Sputum AFB smear, NAA testing and culture in pulmonary tuberculosis

What it is

The assays used in the diagnosis of active tuberculosis answer the same clinical laboratory question, are mycobacteria present in a clinical specimen, and they differ in speed, in sensitivity and in what still has to be done after them.

How it is diagnosed

Collection comes first: three sputum specimens for AFB smear microscopy and culture, at least one of them also tested using an NAA test. Sputum is preferably 3 samples including an early morning sample, induced with nebulised hypertonic saline if the patient is not expectorating.

• Acid-fast stain gives same-day results with simple, inexpensive technology. Direct microscopy of sputum is the most important first step, and the probability of detecting acid-fast bacilli is proportional to the bacillary burden, typically positive when 5000-10 000 organisms are present. Its limits are that it is less sensitive than culture, requiring 10,000 organisms/mL, and it cannot distinguish M. tuberculosis from other mycobacteria. Ziehl-Neelsen and auramine fluorescence are the stains; auramine fluorescence is more sensitive though less specific than Ziehl-Neelsen, and by fluorescence microscopy bacilli appear as yellow-orange on a green background.
• Culture on solid media (Lowenstein-Jensen egg-based or Middlebrook agar-based) is the gold standard for isolating Mycobacterium tuberculosis. It detects 10-100 organisms/mL, shows colony morphology, detects mixed infection, allows quantification of growth, and provides organisms for speciation, strain identification and susceptibility testing. Visible growth takes 3-8 wk, or 4-6 weeks. Culture in liquid broth has similar sensitivity and specificity, automated systems decrease workload, and growth is detected in 7-21 days, or 1-3 weeks, but it does not show colony morphology, detect mixed cultures or quantify growth.
• Nucleic acid amplification gives same-day results with sensitivity intermediate between acid-fast stain and culture, and identifies organisms as members of the M. tuberculosis complex. It requires advanced laboratory techniques, cannot distinguish dead from viable organisms, and culture is still needed for speciation, strain identification and susceptibility testing. GeneXpert MTB/RIF has sensitivity that nearly approximates culture, requires 100 minutes and detects rifampin resistance mutations, at the price of expensive equipment and detection of dead bacilli. PCR is only useful at the initial stage of diagnosis, since it frequently remains positive despite treatment.
• Urinary antigen detection, for example ELISA for LAM, has potential for point-of-care testing and relatively high sensitivity in advanced untreated AIDS, under 50 CD4 T cells, but sensitivity is very low in any other situation.