zyx zyx Journal of Medical Virology 42:405-408 (1994) zyxwvutsrq zyxwvutsrqpo zyxwvutsr zyxwvu Acute Sporadic Hepatitis E in Kuwait Abraham Koshy, Allen L. Richards, Siham Al-Mufti, Saroj Grover, Mohamed A. Shabrawy, Alexander Pacsa, Abdul-Aziz H. Al-Anezi, Basil Al-Nakib, Jim Burans, Mitchell Carl, and Kenneth C. Hyams Thuniyan Al-Ghanim Gastroenterology Center, Al-Amiri Hospital (A.K., B A - N . ) , U.S. Naval Medical Research Unit No. 2, Jakarta, Indonesia (A.L.R.); Public Health Laboratory (S.A.-M.), Infectious Disease Hospital (S.G., M.A.S., A.-A.H.A.-A.), and Faculty of Medicine, Kuwait University (A.P.), Safat, Kuwait; U S . Naval Medical Research Institute, Bethesda, Maryland (J.B., M.C., K.C.H.) Fifty-seven adult patients with acute hepatitis and 34 comparison patients without liver disease were evaluated using a newly developed Western blot assay for IgM antibody to hepatitis E virus. The mean age of patients with hepatitis was 32 years (range, 18-55 years); 88% were male. Among patients with acute hepatitis, hepatitis A (anti-HAV IgM positive) was diagnosed in two (4%),hepatitis B (anti-HBc IgM positive) in three (5%), and hepatitis E (anti-HEV IgM positive) in 34 (60%). One hepatitis patient had CMV IgM, another had EBV IgM, and 16 others (28%) were negative for all serologic markers of acute viral hepatitis. No patient with acute hepatitis A or B and none of the comparison patients without acute hepatitis had anti-HEV IgM. All but one case of acute hepatitis E were found among expatriates of Asian origin, and acute hepatitis E was associated significantly with recent travel to the Indian subcontinent. These data suggest that acute hepatitis E is common among foreign workers in Kuwait but that little HEV transrnission is occurring directly in Kuwait. Dawson et al., 1992; Wong et al., 1980; Kane et al., 1984; Iqbal et al., 1989; Byskov et al., 1989; Purcell and Ticehurst, 19881. Whether hepatitis E is also a major cause of acute sporadic hepatitis is not well understood due t o lack of a specific serologic test [Khuroo et al., 1983; Shamma’a, 1984; Hyams et al., 19901. In a previous study of acute hepatitis in the Persian Gulf [Glynn et al., 19851,NANB hepatitis was found to be common among immigrant workers, but it was not possible t o demonstrate if HCV or HEV was the major cause of infection. The recent development of serologic assays for hepatitis C and E has made it possible to evaluate the specific causes of acute sporadic non-A non-B hepatitis in this region [Alter et al., 1989a,b; Purdy et al., 1992; Goldsmith et al., 1992; Skidmore et al., 19911. zyxwvutsrqp zyxwvut PATIENTS AND METHODS The study was conducted at the Infectious Disease Hospital, Kuwait City, from January to June 1992. This hospital is the primary center for treatment of infectious diseases in Kuwait. Patients over the age of 17, suspected of acute viral hepatitis with jaundice of less than 1 month’s duration and likely to stay in the 0 1994 Wiley-Liss, Inc. country for at least 2 months were entered into the study as cases. Patients found to have alcoholic hepatiKEY WORDS anti-HEV IgM, travel to Indian tis, drug-induced hepatitis, septicemic cholestasis, bilsubcontinent, Middle East iary obstruction, or hemolytic jaundice were excluded. In addition, a group of patients who presented at the Infectious Disease Hospital with acute infections, withINTRODUCTION out evidence of liver disease, were included as compariHepatitis C virus (HCV)and hepatitis E virus (HEV) son patients. Comparison patients were selected on the appear t o be the major causes of what was previously basis of similarity of age and admission date with cases known as non-A non-B (NANB) hepatitis [Khuroo, of acute hepatitis. 