Time Trends in Sputum Mycobacterial Load and Two-Day Bactericidal Activity of Isoniazid-Containing Antituberculosis Therapies.

Recent early bactericidal activity (EBA) studies of isoniazid-based antituberculosis therapies have shown a lower EBA over the first two treatment days than in earlier years. To quantify this trend and evaluate factors contributing to it, we extracted individual data from 18 studies with a total of 182 participants using isoniazid-containing therapies between 1992 and 2015 at a single site and laboratory in Cape Town, South Africa. We recalculated EBA as the daily fall in CFU per milliliter sputum up to day 2 of therapy (EBA0-2) for individual patients and treatment groups and used mixed-effects linear models to investigate the correlation between pretreatment CFU, EBA0-2, and year of study. We found that mean pretreatment CFU and year of study accounted for 46% and 47%, respectively, of the variation in mean EBA0-2 Mean pretreatment CFU differed between the periods 1992 to 2001 and 2007 to 2015 by 0.92 log10 CFU (95% confidence interval [CI], 0.57 to 1.28; P < 0.0001). On average, pretreatment CFU dropped by 0.053 log10 CFU (95% CI, 0.029 to 0.076; P = 0.0004) and EBA0-2 by 0.012 log10 CFU (95% CI, 0.006 to 0.018; P = 0.001) per year. The EBA0-2 of isoniazid-based antituberculosis therapy is strongly correlated with baseline mycobacterial load and shows a declining trend over the past 2 decades.

Population Pharmacokinetics of AZD-5847 in Adults with Pulmonary Tuberculosis.

AZD-5847 is a new oxazolidinone derivative under development for the treatment of tuberculosis (TB). Here we describe the population pharmacokinetics (PK) of AZD-5847 in patients with tuberculosis based on a recently completed phase II study. The study included 60 patients with drug-susceptible TB. Patients were randomized to four dosages (500 mg once daily, 1,200 mg once daily, 500 mg twice daily, and 800 mg twice daily). Patients were intensively sampled on days 1 and 14. AZD-5847 pharmacokinetics were best described with a two-compartment model with lag time (Tlag) for absorption. AZD-5847 bioavailability was nonlinear and plateaued at 800 mg. We performed deterministic simulation to compare the PK/pharmacodynamics (PD) of AZD-5847, linezolid, and sutezolid. AZD-5847 PK/PD in terms of both area under the concentration-time curve for the free, unbound fraction (fAUC)/MIC and time the free concentration was above the MIC (fT>MIC) were less favorable than those for linezolid and sutezolid. This could help explain the poor bactericidal activity of AZD-5847 in the recent phase II study.

MIC of Delamanid (OPC-67683) against Mycobacterium tuberculosis Clinical Isolates and a Proposed Critical Concentration.

The increasing global burden of multidrug-resistant tuberculosis (MDR-TB) requires reliable drug susceptibility testing that accurately characterizes susceptibility and resistance of pathogenic bacteria to effectively treat patients with this deadly disease. Delamanid is an anti-TB agent first approved in the European Union in 2014 for the treatment of pulmonary MDR-TB in adults. Using the agar proportion method, delamanid MIC was determined for 460 isolates: 316 from patients enrolled in a phase 2 global clinical trial, 76 from two phase 2 early bactericidal activity trials conducted in South Africa, and 68 isolates obtained outside clinical trials (45 from Japanese patients and 23 from South African patients). With the exception of two isolates, MICs ranged from 0.001 to 0.05 µg/ml, resulting in an MIC50 of 0.004 µg/ml and an MIC90 of 0.012 µg/ml. Various degrees of resistance to other anti-TB drugs did not affect the distribution of MICs, nor did origin of isolates from regions/countries other than South Africa. A critical concentration/breakpoint of 0.2 µg/ml can be used to define susceptible and resistant isolates based on the distribution of MICs and available pharmacokinetic data. Thus, clinical isolates from delamanid-naive patients with tuberculosis have a very low MIC for delamanid and baseline resistance is rare, demonstrating the potential potency of delamanid and supporting its use in an optimized background treatment regimen for MDR-TB.

