Time to liquid culture positivity can substitute for colony counting on agar plates in early bactericidal activity studies of antituberculosis agents

Abstract

The measurement of early bactericidal activity (EBA) is the first step in the clinical investigation of antituberculosis agents. EBA is determined by quantifying the viable sputum mycobacterial load on consecutive days of treatment. To investigate whether time to positivity (TTP) in mycobacterial liquid culture can substitute for colony forming unit (CFU) counting on agar plates we compared the error variation of TTP and CFU in 2115 pooled sputum samples collected overnight from 250 individuals included in five EBA studies. We found that the technical variation between duplicate laboratory measurements and the within-subject or day-to-day variation were similar for TTP (8.5% and 27.4% of total variation, respectively) and CFU (6.7% and 29.3% of total variation). The ability of the measurements to separate the EBA of 22 treatment arms was determined with group rank correlation of means and one-way analysis of variance. Except for the EBA over 0-2 days, individual and group EBAs measured with TTP and CFU were highly correlated. Treatment group means rank correlation coefficients were r=0.472, r=0.910 and r=0.818, respectively, for EBA 0-2 days, EBA 0-7 days and EBA 0-14 days. Analysis of variance significantly favoured TTP over CFU for discrimination between groups with F values of 6.58 and 1.87, 7.77 and 4.58, and 8.71 and 3.56, respectively. We conclude that TTP is an acceptable alternative to CFU counting for the determination of the viable sputum mycobacterial load in EBA studies of up to 14 days duration.

Phase II Dose-Ranging Trial of the Early Bactericidal Activity of PA-824

“…CFU were counted after 3 to 4 weeks of incubation at 37°C at the dilution that yielded 20 to 200 visible colonies. TTP was measured in a standard liquid culture system (Bactec MGIT 960; Becton Dickinson) (3,4). For TTP, homogenized sputum was decontaminated (MycoPrep; Becton Dickinson), centrifuged, and resuspended, and 0.5 ml was used for incubation in duplicate.…”

Direct Comparison of Xpert MTB/RIF Assay with Liquid and Solid Mycobacterial Culture for Quantification of Early Bactericidal Activity

“…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)(4)(5).…”

Short-Term Storage Does Not Affect the Quantitative Yield of Mycobacterium tuberculosis in Sputum in Early-Bactericidal-Activity Studies

“…This is a considerably larger daily change than the 2.9 h that storage might induce. An additional consideration is the technical variation of the TTP measurement, which we estimated to be 4.3 h from 2,198 duplicate TTP measurements from the same sputum sample (8). This duration is, again, greater than the increase expected from a day of storage.…”

“…Storage of sputum for deferred culture may be considered where decentralized health care centers rely on a central laboratory service, but this may result in loss of viability and false-negative cultures (1)(2)(3)(4) or overgrowth of contaminants, particularly if sensitive liquid media are used (5)(6)(7). Beyond its clinical purpose, sputum culture can quantify the viable M. tuberculosis sputum load expressed as CFU per ml of sputum counted on agar plates or as times to culture positivity (TTP) of a volume of decontaminated sputum inoculated in liquid mycobacterial culture (8). High sputum mycobacterial loads at diagnosis have been associated with treatment failure and recurrence (9,10).…”