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

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.

Acquisition of Rifampin Resistance in Pulmonary Tuberculosis.

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.

Direct comparison of Xpert MTB/RIF assay with liquid and solid mycobacterial culture for quantification of early bactericidal activity.

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.

Suitability of Xpert MTB/RIF and genotype MTBDRplus for patient selection for a tuberculosis clinical trial.

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.

Propidium monoazide and Xpert MTB/RIF to quantify Mycobacterium tuberculosis cells.

Propidium monoazide (PMA) penetrates non-viable cells with compromised membranes. PMA has been proposed to improve the specificity of Xpert MTB/RIF (Xpert) for the detection of viable Mycobacterium tuberculosis. This study assessed the effect of PMA on Xpert cycle thresholds (CT) of M. tuberculosis made non-viable under antibiotic pressure. In vitro, we measured the difference between CT with and without PMA (ΔCT) in liquid cultures treated with one of six anti-tuberculosis drugs (isoniazid, rifampin, pyrazinamide, ethambutol, streptomycin, moxifloxacin) and found significant ΔCT only with isoniazid and ethambutol for pan-susceptible M. tuberculosis and only with ethambutol for extensively drug-resistant M. tuberculosis. In the clinic we assessed ΔCT in sputum samples collected from patients with pulmonary tuberculosis before and at regular intervals over 12 weeks after initiation of treatment. Before treatment start, estimated CT were 19.3 (95% CI: 17.1-21.4) and 19.8 (95% CI: 17.6-22.1) without and with PMA, respectively. Under treatment CT increased by 2.54 per √√day (95% CI: 1.38-3.69) without PMA and an additional 0.55 per √√day (95% CI: 0.37-0.74; p < 0.0001) with PMA. We conclude that PMA increases the specificity of Xpert for viable M. tuberculosis but the effect is small and dependent on the antibiotics used.

Simultaneous staining of sputum smears for acid-fast and lipid-containing Myobacterium tuberculosis can enhance the clinical evaluation of antituberculosis treatments.

Dormant, slow-growing, antibiotic-tolerant Mycobacterium tuberculosis undermine the shortening of tuberculosis treatment to less than 6 months and are thought to be characterised by intracellular lipid bodies. Antibiotic effects on such persisting bacilli escape evaluation as they cannot be readily cultured. We identified cells containing lipid bodies in sputum smears from 86 newly diagnosed pulmonary tuberculosis patients and monitored these cells daily in 42 patients over the first 14 days of treatment with rifampicin, the experimental compound SQ-109, or both agents combined. Counts of Nile-Red-positive lipid-body containing cells were correlated with those of Auramine-O-positive cells and colony forming units of viable Mycobacterium tuberculosis on agar plates. Rifampicin but not SQ-109 significantly reduced colony forming units but all treatments distinctively and significantly changed the proportions of lipid body-containing bacilli and viable Mycobacterium tuberculosis. Monitoring lipid-body containing bacilli in sputum during treatment with experimental antituberculosis regimens may identify putative treatment-shortening regimens.