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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., Competing Interests: C.U.K. is a consultant for Becton Dickinson, the Foundation for Innovative New Diagnostics, the TB Alliance, and the WHO Global TB Programme. C.U.K.'s consulting for Becton Dickinson involves collaboration with Janssen and Thermo Fisher Scientific. C.U.K. is collaborating with PZA Innovation and is an unpaid advisor to Cepheid and GenoScreen (GenoScreen covered related travel and accommodation expenses only). C.U.K. worked as a consultant for the Stop TB Partnership and the WHO Regional Office for Europe. C.U.K. gave a paid educational talk for Oxford Immunotec. C.U.K. was an unpaid advisor to BioVersys.

Globally, tuberculosis (TB) remains the deadliest bacterial infectious disease, and spreading antibiotic resistances is the biggest challenge for combatting the disease. Rapid and comprehensive diagnostics including drug susceptibility testing (DST) would assure early treatment, reduction of morbidity and the interruption of transmission chains. To date, rapid genetic resistance testing addresses only one to four drug groups while complete DST is done phenotypically and takes several weeks. To overcome these limitations, we developed a two-stage workflow for rapid TB diagnostics including DST from a single sputum sample that can be completed within three days. The first stage is qPCR detection of M. tuberculosis complex (MTBC) including antibiotic resistance testing against the first-line antibiotics, isoniazid (Inh) and rifampicin (Rif). The test is automated by centrifugal microfluidics and designed for point of care (PoC). Furthermore, enriched MTBC DNA is provided in a detachable sample tube to enable the second stage: if the PCR detects MTBC and resistance to either Inh or Rif, the MTBC DNA is shipped to specialized facilities and analyzed by targeted next generation sequencing (tNGS) to assess the complete resistance profile. Proof-of-concept testing of the PoC test revealed an analytical sensitivity of 44.2 CFU ml -1 , a diagnostic sensitivity of 96%, and a diagnostic specificity of 100% for MTBC detection. Coupled tNGS successfully provided resistance profiles, demonstrated for samples from 17 patients. To the best of our knowledge, the presented combination of PoC qPCR with tNGS allows for the fastest comprehensive TB diagnostics comprising decentralized pathogen detection with subsequent resistance profiling in a facility specialized in tNGS.

