Seroepidemiology of Hepatitis E virus among the voluntary blood donors in the coastal region of Karnataka, India [version 2; peer review: awaiting peer review]

Background: Hepatitis E is a fecal orally transmitted disease and an emerging transfusion transmissible infection with potential transfusion safety implications. Hepatitis E Virus screening among blood donors is not routinely done despite the fact that it is endemic in India. As there are very few reports and evidence, the justification for mandating HEV screening among blood donors is still only marginally convincing. Therefore, the present study was carried out to determine the incidence and prevalence of HEV infection among blood donors in Coastal Karnataka, India. Methods: This cross-sectional study was conducted in a tertiary care hospital in Udupi district of Karnataka, India. A total of 1939 blood donors were randomly selected between June 2020 to June 2022 to participate in the study, where anti-HEV IgM antibody screening was performed using HEV IgM ELISA test kit (DiaPro, Italy). Further, a randomly selected 588 and 1620 pooled donor samples were screened for anti-HEV IgG antibodies and HEV RNA, respectively. Results: The overall prevalence of HEV IgM antibodies among study population is found


Introduction
Hepatitis E virus (HEV) is the most common causative agent of acute viral hepatitis and is self-limiting in most cases. The World Health Organization (WHO) estimates that, every year there are an estimated 20 million HEV infections worldwide, leading to an estimated 3.3 million symptomatic cases of hepatitis E. 1 Four reported genotypes of HEV are known to infect humans, of which genotype-1 (HEV1) and genotype-2 (HEV2) infect only humans, whereas genotype-3 (HEV3) and genotype-4 (HEV4) are zoonotic and are transmitted particularly by swine populations and their related meat products. 2 To date, HEV1 is the only genotype known to infect humans in India. 3 HEV is considered highly endemic in various parts of India and neighbouring countries. 4 And, it has caused several outbreaks and epidemics of viral hepatitis in India. 5,6 HEV is also one of the emerging infectious diseases associated with blood transfusions. In 2004, the first case of transfusion-transmitted HEV (TT-HEV) infection was reported from Japan, HEV RNA from the recipient and donor were genotype-matched to confirm the transmission of the infection through blood transfusion in this case. 7 The prevalence of HEV in different populations varies, and in recent times, there has been an increasing report of HEV infection among blood donors in developing countries. [8][9][10] Although HEV is endemic in India, there are only a few reports on the prevalence of HEV among blood donors and none from south India. Currently, there are no policies or guidelines for screening for HEV among blood donors in the country. Therefore, it is essential to investigate the burden of the disease and the extent of the threat to blood safety through healthy asymptomatic blood donors, particularly in India.
Important characteristics that have been reported in developed countries include serological and molecular evidence of TT-HEV, prevalence and risk factors among blood donors, and severity of the infection among immunocompromised patients. 11 However, there is little, or no information related to TT-HEV and possible risk factors, common genotype of HEV that is prevalent among healthy blood donors from India. Therefore, this prospective cross-sectional study was carried out to determine the prevalence of HEV in healthy voluntary blood donors and the potential risk factors for a silent infection. To the best of our knowledge, this is the first study of its kind to examine the incremental risk of exposure to asymptomatic HEV infection among healthy volunteer blood donors in India.

