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Manual of Clinical Microbiology, Eleventh Edition
Editors: James H. Jorgensen1, Michael A. Pfaller2, Karen C. Carroll3, Guido Funke4, Marie Louise Landry5, Sandra S. Richter6, David W. Warnock7
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Affiliations:
1: Emeritus, Department of Pathology, University of Texas Health Science Center, San Antonio, Texas;
2: T2 Biosystems, Lexington, Massachusetts, and Professor Emeritus, University of Iowa College of Medicine, Iowa City, Iowa;
3: Department of Pathology, Division of Microbiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland;
4: Risch Laboratories Group, Schaan, Principality of Liechtenstein;
5: Departments of Laboratory Medicine and Internal Medicine, Yale University School of Medicine, New Haven, Connecticut;
6: Department of Laboratory Medicine, Cleveland Clinic, Cleveland, Ohio;
7: Faculty of Medical and Human Sciences, University of Manchester, Manchester, United Kingdom
Content Type: Reference
Format:
Hardcover, Electronic
Publication Year:
2015
Category: Clinical Microbiology
Print ISBN
:
9781555817374
e-ISBN
:
9781555817381
MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.
Revised by a collaborative, international, interdisciplinary team of editors and authors, this edition includes the latest applications of genomics and proteomics and is filled with current findings regarding infectious agents, leading-edge diagnostic methods, laboratory practices, and safety guidelines. This seminal reference of microbiology continues to set the standard for state-of-the-science laboratory practice as the most authoritative reference in the field of microbiology.
“What do you do when your MALDI-TOF reports Sneathia sanguinegens and the doctors is asking what it is, or when you are asked whether a Borrelia recurrentis infection can be treated with ceftriaxone, or whether Coxsackieviruses cause hepatitis? You turn to “THE source” for clinical microbiology information - The Manual of Clinical Microbiology. Whether on your tablet or on the bench; it has what you need.”
—Fred C. Tenover, Vice President, Scientific Affairs, Cepheid
“The Manual of Clinical Microbiology is the key resource for understanding what, why, and how in clinical microbiology. It is truly a must-have document for guiding current practice.”
—Carol A. Rauch, Associate Professor of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center
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Two-Volume Hardcover Set, 2,892 pages, full-color illustrations, index.
Book Summary
VOLUME 1
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DIAGNOSTIC STRATEGIES AND GENERAL TOPICS
Chapter 1 : Introduction to the 11th Edition of the Manual of Clinical Microbiology
- Authors: James H. Jorgensen, Michael A. PFaller
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This chapter by the co-editors in chief summarizes the major changes in the field of clinical microbiology since the last edition and lists highlights of the 11th edition of the Manual. It recognizes the important contributions of the volume editors, section editors, and authors who have contributed to this edition.
Chapter 2 : Microscopy
- Author: Danny L. Wiedbrauk
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This chapter provides a general overview of infectious disease microscopy as practiced in the clinical microbiology laboratory. Additional information about the care and use of the microscope and ergonomic factors is also covered.
Chapter 3 : Laboratory Detection of Bacteremia and Fungemia
- Authors: Michael L. Wilson, Melvin P. Weinstein, L. Barth Reller
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The laboratory detection of bacteremia and fungemia remains one of the most important functions performed by clinical microbiology laboratories. Culture-based methods remain both the gold standard as well as the most commonly used methods. Newer diagnostic technologies do not replace cultures, but are used primarily as adjunct tests to shorten the time needed for final results.
Chapter 4 : Systems for Identification of Bacteria and Fungi*
- Authors: Karen C. Carroll, Robin Patel
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This chapter provides an overview of technologies used for identification of bacteria and fungi recovered from clinical specimens. This includes manual and automated approaches based on biochemical, protein, and/or DNA analysis. Matrix-assisted laser desorption ionization–time-of-flight mass spectrometry, which is being rapidly adopted by clinical laboratories, is highlighted. Regardless of the method used, the scientific approach to identify microorganisms, highlighted in this chapter, relies on the same fundamental principles.
Chapter 5 : Automation and Design of the Clinical Microbiology Laboratory
- Authors: Christopher D. Doern, Martin Holfelder
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This chapter discusses laboratory design and the growing impact of laboratory automation. The chapter is divided into two sections. The first section addresses more traditional considerations of laboratory design such as staffing, workflow, laboratory location, and the role of efficiency programs such as lean and Six Sigma. The second section discusses currently available systems for automating the clinical microbiology laboratory. Individual technologies for specimen processing, organism identification, and susceptibility testing as well as total lab automation are covered. Important considerations for purchase and utilization are outlined to aid readers in deciding which technology is right for their lab and how best to approach implementation.
Chapter 6 : Molecular Microbiology*
- Author: Frederick S. Nolte
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Nucleic acid amplification techniques are now commonly used to diagnose and manage patients with infectious diseases. The growth in the number of FDA-cleared test kits and analyte-specific reagents has facilitated the use of this technology in the clinical laboratory. Technological advances in nucleic acid amplification techniques, automation, nucleic acid sequencing, and multiplex analysis have revitalized the field of molecular microbiology and created new opportunities for growth. Simple, sample-in, answer-out molecular test systems are now available that can be deployed in a variety of laboratory and clinical settings. Molecular microbiology remains the leading area in molecular pathology in terms of both the numbers of tests performed and clinical relevance. Nucleic acid-based tests have reduced the dependency of the clinical microbiology laboratory on more traditional antigen detection and culture-based methods and created new opportunities for the laboratory to affect patient care. This chapter covers nucleic acid probes, signal and target amplification techniques, postamplification detection and analysis (e.g., electrophoresis, hybridization, sequencing, microarrays, and mass spectrometry), clinical applications of these techniques, and the special challenges and opportunities that these techniques provide for the clinical laboratory.
Chapter 7 : Immunoassays for Diagnosis of Infectious Diseases*
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By definition, immunoassays are biochemical assays that detect the presence of an analyte, either antibody or antigen, using labeled antibodies as the analytical reagent. Immunoassays can be adapted for detection of analytes across laboratory disciplines and are often more cost-effective than other diagnostic methods. In the clinical microbiology laboratory, immunoassays often serve as confirmatory tests, and therefore the results are typically not intended to be used as the sole basis on which a diagnosis is made. However, for certain infectious diseases, including Lyme disease, cryptococcal meningitis, and syphilis, antibody and antigen detection by immunoassays is the primary means by which the infection is established. Due to their ease of use, rapid turnaround time, and generally high specificity, immunoassays are increasingly becoming available for point-of-care testing. This chapter summarizes the common immunologic testing methods currently used in clinical microbiology laboratories and their application for the diagnosis of infectious diseases. The discussion emphasizes general assay design, with important caveats relevant to test development and interpretation. While some examples relating to clinical testing are included, for an in-depth, pathogen-focused discussion, the reader is directed to the designated chapters in this Manual.
Chapter 8 : Prevention of Health Care-Associated Infections
- Authors: Daniel J. Diekema, Michael A. Pfaller
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Health care-associated infections (HAIs) are among the most common complications of health care delivery. Every health care facility must have an infection prevention program charged with monitoring and preventing infections in the health care environment. The clinical microbiology laboratory is inextricably linked to any infection prevention program, because preventing infections requires the ability to diagnose those infections when they occur. In this chapter, we discuss the impact of HAIs, outline the organization of the hospital infection prevention program, and describe the important roles of the clinical microbiology laboratory in HAI prevention. These roles include essential support for surveillance, outbreak detection and management, antimicrobial stewardship, risk assessment and planning, and education. In summary, the clinical microbiology laboratory is an essential element of any effective infection prevention program.
Chapter 9 : Investigation of Disease Outbreaks
- Authors: Iona Munjal, Belinda Ostrowsky
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This chapter reviews concepts associated with infectious disease outbreaks, including disease surveillance, mechanisms used to detect outbreaks, and the epidemiological steps used to investigate an outbreak. We catalogue both routine and novel laboratory detection methods that can be used in an outbreak setting and examples in which laboratory testing played a central role. We introduce resources that readers can access when facing a known or novel pathogen in the context of an outbreak, including agencies that perform sophisticated surveillance and outbreak investigation. Emphasis is placed on the importance of thinking about the world health community with the stark rise in globalization, and the epidemiological players at each level from local to international are listed. In past editions of the text, the focus was largely foodborne outbreaks; this chapter expands the focus to include many different types of pathogens and settings for outbreaks.
Chapter 10 : Molecular Epidemiology
- Authors: Eija Trees, Paul A. Rota, Duncan MacCannell, Peter Gerner-Smidt
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Molecular epidemiology may be defined as the application of molecular, i.e., nucleic acid or protein based, methods to study the transmission of microbial pathogens in human populations. In molecular epidemiology, molecular methods are used for detection, identification, virulence characterization, and subtyping, i.e., to generate isolate-specific profiles or fingerprints for assessment of epidemiological relatedness. Molecular methods have gradually replaced old phenotypic methods, starting with the introduction of plasmid profiling in the 1970s and reaching the most recent resolution with the emergence of next-generation sequencing techniques during the latter part of the past decade. This chapter describes the most commonly used methods, together with their strengths, weaknesses, and applications in the context of molecular epidemiology. The appropriate process for method selection, development, and validation is discussed, along with the factors that may affect data interpretation. Finally, we provide examples of the reference libraries that are available for molecular methods.
Chapter 11 : Procedures for the Storage of Microorganisms*
- Authors: Rosemary C. She, Cathy A. Petti
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Preservation of microorganisms for future study has a long tradition in microbiology. Culture collections of microorganisms are valuable resources for scientific research in microbial diversity and evolution, patient care management, epidemiological investigations, and educational purposes. Preserved individual strains of microorganisms serve as permanent records of microorganisms’ unique phenotypic profiles and provide the material for further genotypic characterizations. Effective storage involves maintaining an organism in a viable state free of contamination and without changes in its genotypic or phenotypic characteristics. The organism must also be easily restored to its condition prior to preservation. This chapter presents methods that can be used for the storage of bacteria, protozoa, fungi, and viruses.
Chapter 12 : Prevention of Laboratory-Acquired Infections
- Author: Michael A. Noble
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Laboratory workers are at risk of acquiring infections while at work. This chapter describes the history and epidemiology (including behavioral characteristics) of laboratory-acquired infections and describes the programs, procedures, provisions and practices, and requirements (including risk management) in place to help reduce their frequency and consequence.
Chapter 13 : Decontamination, Disinfection, and Sterilization
- Authors: Constanze Wendt, Reno Frei, Andreas F. Widmer
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Antisepsis of living tissue and decontamination, disinfection, and sterilization of the environment and medical devices are basic components of any infection control program. Patients expect to be treated in a clean environment and that any reusable instrument or device used for diagnosis or treatment has undergone a process to eliminate any risks for cross-infection. Due to the increasing frequency of multiresistant bacterial pathogens at a time when pharmaceutical companies have shifted from developing antimicrobial agents to designing drugs for chronic diseases, reprocessing techniques, disinfectants, and general infection control practice have garnered more attention recently than in the past.This chapter describes substances and procedures used for antisepsis, decontamination, disinfection, and sterilization and highlights some special issues like decolonization of colonized patients, reprocessing of endoscopes or dental equipment, and reprocessing of medical devices contaminated with prions.
Chapter 14 : Biothreat Agents
- Authors: Susan E. Sharp, Michael Loeffelholz
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The ideal qualities for a successful biothreat agent are a high rate of illness in exposed persons/animals, a high case fatality rate, a short incubation period, and a paucity of immunity in the targeted population. Success is also influenced by the availability of treatment, the ability of the agent to transmit from person to person, and the ease with which the agent can be produced and disseminated; in addition, a disease that is, at least initially, difficult to recognize clinically or diagnose contributes to the success of the agent. General clues that one could be dealing with an unrecognized bioterror event include a large outbreak of illness with a high death rate, a recognized case(s) of an uncommon disease, disease in a region of the world where the disease is not endemic, disease out of its usual seasonality, simultaneous outbreaks of the same disease in various part of the country or world, and sick and dying animals. The Centers for Disease Control and Prevention has classified potential biothreat agents into categories on the basis of their threat to national security, with those organisms belonging to category A being the most serious threats. These and other agents are discussed in this chapter.
Chapter 15 : The Human Microbiome
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The total number of bacteria in the human body is at least 10 times greater than the number of human cells, and recent studies, particularly the efforts of the National Institutes of Health Human Microbiome Project consortium, have led to a greater understanding of the identity and distribution of the microorganisms that constitute these populations. In particular, implementation of next-generation sequencing (NGS) has helped illuminate how these bacteria contribute to, and are affected by, human health and disease. Significant progress in cataloging and characterizing these organisms and genes has been made in recent years thanks to NGS approaches, and studies have been expanded beyond those focused solely on the microbes that colonize the gut. The newest DNA sequencing platforms are making it possible to sequence the DNA of the collective genome (or metagenome) of entire communities of microbes from all body sites, at various stages of health and disease, over significant periods of time, effectively enabling the characterization of the “human microbiome.”
Chapter 16 : Microbial Genomics and Pathogen Discovery*
- Authors: Jennifer K. Spinler, Peera Hemarajata, James Versalovic
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In recent years, our understanding of microbial diversity has grown tremendously as many previously unidentified bacterial, archaeal, and viral species have been discovered and sequenced. In the era of the human microbiome and metagenomics (chapter 15), large-scale DNA sequencing projects and advances in bioinformatics have yielded abundant data regarding human-associated microbes. As human microbiology rapidly expands beyond its past framework of cultured pathogens in the medical microbiology laboratory, opportunities for detection and identification of novel human pathogens associated with infectious diseases abound. In this chapter, we focus on specific or defined sets of pathogens associated with human infections, in contrast to microbial components of disease and microbial ecology. We begin with an overview of historical methodologies, followed by a brief description of the evolution of nucleic acid sequencing technologies. Finally, we describe how microarrays, nucleic acid sequencing technologies, and mass spectrometry are profoundly reshaping strategies aimed at pathogen discovery and identification.
BACTERIOLOGY
General
Chapter 17 : Taxonomy and Classification of Bacteria
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This chapter discusses the state of the art in bacterial taxonomy. It addresses criteria for species delineation, the polyphasic species concept, and multilocus sequence- and whole-genome sequence-based approaches for species definition. An overview of classification and identification methods is presented, which includes traditional phenotypic, chemotaxonomic, and genotypic techniques. In addition, the chapter briefly describes major groups of bacteria, the practice of reporting the taxonomy of uncultured bacteria, bacterial nomenclature, the valid publication of bacterial names, and name changes.
Chapter 18 : Specimen Collection, Transport, and Processing: Bacteriology*
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This chapter provides a detailed description of collection, transport, and initial handling of all sample types submitted for bacteriological studies. It includes guidance on specimen rejection, choice of specimen based on syndrome, media, molecular tests available, and Gram stain interpretations.
Chapter 19 : Reagents, Stains, and Media: Bacteriology*
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This chapter describes the media, reagents, and stains used in the isolation and identification of bacteria of medical importance by the clinical microbiology laboratory.
Gram-Positive Cocci
Chapter 20 : General Approaches to Identification of Aerobic Gram-Positive Cocci
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This chapter summarizes the basic phenotypic characteristics that can be employed for identification of aerobic and facultatively aerobic Gram-positive cocci isolated from clinical specimens. The chapter outlines how the appearance of Gram-stained cells and reactions in a relatively small number of key biochemical tests may be used for identification of these bacteria to the genus level.
Chapter 21 : Staphylococcus, Micrococcus, and Other Catalase-Positive Cocci
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This chapter discusses mainly those Gram-positive cocci that were members of the former family Micrococcaceae prior to its redefinition. The genera have now been rearranged in different families: the Staphylococcaceae, the redefined Micrococcaceae, and the newly established Dermacoccaceae. The two last families comprise micrococci, clinically relevant species of which now belong to the genera Micrococcus, Kocuria, and Kytococcus. Staphylococci and micrococci are part of the microbiota of human and animal skin and mucous membranes and/or are found in the environment or on food. Many of these species are opportunists that may become pathogenic in a host- as well as species- and strain-dependent manner following breaks in the cutaneous barrier. Their recovery requires assessment of clinical significance to determine whether an organism is a contaminant, colonizer, or pathogen. Staphylococcus aureus is the clinically most important species, causing a wide range of human and animal diseases with high medical and economic burden. The coagulase-negative Staphylococcus epidermidis and Staphylococcus haemolyticus cause nosocomial infections mainly in patients with predisposing factors such as immunodeficiency and/or indwelling or implanted foreign polymer bodies. The fact that staphylococci have become resistant to many available therapeutic agents has had, particularly in the case of methicillin-resistant S. aureus, profound impact on patient and hygiene management. In this chapter, the taxonomy, phylogeny, biology, and mechanisms of resistance of these pathogens and their infectious diseases are covered, and the consequences for identification, differentiation, genotyping, susceptibility determination, and antibiotic therapy are discussed.
Chapter 22 : Streptococcus
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The genus Streptococcus currently comprises >100 recognized species, and this number can certainly be expected to rise with the increasing availability of next-generation sequencing technologies. More than 10 novel species have been validly published in the last 5 years alone. Most of these were found in the oral cavity and gastrointestinal tract of various mammals and so far have not been shown to play a role in human infections. This chapter focuses on well-known and novel streptococcal species found in human specimens and covers changes that have been applied to the taxonomy of streptococci in recent years.
Chapter 23 : Enterococcus
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No descriptions available.
Chapter 24 : Aerococcus, Abiotrophia, and Other Aerobic Catalase-Negative, Gram-Positive Cocci
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This chapter examines clinically significant catalase-negative, Gram-positive cocci other than streptococci, enterococci, and the obligately anaerobic Gram-positive cocci. These bacteria form a taxonomically diverse collection of organisms that are infrequently isolated from clinical specimens and that may be misidentified as other more well-known Gram-positive cocci. The chapter discusses their taxonomy, their clinical significance, and the phenotypic and molecular methods for identification of these organisms when encountered in clinical microbiology laboratories. The genera included are Abiotrophia, Aerococcus, Dolosicoccus, Dolosigranulum, Facklamia, Gemella, Globicatella, Granulicatella, Helcococcus, Ignavigranum, Lactococcus, Leuconostoc, Pediococcus, Vagococcus, and Weissella.
