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laboratory features and outcome
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Contents lists available at ScienceDirect
Digestive and Liver Disease journal homepage: www.elsevier.com/locate/dld
Liver, Pancreas and Biliary Tract
Locally acquired hepatitis E virus in Marche Italy: Clinical/laboratory features and outcome Giuseppe Tarantino a,∗ , Alessio Ortolani a , Katia Marinelli b , Antonio Benedetti a , Giulia Marconi a , Manuela Calzolari a , Harry R. Dalton c , Marco Marzioni a , Laura Schiadà a , Giammarco Fava a , Alessandro Chiodera d , Giorgio Amadio e , Alessandro Fiorentini f , Alessandra Riva f , Paolo Fraticelli g , Stefano Menzo b , Patrizia Bagnarelli b a
Department of Gastroenterology and Transplants, Clinic of Gastroenterology, Hospital of Ancona, Università Politecnica delle Marche, Italy Department of Biomedical Sciences and Public Health, Virology, Hospital of Ancona, Università Politecnica delle Marche, Italy c Queens Park, London, UK d Unit of Infective diseases, Hospital of Macerata, Italy e Unit of Infective diseases, Hospital of Fermo, Italy f Units of Infective diseases Hospital of Ancona, Università Politecnica delle Marche, Italy g Department of Internal Medicine, Unit of Medical Clinic, Hospital of Ancona, Università Politecnica delle Marche, Italy b
a r t i c l e
i n f o
Article history: Received 20 August 2019 Accepted 18 November 2019 Available online xxx Keywords: Acute on-chronic liver failure Chronic liver disease Diabetes HEV
a b s t r a c t Background and aims: Hepatitis E Virus is endemic in Europe with increasing numbers of cases in recent years, also in Italy where this phenomenon has hitherto been modest. The aim of this study was to document the clinical features/natural history of locally acquired hepatitis E in our territory and explore factors which determine adverse outcome. Methods: Retrospective study of patients with locally-acquired HEV (hepatitis E virus) in Marche, Italy (2011–2019). Results: 1189 patients were tested for HEV with 89 confirmed cases. 81 (6.8%) had locally acquired infection; 54 (66%) were male (mean age 55.5 years) and 32 (39.5%) had active co-morbidities. 41 cases were viraemic (all HEV-3 (HEV genotype 1,2,3,4)); acute infection was found in 79 and chronic infection in 2. Forty-five cases (55%) required admission to hospital, for a total of 785 days. 4 patients developed acute on-chronic liver failure, 6 developed acute kidney injury and 8 died: all had active comorbidities. Univariate analysis showed that bilirubin, INR, immunosuppression, cirrhosis and diabetes were associated with death. On multivariant analysis the only predictor of death was the presence of diabetes (p = 0.04). Conclusions: Hepatitis E in Marche Italy is mostly locally acquired and caused by HEV-3 that impacts on the morbidity and mortality particularly for fragile patients. © 2019 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.
1. Introduction In low-income countries in Asia and Africa, hepatitis E is mainly caused by HEV-1 and 2, which are obligate humans pathogens spread orofecally mainly by contaminated water. Hepatitis E occurs as both sporadic cases and outbreaks, usually resulting in a selflimiting hepatitis in young adults. However, there is adverse outcome in pregnant women, with mortality rates of up to 25%. For many years’ hepatitis E was thought not to be a major issue in high-income countries (including Europe), except for imported
∗ Corresponding author at: Via Conca 71, 60126, Ancona, Italy. E-mail address: [email protected] (G. Tarantino).
cases in travelers returning from countries endemic for HEV-1 and 2 [1–4]. Over the past 15 years, it has become clear that HEV is a global health issue. Many higher income countries are also endemic for HEV. In these areas locally acquired hepatitis E is mainly zoonotic and caused by HEV-3 (Europe and Japan) or 4 (China and Japan) usually resulting in sporadic asymptomatic infection [1–4]. Only a small minority of patients develop acute hepatitis and chronic infection occurs in the immunosuppressed [4–8]. Pigs are considered the primary reservoir. However, human infection with HEV has also been found to originate from other sources, including wild boar, deer, rabbits, camels (genotype 7), rats and blood products [9–11].
https://doi.org/10.1016/j.dld.2019.11.015 1590-8658/© 2019 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.
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In patients with HEV-3 infection, the outcome is usually favorable and deaths in pregnancy have not been observed. Case reports/cohort studies have reported mortality in patients with underlying chronic liver disease (CLD) due to acute-on-chronic liver failure (ACLF) [12,13], and less commonly in patients without liver disease due to acute liver failure (ALF) and chronic infection [8,14]. However, the burden of disease and the factors that predict poor outcome are unknown. In Europe it is estimated that there are > 2 million human infections with HEV per year [15], caused mostly by locally acquired HEV-3, but the incidence varies considerably between countries. In Italy, along with most countries in Europe, the number of locally acquired cases is increasing [16]. However, the number of laboratory-confirmed cases of hepatitis E has hitherto been modest, with more travel-related cases compared to many other European countries. The aim of this study was to document the clinical features and natural history of locally acquired hepatitis E in the Marche region of Italy, and to explore factors which determine adverse outcome. 2. Patients and methods Patients presenting with hepatitis E infection during the period 2011–2019 were studied retrospectively. The setting for the study was the Marche region of Italy (population approximately 1.5 million inhabitants), including the city of Ancona and the towns of Macerata, Fermo, Pesaro, Ascoli e Fabriano. By retrospectively evaluating clinical records, screened patients for HEV were generally those with acute liver injury (ALI) in whom common etiologies such as HAV, HBV, HCV, biliary obstruction and drug-hepatotoxicity, were not found. Moreover, the cases with suspected alimentary risk factors were screened too. In Ancona Hospital, where the awareness of HEV-related disease has grown over the years, fragile patients, due to immune-suppression or chronic liver disease, even if they had mild ALI or those with suspected extrahepatic manifestations, were tested too. Cases of HEV infection were identified from their Microbiology Department records in Ancona, where regional HEV testing was performed throughout the study period. Cases who had recently travelled to areas known to be endemic for HEV-1 and 2 were excluded from the study and/or cases that were found to have HEV infection caused by genotypes 1 and 2. Each laboratory-confirmed locally acquired case underwent retrospective case note review and a pre-defined set of variables were recorded. This included demographic, dietary, clinical and laboratory data; presence or absence of pre-existing CLD, decompensation and/or ACLF; clinical outcome, including death and its cause(s); and length of hospital stay. The contribution of HEV to the cause of death was made by two experienced clinicians (GT and HD). 2.1. Definitions 1. A case of hepatitis E infection was defined as: • Anti-HEV IgM reactive and/or • HEV RNA detected in serum and/or stool
