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.

Multidrug-resistant tuberculosis control in Rwanda overcomes a successful clone that causes most disease over a quarter century.

SUMMARY BACKGROUND: Multidrug-resistant (MDR) tuberculosis (TB) poses an important challenge in TB management and control. Rifampicin resistance (RR) is a solid surrogate marker of MDR-TB. We investigated the RR-TB clustering rates, bacterial population dynamics to infer transmission dynamics, and the impact of changes to patient management on these dynamics over 27 years in Rwanda. METHODS: We analysed whole genome sequences of a longitudinal collection of nationwide RR-TB isolates. The collection covered three important periods: before programmatic management of MDR-TB (PMDT; 1991-2005), the early PMDT phase (2006-2013), in which rifampicin drug-susceptibility testing (DST) was offered to retreatment patients only, and the consolidated phase (2014-2018), in which all bacteriologically confirmed TB patients had rifampicin DST done mostly via Xpert MTB/RIF assay. We constructed clusters based on a 5 SNP cut-off and resistance conferring SNPs. We used Bayesian modelling for dating and population size estimations, TransPhylo to estimate the number of secondary cases infected by each patient, and multivariable logistic regression to assess predictors of being infected by the dominant clone. RESULTS: Of 308 baseline RR-TB isolates considered for transmission analysis, the clustering analysis grouped 259 (84.1%) isolates into 13 clusters. Within these clusters, a single dominant clone was discovered containing 213 isolates (82.2% of clustered and 69.1% of all RR-TB), which we named the "Rwanda Rifampicin-Resistant clone" (R3clone). R3clone isolates belonged to Ugandan sub-lineage 4.6.1.2 and its rifampicin and isoniazid resistance were conferred by the Ser450Leu mutation in rpoB and Ser315Thr in katG genes, respectively. All R3clone isolates had Pro481Thr, a putative compensatory mutation in the rpoC gene that likely restored its fitness. The R3clone was estimated to first arise in 1987 and its population size increased exponentially through the 1990s', reaching maximum size (∼84%) in early 2000 s', with a declining trend since 2014. Indeed, the highest proportion of R3clone (129/157; 82·2%, 95%CI: 75·3-87·8%) occurred between 2000 and 13, declining to 64·4% (95%CI: 55·1-73·0%) from 2014 onward. We showed that patients with R3clone detected after an unsuccessful category 2 treatment were more likely to generate secondary cases than patients with R3clone detected after an unsuccessful category 1 treatment regimen. CONCLUSIONS: RR-TB in Rwanda is largely transmitted. Xpert MTB/RIF assay as first diagnostic test avoids unnecessary rounds of rifampicin-based TB treatment, thus preventing ongoing transmission of the dominant R3clone. As PMDT was intensified and all TB patients accessed rifampicin-resistance testing, the nationwide R3clone burden declined. To our knowledge, our findings provide the first evidence supporting the impact of universal DST on the transmission of RR-TB.

Initial resistance to companion drugs should not be considered an exclusion criterion for the shorter multidrug-resistant tuberculosis treatment regimen.

OBJECTIVES: We investigated whether companion drug resistance was associated with adverse outcomes of the shorter multidrug-resistant tuberculosis (MDR-TB) treatment regimen in Bangladesh after adjustment for fluoroquinolone resistance. METHODS: MDR-TB/rifampicin-resistant tuberculosis patients registered for treatment with a standardized gatifloxacin-based shorter MDR-TB treatment regimen were selected for the study. Drug resistance was determined by the proportion method, gatifloxacin and isoniazid minimum inhibitory concentration testing for selected isolates, and whole-genome sequencing. RESULTS: Low-level fluoroquinolone resistance and high-level fluoroquinolone resistance were the most important predictors of adverse outcomes, with pyrazinamide resistance having a significant yet lower impact. In patients with fluoroquinolone-/second-line-injectable-susceptible tuberculosis, non-eligibility for the shorter MDR-TB treatment regimen (initial resistance to pyrazinamide, ethionamide, or ethambutol) was not associated with adverse outcome (adjusted odds ratio 1.01; 95% confidence interval 0.4-2.8). Kanamycin resistance was uncommon (1.3%). Increasing levels of resistance to isoniazid predicted treatment failure, also in a subgroup of patients with high-level fluoroquinolone-resistant tuberculosis. CONCLUSIONS: Our results suggest that resistance to companion drugs in the shorter MDR-TB treatment regimen, except kanamycin resistance, is of no clinical importance as long as fluoroquinolone susceptibility is preserved. Hence, contrary to current WHO guidelines, exclusions to the standard regimen are justified only in the case of fluoroquinolone resistance. and possibly kanamycin resistance.

