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.

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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...

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