Early Bactericidal Activity of AZD5847 in Patients with Pulmonary Tuberculosis.

AZD5847 is an oxazolidinone antibiotic with in vitro activity against Mycobacterium tuberculosis The objective of this study was to evaluate the antimycobacterial activity, safety, and pharmacokinetics of AZD5847 in patients with pulmonary tuberculosis. Groups of 15 treatment-naive, sputum smear-positive adults with pulmonary tuberculosis were randomly assigned to receive AZD5847 at one of four doses (500 mg once daily, 500 mg twice daily, 1,200 mg once daily, and 800 mg twice daily) or daily standard chemotherapy. The primary efficacy endpoint was the mean daily rate of change in the log10 number of CFU of M. tuberculosis per milliliter of sputum, expressed as the change in log10 number of CFU per milliliter of sputum per day. The mean 14-day activity of the combination of isoniazid, rifampin, ethambutol, and pyrazinamide (-0.163 log10 CFU/ml sputum/day; 95% confidence interval [CI], -0.193, -0.133 log10 CFU/ml sputum/day) was consistent with that found in previous studies. AZD5847 at 500 mg twice daily significantly decreased the number of CFU on solid medium (-0.039; 95% CI, -0.069, -0.009; P = 0.0048). No bactericidal activity was detected at doses of AZD5847 of 500 mg once daily (mean early bactericidal activity [EBA], 0.02 [95% CI, -0.01, 0.05]), 1,200 mg once daily (mean EBA, 0.02 [95% CI, -0.01, 0.05]), and 800 mg twice daily (mean EBA, 0.02 [95% CI, -0.01, 0.05]). AZD5847 at doses of both 500 mg and 800 mg twice daily also showed an increase in the time to a positive culture in MGIT liquid culture medium. Two serious adverse events (grade 4 thrombocytopenia and grade 4 hyperbilirubinemia) occurred in patients receiving AZD5847 at higher doses. AZD5847 dosed twice daily kills tubercle bacilli in the sputum of patients with pulmonary tuberculosis and has modest early bactericidal activity. (This study has been registered at ClinicalTrials.gov under registration no. NCT01516203.).

Efficiency and safety of the combination of moxifloxacin, pretomanid (PA-824), and pyrazinamide during the first 8 weeks of antituberculosis treatment: a phase 2b, open-label, partly randomised trial in patients with drug-susceptible or drug-resistant pulmonary tuberculosis.

