bluuebunny/crossref_metadata_2025 huggingface dataset:
Supplementing Blood Cultures with Chromogenic Media to Expedite Microorganism Identification and/or Antimicrobial Resistance Detection
Authors: Faron Matthew L.
Published: January 2025

Chromogenic agar is medium containing specific combinations of antibiotics and/or other inhibitory substances and chromogen substrates. This allows selective growth of specific organisms and differentiation of various species through colony color and morphology. Chromogenic medium has also been developed for the identification of other types of microorganisms, such as
Candida
species. Chromogenic agar is commonly used for screening polymicrobial specimens to identify resistant organisms, such as carbapenem‐resistant
Enterobacterales
or vancomycin‐resistant
Enterococcus
from rectal swabs or surveillance cultures from the anterior nares to identify methicillin‐resistant
Staphylococcus aureus
. After a Gram stain is performed, chromogenic agar appropriate for the Gram stain morphology can be inoculated to reduce the time to identification of bacterial species and/or key resistance profiles. Differentiation of
Candida
spp. can be improved using chromogenic agar, which may be helpful for laboratories that use rapid molecular methods, as some smaller panels may lack species‐specific identification or identify only a few species.
Diagnosis of Catheter‐Related Bloodstream Infection: Differential‐Time‐To‐Positivity Cultures and Catheter Tip Cultures
Authors: Miller Steve
Published: January 2025

This chapter lists the most common types of vascular and hemodialysis access catheters used for patient care that the laboratory may encounter. Clinical policy should instruct physicians to submit peripheral blood cultures and either paired cultures from the catheter lumen or removed catheter segments to the laboratory for diagnosis of suspected catheter‐related bloodstream infection. Several methods have been used to diagnose catheter‐related sepsis. The submission of differential‐time‐to‐positivity cultures, consisting of two sets of blood cultures, is the preferred method to diagnose catheter‐related bacteremia and does not require removal of the catheter. For diagnosis after the catheter has been removed, semiquantitative cultures are more reliable than qualitative catheter tip cultures. The chapter focuses on the specimen collection, transport, and handling, materials, quality control, interpretation, reporting results and limitations of procedure.
Multiplex Molecular Panels for Positive Blood Cultures
Authors: She Rosemary C. , Van Tam T.
Published: January 2025

This chapter provides microbial analysis of multiplex molecular panels with procedures for the detection, identification, and characterization of microorganisms involved in human infections. This procedure summarizes the major FDA‐cleared methods available for rapid identification of common bloodstream pathogens and antimicrobial resistance markers from positive blood culture bottles. The chapter discusses general considerations for implementation and application of such panels in the clinical laboratory. It covers proper quality control principles for analytical tests. With postanalytical considerations, the chapter provides attention to common test limitations, interpretive challenges, and results reporting. It emphasizes the importance of correlating molecular results with Gram stain and culture to help avoid pitfalls and aid in troubleshooting. The chapter also provides guidance on basic interpretations of molecular panels that include antimicrobial resistance markers. It develops collaboration among laboratory, patient providers, and antimicrobial stewardship stakeholders in order to maximize the clinical benefits of these molecular panels.
Introduction to Antimicrobial Susceptibility Testing Methods
Authors: Humphries Romney M. , Simner Patricia J.
Published: January 2025

Antimicrobial susceptibility testing (AST) is arguably one of the most important roles that clinical microbiology laboratories play, especially in the era of increasing antimicrobial resistance. Several conventional and novel methods are available to perform AST. Traditional quantitative AST methods, such as broth dilution, gradient diffusion, and agar dilution, and automated broth microdilution instruments generate a MIC. Disk diffusion is a method reported qualitatively that generates a zone diameter that is inversely related to the MIC. This chapter describes the different types of phenotypic antimicrobial susceptibility tests, with reference to the methods in CMPH. Breakpoints are based on robust clinical, pharmacokinetic/pharmacodynamic, and microbiological data in order to provide a reasonable prediction of clinical outcome for an infection treated with a given antimicrobial agent.
Extended‐Spectrum Beta‐Lactamase Testing for
Enterobacterales
Authors: Pitout Johan J.
Published: January 2025

The extended‐spectrum β‐lactamases (ESBLs) are enzymes that cause resistance to the oxyimino‐cephalosporins and the monobactams but not the cephamycins or the carbapenems. The detection of ESBLs involves a screening step followed by confirmation tests. ESBLs are inhibited by “classical“ β‐lactamase inhibitors such as clavulanic acid, sulbactam, and tazobactam. Laboratory tests for confirming ESBL‐producing bacteria are based on this property. The phenotypic ESBL confirmatory test can be performed by disk diffusion or by the broth microdilution MIC method. Commercially available ESBL gradient diffusion strips may also be used for ESBL confirmatory testing. 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.
Tests to Assess Bactericidal Activity
Authors: Koeth Laura M.
Published: January 2025

