Original Articles Evaluation of Assays for Antibody to Hepatitis E Virus by a Serum Panel

HEPATOLOGY, March 1998, p. 857-861, Vol. 27, No. 3 Eric E. Mast1, Miriam J. Alter1, Paul V. Holland2, and Robert H. Purcell3 for the for the Hepatitis E Virus Antibody Serum Panel Evaluation Group From the 1 Hepatitis Branch, Centers for Disease Control and Prevention, Atlanta, GA; 2 Sacramento Medical Foundation, Sacramento, CA; and 3 Hepatitis Viruses Section, National Institutes of Health, Bethesda, MD ABSTRACT Few data are available to evaluate the performance of existing assays for antibody to the Hepatitis E virus (anti-HEV). A panel of 164 randomized and coded sera was tested for anti-HEV by 12 different assays. The panel included a dilution series of an early convalescent human serum, known-positive sera (undiluted human sera obtained 2 months to 13 years after acute Hepatitis E, and postinoculation chimpanzee sera), known-negative sera (preinoculation chimpanzee sera; sera from chimpanzees with Hepatitis A virus, heomain Hosting, Domain Registration, Registration, NSI, Network Solutions, InterNIC, Hosting Plans, Web Site Hosting, Project Management, Microsoft Project, Project, Project 98, Project 2000, Project Mentoring, LAN, WAN, Network, Local Area Network, NT, Windows NT, MS Small Business Server, Novell” INTRODUCTION The performance of tests for antibody to the Hepatitis E virus (anti-HEV) is an important factor in assessing the epidemiology of Hepatitis E virus (HEV) infection. The first serologic assays for anti-HEV included immune electron microscopy 1,2 and a fluorescent antibody blocking assay, 3,4 both of which utilize native HEV antigen as a target for detection. Although these assays are specific, they have limited sensitivity: anti-HEV has been detected in only 50% to 70% of patients with acute hepatitis during Hepatitis E outbreaks, and anti-HEV titers decline to subdetectable levels within several months after acute infection. After HEV was cloned and sequenced, a variety of tests, including both Western blot assays and enzyme immunoassays (EIAs), were developed to detect anti-HEV by using recombinant-expressed proteins or synthetic peptides that model immunodominant epitopes of the putative structural regions of HEV [open reading frames (ORFs) 2 and 3].5-16 Several recombinant protein-based tests have demonstrated increased sensitivity compared to prior assays, detecting anti-HEV in 90% to 95% of patients with acute hepatitis during outbreaks of Hepatitis E in HEV-endemic areas. 6,17 Recently, a number of cases of acute Hepatitis E, diagnosed on the basis of serologic testing, have been reported among persons who had no history of travel to HEV-endemic areas.18-23 However, the interpretation of these findings is problematic because few data are available to evaluate the performance of anti-HEV assays for the diagnosis of acute Hepatitis E in this setting. In addition, the performance of these assays in detecting anti-HEV in persons with remote infection is unknown, and several studies have reported unexplained positive anti-HEV results among persons who did not have disease or known exposure to HEV. 7,24-27 We present the findings of a serum panel evaluation conducted to assess the sensitivity and specificity of available tests for anti-HEV and to assess the variability in detecting anti-HEV among tests. MATERIALS AND METHODS A total of 164 sera were included in the panel. Known-positive sera included human sera obtained 2 months to 13 years after acute Hepatitis E (n = 5) and serial sera from chimpanzees inoculated with HEV strains from various geographic regions, including Mexico, Pakistan, and Uzbekistan (n = 24). In addition, the panel contained duplicate serial dilutions of an early-convalescent human serum (diluted with normal human serum; n = 20). Two groups of known-negative sera were included in the panel to evaluate specificity: preinoculation chimpanzee sera (n = 6) and sera from chimpanzees with Hepatitis A virus, Hepatitis B virus, or Hepatitis C virus infection (n = 8). In addition, sera in the dilution series that were consistently nonreactive were considered to be known-negative sera. To evaluate the variability between tests in detecting anti-HEV, the panel contained sera obtained from U.S. blood donors, all of whom denied a history of hepatitis. Sera from blood donors were previously tested by assays 1, 6, and 