Evaluating newly approved drugs in combination regimens for multidrug-resistant tuberculosis with fluoroquinolone resistance (endTB-Q): study protocol for a multi-country randomized controlled trial.

BACKGROUND: Treatment for fluoroquinolone-resistant multidrug-resistant/rifampicin-resistant tuberculosis (pre-XDR TB) often lasts longer than treatment for less resistant strains, yields worse efficacy results, and causes substantial toxicity. The newer anti-tuberculosis drugs, bedaquiline and delamanid, and repurposed drugs clofazimine and linezolid, show great promise for combination in shorter, less-toxic, and effective regimens. To date, there has been no randomized, internally and concurrently controlled trial of a shorter, all-oral regimen comprising these newer and repurposed drugs sufficiently powered to produce results for pre-XDR TB patients. METHODS: endTB-Q is a phase III, multi-country, randomized, controlled, parallel, open-label clinical trial evaluating the efficacy and safety of a treatment strategy for patients with pre-XDR TB. Study participants are randomized 2:1 to experimental or control arms, respectively. The experimental arm contains bedaquiline, linezolid, clofazimine, and delamanid. The control comprises the contemporaneous WHO standard of care for pre-XDR TB. Experimental arm duration is determined by a composite of smear microscopy and chest radiographic imaging at baseline and re-evaluated at 6 months using sputum culture results: participants with less extensive disease receive 6 months and participants with more extensive disease receive 9 months of treatment. Randomization is stratified by country and by participant extent-of-TB-disease phenotype defined according to screening/baseline characteristics. Study participation lasts up to 104 weeks post randomization. The primary objective is to assess whether the efficacy of experimental regimens at 73 weeks is non-inferior to that of the control. A sample size of 324 participants across 2 arms affords at least 80% power to show the non-inferiority, with a one-sided alpha of 0.025 and a non-inferiority margin of 12%, against the control in both modified intention-to-treat and per-protocol populations. DISCUSSION: This internally controlled study of shortened treatment for pre-XDR TB will provide urgently needed data and evidence for clinical and policy decision-making around the treatment of pre-XDR TB with a four-drug, all-oral, shortened regimen. TRIAL REGISTRATION: ClinicalTrials.Gov NCT03896685. Registered on 1 April 2018; the record was last updated for study protocol version 4.3 on 17 March 2023.

Bedaquiline- and clofazimine- selected Mycobacterium tuberculosis mutants: further insights on resistance driven largely by Rv0678.

Drug-resistant tuberculosis is a serious global health threat. Bedaquiline (BDQ) is a relatively new core drug, targeting the respiratory chain in Mycobacterium tuberculosis (Mtb). While mutations in the BDQ target gene, atpE, are rare in clinical isolates, mutations in the Rv0678 gene, a transcriptional repressor regulating the efflux pump MmpS5-MmpL5, are increasingly observed, and have been linked to worse treatment outcomes. Nevertheless, underlying mechanisms of (cross)-resistance remain incompletely resolved. Our study aims to distinguish resistance associated variants from other polymorphisms, by assessing the in vitro onset of mutations under drug pressure, combined with their impact on minimum inhibitory concentrations (MICs) and on protein stability. For this purpose, isolates were exposed in vitro to sub-lethal concentrations of BDQ or clofazimine (CFZ). Selected colonies had BDQ- and CFZ-MICs determined on 7H10 and 7H11 agar. Sanger sequencing and additional Deeplex Myc-TB and whole genome sequencing (WGS) for a subset of isolates were used to search for mutations in Rv0678, atpE and pepQ. In silico characterization of relevant mutations was performed using computational tools. We found that colonies that grew on BDQ medium had mutations in Rv0678, atpE or pepQ, while CFZ-exposed isolates presented mutations in Rv0678 and pepQ, but none in atpE. Twenty-eight Rv0678 mutations had previously been described among in vitro selected mutants or in patients' isolates, while 85 were new. Mutations were scattered across the Rv0678 gene without apparent hotspot. While most Rv0678 mutations led to an increased BDQ- and/or CFZ-MIC, only a part of them surpassed the critical concentration (69.1% for BDQ and 87.9% for CFZ). Among the mutations leading to elevated MICs for BDQ and CFZ, we report a synonymous Val1Val mutation in the Rv0678 start codon. Finally, in silico characterization of Rv0678 mutations suggests that especially the C46R mutant may render Rv0678 less stable.

