bluuebunny/crossref_metadata_2025 huggingface dataset:
Synergy Testing: Broth Microdilution and Broth Macrodilution Checkerboard Methods
Authors: Doern Christopher D. , Van Belkum Alex
Published: January 2025

Although uncommon, microbiology laboratories may use a two‐dimensional, two‐agent broth microdilution checkerboard to evaluate combinations of antimicrobial agents against microorganisms. A broth macrodilution limited series, agar dilution method, gradient diffusion, or disk diffusion method can also be used, although filter‐based methods have been described as well. Test methods are based on Clinical and Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility Testing broth dilution susceptibility methods for evaluating the inhibitory or bactericidal activity of specific concentrations in combination at a fixed time.
In vitro
interactions are calculated algebraically and interpreted as synergistic, indifferent, or antagonistic depending on whether the antibacterial activity of the combination is greater than, equivalent to, or less than the activities of the individual agents, respectively.
In vitro
interactions can also be represented geometrically with isobolograms. Synergism testing between more formal antibiotics and natural compounds is increasingly reported.
Evaluation and Verification of Antimicrobial Susceptibility Test Systems
Authors: Bobenchik April M.
Published: January 2025

A wide variety of antimicrobial susceptibility test systems are available to clinical microbiologists. This procedure provides guidance on evaluating and choosing a system or method appropriate for the laboratory. The primary focus should be on determination of the systems' capabilities, assessment of costs and workflow, and examination of performance data. This chapter describes a procedure for analyzing results generated from evaluating a new system in comparison with those generated from a reference system. Some of the suggestions in this protocol can be considered to fulfill the verification requirements. The chapter covers proper quality control principles for analytical tests. With postanalytical considerations, it provides attention to common test limitations, interpretive challenges, and results reporting.
Selecting Antimicrobial Agents for Testing and Reporting
Authors: Bobenchik April M. , Simner Trish
Published: January 2025

Developing protocols for antimicrobial susceptibility testing (AST) and reporting is best done by the clinical laboratory with input from the antimicrobial stewardship program, infectious disease service, infection control, and the pharmacy and therapeutics committee, as available at a given institution. The goals are to provide clinically relevant information that will support appropriate and cost‐effective utilization of antimicrobial agents to avoid reporting results that may adversely affect patient care or public health. Selective reporting is a reporting strategy that reports certain AST results based on defined criteria such as organism identification, body site, or patient demographics. Cascade reporting involves reporting results for specific agents based on the overall antimicrobial susceptibility profile of an isolate. These reporting techniques may withhold (i.e., suppress) some AST results from the final report provided to clinical end users, in part to support antimicrobial stewardship efforts.
Susceptibility Testing
Authors: Sussland Zdenek , Saubolle Michael A.
Published: January 2025

The Clinical and Laboratory Standards Institute has published guideline M24, and its supplement M62, which describe standard methods for appropriate
in vitro
antimicrobial susceptibility testing against aerobic actinomycetes. The broth microdilution method, providing MICs, is the method recommended for susceptibility testing of the aerobic actinomycetes, including the
Nocardia
spp.,
Rhodococcus
spp.,
Tsukamurella
spp.,
Gordonia
spp.,
Streptomyces
spp., and
Actinomadura
. These procedures have been primarily used for the most commonly isolated species of the
Nocardia
genus but may be cautiously used for other clinically relevant genera of aerobic actinomycetes. Commercially prepared microdilution plates with the appropriate antimicrobial agents are available but have not yet been approved by the Food and Drug Administration. A disk diffusion test with sulfisoxazole may be used only to verify sulfonamide MIC results and to confirm concentration of organisms in the inoculum.
Solid Media Used for Isolation
Authors: Wengenack Nancy L.
Published: January 2025

Both liquid and solid media are recommended for optimal recovery of mycobacteria in culture. Pure cultures of mycobacteria, particularly
Mycobacterium tuberculosis
, are still required, despite the availability of molecular technology, to obtain phenotypic antimicrobial susceptibility test results and for strain typing. The advantage of solid medium is that it enables the detection of mixed cultures and contaminants. Egg‐based or agar‐based media may be used. The main advantage of an egg‐based medium is that it best supports the growth of
M. tuberculosis
and permits niacin testing, if needed. The advantages of agar‐based media include less contamination and earlier and easier visibility of colonial morphology. Colonial morphology can aid in the identification of mycobacteria. Use of both nonselective and selective media is needed for optimal mycobacterial isolation, the latter containing one or more antimicrobial agents to prevent overgrowth by contaminating bacteria or fungi.
Susceptibility Testing by the Agar Proportion Method for
Mycobacterium tuberculosis
Complex
Authors: Escuyer Vincent E. , Salfinger Max
Published: January 2025

Antimicrobial susceptibility testing should be performed on each patient's initial
Mycobacterium tuberculosis
complex isolate, in relapse or retreatment cases and when acquired drug resistance is suspected. The modified agar proportion method is used to determine the susceptibility of
M. tuberculosis
complex, indirectly from a pure culture. Quadrant plates are used that contain either Middlebrook 7H10 agar without antimicrobial agents or 7H10 agar with antimicrobial agents at various concentrations. Agar proportion testing compares the growth of an isolate on solid agar medium with and without antimycobacterial drug. The concentrations of the drugs tested are based on the recommendations of the CLSI. The agar proportion method also allows the determination of the proportion or percentage of resistance among the tested isolates. This chapter covers proper quality control principles for analytical tests. With analytical considerations, it provides attention to common test limitations, interpretive challenges, and results reporting.
Fungal Taxonomy and Nomenclature
Authors: Lockhart Shawn R. , Wiederhold Nathan P.
Published: January 2025

