Publications by authors named "Stefan Niemann"

221 Publications

Microevolution of hetero-resistance subpopulations in a patient receiving 27 years of tuberculosis treatment in Germany.

Antimicrob Agents Chemother 2021 Apr 26. Epub 2021 Apr 26.

Molecular and Experimental Mycobacteriology, Research Centre Borstel, Parkalle 1, 23845 Borstel, Germany

Pre-existing and newly emerging resistant pathogen subpopulations (hetero-resistance) are potential risk factors for treatment failure of multi/extensively drug resistant (MDR/XDR) tuberculosis (TB). Intra-patient evolutionary dynamics of complex (Mtbc) strains and their implications on treatment outcomes are still not completely understood.To elucidate how Mtbc strains escape therapy, we analysed 13 serial isolates by whole genome sequencing from a German patient. Sequencing data was compared to phenotypic drug susceptibility profiles, and the patient's collective 27-year treatment history, to further elucidate factors fostering intra-patient resistance evolution.The patient endured five distinct TB episodes, ending in resistances to 16 drugs and a nearly untreatable XDR-TB infection. The first isolate obtained, during the patient's 5 TB episode, presented fixed resistance mutations to seven anti-TB drugs including isoniazid, rifampicin, streptomycin, pyrazinamide, prothionamide, para-aminosalicyclic acid and cycloserin/terizidone. Over the next 13 years a dynamic evolution with co-existing, heterogeneous subpopulations was observed in six out of 13 sequential bacterial isolates. The emergence of drug-resistant subpopulations coincided with frequent changes in treatment regimens, which often included two or less active compounds. This evolutionary arms race between competing sub-populations, ultimately resulted in the fixation of a single XDR variant.Our data demonstrates the complex intra-patient 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.
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http://dx.doi.org/10.1128/AAC.02520-20DOI Listing
April 2021

Design of multidrug-resistant tuberculosis treatment regimens based on DNA sequencing.

Clin Infect Dis 2021 Apr 26. Epub 2021 Apr 26.

German Center for Infection Research (DZIF) Tuberculosis Unit, Borstel, Germany.

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 investigated if 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 01/01/2015 and 04/30/2019 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 minimum inhibitory drug concentrations (MIC) 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 presence of polymorphisms with yet unknown implications for drug resistance. Importantly, 549/561 (97.9%) predictions of drug susceptibility were phenotypically confirmed in MGIT, and 27/64 (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 pDST.

Conclusions: NGS-based genomic DST can reliably guide the design of effective MDR/RR-TB treatment regimens.
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http://dx.doi.org/10.1093/cid/ciab359DOI Listing
April 2021

A multi-phenotype genome-wide association study of clades causing tuberculosis in a Ghanaian- and South African cohort.

Genomics 2021 Apr 20;113(4):1802-1815. Epub 2021 Apr 20.

DSI-NRF Centre of Excellence for Biomedical Tuberculosis Research, South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa.

Despite decades of research and advancements in diagnostics and treatment, tuberculosis remains a major public health concern. New computational methods are needed to interrogate the intersection of host- and bacterial genomes. Paired host genotype datum and infecting bacterial isolate information were analysed for associations using a multinomial logistic regression framework implemented in SNPTest. A cohort of 853 admixed South African participants and a Ghanaian cohort of 1359 participants were included. Two directly genotyped variants, namely rs529920 and rs41472447, were identified in the Ghanaian cohort as being statistically significantly associated with risk for infection with strains of different members of the MTBC. Thus, a multinomial logistic regression using paired host-pathogen data may prove valuable for investigating the complex relationships driving infectious disease.
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http://dx.doi.org/10.1016/j.ygeno.2021.04.024DOI Listing
April 2021

Evaluation of the Roche cobas MTB and MTB-RIF/INH Assays in Samples from Germany and Sierra Leone.

J Clin Microbiol 2021 Apr 20;59(5). Epub 2021 Apr 20.

National and Supranational Reference Laboratory for Mycobacteria, Research Center Borstel, Leibniz Lung Center, Borstel, Germany

The Roche cobas MTB and MTB-RIF/INH assays allow for detection of complex (MTBC) nucleic acid and rifampicin (RIF) and isoniazid (INH) resistance-associated mutations in an automated, high-throughput workflow. In this study, we evaluated the performance of these assays, employing samples from settings of low and high tuberculosis (TB) burdens. A total of 325 frozen, leftover respiratory samples collected from treatment-naive patients with presumptive TB in Germany ( = 280) and presumptive RIF-resistant TB in Sierra Leone ( = 45) were used in this study. cobas MTB results for detection of MTBC DNA from -acetyl-l-cysteine-sodium hydroxide (NALC-NaOH)-treated samples were compared to culture results. Predictions of RIF and INH resistance by the cobas MTB-RIF/INH assay were compared to a composite reference standard (phenotypic drug susceptibility testing and line probe assay). Whole-genome sequencing was used to resolve discordances. The overall sensitivity of cobas MTB for detection of MTBC DNA in culture-positive samples ( = 102) was 89.2% (95% confidence interval [CI], 81.7 to 93.9%). The specificity of cobas MTB was 98.6% (95% CI, 96.1 to 99.5%). Sensitivity and specificity for detection of RIF and INH resistance were 88.4% (95% CI, 75.5 to 94.9%) and 97.6% (95% CI, 87.4 to 99.6%) and 76.6% (95% CI, 62.8 to 86.4%) and 100.0% (95% CI, 90.8 to 100.0%), respectively. Discordant results for RIF and INH resistance were mainly due to uncommon mutations in samples from Sierra Leone that were not covered by the cobas MTB-RIF/INH assay. In conclusion, cobas MTB and MTB-RIF/INH assays provide accurate detection of MTBC DNA and resistance-associated mutations in respiratory samples. The influence of regional variations in the prevalence of resistance-conferring mutations requires further investigation.
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http://dx.doi.org/10.1128/JCM.02983-20DOI Listing
April 2021