The minimum bactericidal concentration test can be used to assess the ability of an antimicrobial agent to kill a bacterial isolate. Time‐kill assays can be used to study both concentration‐dependent and time‐dependent bactericidal activities of antimicrobial agents. Prior to performance of the time‐kill assay, concentrations of individual agents and combinations most likely to result in synergism are planned from data resulting from a checkerboard synergism test. The time‐kill assay can be used to study the dynamics of synergism or antagonism for a combination of antimicrobial agents by determining the number of viable bacteria remaining over time after exposure to each individual agent and various combinations. This method is used primarily to study organisms from patients with meningitis or endocarditis for whom clinical treatment failed or as a research tool to study novel antimicrobial agents.
Definitive Identification of Aerobic Actinomycetes
Authors: Bell Melissa E. , Mochon A. Brian , Highlander Sarah K.
Published: January 2025

Using technologies such as matrix‐assisted laser desorption ionization–time of flight mass spectrometry (MALDI‐TOF MS) and genetic sequencing, clinical microbiology laboratories can provide definitive identification of most clinically relevant aerobic actinomycetes. Both FDA‐approved MALDI‐TOF MS systems, the VITEK MS and Bruker Biotyper, can identify common
Nocardia
species. Accurate identification of most aerobic actinomycetes to the species level requires the use of molecular methods, specifically, gene sequencing and analysis. 16S rRNA gene sequence analysis can provide genus‐level and often species‐level identification of clinically relevant aerobic actinomycetes. The chapter provides a schematic flowchart for the definitive identification of medically important aerobic actinomycetes. It covers proper quality control principles for analytical tests. With postanalytical considerations, the chapter provides attention to common test limitations, interpretive challenges, and results reporting.
Introduction
Authors: Wengenack Nancy L.
Published: January 2025

This part introduction presents an overview of the key concepts discussed in the subsequent chapters. The part presents the standard operating procedures for technologies that currently constitute standard of care for mycobacterial diseases. Mycobacteria are important human pathogens. Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains a major global public health problem. The gold standard for diagnosing mycobacterial infections involves routine culture methods using both solid and liquid media and acid‐fast smear microscopy. Traditional liquid and agar culture technologies to detect growth in the presence of antimycobacterial drugs remain the standard of practice for susceptibility testing. Next‐generation sequencing molecular technologies are in the pipeline for the early detection of TB and drug‐resistant strains and promise enhanced sensitivity and expanded target product profiles but are not yet routinely available to most diagnostic laboratories.
Susceptibility Testing by TREK Sensititre Microdilution Plates
Authors: Escuyer Vincent E. , Salfinger Max , Elliott Barbara Brown , Parrish Nikki , Armstrong Derek
Published: January 2025

This chapter provides the information on antimicrobial susceptibility testing for rapidly growing nontuberculous mycobacteria, slowly growing nontuberculous mycobacteria, and the
Mycobacterium tuberculosis
complex using broth microdilution. Antimicrobial susceptibility testing is performed on rapidly growing mycobacteria isolated from clinical specimens by using a broth microdilution method with the Thermo Fisher Scientific Sensititre 96‐well plate for rapidly growing mycobacteria. The intended use of the Thermo Fisher Scientific Sensititre SLOWMYCO2 broth microdilution plate is the determination of MICs of antimicrobial agents against slowly growing nontuberculous mycobacteria. This test determines MIC breakpoints via broth microdilution of the NTM in Mueller Hinton broth containing specific concentrations of recommended antimicrobial agents. The MICs of antibiotics used to treat
Mycobacterium tuberculosis
complex infections can be determined by the broth microdilution method. 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.
Specimen Selection, Collection, and Transport
Authors: Abdolrasouli Alireza , Howell Susan
Published: January 2025

Suitable specimen selection, proper specimen collection, and timely specimen transport must be performed to ensure the successful detection and recovery of the etiologic agent of a fungal infection. To establish or confirm the diagnosis of a suspected fungal infection, it is essential for the clinician to provide the laboratory with adequate and relevant specimens for examination. Mycological investigation of clinical specimens usually includes direct microscopic examination, culture, detection of fungal antigens, study of the host's antibody response to a specific fungal pathogen, and molecular assays. Collection and transport of clinical specimens for fungal investigation are similar to those for bacteria; however, a larger volume of sample is usually preferred. Environmental contamination of clinical specimens can occur during collection, transport, and processing in the laboratory. Delay in processing samples may result in an overgrowth of bacterial contaminants and impede recovery of fungal pathogens.
Macroscopic and Microscopic Examination of Fecal Specimens
Authors: Chan Wilson W.
Published: January 2025

In the diagnosis of parasitic infections, the age of the fecal specimen is key. The physical characteristics of a fresh fecal specimen may aid in determining the possible organisms present. Fecal specimens may be described as formed, semiformed, soft, loose, or watery. When the fresh specimen is examined visually in the collection container, macroscopic helminth forms may be seen. The microscopic examination of a direct smear has several purposes: to assess the worm burden of a patient, to provide a quick diagnosis of a heavily infected specimen, to check organism motility, and to observe parasites that may be lost in concentration techniques. Fecal concentration has become a routine procedure as part of the complete ova and parasite examination, particularly for human clinical specimens. The zinc flotation procedure separates protozoan cysts and most helminth eggs from excess debris through the use of a liquid with a high specific gravity.
Special Stains for Coccidia and Microsporidia
Authors: Mathison Blaine A. , Garcia Lynne S. , Leal Sixto M.
Published: January 2025