less than 48 h, or by a percentage increase in serum creatinine of more or equal to 50% (1.5-fold from baseline) in less than seven days. [18]. 2.2. HEV testing The presence of anti-HEV IgM and IgG antibodies was determined by testing patient sera with a commercial ELISA (enzyme linked immunoassay) immunoassay employing a synthetic HEV antigen derived from open reading frame (ORF) 2 and ORF3 regions (Diagnostic Bioprobes Srl, Milan, Italy). The presence of HEV-RNA was determined following RNA extraction from patient faecal and/or serum samples. Nucleic acid extraction was performed on QIA symphony SP instrument (QIAGEN® ) using dedicated kits and protocols. HEV-RNA was detected by reverse transcription nested-PCR using a broad-range primer set amplifying a portion of the ORF1 corresponding to the methyl transferase gene with a HEV 1–4 genotypes specificity. The primer sequences were: ORF1F 5 -CCAYCAGTTYATHAAGGCTCC (forward first round RTPCR (reverse transcription-polymerase chain reaction)) and ORF1R 5 -TACCAVCGCTGRACRTC (reverse first round RT-PCR), product length 348 bp; ORF1FN 5 -CTCCTGGCRTYACWACTGC (forward nested PCR) and ORF1RN 5 -GGRTGRTTCCAIARVACYTC (reverse nested PCR), product length 172 bp. Sanger sequencing of the purified HEV amplicons was carried out with the same internal primer pair used for amplifications and the BigDye Terminator v1.1 cycle sequencing ready reaction kit (Applied Biosystems, Thermo Fisher Scientific Baltics UAB, Vilnius, Lithuania). Sequencing reactions were analysed on the 3130xl Genetic Analyzer (Applied Biosystems). Electropherograms were edited using Bioedit v7.1.3 (Hall 1999) and finally each 133 bp sequence (obtained after subtraction of primers sequence) was used for comparison with similar sequences released in database by using the BLAST algorithm (blast. ncbi.nlm.nih.gov). Phylogenetic and molecular evolutionary analyses of the study sequences, reference sequences for Hepatitis E subtypes and similar sequences identified by BLAST, were conducted using MEGA version 7, and compared to existing sequences of HEV from human and non-human origin by bootstrap analysis [19–22]. 2.3. Statistics Continuous parametric variables were expressed as mean ± standard deviation, indicating where appropriate, the minimum and maximum values observed. Continuous variables with non-parametric distribution were expressed as medians. Categorical variables were expressed as frequency and percentage of the cases. The differences were calculated using the “Student’s t” and the “Mann–Whitney” tests for continuous variables and the “chi-square” test for categorical ones. To investigate associations between outcome and other variables, we performed univariate and multivariate analysis by binary logistic regression. Statistical analysis was performed with SPSS software 15.0® and graphics formulated with Excel 2016® . 2.4. Ethics
2. Acute on chronic liver failure (ACLF) was defined as: acute decompensation in patients with chronic liver disease with one or more extrahepatic organ failure(s) [17]. 3. Acute decompensation in patients with CLD was defined as: the development of jaundice, ascites, variceal bleeding or mild hepatic encephalopathy (grade 1–2) in patients with cirrhosis, (previously diagnosed or not), but with no organ failure [17]. 4. Acute kidney injury (AKI) was defined as either an absolute increase in serum creatinine of more than or equal to 0.3 mg/dl in
The study was performed in compliance with the principles of Good Clinical Practice in accordance with the Declaration of Helsinki. 3. Results During the study period 1189 patients underwent testing for HEV. Eight cases were excluded as they had recently travelled to
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Table 1 HEV testing results in patients with autochthonous hepatitis E infection (n = 81). IgM - Positive - Borderline - Not tested IgG - Positive - Borderline - Negative - Not tested PCR - Positive Stool (22/41) Plasma (8/41) Stool and plasma (11/41) - Negative - Not tested Genotype 3 - Stool (22/41) - Plasma (8/41) - Stool and plasma (11/41)
76 (93.9%) 4 (4.9%) 1 (1.2%) 71 (87.6%) 1 (1.2%) 8 (9.9%) 1 (1.2%) 41 (50.6%)
26 (32.1%) 14 (17.3%) 41 (50.6%)
Table 2 Demographic and clinical characteristics of cases of autochthonous hepatitis E infection (n = 81). Male/female Mean age (years) Region - Ancona - Macerata - Fermo - Other towns Recent blood transfusion Alcohol intake >15 units/week Family members affected Pork consumption Active co-morbidity Immunosuppressed • Transplant recipients Liver Kidney • HIV infection • Iatrogenica Cirrhosis Hepatocellular carcinoma Diabetes Previous neoplasia
54/27 55.5 ± 16.7 48 (59.2%) 18 (22.2%) 8 (9.8%) 7 (8.6%) 4 (5.0%) 5 (6.2%) 4 (4.9%) 46 (56.8%) 32 (39.5%) 8 (9.9%) 6 (7.4%) 5 1 1 1 5 (6.2%) 5 (6.2%) 6 (7.4%) 3 (3.4%)
a This patient was taking mycophenolate mofetil and rituximab for Sjogren’s syndrome.
Asia or Africa and/or were found to have HEV-1 infection on testing. This left 81 (6.8% of the total tested) patients that fulfilled the above case definition for locally acquired hepatitis E infection. One patient had visited Asia but had returned to Italy one month prior to presentation. She was found to be infected with HEV-3 and was considered as a locally acquired case. The virologic testing details are shown in Table 1. Forty-one patients were found to be positive for HEV RNA either on plasma or stool or on both specimens, and in all cases genotyping confirmed infection with HEV-3. The mean age of the cases was 55.5 years and 54 (66.6%) were male. Thirty-two patients (39.5%) had active co-morbidities (Table 2). This included eight patients who were immunosuppressed, five patients with pre-existing cirrhosis, and six patients with diabetes. None of the patients were pregnant. Acute hepatitis E infection was found in 79 cases and chronic infection (viraemia >3 months) in two cases, both of whom were immunosuppressed. The laboratory data are shown in Table 3. Forty-five (55.5%) cases required hospital admission for a median of 10 days (range 1–139 days), and in total required 785 days of in-patient hospital care. One patient was transferred in intensive care for a rapid deterioration with multi organ failure.
