Antibody-enhanced hepatitis E virus nanofiltration during the manufacture of human immunoglobulin.

BACKGROUND: Circulation of hepatitis E virus (HEV) in areas where plasma is sourced for the manufacture of plasma-derived medicinal products (PDMPs) has prompted verification of HEV clearance. HEV exists as quasi lipid-enveloped (LE) and non-lipid-enveloped (NLE) forms, which might be of relevance for HEV clearance from manufacturing processes of antibody-containing PDMPs with solvent/detergent (S/D) treatment upstream of further clearance steps. STUDY DESIGN AND METHODS: Presence of different HEV particles in stocks used in clearance studies was investigated, with nanofilters graded around the assumed HEV particle sizes and by gradient centrifugation. HEV removal by 35-nm nanofiltration was investigated in the presence or absence of HEV antibodies, in buffer as well as in immunoglobulin (IG) manufacturing process intermediates. RESULTS: HEV particles consistent with LE, NLE, and an "intermediate" (IM) phenotype, obtained after S/D treatment, were seen in different HEV stocks. In the absence of HEV antibodies, log reduction factors (LRFs) of 4.0 and 2.5 were obtained by 35-nm nanofiltration of LE and IM HEV, consistent with the larger and smaller sizes of these phenotypes. Addition of HEV antibodies enhanced IM HEV removal around 1000-fold (LRF, 5.6). Effective (LRF, >4.8 and >4.0) HEV removal was obtained for the nanofiltration processing step for IG intermediates with varying HEV antibody content. CONCLUSION: HEV spikes used in clearance studies should be carefully selected, as differences in physicochemical properties might affect HEV clearance. Antibody-mediated enhancement of HEV nanofiltration was demonstrated in IG process intermediates even at low HEV antibody concentration, illustrating the robustness of this manufacturing step.

Seroprevalence of hepatitis E among hospitalized patients in Slovakia: first report.

OBJECTIVE: Hepatitis E infection is one of the most frequent acute hepatitis in the world. Currently five human genotypes with different geographical distributions and distinct epidemiologic patterns are identified. In Slovakia, only rare cases of hepatitis E have been reported in recent years. Therefore, the aim of the study was to evaluate the prevalence of anti-HEV total antibodies and the main risk factors for HEV in the general population in Eastern Slovakia. METHODS: Detection of anti-HEV total antibodies samples was done by a commercial enzyme-linked immunosorbent assay (ELISA) kit. RESULTS: Of 175 hospitalized patients included in the study, 76 (43.5%) showed positivity for anti-HEV total antibodies. No statistically significant differences were found in anti-HEV positivity between men and women or in the groups of different living areas (town/village - urban/rural). CONCLUSION: Prevalence of anti-HEV total antibodies of hospitalised patients was high. The risk factor significantly associated with antibody positivity was eating raw meat. Other factors, such as sex, age, living area and contact with animals were not associated with antibody positivity.

Epidemiology of hepatitis E in South-East Europe in the "One Health" concept.

The significance of hepatitis E virus (HEV) as an important public health problem is rising. Until a decade ago, cases of HEV infection in Eur-ope were mainly confined to returning travelers, but nowadays, hepatitis E represents an emerging zoonotic infection in many European countries. The aim of this manuscript is to perform a systematic review of the published literature on hepatitis E distribution in humans, animals and environmental samples ("One Health" concept) in the South-Eastern European countries. Comparison of the available data showed that the anti-HEV seroprevalence in the South-Eastern Europe varies greatly, depending on the population studied, geographical area and methods used. The IgG seroprevalence rates in different population groups were found to be 1.1%-24.5% in Croatia, up to 20.9% in Bulgaria, 5.9-%17.1% in Romania, 15% in Serbia, up to 9.7% in Greece and 2%-9.7% in Albania. Among possible risk factors, older age was the most significant predictor for HEV seropositivity in most studies. Higher seroprevalence rates were found in animals. HEV IgG antibodies in domestic pigs were detected in 20%-54.5%, 29.2%-50%, 38.94%-50% and 31.1%-91.7% in Serbia, Bulgaria, Romania and Croatia, respectively. In wild boars seroprevalence rates were up to 10.3%, 30.3% and 31.1% in Romania, Slovenia and Croatia, respectively. A high HEV RNA prevalence in wild boars in some countries (Croatia and Romania) indicated that wild boars may have a key role in the HEV epidemiology. There are very few data on HEV prevalence in environmental samples. HEV RNA was detected in 3.3% and 16.7% surface waters in Slovenia and Serbia, respectively. There is no evidence of HEV RNA in sewage systems in this region. The available data on genetic characterization show that human, animal and environmental HEV strains mainly belong to the genotype 3.