Drug-resistant tuberculosis is a substantial health-care concern worldwide. Despite culture-based methods being considered the gold standard for drug susceptibility testing, molecular methods provide rapid information about the Mycobacterium tuberculosis mutations associated with resistance to anti-tuberculosis drugs. This consensus document was developed on the basis of a comprehensive literature search, by the TBnet and RESIST-TB networks, about reporting standards for the clinical use of molecular drug susceptibility testing. Review and the search for evidence included hand-searching journals and searching electronic databases. The panel identified studies that linked mutations in genomic regions of M tuberculosis with treatment outcome data. Implementation of molecular testing for the prediction of drug resistance in M tuberculosis is key. Detection of mutations in clinical isolates has implications for the clinical management of patients with multidrug-resistant or rifampicin-resistant tuberculosis, especially in situations when phenotypic drug susceptibility testing is not available. A multidisciplinary team including clinicians, microbiologists, and laboratory scientists reached a consensus on key questions relevant to molecular prediction of drug susceptibility or resistance to M tuberculosis, and their implications for clinical practice. This consensus document should help clinicians in the management of patients with tuberculosis, providing guidance for the design of treatment regimens and optimising outcomes., Competing Interests: Declaration of interests JD reports a technology licence to GenID (Germany), and honoraria for lectures from Oxford Immunotec (UK). EC reports support for attendance, accommodation, and travel for ECCMID 2022, Lisboa from European Society of Clinical Microbiology and Infectious Diseases (ESCMID), for annual ERL-TB net meetings from ERL-TB net (Network of National Reference Centers for Tuberculosis in Europe), and for the annual congress 2022, Bologna, from the European Society for Mycobacteriology; is a member of the Executive committee of ESCMID; and is chair of the subcommittee for antimycobacterial agents of EUCAST (European Committee on Antimicrobial Susceptibility Testing). MRF reports grants or contracts from NIH/NIAID (5R01AI155765 and 5R21AI154089) and consulting fees (paid to them) from FIND. LG is a member (unpaid) of the data safety monitoring board for the XACT-19 clinical trial in University of Cape Town, Cape Town, South Africa, and is co-principal investigator of two phase 3 clinical trials on shorter treatment for MDR-TB (endTB and endTB-Q), funded by Unitaid. BL reports grants or contracts from European Union, German Ministry for Education and Research (BMBF), Kultusministerkonferenz, German Centre for Infection Research, and Helmholtz Association, and unpaid leadership or fiduciary roles for DZIF IAB, DZIF Steering Committee transplant Cohort, and TBnet chair Epidemiology. SN reports support for this manuscript (eg, funding, provision of study materials, medical writing, and article processing charges) from BMBF (German Center for Infection Research), DFG (Excellenz Cluster Precision Medicine in Chronic Inflammation EXC 2167), and Leibniz Science Campus Evolutionary Medicine of the LUNG (EvoLUNG), and consulting fees from Illumina advisory board in 2022. NV reports grants or contracts for a study on bedaquiline from Janssen. CRH reports grants or contracts from NIH/NIAID (R01AI134430, DAA3-19-65672, R01AI147316 U01AI152980, and R01AI146555), and Centers for Disease Control and Prevention (NU38PS004651); consulting fees from Otsuka Pharmaceuticals; participation on a data safety monitoring board for SODUCU (PanACEA Sutezolid Dose-finding and Combination Evaluation), BEAT-Tuberculosis (Building Evidence for Advancing New treatment for tuberculosis), DECODE (PanACEA Delpazolid Dose-finding and Combination Development), and Médecins Sans Frontières; and a leadership or fiduciary role from the International Union Against Tuberculosis and Lung Diseases. CLa reports support for the present manuscript (eg, funding, provision of study materials, medical writing, and article processing charges) from DZIF (German Center of Infection Research); consulting fees from a consultation service to INSMED, a company that produced liposomal amikacin as an inhalative suspension for the treatment of non-tuberculous mycobacteria pulmonary disease (outside of the scope of this work); speakers' honoraria from Insmed, Gilead, and Janssen (all outside of the scope of this work); is a member of the data safety board of trials from Médecins Sans Frontières (outside of the scope of this work); is supported by the German Center for Infection Research (DZIF); and acknowledges funding from the European Commission (anTBiotic EU-H2020 733079, ClicTB EDCTP2 RIA2017T-2030, stool4TB EDCTP2 RIAD2018-2511, and UNITE4TB EU-IMI 101007873). All other authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: Whole-genome sequencing (WGS) of Mycobacterium tuberculosis complex has become an important tool in diagnosis and management of drug-resistant tuberculosis. However, data correlating resistance genotype with quantitative phenotypic antimicrobial susceptibility testing (AST) are scarce., Methods: In a prospective multicentre observational study, 900 clinical M tuberculosis complex isolates were collected from adults with drug-resistant tuberculosis in five high-endemic tuberculosis settings around the world (Georgia, Moldova, Peru, South Africa, and Viet Nam) between Dec 5, 2014, and Dec 12, 2017. Minimum inhibitory concentrations (MICs) and resulting binary phenotypic AST results for up to nine antituberculosis drugs were determined and correlated with resistance-conferring mutations identified by WGS., Findings: Considering WHO-endorsed critical concentrations as reference, WGS had high accuracy for prediction of resistance to isoniazid (sensitivity 98·8% [95% CI 98·5-99·0]; specificity 96·6% [95% CI 95·2-97·9]), levofloxacin (sensitivity 94·8% [93·3-97·6]; specificity 97·1% [96·7-97·6]), kanamycin (sensitivity 96·1% [95·4-96·8]; specificity 95·0% [94·4-95·7]), amikacin (sensitivity 97·2% [96·4-98·1]; specificity 98·6% [98·3-98·9]), and capreomycin (sensitivity 93·1% [90·0-96·3]; specificity 98·3% [98·0-98·7]). For rifampicin, pyrazinamide, and ethambutol, the specificity of resistance prediction was suboptimal (64·0% [61·0-67·1], 83·8% [81·0-86·5], and 40·1% [37·4-42·9], respectively). Specificity for rifampicin increased to 83·9% when borderline mutations with MICs overlapping with the critical concentration were excluded. Consequently, we highlighted mutations in M tuberculosis complex isolates that are often falsely identified as susceptible by phenotypic AST, and we identified potential novel resistance-conferring mutations., Interpretation: The combined analysis of mutations and quantitative phenotypes shows the potential of WGS to produce a refined interpretation of resistance, which is needed for individualised therapy, and eventually could allow differential drug dosing. However, variability of MIC data for some M tuberculosis complex isolates carrying identical mutations also reveals limitations of our understanding of the genotype and phenotype relationships (eg, including epistasis and strain genetic background)., Funding: Bill & Melinda Gates Foundation, German Centre for Infection Research, German Research Foundation, Excellence Cluster Precision Medicine of Inflammation (EXC 2167), and Leibniz ScienceCampus EvoLUNG., Competing Interests: Declaration of interests MM and SN report grants from the German Center for Infection Research, Excellenz Cluster Precision Medicine in Chronic Inflammation, and Leibniz Science Campus Evolutionary Medicine of the LUNG (EvoLUNG). TR reports personal fees from FIND, grants from the US National Institute of Allergy and Infectious Diseases, and is a board member for Verus Diagnostics; and has a provisional patent (#63/048.989) and a pending patent (#14840432.0) for tuberculosis diagnostics. All other authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Objectives: To develop a robust phenotypic antimicrobial susceptibility testing (AST) method with a correctly set breakpoint for pretomanid (Pa), the most recently approved anti-tuberculosis drug., Methods: The Becton Dickinson Mycobacterial Growth Indicator Tube™ (MGIT) system was used at six laboratories to determine the MICs of a phylogenetically diverse collection of 356 Mycobacterium tuberculosis complex (MTBC) strains to establish the epidemiological cut-off value for pretomanid. MICs were correlated with WGS data to study the genetic basis of differences in the susceptibility to pretomanid., Results: We observed ancient differences in the susceptibility to pretomanid among various members of MTBC. Most notably, lineage 1 of M. tuberculosis, which is estimated to account for 28% of tuberculosis cases globally, was less susceptible than lineages 2, 3, 4 and 7 of M. tuberculosis, resulting in a 99th percentile of 2 mg/L for lineage 1 compared with 0.5 mg/L for the remaining M. tuberculosis lineages. Moreover, we observed that higher MICs (≥8 mg/L), which probably confer resistance, had recently evolved independently in six different M. tuberculosis strains. Unlike the aforementioned ancient differences in susceptibility, these recent differences were likely caused by mutations in the known pretomanid resistance genes., Conclusions: In light of these findings, the provisional critical concentration of 1 mg/L for MGIT set by EMA must be re-evaluated. More broadly, these findings underline the importance of considering the global diversity of MTBC during clinical development of drugs and when defining breakpoints for AST., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

Rationale: Bedaquiline has been classified as a group A drug for the treatment of multidrug-resistant tuberculosis (MDR-TB) by the World Health Organization; however, globally emerging resistance threatens the effectivity of novel MDR-TB treatment regimens., Objectives: We analysed pre-existing and emerging bedaquiline resistance in bedaquiline-based MDR-TB therapies, and risk factors associated with treatment failure and death., Methods: In a cross-sectional cohort study, we employed patient data, whole-genome sequencing (WGS) and phenotyping of Mycobacterium tuberculosis complex (MTBC) isolates. We could retrieve baseline isolates from 30.5% (62 out of 203) of all MDR-TB patients who received bedaquiline between 2016 and 2018 in the Republic of Moldova. This includes 26 patients for whom we could also retrieve a follow-up isolate., Measurements and Main Results: At baseline, all MTBC isolates were susceptible to bedaquiline. Among 26 patients with available baseline and follow-up isolates, four (15.3%) patients harboured strains which acquired bedaquiline resistance under therapy, while one (3.8%) patient was re-infected with a second bedaquiline-resistant strain. Treatment failure and death were associated with cavitary disease (p=0.011), and any additional drug prescribed in the bedaquiline-containing regimen with WGS-predicted resistance at baseline (OR 1.92 per unit increase, 95% CI 1.15-3.21; p=0.012)., Conclusions: MDR-TB treatments based on bedaquiline require a functional background regimen to achieve high cure rates and to prevent the evolution of bedaquiline resistance. Novel MDR-TB therapies with bedaquiline require timely and comprehensive drug resistance monitoring., Competing Interests: Conflict of interest: E. Chesov has nothing to disclose. Conflict of interest: D. Chesov has nothing to disclose. Conflict of interest: F.P. Maurer has nothing to disclose. Conflict of interest: S. Andres has nothing to disclose. Conflict of interest: C. Utpatel has nothing to disclose. Conflict of interest: I. Barilar has nothing to disclose. Conflict of interest: A. Donica has nothing to disclose. Conflict of interest: M. Reimann has nothing to disclose. Conflict of interest: S. Niemann reports grants from EXC 2167 Precision Medicine in Inflammation, grants from Leibniz Science Campus Evolutionary Medicine of the LUNG, grants from German Center for Infection Research, during the conduct of the study. Conflict of interest: C. Lange reports personal fees from Chiesi, Gilead, Janssen, Novartis, Oxfordimmunotec and Insmed, outside the submitted work. Conflict of interest: V. Crudu has nothing to disclose. Conflict of interest: J. Heyckendorf has nothing to disclose. Conflict of interest: M. Merker has nothing to disclose., (Copyright ©The authors 2022.)