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Table 3 Laboratory data of the cases of autochthonous hepatitis E infection (n = 81). Bilirubin (mg/dl) Median (min–max) - Presenting - Peak ALT (U/L) - Presenting - Peak ALKP (U/L) - Presenting - Peak INR - Presenting - Peak Albumin (g/dl) - Presenting - Lowest value EGFR (ml/min) (Cockcroft-Gault) - Presenting - Lowest value
2.58 (0.4–24.6) 4.56 (0.5–36.9) 1,505.9 ± 1,425.6 1693,9 ± 1,454.8 277.3 ± 157.4 303.3 ± 158.0 1.37 ± 0.55 1.51 ± 0.63 3.43 ± 0.54 3.17 ± 0.65
86.5 ± 30.6 76.4 ± 32.1
The variables are expressed as mean ± SD, unless otherwise indicated. ALT = alanine aminotransferase (normal range <40 U/L); ALKP = alkaline phosphatase (normal range <135 U/L). Normal values: bilirubin 0.2–1.2 mg/dl; albumin 3.5-4.5 g/dl. EGFR = estimated glomerular filtration rate.
Table 4 Clinical outcome in cases of autochthonous hepatitis E infection (n = 81). Admitted to hospital Median number of days spent in hospital (range) Required intensive care Median weeks of follow-up (range) Chronic infection Acute on chronic liver failure Hepatic encephalopathy Acute kidney injury Nephropathy due to HEV Neurological manifestations • Parsonage Turner syndrome • Myalgia, paraesthesia Death Median number of days (range) from presentation to death
45 (55.5%) 10 (1–139) 1 (1.2%) 5 (1–220) 2 (2.4%) 4 (4.9%) 3 (3.7%) 6 (7.4%) 1 1 1 8 (9.8%) 90 (30–150)
Four patients developed ACLF, six patients developed AKI and eight patients died (all had acute infection) at a median of 90 days post-infection (Tables 4). The median age of the patients who died was 61 years (range 48–76 years), and all had active comorbidities including CLD, diabetes, immunosuppression and hepatocellular/cholangiocarcinoma. In six (7.4%) of the cases HEV infection was judged to be a contributory cause to the patient’s death, two of whom were liver transplant recipients. Univariate analysis showed that serum bilirubin and INR (at presentation), presence of immunosuppression, cirrhosis or diabetes were associated with death. On multivariant analysis the only predictor of death was the presence of diabetes (p = 0.04; CI 1.14–314.97), (Table 5). During hospital admission there was a nonsignificant fall in renal function. However, the reduction in renal function was significant in patients with diabetes (Fig. 1). Three cases with ACLF developed concurrent AKI, and all of them died. Two patients presented with neurological symptoms (Parsonage Turner Syndrome n = 1, and myalgia/paraesthesia n = 1). Both patients survived with complete resolution of their neurological symptoms. Two immunosuppressed patients (post liver transplant tacrolimus therapy n = 1; corticosteroids and rituximab therapy for Sjogren’s syndrome n = 1) developed chronic infection. Following virologic and histological diagnosis of chronic hepatitis, both patients were treated with ribavirin (13–15 mg/kg) for three months. The first case achieved rapid and sustained viral clearance with normalization of serum transaminases. The second
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Table 5 Univariate and multivariate analysis of variables associated with death in patients with autochthonous hepatitis E. Variables

SE
P
Odds ratio
95%CI

SE
P
Odds ratio
95%CI
ALT Bilirubin INR Albumin EGFR PPI consumption Alcohol >15 units/week Immunosuppression Liver cirrhosis Diabetes
−0.01 0.08 2.14 −1.53 −0.01 2.15 2.23 2.1 4.8 3.6
0.001 0.04 0.88 0.81 0.01 1.21 1.3 0.86 1.24 1.01
0.14 0.03 0.01 0.06 0.45 0.07 0.07 0.01 0.001 0.001
0.99 1.08 8.50 0.22 0.98 8.57 10.4 8.16 120 35.5
0.99–1.00 1.0–1.17 1.52–47.40 0.04-1.06 0.96–1.02 0.80–91.49 0.78–137.83 1.49–44.45 10.5–1373.9 4.93–255.50
2.94
1.43
0.04
19.0
1.14–314.97
ALT = alanine aminotransferase; INR = international normalized ratio; PPI = proton pump inhibitor.
4. Discussion
Fig. 1. Renal function in hepatitis E cases. (a) EGFR* (estimated glomerular filtration rate) at baseline and lowest values during hospitalization in overall hospitalized patients (n = 46) and in diabetics (n = 6) with hepatitis E. (b) EGFR* at baseline and lowest values during hospitalization in nondiabetics (n = 40) compared with diabetics (n = 6) with hepatitis E. (c) Change () of EGFR* estimate at baseline and during hospitalization (lowest value) comparing diabetic and non-diabetic patients. *Mean, standard deviation.
patient relapsed after three months therapy and was re-treated with a further course of ribavirin for six months resulting in sustained response. None of the acute cases received anti-viral therapy.
