RATIONALE: Optimizing pyrazinamide dosing is critical to improve treatment efficacy while minimizing toxicity during tuberculosis treatment. Study 31/ACTG A5349 represents the largest Phase 3 randomized controlled therapeutic trial to date for such investigation. OBJECTIVES: We sought to report pyrazinamide pharmacokinetic parameters, risk factors for lower pyrazinamide exposure, and relationships between pyrazinamide exposure with efficacy and safety outcomes. We aimed to determine pyrazinamide dosing strategies that optimize risks and benefits. METHODS: We analyzed pyrazinamide steady-state pharmacokinetic data using population nonlinear mixed-effects models. We evaluated the contribution of pyrazinamide exposure to long-term efficacy using parametric time-to-event models and safety outcomes using logistic regression. We evaluated optimal dosing with therapeutic windows targeting ≥95% durable cure and safety within the observed proportion of the primary safety outcome. MEASUREMENTS AND MAIN RESULTS: Among 2255 participants with 6978 plasma samples, pyrazinamide displayed 7-fold exposure variability (151-1053 mg·h/L). Body weight was not a clinically relevant predictor of drug clearance and thus did not justify the need for weight-banded dosing. Both clinical and safety outcomes were associated with pyrazinamide exposure, resulting in a therapeutic window of 231-355 mg·h/L for the control and 226-349 mg·h/L for the rifapentine-moxifloxacin regimen. Flat dosing of pyrazinamide at 1000 mg would have permitted an additional 13.1% (n=96) participants allocated to the control and 9.2% (n=70) to the rifapentine-moxifloxacin regimen dosed within the therapeutic window, compared to the current weight-banded dosing. CONCLUSIONS: Flat dosing of pyrazinamide at 1000 mg daily would be readily implementable and could optimize treatment outcomes in drug-susceptible tuberculosis. Clinical trial registration available at www. CLINICALTRIALS: gov, ID: NCT02410772.

BACKGROUND: The optimal dosing strategy for rifampicin in treating drug-susceptible tuberculosis (TB) is still highly debated. In the Phase 3 clinical trial Study 31/ACTG 5349 (NCT02410772), all participants in the control regimen arm received 600 mg rifampicin daily as a flat dose. Here, we evaluated relationships between rifampicin exposure and efficacy and safety outcomes. METHODS: We analyzed rifampicin concentration time profiles using population nonlinear mixed-effects models. We compared simulated rifampicin exposure from flat- and weight-banded dosing. We evaluated the effect of rifampicin exposure on stable culture conversion at 6 months, TB-related unfavorable outcomes at 9, 12, and 18 months using Cox proportional hazard models, and all trial-defined safety outcomes using logistic regression. RESULTS: Our model derived rifampicin exposure ranged from 4.57 mg·h/L to 140.0 mg·h/L with a median of 41.8 mg·h/L. Pharmacokinetic simulations demonstrated that flat-dosed rifampicin provided exposure coverage similar to weight-banded dose. Exposure-efficacy analysis (N=680) showed that participants with rifampicin exposure below the median experienced similar hazards of stable culture conversion and TB-related unfavorable outcomes compared to those with exposure above the median. Exposure-safety analysis (N=722) showed that increased rifampicin exposure was not associated with increased grade 3 or higher adverse events, or serious adverse events. CONCLUSIONS: Flat-dosing of rifampicin at 600 mg daily may be a reasonable alternative to the incumbent weight-banded dosing strategy for the standard of care 6-month regimen. Future research should assess the optimal dosing strategy for rifampicin, at doses higher than the current recommendation.

