ACTINOMYCETES Actinomycetes are a group of filamentous, branching, gram-positive organisms that easily fragment into slender rods (Figure 18.15). Although they superficially resemble fungi on morphologic grounds, they are prokaryotes of bacterial size. They are free-living, mostly soil organisms that are related to corynebacteria and mycobacteria as well as to the streptomycetes that are sources of important antibiotics. A. Actinomyces israelii Actinomyces israelii is part of the normal oral and intestinal flora in humans. The organism is a strict anaerobe. 1. Clinical significance: Actinomycosis is an infection in which a chronic suppurative abscess leads to scarring and disfigurement. The infection is probably initiated by accidental introduction of organisms into the underlying soft tissue during conditions of sufficient anaerobiasis to support their growth. About half of the cases have a cervicofacial location and are associated with poor dental hygiene and/or tooth extraction (“lumpy jaw”). Other cases involve the lung and chest wall, cecum, appendix, abdominal wall, and pelvic organs. The lesion (mycetoma) begins as a hard, red, relatively nontender swelling that develops slowly, becomes filled with liquid, and ruptures to the surface, discharging quantities of pus. It also spreads laterally, draining pus through several sinus tracts. 2. Laboratory identification: The most typical and diagnostic finding in actinomycosis is the presence of “sulfur granules” in the draining pus. These are small, firm, usually yellowish particles, which, in fact, do not contain sulfur. When examined under the microscope, sulfur granules appear as microcolonies composed of filaments of the organism embedded in an amorphous, eosinophilic material thought to be antigen–antibody complexes. The organism can be grown anaerobically on enriched media, such as thioglycollate broth or blood agar. Growth is slow, often requiring 10 to 14 days for visible colonies. 3. Treatment: Penicillin G is the treatment of choice for actinomyco- sis, although a number of antibiotics (clindamycin, erythromycin, and tetracycline) have been shown to have clinical effect. Figure 18.13 A. Leprosy in a 13-year-old Hawaiian boy in 1931. B. Same boy 2 years later. [Note: This patient had the misfortune of contracting leprosy before the era of effective antibiotics.] 194 18. Mycobacteria And Actinomycetes Other • Acid-fast rods Not colored by Gram stain • due to lipid-rich cell walls • Long, slender, nonmotile rods • Aerobic • Resistant to drying Culture Mycobacterium • tuberculosis on specialized Mycobacterium species Mycobacterium tuberculosis (acid-fast stain) Mycobacterium tuberculosis (colonies on LowensteinJensen media) Mycobacterium tuberculosis Mycobacterium leprae • Tuberculosis: • Hansen disease (leprosy) Isoniazid Dapsone Rifampin 1 Pyrazinamide Ethambutol or streptomycin • medium such as Lowenstein-Jensen agar; Mycobacterium leprae does not grow in culture 1 Rifampin Clofazamine Figure 18.14 Summary of Mycobacterium species. Red lines connecting drugs indicates concomitant use of multiple drugs. 1 Indicates first-line drugs. Treatment must be maintained for weeks to months and may be accompanied by surgical debridement and/or drainage. No significant resistance to penicillin G has been reported. [Note: Good oral hygiene is an important preventive measure.] B. Nocardia asteroides and Nocardia brasiliensis Nocardiae are aerobic soil organisms. Infections of humans and domestic animals are opportunistic and not transmissible from person to person. Instead, nocardiae are inhaled or acquired by contamination of skin wounds. 1. Clinical significance: The most common presentation of human nocardiosis is a pneumonia of rather chronic course with Figure 18.15 Actinomycetoma of the foot using a Brown-Brenn stain. Actinomycetoma is a chronic, granulomatous infection of the skin and subcutaneous tissue caused by Actinomycetes. abscesses, extensive necrosis, and cavity formation. The organisms may metastasize, with the brain and kidneys the most common secondary locations. Important predisposing conditions are immunosuppression associated with lymphoma or other malignancy or with drugs. In the United States, Nocardia asteroides is the more common organism associated with this infection. 2. Laboratory identification: Nocardiae are gram-positive but irregu- larly staining, branched filaments (Figure 18.16). They are usually numerous in clinical material and do not form sulfur granules. They stain weakly acid-fast after decolorization with 1 percent sulfuric acid alcohol but fully decolorize with the routine Ziehl- IV. Atypical Mycobacteria 195 Neelsen procedure. Nocardiae are strictly aerobic. They grow slowly on a variety of simple media (such as fungal media without antibiotics) and on standard blood agar. 3. Treatment: Tmethoprim-sulfamethoxazole (TMP-SMX) is consid- ered by most clinicians to be the drug of choice. Given that some isolates are resistant to TMP-SMX, formal antimicrobial susceptibility testing is always necessary to ensure optimal antibiotic therapy. Other antibiotics, such as ceftriaxone and minocycline, may be effective if in vitro susceptibility is demonstrated. The nocardiae are relatively resistant to penicillin. Surgical drainage of the lesions is important, and prolonged therapy may be required to eliminate the infection. IV. ATYPICAL MYCOBACTERIA The atypical mycobacteria are distinct from classical mycobacteria in that they are widespread in the environment and are not pathogenic in rodent animal models. The atypical mycobacteria are classified into four groups (Runyon groups I–IV) based upon several phenotypic characteristics, including pigment production and growth rate. Group I contains the photochromogens, which produce pigment in the light. This group grows very slowly and includes the species Mycobacterium kansasii and Mycobacterium marinum. M. kansasii causes a chronic pulmonary disease, which can spread within the lungs in a manner similar to tuberculosis. The organism is found in tapwater, primarily localized to the Midwestern states and Texas. M. marinum causes a cutaneous infection and is found in fresh- and saltwater habitats. Group II includes Mycobacterium scrofulaceum , which is a slow-growing atypical Mycobacterium species that produces pigment both in the light and in the dark. The pathogen causes cervical adenitis in children and is found in raw milk, dairy products, soil, and water. Clearance of this pathogen requires excision of the affected lymph nodes. Group III contains the slow-growing nonphotochromogens, including Mycobacterium aviumintracellulare complex and Mycobacterium ulcerans. M. avium and M. intracellulare are virtually indistinguishable diagnostically. They are both found ubiquitously in the environment and cause a serious disseminated disease, very similar to tuberculosis, in immunocompromised patients, in particular those with AIDS. These atypical mycobacteria are particularly resistant to antituberculosis drugs. M. ulcerans causes indolent cutaneous infections, known as Buruli ulcers in tropical countries, including those in Africa. The atypical mycobacteria within group IV grow rapidly but do not produce any pigment. This grouping includes three potential pathogens, although all are found ubiquitously in the environment. Mycobacterium abscessus causes a chronic lung disease, which can disseminate to skin, bone, and joints. Mycobacterium fortuitum and Mycobacterium chelonei primarily infect immunocompromised individuals to cause skin and soft tissue infections. Figure 18.16 Nocardiae. 196 18. Mycobacteria And Actinomycetes Study Questions Choose the ONE correct answer 18.1 Which one of the following is characteristic of mycobacteria? A. B. C. D. E. They contain mycolic acids. They are resistant to inactivation by heat. They grow extracellularly. They are anaerobic. They are spore forming. 18.2 An acid-fast smear on a patient’s sputum is positive. The tuberculin test, however, is negative. A more definitive diagnosis could be obtained by A. B. C. D. E. paying attention to the patient’s history. a more extensive physical examination. a chest radiograph. repeat of the sputum smear. laboratory culture and speciation. 18.3 Which of the following statements regarding Actinomyces and Nocardia is true? A. Both organisms have branching growth but are prokaryotes. B. Neither can be cultured in the laboratory C. Nocardia infections are endogenous and often initiated by trauma. D. Actinomyces usually causes infections in systemically compromised patients. E. Neither is sensitive to antibacterial drugs. Correct choice = A. Mycobacteria are unique in that their cell walls contain high concentrations of mycolic acids. Mycobacteria are not particularly heat resistant, as witnessed by their susceptibility to pasteurization. They are aerobic, intracellular organisms that do not form spores. Correct answer = E. Laboratory culture and speciation would best resolve the question, although any of the procedures listed might yield helpful information. The possibility of anergy could be investigated by skin testing for delayed hypersensitivity to unrelated antigens, but the patient might be anergic and still be infected with a mycobacterium. Correct answer = A. Both species appear as filamentous rods or branching forms in stained preparations. Actinomyces are often seen in association with amorphous material from "sulfur granules" in such smears. Both Actinomyces and Nocardia can be cultured. Colonies produced by both genera have aerial hyphae that resemble those produced by fungi. Actinomyces infections can be endogenous, and often initiated by trauma. Nocardia usually causes infections in systemically compromised patients. 18.4 The treatment of tuberculosis A. is initiated with a single “first-line” drug. B. is initiated after the results of sensitivity testing is available. C. is most effective in patients with chronic or arrested tubercles. D. may last 2 to 3 weeks. E. should be directly observed whenever possible. 18.5 Virulence in mycobacteria is most strongly correlated with: A. B. C. D. E. mycotoxin production. slow growth. composition of the cell envelope. small size of cells. dependence on oxygen for growth. Correct answer = E. Where directly observed therapy used, the incidence of new cases falls dramatiacally and success of therapy is much more likely. The standard procedure is to begin treatment with two or more drugs to prevent emergence of resistant strains. Sensitivity tests are an important guide to modifying treatment, but sensitivity data are not required to initiate therapy. In chronic or arrested tubercles, the organisms are nonproliferating, and therefore are not susceptible to many antimicrobial agents. Therapy may last from 6 to 18 months. Correct answer = C: Several cell wall components promote the intracellular growth of the organisms, and their dissemination in the infected host. 19 Rickettsia, Erhlichia, Anaplasma and Coxiella I. OVERVIEW Rickettsia, Ehrlichia, Anaplasma, and Coxiella (Figure 19.1) have a number of features in common. For example: 1) They grow only inside living host cells. [Note: Many pathogenic bacteria grow well inside particular cell types but do not require this environment for multiplication. The organisms discussed here, like chlamydiae, are obligately intracellular parasites.] 2) Most infections are transmitted by infected arthropods vectors (for example, lice, ticks, fleas, and mites). 3) Diseases caused by these organisms, such as typhus, spotted fevers, human ehrlichiosis, and Q fever, are generalized infections, with rash sometimes being a prominent feature. Mortality rates of these diseases are variable but may be high in the absence of appropriate treatment. II. RICKETTSIA Rickettsia have the structural features of typical prokaryotic cells. They are small, rodlike or coccobacillary shaped (Figure 19.2), and have a typical double-layered, gram-negative cell wall. However, they stain poorly and, because of their usual occurrence inside host cells, are best visualized under the light microscope with one of the polychrome stains, such as Giemsa or Macchiavello. A. Physiology The obligate requirement for an intracellular environment for rickettsial replication is not fully understood, but its plasma membrane is leaky and, therefore, easily permeable to host cell nutrients and coenzymes. These intracellular parasites employ host-derived carbon sources, amino acids, and nucleosides for their own metabolism. They lack a glycolytic pathway but retain the enzymes necessary for the Krebs cycle. This genus is closely related to the ancestor of mitochondria, found within eukaryotic cells. The rickettsial electron transpor t chain and adenosine triphosphate–generating machinery closely resemble those found in current-day mitochondria. Rickettsia contain a number of antigens that convey both group and species specificity. B. Pathogenesis Rickettsia are transmitted to humans by arthropods, such as fleas, ticks, mites, and lice. Depending on the rickettsial species, rodents, humans, or arthropods can serve as reservoirs of infectious organisms. Rickettsia species have an affinity for endothelial cells located throughout the circulatory system. Following a bite by an infected Medically Important Bacteria Rigid cell wall Simple Unicellular Filamentous Obligate Intracellular Parasite Freeliving Anaplasma Anaplasma phagocytophilum Chlamydia Coxiella Coxiella burnetii Ehrlichia Ehrlichia chaffeensis Ehrlichia equi Rickettsia Rickettsia akari Rickettsia canadensis Rickettsia conorii Rickettsia prowazekii Rickettsia rickettsii S Rickettsia sibirica Rickettsia typhi Figure 19.1 Classification of obligate intracellular parasites. S See p. 347 for a summary of this organism. 197 198 Double-layered cell wall similar to that of gramnegative bacteria Figure 19.2 Electron micrograph of Rickettsia prowazekii in experimentally infected tick tissue. Figure 19.3 Child’s right hand and wrist displaying the characteristic spotted rash with raised or palpable purpura, which is pathognomonic of vasiculitis (the fundamental lesion of Rocky Mountain spotted fever). 19. Rickettsia, Erhlichia, Anaplasma And Coxiella arthropod, the organisms are taken into cells by a process similar to phagocytosis. The organisms degrade the phagosome membrane by production of a phospholipase C. The Rickettsia in the spotted fever group multiply in both the nucleus and cytoplasm of host cells. They appear to mobilize host cell actin fibrils that facilitate their exit into adjacent cells in a manner similar to that of the genera Listeria and Shigella (see pp. 97, 119). The Rickettsia within the typhus group are not capable of actin-based motility; cannot escape the cell via cytoplasmic extensions; and, therefore, are limited to growth within the cytoplasm until the host cell eventually dies, releasing the bacteria. In both cases, the rickettsiae spread throughout the body via the bloodstream or lymphatics. Focal thrombi are formed in various organs including the skin (Figure 19.3), and a variety of small hemorrhages and hemodynamic disturbances create the symptoms of illness. C. Clinical significance—spotted fever group 1. Rocky Mountain spotted fever: Rocky Mountain spotted fever is a potentially lethal, but usually curable tickborne disease, and is the most common rickettsial infection in the United States. The disease is caused by Rickettsia rickettsii. Human infection is initiated by the bite of an infected wood or dog tick. Ticks can transmit the organism transovarially to their progeny and, thereby, the organism can be maintained without mammalian hosts in specific geographic regions for many years. Currently in the United States, such infected tick populations are prevalent in south-central states and along the mid-Atlantic coast. The disease usually occurs with highest frequency during the warmer months when tick activity is greatest. Symptoms begin to develop an average of 7 days after infection. The disease is characterized by high fever and malaise, followed by a prominent rash that is initially macular but may become petechial or frankly hemorrhagic (see Figure 19.3). The rash typically begins on the extremities, involving the palms and soles, and develops rapidly to cover the body. In untreated cases, vascular disturbances leading to tissue infarction and maryocardial or renal failure may ensue. Two thirds of cases of Rocky Mountain spotted fever occur in children younger than age 15 years, with the peak incidence occurring between ages 5 and 9 years. A potential diagnostic problem occurs in those infected patients (approximately 10 percent) in whom a rash does not occur. These cases of “spotless” Rocky Mountain spotted fever may be severe and end fatally. 2. Other spotted fevers: Tickborne spotted fevers similar to Rocky Mountain spotted fever are found in several regions of the world. They vary in severity and are caused by organisms such as Rickettsia conorii, Rickettsia canadensis, and Rickettsia sibirica. A clinically different disease, rickettsialpox, is caused by Rickettsia akari. It has been reported in the United States and the former Soviet Union. The vector for R. akari is a mite, and its reservoir is the common house mouse or similar small rodents. Rickettsialpox is characterized by scattered papulovesicles that are preceded by an eschar at the site of the mite bite and with mild constitutional symptoms of a few days’ duration. Figure 19.4 illustrates the spotted fevers caused by rickettsial organisms. II. Rickettsia 199
Rickettsia prowazekii. [Note: Epidemic typhus is a different disease from salmonella-induced typhoid fever (see p. 86). Both were originally thought to be variations of the same disease, which was called “typhus” after the Greek word meaning “stupor.” When the two diseases were determined to be caused by different organisms, the salmonella-induced disease was named “typhoid,” meaning “typhuslike.”] R. prowazekii is transmitted from person to person by an infected human body louse that excretes organisms in its feces. Scratching louse bites facilitates the introduction of the pathogen from louse feces into a bite wound. Infected lice are themselves eventually killed by the infecting bacterium. Thus, this disease is not maintained in the louse population, but, rather, lice serve as vectors, transmitting the organism between humans. ROCKY MOUNTAIN SPOTTED FEVER • Rickettsia rickettsii • Maintained in wood and dog tick populations • Causes high fever, malaise, and a prominent rash • Complications (if untreated): Vascular disturbances Myocardial failure Renal failure
demics under conditions of displacement of people, crowding, and poor sanitation. Currently a major focus of such outbreaks is found in northeast Africa. The epidemic form of typhus has not occurred in the United States since early in the 20th century. However, sporadic cases of typhus have occurred in the eastern half of the United States, where the reservoir appears to be flying squirrels. The pathogen is probably transmitted from flying squirrels to humans via the bite of ectoparasites. Clinical symptoms of typhus develop an average of 8 days after infection and include high fever; chills; severe headache; and, often, a considerable degree of prostration and stupor. Although rash may be observed, unlike the rash associated with Rocky Mountain spotted fever, the epidemic typhus rash spreads centrifugally from trunk to extremities. The disease lasts 2 weeks or longer,and tends to be more severe in older individuals. Complications of epidemic typhus may include central nervous system dysfunction, myocarditis, and death. b. Brill-Zinsser disease (recrudescent typhus): This is a usually milder form of typhus that occurs in persons who previously recovered from primary infections (10 to 40 years earlier). Latent infection is thought to be maintained in the reticuloendothelial system and probably serves as a reservoir for the organism in interepidemic periods. 2. Other forms of typhuslike fever: Murine (endemic) typhus, caused by Rickettsia typhi, is a clinically similar but usually milder disease than that caused by R. prowazekii. Human infections are initiated by the bites of infected rat fleas, and a worldwide reservoir for R. typhi exists in urban rodents. Murine typhus was endemic in ratinfested areas, particularly in the southeastern United States and in the Gulf of Mexico region. However, with improving rodent control, it has become rare in this country. [Note: The cat flea, which also resides on skunks, opossums, and raccoons, is still a significant vector of murine typhus in the United States.] RICKETTSIALPOX • Rickettsia akari • Caused by the bite of a mite that has a small rodent reservoir • Eschar (thick crust) at site of bite and papulovesicular rash Figure 19.4 Spotted fevers caused by Rickettsia. 200 19. Rickettsia, Erhlichia, Anaplasma And Coxiella D. Laboratory identification Other Rickettsia species • Gram-negative, but stain poorly • Small, rod-like or coccobacillary in shape • Grow only inside living host cells • Transmitted by infected tick • Not routinely cultured because of obligate intracellularity and hazard to laboratory personnel. A variety of serologic procedures have been developed, most of which rely on the demonstration of a rickettsia-specific antibody response during the course of infection. Suspensions or soluble extracts of rickettsia are used to demonstrate group- and speciesspecific antibodies by indirect immunofluorescence. Alternatively, although not widely available, infected cells can be detected by immunofluorescence or histochemical procedures on some clinical samples such as punch biopsies from areas of rash. Polymerase chain reaction (PCR) amplification can also be employed for the specific diagnosis of rickettsial diseases. E. Treatment Doxycycline is the drug of choice for the treatment of Rocky Mountain spotted fever in both adults and children, except for pregnant women who should be treated with chloramphenicol (Figure 19.5). The risk of dental staining with doxycycline is minimal if a short course is administered. The decision to treat must be made on clinical grounds, together with a history or suspicion of contact with an appropriate arthropod vector, before the seroconversion data are available. Early therapy for Rocky Mountain spotted fever is important, because delay beyond the 5th day of illness is associated with an increased mortality rate. Rash of Rocky Mountain spotted fever Rickettsialpox
Rickettsia rickettsii • Rocky Mountain spotted fever 1 Doxycycline 2 Chloramphenicol1 1Treatment of pregnant women Figure 19.5 Summary of Richettsia disease. 1 Indicates first-line drug; 2 indicates alternative drug. III. EHRLICHIA AND ANAPLASMA Ehrlichia and Anaplasma resemble Rickettsia in appearance and behavior. However, these organisms parasitize monocytes and neutrophils, respectively, and grow exclusively within host-derived cytoplasmic vacuoles, creating characteristic inclusions called morulae. A. Clinical significance Human monocytic ehrlichiosis (HME) is caused by Ehrlichia chaffeensis. Human granulocytic anaplasmosis (HGA) is caused by the organism Anaplasma phagocytophilum (Figure 19.6). The symptoms of HME and HGA are similar and often nonspecific. Common symptoms include fever, chills, headache, myalgia, and arthralgia. HME often presents with nausea, which is rare with HGA. More IV. Coxiella severe manifestations of HME include meningoencephalitis, myocarditis, and acute renal failure. Serious manifestations of HGA include severe leukocytopenia and thrombocytopenia due to damage of the infected cell populations. Rash is seldom seen for either HME or HGA, and deaths from HGA and HME have occurred. HME has been confirmed in some thirty states in the southeastern and south-central United States and has been most commonly associated with bites of the Lone Star tick. HGA has been associated with the bites of deer and dog ticks and has been reported in North and South America, Europe, and Asia. 201 HUMAN MONOCYTIC EHRLICHIOSIS (HME) • Ehrlichia chaffeensis • Lone Star tick vector • Causes acute fever, myalgia, leucocytopenia and thrombocytopenia
IV. COXIELLA Coxiella burnetii, the causal agent of Q fever, is found worldwide (the “Q” stands for “query” because the cause of the fever was unknown for many years). It has several features that distinguish it from other rickettsia. For example: 1) It grows in cytoplasmic vacuoles and seems to be stimulated by the low pH of a phagolysosome, being resistant to the host degradative enzymes within that structure; 2) it is extremely resistant to heat and drying and can persist outside its host for long periods; and 3) it causes disease in livestock, such as cattle and in other mammals, but is not transmitted to humans by arthropods. Although the organism has been reported to be recovered from ticks, human infection usually occurs following inhalation of infected dust in, for example, barnyards and slaughterhouses (a transmission route made possible because of the ability of C. burnetii to withstand drying). [Note: C. burnetii has also been known to enter the body via other mucous membranes, abrasions, and the gastrointestinal tract through consumption of milk from infected animals.] A. Clinical significance
Human granulocytic anaplasmosis (HGA) • Caused by Anaplasma phagocytophilum Deer and dog tick • vectors • Causes a disease similar to HME but more severe Figure 19.6 Diseases caused by Ehrlichia and Anaplasma. 202 19. Rickettsia, Erhlichia, Anaplasma And Coxiella Study Questions Choose the ONE correct answer. 19.1 Pathogens in the genus Rickettsia: A. grow only extracellularly. B. have eukaryotic-type cell organization. C. cause contagious infections because they are disseminated by respiratory droplets. D. are clinically sensitive to penicillin. E. generally invade the endothelial lining of capillaries, causing small hemorrhages. 19.2 The vector of Rocky Mountain spotted fever is the: A. B. C. D. E. human body louse. rat flea. deer tick. dog tick. mosquito. 19.3 Ehrlichiosis and Rocky Mountain spotted fever have all but which of the following clinical features in common? A. B. C. D. E. Both involve parasitized blood cells. Both are acute fevers. Both are transmitted by the same vector. Both can be treated by doxycycline. Both are potentially lethal. 19.4 Coxiella burnetii: A. B. C. D. cannot survive outside its host. has no reservoir other than humans. causes a pneumonitis called Q fever. causes symptomatic disease only in the lower respiratory tract. E. is found only in the United States. 19.5 A 14-year-old male presents to the emergency room in North Carolina with fever and a distinctive rash on his extremities. The rash is most prominent on the palms of his hands, but has spread to his lower arms as well. The patient was well before a camping trip 1 week prior to symptom onset. Which of the following bacterial pathogens is the most likely causative agent of this disease? A. Coxiella burnetii B. Ehrlichia chaffeensis C. Rickettsia rickettsii D. Anaplasma phagocytophilum E. Rickettsia prowazekii Correct answer = E. Most Rickettsia cause rashes resulting from damage to the vascular system. They are obligate intracellular, prokaryotic parasites. Rickettsia species are transmitted by the bite of an arthropod. They are sensitive to tetracyclines but not to penicillin. Correct answer = D. Rocky Mountain spotted fever is initiated by the bite of an infected wood or dog tick. The human louse is involved in the transmission of Rickettsia prowazekii, causing thypus. Deer ticks are involved in the transmission of Lyme disease (see p. 165). Mosquitoborne diseases include dengue fever, malaria, and yellow fever. Correct answer = A. Ehrlichia parasitizes leukocytes, whereas Rickettsia rickettsii invades capillary linings, causing the "spotted" rash of Rocky Mountain spotted fever. Correct answer = C. Whereas lower respiratory tract disease is most characteristic, the organism fairly frequently causes hepatitis, myocarditis or endocarditis, and other visceral infections. It is resistant to drying and heat and infects a variety of animals (including ticks, but they play no role in human disease). Its distribution is worldwide. Correct answer: C. The symptoms and presentation of this disease are most consistent with Rocky Mountain spotted fever, caused by Rickettsia rickettsii. The rash is the most prominent symptom of infection, which typically begins on the extremities and spreads towards the trunk. The infection is transmitted to humans by the bite of a tick, which is common during outdoor activities. Q fever, caused by Coxiella burnetii, generally presents with respiratory symptoms and most likely follows recent contact with livestock. Ehrlichia chaffeensis causes human monocytic ehrlichiosis, which presents with systemic signs but no rash. Presentation of human granulocytic anaplasmosis, caused by Anaplasma phagocytophilum, similarly does not include a distinctive rash. Rickettsia prowazekii causes epidemic typhus, which is rare in the United States. However, if it occurs, the rash is different from that caused by R. rickettsii in that it spreads from the trunk. UNIT III: Fungi and Parasites 20 Fungi I. OVERVIEW Fungi are a diverse group of saprophytic (deriving nourishment from dead organic matter) and parasitic eukaryotic organisms. Although formerly considered to be plants, they are now assigned their own kingdom, Mycota. Virtually all organisms are subject to fungal infection. Of some 200,000 fungal species, only about 100 have pathogenic potential for humans. Of these, only a few species account for most clinically important fungal infections (Figure 20.1). Human fungal diseases (mycoses) are classified by the location on or in the body where the infection occurs. They are called cutaneous when limited to the epidermis, subcutaneous when the infection penetrates significantly beneath the skin, and systemic when the infection is deep within the body or disseminated to internal organs. Systemic mycoses can be further divided into those that are caused by true pathogenic fungi capable of infecting healthy individuals and those that are opportunistic, infecting primarily those individuals who have predisposing conditions, such as immunodeficiency or debilitating diseases (for example, diabetes, leukemia, and Hodgkin and other lymphomas). Fungi produce and secrete a variety of unusual metabolic products, some of which, when ingested, are highly toxic to animals, including humans. Thus, fungi can cause poisoning as well as infection. Lastly, fungal spores, which are critical for dispersal and transmission of the fungus, are also important as human allergenic agents. Pathogenic fungi TRUE PATHOGENS Cutaneous infective agents Epidermophyton species Microsporum species Trichophyton species Subcutaneous infective agents Actinomadura madurae Cladosporium Madurella grisea Phialophora Sporothrix schenckii Systemic infective agents Blastomyces dermatitidis Coccidioides immitis II. CHARACTERISTICS OF MAJOR FUNGAL GROUPS Fungi can be distinguished from other infectious organisms such as bacteria or viruses because they are eukaryotes (that is, they have a membrane-enclosed nucleus and other organelles). Fungi have no chlorophyll or chloroplasts, thus distinguishing them from plants. Their characteristic structures, habitats, and modes of growth and reproduction are used to distinguish different groups among fungi. Histoplasma capsulatum Paracoccidioides brasiliensis OPPORTUNISTIC PATHOGENS (continued on next page) Figure 20.1 Classification of pathogenic fungi (figure continues on next page). 203 204 20. Fungi A. Cell wall and membrane components Pathogenic fungi (continued) OPPORTUNISTIC PATHOGENS Absidia corymbifera Aspergillus fumigatus Candida albicans Cryptococcus neoformans Pneumocystis jiroveci Rhizomucor pusillus Rhizopus oryzae (R. arrhizus) Figure 20.1 (continued) Classification of pathogenic fungi. The fungal cell wall and cell membrane are fundamentally different from those of bacteria and other eukaryotes. Fungal cell walls are composed largely of chitin, a polymer of N-acetylglucosamine, rather than peptidoglycan, which is a characteristic component of bacterial cell walls. Fungi are, therefore, unaffected by antibiotics (for example, penicillin) that inhibit peptidoglycan synthesis. The fungal membrane contains ergosterol rather than the cholesterol found in mammalian membranes. These chemical characteristics are useful in targeting chemotherapeutic agents against fungal infections. Many such agents interfere with fungal membrane synthesis or function. For example, amphotericin B and nystatin bind to ergosterol present in fungal cell membranes. There they form pores that disrupt membrane function, resulting in cell death. Imidazole antifungal drugs (clotrimazole, ketoconazole, miconazole) and triazole antifungal agents (fluconazole and itraconazole) interact with the P450 enzyme 14 α-sterol-demethylase to block demethylation of lanosterol to ergosterol. Because ergosterol is a vital component of fungal cell membranes, disruption of its biosynthesis results in cell death. B. Habitat and nutrition All fungi are chemoheterotrophs, requiring some preformed organic carbon source for growth. Fungi do not ingest food particles as do organisms such as protozoa (see p. 217) but depend upon transport of soluble nutrients across their cell membranes. To obtain these soluble nutrients, fungi secrete degradative enzymes (for example, cellulases, proteases, nucleases) into their immediate environment, which enable them to live saprophytically on organic waste. Therefore, the natural habitat of almost all fungi is soil or water containing decaying organic matter. Some fungi can be parasitic on living organisms. However, these parasitic infections usually originate from the individual’s contact with fungus-contaminated soil, an exception being Candida , which is part of the normal human mucosal flora (see p. 7). A
B Most fungi exist in one of two basic morphologic forms (that is, either as filamentous mold or unicellular yeast). However, some fungi are dimorphic and can switch between these two forms in response to environmental conditions. Bud Parental cell 1. Filamentous (mold-like) fungi: The vegetative body, or thallus, of Figure 20.2 A. Filamentous (mold-like) fungi (light micrograph). B. Budding yeast-like fungi (scanning electron micrograph). mold-like fungi is typically a mass of threads with many branches (Figure 20.2A). This mass is called a mycelium, which grows by branching and tip elongation. The threads (hyphae) are actually tubular cells that, in some fungi, are partitioned into segments (septate), whereas, in other fungi, the hyphae are uninterrupted by crosswalls (nonseptate). Even in septate fungi, however, the septae are perforated so that the cytoplasm of the hyphae is continuous. When hyphal filaments become densely packed, the mycelium may have the appearance of a cohesive tissue (for example, as seen in the body of a mushroom). II. Characteristics Of Major Fungal Groups 205 2. Yeast-like fungi: These fungi exist as populations of single, unconnected, spheroid cells, not unlike many bacteria, although they are some 10 times larger than a typical bacterial cell (see Figure 20.2B). Yeast-like fungi generally reproduce by budding. 3. Dimorphic fungi: Some fungal species, especially those that cause systemic mycoses, are dimorphic, being yeast-like in one environment and mold-like in another. Conditions that can affect morphology include temperature and carbon dioxide level. Examples of dimorphic fungi include Blastomyces dermatiditis and Histoplasma capsulatum. D. Sporulation Sporulation is the principal means by which fungi reproduce and spread through the environment. Fungal spores are metabolically dormant, protected cells, released by the mycelium in enormous numbers. They can be borne by air or water to new sites, where they germinate and establish colonies. Spores can be generated either asexually or sexually (Figure 20.3). 1. Asexual sporulation: Asexual spores (conidia) are formed by mitosis in or on specialized hyphae (conidiophores) as shown in A Asexual sporulation Conidia Conidiophore Sterigmata Vesicle B Sexual sporulation Nuclear fusion Meiosis + Spore Ascus Ascospores – Spore Figure 20.3 Sporulation among Aspergillus nidulans. A. Asexual. B. Sexual. 206 20. Fungi Figure 20.3A. The color of a typical fungal colony seen on bread, fruit, or culture plate is caused by the conidia, which can number tens of millions per cm 3 of surface. Because they are easily detached from their underlying mycelial mats, conidia can become airborne and, therefore, are a major source of fungal infection (see p. 209). 2. Sexual sporulation: This process is initiated when a haploid nucleus from each of two compatible strains of the same species fuse to form a transient diploid (see Figure 20.3B). The products of meiosis of this transient diploid become sexual spores (ascospores). Compared to asexual sporulation, sexual sporulation is relatively rare among human fungal pathogens. Spores, especially sexual spores, often have a characteristic shape and surface ornamentation pattern that may serve as the primary or only means of species identification. E. Laboratory identification Most fungi can be propagated on any nutrient agar surface. The standard medium is Sabouraud dextrose agar, which, because of its low pH (5.0), inhibits bacterial growth while allowing fungal colonies to form (Figure 20.4). Various antibacterial antibiotics can also be added to the medium to further inhibit bacterial colony formation. Cultures can be star ted from spores or hyphal fragments. Identification is usually based on the microscopic morphology of conidial structures. Clinical samples may be pus, blood, spinal fluid, sputum, tissue biopsies, or skin scrapings. These specimens can also be rapidly evaluated histologically by direct staining techniques to identify hyphae or yeast forms. Serologic tests and immunofluorescent techniques are also useful in identification of fungi from clinical isolates. Figure 20.4 Colonies of Candida albicans grown on Sabouraud dextrose agar. III. CUTANEOUS (SUPERFICIAL) MYCOSES Also called dermatophytoses, these common diseases are caused by a group of related fungi, the dermatophytes. Dermatophytes fall into three genera, each with many species: Trichophyton, Epidermophyton, and Microsporum. A. Epidemiology The causative organisms of the dermatophytoses are often distinguished according to their natural habitats: anthropophilic (residing on human skin), zoophilic (residing on the skin of domestic and farm animals), or geophilic (residing in the soil). Most human infections are by anthropophilic and zoophilic organisms. Transmission from human to human or animal to human is by infected skin scales on inanimate objects. Only the pathogenic fungi are capable of humanto-human spread. B. Pathology A defining characteristic of the dermatophytes is their ability to use keratin as a source of nutrition. This ability allows them to infect ker- III. Cutaneous Mycoses atinized tissues and structures, such as skin, hair, and nails. There is some specificity, however. Although all three genera attack the skin, Microsporum does not infect nails, and Epidermophyton does not infect hair. None invades underlying, nonkeratinized tissue. 207
1. Tinea pedis (“athlete’s foot”): Organisms most often isolated from infected tissue are Trichophyton rubrum, Trichophyton mentagrophytes, and Epidermophyton floccosum. The infected tissue is initially between the toes but can spread to the nails, which become yellow and brittle. Skin fissures can lead to secondary bacterial infections with consequent lymph node inflammation (Figure 20.5A).