Kenya is a country with a high tuberculosis (TB) burden. However, knowledge on the genetic diversity of Mycobacterium tuberculosis complex (MTBC) strains and their transmission dynamics is sparsely available. Hence, we used whole-genome sequencing (WGS) to depict the genetic diversity, molecular markers of drug resistance, and possible transmission clusters among MTBC strains in urban and slum settings of Nairobi. We analyzed 385 clinical MTBC isolates collected between 2010 and 2015 in combination with patients' demographics. We showed that the MTBC population mainly comprises strains of four lineages (L1-L4). The two dominating lineages were L4 with 55.8% ( n = 215) and L3 with 25.7% ( n = 99) of all strains, respectively. Genome-based cluster analysis showed that 30.4% (117/385) of the strains were clustered using a ≤5 single-nucleotide polymorphism (SNP) threshold as a surrogate marker for direct patient-to-patient MTBC transmission. Moreover, 5.2% (20/385) of the strains were multidrug-resistant (MDR), and 50.0% ( n = 10) were part of a genome-based cluster (i.e., direct MDR MTBC transmission). Notably, 30.0% (6/20) of the MDR strains were resistant to all first-line drugs and are part of one molecular cluster. Moreover, TB patients in urban living setting had 3.8 times the odds of being infected with a drug-resistant strain as compared to patients from slums ( p -value = 0.002). Our results show that L4 strains are the main causative agent of TB in Nairobi and MDR strain transmission is an emerging concern in urban settings. This emphasizes the need for more focused infection control measures and contact tracing of patients with MDR TB to break the transmission chains.

Preexisting and newly emerging resistant pathogen subpopulations (heteroresistance) are potential risk factors for treatment failure of multi/extensively drug resistant (MDR/XDR) tuberculosis (TB). Intrapatient evolutionary dynamics of Mycobacterium tuberculosis complex (Mtbc) strains and their implications on treatment outcomes are still not completely understood. To elucidate how Mtbc strains escape therapy, we analyzed 13 serial isolates from a German patient by whole-genome sequencing. Sequencing data were compared with phenotypic drug susceptibility profiles and the patient's collective 27-year treatment history to further elucidate factors fostering intrapatient resistance evolution. The patient endured five distinct TB episodes, ending in resistance to 16 drugs and a nearly untreatable XDR-TB infection. The first isolate obtained, during the patient's 5th TB episode, presented fixed resistance mutations to 7 anti-TB drugs, including isoniazid, rifampin, streptomycin, pyrazinamide, prothionamide, para-aminosalicylic acid, and cycloserine-terizidone. Over the next 13 years, a dynamic evolution with coexisting, heterogeneous subpopulations was observed in 6 out of 13 sequential bacterial isolates. The emergence of drug-resistant subpopulations coincided with frequent changes in treatment regimens, which often included two or fewer active compounds. This evolutionary arms race between competing subpopulations ultimately resulted in the fixation of a single XDR variant. Our data demonstrate the complex intrapatient microevolution of Mtbc subpopulations during failing MDR/XDR-TB treatment. Designing effective treatment regimens based on rapid detection of (hetero) resistance is key to avoid resistance development and treatment failure.

Standardised management of tuberculosis may soon be replaced by individualised, precision medicine-guided therapies informed with knowledge provided by the field of systems biology. Systems biology is a rapidly expanding field of computational and mathematical analysis and modelling of complex biological systems that can provide insights into mechanisms underlying tuberculosis, identify novel biomarkers, and help to optimise prevention, diagnosis and treatment of disease. These advances are critically important in the context of the evolving epidemic of drug-resistant tuberculosis. Here, we review the available evidence on the role of systems biology approaches - human and mycobacterial genomics and transcriptomics, proteomics, lipidomics/metabolomics, immunophenotyping, systems pharmacology and gut microbiomes - in the management of tuberculosis including prediction of risk for disease progression, severity of mycobacterial virulence and drug resistance, adverse events, comorbidities, response to therapy and treatment outcomes. Application of the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) approach demonstrated that at present most of the studies provide "very low" certainty of evidence for answering clinically relevant questions. Further studies in large prospective cohorts of patients, including randomised clinical trials, are necessary to assess the applicability of the findings in tuberculosis prevention and more efficient clinical management of patients., Competing Interests: Conflict of Interest: I. Kontsevaya has nothing to disclose. Conflict of interest: C. Lange reports personal fees from Chiesi, Gilead, Janssen, Lucane, Novartis, Oxoid, Berlin Chemie, Thermofisher and Oxfordimmunotec, outside the submitted work. Conflict of interest: P. Comella-del-Barrio has nothing to disclose. Conflict of interest: C. Coarfa has nothing to disclose. Conflict of interest: A.R. DiNardo has nothing to disclose. Conflict of interest: S.H. Gillespie reports non-financial support from LifeArc, during the conduct of the study; and other support from ODx Innovations, outside the submitted work. Conflict of interest: M. Hauptmann has nothing to disclose. Conflict of interest: C. Leschczyk has nothing to disclose. Conflict of interest: A.M. Mandalakas has nothing to disclose. Conflict of interest: A. Martinecz has nothing to disclose. Conflict of interest: M. Merker has nothing to disclose. Conflict of interest: S. Niemann reports grants from German Center for Infection Research, Excellenz Cluster Precision Medicine in Chronic Inflammation EXC 2167, and Leibniz Science Campus Evolutionary Medicine of the LUNG (EvoLUNG), during the conduct of the study. Conflict of interest: M. Reimann has nothing to disclose. Conflict of interest: O. Rzhepishevska has nothing to disclose. Conflict of interest: U.E. Schaible has nothing to disclose. Conflict of interest: K.M. Scheu has nothing to disclose. Conflict of interest: E. Schurr has nothing to disclose. Conflict of interest: P. Abel zur Wiesch has nothing to disclose. Conflict of interest: J. Heyckendorf has nothing to disclose., (Copyright ©The authors 2021.)

