The Forest behind the Tree: Phylogenetic Exploration of a Dominant Mycobacterium tuberculosis Strain Lineage from a High Tuberculosis Burden Country

BACKGROUND: Genotyping of Mycobacterium tuberculosis isolates is a powerful tool for epidemiological control of tuberculosis (TB) and phylogenetic exploration of the pathogen. Standardized PCR-based typing, based on 15 to 24 mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) loci combined with spoligotyping, has been shown to have adequate resolution power for tracing TB transmission and to be useful for predicting diverse strain lineages in European settings. Its informative value needs to be tested in high TB-burden countries, where the use of genotyping is often complicated by dominance of geographically specific, genetically homogeneous strain lineages. METHODOLOGY/PRINCIPAL FINDINGS: We tested this genotyping system for molecular epidemiological analysis of 369 M. tuberculosis isolates from 3 regions of Brazil, a high TB-burden country. Deligotyping, targeting 43 large sequence polymorphisms (LSPs), and the MIRU-VNTRplus identification database were used to assess phylogenetic predictions. High congruence between the different typing results consistently revealed the countrywide supremacy of the Latin-American-Mediterranean (LAM) lineage, comprised of three main branches. In addition to an already known RDRio branch, at least one other branch characterized by a phylogenetically informative LAM3 spoligo-signature seems to be globally distributed beyond Brazil. Nevertheless, by distinguishing 321 genotypes in this strain population, combined MIRU-VNTR typing and spoligotyping demonstrated the presence of multiple distinct clones. The use of 15 to 24 loci discriminated 21 to 25% more strains within the LAM lineage, compared to a restricted lineage-specific locus set suggested to be used after SNP analysis. Noteworthy, 23 of the 28 molecular clusters identified were exclusively composed of patient isolates from a same region, consistent with expected patterns of mostly local TB transmission. CONCLUSIONS/SIGNIFICANCE: Standard MIRU-VNTR typing combined with spoligotyping can reveal epidemiologically meaningful clonal diversity behind a dominant M. tuberculosis strain lineage in a high TB-burden country and is useful to explore international phylogenetical ramifications

Whole-genome sequencing for prediction of Mycobacterium tuberculosis drug susceptibility and resistance : a retrospective cohort study

BACKGROUND : Diagnosing drug-resistance remains an obstacle to the elimination of tuberculosis. Phenotypic drugsusceptibility testing is slow and expensive, and commercial genotypic assays screen only common resistancedetermining mutations. We used whole-genome sequencing to characterise common and rare mutations predicting drug resistance, or consistency with susceptibility, for all fi rst-line and second-line drugs for tuberculosis. METHODS : Between Sept 1, 2010, and Dec 1, 2013, we sequenced a training set of 2099 Mycobacterium tuberculosis genomes. For 23 candidate genes identifi ed from the drug-resistance scientifi c literature, we algorithmically characterised genetic mutations as not conferring resistance (benign), resistance determinants, or uncharacterised. We then assessed the ability of these characterisations to predict phenotypic drug-susceptibility testing for an independent validation set of 1552 genomes. We sought mutations under similar selection pressure to those characterised as resistance determinants outside candidate genes to account for residual phenotypic resistance. FINDINGS : We characterised 120 training-set mutations as resistance determining, and 772 as benign. With these mutations, we could predict 89·2% of the validation-set phenotypes with a mean 92·3% sensitivity (95% CI 90·7–93·7) and 98·4% specifi city (98·1–98·7). 10·8% of validation-set phenotypes could not be predicted because uncharacterised mutations were present. With an in-silico comparison, characterised resistance determinants had higher sensitivity than the mutations from three line-probe assays (85·1% vs 81·6%). No additional resistance determinants were identifi ed among mutations under selection pressure in non-candidate genes. INTERPRETATION : A broad catalogue of genetic mutations enable data from whole-genome sequencing to be used clinically to predict drug resistance, drug susceptibility, or to identify drug phenotypes that cannot yet be genetically predicted. This approach could be integrated into routine diagnostic workfl ows, phasing out phenotypic drugsusceptibility testing while reporting drug resistance early.Wellcome Trust, National Institute of Health Research, Medical Research Council, and the European Union.http://www.thelancet.com/infectionhb201

Marqueurs génétiques du complexe Mycobacterium tuberculosis: études phylogénétiques et épidémiologiques de la tuberculose