1980; Skidmore et al., 1992; Dawson et al., 1992; Alter et al., 1989a,b; Larsen et al., 19921. These two viruses have distinctly different modes of transmission, HCV Accepted for publication July 23, 1993. being parenterally transmitted [Alter et al., 1989a,b; Address reprint requests to Dr. Abraham Koshy, M.D.,Faculty Larsen et al., 19921 and HEV being enterically transmitted [Khuroo, 1980; Skidmore et al., 1992; Dawson et of Medicine, Kuwait University, Post Box 24923, 13110 Safat, Kuwait. al., 19921. Because HEV can be widely and rapidly Consent: Informed consent was obtained from study subjects transmitted through contamination of drinking water, and the research guidelines of the U S . Naval Medical Research large outbreaks of hepatitis E have occurred in develop- Unit No. 2 Committee for the Protection of Human Subjects were ing countries [Khuroo, 1980; Skidmore et al., 1992; followed. ~ 0 1994 WILEY-LISS, INC. 406 All study subjects had a moderate standard of living and resided in homes with electricity and indoor plumbing. At the time this study was initiated, the water and sewage system in Kuwait had been fully restored to operation following the war with Iraq. A standardized questionnaire was administered to all study subjects. The questionnaire elicited basic demographic data and subjects were asked about the occurrence of potential risk factors of viral hepatitis transmission during the six months prior to onset of symptoms. A venous blood sample was obtained from each study participant upon admission. Whenever possible, a follow up sample was obtained 2 weeks to 2 months after the initial sample. Serum samples from patients with acute hepatitis were analyzed by standard methods for alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin, alkaline phosphatase, total protein, and albumin. Only patients with AST and ALT levels greater than 2% times the upper limit of normal were included in the study as cases. Sera from all study subjects were tested for serologic markers of hepatitis A (anti-HAV IgM), hepatitis B (HBsAg and anti-HBc IgM), and hepatitis C (total antiHCV) by enzyme immunoassay (ELISA; Abbott Laboratories, Abbott Park, IL). Only sera repeatedly reactive by ELISA were considered positive. Sera positive for anti-HCV by second generation ELISA were verified using a second generation immunoblot assay (RIBA; Ortho Laboratories, Raritan, NJ). Cytomegalovirus infection was assessed by testing for IgM antibody to cytomegalovirus by indirect fluorescence (CMV IgM; Gull Laboratories, Salt Lake City, UT). Epstein-Barr virus infection was assessed by testing for IgM antibody to Epstein-Barr virus by indirect fluorescence (EBV IgM; Organon Teknika Corporation, Durham, NC). Only samples repeatedly positive after absorption of Rheumatoid factor were considered positive for CMV IgM or EBV IgM. In addition to commercial tests, sera from all cases of acute hepatitis and comparison subjects were tested for IgM and IgG antibody to hepatitis E virus (HEV) using a Western blot assay designed to detect antibodies to the protein encoded by ORF 2 of HEV. In brief, Sfs cells were infected with ORF2-rAcNPV a t a multiplicity of infection of 5 and incubated at 27°C for 48 hrs. The cells were pelleted by centrifugation at 3 , 0 0 0 ~for 10 mins. The cell pellet was then suspended in 0.5 ml of 2% SDS, 0.2 M NaC1, 0.2 M Tris (pH 7.5), and 1.5 mM MgC1,. The suspension was heated to 100°C for 3 min and stored a t -20°C. These cell lysates and supernatants were then diluted with 2 x protein sample buffer and separated by 10% SDS-PAGE. Proteins separated by SDS-PAGE were electroblotted [Sambrook et al., 19891 onto nitrocellulose membranes in transfer buffer containing 25 mM Tris, 190 mM glycine, and 20% methanol. Membranes were blocked with PBS containing 5% skim milk, 0.01% Tween 20, and 0.001% thimersol for 1 hr a t room temperature and cut into 2 mm strips. The nitrocellulose membranes were incubated with a n opti- Koshy et al. ma1 concentration of patient sera (1:200) in PBS containing 1% skim milk, 0.01% Tween 20, and 0.001% thimersol for 8-12 h r at 25°C. After washing four times with PBS containing 0.01% Tween and 0.001% thimersol (wash medium), the strips were then incubated with 1:1,000 dilution of goat horseradish peroxidase labeled anti-human IgG (gamma specific) or IgM (mu specific; Kirkegaard and Perry Laboratories, Gaithersberg, MD) in wash medium for 1 h r a t room temperature. After additional washing, the nitrocellulose membranes were incubated up to 15 min with chloronapthol (Kirkegaard and Perry). Lastly, the strips were evaluated for the presence of the 70.9 kd HEV ORF 2 encoded protein. zyxwv zy zyxw zy Statistical Analysis Comparisons of proportion were made using the x2 test with Yates’ correction or the Fisher’s exact test. Mean values were compared using the