Phylogenomic and genomic analysis reveals unique and shared genetic signatures of Mycobacterium kansasii complex species.

Species belonging to the Mycobacterium kansasii complex (MKC) are frequently isolated from humans and the environment and can cause serious diseases. The most common MKC infections are caused by the species M. kansasii (sensu stricto), leading to tuberculosis-like disease. However, a broad spectrum of virulence, antimicrobial resistance and pathogenicity of these non-tuberculous mycobacteria (NTM) are observed across the MKC. Many genomic aspects of the MKC that relate to these broad phenotypes are not well elucidated. Here, we performed genomic analyses from a collection of 665 MKC strains, isolated from environmental, animal and human sources. We inferred the MKC pangenome, mobilome, resistome, virulome and defence systems and show that the MKC species harbours unique and shared genomic signatures. High frequency of presence of prophages and different types of defence systems were observed. We found that the M. kansasii species splits into four lineages, of which three are lowly represented and mainly in Brazil, while one lineage is dominant and globally spread. Moreover, we show that four sub-lineages of this most distributed M. kansasii lineage emerged during the twentieth century. Further analysis of the M. kansasii genomes revealed almost 300 regions of difference contributing to genomic diversity, as well as fixed mutations that may explain the M. kansasii's increased virulence and drug resistance.

Single nucleotide variation catalog from clinical isolates mapped on tertiary and quaternary structures of ESX-1-related proteins reveals critical regions as putative Mtb therapeutic targets.

Proteins encoded by the ESX-1 genes of interest are essential for full virulence in all Mycobacterium tuberculosis complex (Mtbc) lineages, the pathogens causing the highest mortality worldwide. Identifying critical regions in these ESX-1-related proteins could provide preventive or therapeutic targets for Mtb infection, the game changer needed for tuberculosis control. We analyzed a compendium of whole genome sequences of clinical Mtb isolates from all lineages from >32,000 patients and identified single nucleotide polymorphisms. When mutations corresponding to all non-synonymous single nucleotide polymorphisms were mapped on structural models of the ESX-1 proteins, fully conserved regions emerged. Some could be assigned to known quaternary structures, whereas others could be predicted to be involved in yet-to-be-discovered interactions. Some mutants had clonally expanded (found in >1% of the isolates); these mutants were mostly located at the surface of globular domains, remote from known intra- and inter-molecular protein-protein interactions. Fully conserved intrinsically disordered regions of proteins were found, suggesting that these regions are crucial for the pathogenicity of the Mtbc. Altogether, our findings highlight fully conserved regions of proteins as attractive vaccine antigens and drug targets to control Mtb virulence. Extending this approach to the whole Mtb genome as well as other microorganisms will enhance vaccine development for various pathogens. IMPORTANCE: We mapped all non-synonymous single nucleotide polymorphisms onto each of the experimental and predicted ESX-1 proteins' structural models and inspected their placement. Varying sizes of conserved regions were found. Next, we analyzed predicted intrinsically disordered regions within our set of proteins, finding two putative long stretches that are fully conserved, and discussed their potential essential role in immunological recognition. Combined, our findings highlight new targets for interfering with Mycobacterium tuberculosis complex virulence.

Post-exposure prophylaxis in leprosy (PEOPLE): a cluster randomised trial.

