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[Background & aims] It has been reported that specific killer-cell immunoglobulin-like receptors (KIRs) and HLA genotype combinations, such as KIR2DS4/HLA-C1 with presence of KIRDL2 or KIRDL3, homozygous KIRDL3/HLA-C1 and KIR3DL1/≥2HLA-Bw4, are strongly associated with the lack of active infection and seroconversion after exposition to hepatitis C virus (HCV)., [Objective] To determine whether these KIR-HLA combinations are relevant factors involved in that phenotype., [Patients and methods] In this retrospective case-control study, genotype data from a genome-wide association study previously performed on low susceptibility to HCV-infection carried out on 27 high-risk HCV-seronegative (HRSN) individuals and 743 chronically infected (CI) subjects were used. HLA alleles were imputed using R package HIBAG v1.2223 and KIR genotypes were imputed using the online resource KIR*IMP v1.2.0., [Results] It was possible to successfully impute at least one KIR-HLA genotype combination previously associated with the lack of infection and seroconversion after exposition to HCV in a total of 23 (85.2%) HRSN individuals and in 650 (87.5%) CI subjects. No KIR-HLA genotype combination analyzed was related to the HRSN condition., [Conclusions] Our results suggest that those KIR-HLA genotype combinations are not relevant factors involved in the lack of infection and seroconversion after exposition to HCV. More studies will be needed to completely understand this phenotype.

Rat hepatitis E virus (ratHEV; species Rocahepevirus ratti) is considered a newly emerging cause of acute hepatitis of zoonotic origin. ratHEV infection of people living with HIV (PLWH) might portend a worse, as with hepatitis E virus (HEV; species Paslahepevirus balayani), and consequently this group may constitute a high-risk population. We aimed to evaluate the prevalence of ratHEV by measuring viral RNA and specific IgG antibodies in a large Spanish cohort of PLWH. Multicentre study conducted in Spain evaluating PLWHIV included in the Spanish AIDS Research Network (CoRIS). Patients were evaluated for ratHEV infection using PCR at baseline and anti-ratHEV IgG by dot blot analysis to evaluate exposure to ratHEV strains. Patients with detectable ratHEV RNA were followed-up to evaluate persistence of viremia and IgG seroconversion. Eight-hundred and forty-two individuals were tested. A total of 9 individuals showed specific IgG antibodies against ratHEV, supposing a prevalence of 1.1 (95% CI; 0.5%−2.1%). Of these, only one was reactive to HEV IgG antibodies by ELISA. One sample was positive for ratHEV RNA (prevalence of infection: 0.1%; 95% CI: 0.08%−0.7%). The case was a man who had sex with men exhibiting a slightly increased alanine transaminase level (49 IU/L) as only biochemical alteration. In the follow-up, the patients showed undetectable ratHEV RNA and seroconversion to specific ratHEV IgG antibodies. Our study shows that ratHEV is geographical broadly distributed in Spain, representing a potential zoonotic threat.

[Background & Aim] Hepatitis E virus (HEV) was considered the only member of the Hepeviridae family with zoonotic potential. Nevertheless, this consideration has been reassessed owing to several reported cases of acute and chronic hepatitis linked to the Orthohepevirus C genus. Because the circulation of Orthohepevirus C in rodents has been described worldwide, the risk of zoonotic transmission is plausibly global., [Methods] Orthohepevirus C RNA was retrospectively evaluated in 2 cohorts of patients in Spain. The first cohort included patients with acute hepatitis without etiological diagnosis after screening for hepatotropic virus infection. The second cohort included patients diagnosed with acute HEV infection, defined as positivity for anti-HEV-IgM antibodies and/or detectable HEV RNA in serum., [Results] Cohort 1 comprised 169 patients (64.4% male, median age 43 years) and cohort 2 comprised 98 individuals (68.3% male, median age 45 years). Of the individuals included in Cohort 1, two (1.18%; 95% CI 0.2-3.8) had detectable Orthohepevirus C RNA in serum. In Cohort 2, of the 98 included patients, 58 showed detectable HEV RNA, while 40 only showed positivity for IgM antibodies. Among those bearing only IgM antibodies, Orthohepevirus C RNA was detected in 1 (2.5%; 95% CI 0.06-13.1) individual. All strains were consistent with genotype C1. The infection resulted in mild self-limiting acute hepatitis in 2 patients. Infection caused severe acute hepatitis in the remaining patient who died as a result of liver and renal failure., [Conclusions] We described 3 cases of Orthohepevirus C in patients with acute hepatitis, resulting in the first description of this infection in Europe. The prevalence obtained in our study suggests that Orthohepevirus C could be an emerging disease in Europe.