BACKGROUND: New antituberculosis regimens are urgently needed to shorten tuberculosis treatment. Following on from favourable assessment in a 2 week study, we investigated a novel regimen for efficacy and safety in drug-susceptible and multidrug-resistant (MDR) tuberculosis during the first 8 weeks of treatment. METHODS: We did this phase 2b study of bactericidal activity--defined as the decrease in colony forming units (CFUs) of Mycobacterium tuberculosis in the sputum of patients with microscopy smear-positive pulmonary tuberculosis-at eight sites in South Africa and Tanzania. We enrolled treatment-naive patients with drug-susceptible, pulmonary tuberculosis, who were randomly assigned by computer-generated sequences to receive either 8 weeks of moxifloxacin, 100 mg pretomanid (formerly known as PA-824), and pyrazinamide (MPa100Z regimen); moxifloxacin, 200 mg pretomanid, and pyrazinamide (MPa200Z regimen); or the current standard care for drug-susceptible pulmonary tuberculosis, isoniazid, rifampicin, PZA, and ethambutol (HRZE regimen). A group of patients with MDR tuberculosis received MPa200Z (DRMPa200Z group). The primary outcome was bactericidal activity measured by the mean daily rate of reduction in M tuberculosis CFUs per mL overnight sputum collected once a week, with joint Bayesian non-linear mixed-effects regression modelling. We also assessed safety and tolerability by monitoring adverse events. This study is registered with ClinicalTrials.gov, number NCT01498419. FINDINGS: Between March 24, 2012, and July 26, 2013 we enrolled 207 patients and randomly assigned them to treatment groups; we assigned 60 patients to the MPa100Z regimen, 62 to the MPa200Z regimen, and 59 to the HRZE regimen. We non-randomly assigned 26 patients with drug-resistant tuberculosis to the DRMPa200Z regimen. In patients with drug-susceptible tuberculosis, the bactericidal activity of MPa200Z (n=54) on days 0-56 (0·155, 95% Bayesian credibility interval 0·133-0·178) was significantly greater than for HRZE (n=54, 0·112, 0·093-0·131). DRMPa200Z (n=9) had bactericidal activity of 0·117 (0·070-0·174). The bactericidal activity on days 7-14 was strongly associated with bactericidal activity on days 7-56. Frequencies of adverse events were similar to standard treatment in all groups. The most common adverse event was hyperuricaemia in 59 (29%) patients (17 [28%] patients in MPa100Z group, 17 [27%] patients in MPa200Z group, 17 [29%] patients. in HRZE group, and 8 [31%] patients in DRMPa200Z group). Other common adverse events were nausea in (14 [23%] patients in MPa100Z group, 8 [13%] patients in MPa200Z group, 7 [12%] patients in HRZE group, and 8 [31%] patients in DRMPa200Z group) and vomiting (7 [12%] patients in MPa100Z group, 7 [11%] patients in MPa200Z group, 7 [12%] patients in HRZE group, and 4 [15%] patients in DRMPa200Z group). No on-treatment electrocardiogram occurrences of corrected QT interval more than 500 ms (an indicator of potential of ventricular tachyarrhythmia) were reported. No phenotypic resistance developed to any of the drugs in the regimen. INTERPRETATION: The combination of moxifloxacin, pretomanid, and pyrazinamide, was safe, well tolerated, and showed superior bactericidal activity in drug-susceptible tuberculosis during 8 weeks of treatment. Results were consistent between drug-susceptible and MDR tuberculosis. This new regimen is ready to enter phase 3 trials in patients with drug-susceptible tuberculosis and MDR-tuberculosis, with the goal of shortening and simplifying treatment. FUNDING: Global Alliance for TB Drug Development.

A Multilaboratory, Multicountry Study To Determine MIC Quality Control Ranges for Phenotypic Drug Susceptibility Testing of Selected First-Line Antituberculosis Drugs, Second-Line Injectables, Fluoroquinolones, Clofazimine, and Linezolid.

Our objective was to establish reference MIC quality control (QC) ranges for drug susceptibility testing of antimycobacterials, including first-line agents, second-line injectables, fluoroquinolones, and World Health Organization category 5 drugs for multidrug-resistant tuberculosis using a 7H9 broth microdilution MIC method. A tier-2 reproducibility study was conducted in eight participating laboratories using Clinical Laboratory and Standards Institute (CLSI) guidelines. Three lots of custom-made frozen 96-well polystyrene microtiter plates were used and prepared with 2× prediluted drugs in 7H9 broth-oleic acid albumin dextrose catalase. The QC reference strain was Mycobacterium tuberculosis H37Rv. MIC frequency, mode, and geometric mean were calculated for each drug. QC ranges were derived based on predefined, strict CLSI criteria. Any data lying outside CLSI criteria resulted in exclusion of the entire laboratory data set. Data from one laboratory were excluded due to higher MIC values than other laboratories. QC ranges were established for 11 drugs: isoniazid (0.03 to 0.12 µg/ml), rifampin (0.03 to 0.25 µg/ml), ethambutol (0.25 to 2 µg/ml), levofloxacin (0.12 to 1 µg/ml), moxifloxacin (0.06 to 0.5 µg/ml), ofloxacin (0.25 to 2 µg/ml), amikacin (0.25 to 2 µg/ml), kanamycin (0.25 to 2 µg/ml), capreomycin (0.5 to 4 µg/ml), linezolid (0.25 to 2 µg/ml), and clofazimine (0.03 to 0.25 µg/ml). QC ranges could not be established for nicotinamide (pyrazinamide surrogate), prothionamide, or ethionamide, which were assay nonperformers. Using strict CLSI criteria, QC ranges against the M. tuberculosis H37Rv reference strain were established for the majority of commonly used antituberculosis drugs, with a convenient 7H9 broth microdilution MIC method suitable for use in resource-limited settings.

