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Seroepidemiology of hepatitis E on Japanese expatriates in Southeast Asian countries: a study at a clinic in Singapore
Hepatology Research 9 (1997) 93 – 102
Seroepidemiology of hepatitis E on Japanese expatriates in Southeast Asian countries: a study at a clinic in Singapore Mami Hirota Shields b, Hideki Yamamoto a, Satoru Ikeda a, Nirmal Rimal a, Kazuhisa Taketa a,* a
Department of Public Health, Okayama Uni6ersity Medical School, 2 -5 -1 Shikata-cho, Okayama 700, Japan b Di6ision of Health Ser6ices, Office of Public Health, 325 Loyola A6enue, New Orleans, LA 70112, USA Received 4 August 1997; received in revised form 5 September 1997; accepted 8 September 1997
Abstract A two-phase study was conducted in order to examine anti-hepatitis E virus (HEV) antibody prevalence among Japanese expatriates in Southeast Asian countries at a clinic in Singapore serving Japanese expatriates. In phase I of the study, we identified a 20 month series of adult patients with abnormal liver function tests on routine medical examination or diagnostic evaluation. Serum was collected from the 179 patients from January 1993 through August 1994 and was analyzed for IgG and IgM anti-HEV antibodies with HEV-ELISA kits from Genelabs Diagnostics (GD). Out of 179 (13.4%) patients, 24 were positive for IgG anti-HEV. An IgM anti-HEV ELISA kit (GD) was available for the last 142 patients and five of these (3.5%) were positive for IgM anti-HEV and one case was positive for both. In phase II of the study, we identified a series of 168 adult patients who visited the clinic for routine medical check-up from January to April 1995 and for whom serum was collected and analyzed for liver function tests and IgG anti-HEV. Fifteen (8.9%) were positive for IgG anti-HEV. Thirty-seven (22%) of the 168 had abnormal results of liver function tests. However, abnormal liver function tests were not associated with anti-HEV IgG positivity. Anti-HAV measured in the subjects in phase II of the study showed no significant * Corresponding author. Tel: + 81 862 357179; fax: + 81 862 260715; e-mail: [email protected] 1386-6346/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 1 3 8 6 - 6 3 4 6 ( 9 7 ) 0 0 0 8 3 - 1
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association with anti-HEV seropositivities.The prevalence of HEV infection observed in this study was much higher than reported in Japan and also higher than the Singapore host population (3.4%). © 1997 Elsevier Science Ireland Ltd. Keywords: Hepatitis E; IgG anti-HEV antibodies; Japanese expatriates; Southeast Asia
1. Introduction Hepatitis E virus (HEV) has been a major etiological agent of epidemic and sporadic, ‘non-A non-B’ viral hepatitis in developing countries [1–4]. In recent years, a considerable number of patients with hepatitis E have also been reported in developed countries [5 – 7], particularly among those who had travelled in tropical or subtropical countries. There have been some cases of hepatitis E reported in Japan [8 – 11], including a case without history of travel abroad [11]. We documented [12] a considerably high seroprevalence of hepatitis E among Japanese people living in Southeast Asian countries and visiting a clinic in Singapore as compared with that in the domestic populations of Japan or Singapore. This study was first conducted on a series of all-adult subjects with abnormal results of liver function tests, with or without symptoms related to acute hepatitis (phase I study). This was extended in phase II of the study to a series of asymptomatic adults only, visiting the clinic for routine examination irrespective of the results of liver function tests. In this paper, we report results of the phase I and II studies on seroprevalence of hepatitis E among the Japanese population in Southeast Asian countries served by this clinic. The data were analyzed with respect to age, sex, countries of residence and duration of residence, clinical status (phase I) and liver function tests (phase II). We confirmed the high prevalence of IgG anti-HEV even among apparently healthy individuals visiting this clinic.
2. Patients and methods
2.1. Population demographics Subjects who presented with symptoms consistent with acute hepatitis were classified as ‘symptomatic’ and subjects who presented for routine annual or biennial examination as ‘asymptomatic’. Demographic characteristics of the studied populations are shown in Table 1. Except for ‘symptomatic’ clients in phase I, clients eligible for the study were restricted to employees of certain companies whose health care arrangements with the clinic included periodic routine assessment of liver function tests as part of the regular examination. This selection bias did not appear to correlate with known risk factors for seropositivity.