Mycobacterium tuberculosis retains viability in RNAlater buffer but not in GTC-TCEP and DNA/RNA Shield.

Efficient inactivation of clinical samples containing mycobacteria is crucial for biosafety during shipment and handling. When stored in RNAlater, Mycobacterium tuberculosis H37Ra remains viable, and our results suggest that at -20 °C and 4 °C changes in the mycobacterial transcriptome are possible. Only GTC-TCEP and DNA/RNA Shield inactivate sufficiently for shipment.

Clinical standards for drug-susceptible pulmonary TB.

BACKGROUND: The aim of these clinical standards is to provide guidance on 'best practice´ for diagnosis, treatment and management of drug-susceptible pulmonary TB (PTB).METHODS: A panel of 54 global experts in the field of TB care, public health, microbiology, and pharmacology were identified; 46 participated in a Delphi process. A 5-point Likert scale was used to score draft standards. The final document represents the broad consensus and was approved by all 46 participants.RESULTS: Seven clinical standards were defined: Standard 1, all patients (adult or child) who have symptoms and signs compatible with PTB should undergo investigations to reach a diagnosis; Standard 2, adequate bacteriological tests should be conducted to exclude drug-resistant TB; Standard 3, an appropriate regimen recommended by WHO and national guidelines for the treatment of PTB should be identified; Standard 4, health education and counselling should be provided for each patient starting treatment; Standard 5, treatment monitoring should be conducted to assess adherence, follow patient progress, identify and manage adverse events, and detect development of resistance; Standard 6, a recommended series of patient examinations should be performed at the end of treatment; Standard 7, necessary public health actions should be conducted for each patient. We also identified priorities for future research into PTB.CONCLUSION: These consensus-based clinical standards will help to improve patient care by guiding clinicians and programme managers in planning and implementation of locally appropriate measures for optimal person-centred treatment for PTB.

Effectiveness of GenoType MTBDRsl in excluding TB drug resistance in a clinical trial.

OBJECTIVES: To assess the performance of the GenoType MTBDRsl v1, a line-probe assay (LPA), to exclude baseline resistance to fluoroquinolones (FQs) and second-line injectables (SLIs) in the Standard Treatment Regimen of Anti-tuberculosis Drugs for Patients With MDR-TB 1 (STREAM 1) trial.METHODS: Direct sputum MTBDRsl results in the site laboratories were compared to indirect phenotypic drug susceptibility testing (pDST) results in the central laboratory, with DNA sequencing as a reference standard.RESULTS: Of 413 multidrug-resistant TB (MDR-TB) patients tested using MTBDRsl and pDST, 389 (94.2%) were FQ-susceptible and 7 (1.7%) FQ-resistant, while 17 (4.1%) had an inconclusive MTBDRsl result. For SLI, 372 (90.1%) were susceptible, 5 (1.2%) resistant and 36 (8.7%) inconclusive. There were 9 (2.3%) FQ discordant pDST/MTBDRsl results, of which 3 revealed a mutation and 5 (1.3%) SLI discordant pDST/MTBDRsl results, none of which were mutants on sequencing. Among the 17 FQ- and SLI MTBDRsl-inconclusive samples, sequencing showed 1 FQ- and zero SLI-resistant results, similar to frequencies among the conclusive MTBDRsl. The majority of inconclusive MTBDRsl results were associated with low bacillary load samples (acid-fast bacilli smear-negative or scantily positive) compared to conclusive results (P < 0.001).CONCLUSION: MTBDRsl can facilitate the rapid exclusion of FQ and SLI resistances for enrolment in clinical trials.

Evaluating newly approved drugs for multidrug-resistant tuberculosis (endTB): study protocol for an adaptive, multi-country randomized controlled trial.