Methods
Setting A cross-sectional study was conducted between July 2020 and June 2022 in a blood center of a tertiary care hospital in the coastal belt of Karnataka, India. The center has a yearly collection of approximately 15,000 blood donations, and participants were recruited from this healthy volunteer blood donor pool who were residents of Udupi district, Karnataka. The blood donor participants were from an in-house blood center collection and voluntary blood donation camps held within the district. The study was conducted following the principles of the Helsinki Declaration 2013, 12 and approval was obtained from the Kasturba Medical College and Kasturba Hospital Institutional Ethics Committee (IEC no: 06/2020). Blood donors' registration numbers were systematically randomized, and every fifth donor number was the pre-determined participant included in the study. However, if the 5 th donor did not meet the study's inclusion criteria, the next donor in line was considered for recruitment. Only donors who were eligible for blood donation according to the criteria of the National Blood Transfusion Council (NBTC) and the Drug and Cosmetic Act 1940 of the Government of India were recruited for the study. 13,14 Written informed consent was obtained from each participant after counseling, and a pre-validated questionnaire was given to the participants to gather information about the risk of exposure. The questionnaire included details regarding exposure to a high-risk work environment, dietary choices, the source of drinking water, travel history, floods in the locality, and the type of sanitation used by the donor. Any further responses from the reviewers can be found at the end of the article markers, respectively. Qualisa™ Malaria, Catalogue no. 405010096 (Qualpro Diagnostic, Goa, India) and Carbogen Rapid Plasma Reagin, Catalogue no. 10514100 (RPR) card test (Tulip Diagnostics Pvt Ltd, India) were also routinely used in our blood centre according to the manufacturer's protocol for screening of Malarial parasites and syphilis. Further, the 6 ml vacutainer samples were subjected to a mini pool nucleic acid amplification test (NAT) for TTIs screening with the Roche Cobas® TaqScreen MPX test kit in Roche Cobas® s201 instrument. This NAT test kit can simultaneously detect HIV-1 (group M and O), HIV-2, HBV, and HCV nucleic acid. About 500 μL of plasma was aliquoted from these pilot tubes into 1.5 mL cryotubes for screening HEV.

Screening for anti-HEV IgM antibodies
We have screened the samples of the study subjects for anti-HEV IgM antibodies to determine the prevalence of HEV among blood donors. Anti-HEV IgM antibodies were screened at the same time as other TTIs. Anti-HEV IgM antibodies were screened as per the manufacturer's instructions for the screening kit used (DiaPro HEV IgM ELISA kit, Diagnostic Bioprobe SRL, Milano, Italy). The manufacturer's manual claims a diagnostic sensitivity and specificity of greater than 98%. A sample to cut-off ratio (S/CO) above 1 is considered to be reactive. All the samples that were reactive or in the grey zone (S/CO between 0.9 and 1) were repeated in duplicate in the subsequent run for confirmation (as per the standard operating procedure of the deparmtent).

Screening for anti-HEV IgG antibodies
Screening for anti-HEV IgG antibodies was performed to determine the seroprevalence and exposure to HEV in recent or past infections. A total of 571 samples were estimated as the minimum number of samples required to determine the prevalence of HEV IgG with a reference from the previous study and the current study population, with a confidence level of 96% and a 4% margin of error. 15 To detect HEV IgG antibodies, we used a DiaPro HEV IgG ELISA test kit (Diagnostic Bioprobe Srl, Milan, Italy). The test kit is CE marked and has 100% sensitivity and specificity as per the manufacturer's manual.
Screening of HEV RNA Samples that tested negative for anti-HEV IgM antibodies were randomly selected and grouped into six sample pools. Nucleic acid was manually extracted from the pooled samples with the FavorPrep Viral Nucleic Acid Mini Kit (Favorgen Biotech Corporation, Taiwan) following the manufacturer's protocol. The extracted nucleic acid was screened for the presence of HEV RNA with RT-PCR using the RealStar™ HEV RT-PCR test kit 2.0 (Altona Diagnostic, Hamburg, Germany) in a QuantStudio™ 5 platform (Applied Biosystem, USA) as per manufacturer guidelines.

Data collection and analysis
All data were primarily documented and stored in a spreadsheet in Microsoft Excel, and they were analyzed using SPSS 20.0 software (IBM, US). Quantitative and qualitative descriptive variables were presented with the mean, median, mode, and frequency of nominal variables. The factors influencing HEV seropositivity were examined using a chi-square test and logistic regression analysis (95% confidence range).

