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.

BACKGROUND: 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.

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

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.

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

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.

Keratitis due to Colletotrichu gloeosporioides and Herpesvirus reactivation / Queratiris por Colletotrichum gloeosporioides y reactivación de Herpesvirus

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[Incidence of Bartonella Henselae Infection during the period 2009-2012 in the Valencian Community, Spain]. / Incidencia de la infección por Bartonella henselae en la Comunidad Valenciana durante el período 2009-2012.

BACKGROUND: Bartonella henselae is the causative agent of the disease caused by cat scratches and it has a worldwide distribution. The objective of the study was to investigate its incidence in the Valencian Community during the period 2009-2012. METHODS: A study of the chosen microbiological tests for B. henselae during four years was carried out. Pacients with serology (IgM and IgG), culture, or PCR positive were considered case studies. Data from the Red de Vigilancia Microbiológica Valenciana (RedMIVA) depending on the "General Direction of Public Health" (Dirección General de Salud Pública) was used as the source of information. RESULTS: During the studied period, 14 cases were detected. The incidence rates were: 0,07 per 10(5) inhabitants and year of the Valencian Community, 0,10 per 10(5) inhabitants/year in the province of Alicante, with marked differences in relation to Valencia of 0,06 per 105 inhabitants/year and Castellon (with no cases). The temporal distribution of the cases were: 4 in 2009, 4 in 2010, 3 in 2011, and 3 in 2012. 64% of the cases were women and 36% men. The median age was 21 years (range 1-65 years). Predominant age groups arose between 1-10 years (42%) and 31-40 years (28%). CONCLUSIONS: The incidence of B. henselae in the Valencian Community is low. The data obtained suggest that its distribution varies depending on the geographical area in the Community. There is a predominance of young people.

Incidencia de la infección por bartonella henselae en la comunidad valenciana durante el período 2009-2012 / Incidence of Bartonella Henselae Infection during the period 2009-2012 in the Valencian Community, Spain

Fundamentos: Bartonella henselae es el agente causal de la enfermedad por arañazo de gato y su distribución es mundial. El objetivo del trabajo fue conocer la incidencia de esta infección en la Comunidad Valenciana durante el periodo 2009-2012. Métodos: Se realizó el estudio sobre las pruebas microbiológicas solicitadas para B. henselae durante cuatro años. Se consideró 'caso' al paciente con serología (IgM e IgG), cultivo o PCR positivos. Como fuente de información se utilizaron los datos procedentes de la Red de Vigilancia Microbiológica Valenciana (RedMIVA) dependiente de la Dirección General de Salud Pública. Resultados: Durante el periodo estudiado se detectaron 14 casos. Las tasas de incidencia fueron: 0,07 por 105 habitantes y año en la Comunidad Valenciana, 0,10 por 105 habitantes/año en la provincia de Alicante, 0,06 por 105 habitantes/año en Valencia y ningún caso en la provincia de Castellón. La distribución temporal de los casos fue: 4 en 2009, 4 en 2010, 3 en 2011 y 3 en 2012. El 64% fueron mujeres y el 36% hombres. La mediana de edad fue de 21 años (rango de 1-65 años). Los grupos etarios predominantes se presentaron entre 1-10 años (42%) y 31-40 años (28%). Conclusiones: La incidencia de infección por B. henselae en la Comunidad Valencia es baja. Los datos obtenidos sugieren que varía su distribución según el área geográfica dentro de la Comunidad. Se observa un predominio de casos entre la población infantil (AU)

Background: Bartonella henselae is the causative agent of the disease caused by cat scratches and it has a worldwide distribution. The objective of the study was to investigate its incidence in the Valencian Community during the period 2009-2012. Methods: A study of the chosen microbiological tests for B. henselae during four years was carried out. Pacients with serology (IgM and IgG), culture, or PCR positive were considered case studies. Data from the Red de Vigilancia Microbiológica Valenciana (RedMIVA) depending on the 'General Direction of Public Health' (Dirección General de Salud Pública) was used as the source of information. Results: During the studied period, 14 cases were detected. The incidence rates were: 0,07 per 105 inhabitants and year of the Valencian Community, 0,10 per 105 inhabitants/year in the province of Alicante, with marked differences in relation to Valencia of 0,06 per 105 inhabitants/year and Castellon (with no cases). The temporal distribution of the cases were: 4 in 2009, 4 in 2010, 3 in 2011, and 3 in 2012. 64% of the cases were women and 36% men. The median age was 21 years (range 1-65 years). Predominant age groups arose between 1-10 years (42%) and 31-40 years (28%). Conclusions: The incidence of B. henselae in the Valencian Community is low. The data obtained suggest that its distribution varies depending on the geographical area in the Community. There is a predominance of young people (AU)