Defining the rules for fungal taxonomy is an evolving process. Changes in nomenclature continue to have significant implications for understanding fungi in the environment, fungal disease, and the treatment of infections. Fungi differ from bacteria in that many of them are capable of undergoing a sexual cycle and multiplying through meiotic division. They can exist in two states: an asexual state that divides only through mitotic division, called the anamorph state, and a sexual state that divides by meiotic division, called the teleomorph state. As most fungal infections are caused by a single isolate, mating in the laboratory is rare and the anamorph state is most often identified. However, on rare occasions a homothallic fungus is isolated and the teleomorph state is identified. The Clinical and Laboratory Standards Institute is working on a standard for the incorporation of bacterial and fungal taxonomy in the clinical laboratory.
Specimen Examination
Authors: Abdolrasouli Alireza , Howell Susan
Published: January 2025

The microscopic examination of clinical specimens for the presence of fungi plays an important part in the laboratory diagnosis of most fungal infections. Microscopic examination may provide a rapid indication of the cause of an infection, allowing the prompt initiation of appropriate antifungal therapy. An initial macroscopic examination of the specimen is also important. When the specimen is too small to allow full examination, culture usually takes precedence over microscopic examination due to the higher sensitivity and specificity of culture. Pretreatment of clinical specimens is often required in order to concentrate fungal cells or to see the fungal material more clearly.
Pneumocystis jirovecii
can be detected microscopically in a variety of respiratory specimens including induced sputum, bronchoalveolar lavage fluid, tracheal aspirate, and bronchial brush. Both
P. jirovecii
‐positive and
P. jirovecii
‐negative but yeast‐positive material should be used for immunospecific staining.
Clinical Breakpoints and Epidemiological Cutoff Values
Authors: Dufresne Philippe J. , Fuller Jeff
Published: January 2025

Interpreting the value of
in vitro
susceptibility test results relies not only on the quality and standardization of test methods but also on the quality and standardization of the methods used to derive interpretive thresholds. Clinical breakpoints (CBPs) are developed from a collection of
in vitro
and
in vivo
data so that the interpretation of a susceptibility test result correlates with the likelihood of a good patient outcome. Epidemiological cutoff values, which categorize MIC values as wild‐type or non‐wildtype, are developed strictly from
in vitro
MIC data and correlate with the likelihood of a cultured isolate harboring an acquired mechanism of resistance to the tested antimicrobial agent. Clinical and Laboratory Standards Institute CBPs have been developed for the echinocandins, fluconazole, and voriconazole against the more common species of
Candida
as well as for voriconazole against
Aspergillus fumigatus
. EUCAST provides CBPs for yeasts and molds for susceptibility testing performed according to its broth microdilution protocol.
Additional Techniques for Stool Examination
Authors: Shawar Ribhi , Garcia Lynne S. , Johnson Lisa
Published: January 2025

Agar plate cultures are recommended for the recovery of
Strongyloides stercoralis
larvae and tend to be more sensitive than some of the other diagnostic methods.
Strongyloides stercoralis
larvae are usually the only larvae found in stool specimens. Occasionally, it is necessary to examine stool specimens for scolices and proglottids of cestodes and for adult nematodes and trematodes to confirm the diagnosis and/or to identify a species. AimTab may provide clinically useful information about carbohydrate metabolism by semiquantitatively determining the amount of reducing substance in stool. Harada‐Mori Technique employs a filter paper to which fecal material is added and a test tube into which the filter paper is inserted. The qualitative fecal fat test is used to screen for malabsorption (e.g., due to intestinal inflammation) and maldigestion (e.g., due to pancreatic insufficiency) of dietary fat.
Introduction: Traditional Methods for Laboratory Diagnosis of Viral and Chlamydial Infections
Authors: Hodinka Richard L.
Published: January 2025

This part introduction presents an overview of the key concepts discussed in the subsequent chapters. The part focuses on some of the more traditional culture and rapid immunologic methods for the detection and identification of viruses in a diagnostic laboratory. It includes
Chlamydia
culture given that this method is virtually always performed in a clinical virology laboratory or a distinct section of the clinical microbiology laboratory due to the need for a cell culture system and appropriate facilities. Traditional methods continue to have a place in regional and national reference laboratories to help identify and characterize new viruses or variants of well‐recognized viruses not detected by molecular methods, for active surveillance to detect and monitor disease outbreaks, and in support of research activities for antiviral drug and vaccine development.
Immunoassays for Detection of Infectious Diseases
Authors: Theel Elitza S.
Published: January 2025

