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Manual of Clinical Microbiology, 10th Edition
Editors: James Versalovic1, Karen C. Carroll2, Guido Funke3, James H. Jorgensen4, Marie Louise Landry5, David W. Warnock6
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Affiliations:
1: Department of Pathology, Texas Children's Hospital, and Department of Pathology and Immunology, Baylor College of Medicine, Houston, Texas;
2: Division of Medical Microbiology, Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland;
3: Department of Medical Microbiology and Hygiene, Gärtner & Colleagues Laboratories, Ravensburg, Germany;
4: Department of Pathology, University of Texas Health Science Center, San Antonio, Texas;
5: Department of Laboratory Medicine, Yale University School of Medicine, New Haven, Connecticut;
6: National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia
Content Type: Reference
Format:
Electronic
Publication Year:
2011
Category: Clinical Microbiology
Print ISBN
:
9781555814632
e-ISBN
:
9781555816728
MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.
The 10th edition of the Manual of Clinical Microbiology continues to set the standard for state-of-the-science laboratory practice as the most authoritative reference in the field. This 10th edition represents the collaborative efforts of 22 editors and more than 260 authors from around the world, all experienced researchers and practitioners in medical and diagnostic microbiology. Together, they have brought the manual fully up to date, producing a remarkable work of two volumes, nine sections, and 149 chapters that is filled with the latest research findings, infectious agents, methods, practices, and safety guidelines.
Electronic Only, 2,630 pages, color illustrations, indexes.
Are you interested in purchasing a site license to the Manual of Clinical Microbiology, 10th Edition for your institution or lab? Contact [email protected] for more information!
Book Summary
VOLUME 1
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DIAGNOSTIC STRATEGIES AND GENERAL TOPICS
Chapter 1 : Introduction to the 10th Edition of the Manual of Clinical Microbiology
- Author: James Versalovic
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This introductory chapter of the 10th edition of the Manual of Clinical Microbiology, (MCM10) marks a significant milestone in the evolution of this important work. It talks about the overall organization of the book, presenting insights into the key themes discussed. Now entering its fifth decade, the Manual strives to continue to be the leading, most authoritative reference for the “real-world” practice of clinical microbiology. Hopefully the MCM10 continues to provide a highly respected benchmark and authoritative reference for the entire field of clinical microbiology. The work never stops, and the knowledge base keeps growing, and hence it is essential to continuously enhance the practice and contribute to the evolution of the cherished profession of clinical microbiology.
Chapter 2 : Microscopy
- Author: Danny L. Wiedbrauk
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This chapter describes the basic concepts of light microscopy as they are practiced in the microbiology laboratory. Immersion fluids are used between the condenser and the microscope slide in transmitted light fluorescence microscopy and dark-field microscopy to minimize refraction, increase the numerical aperture of the objective, and improve optical resolution. As the refractive index of a material increases, light beams entering or leaving a material are deflected to a greater extent. Field diaphragm is located in the light path between the light source and the substage condenser. The resolving power of a microscope is the most important feature of the optical system because it defines one's ability to distinguish fine details in a specimen. Reducing the voltage will alter the color of the incoming light, and voltage changes are not recommended for photomicroscopy. Phase microscopy is an important tool for examining living and/or unstained material in wet mounts and cell cultures. In phase-contrast microscopy, structures within living cells appear as hills or craters, depending upon their optical thickness. Today, fluorescence microscopy is used in conjunction with nucleic acid hybridization to visualize the location of fluorescent in situ hybridization and multicolor fluorescent in situ hybridization probes. The availability of digital photomicroscopy has significantly enhanced the microbial identification process, and it has helped to standardize microbe identification. Microscopy still has a central role in the detection of infectious agents despite highly publicized advances in DNA and RNA detection systems.
Chapter 3 : Systems for Detection and Identification of Bacteria and Yeasts *
- Authors: Cathy A. Petti, Melvin P. Weinstein, Karen C. Carroll
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Traditionally, the detection and identification of bacteria were based on conventional tube-based biochemical reactions, and their results were compared to historical charts of expected biochemical reactions. Automation in microbiology first occurred in the early 1970s with the introduction of semiautomated blood culture instruments, followed by instrumented systems for identification and susceptibility testing of bacteria. This chapter reviews the systems used for the detection of bacteria and yeasts from blood and provides an overview of technologies for microorganism identification. The volume of blood obtained for culture is one of the most important variables in the detection of bloodstream infections (BSIs). The chapter describes the identification of microorganisms by nonphenotypic methods from instrument-flagged blood culture bottles, and from pure culture. The approximate turnaround time of the fluorescence in situ hybridization (FISH) procedure is 2.5 to 3 h (without batch testing), compared with >18 to 24 h for identification of bacteria and yeasts by conventional methods. The selection of DNA targets to identify bacteria and fungi relies on the concept that some genes have conserved segments flanked by variable regions. The technology is based on analyzing the protein composition of a bacterial cell, with ribosomal proteins comprising most bacterial proteins being detected. Another technique to characterize bacteria is the application of electrospray ionization-mass spectrometry to analyze products of multilocus, broad-range PCR (PCR/ESI-MS).
Chapter 4 : Molecular Microbiology
- Authors: Frederick S. Nolte, Angela M. Caliendo
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Technological advances in realtime PCR techniques, automation, nucleic acid sequencing, multiplex analysis, and mass spectrometry have reinvigorated the field and created new opportunities for growth. Molecular microbiology is the leading area in molecular pathology in terms of both the numbers of tests performed and clinical relevance. This chapter covers amplified- and nonamplified-probe techniques, postamplification detection and analysis, clinical applications of these techniques. It also discusses the special challenges and opportunities that these techniques provide for the clinical laboratory. Although probes can range from 15 to thousands of nucleotides in size, synthetic oligonucleotides of less than 50 nucleotides are commonly incorporated into commercial kits. In most clinical applications, formalin-fixed, paraffin-embedded tissue sections are used. Nonamplified-probe techniques are used most effectively in culture confirmation assays for mycobacteria and systemic dimorphic fungi. Strategies other than PCR, are based on signal, target, or probe amplification, and have sensitivity unparalleled in laboratory medicine, have created new opportunities for the clinical laboratory to have an effect on patient care, and have become the new “gold standards” for laboratory diagnosis of many infectious diseases. In signal amplification methods, the concentration of the probe or target does not increase. Signal amplification assays have several advantages over target amplification assays. The target amplification systems share certain fundamental characteristics. They use enzyme-mediated processes, in which a single enzyme or multiple enzymes synthesize copies of target nucleic acid. In these techniques, the amplification products are detected by two oligonucleotide primers that bind to complementary sequences on opposite strands of double-stranded targets.
Chapter 5 : Immunoassays for the Diagnosis of Infectious Diseases *
- Author: A. Betts Carpenter
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This chapter summarizes the variety of different assays available and their particular application in the field of infectious diseases. The discussion emphasizes general assay design, with important caveats relevant to test interpretation and development. The first immunoassays available measured milligram to microgram quantities of antibodies and relied primarily upon precipitation reactions between antigen and antibody. There are many classic agglutination assays used in the diagnosis of infectious diseases, such as the Widal test for typhoid fever. The spectrum of immunologic assays is discussed in detail. Enzyme immunoassays (EIAs) can be broadly classified as either homogeneous or heterogeneous assays. Noncompetitive indirect solid-phase ELISA is one of the most frequently employed immunoassays in a clinical laboratory. There are various types of anti-animal antibodies; the most commonly reported are human anti-mouse antibodies (HAMA). There are many different assay designs which combine an array of technologies, including chemiluminescence (CL), fluorescence immunoassay (FIA), flow cytometry, and molecular diagnostics (PCR and use of oligonucleotides and nanoparticles). As these assays provide rapid results and can be performed with very small sample sizes, they have wide applicability to epidemiological studies and vaccine trials. These assays are also especially suited to the assessment of multiple biological agents in a variety of samples.
Chapter 6 : Infection Control Epidemiology and Clinical Microbiology
- Authors: Daniel J. Diekema, Michael A. Pfaller
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This chapter discusses the impact of nosocomial infections, outlines the organization of the hospital infection control program, and describes the important role of the clinical microbiology laboratory in the prevention and control of health care-associated infections. The Study of the Efficacy of Nosocomial Infection Control indicated that the presence of an active surveillance and infection control program was associated with a 32% decrease in nosocomial infection rates while the absence of such a program was associated with an 18% increase in nosocomial infection rate. The hospital infection control program should include surveillance and prevention of nosocomial infections. The chapter focuses on the most important specific roles played by a microbiology laboratory in the day-to-day practice of infection control. Commercial identification and susceptibility testing systems allow most laboratories to identify microorganisms to species level and perform antimicrobial susceptibility testing (AST). However, the expanding spectrum of organisms that colonize and infect seriously ill patients challenges the ability of a clinical microbiology laboratory to identify and characterize nosocomial pathogens accurately. When the infection control team detects a cluster or outbreak of nosocomial infection, they must act promptly to identify the etiologic agent if it is not known, define the extent of the outbreak, learn the mode of transmission for the pathogen, and institute appropriate control measures. Development and application of new technologies in the clinical laboratory can greatly enhance infection control efforts.
Chapter 7 : Investigation of Enteric Disease Outbreaks
- Authors: Timothy F. Jones, William E. Keene
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This chapter talks about acute gastroenteritis and other foodborne diseases that can be caused by bacteria, viruses, protozoa, fungi, helminths, prions, and biological or environmental toxins. Many foodborne and waterborne diseases are self-limited and characterized by gastrointestinal symptoms such as vomiting and diarrhea. In a few situations, such as mushroom poisoning, ciguatera fish poisoning, or other chemical intoxications, it is sufficient to document the clinical syndrome among affected persons. Staphylococcus can also be problematic because the organism may not be viable in stool or food samples, and most laboratories cannot test for enterotoxin. Enteric disease surveillance is generally the province of public health agencies. Surveillance includes the collection and analysis of information about disease occurrence and leads to taking considered action based upon those data. Disease surveillance often is based on mandatory reporting laws, whereby diagnostic laboratories or clinicians are required to notify public health agencies about individuals with specified conditions, e.g., salmonellosis or hepatitis A, as well as unusual clusters of illness. In recent years, however, public health officials and the public have become increasingly concerned that the food supply system is a potential target of intentional acts of contamination, sabotage, or terrorism. Summary information about foodborne and waterborne outbreak investigations is typically reported to the CDC, which periodically summarizes these data. It should be emphasized that the quality of these data is highly variable, which complicates one's ability to summarize them meaningfully.
Chapter 8 : Molecular Epidemiology
- Authors: Peter Gerner-Smidt, Eija Hyytiä-trees, Paul A. Rota
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Molecular epidemiology is often confused with another related but distinct microbiology discipline: molecular taxonomy. The use of nucleic acid hybridization techniques and the analysis of housekeeping gene sequences have greatly improved our understanding of microbial evolution. Compared to taxonomy and phylogeny, molecular epidemiology is the study of more recent population dynamics. In molecular epidemiology, molecular methods are used for detection, identification, virulence characterization, and subtyping, i.e., to generate isolate-specific molecular fingerprints for assessment of epidemiological relatedness. This chapter is an introduction to molecular epidemiology and basic molecular epidemiological concepts. A nonexhaustive list of subtyping methods that are commonly used now or are under development and are anticipated to supplement or replace the currently used ones is given. Subtyping method development including validation and quality control is discussed. The selection of methods appropriate in different contexts and the manner in which the choice of method and the epidemiological context influence the interpretation of data are also dealt with. Since the DNA arrays and sequencing methods are organism specific to a very great extent, traditional methods will still be needed to detect and identify new and reemerging pathogens.
Chapter 9 : Procedures for the Storage of Microorganisms *
- Authors: Cathy A. Petti, Karen C. Carroll
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This chapter presents methods that can be used for the storage of bacteria, protozoa, fungi, and viruses. There are two types of cryoprotective agents: those that enter the cell and protect the intracellular environment and others that protect the external milieu of the organism. Glycerol and dimethyl sulfoxide (DMSO) are most often used for the former; sucrose, lactose, glucose, mannitol, sorbitol, dextran, polyvinylpyrrolidone, polyglycol, and skim milk are used for the latter. To protect microorganisms from damage during the freezing process, during storage, and during thawing, cryoprotective agents are often added to the culture suspension. Whereas most bacteria, fungi, and viruses survive better with such additives, studies have shown that cryoprotective agents significantly damage others. Freeze-drying is considered to be the most effective way to provide long-term storage of most bacteria, yeasts, sporulating molds, and viruses. The preparation is then used in smaller volumes as described above for freezing. All of the material presented in this chapter applies primarily to the preservation of bacteria. Subculturing is the simplest method of maintaining living fungi and involves serial transfer to fresh solid or liquid media. Storage is accomplished usually at room or refrigerator temperature. All of the techniques described have been applied to the storage of yeasts and fungi. Viruses tend to be more stable than other microorganisms because of their small size and simple structure, and the absence of free water. Many viruses can be stored for months at refrigerator temperatures or for years by ultralow-temperature freezing or freeze-drying.
Chapter 10 : Prevention and Control of Laboratory-Acquired Infections
- Author: Michael A. Noble
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This chapter talks about laboratory biosafety and its goals that include prevention of laboratory-acquired infections in workers and accidental releases of live agents which can potentially endanger and have severe negative impact on humans, animals, and plants. Laboratory safety involves all aspects of the laboratory cycle, starting from before microorganisms arrive in the facility and continuing through the training of personnel, the establishment and monitoring of safe working practices, the proper use of reagents, materials, and equipment, the safe storage and transport of agents, and ultimately the terminal sterilization and destruction of microorganisms. Attitudes and work habits are considered to be important contributing factors to laboratory accidents according to a matched case-control study. Scalpels, needles, broken glass, and other sharps are commonly associated with wound injuries and laboratory-acquired infections. Hand washing is a useful technique to stop the transmission of microorganisms and acquisition of infection in medical laboratories. Gloves can provide an important barrier within the laboratory, provided that they are used appropriately. Immunization provides protection against some laboratory acquired infectious diseases but should be considered secondary to mental alertness and good laboratory practices. Finally, personal protective equipment, including M95 respirators, should be considered for additional protection.
Chapter 11 : Decontamination, Disinfection, and Sterilization
- Authors: Andreas F. Widmer, Reno Frei
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Most disinfectants were introduced to the market more than 30 years ago and little is known about their modes of action and the mechanisms of resistance. In addition, a few basic procedures in decontamination, disinfection, and sterilization have been tested in randomized clinical trials. This chapter cites highest level of evidence available. Even the definition of sterilization as the absence of any viable microorganisms must be revised to address the prions responsible for Creutzfeldt-Jakob disease (CJD) and variant CJD (vCJD). Semicritical equipment should be processed with a high-level disinfectant such as glutaraldehyde, stabilized hydrogen peroxide, peracetic acid, or a chlorine compound. The chapter discusses use of peracetic acid for chemical sterilization of instruments and endoscopes. Studies of sheep naturally infected with scrapie demonstrated that the infectious agent first appears in lymphatic tissue of the tonsils and gastrointestinal tract, suggesting the oral route may be the principal mode of transmission. Numerous studies underline the importance of the B cell in transmission of the bovine spongiform encephalopathy (BSE) agent. In May 2005, British officials published an excellent assessment of the risk for contaminating surgical instruments with prions. The key observation in this report is that on average 0.2 mg of protein remains on surgical instruments despite “standard cleaning and disinfection,” which was sufficient to cause an experimental case of CJD. Therefore, more research and new methods of cleaning and disinfection are needed for surgical instruments.
Chapter 12 : Biothreat Agents
- Authors: Susan E. Sharp, Mike Loeffelholz
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Due to their unique features in causing mass destructive diseases, the priority biological agents for use in bioterror events include anthrax, brucellosis, Q fever, tularemia, plague, hemorrhagic fever viruses, and toxins (botulinum, staphylococcal enterotoxins, and T-2 mycotoxins). Each of these is discussed in this chapter with regard to its significance as a biothreat agent, its epidemiology and natural routes of transmission, and appropriate specimens to submit for laboratory diagnosis. The two laboratories considered as Laboratory Response Network (LRN) national laboratories function to perform susceptibility testing on biothreat agents when necessary and can also safely handle highly infectious viral agents. The function of the LRN sentinel laboratories is to recognize possible biothreat agents and submit them to an LRN reference laboratory as soon as possible for definitive identification. A section of the chapter focuses on 16 recently emergent pathogens-some well recognized, others less so-and provides contemporary knowledge, with emphasis on epidemiology and factors responsible for emergence. Most human infections are associated with consumption of beef or direct contact with animals and animal feces. Clostridium difficile is one of the most important health care-associated pathogens, causing diarrhea and pseudomem-branous colitis in hospitalized patients and residents of long-term care facilities. Japanese encephalitis virus (JEV) is endemic in East and Southeast Asia, where it is the most important cause of mosquito-borne encephalitis.
Chapter 13 : The Human Microbiome
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The human microbiome includes bacteria, viruses, and small eukaryotes, such as fungi, and this chapter focuses on the bacterial members of the microbiome. The Human Microbiome Project (HMP) aims at developing tools and resources for characterization of the human microbiota and to relate this microbiota to human health and disease. The goals of the jumpstart phase have been to sequence 900 reference genomes to provide a catalog of genomes for metagenomic studies, to sample at least 300 healthy adults between 18 and 40 years of age at five body sites, and to develop sequencing and analysis protocols for the samples derived from human subjects. The second phase of the HMP includes human microbiome studies that target particular disease states. In a recent study, four phyla comprised 92.3% of bacterial DNA sequences analyzed from multiple human sources, including hair, oral cavity, skin, genitourinary, and gastrointestinal tract. A study by Pei et al. showed that the distal esophageal microbiomes of four adults had compositions similar to that of the oropharynx, with the exception that no spirochetes were found in the esophagus. The chapter concludes by highlighting that pathogen discovery efforts will be enhanced by new metagenomics strategies, and these studies may uncover single etiologic agents of infections as well as relative shifts in groups of bacterial pathogens that may contribute to human disease.
Chapter 14 : Microbial Genomics and Pathogen Discovery
- Authors: Anne M. Gaynor, David Wang
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This chapter discusses the concepts of microbial genomics and pathogen discovery. The discussion begins with an overview of traditional efforts, followed by a brief description of the evolution of sequencing technologies. Finally, the chapter illustrates how the two fields began to intersect in the first few years of the 21st century. The discoveries of Hepatitis C virus (HCV) and Human herpesvirus 8 (HHV-8), also called Kaposi's sarcoma-associated herpesvirus (KSHV), represented two breakthroughs in the application of candidate-independent molecular methods for pathogen discovery. The onset of the 21st century has seen the convergence of the fields of pathogen discovery and microbial genomics. The PhyloChip, a high-density 16S rRNA microarray containing 300,000 probes, was used to examine the microbial diversity in three environmental samples, including urban aerosol, subsurface soil, and subsurface water. This study demonstrated that the array could reveal broader diversity than classical cloning and sequencing of the same sample. The challenge facing the scientific community for the remainder of this century is to develop commensurate approaches to defining the biological relevance of this multitude of novel microbes to human disease.
BACTERIOLOGY
GENERAL
Chapter 15 : Taxonomy and Classification of Bacteria
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The process of species delineation in bacterial systematics has undergone drastic modifications as the species concept evolved in parallel with technical progress. The early classification concept was replaced by theories of so-called natural concepts, which were the phenetic and phylogenetic classifications. In the former, relationships between bacteria were based on the overall similarity of phenotypic and genotypic characteristics. The species is the most important and, at the same time, the central element of bacterial taxonomy. The tree of life, based on comparative small-subunit rRNA studies, comprises three lines of descent that are nowadays referred to as the domains Bacteria, Archaea, and Eucarya. Researchers have reported on the place for 16S rRNA gene sequence analysis and DNA-DNA reassociation in the present species definition in bacteriology. In spite of its limitations, rRNA sequence analysis is now commonly used for the identification of bacteria. The determination of the cell wall composition has traditionally been important for gram-positive bacteria. The majority of bacteria in routine diagnostic laboratories will continue to be identified by classical methods, as these methods are adequate, inexpensive, readily available and easy to handle. The present view of classification reflects the best science of this time. The same was true in the past, when only data from morphological and biochemical analyses were available. The main perspective in bacterial taxonomy is that technological progress will dominate and drastically influence methodology, as it always has.
Chapter 16 : Specimen Collection, Transport, and Processing: Bacteriology
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One of the key principles of good specimen collection is to avoid introduction of colonizing bacteria surrounding the site of infection or on the skin or mucous membranes near the infectious site. A relatively recently introduced type of swab, the flocked swab, has proved to be superior to fiber swabs for collection of nasopharyngeal samples for detection of respiratory viruses, but there were no significant differences between flocked and rayon swabs when throat cultures were evaluated. For abscess contents, body fluids, and other fluid collections below the skin, aspirates obtained through disinfected intact skin are preferred over swabs. Expectorated sputum is the best sample for diagnosis of pneumonia, a disease of the distal lung alveolar spaces. Urine can be collected by midstream collection, catheterization, cystoscopic collection, or suprapubic aspiration. Urine specimens should be transported to the laboratory immediately and processed within 2 h of collection. Testing more than one stool for bacterial pathogens is usually not productive. Fresh stool should be examined visually, and the areas showing blood, pus, or mucus should be sampled preferentially. In spite of acceptable labeling, some specimen collection sites, transport containers, or transport conditions render the specimen unacceptable for processing. Although blood has not yet been used widely for detection of bacteria using nucleic acid amplification (NAA) tests or hybridization, such tests are in development. Swabs should be transported to the laboratory in special media.
Chapter 17 : Reagents, Stains, and Media: Bacteriology *
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A number of classical and rapid tests are used for the identification of medically important bacteria. N-acetyl-L-cysteine-sodium hydroxide (NALC (mucolytic agent)-NaOH), Cetylpuridium chloride-sodium chloride (CPC-NaCl), oxalic acid are used for decontamination agents. A variety of dyes and indicators are used to detect specific reactions such as pH and oxygen production. A variety of stains can then be used to help visualize and differentiate bacteria from the specimen. Gram staining is the differential staining procedure most commonly used for microscopic examination of bacteria. Based upon the staining reaction, bacteria are classified as gram-positive organisms, which retain the primary crystal violet dye and appear deep blue or purple, and gram-negative organisms, which can be decolorized, thereby losing the primary stain and subsequently taking up the counterstain safranin and appearing red or pink. Louis Pasteur in 1860 was the first to use culture media for growing bacteria in the laboratory. Almost immediately, additional media began to be developed by Robert Koch and his colleagues, who used animal and plant tissues as sources of nutrients to support bacterial growth. Using solid media permits the isolation of pure cultures of bacteria. Agar is the most common solidifying agent used in microbiological media. Many media contain selective components that inhibit the growth of nontarget bacteria. Some pathogens are anaerobic, and factors such as thioglycolate are included in some media to reduce the availability of molecular oxygen so that anaerobes may be cultured. Bacteria that reduce nitrate to nitrite turn the reagents red or pink.
GRAM-POSITIVE COCCI
Chapter 18 : General Approaches to Identification of Aerobic Gram-Positive Cocci
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Majority of aerobic, or facultatively aerobic, gram-positive cocci isolated from clinical specimens are distributed among the genera Staphylococcus, Streptococcus, and Enterococcus. This chapter provides tables containing organisms with similar cellular morphologies, either "streptococcal," consisting of gram-positive cocci or coccobacilli arranged primarily in pairs and/or chains, or "staphylococcal," signifying that cells appear as cocci arranged in pairs, tetrads, clusters, and irregular groups. The commonly isolated aerobic gram-positive cocci (staphylococci, streptococci, enterococci) can usually be accurately identified by determining a few basic phenotypic traits (cellular morphology, catalase reaction, and production of pyrrolidonyl arylamidase [PYR]). The chapter highlights the fact that it is increasingly difficult to identify some of the less frequently isolated organisms solely on the basis of phenotypic traits, as new genera and species of aerobic gram-positive cocci are described and characterized. Basic phenotypic tests can usually suggest a possible identity for strains of infrequently encountered aerobic gram-positive cocci, but evaluation with a larger battery of phenotypic tests or molecular identification methods is often valuable, if not indispensible, for accurate identification. Nucleic acid probe tests and amplification methods for identification of some of the commonly isolated aerobic gram-positive cocci are commercially available and designed for use in medium to large-volume clinical microbiology laboratories. The chapter concludes by emphasizing that the comparison of 16S rRNA gene sequences is the most useful method for molecular characterization of the aerobic gram-positive cocci of clinical interest, although sequence comparison of other genes may also be helpful for identification.