Gram-Positive Rods
Chapter 25 : General Approaches to the Identification of Aerobic Gram-Positive Rods
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This chapter summarizes in brief the macroscopic and microscopic features, identification methods, and chapter locations within the 11th edition of this Manual of a wide variety of genera and species of aerobic or facultatively anaerobic Gram-positive rods.
Chapter 26 : Bacillus and Other Aerobic Endospore-Forming Bacteria*
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Aerobic endospore-forming bacteria are ubiquitous in nature. Because endospores are resistant to heat, desiccation, radiation, and disinfectants, they are found in terrestrial and aquatic habitats of all kinds and persist in places where most other organisms cannot. Dissemination of spores, via aerosols, wind, and dust, contributes to contamination of health care facilities, industrial clean rooms, and food production environments. Although commonly encountered in the microbiology laboratory, the majority of aerobic endospore-forming bacteria are nonpathogenic and of no clinical relevance. The few clinically significant species of Bacillus, Geobacillus, Lysinibacillus, Paenibacillus, and Brevibacillus are best described as opportunistic human pathogens. Transmission is restricted to ingestion, injection, injury, inhalation, or other contact with material that has been contaminated with spores or vegetative cells. Several environmental species are professional pathogens of invertebrates, and toxigenic strains of the Bacillus cereus group are an important cause of food poisoning, but only Bacillus anthracis is recognized as an obligate pathogen of animals. Clinical and laboratory expertise is a critical components of rapid anthrax diagnosis. Due to the biothreat potential of B. anthracis, many jurisdictions regulate possession and transportation of this agent, and testing requires enhanced safety precautions and training.
Chapter 27 : Listeria and Erysipelothrix
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Listeria and Erysipelothrix are Gram-positive, rod-shaped bacteria. They occur naturally in a variety of animal species and the environment. Listeria monocytogenes is the causative agent of listeriosis, which manifests mainly as septicemia or meningitis. People with an underlying condition, pregnant women, and individuals ≥60 years of age have a markedly increased risk of acquiring listeriosis. L. monocytogenes can enter the food chain in various manners, and since it is able to grow even at temperatures below 10°C, it is an important cause of food-related outbreaks. Detection of L. monocytogenes is facilitated by culture on selective media, followed by biochemical, molecular, and mass spectrometry identification methods. Typing methods, like pulsed-field gel electrophoresis and also faster molecular methods, are available for investigation of outbreaks and surveillance purposes. Listeriosis is primarily treated with an aminopenicillin, while Listeria is intrinsically resistant to cephalosporins. Erysipelothrix rhusiopathiae causes erysipeloid in humans, a zoonosis which is mostly acquired by exposure to contaminated fish and meat. Erysipeloid is characterized by localized cellulitis accompanied by local lymphadenopathy and sometimes systemic symptoms. E. rhusiopathiae can be differentiated from other Gram-positive rods by biochemical and molecular methods and is typically positive for H2S. Typing of isolates is of limited value, since most cases are caused by two serogroups. Erysipeloid is primarily treated with penicillin or ampicillin, and of note, E. rhusiopathiae is intrinsically resistant to vancomycin. Up-to-date detection, identification, and typing methods for Listeria spp. and E. rhusiopathiae as well as antimicrobial susceptibilities are described in detail in this Manual.
Chapter 28 : Coryneform Gram-Positive Rods
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This chapter deals with aerobically growing, asporogenous, irregularly shaped, non-partially acid-fast, Gram-positive rods generally called coryneforms. Genera mainly covered include Corynebacterium, Turicella, Arthrobacter, Brevibacterium, Dermabacter, Helcobacillus, Rothia, Exiguobacterium, Oerskovia, Cellulomonas, Cellulosimicrobium, Microbacterium, Curtobacterium, Leifsonia, Pseudoclavibacter, Auritidibacter, Arcanobacterium, Trueperella, and Gardnerella. Information is presented on epidemiology and transmission, clinical significance, direct examination, isolation procedures, and identification of coryneforms. Taxa are described in detail with emphasis on identification tools for the clinical microbiologist. Finally, antimicrobial susceptibility testing procedures and resistance patterns of coryneforms are outlined.
Chapter 29 : Nocardia, Rhodococcus, Gordonia, Actinomadura, Streptomyces, and Other Aerobic Actinomycetes
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The aerobic actinomycetes are aerobic Gram-positive rods, most of which exhibit branching on microscopic evaluation. These organisms have been noted to cause significant infections, most frequently in immunocompromised hosts. Because of the large number of species included in these genera, it has become increasingly difficult to identify members of this group to the species level. We present information on the taxonomy, clinical significance, methods of identification, and susceptibility testing of these organisms.
Chapter 30 : Mycobacterium: General Characteristics, Laboratory Detection, and Staining Procedures*
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Many species within the genus Mycobacterium are prominent pathogens, above all members of the M. tuberculosis complex, M. leprae, and M. ulcerans. In addition, there are, at present, more than 150 species of environmental mycobacteria (nontuberculous mycobacteria [NTM]) which exert various degrees of pathogenicity and virulence in humans. This chapter describes clinical, taxonomic, genetic, and epidemiologic aspects of these organisms and includes the characteristics and clinical significance of novel species. Emphasis is put on laboratory diagnosis of mycobacteria, which includes staining procedures, detection by microscopic procedures, protocols for collection and pretreatment of clinical specimens, culture, and quality assurance, as well as safety and transport issues. Furthermore, evaluation, interpretation, and reporting of results are addressed. The practical value and the current state of knowledge of the immunodiagnostic tests for tuberculosis (interferon gamma release assays [IGRAs]) are also discussed.
Chapter 31 : Mycobacterium: Laboratory Characteristics of Slowly Growing Mycobacteria*
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This chapter summarizes the epidemiology, transmission, and clinical significance of the slowly growing mycobacteria, including Mycobacterium tuberculosis complex and Mycobacterium avium complex. In addition, laboratory diagnostics for the species-level identification of slowly growing mycobacteria are discussed, including direct examinations and phenotypic and genotypic methods. Strain typing and antimicrobial susceptibility test methods are also included. Updates to this chapter since the 10th edition of this Manual include the addition of recently identified species; discussion of the lipoarabinomannan urinary antigen; and discussions of new molecular methods of identification, including the Cepheid GeneXpert MTB/RIF test, the Speed-Oligo Line Probe Assay, the Seegene multiplex PCR assay, mass spectrometry for M. tuberculosis and nontuberculous mycobacteria, as well as real-time PCR for Mycobacterium leprae detection.
Chapter 32 : Mycobacterium: Clinical and Laboratory Characteristics of Rapidly Growing Mycobacteria
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Prior to the advent of modern molecular technology in the clinical laboratory, the rapidly growing mycobacteria (RGM) were routinely identified by phenotypic characteristics, including batteries of biochemical tests, growth rates, and colonial morphology. The impact of the molecular revolution in the clinical laboratory has brought more rapid, efficient, and accurate identification than was possible with most of the techniques based solely on phenotypic characteristics. Moreover, these technologies have introduced a large number of new species, including some pathogens and some environmental species with no current claim to pathogenicity. Molecular methods such as DNA gene sequence analysis are now recognized as the gold standard for the identification of nontuberculous mycobacteria (NTM). Newer techniques, such as matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) may, however, soon supplant DNA (sequencing) technology. The current disadvantage of all of the newer systems, including sequencing technology, is that complete databases may be lacking. The Clinical and Laboratory Standards Institute (CLSI) has approved guidelines not only for molecular identification methods but also for antimicrobial susceptibility testing of the RGM, thus allowing clinical laboratories to better standardize testing of the RGM. This chapter details the clinical significance of the RGM species and describes methods of laboratory identification, susceptibility testing, and reporting of these RGM species, with an emphasis on taxonomy and antimicrobial susceptibility changes that have occurred within the last 5 years.
Gram-Negative Bacteria
Chapter 33 : Approaches to the Identification of AerobicGram-Negative Bacteria*
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This chapter describes the approaches used to identify Gram-negative rods, with emphasis on the greater difficulty in identifying non-glucose-fermenting organisms. We amplify what was presented in the 9th edition of this Manual by presenting a scheme to identify these organisms that is centered around three enzymatic activities, i.e., oxidase, trypsin (benzyl-arginine arylamidase or benzyl-arginine aminopeptidase), and pyrrolidonyl aminopeptidase. These enzymatic reactions are fast and easy to interpret, and they are stable markers in almost all taxa discussed; i.e., there are few species for which these tests yield variable intraspecies results.
Chapter 34 : Neisseria
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The genus Neisseria currently consists of 28 species, most of which are commensals of mucous membranes of humans and animals. The two species most commonly associated with disease are Neisseria meningitidis and N. gonorrhoeae. N. meningitidis, also termed meningococcus, is spread by large-droplet oropharyngeal secretions and rarely causes invasive meningococcal disease (IMD), in the form of meningitis and sepsis. On the other hand, N. gonorrhoeae, which is also named gonococcus, is spread by sexual contact and is the causal agent of gonorrhea. In many industrialized countries the incidences of IMD and gonorrhea have decreased over the last decades, with present rates of around 1/100,000 and over 30/100,000, respectively. Microscopic examination of samples remains a cornerstone of laboratory diagnosis, as specificities of detection of Gram-negative diplococci from cerebrospinal fluid and genital swabs are high for the confirmation of meningococcal meningitis and gonorrhea, respectively. Both species are exquisitely susceptible to extreme temperatures and desiccation, necessitating rapid transport of specimens to the laboratory for culture-based detection. Culture remains the standard approach for verifying the presence of Neisseria spp. in human specimens and is the only method allowing antibiotic resistance testing. Nevertheless, molecular assays, notably nucleic acid amplification tests, are frequently employed for the diagnosis of IMD and gonorrhea, due to less stringent sampling requirements. Moreover, DNA sequence-based single- and multilocus approaches, as well as matrix-assisted laser desorption ionization–time of flight mass spectrometry, are gaining acceptance as methods for species assignment within the genus Neisseria.
Chapter 35 : Aggregatibacter, Capnocytophaga, Eikenella, Kingella, Pasteurella, and Other Fastidious or Rarely Encountered Gram-Negative Rods*
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The bacterial genera covered in this chapter are taxonomically diverse—they belong to the families Cardiobacteriaceae, Flavobacteriaceae, Leptotrichiaceae, Neisseriaceae, Pasteurellaceae, and Porphyromonadaceae—but common traits justify their discussion as a group. Most bacteria in this group are part of the microbiota of the nasopharynx and/or the oral cavity of animals and/or humans and are parasitic, with the only environmental genus being Chromobacterium. Transmission from animals occurs by contact (e.g., bites and licking of wounds), from humans to humans by droplets (e.g., directly with Kingella spp. or via human bites with Eikenella corrodens). The epidemiology and clinical significance of the genera Actinobacillus, Aggregatibacter, and Pasteurella, of the family Pasteurellaceae; the genera Cardiobacterium, Eikenella, and Kingella, belonging to the HACEK group; and other less frequently encountered genera are discussed separately. The identification of most genera is difficult by phenotypic methods. Direct microscopy may identify the corresponding bacteria; e.g., the typical morphology of spindle-shaped cells in the Gram stain and the anamnestic history of a dog bite allow the presumptive identification of Capnocytophaga canimorsus. Phenotypic key reactions as well as commercially available systems allow identification of the frequently encountered human isolates. If accurate identification of uncommon isolates is of concern, then molecular methods, e.g., 16S rRNA gene analysis, are necessary. The application of matrix-assisted laser desorption ionization–time-of-flight mass spectrometry shows promising results and is discussed here. Finally, susceptibility testing according to CLSI and EUCAST is described, especially for Pasteurella multocida.
Chapter 36 : Haemophilus
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No descriptions available.
Chapter 37 : Escherichia, Shigella, and Salmonella
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No descriptions available.
Chapter 38 : Klebsiella, Enterobacter, Citrobacter, Cronobacter, Serratia, Plesiomonas, and Other Enterobacteriaceae
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This chapter deals with a number of both major and less prominent members of the Enterobacteriaceae family. The genera Klebsiella, Enterobacter, Citrobacter, Serratia, Plesiomonas, and Cronobacter cause a range of infections in humans. These continue to warrant serious consideration since many of these infections are poorly characterized, are likely to be underreported, and have the capacity to resist antimicrobial agents. The Infectious Diseases Society of America recognizes Klebsiella and Enterobacter spp. as members of the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.), which exhibit antimicrobial resistance and present new challenges in their pathogenesis and transmission. Therefore, this chapter contains a wide breadth of information not only on the taxonomy, detection, and identication of these genera, but also the global issue of antimicrobial resistance.
Chapter 39 : Yersinia*
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The pathogenic Yersinia species, Y. pseudotuberculosis, Y. enterocolitica, and Y. pestis, are zoonotic agents that cause disease in humans. Human clinical infections caused by Y. pseudotuberculosis and Y. enterocolitica occur after the ingestion of contaminated food or water and manifest primarily as mild gastroenteritis, whereas the etiologic agent of plague, Y. pestis, is transmitted to humans by the bite of an infected flea and results in life-threatening illness. Within the Yersinia genus, these three species are joined by 14 lesser-known Yersinia species which are largely considered environmental species and nonpathogenic to humans. Pathogenic Yersinia species share a highly conserved virulence plasmid and a chromosomal high-pathogenicity island (HPI) and show tropism for lymphoid tissue, where their ability to evade host innate immunity enables extracellular proliferation. Plague is a notorious disease, with strikingly high mortality rates of 40 to 100% if untreated; it is the cause of three major pandemics, including the Black Death of the 14th century, in which an estimated 17 to 28 million Europeans died. Y. pestis was weaponized by the United States, Japan, and the former USSR during and after World War II and remains a high-level biothreat agent. In 2012, Y. pestis was classified as a tier 1 agent, one of 6 bacterial agents considered to present the greatest risk of deliberate misuse with the most significant potential for mass casualties or devastating effects to the economy, critical infrastructure, or public confidence.
Chapter 40 : Aeromonas*
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This chapter on the genus Aeromonas is a review and update of basic topics of interest to clinical microbiologists, such as taxonomy, epidemiology, isolation, and identification procedures and antimicrobial susceptibility testing. It includes all new validated species proposed since the 10th edition and any new sources of infection, as well as the latest recommendations for the appropriate antimicrobials to be tested and the appropriate CLSI guidelines.
Chapter 41 : Vibrio and Related Organisms*
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This chapter summarizes the taxonomy, family description, epidemiology, and other details about the family Vibrionaceae. Prevalent and highly virulent organisms, such as Vibrio cholerae, are described in detail. Methods for collection, transport, storage, and isolation and traditional biochemical identification, antibiotic susceptibility testing, and molecular subtyping are described.
Chapter 42 : Pseudomonas*
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Pseudomonas aeruginosa may be associated with colonization or clinically significant infections. Interpretation of the Gram stain often directs the further workup of this organism. The presence of small clusters of Gram-negative organisms surrounded by amorphous material is indicative of biofilm formation compatible with a chronic infection. This finding should be reported to physicians, and incubation should be prolonged, as these isolates usually exhibit slower growth characteristics. Isolation of P. aeruginosa from sterile body sites should always be interpreted as indicative of probable infection. Isolation in mixed culture requires correlation with the direct smear, other organisms isolated, and clinical history. Isolates from sites of chronic infection, such as cystic fibrosis respiratory sites, often exhibit multiple morphotypes that can make identification difficult. Molecular methods increasingly are finding a role in the identification of this organism, especially for epidemiological studies. Susceptibility testing of this organism is difficult, especially for mucoid isolates, due to increasing resistance, lack of reproducibility of results, and lack of clinical correlation. Piperacillin and piperacillin-tazobactam results obtained from automated systems may be unreliable for Pseudomonas spp., and in particular for mucoid isolates, and results should be confirmed by disk diffusion or Etest systems. A basic understanding of the multiple mechanisms of resistance, both intrinsic and acquired, is essential to interpret susceptibility testing results and give therapeutic recommendations to physicians. Other Pseudomonas species are infrequently isolated in the laboratory and are usually not clinically significant. Clinical correlation and correlation with the Gram stain are essential before further workup is undertaken.
Chapter 43 : Burkholderia, Stenotrophomonas, Ralstonia, Cupriavidus, Pandoraea, Brevundimonas, Comamonas, Delftia, and Acidovorax
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Species in the genera Burkholderia, Stenotrophomonas, Ralstonia, Cupriavidus, Pandoraea, Brevundimonas, Comamonas, Delftia, and Acidovorax are unusual human pathogens that are infrequently encountered in the clinical microbiology laboratory. However, the incidence of human infection due to some of these species has increased in recent years. Further, the taxonomy of these genera has been expanded, with several new species being described. Burkholderia gladioli and members of the Burkholderia cepacia complex are important opportunistic pathogens in persons with cystic fibrosis and chronic granulomatous disease, while Burkholderia pseudomallei causes significant human infection in Southeast Asia and northern Australia. Stenotrophomonas maltophilia is an important nosocomial pathogen and, together with certain Ralstonia, Cupriavidus, and Pandoraea species, is also encountered in respiratory specimens from persons with cystic fibrosis. Historically, commercial phenotypic identification systems have performed rather poorly in identifying many of these species. Genetic-based identification methods have provided alternatives to traditional phenotypic analyses. More recently, matrix-assisted laser desorption ionization–time-of-flight mass spectrometry has shown excellent potential for reliable identification, which should continue to improve as libraries of reference spectra expand. A variety of genotyping systems provide for reliable strain typing of most of these species and have enabled a greater appreciation of the epidemiology and natural history of human infection. This group of species is also characterized by limited susceptibility to many currently available antimicrobials. Treatment of human infection is often empiric and relies on combined antimicrobial therapy.
