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Time to detection of Mycobacterium tuberculosis in broth culture was examined for utility as a treatment efficacy end point. Of 146 patients in a phase IIB trial, a decreased mean time to detection was found in 5 with treatment failure. Time to detection in an analysis-of-covariance model was associated with lung cavities, less intensive treatment, and differences in the bactericidal effects of treatment regimens.
Development of new treatments for tuberculosis is hampered by the lack of an accurate surrogate end point and the high degree of efficacy of current 6-month regimens. Sputum culture status after 2 months of therapy, a binary test, is widely used for phase IIB trials but has only moderate accuracy for predicting failure/relapse (12) and requires large sample sizes (4, 8). Changes in the number of colonies found in dilutions of sputum applied to solid medium is an end point that has been used to assess activities of single drugs and doses in phase IIA (early-bactericidal-activity) studies (10) and has also been suggested as an end point for phase IIB trials (15). Though promising, quantitative culture on solid medium involves prolonged sputum collections and intensive laboratory techniques and has been difficult to standardize at multiple sites. Time to detection in broth culture (TTD) is a potential end point that has a good correlation with quantitative culture on solid medium (11, 13). An initial small study had suggested a correlation between a shorter time to detection (an indication of higher numbers of viable bacilli) and poor treatment outcomes (9). In this study, TTD was evaluated as a marker of regimen potency. Preliminary results have been reported elsewhere (16).
Patients for this study were drawn from Tuberculosis Trials Consortium (TBTC) Study 27, a randomized phase IIB trial that compared moxifloxacin to ethambutol and treatment 5 versus 3 times per week during the initial 2 months of treatment among patients with smear-positive, pulmonary tuberculosis (4). After completion of intensive-phase therapy, patients received standard continuation-phase treatment (3). The primary end point of the treatment trial was the dichotomous end point of 2-month culture status.
For this post hoc study, TTD data were available from two African sites. Of 176 African patients in study 27, TTD data were available for 163 (93%). Treatment failure occurred in six of these patients, and although the patients were not routinely followed after therapy, one with a relapse was identified 5 weeks after treatment.
Spontaneously expectorated (spot) sputum samples were cultured every 2 weeks during the first 2 months and monthly thereafter. Standard culture methods were used but differed somewhat between sites. In Uganda, samples were processed using a final concentration of 1% sodium hydroxide, inoculated into Bactec 12B bottles, and monitored with the Bactec 460 system. In South Africa, a final concentration of 1.25% sodium hydroxide, MGIT broth, and the Bactec MGIT 960 instrument were used. Cultures were monitored daily at both sites, with the exception that after 2 weeks, the Bactec 460 protocol used daily monitoring for cultures with a Bactec growth index (GI) of ≥30 and once weekly for cultures with a GI of <30 (no. 444824; Bactec TB System Product & Procedure Manual; Becton Dickinson). TTD was calculated as the number of days between inoculation and detection of a positive culture by the Bactec instruments; the results for specimens not positive by the end of the monitored interval were recorded as 42 days. Most patients had two sputum specimens cultured at each time point. The result of the first culture for each patient and time point was used, with two exceptions. If the first culture was contaminated with bacteria or grew nontuberculosis mycobacteria (NTM), the second culture was used.
The primary objective of this analysis was to compare microbiological end points for predicting treatment failure by TTD and by 2-month culture status. We used test cutoffs for the TTD end point that identified all patients with treatment failure (sensitivity, 100%). The accuracy of the test was calculated as the number of true-positive patients plus the number of true-negative patients for treatment failure with tuberculosis divided by the number of true-positive, false-positive, true-negative, plus false-negative patients.
Data analyses were performed using the SAS program (version 9.1) for the mixed-model analysis of covariance (ANCOVA) and generalized estimating equations (GEE), the StatXact program for nonparametric tests, and the NCSS 2007 program for other tests. For binary data, differences between groups were determined using Fisher's exact test or Pearson's chi-square statistic, and for nonparametric analyses, the Kruskal-Wallis test by the Monte Carlo method was used. Arithmetic means (see Table Table11 and Fig. 2) and adjusted means with the ANCOVA model (see Table 4 and Fig. 3) were used where applicable. Differences between groups were considered statistically significant at the level of a P value of <0.05.
