GUIDE TO

Clinical and Diagnostic Virology

REETI KHARE, PhD, D(ABMM)

Department of Pathology and Laboratory Medicine

Northwell Health Laboratories

Lake Success, New York

Copyright © 2019 American Society for Microbiology. All rights reserved. No part of this publication may be reproduced or transmitted in whole or in part or reused in any form or by any means, electronic or mechanical, including photocopying and recording, or by any information storage and retrieval system, without permission in writing from the publisher.

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Library of Congress Cataloging-in-Publication Data

Names: Khare, Reeti, author.

Title: Guide to clinical and diagnostic virology / Reeti Khare, PhD, D(ABMM), Department of Pathology and Laboratory Medicine, Northwell Health Laboratories, Lake Success, New York.

Description: Washington, DC : ASM Press, [2019] | Includes bibliographical references and index.

Identifiers: LCCN 2018055597 (print) | LCCN 2018057746 (ebook) | ISBN 9781683672920 (ebook) | ISBN 9781555819910 (softcover)

Subjects: LCSH: Medical virology—Handbooks, manuals, etc. | Diagnostic virology—Handbooks, manuals, etc.

Classification: LCC QR201.V55 (ebook) | LCC QR201.V55 K43 2019 (print) | DDC 616.9/101—dc23

LC record available at https://lccn.loc.gov/2018055597

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To my husband, for being my best friend. You are the eye of the storm.

To my children. You are my bubbles of light.

To my parents, for the privilege of your unwavering support.

To my sister, for being my role model.

CONTENTS

Preface

Acknowledgments

Abbreviations

About the Author

SECTION I: Foundations of Clinical Virology

  1   Introduction to Viruses

Virus structure, life cycle, Baltimore classification, transmission, nomenclature

  2   Laboratory Diagnosis of Viral Infections

Differential diagnosis for viral syndromes, specimen collection, general comparison of diagnostic techniques

SECTION II: Viral Pathogens and Clinical Presentation

  3   Respiratory Viruses

Influenza virus, respiratory syncytial virus, parainfluenza virus, human metapneumovirus, rhinovirus, coronaviruses, mumps virus

  4   Viruses with Dermatologic Manifestations

Herpes simplex virus 1 and 2, varicella-zoster virus, measles virus, rubella virus, human herpesviruses 6 and 7, molluscum contagiosum virus, smallpox virus, comparison of herpesviruses 1 to 8

  5   Gastrointestinal and Fecal-Oral Hepatitis Viruses

Rotavirus, norovirus, astrovirus, sapovirus, hepatitis A virus, hepatitis E virus

  6   Viruses That Can Cause Multiple Syndromes

Enteroviruses and parechoviruses, adenoviruses, parvovirus B19, human bocavirus

  7   Opportunistic Viruses Associated with Immunosuppression

Cytomegalovirus, BK virus, JC virus

  8   Blood-Borne Hepatitis Viruses

Hepatitis B, C, and D viruses, and comparison of hepatitis viruses A through E

  9   Human Retroviruses

Human immunodeficiency virus, human T-cell lymphotropic virus

10   Oncogenic Viruses

Human papillomavirus, Epstein-Barr virus, human herpesvirus 8

11   Zoonotic Viruses

Rabies virus, Ebola and Marburg viruses, Lassa virus, Crimean-Congo hemorrhagic fever virus, hantaviruses, lymphocytic choriomeningitis virus, monkeypox virus, herpes B virus, Hendra and Nipah viruses, comparison of zoonotic viruses

12   Arboviruses

Mosquitoes, ticks, dengue virus, yellow fever virus, chikungunya virus, West Nile virus, Zika virus, eastern, western, and Venezuelan equine encephalitis viruses, Japanese encephalitis virus, Powassan virus, tick-borne encephalitis virus, Rift Valley fever virus, comparison of arboviruses

SECTION III: Diagnostic Assays and Techniques

13   Culture and Tissue-Based Diagnostic Techniques

Conventional viral culture and cell lines, cytopathic effect, viral growth rates, shell vial assays, hemadsorption, quantification by plaque-forming units and TCID50, histopathology and cytopathology of viruses, in situ hybridization

14   Diagnostic Techniques Based on Immunological Interactions

Enzyme immunoassays including ELISAs, chemiluminescent immunoassays, and immunoblot assays, immunofluorescence assays, immunochromatographic (lateral flow) assays, hemagglutination inhibition and plaque reduction neutralization assays, serologic assays, kinetics and interpretation of antibody responses, comparison of immunoassays

15   Molecular Techniques: Nucleic Acid Amplification

Importance of nucleic acid structure, sample processing, PCR, reverse transcription-PCR, real-time PCR, quantitative vs. qualitative PCR, melt curve analysis, droplet digital PCR, nested PCR, multiplex PCR, transcription-mediated amplification, PCR controls, minimizing contamination