[Objectives] Real world data on glecaprevir/pibrentasvir (G/P) among active drug users are scarce. We evaluated the sustained virological response (SVR) rates of G/P among individuals with and without active drug use in routine clinical practice., [Methods] Two ongoing prospective multicenter cohorts of individuals starting G/P were analyzed. Overall SVR intention-to-treat (ITT), discontinuations due to adverse effects and dropouts were evaluated. Results in patients with active, past and without active drug use were compared., [Results] Overall, 644 individuals started G/P and have reached the date of SVR evaluation. Of them, 613 (95.2%) individuals achieved SVR. There were two (0.3%) relapses, one (0.2%) discontinuation due to side effects and 35 (5.4%) dropouts. SVR rates for patients with active drug use, past drug use and those who never used drugs were 85.4%(n/N = 70/82), 96.1%(n/N = 320/333) and 97.4%(n/N = 223/229) respectively (p < 0.001). After adjustment by sex, age, HCV genotype and opioid agonist therapy, active drug use was the only factor independently associated with SVR (ITT) [adjusted OR (95%confidence interval): 0.29(0.09–0.99),p = 0.048]., [Conclusions] Active drug use was independently associated with lower SVR rates to G/P, mainly due to voluntary dropout. G/P could be particularly beneficial in this scenario but specific strategies designed to increase the retention in care are needed.

[Introduction] Paslahepevirus balayani (HEV) is an endemic zoonotic disease ranked as a major cause of acute hepatitis in Europe. Most infections occurring in Europe are due to the endemic several subtypes of genotype 3, through the consumption of raw or undercooked pork, observing a genotype geographical distribution pattern among countries Because of global changes in the pig and pork trading markets, subtype distribution might vary. We aimed to evaluate the temporal distribution of HEV genotypes in patients from southern Spain with acute hepatitis to determine whether these changes were related to the pig import trade during the study period between 2018 and 2022., [Methods] Prospective longitudinal study including patients with acute hepatitis from southern Spain between 2018 and 2022. HEV RNA and antibodies was tested in all patients. In patients with detectable HEV RNA, genotype was obtained. To determine the number of imported pigs and their origins, we checked the official data from the Spanish statistics on international trade of Spanish Minister of Industry during by country of origin during the same study period., [Results] A total of 659 patients with acute hepatitis were included in the study. Among them, 162 (24.5%) had at least one marker (IgM or RNA) of acute HEV infection. Among the 71 patients with detectable viral RNA, genotypes could be obtained for 58 (81.6%). The most prevalent HEV genotype was 3f (n = 48; 78.6%), showing a decreasing prevalence of over time, from 100% in 2018 to 70.6% in 2022. Since 2021, the emergence of other genotypes has been determined. A significant increase in the number of animals imported was observed since the beginning of the study. Denmark experienced a significant rise, from 0.03% in 2018 of total imports to 10.4% in 2022., [Conclusions] HEV molecular diversity is changing in Spain, could be linked to changes in fattening pig import origin.

Little information is currently available on the occurrence and molecular diversity of the enteric protozoan parasites Cryptosporidium spp., Giardia duodenalis, and Balantioides coli in wild ungulates and the role of these host species as potential sources of environmental contamination and consequent human infections. The presence of these three pathogens was investigated in eight wild ungulate species present in Spain (genera Ammotragus, Capra, Capreolus, Cervus, Dama, Ovis, Rupicapra, and Sus) by molecular methods. Faecal samples were retrospectively collected from free-ranging (n = 1058) and farmed (n = 324) wild ungulates from the five Spanish bioregions. Overall infection rates were 3.0% (42/1382; 95% CI: 2.1–3.9%) for Cryptosporidium spp., 5.4% (74/1382; 95% CI: 4.2–6.5%) for G. duodenalis, and 0.7% (9/1382; 95% CI: 0.3–1.2%) for B. coli. Cryptosporidium infection was detected in roe deer (7.5%), wild boar (7.0%) and red deer (1.5%), and G. duodenalis in southern chamois (12.9%), mouflon (10.0%), Iberian wild goat (9.0%), roe deer (7.5%), wild boar (5.6%), fallow deer (5.2%) and red deer (3.8%). Balantioides coli was only detected in wild boar (2.5%, 9/359). Sequence analyses revealed the presence of six distinct Cryptosporidium species: C. ryanae in red deer, roe deer, and wild boar; C. parvum in red deer and wild boar; C. ubiquitum in roe deer; C. scrofarum in wild boar; C. canis in roe deer; and C. suis in red deer. Zoonotic assemblages A and B were detected in wild boar and red deer, respectively. Ungulateadapted assemblage E was identified in mouflon, red deer, and southern chamois. Attempts to genotype samples positive for B. coli failed. Sporadic infections by canine- or swine-adapted species may be indicative of potential cross-species transmission, although spurious infections cannot be ruled out. Molecular evidence gathered is consistent with parasite mild infections and limited environmental contamination with (oo)cysts. Free-ranging wild ungulate

