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Epidemiology of Hepatitis E in 2017 in Bavaria, Germany

Epidemiology of Hepatitis E in 2017 in Bavaria, Germany In the last decade, the number of reported hepatitis E virus (HEV) infections in Germany, including Bavaria, has continued to rise. In order to identify risk factors associated with HEV infection, we investigated notified hepatitis E cases from Bavaria during 2017. The project “Intensified Hepatitis E Surveillance in Bavaria” included interviews with questionnaires, collection and genotyping of stool, serum and food samples. In addition, certain risk factors were examined in a sample comparison with healthy population using univariable analysis and logistic regression. In total, 135 hepatitis E cases from Bavaria were included in the analysis. Mean age for women was 46 (range 20–74) years and 47.5 (range 20–85) for men. 56 of the cases (41.5%) were asymptomatic. Among the symptomatic cases, both men and women were equally affected with symptoms like fever (16.3%), jaundice (18.8%) and upper abdominal pain (28.2%). 145 human samples (serum, stool) and 6 food samples were collected. 15.9% of the human samples (n = 23) were positive for HEV RNA by reverse-transcription quantitative real-time PCR (RT-qPCR). Identified risk factors significantly associated with hepatitis E were sausage consumption with odds ratio 9.6 (CI 1.3–70.1), fish with OR 2.2 (CI 1.1–4.4) and cat ownership with OR 1.9 (CI 1.3–3.0) in multivariable analyses. Further investigation is needed to confirm the role of fish in HEV transmission. Autochthonous HEV genotype 3 is prevalent in Bavaria and there could be more transmission routes contributing to the spread of HEV than previously known. Undercooked meat, offal, sausages, fish, shellfish and contact with animals and pets are possible sources for infection. Keywords Hepatitis E · HEV · Genotype 3 · Risk factors · Transmission Introduction subgenotypes, respectively (Smith et al., 2020). The clinical manifestation of an HEV infection varies in severity from Hepatitis E virus (HEV) is a non-enveloped, single-stranded subclinical to fulminant, probably, depending on the geno- RNA virus that belongs to the Hepeviridae family and type and host factors (e.g. immune constitution). Genotypes causes infectious inflammation of the liver. Human patho- 1 and 2 are endemic in developing countries in Asia and genic HEV can be classified in four major genotypes (HEV Africa, as well as in Mexico. They are restricted to humans, 1–4), that can further be classified into subgenotypes. Gen- mostly transmitted through the faecal-oral route and causing otypes 1, 2, 3 and 4 count seven, two, fourteen and nine major outbreaks in tropical and subtropical regions (Khuroo and Khuroo, 2016; Rein et al., 2012). Genotype 3 has been found mainly in European countries, USA and Japan, while genotype 4 is mostly spread in Asia (China, Japan and India) * K. Hriskova katerinahriskova@yahoo.de (Pavio et al., 2010). Genotypes 3 and 4 are zoonoses and have been detected in several animal species (e.g. pigs, wild Institute for Medical Information Processing, Biometry boar, deer and rabbits) (Meng, 2013) and humans. These and Epidemiology - IBE, LMU Munich, Marchioninistr. 15, HEV genotypes can cause sporadic, self-limiting disease 81377 Munich, Germany with clinical symptoms like vomiting, uncolored stools, Pettenkofer School of Public Health, Munich, Germany darkened urine and jaundice (Nan et al., 2017). According Bavarian Public Health and Food Safety Authority (LGL), to the German Infection Protection Act (IfSG) clinical sus- Veterinärstraße 2, 85764 Oberschleißheim, Germany picion, illness or death from acute viral hepatitis and/-or National Consultant Laboratory for HAV and HEV, the direct or indirect detection of hepatitis E virus must be Institute of Clinical Microbiology and Hygiene, University notified. Medical Centre Regensburg, Franz-Josef-Strauß-Allee 11, 93053 Regensburg, Germany Vol.:(0123456789) 1 3 338 Food and Environmental Virology (2021) 13:337–346 Since the introduction of IfSG in 2001, until 2014 the serum samples from patients and food samples if available. HEV incidence per 100.000 inhabitants in Germany was A total of 558 hepatitis E cases were reported to the Local below 1. In recent years, however, the number of reported Health Authorities (LHA) during the study period. The diag- hepatitis E cases has substantially increased. In Bavaria nostic methods for these 558 cases were IgM blood (n = and also nationwide (RKI, 2018), the number of new hep- 530), PCR blood (n = 46) and PCR stool (n = 15), where atitis E cases almost doubled in 2017 compared to 2016 some cases were diagnosed by two methods. The participat- (LGL, 2019). It is uncertain whether the growing number ing health authorities informed the reported cases about the of reported cases is due to an actual increase in the num- study and invited them to join the study. The participation ber of infected humans or due to a diagnostic and notifica- in the study was anonymous and voluntary. Written consent tion biases. Over the last few years, the disease has come was obtained from all recruited participants. Stool sam- into sharp focus as it is no longer perceived only as a travel ples were analysed at the National Consultant Laboratory associated disease. Similar to other European countries, the for Hepatitis A and Hepatitis E Virus, Institute of Clinical autochthonous genotype in Germany is the genotype 3. The Microbiology and Hygiene, Regensburg. If available, food prevalence of antibodies against HEV in the adult population samples from patients’ households, suspected to be risk fac- of Germany is 16.8% (about 5% in people under 30 years of tor for HEV, were collected and analysed at the Laboratory age, up to 25% in those over 60 years of age) (Faber et al., for Food Virology of the Bavarian Public Health and Food 2012). Eating raw or uncooked meat, oa ff l and meat products Safety Authority (LGL) in Erlangen. Inclusion criteria for (e.g. sausages) were considered as a risk factor for hepatitis descriptive analysis of HEV cases were notified hepatitis E in humans (Wichmann et al., 2008; Szabo et al., 2015; E cases with laboratory confirmed HEV infection (IgM or Faber et al., 2018; Said et al., 2017; Meng, 2010). Other PCR positive) with at least 80% completed questionnaire reported risk factors associated with hepatitis E are the con- and no travel within the incubation period to HEV genotype sumption of shellfish ( e.g. oysters, mussels) (Grodzki et al., 1, 2 or 4 endemic countries, according to a resent review 2014; O’Hara et al., 2018; Said et al., 2009; Mesquita et al., article Perez-Gracia et al (2015) (Fig. 1). Plausibility check 2016) and the contact with waste waters (Clemente-Casares, was performed to verify answer consistency. All available 2016). Bavaria is a federal state with a high density of pigs sequenced serum and stool samples were included in the and high seroprevalence for HEV in pig herds. On aver- subgenotype analysis irrespective of the completeness of the age 51.4% of tested pig sera samples in Southern Germany questionnaire. (Bavaria  and Baden-Wuerttemberg) exhibited anti-HEV antibodies (Krumbholz et al., 2013). Direct contact with Laboratory Confirmation and Genotyping of HEV pigs is considered a risk factor for hepatitis E (Chaussade et al., 2013; Krumbholz et al., 2014). Furthermore, IgM and RNA Extraction and Molecular Detection IgG antibodies against HEV were detected further up the food chain in serum and meat juice samples from Bavarian Nucleic acid isolation from human specimens was per- slaughterhouses (Wacheck et al., 2012). In addition, 4% of formed from 200 μl serum or stool suspension on an EZ1 pork livers purchased from butchers in Regensburg, Bavaria, Advanced XL workstation using the EZ1 Virus Mini Kit had detectable levels of HEV RNA (Wenzel et al., 2011). v2.0 (Qiagen, Hilden, Germany). Eluted nucleic acid was Based on the above evidence and the increasing number of analysed by RT-qPCR as previously described (Wenzel new infections in Bavaria, intensified hepatitis E surveillance et al., 2011). The lower limit of detection (95% LoD) for was conducted in 2017. The project had three major objec- HEV RNA was 1200 IU/ml. tives: 1. to describe the population diagnosed with hepatitis E in Bavaria, 2. to identify the most common subgenotypes Sequencing of HEV RNA of HEV circulating in Bavaria, 3. attempt to identify the risk factors associated with transmission of HEV genotype 3. HEV RT-qPCR positive samples were further character- ized by amplicon sequencing. The initial amplification was performed by using specific primers for a fragment Materials and Methods of HEV-ORF1 (418 nt, FJ705359 pos. 54-471) (Wenzel Data Collection of Hepatitis E Cases The number 558 include all HEV cases reported within the study The project “Intensified Hepatitis E Surveillance in Bavaria” period, from the third calendar week of 2017 until the fourth calen- was conducted from the third calendar week of 2017 until dar week of 2018, without consideration of the reference definition of the fourth calendar week of 2018. The project included a RKI. The reference definition of RKI requires the presence of symp- specially designed questionnaire, collection of stool or toms and laboratory diagnosis. 