2. Tinea corporis (“ringworm”): Organisms most often isolated are
and Microsporum have been isolated from scalp ringworm lesions, the predominant infecting species depending on the geographic location of the patient. In the United States, for example, the predominant infecting species is Trichophyton tonsurans . Disease manifestations range from small, scaling patches, to involvement of the entire scalp with extensive hair loss (see Figure 20.5C). The hair shafts can become invaded by Microsporum hyphae, as demonstrated by their green fluorescence in long-wave ultraviolet light (Wood lamp).
4. Tinea cruris (“jock itch”): Causative organisms are E. floccosum and T. rubrum. Disease manifestations are similar to ringworm, except that lesions occur in the moist groin area, where they can spread from the upper thighs to the genitals (see Figure 20.5D). 5. Tinea unguium (onychomycosis): The causative organism is most often T. rubrum. Nails thicken and become discolored and brittle. Treatment must continue for 3 to 4 months until all infected portions of the nail have grown out and are trimmed off (see Figure 20.5E). Figure 20.5 Cutaneous mycoses. 208 20. Fungi D. Treatment
Removal of infected skin, followed by topical application of antifungal antibiotics, such as miconazole and clotrimazole, is the first course of treatment. Refractory infections usually respond well to oral griseofulvin and itraconazole. Infections of the hair and nails usually require systemic (oral) therapy. Terbinafine is the drug of choice for onychomycosis. IV. SUBCUTANEOUS MYCOSES
Subcutaneous mycoses are fungal infections of the dermis, subcutaneous tissue, and bone. Causative organisms reside in the soil and decaying or live vegetation. A. Epidemiology
Subcutaneous fungal infections are almost always acquired through traumatic lacerations or puncture wounds. Sporotrichosis, for example, is often acquired from the prick of a thorn. As expected, these infections are more common in individuals who have frequent contact with soil and vegetation and wear inadequate protective clothing. The subcutaneous mycoses are not transmissible from human to human. B. Clinical significance With the rare exception of sporotrichosis, which shows a broad geographic distribution in the United States, the common subcutaneous mycoses discussed below are confined to tropical and subtropical regions. Figure 20.6 Subcutaneous mycoses. A. Sporotrichosis. The forearm of a gardener exhibiting the cutaneouslymphatic form of sporotrichosis. B. Chromomycosis showing multiple plaques on the lower leg. C. Mycetoma of the arm. 1. Sporotrichosis: This infection, characterized by a granulomatous ulcer at the puncture site, may produce secondary lesions along the draining lymphatics (Figure 20.6A). The causative organism, Sporothrix schenckii, is a dimorphic fungus that exhibits the yeast form in infected tissue (see Figure 20.7) and the mycelial form upon laboratory culture. In most patients, the disease is self-limiting but may persist in a chronic form. Dissemination to distant sites is possible in patients with deficiencies in T-cell function (such as in AIDS and lymphomas). Oral itraconazole is the drug of choice. 2. Chromomycosis: Also called chromoblastomycosis, this infection is characterized by warty nodules that spread slowly along the lymphatics and develop crusty abscesses (see Figure 20.6B). Pathogens causing this mycosis include several species of pigmented soil fungi (for example, Phialophora and Cladosporium), and the infection is most commonly seen in the tropics. Treatment is difficult. Surgical removal of small lesions is effective but must be performed cautiously and with wide margins to prevent dissemination. More advanced stages of the disease are treated with itraconazole and terbinafine.
209 3. Mycetoma (“Madura foot”): Mycetoma appears as a localized abscess, usually on the feet, but is not limited to the lower extremity (see Figure 20.6C). The abscess discharges pus, serum, and blood through sinuses (in this usage, sinus means “abnormal channel”). The infection can spread to the underlying bone and results in crippling deformities . The pathogenic agents are various soil fungi. Most common are Madurella grisea and Exophiala jeanselmei. Mycetomas appear similar to the lesions of chromomycosis, but the defining characteristic of mycetoma is the presence of colored grains, composed of compacted hyphae, in the exudate. The color of the grains (black, white, red, or yellow) is characteristic of the causative organism and, therefore, useful in identifying the particular pathogen. There is no effective chemotherapy for fungal mycetoma. Treatment is usually surgical excision.
cases of coccidioidomycosis occur in the arid areas of southwestern United States (Figure 20.9) and Central and South America. Initial infection with C. immitis can cause fever with varying degrees of respiratory illness (called “Valley fever” because of its prevalence in the San Joaquin Valley of the southwestern United States). In the soil, the fungus generates spores by septation of Figure 20.7 Tissue section showing the budding yeast Sporothrix schenckii. 210 20. Fungi 1 Spore production Fungi are found in soil and the feces of birds and bats. The fungi produce spores that become airborne. 2 Primary pulmonary infection When inhaled, spores cause a primary pulmonary infection. Yeast form of Blastomyces dermatitidis Lung of a patient with acute coccidioidal pneumonia Blastomycosis Coccidioidomycosis Spherule Possible sites of infection: • Central nervous system • Bone Methenamine silver stain Possible sites of infection: 3 Dissemination of infection The fungi may travel from the lungs to other sites where infection can occur. • Skin • Bone • Genitourinary tract Central fungal cell with a series of buds that look like spokes of a wheel Histoplasmosis Paracoccidioidomycosis Possible sites of infection: Possible sites of infection: • Liver • Spleen • Lymph nodes • Bone marrow Figure 20.8 Systemic mycoses. • Mucosa of the mouth and nose
latum. In the soil, the fungus generates conidia, which, when airborne, enter the lungs and germinate into yeast-like cells. These yeast cells are engulfed by macrophages, in which they multiply. Pulmonary infections may be acute but relatively benign and selflimiting, or they can be chronic, progressive, and fatal. Dissemination is rare but can occur in older adults, the very young, and patients with deficiencies in T-cell function. Disseminated disease results in invasion of cells of the reticuloendothelial system, which distinguishes this organism as the only fungus to exhibit intracellular parasitism. Definitive diagnosis is by isolation and culture of the organism, which is a slow process (4 to 6 weeks), or by detection of exoantigen in urine specimens. The disease occurs worldwide but is most prevalent in central North America, especially the Ohio and Mississippi River Valleys (Figure 20.10). Soils that are laden with bird, chicken, or bat droppings are a rich source of H. capsulatum spores. Local epidemics of the disease can occur, in particular, in areas where construction has disturbed bird, chicken, and bat roosts. AIDS patients who live in or travel through endemic areas are especially at risk. The wide range of clinical manifestations of histoplasmosis makes it a particularly complex disease, often resembling tuberculosis. 3. Blastomycosis: Blastomyces dermatitidis causes blastomycosis. Like Histoplasma, the fungus produces microconidia, most often in the soil, which become airborne and enter the lungs. There they germinate into thick-walled yeast cells that often appear with unipolar, broad-based buds. Although initial pulmonary infections (Figure 20.11) rarely disseminate to other sites, when dissemination does occur, secondary sites include skin (70 percent), bone (30 percent), and the genitourinary tract (20 percent), where they manifest as ulcerated granulomas. Definitive diagnosis is accomplished by isolation and culture of the organism. Identifiable colonies can be obtained in 1 to 3 weeks, but identity can be established more rapidly by subjecting the young mycelial colonies to an exoantigen test. Infections are most common in the South Central and South Eastern United States and are much more common in adult males than in females or children. 4. Paracoccidioidomycosis: Also called South American blastomy- cosis, paracoccidioidomycosis is caused by Paracoccidioides brasiliensis. The clinical presentation is much like that of histoplasmosis and blastomycosis except that the most common sec- 211 CA WY NV UT CO NM TX High prevalence Figure 20.9 Geographic prevalence of coccidioidomycosis in the United States. Moderate prevalence High prevalence Figure 20.10 Endemic areas of histoplasmosis in North America. Figure 20.11 Chest radiograph showing a diffuse reticulonodular infiltrate of the lungs in a male landscaper. Bronchoalveolar lavage recovered Blastomyces dermatitidis. 212 20. Fungi ondary site of infection is the mucosa of the mouth and nose, where painful, destructive lesions may develop. Like other dimorphic pathogens, morphologic identification via conidia is slow, but the yeast form observed in infected tissue or exudates has a characteristic appearance resembling a ship’s steering wheel, caused by the presence of multiple buds (see Figure 20.8). The disease is restricted to Central and South America, and over 90 percent of patients with symptomatic disease are mature males. It is speculated that estrogen may inhibit formation of the yeast form. Urinary tract infections Candida albican albicans n Gram (+) cocci Staphylococcus aureus Coagulase-negative staphylococci
Klebsiella pneumoniae Pseudomonas aeruginosa 0 30% Urinary tract infections (UTIs) are commonly caused by Candida albicans. In hospitals, most UTIs are associated with urinary tract catheters. Figure 20.12 Commonly reported pathogens from urinary tract infections in patients in adult medical intensive care units. These diseases are not communicable from one person to another. However, laboratory cultures should be handled cautiously, especially those of C. immitis, because, under culture conditions, the fungi revert to the spore-bearing, infectious form. Because these organisms have slow growth rates, morphologic identification of the characteristic conidia can take several weeks. Histological examination of body fluids (sputum, pus, draining fistulas) for the presence of yeasts, hyphae, or conidia allows for rapid identification of the fungal etiological agent prior to the availability of culture results. A rapid method for identifying the four systemic pathogens discussed above is the exoantigen test in which cell-free antigens produced by young mycelial colonies (or liquid cultures) are detected by immunodiffusion assay. The exoantigen test can also be applied to urine specimens collected from patients suffering from histoplasmosis. Polymerase chain reaction is another rapid, accurate diagnostic method that detects specific fungal DNA sequences. D. Treatment Systemic mycoses are usually treated with amphotericin B, sometimes in combination with flucytosine. Ketoconazole, fluconazole and intraconazole are also used, depending on the infecting organism and the stage and site of the disease. VI. OPPORTUNISTIC MYCOSES Figure 20.13 Candida albicans. Opportunistic mycoses afflict debilitated or immunocompromised individuals but are rare in healthy individuals. The use of immunosuppressive drugs for organ transplantation and chemotherapy in cancer treatment, and the high number of immunodeficient individuals caused by the AIDS epidemic have resulted in significant expansion of the immunocompromised population as well as increased spectrum of opportunistic fungal pathogens. Fungal infections represent approximately 15 percent of all nosocomial infections (infections that are a result of treatment in a hospital) in intensive care units in the United States, with Candida species being the most commonly occurring fungal nosocomial pathogen (Figure 20.12). The opportunistic mycoses most commonly encountered today include the following. VI. Opportunistic Mycoses 213
Figure 20.14 Oral candidiasis (thrush).
Figure 20.15 Cryptococcus neoformans. [Note: Capsules are visible because they do not take up the hematoxylin and eosin stain]. 214 20. Fungi C. Aspergillosis Figure 20.16 Aspergillus species. Aspergillosis is caused by several species of the genus Aspergillus but primarily by Aspergillus fumigatus . Aspergillus is rarely pathogenic in the normal host but can produce disease in immunosuppressed individuals and patients treated with broad-spectrum antibiotics. The disease has a worldwide distribution. Aspergilli are ubiquitous, growing only as filamentous molds (Figure 20.16) and producing prodigious numbers of conidiospores. They reside in dust soil, and decomposing organic matter. In fact, hospital outbreaks affecting neutropenic patients (that is, those with decreased neutrophils in their blood) have been traced to dust from neighboring construction work. Aspergillosis manifests itself in several forms, depending in part on the patient’s immunologic status. 1. Acute aspergillus infections: The most severe, and often fatal, form of aspergillosis is acute invasive infection of the lung, from which the infection can be disseminated to the brain, GI tract, and other organs. A less severe, noninvasive lung infection gives rise to a fungus ball (aspergilloma), a mass of hyphal tissue that can form in lung cavities derived from prior diseases such as tuberculosis (Figure 20.17). Although the lung is the most common primary site of infection, the eye, ear, nasal sinuses, and skin can also be primary sites. 2. Diagnosis and treatment: Definitive diagnosis of an aspergillus Fungus ball Figure 20.17 Fungus ball. infection is afforded by detection of hyphal masses and isolation of the organism from clinical samples. Aspergillus hyphae characteristically form V-shaped branches (septate hyphae that branch at a 45-degree angle, see Figure 20.16) that are distinguished from Mucor species, which form right-angle branches. Also, septa are present in Aspergillus hyphae but absent from those of Mucor. In culture, the spore-bearing structures of the aspergilli are unmistakable, but, because these organisms are so ubiquitous, external contamination of clinical samples can give false-positives. Treatment of Aspergillus infections is typically by amphotericin B and surgical removal of fungal masses or infected tissue. The antifungal drugs miconazole, ketoconazole, and fluconazole have not proven useful, although itraconazole has been used with some effectiveness for Aspergillus osteomyelitis. D. Mucormycosis Figure 20.18 Rhizopus oryzae. Mucormycosis is caused most often by Rhizopus oryzae (also called R. arrhizus), as shown in Figure 20.18, and less often by other members of the order Mucorales, such as Absidia corymbifera and Rhizomucor pusillus. Like the aspergilli, these organisms are ubiquitous in nature, and their spores are found in great abundance on rotting fruit and old bread. Mucor infections occur worldwide but are almost entirely restricted to individuals with some underlying predisposing condition, such as burns, leukemias, or acidotic states such as diabetes mellitus. The most common form of the disease, which can be fatal within 1 week, is rhinocerebral mucormycosis, in which the infection begins in the nasal mucosa or sinuses and pro- VI. Opportunistic Mycoses 215 gresses to the orbits, palate, and brain. Because the disease is so aggressive, many cases are not diagnosed until after death. Treatment is based on high-dose amphotericin B but must be accompanied, when possible, by surgical debridement of necrotic tissue and correction of the underlying predisposing condition. Antifungal drugs other than amphotericin have not proven useful. With early diagnosis and optimal treatment, about half of diabetic patients survive rhinocerebral mucormycosis, but prognosis is very poor for leukemic patients. E. Pneumocystis jiroveci Pneumocystis jiroveci pneumonia is caused by a yeast-like fungus called P. jiroveci (formerly, P. carinii) as shown in Figure 20.19. The disease is still often referred to as PCP, for P. carinii pneumonia. Before the use of immunosuppressive drugs and the onset of the AIDS epidemic, infection with this organism was a rare occurrence. It is one of the most common opportunistic diseases of individuals infected with HIV-1 (see Figure 33.10) and almost 100 percent fatal if untreated. 1. Classification: Previously, P. jiroveci was considered a protozoan, but recent molecular homology studies of both protein and nucleic acid sequences indicate that P. jiroveci is a fungus related to the ascomycetous yeasts. However, ergosterol, which is an essential component of most fungal membranes, is lacking in P. jiroveci. It has so far not been possible to cultivate P. jiroveci in vitro, limiting understanding of its life cycle. Figure 20.19 Silver stain of Pneumocystis jiroveci cysts in tissue from a patient with AIDS. 2. Pathology: The infectious form and the natural reservoir of this organism have not been identified, but they must be ubiquitous in nature because almost 100 percent of children worldwide have antipneumocystis antibodies. The disease is not transmitted from person to person. Instead, development of P. jiroveci in immunodeficient patients is thought to be by activation of preexisting dormant cells in the lungs. The encysted forms induce inflammation of alveoli, resulting in production of an exudate that blocks gas exchange. Figure 20.20 shows typical radiographic findings in Pneumocystis pneumonia. 3. Diagnosis and treatment: Because P. jiroveci cannot be cultivated, diagnosis is based on microscopic examination of biopsied lung tissue or washings. The most effective therapy is a combination of sulfamethoxazole and trimethoprim, which is also used prophylactically to prevent infection in AIDS patients. Aggressive treatment can spare about half of patients. Because the mechanism of action of many antifungal drugs, such as amphotericin, involves interfering with ergosterol synthesis or function, these drugs are useless for fungi that lack ergosterol. Figure 20.20 Pneumocystis pneumonia. 216 20. Fungi Study Questions Choose the ONE correct answer. 20.1 A component of the cell membrane of most fungi is: A. B. C. D. E. cholesterol. chitin. ergosterol. peptidoglycan. keratin. Correct answer = C. Ergosterol in fungi is the functional equivalent of cholesterol in higher organisms. Peptidoglycan is a component of the bacterial cell wall, whereas chitin is a component of the cell wall of fungi. [Note: Chitin also comprises the exoskeletons of insects and crustacea.] Keratin is the major protein of hair and nails . 20.2 A physician visiting a rural Latin American village finds that many mature males but few immature males or females of any age are afflicted by a particular fungal disease. What is likely to be the diagnosis? A. B. C. D. E. Mycetoma Blastomycosis Paracoccidioidomycosis Mucormycosis Histoplasmosis 20.3 A fungus that can attack hair is: A. B. C. D. E. Trichophyton. Rhizopus. Microsporum. Sporothrix. Epidermophyton. 20.4 A farmer in Mississippi presents with a chronic cough. Chest radiograph reveals an opaque mass, and biopsy of the lung shows macrophages with multiple yeast forms. Which one of the following diagnoses is most likely? A. B. C. D. E. Correct answer = C. For some reason, possibly hormonal, this disease favors mature males. Coccidioidomycosis Histoplasmosis Blastomycosis Paracoccidioidomycosis Sporotrichosis Correct answer = C. All attack skin, but only Microsporum attacks hair. Correct answer = B. Histoplasmosis is caused by Histoplasma capsulatum. In the soil, the fungus generates conidia, which, when airborne, enter the lungs and germinate into yeast-like cells. These yeast cells are engulfed by macrophages, in which they multiply. Pulmonary infections may be acute but relatively benign and self-limiting, or it may be chronic, progressive, and fatal. Dissemination is rare but results in invasion of reticuloendothelial system cells, which distinguishes this organism as the only fungus to exhibit intracellular parasitism. The disease occurs worldwide but is most prevalent in central North America, especially the Ohio and Mississippi River Valleys. Protozoa 21
Pathogenic protozoa Intestinal Entamoeba histolytica (Ameba) Giardia lamblia (Flagellate) Cryptosporidium parvum (Sporozoan) Balantidium coli (Ciliate) Urogenital Trichomonas vaginalis (Flagellate) Blood and tissues Plasmodium species II. CLASSIFICATION OF CLINICALLY IMPORTANT PROTOZOA (Sporozoan) Toxoplasma gondii (Sporozoan) Trypanosoma species Among the pathogenic protozoa, there are important common features that are clinically relevant. For example, many protozoa have both a dormant, immotile cyst stage that permits survival when environmental conditions are hostile and a motile, actively feeding and reproducing, vegetative (trophozoite) stage. For convenience, protozoa are classified according to mode of locomotion. The clinically relevant protozoa are divided into four groups (Figure 21.2). (Flagellate) Leishmania species (Flagellate) Naegleria fowleri (Ameba) Acanthamoeba castellanii (Ameba) Balamuthia mandrillaris (Ameba)
Babesia microti (Sporozoan) Amebas move by extending cytoplasmic projections (pseudopodia) outward from the main cell body. A single cell can have several pseudopodia projecting in the same general direction, with the remainder of the cytoplasm flowing into the pseudopodia. Amebas feed by engulfing food particles with their pseudopodia. Some amebas have flagella as well. Figure 21.1 Clinically relevant protozoa, classified according to site of infection. 217 218 21. Protozoa B. Flagellates Pathogenic protozoa Flagellates move by means of two or more whiplike projections (flagella) that rotate and propel the cells through their liquid environment. Some flagellates, for example Trichomonas vaginalis, also have undulating membranes that assist in swimming. Flagellates ingest food particles through an oral groove called a cytostome. Amebas Move by extending cytoplasmic projections.
Ciliates move by means of many hairlike projections (cilia) arranged in rows that cover the cell surface and beat in synchrony, propelling the cell much like a row boat. Most ciliates have cytostomes that pass food particles through a cytopharynyx and finally into vacuoles where digestion takes place. Although there are some 7,000 species of ciliates, only Balantidium coli is pathogenic for humans, and the disease, balantidiasis, is rare. Move by rotating whip-like flagella. Ciliates Move by synchronous beating of hair-like cilia. Sporozoa
Generally have nonmotile adult forms. Sporozoans (also called apicomplexa) are obligate, intracellular parasites. Although they generally have nonmotile adult forms, in some species, male gametes have flagella. An example of a sporozoan is Plasmodium vivax (see p. 221), which causes malaria. Sporozoans can have complex life cycles with more than one host. The definitive host is that which harbors the sexually reproducing stage, whereas the intermediate host provides the environment in which asexual reproduction occurs. Figure 21.2 The four major protozoal groups, classified according to mode of locomotion. A STOMACH LIVER 1 ILEUM Ingestion of cysts 2 3 Penetration of intestinal wall Formation of trophozoites Multiplication of trophozoites within colon wall COLON WALL Cyst formation 4 5 Systemic invasion B COLON Expelled trophozoite ctive) (noninfective) 6 Cysts discarded with feces Expelled cyst (infective) Trophozoite RECTUM Cyst Figure 21.3 A. Life cycle of Entamoeba histolytica. B. Photomicrographs of trophozoite and cyst forms. III. Intestinal Protozoal Infections 219 III. INTESTINAL PROTOZOAL INFECTIONS There are four principal protozoal intestinal parasites: the ameba, Entamoeba histolytica; the flagellate, Giardia lamblia; the sporozoan, Cryptosporidium (several species); and Balantidium coli (the only ciliate protozoan to cause human disease). Each pathogen causes diarrhea, which, although similar, differ in the site of infection, its severity, and secondary consequences. A. Amebic dysentery (Entamoeba histolytica) Ingested cysts from contaminated food or water form trophozoites in the small intestine (Figure 21.3). These pass to the colon, where they feed on intestinal bacteria, and may invade the epithelium, potentially inducing ulceration. The parasite can further spread to the liver and cause abscesses. In the colon, trophozoites form cysts that pass in the feces. Amebic cysts are resistant to chlorine concentrations used in most water treatment facilities. Diagnosis is made by examination of fecal samples for motile trophozoites or cysts (Figure 21.4). Serologic test kits are useful when microscopic examination is negative. Liver abscesses should be biopsied from the abscess edge where the active amebas accumulate. Mild cases of luminal amebic dysentery are treated with iodoquinol, paromomycin, or diloxanide furoate. More severe cases, including liver infections, are treated with metronidazole (which also has antibacterial activity) in combination with chloroquine and/or diloxamide furoate or emetine. Up to 80% of infections due to E. histolytica are asymptomatic. These asymptomatic cyst-passers are a source of infection to others and may not be detected because they are asymptomatic. Figure 21.4 Entamoeba histolytica cysts.
Cryptosporidium is an intracellular parasite that inhabits the epithelial cells of the villi of the lower small intestine. The source of infection is often the feces of domestic animals, and farm run-off has been implicated as a source of Cryptosporidium contamination of drinking water. Asymptomatic to mild cases are common, and, if the immune system of the patient is normal, the disease usually Figure 21.5 Giardia lamblia trophozoite in stool sample. 220 Entamoeba histolytica colon with secondary infection • Infects of liver. patients pass noninfectious • Infected trophozoites as well as infectious cysts in stools. by presence of characteristic • Diagnosis cysts (containing one to four nuclei) in stools. • Therapy: Iodoquinol, metronidazole. Giardia lamblia usually results from • Infection drinking contaminated water. duodenum, with incubation • Infects time of about 10 days. infection shows sudden onset • Acute with foul smelling, watery diarrhea. by presence of cysts or • Diagnosis trophozoites in stools. • Therapy: Metronidazole. Cryptosporidium parvum • Infects lower small intestine. are intracellular parasites • Organisms in epithelial cells of intestinal villi. by modified acid-fast stain • Diagnosis of stool sample. Paromomycin (often • Therapy: not effective). Balantidium coli Causes dysentery by infecting the • large intestine, forming ulcers. • Not invasive. by presence of cysts or • Diagnosis trophozoites in stools. Tetracyclines or • Therapy: metronidazole. Figure 21.6 Summary of intestinal protozoal infections. 21. Protozoa resolves without therapy. However, in immunocompromised individuals (for example, those with AIDS), infection may be severe and intractable, although paromomycin has provided some improvement. Diagnosis is made by acid-fast staining of the tiny (4 to 6 μm) oocysts in fresh stool samples. D. Balantidiasis (Balantidium coli ) Balantidiasis is caused by the ciliate protozoon B. coli, which causes dysentery by infecting the large intestine. B. coli is locally invasive, causing colonic ulcers. Although these may perforate, leading to peritonitis, it is unlike E. histolytica in that it is very rarely associated with spread to distant organs. Manifestations can range from asymptomatic carriage to abdominal discomfort and mild diarrhea to acute dysentery with blood and pus in the stool. The life cycle includes both trophozoite and cyst forms, and identification of either in the stool can be diagnostic. The cysts, which are the infective stage, can be found in contaminated water and are not inactivated by chlorination. Pigs are the natural reservoir of B. coli. The infection can be treated with tetracyclines or metronidizole. A summary of intestinal protozoal infections is shown in Figure 21.6. IV. UROGENITAL TRACT INFECTION: TRICHOMONIASIS Trichomoniasis is caused by Trichomonas vaginalis (Figure 21.7). Trichomoniasis is the most common protozoal urogenital tract infection of humans. The trichomonads are pear-shaped flagellates, with undulating membranes. There is no cyst form in the life cycle of Trichomonas. Several nonpathogenic species, including Trichomonas tenax and Trichomonas hominis, can be found in the human mouth and intestines, respectively. These species, which are part of the normal flora, are not easily distinguished morphologically from the pathogenic species, T. vaginalis. In females, it causes inflammation of the mucosal tissue of the vagina, vulva, and cervix, accompanied by a copious, yellowish, malodorous discharge. Less commonly, it infects the male urethra, prostate, and seminal vesicles, producing a white discharge. The disease is largely sexually transmitted, and both (or all) sexual partners should be treated. Because the optimum pH for growth of this organism is about 6.0, T. vaginalis does not thrive in the normal, acidic vagina, which has a pH of about 4.0. Abnormal alkalinity of the vagina, therefore, favors acquisition of the disease. Diagnosis is made by detection of motile trophozoites in vaginal or urethral secretions. If the concentration of parasites is too low to be observed directly, laboratory culture can be used to obtain observable organisms. Effective treatment is afforded by metronidazole. Figure 21.8 summarizes urogenital infections caused by T. vaginalis.