The European Observatory of Wildlife (EOW) as part of the ENETWILD project aims progressively developing integrated wildlife monitoring (population abundance and pathogens). The present report shows how to link the wildlife population monitoring (by camera trapping) and wildlife disease surveillance at European scale, by using environmental sampling over 15 study areas of the EOW from 10 Countries (4 study areas in 4 countries will be incorporated next). We specifically focused on multi-host pathogens Mycobacterium tuberculosis Complex (bacteria, MTC), and Hepatitis E virus (HEV). The aims of this trial were, first, to evaluate the harmonized implementation of a simple field sampling protocol for detecting zoonotic pathogens in environmental samples (standing water) through a network of wildlife professionals at European level. Secondly, we got insights for future improved strategies of wildlife integrated monitoring through environmental sampling. This trial prioritized the inclusion of a diverse array of study areas and a simple sampling approach rather than complex protocols and illustrated. We evidenced the importance of supporting such a coordinate network of wildlife professionals to progressively improve strategies, protocols, the general design, sampling, target matrix, selected pathogens, preservation and transport of samples, analytical techniques, and sample and data flow. We discuss specific results on pathogens, remarking the detection of the MTC in certain areas.

To evaluate the diagnostic value of the combination of two broad-range PCR assays targeting two different and conserved regions of the viral genome for the diagnosis of acute Hepatitis E virus (HEV) infection. Patients with acute hepatitis were prospectively recruited. In all, HEV-IgM antibodies were tested together with evaluation of HEV viraemia by two PCR assays (ORF3 and ORF1). The number of individuals exhibiting negative IgM antibody results but carrying viral RNA was calculated by each PCR assay. Four-hundred and seventy individuals were included, of whom 145 (30.8%) were diagnosed as having acute HEV. Of them, 122 (84.1%) exhibited HEV-IgM antibodies, and 81 (55.8%) had detectable viral RNA for at least one PCR. Using the ORF3 molecular assay, 70 (48.3%) individuals were identified with HEV infection. When the ORF1 molecular assay was applied, 49 (33.8%) individuals were identified. The ORF3 assay detected viral RNA in 32 patients not detected by the ORF1 assay. In contrast, the ORF1 assay could amplify viral RNA in 11 patients who were not detected by the ORF3 assay. The parallel use of two broad-range PCR assays significantly increased the performance of the molecular diagnosis of HEV.

Rat hepatitis E virus (ratHEV; species Rocahepevirus ratti) is considered a newly emerging cause of acute hepatitis of zoonotic origin. ratHEV infection of people living with HIV (PLWH) might portend a worse, as with hepatitis E virus (HEV; species Paslahepevirus balayani), and consequently this group may constitute a high-risk population. We aimed to evaluate the prevalence of ratHEV by measuring viral RNA and specific IgG antibodies in a large Spanish cohort of PLWH. Multicentre study conducted in Spain evaluating PLWHIV included in the Spanish AIDS Research Network (CoRIS). Patients were evaluated for ratHEV infection using PCR at baseline and anti-ratHEV IgG by dot blot analysis to evaluate exposure to ratHEV strains. Patients with detectable ratHEV RNA were followed-up to evaluate persistence of viremia and IgG seroconversion. Eight-hundred and forty-two individuals were tested. A total of 9 individuals showed specific IgG antibodies against ratHEV, supposing a prevalence of 1.1 (95% CI; 0.5%−2.1%). Of these, only one was reactive to HEV IgG antibodies by ELISA. One sample was positive for ratHEV RNA (prevalence of infection: 0.1%; 95% CI: 0.08%−0.7%). The case was a man who had sex with men exhibiting a slightly increased alanine transaminase level (49 IU/L) as only biochemical alteration. In the follow-up, the patients showed undetectable ratHEV RNA and seroconversion to specific ratHEV IgG antibodies. Our study shows that ratHEV is geographical broadly distributed in Spain, representing a potential zoonotic threat.