1 3 Food and Environmental Virology (2021) 13:337–346 339 All laboratory using CodonCode Aligner software ( http:// codon- code. confirmed infec- com/). tions reported to LGL* (n=558) Phylogenetic Analyses A maximum likelihood phylogenetic consensus tree for HEV patients partial HEV-ORF1 and RdRp nucleotide sequences was Food samplessent Stool andserum filledout the to LGL* (n=6) sample(n=145) questionnaire inferred using RAxML version 8.2.11 (available at: https:// (n=167) sco.h- its. org/ exeli xis/ web/ softw are/ raxml). The sequences Patients with derived from the patients’ samples were put into context <80%incom- pletequestion- with the published reference sequences for HEV subtypes naires (n=5) which are currently known to infect humans (Smith et al., Patients withcom- 2020). All negative (n=6) plete >80%ques- tionnaires (n=162) Population from the DEGS1 Study Patients traveled to non-3genotype Stool/serum endemic countries negative (n=122) within incubation In addition to the recruited HEV cases, a dataset from the period,and PCR Robert Koch Institute (RKI, Berlin) was used in order to negative (n=27) compare alimentary habits of Bavarian hepatitis E cases in 2017 with the general healthy adult population of Germany Numberof participating in the RKI survey. This survey was conducted Genotyped questionnaires stooland serum availablefor between November 2008 and December 2011 called “Study samples, all descriptive belonged to on adult health in Germany” (DEGS1) (DEGS1,  2015). analysis (n=135) genotype3 (n=23) Details about this study are described elsewhere (Scheidt- Nave et al., 2012; Gößwald et al., 2012). Briefly, the study included randomly selected individuals living in Germany between 18 and 79 years, recruited randomly from residents’ Genotyped registration offices. The data collection included interviews stool/serum sample and filled with detailed Food Frequency Questionnaire (FFQ) and out question- naire (n=14) additional measurements and tests carried out in participat- ing health care facilities. A total of 7.009 participants com- pleted the FFQ. Inclusion criteria for our study included Fig. 1 Study population. Bavarian Public Health and Food Safety Authority living in western federal states as federal district per par- ticipant was not available ( n = 4.785 ) and no diagnosis of hepatitis in the past (hepatitis A, B, C, D, E or unknown type) ( n = 4.512). Statistical Methods et al., 2011). Moreover, a fragment of the RNA-dependent RNA polymerase (RdRp, 470 nt, FJ705359 pos. 4181- Descriptive analysis for Bavaria’s hepatitis E cases was 4650) was amplified according to a published protocol performed and the absolute and relative frequencies were (Johne et  al., 2010). The nested PCRs were performed reported. T test and ANOVA were used to test if the differ - with specific primers for HEV-ORF1 (286 nt, FJ705359 ences in the mean age between women and men are signifi- pos. 102-387) and RdRp (332 nt, FJ705359 pos. 4286- cant. Chi Square Test was used to investigate the associa- 4617), respectively. The PCR products were purified by tion between variables with two categories. In addition, the using QIAquick columns (Qiagen, Hilden, Germany) and detailed questionnaire for HEV cases and the FFQ ques- sequenced in both directions with the nested PCR prim- tionnaire completed by the DEGS participants contained ers. Nucleotide sequences of amplicons were determined 6 identical questions on dietary habits and 2 questions on by using the BigDye Terminator cycle sequencing kit pet husbandry. On the basis of these 8 questions the two (Applied Biosystems) and separated on a model 3730xl populations, HEV cases ( n = 135 ) and baseline population genetic analyzer (Applied Biosystems, Waltham, USA). ( n = 4.512 ), could be compared. A univariable analysis with Nucleotide sequences of PCR products were analysed by a total of 8 variables was performed by calculating odds ratios (OR) and 95% confidence intervals (CIs). Thereafter 1 3 340 Food and Environmental Virology (2021) 13:337–346 a logistic regression was conducted. Considering the AIC Table 1 Descriptive analysis of hepatitis E cases as selection criterion for the best model, we decided to keep Variable All cases ( n = 135) % the full model (variables included see Table 2) as it resulted Symptoms & previous diseases Yes in the smallest AIC. In addition, we wanted our final model Symptoms 79 58.5 to include age and sex variables, regardless of significance, Elevated liver enzymes 76 56.3 in order to take into account possible confounding factors. Upper abdominal pain 38 28.2 The odds ratios, adjusted for gender and age, were reported. Icterus 24 17.8 The analysis was performed with SAS University Edition. Fever 22 16.3 Liver disease 21 15.6 Fatty liver 15 11.1 Immunosuppression 12 8.9 Results Liver infection 3 2.2 Hepatitis C 2 1.5 Descriptive Analysis of Bavaria’s Hepatitis E cases Blood transfusion 1 0.7 Liver cirrhosis 1 0.7 The questionnaire was filled out by 167 patients with hepa- Meat consumption Yes titis E, which corresponds to 30% of all reported HEV cases Pork on piece 126 94.7 in Bavaria during the study period. A total of 5 question- Raw ham 120 89.6 naires were filled out less than 80% and therefore excluded Beef not fully cooked 68 51.5 from further evaluation. (Fig.  1). Out of the remaining 162 Wild boar 35 26.1 patients, 35 had travelled abroad. Eight of them visited Pig liver 33 24.6 countries where genotype 3 is prevalent, during the relevant Meat products consumption Yes incubation period. 27 patients visited countries, where other Salami 131 97.0 or unknown HEV genotypes are endemic (e.g. Singapore, Cooked sausage 128 95.5 Nicaragua, Namibia) . Stool samples were available from Liver pate 98 73.1 17 of these 27 patients, but HEV genotype 3 could not be Other risk factor consumption Yes detected by RT-qPCR. As our aim was to focus on risk fac- Fish 123 91.8 tors associated with transmission of HEV genotype 3, these Raw vegetable 121 90.3 27 were not considered in the further analysis. In total, 66 Raw fish 40 30.1 women and 69 men were included in the study. The mean Grill party Yes age was 46 years for women (range 20–74 years) and 47.5 Visited grill party 63 46.7 years for men (range 20–85 years). (Fig. 2). There was no Consume pork/game 55 87.3 significant difference in the mean age between women and Prepared pork/game 44 69.8 men (t test and ANOVA). Pets Yes There were 79 (58%) cases with symptoms. The most Cat 39 28.9 frequent ones were: increased liver values (56%), upper Dog 20 14.8 Rabbits 5 3.7 Other rodents 2 1.5 Direct contact to animals Yes Live/work on a farm 8 5.9 Working in butcher shop 2 1.5 Water contact Yes Wastewater 4 3.0 River/lake 3 2.2 Fountain 3 2.2 abdominal pain (28%), jaundice (17%) and fever (16%). Other symptoms recorded by the patients were fatigue, itching, nausea, aching limbs, muscles and legs. No asso- ciation between gender and the presence of symptoms (Chi Square Test) was detected. One patient reported having Fig. 2 Gender and age distribution of the Bavarian hepatitis E cases 1 3 Food and Environmental Virology (2021) 13:337–346 341 received a blood transfusion during the incubation period. the questionnaire. Nevertheless all available samples were Three patients had a chronic liver disease, and two of them included in the subgenotype analysis. Most of the human reported Hepatitis C. Twenty-one patients reported other samples ( n = 122 ) were negative for HEV RNA by RT- liver disease among which the most common was fatty liver qPCR. A total of 21 stool samples and 2 serum samples were disease ( n = 16 ) (Table 1). genotyped (GenBank accession no. HG998145-HG998188), The specially designed questionnaire contains several 14 of them with an accompanying completed questionnaire. items on the consumption of potential HEV risk food prod- All samples were classified as HEV genotype 3 (Fig.  3). The ucts such as various meat types, cold cuts and sausages. Four most commonly found subgenotype was 3c ( n = 16 ) , fo l- patients indicated to eat halal, one kosher and one vegetar- lowed by 3e ( n = 3 ), 3a ( n = 1 ), 3f ( n = 1 ) and 3 not further ian. The only person who said he was eating kosher also specified ( n = 2 ). Six food samples such as minced meat, checked that he was eating halal. After the check for answer “Kaminwurzen” (smoked pork sausages), beef meat and consistency there were no strictly vegetarian, no strictly deer goulash were collected and tested. All were HEV RNA kosher and three real halal eating patients. Notable is that negative (Fig. 1). 