Alternative sputum preparation to improve polymerase chain reaction assay for Mycobacterium tuberculosis detection

BACKGROUND Tuberculosis (TB), one of the major airborne infectious bacterial diseases, remains an important health problem worldwide. It is estimated that there are 1700 new cases per year in Santa Catarina State, Brazil. OBJECTIVE To improve polymerase chain reaction (PCR) sensitivity in detecting Mycobacterium tuberculosis in sputum samples. METHODS This study proposed the use of glass beads as a modification of the routine protocol for sputum preparation used in the Laboratory of Molecular Biology and Mycobacteria at the Federal University of Santa Catarina, Florianopolis, Brazil. The study comprised 120 sputum samples, 60 of which were treated with the routine protocol, while 60 were treated with the modified protocol using glass beads. RESULTS Samples treated with the routine protocol had a sensitivity of 56.7% (95%CI 44.1-69.2) in 16S rRNA PCR and 81.7% (95%CI 71.9-91.5) in insertion sequence (IS) 6110 PCR, compared with culture. Samples treated with the modified protocol had a sensitivity of 73.3% (95%CI 62.1-84.5) in 16S rRNA PCR and 100% in IS6110 PCR. CONCLUSION The modified protocol using glass beads greatly improved mycobacterial detection in sputum samples compared with the routine protocol.