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Category: Clinical Microbiology
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The past 30 years have seen tremendous developments in the availability of commercial products in microbiology; however, to date there has been no general resource for all sub-disciplines of clinical microbiology to use when evaluating commercial methods, tests, or procedures.
Now the Manual of Commercial Methods in Microbiology reviews all the commercially available tests (both manual and automated) in the discipline of clinical microbiology. A description of the sensitivities, specificities and predictive values from peer-reviewed sources is included. The authors also attempt to predict what new tests or methods may be used in the near future. This reference includes separate chapters devoted to molecular microbiology, information management, emerging infectious diseases, and veterinary clinical microbiology.
Electronic Only, 481 pages, illustrations, index.
As the U.S. Food and Drug Administration (FDA) approval process has a major impact on how and when new in vitro diagnostic devices (IVDs) become available for use by the health care professional or the layperson, it is important to examine how the FDA and the Health Care Finance Administration (HCFA) regulatory processes operate. For the purpose of this chapter, examples and cited regulations will for the most part be confined to the FDA process which permits commercial interstate sale and distribution of clinical microbiology IVDs by the Center for Devices and Radiological Health (CDRH). Detection and identification of microorganisms directly from clinical material were in their infancy in 1976 with the exception of antisera conjugated with a fluorescent dye and directed at fastidious organisms, such as Francisella tularensis, Toxoplasma gondii, rickettsiae, or rabies virus. The study should be carefully designed to provide information and data to establish the clinical and analytical accuracy of the assay. Clinical Laboratory Improvement Amendments of 1988 (CLIA 88) extended federal regulation to cover all laboratories that examine human specimens for the diagnosis, prevention, or treatment of any disease or impairment or for the assessment of the health of human beings. Molecular techniques, such as nucleic acid amplification (NAA), may replace culture as a more sensitive, specific, and rapid method for the identification of specific microorganisms directly from clinical material.
Currently, both manual and automated blood culture systems are available, with the market being dominated increasingly by instrument-based automated systems and methods. This chapter will emphasize those manual and automated systems and media cleared for diagnostic use in the United States at the start of the 21st century as well as comment on possible developments in the rapidly evolving area of clinical microbiology. The fundamental concept of blood culturing has been based on inoculating a defined volume of blood, preferably obtained by a sterile venipuncture, into a broth culture medium that will support the growth of most pathogenic bacteria and fungi. In a study that compared the aerobic FAN bottle to the aerobic 80A bottle, more contaminants were detected in the FAN bottle, and the ESP system had more instrument false-positive readings. Cockerill and colleagues compared the ESP 80A bottle to the Isolator and found that the former recovered more coagulase-negative staphylococci causing bacteremia, whereas the latter recovered more S. aureus, Candida spp., and all microorganisms combined. In this study, the ESP 80A bottle also was compared to the manual Septi-Chek bottle and found to be equivalent in the recovery of microorganisms. Although current blood culture systems have reduced the time to detecting positive cultures, clinicians and patients still would benefit from accurate assay methods that provide results in minutes to hours as opposed to days. Thus, there continues to be a need for further evolution and development of new commercial blood culture systems.
Preliminary detection of bacterial pathogens and isolation in pure culture on solid media was followed by culture confirmation using one of the early-generation kits for bacterial identification. In more recent years, the development and, in some cases, very common use of direct antigen testing has revolutionized the algorithms for specimen testing which are used today in physicians’ office laboratories, community hospitals, and tertiary-care medical centers. Early antigen tests sometimes lacked the sensitivity which was offered by conventional testing. Researchers tested 345 strains of coryneform bacteria and 33 strains of Listeria spp. (representing 49 taxa). In this study, 80.9% were identified to the species level and 12.2% were identified to the genus level, with 3.7% of the strains misidentified and 3.2% of the strains not identified. Prepackaged commercially available kit systems for the identification of clinically significant gram-negative bacteria have been available for a number of years and offer the clinical microbiologist several distinct advantages over previously available methodologies. The identification systems have evolved over time and have been based on either growth dependent or enzyme-mediated substrate utilization or on the detection of specific nucleic acid sequences. A section talks about fastidious bacteria that include Haemophilus spp., Neisseria spp., Gardnerella vaginalis, Bordetella spp., and Legionella spp.