Cryptosporidium
spp.,
Cyclospora cayetanensis
,
Cystoisospora belli
,
Sarcocystis hominis
, and
Sarcocystis suihominis
have been recognized as causes of severe diarrhea in immunocompetent hosts but can also cause severe diarrhea in immunocompromised hosts.
Cryptosporidium
spp.,
C. cayetanensis
, and
C. belli
can also cause ectopic infections in immunocompromised hosts. Oocysts in clinical specimens may be difficult to detect without special staining. Modified acid‐fast stains are recommended for demonstrating these organisms, although oocysts of
Sarcocystis
species will not retain the stain. Modified acid‐fast stains stain the
Cyclospora
oocysts from light pink to deep red, and some of the oocysts will contain granules or have a bubbly appearance, often being described as looking like “wrinkled cellophane.”
Sputum, Aspirates, and Biopsy Material
Authors: Leal Sixto M.
Published: January 2025

A direct smear can be used to detect large or motile organisms from the lung. Although
cryptosporidium
spp. are difficult to see in a direct mount, examination of smears stained with modified acid‐fast stains may provide confirmation of pulmonary cryptosporidiosis. Expectorated sputum specimens are collected after patient instruction in the appropriate measures to take to ensure quality specimens, including mouth wash before expectorating and exclusion of saliva from specimens. If parasite involvement is low on the differential diagnosis, the majority of aspirates and bronchoscopy specimens will be submitted only for standard cytology and microbial culture. Biopsy specimens are sometimes recommended for the diagnosis of tissue parasites. This chapter presents a list of stains for identifying parasites in various tissues. Biopsy specimens are invasive and costly and characterized by a small volume that may not accurately represent the entire infectious lesion.
Culture
Authors: Ali Ibne Karim M. , Visvesvara Govinda , Secor W. Evan , Lawrence Justin J. , Sapp Sarah G.H.
Published: January 2025

Acanthamoeba
,
Naegleria
, and
Balamuthia
are free‐living soil and freshwater amebae. Members of the genera
Acanthamoeba
and
Naegleria
can be easily cultivated in the laboratory either monoxenically or axenically. In contrast,
Balamuthia mandrillaris
cannot be cultivated with bacterial species, although it can be cultured with an associated mammalian cell such as human lung fibroblasts or monkey kidney cells. This chapter focuses on the culture growth of
Naegleria
and
Acanthamoeba
species in clinical specimens only. Cultivation is a more sensitive method than direct wet mounts for the diagnosis of trichomoniasis, but it is less sensitive than tests that detect antigen or nucleic acid.
Trypanosoma cruzi
, which causes Chagas disease, is a major public health problem in Latin America. An important consideration for choosing culture conditions for cultivating
Leishmania
spp. and
Trypanosoma cruzi
is that different media have different effects upon the phenotype, infectivity, and overall culture longevity of the desired organism.
Quality Control and Quality Assurance in Clinical Virology
Authors: Mostafa Heba H.
Published: January 2025

An efficient clinical virology laboratory aims at delivering accurate results in a timely manner. Appropriate procedures must be in place to ensure that this mission is achieved efficiently and with highest quality. Each step in this process is governed systematically by a multifaceted quality management system (QMS) of policies, processes, and procedures used by the laboratory to meet the accreditation and certification requirements and to maintain a high‐quality operation. The scope of QMS in the clinical virology laboratory extends in focus beyond quality control (QC) and quality assurance (QA) as it includes all aspects of laboratory management and the technical operations to ensure sustained monitoring of quality and patient safety. This procedure provides information on the different aspects of QA and QC measures in the clinical virology laboratory as a part of the overall QMS.
Preparation of Cell Culture Media and Supplemental Components
Authors: Hodinka Richard L.
Published: January 2025

At present, most diagnostic virology laboratories that continue to perform some level of cell culture for virus isolation purchase media and supplemental components from commercial sources rather than preparing these materials from basic constituent ingredients. Commercial products of high quality and consistency come premixed and prepackaged and require minimal manipulations by the laboratory to complete the medium before it is ready to use. Many types of synthetic (artificial) media have been developed, modified, and enriched to support the growth of certain cell types, resulting in the availability of a variety of medium formulations for a range of cell culture applications. The basic components of cell culture medium include vitamins, amino acids, carbohydrates, proteins and peptides, fatty acids and lipids, and basic and trace elements as a supply of nutrients needed for cells to survive and grow in culture.
Hemagglutination Inhibition Assay for Quantitative Measurement of Antibody Responses to Influenza Virus
Authors: Young Stephen , Pinsky Benjamin A.
Published: January 2025

