Does One Size Fit All? Drug Resistance and Standard Treatments: Results of Six Tuberculosis Programmes in Former Soviet Countries.

SETTING: After the collapse of the Soviet Union, countries in the region faced a dramatic increase in tuberculosis cases and the emergence of drug resistance. OBJECTIVE: To discuss the relevance of the DOTS strategy in settings with a high prevalence of drug resistance. DESIGN: Retrospective analysis of one-year treatment outcomes of short-course chemotherapy (SCC) and results of drug susceptibility testing (DST) surveys of six programmes located in the former Soviet Union: Kemerovo prison, Russia; Abkhasia, Georgia; Nagorno-Karabagh, Azerbaijan; Karakalpakstan, Uzbekistan; Dashoguz Velayat, Turkmenistan; and South Kazakhstan Oblast, Kazakhstan. Results are reported for new and previously treated smear-positive patients. RESULTS: Treatment outcomes of 3090 patients and DST results of 1383 patients were collected. Treatment success rates ranged between 87% and 61%, in Nagorno-Karabagh and Kemerovo, respectively, and failure rates between 7% and 23%. Any drug resistance ranged between 66% and 31% in the same programmes. MDR rates ranged between 28% in Karakalpakstan and Kemerovo prison and 4% in Nagorno-Karabagh. CONCLUSION: These results show the limits of SCC in settings with a high prevalence of drug resistance. They demonstrate that adapting treatment according to resistance patterns, access to reliable culture, DST and good quality second-line drugs are necessary

Multidrug-resistant tuberculosis in prison inmates, Azerbaijan.

In a tuberculosis (TB) program in the Central Penitentiary Hospital of Azerbaijan, we analyzed 65 isolates of Mycobacterium tuberculosis by IS6110-based restriction fragment-length polymorphism (RFLP) and spoligotyping. From 11 clusters associated with 33 patients, 31 isolates had an IS6110-based banding pattern characteristic of the Beijing genotype of M. tuberculosis. In addition, 15 M. tuberculosis isolates with similar RFLP patterns constituted a single group by spoligotyping, matching the Beijing genotype. Multidrug resistance, always involving isoniazid and rifampin, was seen in 34 (52.3%) of 65 isolates, with 28 belonging to the Beijing genotype

Potential application of digitally linked tuberculosis diagnostics for real-time surveillance of drug-resistant tuberculosis transmission: Validation and analysis of test results

YesBackground: Tuberculosis (TB) is the highest-mortality infectious disease in the world and the main cause of death related to antimicrobial resistance, yet its surveillance is still paper-based. Rifampicin-resistant TB (RR-TB) is an urgent public health crisis. The World Health Organization has, since 2010, endorsed a series of rapid diagnostic tests (RDTs) that enable rapid detection of drug-resistant strains and produce large volumes of data. In parallel, most high-burden countries have adopted connectivity solutions that allow linking of diagnostics, real-time capture, and shared repository of these test results. However, these connected diagnostics and readily available test results are not used to their full capacity, as we have yet to capitalize on fully understanding the relationship between test results and specific rpoB mutations to elucidate its potential application to real-time surveillance. Objective: We aimed to validate and analyze RDT data in detail, and propose the potential use of connected diagnostics and associated test results for real-time evaluation of RR-TB transmission. Methods: We selected 107 RR-TB strains harboring 34 unique rpoB mutations, including 30 within the rifampicin resistance–determining region (RRDR), from the Belgian Coordinated Collections of Microorganisms, Antwerp, Belgium. We subjected these strains to Xpert MTB/RIF, GenoType MTBDRplus v2.0, and Genoscholar NTM + MDRTB II, the results of which were validated against the strains’ available rpoB gene sequences. We determined the reproducibility of the results, analyzed and visualized the probe reactions, and proposed these for potential use in evaluating transmission. Results: The RDT probe reactions detected most RRDR mutations tested, although we found a few critical discrepancies between observed results and manufacturers’ claims. Based on published frequencies of probe reactions and RRDR mutations, we found specific probe reactions with high potential use in transmission studies: Xpert MTB/RIF probes A, Bdelayed, C, and Edelayed; Genotype MTBDRplus v2.0 WT2, WT5, and WT6; and Genoscholar NTM + MDRTB II S1 and S3. Inspection of probe reactions of disputed mutations may potentially resolve discordance between genotypic and phenotypic test results. Conclusions: We propose a novel approach for potential real-time detection of RR-TB transmission through fully using digitally linked TB diagnostics and shared repository of test results. To our knowledge, this is the first pragmatic and scalable work in response to the consensus of world-renowned TB experts in 2016 on the potential of diagnostic connectivity to accelerate efforts to eliminate TB. This is evidenced by the ability of our proposed approach to facilitate comparison of probe reactions between different RDTs used in the same setting. Integrating this proposed approach as a plug-in module to a connectivity platform will increase usefulness of connected TB diagnostics for RR-TB outbreak detection through real-time investigation of suspected RR-TB transmission cases based on epidemiologic linking.KCN was supported by Erasmus Mundus Joint Doctorate Fellowship grant 2016-1346, and BCdJ, LR, and CJM were supported by European Research Council-INTERRUPTB starting grant 311725

