Compensatory evolution in NusG improves fitness of drug-resistant M. tuberculosis.

Drug-resistant bacteria are emerging as a global threat, despite frequently being less fit than their drug-susceptible ancestors1-8. Here we sought to define the mechanisms that drive or buffer the fitness cost of rifampicin resistance (RifR) in the bacterial pathogen Mycobacterium tuberculosis (Mtb). Rifampicin inhibits RNA polymerase (RNAP) and is a cornerstone of modern short-course tuberculosis therapy9,10. However, RifR Mtb accounts for one-quarter of all deaths due to drug-resistant bacteria11,12. We took a comparative functional genomics approach to define processes that are differentially vulnerable to CRISPR interference (CRISPRi) inhibition in RifR Mtb. Among other hits, we found that the universally conserved transcription factor NusG is crucial for the fitness of RifR Mtb. In contrast to its role in Escherichia coli, Mtb NusG has an essential RNAP pro-pausing function mediated by distinct contacts with RNAP and the DNA13. We find this pro-pausing NusG-RNAP interface to be under positive selection in clinical RifR Mtb isolates. Mutations in the NusG-RNAP interface reduce pro-pausing activity and increase fitness of RifR Mtb. Collectively, these results define excessive RNAP pausing as a molecular mechanism that drives the fitness cost of RifR in Mtb, identify a new mechanism of compensation to overcome this cost, suggest rational approaches to exacerbate the fitness cost, and, more broadly, could inform new therapeutic approaches to develop drug combinations to slow the evolution of RifR in Mtb.

Treatment Strategy for Rifampin-Susceptible Tuberculosis.

BACKGROUND: Tuberculosis is usually treated with a 6-month rifampin-based regimen. Whether a strategy involving shorter initial treatment may lead to similar outcomes is unclear. METHODS: In this adaptive, open-label, noninferiority trial, we randomly assigned participants with rifampin-susceptible pulmonary tuberculosis to undergo either standard treatment (rifampin and isoniazid for 24 weeks with pyrazinamide and ethambutol for the first 8 weeks) or a strategy involving initial treatment with an 8-week regimen, extended treatment for persistent clinical disease, monitoring after treatment, and retreatment for relapse. There were four strategy groups with different initial regimens; noninferiority was assessed in the two strategy groups with complete enrollment, which had initial regimens of high-dose rifampin-linezolid and bedaquiline-linezolid (each with isoniazid, pyrazinamide, and ethambutol). The primary outcome was a composite of death, ongoing treatment, or active disease at week 96. The noninferiority margin was 12 percentage points. RESULTS: Of the 674 participants in the intention-to-treat population, 4 (0.6%) withdrew consent or were lost to follow-up. A primary-outcome event occurred in 7 of the 181 participants (3.9%) in the standard-treatment group, as compared with 21 of the 184 participants (11.4%) in the strategy group with an initial rifampin-linezolid regimen (adjusted difference, 7.4 percentage points; 97.5% confidence interval [CI], 1.7 to 13.2; noninferiority not met) and 11 of the 189 participants (5.8%) in the strategy group with an initial bedaquiline-linezolid regimen (adjusted difference, 0.8 percentage points; 97.5% CI, -3.4 to 5.1; noninferiority met). The mean total duration of treatment was 180 days in the standard-treatment group, 106 days in the rifampin-linezolid strategy group, and 85 days in the bedaquiline-linezolid strategy group. The incidences of grade 3 or 4 adverse events and serious adverse events were similar in the three groups. CONCLUSIONS: A strategy involving initial treatment with an 8-week bedaquiline-linezolid regimen was noninferior to standard treatment for tuberculosis with respect to clinical outcomes. The strategy was associated with a shorter total duration of treatment and with no evident safety concerns. (Funded by the Singapore National Medical Research Council and others; TRUNCATE-TB ClinicalTrials.gov number, NCT03474198.).

Trained immunity in recurrent Staphylococcus aureus infection promotes bacterial persistence.

Bacterial persister cells, a sub-population of dormant phenotypic variants highly tolerant to antibiotics, present a significant challenge for infection control. Investigating the mechanisms of antibiotic persistence is crucial for developing effective treatment strategies. Here, we found a significant association between tolerance frequency and previous infection history in bovine mastitis. Previous S. aureus infection led to S. aureus tolerance to killing by rifampicin in subsequent infection in vivo and in vitro. Actually, the activation of trained immunity contributed to rifampicin persistence of S. aureus in secondary infection, where it reduced the effectiveness of antibiotic treatment and increased disease severity. Mechanically, we found that S. aureus persistence was mediated by the accumulation of fumarate provoked by trained immunity. Combination therapy with metformin and rifampicin promoted eradication of persisters and improved the severity of recurrent S. aureus infection. These findings provide mechanistic insight into the relationship between trained immunity and S. aureus persistence, while providing proof of concept that trained immunity is a therapeutic target in recurrent bacterial infections involving persistent pathogens.

