Exposure to hepatitis E virus, hepatitis A virus and Borrelia spp. infections in forest rangers from a single forest district in western Poland

1 Department of Infectious Diseases, Hepatology and Acquired Immunodeficiencies, Poznan University of Medical Sciences, Poland 2 Regional Blood Center in Poznań, Poland 3 Department of Computer Science and Statistics, Poznan University of Medical Sciences, Poland 4 Department of Infectious Diseases, Jozef Strus Multidisciplinary Municipal Hospital in Poznań, Poland 5 Department of Biochemistry and Molecular Biology, Poznan University of Medical Sciences, Poland 6 Department of Anatomy, Poznan University of Medical Sciences, Poland 7 Department of General and Transplant Surgery, Poznan University of Medical Sciences, Poland


Introduction
Hepatitis E virus (HEV) is an important etiologic agent of enterically transmitted hepatitis worldwide. 1In developed European countries, this infection was previously considered only in persons returning from highly endemic areas -some parts of Asia and Africa.][4] The virus belongs to the Hepeviridae family, Orthohepevirus genus. 5Its virions are small (27-34 nm), non-enveloped and icosahedral particles containing positive-sense single-stranded RNA, approx.7.2 kb in length.Four HEV genotypes representing 1 serotype have been identified as a cause of human infections, all of which are classified as members of the Orthohepevirus A species.Genotypes 1 and 2 (HEV-1 and HEV-2) are present in developing areas of the world (Asia and Africa) and can induce large waterborne outbreaks.Genotype 3 (HEV-3), which has worldwide distribution (including Europe), and genotype 4 (HEV-4), predominant in Asia, cause zoonotic infections -pigs, wild boars and deer represent recognized reservoir animals for these variants of the virus.Recently, a single case of HEV-7-related disease resulting from contact with dromedaries has also been reported. 6A broad range of clinical presentations may be related to HEV infection, from an asymptomatic course to severe hepatitis. 7,8][14][15][16] The aim of the present study was to assess the seroprevalence of HEV exposure markers among FRs from a single forest district in western Poland in relation to anti-HAV (hepatitis A virus) IgG and anti-Borrelia spp.antibodies.

Material and methods
The study involved 48 out of 52 FRs from a single forest division in western Poland (Międzychód forest division) who were screened for anti-Borrelia antibodies in the Laboratory of the Department of Infectious Diseases, Jozef Strus Multidisciplinary Municipal Hospital in Poznań in December, 2014, and agreed to participate in this analysis.
Additionally, we recruited 90 unpaid voluntary healthy blood donors (BDs) from the Regional Blood Center in Poznań (west-central Poland) to form the control group.
All the study participants were asked to complete a simple short questionnaire on their demographic, travel and culinary habits, and medical history.

Serologic testing
Anti-Borrelia IgM and IgG detection was performed in a 2-step procedure.First, ELISA tests were used (anti-Borrelia ELISA [IgM] and anti-Borrelia plus VlsE ELISA [IgG]); next, for sera that were positive in this initial screening, confirmation line-blot tests were performed using Anti-Borrelia EUROLINE-RN-AT-adv (IgM) or Anti-Borrelia EUROLINE-RN-AT (IgG) (EUROIMMUN Medizinische Labordiagnostika AG, Luebeck, Germany).Positive results were defined as the presence of the appropriate antibodies (IgM and/or IgG) detected by both screening and confirmatory testing.Anti-Borrelia testing was performed in FRs only (n = 48).
HEV exposure was assessed in all the study participants (n = 138) with IgM and IgG antibody enzyme immunoassay tests (Anti-Hepatitis E Virus ELISA [IgM] and Anti-Hepatitis E Virus ELISA [IgG]; EUROIMMUN Medizinische Labordiagnostika AG, Luebeck, Germany).Additionally, other kits were also used for the detection of anti-HEV IgM in the FRs (MP Diagnostics ASSURE ® HEV IgM Rapid Test, MP Biomedicals Asia Pacific Pte. Ltd., Singapore).
All the serologic tests were carried out according to the manufacturers' instructions.