Tuberculosis is a bacterial infectious disease that is mainly transmitted from human to human via infectious aerosols. Currently, tuberculosis is the leading cause of death by an infectious disease world-wide. In the past decade, the number of patients affected by tuberculosis has increased by ~20 percent and the emergence of drug-resistant strains of Mycobacterium tuberculosis challenges the goal of elimination of tuberculosis in the near future. For the last 50 years, management of patients with tuberculosis has followed a standardized management approach. This standardization neglects the variation in human susceptibility to infection, immune response, the pharmacokinetics of drugs, and the individual duration of treatment needed to achieve relapse-free cure. Here we propose a package of precision medicine-guided therapies that has the prospect to drive clinical management decisions, based on both host immunity and M. tuberculosis strains genetics. Recently, important scientific discoveries and technological advances have been achieved that provide a perspective for individualized rather than standardized management of patients with tuberculosis. For the individual selection of best medicines and host-directed therapies, personalized drug dosing, and treatment durations, physicians treating patients with tuberculosis will be able to rely on these advances in systems biology and to apply them at the bedside., (Copyright © 2020 Lange, Aarnoutse, Chesov, van Crevel, Gillespie, Grobbel, Kalsdorf, Kontsevaya, van Laarhoven, Nishiguchi, Mandalakas, Merker, Niemann, Köhler, Heyckendorf, Reimann, Ruhwald, Sanchez-Carballo, Schwudke, Waldow and DiNardo.)

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., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: The World Health Organization recommends drug-susceptibility testing of Mycobacterium tuberculosis complex for all patients with tuberculosis to guide treatment decisions and improve outcomes. Whether DNA sequencing can be used to accurately predict profiles of susceptibility to first-line antituberculosis drugs has not been clear., Methods: We obtained whole-genome sequences and associated phenotypes of resistance or susceptibility to the first-line antituberculosis drugs isoniazid, rifampin, ethambutol, and pyrazinamide for isolates from 16 countries across six continents. For each isolate, mutations associated with drug resistance and drug susceptibility were identified across nine genes, and individual phenotypes were predicted unless mutations of unknown association were also present. To identify how whole-genome sequencing might direct first-line drug therapy, complete susceptibility profiles were predicted. These profiles were predicted to be susceptible to all four drugs (i.e., pansusceptible) if they were predicted to be susceptible to isoniazid and to the other drugs or if they contained mutations of unknown association in genes that affect susceptibility to the other drugs. We simulated the way in which the negative predictive value changed with the prevalence of drug resistance., Results: A total of 10,209 isolates were analyzed. The largest proportion of phenotypes was predicted for rifampin (9660 [95.4%] of 10,130) and the smallest was predicted for ethambutol (8794 [89.8%] of 9794). Resistance to isoniazid, rifampin, ethambutol, and pyrazinamide was correctly predicted with 97.1%, 97.5%, 94.6%, and 91.3% sensitivity, respectively, and susceptibility to these drugs was correctly predicted with 99.0%, 98.8%, 93.6%, and 96.8% specificity. Of the 7516 isolates with complete phenotypic drug-susceptibility profiles, 5865 (78.0%) had complete genotypic predictions, among which 5250 profiles (89.5%) were correctly predicted. Among the 4037 phenotypic profiles that were predicted to be pansusceptible, 3952 (97.9%) were correctly predicted., Conclusions: Genotypic predictions of the susceptibility of M. tuberculosis to first-line drugs were found to be correlated with phenotypic susceptibility to these drugs. (Funded by the Bill and Melinda Gates Foundation and others.).

Tuberculosis (TB) is a contagious disease with a complex epidemiology. Therefore, molecular typing (genotyping) of Mycobacterium tuberculosis complex (MTBC) strains is of primary importance to effectively guide outbreak investigations, define transmission dynamics and assist global epidemiological surveillance of the disease. Large-scale genotyping is also needed to get better insights into the biological diversity and the evolution of the pathogen. Thanks to its shorter turnaround and simple numerical nomenclature system, mycobacterial interspersed repetitive unit-variable-number tandem repeat (MIRU-VNTR) typing, based on 24 standardized plus 4 hypervariable loci, optionally combined with spoligotyping, has replaced IS6110 DNA fingerprinting over the last decade as a gold standard among classical strain typing methods for many applications. With the continuous progress and decreasing costs of next-generation sequencing (NGS) technologies, typing based on whole genome sequencing (WGS) is now increasingly performed for near complete exploitation of the available genetic information. However, some important challenges remain such as the lack of standardization of WGS analysis pipelines, the need of databases for sharing WGS data at a global level, and a better understanding of the relevant genomic distances for defining clusters of recent TB transmission in different epidemiological contexts. This chapter provides an overview of the evolution of genotyping methods over the last three decades, which culminated with the development of WGS-based methods. It addresses the relative advantages and limitations of these techniques, indicates current challenges and potential directions for facilitating standardization of WGS-based typing, and provides suggestions on what method to use depending on the specific research question.