16   Molecular Techniques: Sequencing

Applications of sequencing, first generation (Sanger, dideoxy chain termination) and next generation (Illumina, Ion Torrent, PacBio, and Oxford Nanopore) sequencing, sample preprocessing, library generation, amplification techniques, quality and depth of coverage, data analysis, comparison of all platforms

SECTION IV: Prevention and Management of Viral Infections

17   Biosafety

Biosafety levels, select agents, reportable diseases, personal protective equipment, biosafety cabinets, isolation precautions

18   Vaccines

Active and passive immunity, types of vaccines, route of administration, risk and other consequences of vaccination, antibody-dependent enhancement, table of available viral and other vaccines, diagram of routine vaccination schedule

19   Antivirals

Antivirals against herpesviruses, human papillomaviruses, influenza virus, respiratory syncytial virus, hepatitis B virus, hepatitis C virus, antiretrovirals and antivirals with broad coverage, immunomodulators, comparison of antivirals, mechanisms of action

SECTION V: The Regulatory Environment for Laboratory Testing

20   Regulatory Requirements

Classification of diagnostic assays, test complexity, role of CMS, CDC, CLSI, CLIA, inspections, proficiency testing, billing and coding

21   Assay Performance and Interpretation

Validation/verification, performance characteristics (precision, accuracy, reportable range, reference range, analytic sensitivity, analytic specificity), diagnostic and clinical sensitivity and specificity, prevalence, predictive value, ROC curves

References

Answers

Index

PREFACE

The field of clinical and diagnostic virology is undergoing a period of remarkable change. Viruses and viral infections are becoming more relevant in the practice of modern medicine, but it is increasingly difficult to keep up with them. Newly discovered viruses with novel clinical presentations, a dizzying array of new diagnostic techniques with unique terminology, and even new antivirals are driving a gap in knowledge. Unlike bacteriology, which has matured over many decades, the explosion in molecular testing has rapidly placed viral etiologies of disease into a prominent light. Now clinicians and microbiologists need to develop a similar level of comfort with virology and its jargon, diagnostic techniques, clinical syndromes, and therapies.

The purpose of this guide is to provide a simple reference for medical and scientific professionals so that we may tread more comfortably in this new era. Its goals are to summarize essential concepts and to highlight important terms in clinical and diagnostic virology. It is designed for a wide range of professionals, including students in medical and laboratory fields (such as infectious disease fellows, pathology residents, medical students, microbiology fellows, medical technologists, technicians, and graduate students), scientists and virologists who need to understand the context for their fields of study, and practicing clinicians who need to understand changes that are occurring in the field.

Comprehensive material in clinical and diagnostic virology can be found in textbooks, primary literature, and on public health sites. This book is a “CliffsNotes” version of all this information. Here critical concepts are carefully selected and concisely summarized in bullet points to make things simple and easy to digest. Key terms are featured in bold, and notable “pearls” are emphasized in the margins. There are also questions at the end of each chapter to help reinforce important ideas.

I hope that this guidebook will be a useful reference and that it helps make clinical and diagnostic virology even more interesting and accessible. I welcome any suggestions, additions, or corrections that would improve the quality of this book.

Reeti Khare

ACKNOWLEDGMENTS

I thank the reviewers who contributed their time and expertise to this book:

• Neil Anderson, MD, D(ABMM), Assistant Medical Director of Clinical Microbiology, Washington University School of Medicine in St. Louis
• Esther Babady, PhD, D(ABMM), Director of Clinical Operations, Microbiology, Memorial Sloan Kettering Cancer Center
• Gregory J. Berry, PhD, D(ABMM), Director of Molecular Diagnostics, Northwell Health
• Matthew J. Binnicker, PhD, D(ABMM), Director of Clinical Virology, Mayo Clinic
• Scott Duong, MD, Assistant Director of Infectious Disease Diagnostics, Northwell Health
• Christine C. Ginocchio, PhD, MT(ASCP), Vice President of Microbiology, BioMérieux, Vice President of Scientific/Global Affairs, BioFire
• Aya Haghamad, PharmD, Clinical Solutions Specialist, Northwell Health
• Stefan Juretschko, PhD, D(ABMM), Senior Director of Infectious Disease Diagnostics, Northwell Health
• Michael J. Loeffelholz, PhD, D(ABMM), Senior Director of Medical Affairs, Cepheid
• Benjamin Pinsky, MD, PhD, Medical Director of Clinical Virology, Stanford Health Care and Stanford Children’s Health
• Elitza Theel, PhD, D(ABMM), Director of Infectious Disease Serology, Mayo Clinic
• Xiomin Zheng, MD, PhD, Pathology Resident, Staten Island University Hospital, Northwell Health

I also thank Drs. Bobbi Pritt, James Crawford, and Megan Angelini for their support and encouragement throughout this project.