A Multilaboratory, Multicountry Study To Determine Bedaquiline MIC Quality Control Ranges for Phenotypic Drug Susceptibility Testing.

The aim of this study was to establish standardized drug susceptibility testing (DST) methodologies and reference MIC quality control (QC) ranges for bedaquiline, a diarylquinoline antimycobacterial, used in the treatment of adults with multidrug-resistant tuberculosis. Two tier-2 QC reproducibility studies of bedaquiline DST were conducted in eight laboratories using Clinical Laboratory and Standards Institute (CLSI) guidelines. Agar dilution and broth microdilution methods were evaluated. Mycobacterium tuberculosis H37Rv was used as the QC reference strain. Bedaquiline MIC frequency, mode, and geometric mean were calculated. When resulting data occurred outside predefined CLSI criteria, the entire laboratory data set was excluded. For the agar dilution MIC, a 4-dilution QC range (0.015 to 0.12 µg/ml) centered around the geometric mean included 95.8% (7H10 agar dilution; 204/213 observations with one data set excluded) or 95.9% (7H11 agar dilution; 232/242) of bedaquiline MICs. For the 7H9 broth microdilution MIC, a 3-dilution QC range (0.015 to 0.06 µg/ml) centered around the mode included 98.1% (207/211, with one data set excluded) of bedaquiline MICs. Microbiological equivalence was demonstrated for bedaquiline MICs determined using 7H10 agar and 7H11 agar but not for bedaquiline MICs determined using 7H9 broth and 7H10 agar or 7H9 broth and 7H11 agar. Bedaquiline DST methodologies and MIC QC ranges against the H37Rv M. tuberculosis reference strain have been established: 0.015 to 0.12 µg/ml for the 7H10 and 7H11 agar dilution MICs and 0.015 to 0.06 µg/ml for the 7H9 broth microdilution MIC. These methodologies and QC ranges will be submitted to CLSI and EUCAST to inform future research and provide guidance for routine clinical bedaquiline DST in laboratories worldwide.

Direct Susceptibility Testing of Mycobacterium tuberculosis for Pyrazinamide by Use of the Bactec MGIT 960 System.

Pyrazinamide (PZA) is a key antituberculosis drug, yet no rapid susceptibility test is commercially available. PZA drug susceptibility testing (DST) was performed directly on sputum samples from 327 patients and compared with the indirect method by using the Bactec MGIT 960 system in the context of patient screening for participation in a drug trial. Compared to standard indirect PZA DST, direct DST was successful in only 59% of cases, but results obtained were highly accurate and available faster. Agreement between the direct and indirect methods varied from 90 to 100% in each laboratory. The median times for obtaining PZA results from the time when the specimen was collected ranged from 11 to 16 days for the direct test and 18 to 95 days for the indirect test across laboratories. The direct method is accurate and reproducible across laboratories. It can be expected to accelerate results in >50% of cases, but it cannot replace indirect DST for PZA. Phenotypic methods remain the gold standard for DST in drug trials. If future studies can optimize the method to decrease the number of uninterpretable results, direct MGIT DST could be the new phenotypic DST standard for clinical trials, providing more rapid detection of resistance to new drugs in experimental regimens.

Bactericidal activity of pyrazinamide and clofazimine alone and in combinations with pretomanid and bedaquiline.

