1 Center for Gastroenterology, Kobayashi Hospital, Hokkaido 090-8567, Japan
2 Department of Internal Medicine, Kin-ikyo Chuo Hospital, Hokkaido 007-0870, Japan
3 Immunology Division and Division of Molecular Virology, Jichi Medical School, Tochigi-Ken 329-0498, Japan
4 Department of Pediatrics, Jichi Medical School, Tochigi-Ken 329-0498, Japan
5 Japanese Red Cross Saitama Blood Center, Saitama-Ken 360-0806, Japan
Correspondence
Hiroaki Okamoto
hokamoto{at}jichi.ac.jp
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ABSTRACT |
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The nucleotide sequences of the 10 human and seven swine HEV isolates reported herein have been assigned DDBJ/EMBL/GenBank accession nos AB105888AB105904 and AB108652AB108666.
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MAIN TEXT |
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Risk factors for HEV infection among patients with sporadic cases of hepatitis E in industrialized countries have not been defined. Recently, we found that nine of ten patients who contracted sporadic acute or fulminant hepatitis E in 2001 and 2002 in Hokkaido, where hepatitis E is most prevalent in Japan, had ingested grilled or undercooked pig liver 2 weeks to 2 months before the disease onset. Therefore, in the present study, we tested packages of raw pig liver sold in grocery stores in Hokkaido as food for the presence of HEV RNA, and determined and analysed the partial nucleotide sequences of HEV isolates obtained from these pig livers and those recovered from patients with sporadic acute or fulminant hepatitis E to determine whether hepatitis E in Japan is likely to be food-borne.
A total of 38 patients with a clinical diagnosis of sporadic acute or fulminant hepatitis were seen at two city hospitals in Sapporo and Kitami on Hokkaido Island, Japan, between January 2001 and December 2002. Hepatitis A was diagnosed in three patients, type B acute hepatitis in five patients and type C acute hepatitis in only one patient. Among the remaining 29 patients with acute or fulminant hepatitis of non-A, non-B, non-C aetiology, ten patients (34 %) had anti-HEV IgG and IgM antibodies detectable by in-house ELISA (Mizuo et al., 2002) and were diagnosed with acute or fulminant hepatitis E (see Table 1
). Serum samples were obtained from the ten patients at admission and stored at -20 °C or below until testing. Informed consent was obtained from each patient. A total of 363 packages of raw pig liver, obtained from domestic pigs raised in Hokkaido and sold as food, was purchased from 25 grocery stores in Hokkaido on 14 different days between 19 December 2002 and 26 February 2003. The pig liver in each package had been processed as slices or a block of 103819 g (mean 207·4 g). Several pieces of tissue specimens (100200 mg) were obtained from each package and stored at -80 °C until testing.
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Of the ten patients studied (Table 1), eight developed acute hepatitis E and had peak total bilirubin levels of 5·626·0 mg dl-1, peak alanine aminotransferase levels of 12764100 IU l-1 and peak aspartate aminotransferase levels of 11934650 IU l-1. The abnormal liver function test values normalized rapidly within 1 month in seven patients, but persisted for longer than 3·5 months in the remaining patient (Patient 9); who was 86 years of age and had severe prolonged jaundice. Two patients (Patients 6 and 8) contracted fulminant hepatitis E; Patient 6 died of hepatic failure 56 days after the disease onset. In Patient 8, the episode of encephalopathy resolved 2 weeks after admission, but severe jaundice and coagulopathy persisted for longer than 2·5 months and the patient died of hepatic failure 105 days after the onset of hepatitis E. The sera from all ten patients at admission were positive for HEV RNA. The HEV isolates obtained from two patients were of genotype III and those from the remaining eight patients were of genotype IV. All ten patients had no history of travel outside Japan or contact with travellers who had been abroad or foreigners, no contact with pigs and no history of blood transfusion in the preceding 6 months. Of particular interest, Patient 9 reported consumption of cooked pig liver 1925 days before the onset of hepatitis E. According to his detailed statement at admission, he lived alone and, for the first time, had purchased processed raw pig liver from a grocery store located near his residence and then fried slices of pig liver at home and ingested it for 7 consecutive days. He became aware of jaundice and felt general malaise 19 days after the last day of consumption of the pig liver. His report prompted us to interview the remaining nine patients to ask whether they had consumed pig liver before the onset of illness. Surprisingly, eight of the nine patients recalled eating grilled pig liver 2 weeks to 2 months before the onset of hepatitis E (Table 1
). For Patient 6, who died due to hepatic failure, in reply to our interview, his wife told us that they had never eaten pig liver at home, but she could not deny that he could have consumed undercooked pig liver while drinking alcohol at Japanese-style bars; he had had a history of alcohol intake for 30 years. When we interviewed 22 randomly selected patients with acute hepatitis of non-E aetiology who were seen at our hospitals between January 2001 and December 2002, they all denied consumption of pig livers before the disease onset, indicating that consumption of pig livers is significantly associated with the occurrence of hepatitis E among patients with acute or fulminant hepatitis (0/22 versus 9/10, P<0·0001; Fisher's exact test).
