Received: 5 November 2019 | Revised: 15 January 2020 | Accepted: 18 February 2020 DOI: 10.1111/jvh.13296 S H O R T C O M M U N I C AT I O N Prevalence and 9-year incidence of hepatitis E virus infection among North Italian blood donors: Estimated transfusion risk Marta Spreafico1 | Livia Raffaele1 | Irene Guarnori1 | Barbara Foglieni1 | Alessandra Berzuini2 | Luca Valenti2,3 | Alessandro Gerosa1 | Agostino Colli4 | Daniele Prati2 Department of Transfusion Medicine and Hematology, Alessandro Manzoni Hospital, ASST-Lecco, Lecco, Italy 1 2 Department of Transfusion Medicine and Hematology, Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, Milan, Italy 3 Department of Pathophysiology and Transplantation, Università degli Studi di Milano, Milan, Italy 4 Department of Internal Medicine, Alessandro Manzoni Hospital, ASST-Lecco, Lecco, Italy Correspondence: Daniele Prati, Department of Transfusion Medicine and Hematology, Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, Via Francesco Sforza, 35 20122 Milano, Italy. Email: daniele.prati@policlinico.mi.it Funding information This study was partially supported by a grant from the European Union (the Blood and Organ Transmissible Infectious Agents (BOTIA) project, No. SP23CT-2006-006487), and a contribution from Confartigianato imprese-Giovani Imprenditori, Lecco. Keywords: hepatitis E virus, incidence, prevalence, transfusion transmission 1 | I NTRO D U C TI O N pressure is not stable over time. This implies that NAT yields determined in the relatively short time frame of a prevalence study Hepatitis E virus (HEV) is mainly spread in humans by contaminated may not be entirely representative of the risk of transfusion-related food and water, but it is increasingly being recognized as a threat to transmission, and serological incidence data may be more useful. blood transfusion safety because of its documented transmission by means of viremic blood components.1,2 Taking advantage of a longitudinal biorepository financed by the European Union,5 we calculated the prevalence and incidence of The risk of transfusion-related infection is generally estimated HEV infection over the last ten years in donors from Northern Italy on the basis of the prevalence of HEV RNA among blood donors. and used these data to estimate the risk of the transfusion-related Nucleic acid testing (NAT) has detected a high rate of viremic do- transmission of HEV infection. nations (up to one in 600) in a number of European countries.1,2 There is some evidence of high prevalence of viremia and anti-HEV reactivity among donors in Abruzzo (Central Italy),3 although a ret- 2 | S U B J EC T S A N D M E TH O DS rospective analysis conducted in plasma pools by the Italian National Blood Centre suggests that the pattern of HEV circulation might be The study was conducted within the framework of the EU-funded different in other Italian regions.4 Blood and Organ Transmissible Infectious Agents (BOTIA) project However, pooling procedures can limit the analytical sensi- (SP23-CT-2006-006487).5 The study protocol was approved by our tivity of NAT and so donations should undergo individual test- local Institutional Review Board and conducted in accordance with ing. Furthermore, a number of reports indicate that the infection Italian Authorisation No. 9/2014 of 11 December 2014 concerning Abbreviations: CIs, confidence intervals; HEV, hepatitis E virus; ID-NAT, individual nucleic acid testing; NAT, nucleic acid testing. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2020 The Authors. Journal of Viral Hepatitis published by John Wiley & Sons Ltd 858 | wileyonlinelibrary.com/journal/jvh J Viral Hepat. 2020;27:858–861. | SPREAFICO Et Al. personal data protection for scientific research purposes. The do- 859 of years of follow-up and expressed as a number per 10 000 per nors underwent biochemical and virological testing as prescribed by year. The risk of receiving an infectious blood unit was estimated Italian regulations. using two methods: HEV RNA yield and serological incidence, as- Frozen plasma samples from donations collected at the Department of Transfusion Medicine and Hematology in Lecco in suming a viremia duration of four weeks in the case of asymptomatic infections.6 2015-2016 were tested for the presence of circulating HEV RNA by means of individual NAT (ID-NAT), using the Procleix HEV assay and Panther instruments (Grifols, Barcelona, Spain), with a 95% limit of 3 | R E S U LT S detection of 7.9 IU/mL. Donors found to be initially ID-NAT reactive were re-tested on a different aliquot, tested for anti-HEV IgG and IgM (DiaPro HEV IgG and HEV IgM kits; Diagnostic BioprobesSrl) and followed up at subsequent donations. Figure 