The current study shows that hepatitis E is not uncommon in the Marche region of Italy, with 89 (7.5%) cases in 1189 patients tested. Over 90% of the cases identified were locally acquired: all were caused by HEV-3, usually resulting in a self-limiting illness most commonly in older males. These observations are congruent with findings from many European countries [16,23] and add weight to the notion that the epidemiology of hepatitis E in Italy is changing. Previous nationwide studies of hepatitis E in Italy showed that infections were mainly imported from Asia/Africa associated with recent travel and/or HEV-1. For example, a study from 1994–2009 showed that 109/134 (81%) of infections were travel-related [24]. Another report (2007–10) found that 12 (70%) of 17 viraemic cases were caused by HEV-1 [25]. A recent study performed in the context of enhanced national surveillance for HEV (2012–16) documented 169 cases of laboratory-confirmed cases. Of 65 viraemic cases with sequencing data, 32% were caused by HEV-1, 66% by HEV-3 and 1% by HEV-4 [26]. The reasons for the apparent temporal increase in locally acquired hepatitis E infection in Italy could have several explanations. One possibility is that the amount of circulating HEV gt3 has increased in the human population, but there is little evidence to support this hypothesis. Over recent years the criteria for HEV testing has changed. Previously, only patients with hepatitis returning from an area endemic for HEV-1 were considered for HEV testing: a diagnostic strategy congruent with previous thinking that HEV in Europe was an imported disease. As it became apparent that HEV is also endemic in many high-income countries, HEV testing strategies have changed and recent European Guidelines recommend testing all patients with hepatitis, irrespective of travel history [15]. Finally, recent data shows that the overall anti-HEV IgG seroprevalence in Italy is 8.7%, which is one of the lowest in Europe [27,28]. Seroprevalence is not uniformly distributed throughout Italy (regional range: 2.2–22.8%) and is higher than average in Marche (13%). Taken together, these data suggest that the most likely reason for the large numbers of locally acquired cases reported in the current paper is a combination of improved case-ascertainment in a region that appears to have moderate amounts of locally acquired HEV-3 affecting the human population. The source and route of infection in the cases described is uncertain. Pork and pork sausage consumption appear to be a risk factor for locally acquired hepatitis E infection in many countries, including Italy [26]. Fifty-seven percent of patients in the current series described recent consumption of pork products, including ‘ciauscolo’ sausage, a local salami stuffed with fresh pork meat which is usually consumed without cooking. Local pork-based dietary preferences might partially explain the high number of locally acquired cases observed [26,29], but sequencing data showed a range of HEV sub genotypes which implies that the source of infection was from multiple sources. The origin of such infection is not necessarily due
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to consumption of infected pork, as HEV-3 has also been found in shellfish [30], fresh fruit and salads [31], in workers with occupational exposure [32] and in blood donors [33]. In addition, HEV-3 has recently been found in sheep [34] in Abruzzo (a region adjacent to Marche), which has the highest seroprevalence in Italy, which in some municipalities is >30% [28]. The burden of disease for HEV-1/2 infections was estimated at 20 million infections and 70,000 deaths per year in 2005 [35]. In contrast, hepatitis E caused by genotype 3 is considered a benign disease usually resulting in an asymptomatic/self-limiting illness. However, published data on outcome are incomplete and the burden of disease is unknown. Deaths due to ACLF have been described in case-reports [36] and small cohort studies [12,13,37] in patients with pre-existing liver disease; less commonly due to ALF in the absence of underlying liver disease [14]; and also, in the immunosuppressed with chronic infection [8]. Reported mortality rates vary considerably. For example, a recent EU-wide study reported 28 deaths in 21,018 cases (0.13%), but the cause of death was not stated [23]. Other cohort studies report mortality rates between 1–4% [12,37], and deaths were mainly due to ACLF in patients with underlying CLD. The current study shows a mortality rate of 8/81 (9.9%), which is much higher than previous estimates on the HEV-related mortality. Therefore, we evaluated HEV role in the deaths of our patients categorizing this as causal, contributory or non-contributory. This approach was adopted independently by two experienced clinicians mindful that the cause of death is often multifactorial, particularly in the elderly. By applying this approach, six (7.4%) cases were found to have HEV as a causal or contributory role in the patients’ death. All the patients who died had significant co-morbidity, including CLD, hepatocellular cancer, diabetes, chronic renal disease, liver transplantation and cholangiocarcinoma. Factors predicting mortality in hepatitis E genotype 3 infection have previously not been described. In the current study univariate analysis showed that serum bilirubin, INR, and presence of immunosuppression, cirrhosis or diabetes were associated with death. On multivariant analysis the only predictor of death was the presence of diabetes. The role of diabetes in the mortality from hepatitis E infection is uncertain. It is noteworthy that renal function deteriorated during hospital admission in patients with diabetes and that three patients with ACLF also developed AKI, two of whom died. Diabetics have an increased risk of fatty liver disease, are prone to develop AKI when exposed to any systemic insult and are more likely to develop severe hepatitis and liver failure in the context of acute viral hepatitis [38]. This may explain, at least in part, why the presence of diabetes was a predictor of death in patients with hepatitis E. The reason that underlying CLD did not feature as a risk factor on multivariant analysis probably relates to the small size of the study population. Another measure of the burden of disease is the requirement for hospitalisation. There are very limited data on the need for hospitalisation in patients with hepatitis E infection. A study of >20,000 cases with data from 14 European countries [23] found that hospitalisations for hepatitis E increased from less than 100 cases in 2005 to more than 1100 in 2015. Over the study period the hospitalisation rate declined from 80% to 55%, but the number of days spent in hospital was not stated. In the current study 45 (55.5%) cases required hospital admission for a median of 10 days and in total required 785 days of in-patient hospital care. This included one patient who required lengthy treatment on the Intensive Care Unit. These observations, together with the mortality data described above suggest that the burden of disease due to HEV-3 could be higher than might have been assumed to be the case. The strengths of our study are that it was performed in a single centre which employed well established and validated diagnostics for HEV using a relatively stable diagnostic algorithm for