7, and the panel included sera reactive by at least two assays (n=17), sera with discordant results (n = 45), and nonreactive sera by at least two assays (n = 39). Among blood donors with reactive sera by at least two assays, 11 had a history of travel to HEV-endemic regions and two had no history of international travel (travel history was unknown for four persons). Among blood donors with discordant results, 27 had a history of travel to HEV-endemic regions and 11 had no history of international travel (travel history was unknown for 6 persons). None of the blood donors with nonreactive sera by at least two assays had a history of international travel (travel history was unknown for 1 person). Informed consent was obtained from each patient who provided sera for the panel. All animals received humane care according to the criteria outlined in Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 86-23, revised 1985). The panel was assembled, randomized, and coded collaboratively by the Hepatitis Viruses Section, National Institutes of Health, and the Hepatitis Branch, Centers for Disease Control and Prevention (CDC); the results were compiled at the CDC. All sera included in the panel were stored at 70°C. Chimpanzee sera and human sera from patients with Hepatitis E were thawed and refrozen an estimated 5 to 10 times prior to distribution. Sera from blood donors were thawed and refrozen 1 to 2 times prior to distribution. The same coded panel containing a 250-µL aliquot of each serum specimen was sent frozen on dry ice to 10 laboratories for anti-HEV testing. The panel was evaluated by 12 different tests (two different test methods were used by two laboratories): 10 were EIAs, and 2 were Western blot assays (table 1). The target for detection was a recombinant protein for 7 assays and synthetic peptides for 4 assays. One of the recombinant protein assays used an artificial mosaic protein consisting of a series of short linear antigenic epitopes. Five assays had epitopes derived only from HEV ORF2, two had epitopes derived only from ORF3, and 5 had epitopes derived from both ORF2 and ORF3. Of the recombinant protein assays, 3 were expressed in insect cells and 5 were expressed in Escherichia coli. Ten of the assays had epitopes derived from a Burmese (Myanmar) HEV strain,28 and 5 of these also had epitopes derived from a Mexican HEV strain.29 One assay had epitopes derived from a Pakistani HEV strain,30 and one from a Chinese strain.31   View This table table 1. Characteristics of 12 Tests to Detect Antibody to HEV Evaluated by the Serum Panel Assay results were determined according to the methods of each participating laboratory.5-15 For EIAs, each serum specimen was tested in duplicate; sera with discordant results were considered to be indeterminate. For Western blot assays, each serum specimen was tested once and a weakly reactive immunoblot result was considered to be indeterminate. In calculations of concordance between tests, sera with indeterminate results were excluded from analysis. RESULTS Six of the eight recombinant protein tests detected anti-HEV in 90% of the undiluted known-positive sera (table 2). A similar proportion of sera obtained less than 6 months and 6 months after onset of illness were anti-HEV reactive for most of the tests (table 2). When sera from the dilution series that were consistently reactive by at least one assay (dilution factor 1:1:60) were included in the analysis, the sensitivity of the 12 assays in detecting anti-HEV ranged from 17% to 100% (data not shown). In the dilution series of an early convalescent serum, the limit of anti-HEV detection by endpoint dilution for the 9 tests ranged from 1:5 to 1:160 (table 3). Two tests (3 and 9) demonstrated seroreactivity at dilutions higher than 1:160; however, there was not a consistent pattern of reactivity with increasing dilutions by these tests. Synthetic peptide-based tests were less sensitive compared to the recombinant protein-based tests, both among known-positive sera and in the dilution series (Tables 2 and 3). Nine tests were nonreactive for all of the known-negative sera (including sera with a dilution factor of >1:160 from the dilution series); however, at least one serum specimen was reactive or indeterminate by 3 of the tests. View This table table 2. Detection of Antibody to HEV in Known-Positive and Known-Negative Sera by 12 Different Tests