Phylogenomics of reveals a new lineage and a complex evolutionary history.

Microb Genom 2021 02;7(2)

University of Basel, Basel, Switzerland.

Human tuberculosis (TB) is caused by members of the complex (MTBC). The MTBC comprises several human-adapted lineages known as , as well as two lineages (L5 and L6) traditionally referred to as . Strains of L5 and L6 are largely limited to West Africa for reasons unknown, and little is known of their genomic diversity, phylogeography and evolution. Here, we analysed the genomes of 350 L5 and 320 L6 strains, isolated from patients from 21 African countries, plus 5 related genomes that had not been classified into any of the known MTBC lineages. Our population genomic and phylogeographical analyses showed that the unclassified genomes belonged to a new group that we propose to name MTBC lineage 9 (L9). While the most likely ancestral distribution of L9 was predicted to be East Africa, the most likely ancestral distribution for both L5 and L6 was the Eastern part of West Africa. Moreover, we found important differences between L5 and L6 strains with respect to their phylogeographical substructure and genetic diversity. Finally, we could not confirm the previous association of drug-resistance markers with lineage and sublineages. Instead, our results indicate that the association of drug resistance with lineage is most likely driven by sample bias or geography. In conclusion, our study sheds new light onto the genomic diversity and evolutionary history of , and highlights the need to consider the particularities of each MTBC lineage for understanding the ecology and epidemiology of TB in Africa and globally.
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http://dx.doi.org/10.1099/mgen.0.000477DOI Listing
February 2021

Survival of hypoxia-induced dormancy is not a common feature of all strains of the Mycobacterium tuberculosis complex.

Sci Rep 2021 Jan 29;11(1):2628. Epub 2021 Jan 29.

Molecular and Experimental Mycobacteriology, Research Center Borstel, Leibniz Lung Center, Parkallee 1-40, 23845, Borstel, Germany.

While persistence in a dormant state is crucial for the life cycle of Mycobacterium tuberculosis, no investigation regarding dormancy survival of different strains across different lineages was performed so far. We analyzed responses to oxygen starvation and recovery in terms of growth, metabolism, and transcription. All different strains belonging to the Euro-American lineage (L4) showed similar survival and resuscitation characteristics. Different clinical isolates from the Beijing (L2), East African-Indian (L3), and Delhi/Central Asian (L1) lineage did not survive oxygen starvation. We show that dormancy survival is lineage-dependent. Recovery from O starvation was only observed in strains belonging to the Euro-American (L4) lineage but not in strains belonging to different lineages (L1, L2, L3). Thus, resuscitation from dormancy after oxygen starvation is not a general feature of all M. tuberculosis strains as thought before. Our findings are of key importance to understand infection dynamics of non-Euro-American vs Euro-American strains and to develop drugs targeting the dormant state.
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http://dx.doi.org/10.1038/s41598-021-81223-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7846770PMC
January 2021

Improving tuberculosis surveillance by detecting international transmission using publicly available whole genome sequencing data.

Euro Surveill 2021 Jan;26(2)

German Center for Infection Research (DZIF), partner site Hannover - Brunswick, Germany.

IntroductionImproving the surveillance of tuberculosis (TB) is especially important for multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB. The large amount of publicly available whole genome sequencing (WGS) data for TB gives us the chance to re-use data and to perform additional analyses at a large scale.AimWe assessed the usefulness of raw WGS data of global MDR/XDR isolates available from public repositories to improve TB surveillance.MethodsWe extracted raw WGS data and the related metadata of isolates available from the Sequence Read Archive. We compared this public dataset with WGS data and metadata of 131 MDR- and XDR isolates from Germany in 2012 and 2013.ResultsWe aggregated a dataset that included 1,081 MDR and 250 XDR isolates among which we identified 133 molecular clusters. In 16 clusters, the isolates were from at least two different countries. For example, Cluster 2 included 56 MDR/XDR isolates from Moldova, Georgia and Germany. When comparing the WGS data from Germany with the public dataset, we found that 11 clusters contained at least one isolate from Germany and at least one isolate from another country. We could, therefore, connect TB cases despite missing epidemiological information.ConclusionWe demonstrated the added value of using WGS raw data from public repositories to contribute to TB surveillance. Comparing the German with the public dataset, we identified potential international transmission events. Thus, using this approach might support the interpretation of national surveillance results in an international context.
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http://dx.doi.org/10.2807/1560-7917.ES.2021.26.2.1900677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7809720PMC
January 2021

MDR M. tuberculosis outbreak clone in Eswatini missed by Xpert has elevated bedaquiline resistance dated to the pre-treatment era.

