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. 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...

The Molecular Bacterial Load Assay Replaces Solid Culture for Measuring Early Bactericidal Response to Antituberculosis Treatment

“…Within-subject variation of Xpert was high, at 56.7%. In contrast, MBL had a 9.6% variance between 2 baseline samples for 16 subjects (18), suggesting that MBL is more reproducible. To corroborate this, within-patient variabilities for solid (17.9%) and liquid (21.6%) culture in our study were compared to those in the study by Kayigire et al, with 16.5% and 22%, respectively (18).…”

“…In contrast, MBL had a 9.6% variance between 2 baseline samples for 16 subjects (18), suggesting that MBL is more reproducible. To corroborate this, within-patient variabilities for solid (17.9%) and liquid (21.6%) culture in our study were compared to those in the study by Kayigire et al, with 16.5% and 22%, respectively (18). Prior to exclusion of outliers, solid culture had invalid results in 11% of samples (18 of 169), whereas MBL had only 1.2% of samples (2 of 169) unreadable.…”

Multicenter Evaluation of Anyplex Plus MTB/NTM MDR-TB Assay for Rapid Detection of Mycobacterium tuberculosis Complex and Multidrug-Resistant Isolates in Pulmonary and Extrapulmonary Specimens

“…The negative predictive value (NPV) and positive predictive value (PPV) of the Anyplex MTB/NTM assay for pulmonary specimens were 98.8% and 88.4%, respectively, and those for extrapulmonary specimens were 98.5% and 100%. Although the Anyplex assay has been licensed as a qualitative test for MTBC detection, the real-time PCR assay can provide semiquantitative results that potentially can be correlated with the bacterial load in the sample and the time to positivity (TTP) of the culture method (14). As shown in Fig.…”

The use of transcriptomics to predict drug efficacy and treatment outcome in tuberculosis

“…This time shift from 2 weeks or more to 2 h is possible due to the switch from microbiological to molecular PCR-based technologies. However, the application of the GeneXpert MTB/RIF assay to monitor treatment success is limited as mycobacterial DNA is a relatively stable molecule, which can remain in tissue months after bacterial sterilization [29] making DNA-based assays not widely applicable for treatment outcome measurements (Table 6.2).…”