Hemagglutination inhibition (HAI) is a traditional laboratory method which continues to be used in public health and research laboratories and by vaccine manufacturers as a primary method for global influenza seroepidemiologic surveillance to determine the distribution and prevalence of influenza in given populations, antigenically characterize influenza viruses that may be selected for use in seasonal influenza vaccines, compare antigenic changes among current and previously circulating influenza viruses, and monitor the effectiveness of annual vaccines and newly developed vaccine candidates to induce an antibody response. In the past, when performing the HAI test, specialized equipment was used in the titration of either virus or antibody. The HAI test is accomplished by making serial 2‐fold dilutions of a patient's serum, mixing and incubating each dilution with a fixed amount of either virus particles or isolated viral hemagglutinin, and then adding a suspension of red blood cells.
Cytomegalovirus Serology
Authors: Prince Harry E.
Published: January 2025

Human cytomegalovirus (CMV), also known as human herpesvirus 5, is a member of the family
Herpesviridae
. CMV infections in healthy children and adults are primarily asymptomatic, although in young healthy adults, they can mimic Epstein‐Barr virus‐mediated infectious mononucleosis syndrome. CMV is shed in a variety of body fluids, including tears, saliva, breast milk, urine, blood, vaginal secretions, and semen; hence, it can be transmitted by intimate personal contact and is more prevalent in conditions of crowding. One of the most serious consequences of CMV infection is congenital infection in infants due to transplacental viral transmission during pregnancy. The laboratory diagnosis of CMV infection can be established by histologic or cytologic detection of classic CMV tissue pathology, isolation of the virus in cell culture, direct detection of viral antigen or nucleic acids, and/or serologic assays. Serologic assays are routinely used for screening purposes and are also valuable adjuncts for CMV diagnosis.
Human Immunodeficiency Virus Serology
Authors: Kaleta Erin J.
Published: January 2025

Human immunodeficiency virus (HIV) is a positive‐sense, enveloped retrovirus in the lentivirus subgroup. Two distinct HIV viruses exist, HIV‐1 and HIV‐2, and among these two groups, there is a great deal of genetic diversity that distinguishes subgroups and clades of HIV viruses. HIV‐1 can be broken down into the subgroup M, the divergent group O, and the more recent groups N and P. Subgroup M accounts for most HIV infections and includes clades A, B, C, D, F, G, H, J, and K. HIV‐2 can be broken down into groups A to H, although A and B are most common. The structure of HIV includes viral RNA contained within the viral capsid, which is composed of core structural proteins; the capsid is surrounded by a lipid bilayer containing protruding surface glycoproteins that are responsible for host cell recognition and binding.
Serologic Diagnosis of Arbovirus Infections
Authors: Collins Matthew H. , Pelc Rebecca S.
Published: January 2025

Arthropod‐borne viruses (ARBV), transmitted by ticks or mosquitoes, are a subset of vectorborne diseases, which in recent years have been on the rise in the United States. New pathogenic ARBV continue to emerge, being identified either in human cases or through vector surveillance over the last few decades. Many ARBV infections are clinically inapparent, but all can cause a nonspecific fever and/or rash illness. Many of the ARBV endemic in the United States are also capable of causing neurologic manifestations, such as meningitis and encephalitis. The possible causes of acute neurologic illness are numerous, some of which are reversible with targeted treatment. The presymptomatic incubation period is typically less than 15 days for most ARBV. The timing and magnitude of antibody responses can be altered by prior infection with the same or similar virus.
Serologic Diagnosis of Chagas Disease
Authors: Whitman Jeffrey D. , Bern Caryn
Published: January 2025

Chagas disease is caused by the protozoan parasite
Trypanosoma cruzi
. The primary route of infection is via host inoculation with feces from infected insect vectors. The infection has acute and chronic phases, which broadly represent active systemic replication and long‐term intracellular parasite persistence. This chapter focuses on serologic testing for chronic Chagas disease, as the acute phase is relatively brief and seldom suspected clinically. There are currently four tests cleared by the U.S. Food and Drug Administration for diagnosis of chronic
T. cruzi
infection. Three of these assays (Hemagen, Wiener, and Ortho) are 96‐well microplate ELISAs, and one is an operator‐interpreted immunochromatographic lateral flow assay. 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.
Serologic Testing for Toxoplasmosis
Authors: Pomares Christelle , Olson Kjerstie , Montoya José G.
Published: January 2025

Toxoplasma gondii is a protozoan intracellular parasite and the etiologic agent of toxoplasmosis. Toxoplasmosis exhibits a wide clinical spectrum, which ranges from an asymptomatic infection state to life‐threatening clinical manifestations. Initial
T. gondii
serologic testing can be performed in local or regional laboratories. Serum is the preferred material for testing. Negative and positive quality controls should be tested before patient samples. The dye test is a functional assay and regarded as the gold standard for the detection of
Toxoplasma
Immunoglobulin G antibodies. The Remington double‐sandwich Immunoglobulin M (IgM) enzyme‐linked immunosorbent assay (ELISA) is more sensitive than the IgM immunofluorescence test in diagnosing recently acquired infections and does not show false‐positive results due to antinuclear antibodies or rheumatoid factor. The IgM immunosorbent agglutination assay is more sensitive than the IgM ELISA and has been used effectively for diagnosis of congenital infection.
Molecular Methods for Epidemiological Typing of Microorganisms
Authors: Fisher Mark A. , Shakir Salika M. , Lopansri Bert K.
Published: January 2025