Chapter 19 : Staphylococcus, Micrococcus, and Other Catalase-Positive Cocci *
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Historically, the genera Staphylococcus and Micrococcus were placed together with the genera Stomatococcusand Planococcus in the family Micrococcaceae containing grampositive, catalase-positive cocci. An unrelated species of gram-positive cocci exhibiting positive catalase reaction and occurring in human specimens is Alloiococcus otitis, the only species of this genus, which is a member of the Carnobacteriaceae family belonging to the order Lactobacillales. In this chapter, the term "micrococci" is used to indicate the members of the genus Micrococcus as understood before the emendation, reflecting most of the clinically relevant species. The Macrococcus genus comprises four hoofed-animal-adapted species including M. caseolyticus, first described as S. caseolyticus. Staphylococcus species can be identified phenotypically on the basis of a variety of conventional characteristics. The diagnosis of staphylococcal toxic shock syndrome (TSS) and staphylococcal scalded skin syndrome (SSSS) is based on clinical signs supplemented by serologic tests and the detection of the toxin production by staphylococcal isolates (S. aureus, rarely other species). Detection of methicillin-resistant S. aureus (MRSA) represents the most important task in determining the antimicrobial susceptibilities of staphylococci. Coagulase-negative staphylococci (CoNS) are an important cause of nosocomial bloodstream infections, but they are also the most common contaminants of blood cultures. The considerations of clinical significance discussed for CoNS are also appropriate for members of the Micrococcaceae and Dermacoccaceae families; however, the criteria used for distinguishing etiologically relevant isolates from contaminants and colonizers, respectively, should be applied much more strictly.
Chapter 20 : Streptococcus
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This chapter presents the information and the identification schemes which adhere in many aspects to the phenotypic classification system. In a study of the genus Streptococcus based on sequence comparisons of small-subunit (16S) rRNA genes, five species groups of viridans group streptococci were demonstrated in addition to the pyogenic group (beta-hemolytic, large-colony formers. Streptococci can cause infections in humans and in many different animal species including mammals and fish. Reflecting the ongoing changes in the epidemiology of group B streptococcal disease, the highest attack rates were observed in patients less than 1 year and adults greater than 65 years of age. The predominant reservoir for S. dysgalactiae subsp. equisimilis strains is the human host, and transmission usually occurs among humans. A rapid method for the detection of S.pyogenes in pharyngeal specimens is based on a single-stranded chemiluminescent nucleic acid probe assay to identify specific rRNA sequences. It is important to distinguish Streptococcus from Enterococcus prior to L-pyrrolidonyl-β-naphthylamide (PYR) testing, and strains of other related genera may be PYR positive (including the genera Abiotrophia, Aerococcus, Enterococcus, Gemella, and Lactococcus). The VP test can be performed for the identification of beta-hemolytic streptococci. In the majority of cases, typing of streptococci has no immediate clinical or therapeutic consequences. It is most often performed by reference laboratories for the purposes of epidemiologic studies and the evaluation of vaccine efficacy. Determination of streptococcal antibodies is indicated for the diagnosis of poststreptococcal disease.
Chapter 21 : Enterococcus
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Current criteria for inclusion in the genus Enterococcus and for the description of new enterococcal species encompass a polyphasic approach resulting from a combination of different molecular techniques (frequently involving DNA-DNA reassociation experiments, 16S rRNA gene sequencing, and whole-cell protein profiling analysis) and phenotypic tests. Several other molecular methods, mostly nucleic acid-based assays, have been used as additional tools to assess the phylogenetic relationships among enterococcal species and to formulate the description of new species, but their use is still limited. Positive catalase testing has also been reported for strains of Enterococcus haemoperoxidus and Enterococcus silesiacus when cultivated on blood-containing agar media. Phenotypic characteristics are used for the identification of Enterococcus species and some physiologically related species of other gram-positive cocci. Serologic tests for detecting antibody responses to different enterococcal antigens have been proposed. In addition to the intrinsic resistance traits, enterococci have acquired different genetic determinants that confer resistance to several classes of antimicrobial agents, including chloramphenicol, tetracyclines, macrolides, lincosamides and streptogramins, aminoglycosides, β-lactams, glycopeptides, quinolones, and even some of the more recently available drugs, such as linezolid, daptomycin, and quinupristin-dalfopristin. Antimicrobial resistance can be classified as either intrinsic or acquired. Molecular methods have been used to detect specific antimicrobial resistance genes and have substantially contributed to the understanding of the spread of acquired resistance among enterococci, especially resistance to vancomycin. However, because of their high specificity, molecular methods do not detect antimicrobial resistance due to mechanisms not targeted by the testing, including emerging resistance mechanisms.
Chapter 22 : Aerococcus, Abiotrophia, and Other Aerobic Catalase-Negative, Gram-Positive Cocci
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The aerobic catalase-negative gram-positive cocci included in this chapter form a taxonomically diverse group of bacteria that are isolated infrequently as opportunistic agents of infection. The genera Abiotrophia and Granulicatella accommodate organisms previously known as nutritionally variant or satelliting streptococci. Most of the genera described here are catalase-negative facultative anaerobes, but Aerococcus viridans are classified as a microaerophile that grows poorly, if at all, under anaerobic conditions. Some strains of Aerococcus may exhibit weakly positive catalase reactions due to nonheme catalase activity. Commercially available identification kits or systems offering a more comprehensive array of phenotypic tests are improving in their ability to identify many of the organisms discussed in the chapter. Serologic response to the organisms described in this chapter has not been extensively investigated. Antimicrobial susceptibility studies on the organisms mentioned in the chapter have generally employed dilution testing methods. The lack of standardized methods and interpretive criteria and the relatively small collections of isolates for some of the genera discussed in the chapter make it difficult to accurately assess antimicrobial susceptibility patterns. Efforts to identify the gram-positive cocci included in the chapter should be made only when isolates are considered to be clinically significant, since the organisms may also appear in clinical cultures as contaminants or constituents of the normal microbiota. More extensive phenotypic testing using commercially available identification systems and molecular methods should be employed for definitive identification.
GRAM-POSITIVE RODS
Chapter 23 : General Approaches to the Identification of Aerobic Gram-Positive Rods
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This chapter emphasizes that the Gram stain (performed on 24- to 48-h-old colonies from rich media) and macroscopic morphologies are initial key features for the differentiation of aerobic gram-positive rods. All strains of aerobic gram-positive rods (except the non-rapidly growing mycobacteria) are initially grown on blood agar plates. Catalase activity should be tested with colonies grown on media lacking heme groups. Type of metabolism can be evaluated using oxidative-fermentative media or in cystine Trypticase agar (CTA) medium. Genera which contain strictly anaerobic gram-positive rods may also contain species, or strains within a species, which grow reasonably well aerotolerantly or aerobically. Some gram-positive rods (e.g., Rhodococcus spp. or Dermabacter might be initially misidentified as gram-positive cocci because of their initial gram stain appearance. Molecular approaches to characterize or subtype pathogens are described in the chapter.
Chapter 24 : Bacillus and Other Aerobic Endospore-Forming Bacteria *
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This chapter discusses isolation and various tests for identification and detection of bacteria. The majority of aerobic endospore-forming species apparently have little or no pathogenic potential and are rarely associated with disease. Human anthrax has traditionally been classified as either (i) nonindustrial, resulting from close contact with infected animals or their carcasses after death from the disease, or (ii) industrial, as acquired by those employed in processing wool, hair, hides, bones, or other animal products. Clinical specimens for isolation of Bacillus species other than Bacillus anthracis can be handled safely on the open bench without special precautions. Sections of tissue, or any blood-stained material, should be collected, and spleen or lymph node specimens should be taken if the animal has been opened. The majority of work on molecular typing of Bacillus spp. has focused on members of the Bacillus cereus group due to their clinical importance and the value of genotyping for molecular epidemiology. Anthraxin does not contain highly specific anthrax antigens and relies on the fact that the only Bacillus species likely to proliferate within and throughout an animal is Bacillus anthracis. Low-level contamination of foodstuffs by aerobic endospore formers is commonplace, as is asymptomatic transient fecal carriage. Therefore, in foodborne illness investigations, qualitative isolation tests are insufficient.
Chapter 25 : Listeria and Erysipelothrix *
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The majority of cases of listeriosis occur in individuals who have an underlying condition that leads to suppression of their cell-mediated immunity. Among veterinarians and abattoir workers, primary cutaneous listeriosis with or without bacteremia has been reported. Transient bacteremia can result in placentitis and/or amnionitis, and since Listeria is able to cross the placenta, it can infect the fetus, causing abortion, stillbirth, or, most commonly, preterm labor. In general, specimens for detection of Listeria do not need special handling during collection. Listeria colonies appear blue, and colonies of other bacteria appear yellowish or orange. The CAMP (Christie, Atkins, Munch-Petersen) test can be used to differentiate among hemolytic Listeria species. Commercially available miniaturized tests considerably speed up biochemical identification of Listeria spp. The matrix-assisted laser desorption ionization-time of flight (mass spectrometry) (MALDI-TOF [MS]) technique has recently been introduced and allows discrimination of the Listeria species by use of the respective software. Treatment with an aminopenicillin (ampicillin or amoxicillin) plus gentamicin is still regarded as the most effective therapeutic regimen for listeriosis. Listeria species are catalase positive, motile, esculin positive, and not alpha-hemolytic. The genus Erysipelothrix has three validly published species, Erysipelothrix rhusiopathiae, Erysipelothrix tonsillarum, and the more recently described Erysipelothrix inopinata. E. rhusiopathiae has been recognized for more than 100 years as the agent of swine erysipelas, an acute or chronic disease. Occurrence of this species in wound or tissue specimen indicates erysipeloid rather than contamination. Species identification is essential in order to ensure adequate antimicrobial therapy.
Chapter 26 : 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." The bacteria discussed in the chapter all belong to the class Actinobacteria, the genera of which are characterized by specific 16S rRNA gene signature nucleotides. The 16S rRNA gene sequencing data demonstrate that the genera Corynebacterium and Turicella are more closely related to the partially acid-fast bacteria and to the genus Mycobacterium than to the other coryneform organisms covered in the chapter. The chapter discusses descriptions of genera and species. Detection of antibodies directed against diphtheria toxin is the only established serologic test for coryneform bacteria. Toxin neutralization assays using a Vero cell culture system have been replaced mainly by enzyme immunoassays. The susceptibility patterns for each taxon were given with the descriptions of each taxon. Since the antimicrobial susceptibility of coryneform bacteria is not predictable in every case, susceptibility testing should always be performed with clinically significant isolates.
Chapter 27 : Nocardia, Rhodococcus, Gordonia, Actinomadura, Streptomyces, and Other Aerobic Actinomycetes
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This chapter talks about aerobic actinomycetes that are now known to be an evolutionarily heterogeneous assemblage of genera. At some stage they all form gram-positive rods, and most of the more commonly isolated species exhibit at least rudimentary branching under certain growth conditions; all grow better under aerobic than anaerobic conditions, a feature distinguishing them from most organisms in the genus Actinomyces. In temperate climates, the respiratory tract is the most frequent portal of entry for the aerobic actinomycetes and therefore the primary site of nocardial infections in the immunocompromised host. PCR paired with restriction endonuclease analysis (REA) has been used for the identification of commonly isolated Nocardia species. With REA of a portion of the HSP gene, Steingrube et al. were able to differentiate among 12 taxonomic groups of Nocardia, in addition to species of Actinomadura, Gordonia, Rhodococcus, Streptomyces, and Tsukamurella. The recommended procedure for Nocardia and the other aerobic actinomycetes is broth microdilution; and panels containing the appropriate dilutions of antimicrobials specifically active against these genera are commercially available. Most isolates of Nocardia species are susceptible to trimethoprim-sulfamethoxazole; one should be careful not to assume too readily that an isolate is resistant to this combination of drugs. The chapter finally points out that sulfonamides or trimethoprim-sulfamethoxazole may not be adequate in certain circumstances, such as patients with central nervous system (CNS) nocardiosis, disseminated disease, or concurrent HIV infection.
Chapter 28 : Mycobacterium: General Characteristics, Laboratory Detection, and Staining Procedures *
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This chapter deals with Mycobacterium the only genus in the family Mycobacteriaceae and related to other mycolic acid-containing genera. The genus Mycobacterium includes obligate pathogens, opportunistic pathogens, and saprophytes. Ben Salah et al. found clinical M. avium complex (MAC) isolates that appear to represent three other new species: M. marseillense, M. timonense, and M. bouchedurhonense. Many different types of clinical specimens may be collected for mycobacteriological analyses. A majority of clinical specimens originate from the respiratory tract (sputum, tracheal and bronchial aspirates, and bronchoalveolar lavage specimens), but urine, gastric aspirates, tissues, biopsy specimens, and normally sterile body fluids such as cerebrospinal fluid and pleural and pericardial aspirates are other commonly submitted specimens. A majority of disseminated mycobacterial infections are due to MAC. Appropriate pretreatment and processing procedures (homogenization, decontamination, concentration, culture media, and conditions of incubation) must be selected to facilitate optimum recovery of mycobacteria. With the advent of molecular techniques designed for molecular epidemiology, cross-contamination either linked to laboratory procedures or, more rarely, to contaminated bronchoscopes can easily be proven. Laboratory aspects of cross-contamination are addressed in the chapter. Quality control (QC) is vital for monitoring a laboratory’s effectiveness in detecting and isolating mycobacteria. Accurate identification of nontuberculous mycobacteria (NTM) will prevent rarely encountered pathogens from being mistaken for nonpathogenic species.
Chapter 29 : Mycobacterium: Laboratory Characteristics of Slowly Growing Mycobacteria *
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This chapter represents that detailed phenotypic methods of identification of mycobacteria. Although some phenotypic characterization remains important, it is now well recognized by experts in mycobacterial taxonomy that current accurate identification of organisms of both the Mycobacterium tuberculosis complex (MTBC) and the nontuberculous mycobacteria (NTM) requires molecular techniques for definitive identification to species level. The MTBC remains the most important group within the genus Mycobacterium from a global and clinical perspective. Mycobacterium species differ in the ability to grow at certain temperatures. For determination of the preferred growth temperature, solid culture media are inoculated with defined suspensions of mycobacteria and incubated at various temperatures. The chapter further highlights that some Mycobacterium species cannot use nicotinic acid, although they produce it in their biosynthetic pathways. Commercially available assays are intended for the detection of some of the most important Mycobacterium species and can be performed from both solid and liquid media. Two tests for the identification of several mycobacterial species are commercially available but are not yet FDA approved. The INNO-LiPA Mycobacteria v2 assay is based on the nucleotide differences in the 16S-23S rRNA gene spacer region. The GenoType Mycobacterium CM/AS test is based on the detection of species-specific sequences in the 23S rRNA gene. The major criterion for molecular strain typing is that the test isolates must all belong to the same species. Thus, definitive identification to the species level is an important prerequisite.
Chapter 30 : Mycobacterium: Clinical and Laboratory Characteristics of Rapidly Growing Mycobacteria
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Rapidly growing mycobacteria (RGM) are generally defined as nontuberculous species that grow within 7 days on laboratory media. The RGM are opportunistic pathogens that produce disease in a variety of clinical settings. Traumatic wound infections, especially open fractures, often involve species within the Mycobacterium fortuitum third biovariant complex. The current proposal for clinical laboratories is that biochemical testing of RGM should be replaced with molecular methods. The INNO LiPA multiplex probe assay is based on the principle of reverse hybridization. Generally, the identification of mycobacteria, including RGM, focuses on two main hypervariable domains known as region A and region B, located on the 5' end of the 16S rRNA gene. A recent Clinical and Laboratory Standards Institute (CLSI) document has recommended guidelines for 16S rRNA gene sequencing in order to identify Mycobacterium sp in a consistently practical manner. Although the 65-kDa heat shock protein gene (hsp65) is highly conserved among species of mycobacteria, it exhibits greater interspecies and intraspecies polymorphism than the 16S rRNA gene sequence. Serotyping has not been suitable for routine species identification of mycobacteria, including RGM, and early studies served to emphasize the complexity of the antigenic compositions of mycobacteria, as many antigens are shared by more than one species. The major species of RGM have different levels of virulence in different clinical settings and different drug susceptibilities.
GRAM-NEGATIVE BACTERIA
Chapter 31 : Approaches to the Identification of Aerobic Gram-Negative Bacteria *
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This chapter describes the approach to the identification of gram-negative rods, with emphasis on the greater difficulty in identifying non-glucose-fermenting organisms. Before presenting the identification scheme, several issues of terminology and methodology are addressed. The chapter uses more precise terminology for the terms utilization, oxidation, and glycolysis. Careful attention to testing methods is critical when describing their application for nonfermenters. Slight variations of reagents or incubation conditions can cause numerous mis-identifications and have caused erroneous descriptions of new species. The chapter provides limited description of assimilation assays for the identification of nonfermenters to that of acetamide and acetate, used solely as additional characteristics to differentiate between some species. Arginine dihydrolase activity by nonfermenters can best be tested by dense inoculation in Moeller broth with 1% arginine. Susceptibility to desferrioxamine, an Fe chelator, can be tested on Mueller-Hinton agar (MHA), or on tryptic soy agar (TSA) for strains growing poorly on MHA, using paper disks loaded with 250 µg of desferrioxamine or using commercially available Rosco tablets. Trypsin or benzyl-arginine arylamidase activity is very discriminative for nonfermenters, since the strains of almost all taxa exhibit an all-or-nothing positive result, providing a test with high discriminatory value. Some gram-negative organisms are vancomycin susceptible, and this can be used as a rapid and distinctive identification tool.
Chapter 32 : Neisseria
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The family Neisseriaceae is currently the only family within the order Neisseriales, which in addition to the genus Neisseria contains Eikenella, Kingella, and 27 other genera. Multiple-locus instead of single-locus approaches might therefore be more suitable for the resolution of species identification within the genus Neisseria. Neisseria species produce acid from carbohydrates by oxidation, not fermentation. Several kits combine carbohydrate utilization tests and direct enzyme detection assays for rapid confirmation of isolates belonging to Neisseria. The Api NH system can be used for the identification of Neisseria, Haemophilus, and Moraxella catarrhalis and uses 13 miniaturized tests. The pan-Neisseria MLST has the capacity to provide sufficient information for accurate species assignment. The two commercially available hybridization assays include Digene CT/GC Dual ID HC2 and Gen-Probe Pace 2, which use RNA probes targeting genomic DNA and DNA probes targeting rRNA, respectively. The difficulties in treatment and control of gonorrhea are aggravated by the ability of N. gonorrhoeae to mount resistance against a wide range of antibiotics. Fluoroquinolones may be used for treatment only if antimicrobial susceptibility can be documented by culture. A definitive diagnosis of N. gonorrhoeae requires (i) isolation of oxidase-positive gram-negative diplococci from sites of exposure by culture on selective media; and (ii) confirmation by biochemical or molecular methods.
Chapter 33 : Actinobacillus, Capnocytophaga, Eikenella, Kingella, Pasteurella, and Other Fastidious or Rarely Encountered Gram-Negative Rods *
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This chapter covers bacterial genera which are taxonomically diverse and belong to the families Cardiobacteriaceae, Flavobacteriaceae, Fusobacteriaceae, Neisseriaceae, Pasteurellaceae, and Porphyromonadaceae, but common traits justify their discussion as a group. It discusses only species that can be isolated from humans. The Pasteurellaceae consist of several genera, of which four are known to contain human pathogens: Actinobacillus, Aggregatibacter, Haemophilus, and Pasteurella. Phenotypic identification of fastidious gram-negative rods presents several challenges. Triple sugar iron or Kligler's agar may not support the growth of fastidious genera (e.g., Eikenella). Detection of antibodies directed against any of the bacteria discussed in the chapter has been tried on a small scale only and does not seem to offer much value. With specimens normally colonized with aerobic and anaerobic bacteria, as well as with specimens from wounds, e.g., bite wounds, the significance of the bacteria discussed in the chapter depends on their predominance and the absence of other potentially pathogenic bacteria. If these conditions are met, identification to the species level is needed for adequate interpretation and reporting as infectious agents and for susceptibility testing. If none of these conditions is present, a repeat culture and close cooperation between the microbiology laboratory and the physician are necessary for interpretation, for identification to the species or genus level, and for susceptibility testing.
Chapter 34 : Haemophilus
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While the number of Haemophilus species described greatly exceeds the number of human pathogens, eight species affecting humans currently included in this genus are H. influenzae, H. aegyptius, H. ducreyi, H. pittmaniae, H. parainfluenzae, H. haemolyticus, H. parahaemolyticus, and H. paraphrohaemolyticus. Aggregatibacter aphrophilus, Aggregatibacter paraphrophilus, and Aggregatibacter segnis were formerly included in the genus Haemophilus but have recently been reclassified into the genus Aggregatibacter (formerly Actinobacillus) based on molecular taxonomy. In the absence of the recently reclassified species, significant genetic diversity still exists in the Haemophilus genus. Haemophilus influenzae may be found as part of the commensal bacterial flora of the mucosal surfaces of the upper respiratory tracts (URT) of many healthy individuals. Several molecular methods, including 16S rRNA gene sequencing, polymerase chain reaction (PCR), and fluorescence in-site hybridization have been described in the literature as being effective tools for the species identification of Haemophilus when performed on organisms recovered in culture. Other molecular methods for typing have also been applied to Haemophilusspecies. In one study evaluating the performance of intergenic dyad sequence (IDS)-PCR with 69 NTHi isolates, the assay demonstrated 65 different banding patterns that were epidemiologically classified as fingerprints similar to those obtained by pulsed-field gel electrophoresis (PFGE).
Chapter 35 : Escherichia, Shigella, and Salmonella
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Members of the genus Shigella are phenotypically similar to Escherichia coli and, with the exception of Shigella boydii serotype 13, would be considered the same species by DNA-DNA hybridization analysis and whole-genome sequence analysis. The dynamic nature of the Shiga toxin-converting phages has implications for diagnostic testing for Shiga toxin-producing E. coli (STEC). Since STEC strains can lose critical virulence genes, some researchers have proposed that multiple virulence-associated genes, as well as conserved genes, be used to diagnose infections by these bacteria. This concept would also apply to other pathotypes of E. coli, as most of them carry critical virulence genes on mobile genetic elements. A preliminary report can be issued as soon as a presumptive identification of Salmonella is obtained. In most situations, a presumptive identification is based on phenotypic traits determined by either traditional or commercial systems or by reactivity with Salmonella O grouping antisera. A confirmed identification requires both phenotypic identification and O group or serotype determination. As national surveillance systems depend on the receipt of serotype information for Salmonella strains isolated in the United States, laboratories should follow the procedures recommended by their state health departments for submitting Salmonella isolates for further characterization, including complete serotyping. The antimicrobial susceptibilities of typhoidal Salmonella strains and strains from normally sterile sites should be determined, and the strains should be forwarded to a reference or public health laboratory for complete phenotypic identification and serotyping.
Chapter 36 : Yersinia *
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The pathogenic Yersinia species, Y. pseudotuberculosis, Y. enterocolitica, and Y. pestis, are zoonotic agents that cause disease in humans ranging from mild gastroenteritis to life-threatening plague. Several dozen virulence genes, their environment- dependent expression control, and the complex mechanisms of their product action and coordination, which enable immune system evasion and disease progression, have been actively investigated and described. Several Yersinia species can be differentiated by a number of phenotypic methods, and the four biovars of Y. pestis can be separated based on differential reactivity with glycerol, nitrate, and arabinose. Recent evidence suggests that biovars based on phenotypic methods do not show a strict correlation to groupings as determined by genotyping methods. Methods used for the evaluation of the relatedness of Yersinia species include a number of different phenotypic methods, including serotyping, biotyping, antibiogram analysis, and bacteriophage typing. Genotyping methods include pulsed-field gel electrophoresis (PFGE), which has long been considered the gold standard for typing of Yersinia species. Isolation rates vary based on geographic locations, with the highest incidence in temperate regions, so the decision to routinely rule out these organisms in stool cultures should be evaluated in individual laboratories after consultation with the infectious disease physicians.
Chapter 37 : Klebsiella, Enterobacter, Citrobacter, Serratia, Plesiomonas, and Other Enterobacteriaceae
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This chapter talks about the new species of Enterobacteriaceae that have been added to or transferred between existing genera. Most common gram-negative organisms isolated from respiratory tract, urinary tract, and bloodstream infections from intensive care unit patients in the United States were K. pneumoniae (15%), E. cloacae (9%), Serratia marcescens (6%), Enterobacter aerogenes (4%), Proteus mirabilis (4%), Klebsiella oxytoca (3%), and Citrobacter freundii (2%). The organisms are, in general, readily isolated from clinical material, and few of the clinically relevant strains covered present difficulties in isolation from sterile body sites. Isolation from nonsterile body or environmental sites may require specialized media such as CHROMagar Orientation and chromID CPS, which perform similarly for the detection of urinary tract pathogens covered in the chapter and can reliably replace MacConkey and blood agars. The chapter also discusses diarrheal pathogens that are easily isolated, and about biochemical tests most useful for separating members. Even when unusual enterobacteria covered in the chapter are included in commercial system databases, the number of strains available to use in challenge studies is very limited; therefore, the ability of these systems to accurately identify these organisms is really unknown. General antimicrobial susceptibility and specialized phenotypic testing procedures are also discussed in the chapter.
