Hepatitis E virus in blood donors in England, 2016 to 2017: from selective to universal screening

Introduction Hepatitis E virus (HEV), the most common cause of acute hepatitis in many European countries, is transmitted through consumption of processed pork but also via blood transfusion and transplantation. HEV infection can become persistent in immunocompromised individuals. Aim We aimed to determine the incidence and epidemiology of HEV infection in English blood donors since the introduction of donation screening in 2016. Methods Between March 2016 and December 2017, 1,838,747 blood donations were screened for HEV RNA. Donations containing HEV RNA were further tested for serological markers, RNA quantification and viral phylogeny. Demographics, travel and diet history were analysed for all infected donors. Results We identified 480 HEV RNA-positive blood donations during the 22-month period, most (319/480; 66%) donors were seronegative. Viral loads ranged from 1 to 3,230,000 IU/ml. All sequences belonged to genotype 3, except one which likely represents a new genotype. Most viraemic donors were over 45 years of age (279/480; 58%), donors aged between 17 and 24 years had a seven-times higher incidence of HEV infection than other donors between March and June 2016 (1:544 donations vs 1:3,830). HEV-infected blood donors were evenly distributed throughout England. Screening prevented 480 HEV RNA-positive blood donations from reaching clinical supply. Conclusion HEV screening of blood donations is a vital step in order to provide safer blood for all recipients, but especially for the immunosuppressed. The unusually high rates of HEV infection in young blood donors may provide some insight into specific risks associated with HEV infection in England.


Introduction
Hepatitis E virus (HEV) is a non-enveloped, singlestranded RNA virus belonging to the Orthohepevirus genus within the family Hepeviridae. Four main HEV genotypes are known to infect humans. Genotypes 1 and 2 are transmitted via the faecal-oral route between humans and cause large waterborne outbreaks in developing countries. Genotypes 3 and 4 can be transmitted to humans zoonotically from infected pigs, deer and wild boar. Transmission usually occurs through consumption of raw or inadequately cooked processed pork meat, or, rarely, by contact with infected animals or their excreta. Transmission of HEV via blood transfusion and transplantation has also been documented [1][2][3][4].
Most cases of acute HEV infections in Europe are currently caused by genotype 3 viruses [5]. Although these infections are usually asymptomatic, HEV is now recognised as the most common cause of acute viral hepatitis in many European countries including France, Germany and the United Kingdom (UK) [5]. Hepatitis E is a concern for those with underlying chronic liver disease and limited hepatic reserve as it can lead to acute-on-chronic liver failure. HEV infection offers a particular risk to persons with compromised immune systems as they may develop persistent infection which often shows a rapid progression to cirrhosis associated with a poor prognosis [6]. It has been estimated that up to two thirds of solid organ transplant recipients with persistent HEV infection develop chronic hepatitis with rapid progression of fibrosis, followed by cirrhosis and even decompensation and death [6][7][8]. Although there is no proven treatment for chronic HEV infection, ribavirin therapy and reduction of immunosuppression have each been successful in achieving HEV RNA clearance in individual cases [8,9].
The first systematic study investigating the potential human-to-human transmission of HEV through blood transfusion was conducted in England in 2012-2013 [4]. As part of the study, 43 recipients of HEV RNAcontaining blood components were followed up; 18 had evidence of infection and a chronic infection was demonstrated in half of the HEV-infected immunosuppressed recipients [4]. Further calculations based on this study demonstrated that a minimum infective dose of 2 × 104 IU is required for efficient HEV transmission by transfusion but noted that the minimum viral load in the donor plasma expected to lead to transmission was influenced by the plasma volume included in the different blood components [10]. However, transfusion risk dominates only in the heavily transfused immunosuppressed patient, whereas dietary exposure to porkderived food has been identified as the most likely route of HEV infections in England [10][11][12]. As consumption of processed pork is likely to be common in the donor population, no specific donor selection criteria can be used to identify donors at enhanced risk of acquiring HEV.
In order to protect specific groups of vulnerable patients from transfusion-acquired HEV infection, blood donation screening for HEV RNA was introduced in the UK in 2016. Initially, at the end of 2015 the Department of Health and Social Care Advisory Committee on the Safety of Blood, Tissues and Organs (SaBTO) recommended the supply of HEV-screened blood components for recipients of allogeneic stem cell transplants and solid organ transplants [13]. In March 2016, NHS Blood and Transplant (NHSBT) introduced HEV NAT screening on pools of 24 for selected blood donations in England. It was anticipated that a minimum of 30% of the blood supply would need to be tested in order to meet clinical demand, including screening of all platelets donated by apheresis. Donations identified as HEV RNA-positive were excluded from the supply; whole blood donors were suspended for 6 months from the date of their HEV RNA-positive donation whereas apheresis platelet donors were re-instated once they had cleared the infection and developed a  high concentration of antibody to HEV (anti-HEV IgG, sample/cutoff (S/CO) > 10).
A change to universal screening of donations in April 2017 followed a further review by SaBTO, which considered that HEV-screened blood should also be supplied for all immunocompromised patients [14]. It was also estimated to be a cost-neutral change, provided that the incidence of HEV infection remained above 1 in 10,000 blood donations. Universal screening of blood for HEV RNA was thought to be as beneficial for patients as selective screening but was estimated to provide an easier and more economical workflow in both hospitals and screening laboratories. In hospitals it would reduce the need for two separate inventories for blood, with a lower risk of errors occurring during the allocation process.
Here we present the results of routine screening of almost two million blood donations for HEV RNA between March 2016 and December 2017 in England (n = 1,838,747). We aimed to determine the incidence of HEV among English blood donors, and to describe the classical and phylogenetic epidemiology of HEV infections in these donors.  [15]. Phylogenetic analysis was performed using MEGA6 [16].