Bedaquiline-Pretomanid-Linezolid Regimens for Drug-Resistant Tuberculosis.

BACKGROUND: The bedaquiline-pretomanid-linezolid regimen has been reported to have 90% efficacy against highly drug-resistant tuberculosis, but the incidence of adverse events with 1200 mg of linezolid daily has been high. The appropriate dose of linezolid and duration of treatment with this agent to minimize toxic effects while maintaining efficacy against highly drug-resistant tuberculosis are unclear. METHODS: We enrolled participants with extensively drug-resistant (XDR) tuberculosis (i.e., resistant to rifampin, a fluoroquinolone, and an aminoglycoside), pre-XDR tuberculosis (i.e., resistant to rifampin and to either a fluoroquinolone or an aminoglycoside), or rifampin-resistant tuberculosis that was not responsive to treatment or for which a second-line regimen had been discontinued because of side effects. We randomly assigned the participants to receive bedaquiline for 26 weeks (200 mg daily for 8 weeks, then 100 mg daily for 18 weeks), pretomanid (200 mg daily for 26 weeks), and daily linezolid at a dose of 1200 mg for 26 weeks or 9 weeks or 600 mg for 26 weeks or 9 weeks. The primary end point in the modified intention-to-treat population was the incidence of an unfavorable outcome, defined as treatment failure or disease relapse (clinical or bacteriologic) at 26 weeks after completion of treatment. Safety was also evaluated. RESULTS: A total of 181 participants were enrolled, 88% of whom had XDR or pre-XDR tuberculosis. Among participants who received bedaquiline-pretomanid-linezolid with linezolid at a dose of 1200 mg for 26 weeks or 9 weeks or 600 mg for 26 weeks or 9 weeks, 93%, 89%, 91%, and 84%, respectively, had a favorable outcome; peripheral neuropathy occurred in 38%, 24%, 24%, and 13%, respectively; myelosuppression occurred in 22%, 15%, 2%, and 7%, respectively; and the linezolid dose was modified (i.e., interrupted, reduced, or discontinued) in 51%, 30%, 13%, and 13%, respectively. Optic neuropathy developed in 4 participants (9%) who had received linezolid at a dose of 1200 mg for 26 weeks; all the cases resolved. Six of the seven unfavorable microbiologic outcomes through 78 weeks of follow-up occurred in participants assigned to the 9-week linezolid groups. CONCLUSIONS: A total of 84 to 93% of the participants across all four bedaquiline-pretomanid-linezolid treatment groups had a favorable outcome. The overall risk-benefit ratio favored the group that received the three-drug regimen with linezolid at a dose of 600 mg for 26 weeks, with a lower incidence of adverse events reported and fewer linezolid dose modifications. (Funded by the TB Alliance and others; ZeNix ClinicalTrials.gov number, NCT03086486.).

Twice-Daily Dosing of Dolutegravir in Infants on Rifampicin Treatment: A Pharmacokinetic Substudy of the EMPIRICAL Trial.

BACKGROUND: We evaluated dolutegravir pharmacokinetics in infants with human immunodeficiency virus (HIV) receiving dolutegravir twice daily (BID) with rifampicin-based tuberculosis (TB) treatment compared with once daily (OD) without rifampicin. METHODS: Infants with HIV aged 1-12 months, weighing ≥3 kg, and receiving dolutegravir BID with rifampicin or OD without rifampicin were eligible. Six blood samples were taken over 12 (BID) or 24 hours (OD). Dolutegravir pharmacokinetic parameters, HIV viral load (VL) data, and adverse events (AEs) were reported. RESULTS: Twenty-seven of 30 enrolled infants had evaluable pharmacokinetic curves. The median (interquartile range) age was 7.1 months (6.1-9.9), weight was 6.3 kg (5.6-7.2), 21 (78%) received rifampicin, and 11 (41%) were female. Geometric mean ratios comparing dolutegravir BID with rifampicin versus OD without rifampicin were area under curve (AUC)0-24h 0.91 (95% confidence interval, .59-1.42), Ctrough 0.95 (0.57-1.59), Cmax 0.87 (0.57-1.33). One infant (5%) receiving rifampicin versus none without rifampicin had dolutegravir Ctrough <0.32 mg/L, and none had Ctrough <0.064 mg/L. The dolutegravir metabolic ratio (dolutegravir-glucuronide AUC/dolutegravir AUC) was 2.3-fold higher in combination with rifampicin versus without rifampicin. Five of 82 reported AEs were possibly related to rifampicin or dolutegravir and resolved without treatment discontinuation. Upon TB treatment completion, HIV viral load was <1000 copies/mL in 76% and 100% of infants and undetectable in 35% and 20% of infants with and without rifampicin, respectively. CONCLUSIONS: Dolutegravir BID in infants receiving rifampicin resulted in adequate dolutegravir exposure, supporting this treatment approach for infants with HIV-TB coinfection.