Doctorat en Sciences médicalesinfo:eu-repo/semantics/nonPublishe

Marqueurs génétiques du complexe Mycobacterium tuberculosis: études phylogénétiques et épidémiologiques de la tuberculose

Doctorat en Sciences médicalesinfo:eu-repo/semantics/nonPublishe

Three-Year Population-Based Evaluation of Standardized Mycobacterial Interspersed Repetitive-Unit-Variable-Number Tandem-Repeat Typing of Mycobacterium tuberculosis▿

Standardized mycobacterial interspersed repetitive-unit-variable-number tandem repeat (MIRU-VNTR) typing based on 15 and 24 loci recently has been proposed for Mycobacterium tuberculosis genotyping. So far, this optimized system has been assessed in a single, 1-year population-based study performed in Germany (M. C. Oelemann, R. Diel, V. Vatin, W. Haas, S. Rusch-Gerdes, C. Locht, S. Niemann, and P. Supply, J. Clin. Microbiol. 45:691-697, 2007). Here, we evaluated these optimized formats in a much larger population-based study conducted during 39 months in the Brussels capital region of Belgium. Isolates from 807 patients were genotyped. The resolution power, cluster, and lineage identification by the standardized MIRU-VNTR sets were compared to those obtained using standardized IS6110-restriction fragment length polymorphism (RFLP), spoligotyping, and a previous 12-MIRU-VNTR-locus set. On a subset representing 77% of the cases during a 16-month period, a high concordance was observed between unique isolates or strain clusters as defined by standardized MIRU-VNTR and IS6110-RFLP (i.e., more than five IS6110 bands). When extended to the entire population-based collection, the discriminatory subset of 15 loci decreased the strain-clustering rate by almost twofold compared to that of the old 12-locus set. The addition of the nine ancillary MIRU-VNTR loci and/or spoligotyping only slightly further decreased this strain-clustering rate. Familial, social, and/or geographic proximity links were found in 48% of the clusters identified, and well-known risk factors for tuberculosis transmission were identified. Finally, an excellent correspondence was determined between our MIRU-VNTR-spoligotyping strain identifications and external reference strain lineages included in the MIRU-VNTRplus database and identified by, e.g., large sequence polymorphisms. Our results reinforce the proposal of standardized MIRU-VNTR typing as a new reference genotyping method for the epidemiological and phylogenetic screening of M. tuberculosis strains

Revision of hospital work organization using nurse and healthcare assistant workload indicators as decision aid tools

International audienceBackground: Historically, governmental hospital organisation consisted in a heterogeneous distribution of staff and a fragmented logistical organisation without cross-functionality or sharing of resources between departments. This organisation could not last in a context of an evolving healthcare environment, changing patient profiles and hospital expenditure constraints. Cost-effective workforce regulation for optimal patient quality of care was urgently needed. The purpose of the study was to describe the reorganization that led to resource management no longer based on what has been achieved but based on a daily measured workload. Methods: This prospective study used nursing intensity indicator, mirroring patient care needs, which was reported daily using VALPAReSO® software. Indirect care activities were recorded in departments of medicine, surgery and obstetrics. Based on data collected in 2012, a new organisation strategy was implemented and evaluated in 2015. Results: Nursing intensity indicator analysis led to a reallocation of workforce per department, and the reinforcement unit (float pool) was managed based on this decision-aid tool for replacement and daily adequate staffing. The healthcare workflow audit resulted in the revision of five working tasks: time spent on handover, working time management, connections between services and the pharmacy, housekeeping, and food management. The reorganization took place at the same time as the transition to the development of very short-term care, resulting in a decrease in the number of full inpatient beds, which were therefore mainly occupied by heavier care profile patients. With the integrated strategy, this transition was achieved with constant staffing, and good overall patient satisfaction and working conditions were maintained. Conclusion: The reorganisation strategy was managed in a context of institutional commitment, coaching leadership built on close manager-employee interaction, a defragmented management between healthcare and all service providers, and a seamlessly dissemination and sharing of indicator information between healthcare managers, nurses and healthcare assistants. The process optimization allowed a better allocation of tasks and enabled nurses to refocus on patient care. Nursing intensity and indirect care indicators, when widely accepted, can be used as decision support tools for daily adequate staffing

Evaluation and Strategy for Use of MIRU-VNTRplus, a Multifunctional Database for Online Analysis of Genotyping Data and Phylogenetic Identification of Mycobacterium tuberculosis Complex Isolates▿