91% of the cases ( n = 123 ) reported having consumed raw or poorly cooked meat or raw mussels or raw fish at least 1 to 3 times per month during the incubation period. Comparison of the HEV cases and the DEGS1 population Human and Food Samples In the DEGS population included in the study ( n = 4.512 ), A total of 145 human samples (143 stools and 2 sera), were 48% of the participants were male ( n = 2.156 ) and 52% tested for the presence of HEV RNA. There were 39 patients female ( n = 2.356 ). The mean age overall was 57.2 range that provided stool or serum samples but did not fill out (24–88), with no significant difference between the sexes (ANOVA and T test). Thus the DEGS participants were con- siderably older than the hepatitis E cases reported in Bavaria in 2017 ( average age = 46.8 years ). The difference in age between the two populations is also significant (T test), while Not detected (n=122) no difference was observed regarding the sex of the partici- pants (Chi-square test). In addition to age and gender, 8 risk factors were tested in the univariable analysis. Six of them 3f (n=1) were about nutrition habits and two risk factors considered 3e (n=3) 3a (n=1) the possession of dogs or cats as pets. The consumption of 3 (n=2) sausages (like salami, liver sausages, raw spread sausages), cooked fish (e.g. pollack, trout) were significantly associated 3c (n=16) with hepatitis E. (Table  2). Owning a cat is significantly associated with hepatitis E, as opposed to owning a dog. Other variables not significantly associated with hepatitis Fig. 3 Detected subgenotypes E infection were the consumption of meat (e.g. pig, wild boar, deer) and raw ham. The variable consumption of raw Table 2 Risk factors (NA—the Risk factors Cases Baseline Univariable analysis Multivariable effect of the variable is not ( n = 135) population analysis significant in the Multivariable ( n = 4.512) Analysis, **Corresponds to significance level 5%, Variable Yes % Yes % Odds ratio 95% CI Odds ratio 95% CI ***Corresponds to significance Sausages consumption (no ham) 133 98.5 4.037 89.7 7.7*** 1.9–31.1 9.6** 1.3–70.1 level 1%) Cat possession 39 31.2 676 15.4 2.5*** 1.7–3.7 1.9*** 1.3–3.0 Fish consumption (cooked) 123 91.8 3.811 84.8 2.0** 1.1–3.7 2.2** 1.1-4.4 Raw vegetable consumption 121 90.3 4.245 95.3 0.5** 0.3–0.8 0.4** 0.2–0.8 Ham consumption 120 89.6 3.828 85.0 1.5 0.9–2.6 NA Meat consumption (no chicken, 131 97.0 4.326 96.2 1.3 0.5–3.6 NA no sausages) Dog possession 20 16.1 608 13.9 1.2 0.7–1.9 NA Vegetarian diet 1 0.8 185 4.1 0.2 0.0–1.3 NA 1 3 342 Food and Environmental Virology (2021) 13:337–346 vegetable seems to be a significant protective factor for hepa- Risk Factors Associated with Food Consumption titis E infection. To the best of our knowledge no study in Europe investi- gated fish consumption as a risk factor for transmission of Discussion autochthonous Hepatitis E. HEV genotype 3 was detected in 32.2% (10 of 31) dolphins at the National Aquarium, Bavarian Hepatitis E Cases and Risk Factors Havana, Cuba. Dolphins generally feed on fish and squid. Infected animals or contaminated surface waters could During 2017 an intensified hepatitis E surveillance was be the source of infection (Villalba et al., 2017). Other conducted in Bavaria. This included a detailed question- seafood such as diverse shellfish—bivalve in Japan (Li naire, stool and serum samples, and food investigations et al., 2007), mussels in UK (Crossan et al., 2012), Spain from notified hepatitis E cases. A total of 135 question- (Mesquita et al., 2016), Italy (La Rosa et al., 2018) and naires, 145 human samples and 6 food samples were inves- 2.9% of shellfish purchased in local supermarkets in Scot- tigated in order to gain deeper understanding of the risk land (O’Hara et al., 2018) have been tested positive for factors for HEV transmission. The recruited hepatitis E HEV genotype 3. An HEV outbreak on a cruise ship was cases from Bavaria ( n = 135 ) were compared with DEGS linked to the consumption of shellfish on board (Said et al., study population from West federal states in Germany 2009). In Italy, apart from the 2.6% HEV positive mussels, ( n = 4.512 ) by univariable and multivariable analyses. 12.8% seawater samples (La Rosa et al., 2018) and raw Case reports and series from Europe suggest that HEV sewage and river samples (Iaconelli et al., 2015) were also genotype 3 is more common in elderly men (Festa et al., tested positive for HEV genotype 3. Since HEV genotype 2014; Dalton et  al., 2008; Chalupa et  al., 2014; Saint- 3 is an enteric pathogen in both human and diverse animal Jacques et al., 2016; Tarantino et al., 2016; Borgen et al., species, human and animal faeces could contaminate the 2008). However, seroprevalence studies are not very con- sewage and coastal water. In countries with high farming clusive. One study concluded that the older (30–39) par- density and shellfish production close to the shore, shell- ticipants and male participants were more often positive for fish can bio-accumulate human and animal enteric viruses anti-HEV IgG than younger (20–29) or female participants (Mesquita et al., 2016; Grodzki et al., 2014). As shellfish in southern Germany. Since the study comprised only young is often consumed raw or undercooked, the virus has not participants aged 20–39, it is unclear whether this trend been deactivated and can pose a risk for HEV infection. holds true also among the older population (Mahrt et al., Fish could also be consumed raw or cooked thoroughly; 2018). In another larger study, with adults aged 18–79 years however, it is not known whether bio-accumulation, sim- from all federal states in Germany no gender difference in ilar to shellfish, can happen in fish. The cutthroat trout the HEV seroprevalence was reported, but an increase in virus (CTV), a virus initially isolated in 1988 shows seroprevalence with age. The authors put forward the idea between 29 and 49% genome sequence similarity to HEV that food frequently consumed by both sexes plays a major genotypes 1–4, rat HEV and avian HEV. Since 2010, a role as a channel of transmission (Faber et al., 2012). The related agent was described from related salmonid fish authors of a systematic review suggested that the higher species. The presence of CTV was confirmed in differ - incidence of clinical cases in men indicates rather gender ent trout species (Johne et al., 2014; Smith et al., 2014). differences in the development of diseases or the use of Recently, the International Committee on Taxonomy of laboratory tests and not difference in infection rates (Lewis Viruses (ICTV) gave a new classification for the family et al., 2010). Our results are in line with Faber et al. (2012), Hepeviridae. All HEV’s have been placed under Hepeviri- since both male and female participants of the study were dae family and further classified under 2 genera namely almost equally represented; however, both male and females Orthohepevirus, which included isolates from mammals were also equally affected with symptoms in our study and, and chicken and Piscihepevirus, containing the fish HEV therefore, not confirming the higher manifestation rates (Purdy et al., 2017). However, the zoonotic potential of among males. However, we had a lower percentage of par- CTV has not yet been clarified and its influence on the ticipation in the study from the population over 60. Among epidemiology of human hepatitis E is largely unknown all cases reported in Bavaria during this period ( n = 558 ), (Johne et al., 2014). Therefore, it is not clear whether we 27% were over 60 years old, while in our study, only 17% see the effect of this HEV-like virus as a risk factor for were over 60 year old. This could indicate that older people HEV infection in humans. Further research is needed to are unable or less motivated to complete the detailed ques- investigate whether fish consumption can be a risk factor tionnaire and therefore less likely to participate in the study. for hepatitis E genotype 3. 1 3 Food and Environmental Virology (2021) 13:337–346 343 In our study the sausages, like salami and liver sausages, that people who consume a lot of vegetables consume less were significantly associated with hepatitis E with OR 9.6 meat. However, this is not conclusive and vegetable-based in multivariable analysis. This is in accordance with other diet is not necessarily excluding a HEV infection, as HEV studies from Germany (Faber et al., 2018; Szabo et al., 2015) has already been detected in vegetables, ready-to-eat salads and other European countries (Di Bartolo et al., 2015; Pavio and irrigation water (Purpari et al., 2019; Terio et al., 2017; et al., 2014; Berto et al., 2013; Di Bartolo et al., 2012; Mooij Kokkinos et al., 2017). et al., 2018; Moor et al., 2018). During the research period six food samples (minced Hepatitis E RNA was detected in several studies in meat, Kaminwurzen—smoked pork sausages, beef meat Europe in different food products such as figatelli (30–58%) and deer goulash) from households of notified hepatitis E (Colson et al., 2010; Pavio et al., 2014), pork liver sausages cases were available for laboratory testing. All were negative (6–29%) (Di Bartolo et al., 2012; Berto et al., 2012; Pavio for HEV RNA. It is possible that these food items were not et al., 2014; Szabo et al., 2015), quenelles (25%) and dried the source of infection, but other food that were no longer salted liver (3%) (Pavio et al., 2014). All HEV sequences available for testing. obtained in these studies were of genotype 3. However, the human HEV infection dose is unknown and Risk Factor Pet Ownership it needs to be investigated whether the viral load in meat and ready-to-eat products is sufficient to infect humans. Our results suggest that possession of a cat is significantly One experiment aiming to answer this question was done associated with HEV infection as opposed to ownership of with pigs. Two out of three pigs were successfully infected dogs. Both cats and dogs have been found seropositive for with HEV genotype 3 after ingesting an inoculum of 10 mL HEV in a study from Brandenburg, Germany (Dähnert et al., with 10 genome equivalents per mL (Andraud et al., 2013). 