Laboratory Diagnosis

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The page below is a sample from the LabCE course Tracking Antibiotic-Resistant Tuberculosis. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

Learn more about Tracking Antibiotic-Resistant Tuberculosis (online CE course)
Laboratory Diagnosis

TB strategies for prevention depend on the efforts 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 the presence of M. tuberculosis.
  • Urine, gastric, pericardial fluid, blood, and other sterile fluids also may be submitted.
  • Specimens from non-sterile sites must be decontaminated prior to culture (eg, respiratory, wound, urine, fecal).
All procedures are performed in a class II biosafety cabinet (BSC), mandated by 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 for the recovery of certain, fastidious mycobacteria. In such cases, C(18)-carboxypropylbetaine may be used to increase recovery but will not prevent bacterial contamination.
  • Note that a rate of media contamination greater than 5% is considered excessive; but a concentration greater than 4% NaOH may inhibit the growth of acid-fast bacteria (AFB).
Digesting procedure
  • 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 misdiagnosis in a patient would have devastating consequences for everyone involved.