BACKGROUND: Post-exposure prophylaxis (PEP) using single-dose rifampicin reduces progression from infection with Mycobacterium leprae to leprosy disease. We compared effectiveness of different administration modalities, using a higher (20 mg/kg) dose of rifampicin-single double-dose rifampicin (SDDR)-PEP. METHODS: We did a cluster randomised study in 16 villages in Madagascar and 48 villages in Comoros. Villages were randomly assigned to four study arms and inhabitants were screened once a year for leprosy, for 4 consecutive years. All permanent residents (no age restriction) were eligible to participate and all identified patients with leprosy were treated with multidrug therapy (SDDR-PEP was provided to asymptomatic contacts aged ≥2 years). Arm 1 was the comparator arm, in which no PEP was provided. In arm 2, SDDR-PEP was provided to household contacts of patients with leprosy, whereas arm 3 extended SDDR-PEP to anyone living within 100 m. In arm 4, SDDR-PEP was offered to household contacts and to anyone living within 100 m and testing positive to anti-phenolic glycolipid-I. The main outcome was the incidence rate ratio (IRR) of leprosy between the comparator arm and each of the intervention arms. We also assessed the individual protective effect of SDDR-PEP and explored spatial associations. This trial is registered with ClinicalTrials.gov, NCT03662022, and is completed. FINDINGS: Between Jan 11, 2019, and Jan 16, 2023, we enrolled 109 436 individuals, of whom 95 762 had evaluable follow-up data. Our primary analysis showed a non-significant reduction in leprosy incidence in arm 2 (IRR 0·95), arm 3 (IRR 0·80), and arm 4 (IRR 0·58). After controlling for baseline prevalence, the reduction in arm 3 became stronger and significant (IRR 0·56, p=0·0030). At an individual level SDDR-PEP was also protective with an IRR of 0·55 (p=0·0050). Risk of leprosy was two to four times higher for those living within 75 m of an index patient at baseline. INTERPRETATION: SDDR-PEP appears to protect against leprosy but less than anticipated. Strong spatial associations were observed within 75 m of index patients. Targeted door-to-door screening around index patients complemented by a blanket SDDR-PEP approach will probably have a substantial effect on transmission. FUNDING: European and Developing Countries Clinical Trials Partnership. TRANSLATION: For the French translation of the abstract see Supplementary Materials section.

The use of Kudoh method for culture of Mycobacterium tuberculosis and Mycobacterium africanum in The Gambia.

BACKGROUND: Mycobacterium tuberculosis culturing remains the gold standard for laboratory diagnosis of tuberculosis. Tuberculosis remains a great public health problem in developing countries like The Gambia, as most of the methods currently used for bacterial isolation are either time-consuming or costly. OBJECTIVE: To evaluate the Kudoh swab method in a West African setting in Gambia, with a particular focus on the method's performance when culturing Mycobacterium africanum West Africa 2 (MAF2) isolates. METHOD: 75 sputum samples were collected in the Greater Banjul Area and decontaminated in parallel with both the standard N-acetyl-L-Cysteine-NaOH (NALC-NaOH) and the Kudoh swab method in the TB diagnostics laboratory in the Medical Research Council Unit The Gambia between 30th December 2017 and 25th February 2018. These samples were subsequently cultured on standard Löwenstein-Jensen and Modified Ogawa media respectively and incubated at 37°C for mycobacterial growth. Spoligotyping was done to determine if the decontamination and culture methods compared could equally detect Mycobacterium tuberculosis, Mycobacterium africanum West Africa 1 and Mycobacterium africanum West Africa 2. RESULT: Among the 50 smear positives, 35 (70%) were culture-positive with Kudoh and 32 (64%) were culture positive with NALC-NaOH, whilst 7(28%) of the 25 smear negative samples were culture positive with both methods (Table 2). There was no significant difference in recovery between both methods (McNemar's test, p-value = 0.7003), suggesting that the overall positivity rate between the two methods is comparable. There were no differences in time-to-positivity or contamination rate between the methods. However, Kudoh yielded positive cultures that were negative on LJ and vice versa. All findings were irrespective of mycobacterial lineages. CONCLUSION: The Kudoh method has comparable sensitivity to the NALC-NaOH method for detecting Mycobacterium tuberculosis complex isolates. It is easy to perform and could be an add on option for mycobacterial culture in the field in The Gambia, since it requires less biosafety equipment.

Newly Identified Mycobacterium africanum Lineage 10, Central Africa.

Analysis of genome sequencing data from >100,000 genomes of Mycobacterium tuberculosis complex using TB-Annotator software revealed a previously unknown lineage, proposed name L10, in central Africa. Phylogenetic reconstruction suggests L10 could represent a missing link in the evolutionary and geographic migration histories of M. africanum.

