Tuberculosis en el Departamento de Salud Valencia-Doctor Peset. Aportación de los métodos de microbiología molecular

La tuberculosis (TB) se mantiene como problema importante de salud pública a pesar de la disminución progresiva de su incidencia en nuestro medio. Los programas de control deben incluir objetivos de mejora en detección precoz de pacientes bacilíferos y la eficacia y rapidez de los métodos de rutina microbiológica para identificación y sensibilidad de Mycobacterium tuberculosis complex, detectando cepas multirresistentes (MDR-TB) de forma rápida y sencilla. En este trabajo se describe el modelo de funcionamiento del Servicio de Microbiología en la detección y control de las TB del Departamento de Salud Valencia- Dr. Peset en los últimos once años, calculando la evolución de tasas de incidencia de TB, influencia de variables demográficas y factores de riesgo (coinfección VIH, inmigración y localización del cuadro). Se estudia la eficacia de la implantación en rutina microbiológica de los métodos moleculares disponibles actualmente en la demora de diagnóstico de caso y detección de resistencias (inmunocromatografía, PCR hibridación, PCR en tiempo real y secuenciación). En el Departamento de Salud Valencia- Dr. Peset se detectó en el periodo 2005- 2015 una media de 43 casos/ año de TB (incidencia media 11,6 x 105 hab.) con disminución progresiva por años. El estudio de las características demográficas y de factores de riesgo nos ofrece un perfil de paciente varón, entre 25- 55 años y/ o mayor de 75 años, con afectación mayoritariamente pulmonar, autóctono en un 64% de los casos y VIH negativo. Un 37,3% fueron inmigrantes (predominio de América del Sur- 43%, seguido de África- 28% y de Europa- 22%). En un 10,2% se asoció coinfección por VIH, con un 27,1% de pacientes en los que no se les solicitó determinación de Ac anti- VIH. No se detectaron coinfecciones TB- VIH en pacientes pediátricos ni en mayores de 65 años. Un 77,4% fueron TB pulmonares. Más de la mitad (52,5%) de los casos de TB confirmados fueron pacientes remitidos desde el Servicio de Urgencias Médicas del Hospital. La rentabilidad de la baciloscopia fue inferior en casos extrapulmonares y en VIH+. No se demostraron diferencias significativas en la asociación TB con inmigración y/ o coinfección VIH. La introducción en la rutina diagnóstica de las técnicas de detección rápida y/ o moleculares conllevó un descenso progresivo de la demora diagnóstica, llegando hasta 3- 4 días en el 75% o incluso a <24h en casi el 60% de los casos. La tasa de MDR-TB fue del 1,9%, sin diferencias entre año de estudio, edad, sexo, nacionalidad ni coinfección VIH de los casos. Sin embargo, el 6% presentaban resistencia a isoniacida. La detección de resistencia a rifampicina mediante PCR en tiempo real contribuyó a la detección precoz (menos de 3h) de todos los casos MDR-TB. Se aplicó la secuenciación genómica en las cepas MDR-TB pudiendo así determinar su linaje, coexistencia de poblaciones, mecanismos de transmisión y resistencias por mutaciones no detectables con métodos convencionales.The tuberculosis (TB) remains as an important problem of public health despite the progressive decrease of his incidence in our society. The control programs should include targets of improvement in precocious detection of smear-positive patients, and the efficiency and promptness of the methods of microbiological routine for identification and sensitivity of Mycobacterium tuberculosis complex, detecting multidrug-resistant strains (MDR-TB) in a fast and simple way. In this study it is described the model of operation of the Service of Microbiology in the detection and control of the TB of the Health Department of Valencia- Dr. Peset in the last eleven years, calculating the evolution of incidence taxes of TB, influence of demographic variables and factors of risk (HIV coinfection, immigration and TB location). It studies the efficiency of the implantation in microbiological routine of the available molecular methods at present in the delay of diagnostic of case and detection of resistances (immunochromatography, PCR hybridisation, Real-Time PCR and sequencing). In the Health Department of Valencia- Dr. Peset were detected between 2005- 2015 an average of 43 cases/ year of TB (half incidence 11,6 x 105 hab.) with progressive decrease by years. The study of the demographic characteristics and factors of risk offers us a patient profile consisted of a man, between 25- 55 years old and/ or older than 75 years old, with affectation mostly pulmonary, autochthonous in 64% of the cases and negative HIV. A 37,3% were immigrant (predominance from South America- 43%, followed of Africa- 28% and of Europe- 22%). In a 10,2% were associated a coinfection by HIV, with 27,1% of patients in which was not requested a determination of Ac anti- HIV. We’re not detected TB- HIV coinfections in pediatric patients neither in older than 65 years old. 77,4% were pulmonary TB. More than half (52,5%) of the confirmed cases of TB were patients remitted from the Service of Medical Urgencies of the Hospital. The profitability of the smear y was inferior in extrapulmonary cases and in HIV+. They did not show significant differences in the association TB with immigration and/ or HIV coinfection. The introduction in the diagnostic routine of the fast detection and/ or molecular techniques comported a progressive decrease of the diagnostic delay, arriving until 3- 4 days in 75% or even to <24h in almost 60% of the cases. The tax of MDR-TB was of 1,9%, without differences between year of study, age, sex, nationality neither HIV coinfection of the cases. However, the 6% presented resistance to isoniazid. The detection of resistance to rifampicin by means of PCR in real time contributed to the precocious detection (less than 3h) of all the cases MDR-TB. It was applied the genomic sequencing in the cases MDR-TB being able to determine his lineage, coexistence of populations, mechanisms of transmission and resistances by no sequencing mutations with conventional methods

