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Laboratory Diagnosis

Although data from 2008 to 2009 indicates a significant decrease in reported cases of M. tuberculosis in the United States, the number of latent infections (potential carriers of disease) includes 14 million US residents and one third of the global population. Multidrug-resistant tuberculosis (MDR TB) and extensively drug-resistant TB (XDR-TB) are inclusive in this data and prevention depends on the effort of both public and private laboratories to isolate, identify, and employ antimicrobial susceptibility testing (AST) against all available anti-tuberculosis antibiotics, according to methods approved by the Centers for Disease Control and Prevention (CDC) and the Food and Drug Administration (FDA).
Specimen collection and processing
Respiratory specimens of sputum, bronchial washings, fine-needle aspirates of pleural fluid, and lung biopsies comprise most of the specimen collection types necessary to rule out presence of M. tuberculosis. However, urine, gastric, pericardial fluid, blood, and other sterile fluids also may be submitted. Specimens form non-sterile sites must be decontaminated prior to culture (eg, respiratory, wound, urine, fecal, etc.). All procedures are performed in a class II biosafety cabinet (BSC), mandated by the CDC to eliminate contamination of the surrounding environment. The cabinet is installed in a self-contained area of the laboratory, designated specifically for isolating Mycobacteria species, where a “negative atmosphere” is maintained. Negative pressure is created inside the cabinet when air is forced out through HEPA filters and ducts to outside the building, thus preventing the flow of infectious materials into the laboratory atmosphere. Regular inspections and servicing are also mandated by the CDC and the Occupational Safety and Health Administration (OSHA) to ensure a high-level functioning cabinet, as well as maintenance of adequate ventilation.
Digestion and decontamination
To liquefy or digest specimens of sputum and bronchial washings, N-Acetyl-L-Cysteine (NALC), dithiothreitol, and enzymes are used for processing with the addition of sodium hydroxide (NaOH) as a decontaminant. The acceptable formula of digestant/decontaminant, NALC-2% NaOH may be considered too harsh a method for recovery of certain, fastidious mycobacteria. In such cases, C(18)-carboxypropylbetaine may be used to increase recovery, but will not prevent bacterial contamination. Technologists are trained in the various techniques and their limitations. For example, a rate of media contamination greater than 5% is considered excessive; yet a concentration greater than 4% NaOH may be inhibitory to the growth of acid-fast bacteria (AFB).
Briefly, the specimen is diluted with an equal volume of digestant/decontaminant and allowed to stand at room temperature. After neutralization with a buffering solution, the specimen is centrifuged to concentrate any AFB present. Centrifugation should be at ≥ 3,000 X g for 15 minutes for maximum recovery.
It should be noted that cross-contamination must be avoided during processing. A false-positive culture leading to mis-diagnosis in a patient would have devastating consequences for everyone involved.