Chapter 38 : Aeromonas *
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Aeromonas is the genera that is pathogenic for humans. The use of frequent reclassifications and constant amended or extended descriptions within Aeromonas taxonomy can often be initially puzzling to microbiologists not working with these organisms on a daily basis. However, information in this chapter should clarify the identification and significance of those species most often associated with human disease. Aeromonads are inhabitants of aquatic ecosystems worldwide such as groundwater, reservoirs, and clean or polluted lakes and rivers. Majority of studies have found a seasonal relationship between the recovery of aeromonads from specimens and the warmer months of the year. Although there have been several DNA probe and realtime PCR methods described for the possible identification of aeromonads from either water, food, or veterinary sources, there are no widely recognized antigen detection and/or nucleic acid detection methods available for detection within clinical specimens. Two of the earliest articles on Aeromonas antimicrobial susceptibilities included only strains well characterized to the species level and expanded previously known susceptibility information on aeromonads isolated less frequently from clinical specimens. Regardless of the site of isolation (intestinal or extraintestinal), aeromonads should be identified either as belonging to the A. hydrophila or A. caviae complex or as A. veronii complex and not "A. sobria", which is now A. veronii bv. sobria. For extraintestinal isolates (from blood or wounds), the general rules should apply to species identification of aeromonads.
Chapter 39 : Vibrio and Related Organisms *
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This chapter focuses on the various Vibrionaceae species that cause disease in humans. Vibrio is the type genus for the family, and Vibrio cholerae, the causative agent of pandemic cholera, is the type species. The genera covered in the chapter are primarily isolated from marine environments. Recent studies have shown that in mixed populations of nonculturable and culturable cells of V. cholerae, the latter appear to be the main contributors to human infections. The majority of persons ingesting toxigenic V. cholerae O1 have asymptomatic infections. Most of the advantages of PCR-based assays over culture methods apply to vibrios and include the ability to freeze stools for epidemiological studies for delayed testing. Molecular identification of vibrios is commonplace in surveys and in research studies. However, it is not commonly employed in clinical laboratories for routine identification because vibrios are relatively rare pathogens in noncoastal areas or regions where cholera is not endemic. The clinical significance of Vibrio strains in other specimens, particularly stool, may be more difficult to determine and requires prompt consultation with the attending physician to better understand the clinical context. Vibrionaceae isolates should also be submitted to public health laboratories, as they are monitored under the CDC’s International Emerging Infections Program and Vibrio Surveillance System; they may also be needed for confirmation and toxin testing. Misidentification of Vibrio species and their relatives can be a problem in the literature unless investigators used methods that are very sensitive in differentiating all of the species in the family Vibrionaceae.
Chapter 40 : Pseudomonas
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As polyphasic taxonomy continues to advance, more changes will doubtlessly arise; a clinical laboratory must keep abreast of such changes, in order to differentiate these isolates from the more clinically important Pseudomonas species. Healthy individuals are resistant to serious infections by all Pseudomonas species, including P. aeruginosa. Immunocompromised hosts are occasionally infected with one of the many non-aeruginosa species, including (but not limited to) P. fluorescens, P. putida, P. stutzeri, P. oryzihabitans, P. luteola, P. alcaligenes, P. mendocina, and P. veronii. Historically, typing of P. aeruginosa for epidemiological purposes has relied upon phenotypic characteristics of the bacteria. The most widely used method was based upon differences in LPS O polysaccharide (LPS serotyping). Several genotypic methods have been developed over the past two decades for typing P. aeruginosa for epidemiological purposes. Isolates from sites of chronic infection, such as cystic fibrosis (CF) 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 P. aeruginosa is difficult, especially for mucoid isolates, due to increasing resistance, lack of reproducibility of results, and lack of clinical correlation. 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 41 : Burkholderia, Stenotrophomonas, Ralstonia, Cupriavidus, Pandoraea, Brevundimonas, Comamonas, Delftia, and Acidovorax *
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This chapter talks about various environmental organisms that include Burkholderia, Ralstonia, Cupriavidus, Pandoraea, Comamonas, Delftia, Acidovorax, Brevundimonas, and Stenotrophomonas spp. in water, soil, the rhizosphere, and in and on plants including fruits and vegetables. Members of these genera are widely recognized as phytopathogens. The species discussed in the chapter grow well on standard laboratory media such as 5% sheep blood and chocolate agars. Genotyping strategy provides robust, reproducible, and portable results and is quickly becoming the preferred method for investigating bacterial epidemiology, evolution, and population structure. Repetitive-sequence PCR using a BOX A1R primer and multilocus variable-number tandem repeat analysis have been developed for B. pseudomallei to exclude a clonal outbreak. Of the organisms discussed in the chapter, B. pseudomallei is the only one for which serologic tests have been used clinically to diagnose the infection. The indirect hemagglutination assay, although not available commercially, is the most widely used test. It is performed by using a prepared antigen from strains of B. pseudomallei sensitized to sheep cells and includes unsensitized cells as a control. This assay can be adapted to a microtiter plate test system. MIC broth microdilution and the Etest are the preferred methodologies for susceptibility testing. For multiresistant strains, consideration could be given to testing for synergy with double or triple combinations of antimicrobial agents in reference laboratories. It is important to note, however, that neither checkerboard MIC broth microdilution testing nor multiple combination bactericidal antibiotic testing is standardized at present.
Chapter 42 : 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. Most of the organisms described in the chapter are found in the environment, i.e., soil and water. The only fastidious species handled in the chapter are Asaia species, Granulibacter bethesdensis, Methylobacterium species, and some Moraxella species. The limited number of tests that have been used to discriminate between the species dealt with in the chapter have been selected because they can be carried out easily and quickly, they mostly yield uniform results per group or species, and also they are highly discriminatory. A recently developed method of bacterial identification is matrix-assisted laser desorption ionization–time-of-flight mass spectrometry, for which commercial systems, with bacterial mass spectrum databases, have become available recently. Decisions about performing antimicrobial susceptibility testing are complicated by the fact that the Clinical and Laboratory Standards Institute (CLSI) interpretive guidelines for disk diffusion testing of the nonfermenting gram-negative bacteria are limited to Pseudomonas species, Burkholderia cepacia, Stenotrophomonas maltophilia, and Acinetobacter species and therefore, except for Acinetobacter species, do not include the organisms covered in the chapter.
Chapter 43 : Bordetella and Related Genera
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The genera Bordetella, Achromobacter, Alcaligenes, Kerstersia, and Advenella belong to the family Alcaligenaceae (order Burkholderiales in the β subclass of the Proteobacteria). The genus Kerstersia was proposed for a set of strains phenotypically resembling A. faecalis that were classified as Kerstersia gyiorum or as belonging to at least one other Kerstersia species. Virulence factors of bordetellae can be classified as adhesins, autotransporters (i.e., filamentous hemagglutinin [FHA], fimbriae [FIM], and pertactin [PRN]), and toxins (i.e., pertussis toxin [PT], adenylate cyclase toxin, and lipopolysaccharide [LPS]). For other bordetellae, normal microbiological transport media seem to be suitable for transport. Similarly, Achromobacter, Alcaligenes, Kerstersia, and Advenella species can survive in a wide range of environments and at various temperatures. Commercial multiplex PCRs for the detection of various respiratory agents, including bordetellae, are available. Members of the genus Advenella can be separated from related species by their inability to assimilate phenyl acetate. B. pertussis and B. parapertussis are susceptible in vitro to a range of antibiotics, including penicillins, macrolides, ketolides, quinolones, and other antibiotics, including tetracyclines, chloramphenicol, and trimethoprim-sulfamethoxazole, whereas they are resistant to most oral cephalosporins. Antimicrobial sensitivity testing of the Bordetella isolates should be interpreted in accordance with criteria for other infrequently isolated and fastidious nonfermentative gram-negative rods.
Chapter 44 : Francisella and Brucella *
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The family Francisellaceae, a member of the gamma subclass of proteobacteria, consists of the single genus Francisella. Francisella tularensis, Francisella novicida, Francisella philomiragia, and Francisella noatunensis as well as unclassified Francisella spp. comprise the genus. Characterization data indicate that these Francisella spp. are distinct from F. tularensis, F. novicida, and F. philomiragia. The genus Francisella comprises tiny gram-negative coccobacilli that can be distinguished from similar genera by several features. A few key differences separate species of the Francisella genus. Under microscopic examination of Gram-stained specimens, Francisella cells (single and pleomorphic) appear tiny and counterstain so faintly with safranin that they can easily be missed. It is important to consider that many F. tularensis PCR assays cross-react with F. novicida. All Francisella isolates examined to date are β-lactamase positive, so penicillins and cephalosporins are not effective and should not be used to treat tularemia. Antimicrobial susceptibility testing of F. tularensis is not usually performed in clinical microbiology laboratories because of safety concerns in working with this organism. Currently, the most effective treatment regimen and optimal duration of treatment remain unclear. Interpretation of serologic test results in relation to exposure, diagnosis, and prognosis of the disease necessitates an accurate assessment of the clinical history and current status of patients and understanding the usefulness and pitfalls of the laboratory tests. Positive cutoff titers in the Brucella agglutination test for diagnosis have generally been considered to be greater than equal to 160 in symptomatic patients.
Chapter 45 : Legionella *
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The majority of community epidemics of Legionnaire's disease (LD) are from Legionella-contaminated cooling towers or other aerosol-generating devices. Expectorated sputum and other lower respiratory specimens are the most common sources of Legionella spp. The identification techniques used by reference laboratories include serotyping using collections of antisera, biochemical characterization, and sequence-based identification. Typing of Legionella spp. is important for public health investigations to help link culture-positive environmental sites with clinical isolates during an epidemic of the disease. Sequence-based typing appears to be the most specific and precise molecular subtyping system for both L. pneumophila and L. pneumophila serogroup 1. A standardized pulsed-field gel electrophoresis method yields reproducible results and is used as a reference typing method by one national laboratory. The antimicrobial susceptibility of L. pneumophila grown in broth or on agar can give results that have no clinical correlation. This is because of the intracellular location of the bacterium in human infection, to which not all antimicrobial agents gain access and retain activity. Multiple laboratory methods have to be used for optimal laboratory diagnosis of LD. Culture of Legionella bacteria from sputum, lung, or other respiratory sites is the most specific (100%) method for diagnosis of the disease, very sensitive in severe untreated disease, and insensitive in those with mild disease.
Chapter 46 : Bartonella *
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Bartonella species are members of the alpha-2 subgroup of the class Alphaproteobacteria, within the Rhizobiales order. There are now more than 22 species or subspecies described, and DNA sequences from numerous other species or strains have been deposited in GenBank. Warthin-Starry silver stain is recommended for microscopic detection of Bartonella organisms in fixed tissue sections but is not highly specific and is insensitive, even with lymph node biopsy samples from cat scratch disease (CSD) patients. In contrast, even when isolation of the infecting species is not possible, PCR amplification of internal transcribed spacer (ITS) DNA directly from diagnostic samples and/or from enrichment cultures followed by nucleic acid sequencing is an invaluable tool for primary identification at the species, subspecies, and genotype levels. The first serologic test for CSD was an immunofluorescence antibody assay (IFA) based on B. henselae bacilli that were cocultivated with Vero cells to inhibit autoagglutination. Antimicrobial susceptibility testing can be performed by agar dilution methods using either blood or chocolate agar or by microdilution techniques using various media supplemented with blood. CSD typically does not respond to antibiotic therapy. Most investigators have observed no or minimal benefit with antibiotic treatment, whereas anecdotal reports indicate that ciprofloxacin, rifampin, and co-trimoxazole may be effective. Diagnosis of Bartonella infection in humans, especially for typical forms of CSD, is mainly based on serologic data, which is the most cost-effective approach.
ANAEROBIC BACTERIA
Chapter 47 : Approaches to Identification of Anaerobic Bacteria
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This chapter presents an adaptable approach to anaerobic bacteriology based on the resources and capabilities of laboratories. Rare urinary tract infections caused by anaerobic bacteria can be detected first by Gram staining. Newer studies using molecular methods have shown that many organisms implicated in bacterial vaginosis cannot be recovered in culture. Clostridium difficile infection is best diagnosed by toxigenic culture, with molecular detection of the toxin B gene yielding the next best results. Analysis of cellular fatty acids has been used to develop extensive databases for anaerobic identification. This method, although mostly being replaced by faster modern methods for clinical use, is still valuable for describing new species. Sequencing of genetic markers, such as portions of the 16S rRNA gene and other useful genetic elements, is the most common method used for anaerobic identification today in clinical laboratories with molecular-assessment capability. It is clear that anaerobic bacterial protocols occupy a separate and distinct place in clinical microbiology laboratories. Laboratories must determine the extent of effort that they can devote to anaerobes and then develop their processes to perform only those protocols that they can guarantee will yield reliable, timely, and accurate results.
Chapter 48 : Peptostreptococcus, Finegoldia, Anaerococcus, Peptoniphilus, Veillonella, and Other Anaerobic Cocci
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The organisms included in this chapter are obligately anaerobic, non-spore-forming, sometimes elongated cocci. The genera Anaerococcus, Anaerosphaera, Finegoldia, Gallicola, Parvimonas, Peptococcus, Peptoniphilus, and Peptostreptococcus, as well as the newly described taxon Murdochiella , are gram-positive, coccobacillary, or, occasionally, coccoid cells. F. magna is the most pathogenic and one of the most frequently isolated gram-positive anaerobic coccal species found in human clinical specimens. Molecular methods, such as nucleic acid probe hybridization and PCR amplification, are not yet standardized or available commercially for the direct demonstration of medically important gram-positive anaerobic cocci from clinical specimens. New information regarding the antimicrobial susceptibilities of gram-positive anaerobic cocci is sparse compared with the information available for other anaerobic species. Bacteria present in pure culture or in large numbers are probably of major importance, as are organisms recovered in multiple cultures and isolated from normally sterile sites. Furthermore, Gram stain results can guide the laboratory in choosing media for optimal recovery of the predicted organisms. The significance of finding anaerobic gram-positive and gram-negative cocci in clinical specimens depends on the specimen and the likelihood that it was contaminated by the microbiota of the skin or mucous membranes. Hence, interpretation of culture results is dependent on the nature and quality of the specimen submitted to the laboratory.
Chapter 49 : Propionibacterium, Lactobacillus, Actinomyces, and Other Non-Spore-Forming Anaerobic Gram-Positive Rods *
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The majority of the organisms described in this chapter are part of the commensal microbiota associated with the mucocutaneous surfaces of the human and animal digestive tract, being found in the mouth, small and large intestines, urogenital tract, and skin. The various features of non-spore-forming anaerobic gram-positive organism including Propionibacterium, Lactobacillus and Actinomyces are described in the chapter. Biochemical characteristics of Actinomyces and related bacteria encountered in human infection are identified. Far-more-precise identifications can be obtained by 16S rRNA gene sequence analysis. DNA can be rapidly and reliably purified from members of this group by using commercially available kits, such as GenElute (Sigma-Aldrich), and the 16S rRNA gene can be amplified using “universal” primers that amplify all members of the domain bacteria. A recent survey revealed that the method most commonly used for testing anaerobes was the Etest, which has been found to be useful and reliable. Etests should be performed on Brucella blood agar and are optimally read after 48 h, to allow sufficient bacterial growth.
Chapter 50 : Clostridium *
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The genus Clostridium comprises obligately anaerobic, gram-positive rods. Although Clostridium species are usually catalase and superoxide dismutase negative, trace amounts of the enzyme activities may be detected in some strains, such as C. perfringens. Common predisposing factors are surgical procedures, trauma, vascular stasis, bowel obstruction, malignancy, immunosuppressive agents, diabetes mellitus, prior aerobic infection, and use of antimicrobial agents with poor activity against clostridia. Strains that carry only the genes for the binary toxin CdtA/B do not cause C. difficile infection (CDI) or pseudomembranous colitis. There are four naturally occurring types of botulism: (i) classical foodborne botulism; (ii) wound botulism; (iii) infant botulism; and (iv) botulism due to intestinal colonization in children and adults. Laboratories with high throughput are increasingly utilizing detection of glutamate dehydrogenase (GDH), as the method of choice for screening stool samples for C. difficile. The clinical hallmark of botulism is an acute flaccid paralysis, which begins with bilateral cranial nerve impairment involving muscles of the eyes, face, head, and pharynx and then descends symmetrically to involve muscles of the thorax and extremities.
Chapter 51 : Bacteroides, Porphyromonas, Prevotella, Fusobacterium, and Other Anaerobic Gram-Negative Rods
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Within the family Bacteroidaceae, the genus Bacteroides consists of saccharolytic, bile-resistant, and nonpigmented species, mainly isolated from the gut. In a study using sequencing of the 16S-23S rRNA gene internal transcribed spacer regions of Fusobacterium species, three phylogenetic clusters were formed. Many anaerobic gram-negative rods, e.g., different species within the genus Fusobacterium, have unique cell morphology. Molecular methods are increasingly used for direct detection of bacteria from clinical specimens. The pigmented Prevotella and Porphyromonas species vary greatly in the degree and rapidity of pigment production (2 to 21 days), which ranges from buff to tan to black, depending primarily on the type of blood and the composition of the base medium used in the agar. Fluorescence under long-wavelength UV light can be helpful in presumptive identification; pigmented Prevotella and Porphyromonas colonies typically fluoresce red, F. nucleatum and F. necrophorum fluoresce yellow-green, and Desulfovibrio and Bilophila species, when tested with a drop of NaOH on a swab of cell paste, fluoresce red due to the presence of desulfoviridin pigment. In a survey, conducted at the National Taiwan University Hospital, the proportion of susceptible isolates of Bacteroides, Prevotella, and/or Fusobacterium species to many antimicrobials, especially cefmetazole, clindamycin, and the combination ampicillin-sulbactam, decreased during the period from 2000 to 2007.
CURVED AND SPIRAL-SHAPED GRAM-NEGATIVE RODS
Chapter 52 : Algorithms for Identification of Curved and Spiral-Shaped Gram-Negative Rods
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This chapter mainly focuses on algorithms used for identification of curved and spiral-Shaped Gram-negative rods. Curved and spiral-shaped bacteria have a common microscopic morphology but represent diverse bacterial pathogens. Helicobacter cinaedi and Helicobacter fennelliae are two important Helicobacter species isolated from fecal specimens. Helicobacter pylori is the most common curved gram-negative rod isolated from gastric tissue, but other Helicobacter species have also been reported in this site. Other less commonly isolated curved gram-negative rods include the anaerobes Desulfovibrio spp., Sutterella wadswortkensis, Wolinella succinogenes, and Anaerobiospirillum succiniciproducens, which may be isolated from blood, abscess material, or other clinical samples. The spirochetes Borrelia spp. and Leptospira spp. cause systemic infections and are infrequently isolated in clinical laboratories, usually only with specialized media. Treponema spp. of clinical importance are diagnosed based on clinical and epidemiologic findings, as well as microscopic, serologic, and molecular test procedures. Various details about curved gram-negative bacilli that may be encountered in clinical specimens are described in the chapter.
Chapter 53 : Campylobacter and Arcobacter
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Three closely related genera, Campylobacter, Arcobacter, and Sulfurospirillum, are included in the family Campylobacteraceae. Originally classified as free-living Campylobacter species, Sulfurospirillum spp. are slender, curved gram-negative rods, 0.1 to 0.5 mm wide and 1 to 3 µm long. These species have no known pathogenicity for humans or animals, and are environmental organisms isolated from water sediments. Arcobacter spp. are aerotolerant, Campylobacter-like organisms frequently isolated from bovine and porcine products of abortion and feces of animals with enteritis. Arcobacter butzleri was reported to be the fourth most common Campylobacter-like organism isolated from patients with diarrhea and was also one of the most common non C. jejuni/coli species isolated over a 10-year period from over 73,000 stool samples. The Gram stain appearance of Arcobacter may differ from that of typical Campylobacter. Unfortunately, Campylobacter species are difficult to differentiate from Arcobacter species based on phenotypic tests. The 16S rRNA gene and 23S rRNA gene are widely used for genus-and species-specific tests. PCR assays based on these targets are described for 12 different Campylobacter species and three Arcobacter species. Broad-range molecular identification schemes involving restriction fragment analysis of PCR-amplified regions of the 16S or 23S rRNA genes have also been described for identification of Campylobacter and Arcobacter species. Special methods including alternative incubation techniques are required and should be performed by special request.
Chapter 54 : Helicobacter *
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The genus Helicobacter is classified in the family "Helicobacteraceae" of the class Epsilonproteobacteria, formerly known as the epsilon subclass of the Proteobacteria, with Helicobacter pylori as the type species. Phylogenetic analyses reliant on 16S rRNA gene sequences alone have sensitivity limitations in investigations of closely related helicobacters, which have a high degree of natural transformation and genetic plasticity. Further insights into the taxonomy of the genus are now possible with the availability of complete genome sequences for five strains of H. pylori, and for strains of H. acinonychis, H. canadensis, and H. hepaticus. Enterohepatic helicobacters inhabit the intestinal and hepatobiliary tracts of various mammal and bird hosts, and several species, such as H. bilis, H. canadensis, H. canis, H. cinaedi, H. fennelliae, H. pullorum, and “H. winghamensis,” infect humans with clinical symptoms. From the microbiology laboratory perspective, microscopic examinations of a smear prepared directly from a biopsy specimen or from imprint cytology provide rapid bacteriological test results for observation of cells of H. pylori. There is currently no ”gold standard” method for use in the clinical laboratory setting for PCR of gastric biopsy specimens, and so it is advised that PCR based assays should not be the sole basis of determining the H. pylori status of a patient. To perform antimicrobial susceptibility testing, bacteriological culture of H. pylori from gastric biopsy specimens is recommended, especially in cases of repeated treatment failure.
Chapter 55 : Leptospira
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The genus Leptospira is comprised of spiral-shaped bacteria with hooked ends. This genus, along with the genera Leptonema and Turneriella, makes up the family Leptospiraceae within the order Spirochaetales and class Spirochaetes of the proposed phylum Spirochaetes. The genus was formerly divided into two species: Leptospira interrogans, comprising all pathogenic strains, and L. biflexa, containing the saprophytic strains isolated from the environment. The molecularly based taxonomy of Leptospira necessitates the identification of isolates to both the species and serovar levels. Species identification is most practically done by sequence analysis, using rrs (16s rRNA), gyrB, rpoB, or secY. The definitive serologic investigation in leptospirosis remains the microscopic agglutination test (MAT), in which patients sera are reacted with live or killed antigen suspensions of leptospiral serovars. Leptospires are susceptible to many antimicrobial agents, including β-lactams, macrolides, tetracyclines, fluoroquinolones, and streptomycin. Despite recent advances in molecular detection and characterization of leptospires and in the development of rapid serologic tests, there are still relatively few laboratories throughout the world with the appropriate capabilities for Leptospira diagnostics.
Chapter 56 : Borrelia *
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Borreliosis, in the form of louse-borne relapsing fever (LBRF), has been the cause of massive epidemics as recently as the early 1900s. Tick-borne relapsing fever (TBRF) and Lyme borreliosis (LB) are caused by over a dozen Borrelia species and were first described about 100 years ago. There is a high prevalence of B.afzelii among human skin isolates from Europe, whereas isolates from cerebrospinal fluid (CSF) in Europe are most often B.garinii. A few studies have reported the detection of Borrelia species (B.valaisiana, B.spielmanii, and B. lusitaniae) in patient samples in Europe. The genomes of the borreliae are unusual among prokaryotes in having a small linear chromosome of approximately 1,000 kb and both linear and circular plasmids. Two colinear plasmids (lp54 and cp26) seem to belong to the basic genome inventory of the Borrelia species that causes Lyme disease. The ecological components that maintain Borrelia species in nature are quite diverse and are spread throughout the world. This chapter talks about collection, transport, and storage of specimens. Direct microscopic visualization of borreliae in clinical samples is applicable only to cases of relapsing fever. The chapter describes molecular techniques and immunological techniques for identifying Borrelia species, and explains about the serologic test. The antimicrobial susceptibility of Borrelia species has been studied intensively in vitro. Standard methods for the determination of the minimal bactericidal concentration have not been established. Clinical criteria (case history and clinical findings) are decisive factors in the diagnosis and ordering of microbiological laboratory testing.