2014). While another study from Germany found that the Another study estimated that figatelli (pig liver sausage possession of pets was a protective factor for hepatitis E and 3 6 from France, commonly eaten uncooked) contains 10 –10 cat ownership was the most protective factor (Wichmann HEV RNA copies per slice (Colson et al., 2010). Figatelli et al., 2008). The authors doubted this and suggested that had already been described as a risk factor for HEV (Capai due to the frequent preparation of raw liver as cat food, a et al., 2019) and had been identified as the cause of infec- pre-existing immunity could exist due to a subclinical infec- tion in case reports and series (Colson et al., 2010; Moal tion among cat owners. Similarly, it was described that cats et al., 2012), suggesting that the infectious dose for humans fed with kitchen leftovers have a higher risk of HEV sero- is comparable to the experimentally proven infectious dose positivity than cats fed with commercial food, suggesting for pigs. In addition, the virus shows relatively high stabil- a common source for HEV infection for both animals and ity in order to be completely inactivated, the meat must be humans (Liang et al., 2014). A case report from Japan also cooked at 71 degrees for 20 minutes (Barnaud et al., 2012). described a patient infected with HEV genotype 4, whose cat Therefore, there is a potential risk to public health consum- was positive for HEV antibodies (Kuno et al., 2003). Rodent ing raw and undercooked products. hunting by cats has been suggested as a possible source of In our analysis the consumption of meat (no chicken and no infection (Mochizuki et al., 2006); however, the literature is sausages) was not a significant risk factor for HEV infection, not consistent as to whether rats are competent hosts of HEV as opposed to findings from other studies and case reports genotype 3 (Shukla et al., 2011; Purcell et al., 2011; Johne (Faber et al., 2018; Wichmann et al., 2008; Slot et al., 2017; et al., 2010; Lack et al., 2012). Legrand-Abravanel et al., 2010; Riveiro-Barciela et al., 2015; Interestingly, in our study women with cat ownership Rivero-Juarez et al., 2017). This might be due to the fact, that have a higher risk (OR 2.7) for hepatitis E than men with cat the question was stated very general in DEGS questionnaire. ownership, and women in the age group over 70 years have Similarly, because no detailed information was available on an even higher risk (OR 9.6). It is tempting to hypothesize pork and pork liver consumption in the questionnaire com- whether women living in a shared household are more likely pleted by the DEGS study population, it was not possible to to care for the pets (feeding and cleaning) and are therefore analyse it separately, although both have been identified as more exposed and have a higher risk of HEV infection. How- risk factors in the literature (Wenzel et al., 2011; Lewis et al., ever, this hypothesis needs further investigation and fuels an 2010; Faber et al., 2018). Our study indicated that there was interesting research aspect that should be considered in the no significant association between consumption of raw ham design of future hepatitis E research studies. and HEV infection. This is in line with the results from case- control study in Germany (Faber et al., 2018), but opposed Limitations of the Study to the findings from England and Wales (Said et al., 2017). The consumption of raw vegetables resulted as some kind The cases analysed in our study are from 2017 and no con- of protective factor for HEV infection. One might speculate trols were recruited. The comparison is based on data from 1 3 344 Food and Environmental Virology (2021) 13:337–346 the DEGS study, which are only partially comparable. DEGS References participants were recruited from 2008 till 2011. To check the Andraud, M., Dumarest, M., Cariolet, R., Aylaj, B., Barnaud, E., Eono, comparability of the healthy population (DEGS study) and F., et al. (2013). Direct contact and environmental contaminations the cases, we looked for trends in the consumption behav- are responsible for hev transmission in pigs. Veterinary Research, iour of the German population. According to the National 44(1), 102. Nutrition Survey (NVS) II and the so-called NEMONIT Barnaud, E., Rogée, S., Garry, P., Rose, N., & Pavio, N. (2012). Ther- mal inactivation of infectious hepatitis E virus in experimentally study, there were no significant changes in the consump- contaminated food. Applied Environmental Microbiology, 78(15), tion of vegetables, meat/meat products and fish/fish products 5153–5159. between 2005–2007 and 2012/2013 (Gose et al., 2016). We Berto, A., Grierson, S., Hakze-van der Honing, R., Martelli, F., Johne, assume that this trend continued further. The DEGS Popu- R., Reetz, J., et al. (2013). Hepatitis E virus in pork liver sausage, France. Emerging Infectious Diseases, 19(2), 264–266. lation is considered healthy, i.e. without HEV infection, we Berto, A., Martelli, F., Grierson, S., & Banks, M. (2012). Hepatitis E had no access to information about IgG or IgM tests in the virus in pork food chain, United Kingdom, 2009–2010. Emerging available dataset, although a subset of the dataset was used Infectious Diseases, 18(8), 1358–1360. and published for a HEV seroprevalence study (Faber et al., Borgen, K., Herremans, T., Duizer, E., Vennema, H., Rutjes, S., Bos- man, A., et al. (2008). Non-travel related hepatitis E virus geno- 2012). We have relied on the information provided by the type 3 infections in the Netherlands; a case series 2004–2006. participants in the questionnaire. Therefore, it is possible BMC Infectious Diseases, 8, 61. that, if there were some asymptomatic acute hepatitis E Capai, L., Masse, S., Gallian, P., Souty, C., Isnard, C., Blanchon, T., cases or cases with HEV IgG (had the disease in the past) in et al. (2019). Seroprevalence study of anti-HEV IGG among dif- ferent adult populations in Corsica, France, 2019. Microorgan- the DEGS population, the actual impact of the risk factors isms, 7(10), 461. would be even greater if they were excluded. The results pre- Chalupa, P., Vasickova, P., Pavlik, I., & Holub, M. (2014). Endemic sented here must be interpreted carefully. Firstly, the cases hepatitis E in the Czech Republic. Clinical Infectious Diseases, and controls used for the statistical analysis were surveyed 58(4), 509–516. Chaussade, H., Rigaud, E., Allix, A., Carpentier, A., Touzé, A., Delz- using different questionnaires, and although risk factors such escaux, D., et al. (2013). Hepatitis E virus seroprevalence and risk as sausages, salami and liver sausages are in line with pub- factors for individuals in working contact with animals. Journal lished research, fish consumption and cat ownership must be of Clinical Virology, 58(3), 504–508. carefully interpreted and considered as possible risk factors Clemente-Casares, P., Ramos-Romero, C., Ramirez-Gonzalez, E., & Mas, A. (2016). Hepatitis E virus in industrialized countries: The in future studies and study designs. silent threat. Biomed Research International. Colson, P., Borentain, P., Queyriaux, B., Kaba, M., Moal, V., Gallian, Acknowledgements This work was supported by the Robert Koch P., et al. (2010). Pig liver sausage as a source of hepatitis E virus Institute and the German Federal Ministry of Health (Grant No. 1369– transmission to humans. Journal of Infectious Diseases, 202(6), 386 to J. Wenzel). 825–834. Crossan, C., Baker, P. J., Craft, J., Takeuchi, Y., Dalton, H. R., & Sco- Funding Open Access funding enabled and organized by Projekt bie, L. (2012). Hepatitis E virus genotype 3 in shellfish, United DEAL. Kingdom. Emerging Infectious Diseases, 18(12), 2085–2087. Dähnert, L., Conraths, F. J., Reimer, N., Groschup, M. H., & Eiden, Data Availability Statement All data about the HEV cases are avail- M. (2014). Molecular and serological surveillance of hepatitis E able from the corresponding author or from durdica.marosevic@lgl. virus in wild and domestic carnivores in Brandenburg, Germany. bayern.de on reasonable request. The DEGS1 Dataset used to support Transboundary and Emerging Diseases, 65(5), 1377–1380. the findings of this study can be requested upon application from the Dalton, H. R., Stableforth, W., Thurairajah, P., Hazeldine, S., Rem- Research Data Centre (Robert Koch Institute, MF4) narace, R., Usama, W., et al. (2008). Autochthonous hepatitis E in Southwest England: Natural history, complications and sea- sonal variation, and hepatitis E virus IGG seroprevalence in blood Open Access This article is licensed under a Creative Commons Attri- donors, the elderly and patients with chronic liver disease. Euro- bution 4.0 International License, which permits use, sharing, adapta- pean Journal of Gastroenterology & Hepatology, 20(8), 784–790. tion, distribution and reproduction in any medium or format, as long DEGS1. (2015). German Health Interview and Examination Survey as you give appropriate credit to the original author(s) and the source, for Adults, (DEGS1). Robert Koch Institute, Department of Epi- provide a link to the Creative Commons licence, and indicate if changes demiology and Health Monitoring. were made. The images or other third party material in this article are Di Bartolo, I., Angeloni, G., Ponterio, E., Ostanello, F., & Ruggeri, F. included in the article’s Creative Commons licence, unless indicated M. (2015). Detection of hepatitis E virus in pork liver sausages. otherwise in a credit line to the material. If material is not included in International Journal of Food Microbiology, 193, 29–33. the article’s Creative Commons licence and your intended use is not Di Bartolo, I., Diez-Valcarce, M., Vasickova, P., Kralik, P., Hernandez, permitted by statutory regulation or exceeds the permitted use, you will M., Angeloni, G., et al. (2012). Hepatitis E virus in pork produc- need to obtain permission directly from the copyright holder. To view a tion chain in Czech Republic, Italy, and Spain, 2010. Emerging copy of this licence, visit http://cr eativ ecommons. or g/licen ses/ b y/4.0/ . 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Update: Proposed reference sequences for subtypes of hepatitis E virus (species orthohepevirus a). Journal of General Virology, 101(7), 692–698. 1 3 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Food and Environmental Virology Springer Journals

Epidemiology of Hepatitis E in 2017 in Bavaria, Germany

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Springer Journals
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Copyright © The Author(s) 2021