Refined understanding of the impact of the Mycobacterium tuberculosis complex diversity on the intrinsic susceptibility to pretomanid.

Previous work reported unprecedented differences in the intrinsic in vitro susceptibility of the Mycobacterium tuberculosis complex (MTBC) to pretomanid (Pa) using the Mycobacteria Growth Indicator Tube (MGIT) system. We tested 125 phylogenetically diverse strains from all known MTBC lineages (1-9) without known Pa resistance mutations and four strains with known resistance mutations as controls. This confirmed that MTBC, unlike most bacteria-antimicrobial combinations, displayed substantial differences in the intrinsic susceptibility relative to the technical variation of Pa MIC testing. This was also the case for the Middlebrook 7H11 (7H11) medium, demonstrating that these differences were not specific to MGIT. Notably, lineage 1 was confirmed to have intrinsically elevated MICs compared with lineages 2, 3, 4, and 7 (L2-4/7), underlining the urgent need for WHO to publish its decision of whether lineage 1 should be deemed treatable by BPaL(M), the now preferred all-oral regimen for treating rifampin-resistant tuberculosis. Lineages 5 and 6, which are most frequent in West Africa, responded differently to Pa, with lineage 5 being more similar to L2-4/7 and lineage 6 being more susceptible. More data are needed to determine whether 7H11 MICs are systematically lower than those in MGIT. IMPORTANCE: This study confirmed that the Mycobacterium tuberculosis complex lineage 1, responsible for 28% of global tuberculosis cases, is less susceptible to pretomanid (Pa). It also refined the understanding of the intrinsic susceptibilities of lineages 5 and 6, most frequent in West Africa, and lineages 8 and 9. Regulators must review whether these in vitro differences affect the clinical efficacy of the WHO-recommended BPaL(M) regimen and set breakpoints for antimicrobial susceptibility testing accordingly. Notably, regulators should provide detailed justifications for their decisions to facilitate public scrutiny.

Ticks are unlikely to play a role in leprosy transmission in the Comoros (East Africa) as they do not harbour M. leprae DNA.

Introduction: Leprosy, one of the oldest known human diseases, continues to pose a global challenge for disease control due to an incomplete understanding of its transmission pathways. Ticks have been proposed as a potential contributor in leprosy transmission due to their importance as vectors for other infectious diseases. Methods: In 2010, a sampling of ticks residing on cattle was conducted on the islands Grande Comore, Anjouan, and Mohéli which constitute the Union of the Comoros where leprosy remains endemic. To investigate the potential role of ticks as a vector in transmission of leprosy disease, molecular analyses were conducted. Results: Out of the 526 ticks analysed, none were found to harbour Mycobacterium leprae DNA, as determined by a quantitative polymerase chain reaction (qPCR) assay targeting a family of dispersed repeats (RLEP) specific to M. leprae. Discussion: Therefore, our results suggest that in the Union of the Comoros, ticks are an unlikely vector for M. leprae.

Community-supported self-administered tuberculosis treatment combined with active tuberculosis screening: a pilot experience in Conakry, Guinea.

Directly observed treatment (DOT) for tuberculosis (TB) is recommended by the World Health Organization. However, DOT does not always meet patients' preferences, burdens health facilities, and is hard to implement in settings where access to healthcare services is regularly interrupted. A model addressing these limitations of DOT is community-supported self-administered treatment (CS-SAT), in which patients who self-administer TB treatment receive regular visits from community members. Guinea is a country with a high TB burden, recurrent epidemics, and periodic socio-political unrest. We piloted a CS-SAT model for drug-susceptible TB patients in Conakry, led by community volunteers, who also conducted active TB case finding among household contacts and referrals for isoniazid preventive treatment (IPT) in children below 5 years old. We aimed to assess TB treatment outcomes of patients on CS-SAT and describe the number of patients identified with TB case finding and IPT provision. Prospectively enrolled bacteriologically confirmed TB patients, presenting to two facilities, received monthly TB medication. Community volunteers performed bi-weekly (initiation phase) and later monthly (continuation phase) home visits to verify treatment adherence, screen household contacts for TB, and assess IPT uptake in children under five. Among 359 enrolled TB patients, 237 (66.0%) were male, and 37 (10.3%) were HIV-positive. Three hundred forty (94.7%) participants had treatment success, seven (1.9%) died, seven (1.9%) experienced treatment failure, and five (1.4%) were lost-to-follow-up. Among 1585 household contacts screened for TB, 26 (1.6%) had TB symptoms, of whom five (19.2%) were diagnosed with pulmonary TB. IPT referral was done for 376 children from 198 households. In a challenging setting, where DOT is often not feasible, CS-SAT led to successful TB treatment outcomes and created an opportunity for active TB case finding and IPT referral. We recommend the Guinean CS-SAT model for implementation in similar settings.