ABBREVIATIONS

Throughout the book, certain abbreviations will be used. The below list highlights some of the most important and frequently used.

ABOUT THE AUTHOR

Reeti Khare, PhD, D(ABMM), is the Director of Microbiology at Northwell Health Laboratories in New York. She received her PhD in Virology and Gene Therapy at Mayo Clinic and did a postdoctoral fellowship at the University of Washington. Her research involved reengineering viral vectors, developing adenoviruses for liver gene therapy, and creating viral vector vaccines against MRSA. She returned to Mayo Clinic for her clinical microbiology fellowship and is a diplomate of the American Board of Medical Microbiology. Reeti enjoys teaching and learning about microbiology and has authored numerous publications, chapters, and reviews. At Northwell Health Labs she continues to pursue clinical research and provide student education, and is responsible for laboratory oversight, improving efficiency, designing workflows, and diagnostic microbiology testing.

SECTION I

FOUNDATIONS OF CLINICAL VIROLOGY

CHAPTER 1

INTRODUCTION TO VIRUSES

I. OVERVIEW. Viruses are obligate intracellular parasites. Unlike all other organisms, they are not “alive” because they are metabolically inactive on their own. They are also not “dead” because they can metabolize and reproduce when associated with a host cell. Instead, they are referred to as being “active” or “inactive.” Viruses are difficult to study because of their minuscule size, but they are even more abundant than bacteria. Most are part of normal environmental or human flora but some viruses are medically relevant and can cause infections that fall anywhere on the spectrum, from asymptomatic to fulminant. Several factors affect the pathogenicity of a virus.

1. Virus-specific factors

• Virulence: Some viruses are more virulent than others. For example, rabies virus is highly pathogenic, while torque teno virus does not cause disease despite being ubiquitous in normal flora.
• Persistence: Some viruses, like herpesviruses, cause mild disease but infect humans for life.
• Indirect effects: Viruses like bacteriophages only infect bacteria but are still indirectly pathogenic to humans. For example, Corynebacterium diphtheriae does not generally cause clinically significant disease unless it is infected with the bacteriophage containing the diphtheria toxin gene.

2. Host-specific factors: Hereditary genetic mutations can allow viruses that are weakly pathogenic to cause significant disease. For example, a specific mutation in CCR5 (a host cell receptor for HIV) has been shown to prevent infection with this virus. On the other hand, other mutations can result in overgrowth of viruses. For example, human papillomavirus 2 (HPV2) typically causes benign warts, but individuals with genetic defects in cell-mediated immunity can demonstrate uncontrolled giant warty overgrowths (“tree man” disease).

3. Immunosuppression: Immunosuppressive drugs, virus-induced immunosuppression, and even pregnancy are all instances in which the immune system is depressed. This can leave patients vulnerable to unique viral infections.

II. VIRUS STRUCTURE. The structure of a virus defines its life cycle, mechanism of pathogenicity, and how it is detected by laboratory assays. A virus particle, or virion, is composed of nucleic acid surrounded by a protective protein coat called a capsid. Together, the nucleic acid and capsid are called the nucleocapsid.

1. Capsids: Occur in three main shapes (Fig. 1.1).

Figure 1.1. Viral capsids come in three main shapes.

• Polyhedral capsids have multiple flat sides that form a rigid shell around the viral nucleic acid. Viruses with lots of flat sides can appear round. Viruses with these kinds of capsids are highly regular and have a rigid shape and size. Most DNA viruses are polyhedral.
• Helical capsids wrap proteins around the strand of nucleic acid to form a spiral, elongated nucleocapsid. These viruses tend to be more variable in size and shape. Most RNA viruses are helical.
• Complex capsids have other shapes or are a combination of helical and polyhedral capsids.

2. Envelopes: Some viruses have an envelope, which is a lipid bilayer that surrounds the nucleocapsid.

Most blood-borne viruses are enveloped because they need to evade the immune system efficiently.

• Envelopes are acquired from cell membranes and act as a shield from the immune system. The disadvantage is that these viruses are susceptible to detergents, drying, and pH changes and typically do not survive for long on external surfaces.
• Nonenveloped, or naked, viruses are more resistant to harsh conditions and tend to be more stable in the environment. They are relatively resistant to disinfectants (e.g., alcohol, dilute bleach, quaternary ammonium compounds, and even water disinfectants like chlorine). This means that they are difficult to eliminate from community and hospital environments.

Most gastrointestinal viruses are naked because they must be highly resistant to the acidic environment of the stomach.

3. Size: Viruses cannot be seen using light microscopes. Medically important viruses range from ∼20 to 500 nm in length (Fig. 1.2).