BACKGROUND: A four-month regimen containing rifapentine and moxifloxacin has non-inferior efficacy compared to the standard 6-month regimen for drug-sensitive tuberculosis. We evaluated the effect of regimens containing daily, high-dose rifapentine on efavirenz pharmacokinetics and viral suppression in patients with HIV-associated TB. METHODS: In the context of a Phase 3 randomized controlled trial, HIV-positive individuals already virally suppressed on efavirenz--containing ART (EFV1), or newly initiating efavirenz (EFV2) received TB treatment containing rifapentine (1200mg), isoniazid, pyrazinamide, and either ethambutol or moxifloxacin. Mid-interval efavirenz concentrations were measured (a) during ART and TB co-treatment (Weeks 4, 8, 12 and 17, different by EFV group) and (b) when ART was taken alone (pre- or post-TB treatment, Weeks 0 and 22). Apparent oral clearance (CL/F) was estimated and compared. Target mid-interval efavirenz concentrations were > 1 mg/L. Co-treatment was considered acceptable if >80% of participants had mid-interval efavirenz concentrations meeting this target. RESULTS: EFV1 and EFV2 included 70 and 41 evaluable participants, respectively. The geometric mean ratio comparing efavirenz CL/F with vs. without TB drugs was 0.79 [90% CI 0.72-0.85] in EFV1 and 0.84 [90% CI 0.69-0.97] in EFV2. The percent of participants with mid-interval efavirenz concentrations >1mg/L in EFV1 at Weeks 0, 4, 8, and 17 was 96%, 96%, 88%, and 89%, respectively. In EFV2, at approximately 4 and 8 weeks post efavirenz initiation, the value was 98%. CONCLUSIONS: TB treatment containing high-dose daily rifapentine modestly decreased (rather than increased) efavirenz clearance and therapeutic targets were met supporting the use of efavirenz with these regimens, without dose adjustment.

The identification of sensitive, specific and reliable biomarkers that can be quantified in the early phases of tuberculosis treatment and predictive of long-term outcome is key for the development of an effective short-course treatment regimen. Time-to-positivity (TTP), a biomarker of treatment outcome against Mycobacterium tuberculosis, measures longitudinal bacterial growth in Mycobacteria Growth Indicator Tube broth culture and may be predictive of standard time-to-stable-culture-conversion (TSCC). In two randomized phase 2b trials investigating dose-ranging rifapentine (Study 29 and 29x), 662 participants had sputum collected over six months where TTP, TSCC and time-to-culture-conversion were quantified. The goals of this post hoc study were to characterize longitudinal TTP profiles and to identify individual patient characteristics associated with delayed time to culture conversion. In order to do so, a nonlinear mixed-effects model describing longitudinal TTP was built. Independent variables associated with increased bacterial clearance (increased TTP), assessed by subject-specific and population-level trajectories, were higher rifapentine exposure, lower baseline grade of sputum acid-fast bacilli smear, absence of productive cough, and lower extent of lung infiltrates on radiographs. Importantly, sensitivity analysis revealed that major learning milestones in phase 2b trials, such as significant exposure-response and covariate relationships, could be detected using truncated TTP data as early as 6 weeks from start of treatment, suggesting alternative phase 2B study designs. The TTP model built depicts a novel phase 2B surrogate endpoint that can inform early assessment of experimental treatment efficacy and treatment failure or relapse in patients treated with shorter and novel TB treatment regimens, improving efficiency of phase 2 clinical trials.

INTRODUCTION: With the growing use of online study management systems and rapid availability of data, timely data review and quality assessments are necessary to ensure proper clinical trial implementation. In this report we describe central monitoring used to ensure protocol compliance and accurate data reporting, implemented during a large phase 3 clinical trial. MATERIAL AND METHODS: The Tuberculosis Trials Consortium (TBTC) Study 31/AIDS Clinical Trials Group (ACTG) study A5349 (S31) is an international, multi-site, randomized, open-label, controlled, non-inferiority phase 3 clinical trial comparing two 4-month regimens to a standard 6 month regimen for treatment of drug-susceptible tuberculosis (TB) among adolescents and adults with a sample size of 2500 participants. RESULTS: Central monitoring utilized primary study data in a five-tiered approach, including (1) real-time data checks & topic-specific intervention reports, (2) missing forms reports, (3) quality assurance metrics, (4) critical data reports and (5) protocol deviation identification, aimed to detect and resolve quality challenges. Over the course of the study, 240 data checks and reports were programed across the five tiers used. DISCUSSION: This use of primary study data to identify issues rapidly allowed the study sponsor to focus quality assurance and data cleaning activities on prioritized data, related to protocol compliance and accurate reporting of study results. Our approach enabled us to become more efficient and effective as we informed sites about deviations, resolved missing or inconsistent data, provided targeted guidance, and gained a deeper understanding of challenges experienced at clinical trial sites. TRIAL REGISTRATION: This trial was registered with ClinicalTrials.gov (Identifier: NCT02410772) on April 8, 2015.

