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% HIV-co-infected) were intensively sampled to determine rifampicin, isoniazid and pyrazinamide plasma concentrations after 7-8 weeks of therapy, and pharmacokinetic parameters determined using non-linear-mixed-effects models. Detailed clinical data and sputum for culture were collected at baseline, 2 and 5-6 months. Minimum inhibitory concentrations (MIC) 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 24-hour-area-under-the-curve (AUC0-24), peak concentration (Cmax), AUC0-24/MIC, Cmax/MIC and % time that concentrations persisted above MIC (%TMIC). Results 26% of patients had Cmax (mg/L) of rifampicin4.6 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

gyrA mutations and phenotypic susceptibility levels to ofloxacin and moxifloxacin in clinical isolates of Mycobacterium tuberculosis

Objectives To compare mutations in the quinolone resistance-determining region of the gyrA gene and flanking sequences with the MICs of ofloxacin and moxifloxacin for Mycobacterium tuberculosis. Methods The presence of mutations in 177 drug-resistant M. tuberculosis isolates was determined by DNA sequencing and the MICs quantified by MGIT 960. Results Single nucleotide polymorphisms were detected at codons 94 (n = 30), 90 (n = 12), 91 (n = 3), 89 (n = 1), 88 (n = 1) and 80 (n = 1). Four isolates with double mutations D94G plus A90V (n = 2) and D94G plus D94N (n = 2) reflect mixed populations. Agreement between genotypic and phenotypic susceptibility was high (≥97%) for both drugs. Mutant isolates had an MIC50 of 8.0 mg/L and an MIC90 of >10 mg/L for ofloxacin compared with an MIC50 and MIC90 of 2.0 mg/L for moxifloxacin. Codons 94 and 88 were linked to higher levels of fluoroquinolone resistance compared with codons 90, 91 and 89. The MIC distributions for the wild-type isolates ranged from ≤0.5 to 2.0 mg/L for ofloxacin and from ≤0.125 to 0.25 mg/L for moxifloxacin. However, 96% of the isolates with genetic alterations had MICs ≤2.0 mg/L for moxifloxacin, which is within its achievable serum levels. Conclusions This study provides quantitative evidence that the addition of moxifloxacin to extensively drug-resistant tuberculosis (XDR-TB) regimens based on a clinical breakpoint of 2.0 mg/L has merit. The use of moxifloxacin in the treatment of multidrug-resistant tuberculosis may prevent the acquisition of additional mutations and development of XDR-T

Primary capreomycin resistance is common and associated with early mortality in patients with extensively drug-resistant tuberculosis in KwaZulu-Natal, South Africa.

CAPRISA, 2015.Abstract available in pdf

Linezolid Pharmacokinetics in South African Patients with Drug-Resistant Tuberculosis and a High Prevalence of HIV Coinfection.

The World Health Organization (WHO) recently recommended that linezolid be prioritized in treatment regimens for drug-resistant tuberculosis (TB), but there are limited data on its pharmacokinetics (PK) in patients with this disease. We conducted an observational study to explore covariate effects on linezolid PK and to estimate the probability of PK/pharmacodynamic target attainment in South African patients with drug-resistant TB. Consecutive adults on linezolid-based regimens were recruited in Cape Town and underwent intensive PK sampling at steady state. Noncompartmental analysis was performed. Thirty participants were included: 15 HIV positive, 26 on the initial dose of 600 mg daily, and 4 participants on 300 mg daily after dose reduction for linezolid-related toxicity. There was a negative correlation between body weight and exposure, with 17.4% (95% confidence interval [CI], 0.1 to 31.7) decrease in area under the concentration-time curve from 0 to 24 h (AUC0-24) per 10-kg weight increment after adjustment for other covariates. Age was an independent predictor of trough concentration, with an estimated 43.4% (95% CI, 5.9 to 94.2) increase per 10-year increment in age. The standard 600-mg dose achieved the efficacy target of free AUC/MIC of >119 at wild-type MIC values (≤0.5 mg/liter), but the probability of target attainment dropped to 61.5% (95% CI, 40.6 to 79.8) at the critical concentration of 1 mg/liter. When dosed at 600 mg daily, trough concentrations were above the toxicity threshold of 2 mg/liter in 57.7% (95% CI, 36.9 to 76.6). This confirms the narrow therapeutic index of linezolid, and alternative dosing strategies should be explored

Serial counts of Mycobacterium tuberculosis in sputum as surrogate markers of the sterilising activity of rifampicin and pyrazinamide in treating pulmonary tuberculosis