Rifampin Based Therapy for Patients With Staphylococcus aureus Native Vertebral Osteomyelitis: A Systematic Review and Meta-analysis.

BACKGROUND: Native vertebral osteomyelitis (NVO) caused by Staphylococcus aureus is associated with high risk of treatment failure and increased morbidity. The role of rifampin-based therapy for the treatment of this condition is controversial. The goal of this systematic review and meta-analysis is to explore the efficacy and safety of rifampin-based therapy for the treatment of S. aureus NVO. METHODS: We searched Cochrane, Embase, Medline, Scopus, and Web of Science databases for studies published up to May 2023, focusing on adults with NVO treated with or without rifampin-containing regimens. A random-effects model meta-analysis estimated relative risks and risk difference with 95% confidence intervals (CI). RESULTS: Thirteen studies (2 randomized controlled trials and 11 comparative cohort studies), comprising 244 patients with S. aureus NVO who received rifampin and 435 who did not, were analyzed. Meta-analysis showed that rifampin-based regimens were associated with lower risk of clinical failure (risk difference, -14%; 95% CI, -19% to -8%; P < .001; I2 = 0%; relative risk, 0.58; 95% CI, .37-.92, P = .02, I2 = 21%). Only 1 study reported on adverse events. All studies had a high or uncertain risk of bias, and the certainty of evidence was rated as very low. CONCLUSIONS: Adjunctive rifampin therapy might be associated with lower risk of S. aureus NVO treatment failure; however, the low certainty of evidence precludes drawing definitive conclusions that would alter clinical practice. A randomized trial is necessary to corroborate these findings.

Monitoring of First-line Drug Resistance Mutations Outside the Scope of Xpert MTB/RIF Ultra is Needed for Successful Control of DR-TB in Southern Mozambique.

Multidrug-resistant(MDR) tuberculosis in Southern Africa is of great concern, exacerbated by the spread of a clone harboring a mutation missed by Xpert Ultra. In Southern Mozambique, the presence of such mutation and rising cases of non-MDR isoniazid resistance highlights the need to ensure accurate detection of antimicrobial-resistance in the country.

Pharmacokinetics and Optimal Dosing of Levofloxacin in Children for Drug-Resistant Tuberculosis: An Individual Patient Data Meta-Analysis.

BACKGROUND: Each year 25 000-32 000 children develop rifampicin- or multidrug-resistant tuberculosis (RR/MDR-TB), and many more require preventive treatment. Levofloxacin is a key component of RR/MDR-TB treatment and prevention, but the existing pharmacokinetic data in children have not yet been comprehensively summarized. We aimed to characterize levofloxacin pharmacokinetics through an individual patient data meta-analysis of available studies and to determine optimal dosing in children. METHODS: Levofloxacin concentration and demographic data were pooled from 5 studies and analyzed using nonlinear mixed effects modeling. Simulations were performed using current World Health Organization (WHO)-recommended and model-informed optimized doses. Optimal levofloxacin doses were identified to target median adult area under the time-concentration curve (AUC)24 of 101 mg·h/L given current standard adult doses. RESULTS: Data from 242 children (2.8 years [0.2-16.8] was used). Apparent clearance was 3.16 L/h for a 13-kg child. Age affected clearance, reaching 50% maturation at birth and 90% maturation at 8 months. Nondispersible tablets had 29% lower apparent oral bioavailability compared to dispersible tablets. Median exposures at current WHO-recommended doses were below the AUC target for children weighing <24 kg and under <10 years, resulting in approximately half of the exposure in adults. Model-informed doses of 16-33 mg/kg for dispersible tablets or 16-50 mg/kg for nondispersible tablets were required to meet the AUC target without significantly exceeding the median adult Cmax. CONCLUSIONS: Revised weight-band dosing guidelines with doses of >20 mg/kg are required to ensure adequate exposure. Further studies are needed to determine safety and tolerability of these higher doses.