BACKGROUND: Rifapentine exposure is associated with bactericidal activity against Mycobacterium tuberculosis, but high interindividual variation in plasma concentrations is encountered. OBJECTIVES: To investigate a genomic association with interindividual variation of rifapentine exposure, SNPs of six human genes involving rifamycin metabolism (AADAC, CES2), drug transport (SLCO1B1, SLCO1B3) and gene regulation (HNF4A, PXR) were evaluated. METHODS: We characterized these genes in 173 adult participants in treatment trials of the Tuberculosis Trials Consortium. Participants were stratified by self-identified race (black or non-black), and rifapentine AUC from 0 to 24 h (AUC0-24) was adjusted by analysis of covariance for SNPs, rifapentine dose, sex, food and HIV coinfection. This study was registered at ClinicalTrials.gov under identifier NCT01043575. RESULTS: The effect on rifapentine least squares mean AUC0-24 in black participants overall decreased by -10.2% for AADAC rs1803155 G versus A allele (Wald test: P = 0.03; false discovery rate, 0.10). Black participants with one G allele in AADAC rs1803155 were three times as likely to have below target bactericidal rifapentine exposure than black participants with the A allele (OR, 2.97; 95% CI: 1.16, 7.58). With two G alleles, the OR was greater. In non-black participants, AADAC rs1803155 SNP was not associated with rifapentine exposure. In both black and non-black participants, other evaluated genes were not associated with rifapentine exposure (P > 0.05; false discovery rate > 0.10). CONCLUSIONS: Rifapentine exposure in black participants varied with AADAC rs1803155 genotype and the G allele was more likely to be associated with below bactericidal target rifapentine exposure. Further pharmacogenomic study is needed to characterize the association of the AADAC rs1803155 with inadequate rifapentine exposure in different patient groups.

More efficacious treatment regimens are needed for tuberculosis, however, drug development is impeded by a lack of reliable biomarkers of disease severity and of treatment effect. We conducted a directed screen of host biomarkers in participants enrolled in a tuberculosis clinical trial to address this need. Serum samples from 319 protocol-correct, culture-confirmed pulmonary tuberculosis patients treated under direct observation as part of an international, phase 2 trial were screened for 70 markers of infection, inflammation, and metabolism. Biomarker assays were specifically developed for this study and quantified using a novel, multiplexed electrochemiluminescence assay. We evaluated the association of biomarkers with baseline characteristics, as well as with detailed microbiologic data, using Bonferroni-adjusted, linear regression models. Across numerous analyses, seven proteins, SAA1, PCT, IL-1beta, IL-6, CRP, PTX-3 and MMP-8, showed recurring strong associations with markers of baseline disease severity, smear grade and cavitation; were strongly modulated by tuberculosis treatment; and had responses that were greater for patients who culture-converted at 8weeks. With treatment, all proteins decreased, except for osteocalcin, MCP-1 and MCP-4, which significantly increased. Several previously reported putative tuberculosis-associated biomarkers (HOMX1, neopterin, and cathelicidin) were not significantly associated with treatment response. In conclusion, across a geographically diverse and large population of tuberculosis patients enrolled in a clinical trial, several previously reported putative biomarkers were not significantly associated with treatment response, however, seven proteins had recurring strong associations with baseline radiographic and microbiologic measures of disease severity, as well as with early treatment response, deserving additional study.

