Simultaneous measurement of 16S-rRNA and pre-16S-rRNA as a strategy to monitor clinical tuberculosis.

Abstract

Culture-based biomarkers of TB treatment response monitoring, e.g., Mycobacterial Growth Indicatory Tube (MGIT), are compromised when bacteria enter a non-replicating persister phase limiting the measurement of antibiotic efficacy and resistance. Understanding how antibiotic exposure to antibiotics alters bacterial physiology could help develop more effective TB therapies. We developed a novel assay with simultaneous measurement of 16S rRNA (bacterial burden) and its precursor, pre-16S rRNA (metabolic activity), and tested it on samples from patients in a trial of optimised-dose rifampicin. We developed a multiplex reverse transcriptase quantitative PCR assay (RT-qPCR) to measure relative gene expression of pre-16S rRNA and 16S rRNA in pre-treatment (control) and sequential samples from patients in the Phase II HIGHRIF2 (NCT00760149) clinical trial. We constructed a mathematical model to assess changes in pre-16S gene expression relative to 16S rRNA over time, facilitating the comparison of rifampicin doses' efficacy. In a retrospective study of 19 patients, pre-16S rRNA and 16S rRNA decreased steadily during the initial 36 days of treatment. This was evidenced by the rising cycle threshold (Cq) values slope 0.404 and 0.212, respectively, however, pre-16S rRNA decreased significantly quicker (P<0.0001). The changes in the relative gene expression of pre-16S rRNA during treatment fitted a double exponential decay curve (R² = 0.996). According to this model, 1200 mg RIF-containing therapy exerted the most potent and rapid impact on pre-16S rRNA expression (Maximum suppression (R_min)=1.694, T (time) =9.78 days), and also resulted in the swiftest daily reduction in bacterial load (-0.072 log₁₀ CFU ml^-1/day). The pre-16S rRNA and 16S rRNA gene expression multiplex PCR reported here provides an easy to use and rapid marker of drug efficacy and has potential to assess the efficacy of existing or novel drug combinations.