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BMC Infect Dis. 2018 Jul 3;18(1):293. doi: 10.1186/s12879-018-3213-7.

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

Author information

1
Pulmonary Section, Denver VA Medical Center, Denver, CO, USA. nicholas.walter@ucdenver.edu.
2
Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Denver, Aurora, CO, USA. nicholas.walter@ucdenver.edu.
3
Division of Biostatistics and Bioinformatics, National Jewish Health, Denver, CO, USA.
4
Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Denver, Aurora, CO, USA.
5
Department of Epidemiology, Colorado School of Public Health, Aurora, CO, USA.
6
Infectious Disease Research Collaboration, Kampala, Uganda.
7
Department of Medicine, Mulago Hospital, Makerere University, Kampala, Uganda.
8
Division of Pulmonary and Critical Care Medicine, San Francisco General Hospital, University of California San Francisco, San Francisco, CA, USA.
9
HIV, Infectious Diseases, and Global Medicine Division, San Francisco General Hospital, University of California San Francisco, San Francisco, CA, USA.
10
Pulmonary Associates, Burlingame, CA, USA.
11
Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA.
12
Yale School of Public Health, New Haven, CT, USA.
13
Division of Pulmonary, Critical Care, and Sleep Medicine Section, Yale School of Medicine, New Haven, CT, USA.

Abstract

BACKGROUND:

According 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.

METHODS:

We 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.

RESULTS:

We 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.

DISCUSSION:

Using 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.

CONCLUSIONS:

A 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.

KEYWORDS:

Differentially culturable; Drug tolerance; Mycobacterium tuberculosis; Sputum; Treatment; Tuberculosis

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