Serologic testing for the detection of antibodies developed against, or antigens released from, human pathogens continues to play an important diagnostic role for numerous infectious agents, ranging from spirochetes to arboviruses, fungi, parasites, and mycobacteria, among others. Serologic testing, including both contemporary methods, such as chemiluminescence or multiplex flow immunoassays, and classic techniques, such as immunodiffusion and complement fixation assays, remains an integral part of the infectious disease diagnostics portfolio. While commercially available serologic assays are numerous, the clinical utility of many of these methods is rather limited. For example, while many manufacturers offer immunoassays for detection of antibodies against
Helicobacter pylori
,
Chlamydia trachomatis
, and
Candida
spp. and anti‐HSV IgM, among others, given the availability of direct detection methods for these pathogens, with high sensitivity and specificity, serologic testing no longer plays a relevant diagnostic role and should be avoided. This chapter focuses on serologic assays where result interpretation may be challenging.
Quality Assessment and Improvement (Quality Assurance)
Authors: Kehl Sue , Harriott Melphine , Samuel Linoj , Culbreath Karissa , Thompson Cecilia
Published: January 2025

A quality management system is an integrated system of laboratory activities to ensure quality and continued improvement throughout the testing process. Quality assessment employs internal and external monitors or audits to verify the quality of laboratory processes. Continual improvement is the process whereby opportunities are identified, either through assessments or other means, and strategies are developed to improve quality. Proficiency testing or external quality assessment is an important component of a laboratory's external assessments. A central component of nonconforming event management is root cause analysis. This is usually limited to high‐risk, high‐cost, high‐volume events or events associated with adverse patient outcome. Clinical microbiology laboratories can benefit from specific feedback on microbiology services and incorporate any documented quality deficiencies into the quality improvement program. A thorough self‐review of the accreditation requirements and examination of protocols can improve quality by identifying areas of nonconformance and can allow the laboratory to correct deficiencies prior to the external inspection.
Verification and Validation of Test System Performance Specifications
Authors: Martin Rebekah M. , Loeffelholz Michael
Published: January 2025

Clinical laboratory testing in the United States is regulated by the Centers for Medicare and Medicaid Services through the Clinical Laboratory Improvement Amendments (CLIA). CLIA provides quality standards for clinical laboratory testing in order to ensure patient safety and improve patient care. Two critical components of quality systems in the laboratory are verification and validation of test system performance. This procedure will provide guidance for clinical microbiology laboratories in the United States for designing and performing verification and validation procedures. CLIA regulations state that nonwaived test systems which are either Food and Drug Administration cleared or approved must undergo verification of performance specifications by the clinical laboratory prior to reporting patient results with that system. The verification process must assess precision, accuracy, reportable range, and reference intervals of the assay where applicable. The validation process must assess precision, accuracy, reportable range, reference intervals, analytical sensitivity, and analytical specificity of the assay where applicable.
Bacillus anthracis
and
Bacillus cereus
biovar anthracis—Anthrax
Authors: Lee Philip A. , Rowlinson Marie‐Claire
Published: January 2025

Bacillus anthracis
, the agent of anthrax, causes a zoonotic disease that is transmissible to humans, most often through consumption or handling of contaminated products. It is an aerobic, spore‐forming, nonmotile, large Gram‐positive bacterium. Phylogenetically related to
B. anthracis
,
Bacillus cereus
biovar anthracis was initially recognized in the early 2000s in Cameroon and Côte d'Ivoire. While rare in the United States,
B. anthracis
causes sporadic outbreaks in wildlife and livestock. This presents an environmental and occupational exposure risk to ranchers and veterinarians. Laboratory in‐house testing includes Gram staining and culturing from the following: lower respiratory tract specimens, wound specimens, and blood cultures. The chapter covers proper quality control principles for analytical tests. With postanalytical considerations, it provides attention to common test limitations, interpretive challenges, and results reporting.
Toxins: Botulinum Toxin (
Clostridium botulinum
) and Staphylococcal Enterotoxin B (
Staphylococcus aureus
)
Authors: Egan Christina , Perry Michael J.
Published: January 2025

Pathogenic microorganisms can contain genes that allow toxin production. Two toxin‐producing organisms are
Clostridium botulinum
and
Staphylococcus aureus
.
C. botulinum
is a group of anaerobic organisms, commonly found in soil and aquatic habitats throughout the world. Staphylococcal enterotoxin B (SEB) is one of several exotoxins that may be produced by
S. aureus
. Depending on the strain,
S. aureus
can produce a variety of extracellular proteins that act as virulence factors, including toxic shock syndrome toxin 1, exfoliative toxins A and B, and a family of structurally related proteins classified as enterotoxins. Testing for SEB may be performed by clinical laboratories with appropriate protocols and safety mitigation. The chapter covers safety considerations, specimen collection, and specimen processing, incubation and examination of cultures for preanalytical considerations. For analytical considerations, it provides quality control and identification. With postanalytical considerations, the chapter provides attention to summary/special considerations.
Procedures for the Storage of Microorganisms
Authors: She Rosemary C. , Butler‐Wu Susan M.
Published: January 2025

