The potential use of rifabutin for treatment of patients diagnosed with rifampicin-resistant tuberculosis.

Background: Use of the Xpert MTB/RIF assay has increased the number of people diagnosed with rifampicin-resistant tuberculosis (RR-TB), especially in South Africa where Xpert is now the initial diagnostic for individuals with TB symptoms. We hypothesized that a proportion of RR-TB patients determined by Xpert can be treated with a rifabutin-containing regimen. Methods: Rifabutin susceptibility by rpoB mutation was assessed in 349 individuals from South Africa and 172 from Belgium. rpoB polymorphisms were identified by Sanger sequencing. Rifampicin and rifabutin susceptibility was assessed phenotypically. A systematic review was performed to comprehensively collate information on rifabutin susceptibility by rpoB polymorphism. Rifabutin susceptibility was assigned to rpoB polymorphisms based on their positive likelihood ratios and ORs. Results: One hundred and twelve rpoB polymorphisms (67.9% from literature) were identified from all 2045 RR-TB patients, of which 17 polymorphisms could be classified as susceptible/resistant to rifabutin. Eleven polymorphisms were associated with rifabutin susceptibility. The 516GTC mutation was the most common, representing 70% (South Africa) and 76% (Belgium) of all rifabutin-susceptible isolates. At a population level, the 11 polymorphisms associated with rifabutin susceptibility occurred in 33.2% and 16.6% of all South African and Belgian patients diagnosed with RR-TB, respectively. Conclusions: Identification of the exact rpoB polymorphism leading to the diagnosis of RR-TB has the potential to allow inclusion of rifabutin in the treatment regimen of a substantial proportion of RR-TB patients. A randomized controlled trial evaluating the efficacy of a rifabutin-containing TB treatment regimen in these selected patients is needed to provide the evidence required for a change in policy.

Low Frequency of Acquired Isoniazid and Rifampicin Resistance in Rifampicin-Susceptible Pulmonary Tuberculosis in a Setting of High HIV-1 Infection and Tuberculosis Coprevalence.

Background: We estimated the incidence of acquired isoniazid and rifampicin resistance in rifampicin-susceptible tuberculosis in a setting of high human immunodeficiency virus type 1 (HIV-1) infection and tuberculosis coprevalence. Methods: GeneXpert MTB/RIF-confirmed patients with rifampicin-susceptible tuberculosis were recruited at antituberculosis treatment initiation in Khayelitsha, South Africa. Liquid culture and adherence assessment were performed at 2 and 5-6 months. MTBDRplus was performed on mycobacteria-positive cultures to ascertain acquired drug resistance (ADR). Spoligotyping and whole-genome sequencing were performed to ascertain homogeneity between baseline isolates and isolates with ADR. Baseline isolates were retrospectively tested for isoniazid monoresistance. An electronic database review was performed to ascertain tuberculosis recurrences. Results: A total of 306 participants (62% with HIV-1 coinfection, of whom 71% received antiretroviral therapy) were recruited. Ascertainment of outcomes was complete for 284 participants. Five acquired a resistant Mycobacterium tuberculosis strain during or subsequent to treatment. One strain was confirmed to have ADR, 2 were confirmed as causing exogenous reinfection, and 2 were unrecoverable for genotyping. Incident ADR was estimated to have ranged from 0.3% (95% confidence interval [CI], .1%-1.9%; 1 of 284 participants) to 1% (95% CI, .2%-3%; 3 of 284 participants). Seventeen of 279 baseline isolates (6.1%; 95% CI, 3.6%-9.6%) had isoniazid monoresistance (13 of 17 had an inhA promoter mutation), but 0 of 17 had amplified resistance. Conclusions: Treatment with standardized antituberculosis regimens dosed daily throughout, high uptake of antiretroviral therapy, and low prevalence of isoniazid monoresistance were associated with a low frequency of ADR.