RATIONALE: New regimens to shorten tuberculosis treatment and manage patients with drug-resistant tuberculosis who are infected with HIV are urgently needed. Experimental and clinical evidence suggests that the new drugs bedaquiline (B) and pretomanid (Pa), combined with an existing drug, pyrazinamide (Z), and a repurposed drug, clofazimine (C), may assist treatment shortening of drug-susceptible and drug-resistant tuberculosis. OBJECTIVES: To evaluate the 14-day bactericidal activity of C and Z in monotherapy and in combinations with Pa and B. METHODS: Groups of 15 treatment-naive, sputum smear-positive patients with pulmonary tuberculosis were randomized to receive combinations of B with Z-C, Pa-Z, Pa-Z-C, and Pa-C, or C or Z alone, or standard combination treatment for 14 days. The primary endpoint was the mean daily fall in log10 Mycobacterium tuberculosis CFU per milliliter sputum estimated by joint nonlinear mixed-effects Bayesian regression modeling. MEASUREMENTS AND MAIN RESULTS: Estimated activities were 0.167 (95% confidence interval [CI], 0.075-0.257) for B-Pa-Z, 0.151 (95% CI, 0.071-0.232) for standard treatment, 0.124 (95% CI, 0.035-0.214) for B-Z-C, 0.115 (95% CI, 0.039-0.189) for B-Pa-Z-C, and 0.076 (95% CI, 0.005-0.145) for B-Pa-C. Z alone had modest activity (0.036; 95% CI, -0.026 to 0.099). C had no activity alone (-0.017; 95% CI, -0.085 to 0.053) or in combinations. Treatments were well tolerated and safe. CONCLUSIONS: B-Pa-Z, including two novel agents without resistance in prevalent M. tuberculosis strains, is a potential new tuberculosis treatment regimen. C had no measurable activity in the first 14 days of treatment. Clinical trial registered with www.clinicaltrials.gov (NCT 01691534).

A dose-ranging trial to optimize the dose of rifampin in the treatment of tuberculosis.

RATIONALE: Rifampin at a dose of 10 mg/kg was introduced in 1971 based on pharmacokinetic, toxicity, and cost considerations. Available data in mice and humans showed that an increase in dose may shorten the duration of tuberculosis treatment. OBJECTIVES: To evaluate the safety and tolerability, the pharmacokinetics, and the extended early bactericidal activity of increasing doses of rifampin. METHODS: Patients with drug-susceptible tuberculosis were enrolled into a control group of eight patients receiving the standard dose of 10 mg/kg rifampin, followed by consecutive experimental groups with 15 patients each receiving rifampin 20, 25, 30, and 35 mg/kg, respectively, for 14 days. In all patients isoniazid, pyrazinamide, and ethambutol were added in standard doses for the second 7 days of treatment. Safety, pharmacokinetics of rifampin, and fall in bacterial load were assessed. MEASUREMENTS AND MAIN RESULTS: Grade 1 and 2 adverse events were equally distributed between the five dose groups; there were five grade 3 events of which one was a possibly related hepatotoxicity. Areas under the time-concentration curves and peak serum concentrations of rifampin showed a more than proportional increase with dose. The daily fall in bacterial load over 14 days was 0.176, 0.168, 0.167, 0.265, and 0.261 log10 colony-forming units/ml sputum in the 10, 20, 25, 30, and 35 mg/kg groups, respectively. CONCLUSIONS: Two weeks of rifampin up to 35 mg/kg was safe and well tolerated. There was a nonlinear increase in exposure to rifampin without an apparent ceiling effect and a greater estimated fall in bacterial load in the higher dosing groups. Clinical trial registered with www.clinicaltrials.gov (NCT 01392911).

Early phase evaluation of SQ109 alone and in combination with rifampicin in pulmonary TB patients.

