Understanding Cofactor F420 dependent mechanism(s) in the activation of bicycle nitroimidazoles and in the physiology of Mycobacterium tuberculosis

Ph.DDOCTOR OF PHILOSOPH

Keeping up with the guidelines: design changes to the STREAM stage 2 randomised controlled non-inferiority trial for rifampicin-resistant tuberculosis

Results from the STREAM stage 1 trial showed that a 9-month regimen for patients with rifampicin-resistant tuberculosis was non-inferior to the 20-month regimen recommended by the 2011 WHO treatment guidelines. Similar levels of severe adverse events were reported on both regimens suggesting the need for further research to optimise treatment. Stage 2 of STREAM evaluates two additional short-course regimens, both of which include bedaquiline. Throughout stage 2 of STREAM, new drug choices and a rapidly changing treatment landscape have necessitated changes to the trial's design to ensure it remains ethical and relevant. This paper describes changes to the trial design to ensure that stage 2 continues to answer important questions. These changes include the early closure to recruitment of two trial arms and an adjustment to the definition of the primary endpoint. If the STREAM experimental regimens are shown to be non-inferior or superior to the stage 1 study regimen, this would represent an important contribution to evidence about potentially more tolerable and more efficacious MDR-TB regimens, and a welcome advance for patients with rifampicin-resistant tuberculosis and tuberculosis control programmes globally.Trial registration: ISRCTN ISRCTN18148631 . Registered 10 February 2016

Evaluation of two short standardised regimens for the treatment of rifampicin-resistant tuberculosis (STREAM stage 2): an open-label, multicentre, randomised, non-inferiority trial.

The STREAM stage 1 trial showed that a 9-month regimen for the treatment of rifampicin-resistant tuberculosis was non-inferior to the 20-month 2011 WHO-recommended regimen. In STREAM stage 2, we aimed to compare two bedaquiline-containing regimens with the 9-month STREAM stage 1 regimen. We did a randomised, phase 3, non-inferiority trial in 13 hospital clinics in seven countries, in individuals aged 15 years or older with rifampicin-resistant tuberculosis without fluoroquinolone or aminoglycoside resistance. Participants were randomly assigned 1:2:2:2 to the 2011 WHO regimen (terminated early), a 9-month control regimen, a 9-month oral regimen with bedaquiline (primary comparison), or a 6-month regimen with bedaquiline and 8 weeks of second-line injectable. Randomisations were stratified by site, HIV status, and CD4 count. Participants and clinicians were aware of treatment-group assignments, but laboratory staff were masked. The primary outcome was favourable status (negative cultures for Mycobacterium tuberculosis without a preceding unfavourable outcome) at 76 weeks; any death, bacteriological failure or recurrence, and major treatment change were considered unfavourable outcomes. All comparisons used groups of participants randomly assigned concurrently. For non-inferiority to be shown, the upper boundary of the 95% CI should be less than 10% in both modified intention-to-treat (mITT) and per-protocol analyses, with prespecified tests for superiority done if non-inferiority was shown. This trial is registered with ISRCTN, ISRCTN18148631. Between March 28, 2016, and Jan 28, 2020, 1436 participants were screened and 588 were randomly assigned. Of 517 participants in the mITT population, 133 (71%) of 187 on the control regimen and 162 (83%) of 196 on the oral regimen had a favourable outcome: a difference of 11·0% (95% CI 2·9-19·0), adjusted for HIV status and randomisation protocol (p<0·0001 for non-inferiority). By 76 weeks, 108 (53%) of 202 participants on the control regimen and 106 (50%) of 211 allocated to the oral regimen had an adverse event of grade 3 or 4; five (2%) participants on the control regimen and seven (3%) on the oral regimen had died. Hearing loss (Brock grade 3 or 4) was more frequent in participants on the control regimen than in those on the oral regimen (18 [9%] vs four [2%], p=0·0015). Of 134 participants in the mITT population who were allocated to the 6-month regimen, 122 (91%) had a favourable outcome compared with 87 (69%) of 127 participants randomly assigned concurrently to the control regimen (adjusted difference 22·2%, 95% CI 13·1-31·2); six (4%) of 143 participants on the 6-month regimen had grade 3 or 4 hearing loss. Both bedaquiline-containing regimens, a 9-month oral regimen and a 6-month regimen with 8 weeks of second-line injectable, had superior efficacy compared with a 9-month injectable-containing regimen, with fewer cases of hearing loss. USAID and Janssen Research & Development

Economic evaluation protocol of a short, all-oral bedaquiline-containing regimen for the treatment of rifampicin-resistant tuberculosis from the STREAM trial