221 sometimes referred to as hemoflagellates. Three free-living amebas cause amoebic encephalitis in humans. Babesia microcoti causes babesiosis, which is transmitted to humans by the bite of an Ixodes tick and results in a red blood cell (RBC) infection, similar to that caused by Plasmodium species. A. Malaria (Plasmodium falciparum and other species) Malaria is an acute infectious disease of the blood, caused by one of five species of the protozoal genus, Plasmodium, which is a sporozoan. P. falciparum accounts for some 15 percent of all malaria cases, and P. vivax for 80 percent of malarial cases. The plasmodial parasite is transmitted to humans through the bite of a female Anopheles mosquito or by an infected, blood-contaminated, needle. Sporozoans reproduce asexually in human cells by a process called schizogony, in which multiple nuclear divisions are followed by envelopment of the nuclei by cell walls producing merozoites. These, in turn, become trophozoites. Sexual reproduction occurs in the mosquito, where new spores (sporozoites) are formed. Plasmodium knowlesi causes malaria, at least in some parts of Asia. In addition to mosquito bites and blood-contaminate needles, blood transfusion is a potentially important mode of transmission, at least in parts of the world in which screening of bank blood may not be as assiduous as it is in the United States. Figure 21.7 Trichomonas vaginalis. 1. Pathology and clinical significance: Plasmodium sporozoites are injected into the bloodstream, where they rapidly migrate to the liver. There they form cyst-like structures containing thousands of merozoites. Upon release, the merozoites invade RBCs, using hemoglobin as a nutrient. Eventually, the infected RBCs rupture, releasing merozoites that can invade other erythrocytes. If large numbers of RBCs rupture at roughly the same time, a paroxysm (sudden onset) of fever can result from the massive release of toxic substances. A predictable consequence of RBC lysis is anemia, which is typical of Plasmodium infections. P. falciparum is the most dangerous plasmodial species. It can cause a rapidly fulminating disease, characterized by persistent high fever and orthostatic hypotension. Infection can lead to capillary obstruction and death if treatment is not prompt. P. malariae, P. vivax, and P. ovale cause milder forms of the disease, probably because they invade either young or old red cells, but not both. This is in contrast to P. falciparum, which invades cells of all ages. Even today, malarial infection is a common and serious disease and in 2010 is estimated to have caused about 655,000 deaths worldwide. A summary of the life cycle of Plasmodium is shown in Figure 21.9. Trichomonas vaginalis vagina, vulva, • Infects and cervix in females. urethra, prostate, • Infects and seminal vesicles in males. pH • Higher-than-normal favors disease. • Therapy: Metronidazole. 2. Diagnosis and treatment: Diagnosis depends on detection of the parasite inside RBCs (Figure 21.10). Thick blood smears stained with Giemsa stain provide the most sensitive visual test. Thin blood smears, in which more detail can be discerned, are used to determine the species involved, which is important in planning a course of therapy. Serologic tests are usually too slow for diagnosis of acute disease. Drug treatment is determined by the Plasmodium species that is causing the infection. Figure 21.8 Summary of urogenital infection. 222 21. Protozoa 1 Infected mosquito injects sporozoites. 7 Sporozoites Infected mosquito The female mosquito picks up gametocytes from an infected human. The sexual cycle occurs in the mosquito, where sporozoites are formed. Female mosquito Gametocytes 2 Infection can also result from use of a bloodcontaminated needle or transfusion of blood. Sporozoites migrate to the liver, where they form merozoites. LIVER 6 Some merozoites become gametocytes. Trophozoite 3 Merozoites are released and invade red blood cells. 4 Red blood cell In the red blood cell, the merozoite becomes a trophozoite. Merozoites 5 In the red blood cell, the trophozoite multiplies, producing new merozoites. These are released when the red blood cell ruptures, and can infect other red blood cells. Figure 21.9 Life cycle of the malarial parasite, Plasmodium falciparum. Because P. falciparum has no exoerythrocytic phase, it needs only to be treated with quinine, artemisin, mefloquine or doxycycline, depending on resistance patterns in the given geographic location. Chloroquine resistance is so prevalent among P. falciparum that it is almost never used for this organism any more. For ovale or vivax infections, after treatment with chloroquine, a two-week course of primaquine is necessar y to achieve a “radical cure” by eliminating exoerythrocytic organisms that persist in the liver. If, in the geographic location of infection, there is chloroquine resistance among P. vivax or P. ovale, then an alternative drug must be used prior to radical cure. Before treatment with primaquine, patients should be screened for glucose 6-phosphate dehydrogenase deficiency as individuals with deficiency of this enzyme develop hemolytic anemia, sometimes very severe, when treated with primaquine. B. Toxoplasmosis (Toxoplasma gondii) Figure 21.10 Ring form of Plasmodium falciparum in red blood cell. Toxoplasma gondii is an intracellular sporozoan, distributed worldwide, that infects all vertebrate species, although the definitive host is the cat. Humans can become infected by the accidental ingestion of oocysts present in cat feces, by eating raw or undercooked meat, congenitally from an infected mother, or from a blood transfusion.
223 1. Pathology and clinical significance: There are two kinds of Toxoplasma trophozoites found in human infections: rapidly growing tachyzoites (“tachy-” = rapid) that are seen in body fluids in early, acute infections, and slowly growing bradyzoites (“brady-” = slow) that are contained in cysts in muscle and brain tissue and in the eye. Tachyzoites directly destroy cells, particularly parenchymal and reticuloendothelial cells, whereas bradyzoites released from ruptured tissue cysts cause local inflammation with blockage of blood vessels and necrosis. Infections of normal human hosts are common and usually asymptomatic. However, they can be very severe in immunocompromised individuals, who may also suffer recrudescence (relapse) of the infection. Congenital infections can also be severe, resulting in stillbirths, brain lesions, and hydrocephaly, and they are a major cause of blindness in newborns. TRYPANOSOMIASIS AMERICAN (also called Chagas disease) caused by Trypanosoma cruzi • Acute infection is common in children. • Chronic infection causes cardiomyopathy. • Transmitted by insect feces contaminating the eye or a break in the skin. • Treated with nifurtimox. 2. Diagnosis and treatment: The initial diagnostic approach involves detection of parasites in tissue specimens, but this may often be inconclusive. With the recent availability of commercial diagnostic kits, serologic tests to identify toxoplasma are now routinely used. These include tests for Toxoplasma-specific immunoglobulin (Ig) G and IgM. The treatment of choice for this infection is the antifolate drug pyrimethamine, given in combination with sulfadiazine. For patients who can not receive sulfa drugs, clindamycin can be added to pyrimethamine. C. Trypanosomiasis (various trypanosome species) Trypanosomiasis refers to two chronic, eventually fatal, diseases (African sleeping sickness and American trypanosomiasis) caused by several trypanosome species. Some of the differences between these diseases and the available chemotherapeutic agents are summarized in Figure 21.11. 1. Pathology and clinical significance: African sleeping sickness is caused by the closely related flagellates, Trypanosoma brucei gambiense or Trypanosoma brucei rhodesiense (Figure 21.12). These parasites are injected into humans by the bite of the tsetse fly, producing a primary lesion, or chancre. The organism then spreads to lymphoid tissue and reproduces extracellularly in the blood. Later, the parasite invades the central nervous system (CNS), causing inflammation of the brain and spinal cord, mediated by released toxins. This inflammation produces the characteristic lethargy and, eventually, continuous sleep and death. American tr ypanosomiasis (Chagas disease), caused by Trypanosoma cruzi, occurs in Central and South America. Unlike African forms of the disease, infection is not transmitted by insect bite but rather by insect feces contaminating the conjunctiva or a break in the skin. The first symptom is a granulomatous lesion at the site of entry by the pathogen, followed by an acute disease characterized by fever and hepatosplenomegaly. Subsequently the disease may go into remission but reappear as digestive system problems. Potential, long term complications include cardiomyopathy and megacolon. AFRICAN caused by Trypanosoma brucei • Transmitted by the bite of the tsetse fly. • Causes “sleeping sickness.” Trypanosoma brucei gambiense • Slow to enter CNS. • Suramin and pentamidine are used only in the early stages of disease. Trypanosoma brucei rhodesiense • Early invasion of CNS. • Usually fatal if not treated. • Melarsoprol used when there is CNS involvement. Figure 21.11 Summary of trypanosomiasis. CNS = central nervous system. Figure 21.12 Trypanosoma brucei. 224 21. Protozoa 2. Diagnosis and treatment: Diagnosis of African trypanosomiasis is 1 Infected sandfly injects promastigotes into the skin. Infected sandfly made primarily by detection of motile trypanosomes in Giemsastained smears of body fluids (for example, blood, cerebrospinal fluid, and lymph node aspirates). Highly specific serologic tests are also available for diagnostic confirmation. Early-stage African tr ypanosomiasis is treated with suramin or pentamidine. Melarsoprol is used in late-stage disease when the CNS is involved. American trypanosomiasis is treated with nifurtimox, but the drug’s effectiveness is limited. D. Leishmaniasis (various Leishmania species) Promastigotes 2 Promastigotes lose their flagella, enter the amastigote stage, and invade macrophages. Amastigote Leishmaniasis refers to a group of infections caused by the flagellate protozoa of the genus Leishmania. About half a million new cases are reported each year, and it is estimated that 12 million people are currently infected with this parasite. There are three clinical types of leishmaniasis: cutaneous, mucocutaneous, and visceral. The various infective organisms are indistinguishable morphologically but can be differentiated biochemically. Two subgenera are recognized (Leishmania leishmania and Leishmania viannia), each with several species. Any species has the potential to cause one of three clinical manifestations. The natural reservoir of the parasite varies with geography and species but is usually wild rodents, dogs, and humans. Transmission to humans is by the bite of the female sandfly of the genus Phlebotomus or Lutzomyia . The life cycle of Leishmania is shown in Figure 21.13. 1. Cutaneous leishmaniasis (local name, “oriental sore”): This dis- Macrophage 3 The amastigotes reproduce, killing the host cell. Following their release, they can invade other cells. ease is caused by Leishmania tropica in North and West Africa, Iran, and Iraq. The cutaneous form of the disease is characterized by ulcerating single or multiple skin sores (Figure 21.14). Most cases spontaneously heal, but the ulcers leave unsightly scars. In Mexico and Guatemala, the cutaneous form is due to Leishmania mexicana, which produces single lesions that rapidly heal. 2. Mucocutaneous leishmaniasis (local name, espundia): This dis- ease is caused by Leishmania viannia brasiliensis in Central and South America, especially the Amazon regions. In this form of the disease, the parasite attacks tissue at the mucosal-dermal junctions of the nose and mouth, producing multiple lesions. Extensive spreading into mucosal tissue can obliterate the nasal septum and the buccal cavity, ending in death from secondary infection. 3. Visceral leishmaniasis (local name, kala-azar): This disease is 4 A sandfly bites an infected person, and acquires amastigotes. These divide in the fly’s gut, producing promastigotes. Figure 21.13 Life cycle of Leishmania. caused by Leishmania donovani in India, East Africa, and China. In the visceral disease, the parasite initially infects macrophages, which, in turn, migrate to the spleen, liver, and bone marrow, where the parasite rapidly multiplies. Symptoms include intermittent fevers and weight loss. The spleen and liver enlarge, and jaundice may develop. Mortality is nearly 100% within 2 years if the disease is untreated. In some cases, complications resulting from secondary infection and emaciation result in death. 4. Diagnosis and treatment: Diagnosis is made by examination of Giemsa-stained tissue and fluid samples for the nonflagellated
225 form (amastigote), which is the only form of the organism that occurs in humans and other mammals. Cutaneous and mucocutaneous disease can be diagnosed from tissue samples taken from the edges of lesions or lymph node aspirates. Visceral disease is more difficult to diagnose, requiring liver, spleen, or bone marrow biopsy. Serologic tests (for example, indirect fluorescent antibody, see p. 28, and complement fixation, see p. 26) are used by the Centers for Disease Control and Prevention. The treatment of leishmaniasis is difficult because the available drugs have considerable toxicity and high failure rates. Pentavalent antimonials, such as sodium stibogluconate, are the conventional therapy, with pentamidine and amphotericin B as second-line agents. E. Amebic encephalitis (Naegleria fowleri, Acanthamoeba castellanii, and Balamuthia mandrillaris) Several environmental amebae are capable of causing fatal CNS infections in humans. Naegleria fowleri can cause primary amebic meningoencephalitis (PAM) in immunocompetent individuals. The ameba exists in one of three morphological forms: flagellate, trophozoite, or cyst. The trophozoite (the infectious form found in fresh water) enters via the nasal cavity, generally infecting swimming children. From the nasal passages, the ameba directly invades the brain by way of the cribriform plate. The pathogen causes necrotic lesions in the brain, and the infection results in death within a few days of symptom onset. Symptoms initially include headache, fever, and nausea. More than 95 percent of cases are fatal, despite appropriate therapy with amphotericin B. Acanthamoeba species, also free-living amebas, cause granulomatous amebic encephalitis (GAE), which is not as rapidly progressing as PAM. However, like PAM, GAE is often fatal. Acanthamoeba species also cause cutaneous acanthamoebiasis, particularly in immunocompromised individuals. Acanthamoeba keratitis is an infection of the cornea, which is most often seen in contact lens wearers who suffer a traumatic eye injury. The source of the ameba is the contact lens solution, but, in immunocompetent persons, damage to the cornea is a prerequisite to infection. Balamuthia mandrillaris is also a free-living ameba capable of causing encephalitis (BAE). Acquisition of the pathogen is thought to be from water or soil with subsequent hematogenous spread to the brain. As with the other amebic encephalitides, infection in both immunocompetent and immunocompromised persons is likely to be fatal. Several cases of BAE were reported in 2010 in recipients of solid organ transplants. Figure 21.14 Skin ulcer due to leishmaniasis, on the hand of a Central American adult.
Figure 21.15 Wright-stained peripheral blood smear from a newborn with probable congenital Babesia microti infection.The smear shows parasites of variable size and morphologic appearance. 226 21. Protozoa debris (hepatosplenomegaly and jaundice). Study Questions Choose the ONE correct answer. 21.1 The protozoal trophozoite phase is characterized by: A. B. C. D. E. metabolic dormancy. toxin production. active feeding and reproduction. flagellar locomotion. residence in the intermediate host. 21.2 The definitive host of a parasite is the host: A. B. C. D. E. in which asexual reproduction occurs. in which sexual reproduction occurs. that is obligatory for the parasite. that is capable of destroying the parasite. that is the vector organism that transports a parasite from an uninfected to an infected host. 21.3 Plasmodium falciparum, which causes malaria, is an example of: A. B. C. D. E. an ameboid protozoan. a sporozoan. a flagellate. a ciliate. a schizont. 21.4 A U.S. businessman who has recently returned home from Haiti suddenly develops a periodic high fever followed by orthostatic hypotension. What is the likely preliminary diagnosis? A. B. C. D. E. Chagas disease Giardiasis Syphilis Malaria Toxoplasmosis 21.5 A 22 year old female visits her gynecologist complaining of a foul-smelling vaginal discharge and severe itching. A specimen was collected and examined it by light microscopy revealing highly motile, nucleated cells with multiple flagella. What is the most likely causative agent of this infection? A. B. C. D. Balantidium coli Plasmodium falciparum Toxoplasma gondii Giardia lamblia Correct answer = C. The trophozoite is, generally speaking, the active phase, in contrast to the cyst, which is the dormant phase. In some species, several varieties of trophozoites are recognized, such as the tachyzoites and bradyzoites of Toxoplasma gondii. Correct answer = B. Sexual reproduction occurs in the definitive host, whereas asexual reproduction occurs in the intermediate host. For example, in the case of malarial Plasmodium, the definitive host is the mosquito, and the intermediate host is the human. In most cases, both hosts are obligatory for propagation of the parasite. Correct answer = B. The sporozoans are also called apicomplexa because of the presence of a complex of organelles at the cell tip that facilitates penetration of the parasite into host tissue. A schizont is not a taxonomic group but a mass of trophozoites. Correct answer = D. All of the signs point to malaria, especially the periodicity of the fever that results from synchronous rupturing of large numbers of red blood cells. Correct answer = E. The symptoms are consistent with the sexually transmitted infection caused by Trichomonas vaginalis. This protozoal flagellate is highly motile and easily distinguished from other sexually transmitted disease pathogens by light microscopy. The other protozoal pathogens listed do not cause diseases that present with genitourinary tract symptoms. 22 Helminths I. OVERVIEW Helmint hs CESTODES (TAPEWORMS) Helminths are worms, some of which are parasitic to humans. These parasites belong to one of three groups: cestodes (tapeworms), trematodes (flukes), or nematodes (roundworms) as shown in Figure 22.1. Although individual species may have preferred primary sites of infestation (often the intestines where they generally do little damage) these organisms may disseminate to vital organs (for example, the brain, lungs, or liver) where they can cause severe damage. It is estimated that at least 70 percent of the world’s population is infected with a parasitic helminth. The mode of transmission to humans varies from species to species but includes ingestion of larvae in raw or undercooked pork, beef, or fish; ingestion of helminth eggs in feces; transmission by insect bites; or by direct skin penetration. In North America, helminthic diseases are becoming rare, whereas they are endemic in regions of the world where community sanitary conditions are poor, and human feceal material is used as fertilizer. Diphyllobothrium latum (Broad fish tapeworm) Echinococcus granulosus (Dog tapeworm) Taenia saginata (Beef tapeworm) Taenia solium (Pork tapeworm) TREMATODES (FLUKES) Clonorchis sinensis (Chinese or Oriental liver fluke) Paragonimus westermani (Lung fluke) Schistosoma mansoni (Blood fluke) Schistosoma haematobium (Blood fluke) Schistosoma japonicum II. CESTODES Intestinal infections NEMATODES (ROUNDWORMS) Ancylostoma duodenale Necator americanus (Old World and New World hookworms) Ascaris lumbricoides (Giant roundworm) Enterobius vermicularis (Pinworm) Strongyloides stercoralis (threadworm) Trichinella spiralis Trichuris trichiura (Whipworm) Tissue infections Cestodes (tapeworms) are ribbon-like, segmented worms that are primarily intestinal parasites. They lack a digestive system and do not ingest particulate matter but, instead, absorb soluble nutrients directly through their cuticles. In the small intestine, some species (for example, the tapeworm Diphyllobothrium latum) can attain enormous lengths of up to 15 meters. Cestodes cause clinical injury by sequestering the host’s nutrients; by excreting toxic waste; and, in massive infestations, by causing mechanical blockage of the intestine. The anterior end of the worm consists of a scolex, a bulbous structure with hooks and suckers that functions to attach the worm to the intestinal wall (Figure 22.2). The body (strobila) is composed of many segments (proglottids), which form continuously in the region just behind the scolex. Each proglottid has a complete set of sexual organs (that, both male and female) that generate fertilized eggs. The mature, egg-filled proglottids are located at the posterior end of the organism. These can break off the chain and pass out of the body in the stool. Characteristics of infections by the four medically important cestodes are summarized in Figure 22.3. Note that Taenia solium has two different disease manifestations, depending on whether transmission is by ingestion of larvae from undercooked pork or by ingestion of its eggs. In the former case, infestation is limited to the intestines, whereas, in the latter case, the eggs develop into larvae that form cysts (cysticerci) in the brain and other tissues. (Blood fluke) Brugia malayi Dracunculus medinensis (Guinea worm) Loa loa (Filarial worm, or African eye worm) Onchocerca volvulus (Filarial worm) Toxocara canis (Dog worm) Wuchereria bancrofti (Filarial worm) Figure 22.1 Clinically important helminths. 227 228 22. Helminths III. TREMATODES Trematodes, commonly called flukes, are small (about 1 cm), flat, leaf-like worms that, depending on the species, infest various organs of the human host (for example, intestinal veins, urinary bladder, liver, or lung). All parasitic trematodes use freshwater snails as an intermediate host. A. Hermaphroditic flukes Figure 22.2 The scolex of Taenia solium is 1 mm wide and has four suckers. Developmental events in the life cycle of a typical fluke begin when the adult fluke, which is hermaphroditic, produces eggs in the human (the definitive host). The eggs are then excreted into the environment. The first larval stage (miracidium) develops inside the eggs. These larvae seek out and infect suitable snail species, which are the first intermediate host. In the snail, asexual reproduction occurs, during which several intermediate developmental forms can be distinguished, including sporocyst; redia (an early larval stage); and, eventually, large numbers of the final larval stage, called cercariae, which leave the snail and seek out a second intermediate host (a fish or crustacean, depending on the species of fluke). In this Cysticercosis is caused by Taenia solium • This larvae. Infection produces cysticerci in Echinococcosis the brain (causing seizures, headache, and vomiting) and in the eyes. disease is caused by • This Echinococcus granulosus (dog disease follows ingestion of eggs • The from human feces. tapeworm). Infection produces large, hydatid cysts in liver, lung, and brain. Anaphylactic reaction to worm antigens can occur if the cyst ruptures. is diagnosed by CT, MRI, • Cysticercosis or biopsy. It is treated with praziquantel, albendazole, and/or surgery. disease follows ingestion of • The eggs in dog feces. Sheep often serve as an intermediate host. Taeniasis is diagnosed by CT • Echinococcosis scan or biopsy of infected tissue and form of the disease is caused by • This the larval form of Taenia saginata (beef is treated with albendazole and surgical excision of intact cysts. tapeworm). The organism primarily infects the intestines and does not produce cysticerci. Most infected individuals are asymptomatic. Taeniasis disease is transmitted by larvae in • The undercooked or raw beef. form of the disease is caused by • This the adult Taenia solium (pork tapeworm). Intestines are the primary site of infection, where the organism can cause diarrhea. Most of these infections, however, are asymptomatic. disease is transmitted by • The ingestion of larvae in undercooked pork. is diagnosed by detection • Taeniasis of proglottids in the stool. It is treated with niclosamide. is diagnosed by detection of • Taeniasis proglottids in the stool. It is treated with niclosamide. Diphyllobothriasis disease is caused by Diphyllobothrium latum (fish tapeworm). • This The adult worm in a host’s intestine can be as long as 15 meters. • The disease is transmitted by larvae in raw or undercooked fish. is diagnosed by detection of characteristic eggs • Diphyllobothriasis in the stool. It is treated with niclosamide. Figure 22.3 Characteristics and therapy for commonly encountered cestode infections. CT = computed tomography; MRI = magnetic resonance imaging. III. Trematodes 229 second intermediate host, the cercariae form cysts called metacercariae that can remain viable indefinitely. Finally, if the infected raw or undercooked fish or crustacean is eaten by a human, the metacercaria excysts (emerges from cysts), and the fluke invades tissues such as the lung or the liver and begins producing eggs, thus completing the life cycle. B. Sexual flukes (schistosomes) The life cycle of schistosomes is similar to that of hermaphroditic flukes. One difference is that schistosomes have only one intermediate host, the snail. Another difference is that schistosomiasis is not acquired by ingestion of contaminated food, but rather from schistosome cercariae directly penetrating the skin of waders or swimmers in contaminated rivers and lakes. After dissemination and development in the human host, adult schistosomes take up residence in various abdominal veins, depending on the species and are, therefore, called “blood flukes.” Also in contrast to the “typical” hermaph Figure 22.4 Male schistosome has a long groove in which the smaller female resides and continuously mates with the male. roditic flukes described above, schistosomes have separate, distinctive sexes. A remarkable anatomic feature is the long groove or schist on the ventral surface of the large male in which the smaller female resides and continuously mates with the male (Figure 22.4). Schistosomiasis Paragonimiasis disease is caused by • This Paragonimus westermani (lung fluke). The organisms reach the lung by penetrating the duodenum, migrating into the peritoneal cavity and through the diaphragm into the pleural cavities. From there they enter the lungs which are the primary site of damage. The inflammatory response to the adults and eggs in the lungs results in cough that often produces bloody sputum. disease is transmitted by • The ingestion of encysted larvae in raw or rare crab meat or crayfish. is diagnosed by • Paragonimiasis identifying eggs in the sputum and disease is caused by Schistosoma • This mansoni and Schistosoma japonicum. The primary site of infection is the gastrointestinal tract. Damage to the intestinal wall is caused by the host’s inflammatory response to eggs deposited at that site. The eggs also secrete proteolytic enzymes that further damage the tissue. presentation includes GI bleeding, • Clinical diarrhea, and liver damage. Periportal fibrosis leads to portal hypertension and massive splenomegaly. disease is transmitted by direct skin • The penetration. form of schistosomiasis is • This diagnosed by identification of characteristic eggs in the stool. It is treated with praziquantel. stool. It is treated with praziquantel. Clonorchiasis disease is caused by Clonorchis • This sinensis (Oriental liver fluke). The primary site of infection is the biliary tract, where the resulting inflammatory response can cause fibrosis and hyperplasia. disease is transmitted by eating • The raw freshwater fish. is diagnosed by • Clonorchiasis identifying eggs in the stool. It is Schistosomiasis disease is caused by Schistosoma • This haematobium. The primary sites of infection are veins of the urinary bladder, where the organism’s eggs can induce fibrosis, granulomas, and hematuria. disease is transmitted by direct • The skin penetration. form of schistosomiasis is • This diagnosed by identifying characteristic treated with praziquantel. Figure 22.5 Characteristics and therapy for commonly encountered trematode infections. eggs in the urine or bladder wall. It is treated with praziquantel. 230 22. Helminths This mating takes place in the human liver. Fertilized eggs penetrate the human host’s vascular walls and enter the intestine or bladder, emerging from the body in feces or urine. In fresh water, the organisms infect snails in which they multiply, producing cercariae (the final, free-swimming larval stage), which are released into the fresh water to complete the cycle. Characteristics of clinically important trematodes are summarized in Figure 22.5. IV. NEMATODES Figure 22.6 Coiled larva of Trichinella spiralis in skeletal muscle. The nematodes (roundworms) are elongated, nonsegmented worms that are tapered at both ends (Figure 22.6). Unlike other helminths, Loiasis Onchocerciasis (river blindness) disease is caused by Loa loa. The • This larvae crawl under the skin, leaving characteristic tracks. They can enter the eye where adult worms are visible in the subconjuctival space around the iris. disease is caused by • This Onchocerca volvulus. It is characterized by subcutaneous nodules, pruritic skin rash, and ocular lesions often causing blindness. disease is transmitted by deer flies. • The There is no animal reservoir, and humans are the only definitive host. disease is transmitted by the bite • The of a blackfly. is diagnosed by detection of • Loiasis microfilariae in blood. It is treated with is diagnosed by • Onchocerciasis detection of microfilariae in skin diethylcarbamazine. biopsy. It is treated with ivermectin and/or surgery. Dracunculiasis disease is caused by Dracunculus • This medinensis. Adult worms cause skin Visceral larva migrans inflammation and ulceration. Adult females can be as long as 100 cm; males are much smaller. disease is caused by Toxocara • This canis. It is primarily a disease of disease is transmitted by drinking • The water containing the intermediate host young children. The larval form matures in the intestines, then migrates to the liver, brain, and eyes. Only the larvae cause disease. copepods in which the larvae live. is diagnosed by finding • Dracunculiasis the head of the worm in a skin lesion or disease is transmitted by • The ingestion of eggs from dog feces. • larvae that are released from a lesion following contact with water. The disease is treated by removing subcutaneous worms (formerly by winding them on a thin stick, now usually by surgery). Visceral larval migrans is diagnosed by detecting larvae in the tissue. It is treated with mebendazole or thiabendazole. Filariasis (elephantiasis) most frequent organisms causing this disease are • The Wuchereria bancrofti and Brugia malayi. These filarial worms block the flow of lymph, causing edematous arms, legs, and scrotum. disease is transmitted by the bite of infected • The female Anopheles and Culex mosquitoes. is diagnosed by detection of microfilariae in • Filariasis blood. It is treated with a combination of diethylcarbamazine and albendazole. Trichinosis disease is caused by Trichinella spiralis, an • This intestinal nematode that encysts in the tissue of human and porcine hosts. disease is transmitted by eating encysted larvae in • The undercooked pork. is diagnosed by locating coiled encysted • Trichinosis larvae in a muscle biopsy. In its early stages, the disease is treated with thiabendazole; no treatment is available for the late stages. Allergic manifestations are treated symptomatically and not with an anthelminthic drug. Figure 22.7 Characteristics and therapy for commonly encountered nematode infections of tissues other than intestine. IV. Nematodes Enterobiasis (pinworm disease) disease is caused by Enterobius • This vermicularis. It is the most common helminthic infection in the United States. Pruritus ani occurs, with white worms visible in the stools or perianal region. disease is transmitted by • The ingesting the organism’s eggs. 231 Trichuriasis (whipworm disease) disease is caused by Trichuris trichiura. • This The infection is usually asymptomatic; however, abdominal pain, diarrhea, flatulence, and rectal prolapse can occur. disease is transmitted by ingestion of soil • The containing the organism’s eggs. disease is diagnosed by • Whipworm identifying characteristic eggs in the stool. It is treated with mebendazole. Humans are the only host. disease is diagnosed by • Pinworm identifying eggs present around the perianal region. It is treated with mebendazole or pyrantel pamoate. Hookworm disease disease is caused by Ancylostoma • This duodenale and Necator americanus. The worm attaches to the intestinal mucosa, causing anorexia; ulcer-like symptoms, and chronic intestinal blood loss, leading to anemia. Ascariasis (roundworm disease) disease is caused by Ascaris • This lumbricoides. It is second only to pinworms as the most prevalent multicellular parasite in the United States. Approximately one third of the world’s population is infected with this worm. disease is transmitted by • The ingestion of soil containing the organism’s eggs. Humans are the sole host. Larvae grow in the intestine, causing abdominal symptoms, including intestinal obstruction. Roundworms may pass to the blood and through the lungs. disease is diagnosed by • Roundworm detection of characteristic eggs in the stool. It is treated with pyrantel pamoate or mebendazole. disease is transmitted through direct skin • The penetration by larvae found in soil. disease is diagnosed by • Hookworm identification of characteristic eggs in the stool. It is treated with pyrantel pamoate or mebendazole. Strongyloidiasis (threadworm disease) disease is caused by Strongyloides stercoralis. It is relatively uncommon • This compared with infections by other intestinal nematodes. It is a relatively benign disease in healthy individuals but can progress to a fatal outcome in immunocompromised patients because of dissemination to the CNS or other deep organs (hyperinfection syndrome) in certain immunocompromised patients. • The disease is transmitted through direct skin penetration by larvae found in soil. disease is diagnosed by identifying larvae in the stool. It is treated • Threadworm with thiabendazole, albendazole or ivermectin. Figure 22.8 Characteristics and therapy for commonly encountered intestinal nematode infections. nematodes have a complete digestive system, including a mouth, an intestine that spans most of the body length, and an anus. The body is protected by a tough, noncellular cuticle. Most nematodes have separate, anatomically distinctive sexes. The mode of transmission varies widely, depending on the species, and includes direct skin penetration by infectious larvae, ingestion of contaminated soil, eating undercooked pork, and insect bites. The parasites can invade almost any part of the body: liver, kidneys, intestines, subcutaneous tissue, and eyes. Generally, nematodes are categorized by whether they infect the intestine or other tissues (Figures 22.7 and 22.8). Alternatively, they can be divided into those for which the eggs are infectious and those for which the larvae are infectious. The most common nematode infection in the United States is enterobiasis (pinworm disease), which causes anal itching (Figure 22.9) but otherwise does little damage. A more serious disease of worldwide occurrence is ascariasis, caused by Ascaris lumbricoides (see Figure 22.8). Figure 22.9 Pinworms leaving the anus of a five-year-old child. 232 22. Helminths Study Questions Choose the ONE correct answer. 22.1 A patient is diagnosed as having a trematode infection. Lacking a more specific identification of the causative organism, which of the following drugs would most likely be effective? A. B. C. D. E. Niclosamide Thiabendazole Praziquantel Diethylcarbamazine Tetracycline 22.2 Which of the following is the most common helminthic infection in the United States? A. B. C. D. E. Filariasis Onchocerciasis Taeniasis Schistosomiasis Visceral larva migrans 22.4 Which of the following helminthic diseases is transmitted by direct skin penetration by helminth larvae? A. B. C. D. E. Correct answer = E. Enterobiasis is known as pinworm disease. Schistosomiasis Diphyllothriasis Clonorchiasis Trichinosis Enterobiasis 22.3 Which of the following helminthic diseases is transmitted by the bite of a mosquito? A. B. C. D. E. Correct answer = C. Praziquantel is the drug of choice for most trematode infections. Filariasis Onchocerciasis Dracunculiasis Schistosomiasis Visceral larva migrans Correct answer = A. Mosquitoes ingest filarial embryos (microfilariae) from infected blood. In the insect, the embryos develop into infective filariform larvae that are injected into the human host. Onchocerciasis is transmitted by the bite of the blackfly. Taeniasis is transmitted by ingestion of larvae in undercooked pork. Schistosomiasis is transmitted by direct skin penetration. Visceral larva migrans is transmitted by ingestion of eggs from dog feces. Correct answer = D. Schistosome cercariae released from snails in fresh water are capable of penetrating human skin. Filariasis is transmitted by mosquitoes. Onchocerciasis is transmitted by the bite of the blackfly. Dracunculiasis is transmitted by drinking water containing the intermediate host copepods in which the larvae live. Visceral larva migrans is transmitted by ingestion of eggs from dog feces. UNIT IV: Viruses 23 Introduction to the Viruses I. OVERVIEW A virus is an infectious agent that is minimally constructed of two components: 1) a genome consisting of either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), but not both, and 2) a protein-containing structure (capsid) designed to protect the genome (Figure 23.1A). Many viruses have additional structural features, for example, an envelope composed of a protein-containing lipid bilayer, whose presence or absence further distinguishes one virus group from another (Figure 23.1B). A complete virus particle combining these structural elements is called a virion. In functional terms, a virion can be envisioned as a delivery system that surrounds a nucleic acid payload. The delivery system is designed to protect the genome and enable the virus to bind to host cells. The payload is the viral genome and may also include enzymes required for the initial steps in viral replication—a process that is obligately intracellular. The pathogenicity of a virus depends on a great variety of structural and functional characteristics. Therefore, even within a closely related group of viruses, different species may produce significantly distinct clinical pathologies. A Nonenveloped virus Capsid Nucleic acid B Enveloped virus Capsid II. CHARACTERISTICS USED TO DEFINE VIRUS FAMILIES, GENERA, AND SPECIES Envelope Nucleic acid Viruses are divided into related groups, or families, and, sometimes into subfamilies based on: 1) type and structure of the viral nucleic acid, 2) the strategy used in its replication, 3) type of symmetry of the virus capsid (helical versus icosahedral), and 4) presence or absence of a lipid envelope. Within a virus family, differences in additional specific properties, such as host range, serologic reactions, amino acid sequences of Proteins in envelope membrane Figure 23.1 General structure: A. nonenveloped; B. enveloped viruses. 233 234 23. Introduction To The Viruses Family (–viridae) For example, Herpesviridae B A viral proteins, degree of nucleic acid homology, among others, form the basis for division into genera (singular, genus) and species (Figure 23.2). Species of the same virus isolated from different geographic locations may differ from each other in nucleotide sequence. In this case, they are referred to as strains of the same species. Subfamily (–virinae) For example, Alphaherpesvirinae Genus (–virus) For example, Herpesvirus Species For example, herpes simplex virus Figure 23.2 Classification of viruses: A. No subfamilies present. B. Subfamilies present.
