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

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.

Fig 1

Individual log CFU, TTP, and C_T values are shown for all patients and time points combined. Rho values were calculated by the Spearman rank sum test for log CFU versus TTP (rho = −0.613; P < 0.0001), log CFU versus C_T (rho = −0.547; P < 0.0001), and TTP versus C_T (rho = 0.632; P < 0.0001).

Fig 2

Individual activities of log CFU, TTP, and C_T from day 0 to day 14 are plotted against each other. A strong correlation was found between log CFU and TTP (rho = −0.726; P < 0.0001), and a moderate correlation was found between log CFU and C_T (rho = −0.385; P = 0.0017) and TTP and C_T (rho = 0.400; P = 0.0004).

Fig 3

The mean change from baseline (SDs are shown as vertical bars) of log CFU, TTP, and C_T after 7 and 14 days of treatment is shown for all six groups. Significantly different measurements for days 0 to 7 or days 0 to 14 were found for TTP in 2 and 3, for log CFU in 1 and 3, and for C_T in 0 and 2 groups, respectively. Statistically significant differences between time points are indicated by asterisks (* = P < 0.05, ** = P < 0.001, *** = P < 0.0001, BL = baseline, D7 = day 7, D14 = day 14).