This chapter presents methods that can be used for the storage of bacteria, protozoa, fungi, and viruses. The simplest method for maintaining the short‐term viability of microorganisms, usually used for bacteria, is periodic subculture to fresh medium. Stability may be longer for non‐fastidious organisms such as
Pseudomonas aeruginosa
and storage at –80 degree Celsius may allow for long‐term preservation using liquid Amies. Archiving of patient specimens containing analytes of interest helps clinical laboratories prepare for future lot‐to‐lot comparisons, quality control needs, and new assay verification studies. For valuable or large collections of microorganisms in long‐term storage, the International Society for Biological and Environmental Repositories provides guidance on emergency preparedness. It is good practice to maintain backup copies of written or electronic data linked to the stored organisms. The field of microbiology continues to rapidly evolve in this era of genomics, transcriptomics, and proteomics.
Prevention of Laboratory‐Acquired Infections
Authors: Lagacé‐Wiens Philippe , Pentella Michael
Published: January 2025

Biosafety is protection of the laboratorian from acquiring illness due to pathogens handled in the laboratory. In contrast, biosecurity is protecting the pathogens from acquisition by those who would use them for harmful purposes. The foundational components of a biorisk management program are the performance of risk assessments, selection of mechanisms to mitigate risk, assessment of staff competency, training, an active biosafety committee, performance of safety audits, collaboration with the occupational health program, and continual improvement of the biosafety practices. The risk assessment is a critical tool to prevent laboratory‐acquired infections. Laboratory preparedness is the ability to recognize and respond to occupational, personal, and public health threats and hazards at all times. The blood‐borne pathogens most commonly associated with laboratory infections are hepatitis B virus, hepatitis C virus, and HIV. The investigation and management of laboratory exposures and laboratory infections vary considerably according to the pathogen and type of exposure.
Approaches to the Identification of Aerobic Gram‐Positive Cocci
Authors: Nielsen Xiaohui Chen , Christensen Jens Jørgen
Published: January 2025

The majority of aerobic, or facultatively anaerobic, Gram‐positive cocci isolated from clinical specimens are distributed among the genera
Staphylococcus
,
Streptococcus
, and
Enterococcus
. As new genera and species of aerobic Gram‐positive cocci are described and characterized, it becomes increasingly difficult to identify some of the less frequently isolated organisms solely on the basis of phenotypic traits. Nucleic acid 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. One of the most useful methods for molecular characterization of aerobic Gram‐positive cocci of clinical interest is comparison of 16S rRNA gene sequences, although sequence comparison of other genes may also be helpful for identification.
Enterococcus
Authors: Teixeira Lúcia Martins , da Glória Siqueira Carvalho Maria , Facklam Richard R. , Shewmaker Patricia Lynn
Published: January 2025

The genus
Enterococcus
includes microorganisms that have a historical connection with the genus
Streptococcus
, as they were initially considered a distinct branch within the genus
Streptococcus
, including most of the serological group D streptococci. Transmission of vancomycin‐resistant enterococci represents a major public health concern. By using molecular typing approaches, the exogenous acquisition of enterococcal strains by direct and indirect contact among patients was demonstrated, breaking the traditional conception that enterococcal infections were endogenous. Serologic tests, in the form of Western blot assays, for detecting antibody responses to different enterococcal antigens have been proposed, but their usefulness in the clinical laboratory setting for the diagnosis or prognosis of enterococcal infections has not been demonstrated. The significance of a particular enterococcal isolate is a major factor in determining when antimicrobial testing should be done.
Bacillus
and Other Aerobic Endospore‐Forming Bacteria
Authors: Turenne Christine Y. , Alexander David C.
Published: January 2025

Historically, most aerobic endospore‐forming bacteria were classified as species of
Bacillus
.
Bacillus
is the type genus of the
Bacillaceae
and contains the largest number of species. Aerobic endospore‐forming bacteria are ubiquitous in nature. Cases of anthrax due to laboratory accidents, biological warfare, and bioterrorism events are also rare, but ongoing vigilance in the clinical microbiology laboratory is critical. This chapter describes the clinically significant infections involving
Bacillus
,
Brevibacillus
,
Lysinibacillus
, and
Paenibacillus
species. Members of the
B. cereus
group, other than
B. anthracis
,have been isolated from diverse ecological niches and are widely distributed in nature. Gastrointestinal anthrax is suspected only if an adequate history of the patient is known. Anthrax meningitis may occur on its own or, more frequently, as a complication of another form of the disease.
Escherichia
and
Shigella
Authors: Humphries Romney M. , Faron Matthew L. , Dekker John P. , Ledeboer Nathan A. , Buchan Blake W.
Published: January 2025

Members of the genera
Escherichia
and
Shigella
are most often associated with infections of the gastrointestinal and urinary tracts, but may also cause more severe systemic or invasive infections including pneumonia, sepsis, and meningitis. This chapter provides an overview of each genus and discusses key aspects of taxonomy, epidemiology, and virulence, along with culture‐dependent and culture‐independent methodologies for the identification and classification of species within these genera. It discusses recent trends in antimicrobial resistance patterns and updates to recommended susceptibility testing modalities and interpretive criteria to understand the current best practice for treating active infections. The epidemiology and clinical impact of EIEC are difficult to ascertain because of its close relationship to
Shigella
and the difficulty in distinguishing EIEC from
Shigella
by using biochemical, phenotypic, and molecular methods. Appropriate transport of fecal specimens is essential for effective culture‐based recovery of bacterial pathogens associated with gastroenteritis.
Vibrio
and Related Organisms
Authors: Glover William A. , Boucher Yann F. , Lee Christine C. , Tarr Cheryl L.
Published: January 2025