Hepatitis E virus RNA testing
It was planned that the search for HEV RNA would be performed only in anti-HEV IgM-positive participants of the study.

RNA extraction and reverse transcription
Total RNA was extracted with the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) from 140 μL of serum according to the manufacturer's instructions, and then stored at −80°C for downstream applications.For reverse transcription (RT), 8.25 μL samples of the extracted RNA were used.First, the RNA was incubated with 1.75 μM oligo d(T)23, 1.25 μM random primers pd(N)6 and 0.5 mM dNTP mix at 70°C for 5 min, then it was reverse-transcribed into cDNA using Invitrogen Moloney Murine Leukemia Virus (M-MLV) Reverse Transcriptase (Thermo Fisher Scientific, Carlsbad, USA).The final volume of 20 μL RT mix contained 4 μL of 5X First Strand Buffer, 10 U of RNaseOUT™ Recombinant Ribonuclease Inhibitor and 100 U M-MLV Reverse Transcriptase (all ingredients by Thermo Fisher Scientific, Carlsbad, USA).The cycling parameters were: 10 min at 25°C, 60 min at 37°C, 15 min at 75°C, followed by a 4°C hold.

Real-time polymerase chain reaction assay
Detection of HEV RNA was performed in a LightCycler ® 480 instrument (Roche Molecular Diagnostics, Pleasanton, USA) using the TaqMan ® approach.Primers and probes for real-time polymerase chain reaction (RT-PCR) were synthesized based on the highly conserved region of the different HEV genotypes in the ORF3 region, where the primers and probe anneal.The forward primer (JVHEVF; 5'-GGTGGTTTCTGGGGTGAC-3'), reverse primer (JVHEVR; 5'-AGGGGTTGGTTGGATGAA-3') and probe (JVHEVP; 5'-TGATTCTCAGCCCTTCGC-3') described by Jothikumar et al. were employed for HEV detection by RT-PCR. 17The TaqMan ® probe was labeled with a 6-carboxy fluorescein fluorophore (6-FAM) at the 5' end and a Black Hole Quencher-1 (BHQ-1) at the 3' end (both primers and probes were provided by DNA Sequencing and Oligonucleotides Synthesis Laboratory, Polish Academy of Science, Warsaw).
The 10 μL reaction mixture contained 5 μL of 2 × Light-Cycler ® 480 Probes Master (Roche Molecular Diagnostics, Basel, Switzerland), 500 nM of primers, 200 nM of probe, and finally 1 μL of cDNA added as a template.Cycling conditions were optimized to 1 cycle of initial denaturation for 10 min at 95°C, followed by 55 amplification cycles at 95°C (10 s), 55°C (25 s) and 72°C (10 s).The products from the TaqMan ® RT-PCR were analyzed on 2% agarose gels.

Ethical issues
Informed consent was signed by all the FRs and BDs.The study was approved by the Bioethics Committee of Poznan University of Medical Sciences (reference No. 155/15).

Statistical analysis
Numerical data were presented as mean values and standard deviations.The comparison of age was performed by Student's t-test.The assumption of normal distribution of the data was checked using the Shapiro-Wilk test.The homogeneity of variances was verified by Levene's test.Nominal data were presented as numbers and percentages.The comparison was done using the χ 2 test of independence.The statistical analysis was performed with STATISTICA v. 12 software (StatSoft Inc., Tulsa, USA).Results were considered significant at p < 0.05.