Impact of Prior Tuberculosis Treatment With New/Companion Drugs on Clinical Outcomes in Patients Receiving Concomitant Bedaquiline and Delamanid for Multidrug- and Rifampicin-Resistant Tuberculosis.

BACKGROUND: There are scarce data on the clinical outcomes of persons retreated with new/companion anti-tuberculosis (TB) drugs for multidrug- and rifampicin-resistant tuberculosis (MDR/RR-TB). We sought to evaluate the efficacy and safety of bedaquiline and delamanid containing regimens among patients with and without prior exposure to the new/companion drugs (bedaquiline, delamanid, linezolid, clofazimine, and fluoroquinolones). METHODS: We conducted a retrospective cohort study among patients with pulmonary MDR/RR-TB in Georgia who received bedaquiline and delamanid combination as a part of a salvage regimen from November 2017 to December 2020 in a programmatic setting. RESULTS: Among 106 persons with a median age of 39.5 years, 44 (41.5%) were previously treated with new/companion TB drugs. Patients with prior exposure to new/companion drugs had higher rates of baseline resistance compared to those without exposure to new/companion TB drugs (bedaquiline 15.2% vs 1.8%, linezolid 22.2% vs 16.7%). Sputum culture conversion rates among patients exposed and not exposed to new/companion drugs were 65.9% vs 98.0%, respectively (P < .001). Among patients with and without prior new/companion TB drug use, favorable outcome rates were 41.0% and 82.3%, respectively (P < .001). Treatment adherence in 32 (30.2%) patients was ≤80%. Five of 21 patients (23.8%) who had a baseline and repeat susceptibility test had acquired bedaquiline resistance. QTC/F prolongation (>500 ms) was rare (2.8%). CONCLUSIONS: Prior exposure to new/companion TB drugs was associated with poor clinical outcomes and acquired drug resistance. Tailoring the TB regimen to each patient's drug susceptibility test results and burden of disease and enhancing adherence support may improve outcomes.

The Impact of Preventive Treatment for Multidrug- and Rifampin-Resistant Tuberculosis Exceeds Trial-Based Estimates.

BACKGROUND: Several clinical trials of tuberculosis preventive treatment (TPT) for household contacts of patients with multidrug- or rifampin-resistant tuberculosis (MDR/RR-TB) are nearing completion. The potential benefits of delivering TPT to MDR/RR-TB contacts extend beyond the outcomes that clinical trials can measure. METHODS: We developed an agent-based, household-structured TB and MDR/RR-TB transmission model, calibrated to an illustrative setting in India. We simulated contact investigation in households of patients with MDR/RR-TB, comparing an MDR/RR-TPT regimen (assuming 6-month duration, 70% efficacy) and associated active case finding against alternatives of contact investigation without TPT or no household intervention. We simulated the TB and MDR/RR-TB incidence averted relative to placebo over 2 years, as measurable by a typical trial, as well as the incidence averted over a longer time horizon, in the broader population, and relative to no contact investigation. RESULTS: Observing TPT and placebo recipients for 2 years as in a typical trial, MDR/RR-TPT was measured to prevent 72% (interquartile range, 45%-100%) of incident MDR/RR-TB among recipients; the median number needed to treat (NNT) to prevent 1 MDR/RR-TB case was 73, compared to placebo. This NNT decreased to 54 with 13-18 years of observation, to 27 when downstream transmission effects were also considered, and to 12 when the effects of active TB screening were included by comparing to a no-household-contact-intervention scenario. CONCLUSIONS: If forthcoming trial results demonstrate efficacy, the long-term population impact of TPT for MDR/RR-TB-including the large effect of increased active TB detection among MDR/RR-TB contacts-could be much greater than suggested by trial outcomes alone.

Treatment of Multidrug-resistant or Rifampicin-resistant Tuberculosis With an All-oral 9-month Regimen Containing Linezolid or Ethionamide in South Africa: A Retrospective Cohort Study.

