Molecular detection of viable Mycobacterium tuberculosis complex in clinical specimens

Tuberculosis (TB) is the global health concern because of the rising incidence and mortality. TB is caused by Mycobacterium tuberculosis complex (MTBC) and transmitted via inhalation of infectious droplet nuclei circulating in the air. The World Health Organization (WHO) recommends the use of Acid-fast (AFB) smear microscopy, MTBC culture, and molecular detection of MTBC DNA for diagnosis of TB. The response to treatment should be acquired as soon as possible to reduce disease transmission and drug resistance. Although MTBC culture is a gold standard, it is laborious, expensive, and may take up to 8 weeks. The AFB staining and molecular detection of MTBC DNA are faster, but they cannot differentiate live and dead MTBC. Ribosomal RNA precursor (pre-rRNA) was previously used as a molecular biomarker to indicate viable bacteria. Therefore, this study aimed to evaluate pre-rRNA MTBC detection by RT-PCR (pre-rRNA MTBC) as a new method to detect viable MTBC in clinical specimens. The total number of 564 clinical specimens were processed by the conventional WHO protocol of decontamination and concentration before RNA extraction. The results showed that pre-rRNA MTBC detection had a sensitivity of 33.33% for all specimens and 52.77% for positive AFB specimens. The specificity and positive predictive value (PPV) is 100%. When 45 specimens were prepared by the modified protocol to remove NaOH in the decontamination solution and washing before used, the sensitivity increased to 57.14% for all specimens and 76.92% for positive AFB specimens and the specificity and positive predictive value (PPV) is100%, which were comparable to those of the Fluorescein diacetate (FDA) vital staining. The modified method of sample preparation (protocol 2) can improve sensitivity of pre-rRNA MTBC for detection viable MTBC, especially in positive AFB specimens. In summary, pre-rRNA MTBC RT-PCR can be used for rapid detection of viable MTBC in clinical specimens.