An optimized method for purifying, detecting and quantifying Mycobacterium tuberculosis RNA from sputum for monitoring treatment response in TB patients

Diagnostics that more accurately detect and quantify viable Mycobacterium tuberculosis (Mtb) in the sputum of patients undergoing therapy are needed. Current culture- and molecular-based tests have shown limited efficacy for monitoring treatment response in TB patients, either due to the presence of viable sub-populations of Mtb which fail to grow under standard culture conditions (termed differentially detectable/culturable Mtb, DD Mtb) or the prolonged half-life of Mtb DNA in sputum. Here, we report an optimized RNA-based method for detecting and quantifying viable Mtb from patient sputum during the course of therapy. We first empirically derived a novel RNA extraction protocol from sputum that improves recovery of Mtb RNA while almost completely eliminating contamination from Mtb DNA and host nucleic acids. Next, we identified five Mtb 16S rRNA primer sets with varying limits of detection that were capable of distinguishing between live versus dead H37Rv Mtb. This combined protocol was then tested on sputa from a longitudinal cohort of patients receiving therapy for drug sensitive (DS) or drug resistant (DR) TB with first-line or second-line regimens, respectively. Results were compared with that of culture, including CFU, BACTEC MGIT, and a limiting dilution assay capable of detecting DD Mtb. The five 16S rRNA primer sets positively identified nearly all (range 94–100%) culture positive sputa, and a portion (19–37%) of culture negative sputa. In comparison, ten highly expressed Mtb mRNAs showed positivity in 72–86% of culture positive sputa, and in 0–13% of culture negative sputa. Two of the five 16S rRNA primer sets were able to positively identify 100% of culture positive sputa, and when tested on culture negative sputa from the DS cohort at 2 months post-initiation of therapy, identified 40% of samples as positive; a percentage that is in line with expected treatment failure rates when first-line therapy is discontinued early. These two primer sets also detected 16S rRNA in 13–20% of sputa at 6 months post-initiation of therapy in the DR cohort. Cycle threshold values for 16S rRNA showed a strong correlation with Mtb numbers as determined by culture (R > 0.87), including as Mtb numbers declined during the course of treatment with first-line and second-line regimens. The optimized molecular assay outlined here may have utility for monitoring treatment response in TB patients.