María del Mar Mosquera

High Genetic Diversity of Measles Virus, World Health Organization European Region, 2005–2006

Importation of viruses from other continents caused prolonged circulation and large outbreaks in the WHO European Region.

Simultaneous Detection of Measles Virus, Rubella Virus, and Parvovirus B19 by Using Multiplex PCR

ABSTRACT We describe here a multiplex reverse transcription-PCR (RTMNPCR) assay designed to detect and differentiate measles virus, rubella virus, and parvovirus B19. Serial dilution experiments with vaccine strains that compared cell culture isolation of measles in B95 cells and rubella in RK13 cells showed sensitivity rates of 0.004 50% tissue culture infective dose (TCID50) for measles virus and 0.04 TCID50 for rubella virus. This RTMNPCR can detect as few as 10 molecules for measles virus and rubella virus and one molecule for parvovirus B19 in dilution experiments with plasmids containing inserts of the primary reaction amplification products. Five pharyngeal exudates from measles patients and 2 of 15 cerebrospinal fluid samples from measles-related encephalitis were found to be positive for measles virus by this RTMNPCR. A total of 3 of 27 pharyngeal exudates from vaccinated children and 2 pharyngeal exudates, plus one urine sample from a case of congenital rubella syndrome, were found to be positive for rubella virus by RTMNPCR, whereas 16 of 19 sera from patients with erythema infectiosum were determined to be positive for parvovirus B19 by RTMNPCR. In view of these results, we can assess that this method is a useful tool in the diagnosis of these three viruses and could be used as an effective surveillance tool in measles eradication programs.

Sensitivity and specificity of immunoglobulin G titer for the diagnosis of mumps virus in infected patients depending on vaccination status

The aim of the study was to evaluate the usefulness of serological detection of mumps IgM and titration of IgG in patients with acute parotitis according to their vaccination status. The detection of mumps virus RNA in saliva by RT‐PCR was used as reference. 116 patients (109 of them previously vaccinated) with mumps RT‐PCR‐negative results and 21 (19 vaccinated) with mumps RT‐PCR‐positive results were studied. Mumps‐specific IgM and IgG were assayed by EIA (Enzygnost, Dade Behring, Germany). IgM results were expressed as positive or negative. For IgG, several cut‐offs were calculated using receiver operating characteristic (ROC) curves. Seven RT‐PCR‐positive and five RT‐PCR‐negative patients showed IgM‐positive results (sensitivity 33.3% and specificity 95.7%). Among vaccinated patients, the sensitivity and specificity of IgM were 26.3% (5/19) and 99.1% (108/109). For IgG, a titer of 5,000 in all the patients showed a sensitivity of 76.2% (16/21) and a specificity of 83.6% (97/116). In vaccinated patients, the corresponding figures for this cut‐off were 84.2% (16/19) and 83.5% (91/109), respectively. Although IgM detection against mumps is highly specific, its sensitivity is very low in immunized subjects. In this group, the titration of IgG could serve as an additional diagnostic tool.

Measles viruses of genotype H1 evade recognition by vaccine-induced neutralizing antibodies targeting the linear haemagglutinin noose epitope

The linear haemagglutinin noose epitope (HNE; aa 379-410) is a protective B-cell epitope and considered to be highly conserved in both the vaccine and the wild-type measles virus (MeV) haemagglutinin (H) proteins. Vaccine virus-derived monoclonal antibodies (mAbs) BH6 and BH216, which target the HNE, neutralized MeVs of genotypes B3, C2, D4, D5, D6, D7 and D8, and the vaccine strain Edmonston Zagreb. In the case of genotype H1, only strain Berlin.DEU/44.01 was neutralized by these mAbs, whereas strains Shenyang.CHN/22.99 and Sofia.BGR/19.05 were not. The H gene sequences of these two strains showed an exchange of proline 397 (P397) to leucine (L397). Mutated H proteins, with P397 exchanged to L and vice versa, were compared with original H proteins by indirect fluorescence assay. H proteins exhibiting P397 but not those with L397 were recognized by BH6 and BH216. This indicates that L397 leads to the loss of the neutralizing HNE. In contrast, human sera obtained from vaccinees (n=10) did not discriminate between genotype H1 variants P397 and L397. This concurs with the epidemiological observation that the live-attenuated vaccine protects against both H1 variants. Furthermore, we demonstrated that MeVs of genotype H1 also lack the neutralizing epitopes defined by the vaccine virus-induced mAbs BH15, BH125 and BH47. The loss of several neutralizing epitopes, as shown for H1 viruses currently circulating endemically in Asia, implies that epitope monitoring should be considered to be included in measles surveillance.