Genome Med 2020 11 25;12(1):104. Epub 2020 Nov 25.

Molecular and Experimental Mycobacteriology, Research Center Borstel, Parkallee 1, 23845, Borstel, Germany.

Background: Multidrug-resistant (MDR) Mycobacterium tuberculosis complex strains not detected by commercial molecular drug susceptibility testing (mDST) assays due to the RpoB I491F resistance mutation are threatening the control of MDR tuberculosis (MDR-TB) in Eswatini.

Methods: We investigate the evolution and spread of MDR strains in Eswatini with a focus on bedaquiline (BDQ) and clofazimine (CFZ) resistance using whole-genome sequencing in two collections ((1) national drug resistance survey, 2009-2010; (2) MDR strains from the Nhlangano region, 2014-2017).

Results: MDR strains in collection 1 had a high cluster rate (95%, 117/123 MDR strains) with 55% grouped into the two largest clusters (gCL3, n = 28; gCL10, n = 40). All gCL10 isolates, which likely emerged around 1993 (95% highest posterior density 1987-1998), carried the mutation RpoB I491F that is missed by commercial mDST assays. In addition, 21 (53%) gCL10 isolates shared a Rv0678 M146T mutation that correlated with elevated minimum inhibitory concentrations (MICs) to BDQ and CFZ compared to wild type isolates. gCL10 isolates with the Rv0678 M146T mutation were also detected in collection 2.

Conclusion: The high clustering rate suggests that transmission has been driving the MDR-TB epidemic in Eswatini for three decades. The presence of MDR strains in Eswatini that are not detected by commercial mDST assays and have elevated MICs to BDQ and CFZ potentially jeopardizes the successful implementation of new MDR-TB treatment guidelines. Measures to limit the spread of these outbreak isolates need to be implemented urgently.
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http://dx.doi.org/10.1186/s13073-020-00793-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7687760PMC
November 2020

Perspective for Precision Medicine for Tuberculosis.

Front Immunol 2020 8;11:566608. Epub 2020 Oct 8.

The Global Tuberculosis Program, Texas Children's Hospital, Immigrant and Global Health, Department of Pediatrics, Baylor College of Medicine, Houston, TX, United States.

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 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 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.
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http://dx.doi.org/10.3389/fimmu.2020.566608DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7578248PMC
October 2020

Phenotypic and Transcriptomic Analyses of Seven Clinical Stenotrophomonas maltophilia Isolates Identify a Small Set of Shared and Commonly Regulated Genes Involved in the Biofilm Lifestyle.

Appl Environ Microbiol 2020 11 24;86(24). Epub 2020 Nov 24.

Department of Microbiology and Biotechnology, Universität Hamburg, Hamburg, Germany

is one of the most frequently isolated multidrug-resistant nosocomial opportunistic pathogens. It contributes to disease progression in cystic fibrosis (CF) patients and is frequently isolated from wounds, infected tissues, and catheter surfaces. On these diverse surfaces lives in single-species or multispecies biofilms. Since very little is known about common processes in biofilms of different isolates, we analyzed the biofilm profiles of 300 clinical and environmental isolates from Europe of the recently identified main lineages Sgn3, Sgn4, and Sm2 to Sm18. The analysis of the biofilm architecture of 40 clinical isolates revealed the presence of multicellular structures and high phenotypic variability at a strain-specific level. Further, transcriptome analyses of biofilm cells of seven clinical isolates identified a set of 106 shared strongly expressed genes and 33 strain-specifically expressed genes. Surprisingly, the transcriptome profiles of biofilm versus planktonic cells revealed that just 9.43% ± 1.36% of all genes were differentially regulated. This implies that just a small set of shared and commonly regulated genes is involved in the biofilm lifestyle. Strikingly, iron uptake appears to be a key factor involved in this metabolic shift. Further, metabolic analyses implied that employs a mostly fermentative growth mode under biofilm conditions. The transcriptome data of this study together with the phenotypic and metabolic analyses represent so far the largest data set on biofilm versus planktonic cells. This study will lay the foundation for the identification of strategies for fighting biofilms in clinical and industrial settings. Microorganisms living in a biofilm are much more tolerant to antibiotics and antimicrobial substances than planktonic cells are. Thus, the treatment of infections caused by microorganisms living in biofilms is extremely difficult. Nosocomial infections (among others) caused by , particularly lung infection among CF patients, have increased in prevalence in recent years. The intrinsic multidrug resistance of and the increased tolerance to antimicrobial agents of its biofilm cells make the treatment of infection difficult. The significance of our research is based on understanding the common mechanisms involved in biofilm formation of different isolates, understanding the diversity of biofilm architectures among strains of this species, and identifying the differently regulated processes in biofilm versus planktonic cells. These results will lay the foundation for the treatment of biofilms.
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http://dx.doi.org/10.1128/AEM.02038-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7688217PMC
November 2020

Insertion and deletion evolution reflects antibiotics selection pressure in a Mycobacterium tuberculosis outbreak.

