Investigating Local Patterns of Mumps Virus Circulation, Using a Combination of Molecular Tools.

Mumps is a vaccine-preventable disease caused by the mumps virus (MuV). However, MuV has re-emerged in many countries with high vaccine coverage. The World Health Organization (WHO) recommends molecular surveillance based on sequencing of the small hydrophobic (SH) gene. Additionally, the combined use of SH and non-coding regions (NCR) has been described in different studies, proving to be a useful complement marker to discriminate general patterns of circulation at national and international levels. The aim of this work is to test local-level usefulness of the combination of SH and MF-NCR sequencing in tracing hidden transmission clusters and chains during the last epidemic wave (2015-2020) in Spain. A database with 903 cases from the Autonomous Community of Madrid was generated by the integration of microbiological and epidemiological data. Of these, 453 representative cases were genotyped. Eight different SH variants and thirty-four SH haplotypes were detected. Local MuV circulation showed the same temporal pattern previously described at a national level. Only two of the thirteen previously identified outbreaks were caused by more than one variant/haplotype. Geographical representation of SH variants allowed the identification of several previously undetected clusters, which were analysed phylogenetically by the combination of SH and MF-NCR, in a total of 90 cases. MF-NCR was not able to improve the discrimination of geographical clusters based on SH sequencing, showing limited resolution for outbreak investigations.

Comparison of circulation patterns of mumps virus in the Netherlands and Spain (2015-2020).

Background: Mumps is a viral infection mainly characterized by inflammation of the parotid glands. Despite of vaccination programs, infections among fully vaccinated populations were reported. The World Health Organization (WHO) recommends molecular surveillance of mumps based on sequencing of the small hydrophobic (SH) gene. The use of hypervariable non-coding regions (NCR) as additional molecular markers was proposed in multiple studies. Circulation of mumps virus (MuV) genotypes and variants in different European countries were described in the literature. From 2010 to 2020, mumps outbreaks caused by genotype G were described. However, this issue has not been analyzed from a wider geographical perspective. In the present study, sequence data from MuV detected in Spain and in The Netherlands during a period of 5 years (2015- March 2020) were analyzed to gain insights in the spatiotemporal spread of MuV at a larger geographical scale than in previous local studies. Methods: A total of 1,121 SH and 262 NCR between the Matrix and Fusion protein genes (MF-NCR) sequences from both countries were included in this study. Analysis of SH revealed 106 different haplotypes (set of identical sequences). Results: Of them, seven showing extensive circulation were considered variants. All seven were detected in both countries in coincident temporal periods. A single MF-NCR haplotype was detected in 156 sequences (59.3% of total), and was shared by five of the seven SH variants, as well as three minor MF-NCR haplotypes. All SH variants and MF-NCR haplotypes shared by both countries were detected first in Spain. Discussion: Our results suggest a transmission way from south to north Europe. The higher incidence rate of mumps in Spain in spite of similar immunization coverage in both countries, could be associated with higher risk of MuV exportation. In conclusion, the present study provided novel insights into the circulation of MuV variants and haplotypes beyond the borders of single countries. In fact, the use of MF-NCR molecular tool allowed to reveal MuV transmission flows between The Netherlands and Spain. Similar studies including other (European) countries are needed to provide a broader view of the data presented in this study.

Utility of MF-non coding region for measles molecular surveillance during post-elimination phase, Spain, 2017-2020.