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HEV testing. Data retrieval was good, and causation of mortality was approached in meaningful ‘real-life’ context. In common with any retrospective study, our study has several weaknesses, including incomplete data capture and possible case-selection bias. Case selection bias probably accounts for the very small number of chronic and neurological cases we observed in our cohort. For example, we documented only two (2.5%) of cases of HEVassociated neurological injury. Previous studies have shown a much higher incidence, with neurological injury observed in 5.5%–7.5% in cohorts from France and England [36,39], and a recent French study which observed neurological injury in 22% of cases of hepatitis E in the immunocompetent [40]. The reason why we found so few cases of neurological damage may relate to the fact that such cases often have only minor liver function test derangement and frequently present to neurologists who may be less likely to request HEV testing. However, the main weakness of the study was the relatively limited number of cases available. This could well have a resulted in a type II error in the statistical analysis of risk factors. For example, underlying CLD was a risk factor for death on univariant but not multivariant analysis: this discrepancy might simply be due to insufficient number of observations. A multi-centre international study of outcomes in locally acquired hepatitis E infection is currently in progress, which will address this issue. In conclusion, hepatitis E in Marche Italy is mostly locally acquired and caused by HEV-3. Eight patients (9.9%) with acute hepatitis E infection died all of whom had significant and often multiple co-morbidity; six cases (7.4%) were found to have HEV as a causal or contributory role in the patients’ death. The presence of diabetes was an independent risk factor for death. 55% of patients required hospitalisation, consuming 785 days of in-patient hospital care. The burden of disease caused by HEV-3 may be higher than previously thought, but this requires confirmation with larger multicentre studies. Conflict of interest None declared. Acknowledgements We must dedicate a special acknowledgement to Dr. Harry R. Dalton, for his decisive role in drafting this work, as he has encouraged us to write it, for his critical re-analysis of data, and his intellectual and linguistic support. References [1] Dalton HR, Bendall R, Ijaz S, Banks M. Hepatitis E: an emerging infection in developed countries. Lancet Infect Dis 2008;8:698–709. [2] Kamar N, Bendall R, Legrand-Abravanel F, Xia NS, Ijaz S, Izopet J, et al. Hepatitis E. Lancet 2012;379:2477–88. [3] Kamar N, Dalton HR, Abravanel F, Izopet J. Hepatitis E virus infection. Clin Microbiol Rev 2014;27:116–38. [4] Kamar N, Izopet J, Pavio N, Aggarwal R, Labrique A, Wedemeyer H, et al. Hepatitis E virus infection. Nat Rev Dis Primers 2017;3(November):17086. [5] Kamar N, Selves J, Mansuy JM, Ouezzani L, Peron JM, Guitard J, et al. Hepatitis E virus and chronic hepatitis in organ-transplant recipients. N Engl J Med 2008;358:811–7. [6] Versluis J, Pas SD, Agteresch HJ, de Man RA, Maaskant J, Schipper ME, et al. Hepatitis E virus: an underestimated opportunistic pathogen in recipients of allogeneic hematopoietic stem cell transplantation. Blood 2013;122(August (6)):1079–86. [7] Dalton HR, Bendall R, Keane F, Tedder R, Ijaz S. Persistent carriage of hepatitis E virus in patients with HIV infection. N Engl J Med 2009;361:1025–7. [8] von Felden J, Alric L, Pischke S, Aitken C, Schlabe S, Spengler U, et al. The burden of hepatitis E among patients with hematological malignancies: a retrospective European cohort study. J Hepatol 2019;(May). [9] Dalton HR, Izopet J. Transmission and epidemiology of hepatitis E virus genotype 3 and 4 infections. Cold Spring Harb Perspect Med 2018;8(November (11)). [10] Lee GH, Tan BH, Teo EC, Lim SG, Dan YY, Wee A, et al. Chronic infection with camelid hepatitis E virus in a liver transplant recipient who regularly consumes camel meat and milk. Gastroenterology 2016;150(February (2)):355–7.
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Digestive and Liver Disease journal homepage: www.elsevier.com/locate/dld
Liver, Pancreas and Biliary Tract
Locally acquired hepatitis E virus in Marche Italy: Clinical/laboratory features and outcome Giuseppe Tarantino a,∗ , Alessio Ortolani a , Katia Marinelli b , Antonio Benedetti a , Giulia Marconi a , Manuela Calzolari a , Harry R. Dalton c , Marco Marzioni a , Laura Schiadà a , Giammarco Fava a , Alessandro Chiodera d , Giorgio Amadio e , Alessandro Fiorentini f , Alessandra Riva f , Paolo Fraticelli g , Stefano Menzo b , Patrizia Bagnarelli b a
Department of Gastroenterology and Transplants, Clinic of Gastroenterology, Hospital of Ancona, Università Politecnica delle Marche, Italy Department of Biomedical Sciences and Public Health, Virology, Hospital of Ancona, Università Politecnica delle Marche, Italy c Queens Park, London, UK d Unit of Infective diseases, Hospital of Macerata, Italy e Unit of Infective diseases, Hospital of Fermo, Italy f Units of Infective diseases Hospital of Ancona, Università Politecnica delle Marche, Italy g Department of Internal Medicine, Unit of Medical Clinic, Hospital of Ancona, Università Politecnica delle Marche, Italy b
a r t i c l e
i n f o
Article history: Received 20 August 2019 Accepted 18 November 2019 Available online xxx Keywords: Acute on-chronic liver failure Chronic liver disease Diabetes HEV
a b s t r a c t Background and aims: Hepatitis E Virus is endemic in Europe with increasing numbers of cases in recent years, also in Italy where this phenomenon has hitherto been modest. The aim of this study was to document the clinical features/natural history of locally acquired hepatitis E in our territory and explore factors which determine adverse outcome. Methods: Retrospective study of patients with locally-acquired HEV (hepatitis E virus) in Marche, Italy (2011–2019). Results: 1189 patients were tested for HEV with 89 confirmed cases. 81 (6.8%) had locally acquired infection; 54 (66%) were male (mean age 55.5 years) and 32 (39.5%) had active co-morbidities. 41 cases were viraemic (all HEV-3 (HEV genotype 1,2,3,4)); acute infection was found in 79 and chronic infection in 2. Forty-five cases (55%) required admission to hospital, for a total of 785 days. 4 patients developed acute on-chronic liver failure, 6 developed acute kidney injury and 8 died: all had active comorbidities. Univariate analysis showed that bilirubin, INR, immunosuppression, cirrhosis and diabetes were associated with death. On multivariant analysis the only predictor of death was the presence of diabetes (p = 0.04). Conclusions: Hepatitis E in Marche Italy is mostly locally acquired and caused by HEV-3 that impacts on the morbidity and mortality particularly for fragile patients. © 2019 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.
1. Introduction In low-income countries in Asia and Africa, hepatitis E is mainly caused by HEV-1 and 2, which are obligate humans pathogens spread orofecally mainly by contaminated water. Hepatitis E occurs as both sporadic cases and outbreaks, usually resulting in a selflimiting hepatitis in young adults. However, there is adverse outcome in pregnant women, with mortality rates of up to 25%. For many years’ hepatitis E was thought not to be a major issue in high-income countries (including Europe), except for imported
∗ Corresponding author at: Via Conca 71, 60126, Ancona, Italy. E-mail address: [email protected] (G. Tarantino).
cases in travelers returning from countries endemic for HEV-1 and 2 [1–4]. Over the past 15 years, it has become clear that HEV is a global health issue. Many higher income countries are also endemic for HEV. In these areas locally acquired hepatitis E is mainly zoonotic and caused by HEV-3 (Europe and Japan) or 4 (China and Japan) usually resulting in sporadic asymptomatic infection [1–4]. Only a small minority of patients develop acute hepatitis and chronic infection occurs in the immunosuppressed [4–8]. Pigs are considered the primary reservoir. However, human infection with HEV has also been found to originate from other sources, including wild boar, deer, rabbits, camels (genotype 7), rats and blood products [9–11].