This chapter talks about the diagnosis of anaerobic bacterial infections involving various steps. Some of the most important considerations involved in the laboratory diagnosis of anaerobic infections include (i) selecting, collecting, and transporting specimens for microbiologic examination and (ii) processing and examining the specimens in the laboratory as rapidly as possible after they are received. Satisfactory commercially available primary isolation media and anaerobic incubation systems are discussed along with detailed recommendations for their use. The current software release (version 3.9) used in the authors' laboratory contains an extensive list of anaerobe genera and species. At present, simple-to-use commercial kits and equipment for specific molecular applications in clinical anaerobic bacteriology have been lacking. It appears that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) could be a powerful tool for rapid identification of bacteria that would otherwise be difficult to differentiate with other methods. This new technology holds promise for markedly decreasing the turnaround time for identification of anaerobes and other slow growing microorganisms, thereby enhancing the clinical relevance of this area of clinical microbiology.
In this chapter, the author reviews the most common commercially available kits and methods for the routine clinical virology laboratory. Outbreaks of influenza occur annually during the winter. The virus infects the columnar epithelial cells of the upper respiratory tract, and in the majority of those infected, the virus causes a disease characterized by fever, pharyngitis, and myalgias. The BD Directigen Flu A assay introduced 10 years ago, detects only influenza A virus, and of all the assays listed, it is the most extensively evaluated. Respiratory syncytial virus (RSV) is a major cause of viral lower respiratory tract infections among infants and young children worldwide. Unlike influenza virus, RSV is relatively difficult to recover in cell culture. Most of the immunoassays for RSV were developed and extensively evaluated in the early 1980s. Gardner and McQuillan comprehensively examined the value of immunofluorescence staining for the diagnosis of respiratory tract infections in the early 1980s. Since influenza antigens are found in the nucleus and cytoplasm and parainfluenza virus antigens are exclusively cytoplasmic, the staining pattern may provide a clue to the identity of the virus and thus direct subsequent staining. Many viruses, including astroviruses, caliciviruses (Norwalk and Norwalk-like viruses), coronaviruses (the genus Torovirus), adenoviruses, and rotaviruses cause gastrointestinal disease in humans, and unfortunately, all are either difficult or impossible to cultivate in routinely used cell cultures. The adenoviruses cause a broad spectrum of human disease, including pharyngitis, pneumonia, conjunctivitis, hemorrhagic cystitis, and diarrhea.
This chapter focuses on the various technologies that are currently available from commercial companies for the diagnosis and monitoring of human immunodeficiency virus (HIV) infections, and it is not meant to duplicate more extensive reviews of HIV. Infection with HIV type 1 (HIV-1) results in the induction of a humoral antibody response specific to viral proteins, with the production of immunoglobulin A (IgA), IgM, and IgG. The majority of the automated immunoassay analyzers provide walk-away simplicity to perform assays from sample processing through interpretation of results. The major advantage of Western blot assays over enzyme immunoassays (EIAs) is that the specific interaction of antibody and antigen can be directly visualized. Immunofluorescence assay (IFA) is a very useful and inexpensive alternative to performing Western blot assays for confirmation of HIVspecific antibody responses. The Fluorognost HIV-1 IFA is based on the specific binding of HIV-1 antibodies in a specimen to HIV-1 antigens expressed on the surfaces of immortalized human T- cells fixed to glass slides. Specific antibody-antigen complexes are then detected using an anti-human antibody conjugated with fluorescein isothiocyanate. The development of molecular assays to quantitate the levels of HIV RNA in infected patients has provided one of the most valuable tools to assess the progression of HIV disease, monitor the impact of antiviral therapy, predict treatment failure and the emergence of drug-resistant viruses, and facilitate our understanding of the natural history and pathogenesis of this virus.
The chapter discusses commercially available methods for identification of Chlamydia trachomatis. Selected molecular assays for Neisseria gonorrhoeae are also included in this chapter, since the evaluation of methods and the performance of tests for C. trachomatis and N. gonorrhoeae are often done concomitantly. The first portion of the chapter describes the epidemiology and clinical manifestation of infections with C. trachomatis. The next portion details the laboratory aspects of detection of C. trachomatis. The chapter details the test performance of the products. The traditional approach to laboratory diagnostic testing for C. trachomatis infections has consisted of cell culture of inocula prepared from urogenital specimens. Several studies have shown that without quality assurance of specimen adequacy, more than 10% of specimens will be unsatisfactory because they contain exudate and lack urethral or endocervical columnar cells.