BACKGROUND: In 2019, the South African tuberculosis program replaced ethionamide with linezolid as part of an all-oral 9-month regimen. We evaluated treatment outcomes for patients assigned to regimens including linezolid in 2019 and ethionamide in 2017. METHODS: This retrospective cohort study included patients treated for multidrug-resistant/rifampicin-resistant tuberculosis throughout South Africa between 1 January and 31 December 2017 and 1 January to 31 December 2019. The cohort treated with a 9-month regimen containing ethionamide for four months, was compared with a cohort treated with a 9-month regimen containing linezolid for 2 months. The regimens were otherwise identical. Inverse probability weighting of propensity scores was used to adjust for potential confounding. A log-binomial regression model was used to estimate adjusted relative risk (aRR) comparing 24-month outcomes between cohorts including treatment success, death, loss to follow up, and treatment failure. Adverse event data were available for the linezolid cohort. FINDINGS: In total, 817 patients were included in the cohort receiving ethionamide and 4244 in the cohort receiving linezolid. No evidence for a difference was observed between linezolid and ethionamide regimens for treatment success (aRR = 0.96, 95% confidence interval [CI] .91-1.01), death (aRR = 1.01, 95% CI .87-1.17) or treatment failure (aRR = 0.87, 95% CI .44-1.75). Loss to follow-up was more common in the linezolid group, although estimates were imprecise (aRR = 1.22, 95% CI .99-1.50). CONCLUSIONS: No significant differences in treatment success and survival were observed with substitution of linezolid for ethionamide as a part of an all-oral 9-month regimen. Linezolid is an acceptable alternative to ethionamide in this shorter regimen for treatment of multidrug-resistant/rifampicin-resistant tuberculosis.

Long-Term Protective Effect of Tuberculosis Preventive Therapy in a Medium/High Tuberculosis Incidence Setting.

BACKGROUND: The duration of the protective effect of tuberculosis preventive therapy (TPT) is controversial. Some studies have found that the protective effect of TPT is lost after cessation of therapy among people with human immunodeficiency virus (HIV) in settings with very high tuberculosis incidence, but others have found long-term protection in low-incidence settings. METHODS: We estimated the incidence rate (IR) of new tuberculosis disease for up to 12 years after randomization to 4 months of rifampin or 9 months of isoniazid, among 991 Brazilian participants in a TPT trial in the state of Rio de Janeiro, with an incidence of 68.6/100 000 population in 2022. The adjusted hazard ratios (aHRs) of independent variables for incident tuberculosis were calculated. RESULTS: The overall tuberculosis IR was 1.7 (95% confidence interval [CI], 1.01- 2.7) per 1000 person-years (PY). The tuberculosis IR was higher among those who did not complete TPT than in those who did (2.9 [95% CI, 1.3-5.6] vs 1.1 [.4-2.3] per 1000 PY; IR ratio, 2.7 [1.0-7.2]). The tuberculosis IR was higher within 28 months after randomization (IR, 3.5 [95% CI, 1.6-6.6] vs 1.1 [.5-2.1] per 1000 PY between 28 and 143 months; IR ratio, 3.1 [1.2-8.2]). Treatment noncompletion was the only variable associated with incident tuberculosis (aHR, 3.2 [95% CI, 1.1-9.7]). CONCLUSIONS: In a mostly HIV-noninfected population, a complete course of TPT conferred long-term protection against tuberculosis.

Impact and cost-effectiveness of the 6-month BPaLM regimen for rifampicin-resistant tuberculosis in Moldova: A mathematical modeling analysis.

BACKGROUND: Emerging evidence suggests that shortened, simplified treatment regimens for rifampicin-resistant tuberculosis (RR-TB) can achieve comparable end-of-treatment (EOT) outcomes to longer regimens. We compared a 6-month regimen containing bedaquiline, pretomanid, linezolid, and moxifloxacin (BPaLM) to a standard of care strategy using a 9- or 18-month regimen depending on whether fluoroquinolone resistance (FQ-R) was detected on drug susceptibility testing (DST). METHODS AND FINDINGS: The primary objective was to determine whether 6 months of BPaLM is a cost-effective treatment strategy for RR-TB. We used genomic and demographic data to parameterize a mathematical model estimating long-term health outcomes measured in quality-adjusted life years (QALYs) and lifetime costs in 2022 USD ($) for each treatment strategy for patients 15 years and older diagnosed with pulmonary RR-TB in Moldova, a country with a high burden of TB drug resistance. For each individual, we simulated the natural history of TB and associated treatment outcomes, as well as the process of acquiring resistance to each of 12 anti-TB drugs. Compared to the standard of care, 6 months of BPaLM was cost-effective. This strategy was estimated to reduce lifetime costs by $3,366 (95% UI: [1,465, 5,742] p < 0.001) per individual, with a nonsignificant change in QALYs (-0.06; 95% UI: [-0.49, 0.03] p = 0.790). For those stopping moxifloxacin under the BPaLM regimen, continuing with BPaL plus clofazimine (BPaLC) provided more QALYs at lower cost than continuing with BPaL alone. Strategies based on 6 months of BPaLM had at least a 93% chance of being cost-effective, so long as BPaLC was continued in the event of stopping moxifloxacin. BPaLM for 6 months also reduced the average time spent with TB resistant to amikacin, bedaquiline, clofazimine, cycloserine, moxifloxacin, and pyrazinamide, while it increased the average time spent with TB resistant to delamanid and pretomanid. Sensitivity analyses showed 6 months of BPaLM to be cost-effective across a broad range of values for the relative effectiveness of BPaLM, and the proportion of the cohort with FQ-R. Compared to the standard of care, 6 months of BPaLM would be expected to save Moldova's national TB program budget $7.1 million (95% UI: [1.3 million, 15.4 million] p = 0.002) over the 5-year period from implementation. Our analysis did not account for all possible interactions between specific drugs with regard to treatment outcomes, resistance acquisition, or the consequences of specific types of severe adverse events, nor did we model how the intervention may affect TB transmission dynamics. CONCLUSIONS: Compared to standard of care, longer regimens, the implementation of the 6-month BPaLM regimen could improve the cost-effectiveness of care for individuals diagnosed with RR-TB, particularly in settings with a high burden of drug-resistant TB. Further research may be warranted to explore the impact and cost-effectiveness of shorter RR-TB regimens across settings with varied drug-resistant TB burdens and national income levels.