KEY: Single stranded Nonenveloped STRANDEDNESS OF GENOME Double stranded Nonenveloped Single-stranded nucleic acid DNA RNA Double-stranded nucleic acid TYPE OF GENOME Hepadnaviridae (26) Herpesviridae (25) Poxviridae (25) PRESENCE OF ENVELOPE DNA viruses Enveloped RNA viruses Nonenveloped Icosahedral symmetry Helical symmetry Arenaviridae (29) Bunyaviridae (29) Filoviridae (29) Orthomyxoviridae (29) Paramyxoviridae (29) Rhabdoviridae (29) Parvovirdae (24) Adenoviridae (24) Papovaviridae (24) Double stranded Enveloped Single stranded Positive strand Icosadedral Nonenveloped Caliciviridae (27) Picornaviridae (27) Single stranded Positive strand Icosadedral or helical Enveloped Coronaviridae (29) Flaviviridae (27) Retroviridae (28) Togaviridae (27) Single stranded Negative strand Helical Enveloped Double stranded, Icosahedral Nonenveloped Reoviridae (30) Figure 23.3 Viral families classified according to type of genome, capsid symmetry, and presence or absence of an envelope. RNA is shown in blue, DNA in red, and viral envelope in green. [Note: Numbers indicate chapters where detailed information is presented.] III. Viral Replication: The One-Step Growth Curve helical (rod shaped or coiled) or icosahedral (spherical or symmetric). The capsid is constructed of multiple copies of a single polypeptide type (found in helical capsids) or a small number of different polypeptides (found in icosahedral capsids), requiring only a limited amount of genetic information to code for these structural components. 235 Several rows of protomers have been removed to reveal nucleic acid surrounded by a hollow protein cylinder. 1. Helical symmetry: Capsids with helical symmetry, such as the paramyxoviridae (see p. 312), consist of repeated units of a single polypeptide species that—in association with the viral nucleic acid—self-assemble into a helical cylinder (Figure 23.4). Each polypeptide unit (protomer) is hydrogen-bonded to neighboring protomers. The complex of protomers and nucleic acid is called the nucleocapsid. Because the nucleic acid of a virus is surrounded by the capsid, it is protected from environmental damage. 2. Icosahedral symmetry: Capsids with icosahedral symmetry are more complex than those with helical symmetry, in that they consist of several different polypeptides grouped into structural subassemblies called capsomers. These, in tur n, are hydrogen-bonded to each other to form an icosahedron (Figure 23.5). The nucleic acid genome is located within the empty space created by the rigid, icosahedral structure. Capsid Figure 23.4 Nucleocapsid of a helical virus. Capsomer Nucleic acid
III. VIRAL REPLICATION: THE ONE-STEP GROWTH CURVE The one-step growth curve is a representation of the overall change, with time, in the amount of infectious virus in a single cell that has been infected by a single virus particle. In practice, this is determined by following events in a large population of infected cells in which the infection is proceeding as nearly synchronously as can be achieved by manipulating the experimental conditions. Whereas the time scale and yield of progeny virus vary greatly among virus families, the basic features of the infectious cycle are similar for all viruses. The one-step growth curve begins with the eclipse period, which is followed by a period of exponential growth (Figure 23.7). Figure 23.5 Structure of a nonenveloped virus showing icosahedral symmetry. Proteins in envelope membrane Envelope Nucleic acid Capsomer Figure 23.6 Structure of an enveloped helical virus. Infectious viruses per cell 236 23. Introduction To The Viruses Exponential growth period 1000 100 Yield per cell 10 1 0 Eclipse period 0 10 Hours
20 Figure 23.7 One-step growth curve of a single cell infected with a single virus particle. Initiation of infection is at zero time. Virus
A IV. STEPS IN THE REPLICATION CYCLES OF VIRUSES Receptor in host cell membrane The individual steps in the virus replication cycle are presented below in sequence, beginning with virus attachment to the host cell and leading to penetration and uncoating of the viral genome. Gene expression and replication are followed by assembly and release of viral progeny. Receptor Attached virus
B Antibody to viral structure required for adsorption Virus fails to bind to host cell receptor 1. Attachment sites on the viral surface: Some viruses have spe- cialized attachment structures such as the glycoprotein spikes found in viral envelopes (for example, rhabdoviruses, see p. 310), whereas, for others, the unique folding of the capsid proteins forms the attachment sites (for example, picornaviruses, see p 284). In both cases, multiple copies of these molecular attachment structures are distributed around the surface of the virion. [Note: In some cases, the mechanism by which antibodies neutralize viral infectivity is through antibody binding to the viral structures that are required for adsorption (Figure 23.8B).] 2. Host cell receptor molecules: The receptor molecules on the host Figure 23.8 A. Attachment of virus to receptor on host cell membrane. B. Antibody prevents adsorption of virus. cell membrane are specific for each virus family. Not surprisingly, these receptors have been found to be molecular structures that usually carry out normal cell functions. For example, cellular membrane receptors for compounds such as growth factors may IV. Steps in the Replication Cycles of Viruses also inadvertently serve as receptors for a particular virus. Many of the compounds that serve as virus receptors are present only on specifically differentiated cells or are unique for one animal species. Therefore, the presence or absence of host cell receptors is one important determinant of tissue specificity within a susceptible host species and also for the susceptibility or resistance of a species to a given virus. Information about the three-dimensional structure of virus-binding sites is being used to design antiviral drugs that specifically interact with these sites, blocking viral adsorption. 237 1 Virion Binding of a virus Host receptor 2 Invagination of the membrane 3 Formation of an endocytotic vesicle
cess by which the cell internalizes compounds, such as growth regulatory molecules and serum lipoproteins, except that the infecting virus particle is bound to the host cell surface receptor in place of the normal ligand (Figure 23.9). The cell membrane invaginates, enclosing the virion in an endocytotic vesicle (endosome). Release of the virion into the cytoplasm occurs by various routes, depending on the virus, but, in general, it is facilitated by one or more viral molecules. In the case of an enveloped virus, its membrane may fuse with the membrane of the endosome, resulting in the release of the nucleocapsid into the cytoplasm. Failure to exit the endosome before fusion with a lysosome generally results in degradation of the virion by lysosomal enzymes. Therefore, not all potentially infectious particles are successful in establishing infection. 2. Membrane fusion: Some enveloped viruses (for example, human immunodeficiency virus, see p. 297) enter a host cell by fusion of their envelope with the plasma membrane of the cell (Figure 23.10). One or more of the glycoproteins in the envelope of these viruses promotes the fusion. The end result of this process is that the nucleocapsid is free in the cytoplasm, whereas the viral membrane remains associated with the plasma membrane of the host cell. Endocytotic vesicle Release of the virion into cytoplasm Figure 23.9 Receptor-mediated endocytosis of virus particle. Enveloped virus 1 2
4 3 Binding of a virus to a host cell membrane receptor Fusion of viral envelope with the host cell membrane Nucleocapsid enters the cell Figure 23.10 Fusion of viral envelope with membrane of host cell. 238 23. Introduction To The Viruses dictably leads to a loss of the ability of that particle to infect other cells, which is the basis for the eclipse period of the growth curve (see Figure 23.7). It is during this phase in the replication cycle that viral gene expression begins. D. Mechanisms of DNA virus genome replication Each virus family differs in significant ways from all others in terms of the details of the macromolecular events comprising the replication cycle. The wide range of viral genome sizes gives rise to great differences in the number of proteins for which the virus can code. In general, the smaller the viral genome, the more the virus must depend on the host cell to provide the functions needed for viral replication. For example, some small DNA viruses, such as Polyomaviruses (see p. 249), produce only one or two replicationrelated gene products, which function to divert host cell processes to those of viral replication. Other larger DNA viruses, such as poxviruses (see p. 270), provide virtually all enzymatic and regulatory molecules needed for a complete replication cycle. Most DNA 1 Synthesis of early proteins Parental DNA Transcription of early genes (fraction of the viral genome transcribed prior to initiation of viral DNA synthesis) Translation Early proteins Early mRNAs Early proteins ( + cell enzymes) 2 Replication of virus DNA 3 Synthesis of late proteins Transcription of late genes (fraction of the viral genome transcribed after initiation of viral DNA synthesis) Translation Late mRNAs Progeny DNA 4 Figure 23.11 Replication of DNA viruses. Assembly of nucleocapids Late proteins IV. Steps in the Replication Cycles Of Viruses viruses assemble in the nucleus, whereas most RNA viruses develop solely in the cytoplasm. Figure 23.11 outlines the essential features of gene expression and replication of DNA viruses. E. Mechanisms of RNA virus genome replication Viruses with RNA genomes must overcome two specific problems that arise from the need to replicate the viral genome and to produce a number of viral proteins in eukaryotic host cells. First, there is no host cell RNA polymerase that can use the viral parental RNA as a template for synthesis of complementary RNA strands. Second, translation of eukaryotic mRNAs begins at only a single initiation site, and they are, therefore, translated into only a single polypeptide. However, RNA viruses, which frequently contain only a single molecule of RNA, must express the genetic information for at least two proteins: an RNA-dependent RNA polymerase and a minimum of one type of capsid protein. Although the replication of each RNA virus family has unique features, the mechanisms evolved to surmount these restrictions can be grouped into four broad patterns (or “types”) of replication. 1. Type I—RNA viruses with a single-stranded genome (ssRNA) of (+) polarity that replicates via a complementary (–) strand intermediate: In Type I viral replication, the infecting parental RNA 239 1 ssRNA serves as mRNA Translation (+) ssRNA Cleavage Polyprotein Viral proteins (+) ssRNA serves as the template for complementary (–) strand synthesis 2 Proteins include RNA-dependant RNA polymerase (–) ssRNA 3 (–) ssRNA serves as the template for complementary (+) strand synthesis RNA-dependant RNA polymerase molecule serves both as mRNA and, later, as a template for synthesis of the complementary (–) strand (Figure 23.12). More viral proteins
genome is of (+), or messenger, polarity, it can be translated directly upon uncoating and associating with cellular ribosomes. The product is usually a single polyprotein from which individual polypeptides, such as RNA-dependent RNA polymerase and various proteins of the virion, are cleaved by a series of proteolytic processing events carried out by a protease domain of the polyprotein (see Figure 23.12). b. Role of (+) ssRNA as the template for complementary (–) strand synthesis: The viral (+) ssRNA functions early in infec- tion, not only as mRNA for translation of polyproteins but also as a template for virus-encoded RNA-dependent RNA polymerase to synthesize complementary (–) ssRNA (see Figure 23.12). The progeny (–) strands, in turn, serve as templates for synthesis of progeny (+) strands, which can serve as additional mRNAs, amplifying the capacity to produce virion proteins for progeny virus. When a sufficient quantity of capsid proteins has accumulated later in the infection, progeny (+) ssRNAs begin to be assembled into newly formed nucleocapsids. 2. Type II—viruses with a ssRNA genome of (–) polarity that replicate via a complementary (+) strand intermediate: Viral genomes with (–) polarity, similar to the (+) strand genomes, also have two functions: 1) to provide information for protein synthesis and 2) to serve as templates for replication. Unlike (+) strand genomes, (+) ssRNA Assembly into nucleocapsid Figure 23.12 Type I virus with a ssRNA genome of (+) polarity replicates via a complementary (–) strand intermediate. 240 1 23. Introduction To The Viruses Transcription of (+) strand mRNAs from the parental viral (–) strand RNA template
(–) ssRNA RNA-dependent RNA polymerase from infecting virus particle (+) ssRNA 2 mRNAs (+) ssRNA serves as a template for complementary (–) strand (viral genome) synthesis- however, the (–) strand genomes cannot accomplish these goals without prior construction of a complementary (+) strand intermediate (Figure 23.13). Viral proteins RNA-dependent RNA polymerase (–) ssRNA Assembly into nucleocapsids Figure 23.13 Type II virus with an ssRNA genome of (–) polarity that replicates via a complementary (+) strand intermediate. The replication problems for these viruses are twofold. First, the (–) strand genome cannot be translated, and, therefore, the required viral RNA polymerase cannot be synthesized immediately following infection. Second, the host cell has no enzyme capable of transcribing the (–) strand RNA genome into (+) strand RNAs capable of being translated. The solution to these problems is for the infecting virus particle to contain viral RNA-dependent RNA polymerase and to bring this enzyme into the host cell along with the viral genome. As a consequence, the first synthetic event after infection is generation of (+) strand mRNAs from the parental viral (–) strand RNA template. b. Mechanisms for multiple viral protein synthesis in Type II viruses: The synthesis of multiple proteins is achieved in one of two ways among the (–) strand virus families: 1) The viral genome may be transcribed into a number of individual mRNAs, each specifying a single, polypeptide. 2) Alternatively, the (–) strand viral genome may be segmented (that is, composed of a number of different RNA molecules, most of which code for a single polypeptide). c. Production of infectious virus particles: Although the details differ, the flow of information in both segmented and unsegmented genome viruses is basically the same. In the Type II replication scheme, an important control point is the shift from synthesis of (+) strand mRNA to progeny (–) strand RNA molecules that can be packaged in the virions. This shift is not a result of activity of a different polymerase, but rather a result of interaction of (+) strand RNA molecules with one or more newly synthesized proteins. This enhances the availability of the (+) strands as templates for the synthesis of genomic (–) strands. 3. Type III—viruses with a dsRNA genome: The dsRNA genome is segmented, with each segment coding for one polypeptide (Figure 23.14). However, eukaryotic cells do not have an enzyme capable of transcribing dsRNA. Type III viral mRNA transcripts are, therefore, produced by virus-coded, RNA-dependent RNA polymerase (transcriptase) located in a subviral core particle. This particle consists of the dsRNA genome and associated virion proteins, including the transcriptase. The mechanism of replication of the dsRNA is unique, in that the (+) RNA transcripts are not only used for translation but also as templates for complementary (–) strand synthesis, resulting in the formation of dsRNA progeny. IV. Steps in the Replication Cycles Of Viruses 241 4. Type IV—viruses with a genome of ssRNA of (+) polarity that is replicated via a DNA intermediate: The conversion of a (+) strand RNA to a double-stranded DNA is accomplished by an RNAdependent DNA polymerase, commonly referred to as a “reverse transcriptase,” that is contained in the virion. The resulting dsDNA becomes integrated into the cell genome by the action of a viral “integrase.” Viral mRNAs and progeny (+) strand RNA genomes are transcribed from this integrated DNA by the host cell RNA polymerase (Figure 23.15). 1 Transcription of (+) strand RNA from virus dsRNA template dsRNA (segmented) RNA-dependent RNA polymerase from infecting virus particle Viral proteins mRNAs
2 (+) RNA strands serve both as mRNA and template for complementary (–) RNA strand synthesis RNA-dependent RNA polymerase Assembly into nucleocapsids 1. Naked viruses: In naked (unenveloped) viruses, the virion is com- plete at this point. Release of progeny is usually a passive event resulting from the disintegration of the dying cell and, therefore, may be at a relatively late time after infection. 2. Enveloped viruses: In enveloped viruses, virus-specific glycopro- teins are synthesized and transported to the host cell membrane (+) ssRNA RNA DNA Viral RNA-dependent DNA polymerase (reverse transcriptase) Viral proteins DNA DNA Viral RNA-dependent DNA polymerase (reverse transcriptase) Host RNA polymerase Translation Figure 23.14 Type III virus with a dsRNA genome. Viral mRNAs Integration into host DNA by viral integrase Host RNA polymerase Assembly into nucleocapsid Viral (+) ssRNA Figure 23.15 Type IV virus with a ssRNA genome of (+) polarity that replicates via a DNA intermediate. 242 1 23. Introduction To The Viruses Virus-specific glycoproteins are synthesized and transported to the host cell membrane. Host cell membrane Viral protein 2 The cytoplasmic domains of membrane proteins bind nucleocapsids. Nucleocapsid in the same manner as cellular membrane proteins. 1 When inserted into the membrane, they displace the cellular glycoproteins, resulting in patches on the cell surface that have viral antigenic specificity. The cytoplasmic domains of these proteins associate specifically with one or more additional viral proteins (matrix proteins) to which the nucleocapsids bind. Final maturation then involves envelopment of the nucleocapsid by a process of “budding” (Figure 23.16). A consequence of this mechanism of viral replication is that progeny virus are released continuously while replication is proceeding within the cell and ends when the cell loses its ability to maintain the integrity of the plasma membrane. A second consequence is that, with most enveloped viruses, all infectious progeny are extracellular. The exceptions are those viruses that acquire their envelopes by budding through internal cell membranes such as those of the endoplasmic reticulum or nucleus. Viruses containing lipid envelopes are sensitive to damage by harsh environments and, therefore, tend to be transmitted by the respiratory, parenteral, and sexual routes. Nonenveloped viruses are more stable to hostile environmental conditions and often transmitted by the fecal–oral route. G. Effects of viral infection on the host cell 3 4 5 A nucleocapsid is enveloped by the host cell membrane. The host cell membrane provides the viral envelope by a process of "budding." The enveloped virion is released from the host cell. The response of a host cell to infection by a virus ranges from: 1) little or no detectable effect; to 2) alteration of the antigenic specificity of the cell surface due to presence of virus glycoproteins; to 3) latent infections that, in some cases, cause cell transformation; or, ultimately, to 4) cell death due to expression of viral genes that shut off essential host cell functions (Figure 23.17). 1. Viral infections in which no progeny virus are produced: In this case, the infection is referred to as abortive. An abortive response to infection is commonly caused by: 1) a normal virus infecting cells that are lacking in enzymes, promoters, transcription factors, or other compounds required for complete viral replication, in which case the cells are referred to as nonpermissive; 2) infection by a defective virus of a cell that normally supports viral replication (that is, by a virus that itself has genetically lost the ability to replicate in that cell type); or 3) death of the cell as a consequence of the infection, before viral replication has been completed. 2. Viral infections in which the host cell may be altered antigenically but is not killed, although progeny virus are released: In this case, the host cell is permissive, and the infection is productive (progeny virus are released from the cell), but viral replication and release neither kills the host cell nor interferes with its ability to multiply and carry out differentiated functions. The infection is, 1See Figure 23.16 Release of enveloped virus from a host cell by the process of “budding.” INFO LINK Chapter 14 in Lippincott’s Illustrated Reviews: Biochemistry for a discussion of the mechanism of insertion of glycoproteins into cell membranes. IV. Steps in the Replication Cycles Of Viruses therefore, said to be persistent. The antigenic specificity of the cell surface may be altered as a result of the insertion of viral glycoproteins. 243 1 Abortive viral infections in which no progeny virus are produced. Virion 3. Viral infections that result in a latent viral state in the host cell: Some viral infections result in the persistence of the viral genome inside a host cell with no production of progeny virus. Such latent viruses can be reactivated months or years in the future, leading to a productive infection. Some latently infected cells contain viral genomes that are stably integrated into a host cell chromosome. This can cause alterations in the host cell surface; cellular metabolic functions; and, significantly, cell growth and replication patterns. Such viruses may induce tumors in animals, in which case they are said to be tumor viruses, and the cells they infect are transformed. Host cell 2 Productive viral infections in which the host cell is not killed, although progeny virus are released. 3 Viral infections that result in a latent viral state in the host cell. 4. Viral infections resulting in host cell death and production of progeny virus: Eliminating host cell competition for synthetic enzymes and precursor molecules increases the efficiency with which virus constituents can be synthesized. Therefore, the typical result of a productive (progeny-yielding) infection by a cytocidal virus is the shutoff of much of the cell’s macromolecular syntheses by one or more of the virus gene products, causing the death of the cell. Such an infection is said to be lytic. The mechanism of the shutoff varies among the viral families. Some viral infections result in the persistence of the viral genome inside a host cell with no production of progeny virus. The viral nucleic acid may or may not be integrated into the host chromosome, depending on the virus. In summary, all viruses: • • • • are small; contain only one species of nucleic acid, either DNA or RNA; attach to their host cell with a specific receptor-binding protein; and express the information contained in the viral genome (DNA or RNA) using the cellular machinery of the host cell Such latent viruses can be reactivated months or years in the future, leading to a productive infection. 4 Viral infections resulting in host cell death and production of progeny. Figure 23.17 Effects of viral infection on a host cell. 244 23. Introduction To The Viruses Study Questions Choose the ONE correct answer. 23.1 Which one of the following statements concerning the viral replication is correct? A. Most RNA viruses assemble in the nucleus, whereas most DNA viruses develop solely in cytoplasm. B. DNA viruses must provide virtually all enzymatic and regulatory molecules needed for a complete replication cycle. C. Viral (+) single-stranded RNA serves as the template for complementary (–) strand synthesis using host RNA-dependant RNA polymerase. D. In a virus with a single-stranded (ss) RNA genome of (–) polarity, (–) ssRNA is translated into viral proteins. E. In a virus with a double-stranded RNA genome, (+) RNA strands serve both as mRNA and template for complementary (–) RNA strand synthesis. 23.2 The term “eclipse period” refers to: A. the period between epidemic outbreaks of diseases that occur in a cyclic pattern. B. the period between recurrences of disease in individuals with latent virus infections. C. the time between exposure of an individual to a virus and the first appearance of disease. D. the time between infection of a susceptible cell by a cytocidal virus and the first appearance of cytopathic effects. E. the time between entry into the cell and disassembly of the parental virus and the appearance of the first progeny virion. 23.3 The early genes of DNA viruses code primarily for proteins whose functions are required for: A. B. C. D. E. transcription of viral mRNA. translation of the capsid proteins. replication of the viral DNA. final uncoating of the infecting virions. processing of the mRNA precursors Correct answer = E. The dual role for (+) RNA strands allows both the synthesis of doublestranded RNA and capsid proteins. Most DNA viruses assemble in the nucleus, whereas most RNA viruses develop solely in cytoplasm. Some DNA viruses may provide only one or two replication-related gene products which function to divert host cell processes to those of viral replication. (–) RNA cannot serve as mRNA. (+) Single-stranded RNA serves as the template for complementary (–) strand synthesis using viral (not host) RNA-dependant RNA polymerase. Correct answer = E. Following initial attachment of a virus to the host cell, the ability of that virus to infect other cells disappears. This is the eclipse period. During this period, active synthesis of virus components is occurring. The time between exposure of an individual to a virus and the first appearance of disease is referred to as the incubation period (choice C). There is no specific term applied to the time periods described by A, B, and D. Correct answer = C. Depending on the virus family, this may consist of a DNA polymerase and other enzymes directly involved in DNA replication or, alternatively, may be a product that stimulates the cell to produce all of the enzymes and precursors needed for DNA synthesis. Transcription, for the most part, is carried out by cellular RNA polymerase. Similarly, translation is done with the cell’s translation system. The poxviruses do code for proteins that are involved in completion of uncoating, but this is an exception. mRNA processing is accomplished by cell enzymes. 24 Nonenveloped DNA Viruses I. OVERVIEW The DNA viruses discussed in this chapter—Papovaviridae, Adenoviridae, and Parvoviridae (Figure 24.1)––share the properties of lacking an envelope and having relatively simple structures and genome organization. However, the diseases commonly associated with these viruses and their mechanisms of pathogenesis are quite different, ranging from upper respiratory infections to tumors. II. INTRODUCTION TO THE PAPOVAVIRDAE Papovaviruses are nonenveloped (naked); have icosahedral nucleocapsids; and contain supercoiled, double-stranded, circular DNA. However, basic differences in genome complexity and regulation of gene expression led to division of this family into two subfamilies: the Papillomavirinae and the Polyomavirinae. Papovaviruses induce both lytic infections and either benign or malignant tumors, depending on infected cell type. DNA viruses Single stranded (Nonenveloped) Double stranded Parvoviridae Parvovirus B19 Nonenveloped Enveloped Adenoviridae Papovaviridae Adenovirus S III. PAPOVAVIRIDAE: SUBFAMILY PAPILLOMAVIRINAE Papillomavirinae All papillomaviruses induce hyperplastic epithelial lesions in their host species. Over 150 types of human papillomaviruses (HPVs) are now recognized, based on differences in the DNA sequences of certain wellcharacterized virus genes. HPVs exhibit great tissue and cell specificity, infecting only surface epithelia of skin and mucous membranes. The HPVs within each of these tissue-specific groups have varying potential for causing malignancies. For example, there are: 1) a small number of virus types (specifically, types 16 and 18) that produce lesions with a high risk of progression to malignancy such as in cervical carcinoma; 2) other virus types produce mucosal lesions that progress to malignancy with lower frequency, causing, for example, anogenital warts (condyloma acuminata, a common sexually transmitted disease) and laryngeal papillomas (the most common benign epithelial tumors of the larynx); and 3) still other virus types that are associated only with benign lesions (for example, common, flat, and plantar warts). Human papillomavirus S Polyomavirinae BK virus JC virus Figure 24.1 Classification of non-enveloped DNA viruses. S See pp. 354, 361 for summaries of these viruses. = Single-stranded, linear DNA = Double-stranded, linear DNA = Double-stranded, circular DNA 245 246 24. Nonenveloped DNA Viruses A. Epidemiology Transmission of HPV infection requires direct contact with infected individuals (for example, sexual contact) or with contaminated surfaces (fomites) such as communal bathroom floors. HPV can also be transmitted from mother to infant during passage down the birth canal. Because the initial phase, as well as the maintenance of infection, occurs in cells of the basal layer of the skin, access to these cells is presumably via epithelial surface lesions such as abrasions. B. Pathogenesis The most striking characteristics of HPV multiplication and pathogenesis are its specificity for epithelial cells and its dependence on the differentiation state of the epithelial host cell. 1. Wart formation: The development of a typical wart results from cell multiplication and delayed differentiation induced by certain papillomavirus early proteins. In cutaneous tissues, for example, infected cells leave the basal layer and migrate toward the surface of the skin. The virus replication cycle proceeds in parallel with the steps of keratinocyte differentiation, which end with the terminally differentiated cornified layer of the growing wart. An important function of two early viral proteins is the activation of host cells, causing them to divide. This activation involves interaction between these viral proteins and cellular proteins (antioncoproteins) that normally function to regulate the cell cycle. Two of these antioncogenic cellular proteins are p53 (cellular growth suppressor protein) and pRb (retinoblastoma gene product). The viral genome is maintained in low copy numbers as an autonomously replicating episome in the nuclei of multiplying basal cells. Expression of only one early gene appears to be required for maintaining this balance between episome persistence and basal cell division. (See Figure 24.2 for a summary of papillomavirus replication and wart formation.) The initially infected basal layer cell multiplies and spreads laterally. In addition, daughter cells begin to migrate toward the surface, but continue to multiply in the prickle cell layer under the influence of the early viral proteins. Increasing epidermal cell differentiation Stratum corneum (horny layer) Stratum spinosum (prickle cells) Basal cells Excess keratin is synthesized and, along with the continued cell proliferation, leads eventually to a thickened cornified layer of dead cells containing infectious virus progeny. The replication cycle begins with the expression of viral early genes, followed by multiplication of the viral genome and, finally, assembly of progeny virus in the most superficial layers of the wart. Viral life cycle Capsid protein Virus particles Replicating viral DNA Expression of early genes Viral DNA (Low copy number) Figure 24.2 Relationship between steps in the development of a skin wart and the life cycle of papilloma virus. III. Papovaviridae: Subfamily Papillomavirinae 247 2. Development of malignancies: Progression to malignancy occurs primarily in warts located on mucosal surfaces, particularly those of the genital tract, and is associated with a limited number of papillomavirus types. The affinity of binding between virus early proteins and cellular antioncoproteins p53 and pRb (which inactivate these cellular regulatory proteins) correlates with a high risk for malignant progression. However, it is clear that this interaction is only the first step in a multistep process involving alterations in expression of other cell oncoproteins and antioncoproteins and including, at some point, the non–site-specific integration of part of the viral genome into a host cell chromosome. Cutaneous warts • • Most common warts are benign and resolve spontaneously. In genetically predisposed individuals, warts can lead to epidermodysplasia and squamous cell carcinoma.
