A..R. Zanetti

C. Galli

C. Tagliacarne

C. Velati

L. Fomiatti

L. Roman&#242

S. Castaldi

AIR Universita degli studi di Milano

502
Blood Transfus 2017; 15: 502-5  DOI 10.2450/2017.0089-17
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B਒ਉਅਆ Cਏ਍਍ਕ਎ਉਃਁਔਉਏ਎
Seroprevalence of hepatitis E virus among blood donors in northern Italy 
(Sondrio, Lombardy) determined by three different assays
Cristina Galli1, Laura Fomiatti2, Catia Tagliacarne1, Claudio Velati3, Alessandro R. Zanetti1, 
Silvana Castaldi1, Luisa Romanò1 
1Department of Biomedical Sciences for Health, University of Milan, Milan; 2Immunohaematology and Transfusion 
Medicine Service, Healthcare Services of Valtellina and Alto Lario, Sondrio; 3Italian National Blood Centre, 
National Institute of Health, Rome, Italy
Introduction
Viral hepatitis E infection is caused by hepatitis E 
virus (HEV), a small non-enveloped virus with a single-
stranded positive-sense RNA. A single serotype and four 
major mammalian genotypes with several HEV subtypes 
have been described: genotype 1 is most prevalent 
in Asia and Africa, genotype 2 is usually detected in 
Mexico and Africa, genotype 3 is distributed globally 
and genotype 4 is found particularly in Asia, although 
it has also recently been isolated in Europe. All four 
genotypes infect humans, but genotypes 3 and 4 have a 
zoonotic reservoir.
For a long time, viral hepatitis E was suspected 
to be endemic only in developing countries where 
sporadic cases and large outbreaks transmitted by the 
faecal-oral route and associated with contamination 
of water supplies have been reported. In non-endemic 
areas, such as European countries, hepatitis E was 
mainly considered an infection associated with travel 
in endemic areas (imported disease). Recently, growing 
numbers of HEV-infected patients with no history of 
travel abroad have been identified in industrialised 
countries. These autochthonous cases have a zoonotic 
origin and may occur through consumption of raw or 
undercooked contaminated meat and meat products or 
through exposure to infected animals1. Although HEV is 
spread mainly by zoonotic and food-borne routes, it can 
also be transmitted parenterally via blood. The recent 
detection of HEV viraemic blood donors may indicate 
a threat to the safety of the blood supply2-4.
Hepatitis E is usually an acute asymptomatic and 
self-limiting infection in industrialised countries. 
However, in the last years an increasing number of 
chronic HEV infections in immunocompromised 
hosts and some cases of transfusion-transmitted HEV 
infections, with a broad variety of outcomes, have been 
described2.
Despite an apparently low number of symptomatic 
cases, probably due to the subclinical course of the 
infection, a wide variability in the prevalence of HEV 
antibodies (anti-HEV) among the general population and 
blood donors has been reported. In fact, in developed 
countries the seroprevalence rates are sometimes higher 
than expected and appear very variable not only from 
country to country, but also in the same geographical 
area and study population. The different sensitivities and 
specificities of the serological assays employed in the 
various studies contribute to this variability5.
Studies carried out in Italy have shown the presence 
of clinical autochthonous hepatitis E cases, with the 
number of cases possibly being underestimated6-8. Data 
concerning the prevalence of anti-HEV in Italy are 
limited and the results differ considerably depending on 
the type of population, geographical area and serological 
assays used in the studies9-12.
The purpose of our research was to assess the 
prevalence of anti-HEV among blood donors in northern 
Italy (Sondrio, Lombardy). In order to do this we used 
three different immunoenzymatic assays. 
Materials and methods
Plasma samples were collected from 685 volunteer 
blood donors who attended the Department of 
Transfusion Medicine and Haematology in Sondrio 
(northern Italy). For each donor, demographic data 
regarding gender, age and place of birth were recorded. 
All collected plasma samples were tested for the presence 
of anti-HEV with three different, commercially available 
enzyme-linked immunosorbent assays (ELISA): (i) HEV 
IgG Dia.Pro (Diagnostic BioProbes Srl, Milan, Italy), (ii) 
HEV IgG Wantai (Biological Pharmacy Enterprise Co., 
Beijing, China) and (iii) HEV Ab Version Ultra Dia.Pro 
(Diagnostic BioProbes Srl).
The first assay uses HEV-specific synthetic antigens 
encoding for conservative and immunodominant 
determinants derived from ORF2 and ORF3 of all the 
four human HEV genotypes; the second uses HEV 
recombinant antigens derived from ORF2 and ORF3 
and able to cross-neutralise human HEV genotypes 1, 2 
and 3. The last assay is a new ELISA for the detection of 
total HEV antibodies using HEV-specific recombinant 
virus-like particles derived from HEV-RNA ORF2 