PLoS Pathog 2020 09 30;16(9):e1008357. Epub 2020 Sep 30.

Institute of General Microbiology, Kiel University, Kiel, Germany.

In genome evolution, genetic variants are the source of diversity, which natural selection acts upon. Treatment of human tuberculosis (TB) induces a strong selection pressure for the emergence of antibiotic resistance-conferring variants in the infecting Mycobacterium tuberculosis (MTB) strains. MTB evolution in response to treatment has been intensively studied and mainly attributed to point substitutions. However, the frequency and contribution of insertions and deletions (indels) to MTB genome evolution remains poorly understood. Here, we analyzed a multi-drug resistant MTB outbreak for the presence of high-quality indels and substitutions. We find that indels are significantly enriched in genes conferring antibiotic resistance. Furthermore, we show that indels are inherited during the outbreak and follow a molecular clock with an evolutionary rate of 5.37e-9 indels/site/year, which is 23 times lower than the substitution rate. Inherited indels may co-occur with substitutions in genes along related biological pathways; examples are iron storage and resistance to second-line antibiotics. This suggests that epistatic interactions between indels and substitutions affect antibiotic resistance and compensatory evolution in MTB.
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http://dx.doi.org/10.1371/journal.ppat.1008357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7549793PMC
September 2020

Evolutionary Approaches to Combat Antibiotic Resistance: Opportunities and Challenges for Precision Medicine.

Front Immunol 2020 27;11:1938. Epub 2020 Aug 27.

Cluster of Excellence Precision Medicine in Chronic Inflammation, Kiel, Germany.

The rise of antimicrobial resistance (AMR) in bacterial pathogens is acknowledged by the WHO as a major global health crisis. It is estimated that in 2050 annually up to 10 million people will die from infections with drug resistant pathogens if no efficient countermeasures are implemented. Evolution of pathogens lies at the core of this crisis, which enables rapid adaptation to the selective pressures imposed by antimicrobial usage in both medical treatment and agriculture, consequently promoting the spread of resistance genes or alleles in bacterial populations. Approaches developed in the field of Evolutionary Medicine attempt to exploit evolutionary insight into these adaptive processes, with the aim to improve diagnostics and the sustainability of antimicrobial therapy. Here, we review the concept of evolutionary trade-offs in the development of AMR as well as new therapeutic approaches and their impact on host-microbiome-pathogen interactions. We further discuss the possible translation of evolution-informed treatments into clinical practice, considering both the rapid cure of the individual patients and the prevention of AMR.
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http://dx.doi.org/10.3389/fimmu.2020.01938DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7481325PMC
April 2021

Two Pandemics, One Challenge-Leveraging Molecular Test Capacity of Tuberculosis Laboratories for Rapid COVID-19 Case-Finding.

Emerg Infect Dis 2020 11 21;26(11):2549-2554. Epub 2020 Sep 21.

In many settings, the ongoing coronavirus disease (COVID-19) pandemic coincides with other major public health threats, in particular tuberculosis. Using tuberculosis (TB) molecular diagnostic infrastructure, which has substantially expanded worldwide in recent years, for COVID-19 case-finding might be warranted. We analyze the potential of using TB diagnostic and research infrastructures for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing. We focused on quality control by adapting the 12 Quality System Essentials framework to the COVID-19 and TB context. We conclude that diagnostic infrastructures for TB can in principle be leveraged to scale-up SARS-CoV-2 testing, in particular in resource-poor settings. TB research infrastructures also can support sequencing of SARS-CoV-2 to study virus evolution and diversity globally. However, fundamental principles of quality management must be followed for both TB and SARS-CoV-2 testing to ensure valid results and to minimize biosafety hazards, and the continuity of TB diagnostic services must be guaranteed at all times.
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http://dx.doi.org/10.3201/eid2611.202602DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7588527PMC
November 2020

Deep amplicon sequencing for culture-free prediction of susceptibility or resistance to 13 anti-tuberculous drugs.

Eur Respir J 2021 Mar 18;57(3). Epub 2021 Mar 18.

Université de Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019-UMR 8204-CIIL (Center for Infection and Immunity of Lille), Lille, France.