Molecular and serological surveillance of Hepatitis E virus in wild and domestic carnivores in Brandenburg, Germany.

Hepatitis E virus (HEV) is a zoonotic virus which circulates in pigs and wild boars as main reservoir species. To reveal the infection rate in carnivores, we have carried out a monitoring study of raccoons, raccoon dogs, dogs and cats sampled in Brandenburg, Germany. In summary, 53.8% (43 of 80) of the raccoons, 34.3% (25 of 73) of the raccoon dogs, 56.6% (47 of 83) of dogs and 32.3% (21 of 65) of cats were tested positive for HEV-specific antibodies. No viral RNA could be detected. This first description of anti-HEV antibodies in raccoons and raccoon dogs worldwide and in dogs and cats in Germany highlights the natural host range expansion of HEV.

Prevalence of HAV Ab, HEV (IgG), HSV2 IgG, and Syphilis Among Sheltered Homeless Adults in Tehran, 2012.

BACKGROUND: This study investigated the prevalence for hepatitis A virus (HAV), hepatitis E virus (HEV), herpes simplex virus type 2 (HSV2) and syphilis among homeless in the city of Tehran. METHODS: In this cross-sectional study, 596 homeless were recruited in Tehran. A researcher-designed questionnaire was used to study demographic data. Using enzyme-linked immunoassay, and rapid plasma reagin (RPR) test, we evaluated the seroprevalence of HAV anti-body, HEV IgG, herpes, HSV2 IgG, and syphilis among sheltered homeless in Tehran. The associations between the participant's characteristics and infections were evaluated using logistic regression and chi-square. RESULTS: A total of 569 homeless, 78 women (13.7%) and 491 men (86.3%) were enrolled into the study from June to August 2012. Their age mean was 42 years and meantime of being homeless was 24 months. Seroprevalence of syphilis, HEV IgG, HSV2 IgG and HAV Ab was 0.55%, 24.37%, 16.48%, and 94.34%, respectively. History of drug abuse was reported in 77.70%; 46.01% of them were using a drug during the study and 26.87% of them had history of intravenous drug abuse. Among people who had intravenous drug abuse, 48.25% had history of syringe sharing. CONCLUSION: The prevalence of HAV, HEV and HSV2 were higher than the general population while low prevalence of syphilis was seen among homeless peoples who are at high risk of sexually transmitted infection (STD). Our findings highlighted that significant healthcare needs of sheltered homeless people in Tehran are unmet and much more attention needs to be paid for the health of homeless people.

Detection of Genotype 4 Swine Hepatitis E Virus in Systemic Tissues in Cross-Species Infected Rabbits.