1 summarizes the study results. A total of 9726 donor samples were collected for the HEV RNA study: 7253 (74.5%) taken from males and 2473 (25.5%) taken from A subset of samples was also analysed for the presence of an- females, with a mean age of 43 years (range: 18-67). ti-HEV IgG in order to study the prevalence of past exposure to HEV. The ID-NAT assay showed that ten of the samples were initially Confirmation of initially reactive samples was based on repeated reactive, but repeated testing confirmed reactivity in only one. testing and, when possible, on follow-up. The donor was a 63-year-old female who had normal alanine ami- Finally, in order to evaluate the dynamics of HEV infection over nostransferase levels (ie 22U/L) and was fully asymptomatic at the time, we identified a subgroup of donors who had given two serial pre-donation examination. She subsequently seroconverted to being samples over a relatively long follow-up: that is the first at the start anti-HEV IgG positive. None of the nine donors whose initial reac- of the BOTIA project in 2007-2010, and the second in 2017. These tivity was not confirmed seroconverted during the follow-up period. samples were tested for anti-HEV IgG, and the incidence of infec- Thus, the prevalence of HEV RNA was 0.01% (95% CI 0.00%-0.06%). tion was calculated as the number of seroconversions by the total In addition, 767 samples (76.7% males, 23.3% females, mean number of initially negative cases, multiplied by the median number age 43 years) were analysed in order to determine the prevalence VIREMIA STUDY 9726 anonymized blood donations (July2015-July2016) Procleix ID HEV Assay (Grifols) If positive SEROPREVALENCE STUDY Prevalence of anti-HEV IgG 767 samples 6.8% Anti-HEV IgG (DiaPro) (95%CI 5.1% – 8.8%) Anti-HEV IgG & IgM (DiaPro) INCIDENCE STUDY 640 samples (320 pairs) Anti-HEV IgG (DiaPro) FOLLOW-UP (1y) Anti-HEV IgG & IgM (DiaPro) 7.6 per 10 000 per year BOTIA donor sample repository (2007-2010) Prevalence of anti-HEV RNA 0.01% (95%CI 0.00% – 0.06%) FIGURE 1 Incidence of HEV infection Flow chart and main results of the study Donor samples (2017) (95%CI 2.1% –27.5%) 860 | SPREAFICO Et Al. of anti-HEV. Anti-HEV IgG reactivity was confirmed in fifty-two do- rates recorded in previous studies is related to methodological nors (45 males and 7 females), thus indicating an overall prevalence differences. of IgG of 6.8% (95% CI 5.1%-8.8%). None of them was concomitantly As expected, the prevalence of anti-HEV increased with age, anti-HEV IgM or HEV RNA positive. The prevalence of anti-HEV IgG thus reflecting a cohort effect and the long-term persistence of anti- increased across age strata, ranging from 1% (95% CI 0.0%-5.7%) body reactivity after primary infection. The fact that the age-related in donors of 18-30 years to 11.1% (95% CI,7%-16.5%) in those of increases in prevalence were relatively uniform (data not shown) 50 years or older. suggests that no major outbreak has occurred since the 1950s. Our Finally, a subset of 320 donors contributed 640 paired samples prospective repository of biological samples allowed us to calcu- collected between 2007 and 2017 (mean interval between sampling: late the incidence of HEV infection over a ten year period, which 9 years, range 8-10). Thirty-one donors (9.7%; 95% CI: 6.7-13.5%) proved to be 7.6/10 000 per year and is substantially lower than the were anti-HEV IgG positive at baseline, and all of them were still calculated incidence in other European countries such as Germany reactive when the second sample was collected. Two of the 289 do- (35/10 000 per year).8 Furthermore, these data allowed us to esti- nors who were initially anti-HEV-IgG negative had seroconverted mate the local transfusion-related risk of infection using a method by the time of the follow-up sample: they were both males and, in based on the assumption that the viremic phase (and therefore, the 2017, were, respectively, 48 and 49 years old. The incidence was 7.6 infectiousness of the blood products collected during active infec- (95% CI 2.1-27.5) per 10 000 per year. The estimated risk of transfu- tion) lasted four weeks in each seroconverting blood donor.6 On the sion-related infection based on HEV RNA yields was 1/10000 blood basis of this calculation, approximately one out of 16 000 donors donations (the upper limit of the 95% CI was 1/1,666); the estimate should be positive at any given time, a figure that is in line with the based on the incidence data was 1/16666 blood donations (95% CI prevalence of HEV RNA found in this study and confirms that in- 1/4350-1/57000). fection pressure remained quite stable in northern Italy during the considered ten years. The limitations