Participation criteria for clinical trials in pulmonary tuberculosis commonly include confirmation of sputum positive for mycobacteria and an indication of drug susceptibility before treatment is initiated. We investigated the suitability of two novel sputum-based nucleic acid amplification methods for patient selection in a recent early bactericidal activity study. Spontaneously expectorated sputum samples of 140 consecutive pulmonary tuberculosis patients were examined with direct fluorescence microscopy, Genotype MTBDRplus assay (MTBDR), Xpert MTB/RIF assay (Xpert), and liquid mycobacterial culture. The methods detected mycobacteria or mycobacterial DNA in 96.8%, 90.5%, 92.9%, and 92.1% of samples, respectively. MTBDR, Xpert, and liquid culture were 100% concordant for detection of resistance to rifampin. Sensitivity and specificity of MTBDR for detection of isoniazid resistance were 83.3% and 100%, respectively. For quantification of mycobacterial sputum load, we found a correlation between Xpert DNA amplification cycle thresholds, time to positivity, and microscopy smear grade. The best correlation was found between Xpert and time to positivity (r ‫؍‬ 0.54), which were both correlated with smear microscopy with r values equal to ؊0.40 and ؊0.48, respectively. We conclude that MTBDR and Xpert are suitable screening tools for determining rifampin resistance in sputum microscopy smear-positive patients before participation in tuberculosis trials. Xpert should be further explored as a surrogate measurement for sputum mycobacterial load.

No abstract

The early bactericidal activity of antituberculosis agents is usually determined by measuring the reduction of the sputum mycobacterial load over time on solid agar medium or in liquid culture. This study investigated the value of a quantitative PCR assay for early bactericidal activity determination. Groups of 15 patients were treated with 6 different antituberculosis agents or regimens. Patients collected sputum for 16 h overnight at baseline and at days 7 and 14 after treatment initiation. We determined the sputum bacterial load by CFU counting (log CFU/ml sputum, reported as mean ؎ standard deviation [SD]), time to culture positivity (TTP, in hours [mean ؎ SD]) in liquid culture, and Xpert MTB/RIF cycle thresholds (C T , n [mean ؎ SD]). The ability to discriminate treatment effects between groups was analyzed with one-way analysis of variance (ANOVA). All measurements showed a decrease in bacterial load from mean baseline (log CFU, 5.72 ؎ 1.00; TTP, 116.0 ؎ 47.6; C T , 19.3 ؎ 3.88) to day 7 (log CFU, ؊0.26 ؎ 1.23, P ‫؍‬ 0.2112; TTP, 35.5 ؎ 59.3, P ‫؍‬ 0.0002; C T , 0.55 ؎ 3.07, P ‫؍‬ 0.6030) and day 14 (log CFU, ؊0.55 ؎ 1.24, P ‫؍‬ 0.0006; TTP, 54.8 ؎ 86.8, P < 0.0001; C T , 2.06 ؎ 4.37, P ‫؍‬ 0.0020). The best discrimination between group effects was found with TTP at day 7 and day 14 (F ‫؍‬ 9.012, P < 0.0001, and F ‫؍‬ 11.580, P < 0.0001), followed by log CFU (F ‫؍‬ 4.135, P ‫؍‬ 0.0024, and F ‫؍‬ 7.277, P < 0.0001). C T was not significantly discriminative (F ‫؍‬ 1.995, P ‫؍‬ 0.091, and F ‫؍‬ 1.203, P ‫؍‬ 0.316, respectively). Culture-based methods are superior to PCR for the quantification of early antituberculosis treatment effects in sputum. Several novel antituberculosis drugs and regimens are currently under clinical investigation. The first step in their evaluation is the measurement of early bactericidal activity (EBA) in sputum over up to 2 weeks of treatment in smear-positive, treatmentnaive pulmonary tuberculosis patients. The EBA is commonly defined as the mean daily decrease in CFU counted on agar plates (log CFU) per ml of expectorated sputum (1, 2). Alternatively, the change in time to culture positivity (TTP) in broth culture can be measured in the mycobacterial growth indicator tube (MGIT) system (Becton, Dickinson, Sparks, MD) (3). This is based on the inverse relationship of the time a culture requires to develop a critical measure of metabolic activity to the number of viable bacteria initially inoculated into the system. TTP in semiautomated liquid culture has been shown to correlate well with CFU counting in the first 2 weeks of treatment (3-5).The Xpert MTB/RIF assay (Xpert; Cepheid, Sunnyvale, CA) is a new nucleic acid amplification test (NAAT) detecting M. tuberculosis complex on sputum specimens with real-time PCR. Xpert is rapid (less than 1 h of hands-on time), standardized, and easy to use (6, 7). Its sensitivity to detect M. tuberculosis in sputum of untreated patients suspected of having tuberculosis in high-burden countries is 98% for AFB-positive samples and 72% for AFBnegat...

No abstract

Mycobacterium tuberculosis strains with spontaneous mutations conferring resistance to rifampin (RIF) are exceedingly rare, and fixed drug combinations typically prevent augmentation of resistance to single drugs. Fourteen newly diagnosed tuberculosis patients were treated with RIF alone for 14 days, and bacterial loads, including mutation frequencies, were determined. A statistical model estimated that 1% of the remaining viable mycobacteria could be RIF resistant after 30 days of monotherapy. This indicates that temporal and spatial windows of RIF monotherapy due to uneven drug distribution within lung lesions could contribute to the acquisition of resistance to RIF.KEYWORDS tuberculosis, rifampin, treatment, resistance D rug resistance is a peculiarity of Mycobacterium tuberculosis in which resistance to antibiotics is genetically encoded. Resistant M. tuberculosis can be transmitted from one person to another, but resistance cannot be transmitted from one bacterium to another (1). Mycobacteria with spontaneous resistance mutations are naturally present in every population of M. tuberculosis (2, 3). The mutation rate, i.e., the probability that a resistance-conferring mutation occurs spontaneously in a bacterium, is very low for rifampin (RIF), the cornerstone of current treatment regimens, with 3.32 ϫ 10 Ϫ9 for M. tuberculosis strain H37Rv (4). However, Beijing strains, which are more prevalent in the Eastern and Western Cape provinces, South Africa, were found to acquire drug resistance in vitro faster than other strains (5-7).It is generally accepted that clinical drug resistance, i.e., the failure of antibiotic treatment to control tuberculosis (TB) in a patient, can emerge through augmentation of a small initial population of resistant mycobacteria that become dominant when susceptible bacteria are eliminated by antibiotic treatment (1, 8). Thus, antibiotic treatment combining different agents can prevent the possibility that a mutant which is resistant to a single antibiotic is able to become clinically relevant, and an effective combination has been shown to provide cure within 6 to 8 months, also in an outpatient setting (9-11). In the absence of a better explanation, patient noncompliance with the prescribed treatment has been blamed for drug resistance, but other lines of evidence, such as the hollow fiber model, have confirmed that noncompliance alone is unlikely to create drug resistance when a fixed-dose combination is used (12). A more convincing theory is pharmacokinetic and pharmacodynamic variability between patients and within patients, referring to differences in drug absorption and metabolism, uneven drug distribution, and bacterial subpopulations that are not equally susceptible. All these factors could conspire to create pockets or temporal windows of monotherapy within patients' lungs where resistance develops (13). Recently published work by Prideaux and coauthors appears to confirm this theory, with

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.

No abstract