Because of its portable data, discriminatory power, and recently proposed standardization, mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing has become a major method for the epidemiological tracking of Mycobacterium tuberculosis complex (MTBC) clones. However, no public MIRU-VNTR database based on well-characterized reference strains has been available hitherto for easy strain identification. Therefore, a collection of 186 reference strains representing the primary MTBC lineages was used to build a database, which is freely accessible at http://www.MIRU-VNTRplus.org. The geographical origin and the drug susceptibility profile of each strain were stored together with comprehensive genetic lineage information, including the 24-locus MIRU-VNTR profile, the spoligotyping pattern, the single-nucleotide- and large-sequence-polymorphism profiles, and the IS6110 restriction fragment length polymorphism fingerprint. Thanks to flexible import functions, a single or multiple user strains can be analyzed, e.g., for lineage identification with or without the use of reference strains, by best-match or tree-based analyses with single or combined marker data sets. The results can easily be exported. In the present study, we evaluated the database consistency and various analysis parameters both by testing the reference collection against itself and by using an external population-based data set comprising 629 different strains. Under the optimal conditions found, lineage predictions based on typing by 24-locus MIRU-VNTR analysis optionally combined with spoligotyping were verified in >99% of the cases. On the basis of this evaluation, a user strategy was defined, which consisted of best-match analysis followed, if necessary, by tree-based analysis. The MIRU-VNTRplus database is a powerful tool for high-resolution clonal identification and has little equivalent in terms of functionalities among the bacterial genotyping databases available so far

Combined Multilocus Short-Sequence-Repeat and Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem-Repeat Typing of Mycobacterium avium subsp paratuberculosis Isolates

International audienceShort-sequence-repeat (SSR) sequencing was applied to 127 Mycobacterium avium subsp. paratuberculosis isolates typed by mycobacterial interspersed repetitive unit-variable-number tandem repeats (MIRU-VNTR) and IS900 restriction fragment length polymorphism (RFLP). Combined MIRU-VNTR and SSR typing followed by secondary IS900 RFLP typing is an improved approach to high-resolution genotyping of this pathogen

Admixed Phylogenetic Distribution of Drug Resistant <em>Mycobacterium tuberculosis</em> in Saudi Arabia

<div><h3>Background</h3><p>The phylogeographical structure of <em>Mycobacterium tuberculosis</em> is generally bimodal in low tuberculosis (TB) incidence countries, where genetic lineages of the isolates generally differ with little strain clustering between autochthonous and foreign-born TB patients. However, less is known on this structure in Saudi Arabia—the most important hub of human migration as it hosts a total population of expatriates and pilgrims from all over the world which is equal to that of its citizens.</p> <h3>Methodology</h3><p>We explored the mycobacterial phylogenetic structure and strain molecular clustering in Saudi Arabia by genotyping 322 drug-resistant clinical isolates collected over a 12-month period in a national drug surveillance survey, using 24 locus-based MIRU-VNTR typing and spoligotyping.</p> <h3>Principal Findings</h3><p>In contrast to the cosmopolitan population of the country, almost all the known phylogeographic lineages of <em>M. tuberculosis</em> complex (with noticeable exception of <em>Mycobacterium africanum</em>/West-African 1 and 2) were detected, with Delhi/CAS (21.1%), EAI (11.2%), Beijing (11.2%) and main branches of the Euro-American super-lineage such as Ghana (14.9%), Haarlem (10.6%) and Cameroon (7.8%) being represented. Statistically significant associations of strain lineages were observed with poly-drug resistance and multi drug resistance especially among previously treated cases (p value of < = 0.001 for both types of resistance), with relative over-representation of Beijing strains in the latter category. However, there was no significant difference among Saudi and non-Saudi TB patients regarding distribution of phylogenetic lineages (p = 0.311). Moreover, 59.5% (22/37) of the strain molecular clusters were shared between the Saudi born and immigrant TB patients.</p> <h3>Conclusions</h3><p>Specific distribution of <em>M. tuberculosis</em> phylogeographic lineages is not observed between the autochthonous and foreign-born populations. These observations might reflect both socially favored ongoing TB transmission between the two population groups, and historically deep-rooted, prolonged contacts and trade relations of the peninsula with other world regions. More vigorous surveillance and strict adherence to tuberculosis control policies are urgently needed in the country.</p> </div