Role of the National Poliovirus Laboratory for the Program of eradication and poliomyelitis surveillance

The Spanish acute flaccid paralysis surveillance network is coordinated by the National Poliovirus Laboratory (NPL), which, since 1998, carries out polioviruses (PV) and other enteroviruses detected characterization by cell culture and molecular techniques. A total of 110,725 (70046+40679) samples were studied between 1998-2012 and enteroviruses were detected in 8% of these. Among these enteroviruses 241 PV were characterized as PV Sabin-like, except samples belong to an imported poliomyelitis case, all of which were characterised as vaccine derived PV type 2. The NPL has carried out the serotyping and the intratypic differentiation of all the isolated PV in Spain of any syndrome. It is shown that wild PV has not circulated in our country during the 15 years studied and that has led to the signing of the Act of the "eradication of poliomyelitis in Spain" by WHO in 2001, and the /"certification of the eradication of wild PV free for European countries" on 21 June 2002. Currently only 3 countries have endemic transmission of wild PV (Pakistan, Afghanistan and Nigeria). Until a complete worldwide eradication, was achieved, Spain will actively continue to participate in the maintenance of the poliomyelitis eradication infrastructure by monitoring and vaccination as well as the wild PV containment plan to avoid the spread of wild PV. El Laboratorio Nacional de Poliovirus (LNP) coordina la Red Española de Vigilancia de Parálisis Flácida Aguda desde 1998 y caracteriza los poliovirus (PV) y otros enterovirus detectados, utilizando métodos de cultivo celular y moleculares. Durante 1998-2012 se estudiaron por la Red un total de 110.725 (70.046+40.679) muestras clínicas, resultando positivas para enterovirus 8.804 (8%), entre las que 241 se caracterizaron como PV. La caracterización intratípica demostró que todos los PV eran vacunales excepto las muestras correspondientes a un caso importado de poliomielitis postvacunal y sus contactos, que fueron caracterizados como PV2 derivado de vacuna. En el LNP se ha realizado el serotipado y la caracterización intratípica de todos los PV aislados en España de cualquier síndrome. Con ello se ha demostrado que el PV salvaje no ha circulado en nuestro país durante los 15 años que recoge este trabajo y eso condujo a la firma del Acta de la “Erradicación de la Poliomielitis en España” por parte de la OMS en 2001 y a la “Certificación de la Erradicación Europea como libre de circulación de PV salvaje” el 21 de junio de 2002. En la actualidad sólo 3 países presentan transmisión endémica de PV salvaje (Pakistán, Afganistán y Nigeria) y hasta que no se haya conseguido la erradicación a nivel mundial, España debe mantener la infraestructura creada en el Plan de Erradicación de la Poliomielitis y continuar con la vigilancia e inmunización. También el Programa de Contención de los PV salvajes en los laboratorios debe seguir en activo para evitar reintroducciones accidentales.S

Método y kit para la detección del virus de la hepatitis E (VHE).

En la presente invención se describe una combinación de cebadores y un método para la detección molecular de las diferentes cepas circulantes del virus de la hepatitis E (VHE) en humanos y reservorios animales distribuidas geográficamente por todo el mundo. La amplificación del genoma del VHE se realiza mediante RT-PCRs anidadas donde los cebadores empleados amplifican dos fragmentos de las regiones ORF1 (Open Reading Frame) y ORF2 del genoma de este virus. Además, la presente invención incluye un kit con la combinación de los cebadores mencionados y otros componentes.REIVINDICACIONES: 1. Uso de los cebadores directos SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 y SEQ ID NO: 7 y los cebadores reversos SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 y SEQ ID NO: 8 para detectar el virus de la hepatitis E (VHE) en una muestra biológica aislada. 2. Método para la detección del virus VHE que comprende: a. obtener una muestra biológica y aislar sus ácidos nucleicos, b. amplificar de forma simultánea dos fragmentos de las regiones ORF1 y ORF2 del ácido nucleico del apartado (a) por medio del par de cebadores SEQ ID NO: 1/SEQ ID NO: 2 y del par de cebadores SEQ ID NO: 5/SEQ ID NO: 6, c. amplificar de forma simultánea dos fragmentos internos de los fragmentos obtenidos en el apartado (b) por medio del par de cebadores SEQ ID NO: 3/SEQ ID NO: 4 y del par de cebadores SEQ ID NO: 7/SEQ ID NO: 8 y d. determinar la desviación de los apartados (b) y (c) con respecto a un control. 3. Método según la reivindicación 2 donde la muestra biológica se obtiene de un mamífero. 4. Método según cualquiera de las reivindicaciones 2 o 3 donde se incluye un control interno para evaluar la presencia de inhibidores de amplificación. 5. Método según cualquiera de las reivindicaciones 2 a 4 que además permite determinar el genotipo del virus VHE mediante la RT-PCR anidada de la región ORF1. 6. Método según cualquiera de las reivindicaciones 2 a 4, para la monitorización de la respuesta a un tratamiento de hepatitis E. 7. Kit para determinar el genotipo del VHE en una muestra biológica aislada mediante RT-PCR anidada de la región ORF1 que comprende, al menos, los cebadores SEQ ID NO: 1/SEQ ID NO: 2 y SEQ ID NO: 3/SEQ ID NO: 4. 8. Kit para la detección del virus VHE en una muestra biológica aislada mediante RT-PCR anidada que comprende, al menos, los cebadores directos SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 y SEQ ID NO: 7 y los cebadores reversos SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 y SEQ ID NO: 8. 9. Kit según la reivindicación 8 para la monitorización de la respuesta a un tratamiento de hepatitis E. 10. Kit según cualquiera de las reivindicaciones 7 a 9 que comprende, además, el control interno para evaluar la presencia de inhibidores de amplificación.Cuando una patente se hace internacional, se puede encontrar en el idioma de cada país en que se ha solicitado. En Espacenet se tiene acceso a los documentos en cada idioma.Instituto de Salud Carlos IIISolicitud de patent