Multidrug-resistant patients receiving treatment in Niger who are infected with M. tuberculosis Cameroon family convert faster in smear and culture than those with M. tuberculosis Ghana family.

In this study, we analyzed the M. tuberculosis complex (MTBc) population structure among multidrug-resistant TB (MDR-TB) patients in Niger and tested whether the Cameroon family displayed a slower response to MDR-TB treatment. We genotyped baseline clinical isolates that had been collected from pulmonary MDR-TB patients recruited consecutively between 2008 and 2016 in Niger. Spoligotyping was used to analyze the genetic diversity of mycobacterial lineages, and Kaplan Meier's analysis to compare treatment outcomes. A total of 222 MTBc isolates were genotyped; 204 (91,9%) were identified as the Euro-American L4 lineage, with the Ghana family (106, 47,4%) and the Cameroon family (63, 28,4%) being predominant. Patients infected by Cameroon family isolates 61(96,8%) showed faster conversion (log-rank p < 0.01) than those infected with Ghana family isolates (91,5%), and were more likely to experience favorable outcome (adjusted odds ratio [aOR] 4.4; 95%CI 1.1-17.9]; p = 0.015). We found no association between MTBc families and second-line drug resistance profiles (p > 0.05). Our findings show that MDR-TB in Niger is caused by major spoligotypes of the Euro-American L4; with more rapid smear and culture conversion in patients infected with the Cameroon family. These first insights may alert clinicians that slow conversion may be associated with the type of infecting strain.

Management of multidrug-resistant tuberculosis with shorter treatment regimen in Niger: Nationwide programmatic achievements.

BACKGROUND: In Niger, the Shorter Treatment Regimen (STR) has been implemented nationwide for rifampicin resistant tuberculosis (RR-TB), since 2008. No previous publication has shown the results from countrywide programmatic implementation using few exclusion criteria, nor exhaustively assessed the effect of initial resistance to companion drugs on outcomes. METHODS: The National Tuberculosis Programme and the Damien Foundation conducted a retrospective observational study to evaluate the management of RR-TB from 2008 to 2016. Baseline resistance to drugs was assessed phenotypically, complemented by screening the inhA, katG and pncA genes. Cured patients were followed-up for a period of one year after cure. FINDINGS: Among 1044 patients tested for rifampicin resistance, mainly previously treated patients, 332 were diagnosed with pulmonary RR/TB, 288 were enrolled on treatment and 255 started on STR. Six patients received a modified STR. Among 249 patients on standardised STR, 207 (83·1%) were cured relapse-free, eight (3·2%) had failure, 23 (9·2%) died, seven (2·8%) were lost to follow-up and four (1·6%) relapsed. The risk of unfavourable outcome was higher in patients with initial resistance to fluoroquinolones (aOR 20·4, 95%CI:5·6-74·6) and very severely underweight (aOR 3·9, 95%CI:1·5-10·1). Successful outcome was not affected by initial resistance to companion drugs. Serious ototoxicity was reported in eight patients (3·2%). INTERPRETATION: A comprehensive nationwide approach to multidrug-resistant tuberculosis management using the STR was feasible and successful. Outcomes were not affected by initial resistance to companion drugs. Our study confirms the effectiveness and safety of the STR. FUNDING: Damien Foundation and Institute of Tropical Medicine-Antwerp.

Bridging the TB data gap: in silico extraction of rifampicin-resistant tuberculosis diagnostic test results from whole genome sequence data.