Mann-Whitney test. Significance was set at 0.05 level. RESULTS A total of 57 cases of acute hepatitis and 34 comparison subjects without liver disease were included in the study. The ages of acute hepatitis and comparison patients were 32 t 7 (M 2 SD, range, 18-55) and 30 & 7 (range, 19-46), respectively. Sixty-two percent of comparison patients and 88% of hepatitis cases were male. Eighty-five percent of comparison patients and 91% cases were from the Indian subcontinent. Patients with hepatitis had been ill with jaundice for a mean of 9 6 days (range, 3-31 days). Among the 57 acute hepatitis cases, hepatitis A (antiHAV IgM positive) was diagnosed in two (4%), acute hepatitis B (anti-HBc IgM positive) in three (5%), and hepatitis E (anti-HEV IgM positive) in 34 (60%). Among cases without markers for hepatitis A, B, or E, one had CMV IgM and another had EBV IgM. Diagnosis of NANB hepatitis was made by exclusion in 16 (28%) cases (Table I). Seroconversion to anti-HCV was not found in the 29 hepatitis follow-up serum samples, but one male Egyptian patient classified as having NANB hepatitis, had anti-HCV when initially evaluated. None of the cases positive for serologic markers of acute hepatitis A or B had anti-HEV IgM (Table I). One patient with hepatitis E had HBsAg without anti-HBc IgM. IgG anti-HEV was detected in 27 of 34 patients with acute hepatitis E (IgM anti-HEV positive). Among the 16 cases classified a s NANB, 10 had anti-HEV IgG. Among the 34 comparison patients, 23 had culture positive typhoid or paratyphoid fever and 9 had acute varicella infection. None of the comparison patients had serologic markers of acute hepatitis A, B, C, or E, but anti-HEV IgG was found in one patient (Table I). This patient was a 31-year-old male Pakistani who had returned from Pakistan 45 days earlier. He did not have a history ofjaundice, had no recent contact with a jaundiced person, had normal serum aminotransferases, and had been admitted for blood culture posi- * zyxwvutsr zyxwvuts zyxwvutsr zyxw zyxwvutsrqpo zyx 407 Acute Hepatitis E in Kuwait TABLE I. Serologic Markers of Viral Hepatitis in Patients With Acute Hepatitis and Comparison Patients Without Evidence of Liver Disease Hepatitis Anti-HAV IgM Anti-HBc IgM HBsAg Anti-HEV IgM Anti-HEV IgG A B E NANB (n = 2) (n = 3) 3 3 - (n = 34) 1 34 27 (n = 16) Comparison patients (n = 34) - - - - 2 - - TABLE 11. Nationality of Comparison Patients and Patients With Acute Hepatitis Nationalitv India Bangladesh Pakistan Nepal Sri Lanka Kuwait Others Total Comparison Datients 16 5 2 0 6 2 3 34 E 18 11 2 1 0 1 1 34 1 1 - 10 TABLE 111. Liver Function Tests Among Cases of Acute Hepatitis E and NANB Hepatitis Hepatitis NANB 7 7 0 0 0 1 1 16 tive paratyphoid fever without signs or symptoms of liver disease. One comparison patient had HBsAg without anti-HBc IgM. None of the comparison patients had CMV IgM, EBV IgM nor anti-HCV when tested initially. The mean age of the 34 acute hepatitis E cases (antiHEV IgM positive) was 30 +- 6 years (range, 18115 years); 94%were male. Four of these patients had history of a previous episode of acute hepatitis. All but two patients with acute hepatitis E, one from Kuwait and one from Syria, were originally from the Indian subcontinent (Table 11). Furthermore, 91%of cases with acute hepatitis E had recently traveled to their home country compared to 56%of non-hepatitis comparison patients (P < 0.001). The mean duration between return from travel and onset of jaundice was 27 23 days for patients with acute hepatitis E. The one native Kuwaiti with acute hepatitis E was a 30-year-old female housewife who had not traveled outside of Kuwait and had no known contact with anyone with acute hepatitis. During the previous 6 months, 2 of 34 patients with acute hepatitis E had a family member with jaundice, 2 had known someone with jaundice, 3 had received a n injection, 3 had been hospitalized, and one had received a blood transfusion. Among 16 acute NANB cases, one had known someone with jaundice and another received an injection during the prior 6 months, but none had a family member with jaundice, had received a blood transfusion, nor been hospitalized. Vomiting was reported by 82% of 34 patients with acute hepatitis E, fever by 68%, arthralgia by 15%, pruritus by 12%, and