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1867-0334
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1867-0342
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10.1007/s12560-021-09474-0
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Abstract

In the last decade, the number of reported hepatitis E virus (HEV) infections in Germany, including Bavaria, has continued to rise. In order to identify risk factors associated with HEV infection, we investigated notified hepatitis E cases from Bavaria during 2017. The project “Intensified Hepatitis E Surveillance in Bavaria” included interviews with questionnaires, collection and genotyping of stool, serum and food samples. In addition, certain risk factors were examined in a sample comparison with healthy population using univariable analysis and logistic regression. In total, 135 hepatitis E cases from Bavaria were included in the analysis. Mean age for women was 46 (range 20–74) years and 47.5 (range 20–85) for men. 56 of the cases (41.5%) were asymptomatic. Among the symptomatic cases, both men and women were equally affected with symptoms like fever (16.3%), jaundice (18.8%) and upper abdominal pain (28.2%). 145 human samples (serum, stool) and 6 food samples were collected. 15.9% of the human samples (n = 23) were positive for HEV RNA by reverse-transcription quantitative real-time PCR (RT-qPCR). Identified risk factors significantly associated with hepatitis E were sausage consumption with odds ratio 9.6 (CI 1.3–70.1), fish with OR 2.2 (CI 1.1–4.4) and cat ownership with OR 1.9 (CI 1.3–3.0) in multivariable analyses. Further investigation is needed to confirm the role of fish in HEV transmission. Autochthonous HEV genotype 3 is prevalent in Bavaria and there could be more transmission routes contributing to the spread of HEV than previously known. Undercooked meat, offal, sausages, fish, shellfish and contact with animals and pets are possible sources for infection. Keywords Hepatitis E · HEV · Genotype 3 · Risk factors · Transmission Introduction subgenotypes, respectively (Smith et al., 2020). The clinical manifestation of an HEV infection varies in severity from Hepatitis E virus (HEV) is a non-enveloped, single-stranded subclinical to fulminant, probably, depending on the geno- RNA virus that belongs to the Hepeviridae family and type and host factors (e.g. immune constitution). Genotypes causes infectious inflammation of the liver. Human patho- 1 and 2 are endemic in developing countries in Asia and genic HEV can be classified in four major genotypes (HEV Africa, as well as in Mexico. They are restricted to humans, 1–4), that can further be classified into subgenotypes. Gen- mostly transmitted through the faecal-oral route and causing otypes 1, 2, 3 and 4 count seven, two, fourteen and nine major outbreaks in tropical and subtropical regions (Khuroo and Khuroo, 2016; Rein et al., 2012). Genotype 3 has been found mainly in European countries, USA and Japan, while genotype 4 is mostly spread in Asia (China, Japan and India) * K. Hriskova katerinahriskova@yahoo.de (Pavio et al., 2010). Genotypes 3 and 4 are zoonoses and have been detected in several animal species (e.g. pigs, wild Institute for Medical Information Processing, Biometry boar, deer and rabbits) (Meng, 2013) and humans. These and Epidemiology - IBE, LMU Munich, Marchioninistr. 15, HEV genotypes can cause sporadic, self-limiting disease 81377 Munich, Germany with clinical symptoms like vomiting, uncolored stools, Pettenkofer School of Public Health, Munich, Germany darkened urine and jaundice (Nan et al., 2017). According Bavarian Public Health and Food Safety Authority (LGL), to the German Infection Protection Act (IfSG) clinical sus- Veterinärstraße 2, 85764 Oberschleißheim, Germany picion, illness or death from acute viral hepatitis and/-or National Consultant Laboratory for HAV and HEV, the direct or indirect detection of hepatitis E virus must be Institute of Clinical Microbiology and Hygiene, University notified. Medical Centre Regensburg, Franz-Josef-Strauß-Allee 11, 93053 Regensburg, Germany Vol.:(0123456789) 1 3 338 Food and Environmental Virology (2021) 13:337–346 Since the introduction of IfSG in 2001, until 2014 the serum samples from patients and food samples if available. HEV incidence per 100.000 inhabitants in Germany was A total of 558 hepatitis E cases were reported to the Local below 1. In recent years, however, the number of reported Health Authorities (LHA) during the study period. The diag- hepatitis E cases has substantially increased. In Bavaria nostic methods for these 558 cases were IgM blood (n = and also nationwide (RKI, 2018), the number of new hep- 530), PCR blood (n = 46) and PCR stool (n = 15), where atitis E cases almost doubled in 2017 compared to 2016 some cases were diagnosed by two methods. The participat- (LGL, 2019). It is uncertain whether the growing number ing health authorities informed the reported cases about the of reported cases is due to an actual increase in the num- study and invited them to join the study. The participation ber of infected humans or due to a diagnostic and notifica- in the study was anonymous and voluntary. Written consent tion biases. Over the last few years, the disease has come was obtained from all recruited participants. Stool sam- into sharp focus as it is no longer perceived only as a travel ples were analysed at the National Consultant Laboratory associated disease. Similar to other European countries, the for Hepatitis A and Hepatitis E Virus, Institute of Clinical autochthonous genotype in Germany is the genotype 3. The Microbiology and Hygiene, Regensburg. If available, food prevalence of antibodies against HEV in the adult population samples from patients’ households, suspected to be risk fac- of Germany is 16.8% (about 5% in people under 30 years of tor for HEV, were collected and analysed at the Laboratory age, up to 25% in those over 60 years of age) (Faber et al., for Food Virology of the Bavarian Public Health and Food 2012). Eating raw or uncooked meat, oa ff l and meat products Safety Authority (LGL) in Erlangen. Inclusion criteria for (e.g. sausages) were considered as a risk factor for hepatitis descriptive analysis of HEV cases were notified hepatitis E in humans (Wichmann et al., 2008; Szabo et al., 2015; E cases with laboratory confirmed HEV infection (IgM or Faber et al., 2018; Said et al., 2017; Meng, 2010). Other PCR positive) with at least 80% completed questionnaire reported risk factors associated with hepatitis E are the con- and no travel within the incubation period to HEV genotype sumption of shellfish ( e.g. oysters, mussels) (Grodzki et al., 1, 2 or 4 endemic countries, according to a resent review 2014; O’Hara et al., 2018; Said et al., 2009; Mesquita et al., article Perez-Gracia et al (2015) (Fig. 1). Plausibility check 2016) and the contact with waste waters (Clemente-Casares, was performed to verify answer consistency. All available 2016). Bavaria is a federal state with a high density of pigs sequenced serum and stool samples were included in the and high seroprevalence for HEV in pig herds. On aver- subgenotype analysis irrespective of the completeness of the age 51.4% of tested pig sera samples in Southern Germany questionnaire. (Bavaria  and Baden-Wuerttemberg) exhibited anti-HEV antibodies (Krumbholz et al., 2013). Direct contact with Laboratory Confirmation and Genotyping of HEV pigs is considered a risk factor for hepatitis E (Chaussade et al., 2013; Krumbholz et al., 2014). Furthermore, IgM and RNA Extraction and Molecular Detection IgG antibodies against HEV were detected further up the food chain in serum and meat juice samples from Bavarian Nucleic acid isolation from human specimens was per- slaughterhouses (Wacheck et al., 2012). In addition, 4% of formed from 200 μl serum or stool suspension on an EZ1 pork livers purchased from butchers in Regensburg, Bavaria, Advanced XL workstation using the EZ1 Virus Mini Kit had detectable levels of HEV RNA (Wenzel et al., 2011). v2.0 (Qiagen, Hilden, Germany). Eluted nucleic acid was Based on the above evidence and the increasing number of analysed by RT-qPCR as previously described (Wenzel new infections in Bavaria, intensified hepatitis E surveillance et al., 2011). The lower limit of detection (95% LoD) for was conducted in 2017. The project had three major objec- HEV RNA was 1200 IU/ml. tives: 1. to describe the population diagnosed with hepatitis E in Bavaria, 2. to identify the most common subgenotypes Sequencing of HEV RNA of HEV circulating in Bavaria, 3. attempt to identify the risk factors associated with transmission of HEV genotype 3. HEV RT-qPCR positive samples were further character- ized by amplicon sequencing. The initial amplification was performed by using specific primers for a fragment Materials and Methods of HEV-ORF1 (418 nt, FJ705359 pos. 54-471) (Wenzel Data Collection of Hepatitis E Cases The number 558 include all HEV cases reported within the study The project “Intensified Hepatitis E Surveillance in Bavaria” period, from the third calendar week of 2017 until the fourth calen- was conducted from the third calendar week of 2017 until dar week of 2018, without consideration of the reference definition of the fourth calendar week of 2018. The project included a RKI. The reference definition of RKI requires the presence of symp- specially designed questionnaire, collection of stool or toms and laboratory diagnosis. 