Figure 1.2. Comparison of sizes. Viruses are ~10 times smaller than a bacterium and ~100 times smaller than a eukaryotic cell.

Rule of thumb: Viruses are about 1/10 the size of a bacterial cell.

III. LIFE CYCLE. The life cycle of the virus is how it binds to a host cell, replicates its nucleic acid, and then spreads to new cells. Knowing each virus’s life cycle is critical to understanding what part of the body will be affected, how long the infection will last, how it can be detected, and which antivirals will work.

Replication: making new genomes.

Transcription: making messenger RNA (mRNA) from the genome.

Translation: making proteins from mRNA.

1. Incubation period: Viruses infect target host cells and replicate. Due to the low level of virus at the beginning of infection, patients are typically asymptomatic.

2. Spread: Viruses spread through the host by infecting adjacent cells, traveling in migratory cells, disseminating through the bloodstream (viremia), and diffusing through body fluids. Viremia can be identified through detection of viral nucleic acids and/or antigens in the blood.

• Viruses infect cells by binding to specific cellular receptors; cells that do not display the correct receptors will not be infected. Tropism is the affinity of a virus for some cell types and not others.
• Once bound, virions enter cells by endocytosis or fusion. During endocytosis, the host cell membrane invaginates and engulfs the virus. During fusion, the viral envelope will fuse with the cell membrane in order to release the viral nucleocapsid into the cell.

3. Prodromal phase: Viruses may produce early, nonspecific symptoms (e.g., fever, aches, pain, and nausea) as they replicate.

• Once inside the cell, viruses replicate, transcribe mRNA, and translate it into viral proteins. These proteins and nucleic acids are assembled into infectious viral particles in the nucleus or cytoplasm.
• These viral aggregates can sometimes be seen as viral inclusions.

4. Active disease: Viruses cause an immediate or long-lived infection in the cell.

• Lytic viruses lyse (destroy) the host cell to get out immediately after replication. This manifests as an acute infection where the patient may show characteristic signs of the viral infection.
• Lysogenic viruses cause a long-lived, latent infection by integrating into the host genome. These infections are often subclinical. Environmental triggers can cause the integrated viruses to excise and enter the lytic cycle. The integrated viral genome is called a provirus. Proviral DNA replicates passively with the cell every time the host cell replicates. This makes them very long-lived and largely undetected by the immune system.

Proviruses = integrated viral genomes

• Pseudolysogenic or episomal viruses persist for a long time in the host cell without integrating. Their nucleic acids remain separate as an episome. These episomes are diluted as cells divide and removed from circulation when cells die (Fig. 1.3).

Episomes = nonintegrated, persistent viral genomes

Figure 1.3. Integration versus episomal persistence. Episomes are diluted when the cell replicates while integrated viruses are multiplied when the cell replicates.

Latent viruses are dormant. They do not actively produce virions or trigger the immune system, and therefore result in a persistent viral reservoir.

• Depending on the type of virus, new viral particles will exit the cell by four main methods (Fig. 1.4).

Figure 1.4. Methods of viral release from a cell. (A) Lysis; (B) budding; (C) exocytosis; (D) cell-to-cell transport within syncytia.

• Lysis: bursting of the host cell
• Budding: Viruses push into the perimeter of the cell and capture part of the cellular membrane. Many enveloped viruses surround their nucleocapsid with the host plasma membrane in order to evade detection by the immune system.
• Exocytosis: Virions are enclosed in a cellular vesicle, which then fuses with the plasma membrane in order to release virus particles outside the cell.
• Cell-to-cell transport: Some viruses can cause host cells to fuse together (syncytia). This allows the new viruses to directly enter neighboring cells without exposing them to the immune system.

5. Resolution of disease: Both the innate and adaptive immune responses clear or suppress the viral infection.

• Innate responses are able to suppress viral infections rapidly.

• Antiviral cytokines, like interferon, are produced. They activate lymphocytes and upregulate proteins that inhibit viral replication.
• Natural killer (NK) cells are specialized lymphocytes that kill virus-infected or cancerous cells.
• CD8+ killer T cells also destroy infected cells, and CD4+ helper T cells activate the memory response.

• Adaptive responses take several weeks to develop but produce immunoglobulins (antibodies) against pathogens that are highly effective, specific, and long-lived. There are 5 different classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM.

• IgM is produced first, within ∼1 to 2 weeks of exposure.
• IgG is produced within 2 to 4 weeks of exposure and provides immunity for life.
• IgA is localized to mucosal membranes and provides immunity against respiratory and gastrointestinal pathogens.
• IgE provides immunity against parasites.
• IgD is present on B cells.