warts may be classified as common (fingers and hands), plantar (sole of foot), or flat (arms, face, and knee). Another category of cutaneous lesion occurs in patients with what appears to be an inherited predisposition for multiple warts that do not regress but, instead, spread to many body sites (epidermodysplasia verruciformis). Of particular interest is that these lesions frequently give rise to squamous cell carcinomas several years after initial appearance of the original warts, especially in areas of skin exposed to sunlight. Mucosal infections • Laryngeal papillomas are benign. • Anogenital warts are usually benign. • Infections with certain types of papillomavirus can lead to cervical carcinoma. 2. Oral infections (caused by types 13 and 32): Oral and nasopha- ryngeal mucosal surfaces can be infected by some HPV types. Most of these infections result in benign papillomas. 3. Genital tract infections: Approximately 30 different types of HPV Figure 24.3 Location and properties of papilloma infections. can infect the genital tract, but types 6 and 11 cause 90 percent of genital papillomas. All of the genital tract HPV infections are acquired via sexual contact. HPV types 6 and 11 can also be spread to the oral mucosa via sexual contact. HPV infections caused by these virus types produce anogenital warts (condyloma acuminata), which are occasionally large (but usually A Common warts Figure 24.4 Warts caused by papillomavirus. B Flat warts C Anogenital warts 248 24. Nonenveloped DNA Viruses A Surgical removal of wart B Destruction of wart • Liquid N2 • Laser vaporization • Cytotoxic chemicals C Drug treatment • Interferon for laryngeal papilloma Figure 24.5 Treatment of papilloma. benign) lesions that often regress spontaneously. Infections with other types of HPV do not lead to overt wart formation but have a high risk of progressing to malignancy. In fact, HPV has been established as the primary cause of cervical cancer in the majority of cases. HPV types 16 and 18 are associated with up to 70 percent of all cervical cancers. There are approximately 13 other, infrequently encountered types of HPV that are also associated with cancer development. Cumulatively, it is estimated that more than 95 percent of all cervical cancers are caused by these highrisk types of HPV. In addition to cervical cancer, the high-risk HPV types are linked to the development of anal, penile, vaginal, vulvar, and oropharyngeal cancers. D. Laboratory identification Diagnosis of cutaneous warts generally involves no more than visual inspection. The major role of laboratory identification in papillomavirus infections is to: 1) determine whether HPV is present in abnormal tissue recovered by biopsy or cervical swab and 2) whether the HPV type detected is one considered a high risk for progression to malignancy (the latter applying primarily to infections of the genital tract). The lack of any tissue culture system for recovery of the virus, and the fact that HPV types are defined by molecular criteria, means that typing is done by quantitative DNA amplification techniques (polymerase chain reaction), using defined, type-specific oligonucleotide primers (see p. 30). In addition, immunohistochemistry can be employed to detect viral protein expression in situ. E. Treatment and prevention Treatment of warts generally involves surgical removal or destruction of the wart tissue with liquid nitrogen, laser vaporization, or cytotoxic chemicals such as podophyllin or trichloroacetic acid (Figure 24.5). Although such treatments remove the wart, HPV often remains present in cells of the surrounding tissue, and recurrence rates of 50 percent have been repor ted. Common war ts often regress spontaneously, and removal is not usually warranted unless there is unusual pain caused by the location or for cosmetic reasons. Cidofovir, an inhibitor of DNA synthesis, appears to be effective when applied topically. Interferon, given orally, has been shown to cause regression of laryngeal papillomas. When injected directly into genital warts, it has given positive results in about one half of patients. Because transmission of the infection is by direct inoculation, avoiding contact with wart tissue is the primary means of prevention. In genital tract warts, all of the procedures for prevention of sexually transmitted diseases are appropriate. In 2006, the Food and Drug Administration approved a vaccine against the four most common HPV types. The vaccine, called Gardasil, contains viral capsids from HPV types 6, 11, 16, and 18. The first two types cause most genital warts, and the latter two types cause the majority of cervical cancers. The vaccine was originally recommended for young females as a protection against cervical cancer. However, the vaccine is now recommended for young males, as well, because it has been demonstrated to protect both males and females against genital warts and specific types of cancer. A second vaccine, Cervarix, contains only two capsid types and is protective against infection with the high-risk HPV types, 16 and 18. IV. Papovaviridae: Subfamily Polyomavirinae. 249 IV. PAPOVAVIRIDAE: SUBFAMILY POLYOMAVIRINAE All members of this virus subfamily have the capacity to transform normal cells in culture and to induce tumors in species other than those in which they are normally found in nature. “Polyoma” means many (poly-) tumor (-oma). However, Polyomavirus has not yet been shown to cause tumors in humans. There are three human polyomaviruses: BK, JC, and Merkel cell polyomaviruses (BKV, JCV, and MCV, respectively). JCV has been associated with progressive multifocal leukoencephalopathy (PML), a rare, fatal, demyelinating disease that occurs only in patients with impaired immune function (for example, those with AIDS). BKV can cause cystitis in this same population. MCV was discovered in 2008 by molecular technologies. MCV DNA can be detected in the majority of cases of Merkel cell carcinoma, a rare and aggressive form of skin cancer. A. Epidemiology and pathogenesis The human polyomaviruses BKV and JCV are transmitted by droplets from the upper respiratory tract of infected persons and, possibly, through contact with their urine. Infection with these viruses usually occurs in childhood. Specific antibody to one or both human polyomaviruses is present in 70 to 80 percent of the adult population. There is evidence that both BKV and JCV spread from the upper respiratory tract to the kidneys, where they may persist in an inactive state in the tubular epithelium of healthy individuals. Polyomaviruses follow the basic pattern of DNA virus genome replication and gene expression in the nucleus. The enzymes and precursors synthesized in preparation for cellular DNA synthesis are made available for synthesis of viral DNA. This productive cycle leads to viral multiplication and, ultimately, to death of the host cell. Progressive multifocal leukoencephalopathy • Rare, fatal demyelinating disease caused by JCV • Occurs in immunocompromised patients Hemorrhagic cystitis • Caused by BKV • Most commonly seen in immunocompromised patients Figure 24.6 Location and properties of polyomavirus infections. JCV = JC polyomavirus; BKV = BK polyomavirus.
Figure 24.7 Electron micrograph of BK virions from the urine of an infected patient. 250 24. Nonenveloped DNA Viruses C. Laboratory identification A Because most people have antibodies to these viruses, serologic techniques are not generally useful in the diagnosis of acute infections. Identification by DNA hybridization of BKV in the urine or JCV in PML lesions in brain tissue is the most sensitive and specific technique for diagnosis of these infections. MCV viral DNA and protein antigens can be detected by molecular techniques in Merkel cell tumors. D. Treatment and prevention B No successful, specific, antiviral therapy is available. Because polyomavirus infection is nearly universal and asymptomatic, and PML represents reactivation of “latent” virus, there are currently no viable preventive measures.
C Adenoviruses are nonenveloped, icosahedral viruses containing double-stranded linear DNA (Figure 24.8). They commonly cause diseases such as respiratory tract infections, gastroenteritis, and conjunctivitis. Adenoviruses were first discovered during screenings of throat washings and cultures of adenoids and tonsils, performed in the search for the common cold virus. They are now recognized as a large group of related viruses commonly infecting humans, other mammals, and birds. Over fifty serotypes of human adenoviruses are known, and antibody surveys have shown that most individuals have been infected by several different types by adulthood. Although some human serotypes are highly oncogenic in experimental animals, none have been associated with human malignancies. A. Epidemiology and pathogenesis Figure 24.8 A. Electron micrograph of an adenovirus virion with fibers. B. Model of adenovirus. C. Crystalline aggregate of adenovirus in the nucleus of a cell. The site of the clinical syndrome caused by an adenovirus infection is generally related to the mode of virus transmission. For example, most adenoviruses are primarily agents of respiratory disease, which are transmitted via the respiratory route. However, most adenoviruses also replicate efficiently and asymptomatically in the intestine, and can be isolated from stool well after respiratory disease symptoms have ended as well as from the stools of healthy persons. Similarly, ocular infections are transmitted by direct inoculation of the eye by virus-contaminated hands, ophthalmologic instruments, or bodies of water in which groups of children swim together. B. Structure and replication The adenovirus capsid is composed of hexon capsomers making up the triangular faces of the icosahedron, with a penton capsomer at each of the vertices (see Figure 24.8). Replication of adenoviruses essentially follows the general model for DNA viruses (see p. 238). Attachment to a host cell receptor occurs via knobs on the tips of the viral fibers, which is followed by entry into the cell by receptormediated endocytosis. The viral genome is then progressively uncoated while it is transported to the nucleus, where all transcription of viral genes, genome replication, and assembly occurs. Two early viral genes have the same function as the early proteins of the Papovaviridae [that is, inactivating cellular regulatory proteins
251 Ocular infections • Follicular conjunctivitis • Keratoconjunctivitis Respiratory infections • Acute febrile pharyngitis • Pharyngoconjunctival fever • Acute respiratory disease • Viral pneumonia Gastrointestinal infections • Infantile gastroenteritis 1. Respiratory tract diseases: The most common manifestation of adenovirus infection of infants and young children is acute febrile pharyngitis, characterized by a cough, sore throat, nasal congestion, and fever. Isolated cases may be indistinguishable from other common viral respiratory infections. Some adenovirus types tend additionally to produce conjunctivitis, in which case the syndrome is referred to as pharyngoconjunctival fever. This entity is more prevalent in school-aged children and occurs both sporadically and in outbreaks, often within family groups or in groups using the same swimming facility (“swimming pool conjunctivitis”). The syndrome referred to as acute respiratory disease occurs primarily in epidemics among new military recruits. It is thought to reflect the lowered resistance brought on by exposure to new strains, fatigue, and crowded living conditions, promoting efficient spread of the infection. Lastly, the respiratory syndromes described above may progress to true viral pneumonia, which has a mortality rate of about 10 percent in infants. 2. Ocular diseases: In addition to the conjunctivitis that sometimes accompanies the upper respiratory syndrome described above, a similar follicular conjunctivitis may occur as a separate disease. It is self-limiting and has no permanent sequelae. A more serious infection is epidemic keratoconjunctivitis, which involves the corneal epithelium, and may be followed by corneal opacity lasting several years. The epidemic nature of this disease partly results from transmission via shared towels or ophthalmic solutions, person-to-person contact, and improperly sterilized ophthalmologic instruments. 3. Gastrointestinal diseases: Most human adenoviruses multiply in the GI tract and can be found in stools. However, these are generally asymptomatic infections. Two serotypes have been associated specifically with infantile gastroenteritis. Adenovirus infections have been estimated to account for 5 to 15 percent of all viral diarrheal disease in children. Urinary tract infections • Hemorrhagic cystitis Figure 24.9 Adenovirus infections. 252 24. Nonenveloped DNA Viruses 4. Less common diseases: Several adenovirus serotypes have been 1 Virus attaches and penetrates host cell. Viral DNA NA associated with an acute, self-limited, hemorrhagic cystitis, which occurs primarily in boys. It is characterized by hematuria, and virus can usually be recovered from the urine. Similarly, adenovirus infection of heart muscle has recently been shown to be one cause of left ventricular dysfunction in both children and adults. In immunocompromised patients, such as those with AIDS, the common respiratory adenovirus infections have a greater risk of proceeding to serious, often fatal, pneumonia. Other disseminated infections leading to a fatal outcome have been reported in patients with a compromised immune system or those immunosuppressed from drug therapy. D. Laboratory identification 2 Replicating dsDNA 3 DNA genome is released into the nucleus. Nonstructural proteins are synthesized. mRNA RNA Nonstructural proteins A mRNA Capsid proteins 4 Structural proteins are synthesized. Isolation of virus for identification is not done on a routine basis but may be desirable in cases of epidemic disease or nosocomial outbreak, especially in the nursery. Identification of the adenovirus serotype can be done by neutralization or hemagglutination inhibition using type-specific antisera. The virus is more commonly detected by direct test of stool specimens by ELISA (enzyme-linked immunosorbent assay). E. Treatment and prevention No antiviral agents are currently available for treating adenovirus infections. Prevention of epidemic respiratory disease by immunization has been used only for protection of the military population. A live, attenuated adenovirus vaccine is used for this purpose that produces a good neutralizing antibody response. In 2011, a new vaccine was licensed for use among U.S. military personnel. This vaccine contains live, unattenuated adenovirus types 4 and 7, formulated for oral administration. VI. PARVOVIRIDAE Viral DNA NUCLEUS 5 Virus assembles and host cell lyses. Figure 24.10 Replication of B19 parvovirus. Parvoviruses are the smallest of the DNA viruses. They are nonenveloped and icosahedral, with single-stranded, linear DNA. A human parvovirus, B19, has been isolated and identified as the cause of transient aplastic crisis in patients with sickle cell disease and implicated in adult acute polyarthritis. This virus is also the cause of the common childhood disease erythema infectiosum and is associated with fetal death in pregnant women experiencing a primary infection. The parvovirus family is divided into two genera, based on whether their ability to replicate requires coinfection with a helper DNA virus, or if they are capable of independent replication (“autonomous parvoviruses”). Members of the first group are referred to as adenoassociated viruses (AAVs), because they are usually found in infected cells in combination with a helper adenovirus. A. Epidemiology and pathogenesis Transmission of parvoviruses is by the respiratory route. A hightitered viremia lasting a few days follows about 1 week after infection, during which time virus is also present in throat secretions. A specific antibody response occurs rapidly, resulting in suppression of the viremia. Replication of parvoviruses requires a host cell in which VI. Parvoviridae 253 DNA synthesis is in progress. Therefore, damage is limited primarily to specific tissues that are mitotically active. [Note: In the case of B19 virus, these are primarily tissues of erythroid origin.] Because of the single-stranded nature of the genome, conversion to a doublestranded DNA molecule by a cellular DNA polymerase must occur before production of additional single-stranded viral DNA genomes or viral mRNA transcription can begin. Despite the limited amount of genetic material, two or three capsid proteins and two nonstructural regulatory proteins are produced by a combination of alternative RNA splicing patterns and posttranslational processing. The parvovirus life cycle is summarized in Figure 24.10. B. Clinical significance The single human pathogen in this family is the autonomous parvovirus, B19. The spectrum of illnesses caused by this virus is related to its unique tropism for cycling erythroid progenitor cells. Although B19 was initially isolated from sickle cell disease patients undergoing a transient aplastic crisis, it has since been recognized that chronic, progressive bone marrow suppression results from B19 infection of immunocompromised patients unable to mount an immune response capable of eliminating the virus. 1. Erythema infectiosum: The observation that 30 to 60 percent of some human populations have antibodies to B19 led eventually to the identification of this virus as the causative agent of the common childhood rash, erythema infectiosum (“fifth disease”) as shown in Figure 24.11. The characteristic rash (“slapped cheek” appearance) occurs about 2 weeks after initial exposure, when the virus is no longer detectable. The rash is apparently immune-system mediated. Another complication accompanying B19 infection is an acute arthritis that usually involves joints symmetrically. This is considerably more frequent in adults than in children and usually resolves within several weeks. 2. Birth defects: Spontaneous abortion rate is elevated in women hav- ing a primary infection during the first trimester, and primary infection during the second or third trimester is associated with some instances of hydrops fetalis. C. Laboratory identification Laboratory identification of B19 infection is not routinely done. The large amount of virus present during the viremic (usually asymptomatic) phase permits detection of viral proteins by immunologic methods or of viral DNA by various amplification techniques. Retrospective diagnosis can be made by any of the usual procedures used to demonstrate a specific antibody response. D. Treatment and prevention No antiviral agent or vaccine is available for treating human B19 infections. Isolation of patients with signs of parvovirus disease is not a useful approach to control because subclinical infections occur, and infected individuals shed virus before symptoms appear. Intraveneously administered immunoglobulin G specific for B19 virus may be helpful in immunocompromised patients with chronic infections. Figure 24.11 Typical “slapped cheek” appearance of a child infected with parvovirus B19 (“fifth disease”). 254 24. Nonenveloped DNA Viruses Study Questions Choose the ONE correct answer. 24.1 An important step in the mechanism proposed for oncogenesis by human papillomaviruses is: A. inactivation of a cellular regulatory gene by human papillomavirus integration into the coding region of the gene. B. transactivation of a normally silent cellular oncogene by a human papillomavirus early protein. C. reversal of keratinocyte differentiation caused by continued active replication and production of progeny human papillomavirus. D. specific binding of certain human papillomavirus early proteins to cellular antioncoproteins. E. induction of a specific chromosome translocation that results in activation of a cellular oncogene. 24.2 The characteristic spectrum of diseases caused by autonomous parvoviruses is related to the fact that they: A. integrate into a specific chromosomal site that disrupts an essential gene and leads to death of the cell. B. require host cells that are actively progressing through the mitotic cycle. C. infect only terminally differentiated cells. D. code for an early protein that shuts off cellular protein synthesis. E. increase the severity of the disease normally caused by their associated helper virus. 24.3 The characteristic rash of erythema infectiosum is due to: A. virion/antibody immune complex formation. B. bone marrow suppression caused by killing of erythrocyte precursors by B19 infection. C. damage to the liver. D. B19 infection of epithelial cells. E. the inflammatory response to B19 infection of capillary endothelium. Correct answer = D. The early proteins of both adenoviruses and Papovaviridae required for immortalization and transformation of normal cells have been shown to bind specifically to cellular proteins p53 and pRb, which are important in maintaining regulation of the mitotic cycle. Interaction with viral proteins is believed to result in loss of their normal functions, as do the mutations that are commonly associated with spontaneously occurring cancers. A, B: Neither gene inactivation by integration nor transcriptional activation by an early protein has been observed. C: Virus replication occurs only in differentiated keratinocytes, but dedifferentiation does not occur. E: Multiple chromosome rearrangements are observed late in progression to malignancy, but none are specific for human papillomavirus–transformed cells. Correct answer = B. The diseases caused by the autonomous parvoviruses all result from the effects of killing multiplying cells that are essential for normal functions. For example, B19 specifically infects erythroblasts, leading to anemia in the fetus or in immunodeficient patients. A, C, D: Parvoviruses are not observed to integrate during the replicative cycle, they cannot replicate in terminally differentiated cells, and they do not shut off cell syntheses. E: By definition, the autonomous parvoviruses do not require a helper virus for replication. Correct answer = A. The appearance of the rash coincides with production of antibodies to B19, which occurs several days after the peak of viremia. B: Infection in immunodeficient individuals can lead to chronic, progressive depletion of erythrocyte precursors and severe anemia but not rash. C: The host range of B19 is restricted to erythroid precursors, including those found in the fetal liver. Although this may be a factor in causing hydrops fetalis due to B19 infection of a pregnant woman, it is not related to the rash. D and E: Again, B19 is not known to infect other than erythroid precursor cells. Enveloped DNA Viruses 25
II. HERPESVIRIDAE: STRUCTURE AND REPLICATION Eight human herpesvirus species are known. All have the ability to enter a latent state following primary infection of their natural host and be reactivated at a later time. However, the exact molecular nature of the latency and the frequency and manifestation of reactivation vary with the type of herpesvirus. A. Structure of herpesviruses Herpesvirus virions consist of an icosahedral capsid enclosed in an envelope derived from the host’s nuclear membrane (Figure 25.2). Between the envelope and the capsid lies an amorphous proteinaceous material called tegument, which contains virus-encoded enzymes and transcription factors essential for initiation of the infectious cycle, although none of these is a polymerase. The genome is a single molecule of linear, double-stranded DNA, encoding from 70 to 200 proteins, depending on the species. Although all members of the family have some genes with homologous functions, there is little nucleotide sequence conservation and little antigenic relatedness between species. DNA viruses Single stranded (Nonenveloped) Double stranded Nonenveloped Enveloped Hepadnaviridae Herpesviridae Alphaherpesvirinae (herpes simplex group) Herpes simplex 1 S Herpes simplex 2 S Varicella-zoster virus S Betaherpesvirinae (cytomegalovirus group) Human cytomegalovirus Human herpesvirus 6 Human herpesvirus 7 S Gammaherpesvirinae (lymphoproliferative group) Epstein-Barr virus S Human herpesvirus 8 S Poxviridae Molluscum contagiosum virus Vaccinia virus Variola virus Figure 25.1 Classification of enveloped DNA viruses. S See p. 356 for summaries of these viruses. 255 256 25. Enveloped DNA Viruses B. Classification of herpesviruses A Capsid Envelope Glycoprotein spikes Herpesviridae cannot readily be differentiated by morphology in the electron microscope, because they all have similar appearances. However, Herpesviridae have been divided into three subfamilies, based primarily on biologic characteristics (see Figure 25.1). 1. Alphaherpesvirinae (herpes simplex virus group): These viruses DNA Tegument B have a relatively rapid, cytocidal or lytic growth cycle and establish dormant or latent infections in nerve ganglia. Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) and varicella-zoster virus (VZV) belong to this group. HSV-1 and HSV-2 share significant nucleotide homology and, therefore, share many common features in replication and pathogenesis. VZV has a smaller genome than HSV, but the two viruses have many genes that share sequence identity. 2. Betaherpesvirinae (cytomegalovirus group): These viruses have a relatively slow replication cycle that results in the formation of characteristic, multinucleated, giant host cells. Latency is established in nonneural tissues, primarily lymphoreticular cells and glandular tissues. Human cytomegalovirus (HCMV) and human herpesviruses types 6 and 7 (HHV-6 and HHV-7) are in this group. 3. Gammaherpesvirinae (lymphoproliferative group): These viruses Figure 25.2 Structure of herpesvirus. A. Schematic drawing. B. Transmission electron micrograph. replicate in mucosal epithelium and establish latent infections primarily in B cells. They induce cell proliferation in and immortalize lymphoblastoid cells. Epstein-Barr virus (EBV) was previously the only well-characterized human gammaherpesvirus. However, genome analysis of a virus recovered from cells of Kaposi sarcoma (KS) revealed it to also be a human member of the Gammaherpesvirinae. It has been designated human herpesvirus type 8 (HHV-8). HHV-8 can also establish latency and immortalize endothelial cells. C. Replication of the herpesviruses Herpesviruses replicate in the nucleus, following the basic pattern of DNA virus replication (see p. 238). Regulation of herpesvirus transcription is referred to as “cascade control,” in that expression of a first set of genes is required for expression of a second set, which, in turn, is required for expression of a third set of genes. [Note: A similar pattern is found in some other DNA virus families in which the genes are referred to as immediate early, delayed early, and late.] The general features of herpesvirus replication are summarized in Figure 25.3. 1. Virus adsorption and penetration: Herpesviruses adsorb to host cell receptors that can differ according to the virus species and the tissue type being infected. Viral envelope glycoproteins promote fusion of the envelope with the cell’s plasma membrane, depositing the nucleocapsid and tegument proteins in the cytosol. One of the tegument proteins is a general RNase that efficiently degrades all mRNAs, effectively shutting off host cell protein syn- III. Herpes Simplex Virus, Types 1 and 2 thesis. Because this protein’s nucleolytic activity occurs prior to the onset of viral mRNA synthesis, it is selective for host RNAs. 257 1 Virus attaches, the viral envelope fuses, and the nucleocapsid penetrates the host cell 2. Viral DNA replication and nucleocapsid assembly: The nucleo- capsid is transported to a nuclear pore, through which viral DNA is released into the nucleus. Another tegument protein is an activator of cellular RNA polymerase that causes the enzyme to initiate transcription of the set of viral immediate early genes, which code for a variety of regulatory functions, including initiation of further gene transcription. Delayed early genes are expressed next, and they code primarily for enzymes that are required for replication of viral DNA, such as viral DNA polymerase, helicase, and thymidine kinase. Because these enzymes are virus specific, they provide excellent targets for antiherpes agents (such as acyclovir), which are relatively nontoxic for the cell. As is the case with other DNA viruses, late genes code for structural proteins of the virion and proteins involved in assembly and maturation of viral progeny. DNA genome released into the nucleus 2 Viral DNA Immediate early mRNA 3. Viral envelope acquisition: Newly synthesized envelope proteins Immediate early proteins accumulate in patches on the nuclear membrane, and nucleocapsids that have been assembled in the nucleus acquire their envelopes by budding through these patches. The completed virus is transported by a vacuole to the surface of the cell. Additional copies of the envelope glycoproteins are also transported to the plasma membrane, which acquires herpesvirus antigenic determinants. These glycoproteins may also cause fusion of neighboring cells, in some cases producing characteristic multinucleated giant cells. The end result of this productive, lytic cycle is cell death because most cellular synthetic pathways are effectively turned off during viral replication. yed Delayed y early NA mRNA Delayed early proteins Late mRNA Late proteins (structural) 4. Latency: All herpesviruses can undergo an alternative infection cycle, entering a quiescent, dormant state (latency) from which they can subsequently be reactivated. The cell type in which this occurs is usually not the same cell type in which productive, lytic infection occurs. For each of the herpesviruses, the mechanism of latency, nature of the host cells, frequency of reactivation, and the nature of the recurrent disease are characteristic. Therefore, the topic of latency is discussed in this chapter in the context of the individual virus species. Viral DNA 3 Transcription and translation of viral • immediate early genes • delayed early genes • late genes Envelope proteins III. Herpes simplex virus, types 1 and 2 HSV-1 and HSV-2 are the only human herpesviruses that have a significant degree of nucleotide sequence identity (about 50 percent). Therefore, they share many common features in replication, disease production, and latency. A. Epidemiology and pathogenesis Transmission of both HSV types is by direct contact with virus-containing secretions or with lesions on mucosal or cutaneous surfaces. Primary or recurrent infections in the oropharyngeal region, caused 4 Virus acquires envelope by budding through nuclear membrane Figure 25.3 Replication of herpesviruses. 258 25. Enveloped DNA Viruses Figure 25.4 Herpes simplex stomatitis. primarily by HSV-1, are accompanied by virus release into saliva, and kissing and saliva-contaminated fingers are major modes of transmission. In genital tract infections, caused primarily by HSV-2, virus is present in genital tract secretions. Consequently, sexual intercourse and passage of newborns through the birth canal of infected mothers are major modes of transmission. Both HSV-1 and HSV-2 multiply in epithelial cells of the mucosal surface onto which they have been inoculated, resulting in production of vesicles or shallow ulcers containing infectious virus. In immunocompetent individuals, epithelial infection remains localized because cytotoxic T lymphocytes recognize the HSV-specific antigens on the surface of infected cells and kill these cells before progeny virus has been produced. A lifelong latent infection is usually established in the regional ganglia as a result of entry of infectious virions into sensory neurons that terminate at the site of the infection. B. Clinical significance A generality (albeit of limited use) is that HSV-1 is most commonly found in lesions above the waist, and HSV-2 is more commonly the cause of lesions below the waist. However, HSV-1 can infect the genital tract, causing similar lesions, and, similarly, HSV-2 can cause lesions in the oral cavity. 1. Primary infections of the upper body: Many primary HSV infec- Male Female tions are subclinical, but the most common symptomatic infections of the upper body are gingivostomatitis in young children (Figure 25.4) and pharyngitis or tonsillitis in adults. The painful lesions typically consist of vesicles and shallow ulcers, which are often accompanied by systemic symptoms, such as fever, malaise, and myalgia. Another clinically important site of infection is the eye, in which keratoconjunctivitis can lead to corneal scarring and eventual blindness. If HSV infection spreads to the central nervous system (CNS), it can cause encephalitis, which, if untreated, has a mortality rate estimated to be 70 percent. Survivors are usually left with neurologic deficits. In the United States, HSV-1 infection of the eye is the second most common cause of corneal blindness (after trauma). HSV infections of the CNS account for up to 20 percent of encephalitis viral infections. 2. Primary infections of the genital tract: Primary genital tract Figure 25.5 Genital herpes simplex infections. lesions are similar to those of the oropharynx. However, based on the frequency of antibody in the population, the majority of these infections are asymptomatic. When symptomatic (genital herpes), local symptoms include painful vesiculo-ulcerative lesions on the vulva, cervix, and vagina in women and the penis in men (Figure 25.5). Systemic symptoms of fever, malaise, and myalgia may be more severe than those that accompany primary oral cavity infections. In pregnant women with a primary genital HSV infection, the risk of infecting the newborn during birth is estimated to be 30 to 40 percent (neonatal herpes). Because such infants have no protective maternal antibody, a disseminated infection, often involving the CNS, may result. There is a high mortality rate if untreated, and survivors are likely to have permanent neurologic sequelae. A newborn is also at risk of acquiring infection from an infected III. Herpes Simplex Virus, Types 1 and 2 mother by transfer on contaminated fingers or in saliva. However, infection in utero appears to occur only rarely. 259
3. Latency: In latently infected cells of the ganglia—HSV-1 in trigemi- nal ganglia and HSV-2 in sacral or lumbar ganglia—from one to thousands of copies of the viral genome are present as nonintegrated, circular molecules of DNA in the nuclei (Figure 25.6). A limited number of viral genes are expressed during latency. These transcripts (called LATS for latency-associated transcripts) suppress production of progeny virus. 1 Virus penetrates into skin, where it replicates. 2 Virus enters cutaneous neurons and migrates to a ganglion, where it remains in a latent state. 4. Reactivation: Several factors, such as hormonal changes, fever, and physical damage to the neurons, are known to induce reactivation and replication of the latent virus (see Figure 25.6). The newly synthesized virions are transported down the axon to the nerve endings from which the virus is released, infecting the adjoining epithelial cells. Characteristic lesions are thus produced in the same general area as the primary lesions. [Note: Virus replication occurs in only a fraction of the latently infected neurons, and these nerve cells eventually die.] The presence of circulating antibody does not prevent this recurrence but does limit the spread of virus to surrounding tissue. Sensory nerve symptoms, such as pain and tingling, often precede and accompany the appearance of lesions. In general, the severity of any systemic symptoms is considerably less than that of a primary infection, and many recurrences are characterized by shedding of infectious virus in the absence of visible lesions. a. Herpes simplex virus type 1: The frequency of oropharyngeal symptomatic recurrences is variable, ranging from none to several a year. The lesions occur as clusters of vesicles at the border of the lips (herpes labialis, or “cold sores” or “fever blisters”) and heal without scarring in 8 to 10 days. Ganglion