Phylogenetic investigations indicate that multiple monophyletic clades (separate or distinct groups in a phylogenetic sense) exist within
Vibrio
, which has led to the reclassification of some species into different genera. The genera covered in this chapter are ubiquitous in aquatic environments, primarily marine waters. Pertinent clinical histories (when known) should accompany specimens to alert the laboratory to include appropriate isolation media for the
Vibrionaceae
in their stool workup; this is especially important in areas where the isolation of
Vibrio
is infrequent. Direct microscopic detection of vibrios in stool is not routinely recommended, since it may not be possible to distinguish pathogenic vibrios from other members of the enteric microbiota. Reagents for the serodiagnosis of cholera are available only in specialized reference laboratories, but titration of acute‐ and convalescent‐phase sera in agglutination, vibriocidal, or antitoxin tests is considered reliable.
Bordetella
and Related Genera
Authors: Kilgore Paul E. , Coenye Tom
Published: January 2025

Bordetellae are catalase positive and oxidize amino acids, but no carbohydrates are fermented.
Achromobacter
species are Gram‐negative, nonsporulating, small bacilli.
Alcaligenes
species are Gram‐negative, strictly aerobic rods or coccobacilli that possess oxidase and catalase activity. Cells of
Kerstersia
and
Advenella
species are Gram‐negative, small, and rod‐shaped or coccoid and occur alone, in pairs, or in short chains. Motility is strain dependent. Cells from
Paenalcaligenes
species are Gram‐negative, small rods. Many
Achromobacter
species have been recovered from clinical samples or from the hospital environment. Direct fluorescent‐antibody staining (DFA) on nasopharyngeal swabs or aspirates to detect
B. pertussis
or
B. parapertussis
is rapid and simple but lacks adequate sensitivity and specificity; for this reason, DFA is not recommended.
Francisella*
Authors: Dietrich Elizabeth A. , Petersen Jeannine M.
Published: January 2025

Francisella tularensis
is the causative agent of tularemia, an acute and fatal illness in animals and humans. Within the genus
Francisella
,
F. tularensis
is joined by several lesser‐known
Francisella
species which are largely environmental organisms.
F. novicida
,
F. philomiragia
,
F. hispaniensis
, and
F. opportunistica
cause rare opportunistic infections in immunocompromised individuals. The clinical spectrum of tularemia depends on the mode of transmission, the virulence of the infecting strain, the immune status of the host, and timely diagnosis and treatment. Once an isolate has been identified as a
Francisella
sp., supplemental tests can be used for additional characterization, including typing of species, subspecies, and strain. Agglutination testing, by either the tube agglutination or the microagglutination method, is a standard serological test for determining the presence of antibodies in tularemia.
Anaerobic Non‐Spore‐Forming Gram‐Positive Rods
Authors: Butler‐Wu Susan M. , She Rosemary C.
Published: January 2025

Anaerobic, Gram‐positive, non‐spore‐forming bacilli are widely distributed among the phyla
Actinomycetota
and
Bacillota
. The traditional genera
Actinomyces
,
Actinobaculum
,
Actinotignum
,
Mobiluncus
,and
Varibaculum
all belong to the family
Actinomycetaceae
. The large degree of taxonomic heterogeneity among members of the genus Actinomyces has been recognized for some time. Many Eubacterium species are closely related to clostridia.
Actinotignum schaalii
is the species most frequently implicated in human disease among the genera
Actinotignum
and
Actinobaculum
. The Gram‐variable, curved Gram‐positive rods of the genus
Mobiluncus
can be detected in samples from the female vaginal tract. The cutaneous group of propionibacteria, now reclassified as
Cutibacterium
species, are members of the skin microbiota.
Leptospira
Authors: Galloway Renee L. , Gulvik Christopher A.
Published: January 2025

The genus
Leptospira
comprises spiral‐shaped bacteria with hooked ends. This genus, along with the genera
Leptonema
and
Turneriella
, makes up the family
Leptospiraceae
in the order
Spirochaetales
and class
Spirochaetes
of the phylum
Spirochaetae
. Leptospires are divided into serovars defined by agglutination after cross‐absorption with homologous antigen. Leptospires are ubiquitous, either free‐living in water or wet soil, or associated with renal colonization of mammals. Knowledge of the prevalent serovars and their maintenance hosts is essential for understanding the epidemiology of the disease. The clinical presentation of leptospirosis is biphasic, with a septicemic phase lasting about a week, followed by the immune phase, characterized by antibody production and excretion of leptospires in the urine. Nucleic acid amplification tests, such as PCR, have been widely used for detection of leptospires. Leptospiral DNA has been amplified from blood, plasma, serum, urine, aqueous humor, cerebrospinal fluid, and tissues obtained at autopsy or biopsy.
Mechanisms of Resistance to Antibacterial Agents
Authors: Bradford Patricia A. , Castanheira Mariana
Published: January 2025