Mycoplasmas and ureaplasmas represent a complex and unique group of microorganisms that has previously been ignored by most diagnostic laboratories. Bacteria commonly referred to as mycoplasmas (fungus form) are included within the class Mollicutes (soft skin), which comprises four orders, five families, eight genera, and over 150 known species. Diagnostic kits for the detection and preliminary characterization of mycoplasmas are generally similar, consisting of strips with wells containing specific dried or lyophilized substrates and inhibitors. Descriptions of some of the most widely used commercial mycoplasma growth media, corresponding diagnostic kits, and their manufacturers are provided. The availability of frozen or freezedried mycoplasma media from commercial sources facilitates such transport or subculture as may be needed on an infrequent basis for low-volume laboratories that do not offer complete diagnostic service for mycoplasmas. There are no automated instruments or procedures available to detect mycoplasmas in clinical specimens. Recent data obtained using the Bact/ALERT instrument have again demonstrated the inability of automated blood culture systems to flag blood cultures containing mycoplasmas. The culture-based procedures and products described in this chapter are suitable for performance in a high-complexity hospital-based general microbiology laboratory or reference laboratory. The availability of complete diagnostic kits, similar to those now sold in Europe and some other areas may further increase the likelihood that hospital-based microbiology laboratories will begin offering diagnostic services for mycoplasmas, provided the kits can be proven to be accurate when compared directly with standard methods.
The methods described in this chapter are meant to represent those that are commercially available, Food and Drug Administration-approved, and published in peer-reviewed journals. The advantages of the nonautomated and automated identification systems discussed in the chapter include the fact that identification is based on databases that include a variety of substrate utilization patterns and that contain many yeast biotypes. Reproducibility was determined as results were compared in an interlaboratory study with broth macrodilution as the reference standard. In this report, the E test quality control limits were 1 dilution greater (4 dilutions overall) than those of the NCCLS broth tube macrodilution method for the following combinations: ketoconazole with C. krusei and amphotericin B and ketoconazole with C. parapsilosis. When the Premier EIA method was compared to the microimmunodiffusion (MID) test and the laboratory CF test for a total of 168 sera, including 68 from proven cases of histoplasmosis, the sensitivity of the EIA for IgG was 97%and that of the MID was 100%, and the specificities were 84 and 100%, respectively. In this study, three sera from histoplasmosis patients which were positive for the histoplasmin antigens tested negative for IgG, specific precipitins, and complement-fixing antibodies. Due to the interest in rapid, cost-effective testing for fungi, the pace and number of publications devoted to diagnostic practices in clinical mycology laboratories are growing.
Currently a number of automated commercially available rapid detection systems that can also perform antimycobacterial susceptibility testing are available to clinical laboratories. Further, several commercially available identification systems that utilize molecular methods are used by reference laboratories for the rapid identification of clinically important species of mycobacteria. One of the most important components of the laboratory diagnosis of mycobacterial infections is the use of the acidfast stain; either it is the auramine-rhodamine fluorescent stain or conventional carbol fuchsin-based stain. The Centers for Disease Control and Prevention emphasized the need for rapid turnaround times, not only for acid-fast smear results and recovery of mycobacteria from clinical specimens but also for subsequent antimicrobial susceptibility testing. The ESP Culture System II is an automated method that was originally devised for blood cultures and subsequently was adapted for the recovery of mycobacteria in clinical specimens. The MTD2 test is an isothermal transcriptionmediated amplification system based on specific mycobacterial rRNA targets using DNA intermediates. MicroSeq program technology promises to be the most accurate identification method and will probably be the standard for mycobacterial identification in the future. Presently, there are three antibodies available; two react with several mycobacterial species, including members of the Mycobacterium tuberculosis complex. One antibody is thought to be specific for M. tuberculosis complex. Currently the gold standard still consists of traditional culture and antimicrobial susceptibility testing procedures. However, the authors feel that molecular methods will allow laboratories to detect, identify, and determine drug resistance of mycobacteria without culturing the organism.
