The study was a randomized, double-blind, double-dummy trial for patients with sputum smear-positive tuberculosis with no prior history of treatment. Eligible patients were age 18 years or older who had clinical signs and symptoms of pulmonary tuberculosis, including an abnormal chest radiograph, and at least one sputum smear with acid fast bacilli visible by Ziehl-Neelsen staining. Potential study subjects underwent baseline screening that included blood counts, liver enzymes and kidney function testing, HIV serology, chest radiograph and culture and drug susceptibility testing for mycobacteria. Patients were excluded if they had a hemoglobin <7 g/dL, aspartate or alanine aminotransferase levels >3 times the upper limits of normal, serum creatinine >2 times the upper limit of normal, an electrocardiogram with a QTc >450 ms, were pregnant or nursing, if they had silicotuberculosis, if they had a history of severe adverse reactions to fluoroquinolones or any other study agent, or if they were HIV seropositive and had a CD4 cell count <200 mm3, as this would be an indication for antiretroviral therapy which could be affected by antituberculosis drug interactions. Initially enrolled patients were subsequently excluded if their baseline culture failed to grow M. tuberculosis or grew a strain of M. tuberculosis that was resistant to isoniazid, rifampin or ethambutol
Consenting patients were stratified by HIV serostatus and randomly assigned to treatment with one of two study regimens using permutated blocked randomization with treatment allocation concealed and allocation slips sealed in opaque envelopes opened after enrollment. Patients received either moxifloxacin 400 mg with an ethambutol placebo or moxifloxacin placebo plus ethambutol 15–20 mg/kg. The study was designed to ensure that all patients received an effective regimen for tuberculosis regardless of the contribution of the two experimental agents, moxifloxacin and ethambutol, and therefore all patients received isoniazid 300 mg, rifampicin 450 mg (weight <50 kg) or 600 mg (weight ≥50 kg), and pyrazinamide 20–25 mg/kg by directly observed therapy. Patients, study clinicians and study staff were unaware of the treatment assignments of patients, with the exception of the pharmacist who dispensed medication packets. All therapy was given by direct supervision either in the hospital clinic or in the community by study personnel, five days per week. At the end of eight weeks, all patients were placed on open-label treatment with isoniazid and rifampin three times weekly to complete another four months of therapy. Moxifloxacin and matching placebo was provided by Bayer Healthcare (West Haven, CT, USA) and ethambutol, ethambutol placebo, isoniazid, rifampin and pyrazinamide were provided by Farmanguinhos, Rio de Janeiro, Brazil. All study agents were produced under Good Manufacturing Process.
Patients had weekly clinic visits to assess clinical status and to monitor for adverse reactions. Liver enzymes, serum creatinine levels and complete blood counts were performed monthly, and an electrocardiogram was obtained at Weeks 2, 4, 6 and 8. A sputum specimen (spontaneous or induced) was obtained weekly for smear and culture. Sputum specimens were digested and decontaminated using the N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) method (9
). Pellets were resuspended in a final volume of 2 ml and used immediately for inoculation of Löwenstein-Jensen culture medium. Löwenstein-Jensen slants were prepared from a commercial powder medium base (BD, Sparks, MD, USA), according to the manufacturer’s instructions. Two hundred microliters of each decontaminated respiratory specimen was inoculated onto each of two slants. Slants were incubated at 37°C in ambient CO2 and examined visually for growth twice weekly for 8 weeks. Growth on slants was quantified by number of visible colonies, and then examined by acid-fast staining to confirm the presence of mycobacteria; isolates were speciated using standard biochemical tests (10
). Drug susceptibility testing of all baseline cultures growing Mycobacterium tuberculosis
was performed using the proportions method (10
The primary endpoint of the study was the proportion of patients with negative sputum cultures after eight weeks of treatment. We assumed that the conversion rate in the control arm (ethambutol) would be 65% (8
), and that moxifloxacin would increase this by 20% to 85%. To achieve a power of 0.8 with an alpha of 0.05, we needed 70 evaluable patients in each arm of the trial. We aimed to enroll 85 per arm to allow for exclusions based on negative initial culture, growth of nontuberculous mycobacteria, or baseline drug resistance. The primary endpoint of the trial, culture conversion, was assessed in a modified intention to treat analysis where those whose baseline cultures were either negative, contaminated or contained drug-resistant M. tuberculosis
were excluded and where all missing week-8 results were considered to be failures (missing=failure). We also analyzed all patients with available Week 8 cultures (evaluable analysis). Multivariate logistic regression was performed using treatment allocation, selected baseline characteristics as covariates and culture conversion as the dependent variable. Time to conversion of sputum cultures to negative was estimated by the Kaplan-Meier method and the difference in median times was compared using the log-rank test. All analyses were performed using STATA Version 9 (STATA Corp., College Station, TX, USA).
Ethical review of the protocol was obtained from the Johns Hopkins Medicine Institutional Review Board and the Ethics Committee of the Federal University of Rio de Janeiro, the National Ethics Committee of the Ministry of Health (CONEP), Brasilia and the Agencia Nacional de Vigilancia Sanitaria (ANVISA), Brazil.
Role of the Funding Source
The study was funded by the Office of Orphan Product Development, United States Food and Drug Administration and was registered as a clinical trial at Clinicaltrials.gov
. Additional support for training was provided by the Fogarty International Center of the National Institutes of Health, and a career development grant helped support Dr. Chaisson’s participation. None of the funding agencies was involved in the design, conduct or analysis of the study. The Investigational New Drug Office of the Food and Drug Administration reviewed the protocol and made suggestions regarding the study design prior to initiation of the trial, but this was unrelated to any funding decisions. The decision to publish was made solely by the authors and the funding agencies did not review or approve the manuscript. Bayer Healthcare donated moxifloxacin and matching placebo for the trial, but had no input into the study design, execution or analysis.