Chapter 44 : Acinetobacter, Chryseobacterium, Moraxella, and Other Nonfermentative Gram-Negative Rods*
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The organisms covered in this chapter belong to a group of taxonomically and phylogenetically diverse, Gram-negative, nonfermentative rods and coccobacilli. Still, several of the genera dealt with belong to the same family; i.e., Acinetobacter, Moraxella, Oligella, and Psychrobacter belong to the family Moraxellaceae (Gammaproteobacteria), and Balneatrix, Bergeyella, Chryseobacterium, Elizabethkingia, Empedobacter, Myroides, Sphingobacterium, Wautersiella, and Weeksella belong to the family Flavobacteriaceae (Bacteroidetes).
Chapter 45 : Bordetella and Related Genera
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No descriptions available.
Chapter 46 : Francisella
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Francisella tularensis is the causative agent of tularemia, an acute and fatal illness in animals and humans. Human infections caused by F. tularensis occur via arthropod bite, handling of infected animals, inhalation of infectious aerosols, and ingestion of contaminated food or water. F. tularensis is highly infectious, with only 10 organisms sufficient to cause illness, and has a notorious reputation for causing laboratory-acquired infections. Within the Francisella genus, F. tularensis is joined by 6 lesser-known Francisella species which are largely considered environmental species and nonpathogenic or opportunistic pathogens of humans. Whereas F. tularensis causes tularemia in animals and humans, F. noatunensis and F. halioticida infect and cause death in fish and abalone, respectively. F. novicida and F. philomiragia are associated with salt or brackish water and are only infrequent causes of opportunistic infections in compromised individuals. F. tularensis was examined by multiple countries for its possible use as both an offensive and retaliatory biological weapon in the years leading up to and following World War II. In 2012, F. tularensis was classified as a Tier 1 agent, one of six bacterial agents considered to present the greatest risk of deliberate misuse with the most significant potential for mass casualties or devastating effects to the economy, critical infrastructure, or public confidence.
Chapter 47 : Brucella*
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Brucellosis is a “stealth” zoonotic disease due to its insidious onset and persistence. Several Brucella spp. can infect humans. They are highly infectious and are categorized as group B biologic weapons. The exact pathophysiology of the disease remains to be elucidated. The epidemiologies of brucellosis differ between regions of endemicity and nonendemicity; the disease occurs among the general community in the former region and in the professional community in the latter region. The multisystem involvement and complications in adults and children and the protean or unusual clinical presentation of the disease masquerade as a wide spectrum of other infectious and noninfectious diseases, causing the disease to merit its label as “the disease of mistakes.” Such difficulty in diagnosing the disease can lead to protracted chronicity and serious complications. Thus, awareness about this disease and the proper use of laboratory resources are indispensable assets in reaching or confirming the diagnosis. Several brucella-specific assays, including conventional and instrument-utilizing culture, serologic tests, and molecular tests, are available. The proper utility and interpretation of these test results during the different presentations and phases of the disease are key factors in making an accurate diagnosis. Handling of the organism for identification to the species level, typing, and susceptibility testing requires high caution in specialized labs. The development of a safe and effective human vaccine is still being sought. The pathogen remains highly susceptible to treatment with doxycycline, rifampicin, gentamicin, and streptomycin. Dual regimens should be used, and the duration of treatment is usually long, depending on the severity of infection and the complications of the disease.
Chapter 48 : Bartonella *
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The genus Bartonella is a branch of alphaproteobacteria in the Rhizobiales order and the Bartonellaceae family. The genus represents a union with bacteria formerly in the genera Rochalimaea and Grahamella, whose members are small facultatively intracellular Gram-negative bacilli and coccobacilli. Members of the genus are zoonotic agents, for which arthropods are often reservoirs and vectors. At least 32 species are validly described, including 12 of which are definite or potential human pathogens. The most important of these are B. bacilliformis, B. quintana, B. elizabethae, and B. henselae. These are the agents for a variety of human illnesses, such as Oroya fever and Carrion’s disease, trench fever, cat scratch disease, and endocarditis among immunocompetent patients and bacillary angiomatosis/peliosis among those who are immunocompromised. Bartonella species infections are also common among domestic pets and wild animals. Diagnostic test methods include culture, serology, and molecularly based assays. The slow growth and fastidious nature of some species necessitates the use of molecular and/or serologic tests for most applications, but commercially available tools are lacking. While approaches to determine antimicrobial resistance are described, most antibiotics that are sensitive are bacteriostatic only, and there is often no correlation between in vitro susceptibility test results and clinical responses.
Chapter 49 : Legionella
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Legionnaires’ disease (LD) is a type of pneumonia caused by the Legionella spp. Legionella pneumophila is the most common cause of the disease and, along with Legionella longbeachae, causes disease in both immunocompetent and immunosuppressed people. The >50 other named Legionella spp. rarely cause LD and do so exclusively in immunocompromised patients. The Legionella spp. are aerobic Gram-negative bacilli that are found mainly in aqueous environments. Humans are accidental hosts of these bacteria, acquiring LD by inhalation or aspiration of environmental bacteria. The bacteria can be grown on buffered yeast extract medium. Bacterial identification can be difficult because of the nonreactivity of the bacteria in biochemical tests. Laboratory diagnosis of LD is based on the use of selective culture media, detection of urinary antigen, antibody detection, and molecular amplification methods, such as PCR. Culture diagnosis is insensitive but very specific, having the highest yield in severely ill and immunosuppressed patients. Urine antigen testing is fast, easy, and relatively sensitive, especially for community-acquired LD, but around 30% of LD patients have negative urine tests. Molecular-amplification diagnosis, such as PCR, appears to be more sensitive than other methods, but the lack of FDA-cleared assays limits its use to large reference laboratories in the United States. Strain typing is mainly sequence based, with monoclonal antibody reactivity being a useful, quick screening test.
Anaerobic Bacteria
Chapter 50 : Approaches to Identification of Anaerobic Bacteria
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This chapter discusses basic sample collection, processing, and an overview of methods for identification and management of clinically important anaerobic bacteria in samples from infected patients.
Chapter 51 : Peptostreptococcus, Finegoldia, Anaerococcus, Peptoniphilus, Veillonella, and Other Anaerobic Cocci
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This chapter describes a group of bacteria that consists of obligately anaerobic non-spore-forming cocci. This group has undergone extensive taxonomic changes during the last decades, including the addition of new genera and species. These bacteria are opportunistic pathogens and can cause various infections involving all areas of the human body. The focus of this chapter is the description of the taxa that are recovered from human clinical materials thought to be medically relevant in terms of their taxonomy, clinical significance, isolation, and identification in clinical settings.
Chapter 52 : Propionibacterium, Lactobacillus, Actinomyces, and Other Non-Spore-Forming Anaerobic Gram-Positive Rods*
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This chapter encompasses non-spore-forming, anaerobic, Gram-positive rods including Propionibacterium, Lactobacillus, Actinomyces, Bifidobacterium, Eubacterium, and various closely related genera. It describes the current taxonomy, epidemiology, and transmission of the bacteria; their clinical significance; and optimal laboratory procedures for isolation and identification of this group of organisms and includes tables to aid the latter.
Chapter 53 : Clostridium*
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Severe infections caused by Clostridium species have been described in the medical literature for centuries—largely because of their fulminant nature, distinctive clinical presentations, and complex management issues. The pathogenesis of these infections is attributable to the production of potent extracellular toxins, many of which are also diagnostically important. This review highlights the microbiological, epidemiological, and clinical characteristics of these infections, with emphasis on both classical and state-of-the-art identification methods and typing systems. The rapid and severe course of these infections mandates clear communication between the clinical microbiology laboratory and the health care provider in order to institute early interventions and appropriate antimicrobial treatment. Current antimicrobial susceptibilities among the clostridia are also presented, as are the evaluation, interpretation, and reporting of clinical microbiology laboratory results. Application of this integrated body of knowledge can reduce the incidence, lessen the severity, and improve outcomes of these devastating clostridial infections.
Chapter 54 : Bacteroides , Porphyromonas , Prevotella , Fusobacterium , and Other Anaerobic Gram-Negative Rods*
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Obligately anaerobic, Gram-negative rods of clinical relevance belong mainly to the phyla Bacteroidetes and Fusobacteria. Anaerobes are detected typically in polymicrobial infections associated with mucosal surfaces close to the site where they reside; e.g., members of the Bacteroides fragilis group are often involved in intra-abdominal infections, with considerable morbidity and mortality. When Gram-negative anaerobes gain entrance to the bloodstream and trigger a systemic inflammatory response, this may result in sepsis or infective endocarditis, with a fatal outcome. Knowledge abou
t the resident microbiota and awareness of their role in disease permit clinicians to anticipate the likely infecting species at different body sites. Considering the current reports of considerable frequencies of anaerobic bacteremia and fusobacterial infections, especially Fusobacterium necrophorum-associated invasive diseases, and increasing rates of resistance of Bacteroides spp. to various antimicrobials, training for anaerobic techniques and introduction of advanced methods for the detection and precise identification of anaerobes are needed in clinical microbiology laboratories. New methods, such as matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) analysis, are increasingly used, but novel Gram-negative anaerobes are rather poorly integrated into the databases. Also, most laboratories neither perform the accurate species-level identification of the isolated organism nor test the susceptibilities of anaerobic isolates. Without knowledge of the local susceptibility patterns, the choice of proper antimicrobial therapy can be hampered and make the treatment outcome of anaerobic infections less predictable.
Curved and Spiral-Shaped Gram-Negative Rods
Chapter 55 : Algorithms for Identification of Curved and Spiral-Shaped Gram-Negative Rods
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Curved and spiral-shaped bacteria have a common microscopic morphology but represent diverse bacterial pathogens. Most bacteria in this group are isolated from patients with gastrointestinal tract-related infections. Campylobacter spp., Helicobacter spp., Arcobacter spp., and Vibrio spp. are the common species in the gastrointestinal tract. Other species, such as Desulfovibrio spp., Sutterella, Wolinella, and Anaerobiospirillum, may be isolated from other sources. The spirochetes Borrelia spp. and Leptospira spp. cause systemic infections and may be isolated in the lab with specialized methods. Treponema spp. of clinical importance are diagnosed using microscopic, serologic, and molecular methods.
Chapter 56 : Campylobacter and Arcobacter
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Campylobacter species and Arcobacter species make up a large group of organisms that may colonize or cause infections in humans and animals. The family Campylobacteraceae currently includes 24 species within the genus Campylobacter and 18 species in the genus Arcobacter. Campylobacter and Arcobacter species have somewhat unique growth and phenotypic characteristics, including microaerobic growth requirements, making this group of organisms challenging to isolate under laboratory conditions. Within the genus Campylobacter, C. jejuni subsp. jejuni remains the most common species isolated from human infections. Arcobacter butzleri appears to be the most common Arcobacter species causing human infection. Except for a few common species, molecular methods are now the preferred techniques for identifying Campylobacter and Arcobacter species.
Chapter 57 : Helicobacter*
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Helicobacter pylori is a major global human pathogen, and this chapter outlines the current state of knowledge regarding its diagnosis, treatment, and epidemiology. In addition, the genus Helicobacter is expanding, with more than 30 species now described. The chapter also covers these where appropriate and their impact on human health. Topics include a brief description of the current taxonomy of the genus, the current approaches to diagnosis, and identification. There is a section on antimicrobial susceptibility testing and the current levels of antimicrobial resistance in different countries around the world. The role of the non-H. pylori gastric Helicobacter species is also discussed.
Chapter 58 : Leptospira
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Leptospira is a genus of spiral-shaped bacteria that comprises two broad groups: a saprophytic group that inhabits fresh water and a pathogenic group that infects animals and may cause chronic renal infection, with intermittent urinary excretion. Infected animals become reservoirs of infection, either through contamination of the environment or by direct contact with other animals. Many animals can become reservoir hosts of Leptospira, but small mammals, especially rodents, are common hosts. Livestock animals can also become chronically infected, and economic losses due to loss of production and abortion are significant. Humans are dead-end hosts in whom a wide spectrum of symptoms may occur, from asymptomatic seroconversion to severe multisystemic disease with high mortality. Human infections are acquired through occupational activities, and increasingly through travel and sporting activities in countries with a warm climate. The genus is divided into 21 species. Several hundred serovars are defined by cross-agglutinin absorption. Major efforts to sequence several hundred genomes will make possible major advances in understanding the genus in the next few years.
Chapter 59 : Borrelia
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Borreliae are divided into relapsing fever (RF) and Lyme borreliosis groups. All species are vectored by blood-feeding arthropods and, with few exceptions, cause zoonotic infections, with humans being rare and dead-end hosts. Human infection is most commonly self-limited, may be asymptomatic, or may cause disease ranging from acute and relapsing fevers to staged illness that may involve the skin and musculoskeletal, neurological, and cardiovascular systems. RF borreliae are vectored by lice or ticks. Louse-transmitted RF has been known to humans for thousands of years and has been the cause of massive epidemics. In contrast, tick-transmitted RF was first described only in the late 1800s, and human disease, which presents in smaller clusters, is now known to occur throughout most of the world. In parts of northwest Africa, it is one of the most common bacterial infections, and in Tanzania, it is a leading cause of prenatal and child mortality. Lyme borreliosis was reported in 19th-century literature from Europe. The disease garnered little notoriety and was largely unknown until 1975, when a group of parents in Lyme, CT, expressed concerns of a seeming epidemic of inflammatory arthritis among local children. Ensuing investigations linked antecedent tick bites with rashes and arthritis among children and adults. The causative agent, Borrelia burgdorferi, and transmission by ticks were described shortly thereafter. Cases reported to the CDC have increased steadily, and it has become and remains the most prevalent vector-borne disease in North America. Lyme borreliosis is thought to be significantly underreported in the United States, Canada, and much of Europe.
Chapter 60 : Treponema and Brachyspira , Human Host-Associated Spirochetes
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Treponema and Brachyspira represent two clusters of the phylum Spirochetes that are either pathogenic or human host associated. Treponema pallidum subsp. pallidum, endemicum, and pertenue are the agents of venereal syphilis, endemic syphilis, and yaws, respectively, while Treponema carateum causes pinta. Numerous treponemal phylotypes in the oral cavity cause gingivitis or periodontitis. Brachyspira aalborgi, Brachyspira pilosicoli, and Brachyspira hominis cause human intestinal spirochetosis. T. pallidum subsp. pallidum is the only treponeme transmitted by sexual contact and vertically from a pregnant woman to her fetus; it is alsoe only pathogenic treponeme that regularly breaches the blood-brain barrier. Venereal syphilis remains a significant public health problem worldwide. Methods of laboratory identification of both Treponema and Brachyspira include direct detection methods such as dark-field microscopy, immunohistochemistry, and PCR. However, while Brachyspira can be cultured, syphilis diagnosis is highly dependent on serologic testing since T. pallidum can only be isolated by inoculation of rabbits (rabbit infectivity testing). Serologic testing for syphilis involves nontreponemal tests that detect antibodies directed against lipoidal antigens and treponemal tests that detect IgM and IgG antibodies directed against T. pallidum protein antigens. Newer treponemal assays now include enzyme immunoassays, point-of-care tests, chemiluminescence assays, immunoblots, and multiplex flow immunoassays, the majority of which use recombinant T. pallidum antigens. In-depth knowledge of these assays and their performance, as well as the application of the reverse syphilis screening algorithm, is critical for both laboratory and clinical practice.
Mycoplasmas and Obligate Intracellular Bacteria
Chapter 61 : General Approaches to Identification of Mycoplasma, Ureaplasma, and Obligate Intracellular Bacteria
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Mycoplasma, Ureaplasma, and obligate intracellular bacteria (Anaplasma, Chlamydia, Coxiella, Ehrlichia, Orientia, Rickettsia, and Tropheryma) differ from others by several characteristics. This includes the lack of efficiency for characterization using Gram stains and, except for Mycoplasma and Ureaplasma species and to a limited extent Coxiella burnetii and Tropheryma whipplei, the obligatory requirement for intracellular growth within a eukaryotic host cell. Thus, the most frequently used tests in clinical microbiology laboratories, Gram staining and axenic culture, are inefficient for detection and diagnosis. As a result, this has often been accomplished by an alternative staining method, such as Giemsa and Wright stains of clinical samples, by histopathologic or direct fluorescent-antibody analysis of biopsy samples; or most often by detection of antibody responses using a variety of serologic tests. The application of nucleic acid tests is now the standard practice for some of these bacteria. Additionally, improvements in axenic and cell culture methods have significantly enhanced their detection and the ability to render laboratory-based diagnoses for the diseases that they cause.
Chapter 62 : Mycoplasma and Ureaplasma
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Class Mollicutes, which includes organisms in the genera Mycoplasma and Ureaplasma, represents a unique group of cell wall-less prokaryotes that are obligate parasites of humans, animals, plants, or insects and represent the smallest known free-living microorganisms. There are at least 16 species that have been isolated from humans, several of which are proven causes of diseases that affect primarily the respiratory or urogenital tracts, where they reside on mucosal surfaces and sometimes invade deeper tissues. The most important pathogens of humans are Mycoplasma pneumoniae, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma urealyticum, and Ureaplasma parvum. Diagnostic test methods include culture, serology, and molecular-based assays. The slow growth rates and the fastidious growth requirements for some species such as M. genitalium and M. pneumoniae limits the use of culture for patient management purposes. However, for rapidly growing species such as M. hominis and Ureaplasma spp., culture is used for detection in clinical specimens, but specialized media and techniques are required that are not widely used in many clinical laboratories. Molecular-based assays are becoming more commonly available for detection of M. pneumoniae and are gradually supplanting serology for primary diagnosis of respiratory infections with this organism. Various methods of molecular strain typing have been described for the most important mycoplasmas of humans, mainly for epidemiological purposes. Antimicrobial resistance to commonly used drugs can occur in all of the human mycoplasmas. Standardized methods and quality control parameters have now been described for in vitro antimicrobial susceptibility testing for human mycoplasmas, making it possible to identify clinically significant drug resistance.