Bacterial pathogens have developed resistance to antibacterial agents via multiple routes. The emergence of antimicrobial resistance phenotypes is directly linked to the use of any given anti microbial agent. Resistance to aminoglycosides occurs by several different mechanisms that can coexist in the same bacterial cell. β‐Lactam antibiotics act by inhibiting penicillin‐binding proteins, which are transpeptidases and transglycosylases that synthesize the peptidogly can that composes the bacterial cell wall. The main bacterial resistance mechanism against fluoroquinolonesis is the alteration of their target in the deoxyribonucleic acid synthesis process. Tetracyclines are bacteriostatic antibiotics that inhibit bacterial growth by binding to the ribosome, thereby preventing protein synthesis. Nitrofurantoin is a synthetic antibacterial agent that is only used in the treatment of urinary tract infections. Rifampin is particularly active against Gram‐positive bacteria and mycobacteria. Daptomycin is a cyclic lipopeptide antibiotic with activity exclusively against Gram‐positive bacteria.
Antimicrobial Susceptibility Test Methods: Dilution and Disk Diffusion Methods
Authors: Koeth Laura M. , Miller Linda A.
Published: January 2025

The performance of antimicrobial susceptibility tests (ASTs) to help guide patient therapy is one of the most important functions of a clinical microbiology laboratory. The most commonly used AST methods to date can be categorized into dilution methods that generate minimum inhibitory concentration values and disk diffusion methods that generate a zone diameter result. Most clinical microbiology laboratories in the United States and in many developed countries utilize manual or automated commercial dilution products that provide AST results which are demonstrated to correspond to the International Organization for Standardization/Clinical and Laboratory Standards Institute broth microdilution reference method. This chapter provides descriptions, context, advantages, and disadvantages for each of the methods: broth microdilution, broth macrodilution, agar dilution, and disk diffusion. The convenience afforded by the availability of sterile microdilution trays with round or conical bottoms for use in dilution susceptibility testing led to the widespread use of broth microdilution methods.
A Practical Guide to the Taxonomy, Classification, and Characterization of Clinically Important Viruses
Authors: Drews Steven D.
Published: January 2025

This chapter focuses on the characterization, classification, and taxonomy of viruses that infect humans, both pathogens that infect humans alone and those that infect humans but may also infect other mammalian or nonmammalian genera and species. While other forms of life encode genetic information within double‐stranded DNA (dsDNA), viruses encode their genetic information within genomes that may be composed of single‐stranded RNA, double‐stranded RNA, single‐stranded DNA, and dsDNA. The Baltimore classification, a nonhierarchical approach, categorizes viruses into seven groups and is based on the genome present in virions and type of replication. Classification of viruses below the level of species is not standardized across all species by the International Committee on Taxonomy of Viruses, but in some cases, subspecies standardization does occur. Between 2016 and 2021, a series of changes have occurred in the taxonomy of viruses that infect humans.
Respiratory Syncytial Virus and Human Metapneumovirus
Authors: Berry Gregory J. , Babady N. Esther
Published: January 2025

Human respiratory syncytial virus (RSV) is a member of the genus
Orthopneumovirus
, family
Pneumoviridae
, order
Mononegavirales
. Most general viral specimen collection principles also apply to specimen collection for RSV detection and culture. Although RSV can be visualized by electron microscopy, its application has been limited to research laboratories. However, characteristic syncytial cytopathic effects in lung biopsies can facilitate the diagnosis. Human metapneumovirus (HMPV) virus belongs to the order
Mononegavirales
, the family
Pneumoviridae
, and the genus
Metapneumovirus
, which also includes avian metapneumovirus, also known as turkey rhinotracheitis virus, a cause of respiratory tract infections in turkeys, ducks, and chickens. HMPV is the only human pathogen within the genus
Metapneumovirus
.
Orthopneumovirus
, the other genus in the family
Pneumoviridae
, includes the closely related human RSV.
Gastroenteritis Viruses
Authors: Hindiyeh Musa , Das Shubhagata , Dunbar Sherry
Published: January 2025

Acute viral gastroenteritis (AVG) is a common self‐limiting illness affecting infants, children, and adults worldwide. In developed countries, AVG is associated with significant morbidity in children and the elderly, while in developing countries these infections have been associated with high morbidity and mortality, especially in infants. The clinical manifestation of AVG is due to the effects that the virus and its specific cytotoxins exert on the enterocytes of the intestine. AVG can be triggered by many viruses, most often rotavirus, caliciviruses, enteric adenoviruses 40/41, and astroviruses. The luciferase immunoprecipitation system assay, a liquid‐phase immunoassay that quantifies antigen‐specific serum antibodies, has also been used for the quantitation of
Norovirus
antibodies. Antibody‐neutralization assays have been used for evaluating antibodies induced post RV vaccination. The major advantage of molecular diagnostic methods is the increased sensitivity and specificity for common viruses, such as rotaviruses and adenoviruses, and the ability to detect uncultivable viruses such as noroviruses, astroviruses, and sapoviruses.
Rabies Lyssavirus
Authors: Orciari Lillian A. , Yager Pamela A. , Satheshkumar P. S.
Published: January 2025