and bearing immunodominant regions of the four 
viral strains. All tests were performed and the results 
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Blood Transfus 2017; 15: 502-5  DOI 10.2450/2017.0089-17
Prevalence of HEV in blood donors
interpreted according to the manufacturers' instructions.
Comparisons between frequencies were performed 
using the chi-squared test; p values <0.05 were 
considered statistically significant.
Results
The 685 blood donors studied were all Italian and 
about three-quarters were male (72% male, 28% female). 
Their median age at the time of blood donation was 48 
years (range, 19-68 years). 
Seventy of the 685 blood donors (10.2%) tested 
positive for anti-HEV immunoglobulins G (IgG) with 
assay 1 while 67 (9.8%) tested positive with assay 2 
(p=n.s.). The prevalence of anti-HEV detected by assay 
3 was 119/685 (17.4%), which was significantly higher 
than the prevalences determined by the other two assays 
(p<0.001). Among the 119 plasma samples found to be 
positive by assay 3, 58 were positive by both the other 
two tests, 17 were positive by assay 1 or 2, while 44 were 
positive only according to assay 3. A small proportion 
(11/685, 1.6%) of samples resulted equivocal when 
tested by assay 2 and/or 3 (Table I).
Overall, the concordance among the three ELISA 
was 89.9% (58 positive and 558 negative results), 
corresponding to a prevalence rate of 8.5% (58/685). 
The concordance between at least two assays was 99.7% 
(616 concordant results with all tests and 67 with two 
tests). In detail, considering test-by-test agreement, the 
concordance was 96.8% (663/685) between assays 1 and 
2, 91.5% (627/685) between assays 1 and 3 and 91.2% 
(625/685) between assays 2 and 3 (Figure 1).
As illustrated in Figure 2, we observed an age-related 
difference in the prevalence of anti-HEV with all three 
Table I - Anti-HEV results obtained by three different 
ELISA used to determine the prevalence among 
blood donors.
Anti-HEV result Assay 1n (%)
Assay 2
n (%)
Assay 3
n (%)
Positive 70 (10.2) 67 (9.8) 119 (17.4)
Negative 615 (89.8) 612 (89.3) 560 (81.7)
Equivocal 0 (0) 6 * (0.9) 6 ** (0.9)
Total 685 (100) 685 (100) 685 (100)
* 4/6 positive by assay 1 and assay 3;  ** 4/6 negative by assay 1 and assay 2.
HEV: hepatitis E virus.
Figure 1 -  Concordance among anti-HEV results obtained by three different ELISA.
 HEV: hepatitis E virus.
tests employed. The lowest anti-HEV prevalence values 
were found in the youngest age groups while the highest 
prevalence rates were recorded in the oldest age groups. 
No significant difference in anti-HEV prevalence was 
observed between genders. 
Discussion
The detailed picture of the global spread of HEV 
infection is still unclear. In developed countries the 
number of clinical cases of viral hepatitis E is limited, 
probably because infection with this virus is frequently 
asymptomatic; however, the anti-HEV seroprevalence in 
the general population and blood donors is very variable 
and sometimes higher than expected. Indeed, studies 
conducted in Europe have found anti-HEV prevalence 
rates ranging from 0.6% to 52.5%. Such broad ranges 
have been observed not only between countries but 
also in the same country and sometimes in the same 
geographical area, in the same study population and 
study period. This variability could be explained in 
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Galli C et al
Blood Transfus 2017; 15: 502-5  DOI 10.2450/2017.0089-17
part by the use of different immunoenzymatic assays, 
with different characteristics in terms of the antigens 
employed, their sensitivity and their specificity5.
In this study, we evaluated the anti-HEV prevalence 
in blood donors in northern Italy (Sondrio, Lombardy) 
using three different assays and we obtained an overall 
anti-HEV prevalence of 8.5% when considering 
concordant results for all three tests. However, the 
percentages of anti-HEV positivity were different when 
considering the three assays individually, being 9.8%, 
10.2% and 17.4%, respectively. We, therefore, confirm 
the variability in anti-HEV seroprevalence depending 
on the different ELISA used, as previously observed 
in Denmark (10.7% vs 19.8%)13 and Spain (10.7% vs 
19.96%)14. 
Given the marked variability of anti-HEV 
seroprevalence in blood donors, we compared our results 
obtained using HEV IgG Wantai with epidemiological 
data recorded using the same assay in the same population 
in other European countries. The anti-HEV seroprevalence 
in our study population was slightly higher than that in 
Austria (13.5%)15 and Norway (14%)16, but lower than 
the anti-HEV level detected in Danish (19.8%)13, Spanish 
(19.96%)14 and French (52.5%)17 blood donors.
When comparing our current results with data 
collected in the same population from the same country, 
we noted that several studies conducted in Italy on 
blood donors have already revealed large differences 