Results
The baseline characteristics of all the study participants are presented in Table 1.
Anti-HEV antibodies were detected in 3 participants (2.2%).In the FR group there was a positive anti-HEV result in a single individual: a 43-year-old man with anti-HEV IgM antibodies only (which were found with both IgM-detecting tests), but no anti-HEV IgG or HEV-RNA were detected.He had never had an icteric disease and was anti-HAV negative, but tested positive for anti-Borrelia IgG antibodies.In the BDs only, anti-HEV IgG was found in 2 men (aged 42 and 55 years) out of 90 persons (2.2%); anti-HAV were not detected in either of them.
The results of the assessment for Borrelia antibodies indicated exposure to these bacteria in 37.5% of the FRs (Table 2).In this group, anti-HEV results were positive much less frequently than anti-Borrelia (p < 0.0001).
The anti-HAV positive individuals were older (51.9 ±7.8 years) than the HAV-seronegative study participants (44.0 ±7.9 years; p = 0.0004); this was also true when the 2 groups were analyzed separately (among the FRs: 52.6 ±9.1 years and 43.3 ±9.0 years, respectively, p = 0.0078; among the BDs: 51.1 ±6.3 years and 44.6 ±6.8 years, respectively, p = 0.0221).Susceptibility to HAV infection (as expressed by a lack of anti-HAV IgG) was more frequent among individuals under 45 years of age (46 out of 48 individuals, 95.8%) than in older individuals (34 out of 48 individuals, 70.8%; p = 0.001).In the study, 6 FRs mentioned having had an icteric disease in the past.Viral hepatitis was recognized in 2 of them, while the diagnostic conclusions were not known in the remaining cases; all but 1 were HAV IgG-seropositives.The BDs had no jaundice in their medical histories.
In total, 9 of the study participants (3 FRs and 6 BDs) declared that they had had vaccinations against hepatitis A; anti-HAV antibodies were found in only 4 of them (2 FRs, including 1 with a history of icteric hepatitis, and 2 BDs).