Clofazimine as a substitute for rifampicin improves efficacy of Mycobacterium avium pulmonary disease treatment in the hollow-fiber model.

Mycobacterium avium complex pulmonary disease is treated with an azithromycin, ethambutol, and rifampicin regimen, with limited efficacy. The role of rifampicin is controversial due to inactivity, adverse effects, and drug interactions. Here, we evaluated the efficacy of clofazimine as a substitute for rifampicin in an intracellular hollow-fiber infection model. THP-1 cells, which are monocytes isolated from peripheral blood from an acute monocytic leukemia patient, were infected with M. avium ATCC 700898 and exposed to a regimen of azithromycin and ethambutol with either rifampicin or clofazimine. Intrapulmonary pharmacokinetic profiles of azithromycin, ethambutol, and rifampicin were simulated. For clofazimine, a steady-state average concentration was targeted. Drug concentrations and bacterial densities were monitored over 21 days. Exposures to azithromycin and ethambutol were 20%-40% lower than targeted but within clinically observed ranges. Clofazimine exposures were 1.7 times higher than targeted. Until day 7, both regimens were able to maintain stasis. Thereafter, regrowth was observed for the rifampicin-containing regimen, while the clofazimine-containing regimen yielded a 2 Log10 colony forming unit (CFU) per mL decrease in bacterial load. The clofazimine regimen also successfully suppressed the emergence of macrolide tolerance. In summary, substitution of rifampicin with clofazimine in the hollow-fiber model improved the antimycobacterial activity of the regimen. Clofazimine-containing regimens merit investigation in clinical trials.

How much should we still worry about QTc prolongation in rifampicin-resistant tuberculosis? ECG findings from TB-PRACTECAL clinical trial.

Regimens for the treatment of rifampicin-resistant tuberculosis currently rely on the use of QT-prolonging agents. Using data from the randomized controlled trial, TB-PRACTECAL, we investigated differences in QTcF among participants in the three interventional arms: BPaL (bedaquiline, pretomanid, and linezolid), BPaLC (BPaL with clofazimine), and BPaLM (BPaL with moxifloxacin). Additionally, we assessed whether age, body mass index, and country were causally associated with QTcF prolongation. The trial included participants from South Africa, Uzbekistan, and Belarus. A post hoc analysis of electrocardiogram data was undertaken. Random effects regression was used to model QTcF longitudinally over 24 weeks and causal frameworks guided the analysis of non-randomized independent variables. 328 participants were included in BPaL-based arms. The longitudinal analysis of investigational arms showed an initial QTcF steep increase in the first week. QTcF trajectories between weeks 2 and 24 differed slightly by regimen, with highest mean peak for BPaLC (QTcF 446.5 ms). Overall, there were 397 QTcF >450 ms (of 3,744) and only one QTcF >500 ms. The odds of QTcF >450 ms among participants in any investigational arm, was 8.33 times higher in Uzbekistan compared to Belarus (95% confidence interval: 3.25-21.33). No effect on QTcF prolongation was found for baseline age or body mass index (BMI). Clinically significant QTc prolongation was rare in this cohort of closely monitored participants. Across BPaL-based regimens, BPaLC showed a slightly longer and sustained effect on QTcF prolongation, but the differences (both in magnitude of change and trajectory over time) were clinically unimportant. The disparity in the risk of QTc prolongation across countries would be an important factor to further investigate when evaluating monitoring strategies. CLINICAL TRIALS: This study is registered with ClinicalTrials.gov as NCT02589782.

