Sputum volume predicts sputum mycobacterial load during the first 2 weeks of antituberculosis treatment.

Disease severity in patients with pulmonary tuberculosis is associated with mycobacterial sputum load. To ascertain whether reduced sputum production during treatment is a useful clinical sign of improvement, we analyzed the mycobacterial loads of 5,552 sputum samples collected from 439 newly diagnosed sputum smear-positive tuberculosis patients who participated in six 14-day studies of antituberculosis treatment. Sputum volumes were categorized as low (<6 ml), medium (6 to 10 ml), or large (>10 ml), and mycobacterial load was measured by the time to positivity in liquid culture and the CFU counts on solid culture. The association of sputum volume with mycobacterial load was estimated with multiple linear regression models adjusted for repeated measures. The predictor variables were sputum volume category, treatment day, specific study , and the interaction of sputum volume category and treatment day. Mycobacterial load was significantly associated only with the day on treatment and sputum volume, which tended to decrease with ongoing treatment. With the volume held constant, each day on treatment decreased the log CFU by 0.082 (P<0.001) and increased the time to positivity (TTP) by 1.04 h (P<0.001). From low to medium and from medium to large sputum volumes, the log CFU/ml increased by 0.265 (P<0.003) and 0.490 (P<0.001), respectively, and the TTP decreased by 1.17 h (P<0.001) and 1.30 h (P<0.001), respectively, for a given day of treatment. The variability of the sputum load measurements increased with the day of treatment and lower sputum volumes. The significant association of sputum volume and mycobacterial load validates decreasing sputum production as a clinical sign of improvement during early antituberculosis treatment.

Quantification of viable bacterial load in artificial sputum spiked with Mycobacterium tuberculosis.

OBJECTIVE: Artificial sputum spiked with Mycobacterium tuberculosis could serve for validation of procedures that determine viable mycobacterial load. DESIGN: Artificial sputum specimens prepared in-house were spiked with low, medium or high concentrations of Mycobacterium tuberculosis H37Rv stock solution. In a first series, a single technologist processed two batches of specimens daily with high load that were stored refrigerated or at room temperature for up to 8 days. In a second series, nine different technologists processed freshly made batches of specimens with low, medium or high loads. We recorded time to positivity (TTP) in duplicate liquid cultures made from each specimen. RESULTS: Specimens were well grouped around the mean TTP (hours; standard deviation) of low: 271.7 (25.9), medium: 233.5 (16.3), and two batches of high load: 186.9 (12.3) and 191.8 (9.0), respectively. A variance component model that included load, storage temperature, days of storage until processing, batch of specimens made, sample ID and technologist ID as random effects in a linear mixed-effects model identified only load, technologist and residual as significant contributors to overall TTP variance. CONCLUSION: Artificial sputum specimens with reproducible and stable viable mycobacterial loads can be made that could serve for training and validation purposes.

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.