BACKGROUND: Treatment of multidrug- and rifampin-resistant tuberculosis (MDR/RR-TB) is expensive, labour-intensive, and associated with substantial adverse events and poor outcomes. While most MDR/RR-TB patients do not receive treatment, many who do are treated for 18 months or more. A shorter all-oral regimen is currently recommended for only a sub-set of MDR/RR-TB. Its use is only conditionally recommended because of very low-quality evidence underpinning the recommendation. Novel combinations of newer and repurposed drugs bring hope in the fight against MDR/RR-TB, but their use has not been optimized in all-oral, shorter regimens. This has greatly limited their impact on the burden of disease. There is, therefore, dire need for high-quality evidence on the performance of new, shortened, injectable-sparing regimens for MDR-TB which can be adapted to individual patients and different settings. METHODS: endTB is a phase III, pragmatic, multi-country, adaptive, randomized, controlled, parallel, open-label clinical trial evaluating the efficacy and safety of shorter treatment regimens containing new drugs for patients with fluoroquinolone-susceptible, rifampin-resistant tuberculosis. Study participants are randomized to either the control arm, based on the current standard of care for MDR/RR-TB, or to one of five 39-week multi-drug regimens containing newly approved and repurposed drugs. Study participation in all arms lasts at least 73 and up to 104 weeks post-randomization. Randomization is response-adapted using interim Bayesian analysis of efficacy endpoints. The primary objective is to assess whether the efficacy of experimental regimens at 73 weeks is non-inferior to that of the control. A sample size of 750 patients across 6 arms affords at least 80% power to detect the non-inferiority of at least 1 (and up to 3) experimental regimens, with a one-sided alpha of 0.025 and a non-inferiority margin of 12%, against the control in both modified intention-to-treat and per protocol populations. DISCUSSION: The lack of a safe and effective regimen that can be used in all patients is a major obstacle to delivering appropriate treatment to all patients with active MDR/RR-TB. Identifying multiple shorter, safe, and effective regimens has the potential to greatly reduce the burden of this deadly disease worldwide. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT02754765. Registered on 28 April 2016; the record was last updated for study protocol version 3.3, on 27 August 2019.

Thin-Layer-Agar-Based Direct Phenotypic Drug Susceptibility Testing on Sputum in Eswatini Rapidly Detects Mycobacterium tuberculosis Growth and Rifampicin Resistance Otherwise Missed by WHO-Endorsed Diagnostic Tests.

Xpert MTB/RIF rapidly detects resistance to rifampicin (RR); however, this test misses I491F-RR conferring rpoB mutation, common in southern Africa. In addition, Xpert MTB/RIF does not distinguish between viable and dead Mycobacterium tuberculosis (MTB). We aimed to investigate the ability of thin-layer agar (TLA) direct drug-susceptibility testing (DST) to detect MTB and its drug-resistance profiles in field conditions in Eswatini. Consecutive samples were tested in parallel with Xpert MTB/RIF and TLA for rifampicin (1.0 µg/ml) and ofloxacin (2.0 µg/ml). TLA results were compared at the Reference Laboratory in Antwerp with indirect-DST on Löwenstein-Jensen or 7H11 solid media and additional phenotypic and genotypic testing to resolve discordance. TLA showed a positivity rate for MTB detection of 7.1% versus 10.0% for Xpert MTB/RIF. Of a total of 4,547 samples included in the study, 200 isolates were available for comparison to the composite reference. Within a median of 18.4 days, TLA detected RR with 93.0% sensitivity (95% confidence interval [CI], 77.4 to 98.0) and 99.4% specificity (95% CI, 96.7 to 99.9) versus 62.5% (95% CI, 42.7 to 78.8) and 99.3% (95% CI, 96.2 to 99.9) for Xpert MTB/RIF. Eight isolates, 28.6% of all RR-confirmed isolates, carried the I491F mutation, all detected by TLA. TLA also correctly identified 183 of the 184 ofloxacin-susceptible isolates (99.5% specificity; 95% CI, 97.0 to 99.9). In field conditions, TLA rapidly detects RR, and in this specific setting, it contributed to detection of additional RR patients over Xpert MTB/RIF, mainly but not exclusively due to I491F. TLA also accurately excluded fluoroquinolone resistance.

Molecular Biomarkers for Celiac Disease: Past, Present and Future.

