The superoxide dismutase gene, a target for detection and identification of mycobacteria by PCR.

The superoxide dismutase gene has been identified as a target in screening for the presence of mycobacteria on the genus level and differentiating relevant mycobacterial species from one another by PCR. Consensus primers deduced from known superoxide dismutase gene sequences allowed the amplification of DNAs from a variety of bacteria, fungi, and protozoa. Selected amplicons from Actinomyces viscosus, Corynebacterium diphtheriae, Corynebacterium pseudodiphtheriticum, Mycobacterium avium, M. fortuitum, M. gordonae, M. intracellulare, M. kansasii, M. scrofulaceum, M. simiae, M. tuberculosis, M. xenopi, and Nocardia asteroides were subsequently cloned and sequenced. The alignment of those sequences facilitated the selection of primers targeting conserved regions present in myobacterial species but absent in nonmycobacterial species and thus allowed the genus-specific amplification of all 28 different mycobacterial species tested. A pool of genus-specific allowed the genus-specific amplification of all 28 different mycobacterial species tested. A pool of genus-specific probes recognized 23 of the 28 mycobacterial species and did not cross-react with any of the 96 nonmycobacterial species tested. In addition, probes recognizing species-specific variable regions within the superoxide dismutase genes of M. avium, M. fortuitum, M. gordonae, M. intracellulare, M. kansasii, M. scrofulaceum, M. simiae, the M. tuberculosis complex, and M. xenopi were identified. All probes recognized only the species from which they were derived and did not cross-react with any other mycobacterial species or with any of the nonmycobacterial species tested. We conclude that the superoxide dismutase gene is a suitable target for amplifying mycobacteria by PCR on the genus level, confirming correct amplification by genus-specific probes, and differentiating relevant species from one another by a set of species-specific probes.

Monitoring treatment of patients with pulmonary tuberculosis: can PCR be applied?

To assess whether PCR is applicable for monitoring the efficacy of antituberculous treatment, respiratory specimens obtained during treatment and follow-up from sputum smear-positive tuberculosis (TB) patients were examined. First, results of smear, culture, and PCR for Mycobacterium tuberculosis complex (MTB) and an internal inhibition control (MCC) were correlated retrospectively on 1,601 respiratory specimens from patients with no previous cultures of MTB. MTB optical density (OD) values increased to a maximum level of 3.5 to 4.0, with both increasing numbers of acid-fast bacilli and CFU. MTB/MCC OD ratios also increased with both smear and culture grading and correlated significantly better with both than the MTB OD value. Second, changes in MTB OD values and MTB/MCC OD ratios were compared with microscopy and culture for MTB in monthly sputa obtained during treatment and follow-up in 22 smear-positive pulmonary TB patients. Declines in MTB/MCC OD ratios during antituberculous treatment and follow-up were observed. Patients with moderate disease reached the baseline after 6 to 8 months of standard antituberculous treatment regimen, whereas patients with extensive disease were predicted to reach the baseline 1 year or more after the initiation of treatment. Although PCR detects both dead and live bacteria, we believe that PCR can be used to assess the efficacy of antituberculous treatment since increases or slow reductions in MTB/MCC OD ratios would indicate nonoptimal treatment, noncompliance, reduced bioavailability of drugs, or resistant strains of MTB and thereby would identify patients at risk for treatment failure or reactivation.