We also evaluated the effect of TTD with clinical and radiographic factors and the randomized study arms. Initially, univariate analysis of each factor, adjusted for missing data (TTD at weeks 2, 4, 6, and 8), was performed. A mixed-model ANCOVA with repeated measures was then evaluated. In the final model, main effects were cavity (absent, <4 cm in total size, ≥4 cm in total size), HIV infection status (infected or not infected), drug (moxifloxacin versus ethambutol), frequency of study treatment (5 versus 3 times per week), site (Uganda or South Africa), baseline TTD, the repeated measure (2, 4, 6, and 8 weeks), and the two-way interactions of the main effects (with the exceptions of study site and the baseline culture). Because of the model's complexity, a parsimonious approach with elimination of nonsignificant higher-level interactions was adopted. Of the 163 patients, 158 patients had complete data for independent factors and baseline TTD (Fig. (Fig.1).1). Homogeneity of variance was supported by plotting of residuals versus predicted values. Adjusted means refers to means adjusted for all model effects, unequal sample sizes, and missing data among the repeated measures. The model takes into account the intrasubject correlation of repeated sampling; serial correlation was estimated to be small, with R2 being <0.05. The significance of pairwise comparisons is reported using Fisher's least significant difference (LSD).
We also performed an alternate GEE analysis with cultures identified as positive or negative for M. tuberculosis and compared the results with those of the comparable ANCOVA model.
In this model, TTDs of 1 to 41 days were scored positive and a TTD of 42 was scored negative. Because of the small sample size for one of the three cavity levels (≥4 cm), cavity was collapsed to a binomial response. Adjusted proportions refer to the proportions adjusted for all model effects, unequal sample sizes, and missing data among the repeated measures.
Of 163 patients, 147 (90%) were enrolled in Uganda and 16 (10%) were enrolled in South Africa. Compared to patients enrolled in the original clinical trial who were not included, patients in this substudy were significantly younger, more often male and of black race, weighed less, and were more likely to be HIV infected (Table (Table1).1). The median baseline CD4+ cell count of HIV-infected subjects in the substudy was 200 μl−1 (range, 110 to 286 μl−1).
Of 163 patients with TTD culture results, 146 patients, including 5 of the 6 patients with failure, had data for four cultures in the 2 months of study therapy. In these 146 patients, there was more rapid detection of growth (TTD of four cultures) in the five patients who failed treatment than in the others who did not fail treatment (mean ± standard deviation, 16.3 ± 2.9 days [range, 13.0 to 20.3 days] versus 25.4 ± 6.7 days [range, 6.0 to 42.0 days]; P = 0.003 by t test) (Tables (Tables22 and and33 and Fig. Fig.2).2). When TTD was assessed as a diagnostic end point for treatment failure, using a mean cutoff of less than 21 days (for specimens for culture obtained 2, 4, 6, and 8 weeks after the beginning of study treatment), the accuracy was 75%, the sensitivity was 100%, the specificity was 74%, the positive predictive value (PPV) was 12%, and the negative predictive value (NPV) was 100% (Table (Table3,3, end point label 1). Improved PPVs (18 to 20%) and similar sensitivities and specificities were obtained when fewer time points were used (two time points at week 6 and 8 cultures or one time point at week 8 culture) (Table (Table3,3, end point labels 2 and 3). By comparison, the alternative end point of culture conversion to negative after 2 months of treatment was less accurate and specific (accuracy, 47%; sensitivity, 100%; specificity, 49%; positive predictive value, 6%; negative predictive value, 100; Table Table3,3, end point label 4).
Among 38 HIV-infected patients at initial testing, 7 (18%) had cavitary lung disease, received ethambutol, and were on intermittent therapy; 4 of the 7 (57%) had treatment failure. The accuracy of the TTD end point (four time points with a mean of <21 days) among the 24 HIV-infected patients with cavitary disease was 71% (sensitivity, 100%; specificity, 65%; PPV, 36%; NPV, 100%).
Significant effects of TTD in univariate analysis were also found in multivariate ANCOVA (Table (Table4).4). TTD was associated with cavitary lung disease; i.e., the greatest decrease (delayed response) was with large cavities, an intermediate decrease was with small cavities, and the lowest decrease was without cavities (Table (Table4;4; Fig. Fig.3).3). The adjusted mean TTD was also significantly decreased among patients treated thrice weekly and with ethambutol (versus moxifloxacin). By comparison, 2-month culture conversion rates were similar among 158 patients treated thrice weekly versus 5 days/week (46% versus 51% culture conversion; P = 0.5, chi-square test) or treated with moxifloxacin versus ethambutol (culture conversion, 54% versus 46%; P = 0.42). Although not significant, the direction of proportions with 2-month culture conversion corresponded to the significant differences observed with TTD.