Face mask sampling (FMS) for tuberculosis shows lower diagnostic sensitivity than sputum sampling in Guinea.

BACKGROUND: Pulmonary tuberculosis (PTB) diagnosis relies on sputum examination, a challenge in sputum-scarce patients. Alternative non-invasive sampling methods such as face mask sampling (FMS) have been proposed. OBJECTIVE: To evaluate the value of FMS for PTB diagnosis by assessing its agreement with sputum samples processed by GeneXpert MTB/RIF (Ultra)(Xpert) testing, and describe FMS sensitivity and specificity. METHODS: This was a prospective study conducted at the Carrière TB clinic in Guinea. Presumptive TB patients willing to participate were asked to wear a surgical mask containing a polyvinyl alcohol (PVA) strip for thirty minutes. Subsequently, two spot sputum samples were collected, of which one was processed by microscopy on site and the other by Xpert in Guinea's National Reference Laboratory of Mycobacteriology (LNRM). The first 30 FMS were processed at the Supranational Reference Laboratory in Antwerp, Belgium, and the following 118 FMS in the LNRM. RESULTS: One hundred fifty patients participated, of whom 148 had valid results for both mask and sputum. Sputum smear microscopy was positive for 47 (31.8%) patients while sputum-Xpert detected MTB in 54 (36.5%) patients. Among the 54 patients testing sputum-Xpert positive, 26 (48.1%) yielded a positive FMS-Xpert result, while four sputum-Xpert negative patients tested positive for FMS and 90 patients were Xpert-negative for both sputum and mask samples, suggesting a moderate level of agreement (k-value of 0.47). The overall mask sensitivity was 48.1%, with 95.7% specificity. CONCLUSION: In our setting, Xpert testing on FMS did not yield a high level of agreement to sputum sample.

Rifampicin resistance conferring mutations among Mycobacterium tuberculosis strains in Rwanda.

Background: The World Health Organization-endorsed phenotypic and genotypic drug-susceptibility testing (gDST/pDST) assays for the detection of rifampicin-resistant (RR) tuberculosis (TB), may miss some clinically relevant rpoB mutants, including borderline mutations and mutations outside the gDST-targeted hotspot region. Sequencing of the full rpoB gene is considered the reference standard for rifampicin DST but is rarely available in RR-TB endemic settings and when done indirectly on cultured isolates may not represent the full spectrum of mutations. Hence, in most such settings, the diversity and trends of rpoB mutations remain largely unknown. Methods: This retrospective study included rpoB sequence data from a longitudinal collection of RR-TB isolates in Rwanda across 30 years (1991-2021). Results: Of 540 successfully sequenced isolates initially reported as RR-TB, 419 (77.6%) had a confirmed RR conferring mutation. The Ser450 Leu mutation was predominant throughout the study period. The Val170Phe mutation, not covered by rapid gDST assays, was observed in only four patients, three of whom were diagnosed by pDST. Along with the transition from pDST to rapid gDST, borderline RR-associated mutations, particularly Asp435Tyr, were detected more frequently. Borderline mutants were not associated with HIV status but presented lower odds of having rpoA-C compensatory mutations than other resistance-conferring mutations. Conclusion: Our analysis showed changes in the diversity of RR-TB conferring mutations throughout the study period that coincided with the switch of diagnostic tools to rapid gDST. The study highlights the importance of rapid molecular diagnostics reducing phenotypic bias in the detection of borderline rpoB mutations while vigilance for non-rifampicin resistance determinant region mutations is justified in any setting.

