Role of the first WHO mutation catalogue in the diagnosis of antibiotic resistance in Mycobacterium tuberculosis in the Valencia Region, Spain: a retrospective genomic analysis

9 páginas, 2 figuras, 1 tablaBackground: In June, 2021, WHO published the most complete catalogue to date of resistance-conferring mutations in Mycobacterium tuberculosis. Here, we aimed to assess the performance of genome-based antimicrobial resistance prediction using the catalogue and its potential for improving diagnostics in a real low-burden setting. Methods: In this retrospective population-based genomic study M tuberculosis isolates were collected from 25 clinical laboratories in the low-burden setting of the Valencia Region, Spain. Culture-positive tuberculosis cases reported by regional public health authorities between Jan 1, 2014, and Dec 31, 2016, were included. The drug resistance profiles of these isolates were predicted by the genomic identification, via whole-genome sequencing (WGS), of the high-confidence resistance-causing variants included in the catalogue and compared with the phenotype. We determined the minimum inhibitory concentration (MIC) of the isolates with discordant resistance profiles using the resazurin microtitre assay. Findings: WGS was performed on 785 M tuberculosis complex culture-positive isolates, and the WGS resistance prediction sensitivities were: 85·4% (95% CI 70·8–94·4) for isoniazid, 73·3% (44·9–92·2) for rifampicin, 50·0% (21·1–78·9) for ethambutol, and 57·1% (34·0–78·2) for pyrazinamide; all specificities were more than 99·6%. Sensitivity values were lower than previously reported, but the overall pan-susceptibility accuracy was 96·4%. Genotypic analysis revealed that four phenotypically susceptible isolates carried mutations (rpoB Leu430Pro and rpoB Ile491Phe for rifampicin and fabG1 Leu203Leu for isoniazid) known to give borderline resistance in standard phenotypic tests. Additionally, we identified three putative resistance-associated mutations (inhA Ser94Ala, katG Leu48Pro, and katG Gly273Arg for isoniazid) in samples with substantially higher MICs than those of susceptible isolates. Combining both genomic and phenotypic data, in accordance with the WHO diagnostic guidelines, we could detect two new multidrug-resistant cases. Additionally, we detected 11 (1·6%) of 706 isolates to be monoresistant to fluoroquinolone, which had been previously undetected. Interpretation: We showed that the WHO catalogue enables the detection of resistant cases missed in phenotypic testing in a low-burden region, thus allowing for better patient-tailored treatment. We also identified mutations not included in the catalogue, relevant at the local level. Evidence from this study, together with future updates of the catalogue, will probably lead in the future to the partial replacement of culture testing with WGS-based drug susceptibility testing in our setting. Funding: European Research Council and the Spanish Ministerio de Ciencia.This project received funding from the European Research Council under the European Union’s Horizon 2020 Research and Innovation Program Grant 101001038 (TB-RECONNECT; awarded to IC), from Ministerio de Ciencia (Spanish Government) Project PID2019-104477RB-I00 (awarded to IC), and from Generalitat Valenciana Project AICO/2018/113 (awarded to IC). AMG-M is funded by a Formación deProfesorado Universitario grant programme (FPU19/04562) from Ministerio de Universidades (Spanish Government). IC is also supported by the European Commission–NextGenerationEU, through Centro Superior de Investigaciones Científicas Global Health Platform (PTI Salud Global). We thank all the members of the Valencia RegionTuberculosis Working Group

Perfil clínico de los pacientes tratados con evolocumab en unidades hospitalarias de nefrología en España

Describir las características clínicas de los pacientes tratados con evolocumab, las razones del inicio de la terapia y los efectos del tratamiento en la fase inicial de disponibilidad de evolocumab en las unidades de nefrología de España

Unravelling the population structure and transmission patterns of Mycobacterium tuberculosis in Mozambique, a high TB/HIV burden country