Evaluation of Diagnostic Markers for Measles Virus Infection in the Context of an Outbreak in Spain

ABSTRACT A measles outbreak occurred from January to July 2003 in Spain, despite the fact that the Plan of Eradication of Measles and its surveillance program had been set up in 2001. Different diagnostic markers for measles virus infection were compared for 246 patients in tests of serum, urine, and pharyngeal exudate specimens. Measles virus immunoglobulin M (IgM) and IgG and rubella virus and parvovirus IgM levels in serum were assayed. Multiplex PCR was done on urine, serum, and pharyngeal exudates, and isolation of measles virus in the B95a cell line from urine was attempted. At least one positive marker for measles virus was obtained from 165 patients (67.1%; total number of patients, 246). A total of 136 cases (82.4% of the patients showing positive markers) were diagnosed by PCR and/or isolation and IgM detection methods. The results for 27 patients (16.4%) were positive only by direct methods. The results for two patients (1.2%) were positive only by IgM detection. In the case of the first group (136 cases), the time elapsed from appearance of the rash was significantly longer than in the case of the group which was only positive by PCR. Besides, 8 out of 27 PCR-positive IgM-negative cases showed specific IgG results, suggesting either secondary vaccine failure or reinfection. Numbers resulting from PCR performed with pharyngeal exudates proved to be significantly higher than those obtained with other specimens. Phylogenetic analysis showed the presence of genotype B3. The results strongly back the World Health Organization recommendation that detection of IgM should be supplemented by PCR and isolation for the diagnosis of measles virus infection.

Comparación de los procedimientos serológicos de los laboratorios del Plan para la Eliminación del Sarampión en el diagnóstico de exantemas víricos

Objetivo Comparar los metodos serologicos empleados por los laboratorios de la Red del Plan de Eliminacion del Sarampion para el diagnostico de la infeccion por virus del sarampion, y para el diagnostico diferencial con otras enfermedades exantematicas. Materiales y metodos Se ha establecido un panel de 20 muestras de suero de casos de enfermedad exantematica (sarampion [12], rubeola [4], parvovirus B19 [2] y dengue [2]), diagnosticados por deteccion de inmunoglobulina M (IgM) especifica. El panel se ha enviado a los laboratorios de la Red. Resultados y discusion Se han recibido respuestas de 20 laboratorios para IgM frente a sarampion, 19 realizadas por enzimoinmunoanalisis (ELISA) y una por inmunofluorescencia indirecta (IFI). La concordancia con el laboratorio de referencia ha sido del 91,5%. Para IgM de rubeola se han recibido respuestas de seis laboratorios (todas obtenidas por ELISA) con una concordancia del 98,7%. Para IgM de parvovirus B19 se ha recibido respuesta de 10 laboratorios (ocho realizaron ELISA y dos IFI), con una concordancia del 94,6%. Ningun laboratorio realizo diagnostico de dengue. Conclusion Algunos de los laboratorios de la Red deberian revisar los metodos empleados para el diagnostico de sarampion. Por otra parte, se considera necesaria la existencia de un laboratorio de referencia que sirva de apoyo para la confirmacion de resultados, asi como para el diagnostico de otros agentes exantematicos, cuando los antecedentes epidemiologicos asi lo requieran.

Appearance of a novel measles G3 strain in multiple European countries within a two month period, 2010

During late 2010, a previously unrecognised strain of measles genotype G3 virus was identified in five different European countries by the World Health Organization Measles and Rubella Laboratory Network.Apart from one, none had a travel history to south-east Asia, the usual source of G3 viruses, although epidemiological links could be established between some of the cases. This case series illustrates the value of genotyping and sequencing in tracking measles infections, and identifying otherwise unrecognised chains of transmission.