Evaluation of dried blood spot samples for screening of hepatitis C and human immunodeficiency virus in a real-world setting

Both hepatitis C virus (HCV) infection and human immunodeficiency virus (HIV) infection are underdiagnosed, particularly in low-income countries and in difficult-to-access populations. Our aim was to develop and evaluate a methodology for the detection of HCV and HIV infection based on capillary dry blood spot (DBS) samples taken under real-world conditions. We carried out a cross-sectional study of 139 individuals (31 healthy controls, 68 HCV-monoinfected patients, and 40 HCV/HIV-coinfected patients). ELISA was used for anti-HCV and anti-HIV antibody detection; and SYBR Green RT-PCR was used for HCV-RNA detection. The HIV serological analysis revealed 100% sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). The HCV serological analysis revealed a sensitivity of 92.6%, specificity of 100%, PPV of 100%, and NPV of 79.5%. Finally, the HCV-RNA detection test revealed a detection limit of 5 copies/µl with an efficiency of 100% and sensitivity of 99.1%, specificity of 100%, PPV of 100%, and NPV of 96.9%. In conclusion, our methodology was able to detect both HCV infection and HIV infection from the same DBS sample with good diagnostic performance. Screening for HCV and HIV using DBS might be a key strategy in the implementation of national programs for the control of both infections.We acknowledge the patients’ involvement in this study. The authors thank Thomas O’Boyle for writing assistance during the preparation of the manuscript. This study was supported by grants from Fondo de Investigación de Sanidad en España (FIS) [Spanish Health Founds for Research] [grant numbers PI14CIII/00011] Red Española de Investigación en SIDA (RIS) [AIDS Research Network] [grant numbers RD16CIII/0002/0002RD16] and a research grant from Merck Sharpe & Dohme (MISP IIS#54846).S

Prevalence of hepatitis E infection in HIV/HCV-coinfected patients in Spain (2012-2014)