Genomic studies of Mycobacterium tuberculosis complex (MTBC) might shed light on the dynamics of its transmission, especially in high-burden settings, where recent outbreaks are embedded in the complex natural history of the disease. We applied Whole-genome sequencing (WGS) to characterize the local population of MTBC, unravel potential transmission links and evaluate associations with host and pathogen factors. Methods A one-year prospective study was conducted in Mozambique, a high HIV/TB burden country. WGS was applied to 295 positive cultures. We combined phylogenetic, geographical and clustering analysis, and investigated associations between risk factors of transmission. Findings A significant high proportion of strains were in recent transmission (45.5%). We fully characterized MTBC isolates by using phylogenetic approaches and dating evaluation. We found two likely endemic clades, comprised of 67 strains, belonging to L1.2, dating from the late XIX century and associated with recent spread among PLHIV. Interpretation Our results unveil the population structure of MTBC in our setting. The clustering analysis revealed an unexpected pattern of spread and high rates of progression, suggesting the failure of control measures. The long-term presence of local strains in Mozambique, which were responsible for large transmission among HIV/TB coinfected patients, hint at possible coevolution with sympatric host populations and challenge the role of HIV in TB transmission.Ministry of Enterprise and Knowledge (Government of Catalonia & European Social Fund, AGAUR fellowship); European Research Council (ERC) European Union’s Horizon 2020.N

XVI Congreso Galego de Estatística e Investigación de Operacións ; I Xornadas Innovación Docente na Estatística e Investigación de Operacións : libro de actas

O presente libro de actas recolle o resumo das catro conferencias plenarias e os 56 traballos presentados: 41 comunicacións orais, das que 9 son traballos que optan ao premio a investigadores novos e 3 son traballos presentados na sesión de biometría que organizan conxuntamente a SGAPEIO e a Sociedade Portuguesa de Estatística (SPE); 11 pósteres e 4 comunicacións orais nas xornadas de innovación docente

Population-based sequencing of Mycobacterium tuberculosis reveals how current population dynamics are shaped by past epidemics

23 páginas, 4 figuras, 1 tabla.Transmission is a driver of tuberculosis (TB) epidemics in high-burden regions, with assumed negligible impact in low-burden areas. However, we still lack a full characterization of transmission dynamics in settings with similar and different burdens. Genomic epidemiology can greatly help to quantify transmission, but the lack of whole genome sequencing population-based studies has hampered its application. Here, we generate a population-based dataset from Valencia region and compare it with available datasets from different TB-burden settings to reveal transmission dynamics heterogeneity and its public health implications. We sequenced the whole genome of 785 Mycobacterium tuberculosis strains and linked genomes to patient epidemiological data. We use a pairwise distance clustering approach and phylodynamic methods to characterize transmission events over the last 150 years, in different TB-burden regions. Our results underscore significant differences in transmission between low-burden TB settings, i.e., clustering in Valencia region is higher (47.4%) than in Oxfordshire (27%), and similar to a high-burden area as Malawi (49.8%). By modeling times of the transmission links, we observed that settings with high transmission rate are associated with decades of uninterrupted transmission, irrespective of burden. Together, our results reveal that burden and transmission are not necessarily linked due to the role of past epidemics in the ongoing TB incidence, and highlight the need for in-depth characterization of transmission dynamics and specifically tailored TB control strategies.European Research Council 638553-TB-ACCELERATE; European Research Council 101001038-TBRECONNECT; Ministerio de Ciencia e Innovación SAF2016-77346-RPeer reviewe

A high resolution picture of tuberculosis transmission obtained from direct-from- sputum whole-genome sequencing

