Journal of Medical Virology 46244-246 (1995)

Hepatitis E Virus Antibodies Among Patients With Hemophilia, Blood Donors, and Hepatitis Patients H.L. Zaaijer, E.P. Mauser-Bunschoten, J.H. ten Veen, H.P. Kapprell, M. Kok, H.M. van den Berg, and P.N. Lelie Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam (H.L.Z., M.K., P.N.L.); Van Creveld Clinic, University Hospital Utrecht, Utrecht (E.P.M.-B., H.M.v.d.B.), Prinsengracht Hospital, Amsterdam (J.2T.t.V.) the Netherlands; Abbott Laboratories, Delkenheim (H.P.K.), Germany

The presence of antibodies to hepatitis E virus (HEV) was studied among hemophiliacs, blood donors, and hepatitis patients. Four of 296 (1.4%) hemophiliacs and 5 of 1,275 (0.4%) donors were confirmed as positive for HEV antibodies (differ- ence was not significant: P = 0.07). Parenteral transmission of HEV to hemophiliacs was thus rare or nonexistent. Seven of 187 hepatitis pa- tients were found with HEV antibodies (IgG and IgM). Six persons fell ill shortly after arriving from HEV-endemic countries. The seventh pa- tient, without a history of travel, represents a case of nontropical hepatitis E. Consequently, hepatitis E should be considered in patients suf- fering from acute non-ABC hepatitis, even in in- dustrialized countries. o 1995 Wiley-Liss, Inc.

KEY WORDS HEV EIA, transfusion, endemic

INTRODUCTION The hepatitis E virus (HEW is transmitted by the

fecal-oral route, and i t causes isolated and epidemic forms of acute hepatitis, mostly in tropical countries [Balayan, 19911. Imported cases have been described in the Western world [Skidmore et al., 19911. A small percentage of blood donors in nontropical countries ap- pears to have HEV antibodies [Paul et al., 19941, but no transmission of HEV by blood has been reported.

Like HEV, the hepatitis A virus (HAV) is a nonenvel- oped virus capable of causing hepatitis by the fecal-oral route. Recently, several cases of hepatitis A among he- mophiliacs have been described [Mannucci et al., 19941. Most likely, HAV was transmitted in these patients through blood products that were inactivated by the solvent/detergent method. This method fails to inacti- vate nonenveloped viruses such as HAV and parvovi- rus B19 [Mannucci et al., 1994; Santagostino et al., 19941. Could HEV, also a nonenveloped virus, be trans- mitted by blood products? Do anti-HEV-positive donors reflect the existence of HEV in nontropical countries? To answer these questions, HEV antibody reactivity was studied among hemophilia patients, volunteer 0 1995 WILEY-LISS, INC.

blood donors, and non-ABC hepatitis patients in the Netherlands.

MATERIALS AND METHODS Samples

Panel A contained serum samples from 296 anonymously coded hemophilia patients (average age: 26 years; range, 2-73 years). All patients were treated with large-pool clotting factor concentrate. One hundred and eighteen (40%) patients had received small pool cryoprecipitate clotting factor in the past. Forty-seven (16%) patients had received clotting factor originating from non-Dutch (i.e., German, Austrian, or American) donors. The PCR test for HCV-RNA con- firmed HCV infection in 202 (68%) patients. Western blotting confirmed HIV-1 infection in 44 (15%) patients (all HCV-RNA positive).

Panel B contained 1,275 random serum samples of unpaid blood donors from different parts of the Netherlands.

Panel C. Panel C contained 187 serum samples of hepatitis patients, submitted to our laboratory for sero- logical testing of HEV antibodies between August

Assays Panels A, B, and C were screened for HEV antibodies

using a n enzyme immunoassay (EIA) which employs 2 recombinant HEV antigens for antibody detection, rep- resenting sequences from open reading frame 2 (ORF2) and open reading frame 3 (ORF3) of a Burmese HEV strain (Abbott Laboratories, Delkenheim, Germany). Repeat reactive samples were tested using 3 additional HEV antibody assays: 1) an anti-HEV EIA from Diag- nostic Biotechnology (DB, Singapore), which employs different recombinant (ORF3) HEV antigens from Bur- mese and Mexican HEV strains; 2) an experimental

Panel A.

Panel B.

1993-Aumst 1994.

Accepted for publication January 28,1995. Address reprint requests to H.L. Zaaijer, M.D., Central Labora-

tory of the Netherlands Red Cross Blood Transfusion Service- Viral Serology, Plesmanlaan 125,1066 CX Amsterdam, the Neth- erlands.

HEV Antibodies Among Donors and Patients

HEV antibody assay (Abbott Laboratories) based on 2 synthetic HEV peptides; and 3) an immunoblot assay with the recombinant HEV antigens from Abbott Labo- ratories. Repeat reactive samples were considered con- firmed if found positive by all 3 additional HEV anti- bodies assays.

Reactive patient samples were tested for the presence of IgM antibodies to HEV, using an EIA based on the DB HEV antigens. The IgM assay procedure included the removal of possible interfering IgG antibodies.