Hepatitis E virus (HEV) has emerged as a relevant pathogen for HIV-infected patients. However, there is scarce data on HEV infection in HIV/HCV-coinfected individuals with advanced fibrosis, which seems to increase the risk of HEV infection and worsen the prognosis of liver disease. We aimed to determine the prevalence of anti-HEV antibodies, acute hepatitis E, resolved hepatitis E, and exposure to HEV in HIV/HCV-coinfected patients and to evaluate associations with clinical and epidemiological characteristics. We performed a cross-sectional study on 198 HIV/HCV-coinfected patients, 30 healthy controls and 36 HIV-monoinfected patients. We found a low concordance between techniques used for detection of anti-HEV antibodies (ELISA versus Immunoblot), particularly in HIV/HCV-coinfected patients. HIV/HCV-coinfected patients showed the highest prevalence of IgG against HEV, resolved hepatitis E, and exposure to HEV (19.2%, 17.2%, and 22.2% respectively). However, we did not find any samples positive for HEV-RNA nor significant differences between groups. Moreover, HIV/HCV-coinfected patients with CD4 T-cells <350 cells/mm3 had higher prevalence for anti-HEV IgG antibodies, resolved hepatitis E, and exposure to HEV than healthy controls or those with CD4 T-cells ≥ 350 cells/mm3 (p = 0.034, p = 0.035, and p = 0.053; respectively). In conclusion, HIV/HCV-coinfected patients in Spain have a high prevalence for IgG anti-HEV antibodies, resolved hepatitis E, and exposure to HEV; particularly patients with CD4+T-cells <350 cells/mm3.The HIV BioBank, integrated in the Spanish AIDS Research Network, is supported by the Institute of Health Carlos III, ISCIII, Spanish Health Ministry (Grant n° RD06/0006/0035 and RD12/0017/0037) as part of the State Plan for Scientific and Technical Research and Innovation and co-financed by ISCIII- Sub-Directorate General for Research Assessment and Promotion and European Regional Development Fund (ERDF) and Foundation for Research and Prevention of AIDS in Spain (FIPSE). The RIS Cohort (CoRIS) is funded by the ISCIII through the Spanish AIDS Research Network (RIS C03/173 and RD12/0017/0018) as part of the State Plan for Scientific and Technical Research and Innovation and co-financed by ISCIII- Sub-Directorate General for Research Assessment and Promotion and European Regional Development Fund (ERDF). This study was supported by grants from Instituto de Salud Carlos III (ISCII; grant numbers grant numbers PI14/01094 and PI17/00657 to JB, PI14/01581, and PI17/00903 to JGG, PI14CIII/00011, and PI17CIII/00003 to SR), Ministerio de Sanidad, Servicios Sociales e Igualdad (grant number EC11–241). The RD16CIII/0002/0002, RD16/0025/0017, and RD16/0025/0018 projects also funded the study as part of the Plan Nacional R + D + I and co-funded by ISCIII- Subdirección General de Evaluación and the Fondo Europeo de Desarrollo Regional (FEDER). JB is an investigator from the Programa de Intensificación de la Actividad Investigadora en el Sistema Nacional de Salud (I3SNS), Refs INT15/00079 and INT16/00100.S

Impact of the COVID-19 pandemic on tuberculosis national reference laboratory services in the WHO European Region, March to November 2020

We assessed the impact of COVID-19 on diagnostic services for tuberculosis (TB) by national reference laboratories in the WHO European Region. Of 35 laboratories, 30 reported declines in TB sample numbers, amounting up to > 50% of the pre-COVID-19 volumes. Sixteen reported reagent or consumable shortages. Nineteen reallocated ressources to SARS-CoV-2 testing, resulting in an overall increase in workload, largely without a concomitant increase in personnel (n = 14). This poses a risk to meeting the 2025 milestones of the End TB Strategy.Financial support for this work has been provided by the German Government.S

Hepatitis A Outbreak Characteristics: A Comparison of Regions with Different Vaccination Strategies, Spain 2010-2018

We compared the cumulative incidence and characteristics of hepatitis A outbreaks in two groups of Spanish autonomous regions according to whether a universal or risk group vaccination strategy was followed. Outbreaks between 2010 and 2018 were analyzed. The cumulative incidence rate of outbreaks was estimated and compared by estimating the rate ratio (RR). The characteristics of the outbreaks and those of the first cases were compared. Adjusted OR (aOR) were calculated using a multivariate logistic regression model. Outbreak incidence was 16.04 per million persons in regions with universal vaccination and 20.76 in those with risk-group vaccination (RR 0.77; 95%CI 0.62-0.94). Imported outbreaks accounted for 65% in regions with universal vaccination and 28.7% in regions with risk-group vaccination (aOR 3.88; 95%CI 2.13-7.09). Adolescents and young adults aged 15-44 years and men who have sex with men were less frequently the first case of the outbreak in regions with a universal vaccination strategy (aOR 0.54; 95%CI 0.32-0.92 and 0.23; 95%CI 0.07-0.82, respectively). The cumulative incidence rate of outbreaks was lower in regions with universal vaccination. In all regions, independently of the vaccination strategy, activities to vaccinate persons belonging to high-risk groups for infection should be emphasized.This study was funded by the Programme of Prevention, Surveillance, and Control of Transmissible Diseases (PREVICET), CIBER de Epidemiología y Salud Pública (CIBERESP), Instituto de Salud Carlos III, Madrid (Grant number ESPC07/2021).S

Detection of hepatitis C virus (HCV) core-specific antibody suggests occult HCV infection among blood donors