Re-evaluation of critical concentrations of antituberculosis fluoroquinolones in the Mycobacteria Growth Indicator Tube 960 system.

Background: Fluoroquinolones (FQs) have substantial activity against the Mycobacterium tuberculosis complex (MTBc) by preventing bacterial DNA synthesis through DNA gyrase inhibition. The reference standard for FQ-resistance testing is phenotypic drug-susceptibility testing (pDST) based on growth inhibition of MTBc in drug-containing Mycobacteria Growth Indicator Tube system (MGIT) media at a critical concentration (CC) that differentiates phenotypically wild-type from nonwild-type MTBc and at a clinical breakpoint that identifies strains that will likely still respond to treatment at higher doses. Despite the recent introduction of powerful new TB drugs, highly sensitive detection of clinically defined FQ resistance remains key. Method: In this study, we re-evaluated the current WHO-recommended CCs of Lfx (1.0 mg/ml), Mfx (0.25 mg/ml), Gfx (0.25 µg/ml), and the nowadays, obsolete CC of Ofx (2.0 mg/ml) for MGIT, using 147 MTBc isolates with known gyrA and gyrB sequences including both high-and low-level FQ resistance-conferring mutants. We tested a wide range of drug concentrations covering the current and former/obsolete WHO-recommended CCs for FQs and some intermediate concentrations to challenge the current WHO-recommended CCs. Results: The specificity of all four CCs was 100%. The sensitivities varied: 92.4% for Ofx and Lfx, 85.7% for Mfx, and 83.2% for Gfx. Lowering the CC of Mfx to 0.125 mg/ml would allow to correctly classify all wild-type and mutant isolates while lowering the CC of Gfx to 0.125 mg/ml would still misclassify some gyrA/gyrB mutants as susceptible. Conclusion: Based on our findings, a minimal inhibitory concentration of 0.125 mg/ml on MGIT medium is a more appropriate CC for Mfx and probably also as a surrogate for overall FQ resistance in the MTBc.

Borderline rpoB mutations transmit at the same rate as common rpoB mutations in a tuberculosis cohort in Bangladesh.

The spread of multidrug-resistant tuberculosis (MDR-TB) is a growing problem in many countries worldwide. Resistance to one of the primary first-line drugs, rifampicin, is caused by mutations in the Mycobacterium tuberculosis rpoB gene. So-called borderline rpoB mutations confer low-level resistance, in contrast to more common rpoB mutations which confer high-level resistance. While some borderline mutations show lower fitness in vitro than common mutations, their in vivo fitness is currently unknown. We used a dataset of 394 whole genome sequenced MDR-TB isolates from Bangladesh, representing around 44 % of notified MDR-TB cases over 6 years, to look at differences in transmission clustering between isolates with borderline rpoB mutations and those with common rpoB mutations. We found a relatively low percentage of transmission clustering in the dataset (34.8 %) but no difference in clustering between different types of rpoB mutations. Compensatory mutations in rpoA, rpoB, and rpoC were associated with higher levels of transmission clustering as were lineages two, three, and four relative to lineage one. Young people as well as patients with high sputum smear positive TB were more likely to be in a transmission cluster. Our findings show that although borderline rpoB mutations have lower in vitro growth potential this does not translate into lower transmission potential or in vivo fitness. Proper detection of these mutations is crucial to ensure they do not go unnoticed and spread MDR-TB within communities.

GeneXpert MTB/RIF Ultra performance to detect uncommon rpoB mutations in Mycobacterium tuberculosis.