To investigate whether raw pig liver as food is contaminated with HEV, a total of 363 packages of raw pig liver, purchased from grocery stores in Hokkaido where the patients lived, were tested for HEV RNA. Pig liver specimens from seven (1·9 %) of the 363 packages had detectable HEV RNA, with the viral load ranging from 102 copies g-1 to 107 copies g-1 (Table 2). The amplification products of ORF2 (412 nucleotides; primer sequences at both ends excluded) from the seven HEV RNA-positive pig liver specimens were sequenced and compared (Table 2
). The seven swine HEV isolates, designated swJL82, swJL97, swJL98, swJL131, swJL145, swJL234 and swJL325, segregated into genotype III or IV, differing by 022·2 % from each other in the 412 nucleotide sequence. Pig livers nos 97 and 98 were purchased from the same store (Store B) where only these two packages of pig liver had been available on 12 January 2003. Of note, the swJL97 and swJL98 isolates had identical sequences to each other and the same viral load of 107 copies g-1, indicating that slices of pig liver in packages nos 97 and 98 were derived from the liver of a single pig. The swJL234 and swJL325 isolates had the same sequence of 412 nucleotides with six common ambiguity codes of Y (T or C), but they were isolated from slices or a block of pig liver purchased from different stores (Stores E or F) on different days (14 or 15 February 2003) and had a distinct viral load of 105 or 103 copies g-1, respectively, suggesting that pig liver packages nos 234 and 325 were derived from the livers of distinct pigs in the same herd.
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As described in a recent review article by Smith (2001), it is likely that foods can act as vehicles for transmission of HEV. The occurrence of acute hepatitis E in an individual in eastern Sicily after consumption of shellfish obtained from faecally contaminated waters was reported by Cacopardo et al. (1997)
. Similarly, Mechnik et al. (2001)
reported an Israeli who acquired acute HEV infection possibly as a result of eating raw shellfish. Hartmann et al. (1998)
attributed acute hepatitis E in a Turkish international living in Germany to ethnic foods brought as a gift by a Turkish visitor. However, no case of hepatitis E has been unequivocally shown to be due to food consumption. In the present study, nine of ten patients who developed sporadic acute or fulminant hepatitis E had consumed grilled pig liver 2 weeks to 2 months before the onset of the illness. Since the pig livers they ingested were not available for testing, we purchased 363 packages of raw pig livers as food from grocery stores near the residences of our subjects in Hokkaido and showed that a certain proportion of packaged pig livers (1·9 % or 7/363) was contaminated with HEV. The incubation period for hepatitis E is considered to be 29 weeks and, therefore, we speculate that inadequately cooked pig liver contaminated with HEV was the source of the patients' infection. Patient 10 in the present study actually reported ingestion of undercooked pig liver. Our speculation is supported by the evidence that one genotype IV HEV isolate (swJL145) obtained from a package of pig liver had a high nucleotide sequence identity, 97·8100 %, with ten HEV isolates recovered from patients living in Hokkaido, including those from eight patients in the present study (Fig. 1
), and that two genotype III HEV isolates (swJL234 and swJL325) from two distinct packages of pig liver had high nucleotide sequence identity, 96·6100 %, with five human HEV isolates obtained from patients who lived in Hokkaido, including those from two patients in the present study (Fig. 1
). With regard to the four patients with hepatitis E living in Hokkaido in our previous study (Mizuo et al., 2002
), in reply to our interview, the HE-JA2 and HE-JA4 patients told us that they ingested undercooked pig liver several times or once a year, respectively; the last consumption date was 1 or 2 months before the onset of hepatitis E. However, the HE-JA1 patient denied consumption of pig liver, but said that he preferred to consume inadequately grilled pig intestine or colon once a month. Patients 2, 8 and 10 in the present study also reported ingestion of undercooked pig intestine/colon together with pig liver. HEV is shed into faeces for transmission by the faecaloral route. Furthermore, it has been shown that both swine HEV and human HEV replicate in the colon and intestines (Williams et al., 2001
). Therefore, undercooked pig intestine or colon contaminated with HEV may be another source of transmission of HEV. To support our speculation further, the following questions have to be answered. (i) Why were family member(s) of Patients 15, 7 and 8 who ingested pig liver with the patient at home not infected with HEV during the same period? (ii) Why did patients who had consumed pig liver in the past (Patients 1, 2, 5, 8 and 10) acquire HEV infection on this occasion? (iii) Do packaged pig livers sold in grocery stores as food contain infectious virus or just viral RNA? The extremely low chance of acquiring HEV infection by ingestion of pig liver may be explained by the observed low frequency and by the low viral load in HEV-contaminated pig livers. Interestingly, a recent experimental transmission study of swine HEV to susceptible pigs showed that the risk of contracting swine HEV infection through consuming uncooked, virus-contaminated pork is extremely small (Kasorndorkbua et al., 2002
). The extent of grilling may affect the risk of acquiring HEV infection via consumption of pig liver. In some patients' families, the patient's spouse ingested only well-cooked pig liver. Detection of HEV RNA by RT-PCR in raw pig liver in grocery stores does not necessarily mean that the contaminated swine HEV in the packaged pig livers is infectious. Therefore, further studies are needed to determine whether the packaged pig livers contain infectious virus.
In conclusion, we would like to speculate that sporadic acute or fulminant hepatitis E in Hokkaido, where clinical HEV infection is most prevalent in Japan, is transmitted by ingestion of inadequately cooked pig liver contaminated with HEV, based on our patients' dietary habits and the evidence that raw pig livers that are available in grocery stores located near our patients' residences were contaminated with HEV having a high nucleotide sequence identity to human HEV isolates recovered from patients living in Hokkaido. Therefore, although our present results may raise further public health concerns for HEV zoonosis, extended studies must be performed to determine to what extent pig liver and intestine/colon available as food in grocery stores are contaminated with HEV, not only in Hokkaido but also in other areas in Japan. Since we unequivocally found the presence of HEV among pig livers for sale as food in the present study, pig livers should be cooked well before ingestion to prevent the occurrence of possible food-borne hepatitis E in Japan.
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ACKNOWLEDGEMENTS |
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Received 20 March 2003;
accepted 23 April 2003.