OBJECTIVES: SQ109, an asymmetrical diamine, is a novel anti-TB drug candidate. This first study in patients was done to determine safety, tolerability, pharmacokinetics and bacteriological effect of different doses of SQ109 alone and in combination with rifampicin when administered over 14 days. PATIENTS AND METHODS: Smear-positive pulmonary TB patients were randomized into six groups of 15 to receive once-daily oral treatment with 75, 150 or 300 mg of SQ109, rifampicin (10 mg/kg body weight), rifampicin plus 150 mg of SQ109, or rifampicin plus 300 mg of SQ109 for 14 days. Patients were hospitalized for supervised treatment, regular clinical, biochemical and electrocardiographic safety assessments, pharmacokinetic profiling and daily overnight sputum collection. RESULTS: SQ109 was safe and generally well tolerated. Mild to moderate dose-dependent gastrointestinal complaints were the most frequent adverse events. No relevant QT prolongation was noted. Maximum SQ109 plasma concentrations were lower than MICs. Exposure to SQ109 (AUC0-24) increased by drug accumulation upon repeated administration in the SQ109 monotherapy groups. Co-administration of SQ109 150 mg with rifampicin resulted in decreasing SQ109 exposures from day 1 to day 14. A higher (300 mg) dose of SQ109 largely outweighed the evolving inductive effect of rifampicin. The daily fall in log cfu/mL of sputum (95% CI) was 0.093 (0.126-0.059) with rifampicin, 0.133 (0.166-0.100) with rifampicin plus 150 mg of SQ109 and 0.089 (0.121-0.057) with rifampicin plus 300 mg of SQ109. Treatments with SQ109 alone showed no significant activity. CONCLUSIONS: SQ109 alone or with rifampicin was safe over 14 days. Upon co-administration with rifampicin, 300 mg of SQ109 yielded a higher exposure than the 150 mg dose. SQ109 did not appear to be active alone or to enhance the activity of rifampicin during the 14 days of treatment.

Randomized dose-ranging study of the 14-day early bactericidal activity of bedaquiline (TMC207) in patients with sputum microscopy smear-positive pulmonary tuberculosis.

Bedaquiline is a new antituberculosis agent targeting ATP synthase. This randomized, double-blinded study enrolling 68 sputum smear-positive pulmonary tuberculosis patients evaluated the 14-day early bactericidal activity of daily doses of 100 mg, 200 mg, 300 mg, and 400 mg bedaquiline, preceded by loading doses of 200 mg, 400 mg, 500 mg, and 700 mg, respectively, on the first treatment day and 100 mg, 300 mg, 400 mg, and 500 mg on the second treatment day. All groups showed activity with a mean (standard deviation) daily fall in log10 CFU over 14 days of 0.040 (0.068), 0.056 (0.051), 0.077 (0.064), and 0.104 (0.077) in the 100-mg, 200-mg, 300-mg, and 400-mg groups, respectively. The linear trend for dose was significant (P = 0.001), and activity in the 400-mg dose group was greater than that in the 100-mg group (P = 0.014). All of the bedaquiline groups showed significant bactericidal activity that was continued to the end of the 14-day evaluation period. The finding of a linear trend for dose suggests that the highest dose compatible with safety considerations should be taken forward to longer-term clinical studies.

14-day bactericidal activity of PA-824, bedaquiline, pyrazinamide, and moxifloxacin combinations: a randomised trial.