The increased availability of molecular methods for comparative analysis of genetic changes across microbial isolates has greatly expanded the ability of the clinical microbiologist to aid in the prevention and control of outbreaks. Pulsed‐field gel electrophoresis strain typing relies on changes in restriction sites, which can arise by single‐nucleotide changes or by large genomic rearrangements. Strain typing in the context of epidemiological investigation is based on several assumptions: that bacterial genomes are not static and that they undergo mutation at a given rate within a strain; that an outbreak is caused by a single strain, and subsequent isolates from different sources will have the same genotype; and that isolates not derived from the outbreak strain will have different genotypes. Ribotyping is a molecular method that was originally developed in 1986 by Grimont and Grimont for genotyping bacteria and adapted for use with PCR by multiple groups in the early 1990s.
Surveillance Cultures for Hospital‐Associated Infections
Authors: Gumbis Stephanie , Aubert Rachel D. , Sievert Dawn , Lainhart William D. , Pitout Johann D. , Bibbi Helen , Bourassa‐Blancette Sam , Lockhart Shawn R. , Green Daniel A. , Imlay Hannah , Hanson Kimberly E.
Published: January 2025

Antibiotic and antifungal resistance (AR)—the ability of bacteria and fungi to defeat the drugs designed to kill them—is one of the greatest global public health challenges. Antibiotics and antifungals are among our most powerful tools for fighting life‐threatening infections. The threat of AR undermines progress in health care, food production, and life expectancy. The Centers for Disease Control and Prevention (CDC) is a leader in the fight against this global threat, driving aggressive action along with partners and facilitating collaborative responses to resistance through a One Health approach. State and local health care‐associated infection (HAI)/AR programs are essential to U.S. efforts to detect, prevent, respond to, and contain HAI and AR pathogens. In 2019, CDC evaluated the sensitivity and specificity of many carbapenem‐resistant
Enterobacterales
and
Pseudomonas aeruginosa
antimicrobial susceptibility testing phenotypes to determine which were most predictive of the presence of carbapenemase genes.
Infection Control in the Laboratory
Authors: Pentella Michael A. , Luethy Paul M. , Church Deirdre L.
Published: January 2025

The immunization of laboratory staff is an important component of the laboratory biosafety plan and occupational health services. Immunization is the primary means of preventing the acquisition of disease in workers who have the potential for exposure to various microbial agents in the laboratory workplace. Personal health status must be considered by the individual in electing to be vaccinated or deferring on vaccination. This chapter describes immunization practices and procedures for specific functions in the clinical and public health laboratory setting. Employees who decline a recommended vaccination may be required to complete a vaccination declination form for retention in their occupational health record. Risk for occupational HIV infection is modified not just by the general type of exposure but also by several specific factors, including the depth of the exposure, the exposure inoculum, and the exposed worker's immunological response.
Risk Assessment
Authors: Buchan Blake W.
Published: January 2025

Working in a clinical microbiology laboratory presents a number of potential hazards in the form of exposure to infectious biological agents, dangerous chemical reagents, and both manual and automated instrumentation capable of causing bodily harm. The collection of safety policies is referred to as a biosafety manual and should be unique to each laboratory, considering the personnel, facilities, test procedures, and biological and other risks inherent to that laboratory. The development of individualized and effective safety policies requires a multistep process formally known as a risk assessment. There are several laboratory‐focused resources that provide recommendations for the design of a risk assessment, as well as important considerations for risk mitigation strategies related to common biological, chemical, and mechanical hazards. These include publications by the U.S. Centers for Disease Control and Prevention, NIH, and the American Society for Microbiology. The primary goal of hazard characterization is to determine if the relative risk is acceptable or unacceptable.
Yersinia pestis
Authors: Sharp Susan
Published: January 2025

Yersinia pestis
is a nonmotile, slow‐growing, facultative organism classified in the order
Enterobacterales
. It appears as plump, Gram‐negative bacilli or coccobacilli that are seen mostly as single cells or pairs, which may exhibit bipolar staining from a direct specimen if stained with Wright stains.
Y. pestis
, the causative agent of plague, has a protracted history, being described in epidemics and pandemics since biblical times. Plague is a zoonotic disease transmitted ordinarily by animals and their infected fleas. Humans can acquire plague through the bite of infected fleas, direct contact with contaminated tissue, or inhalation. The chapter covers safety considerations, materials, specimen collection, and quality control principles for preanalytical considerations. For analytical considerations, it provides specimen processing, incubation and examination of cultures, and culture identification. With postanalytical considerations, the chapter also provides attention to common test limitations, interpretive challenges, and results reporting.
High‐Consequence Viral Pathogens
Authors: Relich Ryan F.
Published: January 2025

High‐consequence viruses comprise a growing list of pathogens that periodically cause outbreaks of infectious diseases that are characterized by severe symptomatology and high case fatality rates. Many of these agents are transmissible from person to person and elicit early signs and symptoms that overlap with more common diseases, resulting in the delayed implementation of infection control measures designed to prevent their spread. This chapter focuses on viruses that cause hemorrhagic fever, central nervous system diseases, and respiratory tract diseases. All known high‐consequence viruses originate in animals, and their spillover from reservoir species, intermediate hosts, and/or arthropod vectors precipitates outbreaks among humans. The expeditious diagnosis of high‐consequence viral diseases is paramount for implementation of appropriate patient treatment measures and containment of outbreaks. Arthropods, primarily ticks, serve as vectors of several dangerous viruses, including Crimean‐Congo hemorrhagic fever virus and tick‐borne encephalitis virus.
Phenotypic Tests and Biochemical Procedures
Authors: Lee Philip A.
Published: January 2025

