Multiplex PCR assay for immediate identification of the infecting species in patients with mycobacterial disease

Rapid identification of infecting mycobacterial species enables appropriate medical care decisions to be made. Our aim was to demonstrate the clinical usefulness of the multiplex PCR assay, a test based on PCR, which permits direct identification of 12 mycobacterial species in clinical specimens. A total of 259 specimens from 177 patients who had clinical symptoms of mycobacterial disease but for whom there were difficulties in diagnosis were tested. Specimens were analyzed within 48 h of receipt of the sample. Mycobacteria were identified in 102 specimens; 66 specimens contained nontuberculous mycobacteria, and 36 specimens contained Mycobacterium tuberculosis complex mycobacteria. The PCR assay identified the mycobacterial species in 43 (97.7%) of 44 microscopy- and culture-positive specimens and in 15 (93.8%) of 16 culture-positive, microscopy-negative specimens. It also permitted species identification in infections caused by more than one mycobacterial species. For 56 (96.5%) of the 58 specimens from patients with infections caused by opportunistic mycobacteria, the organisms were identified with the PCR assay. The test was useful also for the identification of fastidious mycobacteria, e.g., M. genavense, and those that cannot be cultured, e.g., M. leprae. After resolution of discrepant results, the sensitivity of the PCR assay was 97.9%, the specificity was 96.9%, the positive predictive value was 95.0%, and the negative predictive value was 98.7%. For culture these values were 60.8, 100, 100, and 81.0%, respectively. Thus, the multiplex PCR assay enables prompt diagnosis when rapid identification of infecting mycobacteria is necessary

A fast method for the identification of Mycobacterium tuberculosis in sputum and cultures based on thermally assisted hydrolysis and methylation followed by gas chromatography-mass spectrometry

A fast gas chromatography-mass spectrometry (GC-MS) method with minimum sample preparation is described for early diagnosis of tuberculosis (TB). The automated procedure is based on the injection of sputum samples which are then methylated inside the GC injector using thermally assisted hydrolysis and methylation (THM). The THM-GC-MS procedure was optimized for the injection of sputum samples. For the identification of Mycobacterium tuberculosis the known marker tuberculostearic acid (TBSA) and other potential markers were evaluated. Hexacosanoic acid in combination with TBSA was found to be specific for the presence of M. tuberculosis. For validation of the method several sputum samples with different viscosities spiked with bacterial cultures were analyzed. Finally, 18 stored sputum samples collected in Vietnam from patients suspected to suffer from TB were re-analyzed in Amsterdam by microscopy after decontamination/concentration and using the new THM-GC-MS method. No false positives were found by THM-GC-MS and all patients who were diagnosed with TB were also found positive using our newly developed THM-GC-MS method. These results show that the new fast and sensitive THM-GC-MS method holds great potential for the diagnosis of TB