Background: In countries entering the post-elimination phase for measles, the study of variants by sequencing of 450 nucleotides of the N gene (N450) does not always allow the tracing of chains of transmission. Indeed, between 2017 and 2020, most measles virus sequences belonged to either the MVs/Dublin.IRL/8.16 (B3-Dublin) or the MVs/Gir Somnath.IND/42.16 (D8-Gir Somnath) variants. We evaluated the additional use of a non-coding region (MF-NCR) as a tool to enhance resolution and infer case origin, chains of transmission and characterize outbreaks. Methods: We obtained 115 high-quality MF-NCR sequences from strains collected from Spanish patients infected with either B3-Dublin or D8-Gir Somnath variants between 2017 and 2020, performed epidemiological, phylogenetic and phylodynamic analyses and applied a mathematical model to determine relatedness among identified clades. Results: Applying this model allowed us to identify phylogenetic clades potentially derived from concomitant importations of the virus rather than single chain of transmission, inferred based on only N450 and epidemiology data. In a third outbreak, we found two related clades that corresponded to two chains of transmission. Discussion: Our results show the ability of the proposed method to improve identification of simultaneous importations in the same region which could trigger enhanced contact tracing. Moreover, the identification of further transmission chains indicates that the size of import-related outbreaks was smaller than previously found, supporting the interpretation that endemic measles transmission was absent in Spain between 2017 and 2020. We suggest considering the use of the MF-NCR region in conjunction with the study of N450 variants in future WHO recommendations for measles surveillance.

An interregional measles outbreak in Spain with nosocomial transmission, November 2017 to July 2018.

Given sustained high vaccination coverage and enhanced surveillance for measles, Spain has been free of endemic measles transmission since 2014, achieving elimination certification from the World Health Organization in 2017. In November 2017, measles was introduced through an imported case travelling to the Valencian Community, causing an interregional outbreak. Here, we describe the outbreak using data reported to the national epidemiological surveillance network. The outbreak involved 154 cases (67 males, 87 females) notified in four regions; 148 were laboratory-confirmed and six epidemiologically linked. Most cases were adults aged 30-39 (n = 62, 40.3%) years. Sixty-two cases were hospitalised (40.3%) and 35 presented complications (22.7%). Two thirds of the cases (n = 102) were unvaccinated including 11 infants (≤ 1 year) not yet eligible for vaccination. The main route of transmission was nosocomial; at least six healthcare facilities and 41 healthcare workers and support personnel were affected. Sequencing of the viral nucleoprotein C-terminus (N450) identified genotype B3, belonging to the circulating MVs/Dublin.IRL/8.16-variant. Control measures were implemented, and the outbreak was contained in July 2018. The outbreak highlighted that raising awareness about measles and improving the vaccination coverage in under-vaccinated subgroups and personnel of healthcare facilities are key measures for prevention of future outbreaks.

SARS-CoV-2 Infection During the First and Second Pandemic Waves in Spain: the ENE-COVID Study.

Objectives. To describe participant characteristics associated with severe acute respiratory syndrome coronavirus 2 infection in Spain's first 2 COVID-19 waves per the Spanish National Seroepidemiological Survey of SARS-CoV-2 Infection (ENE-COVID). Methods. A representative cohort of the noninstitutionalized Spanish population, selected through stratified 2-stage sampling, answered a questionnaire and received point-of-care testing April to June 2020 (first wave: n = 68 287); previously seronegative participants repeated the questionnaire and test November 2020 (second wave: n = 44 451). We estimated seropositivity by wave and participant characteristics, accounting for sampling weights, nonresponse, and design effects. Results. We found that 6.0% (95% confidence interval [CI] = 5.7%, 6.4%) of Spain's population was infected by June and 3.8% (95% CI = 3.5%, 4.1%) more by November 2020. Both genders were equally affected. Seroprevalence decreased with age in adults 20 years and older in the second wave; socioeconomic differences increased. Health care workers were affected at 11.1% (95% CI = 9.0%, 13.6%) and 6.1% (95% CI = 4.4%, 8.5%) in the first and second waves, respectively. Living with an infected person increased infection risk to 22.1% (95% CI = 18.9%, 25.6%) in the first and 35.0% (95% CI = 30.8%, 39.4%) in the second wave. Conclusions. ENE-COVID characterized the first 2 pandemic waves, when information from surveillance systems was incomplete. (Am J Public Health. 2023;113(5):533-544. https://doi.org/10.2105/AJPH.2023.307233).

Design and Implementation of a Nationwide Population-Based Longitudinal Survey of SARS-CoV-2 Infection in Spain: The ENE-COVID Study.