Chapter 63 : Chlamydiaceae*
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The Chlamydiaceae contain the known human pathogens C. trachomatis, C. pneumoniae, and C. psittaci as well as organisms such as C. abortus and C. felis that have been only rarely associated with human infections. C. trachomatis is currently divided into 18 serovars. Serovars A, B, Ba, and C can be isolated from patients with clinical trachoma, whereas D-K are urogenital serovars, and L1-3 cause lymphogranuloma venereum. C. pneumoniae causes infections of the upper and lower respiratory tracts such as sinusitis, pharyngitis, bronchitis, and pneumonia. C. psittaci can be readily transmitted to humans either by direct contact with infected birds or following inhalation of aerosols from nasal discharges and from infectious fecal or feather dust. Some strains previously designated as C. psittaci have been placed into several animal species such as C. abortus, C. muridarum, C. suis, C. felis, and C. caviae. This chapter covers specimen collection, specimen processing, isolation procedures, diagnostic assays, biosafety, identification procedures, and typing systems, as well as serological procedures, treatment, and nucleic acid amplification tests (NAATs). NAATs are recommended by the Centers for Disease Control and Prevention for the detection of C. trachomatis in urogenital samples. Newly approved specimen types for these assays include vaginal swabs and urines. There are now five United States Food and Drug Administration-cleared NAAT assays that can be used for the diagnosis of C. trachomatis.
Chapter 64 : Rickettsia and Orientia
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Rickettsia and Orientia are obligately intracellular bacteria that are transmitted by ticks, mites, fleas, and lice. Disseminated endothelial infection results in febrile illness, often manifesting as headache, myalgia, and rash and, in severe cases, interstitial pneumonia and encephalitis. Rocky Mountain spotted fever, Rickettsia parkeri infection, murine typhus, and rickettsialpox are endemic in the United States. Scrub typhus, caused by Orientia tsutsugamushi, is a highly prevalent disease in southern and eastern Asia and islands of the western Pacific and Indian Oceans. African tick bite fever occurs frequently in travelers returning from South Africa. Laboratory-confirmed diagnosis is seldom achieved during the acute stage of illness, when therapeutic decisions are crucial, although effective direct detection of rickettsiae by immunohistochemical and molecular methods is possible. Serologic methods, the mainstay of diagnosis, do not usually detect antibodies until the second week of illness. Seroconversion or a 4-fold rise in titer during convalescence emphasizes that this approach provides a retrospective diagnosis. Cultivation of rickettsiae requires antibiotic-free cell culture and a biosafety level 3 biocontainment facility and procedures and does not provide a timely diagnosis.
Chapter 65 : Ehrlichia, Anaplasma, and Related Intracellular Bacteria
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Members of the genera Ehrlichia and Anaplasma are now recognized to be important human pathogens. Ehrlichia and Anaplasma species infect bone marrow-derived cells, such as granulocytes, monocytes, erythrocytes, and platelets, of humans and other mammals. Ehrlichia and Anaplasma spp. are Gram-negative obligate intracellular bacteria that reside and propagate within membrane-lined vacuoles in the cytoplasm of the bone marrow-derived cells. They are zoonotic agents transmitted to animals and humans by ticks. Recognized natural reservoirs for Ehrlichia chaffeensis include deer, domestic dogs, and perhaps other animals that host Amblyomma ticks. The causative agent of human monocytic ehrlichiosis (HME) is E. chaffeensis, a monocytotropic ehrlichia first identified as a human pathogen in a patient with a severe febrile illness after tick bites in 1986. The causative agent of human granulocytic anaplasmosis (HGA) is Anaplasma phagocytophilum. Other human ehrlichioses include Venezuelan human ehrlichiosis, E. ewingii ehrlichiosis, Ehrlichia muris-like agent ehrlichiosis, “Candidatus Neoehrlichia mikurensis” ehrlichiosis, and neorickettsiosis. Currently, there are 3 methods for diagnosis of acute HME or HGA: (i) PCR amplification of nucleic acids from Ehrlichia or Anaplasma species in blood, (ii) detection of morulae in the cytoplasm of infected leukocytes by Romanowsky stains (e.g., Giemsa or Wright) or by specific immunostains using E. chaffeensis or A. phagocytophilum antibodies, and (iii) culture of Ehrlichia or Anaplasma from blood or cerebrospinal fluid. Although disease can be severe or fatal, in retrospective studies and routine clinical practice, patients with either HME or HGA defervesce within 48 h of therapy with doxycycline, the drug of choice.
Chapter 66 : Coxiella*
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Q or “Query” fever is an infection in humans caused by the Gram-negative bacterium Coxiella burnetii. The disease occurs worldwide and most often presents as an acute febrile illness, although chronic disease may occur in the form of endocarditis or vascular infections. Livestock, particularly cattle, goats, and sheep, are considered the primary reservoirs for the agent, although many other veterinary species may be infected. Bacteria shed by infected livestock are infectious, and large numbers of bacteria can be released during parturition. Certain high-risk occupations have been identified, such as abattoir workers and dairy farmers; however, the aerosol transmission route, low infectious dose (<10 organisms), and stability in the environment can result in wind-borne transmission distal to the veterinary source. Antibiotic treatment is effective, with doxycycline the drug of choice for acute infections; treatment of chronic disease requires long-term therapy, with both doxycycline and hydroxychloroquine currently recommended. A human vaccine (Q-VAX) has been licensed but is currently only available in Australia. Diagnostic methods include serology, molecular methods, immunohistochemistry, and isolation, with this last restricted to high-containment (BSL-3) facilities. Genetic analyses based on whole genome sequencing, targeted gene sequencing, and genotyping methods have suggested that C. burnetii strains can vary based on reservoir host and geographic distribution. Although recent advances have improved our understanding of Q fever, further studies are needed to address the epidemiology, control, and prevention of the disease.
Chapter 67 : Tropheryma whipplei
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Classical Whipple’s disease is usually diagnosed by periodic acid-Schiff staining of small-bowel biopsy specimens. These show typical infiltrations of the lamina propria with macrophages containing granular inclusions which represent intracellular or ingested bacteria. PCRs to detect T. whipplei DNA in clinical samples are of growing importance to confirm histological results. Whipple’s disease requires long-term antibiotic treatment. In the case of relapse and failure of co-trimoxazole treatment, therapy with doxycycline plus hydroxychloroquine is an effective alternative. This chapter summarizes the latest findings on the epidemiology, biology, pathogenicity, and immunological control of this fastidious Gram-positive bacterium. One focus is on the clinical appearance, which is crucial for the initial suspicion of Whipple’s disease, the decision to initiate diagnostic tests, and the assessment of laboratory results. The chapter also covers the laboratory methods, presents a hierarchical scheme for the diagnosis of Whipple’s disease, and discusses the current treatment options.
Antibacterial Agents and Susceptibility Test Methods
Chapter 68 : Antibacterial Agents*
- Authors: James S. Lewis,, Karen Bush
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Antimicrobial chemotherapy has played a vital role in the treatment of human infectious diseases since the discovery of penicillin in the 1920s. Hundreds of antimicrobial agents have been developed, and multiple agents in a variety of classes are currently available for clinical use. However, the sheer numbers and continuing development of agents make it difficult for clinicians to keep up with progress in the field. Similarly, this variety presents significant challenges for clinical microbiologists, who must decide which agents are appropriate for inclusion in routine and specialized susceptibility testing. This chapter provides an overview of the antibacterial agents currently marketed in the United States, with major emphasis on their mechanisms of action, spectra of activity, important pharmacologic parameters, and toxicities. Antibiotics that have fallen into disuse or remained investigational are mentioned only briefly.
Chapter 69 : Mechanisms of Resistance to Antibacterial Agents*
- Authors: Jean B. Patel, Sandra S. Richter
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This chapter describes mechanisms of antibiotic resistance in bacteria.
Chapter 70 : Susceptibility Test Methods: General Considerations
- Author: John D. Turnidge
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The main objective of susceptibility testing is to predict the outcome of treatment with the antimicrobial agents tested. The test results are generally reported to the treating physician, using the categories of susceptible, intermediate, and resistant. From the laboratory perspective, the key decisions involve the selection of a susceptibility testing method and the antimicrobial agents to be tested for each specimen and pathogen type. The principal methods used internationally are those of the Clinical and Laboratory Standards Institute (CLSI), based in the United States, and the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Both organizations have a sophisticated approach to the setting of breakpoints (interpretive criteria) which permit the categorization of the susceptibility test results. This approach includes the collation and analysis of MIC distribution data, pharmacokinetic and pharmacodynamic data and associated target values, and clinical and bacteriological response rates from prospective clinical trials. This analysis is followed by the correlation of MICs and zone diameter distributions in order to establish breakpoints for commonly used methods. Modern susceptibility testing methods are often supplemented by specialized phenotypic and/or molecular testing to ensure that critical resistances and resistance mechanisms are detected.
Chapter 71 : Susceptibility Test Methods: Dilution and Disk Diffusion Methods*
- Authors: James H. Jorgensen, John D. Turnidge
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This chapter describes reference dilution (MIC) and disk diffusion antimicrobial susceptibility test methods for nonfastidious, aerobic bacteria. It describes CLSI (United States) and EUcst (European) reference methods and standardized disk diffusion methods. The procedure for performing each test type is described as well as quality control procedures. The advantages and disadvantages of each method are discussed. Finally, the most current interpretive breakpoint criteria for MIC and disk tests from the CLSI are listed.
Chapter 72 : Antimicrobial Susceptibility Testing Systems*
- Authors: James A. Karlowsky, Sandra S. Richter
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This chapter focuses primarily on commercial susceptibility testing systems currently available in the United States. Semiautomated disk diffusion and manual and semiautomated broth microdilution systems are utilized for small volumes of susceptibility testing, while larger laboratories often choose an automated broth microdilution system. The broth microdilution antimicrobial susceptibility testing (AST) systems are manufactured by four companies: bioMérieux (Durham, NC), Siemens Healthcare Diagnostics (Deerfield, IL), Becton Dickinson Diagnostics (Sparks, MD), and Thermo Scientific (Cleveland, OH; formerly TREK Diagnostic Systems). The BIOMIC V3 system (Giles Scientific, Santa Barbara, CA) has FDA clearance to read disk diffusion zone sizes and broth microdilution panel wells. AST systems include data management software that may be interfaced with a laboratory information system and offer various levels of expert system and epidemiological analyses. Expert analysis may improve work flow as well as the quality of reported results. Advantages of automated AST systems include labor savings, reproducibility, data management with expert system analysis, and the opportunity to generate results more rapidly. Disadvantages of automated systems include higher cost for equipment and consumables than with manual methods, predetermined antimicrobial panels, an inability to test all clinically relevant bacteria and antimicrobial agents, and problems with detection of heteroresistant isolates and some resistance phenotypes. The chapter summarizes published literature on the abilities of automated systems to detect antimicrobial resistance in Gram-positive and Gram-negative pathogens (e.g., oxacillin resistance in staphylococci, extended-spectrum β-lactamase-producing Enterobacteriaceae). The provision of more-rapid AST results with a short-incubation system may improve patient care and lower health care costs.
Chapter 73 : Special Phenotypic Methods for Detecting Antibacterial Resistance*
- Authors: Brandi M. Limbago, Jana M. Swenson
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No descriptions available.
Chapter 74 : Susceptibility Test Methods: Fastidious Bacteria*
- Authors: Romney M. Humphries, Janet A. Hindler
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This chapter summarizes key considerations for antimicrobial susceptibility testing of fastidious and infrequently isolated bacteria. Up-to-date data on the global incidence of resistance, as documented by international, national, and local surveillance studies for these organisms, are presented. Important resistance mechanisms are discussed for many organisms, including Streptococcus spp., Haemophilus influenzae, Neisseria gonorrhoeae, and Neisseria meningitidis. This chapter also describes standard methods for testing the infrequently isolated or fastidious bacteria included in the CLSI M100 standard and M45 guideline.
Chapter 75 : Susceptibility Test Methods: Anaerobic Bacteria*
- Authors: Audrey N. Schuetz, David W. Hecht
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Infections due to anaerobic bacteria are common and have the capacity to be life threatening. Anaerobic bacterial resistance is increasing globally among many different anaerobes for a wide range of antibiotics. Thus, clinicians and laboratorians can no longer assume susceptibility of anaerobes without performing the testing. Various methods of susceptibility testing are reviewed in this chapter, including the Clinical and Laboratory Standards Institute (CLSI) agar dilution and broth microdilution methods. Additionally, commercial methods of susceptibility testing for anaerobes are discussed. The advantages and disadvantages of each testing method are emphasized. Specific patterns of resistance are covered for Gram-negative and Gram-positive anaerobes. Finally, specific recommendations for antibiotic testing and reporting, including the use of susceptibility antibiograms, are presented.
Chapter 76 : Susceptibility Test Methods: Mycobacteria, Nocardia, and Other Actinomycetes
- Authors: Gail L. Woods, Shou-Yean Grace Lin, Edward P. Desmond
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This chapter has four major sections. First is a brief review of antimicrobial agents used to treat mycobacterial infections. This is followed by a discussion of methods used to test susceptibility of Mycobacterium tuberculosis complex to first- and second-line agents. Methods described are agar proportion, rapid broth systems, broth microdilution, and molecular techniques, which are being used with increasing frequency. The next topic is susceptibility testing of nontuberculous mycobacteria by broth microdilution, focusing on Mycobacterium avium complex, Mycobacterium kansasii, Mycobacterium marinum, and the rapidly growing mycobacteria. The last section covers susceptibility testing of Nocardia species by broth microdilution and includes a brief discussion of other aerobic actinomycetes.
Chapter 77 : Molecular Detection of Antibacterial Drug Resistance
- Authors: April N. Abbott, Ferric C. Fang
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This chapter provides an overview of current FDA-cleared molecular assays and selected assays in development for the detection of antibiotic resistance.
VOLUME 2
VIROLOGY
General
Chapter 78 : Taxonomy and Classification of Viruses
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This chapter describes the rules and processes utilized for the taxonomic classification of viruses. Taxonomy at its most basic level involves the classification and naming of objects. Living objects have been grouped for hundreds of years according to the Linnaean system, a classification scheme that places living things hierarchically into groups of species followed by groupings into higher-level taxa dependent on common shared characteristics. Taxonomy functions beyond mere categorization. By having information about, and an understanding of, a few of the organisms in a group of closely related taxa, it is often possible to extend that knowledge to other organisms in related taxa for which much less biological information may be available. Virus classification and taxonomic assignment are the responsibility of the International Committee on Taxonomy of Viruses (ICTV), which has been charged with the task of developing, refining, and maintaining a universal viral taxonomy. The ICTV currently recognizes five hierarchical ranks that are used to define the universal viral taxonomy: the order, family, subfamily, genus, and species. The 2012 ICTV viral taxonomy comprises 7 orders, 96 families, 22 subfamilies, 420 genera, and 2,618 species.
Chapter 79 : Specimen Collection, Transport, and Processing: Virology*
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The importance of appropriate specimen collection and handling to ensure accurate laboratory results cannot be overstated. This aspect of the preanalytical phase of laboratory testing is often the most vulnerable part of the testing process and accounts for many of the errors in laboratory diagnostics. The preanalytical steps for viral diagnostics involving specimen selection, collection, transport, and processing are described in this chapter. It is important for the laboratory to ensure that many of these variables are controlled to maintain specimen integrity. The laboratory should serve as a resource for clinicians and those collecting patient specimens to ensure that only quality samples are collected, processed, and tested and that results are reliable and meaningful.
Chapter 80 : Reagents, Stains, Media, and Cell Cultures: Virology
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Despite the integration of nucleic acid-based diagnostic methods into routine clinical virology laboratory practices, direct fluorescent-antibody assays (DFAs) using monoclonal antibodies and culture-based methods are still an integral part of viral diagnostics. DFAs can provide a rapid diagnosis when used directly with clinical samples. In addition, DFAs are used for confirmation of virus identity when cytopathic effect is noted in traditional cell culture and to identify viral proteins by blind staining when rapid centrifugation-enhanced cell culture is used. Cell culture methods are useful for the detection of novel or unsuspected viral agents, for documentation of active infection, to obtain isolates for antiviral susceptibility testing, to assess response to antiviral therapy, for serologic strain typing, and for vaccine and therapeutic clinical trials. This chapter reviews the reagents, stains, tissue culture media, and traditional cell culture methods currently used in diagnostic virology laboratories. Rapid cell culture methods using single cell lines, cocultured cell lines, and transgenic cell lines are described. Quality control and safety regarding the handling and culturing of virology samples is discussed.
Chapter 81 : Algorithms for Detection and Identification of Viruses
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Virology is a dynamic field that in recent decades has moved from the periphery to the mainstream of clinical laboratory practice. Since the last edition of this Manual, the dramatic growth in the development and implementation of clinically useful molecular methods has accelerated. This trend has been facilitated by the increasing availability of commercial reagents and FDA-cleared kits, some requiring minimal molecular expertise. Laboratories without classical virology or molecular expertise can now implement state-of-the-art viral molecular testing. In addition to the advantages of molecular testing, some pitfalls are also becoming apparent as the tests are more widely used. Cost and value are increasing concerns. As we move forward, it is critical that laboratorians communicate with one another to address problems and optimize and standardize methods, as well as communicate with clinicians to guide appropriate usage and interpretation and to encourage input and feedback.