Sputum samples from new tuberculosis (TB) cases were collected over 2 years as part of a prospective study in the northeastern part of Lima, Peru. To measure the contribution of recent transmission to the high rates of multidrug resistance (MDR) in this area, Mycobacterium tuberculosis complex (MTBc) isolates were tested for drug susceptibility to first-line drugs and were genotyped by spoligotyping and 15-locus mycobacterial interspersed repetitive-unit (MIRU-15)-variable-number tandem repeat (VNTR) analysis. MDR was found in 6.8% of 844 isolates, of which 593 (70.3%) were identified as belonging to a known MTBc lineage, whereas 198 isolates (23.5%) could not be assigned to these lineages and 12 (1.4%) represented mixed infections. Lineage 4 accounted for 54.9% (n = 463) of the isolates, most of which belonged to the Haarlem family (n = 279). MIRU-15 analysis grouped 551/791 isolates (69.7%) in 102 clusters, with sizes ranging from 2 to 46 strains. The overall high clustering rate suggests a high level of recent transmission in this population, especially among younger patients (odds ratio [OR], 1.6; P = 0.01). Haarlem strains were more prone to cluster, compared to the other families taken together (OR, 2.0; P < 0.0001), while Beijing (OR, 0.6; P = 0.006) and LAM (OR, 0.7; P = 0.07) strains clustered less. Whereas streptomycin-resistant strains were more commonly found in clusters (OR, 1.8; P = 0.03), clustering rates did not differ between MDR and non-MDR strains (OR, 1.8; P = 0.1). Furthermore, only 16/51 MDR strains clustered with other MDR strains, suggesting that patients with primary MDR infections acquired the infections mostly from index cases outside the study population, such as retreated cases., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Whole-genome sequencing (WGS) allows for effective tracing of Mycobacterium tuberculosis complex (MTBC) (tuberculosis pathogens) transmission. However, it is difficult to standardize and, therefore, is not yet employed for interlaboratory prospective surveillance. To allow its widespread application, solutions for data standardization and storage in an easily expandable database are urgently needed. To address this question, we developed a core genome multilocus sequence typing (cgMLST) scheme for clinical MTBC isolates using the Ridom SeqSphere(+) software, which transfers the genome-wide single nucleotide polymorphism (SNP) diversity into an allele numbering system that is standardized, portable, and not computationally intensive. To test its performance, we performed WGS analysis of 26 isolates with identical IS6110 DNA fingerprints and spoligotyping patterns from a longitudinal outbreak in the federal state of Hamburg, Germany (notified between 2001 and 2010). The cgMLST approach (3,041 genes) discriminated the 26 strains with a resolution comparable to that of SNP-based WGS typing (one major cluster of 22 identical or closely related and four outlier isolates with at least 97 distinct SNPs or 63 allelic variants). Resulting tree topologies are highly congruent and grouped the isolates in both cases analogously. Our data show that SNP- and cgMLST-based WGS analyses facilitate high-resolution discrimination of longitudinal MTBC outbreaks. cgMLST allows for a meaningful epidemiological interpretation of the WGS genotyping data. It enables standardized WGS genotyping for epidemiological investigations, e.g., on the regional public health office level, and the creation of web-accessible databases for global TB surveillance with an integrated early warning system., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Background: Although Ethiopia ranks seventh among the world's 22 high-burden tuberculosis (TB) countries, little is known about strain diversity and transmission. In this study, we present the first in-depth analysis of the population structure and transmission dynamics of Mycobacterium tuberculosis strains from Northwest Ethiopia., Methods: In the present study, 244 M. tuberculosis isolates where analysed by mycobacterial interspersed repetitive unit - variable number tandem repeat 24-loci typing and spoligotyping methods to determine phylogenetic lineages and perform cluster analysis. Clusters of strains with identical genotyping patterns were considered as an indicator for the recent transmission., Results: Of 244 isolates, 59.0% were classified into nine previously described lineages: Dehli/CAS (38.9%), Haarlem (8.6%), Ural (3.3%), LAM (3.3%), TUR (2.0%), X-type (1.2%), S-type (0.8%), Beijing (0.4%) and Uganda II (0.4%). Interestingly, 31.6% of the strains were grouped into four new lineages and were named as Ethiopia&#95;3 (13.1%), Ethiopia&#95;1 (7.8%), Ethiopia&#95;H37Rv like (7.0%) and Ethiopia&#95;2 (3.7%) lineages. The remaining 9.4% of the isolates could not be assigned to the known or new lineages. Overall, 45.1% of the isolates were grouped in clusters, indicating a high rate of recent transmission., Conclusions: This study confirms a highly diverse M. tuberculosis population structure, the presence of new phylogenetic lineages and a predominance of the Dehli/CAS lineage in Northwest Ethiopia. The high rate of recent transmission indicates defects of the TB control program in Northwest Ethiopia. This emphasizes the importance of strengthening laboratory diagnosis of TB, intensified case finding and treatment of TB patients to interrupt the chain of transmission.

Tuberculosis is a respiratory disease that is treatable with antibiotics. An increasing prevalence of resistance means that to ensure a good treatment outcome it is desirable to test the susceptibility of each infection to different antibiotics. Conventionally, this is done by culturing a clinical sample and then exposing aliquots to a panel of antibiotics, each being present at a pre-determined concentration, thereby determining if the sample isresistant or susceptible to each sample. The minimum inhibitory concentration (MIC) of a drug is the lowestconcentration that inhibits growth and is a more useful quantity but requires each sample to be tested at a range ofconcentrations for each drug. Using 96-well broth micro dilution plates with each well containing a lyophilised pre-determined amount of an antibiotic is a convenient and cost-effective way to measure the MICs of several drugs at once for a clinical sample. Although accurate, this is still an expensive and slow process that requires highly-skilled and experienced laboratory scientists. Here we show that, through the BashTheBug project hosted on the Zooniverse citizen science platform, a crowd of volunteers can reproducibly and accurately determine the MICs for 13 drugs and that simply taking the median or mode of 11-17 independent classifications is sufficient. There is therefore a potential role for crowds to support (but not supplant) the role of experts in antibiotic susceptibility testing.

Inappropriately high breakpoints have resulted in systematic false-susceptible AST results to anti-TB drugs. MIC, PK/PD and clinical outcome data should be combined when setting breakpoints to minimise the emergence and spread of antimicrobial resistance. https://bit.ly/3i43wb6

BACKGROUND: Atypical Beijing genotype Mycobacterium tuberculosis strains are widespread in South Africa and have acquired resistance to up to 13 drugs on multiple occasions. It is puzzling that these strains have retained fitness and transmissibility despite the potential fitness cost associated with drug resistance mutations. METHODS: We conducted Illumina sequencing of 211 Beijing genotype M. tuberculosis isolates to facilitate the detection of genomic features that may promote acquisition of drug resistance and restore fitness in highly resistant atypical Beijing forms. Phylogenetic and comparative genomic analysis was done to determine changes that are unique to the resistant strains that also transmit well. Minimum inhibitory concentration (MIC) determination for streptomycin and bedaquiline was done for a limited number of isolates to demonstrate a difference in MIC between isolates with and without certain variants. RESULTS: Phylogenetic analysis confirmed that two clades of atypical Beijing strains have independently developed resistance to virtually all the potent drugs included in standard (pre-bedaquiline) drug-resistant TB treatment regimens. We show that undetected drug resistance in a progenitor strain was likely instrumental in this resistance acquisition. In this cohort, ethionamide (ethA A381P) resistance would be missed in first-line drug-susceptible isolates, and streptomycin (gidB L79S) resistance may be missed due to an MIC close to the critical concentration. Subsequent inadequate treatment historically led to amplification of resistance and facilitated spread of the strains. Bedaquiline resistance was found in a small number of isolates, despite lack of exposure to the drug. The highly resistant clades also carry inhA promoter mutations, which arose after ethA and katG mutations. In these isolates, inhA promoter mutations do not alter drug resistance, suggesting a possible alternative role. CONCLUSION: The presence of the ethA mutation in otherwise susceptible isolates from ethionamide-naïve patients demonstrates that known exposure is not an adequate indicator of drug susceptibility. Similarly, it is demonstrated that bedaquiline resistance can occur without exposure to the drug. Inappropriate treatment regimens, due to missed resistance, leads to amplification of resistance, and transmission. We put these results into the context of current WHO treatment regimens, underscoring the risks of treatment without knowledge of the full drug resistance profile.