Comparative genomics shows differences in the electron transport and carbon metabolic pathways of Mycobacterium africanum relative to Mycobacterium tuberculosis and suggests an adaptation to low oxygen tension

YesThe geographically restricted Mycobacterium africanum lineages (MAF) are primarily found in West Africa, where they account for a significant proportion of tuberculosis. Despite this phenomenon, little is known about the co-evolution of these ancient lineages with West Africans. MAF and M. tuberculosis sensu stricto lineages (MTB) differ in their clinical, in vitro and in vivo characteristics for reasons not fully understood. Therefore, we compared genomes of 289 MAF and 205 MTB clinical isolates from the 6 main human-adapted M. tuberculosis complex lineages, for mutations in their Electron Transport Chain and Central Carbon Metabolic pathway in order to explain these metabolic differences. Furthermore, we determined, in silico, whether each mutation could affect the function of genes encoding enzymes in these pathways. We found more mutations with the potential to affect enzymes in these pathways in MAF lineages compared to MTB lineages. We also found that similar mutations occurred in these pathways between MAF and some MTB lineages. Generally, our findings show further differences between MAF and MTB lineages that may have contributed to the MAF clinical and growth phenotype and indicate potential adaptation of MAF lineages to a distinct ecological niche, which we suggest includes areas characterized by low oxygen tension.European Research CouncilINTERRUPTB starting grant nr. 311725 (to BdJ, FG, CM, LR, BO, MA) and The UK Medical Research Council and the European & Developing Countries Clinical Trials Partnership (EDCTP) Grant No. CB. 2007. 41700.007.Research Development Fund Publication Prize Award winner, January 2020

Primary tooth abscess caused by Mycobacterium bovis in an immunocompetent child

Bovine tuberculosis is a zoonotic disease, and although its incidence has dramatically decreased in developed countries where effective control measures are applied, it still remains a potential health hazard in the developing world. Tuberculosis of the oral cavity is extremely rare and is usually secondary to pulmonary involvement. We present the unusual case of an immunocompetent 6-year-old child residing in an urban area with primary oral tuberculosis due to Mycobacterium bovis, which was confirmed by the application of a molecular genetic approach. M. bovis belongs to Mycobacterium tuberculosis complex which comprises species with close genetic relationship, and for this reason, the use of new molecular techniques is a useful tool for the differentiation at species level of the closely related members of this complex

Frameshift mutations in the mmpR5 gene can have a bedaquiline-susceptible phenotype by retaining a protein structure and function similar to wild-type Mycobacterium tuberculosis

