Does discovery of differentially culturable M tuberculosis really demand a new treatment paradigm? Longitudinal analysis of DNA clearance from sputum

Abstract

BackgroundAccording to the traditional tuberculosis (TB) treatment paradigm, the initial doses of treatment rapidly kill most Mycobacterium tuberculosis (Mtb) bacilli in sputum, yet many more months of daily treatment are required to eliminate a small, residual subpopulation of drug-tolerant bacilli. This paradigm has recently been challenged following the discovery that up to 90% of Mtb bacilli in sputum are culturable only with growth-factor supplementation. These “differentially culturable” bacilli are hypothesized to be more drug-tolerant than routinely culturable bacilli. This hypothesis implies an alternative paradigm in which TB treatment does not rapidly reduce the total Mtb population but only the small, routinely culturable subpopulation. To evaluate these competing paradigms, we developed a culture-independent method for quantifying the viable fraction of Mtb bacilli in sputum during treatment.MethodsWe used GeneXpert MTB/RIF to quantify Mtb DNA in sputa collected longitudinally from Ugandan adults taking standard 4-drug treatment for drug-susceptible pulmonary TB. We modeled GeneXpert cycle thresholds over time using nonlinear mixed-effects regression. We adjusted these models for clearance of DNA from killed-but-not-yet-degraded bacilli, assuming clearance half-lives ranging from 0 to 1.25 days. We used a convolution integral to quantify DNA from viable bacilli only, and converted cycle thresholds to Mtb genomic equivalents. We replicated our results in a South African cohort.ResultsWe enrolled 41 TB patients in Uganda. Assuming a DNA-clearance half-life of 0 days, genomic equivalents of viable sputum bacilli decreased by 0.22 log/day until 8.8 days, then by 0.07 log/day afterwards. Assuming a DNA-clearance half-life of 1.25 days, genomic equivalents of viable bacilli decreased by 0.36 log/day until 5.0 days, then by 0.06 log/day afterwards. By day 7, viable Mtb had decreased by 97.2–98.8%. We found similar results for 19 TB patients in South Africa.DiscussionUsing a culture-independent method, we found that TB treatment rapidly eliminates most viable Mtb in sputum. These findings are incompatible with the hypothesis that differentially culturable bacilli are drug-tolerant.ConclusionsA culture-independent method for measuring viable Mtb in sputum during treatment corroborates the traditional TB treatment paradigm in which a rapid bactericidal phase precedes slow, elimination of a small, residual bacillary subpopulation.Electronic supplementary materialThe online version of this article (10.1186/s12879-018-3213-7) contains supplementary material, which is available to authorized users.

Differentially Detectable Mycobacterium tuberculosis Cells in Sputum from Treatment-Naive Subjects in Haiti and Their Proportionate Increase after Initiation of Treatment

“… M. tuberculosis strains that can be considered viable only by a criterion other than standard culture methods are, by definition, DD M. tuberculosis , whether or not they are demonstrable following an LD assay such as that used here. In contrast, another recent study based on an estimation of M. tuberculosis DNA in sputum samples suggested that the apparent initial efficacy of therapy argues against the relevance of DD M. tuberculosis ( 21 ). The results of that work are difficult to interpret without associated culture-based quantification, given the variability in the presence of DD M. tuberculosis found before treatment in our study.…”

Clinically encountered growth phenotypes of tuberculosis-causing bacilli and their in vitro study: A review

“…Whether these methods of improving culture yield by finding DD Mtb can meaningfully improve rates of diagnosis and whether the more accurate quantification of mycobacterial load in some patients is clinically useful is being studied. It remains unclear and debated ( Walter et al., 2018 ). McAulay et al.…”