3 Virus can subsequently be reactivated and travel through sensory neurons to the epidermis.
infections can occur with considerably greater frequency (for example, monthly) and is often asymptomatic but still results in viral shedding. Consequently, sexual partners or newborn infants may be at increased risk of becoming infected resulting from lack of precautions against transmission. The risk of transmission to the newborn is much less than in a primary infection because considerably less virus is shed and the baby has some maternal anti-HSV antibody. This antibody also lessens the severity of the disease if infection does occur. 4 A recurrent infection results.
Figure 25.6 Primary and recurrent herpes simplex infections. 260 25. Enveloped DNA Viruses G O HO Acyclovir Thymidine kinase coded by the viral genome phosphorylates acyclovir more than a hundred times faster than host cell enzyme. G P O O Acycloguanosine monophosphate (acyclo-GMP) Additional phosphorylations are catalyzed by enzymes of the host cell. G PP P O O O Acycloguanosine triphosphate (acyclo-GTP) P C O lar exanthems (skin eruptions) caused by other viruses or, in some cases, by bacteria or noninfectious, allergy-based reactions. Demonstration of HSV by inoculation of human cell tissue culture with a sample of vesicle scraping, fluid, or genital swab is the definitive method for demonstrating infection. The presence of the virus can result in syncytia formation between cells and the formation of Cowdry type A bodies within the host cell nucleus. Gross cytopathic changes may require several days to appear, but individual infected cells can be detected within 24 hours by use of immunofluorescence (see p. 28) or immunoperoxidase staining with antibodies directed against viral early proteins. Using these same techniques, infected cells can also be demonstrated directly in clinical specimens, although this approach is generally less sensitive than virus isolation in tissue culture. Direct detection of viral DNA by hybridization techniques complements these procedures and, after amplification of the DNA by polymerase chain reaction ([PCR] see p. 29), is considerably more sensitive. For example, in patients with encephalitis, HSV etiology can be confirmed by demonstration of viral DNA in the cerebral spinal fluid (CSF) instead of by brain biopsy. P O G C
O P P O C G O G C O P P Growing strand O Template Incorporation of acyclo-GMP from acyclo-GTP into the growing strand of viral DNA causes chain termination because acyclo-GMP lacks a 3' hydroxyl group. Figure 25.7 Mechanism of action of acyclovir. 1 Acyclovir DNA polymerase 1 Famciclovir 2 Penciclovir (topical) The guanine analog, acycloguanosine (acyclovir), is selectively effective against HSV because it becomes an active inhibitor of DNA synthesis only after initially being phosphorylated by the HSV thymidine kinase (Figure 25.7). The drug of choice for any primary HSV infection, acyclovir is especially important in treating herpes encephalitis, neonatal herpes, and disseminated infections in immunocompromised patients. Other drugs effective in treating herpes simplex infection include famciclovir and topical penciclovir (Figure 25.8). Famciclovir is a prodrug that is metabolized to the active penciclovir. It provides more convenient dosing and greater bioavailability than oral acyclovir. Penciclovir is active against HSV1, HSV-2, and VZV. None of these drugs can cure a latent infection, but they can minimize asymptomatic viral shedding and recurrences of symptoms (Figure 25.9). E. Prevention Prevention of HSV transmission is enhanced by avoidance of contact with potential virus-shedding lesions and by safe sexual practice. Although prevention of neonatal HSV infections is important, genital infection of the mother can be difficult to detect because it is often asymptomatic. When overt genital tract lesions are detected at the time of delivery, cesarean section is usually warranted. Prophylactic therapy of the mother and the newborn with acyclovir can be employed if the presence of HSV is detected just before or at the time of birth. Measures to prevent physical transmission following birth are also important. A vaccine is not currently available. IV. VARICELLA-ZOSTER VIRUS Figure 25.8 Drug therapy for herpes simplex infection. 1 Indicates first-line drugs; 2 indicates alternative drugs. VZV has biologic and genetic similarities to HSV and is classified with HSVs in the Alphaherpesvirinae subfamily. Biologic similarities between VZV and HSV include that latency is established in sensory ganglia and IV. Varicella-Zoster Virus 261 infections are rapidly cytocidal. Primary infections with VZV cause varicella (“chickenpox”), whereas reactivation of the latent virus causes herpes zoster (“shingles”). In both men and women, famciclovir treatment reduces herpes simplex viral shedding.
4 (% of days when patient shows viral shedding) VZV is the only herpesvirus that can be easily spread from person to person by casual contact. Transmission of VZV is usually via respiratory droplets and results in initial infection of the respiratory mucosa, followed by spread to regional lymph nodes (Figure 25.10). Progeny virus enter the bloodstream, undergo a second round of multiplication in cells of the liver and spleen, and are disseminated throughout the body by infected mononuclear leukocytes. Endothelial cells of the capillaries and, ultimately, skin epithelial cells become infected, resulting in the characteristic, virus-containing vesicles of chickenpox that appear from 14 to 21 days after exposure. The infected individual is contagious from 1 to 2 days before the appearance of the exanthem, implying that viruses reinfect cells of the respiratory mucosa near the end of the incubation period. The vesicular fluid from the chickenpox rash is also highly contagious and can be spread to nonimmune individuals if it becomes airborne.” Asymptomatic shedding
Famciclovir treatment Placebo Figure 25.9 Chronic suppressive antiviral therapy reduces the frequency of asymptomatic herpes simplex virus shedding. In contrast to HSV infections, the primary and recurrent diseases (varicella and zoster) due to VZV are quite distinct. Whereas neither is usually life threatening in the normal, healthy individual, both can have severe complications in immunocompromised patients. 1. Primary infection (varicella, or chickenpox): In a normal, healthy child, the incubation period is most commonly from 14 to 16 days. The first appearance of exanthem is often preceded by 1 to 2 days of a prodrome of fever, malaise, headache, and abdominal pain. The exanthem begins on the scalp, face, or trunk as erythe- Infection of upper respiratory mucosa with virus-containing droplets. Virus spreads to regional lymph nodes, where it replicates. Virus spreads to the liver and spleen. Infection of the skin leads to appearance of vesicular rash. Fever Virus enters cutaneous neurons and migrates to ganglia, where it enters a latent state. Rash Virus Vi Ganglion Primary viremia Days 0 5 10 Incubation period Secondary viremia 15 20 Contagious period 25 30 35 40 Viral latency Figure 25.10 Time course of varicella (chickenpox) in children. In adults, the disease shows a longer time course and is more severe. 262 Figure 25.11 Appearance of chickenpox with lesions at all stages of development. 25. Enveloped DNA Viruses matous macules, which evolve into virus-containing vesicles that begin to crust over after about 48 hours (Figure 25.11). Itching is most severe during the early stage of vesicle development. While the first crop of lesions is evolving, new crops appear on the trunk and extremities. In older adults and the immunocompromised, lesions may also appear on mucous membranes, such as in the oropharynx, conjunctivae, and vagina. New lesions continue to appear over a period of up to 6 or 7 days. Healing usually occurs without long-term consequences, but crater-like scars can remain after the lesions heal. Varicella is a more serious disease in both healthy and immunocompromised adults than it is in children. Varicella pneumonia is the most common of the serious complications, but fulminant hepatic failure and varicella encephalitis may also result. Primary infection of a pregnant woman may cause her to contract the more severe adult form of varicella and may affect the fetus or neonate as well. Fetal infection early in pregnancy is uncommon but can result in multiple developmental anomalies. More commonly, a fetus infected near the time of delivery may exhibit typical varicella at birth or shortly thereafter. The severity of the disease depends on whether the mother has begun to produce anti-VZV immunoglobulin (Ig) G by the time of delivery. 2. Reye syndrome: Reye syndrome, an acute encephalopathy accompanied by fatty liver, can sometimes follow VZV or influenza infections in children. Epidemiological evidence suggests that use of aspirin or other salicylate-containing compounds to treat pain and fever during the viral illness is associated with the development of Reye syndrome. It is also important to avoid aspirin following vaccination against chickenpox. 3. Recurrent infection (herpes zoster, or shingles): Due to the dis- seminated nature of the primary infection, latency is established in multiple sensory ganglia, the trigeminal, and thoracic and lumbar dorsal root ganglia being most common. Unlike most of the herpesviruses, asymptomatic virus shedding is a rare event. Herpes zoster results from reactivation of the latent virus, rather than from new, exogenous exposure. Reactivation occurs in up to 30 percent of individuals who have been infected at some point during their lifetime, and the likelihood increases with advancing age. The most striking feature of herpes zoster is that distribution of the clustered vesicular lesions is dermatomal (affecting the area of skin supplied by cutaneous branches from a single spinal nerve) as shown in Figure 25.12. Even after the lesions heal, some individuals continue to suffer debilitating pain for months to years. This postherpetic neuralgia (PHN) is the most significant sequela of herpes zoster, but it can be mitigated by early treatment with antivirals and pain management medications. The incidence of herpes zoster and postherpetic neuralgia can be markedly reduced by using zoster vaccine in appropriate (greater than 50 years old) populations. C. Laboratory identification Laboratory diagnosis of uncomplicated varicella or zoster is generally not necessary and not usually done because of the typical clinical appearance and distribution of lesions. However, in the immunocompromised patient in whom therapy is warranted, it is
263 Vesicles erupt on an erythematous base and eventually dry and scab. The vesicles appear in regions supplied by the peripheral sensory nerves arising in latently infected root ganglia.
Figure 25.12 Cutaneous manifestations of acute herpes zoster in the territory of a cervical dorsal root ganglion (dermatome).
1 Famciclovir DNA polymerase 1 Valacyclovir 1 Acyclovir Figure 25.13 Drug therapy for varicella virus. 1 Indicates first-line drugs. 264 25. Enveloped DNA Viruses 1. Transmission: Infection in children is usually asymptomatic, and Figure 25.14 Cytomegalovirus infection. Lung section showing typical owl-eye inclusions. these children continue to shed virus for months in virtually all body fluids, including tears, urine, and saliva. Transmission is by intimate contact with these fluids, although saliva may be the most common source. In adults, the virus can also be transmitted by: 1) sexual means because it is present in semen and vaginal secretions, 2) organ transplants, and 3) blood transfusions. Similarly, virus is present in breast milk, and neonates can be infected by this route. HCMV can also cross the placenta and infect a fetus in utero. Initial replication of the virus in epithelial cells of the respiratory and gastrointestinal (GI) tracts is followed by viremia and infection of all organs of the body. In symptomatic cases, kidney tubule epithelium, liver, and CNS, in addition to the respiratory and GI tracts, are most commonly affected. 2. Latency and reactivation: A distinctive feature of HCMV latency is the phenomenon of repeated episodes of asymptomatic virus shedding over prolonged periods. Latency is probably established in monocytes and macrophages, but other cell types, such as those of the kidney, are also involved. B. Clinical significance In healthy individuals, primary HCMV infection is usually subclinical (no apparent symptoms). Although most infections occur in childhood, primary infection as an adult may result in a mononucleosis syndrome clinically identical to that caused by EBV (see p. 268). It is estimated that about 8 percent of infectious mononucleosis (IM) cases are caused by HCMV. Persistent fever, muscle pain, and lymphadenopathy are characteristic IM symptoms, as are elevated levels of abnormal lymphocytes and liver enzymes. The major distinguishing feature of HCMV IM is the absence of the heterophile antibodies that characterize IM caused by EBV (see p. 268). Two specific situations have greater clinical significance, namely, congenital infections and infection of immunocompromised patients. 1. Congenital infections: HCMV is the most common intrauterine viral infection. However, there is a great disparity in incidence of fetal infection and severity of outcome, depending on whether the mother is experiencing a primary or recurrent infection. In women experiencing their first HCMV infection during pregnancy (who, therefore, have not yet produced antibodies against HCMV), 35 to 50 percent of fetuses will be infected, and 10 percent of these will be symptomatic (Figure 25.15). It is known as cytomegalic inclusion disease, and the severity of the symptoms is most pronounced when infection occurs during the first trimester. Results of the infection range from varying degrees of damage to liver, spleen, blood-forming organs, and components of the nervous system to fetal death. Damage to the nervous system is a common cause of hearing loss and mental retardation. Even in infants who are asymptomatic at birth, hearing deficits and ocular damage (for example, chorioretinitis) may appear later and continue to progress during the first few years of life. Congenitally and perinatally infected infants may continue to excrete virus for years after birth, serving as an important virus reservoir.
265 2. Infections of immunosuppressed and immunodeficient patients: Immunosuppressed transplant recipients are multiply at risk from: 1) HCMV present in the tissue being transplanted, 2) virus carried in leukocytes in the associated blood transfusions, and 3) reactivation of their own endogenous latent virus. Immune suppression for the transplant can negate any protective advantage of a seropositive recipient. Destruction of GI tract tissue, hepatitis, and pneumonia are common, the latter being a major cause of death in bone marrow transplant recipients. HCMV infection is also associated with decreased survival of solid tissue grafts (i.e., heart, liver, kidney). HCMV coinfection of patients with HIV infection occurs frequently, probably because of their similar modes of transmission (see p. 364). As a common opportunistic infection in AIDS patients, invasive HCMV infections arising from reactivation of latent virus become increasingly important as CD4+ lymphocyte counts and immune competence decline (see p. 300). Although any organ system can be affected, pneumonia and blindness caused by HCMV retinitis are especially common. Encephalitis, dementia, esophagitis, enterocolitis, and gastritis are other significant problems. In addition, coinfection with HCMV may accelerate the progression of the pathology of AIDS (Figure 25.16). Figure 25.15 Newborn with congenital cytomegalovirus disease, showing hepatosplenomegaly and rash. Because the incidence of HCMV infection in the population is so high, and periodic inapparent recurrent infections occur frequently, simple detection of virus or anti-HCMV antibody is not generally useful. Recovery of virus is not usually done. Serologic diagnosis using ELISA (or, enzyme-linked immunosorbent assay) techniques can distinguish primary from recurrent infection by demonstrating IgG seroconversion or the presence of HCMV-specific IgM. Direct determination of the presence and amount of viral DNA or proteins in white blood cells is useful as an indicator of invasive disease, whereas extracellular virus in urine or saliva may simply result from asymptomatic recurrence. Any of these techniques can also be used to screen transplant donors and recipients to determine HCMV status. D. Treatment and prevention Treatment of HCMV infection is indicated primarily in immunocompromised patients (Figure 25.17). Acyclovir is ineffective because HCMV lacks its own thymidine kinase. However, two inhibitors of HCMV DNA polymerase are available: ganciclovir, a guanine analog that is phosphorylated by a virus-coded protein kinase, and cidofovir, a deoxycytidine analog. A third inhibitor of DNA polymerase, unrelated to the two just described, is phosphonoformic acid (foscarnet). Ganciclovir is used for invasive infections of transplant recipients and AIDS patients, but it has considerable toxicity. For retinitis in AIDS patients, toxic adverse effects can be avoided by direct intraocular placement of a ganciclovir-impregnated implant. Following organ transplants, patients are treated prophylactically with gancyclovir or anti-HCMV Ig. Alternatively, patients are monitored for the first sign of HCMV replication and then treated preemptively with antivirals. A vaccine for active immunization is not available. CNS disease (%)
Children infected with both HIV-1 and CMV show a higher incidence of CNS disease (impaired brain growth or progressive motor deficits) 70 60 50 40 30 20 10 0 CMV positive CMV negative 0 10 Age (months) 18 Figure 25.16 Incidence of central nervous system (CNS) disease in HIV-1-infected children, with or without cytomegalovirus (CMV) infection. 266 25. Enveloped DNA Viruses VI. HUMAN HERPESVIRUS TYPES 6 AND 7 DNA polymerase 1 Ganciclovir 1 Cidofovir 2 Foscarnet Figure 25.17 Drug therapy for cytomegalovirus. 1 Indicates first-line drugs; 2 indicates alternative drug. HHV-6 and HHV-7, classified as members of the Betaherpesvirinae, have marked similarities to HCMV in biologic and genome characteristics. Both HHV-6 and HHV-7 are causative agents of roseola infantum (exanthem subitum), although infection with HHV-7 is more frequently asymptomatic. Two variants of HHV-6 have been recognized: HHV-6A and HHV-6B. HHV-6B is virtually ubiquitous and is the causative agent of roseola infantum. HHV-6A has been implicated in the progression of HIV disease to full-blown AIDS. A. Epidemiology and pathogenesis Most infections with HHV-6 and HHV-7 occur during the first 3 years of life, with overall incidence of antibody approaching 90 percent of the population by age 3. Transmission is thought to be via oral secretions because the viruses replicate in the oropharynx as well as in B and T lymphocytes. HHV-7, in particular, is commonly recovered from healthy individuals’ saliva. These viruses also infect peripheral blood lymphocytes and the cells of various solid organs. HHV-6A infection of lymphoid cells induces a number of significant cell responses, including the synthesis of CD4 glycoprotein, interferon-α, tumor necrosis factor-α, and interleukin-1-β. The ability of HHV-6A to induce expression of CD4 in cells not normally expressing it extends the range of cells that can be infected by HIV. In addition, HHV-6A transactivates transcription of HIV, accelerating the rate of cell death in coinfected cells. Latently infected cells are found among the peripheral blood lymphocyte population. HHV-6A was shown to accelerate AIDS progression in an animal model of the disease. B. Clinical significance HHV-6 infections resulting in disease are most common in infants and individuals who are immunocompromised. 1. Primary infections: Symptomatic roseola infantum (exanthem subitum) occurs in roughly one third to one half of infants with a primary HHV-6 infection. It is characterized by a high fever of 3 to 5 days’ duration, after which a characteristic erythematous macular rash appears on the neck and trunk, resolving after several more days without sequelae (Figure 25.18). HHV-7 infection has been shown to produce an identical clinical picture. Of greater clinical significance is that primary HHV-6 infection of infants is the cause of many acute febrile illnesses and febrile seizures in the absence of the characteristic rash. In some of these cases, HHV-7 has been shown to be the causative agent, whereas, in others, the patient was coinfected with both HHV-6 and HHV-7. Over 20 percent of emergency room visits for febrile illness in infants and one third of febrile seizures are caused by primary infection with HHV-6 and/or HHV-7 (Figure 25.19). Figure 25.18 Roseola infantum. 2. Recurrent infections: Following immunosuppression for organ transplantation or immunocompromise related to HIV infection, reactivation of latent HHV-6, frequently together with HCMV, has VI. Human Herpesvirus Types 6 And 7 30 Visits due to HHV-6 (percent of all visits) been associated with sometimes-fatal interstitial pneumonitis, fever, hepatitis, and encephalitis as well as with transplant rejection. The relationship of HHV-6A to AIDS has not been completely elucidated. Three factors may accelerate the progression from early HIV infection to terminal AIDS: 1) HHV-6A broadens the range of cell types infected by HIV by inducing CD4, 2) coinfected cells are killed more rapidly, and 3) extensively disseminated HHV-6A infection frequently occurs in terminal AIDS patients. The most common clinical syndrome associated with HHV-6 in AIDS patients is encephalitis (Figure 25.20). 267 20 10 0
1 4 7 10 13 16 19 22 >24 Age (months) Figure 25.19 Percentage of visits to the emergency department for febrile illness associated with human herpesvirus type 6 (HHV-6).
VII. HUMAN HERPESVIRUS TYPE 8 HHV-8 infection appears not to occur as frequently as the other human herpesviruses in the normal, healthy population. Yet the virus genome and/or viral proteins have been detected in more than 90 percent of patients with KS, but in less than 1 percent of non-KS tissues. The primary method for detection of HHV-8 is PCR amplification. VIII. EPSTEIN-BARR VIRUS EBV is most commonly known as the causative agent of IM in young adults. Its initial discovery in association with the childhood disease Burkitt lymphoma (BL) led to its recognition as the first human virus clearly related to a malignancy. More recently, EBV has been associated with several additional human neoplastic diseases. Early HIV infection Human herpesvirus type 6 coinfection + Terminal HIV infection (particularly encephalitis)
Figure 25.20 Coinfection with human herpesvirus type 6 accelerates the progression of HIV symptoms. 268 25. Enveloped DNA Viruses EBV in saliva Infection of epithelial cells of oropharynx Pharyngitis Infection of B cells Shedding of virus in saliva B cell proliferation Expression of EBV early proteins Heterophile antibody (agglutinates sheep and horse red blood cells) T-cell activation Atypical lymphocytes Enlargement of liver, spleen, and lymph nodes Figure 25.21 Pathogenesis of infectious mononucleosis caused by Epstein-Barr virus (EBV). tained as a circular plasmid-like form called an episome during latency. One protein that is expressed during latency is called EBNA1, and one of its key functions is to segregate the episomes into daughter cells following cell division. EBV infection of B cells also causes the induction of a number of cellular lymphokines, including B-cell growth factors. In contrast to other herpesviruses, the early genes of EBV induce cell multiplication and immortalization, rather than cell death. Thus, infection induces a polyclonal Bcell proliferation and an accompanying nonspecific increase in total IgM, IgG, and IgA. The IgM class contains heterophile antibodies that agglutinate sheep and horse red blood cells. These antibodies are the basis for the classic diagnostic test for EBV-associated IM (see p. 269). B. Clinical significance As stated earlier, primary infection in infancy or childhood is usually asymptomatic, but as many as 50 percent of those infected later in life develop IM. Although B cells are the primary targets of infection as a result of the presence of the EBV-receptor molecule, EBV has more recently been found to be associated with a small number of T-cell malignancies as well. In patients who are immunodeficient or immunosuppressed, the lack of cell-mediated immune control increases the likelihood of lymphoproliferative disorders of various kinds. Throughout life, healthy EBV carriers continue to have episodes of asymptomatic virus shedding. The source of this virus seems to be the productively infected oropharyngeal cells that acquire the virus from latently infected B cells in which the lytic cycle has been activated. 1. Infectious mononucleosis: The manifestations and severity of pri- mary EBV infection vary greatly, but the typical IM syndrome appears after an incubation period of 4 to 7 weeks and includes phar yngitis, lymphadenopathy, fever, splenomegaly, and increased levels of liver enzymes in the blood (Figure 25.22). Headache and malaise often precede and accompany the disease, which may last several weeks. Complete recovery may take much longer. 2. EBV and malignancies: Following the initial discovery of EBV in association with BL, it has been shown to be associated with a number of other human neoplastic diseases. a. Burkitt lymphoma: BL was first described in 1958 as a rather unique malignancy of the jaw, found in an unusually high frequency in children in regions of equatorial Africa. BL cells all contain one of three characteristic chromosome translocations. The breakpoints of these translocations are such that the cmyc proto-oncogene on chromosome 8 is constitutively activated. Malarial infection and HIV infection are known risk factors for development of BL. b. Epstein-Barr–associated nasopharyngeal carcinoma: Nasopharyngeal carcinoma (NPC) is one of the most common cancers in southeast Asia and North Africa and in the Inuit VIII. Epstein-Barr Virus population, but it is less common elsewhere. NPC differs from BL in that there is no characteristic chromosomal alteration, and the cells involved are epithelial in origin. A role for EBV is suggested because all cells of the tumor contain cytoplasmic viral DNA molecules (episomes). c. Epstein-Barr virus infections in immunocompromised and immunosuppressed patients: In BL and NPC, EBV infection appears to be only one step in a multistep, disease-causing process, and its specific role is still not well defined. In contrast, EBV alone appears to be sufficient for induction of B-cell lymphomas in immunocompromised patients, such as transplant recipients and individuals with AIDS, who cannot control the cell multiplication induced by the early proteins. For example, many AIDS patients develop a B-cell malignancy of some type: BL of the sporadic type occurs with high frequency in earlier stages of AIDS progression, whereas non–BL-type lymphoblastic lymphomas are more characteristic in late-stage AIDS patients. Not all of the HIV-associated BL cases contain the EBV genome. AIDS patients infected with EBV may exhibit nonmalignant, white-gray lesions on the tongue (“hairy leukoplakia”) as shown in Figure 25.23. 269 A Classic clinical triad of infectious mononucleosis: • Fever • Pharyngitis • Lymphadenopathy (usually enlargement of anterior and posterior cervical lymph nodes) Note: Acute infection is often asymptomatic in children, whereas adolescents and young adults show the typical symptoms of infectious mononucleosis. B Transmission: • Exposure to oropharyngeal secretions C Clinical manifestations of Epstein-Barr–associated infectious mononucleosis by age-group: Key: Patients < 35 years old Patients > 35 years old
Fever Pharyngitis Lymphadenopathy Jaundice Rash
IX. POXVIRIDAE Poxviruses belong to a family of large, genetically complex viruses having no obvious symmetry. Members of this family are widely distributed in nature. The agent of previous medical importance to humans, variola virus, was the cause of smallpox, the first infectious disease to be declared eradicated from the Earth. Among the factors that led to this 0 D 50% 100% Incidence of Epstein-Barr– associated infectious mononucleosis: • Peak incidence occurs between ages 15 and 19 years. • Incidence is 30 times higher in blacks than whites in the United States. • No difference in incidence between sexes. • Ninety percent of the general population shows evidence of previous infection with Epstein-Barr virus. Figure 25.22 Characteristics of infectious mononucleosis. 270 25. Enveloped DNA Viruses success are: 1) the availability of an effective, attenuated vaccine; 2) variola’s antigenic stability (that is, only a single antigenic type existed); 3) the absence of asymptomatic cases or persistent carriers; 4) the absence of an animal reservoir; and 5) the emotional effect of this highly lethal, disfiguring disease, which helped to galvanize public support of and cooperation in the eradication efforts. The highly effective poxvirus vaccine contains live vaccinia virus (which causes cowpox), and the viral genome is currently being used in attempts to construct vectors carrying immunizing genes from other infectious agents. Finally, the poxvirus, molluscum contagiosum virus (MCV), causes small, wartlike tumors (not to be confused with true warts caused by papilloma virus, see p. 245). Figure 25.23 Hairy leukoplakia caused by EpsteinBarr virus infection.