The key to performance of diagnostic medical parasitology procedures is formal training and experience. The majority of diagnostic parasitology procedures can be performed either within the hospital setting or in an offsite location. The majority of physician office laboratories are not involved in diagnostic parasitology testing; however, as more molecular (nonmicroscopic) methods are developed, they may become more widely used in this setting. The specimen most commonly submitted to the diagnostic parasitology laboratory is the stool specimen, and the most commonly performed procedure in parasitology is the ova and parasite (O&P) examination, which comprises three separate protocols: the direct wet mount, the concentration, and the permanent-stained smear. Both flotation and sedimentation methods are available, the most common procedure being the formalin-ethyl acetate sedimentation method (formerly used was the formalin-ether method). Another simplified culture option has been developed for the isolation and identification of Trichomonas vaginalis. This approach has proven to be much more sensitive than the examination of wet preparations alone and has been incorporated into use in many institutions with dramatic increases in the number of positive specimens identified.
This chapter discusses both home brew assays and commercially available kits and related instrumentation for the detection of infectious agents by molecular methods, and is subdivided into two sections. The first section deals with molecular amplification tests that are performed under home brew assay guidelines using reagents that are commercially available. It is recognized that the use of molecular methods for diagnosis of infectious diseases is a rapidly evolving field, and much of the initial diagnostic work is done with home brew assays developed and validated within each laboratory. The second section of the chapter reviews the commercially available products that make it easier to assemble home brew assays using partial kits and analyte-specific reagents. The integration of commercially available components into home brew molecular assays for detection of pathogens helps to avoid human error and enables standardization of testing procedures, thus increasing the precision and reproducibility of the results. The commercially available nucleic acid detection assays have several important advantages over home brew assays. These assays fall into two general categories: the direct nonamplified nucleic acid detection methods, and the amplified tests. Where possible, the use of commercial Food and Drug Administration (FDA) (or equivalent)-approved nucleic acid tests (NAT) kits and instrumentation is advantageous because of the standardization of results, convenience of use for greater numbers of clinical laboratories, and benefits related to technologist training, support, and savings of hands-on time related to a higher degree of automation.
Enzyme immunoassays (EIA) have great potential for automation in diagnostic microbiology laboratories, and a number of semiautomated and fully automated immunoassay analyzers are now commercially available for the performance of a variety of viral, bacterial, fungal, and parasitic antibody and/or antigen detection tests. The majority of the automated immunoassay analyzers provide walk-away simplicity to perform assays from sample processing through interpretation of results. The instruments can automatically generate work lists of specimens to be tested, pipette specimens from primary tubes and dilute the samples, dispense all reagents, time the incubations at a desired temperature, shake the assay vessels if needed, perform washes, and read and store the final results. The use of automated immunoassay analyzers can be advantageous to the laboratory that has a shortage of trained medical technologists or that needs to reduce costs or improve the throughput and turnaround time for test results.
This chapter describes, and in some cases reports evaluations of, those commercial identification systems that are used by veterinary diagnostic laboratories. Most modern viral diagnostic tests are rapid assays aimed at detection of antiviral antibodies (enzyme-linked immunosorbent assay [ELISA], Western immunoblot analysis, immunofluorescence, serum virus neutralization [SVN], hemagglutination inhibition [HI], agar gel immunodiffusion [AGID], and complement fixation [CF]), detection of viral antigens (immunohistochemistry [IHC], antigen-capture ELISA, and frozen tissue immunofluorescence), or detection (or amplification) of viral nucleic acids (PCR and its related techniques, in situ hybridization [ISH], Southern blot hybridization, and dot blot hybridization). In addition, modern molecular tests and procedures (such as PCR-restriction fragment length polymorphism [PCR-RFLP] analysis, sequencing, and sequence analysis) are being used more commonly for further genetic characterization of viruses and for differential diagnosis. With a known viral antigen, latex agglutination test (LAT) can also be used to detect antiviral antibody, such as that for swine pseudorabies virus. LAT kits are commercially available for a few veterinary viruses. The chapter also focuses on reviews of studies that have evaluated the applicability of bacterial identification systems for identification of bacteria isolated from animal specimens. The identification of dermatophytes from animal specimens is emphasized in the chapter because dermatophytes are causes of zoonotic infections as well as common infections in animals. Identification of mites, lice, ticks, fleas, and fly larvae infecting animals is a common occurrence for veterinary parasitologists.