 

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table 3. Antibody to HEV Endpoint Dilution by 12 Different Tests in Early-Convalescent Human Sera Diluted With Normal Human Serum

Seven of the eight recombinant protein tests detected anti-HEV in 5 convalescent human sera obtained 2 months to 13 years following onset of acute Hepatitis E (data not shown). One recombinant protein test detected anti-HEV in sera obtained 2 months to 2 years after illness onset, but was indeterminate in serum obtained 13 years after illness onset. Only one of the four synthetic peptide tests detected anti-HEV in all four sera obtained 2 months to 2 years after illness onset, and none of the synthetic peptide tests detected anti-HEV in serum obtained 13 years after illness onset.

Six tests detected anti-HEV in more than 85% of serial chimpanzee sera after inoculation with HEV strains from various geographic regions (table 4). However, there was considerable variation in the pattern of anti-HEV detection by these assays, in sera collected both <6 months and 6 months after inoculation. Tests that included ORF3 epitopes from the Mexican HEV strain (tests 6, 7, 8, 11, and 12) did not detect anti-HEV in several chimpanzee sera after inoculation with this HEV strain (table 4).

 

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table 4. Detection of Anti-HEV by 12 Different Tests in Sera From Chimpanzees Inoculated With HEV Strains From Various Geographic Regions

The detection of anti-HEV in blood donor sera varied considerably among assays. The range of anti-HEV detection in 17 sera that were previously reactive by at least two tests was 6% to 100%, the range of detection in 45 sera with prior discordant results was 4% to 71%, and the range of detection in 39 sera with prior negative results by at least two tests was 0% to 31% (Fig. 1). In pairwise comparisons of different tests, the overall concordance in all blood donor sera ranged from 41% to 94% (median, 68%) and the concordance among reactive sera by either test ranged from 0% to 89% (median, 32%) (table 5). Only 23% (15/66) of the pairwise test comparisons had concordant results for >50% of reactive sera.

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Fig. 1. Antibody to HEV reactivity in blood donor sera with prior reactive results by  2 tests, prior discrepant results and prior negative results by  2 tests.

 

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table 5. Pairwise Test Comparisons of Concordant Detection of Antibody to HEV among U.S. Blood Donor Sera

DISCUSSION

Most currently available assays for anti-HEV were designed for the diagnosis of acute HEV infection acquired in HEV-endemic regions. The findings of this study indicate that several of the recombinant protein assays (tests 1, 2, 4, 5, and 7) have an adequate combination of sensitivity and specificity to perform well for this purpose (table 6). The peptide-based assays were generally much less sensitive compared to the recombinant protein assays and are therefore likely to be nonreactive in a high proportion of acute Hepatitis E cases. Further comparative studies that include testing for immunoglobulin M anti-HEV would be useful to validate the performance of the recombinant protein assays for the diagnosis of acute Hepatitis E. In addition, the performance of these assays for the diagnosis of acute Hepatitis E in persons who do not have a history of travel to HEV-endemic regions needs to be determined.

 

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table 6. Overall Performance of 12 Tests for Antibody to HEV

In prior studies, HEV isolates from various geographic regions have been demonstrated to have at least one major cross-reactive epitope by a variety of serologic assays. ^5,10,32,33 However, we found substantial variation in the detection of anti-HEV by these tests in acute and early convalescent-phase sera from chimpanzees infected with HEV isolates from various geographic regions. One possible reason for these findings is differences in the geographic strain-specific antigenic domains included in these tests. However, there is little variation in the RNA sequence of ORF2 among HEV isolates from various geographic regions. Moreover, some assays (tests 6, 7, 8, and 12) did not detect anti-HEV in chimpanzee sera even though these tests included ORF3 epitopes from the same geographic region as the chimpanzee inoculum. The seroreactivity of recombinant proteins may also vary if they are produced in different expression systems or used in different test formats (i.e., Western blot vs. EIA). In addition, all of these assays were designed to detect human antibody, and differences may exist in the ability of assay conjugates to detect chimpanzee antibody. However, if the assay conjugate were the reason for a test’s not detecting anti-HEV, the assay would be expected either to be nonreactive in all the chimpanzee sera or to have a uniform decline in seroreactivity in chimpanzee sera compared to human sera. None of the assays that consistently detected anti-HEV in human sera exhibited either of these patterns of seroreactivity in chimpanzee sera.