Figure 25.24 Abnormal mononuclear cells commonly seen in infectious mononucleosis. Poxviruses follow the basic replication pattern for DNA viruses (see p. 238), with a few notable exceptions. The most striking of these is that the entire replication cycle takes place in the cytoplasm, the virus providing all of the enzymes (including a viral DNA-dependent RNA polymerase) necessary for DNA replication and gene expression. Final maturation by acquisition of a lipoprotein envelope occurs as the virus buds from the cell. The replication cycle is rapid and results in early shut-off of all cell macromolecular syntheses, causing the death of the cell. VIRUS SUBFAMILY CLINICAL MANIFESTATIONS OF PRIMARY INFECTION CLINICAL MANIFESTATIONS OF RECURRENT INFECTION Herpes simplex-1 α Keratoconjunctivitis, gingivostomatitis, pharyngitis, tonsilitis Herpes simplex-2 α Varicellazoster virus Cytomegalovirus VIRUS EpsteinBarr virus SITE OF INITIAL INFECTION SITE OF LATENCY Herpes labialis (“cold sores”) Mucoepithelial Trigeminal sensory ganglia Genital herpes, perinatal disseminated disease Genital herpes Mucoepithelial Lumbar or sacral sensory ganglia α Varicella (“chickenpox”) Herpes-Zoster (“shingles”) Mucoepithelial Trigeminal and dorsal root ganglia β Congenital infection (in utero), mononucleosis-like syndrome Asymptomatic shedding of virus Monocytes, lymphocytes, and epithelial cells Monocytes, lymphocytes Infectious mononucleosis, Burkitt lymphoma Asymptomatic shedding of virus Mucosal epithelium, B lymphocytes B lymphocytes γ Figure 25.25 Properties of common herpesvirus infections. IX. Poxviridae 271
Virus infects the upper respiratory tract and then spreads to the regional lymph nodes and small blood vessels in the skin. Incubation period The stages of smallpox are illustrated in Figure 25.26. Although naturally occurring smallpox is no longer a threat, the mutation of one of the animal poxviruses to a form more virulent for humans has continued to be of concern. Human infections with monkeypox are clinically similar to smallpox and, although somewhat less severe, nevertheless have a mortality rate of about 11 percent. Such infections have only been observed where the human population comes into close contact with infected animals. In its natural state, monkeypox is not readily transmitted among humans. MCV infection occurs only in humans, causing benign wartlike tumors on various body surfaces. Usually spread by direct contact, the virus can be spread among adults via sexual contact. Temperature (oF) 104 101
The unique cellular localization of poxvirus replication has enabled rapid diagnosis by observation of DNA-containing intracytoplasmic inclusion bodies in cells scraped from skin lesions. E. Treatment and prevention Although immunization with vaccinia is no longer done routinely, it is still carried out in certain groups, such as the military and laboratory workers. Although one of the safest vaccines in healthy recipients, individuals with eczema may develop a generalized vaccinia rash covering the surface of the body. Immunocompromised patients are likely to develop progressive vaccinia, which has a high mortality rate. Postvaccinal encephalitis, with a mortality of 40 percent, is a rare secondary hazard accompanying vaccination. Day 5 of rash 15 20
Day 7 of rash 25 30 Approximately one third of the infected individuals die from bleeding, cardiovascular collapse, and secondary infections. Scabbing = Figure 25.26 Time course of smallpox. 272 25. Enveloped DNA Viruses Study Questions Choose the ONE correct answer. 25.1 The initial infection with human cytomegalovirus most commonly occurs: A. during early childhood, by exchange of body fluids. B. in utero, by transplacental transmission from a latently infected pregnant woman. C. by transfer of saliva between young adults. D. by sexual intercourse. E. as a result of blood transfusion or organ transplantation. 25.2 The histological presentation typical of infectious mononucleosis caused by Epstein-Barr virus is due to: A. stimulation of B-cell proliferation by the EpsteinBarr virus early proteins synthesized in the infected cells. B. proliferation of cytotoxic T cells responding to Epstein-Barr virus antigens expressed on the surface of infected B cells. C. a primary humoral immune response to the Epstein-Barr virus infection. D. macrophages responding to the death of EpsteinBarr virus–infected cells. E. activation of an oncogene resulting from a chromosome translocation in Epstein-Barr virus–infected lymphocytes. 25.3 Acyclovir is largely ineffective in the treatment of human cytomegalovirus infections because: A. human cytomegalovirus exhibits a high rate of mutation in the target enzyme. B. human cytomegalovirus depends upon the host cell’s DNA polymerase for replication of its DNA. C. human cytomegalovirus lacks the thymidine kinase required for activation of acyclovir. D. the tissues in which human cytomegalovirus multiplies are largely inaccessible to the drug. E. human cytomegalovirus codes for an enzyme that inactivates the drug. Correct answer = A. Depending on the population, up to 90 percent have antibody by adulthood. B: The most serious complications of infection are those resulting from transplacental transmission, but this is not the common mode of transmission. C and D: Transmission by kissing or sexual intercourse can occur, but most individuals would have already been infected. E: This mode of transmission has serious consequences in antibody-negative recipients, but most recipients had been infected at an earlier age. More common is reactivation of latent HCMV in recipients who have been immunosuppressed for purposes of transplantation. Correct answer = B. The proliferation of cytotoxic T cells results in the increased numbers of atypical lymphocytes detected in blood smears of Epstein-Barr virus (EBV)-infected patients. A: Polyclonal stimulation of B cells by EBV infection occurs and results in the appearance of the characteristic heterophile antibodies, but it is the cytotoxic T-lymphocyte response that results in atypical lymphocytosis of infectious mononucleosis. C: EBV-specific humoral immune response is not related to lymphocytosis. D: B cells are not killed by infection with EBV. E: Although this is the process that results in EBV-associated Burkitt lymphoma, it occurs only years after the initial virus infection. Correct answer = C. The specificity of acyclovir derives from its necessary phosphorylation by the herpes simplex virus or varicella-zoster virus thymidine kinase in order to be an active inhibitor of viral DNA synthesis. Human cytomegalovirus (HCMV) does not have a corresponding enzyme. A: HCMV develops resistance to those drugs that are effective, such as ganciclovir and cidofovir, after long-term therapy, but because their mechanisms of action are different, mutants resistant to one are usually not resistant to the other. B: All herpesviruses code for their own DNA polymerase. D: In those cases where access is a problem for treatment of herpesvirus infections, direct inoculation of the drug has been done. E: Resistance to antiherpesvirus drugs has generally involved mutation of the enzyme interacting with the drug, not inactivation of the drug. Hepatitis B and Hepatitis D (Delta) Viruses 26
RNA Single stranded Positive strand Double stranded Icosahedral Nonenveloped Negative strand Helical Enveloped Nonenveloped Icosahedral Enveloped Icosahedral Helical FLAVIVIRIDAE Hepatitis C DNA Single stranded (nonenveloped) CALICIVIRIDAE Double stranded Hepatitis E PICORNAVIRIDAE Nonenveloped Enveloped Hepatitis A HEPADNAVIRIDAE Hepatitis B S Hepatitis D Figure 26.1 Classification of major viral agents causing hepatitis. [Note: Hepatitis D is a defective virus and is classified in its own “floating” genus. Hepatitis A, C, and E are discussed in Chapter 27.] S See p. 355 for a summary of this virus. 273 274 26. Hepatitis B And Hepatitis D (Delta) Viruses II. HEPADNAVIRIDAE MODE OF TRANSMISSION Fecal–oral HAV HEV Injection or other contact with blood or blood products; heterosexual and homosexual sex HBV Acute infections HCV HDV Acute and/or chronic infections Figure 26.2 Classification of hepatitis viruses based on mode of transmission. HAV, HEV, HBV, HCV, and HDV each refer to the specific hepatitis virus. The family Hepadnaviridae (hepatotropic DNA viruses) consists of hepatitis-causing viruses with DNA genomes. Each hepadnavirus has a narrow host range in which it produces both acute and chronic, persistent infections, but HBV is the only member of this family that infects humans. Because highly infectious virus is present in the blood of both symptomatic and asymptomatic patients, chronically infected individuals pose a serious threat to all healthcare workers, immunization of whom is generally required. A highly effective vaccine produced in genetically engineered yeast cells is available and included among routine childhood immunizations (see p. 39). Biologically, HBV is unique among human disease agents in that replication of the DNA genome proceeds via an RNA intermediate, which, in turn, is “reverse transcribed” by a viral enzyme homologous to the retrovirus reverse transcriptase (see p. 296). However, although retroviruses package an RNA genome, Hepadnaviridae package a DNA genome. A. Structure and replication of hepatitis B virus Incomplete particles Complete viral particle The HBV virion, historically referred to as the “Dane particle,” consists of an icosahedral nucleocapsid enclosed in an envelope (Figure 26.3). 1. Organization of the hepatitis B virus genome: The short HBV DNA genome is unusual in that it is a partly single-stranded, partly double-stranded, noncovalently closed, circular DNA molecule (that is, one strand is longer than the other) as shown in Figure 26.4. The short “plus” strand, which can vary in length, is only 50 to 80 percent as long as its complementary strand, the “minus” strand. The circular structure of the genome is maintained by base-pairing the strands at one end. A summary of HBV replication is shown in Figure 26.4. 2. Viral proteins: The four proteins encoded by viral DNA are: 1) the Figure 26.3 Electron micrograph of a fraction of serum from a patient with severe hepatitis. core protein [hepatitis B nucleocapsid core antigen (HBcAg)]; 2) envelope protein [a glycoprotein referred to as hepatitis B surface antigen (HBsAg)]; 3) multifunctional reverse transcriptase/DNA polymerase, which is complexed with the DNA genome within the capsid; and 4) a nonstructural regulatory protein designated the “X protein.” [Note: HBeAg is produced from an alternate start site upstream of the start for HBcAg, followed by proteolytic processing of the pre-core protein.] B. Transmission Infectious HBV is present in all body fluids of an infected individual. Therefore, blood, semen, saliva, and breastmilk, for example, serve as sources of infection. The titer of infectious virus in the blood of an acutely infected patient can be as high as 108 virus particles per ml but generally is lower in other body fluids. In areas of high endemicity (for example, Southeast Asia, Africa, and the Middle East), the majority of the population becomes infected at or shortly after birth from a chronically infected mother or from infected siblings. Individuals infected at this young age have a significant chance of II. Hepadnaviridae becoming chronic carriers, maintaining the high prevalence of virus in the population. Individuals infected at an early age also have an increased risk of developing hepatocellular carcinoma later in life. In the United States and other Western countries, the carrier rate is much lower, and primary infection rarely occurs in newborns. Hepatitis B is primarily a disease of infants in developing nations, and, in Western countries, it is mostly confined to adults who usually contract HBV infection through sexual intercourse or blood exposure from shared needles during injecting drug use. 275 Envelope Capsid protein
((–) DNA NUCLEUS LEUS CYTOPLASM Vira DNA moves to the Viral nucleus, where the short (+) DNA strand of the viral genome is extended, and the (–) strand is repaired, forming closed, circular DNA. D 1
(+) DNA HBV RNA RNA Transcription by host RNA polymerase II produces four RNAs: three are subgenomic mRNAs, and one is of genomic size that serves both as an mRNA and a template for DNA synthesis. 2 Viral proteins RNA 3 Reverse transcriptase forms a complex with the large RNA and synthesizes the (–) strand. Reverse transcriptase constructs a complementary DNA strand using the (–) DNA strand as template. RNA is degraded as the (+) DNA strand is synthesized. 4 1. Phases in acute hepatitis B virus infections: Following infection, HBV has a long but variable incubation period of between 45 and 120 days. Following this period, a pre-icteric (prejaundice) phase occurs, lasting several days to a week. This is characterized by mild fever, malaise, anorexia, myalgia, and nausea. The acute, icteric phase then follows and lasts for 1 to 2 months. During this phase, dark urine, due to bilirubinuria, and jaundice (a yellowish coloration of mucous membranes, conjunctivae, and skin) are evident. There usually is an enlarged and tender liver as well. In 80 to 90 percent of adults, a convalescent period of several more months is followed by complete recovery (Figure 26.6). The nucleocapsid is enclosed in an envelope as it exits the cell. The extension of the short (+) DNA strand stops immediately as the virus leaves the cell. 5 2. Monitoring the course of acute hepatitis B virus infection: Whereas liver-specific enzymes are important clinical determinants of all of Figure 26.4 Replication of hepatitis B virus (HBV). 276 26. Hepatitis B And Hepatitis D (Delta) Viruses
A B Fulminant hepatitis Limited cellmediated and humoral immune responses HBV HBV HBV Severe chronic hepatitis (chronic active hepatitis) HBV HBV HBV C Cirrhosis Hepatocellular carcinoma Minimal chronic hepatitis (chronic persistent hepatitis) Chronic disease Acute hepatitis B HBV HBV HBV Asymptomatic carrier state Effective cell-mediated and humoral immune responses HBV HBV HBV Resolution Figure 26.5 Clinical outcomes of acute hepatitis B virus (HBV) infection. the viral hepatitides, HBV infection is unusual in that the quantities of virions and virion components in the blood are so great that the time course of their appearance and clearance, along with that of the antibodies directed against them, serve as convenient markers of the stage of the disease and the likely future course. a. Appearance of viral antigens: During the incubation period, Jaundice Dark urine Malaise Anorexia Exposure Nausea RUQ pain Incubation period HBsAg and hepatitis B e antigen (HBeAg) are the first indicators of HBV infection to appear in the blood (Figure 26.7). Their presence indicates an active infection but does not distinguish between acute and chronic infections. Next, viral DNA, viral DNA polymerase, and complete virions become detectable. These continue to increase during the acute disease phase, when a patient’s blood has the highest titer of infectious virus. Preicteric Icteric Convalescent period period period Acute disease Figure 26.6 Symptoms of acute hepatitis B infection. RUQ = right upper quadrant.
concurrently with liver enzymes in the serum, whereas antiHBeAg antibodies and, still later, anti-HBsAg antibodies do not appear until the beginning of convalescence (generally after the respective antigens have disappeared from the blood, see Figure 26.7). In those patients in whom the infection resolves completely, anti-HBcAg and anti-HBsAg antibodies remain present for life, providing immunity to reinfection. Continued presence of HBsAg beyond 6 months and absence of anti-HBsAg indicates that the infection has become chronic (Figure 26.8). A patient suffering chronic HBV infection is capable of eliciting an immune response against HBsAg but the anti-HBs antibody levels are too low to be detectable. All of the antibody that develops is complexed with circulating HBsAg. II. Hepadnaviridae 277 A Acute In some individuals with acute infections, HBsAg becomes undetectable before anti-HBsAg appears. This is referred to as the “window period,” during which a person tested for HBsAg and anti-HBs will appear uninfected. Viral DNA infection Viral shedding Elevated liver enzymes in serum Symptoms 1 s ti-H B e 2 Ant i-HB Anti -HB sAnti eAn Anti -HB c Anti-HB 0 eAnti-HBe B ti-H An An ti-H B e Ag In those patients in whom the infection resolves completely, anti-HBcAg and antiHBsAg antibodies remain present for life, providing immunity to reinfection. c sAn s Ag B i-H nt An ti-H B ti-H B B i-H nt c HB s c Anti-HB -HB HB Anti-HbcAg appears early in the clinical phase. Anti-HB A c A During the incubation period, HBsAg and HBeAg are the first indicators of HBV infection to appear in the blood. c c B ti-H An Jaundice 3 5 4 6 12 24 MONTHS AFTER EXPOSURE MON Anti-HBeAg antibodies appear early in the clinical phase. Later, anti-HBsAg antibodies appear at the beginning of convalescence. B Chronic infection Elevated liver enzymes in serum Symptoms c c B ti-H An c HB Anti- c Anti-HB c Anti-HB c AntiHB B ti-H An The continued presence of HBsAg beyond 6 months and the absence of anti-HBsAg is an indication that the infection has become chronic. c An ti- c HB HB B ti-H e Ag An Be -H 0 1 2 3 4 Months i nt An ti-H B Anti-HB c eA HB s Ag 5 6 1 2 3 4 5 6 Years 7 8 9 10 TIME AFTER EXPOSURE Figure 26.7 Typical course of hepatitis B virus infection. A. Acute infection. B. Chronic infection. HBsAg = hepatitis B surface antigen; HBeAg = hepatitis B e antigen; HBcAg = hepatitis B nucleocapsid core antigen; anti-HBsAg, anti-HBeAg, and anti-HBcAg each refer to antibodies to the corresponding antigen. 278 26. Hepatitis B And Hepatitis D (Delta) Viruses 3. Fulminant hepatitis: In 1 to 2 percent of acute symptomatic SERUM MARKER RESOLVED HBeAg – + – HBsAg – + – Anti-HBcAg + + – Anti-HBsAg + – + CHRONIC1 VACCINATED
The absence of antiHBs is an indication that the infection has become chronic. The currently used vaccine, containing recombinant hepatitis surface antigen, elicits only anti-HBsAg antibody, which is the neutralizing antibody. 1One e year after initial infection infection. Figure 26.8 Interpretation of serologic markers of hepatitis B infection. HBeAg = hepatitis B e antigen; HBsAg = hepatitis B surface antigen; antiHBcAg, and anti-HBsAg each refer to antibodies to the corresponding antigen. Infants born to infected mothers 90% Infected children age 5 years or less 25-50% 5% Infected adults 0 20 40 60 80 cases, much more extensive necrosis of the liver occurs during the first 8 weeks of the acute illness. This is accompanied by high fever; abdominal pain; and eventual renal dysfunction, coma, and seizures. Termed fulminant hepatitis, this condition is fatal in roughly 8 percent of cases. Although it is not clear why the acute disease takes this course, a more highly virulent strain of HBV, coinfection with HDV or another hepatitis virus (for example, HCV), and/or perhaps an uncontrolled immune response by the patient, are thought to play a role. 100 Percentage of HBV-infected individuals in the United States that progress to chronic disease. Figure 26.9 Effect of patient’s age on the tendency of acute hepatitis B virus (HBV) infection to progress to chronic disease. In about two thirds of individuals, the primary infection is asymptomatic, even though such patients may later develop symptomatic chronic liver disease, indicating persistence of the virus. Following resolution of the acute disease (or asymptomatic infection), about 2 to 10 percent of adults and over 25 percent of young children remain chronically infected (Figure 26.9). The high rate of progression to chronic liver disease seen in infants born to HBV-infected mothers is thought to relate to the less competent immune status of newborns. Adults with immune deficiencies also have a considerably higher probability of developing chronic infection than do individuals with normal immune systems. 1. Types of chronic carriers: The asymptomatic carriers of HBsAg are the most common type of persistently infected individuals. They usually have anti-HBeAg antibodies and little or no infectious virus in their blood (see Figures 26.7B and 26.8). Later progression of liver damage or recurrence of acute episodes of hepatitis is rare in such patients. Those carriers with minimal chronic hepatitis (formerly, “chronic persistent hepatitis”) are asymptomatic most of the time but have a higher risk of reactivation of disease, and a small fraction does progress to cirrhosis. Severe chronic hepatitis (formerly, “chronic active hepatitis”) results in more frequent exacerbations of acute symptoms, including progressive liver damage, potentially leading to cirrhosis and/or hepatocellular carcinoma (see below), chronic fatigue, anorexia, malaise, and anxiety. These symptoms are accompanied by active virus replication and the corresponding presence of HBeAg in the blood. Serum levels of liver enzymes and bilirubin are increased to varying degrees, reflecting the extent of necrosis. The risk of developing cirrhosis is highest in those carriers with more frequent recurrences of acute disease and those in whom HBeAg is not cleared from the blood, indicating continuing virus replication. Overall life expectancy is significantly shorter in those individuals with cirrhosis. 2. Development of hepatocellular carcinoma (hepatoma): Hepatocellular carcinoma (HCC) is fairly uncommon in the United States, whereas it is 10 to 100 times more frequent in areas of high HBV endemicity. In all populations, males experience a higher rate of chronic HBV infections; a higher rate of progression to cirrhosis; and, ultimately, a higher rate of HCC, for which the male-to-female ratio is 6:1. HCC typically appears many years II. Hepadnaviridae after the primary HBV infection, and the tumor itself is rather slow growing and only occasionally metastasizes. Clinically, a patient with HCC exhibits weight loss, right-upper-quadrant pain, fever, and intestinal bleeding. Although there is no doubt that chronic HBV infection greatly increases the risk of HCC, the mechanisms relating HBV and HCC are not completely understood. By causing continuing liver necrosis, followed by regeneration of the damaged tissue, chronic HBV infection provides the opportunity for chromosomal rearrangements and mutations. Because HBV is a DNA virus, integration of the viral genome into the host's chromosome can also result in mutation and insertion, with concomitant changes in cell growth control. In fact, recent evidence suggests that the HBV gene product X is actively involved in tumor formation, following integration of the gene into the host's chromosome. HCC is a major cause of death due to malignancy worldwide, and its distribution parallels HBV incidence (approximately 80 percent of primary HCCs occur in HBV-infected individuals). F. Laboratory identification The purpose of diagnostic laboratory studies of patients with clinical hepatitis is to, first, determine which hepatitis virus is the cause of the illness and, second (for HBV), to distinguish acute from chronic infections. The diagnosis of hepatitis is made on clinical grounds, coupled with biochemical tests that evaluate liver damage. Elevations of aminotransferases, bilirubin, and prothrombin time all contribute to the initial evaluation of hepatitis. Commonly known as ELISA, enzyme-linked immunosorbent assay (see p. 27), and other immunologic techniques for detection of viral antigens and antibodies are the primary means to distinguish among HAV, HBV, HCV, and HDV. In addition, identification of the presence or absence of specific antiviral antibodies and viral antigens permits differentiating between acute and chronic HBV infections (see Figure 26.7). 279 Interferon and pegylated interferon • • • • • Many side effects No drug resistance Administered subcutaneously High cost Pegylated interferon, a long acting interferon taken once a week, is given for one year.1 1 This is in contrast to the other hepatitis treatments, which are given by mouth for many years until a desired response is achieved. Lamivudine • High rate of drug resistance • Low cost • Many years of experience confirm its safety, including its use during pregnancy Adefovir • Potential nephrotoxicity • Activity against lamivudineresistant HBV Entecavir • High cost • Has potent antiviral activity and a low rate of drug resistance
not needed, because, in about 95 percent of adults, the immune system controls the infection and eliminates the virus within about 6 months. Although drug therapy is usually only required in chronic hepatitis, it may also be required with the acute severe liver impairment that accompanies fulminant hepatitis. 2. Chronic hepatitis: The goal for treatment in patients with chronic hepatitis is to reduce the risk of progressive chronic liver disease and other long-term complications from chronic HBV, such as cirrhosis and hepatocellular carcinoma. The most commonly used drugs include interferon-α or one of a large number of nucleoside/nucleotide antiviral agents (Figure 26.10). The drug of choice depends on multiple factors, including the antibody and antigen status of the patient. Pegylated interferon-α (if the patient does not have cirrhosis), entecavir, or tenofovir are often preferred for initial treatment. The two most commonly used markers to monitor the efficacy of therapy are seroconversion to anti-HBeAg and sustained suppression of HBV DNA. Telbivudine • High rate of drug resistance • Role as primary therapy is limited Tenofovir • Potential nephrotoxicity • First line treatment in treatmentnaïve patients, and in patients with lamivudine, telbivudine, or entecavir resistance, preferably as additional treatment in these patients Figure 26.10 Drugs used in the treatment of hepatitis B. HBV = hepatitis B virus. 280 26. Hepatitis B And Hepatitis D (Delta) Viruses H. Prevention Routine immunization infants and previously • All unvaccinated children by age 11 years Increased risk of hepatitis B People with multiple sexual partners • Sexual or household • contactspartner of HBsAg-positive people who have sex with men • Men Users of illicit injection drugs • Travelers • disease to regions of endemic People occupationally exposed to • blood or body fluids patients • Hemodialysis receiving clotting-factor • Patients concentrates Figure 26.11 Candidates for hepatitis B virus immunization. HBsAg = hepatitis B surface antigen. The purpose of controlling the spread of HBV infection is to prevent cases of acute hepatitis. An additional goal is to decrease the pool of chronically infected individuals who serve as reservoirs for infectious virus in the population and who are at greatly increased risk for developing cirrhosis and liver cancer. The availability of a highly effective vaccine has led to a several-pronged approach: 1) protection of those adults who are at risk because of lifestyle or occupation, 2) protection of newborns from infection by transmission from HBVpositive mothers (important because of the high rate of resulting chronic infections, see p. 274), and 3) protection of siblings and other children from infection by chronically infected family members. 1. Active immunization: HBsAg is used to prepare vaccines confer- ring protection because antibody to the virion component neutralizes infectivity. HBV vaccination is now recommended as a routine infant immunization, as is the immunization of adolescents who were not given the vaccine as infants. An unusual feature of the recommended vaccination schedule is to initiate an HBV vaccine series at birth. This is possible because infants have adequate antibody response to neonatal vaccination with the HBV vaccine. Other individuals who are candidates for HBV vaccine are shown in Figure 26.11. 2. Passive immunization: Hepatitis B immunoglobulin (HBIG) is pre- HBsAg is supplied by coinfection with HBV, which thus serves as a helper virus for HDV replication. Proteins in envelope membrane pared from the blood of donors having a high titer of anti-HBsAg antibody. Immediate administration of HBIG is recommended as the initial step in preventing infection of individuals accidentally exposed to HBV-contaminated blood by needlestick or other means and of those exposed to infection by sexual contact with an HBV-positive partner. In such cases, this should be accompanied by a course of active immunization with the hepatitis B vaccine. It is also strongly recommended that pregnant women should be screened for HBsAg. Infants born to mothers who are HBV positive are given HBIG plus hepatitis B vaccine at birth, followed by additional doses of vaccine at 1 and 6 months. RNA III. HEPATITIS D VIRUS (DELTA AGENT) Envelope HDAg (delta antigen) complexed with RNA genome. Figure 26.12 Structure of hepatitis D virus. HBsAg = hepatitis B surface antigen. HDV is found in nature only as a coinfection with HBV. It is significant because its presence results in more severe acute disease, with a greater risk of fulminant hepatitis and, in chronically infected patients, a greater risk of cirrhosis and liver cancer. A. Structure and replication HDV does not fall into any known group of animal viruses. It has a circular, single-stranded RNA genome with negative polarity that codes for one protein (delta antigen), with which the genome is complexed in the virion (Figure 26.12). In the infectious particle, the nucleoprotein complex is enclosed within an envelope containing III. Hepatitis D Virus (“Delta Agent”) HBV-coded HBsAg. Thus, HDV requires HBV to serve as a helper virus for infectious HDV production. The HDV RNA genome is replicated and transcribed in the nucleus by cellular enzymes, whose specificity is probably modified by complexing with the delta protein. [Note: This phase of HDV replication is independent of HBV, whose only helper function is to supply HBsAg for the envelope.] 281 A Simultaneous primary coinfection HDV V + HBV HB Incubation
Acute cute disease B Chronic coinfection HDV V + HBV
Incubation us primary coinfection c Simultaneous (acute disease) Incubation Chronic disease C Primary HDV infection of a chronically HBVinfected individual HBV Incubation Chronic HBV infection HD HDV
No treatment specific for HDV infection is available. Because HDV depends on coinfection with HBV, the approaches for preventing HBV infection are also effective in preventing HDV infection. There is no vaccine specifically for HDV. Therefore, those who are chronically infected with HBV can only be protected from HDV infection by limiting chances for exposure. Those who are protected against HBV infection through vaccination will not be affected by HDV. Chronic disease Figure 26.13 Consequences of hepatitis D virus (HDV) infection. HBV = hepatitis B virus. 282 26. Hepatitis B And Hepatitis D (Delta) Viruses Study Questions Choose the ONE correct answer. 26.1 Killing of liver cells infected with hepatitis B virus is primarily caused by: A. shut-off of cellular protein synthesis. B. intracytoplasmic accumulation of hepatitis B virus antigen aggregates. C. degradation of cellular mRNA. D. attack by cytotoxic T lymphocytes directed against hepatitis B virus antigens. E. virus-induced aberrant chromosome rearrangements and deletions. 26.2 The most common natural mode of transmission of infection with hepatitis B virus is via: A. B. C. D. E. contaminated water supply. body fluids, such as urine and semen. respiratory droplets. direct contact. infected insect vectors. Correct answer = D. There is no evidence that hepatitis B virus ( HBV) infection is cytocidal. Protein synthesis is not shut off, and mRNA is not degraded in infected cells. Accumulation of HBV proteins is not seen, rather, they are actively exported. Although chromosome damage is observed in cells of primary hepatocellular carcinoma, it is not characteristic of nonmalignant infected liver cells. Correct answer = B. Hepatitis B virus is found at high levels in all body fluids, which results in transmission from mother to newborn, from sibling to sibling, and through sexual intercourse as well as by infection by virus-containing blood. Contaminated water or food is the typical source of hepatitis A and E infection. 26.3 Hepatitis delta virus is unique in that: A. infectivity requires an envelope protein provided by a helper virus. B. it has an RNA genome that is replicated by a replicase supplied by a coinfecting helper virus. C. its mRNA is transcribed by a transcriptase supplied by a helper virus. D. the virion contains a reverse transcriptase provided by a helper virus. E. it encodes a protein delta antigen (HDAg) that replaces helper virus glycoproteins in the envelopes of helper virus particles. 26.4 A patient suffering from hepatitis underwent a battery of laboratory tests to determine the cause of the disease. The following results were obtained from serological and biochemical testing of the patients serum: HBsAg positive, HBeAg positive, anti-HBcAg IgM positive, anti-HBsAg negative, HDV RNA negative, elevated liver enzymes. From these results, how would you diagnose this patient's infection? A. Acute hepatitis B disease B. Chronic hepatitis B disease C. Chronic hepatitis B disease with hepatitis D superinfection D. Acute hepatitis D disease E. Chronic hepatitis D disease Correct answer = A. The only function of the hepatitis B virus (HBV) helper is to supply the envelope. B: Genome replication requires a cell RNA polymerase, presumably modified by the hepatitis D virus (HDV) delta protein such that it can use the HDV RNA as a template. C: Transcription likewise depends on cell enzymes. D: The virion contains only the delta protein. E: HDAg is complexed with the RNA genome in the HDV virion and is not found in the HBV virion. Correct answer = A. The presence of HBs (surface) and HBe antigens is consistent with early or acute hepatitis B virus (HBV) disease. Antibodies against HBsAg have not yet developed, and immunoglobulin (Ig) M against HBcAg (nucleocapsid core) occurs early in the course of infection. The IgM isotype subsequently switches to IgG during convalescence. The absence of hepatitis D virus (HDV) RNA indicates that this person is not superinfected with HDV. D and E are incorrect because HDV does not infect alone but requires HBV as a helper virus. Thus, a person who has infection caused by HDV must be simultaneously infected with HBV. Positive-strand RNA Viruses 27
RNA Viruses Single stranded Double stranded Icosahedral Nonenveloped Positive strand Nonenveloped Icosadedral II. PICORNAVIRIDAE Picornaviridae Picornaviruses are small, naked (nonenveloped), icosahedral viruses (Figure 27.2), which contain a single-stranded, nonsegmented RNA genome and four structural proteins. Picornaviridae are divided into five genera: enteroviruses, rhinoviruses, cardioviruses, aphthoviruses, and hepatoviruses. Cardiovirus species cause encephalitis and myocarditis in mice, whereas Aphthovirus species is represented by “foot-andmouth” disease virus, which infects cattle. Enterovirus, Rhinovirus, and Hepatovirus species cause a wide variety of clinical syndromes in humans. Although the most intensively studied picornavirus is poliovirus, what has been learned about the structure and replication of poliovirus also applies in large measure to the other viruses in this family. A. Enterovirus Negative strand Helical Enveloped Enveloped Continued on next page Enterovirus Coxsackievirus Echovirus Enterovirus Poliovirus S S Rhinovirus Hepatovirus Hepatitis A virus S Caliciviridae Hepatitis E virus Norwalk virus More than seventy enteroviruses have been identified. Currently, as new enteroviruses are identified, they are not assigned to one of these groups but are simply given numerical designations (for example, enterovirus 68, enterovirus 69, and so forth). 1. Epidemiology: Individuals are infected with enteroviruses by ingestion of contaminated food or water. Enteroviruses are stable at the low pH of the stomach, replicate in the gastrointestinal (GI) tract, and are excreted in the stool. Thus, these viruses are said to be transmitted by the fecal–oral route. The virus can replicate in a Figure 27.1 Classification of positive-strand RNA viruses (continued on next page). S See p. 363 for summaries of these viruses. 283 284 27. Positive-strand RNA Viruses Enveloped Togaviridae (icosahedral) Alphavirus Chikungunya virus Eastern and Western equine encephalitis viruses Venezuelan equine encephalitis virus Rubivirus Rubella virus S Flaviviridae (icosahedral) 2. Viral replication: Enteroviruses bind to specific receptors on host cell surfaces. For example, poliovirus binds to a receptor that is a member of the immunoglobulin (Ig) supergene family of proteins. Cells lacking these specific receptors are not susceptible to infection. a. Mechanism of genome replication: This is the same process Flavivirus Dengue fever virus Japanese encephalitis virus St. Louis encephalitis virus Tickborne encephalitis virus West Nile virus Yellow fever virus Hepatitis C virus variety of tissues. For example, after replicating in the oropharynx and intestinal tract lymphoid tissue, enteroviruses can enter the bloodstream and, thereby, spread to various target organs (for example, poliovirus spreads to the central nervous system [CNS]). Although the great majority of infections are asymptomatic, infection, whether clinical or subclinical, usually results in protective immunity. Enteroviruses account for an estimated 10 to 15 million symptomatic infections per year in the United States. S Coronaviridae (helical) Coronavirus Figure 27.1 (continued) Classification of positive-strand RNA viruses. S See pp. 366, 355 for summaries of these viruses. Figure 27.2 Poliovirus, a type of Picornavirus, is one of the simplest and smallest viruses. as described for Type I RNA viruses (see p. 239): Namely, the incoming parental RNA serves as the template for a genomesize, negative-strand RNA, and this, in turn, serves as a template for multiple copies of progeny positive-strand RNA. b. Translation: Enterovirus RNA contains a single, long, open reading frame. Translation of this message results in the synthesis of a single, long polyprotein, which is processed by viral proteases into structural proteins and nonstructural proteins, including the viral RNA polymerase needed to synthesize additional copies of the viral genome. 3. General clinical significance of enterovirus infections: All enteroviruses can cause CNS disease. For example, enteroviruses are currently the major recognizable cause of acute aseptic meningitis syndrome, which refers to any meningitis (infectious or noninfectious) for which the cause is not clear after initial examination plus routine stains and cultures of the cerebrospinal fluid (CSF). Viral meningitis is a common infection in the United States, with an estimated 75,000 cases each year. Viral meningitis can usually be distinguished from bacterial meningitis because: 1) the viral disease is milder; 2) there is an elevation of lymphocytes in the CSF, rather than the elevated neutrophils seen in bacterial meningitis; and 3) the glucose concentration in the CSF is not decreased. Viral meningitis occurs mainly in the summer and fall, affecting both children and adults. The treatment is symptomatic, and the course of the illness is usually benign. Viruses can be isolated from the stool or from various target organs (CNS in meningitis cases and from conjunctival fluid in conjunctivitis cases). Evidence of infection can also be obtained by demonstration of a rise in antibody titer against a specific enterovirus. No antiviral drugs are available for treatment of infections caused by Enterovirus species. 4. Clinical significance of poliovirus infection: Poliomyelitis is an acute illness in which the poliovirus selectively destroys the lower motor neurons of the spinal cord and brainstem, resulting in flaccid, asymmetric weakness or paralysis. In the United States, no cases of paralytic poliomyelitis caused by wild-type poliovirus II. Picornaviridae 285 have occurred in more than 20 years. The few cases of polio that occur (less than 10 per year) are all caused by the reversion to virulence of the virus in the live-attenuated Sabin polio vaccine (see below). In countries with low immunization rates, paralytic polio continues to occur. There are only three countries that remain polio endemic: Afganistan, Pakistan, and Nigeria. The number of countries is down significantly from 125 in 1988. Notably, India was polio free for the first time in 2011. Although this represents significant progress toward the goal of eradicating polio from the world, in 2009–2010, 23 previously polio-free countries were reinfected due to importation of the virus.