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Table 1 Demographic characteristics of the study population Phase
Age group (%) 20–29 years
I: A I: S II: A II: H
12 5 3 13
(9.3) (10.0) (8.1) (9.9)
Total
33 (9.5)
30–39 years 68 25 15 74
(52.7) (50.0) (40.5) (56.5)
40 – 49 years 39 15 13 41
50 years
(30.2) (30.0) (35.1) (31.3)
182 (52.4)
108 (31.1)
Male
Total
116 45 33 64
129 50 37 131
10 5 6 3
Total (7.8) (10.0) (16.2) (2.3)
24 (6.9)
129 50 37 131
(100) (100) (100) (100)
347 (100)
Gender (%) Female I:A I: S II: A II: H
13 5 4 67
(10.1) (10.0) (10.8) (51.1)
Total
89 (25.6)
(89.9) (90.0) (89.2) (48.9)
258 (74.4)
(100) (100) (100) (100)
347 (100)
Country of Residence (%) Singapore I:A I: S II: A II: H Total
79 40 29 52
(61.2) (80.0) (47.5) (48.6)
200 (57.6)
Malaysia 11 3 11 19
(8.5) (6.0) (18.0) (17.8)
44 (12.7)
Indonesia 29 5 11 25
(22.5) (10.0) (18.0) (23.4)
70 (20.2)
Other
Total 10 2 10 11
(7.8) (4.0) (16.4) (10.3)
33 (9.5)
129 50 61 107
(100) (100) (100) (100)
347 (100)
Duration of residence (%) B1 year I:A I: S II: A II: H
14 4 10 23
(10.9) (8.0) (16.4) (21.5)
Total
51 (14.7)
1–1.9 years 19 7 19 22
(14.7) (14.0) (31.1) (20.6)
67 (19.3)
2 – 3.9 years 39 5 20 40
(30.2) (10.0) (32.8) (37.4)
104 (30.0)
4-years 24 3 12 20
Unknown (18.6) (6.0) (19.7) (18.7)
59 (17.0)
33 31 0 2
(25.6) (62.0) (0.0) (1.9)
66 (19.0)
Total 129 50 61 107
(100) (100) (100) (100)
347 (100)
A, asymptomatic; S, symptomatic; H, apparently healthy.
2.2. Phase I study We examined the sera of 179 adult patients (aged 20 and above) from January 1993 to August 1994, who visited Japan Green Clinic, Singapore, which was established to serve only Japanese residents in Singapore and nearby Southeast
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Asian countries. The patients selected were a de-duplicated series of those who had abnormal results of liver function tests on routine examination (aspartate aminotransferase (AST)\ 40 IU/l, or alanine aminotransferase (ALT)\ 40 IU/l, or g-glutamyltransferase (GGT)\ 85 IU/l) at the routine medical check-up or who were clinically suspected to have acute hepatitis and were subsequently shown to have abnormal liver function test results. IgG class anti-HEV antibodies were analyzed with a HEV-ELISA kit (Genelabs Diagnostics (GD), Singapore), using structure proteins (ORF2 and ORF3) of HEV genome as the antigens and were abbreviated as IgG anti-HEV antibodies (GD). Of the 179 liver dysfunction cases, the last 142 were also examined for IgM anti-HEV using the IgM anti-HEV ELISA kit (GD). Intermediate-positive levels of color production in the IgG anti-HEV (GD) assay were confirmed to be positive by absorption testing with the antigen. The 37 subjects whose sera were tested before the IgM anti-HEV kit became available were examined for acute hepatitis E with a HEV-ELISA system developed by Kaketsuken (The Chemo-Sero-Therapeutic Research Institute, Kumamoto, Japan), using the HT-3 protein as the antigen for detection of IgG antibodies to HEV, which specifically appear in the acute phase of hepatitis E. The antibodies tested by this system were designated acute-phase IgG anti-HEV (KK) in order to differentiate them from the IgG anti-HEV (GD).
2.3. Phase II study We examined the sera of all the patients who visited Japan Green Clinic, Singapore for a routine medical check-up from January to April 1995. The number of cases studied was 168, out of which 37 cases (22%) showed abnormal results of liver function tests (AST\40 IU/l, ALT\ 40 IU/l or GGT\ 85 IU/l). This group of 37 clinically ‘asymptomatic’ subjects with abnormal liver function tests corresponds to the stratum of clinically ‘asymptomatic’ subjects from phase I with abnormal liver function tests. The sera were analyzed with the HEV-ELISA kit (GD) for IgG antibodies to HEV.
2.4. Other hepatitis 6irus markers Sera of patients with suspected acute hepatitis were also analyzed for IgM anti-HAV with an EIA kit, IMx HAVAB Assay System (Dainabot) and for HBsAg, HBeAg, HBeAb and HBcAb as routine laboratory tests. All the subjects in the phase II study were assayed for IgG HAVAB with an EIA kit, IMx HAVAB Assay System (Dainabot).
2.5. Population representation Although the candidates for inclusion in the ‘asymptomatic’ group of this study were limited to those companies who contracted for routine periodic examination including liver function tests, we had no basis to suspect that this group had a different risk profile than employees of other companies. Also, the age and
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geographic distribution of the population under study was not representative of all Japanese expatriates. However, adjustments to the estimates of general HEV seropositivity can be made using the age, country of residence and duration of residence strata-specific rates and applying them to different population proportions as one does with simple age adjusted rates. Nontheless, an accurate demographic profile of the full population of Japanese expatriates in this region would be required.
2.6. Statistical tools Statistical analyses were carried out with EpiInfo Version 6 (CDC, WHO), Health Information Retrieval System (HIRS) Version 2 (CDC) and Microsoft Excel Version 5.0.