Background: Blood transfusion safety is based on reliable donor screening for transmissible infections such as the hepatitis C virus (HCV) infection. Study design and methods: A novel HCV core-specific antibody was assayed on random single donations from 2007 first-time blood donors who tested negative for anti-HCV and HCV RNA on routine screening. Sample collection broke the code between donations and donors for ethical reasons. Results: Forty-two donations (2.1%) displayed reactivity in the novel test. The specificity of the reactivity was evaluated by a peptide inhibition assay, and testing against additional nonoverlapping HCV core peptide epitopes and other HCV antigens was performed on these samples. Six donations (14.3%; 0.30% from the total) were considered to contain anti-HCV after such supplemental testing. HCV RNA detection was also performed in peripheral blood mononuclear cells (PBMNCs) and serum or plasma samples from reactive donors after virus concentration by ultracentrifugation. HCV RNA tested negative in all PBMNCs samples, and a very low amount of viral genome was detected in serum or plasma concentrates from three anti-HCV core-reactive donors (7.1%) but not among concentrates from 100 randomly selected nonreactive donors. Sequencing of these polymerase chain reaction products revealed differences between the isolates that excluded partially sample contamination from a common source. Conclusion: These findings argue in favor of an ongoing occult HCV infection among these blood donors and account for some rather low, but perhaps not negligible, infection risk for such donations. Future studies involving larger samples of donations from traceable donors would enlighten the significance of these findings for the viral safety of the blood supply.This work was supported by research grants from DIATERS.A., Madrid, Spain.S

Hepatitis E virus genotype 3 microbiological surveillance by the Spanish Reference Laboratory: geographic distribution and phylogenetic analysis of subtypes from 2009 to 2019

Background: Hepatitis E virus genotype 3 (HEV-3) is widely distributed throughout Europe, with incidence of infections increasing in many countries. Belgium, Bulgaria, France, Germany, Italy, the Netherlands and the United Kingdom have reported the distribution of HEV-3 subtypes in cohorts of patients with hepatic disease. Aim: To describe the distribution of the HEV-3 subtypes in Spain at national and autonomous community (AC) levels between 2009 and 2019. The study was also extended to Andorra. Methods: Of 5,197 samples received by the National Reference Laboratory during the study, 409 were HEV-RNA-positive. Among these, 294 (71.9%) were further typed based on an ORF2 sequence fragment, or, for a subset of 74, based on the full-coding genome sequence. Results: HEV-3 was detected in 291 samples. The dominant subtype in Spain was HEV-3f (88.3%; 257/291), which occurred in all ACs, with no change in detection level over time. Within this subtype, three subclusters were characterised: HEV-3f-B, HEV-3f-A1 and HEV-3f-A2. The second most common HEV subtype was the recently described HEV-3m (7%; 21/291), with two subclusters identified: HEV-3m-A, which has been known since 2010, and HEV-3m-B, since 2014. The third most encountered subtype was HEV-3c (4.1%; 12/291), with a frequency not increasing over time, unlike observations in some European countries. Conclusion: The importance of the surveillance of HEV-3 subtype and subcluster circulation is yet to be assessed. This surveillance together with the comprehensive epidemiological characterisation of clinical cases, could support the identification of sources of transmission and the establishment of control measures nationally and internationally.CIBERESP contract for the first author.S

Occurrence of Hepatitis E Virus in Pigs and Pork Cuts and Organs at the Time of Slaughter, Spain, 2017

Zoonotic hepatitis E, mainly caused by hepatitis E virus (HEV) genotype (gt) 3, is a foodborne disease that has emerged in Europe in recent decades. The main animal reservoir for genotype 3 is domestic pigs. Pig liver and liver derivates are considered the major risk products, and studies focused on the presence of HEV in pig muscles are scarce. The objective of the present study was to evaluate the presence of HEV in different organs and tissues of 45 apparently healthy pigs from nine Spanish slaughterhouses (50% national production) that could enter into the food supply chain. Anti-HEV antibodies were evaluated in serum by an ELISA test. Ten samples from each animal were analyzed for the presence of HEV RNA by reverse transcription real-time PCR (RT-qPCR). The overall seroprevalence obtained was 73.3% (33/45). From the 450 samples analyzed, a total of 26 RT-qPCR positive samples were identified in the liver (7/45), feces (6/45), kidney (5/45), heart (4/45), serum (3/45), and diaphragm (1/45). This is the first report on detection of HEV RNA in kidney and heart samples of naturally infected pigs. HEV RNA detection was negative for rib, bacon, lean ham, and loin samples. These findings indicate that pig meat could be considered as a low risk material for foodborne HEV infection.This study was partially supported by the RTA2014-00024-C04 from the Spanish Ministry of Economy and Innovation. NG and DR-L received a research grant by INTERPORC.S