Chapter 57 : Treponema and Brachyspira, Human Host-Associated Spirochetes
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Researcher demonstrated using 16S rRNA sequences that spirochetes can be grouped into a phylum of five clusters, Treponema, Spirochaeta, Borrelia, Serpula now Brachyspira, and Leptospira. The four members of the genus Treponema that cause venereal syphilis, endemic syphilis, yaws, and pinta are morphologically identical and, despite advances in molecular differentiation, are distinguished primarily by differences in geographic distribution, epidemiology, clinical manifestations, and host range in experimental animals. In the early 1950s, the WHO and United Nations Children's Fund launched an extremely successful program that decreased the global prevalence of endemic treponematoses by more than 95% over an approximately 10-year period. The frequency of spirochetal colonization of the intestinal tract has declined dramatically in developed countries during the 20th century but remains high in the developing world. Polymerase chain reaction (PCR) can be performed on either fixed or unfixed tissue, although unfixed tissue is recommended. Routine antimicrobial susceptibility testing of T. pallidum is not practical due to the lack of a suitable in vitro cultivation system. Researchers confirmed that the serologic response to therapy is slow and often incomplete. This study, as well as anecdotal experience, brought about a substantial loosening in the accepted criteria for an adequate therapeutic response, currently defined as a fourfold decrease in nontreponemal titer by 1 year.
MYCOPLASMAS AND OBLIGATE INTRACELLULAR BACTERIA
Chapter 58 : General Approaches to Identification of Mycoplasma, Ureaplasma, and Obligate Intracellular Bacteria
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This chapter summarizes the epidemiology of infections and the diagnostic tests most often used for the detection of the causative bacteria such as mycoplasma, ureaplasma, and obligate intracellular bacteria. It talks about the most frequently used tests in clinical microbiology laboratories, the Gram stain and culture on artificial media, which are unable to detect these organisms if present in clinical samples. Molecular diagnostic tools and better culture methods have significantly improved the ability to detect these agents and to diagnose the diseases that they cause. The diagnostic tests most often used for the detection of the causative bacteria are described in the chapter.
Chapter 59 : Mycoplasma and Ureaplasma
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Mollicutes are believed to have evolved from clostridium-like gram-positive cells by gene deletion. In humans, mycoplasmas and ureaplasmas are associated with the mucosa, residing predominantly in the respiratory or urogenital tract, rarely penetrating the submucosa, except in cases of immunosuppression or instrumentation, when they may invade the bloodstream and disseminate to various organs and tissues. In humans, mycoplasmas and ureaplasmas may be transmitted by direct contact between hosts, i.e., venereally through genital-genital or oral-genital contact, vertically from mother to offspring either at birth or in utero, by respiratory aerosols or fomites in the case of M. pneumoniae, or even by nosocomial acquisition through transplanted tissues. Polymerase chain reaction (PCR) systems have been developed for all of the clinically important Mycoplasma and Ureaplasma species that infect humans. Typing of human mycoplasmas or ureaplasmas for diagnostic or epidemiological purposes is not recommended at the present time, and the methods are unavailable except in specialized research or reference laboratories. Fluoroquinolones such as levofloxacin and moxifloxacin are usually active against all human mycoplasmal and ureaplasmal species. Eradication of infection under these circumstances can be extremely difficult, requiring prolonged therapy, even when the organisms are susceptible to the expected agents. This difficulty highlights the facts that mollicutes are inhibited but not killed by most commonly used bacteriostatic antimicrobial agents in concentrations achievable in vivo and that a functioning immune system plays an integral part in their eradication.
Chapter 60 : Chlamydiaceae *
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The Chlamydiaceae contain the known human pathogens Chlamydia trachomatis, Chlamydia pneumoniae, and Chlamydia psittaci as well as organisms such as C. abortus and C. felis that have been associated only rarely with human infections. An overview about ranges of sensitivity and specificity for common diagnostic tests for C. trachomatis in urogenital specimens is provided in this chapter. Problems associated with cell culture isolation of chlamydiae, including technical complexity and long turnaround time, and stringent requirements related to collection, transport, and storage of specimens have driven the development of commercially available nonculture methods that have found widespread application in many routine laboratories. The basic procedure for detection of isolated chlamydiae involves demonstration of intracytoplasmic inclusions by fluorescent-antibody staining that provides both morphological and immunological identification of chlamydiae. Serologic testing may be helpful in the diagnosis of human ornithosis, LGV, neonatal pneumonia caused by C. trachomatis, and respiratory C. pneumoniae infections. The most commonly used serological assay formats include the complement fixation (CF) test, the MIF test, and the EIA to detect immunoglobulin M (IgM), IgA, IgG, or total classes of antibodies, with either family, species, or serotype specificity. Evaluation of antimicrobial resistance and potential clinical treatment failure in chlamydial infection is hampered by the lack of standardized antimicrobial susceptibility tests and the fact that in vitro resistance does not correlate with the patient’s clinical outcome.
Chapter 61 : Rickettsia and Orientia
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The family Rickettsiaceae comprises two genera of small, obligately intracellular bacteria that reside free within the host cell’s cytosol, namely, Rickettsia and Orientia. The genus is divided by the phylogenetic clustering of species into the typhus group (TG) and spotted fever group (SFG), defined originally by their distinctive lipopolysaccharide antigens, and the transitional and other basal groups that are widely distributed in arthropods. Among SFG and TG rickettsiae the genomes have remarkable synteny. Orientia resides free in the cytosol and is maintained in nature by transovarian transmission in trombiculid mites, which transmit the infection to humans during feeding at the larval stage. For immunohistologic detection of rickettsiae, the specimen can be snap-frozen for frozen sectioning or fixed in formaldehyde for the preparation of paraffin-embedded sections. Autopsy tissues can also be examined for rickettsiae by immunohistochemistry or polymerase chain reaction (PCR). The technique of in situ hybridization has been developed but has not been reported for the detection of rickettsiae in clinical samples. Antimicrobial susceptibility studies of rickettsiae are not routinely performed clinical laboratory tests. Detection of three or more rickettsiae in vascular endothelium in biopsy specimens or four or more rickettsiae in captured circulating endothelial cells is diagnostic of rickettsial infection.
Chapter 62 : Ehrlichia, Anaplasma, and Related Intracellular Bacteria *
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Members of the genus Ehrlichia and Anaplasma are now recognized to be important human pathogens. They are obligate intracellular bacteria currently placed in the Proteobacteria phylum (Alphaproteobacteria), order Rickettsiales, and family Anaplasmataceae. Anaplasma phagocytophilum, within the genus Anaplasma, now includes Ehrlichia phagocytophila, Ehrlichia equi, and the human granulocytic ehrlichiosis (HGE) agent. Ehrlichia and Anaplasma spp. are gram-negative obligate intracellular bacteria that reside and propagate within membrane-lined vacuoles found in the cytoplasm of bone marrow-derived cells, such as granulocytes, monocytes, erythrocytes, and platelets. The causative agent of human monocytic ehrlichiosis (HME) is E. chaffeensis, a monocytotropic ehrlichia that was first identified as a human pathogen in a patient with a severe febrile illness after tick bites in 1986. The culture conditions for Ehrlichia and Anaplasma species are still being optimized. The most widely used method is polymerase chain reaction (PCR) amplification of DNA from E. chaffeensis in clinical samples using the HE1/HE3 primer set. In a prospective study, the overall sensitivity and specificity of PCR were 56% and 100%, respectively, using the 16S rRNA subunit, nadA, and 120-kDa protein genes. In this study several samples had high titers of antiehrlichial antibodies by immunofluorescence assay (IFA), suggesting that the pathogen may have already been cleared. Routine antimicrobial susceptibility testing of Ehrlichia or Anaplasma species isolates is unnecessary. The typical morphology of an Ehrlichia or Anaplasma spp. morula is observed, an assessment as to the hematopoietic lineage and the percentage of cells that contain morulae should be made and reported.
Chapter 63 : Coxiella *
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This chapter talks about Coxiella burnetii, Q fever and typing systems. Q fever can be latent and recrudesce during periods of relative immunosuppression, such as late pregnancy, causing fetal infection. Isolation must currently be performed in specialized high-containment biosafety level 3 facilities, as the agent is highly infectious and classified as a select agent and a CDC category B bioterrorism agent. IHC is an excellent method for the detection of C.burnetii antigens in tissue samples particularly cardiac valve tissues that are colonized during chronic Q fever. The detection of antibodies to C. burnetii is the most commonly used and effective method for the diagnosis of Q fever. The primary serologic assays in use today are the indirect immunofluorescent antibody (IFA) assay, the complement fixation (CF) test, and the enzyme-linked immunosorbent assay (ELISA), with the IFA assay being the gold standard and most commonly used method. The diagnosis of chronic Q fever is best performed at reference laboratories that have BSL-3 facilities for the propagation and storage of phase I organisms since C. burnetii is classified as a U.S. category B bioterrorism agent. The recommended treatment for acute Q fever is doxycycline, although strains with partial doxycycline resistance have been reported. Chronic Q fever, especially Q fever endocarditis, requires long-term antibiotics and often valve replacement. Tissue specimens (e.g., heart valve, liver, and bone marrow) are most often positive in chronic Q fever, although peripheral blood leukocytes from such patients can be positive or negative.
Chapter 64 : Tropheryma
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Tropheryma whipplei, a rod-shaped, environmental actinomycete with a distinct genome and typical periodic acid-Schiff (PAS) stain reaction, is the causative bacterium of Whipple’s disease (WD). PCR studies have shown the presence of T. whipplei in sewage plants and in human stools and saliva. WD may occur in the following clinical categories and courses: (i) classical WD, which presents with weight loss, diarrhea, and arthropathy in 75% of patients by the time of diagnosis; (ii) isolated infection of cardiac valves without classical WD; (iii) asymptomatic carriers of T. whipplei. The most frequent diagnostic samples are small bowel biopsy specimens. Endoscopic biopsies, at least four to six, from the distal duodenum and/or the jejunum, even in the absence of intestinal symptoms, should also be obtained systematically in suspected WD, because involvement could be patchy. T. whipplei has only recently been cultured in human fibroblasts based on specific cell culture techniques. The use of parenteral therapy was recently clarified in a randomized controlled trial with 40 WD patients; this study also demonstrated a very high clinical remission rate if antimicrobial therapy was closely monitored.
ANTIBACTERIAL AGENTS AND SUSCEPTIBILITY TEST METHODS
Chapter 65 : Antibacterial Agents
- Authors: Joseph D. C. Yao, Robert C. Moellering
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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 or synthesized to date, and a broad number and variety of agents 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. 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. The major antibacterial action of penicillins is derived from their ability to inhibit a number of bacterial enzymes, namely penicillin-binding proteins (PBPs), that are essential for peptidoglycan synthesis. Cephalosporins are generally very well tolerated. The monobactams are β-lactams with various side chains affixed to a monocyclic nucleus. Since the first aminoglycoside, streptomycin, was introduced, this class of antibiotic has played a vital role in the treatment of serious gram-negative infections. The currently available aminoglycosides are derived from Micromonospora spp. or from Streptomyces spp. Tetracyclines are broad-spectrum bacteriostatic antibiotics with the hydronaphthacene nucleus, which contains four fused rings. Glycylcyclines are a group of semisynthetic tetracycline derivatives containing a glycylamido substitution at position 9. Streptogramins are natural cyclic peptides produced by Streptomyces spp. They are a unique class of antibiotics in which each member is a combination of at least two structurally unrelated components, groups A and B streptogramins.
Chapter 66 : Mechanisms of Resistance to Antibacterial Agents
- Authors: Louis B. Rice, Robert A. Bonomo
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Antimicrobial resistance arises by (i) mutation of cellular genes, (ii) acquisition of exogenous resistance genes, or (iii) mutation of acquired genes. The most completely studied example of regulatory mutation resulting in resistance is the derepression of the chromosomal β-lactamase of Enterobacter spp. As bacteria have responded to the challenge of antimicrobial agents, so have researchers responded to the challenge of antibiotic resistance. The majority of pumps that extrude one or more antibiotic classes from the bacterial cell are located in the cytoplasmic membrane and use proton motive force to drive drug efflux. This chapter describes resistance mechanisms for different antimicrbial classes. In explaining resistance to aminoglycosides (amikacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, streptomycin, and tobramycin), the chapter explains how aminoglycosides reach their target in bacterial cells and then reviews their mechanism of action. The clinical indications for aminoglycoside therapy are also summarized in the chapter. Resistance to aminoglycosides can occur by four mechanisms: (i) loss of cell permeability (decreased uptake), (ii) alterations in the ribosome that prevent binding, (iii) expulsion by efflux pumps, and (iv) enzymatic inactivation by aminoglycoside-modifying enzymes (AMEs). The most common mechanism of resistance to chloramphenicol is the elaboration of CATs. The antibiotics nitrofurazone and nitrofurantoin are used in the treatment of genitourinary infections and as topical antibacterial agents. The ultimate importance of efflux pump activations for clinical resistance to tigecycline awaits more extensive clinical use.
Chapter 67 : Susceptibility Test Methods: General Considerations
- Authors: John D. Turnidge, Mary Jane Ferraro, James H. Jorgensen
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Determination of the antimicrobial susceptibilities of significant bacterial isolates is one of the principal functions of the clinical microbiology laboratory. Currently, routine susceptibility testing methods are best standardized for the common aerobic and facultative bacteria and systemic antibacterial agents. The inherent flexibility in drug selection that is provided by the disk diffusion test is an undeniable asset of the susceptibility testing method. There is general agreement that the minimum inhibitory concentration (MICs) is the most basic laboratory measurement of the activity of an antimicrobial agent against an organism. Response rates of at least 80% may be expected for organisms classified as susceptible, although the rates can be lower depending on the site and type of infection. The newest CLSI approach focuses on the rate of interpretive errors near the proposed breakpoint versus rates of errors with MICs more than a single log2 dilution from the MIC breakpoints. For some key resistances, e.g., carbapenem resistance in Enterobacteriaceae, the most effective confirmatory method is resistance gene detection. Cascade reporting is considered to be an essential part of hospital antimicrobial stewardship programs, as is the production of annual reports that summarize overall susceptibility and resistance patterns (antibiograms).
Chapter 68 : Susceptibility Test Methods: Dilution and Disk Diffusion Methods
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There are a number of methods for antimicrobial susceptibility testing of bacteria, and they are categorized into dilution methods that generate Minimum Inhibitory Concentration (MIC) results and disk diffusion methods that generate zone diameter results. The Clinical and Laboratory Standards Institute (CLSI) reference methods are broth macrodilution, broth microdilution, agar dilution, and disk diffusion. Dilution methods are also readily adaptable to automated test systems. Stock solutions are prepared as discussed in the CLSI document on dilution testing and are the same as those used for agar dilution tests. In fact, the broth microdilution method is now considered the international reference susceptibility testing method. This chapter focuses on in-house preparation and use of broth microdilution panels. Most of the principles and practices discussed in the chapter are pertinent to the broth microdilution method regardless of the source of the antibiotic panels. The dilution scheme for preparing broth microdilution panels is the same as that described for agar and broth macrodilution methods. The definitions of the interpretive categories and the comments concerning the use of the standards for agar and broth macrodilution methods are also applicable to broth microdilution methods. Like full-range dilution testing, breakpoint methods require the use of appropriately adjusted and supplemented Mueller-Hinton broth or agar. In addition, the standard inoculation, incubation, and interpretation procedures recommended for the full-range dilution methods should be followed.
Chapter 69 : Susceptibility Testing Instrumentation and Computerized Expert Systems for Data Analysis and Interpretation
- Authors: Sandra S. Richter, Mary Jane Ferraro
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Commercial antimicrobial susceptibility testing (AST) systems were introduced into clinical microbiology laboratories during the 1980s and have been used in the majority of laboratories since the 1990s. 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 AST systems include data management software that may be interfaced with a laboratory information system (LIS) and offer various levels of expert system and epidemiological analyses. This chapter focuses primarily on commercial susceptibility testing systems currently available in the United States. It discusses advantages and disadvantages of automated systems. Reports of AST performance for detecting problematic resistance phenotypes are also discussed. Expert systems to assist in the critical review of AST results are available for all commercial susceptibility systems currently marketed in the United States. Most expert systems use a rules-based approach focusing on AST results for one drug at a time without considering results for other agents tested simultaneously. Factors to consider when selecting an AST system include cost, performance, work flow, data management capabilities, and manufacturer technical support. Future advances in the development of AST systems may increase their clinical impact with the incorporation of molecular techniques that dramatically shorten the time required for results.
Chapter 70 : Phenotypic Methods for Detecting Antibacterial Resistance
- Authors: Jana M. Swneson, Jean B. Patel, James H. Jorgensen
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This chapter on special phenotypic methods for detecting antibacterial resistance describes details of tests for detection of high-level aminoglycoside resistance and acquired vancomycin resistance in enterococci; tests for detection of inducible clindamycin resistance in streptococci; tests for detection of penicillin, oxacillin, vancomycin, induc-ible clindamycin, and high-level mupirocin resistance in staphylococci; tests for detection of extended-spectrum-β-lactamase (ESBL) and carbapenemase production in Enterobacteriaceae; and tests for detection of β-lactamases in multiple organisms. Other special phenotypic tests described in the chapter are those for determining the bactericidal activity or combined activities of antimicrobial agents; they are different from the tests that screen for a specific resistance mechanism. As some of the phenotypic tests described here for Staphylococcus aureus are not recommended for coagulase-negative staphylococci (CoNS), for detection of oxacillin resistance, the two groups are discussed separately as S. aureus and Staphylococcus lugdunensis and CoNS except S. lugdunensis. In the clinical laboratory, β-lactamase tests can be used for two purposes. The first is to detect an underlying mechanism of resistance that may not be detected using routine susceptibility testing methods, and the second is to detect a mechanism of resistance that is an infection control concern and epidemiologically important.
Chapter 71 : Susceptibility Test Methods: Fastidious Bacteria *
- Authors: Janet A. Hindler, James H. Jorgensen
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Most fastidious bacteria do not grow satisfactorily in standard in vitro susceptibility test systems that use unsupple-mented media. Clinical and Laboratory Standards Institute (CLSI) has also published an approved guideline for testing infrequently isolated or fastidious bacteria including Abiotrophia spp., Granulicatella spp., Aeromonas spp., Plesiomonas spp., Bacillus spp. (not B. anthracis), Campylobacter jejuni/coli, Corynebacterium spp., Erysipelothrix rhusiopathiae, the HACEK group, Lactobacillus spp., Leuconostoc spp., Listeria monocytogenes, Moraxella catarrhalis, Pasteurella spp., Pediococcus spp., and Vibrio spp. In addition to conventional MIC test methods (e.g., agar dilution or broth dilution methods), the Etest MIC determination method has been used to test many types of fastidious bacteria. The limitations of this method include its cost and lack of clearance by the U.S. Food and Drug Administration (FDA) for testing many less commonly encountered fastidious bacteria. This chapter summarizes the standard methods recommended by CLSI for antimicrobial susceptibility testing of Streptococcus spp. (including S. pneumoniae), H. influenzae, N. gonorrhoeae, and N. meningitidis. Methods for testing the infrequently isolated or fastidious bacteria included in the CLSI M45 guideline are summarized to include testing potential agents of bioterrorism. The incidence of resistance, test methods, and indications for testing and the reporting of results are provided.
Chapter 72 : Susceptibility Test Methods: Anaerobic Bacteria
- Authors: Diane M. Citron, David W. Hecht
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This chapter describes the currently available methodologies and their interpretation for susceptibility testing of anaerobes. At present, alternative testing methods include limited agar dilution, broth microdilution (for the B. fragilis group), and the Etest gradient strip method. Tigecycline, a glycylcycline derivative of minocycline, was recently introduced for treatment of intra-abdominal and skin and soft tissue infections. Several studies have found very good activity against anaerobes, with rare strains exhibiting tigecycline MICs of >8 µg/ml. Extensive taxonomic changes have occurred within the non-spore-forming gram-positive bacilli, especially with those previously in the genus Eubacterium. It is important to compare the drug-containing plates to the drug-free control plate when reading the tests, as different species of anaerobic bacteria can have very differently appearing spots, ranging from mucoid-opaque, as with the B. fragilis group, to gray-transparent, as with Bacteroides ureolyticus. Broth microdilution MIC determinations require criteria similar to those for the agar dilution procedure for reading end points: the concentration at which the most significant reduction in growth is observed and interpreted as the MIC. Current methodologies allow for accurate surveillance or individual isolate testing by most laboratories. Future studies comparing broth microdilution to the reference agar method and the anticipated development of an improved microdilution system will result in better standardization of the more-user-friendly method and, possibly, more widespread commercial availability.
Chapter 73 : 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 includes a description of nonradioactive broth culture systems and rapid molecular systems for detection of drug resistance, as well as the standard agar proportion method. The antimicrobial agents that are used in the treatment of mycobacterial infections are discussed in the chapter for the most commonly encountered species. The Clinical and Laboratory Standards Institute (CLSI) document on drug susceptibility testing of mycobacteria currently recommends that first-line testing include ethambutol (EMB), RMP, INH, and pyrazinamide (PZA). The chapter describes drug susceptibility testing of M. tuberculosis complex. Broth microdilution is the method recommended by the CLSI for susceptibility testing of nontuberculous mycobacteria (NTM). The CLSI provides guidelines for testing M. avium complex (MAC), M. kansasii, M. marinum, and the rapidly growing mycobacteria. General recommendations regarding the broth microdilution method and QC that apply to all NTM are discussed in the chapter. The chapter describes specific details related to MAC, M. kansasii, M. marinum, and the rapidly growing mycobacteria. It also talks about incubation temperature and time for each species or group. The chapter explains that Nocardia spp. and other aerobic actinomycetes (Actinomadura, Rhodococcus equi, Gordonia, Tsukamurella, and rarely Streptomyces spp.) can cause serious disease in immunocompromised and occasionally even healthy hosts. The recommended method for testing Nocardia and other aerobic actinomycetes is broth microdilution.
Chapter 74 : Detection and Characterization of Antimicrobial Resistance Genes in Pathogenic Bacteria *
- Authors: J. Kamile Rasheed, Fred C. Tenover
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This chapter focuses specifically on genotypic methods for detecting and characterizing antimicrobial-resistant bacterial pathogens. There are four major reasons to use genetic tests to identify antimicrobial resistance genes or mutations associated with resistance in bacterial isolates. DNA sequence analysis has been particularly helpful for identifying point mutations in genes associated with extended-spectrum-β-lactamases (ESBLs) and resistance to carbapenems, fluoroquinolones, oxazolidinones, and antimycobacterial drugs. There are several potential pitfalls associated with using genetic tests to detect resistant organisms. The plethora of novel resistance mechanisms, as outlined in this chapter, suggests that phenotypic methods to identify resistance in the organism groups will continue to have value at least for the next decade. In sections, the chapter reviews applications of molecular diagnostic methods for specific classes of resistance determinants. The rapid detection of klebsiella pneumoniae carbapenemases (KPC)-containing isolates may also be facilitated by using one of several real-time polymerase chain reaction (PCR)-based assays. Mupirocin is an antistaphylococcal agent that is used to suppress or eliminate nasal carriage of staphylococci among colonized patients and hospital personnel. All PCR assays for antimicrobial resistance genes, whether sold commercially or developed in-house, must be validated before use.
VOLUME 2
VIROLOGY
GENERAL
Chapter 75 : Taxonomy and Classification of Viruses
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This chapter discusses about taxonomy and classification of viruses. For viruses, the process of comparative analysis plays a critical role in increasing our overall knowledge of the molecular biology, pathogenesis, epidemiology, and evolution of poorly understood or newly isolated viruses. This knowledge enhances our ability to respond to new threats by supporting the development of diagnostics, vaccines, and other antiviral therapies. In fact, it is likely that viruses have multiple independent evolutionary origins that cannot be easily or completely separated from the evolution of their hosts, as they cannot reproduce or evolve separately from their hosts. Therefore, viruses might be better represented as individual twigs arising from branches spread throughout the rest of the tree. In addition to distinct evolutionary histories, viruses differ from other domains of life in the variety of possible coding molecules they utilize to store their genetic programs. For viruses, the Virology Division of the International Union of Microbiological Societies has charged the International Committee on Taxonomy of Viruses (ICTV) with the task of developing, refining, and maintaining a universal viral taxonomy. As categorized according to the 2009 ICTV taxonomy, viruses that infect humans fall into 4 orders: the Herpesvirales, Mononegavirales, Nidovirales, and Picornavirales. The ICTV produces an extensive amount of information during the process of classifying and naming viruses that is published regularly in the ICTV reports.