Concentration-Dependent Antagonism and Culture Conversion in Pulmonary Tuberculosis.

BACKGROUND: There is scant evidence to support target drug exposures for optimal tuberculosis outcomes. We therefore assessed whether pharmacokinetic/pharmacodynamic (PK/PD) parameters could predict 2-month culture conversion. METHODS: One hundred patients with pulmonary tuberculosis (65% human immunodeficiency virus coinfected) were intensively sampled to determine rifampicin, isoniazid, and pyrazinamide plasma concentrations after 7-8 weeks of therapy, and PK parameters determined using nonlinear mixed-effects models. Detailed clinical data and sputum for culture were collected at baseline, 2 months, and 5-6 months. Minimum inhibitory concentrations (MICs) were determined on baseline isolates. Multivariate logistic regression and the assumption-free multivariate adaptive regression splines (MARS) were used to identify clinical and PK/PD predictors of 2-month culture conversion. Potential PK/PD predictors included 0- to 24-hour area under the curve (AUC0-24), maximum concentration (Cmax), AUC0-24/MIC, Cmax/MIC, and percentage of time that concentrations persisted above the MIC (%TMIC). RESULTS: Twenty-six percent of patients had Cmax of rifampicin <8 mg/L, pyrazinamide <35 mg/L, and isoniazid <3 mg/L. No relationship was found between PK exposures and 2-month culture conversion using multivariate logistic regression after adjusting for MIC. However, MARS identified negative interactions between isoniazid Cmax and rifampicin Cmax/MIC ratio on 2-month culture conversion. If isoniazid Cmax was <4.6 mg/L and rifampicin Cmax/MIC <28, the isoniazid concentration had an antagonistic effect on culture conversion. For patients with isoniazid Cmax >4.6 mg/L, higher isoniazid exposures were associated with improved rates of culture conversion. CONCLUSIONS: PK/PD analyses using MARS identified isoniazid Cmax and rifampicin Cmax/MIC thresholds below which there is concentration-dependent antagonism that reduces 2-month sputum culture conversion.

Impact of nonlinear interactions of pharmacokinetics and MICs on sputum bacillary kill rates as a marker of sterilizing effect in tuberculosis.

The relationships between antituberculosis drug exposure and treatment effects on humans receiving multidrug therapy are complex and nonlinear. In patients on treatment, an analysis of the rate of decline in the sputum bacillary burden reveals two slopes. The first is the α-slope, which is thought to reflect bactericidal effect, followed by a ß-slope, which is thought to reflect sterilizing activity. We sought to characterize the effects of standard first-line treatment on sterilizing activity. Fifty-four patients receiving combination therapy for pulmonary tuberculosis in a clinical trial had drug concentrations measured and Mycobacterium tuberculosis isolates available for MIC identification. Sputum sample cultures were performed at baseline and weekly for 8 weeks. A time-to-event model based on the days to positivity in the liquid cultures was used to estimate the ß-slope. The pharmacokinetic parameters of rifampin, isoniazid, ethambutol, and pyrazinamide were determined for each patient. Multivariate adaptive regression splines analyses, which simultaneously perform linear and nonlinear analyses, were used to identify the relationships between the predictors and the ß-slope. The potential predictors examined included HIV status, lung cavitation, 24-h area under the concentration-time curve (AUC), peak drug concentration (Cmax), AUC/MIC ratio, Cmax/MIC ratio, and the time that that concentration persisted above MIC. A rifampin Cmax of >8.2 mg/liter and a pyrazinamide AUC/MIC of >11.3 were key predictors of the ß-slope and interacted positively to increase the ß-slope. In patients with a rifampin AUC of <35.4 mg · h/liter, an increase in the pyrazinamide AUC/MIC and/or ethambutol Cmax/MIC increased the ß-slope, while increasing isoniazid Cmax decreased it, suggesting isoniazid antagonism. Antibiotic concentrations and MICs interact in a nonlinear fashion as the main drivers of a sterilizing effect. The results suggest that faster speeds of sterilizing effect might be achieved by omitting isoniazid and by increasing rifampin, pyrazinamide, and ethambutol exposures. However, isoniazid and ethambutol exposures may only be of importance when rifampin exposure is low. These findings need confirmation in larger studies. (This study has been registered at controlled-trials.com under registration no. ISRCTN80852505.).