The adjusted mean TTD differed between study sites (Uganda versus South Africa, 25.5 days versus 20.8 days; P = 0.008), reflecting in part differences among patient groups and in the culture processing methods, the broth media, and the monitoring methodologies used. However, differences in the adjusted mean TTD of other cofactors were similar between models of Ugandan patients alone and all patients (Uganda and South Africa). Further, there were no significant differences by site between drugs or treatment frequency; i.e., there were no site interactions. These findings are consistent with an increase in power from a larger sample size with all patients.
From 161 patients, 591 duplicate sputum cultures (of specimens obtained from the baseline to week 8 of treatment) with results of Mycobacterium tuberculosis or no growth were evaluable. TTDs in the duplicate cultures were significantly correlated (P < 0.0001, R2 = 0.69).
Other analyses of the TTD data were performed to explore the operating characteristics of the ANCOVA model. A frequency distribution of TTD data from 1 to 41 days demonstrated a middle peak and two tails with very low frequencies at days 1 and 2 and days 39, 40, and 41. Because about 30% of cultures did not demonstrate growth by the end of the monitored interval, we performed a sensitivity analysis of the effect of censoring at 42 days. Model term estimates and the significance of terms were insensitive to imputed censoring at 39 or at 45 days compared to 42 days.
We also performed GEE analysis to evaluate the effect of scoring all cultures as either positive or negative and compared the results to those obtained with a parallel TTD ANCOVA model. The significance of model terms and the direction of change between model terms were comparable between the different models, with one substantive exception (Table (Table5).5). The adjusted mean TTD of site differed significantly in the ANCOVA model but not in the GEE model. This demonstrates the similarity of data from the Bactec system, used at the Uganda site, and the MGIT system, used in South Africa, when culture results are analyzed as binary positive or negative results. However, a difference is distinguished between the monitoring systems with TTD data, likely reflecting the greater sensitivity of MGIT medium and monitoring to detect M. tuberculosis.
Identification of an accurate surrogate end point of treatment efficacy will advance trial design and development of new antituberculosis drugs and regimens. In this study, TTD of growth appeared to be a promising end point for phase IIB trials because it identified patients who went on to treatment failure more accurately than 2-month culture status. Moreover, TTD was correlated with the most consistent risk factor for treatment failure or relapse: the presence and extent of pulmonary cavitation (1, 2, 4, 6, 7, 8). Finally, TTD appeared to be a more sensitive measure of differences between the randomized treatment arms than 2-month culture status, suggesting the greater activity of more frequent dosing and of moxifloxacin-based regimens than of ethambutol-based regimens. The efficacies of these treatment interventions have been demonstrated in prior studies (5, 7, 14). Notably, in our study, these differences were shown with a relatively small sample size (about 40 patients for each of four treatment arms) and with spot sputum sample collection (rather than prolonged sputum sample collection). Thus, the TTD end point may be valuable in exploring the pharmacodynamics of moxifloxacin and rifampin therapy (17).
Our study has several limitations. Broth cultures were performed according to the manufacturer's specifications and TTD was adjusted between sites, but we did not standardize laboratory processing or monitoring techniques. That we were able to identify cases of treatment failure and detect differences among the four treatment groups is, therefore, all the more notable. Residual confounding by other (unmeasured) differences among sites is possible. However, we found no interaction of site with study drug, frequency of treatment, or other clinical factors. The effect of censoring cultures at 42 days appeared to be small because differences in adjusted mean estimates of TTD and significance (P) values were similar between the final model with censoring set at 42 days and other models with censoring set at 39 or 45 days. Further, results were comparable in GEE analysis when all culture results were scored as either positive or negative. Finally, because the original trial was a phase II study, patients were not followed for relapse. However, a disease continuum between treatment failure and relapse was suggested, in that five of six cases of treatment failure in this study were detected in (more sensitive) liquid broth culture after 4 months of therapy but not in concurrent cultures on solid media.
In summary, TTD identified treatment failure more accurately than 2-month culture status and differentiated among treatment groups, suggesting superior bactericidal activity with more frequent dosing and with moxifloxacin. These findings support further evaluation of the utility of the TTD end point in treatment trials.
We are grateful to William MacKenzie, Chad Heilig, Elsa Villarino, and Andrew Vernon for reviews of the manuscript and for the support of Kenneth Castro.
None of us reports a conflict of interest.
This work was supported by the Centers for Disease Control and Prevention, the United States Public Health Service, and the U.S. Department of Veterans Affairs. The Frederic C. Bartter General Clinical Research Center at the VAMC San Antonio (supported by NIH grant MO1-RR-01346) provided assistance in evaluation of patients. This study was sponsored by the Tuberculosis Trials Consortium.
Published ahead of print on 6 October 2010.
†The authors have paid a fee to allow immediate free access to this article.