Chapter 76 : Specimen Collection, Transport, and Processing: Virology
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In recent years, there have been many advances in diagnostic virology, including improvements in cell culture methods and the development of viral antigen (Ag) and nucleic acid (NA) assays. Regardless of the detection method chosen, accurate test results rely on preanalytical steps, including specimen selection, collection, transport, and processing, that are described in this chapter. General guidelines for these procedures are described. Collection and processing of specific specimen types are then discussed in detail. A final section on specimen transport containing general information and selected topics of importance is included. The use of viral transport medium (VTM) during specimen collection is highly dependent on specimen source. Important specimen information that should be provided includes patient data, ordering physician, specimen source, specific viruses suspected, time and date of specimen collection, and specific diagnostic tests requested. Manufacturers of commercials assays for NA or Ag detection either supply transport media or make recommendations for transport systems that are compatible with their assays. The manufacturer's package insert should therefore be consulted for information on appropriate collection and transport systems. Currently, the majority of diagnostic or clinical specimens can be shipped as biological substances, category B. Only specimens that have or may have a category A pathogen are classified as infectious substance. However, as the field of diagnostic virology evolves, new collection devices, transport systems, and processing methods that enhance detection will continue to be developed.
Chapter 77 : Reagents, Stains, Media, and Cell Cultures: Virology
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Some molecular assays, for practical reporting purposes, may take as long as overnight cell culture and may not lend themselves to single-specimen testing as well as cell culture does. For these reasons, cell cultures are still an indispensible research and clinical laboratory tool, particularly when combined with the use of highly specific monoclonal antibodies (MAbs) for the detection of common viruses and Chlamydia spp. or when cell lines are engineered to produce virus-induced enzymes, such as beta-galactosidase for the detection of herpes simplex viruses (HSV). This chapter describes the cell lines, reagents, stains, and media used in association with traditional tube and rapid viral culture techniques. Definitive identification of certain viruses or Chlamydia spp. can be directly determined in clinical samples when using MAbs labeled with fluorescein isothiocyanate, methylrhodamine isothiocyanate, or phycoerythrin with an Evans blue and/or propidium iodide counterstain. The lot number and date of use for all media, buffers, reagents, and additives should also be recorded. An important consideration in using cell culture is ensuring collection of cellular material and maintaining the viability of the organisms from the time of sample collection to inoculation in cell culture. Cell culture media are an important part of the production and maintenance of cells.
Chapter 78 : Algorithms for Detection and Identification of Viruses
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This chapter analyses methods focused on conventional cell cultures, classical serologic techniques, and light and electron microscopy. Novel detection methods have permitted the diagnosis of multiple respiratory viruses in a single multiplex PCR test. Quantitative monitoring of viral load in blood has become more widely applied due to implementation of real-time PCR techniques. As tests become more sensitive, low levels of clinically irrelevant or nonviable viruses may be detected and can be misleading to clinicians. Similarly, interpreting the clinical relevance of multiple viral pathogens in the same sample when relative quantification is not available is problematic. Laboratories need to choose which tests to offer. Selecting the appropriate test will depend on the viruses sought, sample site, clinical presentation, clinical purpose, patient characteristics, and disease prevalence. Laboratories must recognize the uses and also the limitations of each test in order to guide clinicians in interpreting the results. Due to the speed of methodological change and the continuing discovery of new viruses and new therapies, keeping abreast of the most recent literature is strongly recommended.
RNA VIRUSES
Chapter 79 : Human Immunodeficiency Viruses *
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The human immunodeficiency virus (HIV) is the etiologic agent of AIDS. HIVs are enveloped plus-stranded RNA viruses. The HIV genome is organized similarly to other retroviruses. It contains the gag, pol, and env genes which encode structural proteins, viral enzymes, and envelope glycoproteins, respectively. The major structural proteins which are encoded by the gag gene include p17, p24, p7, and p9. Replication begins with the attachment of virus to the target cell via the interaction of gp120 and the cellular receptor CD4. Both HIV-1 and HIV-2 have the same modes of transmission. The most common mode of HIV infection is sexual transmission at the genital mucosa through direct contact with infected blood fluids, including blood, semen, and vaginal secretions. Serological testing for HIV antibody is used for various purposes, including primary diagnosis, screening of blood products, management of untested persons in labor and delivery, evaluation of occupational exposures to blood/body fluid, and epidemiological surveillance. The first generation of HIV antibody assays relied on the detection of antibody to HIV viral protein lysates. A test using a sandwich-capture format and significantly more blood than other methods was more sensitive in early seroconversion. HIV-1 RNA load testing is sometimes requested to resolve equivocal serologic findings or to facilitate the diagnosis of HIV-1 infection during the acute phase or in a pediatric setting.
Chapter 80 : Human T-Cell Lymphotropic Virus Types 1 and 2
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Human T-cell lymphotropic virus types 1 and 2 (HTLV-1 and HTLV-2, respectively) are part of the Retroviridae family and members of the Deltaretrovirus genus. However, high-risk populations, such as intravenous drug users (IDUs), in which HTLV-2 infection predominates over HTLV-1 infection, are reported to have a seroprevalence of up to 20%. IDU and sex with IDUs are the most important risk factors for HTLV-2 transmission. Peripheral blood mononuclear cells (PBMCs) are appropriate specimens for nucleic acid detection and virus isolation since HTLV-1 and HTLV-2 are cell-associated viruses. Two qualitative PCR procedures, utilizing primers in the pol or tax gene region, have been used to confirm and differentiate between HTLV-1 and HTLV-2 infections. The first uses HTLV consensus primers that allow amplification of both viruses; typing is achieved either by hybridizing the product to an HTLV-1-specific or HTLV-2-specific probe or by specific restriction digestion pattern analysis. The second approach employs type-specific primers and probes in separate amplifications. Testing for antibodies to HTLV-1 and HTLV-2 should be performed for all blood donors and any patients presenting with relevant clinical signs and symptoms. A typical algorithm for HTLV testing for diagnostic purposes is outlined in this chapter. If the initial screening immunoassay (EIA or ChLIA) is reactive, a repeat assay of the same specimen is performed in duplicate.
Chapter 81 : Influenza Viruses
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This chapter discusses about Influenza viruses that cause annual epidemics in areas with temperate climates, while in tropical climates seasonality is less apparent and influenza viruses can be isolated throughout the year. Influenza viruses are transmitted from person to person primarily via droplets generated by sneezing, coughing, and speaking. Direct or indirect (fomite) contact with contaminated secretions and small-particle aerosols are other potential routes of transmission that have been noted. The relative importance of different routes has not been determined for influenza viruses. Influenza viruses infect the respiratory epithelium and can be found in respiratory secretions of all types. A number of transport media are suitable for influenza viruses, including veal infusion broth, Hanks balanced salt solution, tryptose phosphate broth, sucrose phosphate buffer, and commercially available cell culture medium. Molecular methods are increasingly being used for both the detection and the characterization of influenza viruses. The most commonly used molecular method is reverse transcription-PCR (RT-PCR). The initial step is to identify the isolate as an influenza virus and to distinguish it from other respiratory viruses that have the ability to agglutinate or adsorb red blood cells. Cell culture assays do not reliably identify antiviral susceptibility to the NA inhibitors (NAIs) zanamivir and oseltamivir. Influenza viruses isolated in national and global surveillance systems are characterized antigenically and genetically to identify variants.
Chapter 82 : Parainfluenza and Mumps Viruses
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The human parainfluenza viruses (HPIVs) and mumps virus are members of the Paramyxoviridae family. HPIV-1, -2, and -3 are associated with upper respiratory tract infections in infants, children, and adults. Only respiratory syncytial virus (RSV) causes more lower respiratory tract infections in neonates and young infants than HPIV-3. The indirect fluorescent-antibody (IFA) staining test is appropriate for the same types of samples as for the direct fluorescent-antibody (DFA) staining kit, with the same smear preparation and fixation guidelines. Confirmatory testing is routinely completed by immuno-fluorescence techniques involving the use of HPIV MAbs in DFA or IFA assays, as described for HPIV antigen detection in clinical samples. An overview of the recommended protocol from the manufacturer of R-Mix is shown in "Parainfluenza Viruses" under "Isolation and Identification." As mumps virus infections in previously vaccinated individuals result in decreased levels of virus shedding into the buccal cavity, virus isolation may be difficult. Given the potential lack of serologic test specificity, virus isolation and RNA detection in clinical samples remain the most effective ways to confirm infection in unvaccinated individuals.
Chapter 83 : Respiratory Syncytial Virus and Human Metapneumovirus
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Respiratory syncytial virus (RSV) is one of the most vulnerable pathogens to environmental changes. RSV is the major cause of lower respiratory tract illnesses such as bronchiolitis, tracheobronchitis, and pneumonia among infants and young children worldwide. Human metapneumovirus (hMPV) is an RNA virus of the Paramyxoviridae family and is part of the Pneumovirinae subfamily along with RSV. A study of the association of the virus with respiratory disease was performed using prospectively collected data in a cohort of more than 2,000 subjects. This study showed that hMPV is associated with the common cold and with lower respiratory tract illnesses such as bronchiolitis, pneumonia, croup, and exacerbation of reactive airways disease. The signs and symptoms caused by hMPV are very similar to those caused by RSV. Reports in the literature suggest that the virus can be recovered or detected by reverse transcriptase polymerase chain reaction (RT-PCR) from nasal aspirates, nasal washes, nasal or throat swabs, and bronchoalveolar lavage specimens. The most sensitive test for identification of hMPV in clinical samples to date is RT-PCR. The diagnosis of hMPV infection is most likely when a positive nucleic acid test for hMPV infection is obtained when testing a respiratory secretion during late winter or early spring in temperate climates from a patient with acute respiratory illness and negative tests for other respiratory viruses.
Chapter 84 : Measles and Rubella Viruses
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This chapter combines the current laboratory diagnostic methods for measles virus and rubella virus for convenient review and reference. The measles virus genome is 15,894 nucleotides in length and contains six structural genes organized on the single strand of RNA in a gene order consistent with those of most of the paramyxoviruses, i.e., 3'-N, P. M, F, H, L-5'. A recent review by Rota et al. provides an excellent overview of the current status of the molecular epidemiology of measles and the global distribution of the various genotypes. The most common complications associated with measles virus infection are otitis media (7 to 9%), pneumonia (1 to 6%), and diarrhea (6%). Suitable samples for isolation of measles virus or for detection of viral antigen can be whole blood, serum, throat and nasopharyngeal secretions, urine, and, in special circumstances, brain and skin biopsy samples. Characteristic cytopathic effects (CPE) of measles virus infection include multinucleated cells and cellular inclusions (in-tracytoplasmic and intranuclear). The reverse transcriptase PCR (RT-PCR) should be considered for diagnostic use where IgM testing is compromised by the concurrent or recent use of measles virus-containing vaccine as part of an outbreak response or in settings of recent vaccine distribution, such as supplemental immunization activities. Groups of related viruses within the clades have been classified as genotypes. Time course of rubella virus-specific IgM and IgG detection by enzyme-linked immunosorbent assays (ELISAs) in sera of rubella patients.
Chapter 85 : Enteroviruses and Parechoviruses
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Human enteroviruses (HEVs) and human parechoviruses (HPeVs) are spread mainly by the fecal-oral or oral-oral routes, respiratory droplets, and fomites. Two major patterns of circulating enteroviruses have been observed: epidemic (e.g., E9, E13, E30, and CVB5) and endemic (e.g., CVA9, CVB2, CVB4, and EV71). The five most commonly reported serotypes (E9, E11, E30, E6, and CVB5) accounted for 48.1% of cases. Myocarditis was once an often fatal disease associated with EV. Coxsackievirus B (CVB) are responsible for one-third to one-half of all cases of acute myocarditis and pericarditis, with CVB2 and CVB5 being the most predominant serotypes identified in clinical studies. Specimen selection is important for making a diagnosis of EV infection, as asymptomatic shedding, especially in stool, is common. The development of nucleic acid amplification tests (NAAT) (RT-PCR or nucleic acid sequence-based amplification [NASBA]) has provided sensitive, specific, rapid, versatile, and clinically useful methods for the detection of EVs. A combination of human and primate cell lines is typically used for EV and HPeV isolation, since no single cell line supports the growth of all types. A pyrosequencing method was developed for "molecular serotyping" and used in typing the isolates that could not be neutralized by traditional LBM pools and therefore classified as "nontypeable" EVs and in identifying multiple new EV serotypes and novel HPeVs. An understanding of the sites where asymptomatic shedding and disease-induced replication occur is critical to the interpretation of EV test results.
Chapter 86 : Rhinoviruses
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Human rhinoviruses (HRV) are members of the family Picornaviridae. On the basis of neutralization tests in cell culture, 100 rhinovirus serotypes have been designated: HRV1A, HRV1B, HRV2 to HRV86, and HRV88 to HRV100. The authors found divergence within HRV-A of a distinct clade, clade D, and evidence for recombination as a mechanism for HRV diversity. Rhinovirus has been increasingly detected in lower respiratory tract infections, especially in the very young, school-age children, the elderly, and those with chronic illnesses, cancer, immunosuppressive illnesses or transplants or underlying pulmonary disease. Using primers or probes based on sequences in the 59NCR, all rhinovirus serotypes can theoretically be detected in a single assay. This chapter describes isolating procedures and talks about identification of virus. Rhinovirus serotyping is an expensive, labor-intensive research procedure that is rarely performed. Differentiation of rhinoviruses from enteroviruses, with which their cytopathic effects (CPE) can be confused, is based primarily on acid stability testing of isolates. Although clinical laboratories offer specific molecular to elucidate the important role of rhinoviruses as lower respiratory tract pathogens and as significant causes of asthma and chronic obstructive pulmonary disease exacerbation, the standard diagnostic approach may not change substantially until effective therapy becomes available, the impact of rapid and sensitive rhinovirus diagnosis on the management of hospitalized patients can be demonstrated, or highly multiplexed molecular assays incorporating rhinoviruses are more widely adopted.
Chapter 87 : Coronaviruses *
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The family Coronaviridae includes the genera Torovirus and Coronavirus in the order Nidovirales. Coronaviruses (CoVs) were first identified by electron microscopy (EM) of cultured samples and were named for the distinct crown-like morphology of the long surface spikes. Two types of spikes line the outside of the CoV virion. The majority of severe acute respiratory syndrome (SARS)-CoV isolates identified worldwide are genetically closely linked. In one study, human CoV (HCoV)-OC43 was the most commonly identified HCoV in bronchoalveolar lavage (BAL) samples from hospitalized adults. Nucleic acid extraction procedures have the advantage of inactivating viruses before analysis, making them the most likely front-line test in such circumstances. The most common diagnostic approach for identification of HCoVs is now amplification and detection of virus-specific RNA. Assays utilizing one-step or two-step reverse transcription-polymerase chain reaction (RT-PCR) procedures for the amplification stage have dominated most diagnostic formats. The majority of nucleic acid amplification tests (NAATs) for detection of HCoV-229E, HCoV-OC43, and HCoV-NL63 have targeted the N, M, and pol genes. Broad detection of respiratory viruses is particularly important in analyses of outbreaks, and recent studies have shown the added value of broad respiratory virus detection in such diagnostic situations.
Chapter 88 : Hepatitis A and E Viruses *
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The 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. Acute infections with any of the hepatitis viruses cannot be distinguished on clinical characteristics or pathological examinations, and the diseases caused by HAV and HEV are considered together. Clinical presentation of acute viral hepatitis commonly begins with nonspecific, "flu-like" symptoms such as fever, headache, anorexia, nausea, and abdominal discomfort. The high proportion of window period cases found in this study contrasts with the high sensitivity of HAV IgM detection in other studies and may be related to high-dose infections with short incubation periods, and/ or early case detection due to active surveillance of the outbreak. The authors also reported the detection of HAV RNA in 26% of patients initially classified as having acute, sporadic non-A, non-C hepatitis. The detection of HEV-specific IgM should therefore become the method of choice for diagnosis of acute HEV infection in areas of low prevalence. Early assays for HEV-specific IgM showed false-positive reactivity in 3% of U.S. blood donors, roughly equivalent to the rate of IgG reactivity, which limited their usefulness in areas where HEV is nonendemic.
Chapter 89 : Hepatitis C Virus
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Hepatitis C virus (HCV) is classified within the family Flaviviridae in its own genus, Hepacivirus. Phylogenetic analysis of helicase sequences has been used to probe its relatedness to other viruses in the family. The data suggest that HCV is most closely related to the nonpathogenic human virus GBV-C. Viremia clearance kinetics are also important predictors of virologic response (VR), relapse rate, and sustained virologic response (SVR). Signs of hepatitis during acute infection are actually positive indicators as they represent early, vigorous T-cell responses associated with spontaneous virus clearance; these responses are minimal or absent in individuals who progress to chronicity. Unlike other chronic viral infections such as HIV and hepatitis B virus, virologic parameters including viral load and genotype do not predict disease progression or indicate disease severity in chronic hepatitis C. The cutoff of 400,000 IU/ml is the viral load that optimally differentiates high from low probability of SVR in Gt1-infected individuals as shown by receiver operator characteristic (ROC) analysis of data. Nucleic acid tests (NATs) are the cornerstones of chronic HCV treatment since therapeutic tailoring is based on genotype and viral load determinations. Any discussion of HCV NATs must first address nucleic acid extraction due to its contribution to assay performance characteristics. Assays based on 5' untranslated regions (UTR) sequences are generally acceptable for genotype determination but must be carefully designed for subtyping due to the degree of sequence conservation among different viruses. The diagnosis of chronic HCV infection is usually established with serology assays.
Chapter 90 : Gastroenteritis Viruses *
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Common causes of viral gastroenteritis include rotavirus, calicivirus (norovirus and sapovirus), astrovirus, and enteric adenovirus (EAdV). The major symptoms of rotavirus gastroenteritis include watery diarrhea, vomiting, abdominal pain, and fever. The major symptoms of norovirus gastroenteritis include watery diarrhea, vomiting, anorexia, abdominal pain, and fever. AdV-associated acute gastroenteritis is usually milder but lasts longer (5 to 12 days) than other viral gastroenteritis viruses. The major symptoms of astrovirus gastroenteritis include diarrhea, vomiting, anorexia, abdominal pain, and fever, which are generally milder than those of rota-and norovirus gastroenteritis. Due to the difficulty of propagating many gastroenteritis viruses, reliable antigen detection assays remain unavailable for many viral families because of the lack of specific antibodies. Both antigenic and genetic typing methods are important in understanding the classification and epidemiology of many gastroenteritis viruses and for developing preventive strategies against the diseases caused by pathogens. Typing does not affect clinical treatment. Therefore, typing of gastroenteritis viruses is used mainly in research laboratories. The recently recommended new classification system based on sequence information for all 11 genomic RNA segments is an extension of the previous classification systems. There is no neutralization-based serologic test for most other gastroenteritis viruses. Application of assays in sero-surveillance against gastroenteritis viruses has played an important role in understanding the importance of viruses in different populations.
Chapter 91 : Rabies Virus
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The etiologic agents responsible for the acute, progressive viral encephalomyelitis known as rabies belong to the genus Lyssavirus. The virions are rod shaped, approximately 180 by 75 nm, consisting of five structural proteins: the glycoprotein (G), matrix protein (M), nucleoprotein (N), phosphoprotein (P), and large polymerase (L). Human rabies cases due to nonbite exposures are extremely rare. Nonbite exposures include contamination of scratches, open wounds, and mucous membranes with a source of rabies virus, such as infected saliva or central nervous system (CNS) tissues from a rabid animal. Four antemortem clinical samples are required for diagnosing rabies: saliva for nested reverse transcription-polymerase chain reaction (RT-PCR) and/or virus isolation, nuchal skin biopsy for antigen detection and nested RT-PCR, and serum and cerebrospinal fluid (CSF) for immunoglobulin M (IgM) and IgG antibody determination. The standard test for rabies virus antigen detection in CNS tissues is the direct fluorescent-antibody (DFA) test. The direct rapid immunohistochemistry (IHC) test (DRIT) is an alternate procedure for rabies virus antigen detection. An immunohistochemistry test for rabies virus antigen detection is an alternative protocol for formalin- fixed tissue samples which have been processed, embedded in paraffin, and sectioned. Isolation methods are useful in detecting infectious virus in samples and may be applied as an alternate confirmatory test to the standard DFA. Cell cultures may be useful in the propagation, amplification, and quantification of virus and antibodies, to produce vaccines, to determine the safety of vaccine lots, and to study the pathogenesis of rabies virus in particular cells.
Chapter 92 : Hendra and Nipah Viruses
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Hendra and Nipah viruses are classified as biosafety level 4 (BSL-4) agents in the United States and Australia, as there is a risk of infection of laboratory personnel and appropriate precautions must be taken. Widespread presence of Nipah virus antigens could be seen by immunohistochemistry (IHC) in endothelial and smooth muscle cells of blood vessels and in various parenchymal cells, particularly in neurons. Primer sequences can be designed to detect both Hendra and Nipah viruses or to detect each virus separately. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) assays can facilitate research into the pathogenesis of Hendra and Nipah viruses. African green monkey kidney (Vero) cells inoculated with high-titer samples contain antigen within a few days of inoculation. Cytopathic effect (CPE) is usually seen within 3 to 5 days. The CPE is noticeable by the formation of syncytia containing several nuclei. To confirm the presence and identity of the virus, fixed cells are tested by immunofluorescence assay, or supernatant fluids can be tested for evidence of viral replication by RT-PCR techniques. Hendra and Nipah viruses can be reliably distinguished from each other only by neutralization tests. Other cells, such as rabbit kidney cells (RK-13), are susceptible to Hendra and Nipah viruses. Hyperimmune mouse ascites fluid, which recognizes all of the structural proteins of Nipah or Hendra viruses, is used as the positive control. The enzyme-linked immunosorbent assay (ELISA) immunoglobulin M (IgM) test has been the most important technique for serological confirmation in acute patients.
Chapter 93 : Arboviruses *
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Important antigenic determinants have been elucidated for the most important arboviruses, and virus- and group-specific monoclonal antibodies are available for taxonomic and diagnostic purposes. Serologic cross-relationships are most evident in hemagglutination inhibition and binding assays, e.g., enzyme-linked immunosorbent assay (ELISA) and immunofluorescent antibody tests, and occur to a lesser extent in complement fixation tests. The epidemiologic and clinical characteristics of the medically important arboviruses are summarized in this chapter. Immunoglobulin M capture ELISAs for arboviral central nervous system infections transmitted in the United States are offered by several reference and some state public health laboratories. A number of nucleic acid amplification strategies have been developed for the detection and identification of medically important arboviruses, including standard RT-PCR (with agarose gel analysis), real-time RT-PCR using fluorescent probes, nucleic acid sequence-based amplification (NASBA), and the recently developed reverse transcription-loop-mediated isothermal amplification (LAMP) method. Novel arboviruses continue to be discovered, usually as orphan viruses first identified in vector surveys but, remarkably, also from human clinical specimens. The majority of arboviruses are lethal to suckling (2 to 3-day-old) mice, which exhibit signs of illness, paralysis, and death within days to 2 weeks after intracerebral inoculation. While identification of arboviral isolates previously depended upon their antigenic characterization, PCR and other molecular tests are now available for many of the medically important arboviruses.
Chapter 94 : Hantaviruses
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The 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. Specific rodents (i.e., one or two closely related members of the order Rodentia) are the principal hosts of the hantaviruses known to cause human disease. The reverse transcription-PCR (RT-PCR) assays have been employed extensively to detect hantaviral RNA in clinical samples from HPS and HFRS patients and to obtain amplified products for viral characterization by DNA sequencing. Hantaviruses usually are extremely difficult to isolate from clinical materials. Thus, virus isolation is not commonly used for the diagnosis of hantaviral infections in humans. Neutralization of infectivity in vitro, immunofluorescent-antibody assays (IFAs), enzyme-linked immunosorbent assays (ELISAs), and a host of other serologic methods have been used to characterize hantaviruses isolated from clinical samples and rodents. The use of genetic sequence data to define taxonomic relationships within the genus Hantavirus has become increasingly important, in part because some hantaviruses have never been adapted to growth in cultured cells and because our knowledge of the serologic relationships among some of the hantaviral species is based on one strain per species. Early diagnosis is critical to the successful management of HFRS and HPS and, at least in the case of HPS caused by Andes virus, implementation of appropriate isolation procedures to prevent virus transmission to health care providers and other people.
