Study design and data collection
For this cross-sectional study, the local veterinarian authority recruited study participants during four meetings of hunters in the Wetteraukreis district in Hesse, Central Germany, at the beginning of 2013. After informed written consent of the participants, blood specimens were collected and a questionnaire was completed collecting information on age, sex as well as hunting ground, hunting activities and consumption of wild boar meat. Hunting grounds in the Wetteraukreis district were grouped into three areas: East (E), Northwest (NW) and Southwest (SW) (Fig. 1). Pivotal for the attribution of a hunter to a certain hunting ground was the location of the meeting he was attending assuming that this may reflect his or her preference for a certain region. In accordance with Article 25 paragraph 1 of the “German Infection Protection Act” a formal ethical review process and approval was not required. All study participants were informed of their results by the local authority. The data from the pseudonymized questionnaires were entered into an Excel data sheet and then imported into Stata 12 (Stata Corporation, College Station, TX; USA) for statistical analysis.
Additionally, specimens of wild boars (blood, muscle and liver tissue) were taken during different drive hunts in this district during the season 2012/2013. Human as well as wild boar specimens were tested for HEV-specific antibodies and HEV RNA as described below.
Serological testing
For the serological testing of the human sera three Enzyme Linked Immunosorbent Assays (ELISAs) were used: (1) the recomWell HEV IgG assay, which is an indirect ELISA based on recombinant open reading frame (ORF) 2- and ORF3-derived antigens of gt1 and gt3 (Mikrogen GmbH, Neuried, Germany); (2) the recomWell HEV IgM assay (Mikrogen GmbH), which uses the same antigens as the IgG detecting version of the test; (3) the HEV Ab-ELISA kit (Axiom, Bürstadt, Germany), which is a double-antigen sandwich-ELISA based on the capsid protein of gt1. This test is species-independent and detects all classes of antibodies.
The porcine sera were tested using the HEV Ab-ELISA kit (Axiom). All tests were used according to their manuals provided by the manufacturers.
Reverse transcription - polymerase chain reaction and phylogenetic analysis
Nucleic acids were isolated from serum specimens of hunters and wild boars using the NucliSENS® easyMag® device and reagents (bioMérieux Deutschland GmbH, Nürtingen, Germany) according to the manufacturer’s protocol. Specimens from liver and muscle tissue of wild boars were homogenized with mortar and pestle and subjected to RNA isolation with the RNeasy Mini Kit (Qiagen, Hilden, Germany). HEV-specific RNA was detected by real-time reverse - transcription polymerase chain reaction (RT-PCR) as previously described [21] using the Quantitect Probe RT-PCR Kit (Qiagen) in a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Wild boar liver and muscle specimens tested positive by real-time RT-PCR were subjected to conventional RT-PCR as described by Herremans et al. [22] amplifying a 197 nucleotide (nt) fragment of ORF2. This RT-PCR was performed using the QIAGEN OneStep RT-PCR Kit (Qiagen) in a 2720 Thermal Cycler (Applied Biosystems). The amplification products were purified using the QIAquick DNA purification kit (Qiagen) and directly sequenced using the RT-PCR primers in an ABI 3730 DNA Analyzer (Applied Biosystems) [GenBank: KP127667 – KP127670]. Phylogenetic trees were constructed using a neighbour-joining method implemented in the MegAlign module of the DNASTAR software package (Lasergene, Madison, WI, USA) and bootstrap analysis was performed with 1000 trials and 111 random seeds.
Statistical analysis
Among hunters, an acute HEV case was defined as a person who participated in one of the meetings in the Wetteraukreis district in 2013 and tested positive for HEV RNA and/or anti-HEV IgM. A subject tested positive only for anti-HEV IgG was defined as a case with a previous HEV infection. Accordingly, subjects tested negative for HEV RNA as well as anti-HEV IgM and IgG were defined as “non-cases”.
Subjects (a) less than 18 years old, (b) negating any hunting activity, (c) being a butcher or (d) for which the outcome result was missing were excluded from further analysis. The extent of contact between hunters and wild boars was categorized by the frequency of the use of protective gloves while skinning and/or disembowelling of wild boars. A new binary variable was created to differentiate between frequent (“always” and “nearly always”) or infrequent (“never”, “seldom” and “sometimes”) use of gloves.
Seroprevalences and 95 % confidence intervals (CI) were calculated using the results of both serological assays: recomWell IgG and Axiom, separately. Cohen’s kappa coefficient (κ) served as a measure of concordance between the two assays.
To compare the anti-HEV prevalences of the hunters from this study with that of the German general population, a large subsample (n = 4352) of sera originating from the 2008–2011 German Health Examination Survey for Adults (Deutscher Erwachsenen Gesundheitssurvey [DEGS]; www.degs-studie.de) was used to determine the baseline prevalence of anti-HEV antibodies in healthy adults in Germany [3]. The sera in DEGS were screened with the recomLine HEV-IgG/IgM immunoassay (Mikrogen GmbH). As the recomLine assay has the highest concordance with the recomWell IgG assay used in our study with κ = 0.80 [14] indicating a substantial concordance of the two assays [23], comparisons between the DEGS sera representing the German general population and the hunters from this study were based on these results. Pearson’s chi-square and Fisher’s exact statistics were used for significance testing. Results were considered as statistically significant if p-values were <0.05.
To identify factors that are associated with past or present HEV infection, prevalence ratios (PR) were estimated using univariable and stratified analysis as well as log-binomial regression.
For multivariable analysis, the results for the hunters above 70 years of age were excluded due to the possible presence of an unknown confounder and not being able to adjust for. Serology in the hunters (dichotomic: positive/negative) was defined as the dependent variable. In the final model, age group (in categories of 10 years), the extent of contact between hunters and wild boars and the hunting ground plus the according interaction term were included as independent variables.
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