Association of indicators of extensive disease and rifampin-resistant tuberculosis treatment outcomes: an individual participant data meta-analysis.

BACKGROUND: Indicators of extensive disease-acid fast bacilli (AFB) smear positivity and lung cavitation-have been inconsistently associated with clinical rifampin-resistant/multidrug-resistant tuberculosis (RR/MDR-TB) outcomes. We evaluated the association of these indicators with end-of-treatment outcomes. METHODS: We did an individual participant data meta-analysis of people treated for RR/MDR-TB with longer regimens with documented AFB smear and chest radiography findings. We compared people AFB smear-negative without cavities to people: (1) smear-negative with lung cavities; (2) smear-positive without lung cavities and (3) AFB smear-positive with lung cavities. Using multivariable logistic regression accounting for demographic, treatment and clinical factors, we calculated adjusted ORs (aOR) for any unfavourable outcome (death, lost to follow-up, failure/recurrence), and mortality and treatment failure/recurrence alone. RESULTS: We included 5596 participants; included participants significantly differed from excluded participants. Overall, 774 (13.8%) were AFB smear-negative without cavities, 647 (11.6%) only had cavities, 1424 (25.4%) were AFB smear-positive alone and 2751 (49.2%) were AFB smear-positive with cavities. The median age was 37 years (IQR: 28-47), 3580 (64%) were male and 686 (12.5%) had HIV. Compared with participants AFB smear-negative without cavities, aOR (95% CI) for any unfavourable outcome was 1.0 (0.8 to 1.4) for participants smear-negative with lung cavities, 1.2 (0.9 to 1.5) if smear-positive without cavities and 1.6 (1.3 to 2.0) if AFB smear-positive with lung cavities. Odds were only significantly increased for mortality (1.5, 95% CI 1.1 to 2.1) and failure/recurrence (2.2, 95% CI 1.5 to 3.3) among participants AFB smear-positive with lung cavities. CONCLUSION: Only the combination of AFB smear-positivity and lung cavitation was associated with unfavourable outcomes, suggesting they may benefit from stronger regimens.

Challenging the gold standard: the limitations of molecular assays for detection of Mycobacterium tuberculosis heteroresistance.

OBJECTIVES: Heteroresistant infections are defined as infections in which a mixture of drug-resistant and drug-susceptible populations are present. In Mycobacterium tuberculosis (M. tb), heteroresistance poses a challenge in diagnosis and has been linked with poor treatment outcomes. We compared the analytical sensitivity of molecular methods, such as GeneXpert and whole genome sequencing (WGS) in detecting heteroresistance when compared with the 'gold standard' phenotypic assay: the agar proportion method (APM). METHODS: Using two rounds of proficiency surveys with defined monoresistant BCG strains and mixtures of susceptible/resistant M. tb, we determined the limit of detection (LOD) of known resistance associated mutations. RESULTS: The LOD for rifampin-R (RIF-R) detection was 1% using APM, 60% using GeneXpert MTB/RIF, 10% using GeneXpert MTB/RIF Ultra and 10% using WGS. While WGS could detect mutations beyond those associated with RIF resistance, the LOD for these other mutations was also 10%. Additionally, we observed instances where laboratories did not report resistance in the majority population, yet the mutations were present in the raw sequence data. CONCLUSION: The gold standard APM detects minority resistant populations at a lower proportion than molecular tests. Mycobacterium bovis BCG strains with defined resistance and extracted DNA from M. tb provided concordant results and can serve in quality control of laboratories offering molecular testing for resistance. Further research is required to determine whether the higher LOD of molecular tests is associated with negative treatment outcomes.

Targeted sequencing from cerebrospinal fluid for rapid identification of drug-resistant tuberculous meningitis.