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In patients with HEV-3 infection, the outcome is usually favorable and deaths in pregnancy have not been observed. Case reports/cohort studies have reported mortality in patients with underlying chronic liver disease (CLD) due to acute-on-chronic liver failure (ACLF) [12,13], and less commonly in patients without liver disease due to acute liver failure (ALF) and chronic infection [8,14]. However, the burden of disease and the factors that predict poor outcome are unknown. In Europe it is estimated that there are > 2 million human infections with HEV per year [15], caused mostly by locally acquired HEV-3, but the incidence varies considerably between countries. In Italy, along with most countries in Europe, the number of locally acquired cases is increasing [16]. However, the number of laboratory-confirmed cases of hepatitis E has hitherto been modest, with more travel-related cases compared to many other European countries. The aim of this study was to document the clinical features and natural history of locally acquired hepatitis E in the Marche region of Italy, and to explore factors which determine adverse outcome. 2. Patients and methods Patients presenting with hepatitis E infection during the period 2011–2019 were studied retrospectively. The setting for the study was the Marche region of Italy (population approximately 1.5 million inhabitants), including the city of Ancona and the towns of Macerata, Fermo, Pesaro, Ascoli e Fabriano. By retrospectively evaluating clinical records, screened patients for HEV were generally those with acute liver injury (ALI) in whom common etiologies such as HAV, HBV, HCV, biliary obstruction and drug-hepatotoxicity, were not found. Moreover, the cases with suspected alimentary risk factors were screened too. In Ancona Hospital, where the awareness of HEV-related disease has grown over the years, fragile patients, due to immune-suppression or chronic liver disease, even if they had mild ALI or those with suspected extrahepatic manifestations, were tested too. Cases of HEV infection were identified from their Microbiology Department records in Ancona, where regional HEV testing was performed throughout the study period. Cases who had recently travelled to areas known to be endemic for HEV-1 and 2 were excluded from the study and/or cases that were found to have HEV infection caused by genotypes 1 and 2. Each laboratory-confirmed locally acquired case underwent retrospective case note review and a pre-defined set of variables were recorded. This included demographic, dietary, clinical and laboratory data; presence or absence of pre-existing CLD, decompensation and/or ACLF; clinical outcome, including death and its cause(s); and length of hospital stay. The contribution of HEV to the cause of death was made by two experienced clinicians (GT and HD). 2.1. Definitions 1. A case of hepatitis E infection was defined as: • Anti-HEV IgM reactive and/or • HEV RNA detected in serum and/or stool
less than 48 h, or by a percentage increase in serum creatinine of more or equal to 50% (1.5-fold from baseline) in less than seven days. [18]. 2.2. HEV testing The presence of anti-HEV IgM and IgG antibodies was determined by testing patient sera with a commercial ELISA (enzyme linked immunoassay) immunoassay employing a synthetic HEV antigen derived from open reading frame (ORF) 2 and ORF3 regions (Diagnostic Bioprobes Srl, Milan, Italy). The presence of HEV-RNA was determined following RNA extraction from patient faecal and/or serum samples. Nucleic acid extraction was performed on QIA symphony SP instrument (QIAGEN® ) using dedicated kits and protocols. HEV-RNA was detected by reverse transcription nested-PCR using a broad-range primer set amplifying a portion of the ORF1 corresponding to the methyl transferase gene with a HEV 1–4 genotypes specificity. The primer sequences were: ORF1F 5 -CCAYCAGTTYATHAAGGCTCC (forward first round RTPCR (reverse transcription-polymerase chain reaction)) and ORF1R 5 -TACCAVCGCTGRACRTC (reverse first round RT-PCR), product length 348 bp; ORF1FN 5 -CTCCTGGCRTYACWACTGC (forward nested PCR) and ORF1RN 5 -GGRTGRTTCCAIARVACYTC (reverse nested PCR), product length 172 bp. Sanger sequencing of the purified HEV amplicons was carried out with the same internal primer pair used for amplifications and the BigDye Terminator v1.1 cycle sequencing ready reaction kit (Applied Biosystems, Thermo Fisher Scientific Baltics UAB, Vilnius, Lithuania). Sequencing reactions were analysed on the 3130xl Genetic Analyzer (Applied Biosystems). Electropherograms were edited using Bioedit v7.1.3 (Hall 1999) and finally each 133 bp sequence (obtained after subtraction of primers sequence) was used for comparison with similar sequences released in database by using the BLAST algorithm (blast. ncbi.nlm.nih.gov). Phylogenetic and molecular evolutionary analyses of the study sequences, reference sequences for Hepatitis E subtypes and similar sequences identified by BLAST, were conducted using MEGA version 7, and compared to existing sequences of HEV from human and non-human origin by bootstrap analysis [19–22]. 2.3. Statistics Continuous parametric variables were expressed as mean ± standard deviation, indicating where appropriate, the minimum and maximum values observed. Continuous variables with non-parametric distribution were expressed as medians. Categorical variables were expressed as frequency and percentage of the cases. The differences were calculated using the “Student’s t” and the “Mann–Whitney” tests for continuous variables and the “chi-square” test for categorical ones. To investigate associations between outcome and other variables, we performed univariate and multivariate analysis by binary logistic regression. Statistical analysis was performed with SPSS software 15.0® and graphics formulated with Excel 2016® . 2.4. Ethics
2. Acute on chronic liver failure (ACLF) was defined as: acute decompensation in patients with chronic liver disease with one or more extrahepatic organ failure(s) [17]. 3. Acute decompensation in patients with CLD was defined as: the development of jaundice, ascites, variceal bleeding or mild hepatic encephalopathy (grade 1–2) in patients with cirrhosis, (previously diagnosed or not), but with no organ failure [17]. 4. Acute kidney injury (AKI) was defined as either an absolute increase in serum creatinine of more than or equal to 0.3 mg/dl in
The study was performed in compliance with the principles of Good Clinical Practice in accordance with the Declaration of Helsinki. 3. Results During the study period 1189 patients underwent testing for HEV. Eight cases were excluded as they had recently travelled to
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Table 1 HEV testing results in patients with autochthonous hepatitis E infection (n = 81). IgM - Positive - Borderline - Not tested IgG - Positive - Borderline - Negative - Not tested PCR - Positive Stool (22/41) Plasma (8/41) Stool and plasma (11/41) - Negative - Not tested Genotype 3 - Stool (22/41) - Plasma (8/41) - Stool and plasma (11/41)
76 (93.9%) 4 (4.9%) 1 (1.2%) 71 (87.6%) 1 (1.2%) 8 (9.9%) 1 (1.2%) 41 (50.6%)
26 (32.1%) 14 (17.3%) 41 (50.6%)
Table 2 Demographic and clinical characteristics of cases of autochthonous hepatitis E infection (n = 81). Male/female Mean age (years) Region - Ancona - Macerata - Fermo - Other towns Recent blood transfusion Alcohol intake >15 units/week Family members affected Pork consumption Active co-morbidity Immunosuppressed • Transplant recipients Liver Kidney • HIV infection • Iatrogenica Cirrhosis Hepatocellular carcinoma Diabetes Previous neoplasia
54/27 55.5 ± 16.7 48 (59.2%) 18 (22.2%) 8 (9.8%) 7 (8.6%) 4 (5.0%) 5 (6.2%) 4 (4.9%) 46 (56.8%) 32 (39.5%) 8 (9.9%) 6 (7.4%) 5 1 1 1 5 (6.2%) 5 (6.2%) 6 (7.4%) 3 (3.4%)
a This patient was taking mycophenolate mofetil and rituximab for Sjogren’s syndrome.
