A Diagnostic Algorithm To Investigate Pyrazinamide and Ethambutol Resistance in Rifampin-Resistant Mycobacterium tuberculosis Isolates in a Low-Incidence Setting.

Phenotypic drug susceptibility testing (DST) for the two first-line tuberculosis drugs ethambutol and pyrazinamide is known to yield unreliable and inaccurate results. In this prospective study, we propose a diagnostic algorithm combining phenotypic DST with Sanger sequencing to inform clinical decision-making for drug-resistant Mycobacterium tuberculosis complex isolates. Sequencing results were validated using whole-genome sequencing (WGS) of the isolates. Resistance-conferring mutations obtained by pncA sequencing correlated well with phenotypic DST results for pyrazinamide. Phenotypic resistance to ethambutol was only partly explained by mutations in the embB 306 codon. Additional resistance-conferring mutations were found in the embB gene at codons 354, 406, and 497. In several isolates that tested ethambutol susceptibility by phenotypic DST, well-known resistance-conferring embB mutations were determined. Thus, targeted Sanger sequencing beyond the embB 306 codon or WGS together with phenotypic DST should be employed to ensure reliable ethambutol drug susceptibility testing, as a basis for the rational design of multidrug-resistant tuberculosis regimens with or without ethambutol

Molecular Epidemiology of Mycobacterium tuberculosis Complex Strains in Urban and Slum Settings of Nairobi, Kenya

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 chain

Pathogen-based precision medicine for drug-resistant tuberculosis

The implementation of next generation sequencing techniques, such as whole genome sequencing (WGS), in tuberculosis (TB) research has enabled timely, cost-effective, and comprehensive insights into the genetic repertoire of the human pathogens of the Mycobacterium tuberculosis complex (MTBC). WGS data allow for detailed epidemiological analysis based on genomic distance of the MTBC strains under investigation, e.g. for tracing outbreaks; it can accelerate diagnostics by predicting drug resistance from a mutation catalogue (figure 1). Indeed, specific mutations even permit predictions on the possible clinical treatment course and outcome

Linezolid resistance of Mycobacterium tuberculosis – associated mutations and risk factors: cross-sectional retrospective analytical study