The pyrazinamide susceptibility breakpoint above which combination therapy fails.

OBJECTIVES: To identify the pyrazinamide MIC above which standard combination therapy fails. METHODS: MICs of pyrazinamide were determined for Mycobacterium tuberculosis isolates, cultured from 58 patients in a previous randomized clinical trial in Cape Town, South Africa. The MICs were determined using BACTEC MGIT 960 for isolates that were collected before standard treatment with isoniazid, rifampicin, pyrazinamide and ethambutol commenced. Weekly sputum collections were subsequently made for 8 weeks in order to culture M. tuberculosis in Middlebrook broth medium. Classification and regression tree (CART) analysis was utilized to identify the pyrazinamide MIC predictive of sputum culture results at the end of pyrazinamide therapy. The machine learning-derived susceptibility breakpoints were then confirmed using standard association statistics that took into account confounders of 2 month sputum conversion. RESULTS: The pyrazinamide MIC range was 12.5 to >100 mg/L for the isolates prior to therapy. The epidemiological 95% cut-off value was >100 mg/L. The 2 month sputum conversion rate in liquid cultures was 26% by stringent criteria and 48% by less stringent criteria. CART analysis identified an MIC breakpoint of 50 mg/L, above which patients had poor sputum conversion rates. The relative risk of poor sputum conversion was 1.5 (95% CI: 1.2-1.8) for an MIC >50 mg/L compared with an MIC ≤ 50 mg/L. CONCLUSIONS: We propose a pyrazinamide susceptibility breakpoint of 50 mg/L for clinical decision making and for development of rapid susceptibility assays. This breakpoint is identical to that identified using computer-aided simulations of hollow fibre system output.

The rationale for using rifabutin in the treatment of MDR and XDR tuberculosis outbreaks.

Genetically related Mycobacterium tuberculosis strains with alterations at codon 516 in the rpoB gene were observed amongst a substantial number of patients with drug resistant tuberculosis in the Eastern Cape Province (ECP) of South Africa. Mutations at codon 516 are usually associated with lower level rifampicin (RIF) resistance, while susceptibility to rifabutin (RFB) remains intact. This study was conducted to assess the rationale for using RFB as a substitution for RIF in the treatment of MDR and XDR tuberculosis outbreaks. Minimum inhibitory concentrations (MICs) of 34 drug resistant clinical isolates of M tuberculosis were determined by MGIT 960 and correlated with rpoB mutations. RFB MICs ranged from 0.125 to 0.25 µg/ml in the 34 test isolates thereby confirming phenotypic susceptibility as per critical concentration (CC) of 0.5 µg/ml. The corresponding RIF MICs ranged between 5 and 15 µg/ml, which is well above the CC of 1.0 µg/ml. Molecular-based drug susceptibility testing provides important pharmacogenetic insight by demonstrating a direct correlation between defined rpoB mutation and the level of RFB susceptibility. We suggest that isolates with marginally reduced susceptibility as compared to the epidemiological cut-off for wild-type strains (0.064 µg/ml), but lower than the current CC (≤0.5 µg/ml), are categorised as intermediate. Two breakpoints (0.064 µg/ml and 0.5 µg/ml) are recommended to distinguish between susceptible, intermediate and RFB resistant strains. This concept may assist clinicians and policy makers to make objective therapeutic decisions, especially in situations where therapeutic options are limited. The use of RFB in the ECP may improve therapeutic success and consequently minimise the risk of ongoing transmission of drug resistant M. tuberculosis strains.