Conventional molecular tests for detecting complex (MTBC) drug resistance on clinical samples cover a limited set of mutations. Whole-genome sequencing (WGS) typically requires culture.Here, we evaluated the Deeplex Myc-TB targeted deep-sequencing assay for prediction of resistance to 13 anti-tuberculous drugs/drug classes, directly applicable on sputum.With MTBC DNA tests, the limit of detection was 100-1000 genome copies for fixed resistance mutations. Deeplex Myc-TB captured 97.1-99.3% of resistance phenotypes correctly predicted by WGS from 3651 MTBC genomes. On 429 isolates, the assay predicted 92.2% of 2369 first- and second-line phenotypes, with a sensitivity of 95.3% and a specificity of 97.4%. 56 out of 69 (81.2%) residual discrepancies with phenotypic results involved pyrazinamide, ethambutol and ethionamide, and low-level rifampicin or isoniazid resistance mutations, all notoriously prone to phenotypic testing variability. Only two out of 91 (2.2%) resistance phenotypes undetected by Deeplex Myc-TB had known resistance-associated mutations by WGS analysis outside Deeplex Myc-TB targets. Phenotype predictions from Deeplex Myc-TB analysis directly on 109 sputa from a Djibouti survey matched those of MTBSeq/PhyResSE/Mykrobe, fed with WGS data from subsequent cultures, with a sensitivity of 93.5/98.5/93.1% and a specificity of 98.5/97.2/95.3%, respectively. Most residual discordances involved gene deletions/indels and 3-12% heteroresistant calls undetected by WGS analysis or natural pyrazinamide resistance of globally rare "" strains then unreported by Deeplex Myc-TB. On 1494 arduous sputa from a Democratic Republic of the Congo survey, 14 902 out of 19 422 (76.7%) possible susceptible or resistance phenotypes could be predicted culture-free.Deeplex Myc-TB may enable fast, tailored tuberculosis treatment.
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http://dx.doi.org/10.1183/13993003.02338-2020DOI Listing
March 2021

Antimicrobial Susceptibility and Phylogenetic Relations in a German Cohort Infected with Mycobacterium abscessus.

J Clin Microbiol 2020 11 18;58(12). Epub 2020 Nov 18.

University Center of Infectious Diseases, UCI, University Hospital Frankfurt, Goethe University, Frankfurt am Main, Germany

is a highly antibiotic-resistant opportunistic pathogen causing clinically challenging infections in patients with preexisting lung diseases or under immunosuppression. Hence, reliable antibiotic susceptibility data are required for effective treatment. Aims of this study were to investigate (i) the congruence of genotypic and phenotypic antimicrobial susceptibility testing, (ii) the relationship between resistance profile and clinical course, and (iii) the phylogenetic relations of in a German patient cohort. A total of 39 isolates from 29 patients infected or colonized with underwent genotypic and phenotypic drug susceptibility testing. Clinical data were correlated with susceptibility data. Phylogenetic analysis was performed by means of whole-genome sequencing (WGS) and single-nucleotide polymorphism (SNP) analysis. Macrolide resistance was mainly mediated by functional Erm(41) methyltransferases (T28 sequevars) in subsp. ( = 25) and subsp. ( = 2). It was significantly associated with impaired culture conversion ( = 0.02). According to the core SNP phylogeny, we identified three clusters of closely related isolates with SNP distances below 25. Representatives of all circulating global clones (Absc. 1, Absc. 2, and Mass. 1) were identified in our cohort. However, we could not determine evidence for in-hospital interhuman transmission from clinical data. In our patient cohort, we identified three clusters with closely related isolates and representatives of the previously described international clusters but no human-to-human in-hospital transmission. Macrolide and aminoglycoside susceptibility data are critical for therapeutic decision-making in infections.
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http://dx.doi.org/10.1128/JCM.01813-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7685876PMC
November 2020

Population Structure of Mycobacterium bovis in Germany: a Long-Term Study Using Whole-Genome Sequencing Combined with Conventional Molecular Typing Methods.

J Clin Microbiol 2020 10 21;58(11). Epub 2020 Oct 21.

Friedrich-Loeffler-Institut, Federal Institute for Animal Health, Institute of Molecular Pathogenesis, Jena, Germany

is the primary cause of bovine tuberculosis (bTB) and infects a wide range of domestic animal and wildlife species and humans. In Germany, bTB still emerges sporadically in cattle herds, free-ranging wildlife, diverse captive animal species, and humans. In order to understand the underlying population structure and estimate the population size fluctuation through time, we analyzed 131 strains from animals ( = 38) and humans ( = 93) in Germany from 1999 to 2017 by whole-genome sequencing (WGS), mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing, and spoligotyping. Based on WGS data analysis, 122 out of the 131 strains were classified into 13 major clades, of which 6 contained strains from both human and animal cases and 7 only strains from human cases. Bayesian analyses suggest that the population went through two sharp anticlimaxes, one in the middle of the 18th century and another one in the 1950s. WGS-based cluster analysis grouped 46 strains into 13 clusters ranging in size from 2 to 11 members and involving strains from distinct host types, e.g., only cattle and also mixed hosts. Animal strains of four clusters were obtained over a 9-year span, pointing toward autochthonous persistent bTB infection cycles. As expected, WGS had a higher discriminatory power than spoligotyping and MIRU-VNTR typing. In conclusion, our data confirm that WGS and suitable bioinformatics constitute the method of choice to implement prospective molecular epidemiological surveillance of The population of in Germany is diverse, with subtle, but existing, interactions between different host groups.
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http://dx.doi.org/10.1128/JCM.01573-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7587083PMC
October 2020

Use of a whole genome sequencing-based approach for surveillance in Europe in 2017-2019: an ECDC pilot study.