Mortality from tuberculous meningitis (TBM) remains around 30%, with most deaths occurring within 2 months of starting treatment. Mortality from drug-resistant strains is higher still, making early detection of drug resistance (DR) essential. Targeted next-generation sequencing (tNGS) produces high read depths, allowing the detection of DR-associated alleles with low frequencies. We applied Deeplex Myc-TB-a tNGS assay-to cerebrospinal fluid (CSF) samples from 72 adults with microbiologically confirmed TBM and compared its genomic drug susceptibility predictions to a composite reference standard of phenotypic susceptibility testing (pDST) and whole genome sequencing, as well as to clinical outcomes. Deeplex detected Mycobacterium tuberculosis complex DNA in 24/72 (33.3%) CSF samples and generated full DR reports for 22/24 (91.7%). The read depth generated by Deeplex correlated with semi-quantitative results from MTB/RIF Xpert. Alleles with <20% frequency were seen at canonical loci associated with first-line DR. Disregarding these low-frequency alleles, Deeplex had 100% concordance with the composite reference standard for all drugs except pyrazinamide and streptomycin. Three patients had positive CSF cultures after 30 days of treatment; reference tests and Deeplex identified isoniazid resistance in two, and Deeplex alone identified low-frequency rifampin resistance alleles in one. Five patients died, of whom one had pDST-identified pyrazinamide resistance. tNGS on CSF can rapidly and accurately detect drug-resistant TBM, but its application is limited to those with higher bacterial loads. In those with lower bacterial burdens, alternative approaches need to be developed for both diagnosis and resistance detection.

Rifampicin and protein concentrations in paired spinal versus ventricular cerebrospinal fluid samples of children with tuberculous meningitis.

BACKGROUND: Tuberculous meningitis (TBM) is the most lethal form of TB. To study the disease, drug concentrations in samples obtained from the spinal CSF are usually used to reflect brain concentrations. Emerging data suggest that transport of substances across capillaries in the brain (ventricular CSF) and spinal cord may differ. METHODS: We examined paired, time-linked samples of ventricular CSF (VCSF) and lumbar CSF (LCSF) of 28 patients with TBM and analysed these for rifampicin and total protein concentrations. Clinically indicated samples from procedures to determine the level of CSF block were collected from children being treated for TBM and hydrocephalus. Total protein concentrations were determined using the bicinchoninic acid (BCA) or turbidimetry assay, and rifampicin concentrations were determined using a validated LC coupled with tandem MS method. A paired Wilcoxon signed-rank test was used to determine significance. RESULTS: TBM was confirmed in 19 cases (68%) using TB culture or GeneXpert Mtb/Rifampicin assay. All other cases were classified as probable. The median total protein concentration in LCSF was 6.0 g/L and in VCSF was 1.3 g/L. The median rifampicin concentration in LCSF was 299 ng/mL and 133 ng/mL in VCSF. The median ratio of LCSF/VSCF for protein was 4.23 and 1.57 for rifampicin. CONCLUSIONS: Total protein and rifampicin concentrations differed significantly between the two compartments, both being higher in LCSF than in VCSF samples (P < 0.0001 for total protein and P = 0.0046 for rifampicin). Further studies are required to explore the causative reasons for the observed differences.

Genetic and clinical predictors of rifapentine and isoniazid pharmacokinetics in paediatrics with tuberculosis infection.

OBJECTIVES: Twelve weekly doses of rifapentine and isoniazid (3HP regimen) are recommended for TB preventive therapy in children with TB infection. However, they present with variability in the pharmacokinetic profiles. The current study aimed to develop a pharmacokinetic model of rifapentine and isoniazid in 12 children with TB infection using NONMEM. METHODS: Ninety plasma and 41 urine samples were collected at Week 4 of treatment. Drug concentrations were measured using a validated HPLC-UV method. MassARRAY® SNP genotyping was used to investigate genetic factors, including P-glycoprotein (ABCB1), solute carrier organic anion transporter B1 (SLCO1B1), arylacetamide deacetylase (AADAC) and N-acetyl transferase (NAT2). Clinically relevant covariates were also analysed. RESULTS: A two-compartment model for isoniazid and a one-compartment model for rifapentine with transit compartment absorption and first-order elimination were the best models for describing plasma and urine data. The estimated (relative standard error, RSE) of isoniazid non-renal clearance was 3.52 L·h-1 (23.1%), 2.91 L·h-1 (19.6%), and 2.58 L·h-1 (20.0%) in NAT2 rapid, intermediate and slow acetylators. A significant proportion of the unchanged isoniazid was cleared renally (2.7 L·h-1; 8.0%), while the unchanged rifapentine was cleared primarily through non-renal routes (0.681 L·h-1; 3.6%). Participants with the ABCB1 mutant allele had lower bioavailability of rifapentine, while food prolonged the mean transit time of isoniazid. CONCLUSIONS: ABCB1 mutant allele carriers may require higher rifapentine doses; however, this must be confirmed in larger trials. Food did not affect overall exposure to isoniazid and only delayed absorption time.