Notification and follow-up of hepatitis E virusinfected donors
All viraemic donors were sent a notification letter explaining their test results and an information leaflet about HEV infection. They were given an opportunity to telephone and discuss their test results with a member of the NHSBT clinical team. Details of the donors' HEV infections were also sent to their general practitioners with the donors' consent and to the local health protection teams. Apheresis platelet donors were informed that they would not be able to donate again until further blood sampling confirmed viral clearance; a follow-up sample was requested from all apheresis donors within 6-8 weeks from diagnosis, whereas whole blood donors were asked to wait for 6 months before returning to donate. Geometric mean viral load was significantly higher in the 2012-13 testing period than in 2016-17 (4,740 IU/ml vs 883 IU/ml, p = 7 × 10 -9 ). Viral load data for 2012-13 has already been published [4].
Donor details, including age, sex and postcode, were collected for all HEV RNA-positive donors. R-studio (Free software foundation, established in Auckland, New Zealand) was used to match postcode by PHE region, to be then mapped using ArcGIS (ESRI, Aylesbury, UK) to display viraemic donors.

Archived sample
The archived plasma sample from the most recent negative donation was retrieved for all RNA-positive apheresis donations and tested for HEV RNA and antibodies. Archive samples were available for 35 of 45 HEV-infected apheresis donors. If the most recent archive was identified to contain HEV RNA, the next most recent archive sample was also retrieved for testing. Hospitals which had received the HEV RNA-containing blood components were informed of potential risks and advised to take appropriate action.

Questionnaire
The NHSBT clinical team completed a questionnaire for each confirmed viraemic donor for surveillance purposes. Donors were also given an opportunity to call back and discuss their results with a clinician. This discussion included questions about travel 6 weeks prior to donation, food history (meat, meat excluding pork or vegetarian) as well as signs and symptoms experienced around the time of donation.

Ethical statement
Signed consent was obtained from each donor at the time of donation. Donors consent to NHSBT holding information about them including their health, attendances and donations and using their information for the purposes explained in the donor welcome booklet and data protection leaflet which donors are asked to read at the time of donation. This includes using data for the purposes of clinical audit to assess and improve the service and for research, specifically to improve our knowledge of the donor population.

Statistical analysis
Categorical variables were compared using chi-squared tests in VassarStat (Poughkeepsie, NY, US), and geometric mean viral loads were calculated and further compared using Kruskall-Wallace in Systat version 10.2 (Systat Software Inc., San Jose, California, US).

Virology
The geometric mean viral load was 883 IU/ml (range: 1-3,230,000 IU/ml), which was significantly lower than detected in English blood donors in a previous study (Figure 2 [4]). Of the 480 samples, 150    were successfully genotyped (31%), and all except one sequence belonged to genotype 3 ( Figure 3).

Donor demography
Between March 2016 and December 2017, HEV-infected blood donors did not appear to cluster in England ( Figure 4). Twenty six cases were identified in the London area. The remaining 454 cases were evenly spread between the south of England (n = 158), the middle of England (n = 157) and the north of England (n = 139 between March and July 2016 ( Figure 5). This normalised after July 2016 to the level recorded in other age groups.

Donor risk factors for hepatitis E virus infection
Travel history was available for 355 donors, and 82 (23%) reported travel outside the UK in the 6 weeks before donation. Information on diet was available for 348 donors; most HEV-infected blood donors had consumed meat including pork (339/348; 97%). Seven blood donors had eaten meat but not pork; one reported eating pâté, one had swum in water assumed to be contaminated, one lived near a pig farm in England, one had consumed shellfish and one had recently travelled to Croatia. The remaining two donors were vegetarians; one of them worked at a garden centre in England that used manure/compost and the other ate pre-packed salads.

Self-reported clinical signs and symptoms
A total of 334 (70%) HEV RNA viraemic donors provided information on clinical signs and symptoms and 146 reported one or more (Table). Fatigue was the most commonly reported symptom after donation (n = 85), but some donors also reported joint pain/aches (n = 22). A small number of donors reported jaundice (n = 6) after donation.