BACKGROUND: Since the sterilising activity of new antituberculosis drugs is difficult to assess by conventional phase III studies, surrogate methods related to eventual relapse rates are required. METHODS: A suitable method is suggested by a retrospective analysis of viable counts of Mycobacterium tuberculosis in 12-hr sputum collections from 122 newly diagnosed patients with pulmonary tuberculosis in Nairobi, done pretreatment and at 2, 7, 14 and 28 days. Treatment was with isoniazid and streptomycin, supplemented with either thiacetazone (SHT) or rifampicin + pyrazinamide (SHRZ). RESULTS: During days 0–2, a large kill due to isoniazid occurred, unrelated to treatment or HIV status; thereafter it decreased exponentially. SHRZ appeared to have greater sterilising activity than SHT during days 2–7 (p = 0.044), due to rifampicin, and during days 14–28, probably due mainly to pyrazinamide. The greatest discrimination between SHRZ and SHT treatments was found between regression estimates of kill over days 2–28 (p = 0.0005) in patients who remained positive up to 28 days with homogeneous kill rates. No associations were found between regression estimates and the age, sex, and extent of disease or cavitation. An increased kill in HIV seropositive patients, unrelated to the treatment effect, was evident during days 2–28 (p = 0.007), mainly during days 2–7. CONCLUSIONS: Surrogate marker studies should either be in small groups treated with monotherapy during days 2 to about 7 or as add-ons or replacements in isoniazid-containing standard regimens from days 2 to 28 in large groups

Rifampicin mono-resistant tuberculosis is not the same as multidrug-resistant tuberculosis: a descriptive study from Khayelitsha, South Africa

Rifampicin mono-resistant TB (RMR-TB, rifampicin resistance and isoniazid susceptibility) constitutes 38% of all rifampicin-resistant TB (RR-TB) in South Africa and is increasing. We aimed to compare RMR-TB with multidrug-resistant TB (MDR-TB) within a high TB, RR-TB and HIV burden setting. Patient-level clinical data and stored RR-TB isolates from 2008-2017 with available whole genome sequencing (WGS) data were used to describe risk factors associated with RMR-TB and to compare rifampicin-resistance (RR) conferring mutations between RMR-TB and MDR-TB. A subset of isolates with particular RR-conferring mutations were subjected to semi-quantitative rifampicin phenotypic drug susceptibility testing. Among 2,041 routinely diagnosed RR-TB patients, 463 (22.7%) had RMR-TB. HIV-positive individuals (adjusted Odds Ratio 1.4, 95% CI 1.1-1.9) and diagnosis between 2013-2017 versus 2008-2012 (aOR 1.3, 1.1-1.7) were associated with RMR-TB. Among 1,119 (54.8%) patients with available WGS data showing RR-TB, significant differences in the distribution of rpoB RR-conferring mutations between RMR-TB and MDR-TB isolates were observed. Mutations associated with high-level RR were more commonly found among MDR-TB isolates (811/889, 90.2% versus 162/230, 70.4% among RMR-TB, p<0.0001). In particular, the rpoB L430P mutation, conferring low-level RR, was identified in 32/230 (13.9%) RMR-TB versus 10/889 (1.1%) in MDR-TB (p<0.0001). Among 10 isolates with an rpoB L430P mutation, 7 were phenotypically susceptible using the critical concentration of 0.5 mug/ml (range 0.125-1 mug/ml). The majority (215/230, 93.5%) of RMR-TB isolates showed susceptibility to all other TB drugs, highlighting the potential benefits of WGS for simplified treatment. These data suggest that the evolution of RMR-TB differs from MDR-TB with a potential contribution from HIV infection

Outcomes, infectiousness, and transmission dynamics of patients with extensively drug-resistant tuberculosis and home-discharged patients with programmatically incurable tuberculosis: a prospective cohort study.