3. Results
3.1. Phase I study The results of the phase I study are summarized in Table 2. Twenty-four cases (13.4%) out of 179 subjects with abnormal results of liver function tests were positive for IgG anti-HEV (GD). The anti-HEV IgG-seropositive prevalence by gender was 13.7% (22/161) in males and 11.1% (2/18) in females (not listed in Table 2). None of the first 37 sera in the series which were examined for the acute-phase IgG anti-HEV (KK) showed a positive result. However, among the last 142 subjects of this series examined for IgM anti-HEV, five cases showed a positive result, one of which was positive for both IgG and IgM anti-HEV antibodies (GD). Thus, acute hepatitis E accounted for five (3.5%) of the last 142 cases with liver dysfunction. After complete evaluation using respective virus markers and ultrasonography, the remaining 179 abnormal liver function test results were attributed to the following liver diseases, including suspected diagnoses: seven acute hepatitis A, five acute hepatitis B, three chronic hepatitis B, 47 fatty liver, 30 alcoholic liver injury and undiagnosed or other liver diseases for the remaining 82. These results showed that only 3.5% of the observed liver dysfunction could be explained by acute hepatitis E and that among those with abnormal liver function tests without symptoms, positive anti-HEV IgG antibodies had been acquired in the past in 12.8%. Accordingly, we conducted the phase II study in an attempt to measure the seroprevalence of IgG anti-HEV in the Japanese population as a whole, visiting the clinic irrespective of the results of liver function tests.
3.2. Phase II study The results of the phase II study are summarized in Table 3. Out of the 168 subjects, 37 (22.0%) had abnormal results of liver function tests (AST\ 40 IU/l or
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ALT \ 40 IU/l or GGT \ 85 IU/l), of whom 8.1% (3/37) were positive for anti-HEV IgG. Among the remaining 131 cases (78.0%) with normal liver function tests, 9.2% (12/131) were positive for anti-HEV IgG. In all, 15 cases (8.9%) out of 168 were positive for IgG anti-HEV. The prevalence of positive IgG anti-HEV in the ‘normal liver function test results’ group was 9.2% (12/131) and in the ‘asymptomatic abnormal liver function test results’ group was 8.1%, without a statistically significant difference. The results of the phase II study were evaluated for anti-HEV seropositivity with respect to the differences in age, gender, residence area and duration of residence (the results are summarized in Table 4). No statistically significant difference in IgG anti-HEV positivity was seen between males and females, or among countries of residence or duration of residence, although the odds ratio of males to females was 2.14 (95% CI: 0.60, 9.60).
Table 2 Prevalence of IgG anti-HEV (GD), IgM anti-HEV (GD) and acute phase IgG anti-HEV (KK) in the phase I study Factors
Total 179 tested Symptomatic Asymptomatic Total (%) Last 142 tested Symptomatic Asymptomatic Total (%)
IgG anti-HEV antibodies(GD)
IgM anti-HEV antibodies (GD)
Positive
Negative
Total
Positive
Negative
Total
6 18
44 111
50 129
24 (13.4)
155 (86.6)
179 (100) 34 108
3 2
31 106
34 108
142 (100)
5 (3.5)
137 (96.5)
142 (100)
1 4
15 122
16 126
5 (3.5)
137 (96.5)
142 (100)
5 11 16 (11.3)
29 97 126 (88.7)
IgG anti-HEV Positive Negative Total (%)
First 37 tested Symptomatic Asymptomatic Total (%)
IgG anti-HEV antibodies (GD)
Acute-phase IgG anti-HEV (KK)
Positive
Positive
Negative
Total
Negative
Total
1 7
15 14
16 21
0 0
16 21
16 21
8 (21.6)
29 (78.4)
37 (100)
0 (0)
37 (100)
37 (100)
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Table 3 Number of IgG anti-HEV (GD) positive and -negative cases in relation to ‘normal’ and ‘abnormal’ liver function test groups in phase II Liver function tests
IgG anti-HEV antibodies Positive (%)
Negative (%)
Total (%)
Abnormal Normal
3 (8.1) 12 (9.2)
34 (91.9) 119 (90.8)
37 (100) 131 (100)
Total
15 (8.9)
153 (91.1)
168 (100)
Analysis of the association between the positivities of anti-HAV and anti-HEV antibodies for 168 cases in the phase II study revealed that IgG HEV antibodies were positive in 5% (1/20) in the HAV antibody-positive group and 9.5% (14/148) in the HAV antibody-negative group. The odds ratio was 0.5 (95% CI: 0.01, 3.71) and there was insufficient evidence of significant negative association between the HAV and HEV infections.
4. Discussion The significant results of the present study were that the prevalence of HEV infection among Japanese residing in Southeast Asian countries is exceedingly high as compared with that in the domestic Japanese population, which is estimated to be 0.05% (personal communication, Atsushi Takagi, Dainabot, Japan). The observed prevalence of IgG anti-HEV positivity is apparently higher than that of the native population of Singapore, which is reported to be 3.4% [13]. The results could be interpreted as indicating that Japanese expatriates are likely to be infected with HEV while they stay abroad and probably while they are travelling in Southeast Asian countries. The possibility that the high prevalence of IgG anti-HEV results from a high false-positive rate for the detection kit has been raised for the Abbott anti-HEV test kit [14,15] (not used in this study). However, this false-positive rate concern was addressed in part by observing that cases in this study with intermediate levels of color production in ELISA (possibly ‘borderline’ positive or false positive) were confirmed to be positive by absorption test with the antigen. There was no significant difference in IgG anti-HEV prevalence between ‘normal’ and ‘abnormal’ liver function test groups. Therefore, the observed abnormality in the test results at the time of routine examination is not likely to be related to prior HEV infection and in only a few cases would it be associated with current, asymptomatic HEV infection. In fact, most of the hepatic dysfunctions were accounted for by other hepatitis strains or by or fatty liver.
6 2 6 1 4 4 4 3 0
Country Singapore Malaysia Indonesia Others Duration B1 year 1–1.9 years 2–3.9 years 45years Unknown
a
M-H, Mantel Haentzel test.