a rash by 6%.There was no significant difference in the occurrence of various signs and symptoms between patients with acute hepatitis E and E (M f SD, Test ALT (IU/L, N i65) AST (IUIL, N 40) Bilirubin (pmol/L) Alk Phos (IU/L, N 6 85) Total protein (g/W Albumin ( d L ) n = 34) 1,366 2 664 1,116 f 800 126 2 61 198 113 69 t 6 35 t 3 * NANB (M f SD, n = 16) 1,442 f 607 1,385 2 650 156 2 92 189 f 69 71 5 4 36 t 4 zyx NANB hepatitis. Furthermore, there was no significant difference in the age distribution of patients with hepatitis due to HEV (30 6) and NANB (34 8);and liver function tests were comparable among patients with hepatitis E and NANB (Table 111). None of the 57 patients with acute hepatitis nor the 34 comparison patients was known to have died of their illness. The mean length of illness before evaluation was similar for acute hepatitis E cases with IgM anti-HEV alone (8 ? 3 days), hepatitis E cases with both IgM and IgG anti-HEV (8 5 days), and NANB cases with only IgG anti-HEV (8 2 3 days). * * zyxwv zyxwvut * * DISCUSSION In this study, hepatitis E was found to be a frequent cause of acute sporadic hepatitis among adults living in Kuwait. However, little hepatitis E transmission appeared to be occurring directly in Kuwait. Expatriate workers who had recently traveled to India or Bangladesh were most at risk of contracting acute hepatitis E; only one native Kuwaiti with acute hepatitis had anti-HEV IgM. These results are consistent with findings of a previous study conducted in Qatar which indicated that non-A, non-B hepatitis, occurring within 2 months of travel from their native country, was prevalent among expatriate workers from Asia [Glynn et al., 19851. Most foreign workers were probably infected with HEV while visiting their home country because the time interval since travel for the majority of hepatitis E cases was consistent with the incubation period of enterically transmitted non-A, non-B hepatitis [Purcell and Ticehurst, 19881. The primary mode of HEV transmission when traveling home could not be determined, 408 zyxwvutsrqpon zy Koshy et al. although it is assumed that waterbourne transmission predominated, as in outbreaks of hepatitis E in the Indian subcontinent [Khuroo, 1980; Wong et al., 1980; Kane et al., 19841. The finding t h a t foreign workers who had traveled recently to several countries had acute hepatitis E suggests that HEV transmission is widespread in South Asia. The occurrence of acute hepatitis E in one native Kuwaiti and 2 Bangladeshis who had not recently traveled abroad indicates that HEV transmission is also occurring in Kuwait. However, transmission appears to be infrequent, since Kuwaitis seldom presented with acute hepatitis, although all residents of Kuwait with acute hepatitis were routinely referred to the study hospital. There was no obvious source of HEV infection in these patients; contamination of water supply was unlikely since outbreaks of hepatitis and other enteric diseases were not occurring in Kuwait at this time. As reported previously [Hyams et al., 1990, 19921, the newly developed Western blot assay for IgM antiHEV appears to be specific for the diagnosis of acute hepatitis E. No case with serologic markers of acute hepatitis A or B, and no comparison subject without acute hepatitis was positive for IgM anti-HEV. A more precise characterization of the specificity of the test was not possible since cases could not be followed for extended periods of time to detect seroconversion to antiHCV, although acute hepatitis C was probably uncommon because parenteral risk factors were rare among hepatitis cases. The similarity in presentation between hepatitis E and hepatitis NANB patients and the large percentage of NANB hepatitis cases with IgG antiHEV suggests that many NANB cases were also due to HEV infection but that the Western blot assay failed to detect IgM antibody to HEV in these patients. ACKNOWLEDGMENTS This research was supported in part by the U S . Naval Medical Research and Development Command, NMC, NCR (Bethesda, MD), work unit numbers 162787A870AR8, 162787A870AQ208, and 162770AR122 and Kuwait University project number MM032. The opinions and assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the U S . 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