Caracterización epidemiológica, clínica, microbiológica y genómica de Mycobacterium tuberculosis en el Departamento de Salud La Fe de Valencia

ANTECEDENTES: La tuberculosis (TB) es una enfermedad asociada a importante morbi-mortalidad en todo el mundo. En la Comunidad Valenciana (CV), al igual que en el resto de España, la tasa de TB según datos oficiales ha disminuido entre el año 2011 y 2015, así como el porcentaje de enfermos inmigrantes y coinfectados con el virus de la inmunodeficiencia humana (VIH), descenso que no se ha visto reflejado, sin embargo, en los datos oficiales del Departamento de Salud (DS) La Fe. La inmigración desde países de alta endemia de TB hace necesario revisar la epidemiología de los pacientes atendidos en el Hospital Universitario y Politécnico (HUP) La Fe y el patrón de resistencias de las cepas aisladas, para comprobar la utilidad de las nuevas técnicas moleculares incorporadas, así como la de la secuenciación completa del genoma (SCG) de Mycobacterium tuberculosis, método que está siendo evaluado como herramienta para el diagnóstico precoz de la enfermedad y de predicción de resistencias. OBJETIVO: Conocer las características epidemiológicas y clínicas de los enfermos con TB confirmada por microbiología en el HUP La Fe de Valencia entre enero de 2011 y diciembre de 2015, así como la repercusión de la SCG de los aislados de MTBC en la gestión de los pacientes. METODOLOGÍA: Estudio descriptivo retrospectivo de las características de los pacientes con TB confirmada por microbiología en el HUP La Fe entre 2011 y 2015 y análisis comparativo con los resultados de linaje y resistencias obtenidos tras SCG de las cepas aisladas en 2014 y 2015. RESULTADOS PRINCIPALES: De los 194 casos confirmados en el HUP La Fe, sólo el 39,8% pertenecían al DS La Fe, por lo que las tasas calculadas no se corresponden con los datos oficiales. La tasa media fue de 9,7 casos/100.000 hab, con mortalidad asociada del 5%. La edad media de los enfermos fue 39,6 años, con un 5,7% de casos en edad infantil, un 10% de coinfectados con el VIH y un 51,3% de inmigrantes, no presentando este grupo diferencias importantes respecto al resto. La mayoría de los casos se concentraron en Torrefiel-Rascaña, en Secciones Censales de alta vulnerabilidad. La presentación clínica predominante fue la pulmonar (62,9%), y la existencia de coinfección con VIH se asoció a forma extrapulmonar. El tiempo de crecimiento medio en medio líquido fue 14,87 días frente a 21,4 en medio sólido. Hubo un 16% de resistencia fenotípica a los fármacos de primera línea, con resistencia a isoniacida (INH) del 9,2% y a rifampicina (RIF) del 3,6%. El linaje predominante fue el L4, con linajes distintos al L4 en pacientes extranjeros. La concordancia kappa de la resistencia detectada por SCG con la fenotípica fue para INH 0,95 (IC: 0,83-0,999) y para RIF 0,999, siendo las mutaciones más frecuentes las localizadas en -15 fabG1-inhA y codón 315 del gen katG para el primer fármaco y codón 450 del gen rpoB para el segundo. CONCLUSIONES: A pesar de que la tasa de TB es menor, el número de inmigrantes afectados es mayor al estimado en el resto de la CV y de España. La presencia de enfermedades de base no es determinante para el desarrollo de enfermedad tuberculosa, pero sí para el desarrollo de formas extrapulmonares, en concreto en el caso de coinfección con VIH. Las técnicas de biología molecular ayudan al diagnóstico, siendo más útiles en las formas pulmonares y en la detección precoz de resistencia a RIF. La SCG es una herramienta más precisa que las moleculares, en concreto para la detección precoz de resistencia a INH en nuestra área, y para el estudio epidemiológico de contactos.BACKGROUND: Tuberculosis (TB) is a disease associated with significant morbidity and mortality throughout the world. In the Valencian Community (CV), as in the rest of Spain, the TB rate according to official data has decreased between 2011 and 2015, as well as the percentage of immigrant patients and of those coinfected with the human immunodeficiency virus (HIV), a decline that has not been reflected, however, in the official data of the Health Department (DS) La Fe. Immigration from high endemic TB countries, makes it necessary to review the epidemiology of patients attended at the Hospital Universitario y Politécnico (HUP) La Fe and the resistance pattern of the isolated strains, to verify the usefulness of the recently introduced molecular techniques, as well as that of the whole genome sequencing (WGS) of Mycobacterium tuberculosis, a tool that is being evaluated for early diagnosis of the disease and prediction of resistance to first line drugs. OBJECTIVE: To determine the epidemiological and clinical characteristics of patients with microbiologically confirmed TB at the HUP La Fe of Valencia, Spain, between January 2011 and December 2015, as well as the usefulness of WGS of MTBC isolates in the management of the patients. METHODS: Retrospective study of the characteristics of patients with TB confirmed by microbiology in HUP La Fe between 2011 and 2015 and comparative analysis with the results of lineage and resistance obtained after WGS of isolated strains during 2014 and 2015. MAIN RESULTS: Of the 194 confirmed cases, only 39.8% belonged to DS La Fe, so the calculated rates do not correspond to the official data. The mean incidence rate was 9.7 cases / 100,000 inhabitants, with associated mortality of 5%. The mean age of the patients was 39.6 years, with 5.7% of cases in childhood, 10% of co-infection with HIV and 51.3% of cases in immigrants. Most of the cases were concentrated in Torrefiel-Rascaña, in highly vulnerable Censal Sections. The predominant clinical presentation was pulmonary TB (62.9%), and the existence of HIV co-infection was associated with extrapulmonary forms. The mean growth time in liquid medium was 14.87 days versus 21.4 in solid medium. Phenotypic resistance to first line drugs accounted for 16% of cases - isoniazid (INH) resistance of 9.2% and rifampicin (RIF) of 3.6%. The predominant lineage was L4, with non-L4 lineages in foreign patients. The kappa concordance of phenotypic resistance to the resistance detected by WGS was for INH 0.95 (IC: 0.83-0.999) and for RIF 0.999, with the most frequent mutations being located in -15 fabG1-inhA and codon 315 of the katG gene for INH and codon 450 of the rpoB gene for RIF. CONCLUSIONS: Although the rate of TB is lower in DS La Fe than estimated for the rest of the CV and Spain, the percentage of immigrants affected is higher. The presence of underlying disease is not determinant for the development of TB; however, it is for the development of extrapulmonary TB, specifically in the case of HIV co-infection. Molecular biology techniques aid in the diagnosis, being more useful in pulmonary presentations and in the early detection of RIF resistance. WGS is a more precise tool, specifically for the early detection of INH resistance in our area, and for contact tracing