Increasing evidence demonstrates that hepatitis E virus (HEV) can be transmitted across species. According to previous reports, swine HEV has two genotypes, genotype 3 and 4, and both can infect humans by the fecal-oral route. Thus, it is crucial for the control of HEV zoonotic transmission to evaluate the dynamics of viral shedding and distribution in different tissues during cross-species infection by HEV. In this study, rabbits were infected with genotype 4 swine HEV by the intraperitoneal route. The results showed that HEV RNA not only shed in the feces but also in the saliva of some rabbits during infection with swine HEV. Viremia appeared late after infection, and anti-HEV IgG was not obvious until the appearance of high viremia levels. After the rabbits were euthanized, a histopathological examination showed that the livers developed overt hepatitis accompanied by an elevation of alanine aminotransferase (ALT) and aspartate transaminase (AST). Furthermore, HEV RNA was detected in various tissues, especially in the salivary glands and tonsils. Subsequently, negative-stranded HEV RNA was practiced in tissues with positive HEV RNA, which demonstrated that HEV replicated in the tissues. Next, we harvested additional tissues from the liver, salivary gland, tonsil, spleen, thymus gland, lymph node and intestine, which are known as replication sites of swine HEV. Additionally, we also observed the HEV antigen distributed in the organs above through immunohistochemical staining. These results demonstrate that rabbits could be used as an animal model for researching cross-species infection of genotype 4 HEV. It is also noteworthy that HEV can shed in the saliva and presents the risk of droplet transmission. These new data provide valuable information for understanding cross-species infection by HEV.

Simple and specific method for detection of antibodies against hepatitis E virus in mammalian species.

Hepatitis E virus (HEV) is the causative agent of hepatitis E, a food- and water-borne disease. In developed countries, consumption of meats from pigs, wild boars and deer is a major source of infection. Although HEV and HEV-related viruses have been detected in many animal species, their zoonotic potential and prevalence has not been completely understood. To detect anti-HEV antibody in mammalian species, a simple enzyme-linked immunosorbent assay (ELISA) was established using extract from cells expressing HEV capsid protein and protein A/G as an antigen and a reagent for detection of antibody. Absorbance in the ELISA was compared with those in our previous ELISA using VLPs and anti-swine antibody, suggesting that newly established ELISA was similarly specific and sensitive as the previous ELISA. Seroprevalence of HEV infection among wild boars was examined in Yamaguchi Prefecture, confirming that 111 of 364 wild boars (30.5%) were positive for anti-HEV antibody. Next, this ELISA was applied to humans, dogs, cats, ferrets, raccoons and masked palm civets in Japan, and anti-HEV antibodies were detected in humans, ferrets, dogs and cats. This ELISA is thus useful for serological surveys and comparison of HEV infection among various mammals, including humans.

A Novel Blocking ELISA for Detection of Antibodies against Hepatitis E Virus in Domestic Pigs.

Hepatitis E virus (HEV) infects both humans and animals, with an overall human mortality rate generally less than 1%, but as high as 20% among pregnant women. HEV strains fall into 4 major genotypes. Zoonotic genotypes 3 and 4 associate with sporadic human and animal HEV cases in many industrialized countries. To date, collective evidence implicates pigs as the main HEV reservoir, justifying the importance of monitoring HEV infection rates in pig herds to prevent human illness. Due to the lack of a robust in vitro cell culture system for viral propagation, no "gold standard" assay has yet been developed to detect HEV infection in domestic pigs. 1E4, a monoclonal antibody (mAb) specific for the C-terminal 268 amino acids of HEV genotype 4 ORF2 capsid protein (sORF2-C), was generated and conjugated to horseradish peroxidase (HRP) for use in a blocking ELISA (bELISA). Optimal sORF2-C coating antigen concentration (8 µg/ml), HRP-1E4 dilution (1:1000), and test pig serum dilution (1:20) were determined using a checkerboard titration test. A cut-off value of 16.9% was chosen to differentiate between positive vs. negative sera after mean percent inhibition (PI) testing of 230 negative pig sera. Compared with the indirect ELISA (iELISA), western blot, and a commercial ELISA kit for detecting anti-HEV antibodies in human sera, the bELISA showed no statistical differences and statistically high coincidence of 93.23%, 92%, and 95% with the other tests, respectively. A blocking ELISA (bELISA) for detecting anti-HEV antibodies in pig serum samples was developed with high sensitivity and high specificity comparable to that of the indirect ELISA. The bELISA results exhibited high agreement with iELISA, western blot, and a commercial ELISA kit designed to detect human anti-HEV antibodies. Therefore, bELISA should serve as an ideal method for large-scale serological investigation of anti-HEV antibodies in domestic pigs.