two thirds of patients with paralytic poliomyelitis. Complete recovery is less likely when acute paralysis is severe, and patients requiring mechanical ventilation because of respiratory paralysis rarely recover without some permanent disability. c. Postpoliomyelitis syndrome: Approximately 20 to 30 percent of patients who par tially or fully recover from paralytic poliomyelitis experience a new onset of muscle weakness, pain, atrophy, and fatigue 25 to 35 years after the acute illness. No illness 4% (Asymptomatic) Minor illness ABORTIVE POLIOMYELITIS: Minor, nonspecific symptoms of headache, sore throat, and nausea. 1% Major illness NONPARALYTIC POLIOMYELITIS: Symptoms are indistinguishable from aseptic meningitis caused by other enteroviruses. PARALYTIC POLIOMYELITIS: Muscle paralysis follows myalgia and asymmetric weakness. Respiratory paralysis may also occur. Figure 27.3 Clinical outcomes of infection with poliovirus. Infection PHARYNX low one of several courses: 1) asymptomatic infection, which occurs in 90 to 95 percent of cases and causes no disease and no sequelae; 2) abortive infection; 3) nonparalytic infection; or 4) paralytic poliomyelitis (Figure 27.3). The classic presentation of paralytic poliomyelitis is flaccid paralysis, most often affecting the lower limbs. This is a result of viral replication in, and destruction of, the lower motor neurons in the anterior horn of the spinal cord (Figure 27.4). Respiratory paralysis may also occur, following infection of the brainstem. Poliomyelitis should be considered in any unimmunized person with the combination of fever, headache, neck and back pain, asymmetric flaccid paralysis without sensory loss, and lymphocytic pleocytosis (an increase in the number of lymphocytes in the spinal fluid). 95% INSTESTINE
Poliovirus infections Via blood/lymphatics bl Replication in oropharynx and small intestine Febrile illness (Day 3)
treatment of poliomyelitis are not available. Management, therefore, is supportive and symptomatic. Vaccination is the only effective method of preventing poliomyelitis (see p. 40). Poliomyelitis can be prevented by either live-attenuated (Sabin) or killed (Salk) polio vaccines. These vaccines have led to the elimination of wild-type polio from Western Europe, Japan, and the Americas. Killed polio vaccine has no adverse effects, whereas live polio vaccine may undergo reversion to a virulent form while it multiplies in the human intestinal tract and cause vaccine-associated paralytic poliomyelitis in those receiving the vaccine. Because the small numbers of cases of paralytic poliomyelitis in the United States since 1979 were due to vaccine-derived strains, the CDC changed its recommendation for routine polio vaccination to killed (inactivated) polio vaccine (IPV) in 2000. Virus rus is shed in i feces f Meningitis (rare) (Day 6) Paralysis (Day 8) SPINAL CORD Figure 27.4 Central nervous system invasion by poliovirus. 286 27. Positive-strand RNA Viruses 5. Clinical significance of coxsackievirus and echovirus infections: 1 During infection rhinoviruses invade mucosal tissue. These give rise to a large variety of clinical syndromes, including meningitis, upper respiratory infections, gastroenteritis, herpangina (severe sore throat with vesiculoulcerative lesions), pleurisy, pericarditis, myocarditis, and myositis. 6. Clinical significance of enteroviruses 70 and 71 infections: These have been associated with severe CNS disease. A particularly acute form of extremely contagious hemorrhagic conjunctivitis has also been associated with enterovirus 70. B. Rhinovirus Cilia Nasal epithelium 2 3 Viruses adsorb to nasal epithelium. Viruses replicate and are shed, causing spread of infection. Virus-rich discharge Cell damage 4 Antibodies and interferon facilitate recovery. Infection ends, and epithelium regenerates. Antibodies and interferon Figure 27.5 Pathogenesis of the common cold showing stages from infection to recovery. Rhinoviruses cause the common-cold syndrome (Figure 27.5). They differ in two important respects from enteroviruses. First, whereas enteroviruses are acid stable (they must survive the acid environment of the stomach), rhinoviruses are acid labile. Second, rhinoviruses, which replicate in the nasal passages, have an optimal temperature for replication that is lower than that of enteroviruses. This permits rhinoviruses to replicate efficiently at temperatures several degrees below body temperature. Rhinovirus replication is similar to that of the poliovirus (see p. 284). Because there are more than 100 serotypes of rhinoviruses, development of a vaccine is impractical. Studies have shown that in addition to being spread by respiratory droplets, rhinoviruses can also be spread by hand-tohand contact. Therefore, hand washing at appropriate intervals can be a useful preventive measure. C. Hepatovirus The sole member of this genus is hepatitis A virus (HAV). Although at one time HAV was also known as enterovirus 72, sufficient differences have been found between HAV and the enteroviruses to warrant placing HAV in a genus by itself. HAV, of which there is only one serotype, causes viral hepatitis. As with the enteroviruses, transmission is by the fecal–oral route, and the virus is shed in the feces. For example, a common mode of transmission of the virus is through eating uncooked shellfish harvested from sewage-contaminated water. The main site of replication is the hepatocyte. Viral replication results in severe cytopathology, and liver function is significantly impaired (Figure 27.6). In contrast to most other picornaviruses, HAV grows poorly in tissue culture. The prognosis for patients with acute hepatitis A is generally favorable, and the development of persistent infection and chronic hepatitis is uncommon. HAV infection is most common in developing countries with poor sanitation (Figure 27.7). Prevention depends on taking measures to avoid fecal contamination of food and water. Immune globulin has been used for many years, mainly as postexposure prophylaxis. Vaccines prepared from whole virus inactivated with formalin are now available. HAV vaccination is recommended for children over age 1 year and for persons traveling to developing countries. A combination vaccine (Twinrix) is available to protect against both hepatitis A and hepatitis B virus infection. IV. Togaviridae III. CALICIVIRIDAE Caliciviruses are small, nonenveloped, spherical particles. Each contains a single-stranded, nonsegmented RNA genome, and a single species of capsid protein. In contrast to the picornaviruses, the caliciviruses genome contains three open reading frames. Norovirus is the prototype human calicivirus. There are at least four strains of human caliciviruses. A. Caliciviruses Norovirus (formerly known as Norwalk-like virus) replicates in the GI tract and is shed in the stool. Infection by the Norovirus is by the fecal–oral route, following ingestion of contaminated food or water, by person-to-person contact, or by contact with contaminated surfaces. Norovirus is a major cause of epidemic acute gastroenteritis, particularly at schools, camps, military bases, prisons, and other closed environments such as cruise ships. It affects primarily adults and school-age children but not infants. The clinical presentation is characterized by nausea, vomiting, and diarrhea. Symptoms last 24 to 48 hours, and the disease is self-limited. Radioimmunoassays and ELISA (enzyme-linked immunosorbent assay) tests are available for the detection of antiviral antibodies (see p. 27). No specific antiviral treatment is available. Careful attention to hand washing and measures to prevent contamination of food and water supplies should reduce the incidence of these infections. 287 Elevated serum liver enzymes Virus in blood Virus detectable in liver biopsy Virus in feces Incubation 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Weeks Ingestion of virus Figure 27.6 Time course of hepatitis A infection.
IV. TOGAVIRIDAE The togaviruses are enveloped, icosahedral viruses that contain a positive-sense, single-stranded RNA genome and generally three structural proteins. The capsid (C) protein encloses the viral RNA, forming the nucleocapsid, and the two other proteins (E1 and E2) are glycoproteins that form the hemagglutinin-containing viral spikes that project from the lipid bilayer. The family Togaviridae is divided into two genera: Alphavirus and Rubivirus. Low Intermediate High Figure 27.7 Distribution of hepatitis A virus infection worldwide. 288 27. Positive-strand RNA Viruses A. Alphavirus 1 Rubella enters and infects the nasopharynx and lungs. The virus then spreads to the lymph nodes and reticuloendothelial system. 2 The virus is carried in the blood and spreads to other tissues and skin. The alphaviruses, of which there are approximately 26, are arthropod-borne viruses (arboviruses), which are transmitted to humans and domestic animals by mosquitoes. All alphaviruses share a common group antigen. Some arboviruses were initially isolated from horses, hence, the word “equine” in their names (see below). 1. Epidemiology and pathogenesis: Alphaviruses have a broad host range, being able to replicate in organisms that are widely separated phylogenetically, such as mosquitoes and humans. Following inoculation of an Alphavirus by a mosquito, the patient is observed to have a viremia, following which the virus may be seeded in various target organs (for example, the CNS in the encephalitis viruses). Rub Rubella bella virus rus 2. Viral replication: Following attachment to the cell surface, the virus is internalized by receptor-mediated endocytosis. Like the picornaviruses, genome replication is as described for Type I RNA viruses (see p. 239). 3 In a pregnant woman without protective antibodies, the virus can cross the placenta and spread to the fetus. 4 The classic triad of rubella symptoms in affected neonates is: Cataracts 3. Clinical significance: Several different clinical syndromes are associated with Alphavirus infections of humans. These include: 1) acute encephalitis (Eastern and Western equine encephalitis viruses), 2) acute arthropathy (Chikungunya virus), and 3) a febrile illness with a flulike syndrome (Venezuelan equine encephalitis virus). However, the majority of infections are subclinical and can be diagnosed only by the demonstration of an immune response. 4. Laboratory identification: This is generally accomplished by the Deafness demonstration of a rise in antibody titer (that is, comparing acute and convalescent sera). The virus can also be isolated from CSF, blood, or tissue. IgM antibody specific for the pathogen can be detected in the CSF of patients suffering from acute infection. 5. Prevention: The most important measure for prevention of infec- tions caused by Alphavirus is control of the mosquito vector population. A Venezuelan equine encephalitis vaccine is available. Cardiac abnormalities Figure 27.8 Pathology of rubella virus infection.
289 Areas most commonly at risk Yellow Fever Roughly 15,000 cases occur yearly in the tropical regions of South America and Africa. Symptoms include fever; headache; chills; nausea; vomiting, and, occasionally, jaundice. Serious cases can also affect the liver and kidneys. The members of this family are enveloped viruses that contain a singlestranded RNA genome and three structural proteins. The capsid (C) protein and the viral RNA form the icosahedral nucleocapsid, and the other two proteins are envelope associated. Currently, the family Flaviviridae is divided into three genera: Flavivirus, Hepatitis C virus, and Pestivirus. However, the viruses in the genus Pestivirus (classical swine fever virus and bovine viral diarrhea virus) are only of veterinary interest. A. Flavivirus The genus Flavivirus comprises more than sixty viruses. These include many viruses of medical importance, such as yellow fever, St. Louis encephalitis, Japanese encephalitis, dengue fever viruses, and West Nile virus, all of which are mosquito transmitted. Tickborne encephalitis virus is, of course, transmitted by ticks. [Note: Like the viruses in the Alphavirus genus of the family Togaviridae (see p. 287), most of the viruses in this genus are, therefore, arboviruses.] All of the viruses in the genus Flavivirus share a common group antigen. 1. Epidemiology and pathogenesis: As arboviruses, the medically important members of this genus are transmitted to humans by the bite of an infected mosquito or tick. These viruses are maintained in nature by replicating alternately in an arthropod vector and a vertebrate host. Figure 27.9 shows the global distributions of yellow fever and dengue fever. 2. Replication: Following attachment to the cell surface, the virus is taken up by receptor-mediated endocytosis (see Figure 23.9). Replication of the viral RNA is as described for Type I RNA viruses (see p. 239). Only one species of viral mRNA, the genomic RNA, is found in infected cells. It is translated into a single, long polyprotein, which is processed by virus-coded and cellular proteases, giving rise to three structural and seven nonstructural proteins. Nucleocapsids are formed in the cyto- Dengue Fever Approximately 50 million to 100 million cases occur each year and are characterized by sudden onset of fever, headache, and severe myalgia. Severe dengue may lead to shock, hemorrhaging, and death. Figure 27.9 Global distribution of yellow fever and dengue fever. 290 27. Positive-strand RNA Viruses Infection with hepatitis C virus 25% Subclinical infection Acute hepatitis C 75% Resolution of disease (months) Chronic hepatitis C (10 to 15 years) Cirrhosis Hepatocellular carcinoma in patients with cirrhosis Liver failure Figure 27.10 Natural history of infection with hepatitis C virus. plasm, and maturation of the viral particle occurs by envelopment of the nucleocapsid, not at the plasma membrane as with viruses in the family Togaviridae, but, instead, at cytoplasmic Golgi membranes. Virus particles then accumulate in vesicles and are extruded when the vesicles move to the cell surface. 3. Clinical significance: Viruses in the genus Flavivirus are associ- ated with several different clinical syndromes. These include: encephalitis (St. Louis encephalitis, Japanese encephalitis, and tickborne encephalitis viruses); hemorrhagic fever (yellow fever virus); and fever, myalgia, and rash (dengue viruses). Although there is little mortality associated with classic dengue fever, in certain parts of the world, such as Southeast Asia, a severe form of dengue infection occurs, particularly in infants and young children. Called dengue hemorrhagic fever or dengue shock syndrome, it is associated with a significant mortality (10 percent or higher) if untreated. Like dengue fever, West Nile fever is a mosquito-transmitted, acute, usually self-limited illness that presents chiefly with fever, malaise, lymphadenopathy, and rash. Infection may also result in aseptic meningitis or meningoencephalitis, especially in older adults. The first outbreak of West Nile encephalitis in the United States occurred in the New York City area in the summer of 1999. The outbreak was preceded by a significant die-off of wild crows and exotic birds at the Bronx zoo. The West Nile virus, following bird migration, has now spread to all 48 contiguous states in the United States. 4. Laboratory identification: A specific diagnosis is most often made by serologic means (that is, by demonstrating at least a fourfold rise in antibody titer, when comparing acute and convalescent sera). In some cases, virus isolation or demonstration of specific viral antigens is also feasible. Interferon 5. Prevention: A safe, highly effective, live attenuated vaccine for yelRibavirin Interferon + ribavirin 0 20 40 Percentage of patients showing a virologic response (undetectable serum HCV RNA) Figure 27.11 Combination treatment with interferon and ribavirin for chronic hepatitis C. [Note: Patients receiving ribavirin alone showed a biologic and histologic response, but no decrease in circulating hepatitis C virus.] low fever has been available for many years. In China and Japan, a formalin-inactivated Japanese encephalitis virus vaccine is used, whereas, in central Europe, a formalin-inactivated vaccine is widely used to prevent tickborne encephalitis. Another important method of prevention is vector control. In urban areas, elimination of breeding sites can dramatically reduce the population of Aedes aegypti mosquitoes, which serve as the vector for both yellow fever and dengue viruses. B. Hepatitis C viruses Hepatitis C virus (HCV) was discovered in 1988 in the course of searching for the cause of non-A, non-B, transfusion-associated hepatitis. At that time, HCV accounted for 90 percent of the cases of non-A, non-B hepatitis. The hepatitis C viruses are heterogeneous and can be divided into six types on the basis of their nucleotide sequences. 1. Transmission and pathogenesis: Although HCV was initially iden- tified as a major cause of posttransfusion hepatitis, intravenous VI. Coronaviridae drug users and patients on hemodialysis are also at high risk for infection with HCV. Tattooing is also a leading cause of HCV infection. In addition, there is evidence for sexual transmission of HCV as well as for transmission from mother to infant. In the infected individual, viral replication occurs in the hepatocyte and, probably, also in mononuclear cells (lymphocytes and macrophages). Destruction of liver cells may result both from a direct effect of the activities of viral gene products and from the host immune response, including cytotoxic T cells. Although DNA viruses are associated with chronic infection and cancer development, this is not generally the case for RNA viruses. Nonetheless, certain strains of HCV have been associated with hepatocellular carcinoma development, even in the absence of cirrhosis. Particular alleles of the core gene of HCV have been strongly associated with development of hepatocellular carcinoma. Variant core gene alleles have also been associated with interferon-γ (IFN-γ) treatment failures. 291 Total number chronic infections United States Hepatitis A (Not a chronic infection) Hepatitis B Hepatitis C 0 3. Laboratory identification: A specific diagnosis can be made by demonstration of antibodies that react with a combination of recombinant viral proteins. Sensitive tests are also now available for detection of the viral nucleic acid by RT-PCR (reverse transcription of the viral RNA followed by polymerase chain reaction to amplify the DNA copy, see p. 29). 4. Treatment and prevention: Tests to screen blood for HCV have been available for several years, so that HCV as a cause of transfusion-associated hepatitis is now unusual. Treatment of patients with chronic hepatitis by IFN-γ is sometimes beneficial but, in most cases, only for the period during which the patient is receiving the IFN-γ. Treatment with IFN-γ plus ribavirin provides a significantly improved response, and combination therapy is the treatment of choice (Figure 27.11). Chronic hepatitis resulting in severe liver damage may be an indication for a liver transplant. Figure 27.12 summarizes hepatitis A, B, and C. VI. CORONAVIRIDAE Coronaviruses are large, enveloped, pleomorphic particles, with a distinctive arrangement of spikes (peplomers) projecting from their surfaces. [Note: These projections have the appearance of a solar corona, which gives the virus its name.] The Coronavirus genome is the largest described for any RNA virus thus far. Human coronaviruses have been most commonly implicated in upper respiratory infections, causing 10 percent to 30 percent of cases of the common cold. 4 Millions of people Worldwide Hepatitis A (Not a chronic infection) Hepatitis B Hepatitis C 2. Clinical significance: The majority of infections with HCV are sub- clinical, but about 25 percent of infected individuals present with acute hepatitis, including jaundice (Figure 27.10). More importantly, a significant proportion of infections progress to chronic hepatitis and cirrhosis. Finally, some of these individuals go on to develop hepatocellular carcinoma many years after the primary infection. 2 0 200 400 Millions of people New infections per year (U.S.) Hepatitis A Hepatitis B Hepatitis C 0 200 400 Thousands of people Vaccine available Hepatitis A Yes Hepatitis B Yes Hepatitis C No Most common modes of transmission Hepatitis A: Fecal–oral route; contaminated food Hepatitis B: U.S.: Sexual; IV drug users Worldwide: Maternal–fetal Hepatitis C: IV drug users Figure 27.12 Summary of hepatitis A, B, and C. 292 27. Positive-strand RNA Viruses Study Questions Choose the ONE correct answer. 27.1 A company held an elaborate holiday dinner party for its 42 employees. Within 3 to 4 weeks, many of the banquet attendees complained of experiencing fatigue, fever, nausea, and dark urine and were observed to be jaundiced. The group exhibited no bacterial infections in common. The employees who became ill had all eaten raw oysters at the party. The company doctor assayed a sample of the employees’ blood for anti-hepatitis B antibodies, but all samples were negative for anti-hepatitis B surface antigen immunoglobulin M. The causative agent consistent with this history is most likely: A. B. C. D. E. hepatitis A virus. hepatitis B virus. hepatitis C virus. hepatitis D virus. hepatitis E virus. Questions 27.2 to 27.5: Match the appropriate virus from the following list with the statement to which it most closely corresponds. Each virus can match one, more than one, or none of the statements. 27.2 Intravenous drug users are at high risk for the virus. 27.3 Infection is caused by the bite of an infected mosquito. 27.4 Infection predisposes to hepatocellular carcinoma. 27.5 Infection causes congenital malformations. A. Hepatitis A virus B. Coxsackie viruses C. Hepatitis C virus D. Hepatitis E virus E. Yellow fever virus F. Rubella virus 27.6 Which of the following groups RNA viruses are common causes of viral meningitis? A. B. C. D. E. Rhinoviruses Caliciviruses Hepatitis C virus Flaviviruses Enteroviruses Correct answer = A. Hepatitis A is transmitted by the fecal–oral route and is most frequently acquired by eating contaminated shellfish or by contact with a carrier. The symptoms that were exhibited by the partygoers are consistent with liver damage caused by, for example, hepatitis. Hepatitis B infection is excluded because of the negative test for antibodies. Hepatitis C infection is acquired most commonly by intravenous drug users, patients on dialysis, and tattoo recipients. Hepatitis D infection occurs only in combination with hepatitis B infection. Hepatitis E is a major cause of enterically transmitted, waterborne hepatitis in developing countries. Correct answer = C. Until the recent development of tests for the presence of hepatitis C virus (HCV) in blood, HCV was an important cause of transfusion-associated hepatitis. Intravenous drug users are one of several groups still at high risk for infection with this virus. Correct answer = E. Yellow fever virus is an arthropod-borne virus, which is transmitted by the bite of an infected Aedes aegypti mosquito. The virus does not spread from person to person. Correct answer = C. Unlike hepatitis A virus, hepatitis C virus infection has a strong tendency to lead to chronic hepatitis and cirrhosis, often resulting after many years in hepatocellular carcinoma. Correct answer = F. Infection with rubella virus is generally of little consequence to the adult. The exception is the pregnant woman, in whom rubella virus infection can result in congenital malformations in the fetus. The risk is highest in the first trimester. These malformations can affect the central nervous system, the liver, the heart, and the eye. Correct answer = E. All of the enteroviruses can cause CNS disease. The enteroviruses are the major cause of aseptic meningitis. Rhinoviruses cause the common cold whereas the Caliciviruses cause gastrointestinal diseases. Hepatitis virus C causes hepatitis and cirrhosis. Flaviviruses cause encephalitis and hemorrhagic fever. While West Nile virus can cause meningitis, this manifestation is not typical of the entire Flavivirus group. Retroviruses and AIDS 28
II. RETROVIRUS STRUCTURE Despite their wide range of disease manifestations, all retroviruses are similar in structure, genome organization, and mode of replication. Retroviruses are enveloped particles (Figure 28.2). The viral envelope, formed from the host cell membrane, contains a complex HIV protein that protrudes through the surface of the virus particle and appears as spiked knobs on electron micrographs of the virus. The full-length protein, called gp160, is cleaved into two peptides by a viral protease. [Note: The designation "gp" indicates that the protein is glycosylated.] The resulting transmembrane protein is called gp41, or TM, whereas the surface exposed portion of the protein is called gp120, or SU. Host cell proteins, including the major histocompatibility complex class II proteins, are also found in the envelope. The virion has a cone-shaped, icosahedral core containing the major capsid protein called p24, or CA. Between the capsid and the envelope is an outer matrix protein (p17, or MA), which directs entry of the double-stranded DNA provirus into the nucleus and is later essential for the process of virus assembly. There are two identical copies of the positive-sense, single-stranded RNA RNA viruses Single stranded Positive strand Nonenveloped Icosahedral Icosahedral Double stranded Icosahedral Nonenveloped Negative strand Helical Enveloped Enveloped Helical Retroviridae Human immunodeficiency viruses 1 and 2 S Human T-cell lymphotropic viruses 1 and 2 Flaviviridae Togaviridae Figure 28.1 Classification of retroviruses that cause disease in humans. S See p. 364 for a summary of these viruses. 293 294 28. Retroviruses And AIDS NC = Nucleoprotein (p7) CA = Major capsid protein (p24) MA = Outer matrix protein (p17) Singlestranded RNA Host protein genome in the capsid (that is, unlike other viruses, retroviruses are diploid). The RNA is tightly complexed with a basic protein (p7, or NC) in a nucleocapsid structure that differs in morphology among the different retrovirus genera. Also found within the capsid are the enzymes reverse transcriptase and integrase (which are required for viral DNA synthesis and integration into the host cell chromosome) and protease (essential for virus maturation). |