This chapter provides information on taxonomy, description of the agent, epidemiology and transmission, clinical significance, collection, transport, and storage of specimens, direct detection methods, isolation procedures, typing systems, serologic tests, and antiviral susceptibilities of Rabies Lyssavirus infection. There are numerous etiologic agents in the genus
Lyssavirus
that result in an acute, fatal viral encephalomyelitis known as rabies in both humans and animals. Rabies is a zoonotic disease. All mammals are susceptible. The main route of transmission is introduction of saliva containing RABV into a wound via the bite of a rabid animal. Direct methods may be used to detect histopathological changes and viral antigen (inclusions) and to observe virion morphology. These methods include: microscopy, antigen detection, and nucleic acid detection. Identification of a
Lyssavirus
infection is made typically by direct examination of brain impressions and demonstration of specific viral inclusions by DFA testing.
Arboviruses
Authors: Hunsperger Elizabeth A. , Hughes Holly R. , Ritter Jana M. , Brault Aaron C.
Published: January 2025

Arthropod‐borne viruses are transmitted to vertebrate hosts through the bites of infected arthropods and cause over 80 diseases in humans. The medically important arboviruses are found primarily within the families
Togaviridae
,
Flaviviridae
,
Peribunyaviridae
,
Phenuiviridae
, and
Nairoviridae
. Arboviruses are RNA viruses of either positive or negative polarity. Arbovirus replication takes place in the cytoplasm of susceptible cells following viral entry. Many mosquito‐borne arboviruses are most commonly transmitted in tropical and subtropical regions of the world and are highly dependent on the presence of the vector and an amplifying host. Virus isolation serves as a confirmatory test for reverse transcription‐PCR‐positive specimens, helps to further characterize circulating strains for molecular epidemiology studies, and serves to identify novel or emerging arboviral agents. The hemagglutination inhibition assay is a classic test used for arbovirus diagnostics and was the basis for virus family classification prior to genome sequencing.
Human Papillomaviruses
Authors: Cuschieri Kate , Unger Elizabeth R.
Published: January 2025

Papillomaviruses are in the family
Papillomaviridae
. Human papillomavirus (HPV) initiates infection in primitive basal‐like epithelial cells, accessed through minor trauma or at specialized sites of epithelial transition, particularly the squamocolumnar junction. The epidemiology and transmission of genital and nongenital infections differ significantly. Nongenital infections manifesting as hand and foot warts are most common in children and adolescents. The epidemiology and natural history of genital or mucosal HPV differ by anatomic site, by sex, and by age. Warts are more common in immunocompromised individuals, including organ transplant recipients, those infected with HIV, and pregnant women. Recurrent respiratory papillomatosis is a rare condition characterized by benign papillomatous lesions of the larynx and other parts of the upper respiratory tract that are characteristically resistant to treatment. Nucleic acid amplification technologies are the mainstay of clinical HPV tests, particularly those designed to inform screening and management of cervical disease.
Parvovirus B19V, Bocaviruses, and Other Human Parvoviruses
Authors: Söderlund‐Venermo Maria
Published: January 2025

Parvoviruses are small, nonenveloped icosahedral viruses with a linear single‐stranded‐DNA (ssDNA) genome in the family
Parvoviridae
in the realm
Monodnaviria
. The family
Parvoviridae
is divided into three subfamilies,
Parvovirinae
,
Densovirinae
, and the new
Hamaparvovirinae
, on the basis of their ability to infect vertebrates, invertebrates, or both, respectively, and their NS1 amino acid sequences. The first human parvoviruses identified were the nonpathogenic adeno‐associated viruses of the genus
Dependoparvovirus
, which are increasingly used in gene therapy. Human bocavirus (HBoV1) has the typical structure of a member of the
Parvoviridae
. The most common test for diagnosis of HBoV1 infection is DNA detection by qualitative PCR. Parvoviruses are unable to induce cells to divide, so they require actively dividing cells for their replication.
Antiviral Agents
Authors: Santos Carlos A.Q. , Reyna Quito Manuela Jannet
Published: January 2025

There are five classes of antiviral agents for the treatment of human immunodeficiency virus type 1 (HIV‐1): nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs/NtRTIs), nonucleoside reverse transcriptase inhibitors, protease inhibitors (PIs), entry/fusion inhibitors, and integrase strand transfer inhibitors. PIs are commonly used in combined antiretroviral therapy regimens in combination with NRTI and/or NtRTIs for maximum antiretroviral activity and to minimize the development of resistance. Integrase Strand Transfer Inhibitors a class of antiretroviral drugs, target the HIV‐1 integrase enzyme that mediates transfer of the reverse‐transcribed HIV‐1 DNA into the host chromosome. The common target for antiviral drugs active against both viruses is the reverse transcriptase function of the HIV and hepatitis B virus (HBV) replication enzymes. Chronic HBV infection plays an important role in the morbidity and mortality of HIV‐infected patients. Most of the antiviral compounds that are approved to treat the eight human herpesviruses are nucleoside or nucleotide analogues, which inhibit DNA replication.
Leishmania
and
Trypanosoma
Authors: da Silva Alexandre J. , Ndao Momar
Published: January 2025