in anti-HEV seroprevalence especially according to 
geographical area and different immunoenzymatic test 
used. A recent epidemiological study found anti-HEV 
rates of 7% and 4.5% using the HEV Ab Version Ultra 
Dia.Pro and the HEV IgG Wantai, respectively12. In 
another study on HEV seroprevalence carried out in 
central Italy, 49% of the tested blood donors were anti-
HEV IgG-positive using the HEV IgG Wantai11. Lower 
rates have been observed in southern Italy (1.3%)10 and 
Sardinia (5%)9 using the HEV IgG Dia.Pro.
In accordance with almost all the just mentioned 
studies, we found no difference in anti-HEV prevalence 
between genders and an increase of anti-HEV positivity 
with age. This anti-HEV age-related trend is a typical 
cohort effect that could be explained by a decreased risk 
of HEV infection due to the improvement of sanitary 
conditions or by an increased risk of HEV exposure 
during life18.
Our finding of 89.9% concordance among the three 
ELISA used in this study is quite low. This value, which 
cannot be compared with those of other similar reports, 
suggests the importance of standardising serological 
diagnostic tests for HEV.
In separate comparisons, the concordance between 
assay 3 (HEV Ab Version Ultra Dia.Pro) and assay 1 
(HEV IgG Dia.Pro) or 2 (HEV IgG Wantai) was low 
(91.5% and 91.2%, respectively). The differences may 
be due to a higher sensitivity or lower specificity of the 
assay based on virus-like particles compared to the other 
two assays. A recent study comparing the HEV IgG 
Wantai assay to the HEV Ab Version Ultra Dia.Pro assay 
revealed a low concordance between the results of the 
Figure 2 - Prevalence of anti-HEV, detected by three ELISA, among blood donors according 
to age groups.
 HEV: hepatitis E virus.
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Blood Transfus 2017; 15: 502-5  DOI 10.2450/2017.0089-17
Prevalence of HEV in blood donors
Arrived: 28 March 2017 - Revision accepted: 20 June 2017
Correspondence: Luisa Romanò
Dipartimento di Scienze Biomediche per la Salute
Via C. Pascal 36
20133 Milano, Italy
e-mail: luisa.romano@unimi.it
two, with a slightly higher sensitivity of the latter assay12. 
This finding could justify our anti-HEV seroprevalence 
rate obtained with the same assay (assay 3), which was 
significantly higher than the prevalences obtained with 
the other two anti-HEV IgG assays.
On the other hand, two recent studies carried out to 
assess the diagnostic performance of five commercial HEV 
IgG ELISA found a comparable sensitivity for the detection 
of anti-HEV IgG when using the HEV IgG Dia.Pro assay 
(assay 1) and the HEV IgG Wantai assay (assay 2)19,20. 
Norder et al.20 showed that the two above-mentioned assays 
were the most sensitive ELISA. Moreover, in accordance 
with our results, they found a high concordance in anti-HEV 
IgG detection with the two anti-HEV IgG assays, showing 
a higher reactivity in anti-HEV IgG detection with the Dia.
Pro assay than with the Wantai assay. This finding may 
explain why our anti-HEV seroprevalence was higher with 
the HEV-specific synthetic antigen assay than with the HEV 
recombinant antigen assay.
Conclusions
At present, hepatitis E is not a major public health 
problem in Italy. However, the prevalence of anti-HEV 
in blood donors in northern Italy is higher than expected, 
suggesting that the infection is endemic in our country. 
Globally, the anti-HEV seroprevalence greatly varies, 
depending on the geographical area considered and the 
population studied, as well as the assay used in anti-HEV 
detection. In order to clarify the epidemiology of HEV 
infection, a standard immunoenzymatic assay, avoiding 
variability in terms of sensitivity and specificity, is 
required. Further studies are also needed to evaluate the 
role of HEV transmission by blood to better understand 
whether HEV infection could become a real transfusion-
transmitted issue.
Authorship contributions 
CG, CV, ARZ and LR contributed to study design, 
development and interpretation of data. CG, ARZ and 
LR contributed to the writing of the manuscript drafts. 
LF collected the samples. CT carried out the serological 
test. All authors critically reviewed and revised the 
manuscript drafts, approved the final version of the 
manuscript and take responsibility for the integrity of 
the data and accuracy of data analysis.
 The Authors declare no conflicts of interest. 
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Seroprevalence of hepatitis E virus among blood donors in northern Italy (Sondrio, Lombardy) determined by three different assays

https://air.unimi.it/retrieve/handle/2434/527845/914655/Galli%20C.%20Blood%20Transfusion%202017.pdf

Seroprevalence of hepatitis E virus among blood donors in northern Italy (Sondrio, Lombardy) determined by three different assays

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