IV. VIRAL GENOMES. Viruses carry only a small number of essential genes that are necessary for infection and replication. Unlike prokaryotes and eukaryotes, the genome configurations of viruses vary widely—they have variable shapes, structures, copy numbers, and genome sequences. Most importantly, they can be composed of either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

1. The Baltimore system of classification: This classification scheme categorizes viruses into classes I to VII according to the type of nucleic acids they contain and how they are transcribed into mRNA (Fig. 1.5 and Table 1.1). How viruses generate mRNA is important because the faster they make mRNA, the faster they can make proteins and the faster they can assemble these proteins into new virions.

Figure 1.5. The Baltimore system classifies a virus based on the virus’s nucleic acid and its method of mRNA synthesis. Blue indicates DNA, red indicates RNA.

Table 1.1. Characteristics of viruses in each Baltimore class

2. DNA viruses (Fig. 1.6): Replication of DNA has high fidelity, which means that replication errors are rare. This is an advantage because viruses that contain DNA are highly stable and can persist in the host cell for a long time. They can also redirect the cell’s own polymerases and enzymes towards replication of more viral particles.

Figure 1.6. Organization of clinically relevant DNA viruses. Virus images: blue indicates DNA, black indicates capsid, and double brown line indicates an envelope.

“Be HAPPPPy!” for DNA viruses: hepatitis B virus, herpesvirus group, adenovirus, poxvirus, parvovirus, papillomavirus, polyomavirus

3. RNA viruses (Fig. 1.7): RNA replication machinery is significantly more error prone and mutations are incorporated at a much higher rate. Because of this, viruses with RNA genomes mutate quickly, which is an advantage because it allows them to adapt to new environments. Over many rounds of replication there can be so many new mutations that there are almost distinct viral populations within a single patient. These quasispecies can evade memory immune responses and can be difficult to treat because they are so diverse.

Figure 1.7. Organization of clinically relevant RNA viruses. Virus images: red indicates RNA, black indicates capsid, and double brown line indicates an envelope.

A rapid mutation rate is so useful that the majority of medically relevant viruses contain RNA.

4. Strands and sense: Viral nucleic acids are usually single stranded or double stranded (yielding, for example, ssRNA or dsDNA), but some are partially single and double stranded. Single-stranded genomes are either negative or positive sense.

• Negative-sense (−) sequences are back to front (i.e., they are in 3′-to-5′ orientation, or the “non-sense” direction). This makes them the blueprints for mRNA production.
• Positive-sense (+) sequences are in the correct 5′-to-3′ orientation (i.e., they have the same orientation as mRNA).

Negative-sense nucleic acid is the template for mRNA because they are complementary to mRNA.

Positive-sense nucleic acid is the same orientation as mRNA.

Proteins can be translated directly from (+) ssRNA (class IV) viral genomes. So class IV genomes are directly infectious, even when they are not packaged in a capsid.

5. Structure: Viral nucleic acids also have structure. They can be linear or circularized, can form hairpin loops, and can be segmented. Segmented genomes allow viruses to shuffle segments from multiple strains together (i.e., reassort) and make entirely new viral strains (see chapter 3).

V. VIRAL TRANSMISSION. Understanding the way different viruses spread helps prevent new exposures and contain outbreaks. Viruses can be spread through various routes.

1. Direct contact: infected tissue, contact with mucous membranes during sexual intercourse

2. Contact with body fluids: infected blood, saliva, respiratory secretions, seminal fluid, fecal materials

3. Contact with objects (fomites): contaminated surfaces, personal items (e.g., toothbrushes), and other infected materials

4. Vertically from mother to child directly through the placenta, during passage through the birth canal, and via breast milk

5. Exposure to aerosolized droplets: Most droplets containing virus (e.g., cough) are projected within a radius of 3 feet, but they can be transmitted 10 feet or more.

6. Exposure to air: Minuscule droplets containing virus remain suspended in the air and can spread over much larger distances. However, only a few organisms can be transmitted by this route.

7. Iatrogenic intervention: organ transplant, blood transfusion, and immunosuppression

8. Zoonoses: exposure to animal secretions or bites. Many of these viruses require an animal vector and cannot be transmitted directly from person to person.

Many viruses are shed in body fluids and secretions even before symptoms have begun (i.e., in the incubation period). This has dramatic implications for transmission, since quarantines of patients after symptoms have begun may not always be effective.

VI. VIRAL TAXONOMY. Viral taxonomy and nomenclature are more complicated than classification rules for other organisms. The International Committee on Taxonomy of Viruses classifies viruses into only 5 hierarchical ranks, but not all need to be used. Names are not assigned based on standardized criteria and may be named after places, discoverers, region of the body they were first isolated, or symptoms. Most confusingly, viruses are assigned a formal species name but most people use the common name, which may be the same or different (Table 1.2).