Eur Respir J 2021 Jan 5;57(1). Epub 2021 Jan 5.

European Centre for Disease Prevention and Control, Stockholm, Sweden.

Whole genome sequencing (WGS) can be used for molecular typing and characterisation of complex (MTBC) strains. We evaluated the systematic use of a WGS-based approach for MTBC surveillance involving all European Union/European Economic Area (EU/EEA) countries and highlight the challenges and lessons learnt to be considered for the future development of a WGS-based surveillance system.WGS and epidemiological data of patients with rifampicin-resistant (RR) and multidrug-resistant (MDR) tuberculosis (TB) were collected from EU/EEA countries between January 2017 and December 2019. WGS-based genetic relatedness analysis was performed using a standardised approach including both core genome multilocus sequence typing (cgMLST) and single nucleotide polymorphism (SNP)-based calculation of distances on all WGS data that fulfilled minimum quality criteria to ensure data comparability.A total of 2218 RR/MDR-MTBC isolates were collected from 25 countries. Among these, 56 cross-border clusters with increased likelihood of recent transmission (≤5 SNPs distance) comprising 316 RR/MDR-MTBC isolates were identified. The cross-border clusters included between two and 30 resistant isolates from two to six countries, demonstrating different RR/MDR-TB transmission patterns in Western and Eastern EU countries.This pilot study shows that a WGS-based surveillance system is not only feasible but can efficiently elucidate the dynamics of in-country and cross-border RR/MDR-TB transmission across EU/EEA countries. Lessons learnt from this study highlight that the establishment of an EU/EEA centralised WGS-based surveillance system for TB will require strengthening of national integrated systems performing prospective WGS surveillance and the development of clear procedures to facilitate international collaboration for the investigation of cross-border clusters.
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http://dx.doi.org/10.1183/13993003.02272-2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7784142PMC
January 2021

A publicly accessible database for genome sequences supports tracing of transmission chains and epidemics.

Microb Genom 2020 08 29;6(8). Epub 2020 Jul 29.

Braunschweig Integrated Center of Systems Biology (BRICS), Technical University, Braunschweig, Germany.

is the primary infectious cause of antibiotic-associated diarrhea. Local transmissions and international outbreaks of this pathogen have been previously elucidated by bacterial whole-genome sequencing, but comparative genomic analyses at the global scale were hampered by the lack of specific bioinformatic tools. Here we introduce a publicly accessible database within EnteroBase (http://enterobase.warwick.ac.uk) that automatically retrieves and assembles short-reads from the public domain, and calls alleles for core-genome multilocus sequence typing (cgMLST). We demonstrate that comparable levels of resolution and precision are attained by EnteroBase cgMLST and single-nucleotide polymorphism analysis. EnteroBase currently contains 18 254 quality-controlled genomes, which have been assigned to hierarchical sets of single-linkage clusters by cgMLST distances. This hierarchical clustering is used to identify and name populations of at all epidemiological levels, from recent transmission chains through to epidemic and endemic strains. Moreover, it puts newly collected isolates into phylogenetic and epidemiological context by identifying related strains among all previously published genome data. For example, HC2 clusters (i.e. chains of genomes with pairwise distances of up to two cgMLST alleles) were statistically associated with specific hospitals (<10) or single wards (=0.01) within hospitals, indicating they represented local transmission clusters. We also detected several HC2 clusters spanning more than one hospital that by retrospective epidemiological analysis were confirmed to be associated with inter-hospital patient transfers. In contrast, clustering at level HC150 correlated with -mer-based classification and was largely compatible with PCR ribotyping, thus enabling comparisons to earlier surveillance data. EnteroBase enables contextual interpretation of a growing collection of assembled, quality-controlled genome sequences and their associated metadata. Hierarchical clustering rapidly identifies database entries that are related at multiple levels of genetic distance, facilitating communication among researchers, clinicians and public-health officials who are combatting disease caused by .
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http://dx.doi.org/10.1099/mgen.0.000410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7641423PMC
August 2020

Detection of low-frequency resistance-mediating SNPs in next-generation sequencing data of Mycobacterium tuberculosis complex strains with binoSNP.

Sci Rep 2020 05 12;10(1):7874. Epub 2020 May 12.

Molecular and Experimental Mycobacteriology, Research Center Borstel, Borstel, Germany.

Accurate drug resistance detection is key for guiding effective tuberculosis treatment. While genotypic resistance can be rapidly detected by molecular methods, their application is challenged by mixed mycobacterial populations comprising both susceptible and resistant cells (heteroresistance). For this, next-generation sequencing (NGS) based approaches promise the determination of variants even at low frequencies. However, accurate methods for a valid detection of low-frequency variants in NGS data are currently lacking. To tackle this problem, we developed the variant detection tool binoSNP which allows the determination of low-frequency single nucleotide polymorphisms (SNPs) in NGS datasets from Mycobacterium tuberculosis complex (MTBC) strains. By taking a reference-mapped file as input, binoSNP evaluates each genomic position of interest using a binomial test procedure. binoSNP was validated using in-silico, in-vitro, and serial patient isolates datasets comprising varying genomic coverage depths (100-500×) and SNP allele frequencies (1-30%). Overall, the detection limit for low-frequency SNPs depends on the combination of coverage depth and allele frequency of the resistance-associated mutation. binoSNP allows for valid detection of resistance associated SNPs at a 1% frequency with a coverage ≥400×. In conclusion, binoSNP provides a valid approach to detect low-frequency resistance-mediating SNPs in NGS data from clinical MTBC strains. It can be implemented in automated, end-user friendly analysis tools for NGS data and is a step forward towards individualized TB therapy.
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http://dx.doi.org/10.1038/s41598-020-64708-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7217866PMC
May 2020

The phylogenetic landscape and nosocomial spread of the multidrug-resistant opportunist Stenotrophomonas maltophilia.