The clinical laboratory computer, or as it is now commonly called, the laboratory information systems (LISs), has rapidly become one of the most important features of a modern laboratory. One of the most important criteria for choosing an LIS is selecting a company that will be a reliable long-term business partner that supports and tailors its products to meet a laboratory’s changing needs. The systems profiled in the chapter vary wildly in scope and price. They range from basic single-user products for less than $1,000 to complex multisite systems that cost millions of dollars. Some laboratories may get more benefit from a simple lower-cost system than a complex one that takes years to configure and install and for staff to learn to use. The major cost factor in switching from one large LIS to another is not the purchase or installation fees the vendor charges but the immense amount of work the laboratory must perform to train staff and remap its workload and laboratory description from one information model to a different one.
This chapter is devoted to select emerging and reemerging infectious disease entities and, wherever applicable, typical clinical manifestations and laboratory findings are also described. The majority of cases of human monocytic ehrlichia (HME) and human granulocyte ehrlichia (HGE) are diagnosed by retrospective serologic testing, and the most commonly used serologic test is the indirect fluorescent-antibody test, which is available through commercial laboratories and public health laboratories. The majority of cat scratch disease is caused by Bartonella henselae; the different clinical presentations associated with B. henselae infection appear to predominantly be due to the host immune system although there may be differences in virulence between strains as well. Nucleic acid detection techniques for Bartonella species were first described by Relman based on amplification of the 16S rRNA gene. The hantavirus pulmonary syndrome begins with a prodromal phase consisting of sudden onset of fever, myalgias, headache, and backache. Immunohistochemistry staining of tissue samples can also be used to look for hantavirus antigens. This study is usually done as a confirmatory test in appropriate patients. PCR testing on tissue or blood cells can also be used to detect hantavirus RNA.
Antimicrobial susceptibility testing (AST) can be performed by four basic methods: disk diffusion, broth dilution, agar dilution, and gradient diffusion. The authors recognize that susceptibility testing methods and panels are subject to periodic modifications and that new systems and panels are introduced into various global markets as an ongoing process. This chapter reviews recently published evaluations of currently used commercial systems. The automated and semiautomated systems discussed in the chapter have the capability of producing standardized or customized patient test reports generated by computer software packages that are referred to as data management systems (DMS). In the chapter, five current automated systems are discussed individually and evaluations (advantages and limitations) are presented as an overview of the current literature. The automated Sensititre ARIS system is a broth microdilution method utilizing a standard 96-microwell panel containing serial dilutions of dehydrated antimicrobial agents. Panel configurations are currently available for gram-positive and gram-negative bacteria in either a MIC-only format or a breakpoint format and are listed in the chapter. The chapter describes published evaluations of several commercial MIC methods for Staphylococcus pneumoniae, and reviews several commercial methods for the rapid detection of methicillin-resistant S. aureus (MRSA).
This chapter provides an overview of new and emerging molecular diagnostic technologies. While not all techniques can be described here, examples of relevant technologies for specimen preparation, PCR, post-PCR detection, and analysis are presented. Several non-PCR methods are discussed. Advances in fluorescent chemistry have facilitated fluorescent detection of nucleic acids or hybridized nucleic acid probes and have enabled technology to advance from traditional end product analysis to real-time monitoring of PCR amplicons within a closed system. This strategy may very well be the most exciting advance for clinical microbiology since the advent of PCR itself. An alternative to fluorescent-dye incorporation, fluorescence resonance energy transfer (FRET) technology, allows the detection and quantitation of specific PCR products through the use of nucleic acid probes. Current limitations of microarray technology include the high costs of instrumentation and disposables. In addition, one's current inability to easily analyze the enormous amount of information potentially available through this technology will present challenges. It is quite possible that peptide nucleic acids (PNAs) will have practical applications in molecular diagnostics. Advances in nonisotopic, non-gel-based detection and identification of PCR products may allow for cost-effective integration into the clinical microbiology workplace. Microbiologists must responsibly prepare molecular algorithms to include the ability to identify new pathogens or new presentations of a disease, which may be overlooked if one focuses too closely on only a certain set of molecular parameters for disease diagnosis.
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