Discussion
The detection of HEV RNA in 480 of 1,838,747 blood donations demonstrates a high incidence of HEV infection in England, accounting for an overall prevalence of 1 in 3,830 donations during the period between March 2016 and December 2017. A similar prevalence of viraemia in blood donors has been previously reported in many other European countries, indicating the wide spread of this infection in European human populations [17]. However, over the years it has become noticeable that the rate of HEV RNA-containing donations fluctuates considerably over time. A prevalence of 1 in 2,848 donations was noted in a previous study from England in 2012-13 [4], whereas a higher prevalence of 1 in 1,365 was reported 3 months after selective screening was introduced throughout England in March 2016 ( [17]; Figure 1). Interestingly, the detection rate has decreased since then, and has remained steady around 0.20 per 1,000 donations (1/4,781 donations) in England. However, we have not changed the method used for screening of donation in pools of 24 during this time period, and if anything, it seems to have become more sensitive. It remains to be seen how the prevalence of HEV RNA viraemic blood donors will change in the future.
More than half of HEV-infected blood donors were over 45 years old (279/480), which is in line with previously published data [18,19]. However, younger donors aged 17 -24 years were shown to have a seven-times higher risk of acquiring HEV infection when compared with other individuals who had donated between March and July 2016 (1:544 donations vs 1:3,830, Figure 5). This normalised after July 2016 to the level recorded in other age groups. Interestingly, this was also mirrored in the English HEV surveillance programme where data on laboratory-confirmed cases of HEV infections are collected from reference and local laboratories [20]; 20% of all infections were reported to be in 15-24-yearold individuals between April and September 2016 (94/471) whereas only 4% of HEV infections were seen in this age group between October 2016 and December 2017 (43/1,088). These young donors were distributed throughout England, and they were infected with a variety of HEV 3c subgenotype strains (Figures 3 and  5). In the future, we will aim to analyse our incidence data across the different age groups in order to identify possible ongoing outbreaks and investigate and mitigate possible sources of infection.
As in many previous studies, we identified a dietary exposure to pork-derived foods for most HEV-infected donors. It is interesting that the rate of HEV RNA detection was lowest in the London area (15.2/100,000 donations; Figure 4 Our study also demonstrates the diversity of HEV strains circulating in England, and that we identified a Although selective HEV RNA screening was initially implemented in England, a change to universal screening of donations in April 2017 was driven by its comparable costs and easier logistical performance. In addition to the UK, universal HEV RNA screening of blood donations has been implemented in Ireland [25], the Netherlands [17], six German donor centres [17] and parts of Japan [26]. Other countries are either still investigating or considering whether blood donation screening for HEV RNA is needed [17]. Denmark has deemed HEV screening unnecessary as a low prevalence of viraemic donors was detected and no evidence of transfusion-transmitted infections has been found [27]. Similarly, donor screening for HEV RNA has not been introduced in Australia due to the low prevalence estimates of 1 in 74,313 [28]. We identified a total of 480 HEV RNA-positive blood donors over the 22-month screening period, who presented with median viral load of 883 IU/ml. This was significantly lower than previously detected in English blood donors in 2012-13, the slightly increased reported sensitivity of the testing assays used in between 2016 and 2017 (15%) is unlikely to account for this difference [4]. Large numbers of donors were identified with a very low viral load below the stated sensitivity of the screening assay used. To investigate this further, parallel comparison of screening and confirmatory assays used for HEV testing should be performed using the dilution series of the World Health Organization HEV standards. A total of 42 of 480 HEV RNA-positive donors were reactive for anti-HEV IgG antibodies only (no IgM) at the time of donation, reflecting either late infections or possible re-infection.
No new cases of transfusion-transmitted (TT) HEV were reported in England during the study period. Interestingly, we have only detected two donations missed by initial pooled screening, both with very low viral load (4 and 8 IU/ml). A previous study estimated that around 55% of recipients challenged with a component containing a minimum of 20,000 IU of HEV RNA would become infected [10]; this would mean that a viral load of 1,600 IU/ml in a donor (whole blood donation) and 111 IU/ml (apheresis platelet donor) would be sufficient to transmit infection to recipients, in keeping with previous observational studies [4,29]. Based on the number of donations where HEV RNA viral load exceeded these cut-offs, it can be speculated that with universal HEV screening a total of 146 donations with the potential to transmit HEV infection with severe outcomes particularly in immunocompromised transplant recipients, patients with haematological malignancies and underlying liver disease, have been removed from clinical use.
As well as mitigating the risk of transfusion acquired HEV, the screening of blood donations provides a useful and unique insight into HEV infections at a population level. The data obtained inform on fluctuations in risk and changes in HEV incidence and allow for comment to be made on what is largely an asymptomatic infection. They are essential to the development of guidelines for public health purposes and to inform continued risk assessments around blood safety.