Detection of IgM and IgG against hepatitis E virus in serum and meat juice samples from pigs at slaughter in Bavaria, Germany.

Hepatitis E virus (HEV) is an emerging foodborne pathogen with domestic and wild pigs (and likely other species such as deer or rabbits) recognized as reservoir. Pathogenesis in pigs usually leads to an asymptomatic course of disease. Since there is no enzyme-linked immunosorbent assay (ELISA) kit for the detection of anti-HEV antibodies in pigs commercially available, the objective of this study was to assess the seroprevalence in fattening pigs at slaughter and at herd level using a newly developed ELISA based on genotype (GT) 1 and GT 3 in Bavaria, Germany. Based on 516 serum and 198 meat juice samples collected from different herds at four different Bavarian slaughterhouses, the overall seroprevalence of anti-HEV IgG in serum and meat juice samples was 68.6% and 67.6%, respectively. Analyzing the serum for the presence of anti-HEV IgM, 36/516 (7%) were positive for anti-HEV IgM. At herd level, most of the herds were seropositive for anti-HEV antibodies. The present study shows that HEV is widespread among the Bavarian pig population and that some pigs might test positive for anti-HEV IgM even at the age of slaughter. Also, meat juice serves as an equivalent matrix to serum to test for anti-HEV antibodies in pigs.

Seroprevalence of hepatitis delta virus infection among subjects with underlying hepatic diseases in Chennai, southern India.

Four hundred million people are carriers of hepatitis B virus (HBV) worldwide and approximately 5% of these are reportedly positive for hepatitis delta virus (HDV). Several reports indicate a declining trend in the occurrence of HDV infection in the north of tropical India. To our knowledge, no study has been conducted to evaluate whether a similar epidemiological change is occurring in southern India. Therefore we evaluated the seroprevalence of HDV among 153 individuals with HBV-related liver diseases in Chennai, and assessed any change in epidemiological pattern by comparing the results with seroprevalence figures reported previously. Of the 153 patients screened, nine (5.9%) were reactive to anti-delta antibodies, six (3.9%) presented an evidence of past infection (IgG anti-delta positive) and three (2.0%) showed anti-HDV IgM, suggestive of recent HDV infection. Alanine transaminase elevation was not significant in HDV-associated infection compared with HBV alone-infected acute viral hepatitis (AVH) (P=0.82) and chronic liver disease (P=0.77) patients. The anti-HDV positivity in AVH was considerably low (6.6%), compared with previous Indian reports varying from 10.7% to >30%. HDV infection was relatively low and seems to play a minor determining factor of liver diseases in the tropical south Indian population.

Value of HCV antigen-antibody combined HCV assay in hepatitis C diagnosis.

Reduction of the window period of hepatitis C virus (HCV) infection represents an important goal in the transfusional and diagnostic settings. Currently, the detection of HCV infection relies on the use of immunoassays to detect viral antibodies. A new enzyme immunoassay (Monolisa HCV Ag-Ab ULTRA) designed to simultaneously detect circulating HCV antigen and anti-HCV antibodies has been developed by Bio-Rad and registered by the European Authorities. Several evaluations have been conducted in Europe to determine whether this new assay can improve early detection of HCV infection. Sensitivity studies included 130 HCV RNA positive/anti-HCV negative samples, 21 well documented seroconversion panels and 430 anti-HCV genotyped samples from France and Italy. Specificity has also been assessed in 15,302 non-selected blood donations and hospital samples. Studies have shown that Monolisa HCV Ag-Ab ULTRA assay has been able to detect 40-90 % of HCV RNA positive/anti-HCV negative samples collected in the window period, improving early detection of HCV when antibodies may be undetectable. The mean delay in detecting HCV infection between HCV-RNA and this new test was found to be 5 days, reducing the window period by an average of 37 days. All samples collected after seroconversion were detected with the HCV Ag-Ab ULTRA assay. The specificity analyzed in 15,302 random blood donations and hospital samples was estimated at 99.86 %. Although less sensitive than NAT (71 % of HCV RNA positive/anti-HCV negative in window period), this assay could be a reasonable alternative when NAT cannot be used for reasons such as cost, organization, emergency or logistic difficulties.