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

A surface plasmon resonance based approach for measuring response to pneumococcal vaccine

Incidence of pneumococcal disease has increased worldwide in recent years. Response to pneumococcal vaccine is usually measured using the multiserotype enzyme-linked immunosorbent assay (ELISA) pneumococcal test. However, this approach presents several limitations. Therefore, the introduction of new and more robust analytical approaches able to provide information on the efficacy of the pneumococcal vaccine would be very beneficial for the clinical management of patients. Surface plasmon resonance (SPR) has been shown to offer a valuable understanding of vaccines' properties over the last years. The aim of this study is to evaluate the reliability of SPR for the anti-pneumococcal capsular polysaccharides (anti-PnPs) IgGs quantification in vaccinated. Fast protein liquid chromatography (FPLC) was used for the isolation of total IgGs from serum samples of vaccinated patients. Binding-SPR assays were performed to study the interaction between anti-PnPs IgGs and PCV13. A robust correlation was found between serum levels of anti-PnPs IgGs, measured by ELISA, and the SPR signal. Moreover, it was possible to correctly classify patients into "non-responder", "responder" and "high-responder" groups according to their specific SPR PCV13 response profiles. SPR technology provides a valuable tool for reliably characterize the interaction between anti-PnPs IgGs and PCV13 in a very short experimental time

