Manual of Commercial Methods in Clinical Microbiology

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.

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.

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.

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.

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.

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.

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.

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.

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.