BACKGROUND: New drugs, but also shorter, better-tolerated regimens are needed to tackle the high global burden of tuberculosis complicated by drug resistance and retroviral disease. We investigated new multiple-agent combinations over the first 14 days of treatment to assess their suitability for future development. METHODS: In this prospective, randomised, early bactericidal activity (EBA) study, treatment-naive, drug-susceptible patients with uncomplicated pulmonary tuberculosis were admitted to hospitals in Cape Town, South Africa, between Oct 7, 2010, and Aug 19, 2011. Patients were randomised centrally by computer-generated randomisation sequence to receive bedaquiline, bedaquiline-pyrazinamide, PA-824-pyrazinamide, bedaquiline-PA-824, PA-824-moxifloxacin-pyrazinamide, or unmasked standard antituberculosis treatment as positive control. The primary outcome was the 14-day EBA assessed in a central laboratory from the daily fall in colony forming units (CFU) of M tuberculosis per mL of sputum in daily overnight sputum collections. Bilinear regression curves were fitted for each group separately and groups compared with ANOVA for ranks, followed by pair-wise comparisons adjusted for multiplicity. Clinical staff were partially masked but laboratory personnel were fully masked. This study is registered, NCT01215851. FINDINGS: The mean 14-day EBA of PA-824-moxifloxacin-pyrazinamide (n=13; 0·233 [SD 0·128]) was significantly higher than that of bedaquiline (14; 0·061 [0·068]), bedaquiline-pyrazinamide (15; 0·131 [0·102]), bedaquiline-PA-824 (14; 0·114 [0·050]), but not PA-824-pyrazinamide (14; 0·154 [0·040]), and comparable with that of standard treatment (ten; 0·140 [0·094]). Treatments were well tolerated and appeared safe. One patient on PA-824-moxifloxacin-pyrazinamide was withdrawn because of corrected QT interval changes exceeding criteria prespecified in the protocol. INTERPRETATION: PA-824-moxifloxacin-pyrazinamide is potentially suitable for treating drug-sensitive and multidrug-resistant tuberculosis. Multiagent EBA studies can contribute to reducing the time needed to develop new antituberculosis regimens. FUNDING: The Global Alliance for TB Drug Development (TB Alliance).

Short-term storage does not affect the quantitative yield of Mycobacterium tuberculosis in sputum in early-bactericidal-activity studies.

Early-bactericidal-activity (EBA) studies measure the change in mycobacterial load in sputum over time to evaluate antituberculosis drugs. We investigated whether a delay in sputum processing influences the quantitative results of sputum mycobacterial culture. We identified pretreatment smear-positive sputum samples collected overnight and processed at a single laboratory. Sputum volume, time from sputum collection to processing, CFU counts/ml of sputum, and time to culture positivity (TTP) data were retrieved. We obtained 817 TTP and 794 CFU results from a total of 844 sputum samples. Contamination did not occur more frequently with prolonged storage (TTP, 2.0%; CFU, 2.4%). Sample volumes were <5 ml in 5%, 5 to 10 ml in 46%, and >10 ml in 49%. Delays to processing were 0, 1, 2, and 3 days in 696 (43.2%), 722 (44.8%), 128 (7.9%), and 65 (4.0%) samples, respectively. TTP and CFU did not significantly differ between days of delay to processing (P = 0.098 and P = 0.908, respectively), but there was a nonsignificant trend toward a prolonged TTP over time (P = 0.052, Jonckheere-Terpstra trend test). Sputa of <5 ml in volume showed a significantly prolonged TTP compared to sputum of >5 ml (113 h versus 99 h; P < 0.01) but no significant decrease in CFU. Sputum can be stored under refrigerated conditions for deferred processing for at least 3 days. This means that central laboratories can be used for quantitative mycobacterial study endpoints when delays to processing are not expected to exceed a few days. Care should be taken to collect sputum of sufficient volume.

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

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.

Phase II dose-ranging trial of the early bactericidal activity of PA-824.

PA-824 is a novel nitroimidazo-oxazine under evaluation as an antituberculosis agent. A dose-ranging randomized study was conducted to evaluate the safety, tolerability, pharmacokinetics, and early bactericidal activity of PA-824 in drug-sensitive, sputum smear-positive adult pulmonary-tuberculosis patients to find the lowest dose giving optimal bactericidal activity (EBA). Fifteen patients per cohort received oral PA-824 in doses of 50 mg, 100 mg, 150 mg, or 200 mg per kg body weight per day for 14 days. Eight subjects received once-daily standard antituberculosis treatment with isoniazid, rifampin, pyrazinamide, and ethambutol (HRZE) as a positive control. The primary efficacy endpoint was the mean rate of decline in log CFU of Mycobacterium tuberculosis in sputum incubated on agar plates from serial overnight sputum collections, expressed as log(10) CFU/day/ml sputum (± standard deviation). The mean 14-day EBA of HRZE was consistent with previous studies (0.177 ± 0.042), and that of PA-824 at 50 mg, 100 mg, 150 mg, and 200 mg was 0.063 ± 0.058, 0.091 ± 0.073, 0.078 ± 0.074, and 0.112 ± 0.070, respectively. Although the study was not powered for testing the difference between arms, there was a trend toward significance, indicating a lower EBA at the 50-mg dose. Serum PA-824 levels were approximately dose proportional with respect to the area under the time-concentration curve. All doses were safe and well tolerated with no dose-limiting adverse events or clinically significant QTc changes. A dose of 100 mg to 200 mg PA-824 daily appears to be safe and efficacious and will be further evaluated as a component of novel antituberculosis regimens for drug-sensitive and drug-resistant tuberculosis.