of this study include the fact that we could not 4 | D I S CU S S I O N look back on HEV transmission because all of the samples were coded and blinded to investigators, according to current regula- Our study combined HEV viremia and serological data in order to tions. Secondly, the findings cannot be extended to the Italian estimate transfusion risk over a ten year period. Using high sensi- population as a whole because of the geographical heterogene- tive individual testing, we found that only one of the ten initially ity of the circulation of HEV. However, our study population was HEV RNA reactive samples in our series was found to be truly representative of the local community of blood donors as it in- viremic, accounting for one out of almost ten thousand blood do- cluded donors living in urban, suburban, rural and mountain areas. nations. In addition, the overall seroprevalence of anti-HEV IgG Lombardy is the most highly populated region in Italy, and its was 6.8%. These data indicate that the frequency of current and transfusion system provides 24% of the total Italian blood sup- past HEV infections among blood donors in northern Italy is one ply. Although the circulation of HEV is minimal in comparison with of the lowest so far reported in Europe. According to recent re- other European countries, our data indicate that some tens to views, the prevalence of HEV RNA positivity ranges from 1/762 in hundreds of HEV-infected blood components a year may be trans- the Netherlands to 1/8416 in Austria and that of anti-HEV ranges fused into blood recipients. Some of them, such as immunocom- from 12% in England to 53% in south-western France. 2 Our find- promised patients, are susceptible to the development of acute or ings are also very different from those observed in Abruzzo, a chronic liver failure, or a chronic infection that may rapidly prog- region in central Italy. According to Lucarelli et al , almost half ress to liver cirrhosis and death.1,2 3 of the subjects donating blood in L’Aquila during the first months The findings of this study may be useful for the regional and na- of 2014 showed serological signs of previous HEV exposure, and tional blood authorities responsible for making policy decisions given one out of 166 had detectable viremia. The highly endemic nature that recent European guidelines1 recommend that HEV screening of the infection in this area has been attributed to local dietary policies should be based on local risk assessment studies. Whether habits favouring zoonotic transmissions, but another possibility is or not to introduce HEV NAT screening therefore requires careful contaminated water, as the circulation of some faecal pathogens consideration: donor screening may very effectively minimize iatro- increased in the area of L’Aquila for several years after the cata- genic HEV infection, but it is very costly and can be expected to have strophic earthquake in 2006. a relatively minor impact on the number of HEV infections in the 7 Given that nine of the ten initially NAT-reactive samples were not confirmed by further testing or at serological follow-up, it seems population as a whole because the vast majority of new infections seems to be due to dietary exposure.1,2 that a single determination of HEV RNA has little positive predictive value. The impact of false-positive results might be more evident C O N FL I C T O F I N T E R E S T when testing samples at low risk of infection, like blood donors. It Grifols Italia S.p.A (Milan, Italy) and Diagnostic BioprobesSrl, (Milan, is therefore possible that some of the variability in the prevalence Italy) provided the kits for serological and molecular analyses. | SPREAFICO Et Al. ORCID Luca Valenti https://orcid.org/0000-0001-8909-0345 Daniele Prati https://orcid.org/0000-0002-2281-7498 REFERENCES 1. European Association for the Study of the Liver. EASL Clinical Practice Guidelines on hepatitis E virus infection. J Hepatol. 2018;68:1256-1271. 2. Ankcorn MJ, Tedder RS. Hepatitis E: the current state of play. Transfus Med. 2017;27:84-95. 3. Lucarelli C, Spada E, Taliani G, et al. High prevalence of anti-hepatitis E virus antibodies among blood donors in central Italy, February to March 2014. Euro Surveillance. 2016;21(30):pii:30299. https://doi. org/10.2807/1560-7917.ES.2016.21.30.30299 4. Spada E, Pupella S, Pisani G, et al. A nationwide retrospective study on prevalence of hepatitis E virus infection in Italian blood donors. 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Prevalence and 9-year incidence of hepatitis E virus infection among North Italian blood donors: Estimated transfusion risk. J Viral Hepat. 2020;27:858–861. https://doi. org/10.1111/jvh.13296