A positive tuberculin test result does not prove the presence of active disease caused by tubercle bacilli. Isolation of tubercle bacilli provides such proof.

 A. Specimens

 Specimens consist of fresh sputum, gastric washings, urine, pleural fluid, cerebrospinal fluid, joint fluid, biopsy material, blood, or other suspected material.

B. Decontamination and Concentration of Specimens

 Specimens from sputum and other nonsterile sites should be liquefied with N-acetyl-l-cysteine decontaminated with NaOH (kills many other bacteria and fungi), neutralized with buffer, and concentrated by centrifugation. Specimens processed in this way can be used for acid-fast stains and for culture. Specimens from sterile sites, such as cerebrospinal fluid, do not need the decontamination procedure but can be directly centrifuged, examined, and cultured.

 C. Smears

Sputum, exudate, or other material is examined for acid-fast bacilli by staining. Stains of gastric washings and urine generally are not recommended because saprophytic mycobacteria may be present and yield a positive stain. Fluorescence microscopy with auramine–rhodamine stain is more sensitive than traditional acid-fast stains, such as Ziehl-Neelsen, and is the preferred method for clinical material. If acid-fast organisms are found in an appropriate specimen, this is presumptive evidence of mycobacterial infection.

 D. Culture, Identification, and Susceptibility Testing

 Processed specimens from nonsterile sites and centrifuged specimens from sterile sites can be cultured directly onto selective and nonselective media (see earlier discussion). The selective broth culture often is the most sensitive method and provides results most rapidly. A selective agar media (eg, Löwenstein-Jensen or Middlebrook 7H10/7H11 biplate with antibiotics) should be inoculated in parallel with broth media cultures. Incubation is at 35–37°C in 5–10% CO2 for up to 8 weeks. If culture results are negative in the setting of a positive acid-fast stain or if slowly growing NTM (see later) are suspected, then a set of inoculated media should be incubated at a lower temperature (eg, 24–33°C) and both sets incubated for 12 weeks.

Blood for culture of mycobacteria (usually MAC) should be anticoagulated and processed by one of two methods: (1) commercially available lysis centrifugation system or (2) inoculation into commercially available broth media specifi cally designed for blood cultures. It is medically important to characterize and separate M. tuberculosis complex from all the other species of mycobacteria. Isolated mycobacteria should be identified as to species. Conventional methods for identification of mycobacteria include observation of rate of growth, colony morphology, pigmentation, and biochemical profiles. The conventional methods often require 6–8 weeks for identification and are rapidly becoming of historical interest because they are inadequate to identify the expanding numbers of clinically relevant species. Most laboratories have abandoned reliance on these biochemical tests. Growth rate separates the rapid growers (growth in ≤7 days) from other mycobacteria (Table 1). Photochromogens produce pigment in light but not in darkness, scotochromogens develop pigment when growing in the dark, and nonchromogens (nonphotochromogens) are nonpigmented or have light tan or buff-colored colonies. Molecular probe methods are available for four species  and are much faster than the conventional methods. The probes can be used on mycobacterial growth from solid media or from broth cultures. DNA probes specific for ribosomal RNA (rRNA) sequences of the test organism are used in a hybridization procedure. There are approximately 10,000 copies of the rRNA per mycobacterial cell, providing a natural amplification system, enhancing detection. Double-stranded hybrids are separated from unhybridized single-stranded probes. The DNA probes are linked with chemicals that are activated in the hybrids and detected by chemiluminescence. Probes for the M. tuberculosis complex (M. tuberculosis, M. bovis, M. africanum, M. caprae, M. microti, M. canettii, and M. pinnipedii), MAC (M. avium, M. intracellulare, and closely related mycobacteria), M. kansasii, and Mycobacterium gordonae are available. The use of these probes has shortened the time to identification of clinically important mycobacteria from several weeks to as little as 1 day.

In the United States, these four groups (M. tuberculosis complex, M. avium complex, M. kansasii, and M. gordonae) make up 95% or more of clinical isolates of mycobacteria.

For species that cannot be identified by DNA probes, many laboratories with molecular capabilities have implemented 16S rRNA gene sequencing to rapidly identify probe-negative species or send such organisms to a reference laboratory with sequencing capability.

High-performance liquid chromatography (HPLC) has been applied to the identification of mycobacteria. The method is based on development of profiles of mycolic acids, which vary from one species to another. HPLC is available in reference laboratories to achieve species-level identification for most mycobacteria.

Other methods for species-level identification of mycobacteria recovered from culture include pyrosequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Susceptibility testing of mycobacteria is an important adjunct in selecting drugs for effective therapy. A standardized broth culture technique can be used to test for susceptibility to first-line drugs. The com plex and more arduous conventional agar-based technique usually is performed in reference laboratories; first- and second-line drugs can be tested by this method. A modification of liquid broth cultures involves inoculating mycobacteria on a multi-well plate with and without addition of antibiotics (Microscopic Observation Drug Susceptibility, MODS assay) and examining for cording that is characteristic of M. tuberculosis complex. This method is largely used outside the United States.

E. Nucleic Acid Amplification Tests (NAATs)

 NAATs are available for the rapid and direct detection of M. tuberculosis in clinical specimens. An advance over the in-lab-developed PCR tests and the existing FDA-cleared commercial assays is the GeneXpert MTB/RIF test (Cepheid, Sunnyvale, CA), a real-time multiplex PCR method that both identifies the Mtb complex and also detects genes that encode rifampin resistance. One of the earlier publications on this method (see reference Boehme) reported a sensitivity for smear positive respiratory specimens of 98.2% and for smear negative samples, 72.5%. Overall specificity was 99.2%. In terms of the detection of rifampin resistance, the assay does detect the common mutations, but discrepancies between phenotypic test results and genotypic results still challenge complete reliance on this component of the test. This assay is not yet widely available in the United States but is available in other countries.

The characterization of specific strains of M. tuberculosis can be important for clinical and epidemiologic purposes. It facilitates tracking transmission, analysis of outbreaks of tuberculosis, and demonstration of reactivation versus reinfection in individual patients. DNA fingerprinting is done using a standardized protocol based on restriction fragment length polymorphism. Many copies of the insertion sequence 6110 (IS6110) are present in the chromosome of most strains of M. tuberculosis, and these are located at variable positions. DNA fragments are generated by restriction endonuclease digestion and separated by electrophoresis. A probe against IS6110 is used to determine the genotypes. Other useful methods for strain characterization include spoligotyping, a PCR-based technique that targets the direct repeat locus of M. tuberculosis and mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) analysis. The latter method is slowing replacing IS6110 typing. Genotyping is done at the CDC, at some state health department laboratories, and in research laboratories.

مواضيع ذات صلة

α1-antitrypsin

Albumin

Glucose, blood (Blood sugar, Fasting blood sugar [FBS])

Gliadin, endomysial, and tissue transglutaminase antibodies

Antimicrobial Susceptibility Testing and Therapy of Staphylococcus, Micrococcus, and Similar Organisms

Glucagon

genetic testing (Breast cancer [BRCA] and ovarian cancer, Colon cancer, Cardiovascular disease, Tay–Sachs disease, Cystic fibrosis, Melanoma, Hemochromatosis, Thyroid cancer, Paternity [parentage analysis], Forensic genetic testing)

Tuberculin Test

Clinical Relevance of Plasma Cell Dyscrasias

Liver Cancers

Biomarkers of Alcoholism

Gastrin