BACKGROUND: Mycobacterium tuberculosis rapid diagnostic tests (RDTs) are widely employed in routine laboratories and national surveys for detection of rifampicin-resistant (RR)-TB. However, as next-generation sequencing technologies have become more commonplace in research and surveillance programs, RDTs are being increasingly complemented by whole genome sequencing (WGS). While comparison between RDTs is difficult, all RDT results can be derived from WGS data. This can facilitate continuous analysis of RR-TB burden regardless of the data generation technology employed. By converting WGS to RDT results, we enable comparison of data with different formats and sources particularly for low- and middle-income high TB-burden countries that employ different diagnostic algorithms for drug resistance surveys. This allows national TB control programs (NTPs) and epidemiologists to utilize all available data in the setting for improved RR-TB surveillance. METHODS: We developed the Python-based MycTB Genome to Test (MTBGT) tool that transforms WGS-derived data into laboratory-validated results of the primary RDTs-Xpert MTB/RIF, XpertMTB/RIF Ultra, GenoType MDRTBplus v2.0, and GenoscholarNTM+MDRTB II. The tool was validated through RDT results of RR-TB strains with diverse resistance patterns and geographic origins and applied on routine-derived WGS data. RESULTS: The MTBGT tool correctly transformed the single nucleotide polymorphism (SNP) data into the RDT results and generated tabulated frequencies of the RDT probes as well as rifampicin-susceptible cases. The tool supplemented the RDT probe reactions output with the RR-conferring mutation based on identified SNPs. The MTBGT tool facilitated continuous analysis of RR-TB and Xpert probe reactions from different platforms and collection periods in Rwanda. CONCLUSION: Overall, the MTBGT tool allows low- and middle-income countries to make sense of the increasingly generated WGS in light of the readily available RDT results, and assess whether currently implemented RDTs adequately detect RR-TB in their setting. With its feature to transform WGS to RDT results and facilitate continuous RR-TB data analysis, the MTBGT tool may bridge the gap between and among data from periodic surveys, continuous surveillance, research, and routine tests, and may be integrated within the national information system for use by the NTP and epidemiologists to improve setting-specific RR-TB control. The MTBGT source code and accompanying documentation are available at https://github.com/KamelaNg/MTBGT.

The relationship between transmission time and clustering methods in Mycobacterium tuberculosis epidemiology.

BACKGROUND: Tracking recent transmission is a vital part of controlling widespread pathogens such as Mycobacterium tuberculosis. Multiple methods with specific performance characteristics exist for detecting recent transmission chains, usually by clustering strains based on genotype similarities. With such a large variety of methods available, informed selection of an appropriate approach for determining transmissions within a given setting/time period is difficult. METHODS: This study combines whole genome sequence (WGS) data derived from 324 isolates collected 2005-2010 in Kinshasa, Democratic Republic of Congo (DRC), a high endemic setting, with phylodynamics to unveil the timing of transmission events posited by a variety of standard genotyping methods. Clustering data based on Spoligotyping, 24-loci MIRU-VNTR typing, WGS based SNP (Single Nucleotide Polymorphism) and core genome multi locus sequence typing (cgMLST) typing were evaluated. FINDINGS: Our results suggest that clusters based on Spoligotyping could encompass transmission events that occurred almost 200 years prior to sampling while 24-loci-MIRU-VNTR often represented three decades of transmission. Instead, WGS based genotyping applying low SNP or cgMLST allele thresholds allows for determination of recent transmission events, e.g. in timespans of up to 10 years for a 5 SNP/allele cut-off. INTERPRETATION: With the rapid uptake of WGS methods in surveillance and outbreak tracking, the findings obtained in this study can guide the selection of appropriate clustering methods for uncovering relevant transmission chains within a given time-period. For high resolution cluster analyses, WGS-SNP and cgMLST based analyses have similar clustering/timing characteristics even for data obtained from a high incidence setting.

Isoniazid resistance levels of Mycobacterium tuberculosis can largely be predicted by high-confidence resistance-conferring mutations.

The majority of Mycobacterium tuberculosis isolates resistant to isoniazid harbour a mutation in katG. Since these mutations cause a wide range of minimum inhibitory concentrations (MICs), largely below the serum level reached with higher dosing (15 mg/L upon 15-20 mg/kg), the drug might still remain partly active in presence of a katG mutation. We therefore investigated which genetic mutations predict the level of phenotypic isoniazid resistance in clinical M. tuberculosis isolates. To this end, the association between known and unknown isoniazid resistance-conferring mutations in whole genome sequences, and the isoniazid MICs of 176 isolates was examined. We found mostly moderate-level resistance characterized by a mode of 6.4 mg/L for the very common katG Ser315Thr mutation, and always very high MICs (≥19.2 mg/L) for the combination of katG Ser315Thr and inhA c-15t. Contrary to common belief, isolates harbouring inhA c-15t alone, partly also showed moderate-level resistance, particularly when combined with inhA Ser94Ala. No overt association between low-confidence or unknown mutations, except in katG, and isoniazid resistance (level) was found. Except for the rare katG deletion, line probe assay is thus not sufficiently accurate to predict the level of isoniazid resistance for a single mutation in katG or inhA.