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We evaluated the sensitivity of a DNA amplification test for the detection of Mycobacterium avium in blood samples using different blood components and different DNA extraction methods. M. avium-inoculated blood samples were processed to obtain separate blood components: peripheral blood mononuclear cells (PBMCs), polymorphonuclear cells (PMNCs), and whole-blood sodium dodecyl sulfate (SDS)-lysate pellets. The sensitivity for the detection of the lowest mycobacterial load (1 CFU/ml) was significantly greater (P < 0.01) with DNA extracted from SDS-lysate pellets than with DNA extracted from PBMCs or PMNCs. Subsequently, DNA extraction methods based on guanidine NaOH, and proteinase were compared. The sensitivity of the guanidine-based method was significantly greater (P < 0.01) than those of the others.

Differentiation between Mycobacterium tuberculosis and M. avium is essential for the treatment of mycobacterial infections. We have developed an easy and rapid detection assay for the diagnosis of mycobacterial diseases. This is a PCR-hybridization assay based on selective amplification of a 16S rRNA gene sequence using pan-Mycobacterium primers followed by hybridization of the amplification products to biotinylated M. tuberculosis and M. avium-specific probes. A total of 55 mycobacterial isolates were tested. For all isolates, results concordant with those of conventional identification methods were obtained. Moreover, we developed a method for extraction of DNA from Ziehl-Neelsen-positive smears which allows the recovery of intact target DNA in our PCR-hybridization assay. Our method was able to confirm all culture results for 59 Ziehl-Neelsen-positive smears from clinical specimens (35 sputum, 11 lymph node biopsy, 6 stool, 4 pus, 2 urine, and 1 pericardial fluid specimens). These data suggest that our PCR-hybridization assay, which is simple to perform and less expensive than commercial probe methods, may be suitable for the identification of M. tuberculosis and M. avium. It could become a valuable alternative approach for the diagnosis of mycobacterial infections when applied directly to DNA extracted from Ziehl-Neelsen-positive smears as well.

We compared the sensitivities and specificities of four nested PCR assays for the detection of Mycobacterium tuberculosis from formalin-fixed, paraffin-embedded tissues. Thirty-seven autopsy samples from human immunodeficiency virus-positive patients were analyzed: 15 were M. tuberculosis positive, 11 served as negative controls, and 11 were Ziehl-Neelsen positive without cultural confirmation of M. tuberculosis. Three genomic sequences (mtp40, 65-kDa antigen gene, and IS6110) with different molecular masses and numbers of repetitions within the M. tuberculosis genome were targeted. On the IS6110 sequence, two fragments of different sizes (106 and 123 bp, respectively) were amplified with two separate pairs of primers. The highest sensitivity rates were obtained by amplifying the highly repetitive IS6110 insertion sequence, and the different primers tested showed a sensitivity ranging from 80 to 87%. Amplification of the large 223-bp fragment of the mtp40 sequence present in a single copy in the M. tuberculosis genome yielded a high rate of false-negative results, ranging from 66 to 80%. A poor sensitivity (from 47 to 60%) was also shown by PCR amplification of the 142-bp 65-kDa antigen gene. All the PCRs except that for the 65-kDa antigen gene showed a specificity of 100%. Moreover, different results were obtained with different dilutions of DNA, and DNA concentrations of 1 and 3 microg yielded the highest sensitivities depending upon which protocol was used. Application of the PCRs to the Ziehl-Neelsen-positive, culture-negative samples confirmed the sensitivities of the PCRs obtained with the control samples. In conclusion, PCR can successfully be used to detect M. tuberculosis from paraffin-embedded tissues and can be particularly useful in the validation of a diagnosis of tuberculosis in clinical settings in which the diagnosis is uncertain. However, the efficacy of PCR strictly depends on several amplification parameters such as DNA concentration, target DNA size, and the repetitiveness of the amplified sequence.