RNA Viruses
Chapter 82 : Human Immunodeficiency Viruses*
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HIV is the etiologic agent of AIDS. HIV virions are enveloped positive-strand RNA retroviruses and are classified based on their degree of phylogenetic relatedness into types (HIV-1 and HIV-2), groups, subtypes, sub-subtypes, and recombinant forms. Approximately 75 million people worldwide have become infected with HIV. In the United States, the CDC estimates that 1.1 million persons are living with HIV. Previously, diagnosis was most often made by detection of antibody against HIV. HIV infection can now be expeditiously and more accurately identified during its highly infectious acute phase using a diagnostic algorithm. The algorithm employs an initial HIV-1/2 antibody/p24 antigen combination immunoassay followed by an antibody assay that differentiates HIV-1 from HIV-2 and, if necessary, a nucleic acid test to identify HIV RNA before antibodies develop. Chemiluminescent immunoassays that allow rapid processing on random-access analyzers are now available. Single-use rapid point-of-care antibody and antibody/p24 antigen combination assays that can be used with finger-stick whole blood or with oral fluids facilitate testing outside traditional laboratories. Qualitative DNA and RNA assays can diagnose acute HIV infection before seroconversion and also in infants born to HIV-infected mothers, in whom antibody tests are unreliable because of maternal antibody reactivity. Effective antiretroviral therapy delays progression of HIV disease, improves survival, and reduces infectiousness. Genotypic and phenotypic resistance assays guide the selection of antiretroviral regimens, and quantitative viral load assays that can detect fewer than 50 viral copies per ml are useful for prognosis and for monitoring response to therapy.
Chapter 83 : Human T-Cell Lymphotropic Viruses
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Human T-cell lymphotropic viruses (HTLVs) are complex retroviruses that consist of four major groups (HTLV-1, HTLV-2, HTLV-3, and HTLV-4) of which two (HTLV-1 and -2) have spread globally to infect millions of persons and are known to cause neurological and neoplastic diseases. Phylogenetic analysis with simian T-cell lymphotropic viruses (STLVs) show that HTLV-1, HTLV-2, and HTLV-3 all likely originated from multiple cross-species infections. A simian counterpart of HTLV-4 has recently been found in gorillas in Cameroon. Although an understanding of the epidemiology of HTLV-3 and -4 infection is limited by identification of small numbers of infected persons in Cameroon, HTLV-1 and -2 are transmitted sexually, vertically from mother to-child, and parenterally by blood transfusion and by sharing needles during intravenous drug use. Blood screening has limited the blood-borne transmission of HTLV-1 and -2, except in resource-limited countries like those in Africa where blood screening for these viruses is not done. HTLV testing is performed using standard serological and molecular tools. Disease has been associated mostly with only HTLV-1 infection and occurs in less than 5% of infected persons. Unlike other complex retroviruses, HTLVs encode proteins on the minus strand, called antisense proteins of HTLV (APH), that are believed to play a role in viral replication and disease. Much more research is needed to understand how HTLV causes disease, to determine the geographical distribution of HTLV-3 and -4, and to evaluate whether HTLV-3 and -4 can be transmitted from person to person and cause disease.
Chapter 84 : Influenza Viruses
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Each year, influenza viruses are a major cause of febrile respiratory illness and mortality. Two patterns of infection occur in temperate climates: seasonal influenza, which is associated with annual epidemics, and pandemic influenza. Seasonal influenza is caused by influenza type A or B viruses, while pandemics are caused only by influenza type A strains. Avian strains, including A/H5N1 and A/H7N9 viruses, are a potential source of pandemic viruses. Circulation of influenza in a community should be suspected when school or work absenteeism increases in association with increases in febrile respiratory illness. Molecular assays, including real-time reverse transcriptase PCR (RT-PCR), have become the gold standard for influenza diagnosis. Rapid influenza diagnostic tests (RIDTs) are commonly used point-of-care assays that can provide results in less than 30 minutes. Although these assays have good specificity (greater than 90%), their low sensitivity means that a negative result does not exclude influenza as a diagnosis. Immunofluorescence is another antigen detection method, and it may be combined with culture to identify infection 24 to 48 hours after sample collection. Serodiagnostic methods are used in epidemiological studies but are not generally useful in the management of acute illness. Two classes of antiviral drugs, adamantanes and neuraminidase inhibitors, are licensed for treatment of acute influenza. Resistance to these drugs may be present in circulating strains or may develop during therapy. Genotypic and phenotypic methods are available to identify resistant strains.
Chapter 85 : Parainfluenza and Mumps Viruses
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The human parainfluenza (HPIV) and mumps viruses share the Paramyxoviridae family and are included within the Paramyxovirinae subfamily in the Respirovirus genus (HPIV-1 and HPIV-3) and the Rubulavirus genus (HPIV-2, HPIV-4, and mumps). The HPIVs are commonly encountered in the U.S. as the agent of upper and lower respiratory infections in children and in older adults and the immunocompromised. Virology laboratories routinely isolate the HPIVs in culture and detect their antigens and nucleic acids in clinical samples. New multiplex viral respiratory panels include detection of the HPIVs. Mumps, although sometimes seen in outbreaks in the U.S., is seldom encountered in the routine virology laboratory, and methods for mumps virus detection are not widely available. Serologic testing to determine mumps immune status is performed in most laboratories, but confirmation of acute infection, unless the virus can be isolated in culture, must be referred to reference laboratories that provide these services. Diagnosing acute mumps infection is especially challenging in individuals who have been immunized or have experienced the infection previously. This situation presents a challenge in diagnosis.
Chapter 86 : Respiratory Syncytial Virus and Human Metapneumovirus*
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Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are recognized as the most serious causes of severe acute lower respiratory tract illnesses in young children and infants. Rapid and accurate detection and characterization of RSV is important in the clinical management of individual patients because of the availability of specific antiviral drugs. Early detection of RSV and HMPV infections is also necessary for cohorting of infected patients to prevent nosocomial spread. Tube and shell viral cultures remain the gold standard for RSV and HMPV detection, but they are time consuming and require attention to transport and prompt inoculation. Quite a few of rapid viral antigen tests are commercially available, which yield results within 1 hour after specimen collection. However, their sensitivity is limited and negative results usually require a more sensitive back-up test such as a molecular assay or culture. With the increased number of multiplexed molecular Food and Drug Administration (FDA)-approved assays, simultaneous detection and identification of common viral pathogens is becoming the main tool for detection and identification of RSV and MPHV.
Chapter 87 : Measles and Rubella Viruses
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Clinical differentiation of fever and rash illnesses caused by measles and rubella viruses has become increasingly difficult. The paucity of measles and rubella cases in the United States and other developed countries has led to a decline in clinical diagnostic acumen among physicians and health care workers. Milder forms of measles have been reported to occur in previously vaccinated individuals, further obscuring diagnosis based on the clinical case definition. As global programs to control and eliminate measles expand, the medical and public health communities have become more dependent on laboratory confirmation of clinical diagnosis. Those countries that have controlled and eliminated measles through a variety of vaccination strategies, are now controlling and eliminating rubella via the incorporation of rubella and measles combined vaccines in their programs, in some cases taking advantage of new funding opportunities for rubella immunization activities. Laboratory diagnostic tests and laboratory surveillance activities for measles and rubella are performed in parallel in many clinical and public health laboratories worldwide. Consequently, this chapter combines the current laboratory diagnostic methods for measles and rubella for convenient review and reference. A summary of laboratory tests for confirmation of measles and rubella, including many of the tests described briefly herein, has been published by the World Health Organization.
Chapter 88 : Enteroviruses and Parechoviruses
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Enteroviruses (EV) and human parechoviruses (HPeV) are members of the Picornaviridae family, which are nonenveloped, positive-stranded RNA viruses with a 30-nm icosahedral capsid. This virus family exhibits a considerable amount of genetic variability and continues to evolve rapidly as the result of mutation and recombination. Based on phylogenetic analysis, the Enterovirus genus is divided into 10 species, 7 of which contain human viruses, including 3 Rhinovirus (RV) species. The Parechovirus genus comprises two species, Human parechovirus and Ljungan virus. Currently, there are more than 250 types of EV, including RV types, and 16 types of HPeV. The diversity of these virus families is also reflected in the clinical presentation, ranging from asymptomatic (most common) and acute nonspecific febrile illness, to herpangina and hand-foot-and-mouth disease, to myocarditis and central nervous system diseases including aseptic meningitis and acute flaccid paralysis. Laboratory testing is often essential for accurate diagnosis of EV and HPeV disease, however cell tropism diversity has led to difficulties with traditional culture methodologies. Hence, molecular methods are the recommended diagnostic tools, yet the availability of in vitro diagnostic (IVD)- or CE-marked tests is limited for EV and nonexistent for HPeV. An understanding of the sites where disease-induced replication occur is critical to the interpretation of EV and HPeV test results. Whereas detection of these viruses in the central nervous system, bloodstream, and lower respiratory tract implies true infection, detection from the nasopharynx and the gastrointestinal tract may represent past infection, as asymptomatic shedding for weeks to months is common.
Chapter 89 : Rhinoviruses
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Human rhinoviruses (HRVs) are members of the family Picornaviridae. Previously a separate genus, HRVs have been reclassified into three separate species (A, B, C) within the Enterovirus genus. HRV culture isolates were originally classified into 99 serotypes based on neutralization with type-specific antisera. The wider use of molecular methods has led to the discovery of a novel group of HRVs, classified as species “C.” HRV infections are widespread year-round, with peaks in autumn and late spring in temperate zones. HRVs cause about two-thirds of cases of the common cold and thus are responsible for more episodes of human illness than any other infectious agent. Recently, with the use of molecular methods, more severe consequences of HRV infections have been recognized. HRV has been detected in lower respiratory tract infections in patients of all ages hospitalized with wheezing or pneumonia and in patients with chronic illnesses, cancer, immunosuppressive illnesses, transplants, and underlying pulmonary disease such as asthma. While nucleic acid amplification assays have revolutionized HRV detection, the interpretation of a positive result can be problematic. HRV can be detected in asymptomatic persons, can be shed for weeks after respiratory symptoms resolve, are prone to cause serial infections, and can occur as coinfections with other viruses or bacteria. Thus, interpretation in the individual case often relies on risk factors and recovery of other pathogens. Further research is needed to determine the impact of HRV detection on patient management and develop effective therapies.
Chapter 90 : Coronaviruses*
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Coronaviruses (CoV) are enveloped viruses with large RNA genomes and are found in several animal species, including humans. Four coronaviruses HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1 are endemic in humans and associated with a range of upper and lower respiratory tract disease from the common cold to severe pneumonia. HCoVs make up a small but significant proportion of the viruses found in patients with acute respiratory illness (ARI), particularly in children and during the winter and spring months. The highly pathogenic CoVs responsible for severe acute respiratory syndrome (SARS-CoV) and Middle Eastern respiratory syndrome (MERS-CoV) have only recently emerged in the human population through zoonotic transmission. SARS-CoV has not been reported since early 2004 but caused significant morbidity and mortality during a global outbreak in 2002/2003. Since 2012, MERS-CoV has caused a number of cases of severe ARI linked to the Arabian Peninsula and transmission is still occurring. Isolation of CoVs is not effective as a routine diagnostic tool because many commonly used cell lines are not permissive for growth and cytopathic effect is generally nonspecific. Reagents for immunofluorescent detection are not widely available and serological testing is most useful for epidemiological studies and retrospective diagnosis. Direct detection of CoV nucleic acid in clinical specimens is the most common diagnostic method currently in use. A range of in-house RT-PCR methods have been developed for either pan-CoV or species-specific detection, and commercial nucleic acid amplification tests (NAATs) are also now available.
Chapter 91 : Hepatitis A and E Viruses
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Viral hepatitis is the general term for inflammatory disease of the liver caused by at least five different viruses, with hepatitis A, B, C, D, and E viruses having a definite association with acute viral hepatitis. Of these, only hepatitis A virus (HAV) and hepatitis E virus (HEV) are enterically transmitted, and both cause acute and generally self-limiting infections without significant long-term carrier status. While there are no serious long-term sequelae in patients who recover from hepatitis A or hepatitis E, both viruses are associated with significant risks of acute, fulminant hepatitis and liver failure, and chronic hepatitis E has been reported in immunosuppressed patients. Severe outcomes are most commonly seen in patients with acute hepatitis A who also have chronic hepatitis C or hepatitis B infection, and also in patients with acute hepatitis E during pregnancy, especially during the third trimester. In the absence of these cofactors, the diseases show a general trend towards greater severity with increasing age, with the majority of infections being subclinical in children; however, severe and fulminant hepatitis A and E infections can occur at any age. In many developed countries, HEV infection is rare and HAV infection rates have declined, making recognition and diagnosis more challenging. This chapter discusses recent advances in our understanding of HAV and HEV infections, including diagnostic testing and interpretation.
Chapter 92 : Hepatitis C Virus
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Hepatitis C virus (HCV) is a flavivirus that is endemic worldwide. Six genotypes are commonly recognized; a seventh rare genotype has been identified. Given its relative prevalence, this chapter focuses on genotypes 1 through 4. HCV is transmitted by blood-borne routes. Most acute infections are asymptomatic and progress to chronicity. Chronic infections are also asymptomatic until significant liver damage occurs. Pegylated alpha interferon plus ribavirin was the only treatment for many years. Highly potent, pan-genotypic, interferon-free direct-acting antiviral (DAA) regimens that cure >95% of clinical-trial subjects have been developed and will dramatically improve treatment. Routine diagnostics include serology, HCV RNA detection/quantification assays, and HCV RNA genotyping assays. Newer diagnostic tests include characterization of host genotype at loci upstream of IL28B that are associated with alpha interferon responsiveness, and HCV RNA tests to detect drug resistance mutations. Serology and HCV RNA detection/quantification tests are useful after a known exposure to identify acute infection. Serology is used to screen for chronic infection; seropositive individuals are then tested with HCV RNA detection/quantification assays to identify chronic infection. HCV genotype and viral load are determined prior to treatment initiation. In the pre-DAA era, HCV genotype was used to predict likelihood of response. It was also helpful to determine treatment regimen and therapeutic duration; these two uses will remain relevant for DAAs. Viral load testing during therapy will likely still be performed for assessment of medication compliance. IL28B genotype may retain some importance in predicting response rates in alpha interferon-containing regimens. Drug resistance tests thus far have limited applicability.
Chapter 93 : Gastroenteritis Viruses*
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Acute infectious gastroenteritis is one of the most common illnesses in humans resulting in a substantial burden to public health worldwide. Severe disease leading to hospitalizations, increased morbidity, and even death is observed among infants, children, elderly, and immunocompromised persons. Viruses are the predominant cause of gastroenteritis and include rotaviruses, noroviruses, sapoviruses, astroviruses, and adenovirus types 40 and 41. Laboratory testing is required for a reliable diagnosis of viral gastroenteritis, since the specific etiology of disease can be difficult to distinguish on the basis of clinical features alone. Recent advances in technology have resulted in the development of newer diagnostic assays that offer great promise for accurate detection of gastrointestinal viral infections. This chapter emphasizes the fundamental characteristics and clinical importance of viruses that cause gastroenteritis and highlights the laboratory methods that can be used to make a rapid and definitive diagnosis for the greatest impact on the care and management of ill patients and the prevention and control of hospital-acquired infections and community outbreaks due to enteric viruses.
Chapter 94 : Rabies Virus*
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This chapter contains the latest information on taxonomy of the genus Lyssavirus. Since the previous edition, there are three new Lyssavirus species, Shimoni bat virus, Bokeloh bat lyssavirus, Ikoma lyssavirus, and a proposed new lyssavirus, Lleida bat virus. The genetic diversity within the genus has a significant impact on the success of postexposure prophylaxis with current biologics. A description of the morphology and structure of the agent, the structural proteins of diagnostic significance, and the epidemiology and transmission of the disease are also presented. Although rare in affluent countries, more than 55,000 human cases of rabies occur annually. A section on the clinical significance of disease describes signs and symptoms at different stages. Although the successful treatment of a teenager from Wisconsin in 2004 has broadened treatment options, specific rabies antiviral treatment does not exist, and prognosis remains poor. Electronic resources for physicians and public health professionalsare provided, including the most current information concerning rabies diagnosis, experimental treatment protocols and consultation. Rabies prevention, including pre- and postexposure prophylaxis, is discussed according to the Advisory Committee on Immunization Practices (ACIP) guidelines for the United States. Presented are laboratory methods to confirm or rule out rabies in antemortem and postmortem humans and animals. Descriptions of the tests for detection of virus, antigen, antibodies, and nucleic acids and methods for virus (antigenic and genetic) typing are included. The interpretation of test results, sample requirements, and storage and transport conditions are mentioned. The information is provided to laboratory and public health professionals as a resource for information concerning rabies and other lyssaviruses.
Chapter 95 : Arboviruses
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This chapter provides a comprehensive overview of clinical diagnostic testing for most arboviruses as a general guide for clinical laboratory professionals. Arboviruses are transmitted to their vertebrate host via an arthropod vector. These unique viruses span seven distinct families of viruses, encompassing more than 500 viruses. The primary methods for arbovirus laboratory diagnosis are discussed. For many arboviruses, direct detection of virus by nucleic acid amplification tests (NAAT) and/or virus isolation are the most specific and sensitive tests available. However not all patients present for diagnostic testing during their viremic phase and not all arboviruses are detectable by the current tests during the acute phase of infection. Detection of specific immunoglobulin M and G by various methodologies is sensitive but less specific than viral detection methods due to cross-reactive epitopes within some families of arboviruses, thus confirmatory testing is usually necessary. The most effective test for laboratory diagnosis of an arbovirus infection is determined by the days post onset of symptoms of the patient and can be simplified to a single algorithm for all arboviruses. Since many of these virus or immunoglobulin detection tests have been commercialized, an overview of current tests available is included. This chapter summarizes the current laboratory methods for the diagnosis of human-acquired arbovirus infections, their epidemiology and transmission, and clinical significance.