Asia or Africa and/or were found to have HEV-1 infection on testing. This left 81 (6.8% of the total tested) patients that fulfilled the above case definition for locally acquired hepatitis E infection. One patient had visited Asia but had returned to Italy one month prior to presentation. She was found to be infected with HEV-3 and was considered as a locally acquired case. The virologic testing details are shown in Table 1. Forty-one patients were found to be positive for HEV RNA either on plasma or stool or on both specimens, and in all cases genotyping confirmed infection with HEV-3. The mean age of the cases was 55.5 years and 54 (66.6%) were male. Thirty-two patients (39.5%) had active co-morbidities (Table 2). This included eight patients who were immunosuppressed, five patients with pre-existing cirrhosis, and six patients with diabetes. None of the patients were pregnant. Acute hepatitis E infection was found in 79 cases and chronic infection (viraemia >3 months) in two cases, both of whom were immunosuppressed. The laboratory data are shown in Table 3. Forty-five (55.5%) cases required hospital admission for a median of 10 days (range 1–139 days), and in total required 785 days of in-patient hospital care. One patient was transferred in intensive care for a rapid deterioration with multi organ failure.
3
Table 3 Laboratory data of the cases of autochthonous hepatitis E infection (n = 81). Bilirubin (mg/dl) Median (min–max) - Presenting - Peak ALT (U/L) - Presenting - Peak ALKP (U/L) - Presenting - Peak INR - Presenting - Peak Albumin (g/dl) - Presenting - Lowest value EGFR (ml/min) (Cockcroft-Gault) - Presenting - Lowest value
2.58 (0.4–24.6) 4.56 (0.5–36.9) 1,505.9 ± 1,425.6 1693,9 ± 1,454.8 277.3 ± 157.4 303.3 ± 158.0 1.37 ± 0.55 1.51 ± 0.63 3.43 ± 0.54 3.17 ± 0.65
86.5 ± 30.6 76.4 ± 32.1
The variables are expressed as mean ± SD, unless otherwise indicated. ALT = alanine aminotransferase (normal range <40 U/L); ALKP = alkaline phosphatase (normal range <135 U/L). Normal values: bilirubin 0.2–1.2 mg/dl; albumin 3.5-4.5 g/dl. EGFR = estimated glomerular filtration rate.
Table 4 Clinical outcome in cases of autochthonous hepatitis E infection (n = 81). Admitted to hospital Median number of days spent in hospital (range) Required intensive care Median weeks of follow-up (range) Chronic infection Acute on chronic liver failure Hepatic encephalopathy Acute kidney injury Nephropathy due to HEV Neurological manifestations • Parsonage Turner syndrome • Myalgia, paraesthesia Death Median number of days (range) from presentation to death
45 (55.5%) 10 (1–139) 1 (1.2%) 5 (1–220) 2 (2.4%) 4 (4.9%) 3 (3.7%) 6 (7.4%) 1 1 1 8 (9.8%) 90 (30–150)
Four patients developed ACLF, six patients developed AKI and eight patients died (all had acute infection) at a median of 90 days post-infection (Tables 4). The median age of the patients who died was 61 years (range 48–76 years), and all had active comorbidities including CLD, diabetes, immunosuppression and hepatocellular/cholangiocarcinoma. In six (7.4%) of the cases HEV infection was judged to be a contributory cause to the patient’s death, two of whom were liver transplant recipients. Univariate analysis showed that serum bilirubin and INR (at presentation), presence of immunosuppression, cirrhosis or diabetes were associated with death. On multivariant analysis the only predictor of death was the presence of diabetes (p = 0.04; CI 1.14–314.97), (Table 5). During hospital admission there was a nonsignificant fall in renal function. However, the reduction in renal function was significant in patients with diabetes (Fig. 1). Three cases with ACLF developed concurrent AKI, and all of them died. Two patients presented with neurological symptoms (Parsonage Turner Syndrome n = 1, and myalgia/paraesthesia n = 1). Both patients survived with complete resolution of their neurological symptoms. Two immunosuppressed patients (post liver transplant tacrolimus therapy n = 1; corticosteroids and rituximab therapy for Sjogren’s syndrome n = 1) developed chronic infection. Following virologic and histological diagnosis of chronic hepatitis, both patients were treated with ribavirin (13–15 mg/kg) for three months. The first case achieved rapid and sustained viral clearance with normalization of serum transaminases. The second
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Table 5 Univariate and multivariate analysis of variables associated with death in patients with autochthonous hepatitis E. Variables

SE
P
Odds ratio
95%CI

SE
P
Odds ratio
95%CI
ALT Bilirubin INR Albumin EGFR PPI consumption Alcohol >15 units/week Immunosuppression Liver cirrhosis Diabetes
−0.01 0.08 2.14 −1.53 −0.01 2.15 2.23 2.1 4.8 3.6
0.001 0.04 0.88 0.81 0.01 1.21 1.3 0.86 1.24 1.01
0.14 0.03 0.01 0.06 0.45 0.07 0.07 0.01 0.001 0.001
0.99 1.08 8.50 0.22 0.98 8.57 10.4 8.16 120 35.5
0.99–1.00 1.0–1.17 1.52–47.40 0.04-1.06 0.96–1.02 0.80–91.49 0.78–137.83 1.49–44.45 10.5–1373.9 4.93–255.50
2.94
1.43
0.04
19.0
1.14–314.97
ALT = alanine aminotransferase; INR = international normalized ratio; PPI = proton pump inhibitor.