Results
A total of 1939 voluntary blood donors were recruited for the study and among them, 1711 (88.2%) were males and 228 (11.8%) were females, with a male-to-female ratio of 7.5:1. Participants include 871 (44.9%) first-time donors and 1068 (55.1%) repeat blood donors. The mean age of donors was 29.6 years (SD AE9.3 years) within a range of 18 to 64 years, and the predominant participants were (53%) between 18 and 28 years old. Table 1 provides an overview of the demographic characteristics of the participants and the results of serological screening for HEV. On routine serological screening tests for TTIs, 7(0.36%), 8(0.41%), 3(0.15%), and 2(0.10%) participants were reactive to HIV, HBV, HCV, and syphilis, respectively. None were reactive on NAT screening, and for malarial antigen.
Prevalence of anti-HEV IgM antibodies among blood donors A total of 27 participants were reactive to anti-HEV IgM antibodies, including 20 males and seven females, representing a prevalence of 1.39% in our study population. The prevalence of HEV IgM antibodies in different age groups is shown in Table 1. Except for one donor who was reactive for syphilis in the RPR test, none of the HEV IgM reactive donors were reactive to the other TTI markers.

Prevalence of anti-HEV IgG antibodies among blood donors
Irrespective of the anti-HEV IgM status, anti-HEV IgG antibody screening was performed on 588 randomly selected samples, and a total of 67(11.39%) samples were reactive to IgG antibodies. Of the anti-HEV IgG reactive samples, 7 (10.4%) of them were reactive for IgM antibodies as well. In this group, the coexisting HCV and HIV infection was seen in 2 and 4 donors, respectively.

Hepatitis E viral RNA in pooled samples
None of the 270 pools, which included 1620 samples, showed HEV viral RNA.

Assessment of risk factors for HEV infection
A pre-validated questionnaire was used to assess epidemiological risk factors among blood donors. The epidemiological features and risk factors are shown in Table 2. None of the participants had a jaundice-like illness in the past year, and  there was no statistically significant correlation between any of the risk factors assessed and reactive status for anti-HEV IgM antibodies. A significantly higher number of participants reactive to anti-HEV IgG antibodies were exposed to agricultural or veterinary settings (p<0.05). The positivity of IgG antibodies was significantly higher among participants who did not follow regular handwashing with soap and water before eating food.