15
11 4
Gender Male Female
Total
0 6 8 1
Positive
153
29 37 56 29 2
75 28 30 20
86 67
16 83 46 8
Negative
IgG anti-HEV antibodies
Age B30 years 30–39 years 40–49 years 505years
Factors
168
33 41 60 32 2
81 30 36 21
97 71
16 89 54 9
Total
8.9
12.1 9.8 6.7 9.4 0.0
7.4 6.7 16.7 4.8
11.3 5.6
0.0 6.7 14.8 11.1
Prevalence (%)
x 2:1.03; (3 d.f., P= 0.91)
x 2:3.52; (3 d.f., P= 0.32)
Odds ratio = 2.14; (95%CI:0.60, 9.60); x 2:1.64 (M-H)a; P= 0.20
x 2: 4.45; (3 d.f., P= 0.22)
Statistical analysis
Table 4 Prevalence of IgG anti-H EV (GD) at their first visit by age, gender, country of residence and duration of residence in the population of Phase II
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Since anti-HEV IgG positive subjects rarely had a history of any particular symptoms related to acute hepatitis, the acute hepatitis E in this study was subclinical or mild. Although most of the infected subjects were asymptomatic in the study population, a fulminant course of hepatitis E has been reported for pregnant women [1 – 3], therefore, precautions should be taken for pregnant women residing in those countries. The age distribution of anti-HEV IgG among Japanese population in this study was different from that generally reported, in that the highest attack rate has been observed among young adults [16], while the present study revealed a low prevalence of IgG anti-HEV in the 30 –39 years age group, the tendency increasing with age. A significantly higher prevalence of IgG anti-HEV in the 46–55 years age group was reported for healthy individuals in Southern Taiwan [4]. This would be accounted for by a cumulative effect of IgG anti-HEV positive individuals, assuming that antibodies persist over at least two years [17,18], although no significant difference was found in relation to the duration of residence in this study. No significant association of IgG anti-HEV with IgG anti-HAV was shown in this study, despite both of them being enterically transmitted. Tan et al. [19] reported a similarly low superinfection rate of HEV in acute hepatitis A. Instead, an extraordinarily high superinfection rate of HEV with HCV is noted, despite different infection routes [19], this being supported by the report of Thomas et al. [20], although possible parenteral transmission of HEV has been reported from different sources [21,22]. Considering the fact that those who travel or have lived overseas might transmit the disease into their own countries, HEV assay kits should be available not only in tropical countries but also in the northern countries including Japan. It is also desirable, especially for the benefit of pregnant women who would develop severe hepatitis [1 – 3], to establish a surveillance system on HEV transmission for community practices [23].
Acknowledgements We appreciate the cooperation of Japan Green Clinic, Singapore and the active support of this study by Dr Ginnohyoe Suhara, the Center for Adult Diseases, Kurashiki, Japan and also to Dr Tetsuji Rikihisa, for analysis of IgG class anti-HEV appearing in the acute phase of hepatitis E.
References [1] Shrestha SM, Enteric non-A, non-B hepatitis in Nepal: clinical and epidemiological observations. In: Shikata T, Purcell RH, Uchida T, editors. Viral Hepatitis C, D and E. Amsterdam: Excerpta Medica, 1991:265–275. [2] Khuroo MS. Study of an epidemic of non-A, non-B hepatitis: possibility of another human hepatitis virus distinct from post-transfusion non-A, non-B type. Am J Med 1980;78:818 – 24.
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[3] Public Health Service, Center for Disease Control. Enterically transmitted non-A non-B hepatitis — East Asia. MMWR 1987;36:241–244. [4] Peng C-F, Lin M-R, Chue P-Y, Tsai JF, Shih CH, Chen IL, He J, Carl M. Prevalence of antibody to hepatitis E virus among healthy individuals in Southern Taiwan. Microbiol Immunol 1995;39:733–6. [5] Zaaijer HL, Kok M, Lelie PN, Timmerman RJ, Chau K, Van der Pal HJ. Hepatitis E in the Netherlands: imported and endemic. Lancet 1993;341:826. [6] Skidmore SJ, Yarbough PO, Gabor KA, Tam AW, Reyes GR, Flower AJ. Imported hepatitis E in UK. Lancet 1991;337:1541. [7] Public Health Service, Center for Disease Control. Hepatitis E among US travelers, 1989 – 1992. MMWR 1993;42:1–4. [8] Hino K, Kondo T, Niwa H, Uchida T, Shikata T, Rikahisa T, Mizuno K. A small epidemic of enterically transmitted non-A, non-B acute hepatitis. Gastroenterol Jpn 1991;26:139 – 41. [9] Fujiyoshi M, Shoji S, Kondo T, Takikawa H, Miyake K, Yamanaka M. A case of acute hepatitis type E with cholestasis complicated with asymptomatic Endolimax nana infection. Jpn J Gastroenterol 1992;89:2804–7. [10] Kaku H, Hino K, Sainokami S, Shimoda K, Niwa H, Yasuda K, Iino S. Clinical studies of hepatitis E. Jpn J Hepatol 1993;34(1):330. [11] Ando B, Koyanagi T, Sakai K, Yatsuhashi H, Koga M, Yano M. A case of domestically infected acute sporadic hepatitis E demonstrated by enzyme-linked immunoadsorbent assay (ELISA). Jpn J Hepatol 1994;35:560–5. [12] Yamamoto H, Hirota M, Taketa K, Rimal N, Liu M, Ikeda S. Anti-hepatitis E virus antibody prevalence in Japanese residents abroad visiting a clinic in Singapore and having abnormal results of liver function tests. Jpn J Hepatol 1995;36:111 – 2. [13] Oon C-J. Hepatitis E virus infection in Singapore. Epidemiol News Bull 1992;18:45 – 6. [14] Uchida T. Molecular study of hepatitis E virus. Nippon