OBJECTIVE: To investigate the performance of GeneXpert MTB/RIF Ultra to accurately detect rifampicin resistance for less common rpoB mutations that potentially confer phenotypic resistance, we tested 28 such Mycobacterium tuberculosis cultures with Xpert Ultra. RESULTS: They represented 22 different (combinations of) rpoB mutations. Of 28 isolates tested, one was reported by Xpert Ultra as "No rifampicin resistance detected", 8 yielded a "Rifampicin indeterminate" result, and 19 were identified as rifampicin resistant. Overall, our results corroborate previous observations on the "Indeterminate" results for mutations at codon 432, while we add Lys446Gln as additional "Indeterminate" result and Pro439Leu as a false rifampicin-susceptible result. Furthermore, we document other uncommon point mutations and indels across the rpoB gene that are mostly correctly identified as rifampicin resistant by Xpert ultra (V3). Taken together, "Indeterminate" results in Xpert Ultra may indicate underlying rpoB mutations within the rifampicin-resistance determining region and thus increase the post-test probability of rifampicin resistance, albeit to an unknown extent.

Hi-plex deep amplicon sequencing for identification, high-resolution genotyping and multidrug resistance prediction of Mycobacterium leprae directly from patient biopsies by using Deeplex Myc-Lep.

BACKGROUND: Expansion of antimicrobial resistance monitoring and epidemiological surveillance are key components of the WHO strategy towards zero leprosy. The inability to grow Mycobacterium leprae in vitro precludes routine phenotypic drug susceptibility testing, and only limited molecular tests are available. We evaluated a culture-free targeted deep sequencing assay, for mycobacterial identification, genotyping based on 18 canonical SNPs and 11 core variable-number tandem-repeat (VNTR) markers, and detection of rifampicin, dapsone and fluoroquinolone resistance-associated mutations in rpoB/ctpC/ctpI, folP1, gyrA/gyrB, respectively, and hypermutation-associated mutations in nth. METHODS: The limit of detection (LOD) was determined using DNA of M. leprae reference strains and from 246 skin biopsies and 74 slit skin smears of leprosy patients, with genome copies quantified by RLEP qPCR. Sequencing results were evaluated versus whole genome sequencing (WGS) data of 14 strains, and versus VNTR-fragment length analysis (FLA) results of 89 clinical specimens. FINDINGS: The LOD for sequencing success ranged between 80 and 3000 genome copies, depending on the sample type. The LOD for minority variants was 10%. All SNPs detected in targets by WGS were identified except in a clinical sample where WGS revealed two dapsone resistance-conferring mutations instead of one by Deeplex Myc-Lep, due to partial duplication of the sulfamide-binding domain in folP1. SNPs detected uniquely by Deeplex Myc-Lep were missed by WGS due to insufficient coverage. Concordance with VNTR-FLA results was 99.4% (926/932 alleles). INTERPRETATION: Deeplex Myc-Lep may help improve the diagnosis and surveillance of leprosy. Gene domain duplication is an original putative drug resistance-related genetic adaptation in M. leprae. FUNDING: EDCTP2 programme supported by the European Union (grant number RIA2017NIM-1847 -PEOPLE). EDCTP, R2Stop: Effect:Hope, The Mission To End Leprosy, the Flemish Fonds Wetenschappelijk Onderzoek.

Protocol, rationale and design of BE-PEOPLE (Bedaquiline enhanced exposure prophylaxis for LEprosy in the Comoros): a cluster randomized trial on effectiveness of rifampicin and bedaquiline as post-exposure prophylaxis of leprosy contacts.