Nat Commun 2020 04 27;11(1):2044. Epub 2020 Apr 27.

Molecular and Experimental Mycobacteriology, Research Center Borstel, Borstel, Germany.

Recent studies portend a rising global spread and adaptation of human- or healthcare-associated pathogens. Here, we analyse an international collection of the emerging, multidrug-resistant, opportunistic pathogen Stenotrophomonas maltophilia from 22 countries to infer population structure and clonality at a global level. We show that the S. maltophilia complex is divided into 23 monophyletic lineages, most of which harbour strains of all degrees of human virulence. Lineage Sm6 comprises the highest rate of human-associated strains, linked to key virulence and resistance genes. Transmission analysis identifies potential outbreak events of genetically closely related strains isolated within days or weeks in the same hospitals.
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http://dx.doi.org/10.1038/s41467-020-15123-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7184733PMC
April 2020

Comparative analysis of phenotypic and genotypic antibiotic susceptibility patterns in Mycobacterium avium complex.

Int J Infect Dis 2020 Apr 5;93:320-328. Epub 2020 Mar 5.

Institute of Medical Microbiology and Infection Control, University Hospital Frankfurt, Goethe University, Frankfurt am Main, Germany.

Objective: Phenotypic (Sensititre Myco, pDST) and genotypic drug susceptibility testing (GenoType NTM DR, gDST) in M. avium complex (MAC) have become available as standardized assays, but comparable data is needed. This study aimed to investigate the phenotypic and genotypic drug susceptibility patterns in MAC clinical isolates.

Methods: Overall, 98 isolates from 85 patients were included. pDST and gDST were performed on all isolates and results compared regarding specificity and sensitivity using pDST as a reference method. The impact of drug instability on pDST results was studied using a biological assay over 14 days. In addition, the evolution of antimicrobial resistance was investigated in sequential isolates of 13 patients.

Results: Macrolide resistance was rare, 1.2% (95% CI 0.7-7.3) of isolates in the base cohort. No aminoglycoside resistances were found, but 14.1% of the studied isolates (95% CI 7.8-23.8) showed intermediate susceptibility. The GenoType NTM DR identified two out of four macrolide-resistant isolates. Antibiotic stability was demonstrated to be poor in rifampicin, rifabutin, and doxycycylin.

Conclusions: pDST results in NTM for unstable antibiotics must be interpreted with care. A combination of pDST and gDST will be useful for the guidance of antimicrobial therapy in MAC-disease.
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http://dx.doi.org/10.1016/j.ijid.2020.02.059DOI Listing
April 2020

Phylogenetically informative mutations in genes implicated in antibiotic resistance in Mycobacterium tuberculosis complex.

Genome Med 2020 03 6;12(1):27. Epub 2020 Mar 6.

German Center for Infection Research (DZIF), Partner site Hamburg-Lübeck-Borstel-Riems, Borstel, Germany.

Background: A comprehensive understanding of the pre-existing genetic variation in genes associated with antibiotic resistance in the Mycobacterium tuberculosis complex (MTBC) is needed to accurately interpret whole-genome sequencing data for genotypic drug susceptibility testing (DST).

Methods: We investigated mutations in 92 genes implicated in resistance to 21 anti-tuberculosis drugs using the genomes of 405 phylogenetically diverse MTBC strains. The role of phylogenetically informative mutations was assessed by routine phenotypic DST data for the first-line drugs isoniazid, rifampicin, ethambutol, and pyrazinamide from a separate collection of over 7000 clinical strains. Selected mutations/strains were further investigated by minimum inhibitory concentration (MIC) testing.

Results: Out of 547 phylogenetically informative mutations identified, 138 were classified as not correlating with resistance to first-line drugs. MIC testing did not reveal a discernible impact of a Rv1979c deletion shared by M. africanum lineage 5 strains on resistance to clofazimine. Finally, we found molecular evidence that some MTBC subgroups may be hyper-susceptible to bedaquiline and clofazimine by different loss-of-function mutations affecting a drug efflux pump subunit (MmpL5).

Conclusions: Our findings underline that the genetic diversity in MTBC has to be studied more systematically to inform the design of clinical trials and to define sound epidemiologic cut-off values (ECOFFs) for new and repurposed anti-tuberculosis drugs. In that regard, our comprehensive variant catalogue provides a solid basis for the interpretation of mutations in genotypic as well as in phenotypic DST assays.
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http://dx.doi.org/10.1186/s13073-020-00726-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7060619PMC
March 2020

Multidrug- and Extensively Drug-Resistant Mycobacterium tuberculosis Beijing Clades, Ukraine, 2015.