Discussion
The prevalence of anti-HEV IgM and IgG antibodies among blood donors in the study population was 1.39% and 11.39%, respectively. Seven donors were reactive to both anti-IgM and anti IgG HEV antibodies, which may represent the late icteric phase of infection. The prevalence of anti-HEV IgM antibodies is significantly higher in females than in males (1.16% vs. 3.16%; p<0.05). However, there was no significant difference in the prevalence of anti-HEV IgG antibodies between male (54 of 512) and female (13 of 76) participants, this result is in consistent with the study by Tripathy et al., in Pune, India. 16 In India, particularly in south India, there is a paucity of data pertaining to risk factors and HEV infections among the healthy population. As per our knowledge, the present study is the first of its kind on this subject. The prevalence of anti-HEV IgM antibodies in our study (1.39%) is comparatively lower than that mentioned in the study by Gajjar et al., from Gujarat (4.78%) and Shrestha et al., from Nepal (3.0%) but higher than that of the study by Tripathy et al., from Pune, Mumbai (0.20%). The HEV seroprevalence report varies across the Indian subcontinent, although the disease is considered endemic in the region (in Table 3 and Table 4). The anti-HEV IgM prevalence of our study is in accordance with the reports from Poland (1.27%), however, few other international studies report a lower prevalence of anti-HEV IgM (  15,17,18 Additionally, it was noted that none of the countries on the Indian subcontinent had any guidelines for HEV screening blood donors. Nevertheless, Tendulkar et al., (2015, Mumbai) reported a case in which a blood donor self-reported developing jaundice a day after the donation and eventually he was diagnosed to have HEV infection However, the transmission of HEV viremia to the blood recipient was not confirmed. 19 Blood donors reactive to HEV must postpone blood donation for 12 months, as per the blood donor selection and deferrals guidelines in India (published in 2017). 13 The high endemicity of HEV in the area and the high prevalence of anti-HEV antibodies among blood donors may be the key criteria in deciding the necessity for HEV screening in India. [20][21][22]   The majority of HEV-infected people experience asymptomatic, self-limiting infections or a brief sickness without signs of liver damage. The typical infection has an incubation period of up to 2 to 6 weeks, with a prodrome phase and viremia peaking in the fourth week. 23 Anti-HEV IgM antibodies begin to increase in the late incubation stage and reach their peak in the middle of the icteric phase, its concentration declines later with the raise in anti-HEV IgG antibodies. 24 The presence of anti-HEV IgG antibodies among the anti-HEV IgM positive participants could be an indication of late icteric phase stage of infection. 25 Anti-HEV IgG antibodies are detectable for a longer period of time. In our study, there were 61 participants reactive solely to anti-HEV IgG antibodies which indicates either the past infection, or they are in convalescent phase.
There is a chance of iatrogenic HEV transmission throughout the incubation phase and the prodrome stage since HEV viremia is evident even before an anti-HEV antibody response and lasts for about three weeks. 26 HEV viremia, however, might be regarded as blood transfusion transmissible at any stage.
In the current study, the participants were mainly students (1031/1939) and the majority of them were between 18 and 28 years of age. The majority (34.6%) of participants were undergraduate students with the highest degree of education, either Pre-University (PUC) or Higher secondary education. Our results are in line with a longitudinal research conducted in rural Bangladesh among healthy individuals, which found no statistically significant association between anti-HEV IgM antibody reactivity and demographic variables including age and educational qualification. 27 The analysis of the different demographic variables and prevalence of anti HEV antibodies (IgM and IgG) is shown in Table 1.
Since eating with the hands is preferred in India, maintaining good hand cleanliness is crucial in preventing the transmission of HEV infection. Among participants who regularly wash their hands with soap before eating food, the prevalence of HEV IgG was significantly lower (p<0.05) than in other group. Therefore, hand washing with soap could be essential in preventing the transmission of fecal-orally transmitted diseases such as HEV. The anti HEV IgM antibody reactivity rate among donors who wash hands before eating and who did not wash were 1.3% and 2.3% respectively.
There is an increased report of autochthonous HEV cases in developed countries, where HEV is primarily transmitted zoonotically, and it may also be associated with travel. Thus, for a traveller in HEV endemic countries food sources and dietary choice are risk factors for exposure, hence, it is also referred to as a travel-associated disease. However, travel to other districts, states, or other countries, length of stay, and dietary habits were not significant risk factors in the present study. Exposure to agricultural work or animal husbandry was a significant (p < 0.05) risk factor associated with anti-HEV IgG reactivity (OR 2.016; 95% CI 1.149, 3.525) but not with anti-HEV IgM reactivity in the study population, which is consistent with the results from Nigeria. 28 This implies that agricultural and livestock exposures, particularly the swine population, pose a significant occupational risk for HEV infection, which has been noted in several epidemiological studies. 29 The zoonotically transmitted HEV4 and HEV3 genotype has been reported to cause chronic diseases in immunocompromised patients, and HEV4 has been detected among pigs in India. 30 Chronic HEV infections due to HEV3 and HEV4 is well known in developed countries. The sporadic cases of HEV were due to the consumption of undercooked pork and wild boar meat, However, given that outbreaks and epidemics caused by HEV1 are frequently recorded in India, this is an unlikely risk factor for exposure to HEV infection there. 6 A chronic HEV-1 is an emerging infection in immunocompromised patients and was documented by Singh et al., in 2018. 31 Therefore, a critical analysis to perform selective HEV screening in blood donation for high-risk groups such as pregnant women and immunocompromised patients might be helpful.

Conclusion
The present study shows that the prevalence of anti-HEV antibodies among blood donors is found to be high and it appears to be a hidden threat to blood safety. This study emphasizes the importance of a larger multicentric study to estimate the actual burden the disease. The development of policies on universal or selective screening of blood donors for HEV infections will benefit from a review of the cost-effectiveness of HEV testing. This project contains the following underlying data:

Data availability
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