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The adaptation of microbiological techniques to the study of the etiological agents of infectious disease. Clinical microbiologists determine the nature of infectious disease and test the ability of various antibiotics to inhibit or kill the isolated microorganisms. In addition to bacteriology, a contemporary clinical microbiologist is responsible for a wide range of microscopic and cultural studies in mycology, parasitology, and virology. The clinical microbiologist is often the most competent person available to determine the nature and extent of hospital-acquired infections, as well as public-health problems that affect both the hospital and the community. See Animal virus, Medical bacteriology, Medical mycology, Medical parasitology, Virus
Historically, the diagnosis of bacterial disease has been the primary job of clinical microbiology laboratories. Many of the common ailments of humans are bacterial in nature, such as streptococcal sore throat, diphtheria, and pneumococcal pneumonia. The bacteriology laboratory accepts specimens of body fluids, such as sputum, urine, blood, and respiratory or genital secretions, and inoculates the specimens onto various solid and liquid growth media. Following incubation at body temperature, the microbiologist examines these agar plates and tubes and makes a determination as to the relative numbers of organisms growing from the specimen and their importance in the disease process. The microbiologist then identifies these alleged causes of disease and determines their pattern of antibiotic susceptibility to a few chosen agents.
Clinical microbiologists also microscopically examine these body fluids. They report on the presence of bacteria in body fluids and the cellular response to infection, such as the numbers or types of white blood cells observed in the specimen.
While direct microscopy and culture continue to be methodological mainstays in diagnostic microbiology laboratories, nonculture methods are growing in the variety of applications and the sophistication of the technology. For example, polyclonal antibodies raised in animals such as mice, sheep, goats, and rabbits, and monoclonal antibodies produced by hybridization technology are used to detect bacteria, fungi, parasites, or virus-infected cells by using direct or indirect fluorescent techniques. Additional methods include latex agglutination tests to detect particulate antigens and enzyme immunoassays to detect soluble antigens. See Immunoassay, Monoclonal antibodies
Probes for deoxyribonucleic acid (DNA) or messenger ribonucleic acid (mRNA) are available for various applications. Probes are used for direct detection of organisms in clinical material and for culture confirmation.
Further increases in analytical sensitivity have been achieved by nucleic acid amplification techniques. In polymerase chain reaction (PCR), double-stranded DNA is denatured; oligonucleotide probes bind to homologous strands of single-stranded DNA, and the enzyme polymerase extends the probes using deoxyribonucleotides in the milieu. In ligase chain reaction (LCR), the enzyme ligase fills the 3-nucleotide gap between two probes that attach to homologous, target, single-stranded DNA. In nucleic acid sequence–based amplification (NASBA), reverse transcriptase is used to make double-stranded complementary DNA (cDNA) and the target RNA is digested by ribonuclease H.
Analysis of lipopolysaccharides and proteins by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and cellular fatty acid analysis by gas-liquid chromatography have given way to nucleic acid–based methods. Restriction enzymes, which cut DNA at a constant position within a specific recognition site usually composed of four to six base pairs, are used to cut chromosomal DNA; the resulting fragments are compared by pulse field gel electrophoresis (PFGE) or ribotyping. Electrophoresis of isolated plasmid DNA is another method for comparing organisms. DNA sequencing can also compare segments of the DNA of organisms from the same genus and species.
DNA chip or microarray technology is expected to have a greater effect on medicine than either DNA sequencing or PCR. Over 30,000 small cDNA clones of expressed fragments of individual genes are spotted onto a thumbnail-sized glass chip. Fluorescein-labeled genomic or cDNA from the sample being evaluated is passed over the chip to allow hybridization. A laser measures the fluorescent emissions and a computer analyzes the data.