Data System. The Spanish National Seroepidemiological Survey of SARS-CoV-2 (or ENE-COVID; SARS-CoV-2 [severe acute respiratory syndrome coronavirus 2] is the causative agent of COVID-19) was funded by the Spanish Ministry of Health, the Instituto de Salud Carlos III, and the Spanish National Health System. Data Collection/Processing. A stratified 2-stage probability sampling was used to select a representative cohort of the noninstitutionalized population of Spain. ENE-COVID collected longitudinal data from epidemiological questionnaires and 2 SARS-CoV-2 IgG antibody tests. From April 27 to June 22, 2020, 68 287 participants (77.0% of contacted persons) received a point-of-care test and 61 095 (68.9%) also underwent a laboratory immunoassay. A second follow-up phase was conducted between November 16 and 30, 2020. Data Analysis/Dissemination. Analyses use weights to adjust for oversampling and nonresponse and account for design effects of stratification and clustering. ENE-COVID data for research purposes will be available upon request from the official study Web page. Public Health Implications. ENE-COVID, a nationwide population-based study, allowed monitoring seroprevalence of antibodies against SARS-CoV-2 at the national and regional levels, providing accurate figures by gender, age (from babies to nonagenarians), and selected risk factors; characterizing symptomatic and asymptomatic infections; and estimating the infection fatality risk during the first pandemic wave. (Am J Public Health. 2023;113(5):525-532. https://doi.org/10.2105/AJPH.2022.307167).

Brote de sarampión en el área sanitaria de Guadalajara (España): dificultad en el diagnóstico microbiológico en la era de su eliminación / Measles outbreak in the sanitary area of Guadalajara (Spain): Difficulty in microbiological diagnosis in the era of its elimination

Introducción: En España, al igual que en otros países donde el sarampión endémico ha sido eliminado, es necesario utilizar de forma rutinaria las herramientas diagnósticas que confirmen los casos para su prevención y control de la diseminación. Se describen los diferentes ensayos microbiológicos utilizados para su diagnóstico durante un brote de sarampión en 2019 en la provincia de Guadalajara (España). Métodos: Las pruebas serológicas y moleculares se realizaron en el laboratorio de Microbiología del Hospital Universitario de Guadalajara y en el Centro Nacional de Microbiología del Instituto de Salud Carlos III (Majadahonda, España). Los datos de los pacientes se obtuvieron del sistema epidemiológico de vigilancia. Resultados: Se diagnosticaron de sarampión un total de 43 pacientes por métodos microbiológicos: 29 casos por PCR (exudado faríngeo u orina) junto con IgM específica positiva, 11 pacientes solamente por PCR, y 3 pacientes exclusivamente por presencia de IgM. El genotipo D8 fue identificado en 35 pacientes y el genotipo A en 2 casos descartados como postvacunal. La PCR en suero fue positiva en 11 de 14 pacientes con ausencia de IgM en su primera muestra recogida de suero. Once casos confirmados habían recibido una o 2 dosis de la vacuna. Doce adultos fueron ingresados, todos diagnosticados de hepatitis. Conclusiones: La combinación de pruebas moleculares y la presencia de IgG e IgM específicas son necesarias para un diagnóstico correcto y la clasificación de los pacientes como fallo vacunal (primario o secundario). El genotipado es una herramienta fundamental para la correcta clasificación de los pacientes en el contexto de un programa de eliminación del sarampión.(AU)

Introduction: In Spain, like in other countries where endemic measles has been eliminated, there is a need for available diagnostic tools for confirming any cases in order to prevent and control its transmission. We describe the different microbiological tests used for the diagnosis of measles during an outbreak that occurred in 2019 in the province of Guadalajara (Spain). Methods: Serological and molecular tests were performed at the Microbiology laboratory of the Guadalajara University Hospital and at the National Center for Microbiology of the Carlos III Health Institute (Majadahonda, Spain). Patient data were obtained from the surveillance system. Results: A total of 43 patients had a laboratory diagnosis of measles: 29 cases by PCR (pharyngeal exudate or urine) and positive specific IgM, 11 cases by PCR, and 3 cases only by a positive IgM. Genotype D8 was identified in 35 confirmed cases and genotype A in 2 that were discarded as post-vaccination cases. PCR was positive in the acute sera of 11 out of 14 patients with a negative IgM. Eleven confirmed cases had recieved one or 2 vaccine doses. Twelve adult patients were hospitalizated, all of them with a diagnostic of hepatitis. Conclusions: The combination of molecular tests and the presence of specific IgG and IgM are necessary for a correct diagnosis of measles and also to classify patients with a breakthrough infection or vaccine failures (primary or secondary). Genotyping is essential for the correct classification of the patients in the context of a measles elimination program.(AU)

Measles outbreak in the sanitary area of Guadalajara (Spain): difficulty in microbiological diagnosis in the era of its elimination.