Accuracy of Xpert Ultra for the diagnosis of paediatric tuberculosis in a low TB burden country: a prospective multicentre study

[Introduction] Childhood pulmonary tuberculosis (TB) remains a diagnostic challenge. This study aimed to evaluate the performance of Xpert Ultra for the diagnosis of pulmonary TB in children in a low TB prevalence setting.[Methods] Prospective, multicentre, diagnostic accuracy study. Children with clinical or radiological suspicion of pulmonary TB were recruited at 11 paediatric units in Spain. Up to three gastric or sputum specimens were taken on 3 consecutive days, and analysed by Xpert MTB/RIF, Xpert Ultra and culture in parallel.[Results] 86 children were included (median age 4.9 years, IQR 2.0–10.0; 51.2% male). The final diagnosis was pulmonary TB in 75 patients (87.2%); 33 (44.0%) were microbiologically confirmed. A total of 219 specimens, comprising gastric aspirates (n=194; 88.6%) and sputum specimens (n=25; 11.4%), were analysed. Using culture as reference standard and comparing individual specimens, the sensitivity was 37.8% (14/37) for Xpert MTB/RIF and 81.1% (30/37) for Xpert Ultra (p<0.001); specificity was 98.4% (179/182) and 93.4% (170/182), respectively (p=0.02). In the per-patient analysis, considering positive results on any specimen, the sensitivity was 42.9% (9/21) for Xpert MTB/RIF and 81.0% for Xpert Ultra (17/21, p=0.01); specificity was 96.9% (63/65) and 87.7% (57/65, p=0.07), respectively.[Conclusions] In children with pulmonary TB in a low burden setting, Xpert Ultra has significantly higher sensitivity than the previous generation of Xpert assay and only marginally lower specificity. Therefore, in children undergoing evaluation for suspected pulmonary TB, Xpert Ultra should be used in preference to Xpert MTB/RIF whenever possible.This study did not receive any project-specific funding. DA-A was supported by the Spanish Ministry of Health – Instituto de Salud Carlos III (ISCIII) and cofunded by the European Union (FEDER) (Contrato Río Hortega CM18/00100). AN-J was supported by 'Subvencions per a la Intensificació de Facultatius Especialistes' (Departament de Salut de la Generalitat de Catalunya, Programa PERIS 2016-2020) (SLT008/18/00193). DBG was supported by the Spanish Ministry of Science and Innovation – Instituto de Salud Carlos III and Fondos FEDER by 'Contratos para la intensificación de la actividad investigadora en el Sistema Nacional de Salud, 2020 (INT20/00086)'. BS-G was supported by the Spanish Ministry of Health – Instituto de Salud Carlos III (ISCIII) and cofunded by the European Union (FEDER) (Contrato Juan Rodés JR16/00036).Peer reviewe

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

Mycobacterium tuberculosis lineage 4 comprises globally distributed and geographically restricted sublineages