Association of hepatitis E virus (HEV) and postweaning multisystemic wasting syndrome (PMWS) with lesions of hepatitis in pigs.

The aim of the study was to determine the presence of swine hepatitis E virus (HEV) RNA and antibodies in postweaning multisystemic wasting syndrome-affected (n=114) and non-affected (n=46) pigs and the possible association with hepatitis lesions. Forty-four pigs were RT-PCR positive (28.2%); 25 of them were PMWS cases, while 19 were non-PMWS pigs. In both groups, HEV RT-PCR results were associated with hepatitis (OR=5.61 for PMWS-affected pigs and OR=5.17 for non-PMWS affected pigs; p=0.01). No interaction was detected in a logistic regression between PMWS occurrence and HEV infection for the development of hepatitis lesions. Seropositivity to HEV was more likely to occur in pigs with hepatitis (51.9%) compared to pigs without hepatitis (36.1%; p=0.03). Significant differences in optical densities were notices comparing the lesional stage of pigs (p=0.009). While pigs with slight or moderate hepatitis were seropositive, pigs with more severe lesions were seronegative to HEV. These results indicate that swine HEV infection can be a significant contributor to the development of moderate hepatitis in pigs regardless of the PMWS status.

Prevalence of anti-HAV and anti-HEV antibodies in Konya, Turkey.

OBJECTIVE: To determine the prevalence of antibodies to hepatitis A (HAV) and E (HEV) viruses in the different areas of Konya. METHODS: Anti-HAV and anti-HEV antibodies were investigated in 210 healthy children randomly selected (100 from rural areas and 110 from urban areas of Konya). None gave a history of previous icterus nor other signs of hepatitis, had received blood transfusion and HAV vaccine, or had been on hemodialysis. RESULTS: Evidence of HAV infection occurred in children under the age of 6 years. The seroprevalence rate was 67.8% in rural areas and 25.8% in urban areas. This increased rapidly with age and became universal after 11 years of age in both areas. In contrast, HEV infections were not detected until children were 6-11 year olds, and the 5.2% seroprevalence rate in urban areas and 8.5% seroprevalence rate in rural areas in this age group did not significantly increase in older age group. The prevalence of anti-HAV as well as anti-HEV was significantly higher in children with poor socio-economic conditions in both areas. CONCLUSIONS: These results suggest that HAV infection in rural areas of Konya is widespread and that environmental and socio-economic factors play a major role in its transmission. In contrast, hepatitis E is not a public health problem in Konya.

The prevalence of hepatitis C in England and Wales.

OBJECTIVES: To estimate the background population prevalence of hepatitis C in England and Wales, observe the prevalence over time and assess the extent of infection outside of known risk groups. METHODS: Sera from residual specimens from adult patients submitted to laboratories in England and Wales were tested for anti-HCV. Testing was carried out using a cost-effective pooling strategy. RESULTS: Although the prevalence of anti-HCV was highest in 1986 (1.07%), in the multivariable analysis, prevalence did not vary significantly between the 3 periods 1986, 1991 and 1996 (P=0.14). The prevalence of infection was higher in males than in females (P=0.0013). An age-period-cohort analysis revealed a cohort effect due to a lower HCV prevalence in the most recent birth cohorts, that is, those born between the calendar years 1971-1975 and 1976-1980. CONCLUSIONS: The majority of HCV infections in England and Wales were probably acquired before 1986. Infections in younger males identified in 1996 may signify more recent acquisition by injecting drug use.

The epidemiology of hepatitis A infection in Palestine: a universal vaccination programme is not yet needed.