Chapter 96 : Hantaviruses
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Hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) are rodent-borne zoonoses caused by certain members of the virus family Bunyaviridae, genus Hantavirus. Both syndromes are characterized by a febrile prodrome, thromobocytopenia, leakage of fluid from capillaries, and shock. Early diagnosis is critical to the successful management of HFRS and HPS, and—in the case of HPS caused by Andes virus—implementation of appropriate isolation procedures to prevent virus transmission to health care providers. Laboratory assays for evidence of hantaviral infections in HFRS cases and HPS cases during the acute phase of illness should include μ-capture enzyme-linked immunosorbent assay (ELISA) for anti-hantavirus IgM, ELISA for anti-hantavirus IgG, and RT-PCR assays for hantaviral RNA. Immunohistochemistry assays for hantaviral antigens in lung and other solid tissues often reveal widespread infection in the endothelium of the microvasculature. The results of assays for hantaviral RNA should be supported by the results of tests for hantavirus-specific antibodies or antigen. The proper controls for μ-capture ELISA, IgG ELISA, and immunohistochemistry assays are essential for correct interpretation of the results of these assays. Local laboratories usually do not have direct access to these materials. Thus, diagnostic testing is often limited to federal laboratories, reference laboratories, and a small number of commercial laboratories. Recovery from severe disease is absolutely dependent upon carefully managed supportive care, in particular the judicious administration of intravenous fluids during the hypotensive phase in HFRS and the cardiopulmonary phase in HPS.
Chapter 97 : Arenaviruses and Filoviruses
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Arenaviruses and Filoviruses are zoonotic viruses associated with one or more nonhuman host or vector species, mostly rodent and bats. Vascular leak, shock, and multi-organ dysfunction are prominent features and lead frequently to fatal hemorrhagic fever. However, confirming early clinical impressions requires further, specific virologic and serologic testing. A combination of several laboratory techniques should be used to confirm any clinical suspicion, keeping in mind that the manipulation of these dangerous viruses requires specific biosafety requirements. A correct understanding of the order of appearance of RNA, antigen, virus, antibodies in blood, secretions, tissues over the period of the infectious process, as well as accurate knowledge of a patient’s clinical status, is important to understand and interpret laboratory results. During the acute phase of the disease, the amplification and sequence of viral genome, PCR-based assays, are certainly the most sensitive assays, assuming that the primers match with an eventual new virus. The antigen capture ELISA seems to be less virus-specific and readily detects antigen during the obvious clinical phase of disease. Because of the time required for culture and the associated biohazard, virus isolation data for these viruses are usually available only retrospectively. A rising IgM or IgG ELISA titer constitutes a strong presumptive diagnosis. Early recognition of viral hemorrhagic fever infection is valuable, as it can trigger strict isolation procedures, thus preventing or limiting the spread of disease, and allowing specific treatment, when available, to be initiated early in the infectious process.
DNA Viruses
Chapter 98 : Herpes Simplex Viruses and Herpes B Virus
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Herpes simplex virus (HSV) types 1 and 2, formally designated human herpesvirus 1 and human herpesvirus 2, most commonly cause ulcerative orofacial or anogenital disease. Rare but more serious manifestations include herpes keratitis, encephalitis, and neonatal herpes. Primary HSV infection establishes latency within neurons of the trigeminal or dorsal root ganglia, from which HSV can reactivate causing recurrent viral shedding with or without symptoms. HSV asymptomatic shedding represents a major source of transmission. HSV can be cultured, but in recent years, DNA amplification techniques such as PCR have supplanted culture diagnosis in many labs. DNA amplification techniques are more sensitive than culture. This improved sensitivity is critical in certain settings such as HSV encephalitis, in which the viral load is very low. Several FDA-cleared amplification assays for HSV are now available, as are laboratory-developed tests from reference laboratories. Serologic testing can be useful in establishing serostatus in the absence of symptoms. Serologic tests should be type specific and target the HSV glycoprotein gG; other tests show poor discrimination between HSV-1 and HSV-2. Several FDA-cleared gG-based tests are available; these generally show good sensitivity and specificity relative to the gold standard Western blot. Finally, herpes B virus, also known as macacine (formerly cercopithecine) herpesvirus 1 or monkey B virus, can infect humans and cause lethal disease. Specimens suspected of containing herpes B virus should be handled carefully in consultation with CDC or the National B Virus Resource Laboratory.
Chapter 99 : Varicella-Zoster Virus
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Varicella-zoster virus (VZV) is a human herpesvirus and the cause of varicella (chicken pox). After primary infection, the virus remains latent in the human host and may later reactivate and cause herpes zoster (shingles). Although the diagnosis of uncomplicated VZV infection is often based on the typical clinical picture, application of laboratory diagnostic methods is of major importance in specific clinical situations, as for the diagnosis of neurological complications of VZV during infection and reactivation, especially in cases of zoster “sine herpete,” for diagnosis in the immunocompromised host or for the identification of the VZV serostatus, which may be especially important in pregnancy or prior to transplantation. In this chapter, various approaches to identify and quantify the virus directly in the human host are presented, as well as current methods to detect the antibody response against VZV. In addition, other special aspects of VZV diagnostics, such as the detection of cellular immune response against VZV, the detection of antibody avidity, and the characterization of VZV strains and discrimination between vaccination and wild-type strains, are described. The rational application of these methods and the correct interpretation of the test results in specific clinical situations are of high importance and are also discussed, as this has major implications for the clinical management of the patients.
Chapter 100 : Human Cytomegalovirus
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Cytomegalovirus (CMV) belongs to a large family of herpesviruses that commonly infect humans of all ages. Although infections with this virus are normally asymptomatic in healthy children and adults, CMV is one of the most notable viral pathogens causing serious diseases in newborns and individuals with compromised immune systems. This chapter emphasizes the fundamental characteristics and clinical importance of CMV and focuses on the laboratory tests that can be used to identify the virus and monitor and predict disease and susceptibility to antiviral therapy and the philosophy behind the interpretation and reporting of test results in various defined patient populations.
Chapter 101 : Epstein-Barr Virus
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Epstein-Barr virus (EBV) is associated with a wide variety of disease states in immunocompetent and immunosuppressed patients, ranging from infectious mononucleosis (IM) to malignant disorders. Tests for EBV infection diagnosis are used primarily for patients with suspected IM, for which antibody assays are the method of choice. A variety of immunoassays, blots, and flow cytometry tests have been used for EBV serology assays; assays targeting vial capsid antigen (VCA) IgG, VCA IgM, and EBV nuclear antigen 1 (EBNA 1) IgG are favored. Additional assays such as avidity testing or viral load measurement are only rarely necessary to establish the diagnosis of IM. The detection of heterophile antibodies is sometimes useful when a young adult with typical IM symptoms is being tested, but this test often lacks sensitivity and specificity, particularly when testing young children and older adults and when symptoms are atypical. In these settings, EBV-specific assays are preferred. Many EBV antibody-specific point-of-care tests that combine high sensitivity and specificity with rapid detection are currently available. Antibody assays detecting early antigen (EA) are rarely useful because of their heterogeneity with respect to the specific target detected and poor specificity. Assays that measure EBV in peripheral blood using quantitative nucleic acid amplification tests (QNAT) are increasing being used for immunosuppressed hematopoietic stem cell (HSCT) and solid-organ transplant (SOT) recipients and for patients with EBV-associated malignant disorders to prevent and diagnose disease and to monitor response to therapy. In most of these settings, the interpretation of assay results in relationship to patient outcomes is uncertain, and the usefulness of these assays has not been clearly proven. The best evidence exists for EBV DNA monitoring to trigger preemptive therapy for the prevention of early posttransplant lymphoproliferative disorders (PTLD) in high-risk HSCT and SOT patients, as well as for the diagnosis and treatment monitoring of nasopharyngeal carcinoma. Quantitative EBV DNA assays lack standardization, and recently an international reference standard for EBV DNA has been developed that may improve interassay result harmonization. QNAT values that could be used as trigger points for intervention remain undefined and may also vary by specimen type, clinical setting, and patient group. Dynamic changes in viral load, rather than absolute QNAT values alone, may be important in patient management.
Chapter 102 : Human Herpesviruses 6, 7, and 8
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Human herpesviruses 6A and 6B (HHV-6A and HHV-6B) and human herpesvirus 7 (HHV-7) are viruses of the Roseolovirus genus in the betaherpesvirus subfamily, along with human cytomegalovirus. Roseoloviruses share many features of their genomic architecture and genetic content and the ability to replicate and establish latent infections in lymphocytes. Clinically, they cause febrile rash illnesses in young children and are associated with a variety of neurologic disorders. They are opportunistic pathogens in immunocompromised patients. HHV-6A has been identified during early-life febrile disease in Africa and in Hashimoto’s thyroiditis; its clinical spectrum remains to be fully defined. HHV-6B is the main cause of roseola and related febrile rash illnesses in young children and is often active during posttransplant acute limbic encephalitis. HHV-7 causes a subset of roseola cases, and along with HHV-6B, has been identified in children with febrile status epilepticus. Human herpesvirus 8 (HHV-8), or Kaposi’s sarcoma-associated herpesvirus, belongs to the gammaherpesvirus subfamily (as does Epstein-Barr virus). HHV-8 causes Kaposi’s sarcoma, multicentric Castleman’s disease, and primary effusion lymphoma, all of which are more likely to occur in HHV-8-seropositive individuals who are immunocompromised. Diagnosis of these viruses most often involves detection of their genomes by PCR, complemented by immunohistochemistry. Roseolovirus diagnosis is most often employed in the context of acute neurologic illnesses in young children and in organ and hematopoietic stem cell transplant recipients. HHV-8 diagnosis is employed in distinguishing Kaposi’s sarcoma from its mimickers and for definitive diagnosis of its associated lymphoproliferative disorders.
Chapter 103 : Adenoviruses
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Adenoviruses are large, nonenveloped, icosahedral viruses with a worldwide distribution and a wide degree of genetic heterogeneity. They are responsible for a spectrum of diseases, both end-organ and systemic, affecting both immunocompetent and immunocompromised hosts. Signs and symptoms vary in severity as well, from mild upper respiratory tract infection to life-threatening pulmonary involvement. Rates of infection, morbidity, and mortality are especially high in pediatric hematopoietic stem cell transplantation patients. Diagnostic challenges reflect both the diversity of this agent and its myriad effects and sites of involvement. Methodologies historically have included phenotypic methods, such as cell culture, electron microscopy, antigen detection, and tissue pathology. These have been supplemented more recently by both qualitative and quantitative molecular amplification assays that may be applied both diagnostically and as screening modalities (particularly in high-risk populations). As more-effective therapeutics become available, the early recognition of such infections becomes more critical to optimal patient care.
Chapter 104 : Human Papillomaviruses
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Papillomaviruses (PVs) are small, nonenveloped DNA viruses that contain a circular (or episomal) double-stranded genome and an icosahedral capsid structure. The majority of identified human PVs (HPVs) with clinical significance are found in the genus Alphapapillomavirus, which includes types infecting the genital and nongenital mucosal and genital cutaneous surfaces as well as the genotypes associated with human cancers. The vast majority of HPV infections at all sites are subclinical and asymptomatic but can also cause a variety of cancers, including cervical, vaginal, penile, anal, and oropharyngeal cancer. This chapter will describe the epidemiology, pathogenesis, and clinical syndromes associated with HPV infections. The role of HPV testing in cervical cancer screening, current guidelines for HPV testing, and the methodologies available for both detection and genotyping of HPV will be explained.
Chapter 105 : Human Polyomaviruses
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Novel human polyomaviruses (HPyVs) continue to be reported, with a total of 12 HPyVs now reported in the literature. The original HPyVs, BK polyomoavirus (BKPyV) and JC polyomavirus (JCPyV), are important human pathogens, causing infections in immunocompromised patients. Merkel cell polyomavirus (MCPyV) appears to be the causative agent of some forms of Merkel cell carcinoma, a rare form of skin cancer. Another novel HPyV has been linked to trichodysplasia spinulosa, an uncommon skin disease seen in immunocompromised patients. The remaining novel HPyVs have been isolated from respiratory, stool, and skin samples and have not yet been linked to any specific human diseases. Clinical microbiology laboratories are therefore generally not concerned with testing for most of the newly identified HPyVs. Rather, it is molecular-based testing for BKPyV and JCPyV that is of greatest importance. While there are still no FDA-approved assays for these viruses, there are several analyte-specific and research use only reagents available. Testing for BKPyV is performed to monitor kidney transplant recipients for the development of polyomavirus-associated nephropathy and less commonly when searching for a cause of hemorrhagic cystitis. Detection of JCPyV is undertaken to make a diagnosis of progressive multifocal leukoencephalopathy, a disease of the central nervous system that occurs in patients with certain types of underlying immune dysfunction. Looking to the future, it is likely that more HPyVs will be identified. It will be interesting to see if any are linked to specific diseases or if they are simply part of what is being recognized as the human virome.
Chapter 106 : Parvovirus B19 and Bocaviruses
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Parvoviruses are small (∼22-nm-diameter), nonenveloped, icosahedral viruses with a linear, single-stranded DNA genome. Four different parvoviruses are known to infect humans, with parvovirus B19 (B19V) and human bocavirus type 1 (HBoV1) causing clinical disease. The significance of infection with other members of the Bocavirus genus or parvovirus 4 is less clear, but adeno-associated virus infections appear to be nonpathogenic. B19V is highly erythrotropic and replicates efficiently in bone marrow progenitor cells. Infection may lead to transient aplastic crisis, chronic anemia, fetal loss, or rash illness with or without arthropathy, depending on the host. Diagnosis is predominantly by either detection of the serologic response (IgM) or quantitative PCR, depending on the presentation. HBoV1 infects the airway epithelia and is associated with respiratory infection in young children. Diagnosis is also by detection of the serologic response (IgM or low-avidity IgG) or quantitative PCR.
Chapter 107 : Poxviruses
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Human infections by poxviruses are caused by species from the following genera: Orthopoxvirus, Parapoxvirus, Yatapoxvirus, and Molluscipoxvirus. Some poxviruses, such as variola virus (an orthopoxvirus) and molluscum contagiosum virus (a molluscipoxvirus), have very limited host ranges, causing disease only in humans. Epizoonotic poxviruses, such as monkeypox virus, vaccinia virus, and cowpox virus (all orthopoxviruses) and orf virus (a parapoxvirus), can cause disease in humans but can also infect a variety of other mammals. Poxviruses can differ in mode of transmission, disease severity, and clinical manifestations. For example, variola virus can be transmitted by large-droplet respiratory particles, in addition to direct contact with lesions or scab materials, whereas other poxviruses are transmitted mostly through direct contact. Variola virus has the greatest severity in humans, while other poxviruses such as orf simply cause a localized lesion. Each of the individual viruses is presented in detail, with similarities and differences highlighted. Although there are differences across all poxviruses, their great degree of similarity makes cross-species protection possible through vaccination. This high level of similarity can also make diagnosis through clinical symptoms or common laboratory diagnostic methods difficult. In this chapter, selected biologic-, DNA-, and antigen-based diagnostic techniques are discussed in detail.
Chapter 108 : Hepatitis B and D Viruses*
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Hepatitis B virus (HBV) is a DNA virus in the Hepadnaviridae family that is transmitted by percutaneous, sexual, and perinatal transmission. Currently, approximately 1.25 million individuals in the United States and approximately 400 million persons worldwide are infected with HBV. Detection of HBV-specific proteins, antibodies, and nucleic acid is used as key diagnostic tests to detect infection and determine the stage of disease. These include the HBV surface antigen (HBsAg) and a soluble nucleocapsid protein, HBV e antigen (HBeAg), as well as the detection and quantitation of HBV DNA. Additionally, there are specific tests for detection of antibodies to HBsAg and the HB core antigen. Several effective vaccines are available for preventing infection with HBV, and the Centers for Disease Control and Prevention recommend that all infants receive the HBV vaccine at birth. A variety of effective antiviral therapies are available for treatment of chronic HBV. These drugs are antiviral agents which target and prevent HBV replication. With the effective vaccine and therapy, HBV infections can be controlled and prevented.
Subviral Agents
Chapter 109 : Transmissible Spongiform Encephalopathies
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Prion diseases or transmissible spongiform encephalopathies are neurodegenerative disorders affecting both humans and a broad range of animals. Prion diseases are unique transmissible entities where a misfolded, highly stable conformer (PrPSc) of the host-encoded cellular prion protein (PrPC) represents an essential component of infectious prions. Detection of PrPSc and prion infectivity by morphological and/or biochemical assays is key to diagnosis of these diseases. In this review, we summarize and discuss the current state-of-the-art reading diagnostic procedures and principles to diagnose and treat these diseases.
Antiviral Agents and Susceptibility Test ethods
Chapter 110 : Antiviral Agents
- Authors: Aimee C. Hodowanec, Kenneth D. Thompson, Nell S. Lurain
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The use of antiviral agents for the treatment of viral diseases has rapidly increased over the past 2 decades. In this chapter, antiviral therapy for the most common, clinically relevant viral infections is reviewed. The chapter is organized by virus type and covers human immunodeficiency virus types 1 and 2 (HIV-1, HIV-2), hepatitis viruses B and C (HBV and HCV), influenza A and B viruses, and the human herpesviruses with a focus on herpes simplex virus, varicella-zoster virus, and human cytomegalovirus. For each virus, an overview of available therapies is provided, followed by an in-depth description of individual antiviral agents. This description includes information on the agent’s chemical structure, mechanism of action, bioavailability, adverse effects, and significant drug interactions. In addition, current treatment guideline recommendations are incorporated when available. References for these guidelines allow for access to future updates to recommended drug therapies. This chapter strives to provide readers with an up-to-date overview of antiviral therapy. Newly approved agents as well as promising agents in the late stages of clinical trials have been included. Specific new additions to this edition include the antiretroviral agent dolutegravir and newly approved directly acting antiviral agents for hepatitis C virus.
