Laboratory Diagnosis and Therapy of Infectious Diseases

The laboratory diagnosis of infection requires the demonstration either direct or indirect of viral, bacterial, fungal, or parasitic agents in tissues, fluids, or excreta of the host. Clinical microbiology laboratories are responsible for processing these specimens and also for determining the antibiotic susceptibility of bacterial and fungal pathogens. Traditionally, detection of pathogenic agents has relied largely on either the microscopic visualization of pathogens in clinical material or the growth of microorganisms in the laboratory. Identification has been and often still is based on phenotypic characteristics such as fermentation profiles for bacteria, cytopathic effects in tissue culture for viral agents, and microscopic morphology for fungi and parasites. These techniques are reliable but are often time-consuming. Increasingly, protein analysis or genotypic techniques are becoming standard methods for detection, quantitation, and/or identification of microorganisms in the clinical microbiology laboratory, replacing phenotypic characterization and microscopic visualization methods. This chapter discusses general concepts of diagnostic testing, with an emphasis on detection of bacteria. Detection of viral.

Microorganisms can be detected by automated or manual methods that depend on the presence of molecules unique to the specific pathogen. Molecules useful for detection of microorganisms include structural components of bacteria, fungi, and viruses; specific antigens; metabolic end products; unique DNA or RNA sequences; enzymes; toxins or other proteins; and surface polysaccharides. These are detected by a variety of methods, including immunofluorescence; chemiluminescence for DNA/RNA probes; flame ionization detection of short- or long-chain fatty acids; and detection of substrate use or end-product formation as color changes, of enzyme activity as a change in light absorbance, of turbidity changes as a measure of growth, of cytopathic effects in cell lines, and of particle agglutination as a measure of antigen presence. Amplification enhances the sensitivity with which weak signals can be detected.

Precipitation tests

Precipitation tests measure an antigen or antibody in body fluids by the degree of visible precipitation of antigen-antibody complexes within a gel (agarose) or in solution. There are many types of precipitation tests (eg, Ouchterlony double diffusion, counterimmunoelectrophoresis), but their applications are limited.

Usually, a blood specimen is mixed with test antigen to detect patient antibodies, most often in suspected fungal infection or pyogenic meningitis. Because a positive result requires a large amount of antibody or antigen, sensitivity is low.

Western blot test

The Western blot test detects antimicrobial antibodies in the patient’s sample (eg, serum, other body fluid) by their reaction with target antigens (eg, viral components) that have been immobilized onto a membrane by blotting.The Western blot typically has good sensitivity, although often less than that of screening tests such as ELISA, but generally is highly specific. Thus, it is usually used to confirm a positive result obtained with a screening test.