Rinsho (Jpn J Clin Med, Osaka) 1995;53:901–5. [15] Nishiyama Y, Kaku K, Hino K. Clinical medicine of hepatitis E. Nippon Rinsho (Jpn J Clin Med, Osaka) 1995;53:906–10. [16] Zhuang H. Epidemiological and clinical features of enterically transmitted non-A, non-B hepatitis. Kan-Tan-Sui 1990;20:83–93. [17] Ke W-M, Tan D, Li J-G, Izumi S, Shinji Y, Hotta H, Yao JL. Consecutive evaluation of immunoglobulin M and G antibodies against hepatitis E virus. J Gastroenterol 1996;31:818 – 22. [18] Lee SD, Wang YJ, Lu RH, Chan CY, Lo KJ, Moeckli R. Seroprevalence of antibody to hepatitis E virus in Chinese subjects in Taiwan. Hepatology 1994;19:866 – 70. [19] Tan D, Im SWK, Yao JL, Ng MH. Acute sporadic hepatitis E virus infection in Southern China. J Hepatol 1995;23:239–45. [20] Thomas DL, Mahley RW, Badur S, Palaoglu KE, Quinn TC. Epidemiology of hepatitis E virus infection in Turkey. Lancet 1993;341:1561 – 2. [21] Chauhan A, Jameel S, Dilawari JB, Chawla YK, Kaur U, Ganguly NK. Hepatitis E virus transmission to a volunteer. Lancet 1993;341:149 – 50. [22] Buti M, Jardi R, Cotrina M, Rodriguez-Frias F, Troonen H, Viladomiu L, Esteban JI, Esteban R, Guardia J. Hepatitis E virus infection in acute hepatitis in Spain. J Virol Methods 1995;55:49 – 54. [23] Singh J, Aggarwal NR, Bhattacharjee J, Prakash C, Bora D, Jain DC, Sharma RS, Datta KK. An outbreak of viral hepatitis E: role of community practices. J Com Dis 1995;27:92 – 6.
.
Seroepidemiology of hepatitis E on Japanese expatriates in Southeast Asian countries: a study at a clinic in Singapore Mami Hirota Shields b, Hideki Yamamoto a, Satoru Ikeda a, Nirmal Rimal a, Kazuhisa Taketa a,* a
Department of Public Health, Okayama Uni6ersity Medical School, 2 -5 -1 Shikata-cho, Okayama 700, Japan b Di6ision of Health Ser6ices, Office of Public Health, 325 Loyola A6enue, New Orleans, LA 70112, USA Received 4 August 1997; received in revised form 5 September 1997; accepted 8 September 1997
Abstract A two-phase study was conducted in order to examine anti-hepatitis E virus (HEV) antibody prevalence among Japanese expatriates in Southeast Asian countries at a clinic in Singapore serving Japanese expatriates. In phase I of the study, we identified a 20 month series of adult patients with abnormal liver function tests on routine medical examination or diagnostic evaluation. Serum was collected from the 179 patients from January 1993 through August 1994 and was analyzed for IgG and IgM anti-HEV antibodies with HEV-ELISA kits from Genelabs Diagnostics (GD). Out of 179 (13.4%) patients, 24 were positive for IgG anti-HEV. An IgM anti-HEV ELISA kit (GD) was available for the last 142 patients and five of these (3.5%) were positive for IgM anti-HEV and one case was positive for both. In phase II of the study, we identified a series of 168 adult patients who visited the clinic for routine medical check-up from January to April 1995 and for whom serum was collected and analyzed for liver function tests and IgG anti-HEV. Fifteen (8.9%) were positive for IgG anti-HEV. Thirty-seven (22%) of the 168 had abnormal results of liver function tests. However, abnormal liver function tests were not associated with anti-HEV IgG positivity. Anti-HAV measured in the subjects in phase II of the study showed no significant * Corresponding author. Tel: + 81 862 357179; fax: + 81 862 260715; e-mail: [email protected] 1386-6346/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 1 3 8 6 - 6 3 4 6 ( 9 7 ) 0 0 0 8 3 - 1
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association with anti-HEV seropositivities.The prevalence of HEV infection observed in this study was much higher than reported in Japan and also higher than the Singapore host population (3.4%). © 1997 Elsevier Science Ireland Ltd. Keywords: Hepatitis E; IgG anti-HEV antibodies; Japanese expatriates; Southeast Asia
1. Introduction Hepatitis E virus (HEV) has been a major etiological agent of epidemic and sporadic, ‘non-A non-B’ viral hepatitis in developing countries [1–4]. In recent years, a considerable number of patients with hepatitis E have also been reported in developed countries [5 – 7], particularly among those who had travelled in tropical or subtropical countries. There have been some cases of hepatitis E reported in Japan [8 – 11], including a case without history of travel abroad [11]. We documented [12] a considerably high seroprevalence of hepatitis E among Japanese people living in Southeast Asian countries and visiting a clinic in Singapore as compared with that in the domestic populations of Japan or Singapore. This study was first conducted on a series of all-adult subjects with abnormal results of liver function tests, with or without symptoms related to acute hepatitis (phase I study). This was extended in phase II of the study to a series of asymptomatic adults only, visiting the clinic for routine examination irrespective of the results of liver function tests. In this paper, we report results of the phase I and II studies on seroprevalence of hepatitis E among the Japanese population in Southeast Asian countries served by this clinic. The data were analyzed with respect to age, sex, countries of residence and duration of residence, clinical status (phase I) and liver function tests (phase II). We confirmed the high prevalence of IgG anti-HEV even among apparently healthy individuals visiting this clinic.