Celiac disease (CeD) is a complex immune-mediated disorder that is triggered by dietary gluten in genetically predisposed individuals. CeD is characterized by inflammation and villous atrophy of the small intestine, which can lead to gastrointestinal complaints, malnutrition, and malignancies. Currently, diagnosis of CeD relies on serology (antibodies against transglutaminase and endomysium) and small-intestinal biopsies. Since small-intestinal biopsies require invasive upper-endoscopy, and serology cannot predict CeD in an early stage or be used for monitoring disease after initiation of a gluten-free diet, the search for non-invasive biomarkers is ongoing. Here, we summarize current and up-and-coming non-invasive biomarkers that may be able to predict, diagnose, and monitor the progression of CeD. We further discuss how current and emerging techniques, such as (single-cell) transcriptomics and genomics, can be used to uncover the pathophysiology of CeD and identify non-invasive biomarkers.

Fluorescein diacetate and rapid molecular testing for the early identification of rifampicin resistance in Mali.

BACKGROUND: Non-conversion on auramine smear microscopy indicates a lack of treatment response, possibly associated with initial rifampicin-resistant tuberculosis (RR-TB). However, dead bacteria still stain positive and may be detected. Fluorescein diacetate smear microscopy (FDA) shows live mycobacteria only. Therefore, we studied the potential of 2-month (2M) FDA for the identification of initial RR-TB.METHODS: Between 2015 and 2018, we enrolled new smear-positive pulmonary TB patients from five local centres in Bamako, Mali. After baseline screening, sputum samples were collected at 1M, 2M, 5M and 18M. We used rpoB sequencing to identify initial RR-TB.RESULTS: Of 1359 patients enrolled, 1019 (75%) had rpoB sequencing results. Twenty-six (2.6%, 95%CI: 1.7-3.7) had mutations conferring rifampicin resistance. Most frequent rpoB mutations were located at the codons Asp435Val (42.4%) and Ser450Leu (34.7%). Among patients with initial RR-TB, 72.2% were FDA-negative at 2M (P = 0.2). The positive and negative predictive value of 5M FDA for culture-based failure was respectively 20.0% and 94.7%.CONCLUSION: FDA did not identify the majority of patients with initial RR-TB or culture-based failure. As the full spectrum of mutations identified on sequencing was identified using Xpert, our data support its rapid universal implementation in Mali.

First-line tuberculosis treatment with double-dose rifampicin is well tolerated.

OBJECTIVE: To compare the occurrence of unfavourable treatment and safety outcomes of double-dose rifampicin (RMP; 20 mg/kg/d, intervention) with standard dose (10 mg/kg/d, control) in a first-line tuberculosis (TB) treatment regimen for smear-positive TB patients in Bangladesh.DESIGN: This was a randomised clinical trial. The primary efficacy and safety endpoints were the occurrence of an unfavourable treatment outcome (death, failure, relapse or loss to follow-up) and the occurrence of any serious drug-related adverse event (SAE).RESULTS: In primary efficacy analysis, among 343 control and 347 intervention patients, respectively 15.5% and 11.8% had an unfavourable outcome. In safety analysis, among 349 intervention and 352 control patients, respectively 4.3% and 2.6% experienced an SAE. These differences were not significant. There was a significantly lower occurrence of SAEs, explained by a lower occurrence of hepatic toxicity, in a RMP double-dosed but erroneously HZE (isoniazid+pyrazinamide+ethambutol) under-dosed subgroup.CONCLUSIONS: Our findings show that there is no statistically significant difference in terms of efficacy and safety between standard and double-dose RMP. An accidental finding (related to dosage levels of the standard regimen) suggests that high-dose RMP is potentially a lesser cause of hepatotoxicity. Larger trials with more power, or trials with at least a triple-dose might be needed to clearly see the effect of high-dose RMP on unfavourable outcomes.

Reduction of diagnostic and treatment delays reduces rifampicin-resistant tuberculosis mortality in Rwanda.