Nicolae Testemitanu State University of Medicine and Pharmacy, Chisinau, Republic of Moldova, Chiril Draganiuc Phthisiopneumology Institute, Chisinau, Republic of Moldova, German Centre for Infection Research (DZIF), Partner site Hamburg-Lübeck-Borstel- Riems, Clinical Infectious Diseases, Research Center Borstel, Borstel Germany, National and Supranational Reference Center for Mycobacteria, Research Center Borstel, Borstel Germany, Molecular and Experimental Mycobacteriology, Research Center Borstel, Borstel, Germany, Respiratory Medicine & International Health, University of Lübeck, Lübeck, Germany, Department of Medicine, Umeå University, Umeå, Sweden, Global TB Program, Baylor College of Medicine and Texas Children´s Hospital, Houston, TX, USAIntroducere. Linezolidul (LNZ) este unul dintre principalele medicamente utilizate în tratamentul tuberculozei multidrog- rezistente (TB-MDR). La moment determinantele genetice asociate cu rezistenţa la LNZ nu sunt pe deplin caracterizate. Scopul studiului a fost de a descrie mutaţiile asociate cu rezistenţa la LNZ prin aplicarea secvenţierii întregului genom (WGS) și evaluarea factorilor de risc asociaţi cu rezistenţa la LNZ pe un set de tulpini de Mycobacterium tuberculosis (MTB) izolate de la bolnavii de TB-MDR din Republica Moldova. Material și metode. A fost realizat un studiu retrospectiv transversal pe un set de tulpini de MTB preluate din biobanca Laboratorului Naţional de Referinţă în Microbiologia Tuberculozei (LNRM). Au fost identificate toate tulpinile MTB stocate în perioada 2017-2018, obţinute din sputa pacienţilor cu TB-MDR, care au administrat LNZ în schema de tratament pe parcursul ultimilor doi ani, indiferent de durata administrării LNZ. Au fost considerate eligibile pentru studiu doar tulpinile MTB izolate de la pacienţii cu o expunere cumulativă la LNZ mai mare de 30 de zile. Datele WGS ale tulpinilor de MTB incluse în studiu au fost comparate cu rezultatele testelor fenotipice de sensibilitate pe medii lichide pentru 3 concentraţii de LNZ (0,5 mg/L; 1,0 mg/L; 2,0 mg/L). Caracteristicile clinice ale pacienţilor din studiu au fost preluate din Registrul naţional electronic al bolnavilor cu TB (SIME-TB). Regresia logistică multiplă a fost efectuată pentru a determina factorii de risc pentru rezistenţa la LNZ. Rezultate. 52/74 (70,3%) tulpini MTB au fost incluse în studiu, dintre acestea 15 (28,8%) au fost fenotipic rezistente la LNZ, dintre care în cazul a 12/52 (23,1%) tulpini a fost găsită o corelarea genotipică cu rezistenţa la LNZ. În 8 cazuri au fost găsite mutaţii în gena rplC (460T>C). În alte 4 cazuri, au fost identificate mutaţii în gena rrl (2746G>A; 2814G>T; 2810A>C; 2270G>T). S-a determinat o asociere semnificativă între rezistenţa la LNZ și numărul de medicamente active din regimul de tratament TB-MDR (OR 0,23; 95%CI -0,03 - 0,70; p = 0,04). O asociere mai slabă s-a stabilit cu numărul de doze de LNZ administrate anterior (OR 1,01; 95%CI 1,004-1,03; p = 0,03). Concluzii. La majoritatea tulpinilor de MTB studiate, rezistenţa la LNZ a fost asociată cu mutaţii în genele rrl și rplC. Numărul insuficient de medicamente active în regimul de tratament al TB-MDR crește șansele de apariţie a rezistenţei la LNZ.Introduction. Linezolid (LNZ) is one of the main drugs used for multidrug-resistant tuberculosis (MDR-TB) treatment. Genetic targets associated with resistance to LNZ are not fully characterized. The aim of the study was to describe mutations associated with LNZ resistance by applying whole genome sequencing (WGS) and to assess the risk factors for LNZ resistance in a set of M. tuberculosis clinical isolates from the Republic of Moldova. Material and methods. We conducted a retrospective cross-sectional study on a set of MTB isolates retrieved from the biobank of National Reference Laboratory for Tuberculosis Microbiology (NRLM). We identified all sputum culture isolates stored during 2017-2018, obtained from adult MDR-TB patients who had LNZ as part of their MDR-TB treatment at any time during two years before the collection corresponding sputum sample. Only isolates from patients with a cumulative exposure to LNZ longer than 30 days were considered eligible for the study. We performed WGS of the MTB strains and compared these results with liquid culture-based susceptibility tests on 3 concentrations of LNZ (0.5 mg/L; 1.0 mg/L; 2.0 mg/L). The clinical characteristics of the study patients were retrieved form the National TB Data Base (SIME-TB). The multiple regression analysis was performed to assess risk factors for LNZ resistance. Results. 52/74 (70,3%) isolates were included into the study of them 15 (28,8%) were phenotypically LNZ resistant. However only in 12/52 (23,1%) isolates genotypic correlates of LNZ resistant was found. In 8 cases mutations were detected in the rplC gene (460T>C). In other 4 cases the identified mutation implied rrl gene (2746G>A; 2814G>T; 2810A>C; 2270G>T). In a multivariate logistic regression model, significant association between LNZ resistance and the number of the active drugs in the MDR-TB treatment regimen (OR 0,23; 95%CI 0,03 – 0,70; p = 0,04) was found. A weaker association was found with the number of the previously taken LNZ doses (OR 1,01; 95%CI 1,004 – 1,03; p=0,03). Conclusions. In most of the studied MTB isolates LNZ resistance was associated with mutations in the rrl and rplC genes. A reduce number of the active drugs in the LNZ containing treatment regimen increase the chance of LNZ resistance emergence