Objectives Real world data on glecaprevir/pibrentasvir (G/P) among active drug users are scarce. We evaluated the sustained virological response (SVR) rates of G/P among individuals with and without active drug use in routine clinical practice. Methods Two ongoing prospective multicenter cohorts of individuals starting G/P were analyzed. Overall SVR intention-to-treat (ITT), discontinuations due to adverse effects and dropouts were evaluated. Results in patients with active, past and without active drug use were compared. Results Overall, 644 individuals started G/P and have reached the date of SVR evaluation. Of them, 613 (95.2%) individuals achieved SVR. There were two (0.3%) relapses, one (0.2%) discontinuation due to side effects and 35 (5.4%) dropouts. SVR rates for patients with active drug use, past drug use and those who never used drugs were 85.4%(n/N = 70/82), 96.1%(n/N = 320/333) and 97.4%(n/N = 223/229) respectively (p < 0.001). After adjustment by sex, age, HCV genotype and opioid agonist therapy, active drug use was the only factor independently associated with SVR (ITT) [adjusted OR (95%confidence interval): 0.29(0.09–0.99),p = 0.048]. Conclusions Active drug use was independently associated with lower SVR rates to G/P, mainly due to voluntary dropout. G/P could be particularly beneficial in this scenario but specific strategies designed to increase the retention in care are needed.

Background & Aim Hepatitis E virus (HEV) was considered the only member of the Hepeviridae family with zoonotic potential. Nevertheless, this consideration has been reassessed owing to several reported cases of acute and chronic hepatitis linked to the Orthohepevirus C genus. Because the circulation of Orthohepevirus C in rodents has been described worldwide, the risk of zoonotic transmission is plausibly global. Methods Orthohepevirus C RNA was retrospectively evaluated in 2 cohorts of patients in Spain. The first cohort included patients with acute hepatitis without etiological diagnosis after screening for hepatotropic virus infection. The second cohort included patients diagnosed with acute HEV infection, defined as positivity for anti-HEV-IgM antibodies and/or detectable HEV RNA in serum. Results Cohort 1 comprised 169 patients (64.4% male, median age 43 years) and cohort 2 comprised 98 individuals (68.3% male, median age 45 years). Of the individuals included in Cohort 1, two (1.18%; 95% CI 0.2-3.8) had detectable Orthohepevirus C RNA in serum. In Cohort 2, of the 98 included patients, 58 showed detectable HEV RNA, while 40 only showed positivity for IgM antibodies. Among those bearing only IgM antibodies, Orthohepevirus C RNA was detected in 1 (2.5%; 95% CI 0.06-13.1) individual. All strains were consistent with genotype C1. The infection resulted in mild self-limiting acute hepatitis in 2 patients. Infection caused severe acute hepatitis in the remaining patient who died as a result of liver and renal failure. Conclusions We described 3 cases of Orthohepevirus C in patients with acute hepatitis, resulting in the first description of this infection in Europe. The prevalence obtained in our study suggests that Orthohepevirus C could be an emerging disease in Europe. Lay summary We describe the first cases of acute hepatitis related to rat hepatitis E virus in Europe. The prevalence found in our study suggest that rat hepatitis E virus could be considered an em

The objective of this study was to evaluate the spatial risk of exposure to Crimean–Congo haemorrhagic fever virus (CCHFV) infection of healthy blood donors in an enzootic region with a predicted risk gradient based on a virus–animal interaction risk model. We designed a cross-sectional study to test if the exposure pattern of the human population to CCHFV spatially matches the predicted risk. We randomly selected 1384 donors from different risk gradients and analyzed their sera searching for CCHFV antibodies. None of the selected blood donors showed exposure to CCHFV. This study shows that exposure risk spatial patterns, as predicted from animal-tick-virus models, does not necessarily match the pattern of human-infected tick interactions leading to CCHFV infection and CCHF cases, at least in a region of predicted moderate infection risk. The findings suggest that future studies should bear the potential drivers of tick-human encounter rates into account to more accurately predict risks.