SETTING: In 2005, in response to the increasing prevalence of rifampicin-resistant tuberculosis (RR-TB) and poor treatment outcomes, Rwanda initiated the programmatic management of RR-TB, including expanded access to systematic rifampicin drug susceptibility testing (DST) and standardised treatment.OBJECTIVE: To describe trends in diagnostic and treatment delays and estimate their effect on RR-TB mortality.DESIGN: Retrospective analysis of individual-level data including 748 (85.4%) of 876 patients diagnosed with RR-TB notified to the World Health Organization between 1 July 2005 and 31 December 2016 in Rwanda. Logistic regression was used to estimate the effect of diagnostic and therapeutic delays on RR-TB mortality.RESULTS: Between 2006 and 2016, the median diagnostic delay significantly decreased from 88 days to 1 day, and the therapeutic delay from 76 days to 3 days. Simultaneously, RR-TB mortality significantly decreased from 30.8% in 2006 to 6.9% in 2016. Total delay in starting multidrug-resistant TB (MDR-TB) treatment of more than 100 days was associated with more than two-fold higher odds for dying. When delays were long, empirical RR-TB treatment initiation was associated with a lower mortality.CONCLUSION: The reduction of diagnostic and treatment delays reduced RR-TB mortality. We anticipate that universal testing for RR-TB, short diagnostic and therapeutic delays and effective standardised MDR-TB treatment will further decrease RR-TB mortality in Rwanda.

Diabetes Mellitus among new tuberculosis patients in Bamako, Mali.

INTRODUCTION: Diabetes Mellitus (DM) increases worldwide, mostly in low- and middle-income countries. In Mali, the prevalence in the adult population is estimated at 1.8%, but tuberculosis (TB) patients are not systematically screened. The goal of our study was to determine the prevalence of DM among newly diagnosed TB patients. METHODS: We conducted a cross sectional study and a pilot prospective cohort study in four health centers in Bamako. All patients underwent fasting capillary-blood glucose (FCBG) test at Day 0, and repeated after one-week of TB treatment. Venous FBG test was performed for discrepancies between the two FCBG results. Thereafter, FCBG was performed for pilot study at month-2 (M2) and M5 of TB treatment. RESULTS: Two hundred and one patients were enrolled in this study. Impaired fasting blood glucose was identified in 17 (8.5%), of whom 11 (5.5%) had DM (VFBG >7 mmol/L). Among patients with DM, seven (63.6%) had successful TB treatment outcome, versus 142 (74.7%) of those without DM (p = 0.64), and (OR: 1.69, 95%CI 0.47-6.02). CONCLUSION: The prevalence of DM among TB patients in Bamako exceeds that of the general population and screening at TB diagnosis suffices to identify those with DM. Systematic screening of both diseases will allow better treatment.

Fluoroquinolone heteroresistance in Mycobacterium tuberculosis: detection by genotypic and phenotypic assays in experimentally mixed populations.

Heteroresistance - the simultaneous presence of drug-susceptible and -resistant organisms - is common in Mycobacterium tuberculosis. In this study, we aimed to determine the limit of detection (LOD) of genotypic assays to detect gatifloxacin-resistant mutants in experimentally mixed populations. A fluoroquinolone-susceptible M. tuberculosis mother strain (S) and its in vitro selected resistant daughter strain harbouring the D94G mutation in gyrA (R) were mixed at different ratio's. Minimum inhibitory concentrations (MICs) against gatifloxacin were determined, while PCR-based techniques included: line probe assays (Genotype MTBDRsl and GenoScholar-FQ + KM TB II), Sanger sequencing and targeted deep sequencing. Droplet digital PCR was used as molecular reference method. A breakpoint concentration of 0.25 mg/L allows the phenotypic detection of ≥1% resistant bacilli, whereas at 0.5 mg/L ≥ 5% resistant bacilli are detected. Line probe assays detected ≥5% mutants. Sanger sequencing required the presence of around 15% mutant bacilli to be detected as (hetero) resistant, while targeted deep sequencing detected ≤1% mutants. Deep sequencing and phenotypic testing are the most sensitive methods for detection of fluoroquinolone-resistant minority populations, followed by line probe assays (provided that the mutation is confirmed by a mutation band), while Sanger sequencing proved to be the least sensitive method.

Sputum smear microscopy in the Xpert® MTB/RIF era.