RESULTS Twenty-five of 296 (8%) patients with hemophilia

had HEV antibodies by the Abbott assay, but the ma- jority of these samples showed assay signals barely above the negativeipositive cutoff limit (average sam- ple-to-cutoff ratio, 1.48; range, 1.01-2.83). Four (1.4%) hemophilia patients were positive by the additional HEV antibody assays, thus fulfilling the “confirmed positive” criterion. All 4 patients were infected with HCV but not with HIV-1. Three of the 4 patients re- ceived only clotting factor originating from Dutch do- nors.

Fourteen of 1,275 (1.1%) donor samples were repeat- edly reactive by the Abbott assay, of which 5 (0.4%) were confirmed by the additional assays. These 5 do- nors did not have a history of jaundice. The difference in HEV seroprevalence between the hemophiliacs (4/ 296, 1.4%) and the donors (511,275, 0.4%), was not sig- nificant by Fisher’s exact test (P = 0.07).

Seven of 187 (4%) samples of patients with hepatitis had IgG and IgM HEV antibodies. It appeared that 6 patients (5 immigrants, 1 Dutch tourist) developed acute non-ABC hepatitis a few weeks after they arrived in the Netherlands; sexlagelorigin: Ml38lIndia; Mi381 Pakistan; Ml28iBangladesh; M/30/Bangladesh; MI281 Sudan; Fl35lIndia. The seventh sample belonged to a native Dutch patient (female, age 45 years) who had not travelled. She reacted strongly positive by both HEV EIAs, by the confirmatory assays, by the IgM assay based on HEV antigens from DB, and by an addi- tional IgM assay using the 2 recombinant HEV anti- gens from Abbott Laboratories.

The hepatitis patient who had not travelled lives and works in Amsterdam. During the 2 weeks before hospi- talization, she complained of malaise and loss of appe- tite. One day before admission to hospital, jaundice de- veloped (ALT: 1,670 Ull). During the 3 weeks in hospital, her condition improved slowly; the jaundice disappeared, and liver functions returned to normal (ALT: 13 Ull). Serological markers for hepatitis A, B, and C, and recent EBV and CMV infection, were nega- tive. The course of IgG and IgM HEV antibodies sug- gested recent HEV infection (Fig. 1). Approximately 3 weeks before the onset of the illness, she visited a res- taurant serving oysters, but she had fish for dinner.

DISCUSSION The cloning of part of the HEV genome in 1989 and

the subsequent characterization of the complete ge-

245

ALT (U/L) IgG, IgM (S /CO) 2000 I 17

malaise jaundice v -

r\

IgM anti-HEV \ 500 1 I \ ,i -1- J A ‘ 0 0 10 20 30 40 50 60 70 80 90

days

Fig. 1. Course of ALT and anti-HEV antibodies in a Dutch hepatitis patient (female, aged 45 years) who had not travelled. Normal ALT is <30 Uil. Anti-HEV is considered positive if sampleicutoff (SICO) is >l.

nome facilitated the development of assays for the de- tection of HEV antibodies [Reyes et al., 19901. Unfortu- nately, the interpretation of serological results is hampered by the lack of true confirmatory assays. The detection of HEV RNA by cDNA polymerase chain re- action (PCR) would confirm HEV infection elegantly, but a fresh-frozen sample from the early, viremic phase is usually not available [Jameel et al., 1992; Chauhan et al., 19931. The presence of HEV antibodies was stud- ied using several HEV antigens. To avoid false-positive test results, a sample was “confirmed positive” only if positive by 4 different assays. Nevertheless, the true nature of the “confirmed” presence of HEV antibodies in healthy persons is uncertain. Probably these persons experienced (subclinical) infection with HEV or with an HEV-related virus, but crossreactive antibodies causing false positive results cannot be excluded.

Six cases of imported hepatitis E were detected. On first sight, endemic hepatitis E in a nontropical region such as the Netherlands is unlikely, and the limited specificity of antibody assays might account for the finding of some HEV antibody reactivity among Dutch blood donors. However, an Amsterdam woman who had not travelled suffered from acute, resolving non-ABC hepatitis. She reacted strongly positive by all HEV an- tibody tests available, including 2 assays for presence of IgM anti-HEV. If HEV indeed exists in Western Eu- rope, the HEV antibody reactivity in some of the blood donors might reflect silent HEV infections in the past; and transmission of HEV by large-pool blood products would not be impossible. Fortunately, the prevalence of HEV antibody reactivity among hemophiliacs (1.4%) was low, albeit slightly higher than among donors (0.4%). Even if this difference was not caused by coinci- dence but by true transmission of HEV from donor to recipient, the risk that HEV poses to hemophiliacs is small.

246 Zaaijer et a].

Earlier, another case of apparent nontropical hepati- tis-E was reported in the Netherlands (male, age 20 years) [Zaaijer et al., 19931. The route of HEV transmis- sion both to this patient and to the patient in the present study remains unclear. Several possibilities may be considered: HEV infection might occur through contaminated food imported from HEV-endemic areas; an avirulent HEV strain might be present in Europe, normally causing only subclinical infections; and fi- nally, HEV could exist in an unknown animal reser- voir. Until more is known about the global distribution of HEV, it seems wise to consider hepatitis E in pa- tients suffering from acute non-ABC hepatitis, even in nontropical countries.

ACKNOWLEDGMENTS The authors thank Diagnostic Biotechnology/

Genelabs (Singapore), for providing reagents.

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