BACKGROUND: Leprosy is an ancient infectious disease with an annual global incidence of around 200,000 over the past decade. Since 2018, the World Health Organization (WHO) recommends single-dose rifampicin as post-exposure prophylaxis (SDR-PEP) for contacts of leprosy patients. The Post ExpOsure Prophylaxis for Leprosy (PEOPLE) trial evaluated PEP with a double dose of rifampicin in Comoros and Madagascar. Preliminary results of this trial show some reduction in leprosy incidence in intervention villages but a stronger regimen may be beneficial. The objective of the current Bedaquiline Enhanced ExpOsure Prophylaxis for LEprosy trial (BE-PEOPLE) is to explore effectiveness of a combination of bedaquiline and rifampicin as PEP. METHODS: BE-PEOPLE is a cluster-randomized trial in which 44 clusters in Comoros will be randomized to two study arms. Door-to-door screening will be conducted annually during four years, leprosy patients identified will be offered standard of care treatment. Based on study arm, contacts aged five years and above and living within a 100-meter radius of an index case will either receive bedaquiline (400-800 mg) and rifampicin (150-600 mg) or only rifampicin (150-600 mg). Contacts aged two to four years will receive rifampicin only. Household contacts randomized to the bedaquiline plus rifampicin arm will receive a second dose four weeks later. Incidence rate ratios of leprosy comparing contacts who received either of the PEP regimens will be the primary outcome. We will monitor resistance to rifampicin and/or bedaquiline through molecular surveillance in all incident tuberculosis and leprosy patients nationwide. At the end of the study, we will assess anti-M. leprae PGL-I IgM seropositivity as a proxy for the population burden of M. leprae infection in 8 villages (17,000 individuals) that were surveyed earlier as part of the PEOPLE trial. DISCUSSION: The COLEP trial on PEP in Bangladesh documented a reduction of 57% in incidence of leprosy among contacts treated with SDR-PEP after two years, which led to the WHO recommendation of SDR-PEP. Preliminary results of the PEOPLE trial show a lesser reduction in incidence. The BE-PEOPLE trial will explore whether reinforcing SDR-PEP with bedaquiline increases effectiveness and more rapidly reduces the incidence of leprosy, compared to SDR-PEP alone. TRIAL REGISTRATION: NCT05597280. Protocol version 5.0 on 28 October 2022.

High rate of adverse drug reactions with a novel tuberculosis re-treatment regimen combining triple doses of both isoniazid and rifampicin.

OBJECTIVES: High-dose rifampicin (R) and isoniazid (H) are known to be safe but were not yet combined in a single regimen. The primary objective of the TRIple-DOse RE-treatment (TRIDORE) study is to determine whether a 6-month firstline regimen with triple dose of both R and H (intervention arm; 6R3H3ZE) is non-inferior in terms of safety compared to a normal-dose regimen (6RHZE) in previously treated patients with R-susceptible (Rs) recurrent tuberculosis (TB). DESIGN/METHODS: TRIDORE is an ongoing pragmatic open-label multi-stage randomized clinical trial. RESULTS: Between March 2021 and February 2022, 127 consenting patients were randomly assigned to either the intervention or control arm: 62 and 65 were treated with 6R3H3ZE and 6RHZE, respectively. Of 127, 111 (87.4%) were male and the median age (interquartile range) was 37 (30-48) years. The median body mass index at enrollment was 18.1 (16.3-19.7) kg/m2. Drugrelated severe adverse events (AEs) (grade III-V) were significantly more frequent when 6R3H3ZE was used (5/62 vs 0/65, P = 0.03, difference weighted for site 8% [95% confidence interval: 1.0,14.3]). The Data and Safety Monitoring Board recommended publishing our interim safety data analysis. CONCLUSION: We show that the combination of triple-dose R with triple-dose H in a re-treatment regimen for patients with Rs-TB causes excess drug-related AEs.

Back-to-Africa introductions of Mycobacterium tuberculosis as the main cause of tuberculosis in Dar es Salaam, Tanzania.

In settings with high tuberculosis (TB) endemicity, distinct genotypes of the Mycobacterium tuberculosis complex (MTBC) often differ in prevalence. However, the factors leading to these differences remain poorly understood. Here we studied the MTBC population in Dar es Salaam, Tanzania over a six-year period, using 1,082 unique patient-derived MTBC whole-genome sequences (WGS) and associated clinical data. We show that the TB epidemic in Dar es Salaam is dominated by multiple MTBC genotypes introduced to Tanzania from different parts of the world during the last 300 years. The most common MTBC genotypes deriving from these introductions exhibited differences in transmission rates and in the duration of the infectious period, but little differences in overall fitness, as measured by the effective reproductive number. Moreover, measures of disease severity and bacterial load indicated no differences in virulence between these genotypes during active TB. Instead, the combination of an early introduction and a high transmission rate accounted for the high prevalence of L3.1.1, the most dominant MTBC genotype in this setting. Yet, a longer co-existence with the host population did not always result in a higher transmission rate, suggesting that distinct life-history traits have evolved in the different MTBC genotypes. Taken together, our results point to bacterial factors as important determinants of the TB epidemic in Dar es Salaam.