2. Patients and methods
2.1. Population demographics Subjects who presented with symptoms consistent with acute hepatitis were classified as ‘symptomatic’ and subjects who presented for routine annual or biennial examination as ‘asymptomatic’. Demographic characteristics of the studied populations are shown in Table 1. Except for ‘symptomatic’ clients in phase I, clients eligible for the study were restricted to employees of certain companies whose health care arrangements with the clinic included periodic routine assessment of liver function tests as part of the regular examination. This selection bias did not appear to correlate with known risk factors for seropositivity.
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Table 1 Demographic characteristics of the study population Phase
Age group (%) 20–29 years
I: A I: S II: A II: H
12 5 3 13
(9.3) (10.0) (8.1) (9.9)
Total
33 (9.5)
30–39 years 68 25 15 74
(52.7) (50.0) (40.5) (56.5)
40 – 49 years 39 15 13 41
50 years
(30.2) (30.0) (35.1) (31.3)
182 (52.4)
108 (31.1)
Male
Total
116 45 33 64
129 50 37 131
10 5 6 3
Total (7.8) (10.0) (16.2) (2.3)
24 (6.9)
129 50 37 131
(100) (100) (100) (100)
347 (100)
Gender (%) Female I:A I: S II: A II: H
13 5 4 67
(10.1) (10.0) (10.8) (51.1)
Total
89 (25.6)
(89.9) (90.0) (89.2) (48.9)
258 (74.4)
(100) (100) (100) (100)
347 (100)
Country of Residence (%) Singapore I:A I: S II: A II: H Total
79 40 29 52
(61.2) (80.0) (47.5) (48.6)
200 (57.6)
Malaysia 11 3 11 19
(8.5) (6.0) (18.0) (17.8)
44 (12.7)
Indonesia 29 5 11 25
(22.5) (10.0) (18.0) (23.4)
70 (20.2)
Other
Total 10 2 10 11
(7.8) (4.0) (16.4) (10.3)
33 (9.5)
129 50 61 107
(100) (100) (100) (100)
347 (100)
Duration of residence (%) B1 year I:A I: S II: A II: H
14 4 10 23
(10.9) (8.0) (16.4) (21.5)
Total
51 (14.7)
1–1.9 years 19 7 19 22
(14.7) (14.0) (31.1) (20.6)
67 (19.3)
2 – 3.9 years 39 5 20 40
(30.2) (10.0) (32.8) (37.4)
104 (30.0)
4-years 24 3 12 20
Unknown (18.6) (6.0) (19.7) (18.7)
59 (17.0)
33 31 0 2
(25.6) (62.0) (0.0) (1.9)
66 (19.0)
Total 129 50 61 107
(100) (100) (100) (100)
347 (100)
A, asymptomatic; S, symptomatic; H, apparently healthy.
2.2. Phase I study We examined the sera of 179 adult patients (aged 20 and above) from January 1993 to August 1994, who visited Japan Green Clinic, Singapore, which was established to serve only Japanese residents in Singapore and nearby Southeast
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Asian countries. The patients selected were a de-duplicated series of those who had abnormal results of liver function tests on routine examination (aspartate aminotransferase (AST)\ 40 IU/l, or alanine aminotransferase (ALT)\ 40 IU/l, or g-glutamyltransferase (GGT)\ 85 IU/l) at the routine medical check-up or who were clinically suspected to have acute hepatitis and were subsequently shown to have abnormal liver function test results. IgG class anti-HEV antibodies were analyzed with a HEV-ELISA kit (Genelabs Diagnostics (GD), Singapore), using structure proteins (ORF2 and ORF3) of HEV genome as the antigens and were abbreviated as IgG anti-HEV antibodies (GD). Of the 179 liver dysfunction cases, the last 142 were also examined for IgM anti-HEV using the IgM anti-HEV ELISA kit (GD). Intermediate-positive levels of color production in the IgG anti-HEV (GD) assay were confirmed to be positive by absorption testing with the antigen. The 37 subjects whose sera were tested before the IgM anti-HEV kit became available were examined for acute hepatitis E with a HEV-ELISA system developed by Kaketsuken (The Chemo-Sero-Therapeutic Research Institute, Kumamoto, Japan), using the HT-3 protein as the antigen for detection of IgG antibodies to HEV, which specifically appear in the acute phase of hepatitis E. The antibodies tested by this system were designated acute-phase IgG anti-HEV (KK) in order to differentiate them from the IgG anti-HEV (GD).