INTRODUCTION: In Spain, like in other countries where endemic measles has been eliminated, there is a need for available diagnostic tolos for confirming any cases in order to prevent and control its transmission. We describe the different microbiological tests used for the diagnosis of measles during an outbreak that occurred in 2019 in the province of Guadalajara (Spain). METHODS: Serological and molecular tests were performed at the Microbiology laboratory of the Guadalajara University Hospital and at the National Center for Microbiology of the Carlos III Health Institute (Majadahonda, Spain). Patient data were obtained from the surveillance system. RESULTS: A total of 43 patients had a laboratory diagnosis of measles: 29 cases by PCR (pharyngeal exudate or urine) and positive specific IgM, 11 cases by PCR, and 3 cases only by a positive IgM. Genotype D8 was identified in 35 confirmed cases and genotype A in two that were discarded as post-vaccination cases. PCR was positive in the acute sera of 11 out of 14 patients with a negative IgM. Eleven confirmed cases had recieved one or two vaccine doses. Twelve adult patients were hospitalizated, all of them with a diagnostic of hepatitis. CONCLUSIONS: The combination of molecular tests and the presence of specific IgM is necessary for a correct diagnosis of measles and also to classify patients with a breakthrough infection or vaccine failures (primary or secondary). Genotyping is essential for the correct classification of the patients in the context of a measles elimination program.

Mind your Ps: A probabilistic model to aid the interpretation of molecular epidemiology data.

BACKGROUND: Assessing relatedness of pathogen sequences in clinical samples is a core goal in molecular epidemiology. Tools for Bayesian analysis of phylogeny, such as the BEAST software package, have been typically used in the analysis of sequence/time data in public health. However, they are computationally-, time-, and knowledge-intensive, demanding resources that many laboratories do not have available or cannot allocate frequently. METHODS: To evaluate a faster and simpler alternative method to support the routine interpretation of sequence data for epidemiology, we obtained sequences for two regions in the measles virus genome, N-450 and MF-NCR, from patient samples of genotypes B3, D4 and D8 taken between 2011 and 2017 in the UK and Romania. A mathematical model incorporating time, possible shared ancestry and the Poisson distribution describing the number of expected substitutions at a given time point was developed to exclude epidemiological relatedness between pairs of sequences. The model was validated against the commonly used Bayesian phylogenetic method using an independent dataset collected in 2017-19. FINDINGS: We demonstrate that our model, using time and sequence information to predict whether two samples may be related within a given time frame, minimises the risk of erroneous exclusion of relatedness. An easy-to-use implementation in the form of a guide and spreadsheet is provided for convenient application. INTERPRETATION: The proposed model only requires a previously calculated substitution rate for the locus and pathogen of interest. It allows for an informed but quick decision on the likelihood of relatedness between two samples within a time frame, without the need for phylogenetic reconstruction, thus facilitating rapid epidemiological interpretation of sequence data. FUNDING: This work was funded by the United Kingdom Health Security Agency (UKHSA). The World Health Organization European Regional Office funded Aurora Fernández-García and Mihaela Lazar training visits to UKHSA.

Evolution of antibodies against SARS-CoV-2 over seven months: Experience of the nationwide seroprevalence ENE-COVID study in Spain.