Chapter 95 : Arenaviruses and Filoviruses
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This chapter focuses on the viral hemorrhagic fever (VHF) viruses from two taxa, the families Arenaviridae and Filoviridae. The family Arenaviridae comprises 29 named viruses, which have unique morphologic and physiochemical characteristics. Antigenic relationships are established mainly on the basis of broadly reactive antibody binding assays: historically, the complement fixation test and the indirect fluorescent-antibody (IFA) test, and more recently, the enzyme-linked immunosorbent assay (ELISA). The morphology of arenaviruses is distinctive in thin-section electron microscopy and was the basis for first associating lymphocytic choriomeningitis (LCM) virus with Machupo virus and ultimately associating these viruses with all the viruses in the present family. Immunoelectron microscopy techniques also work well for diagnosis of arenavirus infections, although the morphology of the virions is less striking for arenaviruses than filoviruses. The reverse transcriptase polymerase chain reaction (RT-PCR) followed by genome analysis is rapidly replacing identification methods based on antigen-antibody methods criteria and has the advantage of complete inactivation of the samples in the first extraction step. This chapter emphasizes on the application of immunohistochemical techniques for detecting arenaviruses and filoviruses with a variety of chromogens. Western blotting is feasible for demonstrating antibodies to arenaviruses and filoviruses. However, it has never been applied systematically or routinely to diagnosis, although it was proposed as a confirmatory test to supplement the IFA test for filovirus antibodies. The highest priority for future development is refinement of the available diagnostic tools to permit definitive virus identification in the field.
DNA VIRUSES
Chapter 96 : Herpes Simplex Viruses and Herpes B Virus
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This chapter focuses on the herpes simplex viruses (HSV) and herpes B virus, describing their transmission, clinical significance, and detection mechanisms. Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), formally designated human herpesvirus 1 and human herpesvirus 2, respectively, are members of the family Herpesviridae. Primary infection with HSV-1 or HSV-2 is followed by the establishment of latency in the dorsal root ganglia, typically the trigeminal ganglia for orolabial disease and the lumbosacral ganglia for genital disease. Immunostaining methods to detect antigen require less expertise than cytopathic effect (CPE)-based culture methods and are usually less expensive than culture. Polymerase chain reaction (PCR) provides the best sensitivity of the direct detection approaches. Tests based on crude antigen mixtures are still marketed, but they have unacceptably low sensitivity and specificity, especially for detecting new HSV-2 infections in those with prior HSV-1 infection. The HSV Western blot assay used by the University of Washington laboratory uses nitrocellulose blots prepared with human diploid fibroblast-infected cell proteins. Western blotting detects antibodies to multiple viral proteins, including those to the type-specific glycoproteins gG-1 and gG-2. Simple gG-based lateral-flow assays are available that are designed for point-of-care testing. Type-specific serology is critical for identifying pregnant women with new HSV infections. Serodiagnosis of herpes B virus infections has been complicated by extensive cross-reactivity with HSV-1 and HSV-2. Serologic testing can be useful for evaluation of potentially infected animals involved in human exposures and for screening of research animals.
Chapter 97 : Varicella-Zoster Virus *
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Varicella-zoster virus (VZV) belongs to the family Herpesviridae, based on morphological criteria, and is one of the eight human-pathogenic herpesviruses identified so far. The viral genome has an approximate length of 125,000 bp, making it the smallest of the human herpesviruses, and it encodes at least 70 viral genes. Prevaccination seroepidemiological studies in 11 European countries have shown that in most areas, more than 90% of 10 to 15-year-olds were already seropositive for VZV. The association between the clinical syndrome and VZV has been confirmed by detection of viral DNA by polymerase chain reaction (PCR) in fetal tissue. Real-time PCR assays based on sequence polymorphisms, especially in open reading frame (ORF) 62 or ORF 38, have also been established for detection of VZV. The enzyme-linked immunosorbent assay (ELISA) is usually used to determine VZV-specific antibodies (Abs) in blood, but it may also serve to detect Abs in cerebrospinal fluid (CSF). Screening for drug-resistant virus strains is rarely performed. Antiviral susceptibility of VZV strains can be determined. Changes in virus replication in the presence of different concentrations of various drugs are measured in most cases by plaque reduction assay. The major importance of Ab assays, of which ELISAs are currently the most commonly used, lies in their ability to identify previously infected individuals, as indicated by anti-VZV IgG Abs in the absence of immunoglobulin M (IgM). This information can be used to guide further vaccination decisions.
Chapter 98 : Human Cytomegalovirus
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The human cytomegalovirus (CMV), formally designated human herpesvirus 5 (HHV-5) by the International Committee on Taxonomy of Viruses, is a member of the family Herpesviridae, which includes herpes simplex virus types 1 (HHV-1) and 2 (HHV-2), varicella-zoster virus (HHV-3), Epstein-Barr virus (HHV-4), and human herpesviruses 6, 7, and 8. Purified blood leukocytes are used when performing the CMV antigenemia assay, while whole blood, plasma obtained from anticoagulated whole blood, serum obtained from clotted blood, or purified peripheral blood leukocytes have all been used to quantify CMV DNA in molecular amplification assays. The antigenemia assay is relatively simple to perform and is based on immunocytochemical detection of the 65-kDa lower-matrix phosphoprotein (pp65) in the nuclei of peripheral blood leukocytes. PCR is currently the most widely used molecular method for the detection of CMV DNA and mRNAs, and the sensitivity and specificity of PCR for diagnosis of active CMV infection have been evaluated. The spin-amplification shell vial assay is based on the amplification of virus in cell cultures after low-speed centrifugation and detects viral antigens produced early in the replication of CMV, before the development of cytopathic effect (CPE). Confirmation of herpes B virus in culture can be done using monoclonal antibodies or molecular techniques. PCR for herpes B virus is generally preferred over culture for diagnostic purposes, since it is comparatively rapid, is highly sensitive and specific, and avoids the need to amplify infectious virus to high titers.
Chapter 99 : Epstein-Barr Virus *
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This chapter focuses on Epstein-Barr virus (EBV) that is a member of the Herpesviridae and belongs to the subfamily Gammaherpesvirinae, replicating in epithelial cells and establishing long-term latency in lymphocytes like its closest human-pathogenic relative, human herpesvirus 8. EBV nuclear antigen 1 (EBNA1), EBNA2, EBNA3 (also referred to as EBNA3a), EBNA4 (EBNA3b), EBNA5 (EBNALP), EBNA6 (EBNA3c), and latent membrane proteins (LMP1, -2A, and -2B) may be expressed in B cells. Four types of B-cell latency have been defined, based on various levels of expression of the latency-associated proteins. During lytic replication more than 70 proteins are expressed, including the virus capsid antigens (VCA) and the early antigens used in diagnostics. Nucleic acid detection techniques (NAT) might be applied to blood, cerebrospinal fluid (CSF), or biopsy samples. A wide variety of detection methods are available, ranging from in situ hybridization on frozen or paraffin sections through cytohybridization on cell suspensions, dot blot hybridization, Southern blot hybridization, and nucleic acid amplification techniques (NAAT). The most commonly used clinical diagnostic tools for direct detection of EBV are NAAT. Quantitative real-time PCR is the most popular method today for EBV monitoring in patients at risk for EBV-associated disorders. Individual viral isolates can be characterized by molecular techniques on the basis of polymorphism of the EBNA1 and EBNA3 genes. Patients with X-linked lymphoproliferative syndrome have typically a high viral load and do not develop EBNA antibodies. The ultimate diagnosis, however, is based on genetic analysis for a variety of mutations in the SH2D1A gene domain.
Chapter 100 : Human Herpesviruses 6, 7, and 8
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The viruses discussed in this chapter are Human herpesvirus 6 variants A and B (HHV-6A and HHV-6B), Human herpesvirus 7 (HHV-7), and Human herpesvirus 8 (HHV-8; Kaposi's sarcoma [KS]-associated herpesvirus). These viruses cause diseases that are clinically significant primarily in small children and in immunocompromised patients. HHV-6A and HHV-6B, along with HHV-7, comprise the Roseolovirus genus of the betaherpesvirus subfamily. Roseoloviruses share many features of their genomic architecture and genetic content, the ability to replicate and establish latent infections in lymphocytes, and associations with febrile rash illnesses in young children, and they are opportunistic pathogens in immunocompromised patients. Collectively, HHV-6A and HHV-6B are highly prevalent, with seroprevalences in many populations exceeding 90%. During primary infection in young children in the United States, HHV-6A was found by polymerase chain reaction (PCR) in 2.5% of peripheral blood mononuclear cell (PBMC) and 17% of cerebrospinal fluid (CSF) specimens, while HHV-6B was found in 99% of PBMC and 86% of CSF specimens (some specimens were coinfected). HHV-6 primary infection causes roseola in approximately one-quarter of children. Less common but more severe forms of primary HHV-6 infection may include fever of >40°C, respiratory tract distress, tympanic inflammation, diarrhea, and convulsions. Antiviral susceptibility is evaluated by measuring the inhibition of viral replication by immunofluorescence, by antigen slot blots, and by inhibition of virus-induced cytotoxicity.
Chapter 101 : Adenoviruses
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Adenoviruses are of considerable interest as vectors for gene delivery and as emerging human pathogens. Adenovirus infections are common and ubiquitous. Adenovirus infections can be epidemic, endemic, or sporadic, with the pattern of circulation, specific syndrome, and severity varying by serotype, population, and route of exposure. Adenoviruses are best detected from affected sites early in the course of illness. Adenovirus-infected cells can be visualized by light microscopy as “smudge cells” in hematoxylin-and-eosinor Wright-Giemsa-stained tissues, fluid sediments, or cultures. Antigen detection can be used for the rapid detection of adenoviruses in respiratory, ocular, and gastrointestinal tract specimens. Adenoviruses in virtually all specimen types have been detected by PCR or other molecular methods, so the appropriate specimen depends largely on the associated disease. Cultivation of enteric adenoviruses is most successful in Graham 293 cells. Adenoviruses can be isolated by conventional tube culture or shell vial centrifugation culture (SVCC). Enteric adenoviruses can be detected in SVCC if appropriate cells lines are used, but other approaches are more sensitive and convenient. Detection of nonenteric adenoviruses in stools of immunocompromised patients can be achieved by generic enzyme immunoassay (EIA), generic PCR, or culture. Detection of adenoviruses from sites other than the respiratory and gastrointestinal tracts is more straightforward for interpretation because virus is infrequently detected in the absence of disease.
Chapter 102 : Human Papillomaviruses
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The majority of identified human papillomaviruses (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 natural history of oral HPV less is only beginning to be evaluated, though it is clear that while high-risk (HR) HPV infection is associated with a subset of head and neck squamous cell cancers, the prevalence of oral HPV infection in the general population is substantially lower than the HPV prevalence in the genital tracts of men and women. Sample collection, transport, and storage recommendations of specimens vary based on the purpose and method of HPV testing. The morphological cellular changes characteristic of HPV infection and associated neoplasia are the basis for standard cervical cytology and are classified based on the Bethesda system. HPV DNA tests are typically performed on one to three 10-µm sections of formalin-fixed paraffin-embedded (FFPE) tissue. Sections are deparaffinized with octane and digested with a buffer containing proteinase K and a nonionic detergent. The number of commercially available assays for HPV detection has increased dramatically in recent years. HPV RNA:DNA hybrids are detected using a sandwich enzyme-linked immunosorbent assay-type reaction. One of the most characteristic features of HPV infection is the multiplicity of genotypes known to infect the anogenital tract and oral cavity. Complete genotyping has been incredibly useful as a research tool to evaluate the natural history of all genotypes in epidemiological studies.
Chapter 103 : Human Polyomaviruses
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Lymphotropic polyomavirus (LPyV), a nonhuman primate polyomavirus, is classified as a strain of African green monkey polyomavirus. The newly identified putative human polyomaviruses (HPyV) WU polyomavirus (WUPyV), KI polyomavirus (KIPyV), and Merkel cell polyomavirus (MCPyV) have yet to be formally classified. Antemortem direct microscopic examination of tissue is performed less commonly for progressive multifocal leukoencephalopathy (PML) than for polyomavirus-associated nephropathy (PVAN) due to the risks involved in obtaining brain biopsy material. PCR methods can be classified as having either conventional or real-time formats. For JCPyV and BKPyV, two types of serological tests have been described, including hemagglutination inhibition (HAI) and enzyme immunoassays (EIAs) employing either crude antigens or the use of virus-like particles (VLPs). Detection of an antibody response by HAI is based on the capacity of specific antiviral antibodies to inhibit the agglutination of human erythrocytes mediated by the viral structural VP1 proteins of JCPyV and BKPyV. Current approaches to therapy include having optimal highly active antiretroviral therapy (HAART) for HIV-infected patients, with the aim of increasing CD4 cell counts and decreasing the HIV RNA level. The current trend in laboratory testing for PVAN is to monitor levels of the BKPyV in urine and blood and to reduce immunosuppression when viruria or viremia reaches levels that predict progression to disease.
Chapter 104 : Human Parvoviruses *
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The current classification of parvoviruses is based upon their host range and dependence on other viruses for their replication. This chapter focuses primarily on Erythrovirus but does contain brief updates on human bocavirus (HBoV) and the newly described human parvovirus 4 (PARV4). Subsequent evaluation of blood unit, using immune electron microscopy, revealed viral particles consistent in size and morphology with parvovirus. Many important observations regarding the B19 virion have been made using electron microscopy and X-ray crystallography. The major nonstructural protein is NS1 (71 kDa), a DNA binding protein involved in viral replication. The VP1 and VP2 capsid proteins originate from the same ORF and are identical in sequence, except for an additional 227 amino acids at the amino terminus of VP1. Using nucleic acid amplification testing (NAT), B19 DNA has been detected in numerous batches of albumin, factor VIII, factor IX, clotting factor concentrates, and immunoglobulin. Serum from whole blood, collected in a sterile tube lacking anticoagulant, is most suitable for serologic testing, although plasma, containing either EDTA or sodium citrate, can also be used. Capture enzyme immunoassays employing native or recombinant antigens are excellent choices for detecting B19-specific immunoglobulin. Testing sera for the presence of B19-specific IgG antibodies can determine past or previous infection in the immunocompetent individual.
Chapter 105 : Poxviruses
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This chapter deals with poxviruses belonging to the family Poxviridae and subfamily Chordopoxvirinae. The G+C contents of orthopoxviruses, yatapoxviruses, Molluscum contagiosum virus (MCV), and parapoxviruses are ~33, ~32, ~60, and ~63%, respectively. The zoonotic poxviruses include members of the genera Orthopoxvirus (monkeypox virus, cowpox virus, and the vaccinia virus subspecies, including buffalopox virus), Parapoxvirus (orf, pseudocowpox, sealpox, and papulosa stomatitis viruses), and Yatapoxvirus (tanapox virus [TPV], Yaba monkey tumor virus [YMTV], and Yaba-like disease virus [YLDV]). Poxviruses produce inclusions that have characteristic appearances when stained with May-Grunwald Giemsa and hematoxylin-eosin stains. Perinuclear basophilic or B-type cytoplasmic inclusions (virus factories or viroplasm) are observed with cells infected with any of the poxviruses and represent sites of virus replication. Polymerase chain reaction (PCR) analysis is used by the WHO collaborating center (WHOCC) at the center for disease control (CDC) to detect poxvirus DNA in samples. Orthopoxviruses are the only human poxviruses that produce pocks on the chorioallantoic membranes (CAMs) of fertile chicken eggs; pock morphology is useful for biologic species and variant differentiation. Parapoxviruses, yatapoxviruses, and MCV do not form pocks on the CAM, although avipoxviruses, leporipoxviruses, and capripoxviruses do so. Serologic methods currently used to detect antibodies against human orthopoxviruses include enzyme-linked immunosorbent assays (ELISAs), the virus neutralization test (NT), Western blotting, and hemagglutination inhibition. In the current state of bioterrorism response awareness, tests to evaluate residual protection from previous vaccination are being requested.
Chapter 106 : Hepatitis B and D Viruses
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Hepatitis B virus (HBV) was identified and characterized after the discovery of the Australia antigen by Blumberg and colleagues in 1965. The Australia antigen, now designated hepatitis B surface antigen (HBsAg), is detected in the sera of patients with both acute and chronic HBV. HBV infects hepatocytes, leading to an acute infection that resolves or a chronic infection lasting years. Safe and effective vaccines against HBV have been available since 1982. HBV infection is diagnosed by serological and molecular markers using serum or plasma. The laboratory diagnosis of HBV uses a combination of tests that detect virus-specific protein and nucleic acid as well as the host immune response to infection. The methods for identification of HBV infection use a combination of molecular, antigenic, and serological methods. Serologic tests for HBV-specific antibodies are used to determine the stage of disease and to establish immunity due to vaccination. Patients with chronic hepatitis that are hepatitis B e antigen (HBeAg) negative are more likely to have more advanced liver disease in spite of lower serum HBV DNA levels. The majority of individuals vaccinated for HBV have detectable levels of anti-HBs, but some test negative due to waning levels of anti-HBs. Hepatitis D virus (HDV) is a defective RNA virus that requires the presence of HBV for its replication. The laboratory diagnosis of HDV depends on the detection of specific antibodies, HDAg, and HDV RNA.
SUBVIRAL AGENTS
Chapter 107 : Transimissible Spongiform Encephalopathies
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The most widely accepted hypothesis on the nature of the infectious agent causing transmissible spongiform encephalopathies (TSEs), which is termed a prion (for proteinaceous infectious particle), predicates that it consists of a scrapie-like prion protein (PrPSc), an abnormally folded, protease-resistant, beta-sheet-rich isoform of a normal cellular prion protein (PrPC). Human prion diseases manifest as sporadic, genetic, and acquired disorders. They are referred to as sporadic Creutzfeldt-Jakob disease (sCJD), genetic CJD (gCJD), variant CJD (vCJD), and iatrogenic CJD (iCJD). Direct intracerebral exposure to prions and implantation of prion-contaminated dura, for example, are associated with short incubation periods (16 to 28 months), whereas exposure to prions at sites outside the central nervous system (CNS) results in long incubation times ranging from 5 to 30 years. The diagnosis of human prion diseases is based on the evaluation of clinical signs and auxiliary examinations. Whole blood may be used for isolation of DNA for genetic analysis (exclusion of gCJD). Tissue should be fixed in formalin for histologic assessment and snap-frozen for Western blotting. Western blotting is routinely performed on unfixed tissue originating from the CNS. The entire open reading frame may be amplified for sequencing, using PCR. All testing other than detection of surrogate markers in the cerebrospinal fluid (CSF), sequencing of PRNP, and neuropathology is usually performed on a research basis.
ANTIVIRAL AGENTS AND SUSCEPTIBILITY TEST METHODS
Chapter 108 : Antiviral Agents
- Authors: Nell S. Lurain, Kenneth D. Thompson
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The use of antiviral agents for the treatment of viral diseases continues to expand. Most of the agents currently approved by the Food and Drug Administration (FDA) are active against one or more of the following viruses: human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2), hepatitis B and C viruses (HBV and HCV, respectively), the human herpesviruses, and influenza A and B viruses. This chapter is organized according to these virus groups, with cross-referencing for agents with activity against more than one group of viruses. There are now five classes of antiviral agents for treatment of HIV-1: (i) nucleoside and nucleotide reverse transcriptase inhibitors (NRTIs and NtRTIs), (ii) nonnucleoside reverse transcriptase inhibitors (NNRTIs), (iii) protease inhibitors (PIs), (iv) entry/fusion inhibitors, and (v) integrase strand transfer inhibitors (INSTIs). The only antiviral agents that have shown any activity in achieving a sustained virologic response against chronic HCV infection are standard interferon (IFN)-α; 2a and 2b, pegylated IFN-α; (PEG-IFN) 2aµ and 2b, and combinations of these IFNs with ribavirin (RBV). There are two major classes of drugs available to treat HBV: nucleoside and nucleotide analogues and IFNs. Most of the antiviral compounds that are approved to treat the eight human herpesviruses are nucleoside or nucleotide analogues which inhibit DNA replication. The two classes of antiviral agents for the treatment of influenza are M2 protein inhibitors (active only against type A influenza viruses) and neuraminidase inhibitors (activity against both type A and B viruses).
Chapter 109 : Mechanisms of Resistance to Antiviral Agents *
- Authors: Robert W. Shafer, Isabel Najera, Sunwen Chou
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Understanding the mechanisms of viral drug resistance is critical to the clinical management of individuals receiving antiviral therapy, the development of new antiviral drugs, and the surveillance of drug resistance. This chapter reviews the mechanisms of resistance to antiviral agents used to treat seven common viral infections, i.e., infections with herpes simplex virus (HSV), cytomegalovirus (CMV), varicella-zoster virus (VZV), human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2), influenza A and B viruses, hepatitis B virus (HBV), and hepatitis C virus (HCV). Antiviral drug resistance is usually mediated by mutations in the molecular targets of drug therapy, and the development of drug resistance is the most compelling evidence that an antiviral drug acts by specifically inhibiting a virus rather than its cellular host. Drug-resistant viruses are identified by in vitro passage experiments with increasing concentrations of an inhibitory drug and by ex vivo analysis of virus isolates obtained from individuals receiving antiviral therapy. Antiviral agents, mechanisms of resistance, and mutations involved in drug resistance in herpesviruses are summarized in a tabular form. Most TK mutants are classified as TK negative, usually arising from frameshift or stop mutations that delete important functional domains, or TK partial, when a mutation reduces the phosphorylation of both natural nucleosides and antiviral drugs. Nucleoside analog RdRp inhibitors appear to be equally active against the different HCV genotypes. The genetic barrier to nucleoside analog resistance also appears to be higher than that for the other two main classes of HCV inhibitors.
Chapter 110 : Susceptibility Test Methods: Viruses *
- Authors: Max Q. Arens, Ella M. Swierkosz
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This chapter discusses the clinical situations in which antiviral resistance has emerged, thus necessitating in vitro susceptibility testing. It provides an overview of the phenotypic and genotypic susceptibility testing methods that have been employed to detect resistance. Persistent or worsening herpes simplex virus type 1 (HSV-1) or varicella zoster virus (VZV) infection while on acyclovir (ACV) may indicate drug resistance. Persistent or worsening human cytomegalovirus (HCMV) retinitis, pneumonitis, or colitis unresponsive to ganciclovir may also indicate drug-resistant virus. Monitoring for Human Immunodeficiency Virus type 1 (HIV-1) antiretroviral resistance is essential when beginning antiretroviral therapy (ART), for assessing failure of a particular regimen to suppress HIV replication and to test for cross-resistance to alternative antiretroviral drugs to aid in selection of appropriate salvage therapy. The plaque reduction assay (PRA) has been the "standard" method of antiviral susceptibility testing. A number of phenotypic assays are in use for testing of HIV-1 isolate susceptibility to nucleoside analog RT inhibitors. The major advantage of genotypic assays is the relatively rapid turnaround time compared to phenotypic assays. Multicenter studies were carried out to compare sequencing results among participating laboratories for detection of reverse transcriptase (RT) mutations. In this study, sequence concordance was high, even though different editing strategies were used by different labs, but 12% of the resistance mutations present in the 10 electronic files that were distributed and analyzed were not identified in some labs.
MYCOLOGY
GENERAL
Chapter 111 : Taxonomy and Classification of Fungi
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Fungal identification can be challenging and sometimes frustrating because of the importance placed on the morphological characteristics of the organisms, and the need to become familiar with a range of different structures and terms. Investing time to learn the basic structures and principles of taxonomy, classification, and nomenclature can result in the ability to recognize and identify correctly many medically important fungi. The taxonomy and nomenclature of fungi that have both asexual and sexual stages are challenging. A simplified taxonomic scheme illustrating the major groups of medically important fungi is described in this chapter. Identification of yeast relies on a combination of morphological, physiological, and biochemical characteristics. Two artificial form classes of anamorphic or “mitosporic” molds are currently recognized, based on the mode of conidium formation. The Hyphomycetes produce their conidia directly on the hyphae or on specialized conidiophores, while the Coelomycetes have more elaborate reproductive structures, termed conidiomata. Most molds can be identified after growth in culture, but the criteria for recognition often differ from the fundamental characteristics that are used as a basis for classification. Many clinical laboratories today employ DNA sequencing as part of their routine protocol for fungal identification.
