HCV, HIV AND HBV rapid test diagnosis in non-clinical outreach settings can be as accurate as conventional laboratory tests.

Point of care rapid diagnostic tests (POC-RDT) for Hepatitis C virus (HCV), Human Immunodeficiency virus (HIV) and Hepatitis B virus (HBV), are ideal for screening in non-clinical outreach settings as they can provide immediate results and facilitate diagnosis, allowing high risk population screening. The aim of this study was to compare POC-RDT with laboratory conventional tests. A total of 301 vulnerable evaluable subjects (drug users, migrants and homeless population) were recruited at a mobile screening unit in outreach settings in Madrid. Fingerprick whole blood capillary samples were tested using the SD BIOLINE HCV POC-RDT, Determine HIV Early Detect and Determine HBsAg 2, and the results were assessed against the LIAISON XL HCV, HIV and Murex-HBsAg-Quant, reference assays, respectively. The feasibility and user satisfaction of the POC-RDT were evaluated through a questionnaire. The resolved sensitivity and resolved specificity and their 95% confidence intervals (95% CI) were as follows, respectively: SD-BIOLINE-HCV: 98.8% (95% CI 93.4, 100.0) and 100.0% (95% CI 98.3, 100.0); Determine HIV Early Detect: 100% (95% CI 85.2, 100.0) and 100% (95% CI 98.7, 100); and Determine HBsAg 2: 66.7% (95% CI 9.4, 99.2) and 100.0% (95% CI 98.7, 100.0). As expected, the number of subjects with a confirmed positive result for HBsAg was very low (n = 4). Therefore, the analytical sensitivity has been evaluated in addition: The Determine HBsAg 2 test demonstrated 100% sensitivity for standard concentrations ≥ 0.125 IU/mL. The subject questionnaire yielded positive feedback for most subjects. The POC-RDT fingerprick blood collection method was well received, and the tests demonstrated a comparable clinical performance with conventional tests in outreach settings and vulnerable high-risk populations.

Genomic Analysis of West Nile Virus Lineage 1 Detected in Mosquitoes during the 2020-2021 Outbreaks in Andalusia, Spain.

Emerging infectious diseases are one of the most important global health challenges because of their impact on human and animal health. The vector-borne West Nile virus (WNV) is transmitted between birds by mosquitos, but it can also infect humans and horses causing disease. The local circulation of WNV in Spain has been known for decades, and since 2010, there have been regular outbreaks in horses, although only six cases were reported in humans until 2019. In 2020, Spain experienced a major outbreak with 77 human cases, which was followed by 6 additional cases in 2021, most of them in the Andalusian region (southern Spain). This study aimed to characterize the genomes of the WNV circulating in wild-trapped mosquitoes during 2020 and 2021 in Andalusia. We sequenced the WNV consensus genome from two mosquito pools and carried out the phylogenetic analyses. We also compared the obtained genomes with those sequenced from human samples obtained during the outbreak and the genomes obtained previously in Spain from birds (2007 and 2017), mosquitoes (2008) and horses (2010) to better understand the eco-epidemiology of WNV in Spain. As expected, the WNV genomes recovered from mosquito pools in 2020 were closely related to those recovered from humans of the same outbreak. In addition, the strain of WNV circulating in 2021 was highly related to the WNV strain that caused the 2020 outbreak, suggesting that WNV is overwintering in the area. Consequently, future outbreaks of the same strain may occur in in the future.

Hepatitis E genotype 3 genome: A comprehensive analysis of entropy, motif conservation, relevant mutations, and clade-associated polymorphisms.

Hepatitis E virus genotype 3 (HEV-3) is an EU/EEA emergent zoonosis. HEV-3 clades/subtypes have been described. Its genome contains ORF1, which encodes nonstructural proteins for virus replication, ORF2, the capsid protein, and ORF3, a multifunctional protein involved in virion pathogenesis. The study aims with respect to HEV-3 are to: (1) calculate genome entropy (excluding hypervariable region); (2) analyze the described motifs/mutations; (3) characterize clade/subtype genome polymorphisms. Seven hundred and five sequences from the GenBank database were used. The highest entropies were identified in zoonotic genotypes (HEV-3 and HEV-4) with respect to HEV-1 in X domain, RdRp, ORF2, and ORF3. There were statistically significant differences in the entropy between proteins, protease and ORF3 being the most variable and Y domain being the most conserved. Methyltransferase and Y domain motifs were completely conserved. By contrast, essential protease H581 residue and catalytic dyad exhibited amino acid changes in 1.8% and 0.4% of sequences, respectively. Several X domain amino acids were associated with clades. We found sequences with mutations in all helicase motifs except number IV. Helicase mutations related to increased virulence and/or fulminant hepatitis were frequent, the 1,110 residue being a typical HEV-3e and HEV-3f-A2 polymorphism. RdRp motifs III, V, VII also had high mutation rates. Motif III included residues that are polymorphisms of HEV-3e (F1449) and HEV-3 m (D1451). RdRp ribavirin resistance mutations were frequent, mainly 1479I (67.4, 100% in HEV-3efglmk) and 1634R/K (10.0%, almost 100% in HEV-3e). With respect to ORF2, 19/27 neutralization epitopes had mutations. The S80 residue in ORF3 presented mutations in 3.5% of cases. Amino acids in the ORF3-PSAP motif had high substitution rates, being more frequent in the first PSAP (44.8%) than in the second (1.5%). This is the first comprehensive analysis of the HEV-3 genome, aimed at improving our knowledge of the genome, and establishing the basis for future genotype-to-phenotype analysis, given that viral features associated with severity have not been explored in depth. Our results demonstrate there are important genetic differences in the studied genomes that sometimes affect significant viral structures, and constitute clade/subtype polymorphisms that may affect the clinical course or treatment efficacy.