BACKGROUND: The emergence of programmatically incurable tuberculosis threatens to destabilise control efforts. The aim of this study was to collect prospective patient-level data to inform treatment and containment strategies. METHODS: In a prospective cohort study, 273 South African patients with extensively drug-resistant tuberculosis, or resistance beyond extensively drug-resistant tuberculosis, were followed up over a period of 6 years. Transmission dynamics, infectiousness, and drug susceptibility were analysed in a subset of patients from the Western Cape using whole-genome sequencing (WGS; n=149), a cough aerosol sampling system (CASS; n=26), and phenotypic testing for 18 drugs (n=179). FINDINGS: Between Oct 1, 2008, and Oct 31, 2012, we enrolled and followed up 273 patients for a median of 20·3 months (IQR 9·6-27·8). 203 (74%) had programmatically incurable tuberculosis and unfavourable outcomes (treatment failure, relapse, default, or death despite treatment with a regimen based on capreomycin, aminosalicylic acid, or both). 172 (63%) patients were discharged home, of whom 104 (60%) had an unfavourable outcome. 54 (31%) home-discharged patients had failed treatment, with a median time to death after discharge of 9·9 months (IQR 4·2-17·4). 35 (20%) home-discharged cases were smear-positive at discharge. Using CASS, six (23%) of 26 home-discharged cases with data available expectorated infectious culture-positive cough aerosols in the respirable range (<5 μm), and most reported inter-person contact with suboptimal protective mask usage. WGS identified 17 (19%) of the 90 patients (with available sequence data) that were discharged home before the diagnosis of 20 downstream cases of extensively drug-resistant tuberculosis with almost identical sequencing profiles suggestive of community-based transmission (five or fewer single nucleotide polymorphisms different and with identical resistance-encoding mutations for 14 drugs). 11 (55%) of these downstream cases had HIV co-infection and ten (50%) had died by the end of the study. 22 (56%) of 39 isolates in patients discharged home after treatment failure were resistant to eight or more drugs. However, five (16%) of 31 isolates were susceptible to rifabutin and more than 90% were likely to be sensitive to linezolid, bedaquiline, and delamanid. INTERPRETATION: More than half of the patients with programmatically incurable tuberculosis were discharged into the community where they remained for an average of 16 months, were at risk of expectorating infectious cough aerosols, and posed a threat of transmission of extensively drug-resistant tuberculosis. Urgent action, including appropriate containment strategies, is needed to address this situation. Access to delamanid, bedaquiline, linezolid, and rifabutin, when appropriate, must be accelerated along with comprehensive drug susceptibility testing. FUNDING: UK Medical Research Council, South African Medical Research Council, South African National Research Foundation, European & Developing Countries Clinical Trials Partnership, Oppenheimer Foundation, Newton Fund, Biotechnology and Biological Sciences Research Council, King Abdullah University of Science & Technology

Ancient and recent differences in the intrinsic susceptibility of Mycobacterium tuberculosis complex to pretomanid

OBJECTIVES: To develop a robust phenotypic antimicrobial susceptibility testing (AST) method with a correctly set breakpoint for pretomanid (Pa), the most recently approved anti-tuberculosis drug. METHODS: The Becton Dickinson Mycobacterial Growth Indicator Tube™ (MGIT) system was used at six laboratories to determine the MICs of a phylogenetically diverse collection of 356 Mycobacterium tuberculosis complex (MTBC) strains to establish the epidemiological cut-off value for pretomanid. MICs were correlated with WGS data to study the genetic basis of differences in the susceptibility to pretomanid. RESULTS: We observed ancient differences in the susceptibility to pretomanid among various members of MTBC. Most notably, lineage 1 of M. tuberculosis, which is estimated to account for 28% of tuberculosis cases globally, was less susceptible than lineages 2, 3, 4 and 7 of M. tuberculosis, resulting in a 99th percentile of 2 mg/L for lineage 1 compared with 0.5 mg/L for the remaining M. tuberculosis lineages. Moreover, we observed that higher MICs (≥8 mg/L), which probably confer resistance, had recently evolved independently in six different M. tuberculosis strains. Unlike the aforementioned ancient differences in susceptibility, these recent differences were likely caused by mutations in the known pretomanid resistance genes. CONCLUSIONS: In light of these findings, the provisional critical concentration of 1 mg/L for MGIT set by EMA must be re-evaluated. More broadly, these findings underline the importance of considering the global diversity of MTBC during clinical development of drugs and when defining breakpoints for AST

Linezolid Population Pharmacokinetics in South African Adults with Drug-Resistant Tuberculosis.

Linezolid is widely used for drug-resistant tuberculosis (DR-TB) but has a narrow therapeutic index. To inform dose optimization, we aimed to characterize the population pharmacokinetics of linezolid in South African participants with DR-TB and explore the effect of covariates, including HIV coinfection, on drug exposure. Data were obtained from pharmacokinetic substudies in a randomized controlled trial and an observational cohort study, both of which enrolled adults with drug-resistant pulmonary tuberculosis. Participants underwent intensive and sparse plasma sampling. We analyzed linezolid concentration data using nonlinear mixed-effects modeling and performed simulations to estimate attainment of putative efficacy and toxicity targets. A total of 124 participants provided 444 plasma samples; 116 were on the standard daily dose of 600 mg, while 19 had dose reduction to 300 mg due to adverse events. Sixty-one participants were female, 71 were HIV-positive, and their median weight was 56 kg (interquartile range [IQR], 50 to 63). In the final model, typical values for clearance and central volume were 3.57 liters/h and 40.2 liters, respectively. HIV coinfection had no significant effect on linezolid exposure. Simulations showed that 600-mg dosing achieved the efficacy target (area under the concentration-time curve for the free, unbound fraction of the drug [[Formula: see text] at a MIC level of 0.5 mg/liter) with 96% probability but had 56% probability of exceeding safety target ([Formula: see text]. The 300-mg dose did not achieve adequate efficacy exposures. Our model characterized population pharmacokinetics of linezolid in South African patients with DR-TB and supports the 600-mg daily dose with safety monitoring