1 3 Food and Environmental Virology (2021) 13:337–346 339 All laboratory using CodonCode Aligner software ( http:// codon- code. confirmed infec- com/). tions reported to LGL* (n=558) Phylogenetic Analyses A maximum likelihood phylogenetic consensus tree for HEV patients partial HEV-ORF1 and RdRp nucleotide sequences was Food samplessent Stool andserum filledout the to LGL* (n=6) sample(n=145) questionnaire inferred using RAxML version 8.2.11 (available at: https:// (n=167) sco.h- its. org/ exeli xis/ web/ softw are/ raxml). The sequences Patients with derived from the patients’ samples were put into context <80%incom- pletequestion- with the published reference sequences for HEV subtypes naires (n=5) which are currently known to infect humans (Smith et al., Patients withcom- 2020). All negative (n=6) plete >80%ques- tionnaires (n=162) Population from the DEGS1 Study Patients traveled to non-3genotype Stool/serum endemic countries negative (n=122) within incubation In addition to the recruited HEV cases, a dataset from the period,and PCR Robert Koch Institute (RKI, Berlin) was used in order to negative (n=27) compare alimentary habits of Bavarian hepatitis E cases in 2017 with the general healthy adult population of Germany Numberof participating in the RKI survey. This survey was conducted Genotyped questionnaires stooland serum availablefor between November 2008 and December 2011 called “Study samples, all descriptive belonged to on adult health in Germany” (DEGS1) (DEGS1,  2015). analysis (n=135) genotype3 (n=23) Details about this study are described elsewhere (Scheidt- Nave et al., 2012; Gößwald et al., 2012). Briefly, the study included randomly selected individuals living in Germany between 18 and 79 years, recruited randomly from residents’ Genotyped registration offices. The data collection included interviews stool/serum sample and filled with detailed Food Frequency Questionnaire (FFQ) and out question- naire (n=14) additional measurements and tests carried out in participat- ing health care facilities. A total of 7.009 participants com- pleted the FFQ. Inclusion criteria for our study included Fig. 1 Study population. Bavarian Public Health and Food Safety Authority living in western federal states as federal district per par- ticipant was not available ( n = 4.785 ) and no diagnosis of hepatitis in the past (hepatitis A, B, C, D, E or unknown type) ( n = 4.512). Statistical Methods et al., 2011). Moreover, a fragment of the RNA-dependent RNA polymerase (RdRp, 470 nt, FJ705359 pos. 4181- Descriptive analysis for Bavaria’s hepatitis E cases was 4650) was amplified according to a published protocol performed and the absolute and relative frequencies were (Johne et  al., 2010). The nested PCRs were performed reported. T test and ANOVA were used to test if the differ - with specific primers for HEV-ORF1 (286 nt, FJ705359 ences in the mean age between women and men are signifi- pos. 102-387) and RdRp (332 nt, FJ705359 pos. 4286- cant. Chi Square Test was used to investigate the associa- 4617), respectively. The PCR products were purified by tion between variables with two categories. In addition, the using QIAquick columns (Qiagen, Hilden, Germany) and detailed questionnaire for HEV cases and the FFQ ques- sequenced in both directions with the nested PCR prim- tionnaire completed by the DEGS participants contained ers. Nucleotide sequences of amplicons were determined 6 identical questions on dietary habits and 2 questions on by using the BigDye Terminator cycle sequencing kit pet husbandry. On the basis of these 8 questions the two (Applied Biosystems) and separated on a model 3730xl populations, HEV cases ( n = 135 ) and baseline population genetic analyzer (Applied Biosystems, Waltham, USA). ( n = 4.512 ), could be compared. A univariable analysis with Nucleotide sequences of PCR products were analysed by a total of 8 variables was performed by calculating odds ratios (OR) and 95% confidence intervals (CIs). Thereafter 1 3 340 Food and Environmental Virology (2021) 13:337–346 a logistic regression was conducted. Considering the AIC Table 1 Descriptive analysis of hepatitis E cases as selection criterion for the best model, we decided to keep Variable All cases ( n = 135) % the full model (variables included see Table 2) as it resulted Symptoms & previous diseases Yes in the smallest AIC. In addition, we wanted our final model Symptoms 79 58.5 to include age and sex variables, regardless of significance, Elevated liver enzymes 76 56.3 in order to take into account possible confounding factors. Upper abdominal pain 38 28.2 The odds ratios, adjusted for gender and age, were reported. Icterus 24 17.8 The analysis was performed with SAS University Edition. Fever 22 16.3 Liver disease 21 15.6 Fatty liver 15 11.1 Immunosuppression 12 8.9 Results Liver infection 3 2.2 Hepatitis C 2 1.5 Descriptive Analysis of Bavaria’s Hepatitis E cases Blood transfusion 1 0.7 Liver cirrhosis 1 0.7 The questionnaire was filled out by 167 patients with hepa- Meat consumption Yes titis E, which corresponds to 30% of all reported HEV cases Pork on piece 126 94.7 in Bavaria during the study period. A total of 5 question- Raw ham 120 89.6 naires were filled out less than 80% and therefore excluded Beef not fully cooked 68 51.5 from further evaluation. (Fig.  1). Out of the remaining 162 Wild boar 35 26.1 patients, 35 had travelled abroad. Eight of them visited Pig liver 33 24.6 countries where genotype 3 is prevalent, during the relevant Meat products consumption Yes incubation period. 27 patients visited countries, where other Salami 131 97.0 or unknown HEV genotypes are endemic (e.g. Singapore, Cooked sausage 128 95.5 Nicaragua, Namibia) . Stool samples were available from Liver pate 98 73.1 17 of these 27 patients, but HEV genotype 3 could not be Other risk factor consumption Yes detected by RT-qPCR. As our aim was to focus on risk fac- Fish 123 91.8 tors associated with transmission of HEV genotype 3, these Raw vegetable 121 90.3 27 were not considered in the further analysis. In total, 66 Raw fish 40 30.1 women and 69 men were included in the study. The mean Grill party Yes age was 46 years for women (range 20–74 years) and 47.5 Visited grill party 63 46.7 years for men (range 20–85 years). (Fig. 2). There was no Consume pork/game 55 87.3 significant difference in the mean age between women and Prepared pork/game 44 69.8 men (t test and ANOVA). Pets Yes There were 79 (58%) cases with symptoms. The most Cat 39 28.9 frequent ones were: increased liver values (56%), upper Dog 20 14.8 Rabbits 5 3.7 Other rodents 2 1.5 Direct contact to animals Yes Live/work on a farm 8 5.9 Working in butcher shop 2 1.5 Water contact Yes Wastewater 4 3.0 River/lake 3 2.2 Fountain 3 2.2 abdominal pain (28%), jaundice (17%) and fever (16%). Other symptoms recorded by the patients were fatigue, itching, nausea, aching limbs, muscles and legs. No asso- ciation between gender and the presence of symptoms (Chi Square Test) was detected. One patient reported having Fig. 2 Gender and age distribution of the Bavarian hepatitis E cases 1 3 Food and Environmental Virology (2021) 13:337–346 341 received a blood transfusion during the incubation period. the questionnaire. Nevertheless all available samples were Three patients had a chronic liver disease, and two of them included in the subgenotype analysis. Most of the human reported Hepatitis C. Twenty-one patients reported other samples ( n = 122 ) were negative for HEV RNA by RT- liver disease among which the most common was fatty liver qPCR. A total of 21 stool samples and 2 serum samples were disease ( n = 16 ) (Table 1). genotyped (GenBank accession no. HG998145-HG998188), The specially designed questionnaire contains several 14 of them with an accompanying completed questionnaire. items on the consumption of potential HEV risk food prod- All samples were classified as HEV genotype 3 (Fig.  3). The ucts such as various meat types, cold cuts and sausages. Four most commonly found subgenotype was 3c ( n = 16 ) , fo l- patients indicated to eat halal, one kosher and one vegetar- lowed by 3e ( n = 3 ), 3a ( n = 1 ), 3f ( n = 1 ) and 3 not further ian. The only person who said he was eating kosher also specified ( n = 2 ). Six food samples such as minced meat, checked that he was eating halal. After the check for answer “Kaminwurzen” (smoked pork sausages), beef meat and consistency there were no strictly vegetarian, no strictly deer goulash were collected and tested. All were HEV RNA kosher and three real halal eating patients. Notable is that negative (Fig. 1). 