A balanced perspective is advocated for the assessment and application of the most recent and the oldest diagnostic methods for pulmonary tuberculosis (TB)-the molecular Xpert® MTB/RIF assay and microscopy for acid-fast bacilli. We discuss their respective merits and shortcomings and identify threats that may hamper their use in TB control. Neither test on its own provides all the information needed for diagnosis and treatment monitoring. Considering all aspects important for both individual patient care and disease control, neither seems 'better' than the other. The required advancement of microscopy had already been hampered before the introduction of the GeneXpert technology by unsuccessful and probably misguided attempts to decentralise culture-based diagnosis and drug susceptibility testing. It seems evident that systematic replacement of microscopy by Xpert is not a viable option for the foreseeable future. Instead, the two methods should complement each other to arrive at a comprehensive, accessible and continuous service for a maximum number of patients. This will intrinsically prioritise targeting the most potent transmitters with the worst prognosis, simultaneously offering optimised prospects for efficient TB control. New microscopy and Xpert applications are expected to ultimately make control programmes independent of culture-based methods in diagnosis, treatment monitoring and outcome assessment.

Xpert MTB/RIF assay for the diagnosis of extrapulmonary tuberculosis: a diagnostic evaluation study.

OBJECTIVES: The diagnosis of extrapulmonary tuberculosis (EPTB) is often made on clinical suspicion alone, resulting in both under- and overdiagnosis and relatively poor outcomes. In this study, we evaluated the clinical utility of the Xpert MTB/RIF on routinely collected extrapulmonary specimens in Ethiopia. METHODS: This study was carried out at Jimma University Specialized Hospital, Southwest Ethiopia. Extrapulmonary specimens were collected from 572 patients clinically suspected of suffering from EPTB. All specimens were tested for TB by smear microscopy, culture, and Xpert MTB/RIF. The diagnostic accuracy of Xpert MTB/RIF was calculated and compared to a composite reference standard (CRS), comprising clinical and laboratory results. RESULTS: In total, 572 extrapulmonary specimens (279 lymph node, 159 pleural, 80 peritoneal, 45 cerebrospinal, and nine pericardial fluids) were tested. The pooled sensitivity and specificity of Xpert MTB/RIF were calculated to be 75% (95% CI 70-80) and 98% (95% CI 97-100) respectively when compared to the CRS. The highest sensitivity was documented for lymph node specimens (90%; 95% CI 86-94), moderate sensitivity for cerebrospinal fluid (53%; 95% CI 28-79), while the sensitivity was lowest for pleural (30%; 95% CI 17-44) and peritoneal (32%; 95% CI 12-51) fluids. Xpert MTB/RIF in addition detected rifampicin resistance in 13 patients, in perfect agreement with results from the line probe assay. CONCLUSIONS: Xpert MTB/RIF may be used as initial diagnostic tool for testing of lymph node specimens from patients suspected of having TB lymphadenitis. The added value of Xpert MTB/RIF to diagnose pleural or peritoneal TB is limited by its poor sensitivity.

Principles for constructing a tuberculosis treatment regimen: the role and definition of core and companion drugs.

Current World Health Organization guidelines for the formulation of treatment regimens for multidrug-resistant tuberculosis (MDR-TB) pay too little attention to the microbiological activity of anti-tuberculosis drugs. Here, we draw lessons from the pioneering work done on shorter MDR-TB treatment regimens and the current knowledge of the bactericidal and sterilizing properties of the drugs to inform the composition of treatment regimens for MDR-TB. We propose to reserve the term 'core drug' for the one drug in a regimen that contributes most to relapse-free cure. The core drug has both moderate to high bactericidal and sterilizing activity, is given throughout treatment, is well tolerated, and has no cross-resistance with the core drug used in the previous regimen. Currently used core drugs include rifampicin in the first-line 6-month regimen, and fourth-generation fluoroquinolones and bedaquiline in regimens for drug-resistant TB. All other drugs are 'companion drugs', used to avert treatment failure due to acquired drug resistance against the core drug. Some also help further reduce the risk of relapse. Moreover, toxic drugs should be avoided if there is an alternative. A regimen must always include the core drug, plus at least one companion drug with high bactericidal activity, a second bactericidal companion drug, plus two sterilizing companion drugs.