Anti-HEV assays used in seroprevalence studies must have a high level of sensitivity in detecting remote infection. Several tests detected anti-HEV in >90% of chimpanzee and human sera obtained 6 months to 2 years after infection, and all but one of the recombinant protein assays detected anti-HEV in a human serum obtained 13 years after infection. However, our ability to assess the sensitivity of these assays in detecting remote infection was limited because only a small number of such sera were included in the panel. Further studies are needed to determine the ability of these tests to detect anti-HEV in patients with remote infection.

Anti-HEV tests used in seroprevalence studies must have a high level of specificity; this is particularly important to prevent false-positive tests in populations with a low prevalence of infection. Most of these assays did not detect anti-HEV in any of the 22 known-negative sera in the panel. However, among the 101 selected blood donor sera included in the panel, anti-HEV seroreactivity was highly variable, and the concordance in detection of anti-HEV between tests was low. Possible reasons for these findings include nonspecific reactivity and differences in the sensitivity of these assays in detecting antibodies to different HEV strains and in detecting remote infection.

Seroprevalence studies among blood donors in some non-HEV-endemic countries have found an anti-HEV prevalence of 1% to 20%, which is relatively high compared to the low rate of clinically evident disease associated with HEV in these areas. ^7,24-27 In one study, anti-HEV seroreactivity among persons living in nonendemic regions increased with increasing age and was associated with travel to endemic regions, findings that are consistent with prior HEV infection.²⁴ However, in another study, there was no evidence that anti-HEV seroreactivity was related to subclinical infection in high risk populations for Hepatitis A virus, Hepatitis B virus, and Hepatitis C virus infections.²⁷ Thus, the interpretation of seroreactivity among persons living in nonendemic regions is currently problematic. Moreover, the discrepant results among blood donor sera in this study indicate that anti-HEV seroprevalence data in nonendemic countries may be unreliable and should be interpreted with caution. Further studies are needed to determine reasons for the highly discrepant results among blood donor sera. Studies are also needed to determine the significance of anti-HEV seroreactivity among persons living in non-HEV-endemic areas, including the relation of seroreactivity to exposure to the recently discovered virus in pigs that is closely related to human HEV isolates.³⁴ In addition, improved tests are needed for use in seroprevalence studies in nonendemic regions and confirmation tests are needed to verify the specificity of these assays.

References

Footnotes

Acknowledgement: The authors thank Stephen Lambert, Karen McCaustland, John Spelbring, and Doris Wong for assistance in constructing the coded serum panel.

Abbreviations: anti-HEV, antibody to Hepatitis E virus; HEV, Hepatitis E virus; EIA, enzyme immunoassay; ORF, open reading frame.

The Hepatitis E Virus Antibody Serum Panel Evaluation Group included H. J. Alter, National Institutes of Health, Bethesda, MD; D. Anderson, McFarlane Burnet Centre for Medical Research, Fairfield, Victoria, Australia; M. S. Balayan, Institute of Poliomyelitis and Viral Encephalitis, Moscow, Russia; A. N. Burkov, NPO-Diagnostic Systems, Nizhny Novgorod, Russia; L. Chan, Genelabs Diagnostics, Singapore; P. Coursaget, Laboratoire d’Immunologie des Maladies Infectieuses, Tours, France; M. O. Favorov, Centers for Disease Control and Prevention, Atlanta, GA; K. Krawczynski, Centers for Disease Control and Prevention, Atlanta, GA; I. K. Mushahwar, Abbott Laboratories, North Chicago, IL; S. K. Panda, All India Institute of Medical Sciences, New Delhi, India; J. Pillot, Institute Pasteur, Paris, France; S. A. Tsarev, National Institutes of Health, Bethesda, MD; T. Uchida, Nihon University School of Medicine, Tokyo, Japan; and P. O. Yarbough, Genelabs Technologies, Inc., Redwood City, CA.

Received October 28, 1996; accepted November 5, 1997.

Address reprint requests to: Eric E. Mast, M.D., M.P.H., Hepatitis Branch, Mailstop G37, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30333. Fax: (404) 639-1538.

Copyright © 1998 by the American Association for the Study of Liver Diseases.