Leishmania
spp. and
Trypanosoma
spp. belong to the Trypanosomatida in the eukaryote supergroup Excavata. Although microcopy techniques cannot be used to identify
Leishmania
spp. to the species level, the microscopic detection of
Leishmania
amastigotes in clinical specimens continues to be invaluable. Serologic tests including indirect fluorescent‐antibody testing, enzyme‐linked immunoassays, and immunoblot tests may give mixed results when applied to the diagnosis of leishmaniasis. The likelihood of a positive skin test increases with parasite burden and the time the patient has been infected with
Leishmania
parasites. Although vectorial transmission is considered the most important mode of transmission, leishmaniasis can also be transmitted by direct contact with an infected lesion or mechanically through bites by stable flies/dog flies. Molecular techniques for the detection of leishmanial nucleic acid have been used for diagnosis, predicting prognosis, and species identification.
Intestinal and Urogenital Amebae, Flagellates, and Ciliates
Authors: Mathison Blaine A. , Couturier Marc R.
Published: January 2025

Entamoeba histolytica
and
Giardia duodenalis
infections are two of the most common protozoal infections seen worldwide and are of serious concern on a global scale due to their prevalence and the pathogenicity of these causative agents. Microscopic examination of stool specimens continues to be one of the main tools used in the laboratory diagnosis of intestinal amebic, flagellate, and ciliate infections. Antigen detection methods such as enzyme immunoassays, immunochromatographic assays, and direct fluorescent‐antibody assays are available for the detection of pathogens such as the
E. histolytica
/
Entamoeba dispar
group,
E. histolytica
,
Cryptosporidium
spp. and
G. duodenalis
. One of the most important developments related to the molecular detection of parasites is the advent of panels for detection of multiple agents of gastrointestinal disease, including combinations of bacterial, viral, and parasitic agents.
Cystoisospora
,
Cyclospora
, and
Sarcocystis
Authors: Qvarnstrom Yvonne , Arrowood Michael J.
Published: January 2025

The parasites traditionally referred to as coccidia that develop in the intestines of humans—
Cystoisospora belli
,
Cyclospora cayetanensis
, and
Sarcocystis
spp.—are in the Stramenopiles, Alveolates, Rhizaria supergroup in the current taxonomic scheme used by protozoologists. Oocysts of these coccidia are found in the feces of humans, and diagnosis is based ultimately on demonstrating oocysts in human stool samples. The use of molecular methods to evaluate the phylogenetics and genetic diversity of parasites has resulted in a revision of the coccidian parasites.
In vitro
culture of intestinal coccidian parasites is most often used as a tool to study developmental biology or to identify active chemotherapeutic agents. Differentiation of the intestinal coccidia to the subspecies/strain level—genotyping—can be important for epidemiologic reasons, for example to link cases in outbreak investigations or to better understand clinical outcome differences.
Nematodes
Authors: Sheorey Harsha , Biggs Beverley‐Ann , Bradbury Richard S.
Published: January 2025

Nematodes are unsegmented cylindrical worms with a body cavity containing an alimentary canal and genital system; the sexes are separate. They have longitudinal muscles, which allow them to move by flexing their bodies. Molecular diagnosis for intestinal nematodes is increasingly being introduced in diagnostic laboratories. There are a variety of morphological features and DNA sequences which form the basis for taxonomy of nematodes. The primary diagnosis of infection is by demonstration of
A. lumbricoides
eggs in feces. Albendazole is the first choice for treating
A. lumbricoides
, and mebendazole is also effective. Any helminth eggs or larvae found in feces are significant, and treatment is recommended even if the patient is asymptomatic. There is no need to quantitate helminth parasites in a diagnostic context, as this does not necessarily correlate with clinical illness.
Filarial Nematodes
Authors: Showler Adrienne J. , Nutman Thomas B.
Published: January 2025

Filarial worms are arthropod‐transmitted nematodes that dwell, as adults, in the subcutaneous tissues and the lymphatics. Although eight filarial species infect humans, four are responsible for most of the pathology associated with these infections:
Wuchereria bancrofti
,
Brugia malayi
,
Onchocerca volvulus
, and
Loa loa
. The taxonomy of these parasites follows a newly adopted molecular approach. All filariae go through a complex life cycle that includes an infective larval stage carried by the insects and an adult worm stage that resides in humans, either in the lymph nodes, the adjacent lymphatics, the subcutaneous tissue, or the pleural/peritoneal cavities. There are significant differences in the clinical manifestations of filarial infections, or at least in the period over which the manifestations present, between patients native to the areas of endemicity and those who are travelers or recent arrivals in these same areas.
Trematodes
Authors: Jones Malcolm K. , A. Gordon Catherine , Mcmanus Donald P.
Published: January 2025

This chapter focuses on members of the clade Digenea of the phylum Platyhelminthes. At least 70 species of digenean trematodes have been recorded as adult parasites in humans. These species are endoparasitic, occupying a variety of tissue sites. Members of the genus
Schistosoma
are blood flukes responsible for the important human disease schistosomiasis. The second strategy, evident in the remainder of the human‐infecting species, is foodborne transmission, and parasites that use this strategy can be termed collectively the foodborne digeneans. For these species, the human‐infective stage, a metacercaria, is ingested with food. The impact of these foodborne trematodes on human health is large. The primary epidemiological features governing human infection include the distribution of suitable snail intermediate hosts, human food consumption behaviors, the presence of suitable zoonotic hosts, and the potential for water contamination with human or animal excreta.