Estudio multicéntrico sobre la actividad in vitro de ceftarolina frente a Staphylococcus aureus aislados en España / In vitro activity of ceftaroline against Spanish isolates of Staphylococcus aureus: A multicenter study

INTRODUCCIÓN: Ceftarolina fosamil es un nuevo antibiótico de última generación del subgrupo de las cefalosporinas. Es el primer beta-lactámico comercializado que presenta actividad frente a Staphylococcus aureus resistente a la meticilina (SARM). El objetivo del presente estudio es determinar los valores in vitro de la concentración mínima inhibitoria (CMI) y de la concentración mínima bactericida (CMB) de ceftarolina frente a cepas de S. aureus, tanto sensible a la meticilina (SASM) como resistente. MATERIAL Y MÉTODOS: Se realizó un estudio multicéntrico en el que participaron 4 hospitales representativos de la geografía española. Mediante el método de microdilución en caldo se determinaron los valores de CMI y CMB de la ceftarolina frente a cepas de S. aureus (SARM y SASM). RESULTADOS: Se analizaron un total de 266 cepas de S. aureus (95 SARM y 171 SASM). En las 266 cepas analizadas, todos los valores de CMI se encontraron dentro de la categoría de sensible (valor ≤ 1 μg/ml), no detectándose ninguna cepa intermedia ni ni resistente. Las CMI50 y CMI90 para SAMR fueron de 0,25 y 0,5 μg/ml, respectivamente, con un rango de 0,125 a 1 μg/ml. Las CMI50 y CMI90 para SASM fueron de 0,125 y 0,25 μg/ml, con un rango de 0,125 a 0,5 μg/ml. Las CMB50 y CMB90 para SAMR fueron de 0,5 y 1 μg/ml, respectivamente, con un rango de 0,125 a 1 μg/ml. Las CMB50 y CMB90 para SASM fueron de 0,25 y 0,25 μg/ml, con un rango de 0,125 a 0,5 μg/ml. CONCLUSIÓN: Ceftarolina muestra una excelente actividad in vitro frente a S.aureus, incluyendo cepas SARM, por lo que podría presentarse como una alternativa prometedora en el tratamiento de infecciones causadas por esta bacteria

INTRODUCTION: Ceftaroline fosamil is a new-generation antimicrobial agent of cephalosporins subgroup. It is the first commercially available beta-lactam antibiotic that exhibits activity against methicillin-resistantStaphylococcus aureus (MRSA). The aim of this study is to determine the in vitro Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) values of ceftar oline against S. aureus strains (including MRSA). MATERIAL AND METHODS: A multicenter study involving four hospitals representative of the Spanish geography was performed. MIC and MBC values against both the methicillin-resistant and sensitive strains of S. aureus (MRSA and methicillin-sensitive S. aureus [MSSA]) were determined using a broth microdilution method. RESULTS: A total of 266 S. aureus strains were analyzed (95 MRSA and 171 MSSA). Ceftaroline bacterial sensitivity showed a mean MIC of 0.227 μg/ml (SD=0.146; range, 0.06 to 1μg/ml). All MIC values of the 266 strains tested belonged to the sensitive category (value ≤1μg/ml). Intermediate or resistant strains were not detected. MIC50 and MIC90 values for MRSA were 0.25 and 0.5μg/ml, respectively (range = 0.125-1 μg/ml). MSSA strains showed MIC50 and MIC90 values of 0.125 and 0.25 μg/ml, respectively (range = 0.125-0.5 μg/ml). MBC50 and MBC90 values for MRSA were 0.5 and 1μg/ml, respectively (range = 0.125-1μg/ml). MSSA strains showed MBC50 and MBC90 values of 0.25 and 0.25 μg/ml, respectively (range = 0.125-0.5 μg/ml). CONCLUSIÓN: Ceftaroline shows excellent in vitro activity against S.aureus, including MRSA strains. Therefore, this antibiotic may be a promising alternative for the treatment of infections caused by this bacterium

[In vitro activity of ceftaroline against Spanish isolates of Staphylococcus aureus: a multicenter study]. / Estudio multicéntrico sobre la actividad in vitro de ceftarolina frente a Staphylococcus aureus aislados en España.

