Rapid detection of rifampin-resistant Mycobacterium tuberculosis directly from stained sputum smears using single-tube nested polymerase chain reaction deoxyribonucleic acid sequencing.

Microscopy is the mainstay of laboratory diagnosis of tuberculosis especially in resource poor countries. The World Health Organization has also recommended microscopy as the mainstay of diagnosis for directly observed treatment, short course. Using DNA extracts from Ziehl-Neelsen (ZN)-stained sputum smears, a single-tube nested polymerase chain reaction was optimized to confirm Mycobacterium tuberculosis complex and detect rifampin (RIF) resistance by sequencing, using a combination of novel (rpoB47 and rpoB158) and previously described (rpoB105 and rpoB293) primers. Carryover DNA was strictly monitored using several negative controls, and inhibition was ruled out by spiked controls. No such target was detected from negative controls and purified genomic DNA from other nontubercular mycobacteria. Resistance could be detected in 91.1% (51/56) slides. The results obtained were concordant with the 1% proportion method and DNA sequencing performed on culture isolates. Our results demonstrate that the method is suitable for rapid detection of susceptibility to RIF in acid-fast bacillus-positive ZN-stained slides obtained from patients suspected of harboring drug-resistant M. tuberculosis.