Introduction: A December 2019 WHO rapid communication recommended the use of 9-month all-oral regimens for treating multidrug-resistant tuberculosis (MDR-TB). Besides the clinical benefits, they are thought to be less costly than the injectable-containing regimens, for both the patient and the health system. STREAM is the first randomised controlled trial with an economical evaluation to compare all-oral and injectable-containing 9–11-month MDR-TB treatment regimens. Methods and analysis: Health system costs of delivering a 9-month injectable-containing regimen and a 9-month all-oral bedaquiline-containing regimen will be collected in Ethiopia, India, Moldova and Uganda, using ‘bottom-up’ and ‘top-down’ costing approaches. Patient costs will be collected using questionnaires that have been developed based on the STOP-TB questionnaire. The primary objective of the study is to estimate the cost utility of the two regimens, from a health system perspective. Secondary objectives include estimating the cost utility from a societal perspective as well as evaluating the cost-effectiveness of the regimens, using both health system and societal perspectives. The effect measure for the cost–utility analysis will be the quality-adjusted life years (QALY), while the effect measure for the cost-effectiveness analysis will be the efficacy outcome from the clinical trial. Ethics and dissemination: The study has been evaluated and approved by the Ethics Advisory Group of the International Union Against Tuberculosis and Lung Disease and also approved by ethics committees in all participating countries. All participants have provided written informed consent. The results of the economic evaluation will be published in a peer-reviewed journal. Trial registration number: ISRCTN18148631

Economic evaluation of shortened, bedaquiline-containing treatment regimens for rifampicin-resistant tuberculosis (STREAM stage 2) : a within-trial analysis of a randomised controlled trial

Background: The STREAM stage 2 trial assessed two bedaquiline-containing regimens for rifampicin-resistant tuberculosis: a 9-month all-oral regimen and a 6-month regimen containing an injectable drug for the first 2 months. We did a within-trial economic evaluation of these regimens. Methods: STREAM stage 2 was an international, phase 3, non-inferiority randomised trial in which participants with rifampicin-resistant tuberculosis were randomly assigned (1:2:2:2) to the 2011 WHO regimen (terminated early), a 9-month injectable-containing regimen (control regimen), a 9-month all-oral regimen with bedaquiline (oral regimen), or a 6-month regimen with bedaquiline and an injectable for the first 2 months (6-month regimen). We prospectively collected direct and indirect costs and health-related quality of life data from trial participants until week 76 of follow-up. Cost-effectiveness of the oral and 6-month regimens versus control was estimated in four countries (oral regimen) and two countries (6-month regimen), using health-related quality of life for cost-utility analysis and trial efficacy for cost-effectiveness analysis. This trial is registered with ISRCTN, ISRCTN18148631. Findings: 300 participants were included in the economic analyses (Ethiopia, 61; India, 142; Moldova, 51; Uganda, 46). In the cost-utility analysis, the oral regimen was not cost-effective in Ethiopia, India, Moldova, and Uganda from either a provider or societal perspective. In Moldova, the oral regimen was dominant from a societal perspective. In the cost-effectiveness analysis, the oral regimen was likely to be cost-effective from a provider perspective at willingness-to-pay thresholds per additional favourable outcome of more than US$4500 in Ethiopia,$1900 in India, $3950 in Moldova, and$7900 in Uganda, and from a societal perspective at thresholds of more than $15 900 in Ethiopia,$3150 in India, and $4350 in Uganda, while in Moldova the oral regimen was dominant. In Ethiopia and India, the 6-month regimen would cost tuberculosis programmes and participants less than the control regimen and was highly likely to be cost-effective in both cost-utility analysis and cost-effectiveness analysis. Reducing the bedaquiline price from$1·81 to $1·00 per tablet made the oral regimen cost-effective in the provider-perspective cost-utility analysis in India and Moldova and dominate over the control regimen in the provider-perspective cost-effectiveness analysis in India. Interpretation: At current costs, the oral bedaquiline-containing regimen for rifampicin-resistant tuberculosis is unlikely to be cost-effective in many low-income and middle-income countries. The 6-month regimen represents a cost-effective alternative if injectable use for 2 months is acceptable. Funding: USAID and Janssen Research & Development

An F420 dependent anti-oxidant mechanism protects Mycobacterium against oxidative stress and bactericidal agents