The oxidase test is used to determine if an organism possesses the enzyme cytochrome c oxidase as a component of the bacterial electron transport chain. During aerobic cellular respiration, bactericidal reactive oxygen species (ROS) are produced, including peroxide, superoxide, hydroxyl radical, and singlet oxygen. Catalase is an enzyme that protects against oxidative damage from ROS by catalyzing hydrogen peroxide into water and oxygen. The ability of an organism to split indole from the amino acid tryptophan is due to the presence of tryptophanase. In the rapid spot test, indole is detected directly from the colony growing on a medium rich in tryptophan. Beta‐lactamases are enzymes produced by many clinically significant bacteria and are major mediators of bacterial resistance to beta‐lactam agents. The motility test is used to detect the presence of flagella on bacteria, which allow them to travel in and out of the microscopic field or beyond their initial inoculation in agar.
Laboratory Accreditation and Compliance
Authors: Loeffelholz Michael , Samuel Linoj , Sharp Susan E.
Published: January 2025

The current laboratory regulatory framework for accreditation in the United States began with the Clinical Laboratory Improvement Act of 1967. The Clinical Laboratory Improvement Amendments (CLIA) program is administered by three agencies within the Department of Health and Human Services: Centers for Medicare and Medicaid Services, Centers for Disease Control and Prevention, and Food and Drug Administration. CLIA requires that all laboratories have a valid CLIA certificate unless they are CLIA exempt. CLIA inspectors may cite the laboratory for deficiencies based on the nature of the noncompliance. A quality management system is comprehensive; it involves all personnel and includes all phases of the testing workflow: preanalytical, analytical, and postanalytical. Quality control testing must be performed and reviewed after performance of significant maintenance on an instrument or replacement of any important part of the test system, including reagents.
Laboratory Detection of Bacteremia and Fungemia
Authors: T. Harrington Amanda , Jean Sophonie , Hilt Evann E.
Published: January 2025

Clinical microbiology laboratories play an essential role in establishing an etiologic diagnosis for sepsis and bloodstream infection. Diagnosis requires multiple steps, including one or more blood cultures drawn from different sites, recovery and identification of microorganisms detected in blood culture, antimicrobial susceptibility testing of clinically significant organisms, and interpretation of results in conjunction with other clinical, radiographic, and laboratory findings. This chapter provides a summary of the clinical importance of bacteremia and fungemia, the scientific and medical principles underlying current diagnostic methods, and diagnostic approaches. Determining the clinical significance can be difficult for coagulase‐negative staphylococci, as the identity of this organism group is of little or no help. Preparation for blood culture collection and transport to the laboratory includes a number of stepwise processes. Matrix‐assisted laser desorption ionization–time‐of‐flight mass spectrometry is an extensively studied approach to rapid microbial identification of blood culture isolates.
Molecular Techniques
Authors: Parikh Bijal A. , Hughes Andrew E. O. , Anderson Neil W.
Published: January 2025

Molecular methods for the diagnosis of infectious diseases can be broadly grouped into three categories: probe‐based methods without amplification, signal amplification methods, and target amplification methods. These methods differ significantly in terms of their technical requirements, analytical sensitivity, and specificity, which inform the role of individual techniques in specific clinical applications. Probe‐based methods without amplification are among the earliest‐described and simplest molecular tests. The analytical sensitivity of nucleic acid probes can be greatly enhanced through signal amplification, which allows the detection of low quantities of nucleic acid directly from clinical specimens. Transcription‐mediated amplification is a commonly utilized form of PCR‐free target amplification. Molecular assays all have some degree of susceptibility to inhibition by external substances. Quality assurance plans should be designed with consideration of the challenges and potential pitfalls of molecular testing. Molecular methods are exquisitely sensitive, making them very susceptible to false positivity due to environmental contamination.
Prevention of Health Care‐Associated Infections
Authors: Sullivan Kaede V. , Pfaller Michael A. , Diekema Daniel J.
Published: January 2025

This chapter discusses the impact of health care‐associated infections (HAIs) on patient outcomes and health care costs and the organization of the hospital infection prevention program (IPP) and describes the important role that the clinical microbiology laboratory (CML) plays in HAI prevention. An HAI is an infection that is acquired in a hospital or health care facility. The incidence of HAIs involving staphylococci and enterococci increased as these organisms became increasingly resistant to antibiotics. The responsibilities of the hospital IPP include surveillance and prevention of HAIs, continuing education of medical staff, outbreak control, protection of employees and visitors from infection, and providing advice on introduction of new products and procedures. The development and application of new technologies in the CML can greatly enhance infection prevention efforts. Close collaboration between CML and personnel from infection prevention and antimicrobial stewardship programs is required to ensure optimal HAI prevention, which saves money and lives.
Biothreat Agents
Authors: Mahlen Steven D. , Snyder James W.
Published: January 2025