Abstract de la comunicación oral presentada al Scientific Meeting on Mycobacteria. MycoPORTO 2019 Porto (Portugal), 19-20 de septiembre de 2019Whole-genome sequencing of M. tuberculosis (MTB) directly from clinical specimens (dWGS) will suppose a major breakthrough in tuberculosis diagnosis and control. To date, different strategies have been followed to sequence MTB from sputum samples and accurately predict drug-resistance. However, dWGS of MTB has never been used in genomic epidemiology. Here, we test and optimize both the laboratory and computational protocols to implement a dWGS pipeline able to produce a detailed genomic analysis from sputum samples in a week. We used dWGS on clinical specimens of 27 TB patients to evaluate its performance for drugresistance prediction and genomic epidemiology. We were able to predict full drug resistance profiles and epidemiological links for the 28 out of the 37 specimens analyzed. In these samples, the agreement between dWGS and WGS from matching cultures was of 100% for both drug-resistance prediction and epidemiological clusters. In our work, we evaluate the state-of-the-art of dWGS for MTB and provide a cost-effective diagnostic algorithm for tuberculosis based on dWGS. We demonstrate that dWGS is a powerful tool to conduct highprecision genomic epidemiology in real time.Peer reviewe

An evolutionary functional genomics approach identifies novel candidate regions involved in isoniazid resistance in Mycobacterium tuberculosis

12 páginas, 4 figuras, 1 tablaEfforts to eradicate tuberculosis are hampered by the rise and spread of antibiotic resistance. Several large-scale projects have aimed to specifically link clinical mutations to resistance phenotypes, but they were limited in both their explanatory and predictive powers. Here, we combine functional genomics and phylogenetic associations using clinical strain genomes to decipher the architecture of isoniazid resistance and search for new resistance determinants. This approach has allowed us to confirm the main target route of the antibiotic, determine the clinical relevance of redox metabolism as an isoniazid resistance mechanism and identify novel candidate genes harboring resistance mutations in strains with previously unexplained isoniazid resistance. This approach can be useful for characterizing how the tuberculosis bacilli acquire resistance to new antibiotics and how to forestall them.This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programs 638553 (TB-ACCELERATE), SAF2016-77346-R from Ministerio de Economía y Competitividad (Spanish Government) and AICO/2018/113 from Generalitat Valenciana (to I.C.). M.M.-M. is recipient of a FPI grant from the Ministerio de Economía y Competitividad (Spanish Government, code BES-2017-079656). V.F. was recipient of a post-doctoral research grant from the Ministerio de Economía y Competitividad (Spanish Government, code FPDI-2013-18757). Action cofinanced by the European Union through the Operational Program of European Regional Development Fund (ERDF) of Valencia Region (Spain) 2014-2020.Peer reviewe

Assessment of whole genome sequencing technology applied to drugresistant tuberculosis diagnosis

Conferencia (LB-2082-21) presentada al: 51st World Conference on Lung Health of the International Union Against Tuberculosis and Lung Disease (The Union) Virtual event, 20-24 october 2020LB-2082-21 Background: The constant rise and global spreading of drug-resistant tuberculosis is a major threat to Global Health. Drug susceptibility testing (DST) is the reference method for the diagnosis of resistances in Mycobacterium tuberculosis. However, this technique has significant drawbacks such as the requirement of complex infrastructure and expertise, a long period to obtain results and low accuracy and reproducibility for certain first-line drugs. In recent years, Whole Genome Sequencing (WGS) of Mycobacterium tuberculosis has emerged as a fast and reliable tool to predict the drug susceptibility profile of the bacteria. Methods: We have performed a retrospective study of 735 isolates belonging to the Valencia Region (Spain) to assess the performance of WGS resistance prediction in a low burden setting. We compared our bioinformatics prediction with the phenotypic DST data to obtain the sensitivity and specificity. Additionally, we re-tested the clinical isolates with discordant DST-WGS results using the REMA assay

Whole-genome sequencing of Mycobacterium tuberculosis directly from clinical samples for high-resolution genomic epidemiology and drug resistance surveillance: an observational study