Tuberculosis (TB) remains a major global health problem; 10 million new cases were reported in 2018 (1). In Sudan, the estimated national TB incidence in 2018 was 71/100,000 persons; a [...]

Applying whole-genome-sequencing, we aimed to detect transmission events of multidrug-resistant/rifampinresistant strains of Mycobacterium tuberculosis complex at a tuberculosis hospital in Chisinau, Moldova. We recorded ward, room, and bed information for each patient and monitored in-hospital transfers over 1 year. Detailed molecular and patient surveillance revealed only 2 nosocomial transmission events. [ABSTRACT FROM AUTHOR]

Transmission-driven multi-/extensively drug resistant (M/XDR) tuberculosis (TB) is the largest single contributor to human mortality due to antimicrobial resistance. A few major clades of the Mycobacterium tuberculosis complex belonging to lineage 2, responsible for high prevalence of MDR-TB in Eurasia, show outstanding transnational distributions. Here, we determined factors underlying the emergence and epidemic spread of the W148 clade by genome sequencing and Bayesian demogenetic analyses of 720 isolates from 23 countries. We dated a common ancestor around 1963 and identified two successive epidemic expansions in the late 1980s and late 1990s, coinciding with major socio-economic changes in the post-Soviet Era. These population expansions favored accumulation of resistance mutations to up to 11 anti-TB drugs, with MDR evolving toward additional resistances to fluoroquinolones and second-line injectable drugs within 20 years on average. Timescaled haplotypic density analysis revealed that widespread acquisition of compensatory mutations was associated with transmission success of XDR strains. Virtually all W148 strains harbored a hypervirulence-associated ppe38 gene locus, and incipient recurrent emergence of prpR mutation-mediated drug tolerance was detected. The outstanding genetic arsenal of this geographically widespread M/XDR strain clade represents a "perfect storm" that jeopardizes the successful introduction of new anti-M/XDR-TB antibiotic regimens. An outbreak of the multidrug-resistant Mycobacterium tuberculosis lineage W148 has spread widely across Russia, Central Asia and Europe. Here, the authors use whole genome sequences of ~700 isolates of this lineage collected over ~20 years to analyze its spread, evolution of drug resistance, and impact of compensatory mutations. [ABSTRACT FROM AUTHOR]

Background: The AID line probe assay has shown promising evaluation data on the detection of Mycobacterium tuberculosis as well as 1st- and 2nd-line drug resistance, using isolates and selected clinical samples in previous studies. Methods: The diagnostic performance of three AID-modules (AID INH/RIF, AID FQ/EMB and AID AG) was analyzed in sputum samples from patients with presumed tuberculosis against culture methods and phenotypic drug resistance as reference standards. Results: 59 patients had culture-confirmed tuberculosis. All AID modules showed moderate sensitivity (46/59, 78.0%, 65.3–87.7) and very good specificity (100%, 95.5%, 93.7%). There was a high proportion of invalid tests, resulting in 32.6%, 78.3% and 19.6% of 46 AID-positive tuberculosis cases, who could not be assessed for drug resistance by the AID INH/RIF-, AID FQ/EM- and AID AG-module, respectively. A small number of patients showed drug resistance by reference standards: Three MDR-TB cases plus three, one and one patients with resistance to streptomycin, fluoroquinolones and aminoglycosides, respectively. The AID-assay detected all MDR-TB cases, two of three streptomycin-resistant TB cases, one of one of fluoroquinolone-resistant and missed one aminoglycoside-resistant TB case. Discussion: The high proportion of invalid results precludes the use of the AID-assay from direct sputum-based tuberculosis and drug-resistance testing. [ABSTRACT FROM AUTHOR]

The emergence of drug-resistant tuberculosis is a major global public health concern that threatens the ability to control the disease. Whole-genome sequencing as a tool to rapidly diagnose resistant infections can transform patient treatment and clinical practice. While resistance mechanisms are well understood for some drugs, there are likely many mechanisms yet to be uncovered, particularly for new and repurposed drugs. We sequenced 10,228 Mycobacterium tuberculosis (MTB) isolates worldwide and determined the minimum inhibitory concentration (MIC) on a grid of 2-fold concentration dilutions for 13 antimicrobials using quantitative microtiter plate assays. We performed oligopeptide- and oligonucleotide-based genome-wide association studies using linear mixed models to discover resistance-conferring mechanisms not currently catalogued. Use of MIC over binary resistance phenotypes increased sample heritability for the new and repurposed drugs by 26% to 37%, increasing our ability to detect novel associations. For all drugs, we discovered uncatalogued variants associated with MIC, including in the Rv1218c promoter binding site of the transcriptional repressor Rv1219c (isoniazid), upstream of the vapBC20 operon that cleaves 23S rRNA (linezolid) and in the region encoding an α-helix lining the active site of Cyp142 (clofazimine, all p < 10−7.7). We observed that artefactual signals of cross-resistance could be unravelled based on the relative effect size on MIC. Our study demonstrates the ability of very large-scale studies to substantially improve our knowledge of genetic variants associated with antimicrobial resistance in M. tuberculosis. The emergence of drug resistant tuberculosis is a major global public health concern that threatens the ability to control the disease. Phenotyping and sequencing 10,000 Mycobacterium tuberculosis genomes identifies previously uncatalogued genetic variants associated with resistance to thirteen new and repurposed, second line and first line drugs. [ABSTRACT FROM AUTHOR]

Background: Ethiopia is one of the high multidrug-resistant tuberculosis (MDR-TB) burden countries. However, phenotypic drug susceptibility testing can take several weeks due to the slow growth of Mycobacterium tuberculosis complex (MTBC) strains. In this study, we assessed the performance of a Sanger sequencing approach to predict resistance against five anti-tuberculosis drugs and the pattern of resistance mediating mutations. Methods: We enrolled 226 MTBC culture-positive MDR-TB suspects and collected sputum specimens and socio-demographic and TB related data from each suspect between June 2015 and December 2016 in Addis Ababa, Ethiopia. Phenotypic drug susceptibility testing (pDST) for rifampicin, isoniazid, pyrazinamide, ethambutol, and streptomycin using BACTEC MGIT 960 was compared with the results of a Sanger sequencing analysis of seven resistance determining regions in the genes rpoB, katG, fabG-inhA, pncA, embB, rpsL, and rrs. Result: DNA isolation for Sanger sequencing was successfully extracted from 92.5% (209/226) of the MTBC positive cultures, and the remaining 7.5% (17/226) strains were excluded from the final analysis. Based on pDST results, drug resistance proportions were as follows: isoniazid: 109/209 (52.2%), streptomycin: 93/209 (44.5%), rifampicin: 88/209 (42.1%), ethambutol: 74/209 (35.4%), and pyrazinamide: 69/209 (33.0%). Resistance against isoniazid was mainly mediated by the mutation katG S315T (97/209, 46.4%) and resistance against rifampicin by rpoB S531L (58/209, 27.8%). The dominating resistance-conferring mutations for ethambutol, streptomycin, and pyrazinamide affected codon 306 in embB (48/209, 21.1%), codon 88 in rpsL (43/209, 20.6%), and codon 65 in pncA (19/209, 9.1%), respectively. We observed a high agreement between phenotypic and genotypic DST, such as 89.9% (at 95% confidence interval [CI], 84.2%–95.8%) for isoniazid, 95.5% (95% CI, 91.2%–99.8%) for rifampicin, 98.6% (95% CI, 95.9–100%) for ethambutol, 91.3% (95% CI, 84.6–98.1%) for pyrazinamide and 57.0% (95% CI, 46.9%–67.1%) for streptomycin. Conclusion: We detected canonical mutations implicated in resistance to rifampicin, isoniazid, pyrazinamide, ethambutol, and streptomycin. High agreement with phenotypic DST results for all drugs renders Sanger sequencing promising to be performed as a complementary measure to routine phenotypic DST in Ethiopia. Sanger sequencing directly from sputum may accelerate accurate clinical decision-making in the future. [ABSTRACT FROM AUTHOR]