Emerg Infect Dis 2020 03;26(3):481-490

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.
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http://dx.doi.org/10.3201/eid2603.190525DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7045844PMC
March 2020

Mycobacterium tuberculosis Complex Lineage 3 as Causative Agent of Pulmonary Tuberculosis, Eastern Sudan.

Emerg Infect Dis 2020 03;26(3):427-436

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.
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http://dx.doi.org/10.3201/eid2603.191145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7045825PMC
March 2020

Antibiotic resistance prediction for from genome sequence data with Mykrobe.

Wellcome Open Res 2019 2;4:191. Epub 2019 Dec 2.

European Bioinformatics Institute, Cambridge, UK.

Two billion people are infected with , leading to 10 million new cases of active tuberculosis and 1.5 million deaths annually. Universal access to drug susceptibility testing (DST) has become a World Health Organization priority. We previously developed a software tool, , which provided offline species identification and drug resistance predictions for from whole genome sequencing (WGS) data. Performance was insufficient to support the use of WGS as an alternative to conventional phenotype-based DST, due to mutation catalogue limitations.  Here we present a new tool, , which provides the same functionality based on a new software implementation. Improvements include i) an updated mutation catalogue giving greater sensitivity to detect pyrazinamide resistance, ii) support for user-defined resistance catalogues, iii) improved identification of non-tuberculous mycobacterial species, and iv) an updated statistical model for Oxford Nanopore Technologies sequencing data. is released under MIT license at https://github.com/mykrobe-tools/mykrobe. We incorporate mutation catalogues from the CRyPTIC consortium et al. (2018) and from Walker et al. (2015), and make improvements based on performance on an initial set of 3206 and an independent set of 5845 Illumina sequences. To give estimates of error rates, we use a prospectively collected dataset of 4362 . Using culture based DST as the reference, we estimate to be 100%, 95%, 82%, 99% sensitive and 99%, 100%, 99%, 99% specific for rifampicin, isoniazid, pyrazinamide and ethambutol resistance prediction respectively. We benchmark against four other tools on 10207 (=5845+4362) samples, and also show that gives concordant results with nanopore data.  We measure the ability of -based DST to guide personalized therapeutic regimen design in the context of complex drug susceptibility profiles, showing 94% concordance of implied regimen with that driven by phenotypic DST, higher than all other benchmarked tools.
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http://dx.doi.org/10.12688/wellcomeopenres.15603.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7004237PMC
December 2019

A landscape of genomic alterations at the root of a near-untreatable tuberculosis epidemic.

BMC Med 2020 02 4;18(1):24. Epub 2020 Feb 4.

South African Medical Research Council Centre for Tuberculosis Research, DST NRF Centre of Excellence for Biomedical Tuberculosis research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa.

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.
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http://dx.doi.org/10.1186/s12916-019-1487-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6998097PMC
February 2020

Towards standardisation: comparison of five whole genome sequencing (WGS) analysis pipelines for detection of epidemiologically linked tuberculosis cases.

Euro Surveill 2019 Dec;24(50)

Tuberculosis Reference Laboratory, National Institute for Public Health and the Environment (RIVM), Bilthoven, the Netherlands.

BackgroundWhole genome sequencing (WGS) is a reliable tool for studying tuberculosis (TB) transmission. WGS data are usually processed by custom-built analysis pipelines with little standardisation between them.AimTo compare the impact of variability of several WGS analysis pipelines used internationally to detect epidemiologically linked TB cases.MethodsFrom the Netherlands, 535 complex (MTBC) strains from 2016 were included. Epidemiological information obtained from municipal health services was available for all mycobacterial interspersed repeat unit-variable number of tandem repeat (MIRU-VNTR) clustered cases. WGS data was analysed using five different pipelines: one core genome multilocus sequence typing (cgMLST) approach and four single nucleotide polymorphism (SNP)-based pipelines developed in Oxford, United Kingdom; Borstel, Germany; Bilthoven, the Netherlands and Copenhagen, Denmark. WGS clusters were defined using a maximum pairwise distance of 12 SNPs/alleles.ResultsThe cgMLST approach and Oxford pipeline clustered all epidemiologically linked cases, however, in the other three SNP-based pipelines one epidemiological link was missed due to insufficient coverage. In general, the genetic distances varied between pipelines, reflecting different clustering rates: the cgMLST approach clustered 92 cases, followed by 84, 83, 83 and 82 cases in the SNP-based pipelines from Copenhagen, Oxford, Borstel and Bilthoven respectively.ConclusionConcordance in ruling out epidemiological links was high between pipelines, which is an important step in the international validation of WGS data analysis. To increase accuracy in identifying TB transmission clusters, standardisation of crucial WGS criteria and creation of a reference database of representative MTBC sequences would be advisable.
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http://dx.doi.org/10.2807/1560-7917.ES.2019.24.50.1900130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6918587PMC
December 2019