9 páginas, 3 figurasBackground Direct whole-genome sequencing of Mycobacterium tuberculosis from clinical specimens will be a major breakthrough in tuberculosis diagnosis and control. To date, direct whole-genome sequencing has never been used in genomic epidemiology, and its accuracy in transmission inference remains unknown. We investigated the technical challenges imposed by direct whole-genome sequencing, and used it to infer transmission clusters and predict drug resistance. Methods Using an optimised workflow, we did direct whole-genome sequencing for 37 clinical specimens from 23 tuberculosis patients. Nine sputum samples from nine patients who were infected with different non-tuberculous mycobacteria and culture-negative for tuberculosis were used as controls in the qPCR assays and pre-sequencing runs. Additionally, 780 clinical isolates in the region of Comunidad Valenciana (Spain) were whole-genome sequenced between Jan 1, 2014, and Dec 31, 2016. We analysed the genomic variants to build a tuberculosis transmission network for the region, including the clinical specimens, and to predict drug susceptibility profiles. Findings After sequencing 37 clinical specimens, 28 specimens (22 [85%] of 26 smear-positive and six [55%] of 11 smear-negative) met the quality criteria for downstream analysis. All 28 clinical specimens clustered with their matching culture isolates, with a median distance of 0 single nucleotide polymorphisms. Of the 28 clinical specimens, 16 (57%) were accurately assigned to ten transmission clusters in the region, and 12 (43%) were unique cases. Transmission inferences and drug-susceptibility predictions from direct whole-genome sequencing data were concordant with sequences from corresponding cultures and phenotypic drug-susceptibility testing. Complete genomic analysis, within a week of specimen receipt, cost €217 per sample (excluding personnel costs). Interpretation Direct whole-genome sequencing could be used to accurately delineate transmission clusters of tuberculosis and conduct culture-independent surveillance. Compared with conventional approaches, direct wholegenome sequencing allows researchers to do real-time genomic epidemiology and drug resistance surveillance in settings where culture and drug susceptibility testing are not available.This work was supported by projects of the European Research Council (638553-TB-ACCELERATE), Ministerio de Economía y Competitividad, and Ministerio de Ciencia, Innovación y Universidades (Spanish Government; SAF2013-43521-R, SAF2016-77346-R, and SAF2017-92345-EXP [to IC], and BES-2014-071066 [to GAG])Peer reviewe

Tuberculosis in Liberia: high multidrug-resistance burden, transmission and diversity modelled by multiple importation events

12 páginas, 5 figuras, 2 tablas. DNA sequences generated as part of this study have been deposited at the European Nucleotide Archive under study accession number PRJEB32589 (Table S1) (2019)Tuberculosis (TB) surveillance is scarce in most African countries, even though it is the continent with the greatest disease incidence according to the World Health Organization. Liberia is within the 30 countries with the highest TB burden, probably as a consequence of the long civil war and the recent Ebola outbreak, both crippling the health system and depreciating the TB prevention and control programmes. Due to difficulties working in the country, there is a lack of resistance surveys and bacillus characterization. Here, we use genome sequencing of Mycobacterium tuberculosis clinical isolates to fill this gap. Our results highlight that the bacillus population structure is dominated by lineage 4 strains that harbour an outstanding genetic diversity, higher than in the rest of Africa as a whole. Coalescent analyses demonstrate that strains currently circulating in Liberia were introduced several times beginning in the early year 600 CE until very recently coinciding with migratory movements associated with the civil war and Ebola epidemics. A higher multidrug-resistant (MDR)-TB frequency (23.5 %) than current estimates was obtained together with non-catalogued drug-resistance mutations. Additionally, 39 % of strains were in genomic clusters revealing that ongoing transmission is a major contribution to the TB burden in the country. Our report emphasizes the importance of TB surveillance and control in African countries where bacillus diversity, MDR-TB prevalence and transmission are coalescing to jeopardize TB control programmes.This work was supported by the European Research Council (638553-TB-ACCELERATE) and the Ministerio de Economía y Competitividad (SAF2013-43521-R and SAF2016-77346-R to I.C.). Funding support from the John D. and Catherine T. MacArthur Foundation, USA, under the Higher Education Initiative in Africa (grant no. 97944-0-800/406/99) for the establishment of the Center for Control and Prevention of Zoonoses, University of Ibadan, Nigeria, is appreciated.Peer reviewe