Background Comprehensive and reliable drug susceptibility testing (DST) is urgently needed to provide adequate treatment regimens for patients with multidrug-resistant/rifampicin-resistant tuberculosis (MDR/RR-TB). We determined whether next-generation sequencing (NGS) analysis of Mycobacterium tuberculosis complex isolates and genes implicated in drug resistance can guide the design of effective MDR/RR-TB treatment regimens. Methods NGS-based genomic DST predictions of M. tuberculosis complex isolates from MDR/RR-TB patients admitted to a TB reference center in Germany between 1 January 2015 and 30 April 2019 were compared with phenotypic DST results of mycobacteria growth indicator tubes (MGIT). Standardized treatment algorithms were applied to design individualized therapies based on either genomic or phenotypic DST results, and discrepancies were further evaluated by determination of minimal inhibitory drug concentrations (MICs) using Sensititre MYCOTBI and UKMYC microtiter plates. Results In 70 patients with MDR/RR-TB, agreement among 1048 pairwise comparisons of genomic and phenotypic DST was 86.3%; 76 (7.2%) results were discordant, and 68 (6.5%) could not be evaluated due to the presence of polymorphisms with yet unknown implications for drug resistance. Importantly, 549 of 561 (97.9%) predictions of drug susceptibility were phenotypically confirmed in MGIT, and 27 of 64 (42.2%) false-positive results were linked to previously described mutations mediating a low or moderate MIC increase. Virtually all drugs (99.0%) used in combination therapies that were inferred from genomic DST were confirmed to be susceptible by phenotypic DST. Conclusions NGS-based genomic DST can reliably guide the design of effective MDR/RR-TB treatment regimens. [ABSTRACT FROM AUTHOR]

Multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) is an emerging threat to TB control in Ukraine, a country with the third highest XDR TB burden globally. We used whole-genome sequencing of a convenience sample to identify bacterial genetic and patient-related factors associated with MDR/XDR TB in this country. MDR/XDR TB was associated with 3 distinct Mycobacterium tuberculosis complex lineage 2 (Beijing) clades, Europe/Russia W148 outbreak, Central Asia outbreak, and Ukraine outbreak, which comprised 68.9% of all MDR/XDR TB strains from southern Ukraine. MDR/XDR TB was also associated with previous treatment for TB and urban residence. The circulation of Beijing outbreak strains harboring broad drug resistance, coupled with constraints in drug supply and limited availability of phenotypic drug susceptibility testing, needs to be considered when new TB management strategies are implemented in Ukraine. [ABSTRACT FROM AUTHOR]

Pathogen-based factors associated with tuberculosis (TB) in eastern Sudan are not well defined. We investigated genetic diversity, drug resistance, and possible transmission clusters of Mycobacterium tuberculosis complex (MTBC) strains by using a genomic epidemiology approach. We collected 383 sputum specimens at 3 hospitals in 2014 and 2016 from patients with symptoms suggestive of TB; of these, 171 grew MTBC strains. Whole-genome sequencing could be performed on 166 MTBC strains; phylogenetic classification revealed that most (73.4%; n = 122) belonged to lineage 3 (L3). Genome-based cluster analysis showed that 76 strains (45.9%) were grouped into 29 molecular clusters, comprising 2-8 strains/patients. Of the strains investigated, 9.0% (15/166) were multidrug resistant (MDR); 10 MDR MTBC strains were linked to 1 large MDR transmission network. Our findings indicate that L3 strains are the main causative agent of TB in eastern Sudan; MDR TB is caused mainly by transmission of MDR L3 strains. [ABSTRACT FROM AUTHOR]

A retrospective population-based molecular epidemiologic study of multidrug-resistant Mycobacterium tuberculosis complex strains in Serbia (2008-2014) revealed an outbreak of TUR genotype strains in a psychiatric hospital starting around 1990. Drug unavailability, poor infection control, and schizophrenia likely fueled acquisition of additional resistance and bacterial fitness-related mutations over 2 decades. [ABSTRACT FROM AUTHOR]

The article offers information on pathogen based precision medicine for drug resistant tuberculosis. Topics discussed include prevention and treatment strategies take information from systems biology and individual variability into account; model example of precision medicine in infectious diseases is multidrug-resistant tuberculosis (MDR-TB); and molecular tests to quickly identify genotypic drug resistance successfully implemented in daily practice.