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

BackgroundHepatitis 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.AimTo 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.MethodsOf 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.ResultsHEV-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.ConclusionThe 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.

Proline-Rich Hypervariable Region of Hepatitis E Virus: Arranging the Disorder.

The hepatitis E virus (HEV) hypervariable region (HVR) presents the highest divergence of the entire HEV genome. It is characteristically rich in proline, and so is also known as the "polyproline region" (PPR). HEV genotype 3 (HEV-3) exhibits different PPR lengths due to insertions, PPR and/or RNA-dependent RNA polymerase (RdRp) duplications and deletions. A total of 723 PPR-HEV sequences were analyzed, of which 137 HEV-3 sequences were obtained from clinical specimens (from acute and chronic infection) by Sanger sequencing. Eight swine stool/liver samples were also analyzed. N- and C-terminal fragments were confirmed as being conserved, but they harbored differences between genotypes and were not proline-plentiful regions. The genuine PPR is the intermediate region between them. HEV-3 PPR contains a higher percentage (30.4%) of prolines than other genotypes. We describe for the first time: (1) the specific placement of HEV-3 PPR rearrangements in sites 1 to 14 of the PPR, noting that duplications are more frequently attached to sites 11 and 12 (AAs 74-79 and 113-118, respectively); (2) the cadence of repetitions follows a circular-like pattern of blocks A to J, with F, G, H, and I being the most frequent; (3) a previously unreported insertion homologous to apolipoprotein C1; and (4) the increase in frequency of potential N-glycosylation sites and differences in AAs composition related to duplications.

Clinical performance of Determine HBsAg 2 rapid test for Hepatitis B detection.

Hepatitis B virus (HBV) infection is estimated to affect 292 million people worldwide, 90% of them are unaware of their HBV status. The Determine HBsAg 2 (Alere Medical Co, Ltd Chiba Japan [Now Abbott]) is a rapid test that meets European Union (EU) regulatory requirements for Hepatitis B surface antigen 2 (HBsAg) analytical sensitivity, detecting the 0.1 IU/mL World Health Organization (WHO) International HBsAg Standard. This prospective, multicentre study was conducted to establish its clinical performance. 351 evaluable subjects were enrolled, 145 HBsAg-positive. The fingerstick whole blood sensitivity and specificity were 97.2% and 98.5% (15' reading, reference assay cut-off 0.05 IU/mL), sensitivity increasing to 97.9% with the prespecified cut-off 0.13 IU/mL (EU regulations). The venous whole blood, serum and plasma sensitivity was 97.2%, 97.9%, and 98.6%, respectively (15' reading); reaching 99%, 99.5% and 100% specificity. A testing algorithm following up an initial positive fingerstick test result with plasma/serum test demonstrates 100% specificity. The Determine HBsAg 2 test gives 15-minute results with high sensitivity and specificity, making it an ideal tool for point-of-care testing, with the potential to enable large-scale population-wide screening to reach the WHO HBV diagnostic targets. The evaluated test improves the existing methods as most of the reviewed rapid tests do not meet the EU regulatory requirements of sensitivity.

Brote de hepatitis A asociado a un manipulador de alimentos en Bizkaia, 2017 / Hepatitis A outbreak associated with a food handler in Bizkaia, 2017

INTRODUCCIÓN: La notificación de un caso de hepatitis A en un manipulador de alimentos de una pastelería y de 5 casos en trabajadores de una empresa tras consumir productos de la misma dio lugar a una investigación de brote. MÉTODOS: Se definieron como casos las personas con infección por el virus de la hepatitis A (VHA) confirmada por el laboratorio, con comienzo de síntomas en junio y que, durante el periodo de incubación, trabajaron con el manipulador y/o tuvieron contacto estrecho con él y/o consumieron productos de la pastelería. Se realizó una encuesta epidemiológica y se tomaron muestras de sangre para analizar la presencia de anticuerpos anti-hepatitis A. Se realizó la caracterización molecular por PCR, secuenciación de la región VP1/2A y análisis filogenético con el método de máxima verosimilitud, bootstrap 1000 (software MEGA 7.0). RESULTADOS: Se identificaron 14 casos primarios: 11 relacionados con el consumo de productos de la pastelería, 2 compañeros de trabajo del manipulador y un contacto familiar. Los 12 virus secuenciados eran genotipo IA, coincidiendo con una de las cepas (RIVM-HAV16-090) responsable de los brotes producidos en ese momento en Europa y que afectaban fundamentalmente a hombres que tienen sexo con hombres. CONCLUSIONES: Se debería reforzar la vacunación frente al VHA de grupos de riesgo para prevenir brotes futuros. La implementación del uso del tipado molecular en casos de hepatitis A podría mejorar la investigación de brotes, que se puede esperar que aumenten en el futuro debido al descenso de inmunidad en la población