Bedaquiline and clofazimine resistance in Mycobacterium tuberculosis: an in-vitro and in-silico data analysis

Background: Bedaquiline is a core drug for the treatment of multidrug-resistant tuberculosis; however, the understanding of resistance mechanisms is poor, which is hampering rapid molecular diagnostics. Some bedaquiline-resistant mutants are also cross-resistant to clofazimine. To decipher bedaquiline and clofazimine resistance determinants, we combined experimental evolution, protein modelling, genome sequencing, and phenotypic data. Methods: For this in-vitro and in-silico data analysis, we used a novel in-vitro evolutionary model using subinhibitory drug concentrations to select bedaquiline-resistant and clofazimine-resistant mutants. We determined bedaquiline and clofazimine minimum inhibitory concentrations and did Illumina and PacBio sequencing to characterise selected mutants and establish a mutation catalogue. This catalogue also includes phenotypic and genotypic data of a global collection of more than 14 000 clinical Mycobacterium tuberculosis complex isolates, and publicly available data. We investigated variants implicated in bedaquiline resistance by protein modelling and dynamic simulations. Findings: We discerned 265 genomic variants implicated in bedaquiline resistance, with 250 (94%) variants affecting the transcriptional repressor (Rv0678) of the MmpS5–MmpL5 efflux system. We identified 40 new variants in vitro, and a new bedaquiline resistance mechanism caused by a large-scale genomic rearrangement. Additionally, we identified in vitro 15 (7%) of 208 mutations found in clinical bedaquiline-resistant isolates. From our in-vitro work, we detected 14 (16%) of 88 mutations so far identified as being associated with clofazimine resistance and also seen in clinically resistant strains, and catalogued 35 new mutations. Structural modelling of Rv0678 showed four major mechanisms of bedaquiline resistance: impaired DNA binding, reduction in protein stability, disruption of protein dimerisation, and alteration in affinity for its fatty acid ligand. Interpretation: Our findings advance the understanding of drug resistance mechanisms in M tuberculosis complex strains. We have established an extended mutation catalogue, comprising variants implicated in resistance and susceptibility to bedaquiline and clofazimine. Our data emphasise that genotypic testing can delineate clinical isolates with borderline phenotypes, which is essential for the design of effective treatments. Funding: Leibniz ScienceCampus Evolutionary Medicine of the Lung, Deutsche Forschungsgemeinschaft, Research Training Group 2501 TransEvo, Rhodes Trust, Stanford University Medical Scientist Training Program, National Institute for Health and Care Research Oxford Biomedical Research Centre, Oxford University Hospitals NHS Foundation Trust, Bill & Melinda Gates Foundation, Wellcome Trust, and Marie Skłodowska-Curie Actions

Evaluation of whole- genome sequence data analysis approaches for short- and long- read sequencing of Mycobacterium tuberculosis

Whole-genome sequencing (WGS) of Mycobacterium tuberculosis (MTB) isolates can be used to get an accurate diagnosis, to guide clinical decision making, to control tuberculosis (TB) and for outbreak investigations. We evaluated the performance of long-read (LR) and/or short-read (SR) sequencing for anti-TB drug-resistance prediction using the TBProfiler and Mykrobe tools, the fraction of genome recovery, assembly accuracies and the robustness of two typing approaches based on core-genome SNP (cgSNP) typing and core-genome multi-locus sequence typing (cgMLST). Most of the discrepancies between phenotypic drug-susceptibility testing (DST) and drug-resistance prediction were observed for the first-line drugs rifampicin, isoniazid, pyrazinamide and ethambutol, mainly with LR sequence data. Resistance prediction to second-line drugs made by both TBProfiler and Mykrobe tools with SR- and LR-sequence data were in complete agreement with phenotypic DST except for one isolate. The SR assemblies were more accurate than the LR assemblies, having significantly (P<0.05) fewer indels and mismatches per 100 kbp. However, the hybrid and LR assemblies had slightly higher genome fractions. For LR assemblies, Canu followed by Racon, and Medaka polishing was the most accurate approach. The cgSNP approach, based on either reads or assemblies, was more robust than the cgMLST approach, especially for LR sequence data. In conclusion, anti-TB drug-resistance prediction, particularly with only LR sequence data, remains challenging, especially for first-line drugs. In addition, SR assemblies appear more accurate than LR ones, and reproducible phylogeny can be achieved using cgSNP approaches