Rifapentine is a highly active antituberculosis antibiotic with treatment-shortening potential; however, exposure-response relations and the dose needed for maximal bactericidal activity have not been established. We used pharmacokinetic/pharmacodynamic data from 657 adults with pulmonary tuberculosis participating in treatment trials to compare rifapentine (n = 405) with rifampin (n = 252) as part of intensive-phase therapy. Population pharmacokinetic/pharmacodynamic analyses were performed with nonlinear mixed-effects modeling. Time to stable culture conversion of sputum to negative was determined in cultures obtained over 4 months of therapy. Rifapentine exposures were lower in participants who were coinfected with human immunodeficiency virus, black, male, or fasting when taking drug. Rifapentine exposure, large lung cavity size, and geographic region were independently associated with time to culture conversion in liquid media. Maximal treatment efficacy is likely achieved with rifapentine at 1200 mg daily. Patients with large lung cavities appear less responsive to treatment, even at high rifapentine doses.

The Xpert(R) MTB/RIF assay is both sensitive and specific as a diagnostic test. Xpert also reports quantitative output in cycle thresholds (Ct), which may provide a dynamic measure of sputum bacillary burden when used longitudinally. We evaluated the relationship between Xpert Ct trajectory and drug exposure during TB treatment to evaluate the potential utility of Xpert Ct for treatment monitoring. We obtained serial sputum samples from patients with smear-positive pulmonary TB consecutively enrolled at ten international clinical trial sites participating in Study 29X, a CDC-sponsored TB Trials Consortium study evaluating the tolerability, safety, and antimicrobial activity of rifapentine at daily doses up to 20 mg/kg. Xpert was performed at weeks 0, 2, 4, 6, 8, and 12. Longitudinal Ct data were modeled using a nonlinear mixed effects model, in relation to rifapentine exposure (area under the concentration-time curve [AUC]). The rate of change of Ct was higher in subjects receiving rifapentine compared to that in subjects receiving standard dose rifampin. Moreover, rifapentine exposure, but not assigned dose, was significantly associated with rate of change in Ct (p = 0.02). The estimated increase in Ct slope for every additional 100 mcg*h/mL of rifapentine drug exposure (as measured by AUC) was 0.11 Ct/week (95% CI 0.05 - 0.17). Increasing rifapentine exposure is associated with faster rate of change of Xpert Ct, indicating faster clearance of MTB DNA. These data suggest that the quantitative outputs of the Xpert MTB/RIF assay may be useful as a dynamic measure of TB treatment response.

SETTING: Two-month solid medium culture conversion is a commonly used, if suboptimal, endpoint for phase 2 anti-tuberculosis treatment trials. OBJECTIVE AND DESIGN: To model the effect of the performance characteristics (sensitivity and contamination rate) of solid medium on required sample size for a two-arm clinical trial with 85% true (gold standard) culture conversion in the control and 95% in the experimental arm. RESULTS: Increasing sensitivity and decreasing contamination reduced the sample size from 239 subjects/arm (60% sensitivity, 30% contamination) to 138 subjects/arm (95% sensitivity, 1% contamination). CONCLUSION: Optimizing solid medium has significant potential to reduce sample size and increase the efficiency of tuberculosis clinical trials.

OBJECTIVE: Use of tuberculin skin tests (TSTs) and interferon gamma release assays (IGRAs) as part of tuberculosis (TB) screening among immigrants from high TB-burden countries has not been fully evaluated. METHODS: Prevalence of Mycobacterium tuberculosis infection (MTBI) based on TST, or the QuantiFERON-TB Gold test (QFT-G), was determined among immigrant applicants in Vietnam bound for the United States (US); factors associated with test results and discordance were assessed; predictive values of TST and QFT-G for identifying chest radiographs (CXRs) consistent with TB were calculated. RESULTS: Of 1,246 immigrant visa applicants studied, 57.9% were TST positive, 28.3% were QFT-G positive, and test agreement was 59.4%. Increasing age was associated with positive TST results, positive QFT-G results, TST-positive but QFT-G-negative discordance, and abnormal CXRs consistent with TB. Positive predictive values of TST and QFT-G for an abnormal CXR were 25.9% and 25.6%, respectively. CONCLUSION: The estimated prevalence of MTBI among US-bound visa applicants in Vietnam based on TST was twice that based on QFT-G, and 14 times higher than a TST-based estimate of MTBI prevalence reported for the general US population in 2000. QFT-G was not better than TST at predicting abnormal CXRs consistent with TB.