Table 1.2. Nomenclature for viral taxonomic categories

1. Formal species names: Formal names are usually used when referring specifically to a virus’s taxonomy. This name is italicized and the first letter is capitalized (as well as other proper nouns), just like species names for bacteria (1). For example, the species Human respiratory syncytial virus belongs to the order Paramyxoviridae. All higher viral taxon names are also italicized (e.g., Flaviviridae).

2. Common names: These are used more often than formal species names. They are not italicized and are capitalized only if they are proper nouns. For example, Epstein-Barr virus is capitalized because it is named after people, while herpes simplex virus is not. The commonly used name is not uniform and may or may not be the same as the species name. For example, the common name for Mumps virus is mumps virus while the common name for Hepatovirus A is hepatitis A virus.

3. Abbreviations: Abbreviations for viruses are also not uniform. Common names are often abbreviated by capitalizing the first letter of each word, including the word “virus” (e.g., HSV for herpes simplex virus). Sometimes part of the name is used for more clarity (e.g., CHIKV for chikungunya virus), with or without capitalization (e.g., Ad or AdV for adenovirus).

4. Subtypes: Viral species can be further divided into subtypes. Serologic techniques were originally used to differentiate the strains into categories called serotypes. Newer techniques based on sequencing identify them as genotypes. Genotypes and serotypes tend to coincide, but not always.

Most of the time people use common virus names. These are not italicized, and only the proper nouns should be capitalized.

5. Other descriptors: Some viruses, like influenza virus, have additional naming conventions that convey extra information (see chapter 3).

Multiple-Choice Questions

  1. Some viruses have a lipid layer around them. What is it called?

a. A capsule

b. A capsid

c. An envelope

d. A tegument

  2. What do all viruses contain?

a. Envelope and capsid

b. Viral polymerase and nucleic acid

c. Capsule and envelope

d. Capsid and nucleic acid

  3. Which of the following is true about Baltimore class I viruses?

a. They contain DNA and are able to replicate outside of a cell.

b. They contain DNA and are able to use the host cell’s replication machinery.

c. They contain RNA and are able to reassort.

d. They contain RNA and are able to mutate rapidly.

  4. Which of the following is an advantage of Baltimore class IV viruses?

a. Their nucleic acid is directly infectious.

b. Their nucleic acid is able to integrate.

c. They do not need to carry any viral proteins.

d. They are nonmutagenic.

  5. Which viruses are most stable in the environment?

a. Enveloped viruses, due to the extra layer of protection

b. Naked viruses

c. RNA viruses

d. Integrating viruses

  6. Many medically relevant viruses belong to Baltimore class V. This is because they

a. Can all reassort to create novel, highly virulent strains

b. Are highly stable and are able to integrate

c. Can be replicated rapidly and are highly mutable

d. Are transmitted by direct contact

  7. Which of the following terminology is correct?

a. There are many human papillomaviruses.

b. There are many Human Papilloma Viruses.

c. There are many Human Papillomaviruses.

d. There are many Human papilloma Viruses.

  8. Which of the following mechanisms does NOT hide viruses from the immune system?

a. Syncytia

b. Envelopes

c. Provirus

d. Lysis of the host cell

  9. How do Baltimore class VI viruses differ from class IV viruses?

a. They are intrinsically more pathogenic.

b. They package both RNA and DNA within the capsid.

c. They encode reverse transcriptase.

d. They are transmitted by direct contact.

10. Which nucleic acid do most episomally persistent viruses contain?

a. dsDNA

b. dsRNA

c. (+) ssRNA

d. (−) ssRNA

11. Which of the following viruses are in the same family?

a. Dengue and chikungunya viruses

b. Parainfluenza and influenza viruses

c. Lassa and Marburg viruses

d. West Nile and hepatitis C viruses

12. Which of the following viruses are NOT in the same family?

a. Respiratory syncytial and mumps viruses

b. Hepatitis A and rhinovirus viruses

c. Hepatitis B and hepatitis C viruses

d. Herpes simplex and varicella-zoster viruses

Match the following. Use each answer only once.

13. Virus shape

True or False

CHAPTER 2

LABORATORY DIAGNOSIS OF VIRAL INFECTIONS

I. OVERVIEW. Viruses can cause a wide range of manifestations, from asymptomatic infection to latent, acute, localized, and systemic infections or even cancer. Different viruses are associated with different diseases (Fig. 2.1). To diagnose the etiology of an infection, it is essential that the correct specimen is collected and the right tests are ordered. The appropriate specimen type depends on the location of the infection, the type of patient, the collection device, the stage of infection, and the pretest probability of disease. It is also essential that the correct assay is chosen because it will affect how likely it is that the pathogen will be detected accurately, the turnaround time of results, and the type of specimen that needs to be collected.

Figure 2.1. Viruses associated with various clinical syndromes. See abbreviations list at the front of this book.