91% of the cases ( n = 123 ) reported having consumed raw or poorly cooked meat or raw mussels or raw fish at least 1 to 3 times per month during the incubation period. Comparison of the HEV cases and the DEGS1 population Human and Food Samples In the DEGS population included in the study ( n = 4.512 ), A total of 145 human samples (143 stools and 2 sera), were 48% of the participants were male ( n = 2.156 ) and 52% tested for the presence of HEV RNA. There were 39 patients female ( n = 2.356 ). The mean age overall was 57.2 range that provided stool or serum samples but did not fill out (24–88), with no significant difference between the sexes (ANOVA and T test). Thus the DEGS participants were con- siderably older than the hepatitis E cases reported in Bavaria in 2017 ( average age = 46.8 years ). The difference in age between the two populations is also significant (T test), while Not detected (n=122) no difference was observed regarding the sex of the partici- pants (Chi-square test). In addition to age and gender, 8 risk factors were tested in the univariable analysis. Six of them 3f (n=1) were about nutrition habits and two risk factors considered 3e (n=3) 3a (n=1) the possession of dogs or cats as pets. The consumption of 3 (n=2) sausages (like salami, liver sausages, raw spread sausages), cooked fish (e.g. pollack, trout) were significantly associated 3c (n=16) with hepatitis E. (Table  2). Owning a cat is significantly associated with hepatitis E, as opposed to owning a dog. Other variables not significantly associated with hepatitis Fig. 3 Detected subgenotypes E infection were the consumption of meat (e.g. pig, wild boar, deer) and raw ham. The variable consumption of raw Table 2 Risk factors (NA—the Risk factors Cases Baseline Univariable analysis Multivariable effect of the variable is not ( n = 135) population analysis significant in the Multivariable ( n = 4.512) Analysis, **Corresponds to significance level 5%, Variable Yes % Yes % Odds ratio 95% CI Odds ratio 95% CI ***Corresponds to significance Sausages consumption (no ham) 133 98.5 4.037 89.7 7.7*** 1.9–31.1 9.6** 1.3–70.1 level 1%) Cat possession 39 31.2 676 15.4 2.5*** 1.7–3.7 1.9*** 1.3–3.0 Fish consumption (cooked) 123 91.8 3.811 84.8 2.0** 1.1–3.7 2.2** 1.1-4.4 Raw vegetable consumption 121 90.3 4.245 95.3 0.5** 0.3–0.8 0.4** 0.2–0.8 Ham consumption 120 89.6 3.828 85.0 1.5 0.9–2.6 NA Meat consumption (no chicken, 131 97.0 4.326 96.2 1.3 0.5–3.6 NA no sausages) Dog possession 20 16.1 608 13.9 1.2 0.7–1.9 NA Vegetarian diet 1 0.8 185 4.1 0.2 0.0–1.3 NA 1 3 342 Food and Environmental Virology (2021) 13:337–346 vegetable seems to be a significant protective factor for hepa- Risk Factors Associated with Food Consumption titis E infection. To the best of our knowledge no study in Europe investi- gated fish consumption as a risk factor for transmission of Discussion autochthonous Hepatitis E. HEV genotype 3 was detected in 32.2% (10 of 31) dolphins at the National Aquarium, Bavarian Hepatitis E Cases and Risk Factors Havana, Cuba. Dolphins generally feed on fish and squid. Infected animals or contaminated surface waters could During 2017 an intensified hepatitis E surveillance was be the source of infection (Villalba et al., 2017). Other conducted in Bavaria. This included a detailed question- seafood such as diverse shellfish—bivalve in Japan (Li naire, stool and serum samples, and food investigations et al., 2007), mussels in UK (Crossan et al., 2012), Spain from notified hepatitis E cases. A total of 135 question- (Mesquita et al., 2016), Italy (La Rosa et al., 2018) and naires, 145 human samples and 6 food samples were inves- 2.9% of shellfish purchased in local supermarkets in Scot- tigated in order to gain deeper understanding of the risk land (O’Hara et al., 2018) have been tested positive for factors for HEV transmission. The recruited hepatitis E HEV genotype 3. An HEV outbreak on a cruise ship was cases from Bavaria ( n = 135 ) were compared with DEGS linked to the consumption of shellfish on board (Said et al., study population from West federal states in Germany 2009). In Italy, apart from the 2.6% HEV positive mussels, ( n = 4.512 ) by univariable and multivariable analyses. 12.8% seawater samples (La Rosa et al., 2018) and raw Case reports and series from Europe suggest that HEV sewage and river samples (Iaconelli et al., 2015) were also genotype 3 is more common in elderly men (Festa et al., tested positive for HEV genotype 3. Since HEV genotype 2014; Dalton et  al., 2008; Chalupa et  al., 2014; Saint- 3 is an enteric pathogen in both human and diverse animal Jacques et al., 2016; Tarantino et al., 2016; Borgen et al., species, human and animal faeces could contaminate the 2008). However, seroprevalence studies are not very con- sewage and coastal water. In countries with high farming clusive. One study concluded that the older (30–39) par- density and shellfish production close to the shore, shell- ticipants and male participants were more often positive for fish can bio-accumulate human and animal enteric viruses anti-HEV IgG than younger (20–29) or female participants (Mesquita et al., 2016; Grodzki et al., 2014). As shellfish in southern Germany. Since the study comprised only young is often consumed raw or undercooked, the virus has not participants aged 20–39, it is unclear whether this trend been deactivated and can pose a risk for HEV infection. holds true also among the older population (Mahrt et al., Fish could also be consumed raw or cooked thoroughly; 2018). In another larger study, with adults aged 18–79 years however, it is not known whether bio-accumulation, sim- from all federal states in Germany no gender difference in ilar to shellfish, can happen in fish. The cutthroat trout the HEV seroprevalence was reported, but an increase in virus (CTV), a virus initially isolated in 1988 shows seroprevalence with age. The authors put forward the idea between 29 and 49% genome sequence similarity to HEV that food frequently consumed by both sexes plays a major genotypes 1–4, rat HEV and avian HEV. Since 2010, a role as a channel of transmission (Faber et al., 2012). The related agent was described from related salmonid fish authors of a systematic review suggested that the higher species. The presence of CTV was confirmed in differ - incidence of clinical cases in men indicates rather gender ent trout species (Johne et al., 2014; Smith et al., 2014). differences in the development of diseases or the use of Recently, the International Committee on Taxonomy of laboratory tests and not difference in infection rates (Lewis Viruses (ICTV) gave a new classification for the family et al., 2010). Our results are in line with Faber et al. (2012), Hepeviridae. All HEV’s have been placed under Hepeviri- since both male and female participants of the study were dae family and further classified under 2 genera namely almost equally represented; however, both male and females Orthohepevirus, which included isolates from mammals were also equally affected with symptoms in our study and, and chicken and Piscihepevirus, containing the fish HEV therefore, not confirming the higher manifestation rates (Purdy et al., 2017). However, the zoonotic potential of among males. However, we had a lower percentage of par- CTV has not yet been clarified and its influence on the ticipation in the study from the population over 60. Among epidemiology of human hepatitis E is largely unknown all cases reported in Bavaria during this period ( n = 558 ), (Johne et al., 2014). Therefore, it is not clear whether we 27% were over 60 years old, while in our study, only 17% see the effect of this HEV-like virus as a risk factor for were over 60 year old. This could indicate that older people HEV infection in humans. Further research is needed to are unable or less motivated to complete the detailed ques- investigate whether fish consumption can be a risk factor tionnaire and therefore less likely to participate in the study. for hepatitis E genotype 3. 1 3 Food and Environmental Virology (2021) 13:337–346 343 In our study the sausages, like salami and liver sausages, that people who consume a lot of vegetables consume less were significantly associated with hepatitis E with OR 9.6 meat. However, this is not conclusive and vegetable-based in multivariable analysis. This is in accordance with other diet is not necessarily excluding a HEV infection, as HEV studies from Germany (Faber et al., 2018; Szabo et al., 2015) has already been detected in vegetables, ready-to-eat salads and other European countries (Di Bartolo et al., 2015; Pavio and irrigation water (Purpari et al., 2019; Terio et al., 2017; et al., 2014; Berto et al., 2013; Di Bartolo et al., 2012; Mooij Kokkinos et al., 2017). et al., 2018; Moor et al., 2018). During the research period six food samples (minced Hepatitis E RNA was detected in several studies in meat, Kaminwurzen—smoked pork sausages, beef meat Europe in different food products such as figatelli (30–58%) and deer goulash) from households of notified hepatitis E (Colson et al., 2010; Pavio et al., 2014), pork liver sausages cases were available for laboratory testing. All were negative (6–29%) (Di Bartolo et al., 2012; Berto et al., 2012; Pavio for HEV RNA. It is possible that these food items were not et al., 2014; Szabo et al., 2015), quenelles (25%) and dried the source of infection, but other food that were no longer salted liver (3%) (Pavio et al., 2014). All HEV sequences available for testing. obtained in these studies were of genotype 3. However, the human HEV infection dose is unknown and Risk Factor Pet Ownership it needs to be investigated whether the viral load in meat and ready-to-eat products is sufficient to infect humans. Our results suggest that possession of a cat is significantly One experiment aiming to answer this question was done associated with HEV infection as opposed to ownership of with pigs. Two out of three pigs were successfully infected dogs. Both cats and dogs have been found seropositive for with HEV genotype 3 after ingesting an inoculum of 10 mL HEV in a study from Brandenburg, Germany (Dähnert et al., with 10 genome equivalents per mL (Andraud et al., 2013). 