Definitive outcomes in patients with rifampicin-resistant tuberculosis treated in Niger from 2012 to 2019: A retrospective cohort study.

BACKGROUND: Outcomes of retreatment for rifampicin-resistant tuberculosis (RR-TB) are rarely reported. We report 'definitive outcomes' after a cascade approach to RR-TB treatment. After a bacteriologically adverse outcome for the 9-months fluoroquinolone-based Short Treatment Regimen (STR), patients were retreated with a bedaquiline-based regimen (BDQ-regimen). METHODS: A Retrospective cohort study of RR-TB patients treated with the STR during 2012-2019 and retreated with a BDQ-regimen in case of failure or relapse was conducted. Definitive relapse-free cure took into account BDQ-regimen outcomes. RESULTS: Of 367 patients treated with the STR, 20 (5.4%) experienced failure or relapse. Out of these 20 patients, 14 started a BDQ-regimen, of whom none experienced failure or relapse. Definitive end of treatment outcomes of STR after revising with third-line BDQ-regimen outcomes, 84.7% (311/367) were cured relapse-free, 10.6% (39/367) died during treatment and 3.0% (11/367) were lost to follow-up during treatment with either the STR or BDQ-regimen. Six patients (1.6%; 6/367) with STR failure/relapse died before starting a BDQ-regimen. No patient had definitive treatment failure or relapse and remained without treatment. CONCLUSIONS: If fluoroquinolone resistance is excluded or rare, it is beneficial to use fluoroquinolone as the core drug for a first RR-TB treatment regimen and to safeguard bedaquiline for those in need of retreatment.

Strain diversity and gene mutations associated with presumptive multidrug-resistant Mycobacterium tuberculosis complex isolates in Northwest Ethiopia.

OBJECTIVES: In this study, we assessed the genetic diversity and gene mutations that confer resistance to rifampicin (RIF), isoniazid (INH), fluoroquinolone (FQ), and second-line injectable (SLI) drugs in RIF-resistant (RR)/multidrug-resistant tuberculosis (MDR-TB) isolates in Northwest Ethiopia. METHODS: Spoligotyping was used to assign isolates to TB lineages (Ls), and Hain line probe assays were used to detect resistance to RIF, INH, and FQs, and SLIs. RESULTS: Among 130 analyzed strains, 68.5% were RR, and four major Mycobacterium tuberculosis complex lineages (L1, L3, L4, and L7) were identified with a predominance of the Euro-American L4 (72, 54.7%), while L7 genotypes were less common (3, 2.3%). Overall, the L4-T3-ETH (41, 32.0%), L3-CAS1-Delhi (29, 22.7%), and L3-CAS1-Killi (19, 14.8%) families were most common. Line probe analysis showed that among rpoB mutants, 65.2% were S450L, while 87.8% of katG mutants were S315T. Only three isolates showed mutation (c-15t) at the inhA gene, and no double mutation with katG and inhA genes was found. Six strains, two each of L1, L3, and L4, were resistant to FQs, having gyrA mutations (D94G, S91P), of which three isolates had additional resistance to SLI (rrs A1401G or C1402T mutations) including one isolate with low-level kanamycin (KAN) resistance. CONCLUSIONS: This study showed a predominance of L4-T3-ETH, L3-CAS1-Delhi, and L3-CAS1-Killi families, with a high rate of rpoB_S450L and katG_S315T mutations and a low proportion of gyrA and rrs mutations. L7 was less frequently observed in this study. Further investigations are, therefore, needed to understand L7 and other lineages with undefined mutations.