Discussion
In this study, HEV exposure markers (as expressed by anti-HEV positivity) were found in only a few participants (2.2%), less frequently than anti-HAV and anti-Borrelia antibodies.Moreover, in spite of the detection of anti-HEV IgM in 1 FR (confirmed by 2 different assays), further tests for anti-HEV IgG and HEV-RNA in this asymptomatic individual proved to be negative.The lack of clinical symptoms characteristic of acute hepatitis does not exclude the possibility of infection, because contact with HEV is usually subclinical. 7n the other hand, it is also possible that this FR had a primary infection with Epstein-Barr virus (EBV) or cytomegalovirus (CMV) at the time of the HEV testing.It has been shown that the polyclonal stimulation of B cells caused by these viruses can be a source of false anti-HEV IgM positivity; due to the limited volume of available serum, the further investigation of this possibility was impossible. 18or this reason, we postulate that the correlation between the presence of IgM antibodies and a recent HEV infection in this case is arguable -it could be a false positive result.
We are going to discuss the results of this study in the context of the particular study population and in view of the available knowledge regarding HEV seroprevalence in Poland.
Data from a few existing reports suggest that FRs constitute an increased-risk group for contact with HEV.Dremsek et al. proved the presence of anti-HEV IgG in an average of 17.8-21.4%(range: 5.6-28%) of FRs from eastern Germany, depending on the diagnostic test used (commercial vs in-house, respectively), compared to 11.1-12.3%(p < 0.01) in the control group. 13Even higher values (in control populations as well) were found by French researchers: Carpentier et al. reported HEV seroprevalence of 31.2% (compared to 19% in the control group) and Chaussade et al. reported 36.4% (compared to 26.1% in the control group). 14,15Similar data were quoted by Yoon et al. (31.3% in a mixed population of skilled agricultural, forestry, and fishery workers, odds ratio 6.6) in a South Korean analysis. 16On the other hand, lower seroprevalence was observed in FRs from Iowa, USA (5.7% vs 0% in the control group). 12It is believed that higher HEV seroprevalence in FRs may be caused by the following factors: contact with reservoir animals, some culinary habits more common in this professional group (eating raw/undercooked meat, including game meat) and common membership in hunting communities.
Significantly, in spite of much more frequent consumption of food containing raw meat (including game meat) by FRs than by the control group, a high percentage of study participants who had travelled abroad (all but 1 person) and considerable exposure to the forest environment (anti-Borrelia IgG antibody seropositivity of 37.5%, coinciding with other Polish data on this subject), our study was unable to unequivocally establish the features characteristic of contact with HEV (anti-HEV IgG) in this professional group. 19n a recent publication on the HEV exposure of 1027 hunters from all over the country, the presence of anti-HEV IgG was confirmed in 20.3% of cases. 20According to 2 other reports, HEV seroprevalence among the patients of the Department of Infectious Diseases in Poznań (n = 182), and the Department of Internal Medicine in Łódź, central Poland (n = 212), as well as HCV-positive patients (n = 149) from the Department of Infectious Diseases and Hepatology in Łódź was 15.9%, 7.5% and 10%, respectively. 21,22n the context of these data, the low values in the present study are surprising.In our opinion, there are at least 2 possible causes of these findings.Firstly, the available serologic tests have variable and, unfortunately, imperfect accuracy in the detection of anti-HEV markers.Despite the fact that the EUROIMMUN tests used in this analysis were compared with the assays of other manufacturers, knowledge about the diagnostic performance of these tests is very limited. 23,24Moreover, in our recent investigation of the seroprevalence of anti-HEV IgG among 105 HIV patients and 105 age-and sex-matched BDs, using the EUROIMMUN assay, we reported similar low rates of this HEV exposure marker: 0.95% and 3.8%, respectively. 25Additionally, it should be stressed that anti-HEV IgG tests in general have suboptimal sensitivity. 26econdly, exposure to HEV can differ depending on the geographical region (even within the same country) and related elements, such as environmental factors, climate, socioeconomic status, hygienic and sanitary conditions, culinary habits, and agricultural traditions (especially related to livestock husbandry).For example, in Cornwall (United Kingdom), a coastal clustering of hepatitis E cases was observed. 27In a large study among French BDs, HEV seroprevalence varied significantly -from 8% to 86% -depending on the area. 28Similar conclusions were drawn in the previously mentioned study by Sadkowska-Todys et al., according to which HEV seroprevalence differed significantly depending on the region: from 3.85% in the Kuyavia-Pomerania region (mid-northern Poland) to 41.7% in the Opole Province (south-western Poland). 20nfortunately, values for other provinces were not given.Additionally, these considerations are complicated by the fact that anti-HEV IgG prevalence in wild boars (recognized reservoir animals) in the aforementioned regions of Poland was inversely proportional to values for hunters from these regions: 29% for the Opole Province and 68% for the Kuyavia-Pomerania region (unfortunately, data for Greater Poland are not available). 29etermining the factors influencing these discrepancies is challenging.
The HAV seroprevalence found in the present small study reflects the epidemiological situation in countries with very low hepatitis A endemicity, which currently include Poland. 30Moreover, there was no difference in this respect between FRs and the control group.
In view of the lack of medical documentation confirming the vaccination of the study participants against hepatitis A, we believe that at least 5 out of 9 individuals who declared active HAV immunization (HAV seronegative) confused it with hepatitis B vaccinations, even though the question they had to answer was unequivocal and emphasized the differences between vaccinations against hepatitis A and B. This corresponds to what we frequently observe in daily real-life practice.
The common susceptibility to HAV infection found in this study, including among individuals over 45 years of age (70%), suggests that active hepatitis A immunoprophylaxis can also be recommended for these persons, especially when a higher risk of HAV exposure exists.For the FRs participating in the present analysis, the rationale for such an action could be travelling abroad (reported by all but 1 person in this group) and frequent consumption of seafood (56%).

Conclusions
In the present HEV seroprevalence study among FRs from a single forest district in western Poland, we failed to unequivocally prove exposure to the virus in this population.For more in-depth understanding of this issue, further research is necessary among larger populations of FRs in various regions of the country, using diagnostic tests with established reliability.The HAV seroprevalence among the FRs in this study paralleled the situation in the general population and can justify vaccination against hepatitis A in this professional group.Exposure of FRs to Borrelia spp. was considerable.

Table 1 .
Baseline characteristics of the study participants (n = 138) * All ELISA IgM-positive patients were also blot-IgM, ELISA-IgG and blot IgG-positive.