BACKGROUND: The main aims of this study were to analyze trends of SARS-CoV-2 anti-nucleocapsid IgG throughout the four rounds of the seroepidemiologic study ENE-COVID, and compare the fourth-round results of two immunoassays detecting anti-nucleocapsid and anti-RBD IgG. METHODS: ENE-COVID was developed in 2020 (two phases). Phase one included three rounds carried out in April 27-May 11, May 18-June 1, and June 8-June 22. Phase two included a fourth round in the same cohort (November 16-29). A chemiluminescent microparticle immunoassay was offered to participants in the first three rounds (Abbott; anti-nucleocapsid IgG). In the fourth round, we offered this test and a chemiluminescence immunoassay (Beckman; anti-RBD IgG) to i) a randomly selected sub-cohort, ii) participants who were IgG-positive in any of the three first rounds; and iii) participants who were IgG-positive in the fourth round by point-of-care immunochromatography. RESULTS: 10,153 individuals (82.2% of people invited) participated in the fourth round. Of them, 2595 (35.1% of participants with results in the four rounds) were positive for anti-nucleocapsid IgG in at least one round. Anti-nucleocapsid IgG became undetectable in 43.3% of participants with positive first-round results. In fourth round, anti-nucleocapsid and anti-RBD IgG were detected in 5.5% (321/5827) and 5.4% (315/5827) participants of the randomly selected sub-cohort, and in 26.6% (867/3261) and 25.9% (846/3261) participants with at least one previous positive result, respectively. CONCLUSIONS: The IgG response is heterogeneous and conditioned by infection severity. A proportion of SARS-CoV-2 infected population may have negative serologic results in the post-infection months.

Comparative evaluation of assays for IgM detection of rubella and measles infections.

BACKGROUND: Serological diagnosis of infections due to measles and rubella viruses is done by IgM detection. The aim of this study was to comparatively evaluate commercial systems for detecting IgM against both viruses, including those of ELISA, in indirect and capture formats, chemiluminescence and electrochemiluminescence. METHODS: Seven (for rubella) and six (for measles) assays were studied. One hundred and sixty two samples were included in the study (from 90 rubella and 72 measles cases), and all were analyzed in all the assays. RESULTS: The ranges of sensitivity, specificity and agreement for rubella were 94.8-100%, 52.4-100% and 75.5-98.1%, respectively. The corresponding ranges for measles assays were 87.0-100%, 53.3-100%, and 73.0-99.4%. CONCLUSION: The best-performing assays were chemiluminescence (for measles and rubella IgM), and electrochemiluminescence (for rubella IgM).

Comparative evaluation of assays for IgM detection of rubella and measles infections / Evaluación comparativa de ensayos para la detección de IgM en infecciones por rubéola y sarampión

BackgroundSerological diagnosis of infections due to measles and rubella viruses is done by IgM detection. The aim of this study was to comparatively evaluate commercial systems for detecting IgM against both viruses, including those of ELISA, in indirect and capture formats, chemiluminescence and electrochemiluminescence.MethodsSeven (for rubella) and six (for measles) assays were studied. One hundred and sixty two samples were included in the study (from 90 rubella and 72 measles cases), and all were analyzed in all the assays.ResultsThe ranges of sensitivity, specificity and agreement for rubella were 94.8–100%, 52.4–100% and 75.5–98.1%, respectively. The corresponding ranges for measles assays were 87.0–100%, 53.3–100%, and 73.0–99.4%.ConclusionThe best-performing assays were chemiluminescence (for measles and rubella IgM), and electrochemiluminescence (for rubella IgM).

El diagnóstico serológico de las infecciones por los virus de la rubéola y del sarampión se realiza por detección de IgM específica. El objetivo de este estudio fue evaluar comparativamente sistemas comerciales para la detección de IgM frente a ambos virus, incluyendo ensayos de ELISA, tanto con metodologías indirectas como de captura, así como quimioluminiscencia y electroquimioluminiscencia.MétodosSe estudiaron 7 ensayos para rubéola y 6 para sarampión. Se emplearon 162 muestras (de 90 casos de rubéola y de 72 de sarampión) que se analizaron en todos los ensayos.ResultadosLos rangos de sensibilidad, especificidad y concordancia para los ensayos de rubéola fueron 94,8-100%, 52,4-100% y 75,5-98,1%, respectivamente. Los rangos correspondientes para los ensayos de sarampión fueron 87-100%, 53,3-100% y 73-99,4%, respectivamente.ConclusiónLos mejores ensayos fueron quimioluminiscencia (para IgM frente a rubéola y a sarampión) y electroquimioluminiscencia (para IgM frente a rubéola).