2.3. Phase II study We examined the sera of all the patients who visited Japan Green Clinic, Singapore for a routine medical check-up from January to April 1995. The number of cases studied was 168, out of which 37 cases (22%) showed abnormal results of liver function tests (AST\40 IU/l, ALT\ 40 IU/l or GGT\ 85 IU/l). This group of 37 clinically ‘asymptomatic’ subjects with abnormal liver function tests corresponds to the stratum of clinically ‘asymptomatic’ subjects from phase I with abnormal liver function tests. The sera were analyzed with the HEV-ELISA kit (GD) for IgG antibodies to HEV.
2.4. Other hepatitis 6irus markers Sera of patients with suspected acute hepatitis were also analyzed for IgM anti-HAV with an EIA kit, IMx HAVAB Assay System (Dainabot) and for HBsAg, HBeAg, HBeAb and HBcAb as routine laboratory tests. All the subjects in the phase II study were assayed for IgG HAVAB with an EIA kit, IMx HAVAB Assay System (Dainabot).
2.5. Population representation Although the candidates for inclusion in the ‘asymptomatic’ group of this study were limited to those companies who contracted for routine periodic examination including liver function tests, we had no basis to suspect that this group had a different risk profile than employees of other companies. Also, the age and
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geographic distribution of the population under study was not representative of all Japanese expatriates. However, adjustments to the estimates of general HEV seropositivity can be made using the age, country of residence and duration of residence strata-specific rates and applying them to different population proportions as one does with simple age adjusted rates. Nontheless, an accurate demographic profile of the full population of Japanese expatriates in this region would be required.
2.6. Statistical tools Statistical analyses were carried out with EpiInfo Version 6 (CDC, WHO), Health Information Retrieval System (HIRS) Version 2 (CDC) and Microsoft Excel Version 5.0.
3. Results
3.1. Phase I study The results of the phase I study are summarized in Table 2. Twenty-four cases (13.4%) out of 179 subjects with abnormal results of liver function tests were positive for IgG anti-HEV (GD). The anti-HEV IgG-seropositive prevalence by gender was 13.7% (22/161) in males and 11.1% (2/18) in females (not listed in Table 2). None of the first 37 sera in the series which were examined for the acute-phase IgG anti-HEV (KK) showed a positive result. However, among the last 142 subjects of this series examined for IgM anti-HEV, five cases showed a positive result, one of which was positive for both IgG and IgM anti-HEV antibodies (GD). Thus, acute hepatitis E accounted for five (3.5%) of the last 142 cases with liver dysfunction. After complete evaluation using respective virus markers and ultrasonography, the remaining 179 abnormal liver function test results were attributed to the following liver diseases, including suspected diagnoses: seven acute hepatitis A, five acute hepatitis B, three chronic hepatitis B, 47 fatty liver, 30 alcoholic liver injury and undiagnosed or other liver diseases for the remaining 82. These results showed that only 3.5% of the observed liver dysfunction could be explained by acute hepatitis E and that among those with abnormal liver function tests without symptoms, positive anti-HEV IgG antibodies had been acquired in the past in 12.8%. Accordingly, we conducted the phase II study in an attempt to measure the seroprevalence of IgG anti-HEV in the Japanese population as a whole, visiting the clinic irrespective of the results of liver function tests.
3.2. Phase II study The results of the phase II study are summarized in Table 3. Out of the 168 subjects, 37 (22.0%) had abnormal results of liver function tests (AST\ 40 IU/l or
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ALT \ 40 IU/l or GGT \ 85 IU/l), of whom 8.1% (3/37) were positive for anti-HEV IgG. Among the remaining 131 cases (78.0%) with normal liver function tests, 9.2% (12/131) were positive for anti-HEV IgG. In all, 15 cases (8.9%) out of 168 were positive for IgG anti-HEV. The prevalence of positive IgG anti-HEV in the ‘normal liver function test results’ group was 9.2% (12/131) and in the ‘asymptomatic abnormal liver function test results’ group was 8.1%, without a statistically significant difference. The results of the phase II study were evaluated for anti-HEV seropositivity with respect to the differences in age, gender, residence area and duration of residence (the results are summarized in Table 4). No statistically significant difference in IgG anti-HEV positivity was seen between males and females, or among countries of residence or duration of residence, although the odds ratio of males to females was 2.14 (95% CI: 0.60, 9.60).
Table 2 Prevalence of IgG anti-HEV (GD), IgM anti-HEV (GD) and acute phase IgG anti-HEV (KK) in the phase I study Factors
Total 179 tested Symptomatic Asymptomatic Total (%) Last 142 tested Symptomatic Asymptomatic Total (%)
IgG anti-HEV antibodies(GD)
IgM anti-HEV antibodies (GD)
Positive
Negative
Total
Positive
Negative
Total
6 18
44 111
50 129
24 (13.4)
155 (86.6)
179 (100) 34 108
3 2
31 106
34 108
142 (100)
5 (3.5)
137 (96.5)
142 (100)
1 4
15 122
16 126
5 (3.5)
137 (96.5)
142 (100)
5 11 16 (11.3)
29 97 126 (88.7)
IgG anti-HEV Positive Negative Total (%)
First 37 tested Symptomatic Asymptomatic Total (%)
IgG anti-HEV antibodies (GD)
Acute-phase IgG anti-HEV (KK)
Positive
Positive
Negative
Total
Negative
Total
1 7
15 14
16 21
0 0
16 21
16 21
8 (21.6)
29 (78.4)
37 (100)
0 (0)
37 (100)
37 (100)
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Table 3 Number of IgG anti-HEV (GD) positive and -negative cases in relation to ‘normal’ and ‘abnormal’ liver function test groups in phase II Liver function tests
IgG anti-HEV antibodies Positive (%)
Negative (%)
Total (%)
Abnormal Normal
3 (8.1) 12 (9.2)
34 (91.9) 119 (90.8)
37 (100) 131 (100)
Total
15 (8.9)
153 (91.1)
168 (100)
Analysis of the association between the positivities of anti-HAV and anti-HEV antibodies for 168 cases in the phase II study revealed that IgG HEV antibodies were positive in 5% (1/20) in the HAV antibody-positive group and 9.5% (14/148) in the HAV antibody-negative group. The odds ratio was 0.5 (95% CI: 0.01, 3.71) and there was insufficient evidence of significant negative association between the HAV and HEV infections.