Chapter 112 : Specimen Collection, Transport, and Processing: Mycology
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This chapter offers guidelines for specimen collection and transport, specimen handling, specimen pretreatment and processing in the laboratory, medium selection, and incubation of cultures. Abscess specimens should be collected from the active peripheral edge of open abscesses or aspirated from closed abscesses by use of a syringe. Fungemia is a major cause of morbidity and mortality in hospitalized patients, with Candida species being the major cause. Early detection of organisms in the bloodstream is incredibly important because it is an indicator of disseminated disease. As in bacteriology, the volume of blood, the blood-to-broth ratio, and the number of blood cultures are all critical factors, with the volume of blood being the most important variable. Bone marrow is most useful for the diagnosis of disseminated candidiasis, cryptococcosis, and histoplasmosis. Subcutaneous tissue should be examined for the presence of granules. The chapter contains a table listing various media used for the recovery of fungi from clinical specimens, including primary media, selective and/or differential media, and specialized media. A wide variety of media are available for primary isolation, and in many laboratories, the choice is based on personal experience and the technologist's preferences. Mycology laboratories that are responsible for culturing Nocardia and aerobic actinomycetes must include other media for these pathogens, such as Sabouraud agar.
Chapter 113 : Reagents, Stains, and Media: Mycology
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A variety of stains, media, and reagents are available to the mycology laboratory for the detection, isolation, characterization, and identification of yeasts and moulds. Media should be carefully selected based on specimen type and suspected fungal agents. These media include cornmeal agar, inhibitory mould agar, inhibitory mould agar with gentamicin, soy peptone agar with cycloheximide/chloramphenicol without pH indicators, potato dextrose agar, brain heart infusion agar with 5% sheep blood containing chloramphenicol/gentamicin, Sabouraud's dextrose agar, and Sabouraud's dextrose agar with chloramphenicol/gentamicin. Nonexempt media that require specific quality assurance testing include cornmeal agar with Tween, brain heart infusion agar with 5% sheep blood and cycloheximide/chloramphenicol, bismuth sulfite-glucose-glycine-yeast (BiGGY) agar, birdseed agar, brain heart infusion with 5% sheep blood and penicillin/streptomycin, dermatophyte test medium, and potato flakes agar with or without cycloheximide/chloramphenicol. The stains, media, and reagents listed in this chapter include those commonly used and a few specialized items. Methenamine silver stains are perhaps the most useful stains for visualizing fungi in tissue. Specific colonial morphologies and growth patterns of the different Candida species are also detected. Assimilation broth media for yeasts are used for the detection of assimilation, i.e., carbohydrate utilization by yeasts in the presence of oxygen. A brief description of selected chromogenic agar products that are FDA approved for use in U.S. laboratories is provided as an overview of how different yeasts react with chromogenic substrates and the resultant characteristic colony color.
Chapter 114 : General Approaches for Direct Detection of Fungi *
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This chapter on general approaches for direct detection of fungi reviews non-culture-based detection methods that can be performed directly with clinical materials in a clinical laboratory. These methods include direct microscopic examination, antigen detection, detection of fungus-specific metabolites, detection of cell wall components, detection of fungus-specific nucleic acids, and serologic diagnosis. Differentiating yeast from mold is important because yeast may represent normal colonization, while mold can indicate a deeper infectious process. Distinguishing colonization from disease complicates the diagnosis of candidiasis by antigen detection. Mannan, which is highly immunogenic, is one of the major cell wall components of Candida species and is shed into the blood during infection. The glucuronoxylomannan component of the Cryptococcus neoformans capsular polysaccharide is used for diagnosis of cryptococcosis. Strains of A, D, and AD serotypes are now classified as C. neoformans, while B and C serotype strains are classified as Cryptococcus gattii. Studies are in progress to determine the potential of direct identification of fungi in positive blood culture broths and patient samples. White et al. summarizes an update on the Aspergillus PCR standardization project. In this review it was found that the efficiency of the Aspergillus PCR was limited by the extraction procedures utilized and the compliance of some centers in the study.
FUNGI
Chapter 115 : Candida, Cryptococcus, and Other Yeasts of Medical Importance
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The taxonomy of yeasts is continually evolving, and currently yeasts that are of medical importance belong to two classes: the Saccharomycetes (previously Hemiascomycetes or Endomycetes), which contains Candida species, and the Tremellomycetes (previously Heterobasidiomycetes), which contains the basidiomycetous fungi Trichosporon and Cryptococcus. A number of species have been merged into Candida albicans, including Candida claussenii and Candida langeronii. There is evidence that C. albicans var. africana may be a new cause of vaginitis that is often misidentified as C. albicans on the Vitek 2 ID-YST system. Candida species are ubiquitous yeasts, being found on many plants and as the normal biota of the alimentary tract of mammals and mucocutaneous membranes of humans. In nearly 45% of AIDS patients, cryptococcosis was reported as the first AIDS-defining illness. Because none of the presenting signs or symptoms of cryptococcal meningitis (such as headache, fever, and malaise) are sufficiently characteristic to distinguish it from other infections that occur in patients with AIDS, determining cryptococcal antigen titers and culturing blood and cerebrospinal fluid (CSF) are useful in making a diagnosis. Not surprisingly, little effort has been undertaken to develop serologic tests for detection of invasive infections caused by yeasts other than Candida and Cryptococcus species. In order to develop a universal genetic identification system for yeasts, the most reproducible, specific, and sensitive sequence for comparison must be identified, and the majority of research has focused on the rDNA regions.
Chapter 116 : Pneumocystis
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Once known collectively by the single genus and species, "Pneumocystis carinii," it is now understood that distinct species of Pneumocystis infect different mammalian hosts. A recent report of the ability of Pneumocystis spp. to form biofilms outside the lung holds promise for development of an in vitro system. Almost every mammal examined to date appears to harbor at least one species of Pneumocystis that is not found in any other mammal. Human- and rat-derived Pneumocystis species have been the most extensively studied of the species in this group, and discussion of the life cycle in this chapter is based on these findings. The chapter discusses morphological criteria for recognition of Pneumocystis jirovecii stained by various procedures. Recent studies of humans reported the ability to detect P. jirovecii-specific DNA in nasopharyngeal aspirates and oropharyngeal washes after amplification by PCR with P. jirovecii-specific primers. There is currently only one recognized species of Pneumocystis that causes pneumonia in humans, P. jirovecii. It may become desirable in the future to track the emergence of P. jirovecii organisms that are resistant to trimethoprim-sulfamethoxazole (TMP-SMX), or evaluate the potential for therapeutic response. The microscopic demonstration of P. jirovecii in tissue and fluids by staining with Gomori methenamine silver (GMS) or a rapid variant of the Wright-Giemsa stain, by immunofluorescent assay (IFA), or by other stains such as Papanicolaou should be considered sufficient for diagnosis.
Chapter 117 : Aspergillus and Penicillium
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The taxonomy of Aspergillus has been in flux, and this genus has now been subdivided into subgenera and sections. In practice, Aspergillus species are identified mainly on the basis of phenotypic characteristics of the anamorph. Early monographic treatments also included descriptions of the teleomorph. In general, Aspergillus colonies usually grow very rapidly, producing powdery white, green, yellowish, brown, or black colonies. Several Aspergillus species also produce mycotoxins which are harmful to humans and animals when ingested. Aspergillus fumigatus still accounts for most cases of aspergillosis, with A. flavus and A. niger being the other more common pathogenic species worldwide. There has also been an increase in reports demonstrating the recovery of previously recognized Aspergillus species, such as A. udagawae and A. viridinutans, from human infections. Importantly, these species appear to have different in vitro susceptibility patterns and show differences in clinical disease. Enzyme-linked immunosorbent assay (ELISA) reactivity was reported for certain beta-lactam antibacterial agents and, more recently, for patients treated with piperacillintazobactam and amoxicillin-clavulanic acid. The ELISA reactivity observed in batches of these antibiotics might be due to the use of Penicillium fungi for the production of the antibacterial agents. The internal transcribed spacer regions of rDNA have been found to be more discriminatory than the 28S ribosomal subunit for identification of 13 clinically important Aspergillus species. PCR-based assays utilizing these sequences have also been developed. Penicillium marneffei, a member of the subgenus Biverticillium, is considered as the only true pathogen.
Chapter 118 : Fusarium and Other Opportunistic Hyaline Fungi
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The opportunistic hyaline or lightly colored moulds constitute a phylogenetically diverse group of common to rare anamorphic and teleomorphic fungi that typically occur as saprobes in soil, in air, or on plant litter or as facultative plant pathogens. Some may be recovered from specimens without having any clinical significance. While several of the genera treated in this chapter include species having either lightly colored or dark (melanized) conidia, the emphasis is on those fungi that grow in tissue in the form of hyaline or lightly colored, septate hyphal elements. The term fusariosis is used to define infections caused by species of Fusarium, but the practice of coining disease names based on the genus of fungus involved is disadvantageous for infections caused by uncommon or rare fungal pathogens. In the chapter, the name of the teleomorph is used for species that are identified mainly by their sexual structures. Most of the pathogenic moulds considered in the chapter are classified in the form-class Hyphomycetes (genera which bear their conidia free). The conidiogenous cell produces a single conidium or multiple conidia. The number of hyaline fungal species that have been reported to cause opportunistic infection in humans and animals is increasing. The chapter also describes the salient colonial and microscopic features of the medically important species in the genus Fusarium and other selected currently recognized hyaline opportunists.
Chapter 119 : Agents of Systemic and Subcutaneous Mucormycosis and Entomophthoromycosis
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Mucormycosis and entomophthoromycosis are invasive fungal infections caused by environmental nonseptate filamentous fungi. Mucormycosis is caused by the ubiquitous Mucorales fungi and occurs mostly in immunocompromised patients or those with diabetes mellitus. Entomophthoromycosis is caused by the Entomophthorales fungi, found mostly in warm climates. According to the classification proposed by Hibbet et al. the subphylum Entomophthoromycotina contains the order Entomophthorales, which is subdivided into two families: the Ancylistaceae and Basidiobolaceae, containing the genera Conidiobolus and Basidiobolus, respectively. There are 27 species in the genus Conidiobolus, but only two (C. coronatus and C. incongruus) have been recovered from clinical specimens, while in the genus Basidiobolus, B. ranarum is the only species known to cause human disease. Several recent reports have suggested that the incidence of mucormycosis is increasing, based on single-center studies. Mucormycosis represents 2% of invasive fungal infections following solid organ transplantion (SOT), mostly after kidney transplantation. Mucormycosis can also develop in human immunodeficiency virus-infected patients or intravenous drug abusers. Blood cultures have no diagnostic value, as they are almost always negative, despite the fact that mucormycosis is an angioinvasive disease. Demonstration of hyphae in clinical samples is important for the diagnosis of mucormycosis. In summary, new molecular tools have been developed for the diagnosis of mucormycosis and the identification of Mucorales fungi in tissues. Culture results should always be interpreted in light of the clinical presentation and along with the results of direct examination and histopathology.
Chapter 120 : Histoplasma, Blastomyces, Coccidioides, and Other Dimorphic Fungi Causing Systemic Mycoses
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This chapter covers the dimorphic members of the families Onygenaceae and Ajellomycetaceae, which include Blastomyces dermatitidis, Histoplasma capsulatum, Paracoccidioides brasiliensis, and Coccidioides immitis and C. posadasii as well as Emmonsia species. B. dermatitidis is thermally dimorphic, converting from the mold phase to the yeast phase under appropriate conditions of temperature and nutrition. In the environment and in culture at room temperature, the Coccidioides fungus exists as a mold producing septate hyphae and arthroconidia that usually develop in alternate hyphal cells. Inhalation of conidia is the usual mode of infection leading to blastomycosis. The incubation period has been estimated to be 4 to 6 weeks. Inhalation of arthroconidia is the usual mode of infection leading to coccidioidomycosis in humans. The incubation period is 1 to 3 weeks. Paracoccidioidomycosis predominates in adults, who display 85% to 95% of cases, and in persons in agriculture-related occupations. The disease is more often diagnosed in males than in females. There is a wide spectrum of clinical manifestations of histoplasmosis, ranging from a transient pulmonary infection that subsides without treatment to chronic pulmonary infection or to more widespread disseminated disease. Tissue sections should be stained with periodic acid-Schiff, Grocott-Gomori methenamine silver, or hematoxylin and eosin to permit the detection of the characteristic large thick-walled spherules of Coccidioides species.
Chapter 121 : Trichophyton, Microsporum, Epidermophyton, and Agents of Superficial Mycoses *
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The etiologic agents of dermatophytosis are classified, along with some nonpathogenic relatives, in the anamorphic genera Trichophyton, Microsporum, and Epidermophyton. The recorded connections between the teleomorphic (sexual) and anamorphic (asexual) states of the dermatophytes as well as the dermatophytoids are discussed in this chapter. As part of the ongoing molecular revolution in biology, fungal taxonomy is ever more strongly influenced by one's greatly increased understanding of population genetics. The chapter retains a relatively cautious approach to the ongoing debates in this area and synonymizes traditionally recognized species primarily when there is unequivocal, multigene molecular evidence that they are not supported at the species level. 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. Infections with geophilic dermatophytes involve transmission of soil-borne inoculum to humans or other mammals. At present, the great majority of dermatophytes are identified phenotypically. Identification is often based on (i) colony characteristics in pure culture on sabouraud glucose agar (SGA) and (ii) microscopic morphology. Dermatophytes can in principle be tested for susceptibility to antifungal drugs using the Clinical and Laboratory Standards Institute (CLSI) M38-A3 standard procedure for molds. In the superficial mycoses, the causative fungi colonize the cornified layers of the epidermis or the suprafollicular portion of the hair. There is little tissue damage, and lack in cellular response from the host.
Chapter 122 : Bipolaris, Exophiala, Scedosporium, Sporothrix, and Other Melanized Fungi *
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The chapter on Bipolaris, Exophiala, Scedosporium, Sporothrix, and Other Melanized Fungi covers most of the agents of phaeohyphomycosis, chromoblastomycosis, and sporotrichosis, as well as a number of agents of superficial and cutaneous disease. In this chapter, the genera are treated according to their ordinal relationships. Phaeohyphomycoses are usually subcutaneous, but they can also be superficial or even systemic. Members of the order Pleosporales (most commonly Bipolaris, Curvularia, and Exserohilum) are able to cause subcutaneous infections, although more commonly they produce allergic sinusitis with occasional cerebral involvement in otherwise healthy individuals. A panfungal PCR assay has been developed that targets the ITS1 region of the rDNA gene cluster for detecting fungal DNA in fresh and paraffinembedded tissue specimens. This method was useful for the identifying species of Scedosporium, Exophiala, and Exserohilum. The available in vitro data for dematiaceous fungi are increasing every day, and in general the antifungal susceptibility of the most clinically relevant species is known. Posaconazole activity is higher than voriconazole activity against Alternaria spp., Exophiala spp., and Sporothrix spp., although against the last the activity of both drugs was very poor. In the future, direct identification of fungal genera from tissue blocks using immunohistochemistry, in situ DNA hybridization, or DNA sequencing will be a promising approach to rapid detection and identification of these agents.
Chapter 123 : 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. The etiologic agents described in this chapter are species that are isolated most commonly from human or lower-animal mycetoma. F. falciforme is occasionally found as a cause of mycetoma in the United States and Argentina. It was also reported as the cause of an opportunistic mycetoma infection in a renal transplant recipient. P. boydii is the most common agent of mycetoma in humans as well as lower animals in temperate climates, occurring mostly in the limbs. E. jeanselmei identification was confirmed in all mycetoma or mycetoma-like infections, while other environmental strains were found to belong to other Exophiala species, suggesting some predilection for human invasion with particular species. Certain species (M. grisea, M. mycetomatis, N. rosatii, P. mackinnonii, and P. romeroi) do not sporulate readily but can be recognized by molecular techniques. The major difference between the two species is found in the ascospores, which differ in size, shape, septation, and the nature of the gelatinous sheath that surrounds them. With the exception of P. boydii and Fusarium species, most fungi causing eumycotic mycetoma are susceptible in vitro to ketoconazole, itraconazole, voriconazole, and posaconazole. Since agents of eumycotic mycetoma are soil or plant saprobes, their etiologic role in mycetoma must be carefully established.
Chapter 124 : Mycotoxins
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This chapter reviews various mycotoxins and their relevance to sick building syndrome (SBS), veterinary problems, bioterrorism, and food safety. There are some 300 to 400 compounds, toxic to vertebrates in low concentrations, which are currently recognized as mycotoxins. Aflatoxins are produced by several species of Aspergillus, in particular Aspergillus flavus and A. parasiticus. Aflatoxin contamination can be the cause of a variety of economic and health problems. The most common organisms producing citrinin are Penicillium citrinum, Penicillium expansum, Penicillium viridicatum, Penicillium camemberti. Citrinin has demonstrated nephrotoxic effects on all animal species tested and was shown to inhibit dehydrogenase activity in rats’ kidneys, liver, and brain. Ergot alkaloids, produced by the ergot fungus Claviceps purpurea, are the causative agent of ergotism which can manifest in either a gangrenous or convulsive condition following ingestion of contaminated grains. Ochratoxins A and B are produced by Penicillium verrucosum and many species of Aspergillus, especially A. ochraceus. Deoxynivalenol (DON) is one of the most common mycotoxins found in grain, including barley, oats, rye, and wheat. Zearalenone is a nonsteroidal estrogen or phytoestrogen produced by various species of Fusarium. It is estimated that one-quarter of the world’s crops are contaminated to some extent with mycotoxins. There are several classes of mycotoxins, produced by a wide range of fungal species, which have been linked to various environmental issues such as SBS, veterinary problems, bioterrorism, and food safety.
Chapter 125 : Lacazia, Pythium, and Rhinosporidium
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In the past 100 years the microbial pathogens described in this chapter have been classified as fungal and/or parafungal protistan pathogens. Lacazia loboi, Pythium insidiosum and Rhinosporidium seeberi are the most prominent features of the pathogens covered in this chapter. Clinical samples from patients suspected of having lacaziosis submitted to the laboratory comprise deep-skin scrapings and tissue biopsy samples. The samples have to be processed and evaluated for the presence of uniform yeast like cells connected by small tubules forming short chains. In contrast with the other hydrophilic pathogens covered in the chapter, P. insidiosum can be cultured on various media. The first molecular approach for the diagnosis of P. insidiosum from clinical specimens was carried out with a patient with keratitis. The hyphal elements present in the specimen were identified by sequencing part of the 18S ribosomal DNA region using the NS1 and NS2 and internal transcribed spacer (ITS) universal primers. The identification of P. insidiosum using molecular methods are described in this chapter. The first two cases of rhinosporidiosis were reported in 1900 by Guillermo Rodolfo Seeber in his M.D. thesis in Argentina. Ashworth in 1923 stated that the genus Rhinosporidium proposed by Minchin and Fanthan should be adopted and that, based on the description of Seeber and the name Coccidioides seeberia reintroduced by Belou, the binomial R. seeberi has priority.
ANTIFUNGAL AGENTS AND SUSCEPTIBILITY TEST METHODS
Chapter 126 : Antifungal Agents *
- Author: David W. Warnock
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This chapter reviews the four major families of antifungal drugs that are available for systemic administration: the allylamines, the azoles, the echinocandins, and the polyenes. It discusses the characteristics of other agents that can be used for the oral or parenteral treatment of superficial, subcutaneous, or systemic fungal infections. The allylamines are a group of synthetic antifungal compounds effective in the topical and oral treatment of dermatophytoses. The azoles constitute a large group of synthetic agents containing many compounds that are effective in the topical treatment of dermatophyte infections and superficial forms of candidiasis. The activity is essentially fungistatic, although some of the newer triazoles can exert fungicidal effects against some mold species at the concentrations achieved with recommended dosages. Three echinocandins have been approved for the treatment of serious fungal infections: anidulafungin, caspofungin, and micafungin. Acquired resistance to echinocandins is rare at present, but resistant strains of several Candida spp. have been recovered from patients failing caspofungin treatment. The echinocandins do not interact with the human hepatic cytochrome P-450 system, and their use has been associated with very few significant drug interactions. The polyenes bind to sterols, principally ergosterol, in the membranes of susceptible fungal cells causing impairment of membrane barrier function, leakage of cell constituents, metabolic disruption, and cell death. As more compounds have become licensed, the number of novel antifungal drugs entering preclinical development appears to have diminished.
Chapter 127 : Mechanisms of Resistance to Antifungal Agents
- Authors: Theodore C. White, Samantha J. Hoot
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This chapter describes the factors that contribute to a recalcitrant or resistant clinical infection. It focuses on the resistance of fungal isolates, as determined by their MIC. There are two types of resistance: intrinsic resistance, which is an inherited characteristic of a species or strain, and acquired resistance, which occurs when a previously susceptible isolate develops a resistant phenotype, usually as a result of prolonged treatment with antifungals. A section of the chapter concentrates on the mechanisms identified in C. albicans, with discussion of mechanisms identified in other fungi when applicable. Several alterations in ERG11 have been associated with resistance in C. albicans, including (i) point mutations in the coding regions, (ii) overexpression of the gene, (iii) gene amplification, and (iv) gene conversion or mitotic recombination. The interaction between the azoles and Erg11p can be altered by mutation or overexpression of the ERG11 gene. Alterations in other enzymes in ergosterol biosynthesis can also affect azole susceptibility. New azole drugs, such as posaconazole, need to be carefully monitored for their effect on strains that are resistant to fluconazole. An increased understanding of antifungal drug resistance should allow for the development of new diagnostic strategies to identify resistant clinical isolates in a patient, new treatment strategies to treat these resistant infections, and new prevention strategies that would forestall the development of antifungal drug resistance in these patient populations.
Chapter 128 : Susceptibility Test Methods: Yeasts and Filamentous Fungi
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This chapter deals with systemic infections caused by Candida spp. other than C. albicans, Aspergillus spp., and other filamentous fungi (molds). The development of the Clinical and Laboratory Standards Institute (CLSI) reference method M27-A3 has improved the reproducibility of in vitro antifungal susceptibility data and facilitated the establishment of interpretive breakpoints for the triazoles fluconazole, itraconazole, and voriconazole and the echinocandins. Based on historical data and the pharmacokinetics of flucytosine, interpretive breakpoints for flucytosine and Candida spp. also have been established. Some correlation has been suggested between amphotericin B Minimal Inhibitory Concentration (MIC) results obtained by nonstandardized methods and clinical outcome. Unfortunately, most M27-A amphotericin B MICs for yeasts are within a very narrow range, precluding a clear discrimination between susceptible and potentially resistant isolates. The CLSI Subcommittee on Antifungal Susceptibility Testing has developed reference methods for broth macro- and microdilution susceptibility testing of yeasts and mold and more recently a disk diffusion method for yeasts and a proposed disk diffusion method for molds. The European Committee on Antifungal Susceptibility Testing (EUCAST) has developed a modified broth microdilution method for yeast and has developed breakpoints for itraconazole and fluconazole to be applied to this method. Some commercial methods have been approved for the antifungal susceptibility testing of Candida spp. and there is a move towards consensus in the standardized methodology employed in the United States and Europe. This should help to improve surveillance of resistance patterns worldwide and help in the development of universal clinically relevant breakpoints.
PARASITOLOGY
GENERAL
Chapter 129 : Taxonomy and Classification of Human Parasitic Protozoa and Helminths
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This chapter deals with the taxonomy and classification of the parasitic protozoa and helminths that are commonly encountered in humans together with a number that are only occasionally encountered. The concept of species as the basic unit that underlies the logical classification of all eukaryotes is scientifically sound but does not necessarily meet the needs of those whose interests are in the diseases caused by parasites and not the parasites themselves. This subject is discussed in some detail by Tibayrenc, who points out that there have been some 24 concepts of species and that "Biological researchers need more-pragmatic approaches that can be understood by non-specialists. Decision makers need precise answers for cost-effective and efficient control measures against transmissible diseases.” Classification of parasitic protozoa and helminths that infect human are listed in this chapter. The levels of the taxa used differ between the protozoa and helminths, and this is deliberate. In the classification of the Protozoa, the higher taxa, subkingdoms and infrakingdoms, have been omitted for simplicity and protozoans are classified as far as orders, as this is the lowest taxonomic level normally used by parasitologists.
Chapter 130 : Specimen Collection, Transport, and Processing: Parasitology
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This chapter deals with various specimen collection, transport, and processing methods for detection of parasites. Various specimen collection methods are available for specimens suspected of containing parasites or parasitic elements. The most common specimen submitted to the diagnostic laboratory is the stool specimen, and the most commonly performed procedure in parasitology is the ova and parasite (O&P) examination, which is composed of three separate protocols: the direct wet mount, the concentration, and the permanent stained smear. The examination of aspirated material for the diagnosis of parasitic infections may be extremely valuable, particularly when routine testing methods have failed to demonstrate the organisms. These specimens should be transported to the laboratory immediately after collection. Detection of parasites in tissue depends in part on specimen collection and on having sufficient material to perform the recommended diagnostic procedures. The nucleic acid-based diagnostic tests have been developed for almost all species of parasites. The main reason for the minor role of diagnostic PCR in parasitology is the fact that many parasite stages can be adequately diagnosed using established, more traditional techniques that are generally less expensive than PCR and technically less demanding. Diagnostic PCR may become more widespread when simple, fully standardized test kits are available and costs are reduced through the implementation of pre- and post-PCR automated techniques. Furthermore, the possibilities to not only detect and identify but also quantify organisms and determine their genotypes by analyzing the diagnostic PCR product extend the diagnostic power of PCR.