Brote de parotiditis relacionado con el fumar en un narguile de uso público / Mumps virus outbreak related to a water pipe (narghile) shared smoking

Introducción: En este estudio se describe un brote de parotiditis que afectó a un grupo de jóvenes que compartieron un narguile para fumar. Métodos: La notificación de un caso de parotiditis dio lugar a una investigación epidemiológica. Se recabó información de otras 6personas sintomáticas que se habían reunido en una discoteca en la que habían fumado en un mismo narguile. Se obtuvieron muestras de saliva para RT-PCR y sangre para serología de parotiditis de otros 3de estos casos. Las muestras RT-PCR positivas se genotipificaron por secuenciación. Resultados: Los 7 pacientes residían en 3municipios diferentes. Hacía más de un mes que no habían coincidido hasta que estuvieron en la discoteca. Cuatro casos se confirmaron por RT-PCR o IgM específica. La investigación genómica mostró secuencias idénticas. Conclusiones: Este brote es consecuencia de un uso compartido de un narguile. Consideramos que se debería regular la utilización pública de estas pipas.(AU)

Introduction: This study describes a mumps outbreak among a group of young people who shared a same narghile to smoking. Saliva and blood samples were obtained from 3cases for RT-PCR and serology respectively. Methods: The notification of a mumps case started an epidemiological investigation. Information of other 6additional symptomatic persons who had gathered with the case in a discotheque where they smoking in a same narghile was achieved. RT-PCR positive samples were genotyped by sequencing. Results: The 7patients resided in 3different municipalities, and they do not have get together for more than a month until the meeting in the discotheque. Four cases were confirmed by RT-PCR and/or IgM determinations. The genomic investigation showed identical nucleic sequences. Conclusions: This outbreak is consequence of the common use of a narghile to smoking. The public usage of these water pipes should be regulated.(AU)

Measles in Vaccinated People: Epidemiology and Challenges in Surveillance and Diagnosis in the Post-Elimination Phase. Spain, 2014-2020.

The MMR vaccination program was introduced in Spain in 1981. Consistently high vaccination coverage has led to Spain being declared free of endemic measles transmission since 2014. A few imported and import-related cases were reported during the post-elimination phase (2014 to 2020), with very low incidence: three cases per million of inhabitants a year, 70% in adults. In the post-elimination phase an increasing proportion of measles appeared in two-dose vaccinated individuals (up to 14%), posing a challenge to surveillance and laboratory investigations. Severity and clinical presentation were milder among the vaccinated. The IgM response varied and the viral load decreased, making the virus more difficult to detect. A valid set of samples (serum, urine and throat swab) is strongly recommended for accurate case classification. One third of measles in fully vaccinated people was contracted in healthcare settings, mainly in doctors and nurses, consistent with the important role of high intensity exposure in measles breakthrough cases. Surveillance protocols and laboratory algorithms should be adapted in advanced elimination settings. Reinforcing the immunity of people working in high exposure environments, such as healthcare settings, and implementing additional infection control measures, such as masking and social distancing, are becoming crucial for the global aim of measles eradication.

Mumps virus outbreak related to a water pipe (narghile) shared smoking.

INTRODUCTION: This study describes a mumps outbreak among a group of young people who shared a same narghile to smoking. Saliva and blood samples were obtained from 3 cases for RT-PCR and serology respectively. METHODS: The notification of a mumps case started an epidemiological investigation. Information of other 6 additional symptomatic persons who had gathered with the case in a discotheque where they smoking in a same narghile was achieved. RT-PCR positive samples were genotyped by sequencing. RESULTS: The 7 patients resided in 3 different municipalities, and they do not have get together for more than a month until the meeting in the discotheque. Four cases were confirmed by RT-PCR and/or IgM determinations. The genomic investigation showed identical nucleic sequences. CONCLUSIONS: This outbreak is consequence of the common use of a narghile to smoking. The public usage of these water pipes should be regulated.