The ideal qualities of a successful biothreat agent area high rate of illness in exposed persons/animals, a high case fatality rate, a short incubation period, and a paucity of immunity in the target population. The Laboratory Response Network forms an integrated network of more than 120 local and state public health, federal, and military laboratories that can respond to bioterrorism, chemical terrorism, and other public health emergencies. The Federal Select Agent Program was established in response to a heightened concern about the ease with which disease‐causing agents could be obtained. Select agent regulations were revised in October 2014 to designate “tier 1” agents with a documented risk of causing a high‐consequence event greater than those with other biological select agents and toxins. Natural forms of botulism include foodborne, wound, and intestinal botulism. Smallpox has a long history of use as a biothreat weapon.
Staphylococcus
,
Micrococcus
, and Other Catalase‐Positive Cocci
Authors: Becker Karsten , David Michael Z. , Skov Robert L.
Published: January 2025

Historically, the genera
Staphylococcus
and
Micrococcus
were placed together with the genera
Stomatococcus
and
Planococcus
in the family
Micrococcaceae
, containing Gram‐positive, catalase‐positive cocci. As staphylococci and micrococci are part of the microbiota of the skin and mucous membranes, careful procedures should be used to isolate organisms from the presumed focus of infection without collecting surrounding microbiota. Because serological testing for antistaphylococcal antibodies lacks specificity and predictive accuracy, it plays no role in the diagnosis of most staphylococcal diseases. A number of studies and reviews describing the antimicrobial and biocide susceptibilities of clinically important staphylococcal species have been published. Distinguishing contaminants and colonizers from staphylococcal and micrococcal isolates causing infection continues to be an important challenge for laboratorians and clinicians.
Aerococcus
, Abiotrophia, and Other Aerobic Catalase‐Negative, Gram‐Positive Cocci
Authors: Nielsen Xiaohui Chen , Christensen Jens Jørgen
Published: January 2025

The catalase‐negative, Gram‐positive cocci form a taxonomically diverse group of bacteria that are isolated infrequently as opportunistic agents of infection. Most of these organisms resemble other, better‐known clinical isolates and consequently may be mistaken for members of those genera. Some strains of
Aerococcus
may exhibit weakly positive catalase reactions due to nonheme catalase activity. Organisms in the genera
Abiotrophia
and
Granulicatella
are normal residents of the oral cavity and are recognized as agents of endocarditis involving both native and prosthetic valves.
Pediococcus
strains have been isolated from bacteremia and cases of sepsis and hepatic abscess in compromised patients.
Vagococci
are motile organisms that, like lactococci, express Lancefield group N antigen.
Weissella
strains are usually isolated in polymicrobial infections. Antimicrobial susceptibility studies on the organisms have generally employed dilution testing methods.
General Approaches to the Identification of Aerobic Gram‐Positive Rods
Authors: Bernard Kathryn A.
Published: January 2025

All strains of Gram‐positive rods can be grown on blood agar plates, with specific descriptions of growth requirements. Gram‐positive organisms demonstrating “regular” rods are those cells with usually parallel longitudinal edges. “Irregular” rods are those organisms with cells observed to have curved or coccoidal longitudinal edges which are not parallel; these taxa can produce yellowish‐pigmented, yellow‐pigmented, or orange‐pigmented colonies. Molecular methods may be required for definitive identification to genus and species level for bacteria which are poorly reactive biochemically and/or are under represented or absent from databases of commercial matrix‐assisted laser desorption ionization–time‐of‐flight mass spectrometry (MALDI‐TOF MS) systems. MALDI‐TOF MS technology is widely used in clinical microbiology, because that method has been found to provide rapid, cost‐effective, and accurate identifications of both common and rare pathogens.
Coryneform Gram‐Positive Rods
Authors: Mattos‐Guaraldi Ana Luíza , Santos Louisy Sanches dos , Vieira Verônica Viana
Published: January 2025

This chapter presents Coryneform bacteria comprising aerobically or facultatively anaerobically growing, asporogenous, non‐acid fast Gram‐positive rods with irregular morphology. Establishing the clinical significance of coryneform bacteria can be difficult for caregivers and microbiologists.
Corynebacterium
spp. form the predominant group and include diphtheria toxin (DT)‐producing
Corynebacterium diphtheriae
, the causative agent of classic diphtheria, which was once a major cause of illness and death among children. Laboratory procedures for identification of
Corynebacterium
spp. and other coryneform bacteria include collection and transport of clinical samples, isolation practices, culture conditions, phenotypic and molecular assays, as well as antimicrobial susceptibility testing. Molecular techniques have been increasingly used in microbiology laboratories for the identification of
Corynebacterium
species and other coryneform bacteria. Detection of antibodies directed against DT is the only established serologic test for coryneform bacteria.
Nocardia
,
Rhodococcus
,
Gordonia
,
Actinomadura
,
Streptomyces
, and Other Aerobic Actinomycetes
Authors: Brown‐Elliott Barbara A. , Zelazny Adrian M. , Conville Patricia S.
Published: January 2025