Chapter 111 : Mechanisms of Resistance to Antiviral Agents
- Authors: Robert W. Shafer, Sunwen Chou
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Understanding the mechanisms of viral drug resistance is critical for clinical management of individuals receiving antiviral therapy, for developing new antiviral drugs, and for drug resistance surveillance. This chapter reviews the mechanisms of resistance to antiviral drugs used to treat seven common viral infections: herpes simplex virus, cytomegalovirus, varicella-zoster virus, human immunodeficiency virus type 1, influenza A and B viruses, hepatitis B virus, and hepatitis C virus.
Chapter 112 : Susceptibility Test Methods: Viruses*
- Authors: Diana D. Huang, Matthew J. Bankowski
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This chapter describes the phenotypic and genotypic methods that are being used to perform viral susceptibility tests and situations when testing should be considered. The phenotypic method section consists of classic methods that are still in use, such as plaque assays. Newer methods such as recombinant virus assays (RVAs) that use both a molecular and phenotypic approach are also described. The genotypic methods section describes the various sequencing platforms and reverse-hybridization assays that are commonly used. There is a paucity of FDA-approved/cleared kits available. Therefore, other kits/reagents including research-use only (RUO) and laboratory-developed tests (LDTs) using analyte-specific reagents (ASRs) are also included in the discussion. Descriptions of the genotypic assays include the reported mutations associated with antiviral resistance for selected viruses. Finally, there is a discussion of the knowledge-based expert systems available for the clinical interpretation of sequence data. This document should be useful if a clinical diagnostic laboratory chooses to develop an LDT antiviral genotypic assay.
MYCOLOGY
General
Chapter 113 : Taxonomy and Classification of Fungi
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This chapter summarizes current information on the taxonomy and classification of fungi. It offers thumbnail descriptions of the major taxa for true fungi and related organisms.
Chapter 114 : Specimen Collection, Transport, and Processing: Mycology
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Successful laboratory diagnosis of fungal infections requires attentiveness on the part of physicians and nurses, proper collection and transport of appropriate specimens, and comprehensive procedures in the laboratory. This chapter offers guidelines for specimen collection and transport, specimen handling, specimen pretreatment and processing in the laboratory, medium selection, and incubation of cultures.
Chapter 115 : Reagents, Stains, and Media: Mycology*
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This chapter describes the reagents, stains, and media that are available and commonly used for the identification of fungi, ranging from direct detection of fungal structures in patient specimens to selection of media for the culture and identification of fungal isolates.
Chapter 116 : General Approaches for Direct Detection and Identification of Fungi*
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This chapter summarizes general approaches to the direct detection and identification of fungi in clinical samples. Techniques such as direct microscopy, histopathology, antigen and antibody detection, mass spectrometry, and nucleic acid detection are reviewed. The availability of commercial kits and the sensitivity and specificity of the various methods are detailed. Applications and limitations for each area are reviewed.
Fungi
Chapter 117 : Candida, Cryptococcus, and Other Yeasts of Medical Importance
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The chapter on Candida, Cryptococcus, and other yeasts of medical importance outlines the taxonomy, epidemiology, and clinical significance of species from as many as 12 yeast genera. The routine diagnostic methods of microscopic examination, culture, and identification are outlined and include commonly used tests such as germ tubes and biochemical profiling kits, as well as discussion on the application, advantages, and disadvantages of molecular methods such as peptide nucleic acid fluorescent in situ hybridization, real-time PCR, sequencing, and matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF). Antigen/antibody tests provide rapid and clinically important results for species such as Candida and Cryptococcus, and these are also reviewed. The chapter contains five tables containing key identification characteristics of yeasts, including the difficult-to-identify Malassezia and Tichosporon species, a table listing the currently available rapid tests, and a table of antifungal susceptibility profiles.
Chapter 118 : Pneumocystis
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The clinical features, diagnosis, and biology of the fungal pathogen Pneumocystis jirovecii, which causes pneumonia in immunocompromised patients and can colonize healthy individuals with an intact immune system.
Chapter 119 : Aspergillus and Penicillium*
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Aspergillus and Penicillium (and Talaromyces) are environmental moulds, which include a number of species that are human pathogens. Each of these genera is described with regard to assisting in their detection and identification in the diagnostic mycology laboratory. Their taxonomic placement, description, epidemiology and transmission, clinical significance, collection, transport and specimen storage procedures, diagnostic approaches (direct microscopy, isolation, identification procedures [phenotypic, proteomic and genomic methods]), genotyping, serological testing, antimicrobial susceptibilities, and interpretation when isolated are covered. Polyphasic phylogenetic concepts and the recent “one fungus, one name” dictum have led to taxonomic rearrangements of Aspergillus and Penicllium. Consensus to adopt the anamorph term of “Aspergillus” currently holds for members of Aspergillus genus, while the most notable Penicillium pathogen is now termed Talaromyces marneffei. Both genera are soil and environmental fungi but cause a diverse range of human disease ranging from localized lung to disseminated infection. Immunocompromised individuals are especially susceptible. Identification of medically relevant members to species level still requires morphological techniques, but proteomic approaches such as matrix-assisted laser desorption time-of-flight mass spectrometry, direct antigen detection in body fluids (e.g., Aspergillus galactomannan), and PCR-based tools are increasingly used, especially to resolve ambiguous results. Molecular tools are essential to identify cryptic species, e.g., the A. fumigatus complex. Antifungal susceptibility testing is helpful in invasive infection and in monitoring for drug resistance; susceptibility may vary with species. Several genotyping tools are developed for both Aspergillus and Talaromyces, while methods that employ analysis of at least one genetic locus are most useful.
Chapter 120 : Fusarium and Other Opportunistic Hyaline Fungi*
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This chapter focuses on those fungi that grow in tissue in the form of hyaline or lightly colored septate hyphae. These fungi include Fusarium and other hyaline fungi. Disease caused by hyaline fungi is referred to as hyalohyphomycosis. Hyaline fungi described in this chapter include the anamorphic, asexual hyphomycetes and coelomycetes, as well as homothallic ascomycetes that produce sexual structures and ascospores in culture. Phenotypic/morphologic identification of hyaline moulds is based on methods of conidiogenesis and spore formation; however, accurate species-level identification frequently requires DNA sequence data from one or more informative loci. Molecular phylogenetic analysis has revealed that several Fusarium morphospecies actually represent species-rich species complexes, including the following six species complexes: F. solani, F. oxysporum, F. fujikuroi, F. incarnatum-F. equiseti, F. chlamydosporum, and F. dimerum. Fusarium species are cosmopolitan soil saprobes that can cause toxicosis or infection in humans. A frequent infection in immunocompetent humans is keratitis resulting from trauma or contamination of contact lenses/solutions. Severe disseminated fusarial infections are seen in patients with hematological malignancy or an allogenic hematopoietic stem cell transplant. The portal of entry is unknown in most cases of invasive fusarial infections; however, inhalation of airborne conidia appears to be the most common mode of transmission. Other hyaline fungi can cause human infection as well, ranging from cutaneous to disseminated systemic invasive infections. In general, the recovery of a hyaline fungus from a normally sterile site and microscopic evidence of tissue damage provide the most convincing evidence of invasive disease.
Chapter 121 : Agents of Systemic and Subcutaneous Mucormycosis and Entomophthoromycosis
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This chapter reviews current taxonomic changes related to the agents of mucormycosis and entomophthoromycosis and describes epidemiological and clinical features as well as modern diagnostic and therapeutic strategies related to these diseases
Chapter 122 : Histoplasma, Blastomyces, Coccidioides, and Other Dimorphic Fungi Causing Systemic Mycoses*
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Several of the clinically important thermal dimorphic fungi are closely related and belong to the order Onygenales, which includes the causal agents of histoplasmosis, blastomycosis, coccidioidomycosis, paracoccidioidomycosis, and adiaspiromycosis. This chapter provides a review, with updates on recent developments, of our understanding of the pathogenic fungi responsible for these diseases. Aspects relevant to their detection and characterization are emphasized, including taxonomy and genome sequencing updates, morphology of the pathogens at room temperature and at 37°C, epidemiology and transmission, clinical manifestations of the diseases, and diagnostic methods. Diagnostic topics covered include microscopy identification; special stains; immunological tests for detecting antibodies, antigen(s), and metabolites; nucleic acid detection using molecular methods; identification or typing via examination and phenotypic properties; and interpretation of reports. The chapter also describes isolation of the pathogens, including biosafety and biosecurity recommendations; briefly mentions the antifungal agents available for treatment; and reviews the pathogens’ in vitro antifungal susceptibilities.
Chapter 123 : Trichophyton, Microsporum, Epidermophyton, and Agents of Superficial Mycoses
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Superficial fungal infections constitute some of the most common infectious conditions and include dermatophytosis (tinea corporis, tinea capitis, tinea pedis, and tinea unguium) and pityriasis versicolor, as well as rarer disorders like tinea nigra and black and white piedra. The etiologic agents of dermatophytosis are classified, along with some nonpathogenic relatives, in three genera: Trichophyton, Microsporum, and Epidermophyton. Dermatophytes are keratinophilic fungi that are capable of invading the keratinous tissues of living animals. They are grouped into three categories based on host preference and natural habitat: (i) anthropophilic species almost exclusively infect humans, with animals being rarely infected; (ii) geophilic species are soil-associated organisms that can occasionally cause infections in humans and other animals; and (iii) zoophilic species are essentially pathogens of nonhuman mammals, although animal-to-human transmission is not uncommon. This chapter describes the taxonomy, epidemiology, transmission, and clinical significance of dermatophytes. The methods of diagnosis of dermatophyte infections, including specimen collection, transport, and processing; the microscopic examination and culture of clinical specimens from suspected cases of dermatophytosis; and the conventional (phenotypic) and molecular methods of identification of the causative agents, are discussed in detail. Attention is also given to the clinical presentation, diagnosis, and identification of the causative agents for those superficial infections in which the causative fungi colonize the cornified layers of the epidermis or the suprafollicular portion of the hair (tinea versicolor, tinea nigra, black piedra, and white piedra).
Chapter 124 : Curvularia, Exophiala, Scedosporium, Sporothrix , and Other Melanized Fungi
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The taxonomy of the melanized fungi and the most relevant epidemiological and clinical aspects, and the laboratory procedures for the diagnosis of infections caused by these agents, are discussed in this chapter. This chapter covers most of the agents of phaeohyphomycosis, chromoblastomycosis, and sporotrichosis, as well as a number of agents of superficial and cutaneous disease. The genera discussed in this chapter belong to the ascomycetous orders Botryosphaeriales (Lasiodiplodia and Neoscytalidium), Chaetothyriales (Cladophialophora, Exophiala, Fonsecaea, Knufia, Phialophora, Rhinocladiella, and Veronaea), Calosphaeriales (Pleurostomophora), Diaporthales (Phaeoacremonium), Dothideales (Aureobasidium and Hormonema), Microascales (Scedosporium), Ophiostomatales (Sporothrix), Pleosporales (Alternaria, Curvularia, and Exserohilum), and Venturiales (Ochroconis). The most recent data on the in vitro and in vivo experimental antifungal susceptibility of melanized fungi to the available antifungal drugs are also provided.
Chapter 125 : Fungi Causing Eumycotic Mycetoma*
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Eumycetoma is a chronic, granulomatous, progressive subcutaneous fungal disease characterized by the production of large masses of fungal organisms called grains, which are discharged through sinus tracts. Eumycetoma is more frequently seen in tropical and subtropical regions and less frequently in temperate countries. Several hyaline and dematiaceous fungi can cause eumycetoma, and their distributions are greatly affected by climate, especially rainfall. The identification of these fungal species is not always easy, and in many cases species identification needs experience and access to special laboratory testing such as PCR and DNA sequencing. Unfortunately, these tests are not always available in areas of endemicity. The management of eumycetoma is difficult, and response to antifungal drugs is always poor. In this chapter, fungi frequently reported from eumycetoma cases are described. The taxonomy of some previously reported species is updated, and new species from well-confirmed cases are also described. In this chapter, the available information about the natural habitat of causative agents, pathogenesis, isolation, identification procedures, and antifungal susceptibility data are discussed.
Chapter 126 : Mycotoxins*
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Current knowledge of mycotoxins is reviewed in light of the developments of the last decade. We present information on common chemical classes of mycotoxins, including aflatoxins, citrinin, cyclopiazonic acid, ergot alkaloids, fumonisins, ochratoxins, patulin, trichothecenes, and zearalenone, with mention that the genes involved in their synthesis are clustered. Mycotoxins are discussed in the context of relevance to human and veterinary health concerns and food safety. Detection, biological and environmental contributing factors, prevention, and prospects for amelioration of contamination of feed and food products are addressed. We speak to potential and historical application of mycotoxins to biological warfare and bioterrorism. Finally, sick-building syndrome (SBS) and the case for its explanation as being caused by mycotoxins and mold are considered.
Chapter 127 : Lacazia, Lagenidium, Pythium, and Rhinosporidium
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In the past 100 years the microbial pathogens described in this chapter have been classified either as fungal and/or para-fungal protistan pathogens. Based on their apparent epidemiological connection with water, they were at one point also placed in a new category of hydrophilic infectious agents. However, based on taxonomic and other morphological characteristics, these three anomalous species were not well understood. This frustrating situation fueled a strong controversy that has only recently been solved with the advent of molecular methodologies. Despite the recent finding that both Pythium insidiosum and Rhinosporidium seeberi are protistan pathogens, they are still studied by medical mycologists, continuing a historical tradition. More recently, the finding of an oomycete in the genus Lagenidium affecting mammalian hosts alerted the medical community to the presence of a novel pathogen phenotypically similar to the fungi and indistinguishable from the clinical and pathological features displayed by P. insidiosum during infection. Based on rDNA phylogenetic analysis, the evolutionary location of the microbial pathogens discussed in this chapter is illustrated.
Chapter 128 : Microsporidia
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Microsporidia are obligate intracellular, unicellular, spore-forming eukaryotes that belong to the kingdom of the Fungi. Their host range is extensive, including most invertebrates and all classes of vertebrates. More than 160 microsporidial genera and 1,300 species have been identified. Nine genera (Anncaliia, Encephalitozoon, Endoreticulatus, Enterocytozoon, Nosema, Pleistophora, Vittaforma, Tubulinosema, and Trachipleistophora) and unclassified microsporidia have been implicated in human infections. Microsporidiosis appears to occur most frequently in HIV-infected patients, but it is emerging in otherwise immunocompromised and also in immunocompetent persons. The sources of microsporidia infecting humans and their modes of transmission are uncertain but ingestion of the environmentally resistant spores is probably the most important mode of transmission; transmission by dust or aerosol may also occur. Waterborne, foodborne, and zoonotic infections have been reported. In the immunocompromised host, microsporidial infections were found in organ-transplant recipients, in patients with hematologic malignancies receiving monoclonal antibody therapy, in patients with rheumatic disease undergoing anti-tumor necrosis factor therapy, in the elderly, and in malnourished children. The clinical manifestations are diverse, including intestinal, pulmonary, ocular, muscular, cerebral, renal disease, and disseminated infection. In immunocompetent persons, microsporidia are associated with keratoconjunctivitis, self-limiting diarrhea, and rarely cerebral infection. Microsporidia are also detected in asymptomatic persons. Diagnosis is made by morphological or molecular detection of the organisms. Immunofluorescence technique, electron microscopy, or genetic analyses allow species identification. Treatment options are limited; some microsporidial species respond to albendazole. The most frequent HIV-associated opportunistic species, Enterocytozoon bieneusi, causing diarrhea, is not susceptible to albendazole but may be treated with the experimental drug fumagillin.
Antifungal Agents and Susceptibility Test Methods
Chapter 129 : Antifungal Agents*
- Authors: Shawn R. Lockhart, David W. Warnock
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This chapter reviews the four major families of antifungal drugs that are currently available for systemic administration: the allylamines (terbinafine), the azoles (ketoconazole, itraconazole, fluconazole, voriconazole, and posaconazole), the echinocandins (caspofungin, micafungin, and anidulafungin) and the polyenes (amphotericin B). The comparative activities of the major systemic antifungal agents against important groups of fungi are summarized. Discussion includes spectrum of activity, acquired resistance, pharmacokinetics, clinical use, drug interactions, and toxicity and adverse events. This chapter will also discuss the characteristics of the several other agents that can be used for the oral or parenteral treatment of superficial, subcutaneous, or systemic fungal infections. Novel agents that are currently in clinical trials are briefly reviewed.
Chapter 130 : Mechanisms of Resistance to Antifungal Agents*
- Author: David S. Perlin
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Antifungal therapy is an important element of patient management for acute and chronic diseases. Yet, as the global burden of fungal infections rises, treatment choices are constrained due to limited classes of antifungal agents. Furthermore, clinical management of fungal diseases is made even more tenuous by the emergence of antifungal drug resistance. More recently, the evolution of multidrug resistant organisms refractory to several different classes of antifungal agents is alarming. The resistance mechanisms responsible are largely shared by strains displaying inherently reduced susceptibility to specific antifungal agents and strains acquiring resistance during therapy. The principal molecular mechanisms are well characterized and include diminished drug-target interactions through changes in affinity and target abundance, and reduction in the intracellular level of drug through expression of high-capacity efflux pumps and biofilm formation. In some strains, high-level resistance occurs through a stepwise evolution of multiple resistance mechanisms. In recent years, a great deal has been learned about the genetic factors that regulate these mechanisms and the effectors and modulators of cellular stress, which facilitate the emergence of resistance. Understanding the primary molecular mechanisms and their regulation, along with the cellular adaptation factors that promote their emergence, provides an opportunity to develop better diagnostic tools and therapeutic strategies to overcome and prevent the emergence of antifungal resistance.