INTRODUCTION: Ceftaroline fosamil is a new-generation antimicrobial agent of cephalosporins subgroup. It is the first commercially available beta-lactam antibiotic that exhibits activity against methicillin-resistant Staphylococcus aureus (MRSA). The aim of this study is to determine the in vitro Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) values of ceftaroline against S.aureus strains (including MRSA). MATERIAL AND METHODS: A multicenter study involving four hospitals representative of the Spanish geography was performed. MIC and MBC values against both the methicillin-resistant and sensitive strains of S.aureus (MRSA and methicillin-sensitive S.aureus [MSSA]) were determined using a broth microdilution method. RESULTS: A total of 266 S.aureus strains were analyzed (95 MRSA and 171 MSSA). Ceftaroline bacterial sensitivity showed a mean MIC of 0.227 µg/ml (SD=0.146; range, 0.06 to 1 µg/ml). All MIC values of the 266 strains tested belonged to the sensitive category (value ≤ 1 µg/ml). Intermediate or resistant strains were not detected. MIC50 and MIC90 values for MRSA were 0.25 and 0.5 µg/ml, respectively (range=0.125-1 µg/ml). MSSA strains showed MIC50 and MIC90 values of 0.125 and 0.25 µg/ml, respectively (range=0.125-0.5 µg/ml). MBC50 and MBC90 values for MRSA were 0.5 and 1 µg/ml, respectively (range=0.125-1 µg/ml). MSSA strains showed MBC50 and MBC90 values of 0.25 and 0.25 µg/ml, respectively (range=0.125-0.5 µg/ml). CONCLUSION: Ceftaroline shows excellent in vitro activity against S.aureus, including MRSA strains. Therefore, this antibiotic may be a promising alternative for the treatment of infections caused by this bacterium.

Réplica a "Situación de la tos ferina en la Comunidad Valenciana: ¿asistimos a una reactivación epidémica?" / Reply to "Situation of pertussis in the Community of Valencia: An epidemic revival?"

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[Situation of pertussis in Valencian Community: an epidemic revival?]. / Situación de la tos ferina en la Comunidad Valenciana: ¿asistimos a una reactivación epidémica?

INTRODUCTION: Pertussis is a highly contagious vaccine-preventable disease. An increasing incidence has been reported in several countries around the world in the last few years. The aim of this study is to analyze the situation of pertussis in the Valencian Community (Spain), with the purpose of verifying the increased incidence of the illness. METHODS: A descriptive analysis of probable and confirmed cases detected during 2011 was conducted. Methods used for Bordetella pertussis detection from clinical samples were: culture isolation, polymerase chain reaction (PCR)-based detection of bacterial DNA, and/or detection of specific IgM antibodies. The disease incidence and other epidemiological variables were estimated. These results were compared to data collected in previous years (2008-2010). The Epidemiological Surveillance Analysis and Microbiological Surveillance Network integrated systems of the regional Department of Health were used as sources of information. RESULTS: In 2011, 249 cases of pertussis were detected (incidence rate of 4.89×10(5) inhabitants). This rate is statistically significantly higher than those reported in 2008 (0.73×10(5) inhabitants), 2009 (0.53×10(5) inhabitants), and 2010 (0.53×10(5) inhabitants). The highest incidence rate was observed in cases younger than one year old (252.97×10(5) inhabitants), with marked differences compared to the rest of age groups. More than two-thirds (69%) of reported cases were confirmed by a laboratory test. Detection of specific serum IgM antibodies was positive in 10% of cases, culture isolation was positive in 17%, and PCR-based detection of bacterial DNA in 35% of cases. CONCLUSIONS: The results of this study show a clear increase of pertussis incidence in the Valencian Community during 2011.