II. SPECIMEN TYPE. Viruses are intracellular microbes, so the best specimens for virus identification depend on where the infection occurs. This is important because some viruses may be present and detectable in a sample but are not the cause of active symptoms.

1. Tissue: Viruses live in cells, so tissues and biopsy specimens are often preferred samples for viral detection using PCR, culture, shell vial, and histology. Formalin-fixed paraffin-embedded tissue is useful for histology but cannot be cultured because all the organisms are inactivated. Fixing also cross-links DNA, which inhibits PCR, although it can be done in some cases.

2. Cerebrospinal fluid (CSF): Used to diagnose central nervous system (CNS) infections, like meningitis. Viral meningitis is sometimes called “aseptic meningitis” to differentiate it from bacterial meningitis. However, this term is no longer used because there may be other non-bacterial causes of meningitis.

• Overall, viral culture has poor sensitivity from CSF.
• PCR or serology is often the preferred diagnostic approach.
• Other lab findings can also indicate infection (Table 2.1).

Table 2.1. Lab findings in CSF associated with different types of pathogens

3. Blood: It is important to consider the life cycle of viruses before trying to identify them in blood because not all viruses are blood borne.

• Blood is not always a good source for diagnosis because viruses are intracellular. Many viruses have only a short viremic period (e.g., arboviruses are viremic only for a few days), while others are spread from cell to cell instead of hematogenously (e.g., herpesvirus).
• Blood is a good source for diagnosing and monitoring certain blood-borne viral infections, for example, HIV, hepatitis B and C viruses (HBV and HCV), and systemic (instead of localized) cytomegalovirus (CMV) or Epstein-Barr virus (EBV) infection.
• Serum is typically the preferred specimen for serology, although plasma or whole blood can be used in some situations. Serology is not the preferred diagnostic test for many viruses that are common among humans. However, it is very useful for viruses that are rare (e.g., Zika virus) or if a positive result always necessitates some follow-up (e.g., HCV).

4. Respiratory specimens: Are used to detect and diagnose respiratory pathogens. Specimens include nasal, nasopharyngeal, buccal and throat swabs, as well as bronchoalveolar lavage fluid and sputum specimens.

5. Urine and stool: Gastrointestinal (e.g., norovirus) and genitourinary viruses (e.g., BK virus) are often excreted in stool and urine specimens at very high titers. However, identification of viruses from these specimens can be difficult to interpret for several reasons.

• Viruses that cause disease in other areas of the body may be excreted in the urine and/or stool (e.g., enteroviruses causing respiratory disease may be detected in the stool).
• Urine and stool contain bacterial flora that overgrow in culture, components that are toxic to cell monolayers, and PCR inhibitors.
• Several gastrointestinal or genitourinary pathogens cannot be identified by some methods (e.g., norovirus is difficult to grow in cell culture). On the other hand, these pathogens may be excreted into urine and stool even in healthy individuals (e.g., BK virus).
• Some viruses may be shed for days to months after resolution of symptoms, so detection from these specimens may not correlate with disease.

6. Other common specimens: Includes body fluids (vaginal secretions, semen, saliva, and ocular, joint, and amniotic fluid) and cells from lesions.

III. COLLECTION DEVICES AND TRANSPORT.

1. Swabs: These are convenient collection devices, but they gather very little specimen.

• Traditional swabs have long fibers wrapped around the end of a shaft.
• Flocked swabs are made of nylon. They are brush-like and increase the surface area of collection. These are preferred for collection.
• Wooden-shafted swabs and swabs with calcium alginate or cotton tips should not be used because they can inhibit viral recovery.

2. Universal viral transport medium: This is a pH-buffered medium containing antibiotics and antimycotics to inhibit bacterial and fungal overgrowth.

• Specimens such as swabs, tissue, and scrapings are commonly collected into transport media to preserve the integrity of the sample.
• Liquid specimens, such as blood and CSF, should not be added to transport media to prevent dilution of the specimen.

3. Storage conditions:

• For transport and short-term storage (<30 days), specimens are typically placed at refrigerated temperature (2 to 8°C).
• For long-term storage, specimens may be frozen at −70°C, depending on the assay’s storage requirements. Nucleic acid extracts can be frozen at −70oC or refrigerated (∼4°C).

IV. COMPARISON OF ASSAYS USED FOR DIAGNOSIS OF VIRAL INFECTIONS. Currently, the most common methods of viral detection are antigen or antibody detection, serology, culture, and nucleic acid amplification. These tests have different advantages and limitations, such as differences in sensitivity/specificity, ease of use, and cost. Table 2.2 shows a general comparison of these tests, but they are covered in greater detail in section III of this book

Table 2.2. Comparison between commonly used viral identification techniques

Multiple-Choice Questions

  1. Which of the following is true of diagnostic testing for viruses?

a. It is always necessary.

b. Testing can differentiate between viruses that cause similar diseases.

c. Testing does not depend on preanalytic factors.

d. All of the above.