FAM222B Is Not a Likely Novel Candidate Gene for Cerebral Cavernous Malformations

Cerebral cavernous malformations (CCMs) are prevalent slow-flow vascular lesions which harbour the risk to develop intracranial haemorrhages, focal neurological deficits, and epileptic seizures. Autosomal dominantly inherited CCMs were found to be associated with heterozygous inactivating mutations in 3 genes, CCM1(KRIT1), CCM2(MGC4607), and CCM3(PDCD10) in 1999, 2003 and 2005, respectively. Despite the availability of high-throughput sequencing techniques, no further CCM gene has been published since. Here, we report on the identification of an autosomal dominantly inherited frameshift mutation in a gene of thus far unknown function, FAM222B(C17orf63), through exome sequencing of CCM patients mutation-negative for CCM1-3. A yeast 2-hybrid screen revealed interactions of FAM222B with the tubulin cytoskeleton and STAMBP which is known to be associated with microcephaly-capillary malformation syndrome. However, a phenotype similar to existing models was not found, neither in fam222bb/fam222ba double mutant zebrafish generated by transcription activator-like effector nucleases nor in an in vitro sprouting assay using human umbilical vein endothelial cells transfected with siRNA against FAM222B. These observations led to the assumption that aberrant FAM222B is not involved in the formation of CCMs

Operationalizing multimorbidity and autonomy for health services research in aging populations - the OMAHA study

<p>Abstract</p> <p>Background</p> <p>As part of a Berlin-based research consortium on health in old age, the OMAHA (Operationalizing Multimorbidity and Autonomy for Health Services Research in Aging Populations) study aims to develop a conceptual framework and a set of standardized instruments and indicators for continuous monitoring of multimorbidity and associated health care needs in the population 65 years and older.</p> <p>Methods/Design</p> <p>OMAHA is a longitudinal epidemiological study including a comprehensive assessment at baseline and at 12-month follow-up as well as brief intermediate telephone interviews at 6 and 18 months. In order to evaluate different sampling procedures and modes of data collection, the study is conducted in two different population-based samples of men and women aged 65 years and older. A geographically defined sample was recruited from an age and sex stratified random sample from the register of residents in Berlin-Mitte (Berlin OMAHA study cohort, n = 299) for assessment by face-to-face interview and examination. A larger nationwide sample (German OMAHA study cohort, n = 730) was recruited for assessment by telephone interview among participants in previous German Telephone Health Surveys. In both cohorts, we successfully applied a multi-dimensional set of instruments to assess multimorbidity, functional disability in daily life, autonomy, quality of life (QoL), health care services utilization, personal and social resources as well as socio-demographic and biographical context variables. Response rates considerably varied between the Berlin and German OMAHA study cohorts (22.8% vs. 59.7%), whereas completeness of follow-up at month 12 was comparably high in both cohorts (82.9% vs. 81.2%).</p> <p>Discussion</p> <p>The OMAHA study offers a wide spectrum of data concerning health, functioning, social involvement, psychological well-being, and cognitive capacity in community-dwelling older people in Germany. Results from the study will add to methodological and content-specific discourses on human resources for maintaining quality of life and autonomy throughout old age, even in the face of multiple health complaints.</p

Mycobacterium tuberculosis Complex Lineage 3 as Causative Agent of Pulmonary Tuberculosis, Eastern Sudan1.

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

Perspectives for systems biology in the management of tuberculosis

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.Publisher PDFPeer reviewe