4. Discussion The significant results of the present study were that the prevalence of HEV infection among Japanese residing in Southeast Asian countries is exceedingly high as compared with that in the domestic Japanese population, which is estimated to be 0.05% (personal communication, Atsushi Takagi, Dainabot, Japan). The observed prevalence of IgG anti-HEV positivity is apparently higher than that of the native population of Singapore, which is reported to be 3.4% [13]. The results could be interpreted as indicating that Japanese expatriates are likely to be infected with HEV while they stay abroad and probably while they are travelling in Southeast Asian countries. The possibility that the high prevalence of IgG anti-HEV results from a high false-positive rate for the detection kit has been raised for the Abbott anti-HEV test kit [14,15] (not used in this study). However, this false-positive rate concern was addressed in part by observing that cases in this study with intermediate levels of color production in ELISA (possibly ‘borderline’ positive or false positive) were confirmed to be positive by absorption test with the antigen. There was no significant difference in IgG anti-HEV prevalence between ‘normal’ and ‘abnormal’ liver function test groups. Therefore, the observed abnormality in the test results at the time of routine examination is not likely to be related to prior HEV infection and in only a few cases would it be associated with current, asymptomatic HEV infection. In fact, most of the hepatic dysfunctions were accounted for by other hepatitis strains or by or fatty liver.
6 2 6 1 4 4 4 3 0
Country Singapore Malaysia Indonesia Others Duration B1 year 1–1.9 years 2–3.9 years 45years Unknown
a
M-H, Mantel Haentzel test.
15
11 4
Gender Male Female
Total
0 6 8 1
Positive
153
29 37 56 29 2
75 28 30 20
86 67
16 83 46 8
Negative
IgG anti-HEV antibodies
Age B30 years 30–39 years 40–49 years 505years
Factors
168
33 41 60 32 2
81 30 36 21
97 71
16 89 54 9
Total
8.9
12.1 9.8 6.7 9.4 0.0
7.4 6.7 16.7 4.8
11.3 5.6
0.0 6.7 14.8 11.1
Prevalence (%)
x 2:1.03; (3 d.f., P= 0.91)
x 2:3.52; (3 d.f., P= 0.32)
Odds ratio = 2.14; (95%CI:0.60, 9.60); x 2:1.64 (M-H)a; P= 0.20
x 2: 4.45; (3 d.f., P= 0.22)
Statistical analysis
Table 4 Prevalence of IgG anti-H EV (GD) at their first visit by age, gender, country of residence and duration of residence in the population of Phase II
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Since anti-HEV IgG positive subjects rarely had a history of any particular symptoms related to acute hepatitis, the acute hepatitis E in this study was subclinical or mild. Although most of the infected subjects were asymptomatic in the study population, a fulminant course of hepatitis E has been reported for pregnant women [1 – 3], therefore, precautions should be taken for pregnant women residing in those countries. The age distribution of anti-HEV IgG among Japanese population in this study was different from that generally reported, in that the highest attack rate has been observed among young adults [16], while the present study revealed a low prevalence of IgG anti-HEV in the 30 –39 years age group, the tendency increasing with age. A significantly higher prevalence of IgG anti-HEV in the 46–55 years age group was reported for healthy individuals in Southern Taiwan [4]. This would be accounted for by a cumulative effect of IgG anti-HEV positive individuals, assuming that antibodies persist over at least two years [17,18], although no significant difference was found in relation to the duration of residence in this study. No significant association of IgG anti-HEV with IgG anti-HAV was shown in this study, despite both of them being enterically transmitted. Tan et al. [19] reported a similarly low superinfection rate of HEV in acute hepatitis A. Instead, an extraordinarily high superinfection rate of HEV with HCV is noted, despite different infection routes [19], this being supported by the report of Thomas et al. [20], although possible parenteral transmission of HEV has been reported from different sources [21,22]. Considering the fact that those who travel or have lived overseas might transmit the disease into their own countries, HEV assay kits should be available not only in tropical countries but also in the northern countries including Japan. It is also desirable, especially for the benefit of pregnant women who would develop severe hepatitis [1 – 3], to establish a surveillance system on HEV transmission for community practices [23].
Acknowledgements We appreciate the cooperation of Japan Green Clinic, Singapore and the active support of this study by Dr Ginnohyoe Suhara, the Center for Adult Diseases, Kurashiki, Japan and also to Dr Tetsuji Rikihisa, for analysis of IgG class anti-HEV appearing in the acute phase of hepatitis E.
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