  2. A patient with respiratory symptoms is seen in the emergency room. An antigen test for influenza virus is negative but the culture is positive. Which of the following is true?

a. The patient is infected with influenza virus.

b. Serology should be performed for confirmation.

c. PCR should be performed for confirmation.

d. The patient should probably be tested for more viral and bacterial pathogens because of the negative antigen test.

  3. Which of the following is most accurate regarding a patient that is shedding virus in their respiratory secretions?

a. The patient will be symptomatic for as long as they are shedding virus.

b. The patient is likely contagious.

c. The patient will not produce antibodies to the virus until the shedding stops.

d. Any patient shedding virus definitely has active disease.

  4. Which of the following is a limitation of PCR testing?

a. It cannot be performed on serum.

b. It is inexpensive.

c. It is often falsely negative.

d. A positive result does not always mean a patient has active disease.

  5. Which of the following is an advantage of viral culture?

a. It can detect multiple viruses from a single specimen.

b. It is highly sensitive for most specimens.

c. It is easy to perform.

d. All of the above

  6. What is the danger of identifying respiratory viruses in stool?

a. Detection in stool may not correlate with the virus causing respiratory disease.

b. False positivity due to the presence of hemin

c. Respiratory viruses cannot be present in stool.

d. All of the above

True or False

SECTION II

VIRAL PATHOGENS AND CLINICAL PRESENTATION

CHAPTER 3

RESPIRATORY VIRUSES

I. OVERVIEW. Many viruses can cause respiratory symptoms, from mild cough and cold to severe lower respiratory tract infections.

1. Background: Most respiratory infections are caused by RNA viruses. Most of them are also enveloped (Box 3.1).

Most respiratory viruses contain RNA genomes.

Box 3.1. Common and/or important respiratory viruses

2. Transmission: Respiratory viruses are highly contagious and are transmitted primarily by respiratory secretions. Some individuals shed viruses asymptomatically for a prolonged period and others (e.g., children) shed large amounts of virus. Common modes of transmission include the following.

• Inhalation of respiratory droplets (e.g., cough or sneeze)
• Direct contact with respiratory secretions
• Contact with contaminated objects (fomites). Viruses can survive for days to weeks on environmental surfaces, especially hard, nonporous surfaces like metal and plastic (2).
• Many respiratory viruses have a distinct pattern of seasonality (Fig. 3.1). Seasonality is affected by various factors, such as the amount of close contact, temperature, humidity, and precipitation. For example, seasonal influenza outbreaks in temperate climates occur in the winter months, likely because people spend more time indoors (e.g., transmission via close contact). However, in tropical climates, influenza can occur all year-round, or during the rainy season (3).

Figure 3.1. Seasonality of respiratory viruses in temperate regions.

3. Clinical presentation: Respiratory viruses can cause overlapping clinical symptoms. Disease ranges from mild to severe upper and lower respiratory tract symptoms such as nasal discharge, cough, cold, fever, croup, bronchiolitis, pneumonia, and acute respiratory distress syndrome (4)

• Viruses have different incubation periods before time to onset of symptoms (Fig. 3.2).

Figure 3.2. Respiratory viruses have different incubation periods (time before symptom onset). Colored squares represent the range; dark orange shows the most frequent incubation period.

• The duration of symptoms for most respiratory viruses is typically 7 to 14 days.
• Immunosuppressed patients (e.g., transplant recipients) or patients with underlying pulmonary disorders (e.g., individuals with chronic obstructive pulmonary disease or asthma) have a high risk of prolonged infection, serious respiratory disease, and persistent shedding.

Croup (laryngotracheobronchitis) exhibits a characteristic inspiratory stridor, or “seal bark cough.”

4. Diagnostic testing: In cases where there are underlying risk factors or very severe infection, broad “syndromic” testing can be done to identify multiple pathogens that can cause overlapping symptoms, or narrow testing can be done for agents that are treatable (such as bacterial infection or infection with influenza virus or respiratory syncytial virus [RSV]).

• No testing: Most uncomplicated respiratory infections are self-limited and testing is not necessary.
• Nucleic acid amplification tests (NAATs): a commonly used method because it provides rapid results. Some newer PCR assays can be used to detect multiple respiratory pathogens simultaneously.
• Culture: can detect several respiratory pathogens simultaneously, but it is labor-intensive and has a long turnaround time. This makes diagnosis of acute infections difficult. Shell vials are also labor-intensive but have a shorter turnaround time.
• Antigen tests: Rapid antigen detection tests and direct fluorescence antigen (DFA) testing have relatively low sensitivity but are sometimes used to rapidly screen for some respiratory pathogens.