BACKGROUND: The QuantiFERON(R)-TB Gold In-Tube test (QFT-GIT) detects Mycobacterium tuberculosis (Mtb) infection by measuring release of interferon gamma (IFN-gamma) when T-cells (in heparinized whole blood) are stimulated with specific Mtb antigens. The amount of IFN-gamma is determined by enzyme-linked immunosorbent assay (ELISA). Automation of the ELISA method may reduce variability. To assess the impact of ELISA automation, we compared QFT-GIT results and variability when ELISAs were performed manually and with automation. METHODS: Blood was collected into two sets of QFT-GIT tubes and processed at the same time. For each set, IFN-gamma was measured in automated and manual ELISAs. Variability in interpretations and IFN-gamma measurements was assessed between automated (A1 vs. A2) and manual (M1 vs. M2) ELISAs. Variability in IFN-gamma measurements was also assessed on separate groups stratified by the mean of the four ELISAs. RESULTS: Subjects (N = 146) had two automated and two manual ELISAs completed. Overall, interpretations were discordant for 16 (11%) subjects. Excluding one subject with indeterminate results, 7 (4.8%) subjects had discordant automated interpretations and 10 (6.9%) subjects had discordant manual interpretations (p = 0.17). Quantitative variability was not uniform; within-subject variability was greater with higher IFN-gamma measurements and with manual ELISAs. For subjects with mean TB Responses +/-0.25 IU/mL of the 0.35 IU/mL cutoff, the within-subject standard deviation for two manual tests was 0.27 (CI95 = 0.22-0.37) IU/mL vs. 0.09 (CI95 = 0.07-0.12) IU/mL for two automated tests. CONCLUSION: QFT-GIT ELISA automation may reduce variability near the test cutoff. Methodological differences should be considered when interpreting and using IFN-gamma release assays (IGRAs).

BACKGROUND: The QuantiFERON(R)-TB Gold In-Tube test (QFT-GIT) is a viable alternative to the tuberculin skin test (TST) for detecting Mycobacterium tuberculosis infection. However, within-subject variability may limit test utility. To assess variability, we compared results from the same subjects when QFT-GIT enzyme-linked immunosorbent assays (ELISAs) were performed in different laboratories. METHODS: Subjects were recruited at two sites and blood was tested in three labs. Two labs used the same type of automated ELISA workstation, 8-point calibration curves, and electronic data transfer. The third lab used a different automated ELISA workstation, 4-point calibration curves, and manual data entry. Variability was assessed by interpretation agreement and comparison of interferon-gamma (IFN-gamma) measurements. Data for subjects with discordant interpretations or discrepancies in TB Response >0.05 IU/mL were verified or corrected, and variability was reassessed using a reconciled dataset. RESULTS: Ninety-seven subjects had results from three labs. Eleven (11.3%) had discordant interpretations and 72 (74.2%) had discrepancies >0.05 IU/mL using unreconciled results. After correction of manual data entry errors for 9 subjects, and exclusion of 6 subjects due to methodological errors, 7 (7.7%) subjects were discordant. Of these, 6 (85.7%) had all TB Responses within 0.25 IU/mL of the manufacturer's recommended cutoff. Non-uniform error of measurement was observed, with greater variation in higher IFN-gamma measurements. Within-subject standard deviation for TB Response was as high as 0.16 IU/mL, and limits of agreement ranged from -0.46 to 0.43 IU/mL for subjects with mean TB Response within 0.25 IU/mL of the cutoff. CONCLUSION: Greater interlaboratory variability was associated with manual data entry and higher IFN-gamma measurements. Manual data entry should be avoided. Because variability in measuring TB Response may affect interpretation, especially near the cutoff, consideration should be given to developing a range of values near the cutoff to be interpreted as "borderline," rather than negative or positive.