The diarylquinoline TMC207 for multidrug-resistant tuberculosis.

BACKGROUND: The diarylquinoline TMC207 offers a new mechanism of antituberculosis action by inhibiting mycobacterial ATP synthase. TMC207 potently inhibits drug-sensitive and drug-resistant Mycobacterium tuberculosis in vitro and shows bactericidal activity in patients who have drug-susceptible pulmonary tuberculosis. METHODS: In the first stage of a two-stage, phase 2, randomized, controlled trial, we randomly assigned 47 patients who had newly diagnosed multidrug-resistant pulmonary tuberculosis to receive either TMC207 (400 mg daily for 2 weeks, followed by 200 mg three times a week for 6 weeks) (23 patients) or placebo (24 patients) in combination with a standard five-drug, second-line antituberculosis regimen. The primary efficacy end point was the conversion of sputum cultures, in liquid broth, from positive to negative. RESULTS: The addition of TMC207 to standard therapy for multidrug-resistant tuberculosis reduced the time to conversion to a negative sputum culture, as compared with placebo (hazard ratio, 11.8; 95% confidence interval, 2.3 to 61.3; P=0.003 by Cox regression analysis) and increased the proportion of patients with conversion of sputum culture (48% vs. 9%). The mean log(10) count of colony-forming units in the sputum declined more rapidly in the TMC207 group than in the placebo group. No significant differences in average plasma TMC207 concentrations were noted between patients with and those without culture conversion. Most adverse events were mild to moderate, and only nausea occurred significantly more frequently among patients in the TMC207 group than among patients in the placebo group (26% vs. 4%, P=0.04). CONCLUSIONS: The clinical activity of TMC207 validates ATP synthase as a viable target for the treatment of tuberculosis. (ClinicalTrials.gov number, NCT00449644.)

Suitability of Xpert MTB/RIF and genotype MTBDRplus for patient selection for a tuberculosis clinical trial.

Participation criteria for clinical trials in pulmonary tuberculosis commonly include confirmation of sputum positive for mycobacteria and an indication of drug susceptibility before treatment is initiated. We investigated the suitability of two novel sputum-based nucleic acid amplification methods for patient selection in a recent early bactericidal activity study. Spontaneously expectorated sputum samples of 140 consecutive pulmonary tuberculosis patients were examined with direct fluorescence microscopy, Genotype MTBDRplus assay (MTBDR), Xpert MTB/RIF assay (Xpert), and liquid mycobacterial culture. The methods detected mycobacteria or mycobacterial DNA in 96.8%, 90.5%, 92.9%, and 92.1% of samples, respectively. MTBDR, Xpert, and liquid culture were 100% concordant for detection of resistance to rifampin. Sensitivity and specificity of MTBDR for detection of isoniazid resistance were 83.3% and 100%, respectively. For quantification of mycobacterial sputum load, we found a correlation between Xpert DNA amplification cycle thresholds, time to positivity, and microscopy smear grade. The best correlation was found between Xpert and time to positivity (r = 0.54), which were both correlated with smear microscopy with r values equal to -0.40 and -0.48, respectively. We conclude that MTBDR and Xpert are suitable screening tools for determining rifampin resistance in sputum microscopy smear-positive patients before participation in tuberculosis trials. Xpert should be further explored as a surrogate measurement for sputum mycobacterial load.