Urinary incontinence and sedentary behaviour in nursing home residents in Osona, Catalonia: protocol for the OsoNaH project, a multicentre observational study

Introduction Several studies have shown that physical activity (PA) levels and sedentary behaviour (SB) are independent risk factors for many health-related issues. However, there is scarce evidence supporting the relationship between SB and urinary incontinence (UI) in community-dwelling older adults, and no information on any possible association in institutionalised older adults. Stage I of this project has the main objective of determining the prevalence of UI and its associated factors in nursing home (NH) residents, as well as analysing the association between UI (and its types) and SB. Stage II aims to investigate the incidence and predictive factors of functional and continence decline, falls, hospitalisations, mortality and the impact of the COVID-19 pandemic among NH residents.Methods and analysis Stage I is an observational, multicentre, cross-sectional study with mixed methodology that aims to explore the current status of several health-related outcomes in NH residents of Osona (Barcelona, Spain). The prevalence ratio will be used as an association measure and multivariate analysis will be undertaken using Poisson regression with robust variance. Stage II is a 2-year longitudinal study that aims to analyse functional and continence decline, incidence of falls, hospitalisations, mortality and the impact of the COVID-19 pandemic on these outcomes. A survival analysis using the actuarial method for functional decline and continence, evaluated every 6 months, and the Kaplan-Meier method for falls, hospitalisations and deaths, and Cox regression for multivariate analysis will be undertaken.Ethics and dissemination The study received the following approvals: University of Vic - Central University of Catalonia Ethics and Research Committee (92/2019 and 109/2020), Clinical Research Ethics Committee of the Osona Foundation for Health Research and Education (FORES) (code 2020118/PR249). Study results will be disseminated at conferences, meetings and through peer-reviewed journals.Trial registration number NCT04297904

Transcriptomic profiling of TK2 deficient human skeletal muscle suggests a role for the p53 signalling pathway and identifies growth and differentiation factor-15 as a potential novel biomarker for mitochondrial myopathies

BACKGROUND: Mutations in the gene encoding thymidine kinase 2 (TK2) result in the myopathic form of mitochondrial DNA depletion syndrome which is a mitochondrial encephalomyopathy presenting in children. In order to unveil some of the mechanisms involved in this pathology and to identify potential biomarkers and therapeutic targets we have investigated the gene expression profile of human skeletal muscle deficient for TK2 using cDNA microarrays. RESULTS: We have analysed the whole transcriptome of skeletal muscle from patients with TK2 mutations and compared it to normal muscle and to muscle from patients with other mitochondrial myopathies. We have identified a set of over 700 genes which are differentially expressed in TK2 deficient muscle. Bioinformatics analysis reveals important changes in muscle metabolism, in particular, in glucose and glycogen utilisation, and activation of the starvation response which affects aminoacid and lipid metabolism. We have identified those transcriptional regulators which are likely to be responsible for the observed changes in gene expression. CONCLUSION: Our data point towards the tumor suppressor p53 as the regulator at the centre of a network of genes which are responsible for a coordinated response to TK2 mutations which involves inflammation, activation of muscle cell death by apoptosis and induction of growth and differentiation factor 15 (GDF-15) in muscle and serum. We propose that GDF-15 may represent a potential novel biomarker for mitochondrial dysfunction although further studies are required

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

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

Transcriptomic profiling of TK2 deficient human skeletal muscle suggests a role for the p53 signalling pathway and identifies growth and differentiation factor-15 as a potential novel biomarker for mitochondrial myopathies

BACKGROUND: Mutations in the gene encoding thymidine kinase 2 (TK2) result in the myopathic form of mitochondrial DNA depletion syndrome which is a mitochondrial encephalomyopathy presenting in children. In order to unveil some of the mechanisms involved in this pathology and to identify potential biomarkers and therapeutic targets we have investigated the gene expression profile of human skeletal muscle deficient for TK2 using cDNA microarrays. RESULTS: We have analysed the whole transcriptome of skeletal muscle from patients with TK2 mutations and compared it to normal muscle and to muscle from patients with other mitochondrial myopathies. We have identified a set of over 700 genes which are differentially expressed in TK2 deficient muscle. Bioinformatics analysis reveals important changes in muscle metabolism, in particular, in glucose and glycogen utilisation, and activation of the starvation response which affects aminoacid and lipid metabolism. We have identified those transcriptional regulators which are likely to be responsible for the observed changes in gene expression. CONCLUSION: Our data point towards the tumor suppressor p53 as the regulator at the centre of a network of genes which are responsible for a coordinated response to TK2 mutations which involves inflammation, activation of muscle cell death by apoptosis and induction of growth and differentiation factor 15 (GDF-15) in muscle and serum. We propose that GDF-15 may represent a potential novel biomarker for mitochondrial dysfunction although further studies are required