BACKGROUND: Mycobacterium tuberculosis (Mtb) infection may cause overt disease or remain latent. Interferon gamma release assays (IGRAs) detect Mtb infection, both latent infection and infection manifesting as overt disease, by measuring whole-blood interferon gamma (IFN-gamma) responses to Mtb antigens such as early secreted antigenic target-6 (ESAT-6), culture filtrate protein 10 (CFP-10), and TB7.7. Due to a lack of adequate diagnostic standards for confirming latent Mtb infection, IGRA sensitivity for detecting Mtb infection has been estimated using patients with culture-confirmed tuberculosis (CCTB) for whom recovery of Mtb confirms the infection. In this study, cytokines in addition to IFN-gamma were assessed for potential to provide robust measures of Mtb infection. METHODS: Cytokine responses to ESAT-6, CFP-10, TB7.7, or combinations of these Mtb antigens, for patients with CCTB were compared with responses for subjects at low risk for Mtb infection (controls). Three different multiplexed immunoassays were used to measure concentrations of 9 to 20 different cytokines. Responses were calculated by subtracting background cytokine concentrations from cytokine concentrations in plasma from blood stimulated with Mtb antigens. RESULTS: Two assays demonstrated that ESAT-6, CFP-10, ESAT-6+CFP-10, and ESAT-6+CFP-10+TB7.7 stimulated the release of significantly greater amounts of IFN-gamma, IL-2, IL-8, MCP-1 and MIP-1beta for CCTB patients than for controls. Responses to combination antigens were, or tended to be, greater than responses to individual antigens. A third assay, using whole blood stimulation with ESAT-6+CFP-10+TB7.7, revealed significantly greater IFN-gamma, IL-2, IL-6, IL-8, IP-10, MCP-1, MIP-1beta, and TNF-alpha responses among patients compared with controls. One CCTB patient with a falsely negative IFN-gamma response had elevated responses with other cytokines. CONCLUSIONS: Multiple cytokines are released when whole blood from patients with CCTB is stimulated with Mtb antigens. Measurement of multiple cytokine responses may improve diagnostic sensitivity for Mtb infection compared with assessment of IFN-gamma alone.

INTRODUCTION: Interferon gamma (IFN-gamma) release assays, such as QuantiFERON(R)-TB Gold test (QFT-G) and QuantiFERON(R)-TB Gold In-Tube test (QFT-GIT) are designed to detect M. tuberculosis (Mtb) infection. Recognition of unusual IFN-gamma measurements may help indicate inaccurate results. METHODS: We examined QFT-G and QFT-GIT results from subjects who had two or more tests completed. We classified unusual IFN-gamma measurements as: 1) High Nil Concentration (HNC) when IFN-gamma concentration in plasma from unstimulated blood exceeded 0.7 IU/mL; 2) Low Mitogen Response (LMR) when Mitogen Response was <0.5 IU/mL; 3) Very Low Mitogen Response (VLMR) when Mitogen Response was ≤-0.5 IU/mL; and 4) Very Low Antigen Response (VLAR) when the response to a Mtb antigen was ≤-0.35 IU/mL and ≤-0.5 times the IFN-gamma concentration in plasma from unstimulated blood. RESULTS: Among 5,309 results from 1,728 subjects, HNC occurred in 234 (4.4%) tests for 162 subjects, LMR in 108 (2.0%) tests for 85 subjects, VLMR in 22 (0.4%) tests for 21 subjects, and VLAR in 41 (0.8%) tests for 39 subjects. QFT-GIT had fewer HNC, VLMR, and VLAR (p = 0.042, 0.004, and 0.067 respectively); QFT-G had fewer LMR (p = 0.005). Twenty-four (51.6%) of 47 subjects with positive results and HNC were negative or indeterminate by all other tests. Thirteen (61.9%) of 21 subjects with positive results and LMR were negative or indeterminate by all other tests. CONCLUSION: Unusual IFN-gamma measurements including HNC, LMR, VLMR, and VLAR were encountered in small numbers, and in most instances were not seen on simultaneously or subsequently performed tests. To avoid erroneous diagnosis of Mtb infection, IGRAs with unusual IFN-gamma measurements should be repeated with another blood sample and interpreted with caution if they recur.