Bedaquiline (BDQ) is crucial for the treatment of rifampicin-resistant tuberculosis, yet resistance threatens its effectiveness, mainly linked to mutations in the mmpR5 (Rv0678) gene. While frameshift mutations are thought to produce non-functional proteins, we hypothesize that they can result in conserved proteins through late-stop codons or alternative reading frames and remain BDQ susceptible. We extracted 512 isolates harboring frameshift mutations in mmpR5 from the World Health Organization (WHO) catalog and 68 isolates with minimum inhibitory concentration (MIC) in mycobacterial growth indicator tube (MGIT) through a literature review. Using BioPython and AlphaFold2 we computed open (ORF) and alternative reading frames (ARFs) sequences and protein structures and assessed similarity to the wild type using an alignment and template modeling (TM)-score. Among the WHO 512 isolates, 24.8% were BDQ-sensitive. Out of 184 unique frameshift mutations with available nucleotide information, a late-stop codon in the ORF occurred for 32% of the mutations. Also, 40.7% resulted in a conserved sequence, through the ORF or one of the forward ARFs. In 68 isolates with available MGIT MIC data, the presence of late-stop codons in the ORF (OR 4.71, 95% CI 1.36–19.3) or a conserved reading frame (OR 10.4, 95% CI 2.07–102.9) were associated with BDQ sensitivity. Protein structures from the conserved sequences showed high similarity (TM > 0.8). We show that frameshift mutations may retain BDQ susceptibility through late-stop codons in the ORF or conserved ARFs. These findings could improve the prediction of the BDQ phenotype from genomic data and have important implications for treatment decisions. Research Foundation—Flanders, Academy of Medical Sciences, the Wellcome Trust, the Government Department of Business, Energy and Industrial Strategy, the British Heart Foundation and Diabetes UK, and the Global Challenges Research Fund

Mycobacterium tuberculosis whole genome sequencing and protein structure modelling provides insights into anti-tuberculosis drug resistance.

BACKGROUND: Combating the spread of drug resistant tuberculosis is a global health priority. Whole genome association studies are being applied to identify genetic determinants of resistance to anti-tuberculosis drugs. Protein structure and interaction modelling are used to understand the functional effects of putative mutations and provide insight into the molecular mechanisms leading to resistance. METHODS: To investigate the potential utility of these approaches, we analysed the genomes of 144 Mycobacterium tuberculosis clinical isolates from The Special Programme for Research and Training in Tropical Diseases (TDR) collection sourced from 20 countries in four continents. A genome-wide approach was applied to 127 isolates to identify polymorphisms associated with minimum inhibitory concentrations for first-line anti-tuberculosis drugs. In addition, the effect of identified candidate mutations on protein stability and interactions was assessed quantitatively with well-established computational methods. RESULTS: The analysis revealed that mutations in the genes rpoB (rifampicin), katG (isoniazid), inhA-promoter (isoniazid), rpsL (streptomycin) and embB (ethambutol) were responsible for the majority of resistance observed. A subset of the mutations identified in rpoB and katG were predicted to affect protein stability. Further, a strong direct correlation was observed between the minimum inhibitory concentration values and the distance of the mutated residues in the three-dimensional structures of rpoB and katG to their respective drugs binding sites. CONCLUSIONS: Using the TDR resource, we demonstrate the usefulness of whole genome association and convergent evolution approaches to detect known and potentially novel mutations associated with drug resistance. Further, protein structural modelling could provide a means of predicting the impact of polymorphisms on drug efficacy in the absence of phenotypic data. These approaches could ultimately lead to novel resistance mutations to improve the design of tuberculosis control measures, such as diagnostics, and inform patient management

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

The Repetitive Element RLEP Is a Highly Specific Target for Detection of Mycobacterium leprae

YesDamien Foundation, Belgium, and R2Stop, Canada. B.C.D.J. and C.J.M. were supported by the European Research Council-INTERRUPTB starting grant 311725

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

YesSETTING: 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