In Palestine, there has been an increase in the reported incidence of acute hepatitis A virus (HAV) infection since 1995. Since overt clinical disease occurs only among adults, questions were raised whether or not a shift in the epidemiology of HAV has occurred. This is generally characterized by a decrease in the overall incidence rate and a shifting in the mean age of infection towards adolescence and early adulthood. The need for a vaccination programme is being discussed. To resolve this issue, we examined the prevalence of anti-HAV in a representative sample of 396 school children in the Gaza Strip. The prevalence of anti-HAV was 93.7% (95% CI: 91.3, 96.1%). Stratifying the prevalence by age showed that 87.8% (95% CI: 78.6, 97%) were HAV antibody positive by the age of 6. By the age of 14, almost 98% (95% CI: 92.7, 100%) were HAV antibody positive. This means that the majority of HAV infection is still taking place in early childhood, when it is usually asymptomatic and of little clinical significance. The results refuted the shifting epidemiology theory and we recommend that a vaccination programme against HAV infection is not yet needed. Alternative explanations for the increase in reported cases are discussed.

The epidemiology of hepatitis A in Rio de Janeiro: environmental and domestic risk factors.

A serological study of hepatitis A was carried out in low-income areas scheduled for a major sanitation programme in Rio de Janeiro, Brazil. Blood spots were collected by finger puncture and transported on filter paper, and total antibodies to hepatitis A virus were detected by ELISA. Households were also interviewed to collect information on their environmental conditions and socio-economic status. A generalized linear model using a complementary log-log function was fitted to the data, using the logarithm of age as an explanatory variable to derive adjusted rate ratios (RR). The risk of infection was greater among households with 2-3 members per room (RR = 1.4; 95% CI = 1.04-1.8) or more than three per room (RR = 1.5; 95% CI = 1.2-2.0). People living on hilltops (RR = 1.5; 95% CI = 1.02-2.2), near to open sewers (RR = 1.2; 95% CI = 1.03-1.5) or lacking a kitchen (RR = 1.4; 95% CI = 1.08-1.9) were also at greater risk than others. The number of taps and water-using fittings in the house was associated with a protective effect (RR = 0.9 for each tap; 95% CI = 0.9-0.98). A significant protective association was found with maternal education but not with gender or household income. The results do not suggest a strong association with water quality. Ownership of a ceramic water filter was associated with a protective effect on the margin of significance, but the practice of boiling drinking-water was not, nor was the type of water source used. The results suggest that that the risk of infection with hepatitis A is determined by environmental variables in the domestic and public domains.

Antibodies to hepatitis A virus in Italian patients with chronic liver disease.

To improve our knowledge for future hepatitis A virus (HAV) vaccination strategies we carried out a multicentre study on naturally acquired immunological protection against HAV in patients with chronic hepatitis in Italy. We enrolled 830 consecutive patients with chronic hepatitis on their first observation at one of the six Italian liver units participating in the study. Six hundred and fifty-eight patients (79.3%) were positive for total anti-HAV and 172 (20.7%) were negative. The anti-HAV negative patients were younger (median age 33, range 11-78) than the anti-HAV positive (median age 56, 18-87). There was a higher prevalence of cases with circulating anti-HAV among the 508 patients residing in southern Italy than in the 322 residing in northern Italy (88.8% vs. 64%, P < 0.001). No significant difference in the anti-HAV prevalence was observed between patients from northern Italy and those from southern Italy aged 0-30 years or in those over 60 years, while in those 31-60 years old there was a higher prevalence of anti-HAV positive patients from southern Italy (90.2% vs. 65.8%, P < 0.0001). Of the patients with liver cirrhosis in this study, only 3 of the 26 (11.5%) from northern Italy and 8 of the 228 (3.5%) from southern Italy had no immunological protection against HAV infection. The data suggest that the number of patients with chronic liver disease without naturally acquired immunity against HAV is substantial in Italy, particularly in the north of the country, and that new vaccination strategies are needed.