Microsporidia comprises a diverse group of obligate, intracellular, and spore-forming parasites that infect a wide range of animals. Among them, Enterocytozoon bieneusi is the most frequently reported species in humans and other mammals and birds. Data on the epidemiology of E. bieneusi in wildlife are limited. Hence, E. bieneusi was investigated in eight wild ungulate species present in Spain (genera Ammotragus, Capra, Capreolus, Cervus, Dama, Ovis, Rupicapra, and Sus) by molecular methods. Faecal samples were collected from free-ranging (n = 1058) and farmed (n = 324) wild ungulates from five Spanish bioregions. The parasite was detected only in red deer (10.4%, 68/653) and wild boar (0.8%, 3/359). Enterocytozoon bieneusi infections were more common in farmed (19.4%, 63/324) than in wild (1.5%, 5/329) red deer. A total of 11 genotypes were identified in red deer, eight known (BEB6, BEB17, EbCar2, HLJD-V, MWC_d1, S5, Type IV, and Wildboar3) and three novel (DeerSpEb1, DeerSpEb2, and DeerSpEb3) genotypes. Mixed genotype infections were detected in 15.9% of farmed red deer. Two genotypes were identified in wild boar, a known (Wildboar3) and a novel (WildboarSpEb1) genotypes. All genotypes identified belonged to E. bieneusi zoonotic Groups 1 and 2. This study provides the most comprehensive epidemiological study of E. bieneusi in Spanish ungulates to date, representing the first evidence of the parasite in wild red deer populations worldwide. Spanish wild boars and red deer are reservoir of zoonotic genotypes of E. bieneusi and might play an underestimated role in the transmission of this microsporidian species to humans and other animals.

The hepatitis E virus (HEV) is the main cause of viral acute hepatitis in the world, affecting more than 20 million people annually. During the acute phase of infection, HEV can be detected in various body fluids, which has a significant impact in terms of transmission, diagnosis or extrahepatic manifestations. Several studies have isolated HEV in the genitourinary tract of humans and animals, which could have important clinical and epidemiological implications. So, our main objective was to evaluate the presence of HEV in testis of naturally infected wild boars (Sus scrofa). For it, blood, liver, hepatic lymph node and testicle samples were collected from 191 male wild boars. The presence of HEV was evaluated in serum by PCR, as well as in tissues by PCR and immunohistochemistry. Four animals (2.09%; 95%CI: 0.82–5.26) showed detectable HEV RNA in serum, being confirmed the presence of HEV-3f genotype in three of them by phylogenetic analysis. HEV was also detected in liver and/or hepatic lymph nodes of the four animals by RT-PCR, as well as by immunohistochemistry analysis. Only one of these wild boars also showed detectable viral load in testis, observing HEV-specific labelling in a small number of fibroblasts and some Sertoli cells. Our results confirm the presence of HEV genotype 3 in naturally infected wild boar testis, although no associated tissue damage was evidenced. This study does not allow us to discard semen as a possible source of HEV transmission in suids. Future experimental studies are necessary to evaluate the impact of HEV genotype 3 on fertility and the possibility of transmission through sexual contact in this specie.

Autochthonous cases of hepatitis E (HE) associated with zoonotic genotypes HEV-3 and HEV-4 have significantly increased in industrialized countries over the last decade. Suidae are generally recognized as the main reservoirs of these genotypes. Susceptibility to HE virus (HEV) infection and zoonotic potential have also been confirmed in other species, including sheep and goat. However, the information about their role in the epidemiology of HEV remains very scarce. The objective of this study was to assess the prevalence, spatial distribution and risk factors associated with HEV exposure in sheep and goats in southern Spain, the country with the highest census of small domestic ruminants in the European Union. Blood samples from 240 sheep and 240 goats were collected between 2015 and 2017. Sera were analysed in parallel using a commercial double-antigen ELISA and real-time PCR. A total of 38 (7.9%; 95%CI: 5.5–10.3) out of 480 sampled animals showed anti-HEV antibodies. By species, the seroprevalences found in sheep and goats were 2.1% (5/240; 95%CI: 0.3–3.9) and 13.8% (33/240; 95%CI: 9.4–18.1) respectively. Anti-HEV antibodies were found on 19 (59.4%; 95%CI: 42.4–76.4) of the 32 sampled farms. The GEE model showed that species (goat) and number of small ruminants in the farm (≤348 animals and ≥538 animals) were risk factors potentially associated with HEV exposure in small ruminants in the study area. HEV RNA was not detected in any of the 480 (0.0%; 95%CI: 0.0–0.8) tested animals. Our results confirm that sheep and goats are naturally, but not equally exposed to HEV and indicate the widespread spatial distribution of HEV among small ruminant populations in southern Spain. Further studies are required to elucidate the role of sheep and goat in the epidemiology of HEV and their potential implications for public health.