Background: Previous studies suggest the burden of pulmonary tuberculosis (PTB) in Ethiopia may be greater in university students relative to the overall population. However, little is known about the transmission dynamics of PTB among students and members of the communities surrounding university campuses in Eastern Ethiopia. Methods: A cross sectional study was conducted in Eastern Ethiopia among prevalent culture-confirmed PTB cases from university students (n = 36) and community members diagnosed at one of four hospitals (n = 152) serving the surrounding area. Drug susceptibility testing (DST) was performed on Mycobacterium tuberculosis complex (MTBC) isolates using BD Bactec MGIT 960 and molecular genotyping was performed using spoligotyping and 24-loci MIRU-VNTR. MTBC strains with Identical genotyping patterns were assigned to molecular clusters as surrogate marker for recent transmission and further contact tracing was initiated among clustered patients. Results: Among all study participants, four MTBC lineages and 11 sub-lineages were identified, with Ethiopia_3 (Euro-American lineage) being most common sub-lineage (29.4%) in both cohorts and associated with strain clustering (P = 0.016). We further identified 13 (8.1%) strains phylogenetically closely related to Ethiopia_3 but with a distinct Spoligotyping pattern and designated as Ethiopia_4. The clustering rate of MTBC strains was 52.9% for university students and 66.7% for community members with a Recent Transmission Index (RTI) of 17.6% and 48.4%, respectively. Female gender, urban residence, and new TB cases were significantly associated with strain clustering (P<0.05). Forty-eight (30%) of the study participants were resistant to one or more first line anti TB drugs, three patients were classified as multidrug resistant (MDR). Conclusion: We found evidence for recent transmission of PTB among Ethiopian university students and the local community in Eastern Ethiopia, mainly linked to strains classified as Ethiopia_3 sub lineage. Drug resistance didn’t have a major impact on recent transmission but comprehensive molecular surveillance in combination with drug resistance profiling of MTBC strains is desirable to better characterize TB transmission dynamics in high risk congregate living environments such as university campuses and guide regional TB control programs. [ABSTRACT FROM AUTHOR]

Background: Drug-resistant tuberculosis (TB) is a major public health concern threathing the success of TB control efforts, and this is particularily problematic in Central Asia. Here, we present the first analysis of the population structure of Mycobacterium tuberculosis complex isolates in the Central Asian republics Uzbekistan, Tajikistan, and Kyrgyzstan.Methods: The study set consisted of 607 isolates with 235 from Uzbekistan, 206 from Tajikistan, and 166 from Kyrgyzstan. 24-loci MIRU-VNTR (Mycobacterial Interspersed Repetitive Units - Variable Number of Tandem Repeats) typing and spoligotyping were combined for genotyping. In addition, phenotypic drug suceptibility was performed.Results: The population structure mainly comprises strains of the Beijing lineage (411/607). 349 of the 411 Beijing isolates formed clusters, compared to only 33 of the 196 isolates from other clades. Beijing 94-32 (n = 145) and 100-32 (n = 70) formed the largest clusters. Beijing isolates were more frequently multidrug-resistant, pre-extensively resistant (pre-XDR)- or XDR-TB than other genotypes.Conclusions: Beijing clusters 94-32 and 100-32 are the dominant MTB genotypes in Central Asia. The relative size of 100-32 compared to previous studies in Kazakhstan and its unequal geographic distribution support the hypothesis of its more recent emergence in Central Asia. The data also demonstrate that clonal spread of resistant TB strains, particularly of the Beijing lineage, is a root of the so far uncontroled MDR-TB epidemic in Central Asia. [ABSTRACT FROM AUTHOR]

A letter to the editor about the Xpert MTB/RIF-based diagnosis of drug-resistant tuberculosis in Swaziland is presented.

Aims and objectives Resistant-, multidrug resistant- (MDR) and even extensively drug resistant- (XDR) Mycobacterium tuberculosis complex (MTBC) strains have emerged worldwide and represent a serious challenge for global tuberculosis (TB) control. Interestingly, high rates of MDR TB have been associated with particular phylogenetic lineages of the MTBC such as the Beijing lineage and the strong clonal expansion of particular MDR strains in Eastern Europe. However, a systematic investigation of the evolution and cross border transmission of particular highly transmissible MDR strains in Eastern Europe has not been performed so far. Methods To address this question, an in-depth analysis was performed of the population structure (based on 24 loci MIRU-VNTR-typing and spoligotyping) of strains from different settings in Eastern Europe, such as Uzbekistan, Turkmenistan, and Kazakhstan. Results The results showed that two major clones of the Beijing lineage are the drivers of the MDR epidemic in Eastern Europe. In addition, retrospective investigation of an MDR strain collection from Germany using MIRU-VNTR and IS 6110 RFLP patterns revealed that both clones were circulating for more than 15 years. In-depth analysis of genome characteristics confirmed the longitudinal transmission of the two major MDR clones and allowed the identification of particular beneficial combinations of mutations potentially compensating for the fitness defect of the MDR/XDR strains. Moreover, these allele combinations coincide with an enhanced transmissibility compared with other circulating MDR/XDR strain types and promote the selection of extremely drug resistant but also highly virulent MTBC strain variants. Conclusion Taken together, these data argue strongly for an expansion of particular MDR Beijing clones in Eastern Europe that started at least 15 years ago. Evolution and selection over the past years has resulted in “hyper transmissible” MDR-TB strains that have the potential to spread rapidly and, thus, further complicate TB control. [ABSTRACT FROM AUTHOR]

Pathogen-based factors associated with tuberculosis (TB) in eastern Sudan are not well defined. We investigated genetic diversity, drug resistance, and possible transmission clusters of Mycobacterium tuberculosis complex (MTBC) strains by using a genomic epidemiology approach. We collected 383 sputum specimens at 3 hospitals in 2014 and 2016 from patients with symptoms suggestive of TB; of these, 171 grew MTBC strains. Whole-genome sequencing could be performed on 166 MTBC strains; phylogenetic classification revealed that most (73.4%; n = 122) belonged to lineage 3 (L3). Genome-based cluster analysis showed that 76 strains (45.9%) were grouped into 29 molecular clusters, comprising 2-8 strains/patients. Of the strains investigated, 9.0% (15/166) were multidrug resistant (MDR); 10 MDR MTBC strains were linked to 1 large MDR transmission network. Our findings indicate that L3 strains are the main causative agent of TB in eastern Sudan; MDR TB is caused mainly by transmission of MDR L3 strains. [ABSTRACT FROM AUTHOR]

Pyrazinamide (PZA) is a key antibiotic for the treatment of drug susceptible tuberculosis. PZA-resistance is mainly mediated by mutations in the pncA gene; however the current gold standard is a phenotypic drug susceptibility test requiring a well-adjusted pH-value for reliable results. Our melting curve assay detects a non-wild type genotype in selected pncA regions in at least 3750 gene copies/mL within 2.5 hours. The prototype assay was further evaluated by analyzing 271 Mycobacterium tuberculosis complex isolates from Swaziland originating from a previously published drug resistance survey and including 118 isolates with pncA mutations. Sensitivity was 83% (95% CI 75–89%) and specificity was 100% (95% CI 98–100%). Under consideration of further improvements with regard to the target range our melting curve assay has the potential as a rapid rule-in test for PZA susceptibility (wild type pncA), however false resistant results (mutant pncA , but PZA susceptible) cannot be ruled out completely. • Cartridge-based automated detection of M. tuberculosis complex pncA mutants • High specificity could allow for the prediction of PZA susceptibility • Works with heat inactivated culture material • Target range to be improved and applicability on direct patient samples to be shown [ABSTRACT FROM AUTHOR]