Chapter 131 : 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 culture media, the most common of which are discussed in this chapter. Formalin has always been used in parasitology as an allpurpose preservative and in concentration procedures. The accompanying compound with the polyvinyl alcohol (PVA), specifically, mercuric chloride, zinc sulfate, or cupric sulfate, acts as the preservative and allows fixation of protozoan cysts and trophozoites for use with trichrome or iron hematoxylin stains for permanent smears. Specimens treated with Zn-PVA may also be stained with trichrome or iron hematoxylin stains for permanent smears. Specimens treated with Cu-PVA may also be stained with trichrome or iron hematoxylin stains for permanent smears. Examination of blood films for parasites includes the use of two common stains, the Giemsa stain and Wright's stain, both derivatives of the original Romanowsky stain. These stains are very similar, differing primarily in that no fixative is included in the Giemsa stain and the blood film must be fixed with absolute methanol prior to staining. In a basic staining procedure, thick films must be laked in distilled water or treated with saponin prior to performance of the staining procedure.
Chapter 132 : General Approaches for Detection and Identification of Parasites
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This chapter discusses various approaches and diagnostic methods currently in use for the diagnosis of parasitic infections. Examination of prepared wet mounts, concentrated specimens, permanent stained smears, blood films, and various culture materials can provide critical information leading to organism identification and confirmation of the suspected cause of clinical disease. The purpose of the concentration method is to separate parasites from fecal debris and to concentrate any parasites present through either sedimentation or flotation. Wet mounts prepared from concentrated stool are examined in the same manner as that used for the direct wet mount method. Although the routine ova and parasite examination consisting of the direct wet mount, the concentration, and the permanent stained smear is an excellent procedure recommended for the detection of most intestinal parasites, several other diagnostic techniques are available for the recovery and identification of specific parasitic organisms. The only human parasites for which it is reasonably possible to correlate egg production with adult worm burdens are Ascaris lumbricoides, Trichuris trichiura, and the hookworms (Necator americanus and Ancylostoma duodenale). Several parasites may be recovered and identified from urogenital specimens. Although, the most common pathogens are probably Trichomonas vaginalis and Schistosoma haematobium, other organisms such as the microsporidia are becoming much more important. Although other tests such as immunoassay 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 133 : Plasmodium and Babesia
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The genus Plasmodium includes at least 172 named species of intraerythrocytic parasites infecting a wide range of mammals, birds, reptiles, and amphibians. Malaria imposes an enormous burden of illness and substantial mortality on the tropical world and in many subtropical regions. There are an estimated 515 (95% confidence interval, 330 to 660) million clinical episodes of malaria due to Plasmodium falciparum, the most common cause of malaria, and 1.5 million to 2.7 million deaths due to malaria each year. Gastrointestinal complaints, nausea, vomiting, and diarrhea, which may be bloody, are also not uncommon and should not distract the clinician from the diagnosis of malaria. In the face of rapidly developing drug resistance, development of new antimalarial drugs and, ultimately, an effective malaria vaccine are high priorities. The genus Babesia includes approximately 100 species that are transmitted by ticks of the genus Ixodes and infect a variety of wild and domestic animals. The diagnosis of babesiosis should be considered for a patient with the appropriate clinical symptoms and a history of travel to areas where the disease is endemic, exposure to ticks, or recent blood transfusion. Examination of Giemsastained thin blood smears is the most direct approach to diagnosis.
Chapter 134 : Leishmania and Trypanosoma
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Leishmania spp. and Trypanosoma spp. are protozoa belonging to the family Trypanosomatidae. Recent estimates suggest that there are approximately 350 million people at risk of acquiring leishmaniasis, with 112 million currently infected. All adult female sand flies transmitting leishmaniasis belong to the genus Phlebotomus in the Old World and Lutzomyia in the New World. There are more than 30 species of sand flies that can transmit leishmaniasis. Mucocutaneous leishmaniasis is produced most often by the L. braziliensis complex. The majority of infected individuals are asymptomatic or have very few or minor symptoms that resolve without therapy. PCR-based methods for the diagnosis of leishmaniasis have used a variety of specimens, including urine. Molecular techniques for the detection of leishmanial DNA or RNA have been used for diagnosis, prognosis, and species identification. These methods are considered more sensitive than slide examination or culture, particularly for the detection of mucocutaneous leishmaniasis. Promastigote stages can be detected microscopically in wet mounts and then stained with Giemsa stain to observe their morphology. Positive reactions are usually seen in cutaneous and mucocutaneous leishmaniasis; however, patients with active visceral and diffuse cutaneous leishmaniasis exhibit negative reactions. American trypanosomiasis (Chagas' disease) is a zoonosis caused by Trypanosoma cruzi. Population-screening programs have been used to control T. brucei gambiense infections. The most effective control measures include an integrated approach to reduce the human reservoir of infection and the use of insecticide and fly traps.
Chapter 135 : Toxoplasma *
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Serologic prevalence data indicate that toxoplasmosis is one of the most common infections of humans throughout the world. Toxoplasmosis can be categorized into four groups: (i) acquired in the immunocompetent patient; (ii) acquired or reactivated in the immunodeficient patient; (iii) congenital; and (iv) ocular. Acquired infection with Toxoplasma in immunocompetent individuals is generally an asymptomatic infection. Secretions, excretions, body fluids, and tissues are potential specimens for direct observation of parasites but are generally unrewarding. The most important advantage of the immunoglobulin M (IgM) capture enzyme immunoassay (EIA) compared to indirect fluorescent antibody (IFA) is the increased detection of congenital infections: the IgM enzyme-linked immunosorbent assay (ELISA) was positive for 73% of serum samples from newborn infants with proven congenital toxoplasmosis, whereas only 25% of the same serum samples were found positive by an IFA IgM test. The currently recommended drugs work primarily against the actively dividing tachyzoite form of T. gondii and do not eradicate encysted organisms (bradyzoites). T. gondii infection is one of the most common parasitic infections worldwide and, in most instances, is of little clinical significance. The laboratory will need to use additional testing in an attempt to define the timing of infection in the case of pregnant women or the presence of actively replicating parasites in the case of the fetus, neonate, or immunosuppressed host.
Chapter 136 : Pathogenic and Opportunistic Free-Living Amebae
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Small, free-living amebae belonging to the genera Naegleria, Acanthamoeba, and Balamuthia have been identified as agents of central nervous system (CNS) infections of humans and other animals. The concept that these small, free-living amebae may occur as human pathogens was proposed by Culbertson and colleagues, who isolated Acanthamoeba sp. The genus Acanthamoeba contains as many as 24 species in three groups, with groupings based largely on morphologic characteristics. The chapter talks about clinical significance of Naegleria Meningoencephalitis, Acanthamoeba Encephalitis, Balamuthia (Leptomyxid) Encephalitis, and Acanthamoeba Keratitis. It outlines the recommended procedure for isolating free-living pathogenic amebae from biological specimens. Identification of living organisms to the genus level is based on characteristic patterns of locomotion, morphologic features of the trophozoite and cyst forms, and results of enflagellation experiments. Acanthamoeba spp can easily be cultivated axenically, without the addition of serum or host tissue, in many different types of nutrient media, e.g., proteose peptone-yeast extract-glucose medium, Trypticase soy broth medium, and chemically defined medium. The serologic techniques discussed in this chapter have been developed as research tools and are not routinely available to clinical laboratories. Most clinical laboratories rely on the agar plate technique for the isolation and identification of these small, free-living, and pathogenic amebae, as other techniques, like PCR, are not available and sometimes not even feasible. The laboratories usually send the specimens to an outside laboratory like the Centers for Disease Control (CDC) for identification and interpretation.
Chapter 137 : Intestinal and Urogenital Amebae, Flagellates, and Ciliates
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This chapter talks about the intestinal amebae and urogenital amebae, flagellates, and ciliates. It is noteworthy that Dientamoeba fraglis, once classified as an ameba, is now grouped with the flagellates. All diagnostic stages of the amebae (trophozoite and cyst) can be detected in fecal specimens, the most common specimen submitted to the laboratory. Entamoeba histolytica and the other Entamoeba spp. are classified as belonging to the phylum Amoebozoa, subphylum Conosa, class Archamoebea, order Euamoebida. Some flagellates are commensals that reside in the intestinal tract and are harmless to the individual. For the detection of flagellates and ciliates, laboratories predominantly receive stool specimens for microscopic examination. The flagellates have greater morphologic diversity relative to one another than do the amebae, making determination of the genus easier. Despite the lack of external flagella, this parasite is currently classified as a flagellate but has historically been grouped with the amebae. Two additional nonpathogenic intestinal flagellates are E. hominis and Retortamonas intestinalis. Clinical laboratories are now given more choices for testing in diagnostic parasitology, with assays ranging from microscopy, culture, antigen detection, and nucleic acid amplification techniques. Molecular biology has the promise to deliver more sensitive and specific methods but to date, these methods have not been fully adapted to the clinical diagnostic laboratory.
Chapter 138 : Isospora, Cyclospora, and Sarcocystis *
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The Isospora (Cystoisospora), Cyclospora, and Sarcocystis are intestinal coccidia of humans, and oocysts of these coccidia are found in the feces of humans, and diagnosis is based ultimately on demonstrating oocysts (Isospora or Cyclospora) or sporocysts (Sarcocystis) in human stool samples. Cyclospora cayetanensis is in the family Eimeriidae, while Sarcocystis hominis and Sarcocystis suihominis are in the family Sarcocystidae. The oocysts are Isospora-like and contain two sporocysts, each with four sporozoites. In areas of endemicity, there is an increased risk of Cyclospora infection with contact with soil and water. Most of the produce items implicated as transmitting Cyclospora are consumed raw, which does not lend itself to prevention by thermal means. Cyclospora species infecting mammals other than humans may also be present in water samples or on produce, and proofs of specificity are needed for these tests designed to look at environmental sources of C. cayetanensis and to detect C. cayetanensis oocysts on produce. Nitazoxanide has been evaluated for activity against C. cayetanensis, and in these studies, its efficacy for the treatment of cyclosporiasis was about 70%. Both I. belli and C. cayetanensis are usually identified by stool examination and are rarely misidentified in human feces. Computer report notes can indicate that trimethoprim-sulfamethoxazole is the drug of choice for treatment of these parasites. PCR can be used for the identification of organisms but is not commercially available and should be indicated as an experimental test.
Chapter 139 : Cryptosporidium
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Recently, the taxonomy of Cryptosporidium had gone through revisions as the result of extensive molecular genetic studies and biologic characterizations of parasites from various animals. Within the epithelial cells, trophozoites undergo two or three generations of asexual amplification called merogony, leading to the formation of different types of meronts containing four to eight merozites. Studies in the United States and Europe have shown that cryptosporidiosis is more common among homosexual men than among persons in other human immunodeficiency virus (HIV) transmission categories, indicating that direct person-to-person or anthroponotic transmission of cryptosporidiosis is common. Seasonal variations in the incidence of human Cryptosporidium infection in industrialized nations have also been partially attributed to waterborne transmission. As for any pathogens that are transmitted by the fecal-oral route, good hygiene is the key in preventing the acquisition of Cryptosporidium infection. There are significant differences among different Cryptosporidium species and C. hominis subtype families in clinical manifestations of pediatric cryptosporidiosis. In industrialized nations, the most effective treatment and prophylaxis for cryptosporidiosis in AIDS patients is the use of highly active antiretroviral therapy (HAART). From a public health point of view, the reporting of a significant number of cases above background levels in industrialized nations indicates the likely occurrence of outbreaks of cryptosporidiosis or false positivity of diagnostic kits. In situations like this, it is crucial to have the test results verified with a confirmatory test such as direct immunofluorescence assays (DFA) or PCR and to report them to the state or local public health department.
Chapter 140 : Microsporidia *
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Recent genome-wide sequence and synteny analyses indicate that the parasites of the phylum Microsporidia belong to the kingdom of the Fungi. Direct zoonotic transmission of microsporidia infecting humans has not been verified but appears likely because many microsporidial species can infect both humans and animals. Microsporidiosis has been associated with abnormalities in structures and functions of infected organs, but the mechanisms of pathogenicity of the different microsporidial species are not sufficiently understood. The most robust technique for the diagnosis of microsporidial infection is light microscopic detection of the parasites themselves. Evaluation of patients with suspected intestinal microsporidiosis should begin with light microscopic examination of stool specimens, and microsporidia which cause systemic infection are best detected in urine sediments or other body fluids. Microsporidial species causing disseminated infection have been found in almost every organ system. Only highly experienced pathologists have reliably and consistently identified microsporidia in tissue sections by using routine techniques such as hematoxylin and eosin staining. The isolation of microsporidia has no relevance for diagnostic purposes but is an important research tool. Microsporidial ultrastructure is unique and pathognomonic for the phylum, and ultrastructural features can distinguish all microsporidial genera. Observational studies showed that an improvement of immune functions by potent antiretroviral combination therapy results in complete clinical response and normalization of intestinal architecture, which parallels the clearance of intestinal microsporidia. Furthermore, various microsporidial species may cause self-limited diarrhea or keratoconjunctivitis in immunocompetent and otherwise healthy persons.
Chapter 141 : Nematodes
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Soil-transmitted helminths (intestinal nematodes) are the most common infections globally with more than one billion people infected, especially in resource-poor settings where sanitation is inadequate. Studies strongly suggest that nematodes are actually related to the arthropods and priapulids in a newly recognized group, the Ecdysozoa. The Ascaris lumbricoides adult worm is the largest of the human pathogenic nematodes, 15 to 35 cm in length. Ancylostoma and Necatorare the two genera of Ancylostomatidae that infect humans. The major clinical manifestation of hookworm infection is iron deficiency anemia due to intestinal blood loss and depletion of iron stores. Some rhabditiform larvae develop into infective filariform larvae in the bowel lumen, penetrate the intestinal mucosa or perianal skin, and repeat the cycle of maturation within the same host. This process of autoinfection, albeit uncommon among intestinal nematodes, results in chronic infections that may persist for 40 years or more. The clinical features are related to the intensity of infection, as is the case with the other intestinal nematodes. In developed countries, any helminth eggs or larvae found in feces are significant and treatment is recommended, even if the patient is asymptomatic. However, treatment of asymptomatic cases is not necessary in developing countries as many of these parasites are endemic and reinfection is common.
Chapter 142 : Filarial Nematodes *
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This chapter talks about three lymphatic-dwelling filarial parasites of humans: Wuchereria bancrofti, Brugia malayi, and Brugia timori. Lymphatic filariasis (LF) is associated with a variety of clinical manifestations. The four most common presentations are asymptomatic (or subclinical) microfilaremia, lymphedema, hydrocele, and acute attacks. Lymphedema most commonly affects the lower extremities but can also affect arms, breasts in females, and the scrotum in males. Diagnosis of bancroftian and brugian filarial infection can be made noninvasively in some cases by ultrasound. Recent developments in immunodiagnostic and molecular biology techniques give further options for diagnosis. The current global elimination campaign uses three drugs: DEC, ivermectin, and albendazol, in various combinations for mass treatment of communities where the parasites are endemic. Next, the chapter talks about Onchocerca volvulus. The major disease manifestations of onchocerciasis are localized to the skin, lymph nodes, and eyes. Finally, the chapter discusses Loa loa, and Mansonella infections. The adult worms of Mansonella perstans reside in the body cavities (pericardial, pleural, and peritoneal) as well as in the mesentery and the perirenal and retroperitoneal tissues. The major clinical manifestations of M. streptocerca infections are related to the skin: pruritus, papular rashes, and pigmentation changes. For M. perstans infections, diagnosis is made parasitologically by finding the microfilariae in the blood or in other body fluids. The diagnosis of streptocerciasis can be made by finding the characteristic microfilariae on skin-snip examination. The chapter describes treatment and prevention methods for M. perstans, M. streptocerca, and M. ozzardi.
Chapter 143 : Cestodes
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Four species of cestode tapeworms inhabit the human intestine: Diphyllobothrium latum, Taenia saginata, Taenia solium, and Hymenolepis nana. Rarer larval cestode infections affecting humans include coenurosis (Taenia multiceps), sparganosis (Spirometra mansonoides), and cysticercosis by Taenia crassiceps. Eggs can be easily seen by microscopical examination of stools. Eggs should be reported as Taenia sp. because direct observation does not confirm the species. T. solium taeniasis and cysticercosis are highly endemic to all parts of the developing world where pigs are raised as a food source. The infection is now also increasingly diagnosed in industrialized countries due to immigration of tapeworm carriers from zones of endemicity. H. nana is the smallest of the intestinal tapeworms of humans and also the most common tapeworm infection throughout the world. H. nana is normally a parasite of mice, and its life cycle characteristically involves a beetle as intermediate host. In humans, transmission is usually accomplished by direct ingestion of infective eggs containing oncospheres. Diagnosis of the infection rests on finding the spherical eggs in feces by microscopy. Eggs are characteristic and should be reported as H. nana. Beside physical examination, diagnosis usually is based on imaging techniques including ultrasound, computed tomography (CT), and magnetic resonance imaging (MRI) supported by serology. Taenia multiceps and Taenia serialis have canids as definitive hosts and sheep as their normal intermediate host, harboring the larvae or coenurus. Diagnosis is based on pathological demonstration of the typical larval membrane and multiple scolices.
Chapter 144 : Trematodes
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Adult trematodes have distinctive morphology, often with a leaf-like body plan. Despite the diverse range of body sites infected by adult trematodes, the eggs of most digenean flukes are voided with feces. There are many subtle variations in life cycle patterns, but two predominant life cycle strategies exist for these trematodes. Two features of the digenean life cycle are noteworthy. First, digeneans often display high specificity in their choice of first intermediate host. Second, most human parasites are zoonotic, requiring the cooccurrence of other mammalian or avian hosts in an area of endemicity to maintain human infection. In urinary schistosomiasis, granulomatous inflammatory response to embolized eggs gives rise to dysuria, hematuria, and proteinuria, calcifications in the bladder, obstruction of the ureter, renal colic, hydronephrosis, and renal failure. Examination of stools with the Kato-Katz technique is used in field studies for quantification of fecal egg burdens. An interesting development is that of the use of real-time PCR to detect cell-free schistosome DNA in host plasma. This method utilizes a 121-bp tandem repeat sequence that represents approximately 12% of the S. mansoni genome as the target sequence for amplification. Members of the families Echinostomatidae, Heterophyidae, Fasciolidae, and Troglotrematidae are commonly encountered in some countries. Many species of heterophyid trematodes are known to infect humans. Diagnosis of heterophyids is facilitated by observation of eggs in feces.
Chapter 145 : Less Common Helminths
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This chapter covers the less common causes of helminthic parasitic infections, particularly those caused by the less common nematodes and cestodes. It includes some of the most interesting and challenging of parasitic diseases. The collection, transport, and storage of specimens are similar regardless of the helminth present, so these guidelines are consolidated in this chapter to reduce duplication. The chapter presents additional information about serologic tests for the diagnosis of parasites. The diseases caused by less commonly encountered helminthic parasites are interesting and demonstrate their highly evolved life cycles and the complex interactions with their 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 146 : Arthropods of Medical Importance *
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Arthropods comprise a diverse group of invertebrate animals, united in a common body theme (bauplan) of a jointed, chitinous exoskeleton. Medically important arthropods have long been considered to mainly comprise ectoparasites, parasites that limit their activities to the skin. Parasitism, however, is only one of several associations that comprise the interaction of arthropods of medical importance with humans. Arthropods may also be medically important due to indirect effects: fear of insects, delusional parasitosis, or allergy due to dust mites. The various modes by which arthropods may affect human health thus reflect the diversity of these animals, but there are very few instances in which it may be argued that natural selection favored the offspring of those that focused on causing misery. Arthropods are thought of by many in clinical settings with respect to their role as vectors, transmitters of infectious agents including viruses, bacteria, protozoa, and helminths. There are five major groups of vectors: the diptera (flies and mosquitoes), hemiptera (kissing bugs), siphonaptera (fleas), anoplura (lice), and acarines (ticks and mites). The general life history strategies for each group provide the basis for understanding vectorial capacity, which is the sum of physiological and ecological attributes that allow transmission. Vectors impart directionality to a pathogen. In contrast, there are arthropod-pathogen relationships that are not characterized by directionality, and analogous to mathematical terminology, arthropods that inadvertently serve as a source of infection are called scalars.
ANTIPARASITIC AGENTS AND SUSCEPTIBILITY TEST METHODS
Chapter 147 : Antiparasitic Agents
- Authors: Karin Leder, Peter F. Weller
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There are a number of effective antiprotozoal and anthelmintic drugs currently available. These antiparasitic agents are important both for therapy of individual patients and for control of parasitic infections at the community level. This chapter focuses on the mechanisms of action, pharmacology, clinical utility, and adverse effects of common first-line antiparasitic therapies and newer drug alternatives. Most helminth infections in humans can be treated with one of five drugs, namely, albendazole, mebendazole, praziquantel, ivermectin, and diethylcarbamazine (DEC), so these five drugs are reviewed. A newer agent, nitazoxanide, which has both anthelmintic and antiprotozoal activity, is discussed. The chapter also reviews other major antiprotozoal drugs, including those used for malaria, gastrointestinal protozoal infection, leishmaniasis, and trypanosomiasis. The quinoline derivatives can be divided into the following four groups: the 4-aminoquinolines; the cinchona alkaloids; synthetic compounds, such as mefloquine and halofantrine; and the 8-aminoquinolines. The two agents used for treatment of American trypano-somiasis are nifurtimox and benznidazole. Nifurtimox has significant side effects that preclude the completion of therapy in many patients. Less frequent but more-severe toxicities include psychosis and convulsions. Benznidazole crosses the placenta, but there are minimal data regarding teratogenic effects of either agent in either animals or humans.
Chapter 148 : Mechanisms of Resistance to Antiparasitic Agents *
- Authors: W. Evan Secor, Jacques Le Bras
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The chapter talks about the mechanisms of resistance to antiparasitic agents. Plasmodium falciparum, the most virulent species, which adapts more easily to environmental constraints in tropical areas, has become the dominant species, and its resistance to chloroquine, to sulfadoxine-pyrimethamine, and, further, to all known drugs has developed to various degrees. True resistance is more likely in anthroponotic forms of leishmaniasis, such as those caused by Leishmania donovani and Leishmania tropica, because the zoonotic species that infect primarily animals, with humans as an occasional host, rarely encounter drugs. Laboratory or field isolates with resistance to pentavalent antimonials are also killed effectively by paromomycin and miltefosine. Several factors contribute to the emergence of drug- resistant parasites. Public health interventions to correct these factors have not always been successful and would benefit from a better understanding of the drug resistance mechanisms used by parasites. These mechanisms are very diverse and have been difficult to study, but recent technological advances now provide long-awaited tools that will facilitate the task. The genome sequences for P. falciparum, T. vaginalis, L. major, T. brucei, and S. mansoni have been compiled. When these data are used, for example, in combination with microarray technology, where the DNA of drug-resistant parasite strains is compared to that of drugsusceptible strains, identification of the genes that confer resistance should proceed even more rapidly than in the last few years.
Chapter 149 : Susceptibility Test Methods: Parasites *
- Authors: Jacques Le Bras, W. Evan Secor
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Drug susceptibility tests fall into four broad categories: in vivo tests, in vitro tests, tests with experimental animals, and molecular tests. These different categories of tests provide complementary information. At one end of the spectrum, molecular tests analyze parasites at their most basic biologic level, without any outside interference. Most drug resistance tests in malaria concern Plasmodium falciparum, the most prevalent and virulent species and the most prone to the development of resistance. Incorporating pharmacokinetic parameters is now considered essential for true resistance identification within partners of artemisinins, which are frequently poorly absorbed or slowly eliminated drugs. While treatment failures for Trichomonas vaginalis infections have often been disregarded as being due to poor patient compliance or rapid reinfection, there is an increasing recognition that true clinical resistance exists and may be increasing, thus necessitating accurate drug susceptibility testing of patient isolates. Treatment failure in leishmaniasis may result from either true drug resistance or patient immunodeficiencies that preclude effective chemotherapeutic action. Differentiation of these two possibilities is important for both individual patient care and general public health. A standardized in vivo method for detection of drug resistance that was designed for testing trypanosomes that infect cattle can also be applied to those that infect humans. Investigation of drug resistance mechanisms of schistosomes is more difficult and time-consuming than those associated with protozoan infections.
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