2014). While another study from Germany found that the Another study estimated that figatelli (pig liver sausage possession of pets was a protective factor for hepatitis E and 3 6 from France, commonly eaten uncooked) contains 10 –10 cat ownership was the most protective factor (Wichmann HEV RNA copies per slice (Colson et al., 2010). Figatelli et al., 2008). The authors doubted this and suggested that had already been described as a risk factor for HEV (Capai due to the frequent preparation of raw liver as cat food, a et al., 2019) and had been identified as the cause of infec- pre-existing immunity could exist due to a subclinical infec- tion in case reports and series (Colson et al., 2010; Moal tion among cat owners. Similarly, it was described that cats et al., 2012), suggesting that the infectious dose for humans fed with kitchen leftovers have a higher risk of HEV sero- is comparable to the experimentally proven infectious dose positivity than cats fed with commercial food, suggesting for pigs. In addition, the virus shows relatively high stabil- a common source for HEV infection for both animals and ity in order to be completely inactivated, the meat must be humans (Liang et al., 2014). A case report from Japan also cooked at 71 degrees for 20 minutes (Barnaud et al., 2012). described a patient infected with HEV genotype 4, whose cat Therefore, there is a potential risk to public health consum- was positive for HEV antibodies (Kuno et al., 2003). Rodent ing raw and undercooked products. hunting by cats has been suggested as a possible source of In our analysis the consumption of meat (no chicken and no infection (Mochizuki et al., 2006); however, the literature is sausages) was not a significant risk factor for HEV infection, not consistent as to whether rats are competent hosts of HEV as opposed to findings from other studies and case reports genotype 3 (Shukla et al., 2011; Purcell et al., 2011; Johne (Faber et al., 2018; Wichmann et al., 2008; Slot et al., 2017; et al., 2010; Lack et al., 2012). Legrand-Abravanel et al., 2010; Riveiro-Barciela et al., 2015; Interestingly, in our study women with cat ownership Rivero-Juarez et al., 2017). This might be due to the fact, that have a higher risk (OR 2.7) for hepatitis E than men with cat the question was stated very general in DEGS questionnaire. ownership, and women in the age group over 70 years have Similarly, because no detailed information was available on an even higher risk (OR 9.6). It is tempting to hypothesize pork and pork liver consumption in the questionnaire com- whether women living in a shared household are more likely pleted by the DEGS study population, it was not possible to to care for the pets (feeding and cleaning) and are therefore analyse it separately, although both have been identified as more exposed and have a higher risk of HEV infection. How- risk factors in the literature (Wenzel et al., 2011; Lewis et al., ever, this hypothesis needs further investigation and fuels an 2010; Faber et al., 2018). Our study indicated that there was interesting research aspect that should be considered in the no significant association between consumption of raw ham design of future hepatitis E research studies. and HEV infection. This is in line with the results from case- control study in Germany (Faber et al., 2018), but opposed Limitations of the Study to the findings from England and Wales (Said et al., 2017). The consumption of raw vegetables resulted as some kind The cases analysed in our study are from 2017 and no con- of protective factor for HEV infection. One might speculate trols were recruited. The comparison is based on data from 1 3 344 Food and Environmental Virology (2021) 13:337–346 the DEGS study, which are only partially comparable. DEGS References participants were recruited from 2008 till 2011. To check the Andraud, M., Dumarest, M., Cariolet, R., Aylaj, B., Barnaud, E., Eono, comparability of the healthy population (DEGS study) and F., et al. (2013). Direct contact and environmental contaminations the cases, we looked for trends in the consumption behav- are responsible for hev transmission in pigs. Veterinary Research, iour of the German population. According to the National 44(1), 102. Nutrition Survey (NVS) II and the so-called NEMONIT Barnaud, E., Rogée, S., Garry, P., Rose, N., & Pavio, N. (2012). Ther- mal inactivation of infectious hepatitis E virus in experimentally study, there were no significant changes in the consump- contaminated food. Applied Environmental Microbiology, 78(15), tion of vegetables, meat/meat products and fish/fish products 5153–5159. between 2005–2007 and 2012/2013 (Gose et al., 2016). We Berto, A., Grierson, S., Hakze-van der Honing, R., Martelli, F., Johne, assume that this trend continued further. The DEGS Popu- R., Reetz, J., et al. (2013). Hepatitis E virus in pork liver sausage, France. Emerging Infectious Diseases, 19(2), 264–266. lation is considered healthy, i.e. without HEV infection, we Berto, A., Martelli, F., Grierson, S., & Banks, M. (2012). Hepatitis E had no access to information about IgG or IgM tests in the virus in pork food chain, United Kingdom, 2009–2010. Emerging available dataset, although a subset of the dataset was used Infectious Diseases, 18(8), 1358–1360. and published for a HEV seroprevalence study (Faber et al., Borgen, K., Herremans, T., Duizer, E., Vennema, H., Rutjes, S., Bos- man, A., et al. (2008). Non-travel related hepatitis E virus geno- 2012). We have relied on the information provided by the type 3 infections in the Netherlands; a case series 2004–2006. participants in the questionnaire. Therefore, it is possible BMC Infectious Diseases, 8, 61. that, if there were some asymptomatic acute hepatitis E Capai, L., Masse, S., Gallian, P., Souty, C., Isnard, C., Blanchon, T., cases or cases with HEV IgG (had the disease in the past) in et al. (2019). Seroprevalence study of anti-HEV IGG among dif- ferent adult populations in Corsica, France, 2019. Microorgan- the DEGS population, the actual impact of the risk factors isms, 7(10), 461. would be even greater if they were excluded. The results pre- Chalupa, P., Vasickova, P., Pavlik, I., & Holub, M. (2014). Endemic sented here must be interpreted carefully. Firstly, the cases hepatitis E in the Czech Republic. Clinical Infectious Diseases, and controls used for the statistical analysis were surveyed 58(4), 509–516. Chaussade, H., Rigaud, E., Allix, A., Carpentier, A., Touzé, A., Delz- using different questionnaires, and although risk factors such escaux, D., et al. (2013). Hepatitis E virus seroprevalence and risk as sausages, salami and liver sausages are in line with pub- factors for individuals in working contact with animals. Journal lished research, fish consumption and cat ownership must be of Clinical Virology, 58(3), 504–508. carefully interpreted and considered as possible risk factors Clemente-Casares, P., Ramos-Romero, C., Ramirez-Gonzalez, E., & Mas, A. (2016). Hepatitis E virus in industrialized countries: The in future studies and study designs. silent threat. Biomed Research International. Colson, P., Borentain, P., Queyriaux, B., Kaba, M., Moal, V., Gallian, Acknowledgements This work was supported by the Robert Koch P., et al. (2010). Pig liver sausage as a source of hepatitis E virus Institute and the German Federal Ministry of Health (Grant No. 1369– transmission to humans. Journal of Infectious Diseases, 202(6), 386 to J. Wenzel). 825–834. Crossan, C., Baker, P. J., Craft, J., Takeuchi, Y., Dalton, H. R., & Sco- Funding Open Access funding enabled and organized by Projekt bie, L. (2012). Hepatitis E virus genotype 3 in shellfish, United DEAL. Kingdom. Emerging Infectious Diseases, 18(12), 2085–2087. Dähnert, L., Conraths, F. J., Reimer, N., Groschup, M. H., & Eiden, Data Availability Statement All data about the HEV cases are avail- M. (2014). Molecular and serological surveillance of hepatitis E able from the corresponding author or from durdica.marosevic@lgl. virus in wild and domestic carnivores in Brandenburg, Germany. bayern.de on reasonable request. The DEGS1 Dataset used to support Transboundary and Emerging Diseases, 65(5), 1377–1380. the findings of this study can be requested upon application from the Dalton, H. R., Stableforth, W., Thurairajah, P., Hazeldine, S., Rem- Research Data Centre (Robert Koch Institute, MF4) narace, R., Usama, W., et al. (2008). Autochthonous hepatitis E in Southwest England: Natural history, complications and sea- sonal variation, and hepatitis E virus IGG seroprevalence in blood Open Access This article is licensed under a Creative Commons Attri- donors, the elderly and patients with chronic liver disease. Euro- bution 4.0 International License, which permits use, sharing, adapta- pean Journal of Gastroenterology & Hepatology, 20(8), 784–790. tion, distribution and reproduction in any medium or format, as long DEGS1. (2015). German Health Interview and Examination Survey as you give appropriate credit to the original author(s) and the source, for Adults, (DEGS1). Robert Koch Institute, Department of Epi- provide a link to the Creative Commons licence, and indicate if changes demiology and Health Monitoring. were made. The images or other third party material in this article are Di Bartolo, I., Angeloni, G., Ponterio, E., Ostanello, F., & Ruggeri, F. included in the article’s Creative Commons licence, unless indicated M. (2015). Detection of hepatitis E virus in pork liver sausages. otherwise in a credit line to the material. If material is not included in International Journal of Food Microbiology, 193, 29–33. the article’s Creative Commons licence and your intended use is not Di Bartolo, I., Diez-Valcarce, M., Vasickova, P., Kralik, P., Hernandez, permitted by statutory regulation or exceeds the permitted use, you will M., Angeloni, G., et al. (2012). Hepatitis E virus in pork produc- need to obtain permission directly from the copyright holder. To view a tion chain in Czech Republic, Italy, and Spain, 2010. Emerging copy of this licence, visit http://cr eativ ecommons. or g/licen ses/ b y/4.0/ . 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Update: Proposed reference sequences for subtypes of hepatitis E virus (species orthohepevirus a). Journal of General Virology, 101(7), 692–698. 1 3

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Food and Environmental VirologySpringer Journals

Published: Sep 1, 2021

Keywords: Hepatitis E; HEV; Genotype 3; Risk factors; Transmission

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