Generalist and specialist species differ in the breadth of their ecological niches. Little is known about the niche width of obligate human pathogens. Here we analyzed a global collection of Mycobacterium tuberculosis lineage 4 clinical isolates, the most geographically widespread cause of human tuberculosis. We show that lineage 4 comprises globally distributed and geographically restricted sublineages, suggesting a distinction between generalists and specialists. Population genomic analyses showed that, whereas the majority of human T cell epitopes were conserved in all sublineages, the proportion of variable epitopes was higher in generalists. Our data further support a European origin for the most common generalist sublineage. Hence, the global success of lineage 4 reflects distinct strategies adopted by different sublineages and the influence of human migration.We thank S. Lecher, S. Li and J. Zallet for technical support. Calculations were performed at the sciCORE scientific computing core facility at the University of Basel. This work was supported by the Swiss National Science Foundation (grants 310030_166687 (S.G.) and 320030_153442 (M.E.) and Swiss HIV Cohort Study grant 740 to L.F.), the European Research Council (309540-EVODRTB to S.G.), TB-PAN-NET (FP7-223681 to S.N.), PathoNgenTrace projects (FP7-278864-2 to S.N.), SystemsX.ch (S.G.), the German Center for Infection Research (DZIF; S.N.), the Novartis Foundation (S.G.), the Natural Science Foundation of China (91631301 to Q.G.), and the National Institute of Allergy and Infectious Diseases (5U01-AI069924-05) of the US National Institutes of Health (M.E.)

Prediction of Susceptibility to First-Line Tuberculosis Drugs by DNA Sequencing

Background: The World Health Organization recommends drug-susceptibility testing of Mycobacterium tuberculosis complex for all patients with tuberculosis to guide treatment decisions and improve outcomes. Whether DNA sequencing can be used to accurately predict profiles of susceptibility to first-line antituberculosis drugs has not been clear. Methods: We obtained whole-genome sequences and associated phenotypes of resistance or susceptibility to the first-line antituberculosis drugs isoniazid, rifampin, ethambutol, and pyrazinamide for isolates from 16 countries across six continents. For each isolate, mutations associated with drug resistance and drug susceptibility were identified across nine genes, and individual phenotypes were predicted unless mutations of unknown association were also present. To identify how whole-genome sequencing might direct first-line drug therapy, complete susceptibility profiles were predicted. These profiles were predicted to be susceptible to all four drugs (i.e., pansusceptible) if they were predicted to be susceptible to isoniazid and to the other drugs or if they contained mutations of unknown association in genes that affect susceptibility to the other drugs. We simulated the way in which the negative predictive value changed with the prevalence of drug resistance. Results: A total of 10,209 isolates were analyzed. The largest proportion of phenotypes was predicted for rifampin (9660 [95.4%] of 10,130) and the smallest was predicted for ethambutol (8794 [89.8%] of 9794). Resistance to isoniazid, rifampin, ethambutol, and pyrazinamide was correctly predicted with 97.1%, 97.5%, 94.6%, and 91.3% sensitivity, respectively, and susceptibility to these drugs was correctly predicted with 99.0%, 98.8%, 93.6%, and 96.8% specificity. Of the 7516 isolates with complete phenotypic drug-susceptibility profiles, 5865 (78.0%) had complete genotypic predictions, among which 5250 profiles (89.5%) were correctly predicted. Among the 4037 phenotypic profiles that were predicted to be pansusceptible, 3952 (97.9%) were correctly predicted. Conclusions: Genotypic predictions of the susceptibility of M. tuberculosis to first-line drugs were found to be correlated with phenotypic susceptibility to these drugs. (Funded by the Bill and Melinda Gates Foundation and others.

Mycobacterium genavense Infections in a Tertiary Hospital and Reviewed Cases in Non-HIV Patients

Mycobacterium genavense is a relatively new species of nontuberculous mycobacterium reported to cause disseminated infections in patients with AIDS and later on in non-HIV immunosuppressed patients. We describe clinical and laboratory features and response to therapy in 7 patients, three of them with HIV infection and four non-HIV—three organ transplant recipients and one with hyper-IgE syndrome—in Valencia, Spain, in a ten years period. We then summarize the published cases of M. avium complex infection, with invasion of peripheral blood, liver, spleen, bone marrow, lymph nodes, and lungs. In clinical samples a large number of acid-fast bacilli were observed. M. genavense grew only from liquid media and after a prolonged incubation period. Its identification was accomplished through molecular methods. Patients were treated with prolonged combinations of antimicrobial agents. There was clinical favourable outcome in 4 patients