Chapter 131 : Susceptibility Test Methods: Yeasts and Filamentous Fungi*
- Authors: Elizabeth M. Johnson, Maiken Cavling-Arendrup
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While antifungal drug resistance has not proved to be as problematic as that experienced with antibacterial agents, both intrinsic and emergent resistance are encountered and antifungal susceptibility testing can help in the guidance of prescribing practices. Moreover, susceptibility testing is of great importance in detecting intrinsic resistance in a species and therefore establishing the spectrum of activity of new and developmental agents. A great deal of progress has been achieved in the field of antifungal susceptibility testing with both yeasts and filamentous fungi since testing began in earnest in the early 1980s. Standardized broth macrodilution and microdilution methods are available for testing moulds and yeasts, as are standardized disk diffusion methods for systemically active antifungal drugs. Progress is also being made in establishing the relationship between test results and patient responses to therapy in varied clinical settings and with many of the currently available antifungal agents. There has been a strong move towards consensus in the standardization of the methodology employed and the principles by which breakpoints are selected in the USA and Europe, which has meant that internationally agreed epidemiological cutoff value breakpoints can be applied for some drug–fungus combinations. This chapter addresses the methodological issues surrounding antifungal susceptibility testing and breakpoint setting.
Parasitology
General
Chapter 132 : Taxonomy and Classification of Human Parasitic Protozoa and Helminths
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This chapter provides an outline classification of the parasitic protozoa and helminths found in humans. There are six sections: Principles of taxonomy as applied to parasites with special reference to the protozoa, Protozoa, Nematodes, Trematodes, Cestodes, and Acanthocephalans. Any differences between different interpretations of the classification of a particular group are discussed. Each section includes a table listing the medically important species and less important species in separate columns for easy reference.
Chapter 133 : Specimen Collection, Transport, and Processing: Parasitology
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This chapter covers various approaches and diagnostic methods currently in use for diagnosis of parasitic infections. If clinical specimens have been properly collected and processed according to specific specimen rejection and acceptance criteria, the examination of prepared wet mounts, concentrated specimens, permanent stained smears, blood films, and various culture materials provides detailed information leading to parasite identification and confirmation of the suspected etiologic agent. Although other tests such as immunoassay and amplification-based diagnostic kits continue to become available commercially, most medical parasitology diagnostic work depends on the knowledge and microscopy skills of the microbiologist.
Chapter 134 : Reagents, Stains, and Media: Parasitology
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The evaluation of clinical specimens for ova and parasites in the clinical laboratory can involve the use of direct macroscopic examination of the specimen and microscopic examination of fresh and preserved specimens, as well as culture for some parasitic organisms. These examinations necessitate the use of a variety of stains, reagents, and media. This chapter discusses the most common reagents, stains, and media used for diagnostic parasitology, including directions for making reagents and stains and listings of commercially available reagents and stains.
Chapter 135 : General Approaches for Detection and Identification of Parasites
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This chapter covers various approaches and diagnostic methods currently used for the diagnosis of parasitic infections. If clinical specimens have been properly collected and processed according to specific specimen rejection and acceptance criteria, the examination of prepared wet mounts, concentrated specimens, permanent stained smears, blood films, and various culture materials provides detailed information leading to parasite identification and confirmation of the suspected etiologic agent. Although other tests, such as immunoassay and amplification-based diagnostic kits, continue to become available commercially, the majority of medical parasitology diagnostic work depends on the knowledge and microscopy skills of the microbiologist.
Parasites
Chapter 136 : Plasmodium and Babesia*
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Plasmodium and Babesia are intraerythrocytic apicomplexan parasites that cause malaria and babesiosis, respectively. Both organisms can cause severe, life-threatening disease. Malaria is transmitted through the bite of an Anopheles mosquito primarily in the tropics and subtropics while babesiosis is transmitted through the bite of an ixodid tick and is mostly found in temperate regions. The most common Plasmodium species to infect humans are Plasmodium falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. Of these, P. falciparum is responsible for the greatest morbidity and mortality worldwide. Babesia microti and B. divergens are the most common causes of human babesiosis in the United States and Europe, respectively. Diagnosis of both is traditionally accomplished through microscopic examination of conventional Giemsa-stained thick and thin blood films. The thick film provides the greatest sensitivity for parasite detection and is preferred for screening, while the thin film provides the best morphology for differentiation of Babesia and Plasmodium parasites and Plasmodium species determination. Antigen detection methods are also widely used for detection of malaria and molecular amplification methods have been described for both organisms.
Chapter 137 : Leishmania and Trypanosoma
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Human leishmaniasis is caused by Leishmania parasites transmitted primarily by sand fly bites. The parasite resides and multiplies within macrophages of the host. The infection is categorized into cutaneous, mucocutaneous, and visceral leishmaniasis. Diagnosis can be made from the clinical presentation, microscopically, and by culture, real-time PCR, serology, and antigen detection, depending on the resources available. Clinicians may have to use many different diagnostic methods to detect the infection due to the low number of parasites in specimens. Some infections may self-cure, while others will need to be treated depending on the number of lesions and whether the leishmaniasis is mucocutaneous or visceral. Trypanosomiasis is limited to the Americas and Africa. African trypanosomiasis is caused by Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense, with 10,000 new cases each year. The infection is acquired from tsetse fly bites. Diagnosis is similar to that for Leishmania infections. Individuals infected with T. brucei gambiense may be asymptomatic, whereas infections with T. brucei rhodesiense have greater pathogenicity, especially if the infection enters the central nervous system. Therapy can be successful if administered early in the disease, although the drugs can be quite toxic. American trypanosomiasis is caused by Trypanosoma cruzi acquired from a reduviid bug, although the infection can be food or blood borne. There are three phases of disease: acute, indeterminate, and chronic. Diagnostic methods are similar to those for Leishmania infections. The blood supply and organ donors are screened for the presence of T. cruzi antibody to prevent infections.
Chapter 138 : Toxoplasma
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Toxoplasma gondii is a protozoan parasite that infects most species of warm-blooded animals, including humans. Members of the cat family Felidae are the only known definitive hosts for the sexual stages of T. gondii and thus are the main reservoirs of infection. Serologic prevalence data indicate that toxoplasmosis is one of the most common infections of humans throughout the world. However, for the majority of persons, infection is asymptomatic. Primary infection in pregnant women can lead to severe disease in the fetus. Reactivation can cause significant morbidity in immunosuppressed persons, particularly persons with advanced AIDS. Prevention and treatment are available for each of these clinical situations; thus, the major diagnostic issues in toxoplasmosis are to correctly diagnose acute primary infection in pregnant women, diagnose congenital infection in newborns, and identify prior infection in immunosuppressed hosts. Serology is the main tool for each of the above-named clinical situations. Most recently, IgG avidity testing has emerged as an important tool for identifying recent infection in pregnant women, as IgM can be detected for over 6 months following primary infection. PCR is an important tool for identifying the parasite in the fetus. Interpretation of serology for the identification of acute infection in pregnant women can be complicated and often requires multiple tests—IgG, IgM, and IgA avidity testing. Use of a reference laboratory is often necessary in this setting.
Chapter 139 : Pathogenic and Opportunistic Free-Living Amebae
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Pathogenic and opportunistic free-living amebae, such as Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri, and Sappinia pedata, cause devastating central nervous system and disseminated infections in humans, leading to death. A brief review of the types of infections caused, clinical characteristics, pathologic manifestations, morphologic and molecular diagnoses, treatment, and prevention, if any, are discussed.
Chapter 140 : Intestinal and Urogenital Amebae, Flagellates, and Ciliates
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This chapter describes the intestinal and urogenital amebae, flagellates, and ciliates in terms of taxonomy, epidemiology, clinical significance, and diagnostic methods for the clinical microbiologist.
Chapter 141 : Cystoisospora, Cyclospora, and Sarcocystis*
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“Cystoisospora, Cyclospora, and Sarcocystis” provides an overview of the life cycles, pathogenesis, diagnosis, and treatment of these important parasites. These coccidial parasites have an environmentally resistant oocyst stage in their life cycles. They remain an important cause of diarrhea in patients in developing countries, and foodborne outbreaks still occur in developed countries. Humans are the only definitive hosts for Cystoisospora belli and Cyclospora cayetanensis and pass unsporulated oocysts in their stools. Sarcocystis requires two hosts. Humans are definitive hosts for S. hominis and S. suihominis and become infected after ingesting undercooked meat of bovids (cattle, buffalo) and pigs, respectively. Humans pass sporulated oocysts and sporocysts in their stools. Humans are also accidental intermediate hosts for one or more Sarcocystis species, and most cases are observed in Southeast Asia. Muscle sarcocysts are associated with fever and muscle pain. Diagnosis of Cystoisospora, Cyclospora, and Sarcocystis is based on microscopic examination of stool samples using acid-fast-stained slides or with UV microscopy. PCR-based methods of stool examination for oocysts have been developed. There are currently no serologic tests available to detect antibodies to these parasites and aid in their diagnosis. Combination antiretroviral therapy (cART) is associated with a better prognosis in AIDS patients, but clinical episodes still occur even in some patients on cART. Treatments for acute Cystoisospora and Cyclospora infections are available and effective. Apparent drug failures are most likely related to poor drug absorption or distribution rather than to true drug resistance.
Chapter 142 : Cryptosporidium
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Cryptosporidium spp. are important zoonotic pathogens, causing enterocolitis and diarrhea in children and immunocompromised persons. In developing countries, cryptosporidiosis is one of the most important causes of moderate to severe diarrhea and diarrhea-associated death. In industrialized nations, Cryptosporidium spp. are well recognized waterborne, foodborne, and zoonotic pathogens, having caused many outbreaks of human illness. In the United States, the number of annual reported cases of cryptosporidiosis has increased more than 2-fold in recent years. In this chapter, various laboratory techniques for the detection and diagnosis of Cryptosporidium spp. and the most recent progress in Cryptosporidium taxonomy and the molecular epidemiology and treatment of cryptosporidiosis are reviewed.
Chapter 143 : Nematodes*
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This chapter covers an update on laboratory diagnosis of the common human nematodes: Ascaris, Enterobius, hookworms, Strongyloides, and Trichuris. Some information on taxonomy, epidemiology, and treatment is also included. The figures will aid laboratory personnel in diagnosis of these nematodes. The key references are updated.
Chapter 144 : Filarial Nematodes*
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This chapter reviews the different filarial parasites that cause human infection and describes the epidemiology, clinical features, and complications of the diseases caused by the individual species as well as their treatment. Updated information is provided on current diagnostic tools used as well as tests that provide greater sensitivity, which might be used in the near future.
Chapter 145 : Cestodes
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Cestodes are segmented flatworms and have as their key characteristic a flattened body composed of the head or scolex (bearing the fixation organs—suckers, hooks, and bothria), the neck (where the cellular reproduction occurs, to form the strobila), and the strobila, formed by numerous segments or proglottids. Cestode tapeworms live in the lumen of the small intestine with the head or scolex as the only fixation organ, attached to the mucosa, so they develop cephalic fixation organs like hooks, suckers, or shallow grooves as longitudinal suction sulci (bothria). They absorb nutrients from the host’s intestine both at the head and through their tegument, and they thus also develop a specialized tegument. Four species of cestode tapeworms inhabit the human intestine with frequency: Diphyllobothrium latum, Taenia saginata, Taenia solium, and Hymenolepis nana. They differ widely in size, intermediate host, and other characteristics, from the 12-m D. latum to the 3-cm H. nana. In addition, a number of cestode larvae can produce human disease if infective tapeworm eggs are ingested, mainly cysticercosis (Taenia solium), cystic hydatid disease (Echinococcus granulosus), and alveolar hydatid disease (Echinococccus multilocularis). Rarer larval cestode infections affecting humans include coenurosis (Taenia multiceps), sparganosis (Spirometra mansonoides), and cysticercosis by Taenia crassiceps. Tapeworms and especially tapeworm larval infections still represent an important cause of morbidity and mortality, not only in most underdeveloped countries but also in industrialized countries, particularly in rural areas or among immigrants from areas of endemicity.
Chapter 146 : Trematodes
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The chapter provides information on trematodes (digeneans) infecting humans. The trematodes are considered systematically by sites of infection, and information on blood flukes, lung flukes, live flukes, and intestinal flukes is provided.
Chapter 147 : Less Common Helminths
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There are a wide variety of less commonly encountered helminthic parasites, which may be nematodes, cestodes, or trematodes. The diseases caused by these parasites are interesting and demonstrate their highly evolved life cycles and the complex interactions with their hosts. These diseases range from subclinical, e.g., dipylidiasis, to possibly life-threatening, e.g., baylisascariasis. In many instances, the disease occurs only in a particular geographic area, which is largely determined by the biological ranges of the definitive and intermediate hosts. Dietary customs are also important in the prevalence of human disease, as many of these are associated with the ingestion of raw animal products. The treatment of these parasites varies depending on the infectious agent, but common preventive measures may significantly diminish the transmission of many of these parasitic diseases. These measures include the zoonotic control of parasitic disease in animal hosts and the vectors of transmission, washing of fruits and vegetables, access to clean drinking water, and thorough cooking of meats before consumption.
Chapter 148 : Arthropods of Medical Importance
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In addition to their roles as vectors of infectious agents, the diverse group of animals that comprise arthropods may indirectly or directly cause injury that requires diagnosis and treatment. Such injuries include itching, dermal necrosis, anaphylaxis, and systemic toxicosis. The ubiquity of arthropods implies that they may be a frequent cause for clinical attention; reducing exposure is often sufficient to prevent or treat disease.
Antiparasitic Agents and Susceptibility Test Methods
Chapter 149 : Antiparasitic Agents
- Authors: Karin Leder, Peter F. Weller
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This chapter discusses the mechanisms of action, pharmacology, clinical utility, and adverse effects of common first-line antiparasitic therapies and newer drug alternatives. Agents discussed in detail include albendazole, mebendazole, praziquantel, ivermectin, diethylcarbamazine, nitazoxanide, antimalarials, and those used to treat gastrointestinal protozoal infections, leishmaniasis, and trypanosomiasis.
Chapter 150 : Mechanisms of Resistance to Antiparasitic Agents
- Authors: W. Evan Secor, Jacques Le Bras, JrÔme Clain
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Parasitic diseases rank among the most prevalent and severe diseases worldwide and yet their control relies heavily on a single tool: the drugs used for chemotherapy or prophylaxis. This dependence on drugs is compounded by the relative paucity of the current armamentarium of antiparasitic products and the selection of drug-resistant parasites. Mechanisms for true resistance are varied and include a decrease in drug accumulation within the parasite or modifications in parasite enzyme structure or metabolic pathways. However, various host factors modulate the clinical and parasitologic responses to drug treatment, and the observed responses do not necessarily reflect true parasite resistance or susceptibility. A selective review of drug resistance in five parasitic diseases (malaria, trichomoniasis, leishmaniasis, African trypanosomiasis, and schistosomiasis) will illustrate the existing problems and their potential solutions.
Chapter 151 : Susceptibility Test Methods: Parasites
- Authors: Jacques Le Bras, JrÔme Clain, W. Evan Secor
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Accurate methods for ascertaining responses of parasites to antiparasitic drugs can prove useful at several levels. They can assist in the clinical management of individual patients, they can yield epidemiologic information that may guide drug use policies and public health interventions, and they offer crucial research tools for the development of new and better drugs. Drug susceptibility tests fall into three broad categories: in vivo tests, in vitro tests, and molecular tests. In vivo tests with patients directly assess the clinical efficacies of existing compounds. Their interpretation is limited by potential interference by factors related to the host or to the environment. In vitro tests circumvent these interferences by isolating the parasites from their hosts and investigating them in culture under controlled laboratory conditions, with opportunities for repeated assessments against multiple compounds, including experimental compounds. Molecular tests detect genetic variations that are potentially linked with resistance. Such tests offer unique advantages. PCRs can be performed with minute amounts of nonviable parasite genetic material. They can be run in batches, allowing large-scale epidemiologic studies. Because of their short duration (hours), molecular diagnostic procedures can potentially be used to guide patient management. These different categories of tests provide complementary information. They are discussed for five parasitic diseases, selected for their particular chemotherapeutic challenges: malaria, trichomoniasis, leishmaniasis, African trypanosomiasis, and schistosomiasis.
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Doody's Review Service
31 August 2015
Description
This two-volume, 2,500+ page book is the 11th edition of this well respected authoritative reference in clinical microbiology. The previous edition was published in 2011.
Purpose
The purpose is to provide updates to the ever growing field of clinical microbiology. These are worthy objectives well met by the authors.
Audience
This book is intended for practicing clinical microbiologists. However, it has been, and will continue to be, useful to clinical laboratory scientists (in training or in practice) at any level (e.g., bench scientist, specialist, supervisor, manager). It is also useful to doctoral level laboratory directors and infectious disease practitioners (MD, DO, PharmDs, etc.) who rely heavily on microbiology laboratory results. Finally, it would be of interest to anyone in healthcare interested in clinical microbiology, including providers in virtually all specialties or primary care, and other allied health practitioners (e.g. nurses, physician assistants, etc.).
Features
This edition continues its historical and well-deserved reputation as the authoritative reference for clinical microbiology. Simply put, it's everything you ever wanted or might want to know about clinical microbiology. Updates include new molecular technologies (e.g., MALDI-TOF, nucleic acid sequencing) as well as newly emerged diseases (e.g., carbapenem resistant enterobacteriaceae, Ebola virus, etc.). To get a sense of its enormity, the subject index alone is at least 120 pages! The chief editors comment this is only the second edition to have a fully searchable, web-based electronic edition. However, there is no obvious relationship between the print version and the ebook version, as there is no online access key. Given the sheer weight of this book, an ebook would be most appreciated, especially if access is automatic with purchase of the print copy. Two small areas of nitpickiness. First, given the complexity of clinical microbiology, a cohesive and broad-based discussion of the overarching principles of a quality management system would have been useful. If readers are truly interested in this subject, Clinical Laboratory Management, 2nd edition, Garcia (ASM Press, 2014), is a great reference. Second, the chapter on prevention of laboratory-acquired infections has no discussion of laboratory-acquired Neisseria meningitides -- a serious omission given the disproportionately increased lethality of these infections, the need to modify workflow processes to avoid exposure, and the availability of a vaccine.
Assessment
Overall, however, this is truly the authoritative reference for clinical microbiology. Get it.
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