Mycobacterium tuberculosis, an aerobic bacterium that persists intracellularly in host macrophages, has evolved diverse mechanisms to combat and survive oxidative stress. Here we show a novel F420 dependent anti-oxidant mechanism that protects mycobacterium against oxidative stress. Inactivation of fbiC gene in Mtb results in a cofactor F420 deficient mutant; Mtb F420- mutants are hypersensitive to oxidative stress and exhibit an enhanced reduction in NADH/NAD+ ratios upon treatment with menadione. In agreement with the recent hypothesis on oxidative stress being the common pathway resulting in cell death by bactericidal agents, F420- mutants are hypersensitive to mycobactericidal agents such as isoniazid and moxifloxacin. Further we show that Mtb Deazaflavin dependent nitroreductase (Ddn) and its two homologues Rv1261c and Rv1558 encode for an F420H2 dependent quinone reductase (Fqr) function. At the expense of F420H2, Fqr enzymes utilize menaquinone and ubiquinone analogues as efficient substrates catalyzing the formation of their respective dihydroquinones. Our results demonstrate that Ddn and its homologues catalyze an F420H2 specific obligate two electron reduction of endogenous quinones, thereby competing with the one electron reduction pathway and preventing the formation of harmful cytotoxic semiquinones. Fqr thus emerges as a new class of proteins that protects mycobacteria against oxidative stress and bactericidal agents. Further, these findings open up an avenue for the inhibition of F420 biosynthesis pathway or Fqr class as a mechanism to potentiate the action of bactericidal agents

Patient adherence to tuberculosis treatment in the Indian subcontinent: systematic review and meta-synthesis of qualitative research

Objectives How well patients adhere to their tuberculosis (TB) treatment influences their recovery and development of drug resistance, but influences on adherence are multiple and often competing. We synthesised qualitative studies from our setting in the Indian subcontinent to understand the dimensions and dynamics involved to help inform service provision.Design Qualitative synthesis comprising inductive coding, thematic analysis and forming a conceptual framework.Data sources Medline (OVID), Embase (OVID), CINAHL (EBSCOHost), PsycINFO (EBSCOHost), Web of Science Core Collection, Cochrane Library and Epistemonikos were databases searched on 26 March 2020 for studies published since 1 January 2000.Eligibility criteria for selecting studies We included reports in English from the Indian subcontinent that used qualitative or mixed-methodology designs and reported findings around adherence to TB treatment. Full texts meeting eligibility were sampled based on ‘thickness’ (the richness of the qualitative data reported).Data extraction and synthesis Two reviewers used standardised methods to screen abstracts and code. Included studies were assessed for reliability and quality using a standard tool. Qualitative synthesis was performed by inductive coding, thematic analysis and developing conceptual framework.Results Of 1729 abstracts screened from initial search, 59 were shortlisted for full-text review. Twenty-four studies that qualified as ‘thick’ were included in the synthesis. Studies were set in India (12), Pakistan (6), Nepal (3), Bangladesh (1) or in two or more of these countries (2). Of the 24 studies, all but one included people who were taking TB treatment (1 study included only healthcare providers), and 17 included healthcare workers, community members or both.We identified three themes: (1) personal influences on the people with TB include interconnections between their social role in the family unit, their own priorities in day-to-day living and their experience to date with the disease; (2) adherence is profoundly influenced by how individual healthcare providers interact with patients on treatment and address their needs; (3) adherence is influenced across communities by structural, social, economic and cultural factors related to treatment.Conclusion Staff in TB programmes require an understanding of the various competing influences on individuals undergoing treatment. Programmes need to have more flexible and people-centred approaches to service provision in order to achieve adherence, and thus improve treatment outcomes.PROSPERO registration number CRD42020171409

Coadministration of Allopurinol To Increase Antimycobacterial Efficacy of Pyrazinamide as Evaluated in a Whole-Blood Bactericidal Activity Model

10.1128/AAC.00482-17ANTIMICROBIAL AGENTS AND CHEMOTHERAPY611

XBP-1, a Novel Human T-Lymphotropic Virus Type 1 (HTLV-1) Tax Binding Protein, Activates HTLV-1 Basal and Tax-Activated Transcription▿

X-box binding protein 1 (XBP-1), a basic leucine zipper transcription factor, plays a key role in the cellular unfolded protein response (UPR). There are two XBP-1 isoforms in cells, spliced XBP-1S and unspliced XBP-1U. XBP-1U has been shown to bind to the 21-bp Tax-responsive element of the human T-lymphotropic virus type 1 (HTLV-1) long terminal repeat (LTR) in vitro and transactivate HTLV-1 transcription. Here we identify XBP-1S as a transcription activator of HTLV-1. Compared to XBP-1U, XBP-1S demonstrates stronger activating effects on both basal and Tax-activated HTLV-1 transcription in cells. Our results show that both XBP-1S and XBP-1U interact with Tax and bind to the HTLV-1 LTR in vivo. In addition, elevated mRNA levels of the gene for XBP-1 and several UPR genes were detected in the HTLV-1-infected C10/MJ and MT2 T-cell lines, suggesting that HTLV-1 infection may trigger the UPR in host cells. We also identify Tax as a positive regulator of the expression of the gene for XBP-1. Activation of the UPR by tunicamycin showed no effect on the HTLV-1 LTR, suggesting that HTLV-1 transcription is specifically regulated by XBP-1. Collectively, our study demonstrates a novel host-virus interaction between a cellular factor XBP-1 and transcriptional regulation of HTLV-1