The anaerobic organisms now in the genus
Actinomyces
and the aerobic organisms grouped together as the “aerobic actinomycetes” were previously presumed to be related to one another on the basis of shared features of microscopic and colonial morphology. The microscopic and colonial morphologies of organisms in the genus
Actinomadura
, which contains more than 100 validly named species or subspecies, are very similar to those in the genus
Streptomyces
. Species in the genus
Saccharopolyspora
were initially thought to be related to
Nocardia
and
Streptomyces
by biochemical characteristics but are phylogenetically distinct from those genera. However, an unknown number of
Gordonia
infections may be missed, either because the isolate is considered an insignificant coryneform Gram‐positive rod or because the isolate is misidentified by biochemical methods as belonging to another genus, such as
Nocardia
or
Rhodococcus
.
Mycobacterium tuberculosis
Complex
Authors: Rowlinson Marie‐Claire , Musser Kimberlee A. , Khare Reeti
Published: January 2025

Tuberculosis (TB) is caused by a group of closely related slowly growing mycobacteria, collectively named the
Mycobacterium tuberculosis
complex (MTBC). Members of the MTBC have undergone several taxonomic reclassifications, which has resulted in confusing nomenclature. Mycobacterial culture is currently considered the reference standard for MTBC detection. Until the early 1990s, typing of MTBC strains was limited to mycobacterial phage typing and drug resistance patterns, methods which have low discriminatory power that have been replaced by molecular genotypic methods. Antimicrobial susceptibility testing is a critical element of the MTBC laboratory workup. While cell‐based immunodiagnostics, such as tuberculin skin testing or interferon‐gamma release assays, can be used as part of establishing a diagnosis of active TB disease, they are mainly used as tests for latent TB infection and to determine exposure to TB.
Approaches to the Identification of Aerobic Gram‐Negative Bacteria
Authors: Nørskov‐Lauritsen Niels , Chuvochina Maria
Published: January 2025

The description of aerobic Gram‐negative bacteria is long established, based on culture conditions and the failure to retain crystal violet‐iodine complex by Gram's staining method. This chapter shows dichotomous phenotypic and biochemical identification algorithms for Gram‐negative bacteria that are fastidious and grow poorly or not at all on sheep blood agar. The use of KIA/TSI slants as part of the workup of Gram‐negative organisms has largely been discontinued due to the introduction of matrix‐assisted laser desorption ionization‐time of flight mass spectrometry, which provides more rapid and specific identification. The major limitation of analyzing the 16S‐23S ribosomal RNA (rRNA) region for bacterial identification and community profiling is the current lack of a reference database containing combined 16S rRNA and 23S rRNA gene sequences.
Salmonella
Authors: Dekker John P. , Faron Matthew L. , Humphries Romney M. , Buchan Blake W. , Ledeboer Nathan A.
Published: January 2025

Salmonella typhi
has one of the largest sets of universal genetic markers common to
Salmonella enterica
serotypes but also contains among the fewest genetic markers defining a subspecies. This chapter discusses traditional classification schemes based on antigenic typing, pulsed‐field gel electrophoresis, and multilocus sequence typing (MLST) and covers newer molecular and next‐generation sequencing‐based approaches, including ribosomal MLST, whole‐genome and core‐genome MLST, and whole‐genome single‐nucleotide variant‐based strain tracking. Nontyphoidal
Salmonella
is a common inhabitant of the gastrointestinal tract of wild and domestic poultry, which serves as an important reservoir for infection, but is also frequently isolated from warm‐blooded mammals and reptiles. Use of appropriate isolation media along with novel diagnostic techniques is improving understanding of the epidemiology of the genus
Salmonella
and will ultimately help meet the goal of improving patient care. Determination of antimicrobial susceptibility is valuable for surveillance purposes to monitor the development and spread of antimicrobial resistance among
Salmonella
isolates.
Peptostreptococcus
,
Finegoldia
,
Anaerococcus
,
Peptoniphilus
,
Parvimonas
,
Murdochiella
,
Veillonella
, and Other Anaerobic Cocci
*
Authors: Veloo Alida C. M. , Johnson Crystal N.
Published: January 2025

Gram‐positive anaerobic cocci (GPAC) belong to the Firmicutes phylum and have been known to most bacteriologists as peptococci or peptostreptococci. The genera
Anaerococcus
,
Anaerosphaera
,
Fastidiophila
,
Finegoldia
,
Gallicola
,
Murdochiella
,
Parvimonas
,
Peptococcus
,
Peptoniphilus
, and
Peptostreptococcus
are Gram‐positive, coccobacillary or occasionally coccoid cells. The genera
Veillonella
, Acidaminococcus,
Megasphaera
,
Anaeroglobus
, and
Negativicoccus
are Gram‐negative cocci. The polymicrobial nature of GPAC infections, in addition to inadequate classification, has most likely contributed to neglect the clinical significance of individual species of GPAC. A variety of microscopic, immunologic, and molecular techniques have been developed for direct examination of GPAC. New information regarding the antimicrobial susceptibilities of individual GPAC is increasing due to the improvement in classification and identification.