Estudio del rendimiento diagnóstico de la detección de IgM específica y de la amplificación genómica de rubéola

En la actualidad la mayoria de los casos de enfermedades exantematicas prevenibles por inmunizacion afectan a jovenes. Ademas, una elevada proporcion de estos casos se confirman como rubeola. El objetivo de este estudio es evaluar el rendimiento de la IgM especifica y una tecnica de reaccion en cadena de la polimerasa en tiempo real (RT-PCR) en el diagnostico de la infeccion por virus de la rubeola. Se estudiaron 59 pacientes bajo sospecha clinica de sarampion o rubeola de los cuales se disponia de muestras de suero, sangre completa, orina y exudado faringeo. Se comprobo que la RT-PCR en exudado faringeo fue el marcador diagnostico mas eficaz en los primeros dias de la enfermedad (2,5 dias de media). Sin embargo, la deteccion de IgM mostro un mayor rendimiento (76,2%), aunque mas tardiamente (3,7 dias).

Comparative evaluation of tests for detection of parvovirus B19 IgG and IgM

The aim of this study was to evaluate enzyme immunoassays (EIA) (Euroimmun, Lübeck, Germany) and chemiluminiscent immunoassays (CLIA) (Diasorin, Saluggia, Italy) in their application to detect B19V‐IgM and ‐IgG. For this purpose, one hundred and ninety samples were studied. Of them, 101 came from recent infection cases (B19V‐specific IgM (86) and/or PCR (87), 42 from past infections, 18 from non‐infected, and 29 from other viral recent infections (Epstein‐Barr virus, measles, and rubella). Samples were characterized by capture (for IgM), or indirect (for IgG) EIA (Biotrin, Dublin, Ireland); indeterminate samples were classified by indirect immunofluorescence (IIF) (Biotrin). All the samples were used for testing IgM assays, and all but the cases from other viral infections were used for IgG tests. For IgM, CLIA, and EIA identified 76 and 62 of 86 IgM positives, respectively (sensitivity 88.4% and 72.1%). Considering B19V IgM negative samples, negative result was obtained in 95 and 92 of 104, being the specificity values of CLIA and EIA 91.3% and 88.5%, respectively. For IgG, CLIA and EIA identified correctly 114 and 115 of the 122 positive samples (sensitivity 93.4% and 94.3%, respectively), and 39 and 36 of 39 negative samples (specificity 100% and 92.3%). As conclusion, CLIA methods can be used in clinical laboratories as adequate alternatives to the well‐established Biotrin EIAs.

Genetic Characterization of Rubella Virus Strains Detected in Spain, 1998-2014.

The National Plan for the Elimination of Rubella was implemented in Spain in 2008 using the logistics of the National Plan for the Elimination of Measles that have been employed since year 2000. Molecular characterization of rubella virus (RUBV) is important for disease surveillance and for monitoring elimination of the disease throughout the world. We describe the first complete series of data regarding the circulation of RUBV genotypes in Spain. The 739-nucleotide fragment designated by the WHO for RUBV genotyping was sequenced in 88 selected cases collected from 1998 to 2014. Five genotypes were identified: 1E, 2B, 1J, 1I, and 1a. Genotype 1E was predominant between 1998 and 2003 but was replaced by genotype 2B, which was detected in sporadic cases in 2004, 2006, 2008, 2012, 2013 and 2014. There was an outbreak of genotype 2B in Algeciras (Andalusia) in 2008. Genotype 1J caused an outbreak in Madrid in 2004/2005 and sporadic cases in 2005 and 2007. Genotype 1I was found to have infected an immune-suppressed patient with neurological symptoms in 2008. Finally, vaccine strain RA 27/3 was detected in three sporadic cases, two of them immune-suppressed and without a recent history of vaccination. This suggests that during these years there were a series of imported sporadic cases and outbreaks, confirming the findings of epidemiological data analysis. The importation sources were generally consistent with our geographic and cultural ties, mainly with Europe (genotypes 1E, 2B, 1I) and Latin America (1J).