32 I. Introduction to Clinical Infectious Diseases
spp). Some bacteria are extremely difficult to cultivate or cannot be grown in vitro (chlamydia, chlamydophila, rickettsia, anaplasma, orientia, ehrlichia, coxiella, the spirochetes, and M. leprae). For these organisms, alternate diagnostic approaches must be used, such as immunologic methods, cell culture, and molecular diagnostics (see below). Viruses and some other microorganisms are obligate intracellular pathogens and, as such, cannot be cultivated using the techniques described above. Because growth and replication of these pathogens require living cells, three techniques have been used: inoculation of tissue or cell culture, embryonated hens’ eggs, and experimental animals. Tissue culture is the most common method to culture viruses. Visualization of growth can be detected by recognition of the cytopathic effect (CPE) that the virus has on the cell culture. For instance, respiratory syncytial viruses cause fusion of cells to produce multinucleated giant cells, termed syncytia. Some viruses produce proteins that are expressed on the membrane of infected cells. These viral proteins bind erythrocytes, which can be detected by testing for hemadsorption or hemagglutination. Detection and visualization of viruses that produce little to no CPE, do not possess hemagglutinins, or do not completely replicate in cell culture can be achieved through immunologic or nucleic-acid probes. Both indirect fluorescent antibody (IFA) methods and direct fluorescent antibody (DFA) techniques are used for detection of specific agents (see following section). C. Diagnostic Immunology. It is not always possible to isolate a microorganism in culture or visualize it microscopically. In such cases, immunoassays are often used to detect the presence of a particular agent. In general, immunoassays involve one of two main principles: testing for the presence of specific microbial antigens or testing for specific microbial-antigen antibodies. These assays may involve the detection of a microbial antigen directly from a clinical specimen or the detection of a specific antigen once an organism is cultured in vitro. Fluorescent antibody (FA) techniques such as DFA and IFA are commonly used for detection of specific agents. For DFA, a fluorescein-labeled antibody specific for a particular antigen is incubated with a test specimen fixed on a glass microscope slide. If the antigen is present in the specimen, a bright yellow-green fluorescence will be seen under a fluorescent microscope. For IFA, a primary, unlabeled, antigen-specific antibody and a fluorescein-labeled anti-immunoglobulin specific for the primary antibody are used. Both are incubated with the test specimen, and results are interpreted the same as for DFA. D. Molecular Diagnostics. While microscopy, culture, and phenotypic characterization remain the mainstay for microbial identification in most laboratories, advances in molecular techniques have resulted in improved speed, sensitivity, and specificity for identification of some infectious microorganisms. Despite improving the ability to make some diagnoses, most molecular techniques are used more as research tools rather than as a standard-of-care test. Applications of molecular methods for infectious disease testing include the identification of microorganisms or the detection of factors used to monitor disease or predict outcome. Such factors include antimicrobial resistance genes, virulence factors, and quantitation of microorganisms (eg, viral load testing). Traditionally, molecular testing has been widely used for the detection