Transcriptomic profiling of TK2 deficient human skeletal muscle suggests a role for the p53 signalling pathway and identifies growth and differentiation factor-15 as a potential novel biomarker for mitochondrial myopathies

BACKGROUND: Mutations in the gene encoding thymidine kinase 2 (TK2) result in the myopathic form of mitochondrial DNA depletion syndrome which is a mitochondrial encephalomyopathy presenting in children. In order to unveil some of the mechanisms involved in this pathology and to identify potential biomarkers and therapeutic targets we have investigated the gene expression profile of human skeletal muscle deficient for TK2 using cDNA microarrays. RESULTS: We have analysed the whole transcriptome of skeletal muscle from patients with TK2 mutations and compared it to normal muscle and to muscle from patients with other mitochondrial myopathies. We have identified a set of over 700 genes which are differentially expressed in TK2 deficient muscle. Bioinformatics analysis reveals important changes in muscle metabolism, in particular, in glucose and glycogen utilisation, and activation of the starvation response which affects aminoacid and lipid metabolism. We have identified those transcriptional regulators which are likely to be responsible for the observed changes in gene expression. CONCLUSION: Our data point towards the tumor suppressor p53 as the regulator at the centre of a network of genes which are responsible for a coordinated response to TK2 mutations which involves inflammation, activation of muscle cell death by apoptosis and induction of growth and differentiation factor 15 (GDF-15) in muscle and serum. We propose that GDF-15 may represent a potential novel biomarker for mitochondrial dysfunction although further studies are required

High-resolution mapping of tuberculosis transmission: Whole genome sequencing and phylogenetic modelling of a cohort from Valencia Region, Spain

Artículo con 20 páginas, 5 figuras, 1 tabla. All the sequence data are deposited in the European Nucleotide Archive under the Bioproject number PRJEB29604 (https://www.ebi.ac.uk/ena/data/view/PRJEB29604) and the accession numbers ERR2099780 (https://www.ebi.ac.uk/ena/data/search?query=ERR2099780) and ERR2099784 (https://www.ebi.ac.uk/ena/data/search?query=ERR2099784).BACKGROUND: Whole genome sequencing provides better delineation of transmission clusters in Mycobacterium tuberculosis than traditional methods. However, its ability to reveal individual transmission links within clusters is limited. Here, we used a 2-step approach based on Bayesian transmission reconstruction to (1) identify likely index and missing cases, (2) determine risk factors associated with transmitters, and (3) estimate when transmission happened. METHODS AND FINDINGS: We developed our transmission reconstruction method using genomic and epidemiological data from a population-based study from Valencia Region, Spain. Tuberculosis (TB) incidence during the study period was 8.4 cases per 100,000 people. While the study is ongoing, the sampling frame for this work includes notified TB cases between 1 January 2014 and 31 December 2016. We identified a total of 21 transmission clusters that fulfilled the criteria for analysis. These contained a total of 117 individuals diagnosed with active TB (109 with epidemiological data). Demographic characteristics of the study population were as follows: 80/109 (73%) individuals were Spanish-born, 76/109 (70%) individuals were men, and the mean age was 42.51 years (SD 18.46). We found that 66/109 (61%) TB patients were sputum positive at diagnosis, and 10/109 (9%) were HIV positive. We used the data to reveal individual transmission links, and to identify index cases, missing cases, likely transmitters, and associated transmission risk factors. Our Bayesian inference approach suggests that at least 60% of index cases are likely misidentified by local public health. Our data also suggest that factors associated with likely transmitters are different to those of simply being in a transmission cluster, highlighting the importance of differentiating between these 2 phenomena. Our data suggest that type 2 diabetes mellitus is a risk factor associated with being a transmitter (odds ratio 0.19 [95% CI 0.02-1.10], p < 0.003). Finally, we used the most likely timing for transmission events to study when TB transmission occurred; we identified that 5/14 (35.7%) cases likely transmitted TB well before symptom onset, and these were largely sputum negative at diagnosis. Limited within-cluster diversity does not allow us to extrapolate our findings to the whole TB population in Valencia Region. CONCLUSIONS: In this study, we found that index cases are often misidentified, with downstream consequences for epidemiological investigations because likely transmitters can be missed. Our findings regarding inferred transmission timing suggest that TB transmission can occur before patient symptom onset, suggesting also that TB transmits during sub-clinical disease. This result has direct implications for diagnosing TB and reducing transmission. Overall, we show that a transition to individual-based genomic epidemiology will likely close some of the knowledge gaps in TB transmission and may redirect efforts towards cost-effective contact investigations for improved TB control.IC was supported by European Research Council (638553-TB-ACCELERATE), the Ministerio de Economía y Competitividad (SAF2016-77346-R). CC and YX were supported by the Engineering and Physical Sciences Research Council of the UK (EPSRC EP/K026003/1 (CC) and EPSRC EP/N014529/1 (CC and YX).Peer reviewe