INTRODUCTION: The reporting of one case of hepatitis A in a food handler at a bakery and five cases in employees of a company after consuming products from the same bakery prompted an outbreak investigation. METHODS: Outbreak cases were defined as individuals with laboratory-confirmed hepatitis A (HAV) infection, with symptoms which started in June and who, during the incubation period, worked with the food handler and/or had close contact with him and/or consumed products from the bakery. Epidemiologic questionnaires were performed and blood samples were obtained to be tested for the presence of anti-hepatitis A antibodies. Molecular characterisation was carried out by PCR, sequencing of the VP1/2A region and phylogenetic analysis with the maximum likelihood estimation method, bootstrap 1000 (MEGA 7.0 software). RESULTS: A total of 14 primary hepatitis A cases were identified: eleven cases related to the consumption of products from the bakery, two cases among co-workers of the food handler, and one case was a household contact. All 12 sequenced viruses were genotype IA, matching one of the strains (RIVM-HAV16-090) responsible for the outbreaks occurring at that time in Europe, mostly affecting men who have sex with men. CONCLUSIONS: HAV vaccination of at-risk groups should be reinforced in order to prevent future outbreaks. Increasing the use of molecular typing in hepatitis A cases could improve the investigation of outbreaks, which can be expected to increase in the future because of decreasing immunity in the population

Improving preparedness to respond to cross-border hepatitis A outbreaks in the European Union/European Economic Area: towards comparable sequencing of hepatitis A virus.

IntroductionSequence-based typing of hepatitis A virus (HAV) is important for outbreak detection, investigation and surveillance. In 2013, sequencing was central to resolving a large European Union (EU)-wide outbreak related to frozen berries. However, as the sequenced HAV genome regions were only partly comparable between countries, results were not always conclusive.AimThe objective was to gather information on HAV surveillance and sequencing in EU/European Economic Area (EEA) countries to find ways to harmonise their procedures, for improvement of cross-border outbreak responses.MethodsIn 2014, the European Centre for Disease Prevention and Control (ECDC) conducted a survey on HAV surveillance practices in EU/EEA countries. The survey enquired whether a referral system for confirming primary diagnostics of hepatitis A existed as well as a central collection/storage of hepatitis A cases' samples for typing. Questions on HAV sequencing procedures were also asked. Based on the results, an expert consultation proposed harmonised procedures for cross-border outbreak response, in particular regarding sequencing. In 2016, a follow-up survey assessed uptake of suggested methods.ResultsOf 31 EU/EEA countries, 23 (2014) and 27 (2016) participated. Numbers of countries with central collection and storage of HAV positive samples and of those performing sequencing increased from 12 to 15 and 12 to 14 respectively in 2016, with all countries typing an overlapping fragment of 218 nt. However, variation existed in the sequenced genomic regions and their lengths.ConclusionsWhile HAV sequences in EU/EEA countries are comparable for surveillance, collaboration in sharing and comparing these can be further strengthened.

Hepatitis A outbreak associated with a food handler in Bizkaia, 2017. / Brote de hepatitis A asociado a un manipulador de alimentos en Bizkaia, 2017.

INTRODUCTION: The reporting of one case of hepatitis A in a food handler at a bakery and five cases in employees of a company after consuming products from the same bakery prompted an outbreak investigation. METHODS: Outbreak cases were defined as individuals with laboratory-confirmed hepatitis A (HAV) infection, with symptoms which started in June and who, during the incubation period, worked with the food handler and/or had close contact with him and/or consumed products from the bakery. Epidemiologic questionnaires were performed and blood samples were obtained to be tested for the presence of anti-hepatitis A antibodies. Molecular characterisation was carried out by PCR, sequencing of the VP1/2A region and phylogenetic analysis with the maximum likelihood estimation method, bootstrap 1000 (MEGA 7.0 software). RESULTS: A total of 14 primary hepatitis A cases were identified: eleven cases related to the consumption of products from the bakery, two cases among co-workers of the food handler, and one case was a household contact. All 12 sequenced viruses were genotype IA, matching one of the strains (RIVM-HAV16-090) responsible for the outbreaks occurring at that time in Europe, mostly affecting men who have sex with men. CONCLUSIONS: HAV vaccination of at-risk groups should be reinforced in order to prevent future outbreaks. Increasing the use of molecular typing in hepatitis A cases could improve the investigation of outbreaks, which can be expected to increase in the future because of decreasing immunity in the population.

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.