4. Discussion
Fig. 1. Renal function in hepatitis E cases. (a) EGFR* (estimated glomerular filtration rate) at baseline and lowest values during hospitalization in overall hospitalized patients (n = 46) and in diabetics (n = 6) with hepatitis E. (b) EGFR* at baseline and lowest values during hospitalization in nondiabetics (n = 40) compared with diabetics (n = 6) with hepatitis E. (c) Change () of EGFR* estimate at baseline and during hospitalization (lowest value) comparing diabetic and non-diabetic patients. *Mean, standard deviation.
patient relapsed after three months therapy and was re-treated with a further course of ribavirin for six months resulting in sustained response. None of the acute cases received anti-viral therapy.
The current study shows that hepatitis E is not uncommon in the Marche region of Italy, with 89 (7.5%) cases in 1189 patients tested. Over 90% of the cases identified were locally acquired: all were caused by HEV-3, usually resulting in a self-limiting illness most commonly in older males. These observations are congruent with findings from many European countries [16,23] and add weight to the notion that the epidemiology of hepatitis E in Italy is changing. Previous nationwide studies of hepatitis E in Italy showed that infections were mainly imported from Asia/Africa associated with recent travel and/or HEV-1. For example, a study from 1994–2009 showed that 109/134 (81%) of infections were travel-related [24]. Another report (2007–10) found that 12 (70%) of 17 viraemic cases were caused by HEV-1 [25]. A recent study performed in the context of enhanced national surveillance for HEV (2012–16) documented 169 cases of laboratory-confirmed cases. Of 65 viraemic cases with sequencing data, 32% were caused by HEV-1, 66% by HEV-3 and 1% by HEV-4 [26]. The reasons for the apparent temporal increase in locally acquired hepatitis E infection in Italy could have several explanations. One possibility is that the amount of circulating HEV gt3 has increased in the human population, but there is little evidence to support this hypothesis. Over recent years the criteria for HEV testing has changed. Previously, only patients with hepatitis returning from an area endemic for HEV-1 were considered for HEV testing: a diagnostic strategy congruent with previous thinking that HEV in Europe was an imported disease. As it became apparent that HEV is also endemic in many high-income countries, HEV testing strategies have changed and recent European Guidelines recommend testing all patients with hepatitis, irrespective of travel history [15]. Finally, recent data shows that the overall anti-HEV IgG seroprevalence in Italy is 8.7%, which is one of the lowest in Europe [27,28]. Seroprevalence is not uniformly distributed throughout Italy (regional range: 2.2–22.8%) and is higher than average in Marche (13%). Taken together, these data suggest that the most likely reason for the large numbers of locally acquired cases reported in the current paper is a combination of improved case-ascertainment in a region that appears to have moderate amounts of locally acquired HEV-3 affecting the human population. The source and route of infection in the cases described is uncertain. Pork and pork sausage consumption appear to be a risk factor for locally acquired hepatitis E infection in many countries, including Italy [26]. Fifty-seven percent of patients in the current series described recent consumption of pork products, including ‘ciauscolo’ sausage, a local salami stuffed with fresh pork meat which is usually consumed without cooking. Local pork-based dietary preferences might partially explain the high number of locally acquired cases observed [26,29], but sequencing data showed a range of HEV sub genotypes which implies that the source of infection was from multiple sources. The origin of such infection is not necessarily due
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to consumption of infected pork, as HEV-3 has also been found in shellfish [30], fresh fruit and salads [31], in workers with occupational exposure [32] and in blood donors [33]. In addition, HEV-3 has recently been found in sheep [34] in Abruzzo (a region adjacent to Marche), which has the highest seroprevalence in Italy, which in some municipalities is >30% [28]. The burden of disease for HEV-1/2 infections was estimated at 20 million infections and 70,000 deaths per year in 2005 [35]. In contrast, hepatitis E caused by genotype 3 is considered a benign disease usually resulting in an asymptomatic/self-limiting illness. However, published data on outcome are incomplete and the burden of disease is unknown. Deaths due to ACLF have been described in case-reports [36] and small cohort studies [12,13,37] in patients with pre-existing liver disease; less commonly due to ALF in the absence of underlying liver disease [14]; and also, in the immunosuppressed with chronic infection [8]. Reported mortality rates vary considerably. For example, a recent EU-wide study reported 28 deaths in 21,018 cases (0.13%), but the cause of death was not stated [23]. Other cohort studies report mortality rates between 1–4% [12,37], and deaths were mainly due to ACLF in patients with underlying CLD. The current study shows a mortality rate of 8/81 (9.9%), which is much higher than previous estimates on the HEV-related mortality. Therefore, we evaluated HEV role in the deaths of our patients categorizing this as causal, contributory or non-contributory. This approach was adopted independently by two experienced clinicians mindful that the cause of death is often multifactorial, particularly in the elderly. By applying this approach, six (7.4%) cases were found to have HEV as a causal or contributory role in the patients’ death. All the patients who died had significant co-morbidity, including CLD, hepatocellular cancer, diabetes, chronic renal disease, liver transplantation and cholangiocarcinoma. Factors predicting mortality in hepatitis E genotype 3 infection have previously not been described. In the current study univariate analysis showed that serum bilirubin, INR, and presence of immunosuppression, cirrhosis or diabetes were associated with death. On multivariant analysis the only predictor of death was the presence of diabetes. The role of diabetes in the mortality from hepatitis E infection is uncertain. It is noteworthy that renal function deteriorated during hospital admission in patients with diabetes and that three patients with ACLF also developed AKI, two of whom died. Diabetics have an increased risk of fatty liver disease, are prone to develop AKI when exposed to any systemic insult and are more likely to develop severe hepatitis and liver failure in the context of acute viral hepatitis [38]. This may explain, at least in part, why the presence of diabetes was a predictor of death in patients with hepatitis E. The reason that underlying CLD did not feature as a risk factor on multivariant analysis probably relates to the small size of the study population. Another measure of the burden of disease is the requirement for hospitalisation. There are very limited data on the need for hospitalisation in patients with hepatitis E infection. A study of >20,000 cases with data from 14 European countries [23] found that hospitalisations for hepatitis E increased from less than 100 cases in 2005 to more than 1100 in 2015. Over the study period the hospitalisation rate declined from 80% to 55%, but the number of days spent in hospital was not stated. In the current study 45 (55.5%) cases required hospital admission for a median of 10 days and in total required 785 days of in-patient hospital care. This included one patient who required lengthy treatment on the Intensive Care Unit. These observations, together with the mortality data described above suggest that the burden of disease due to HEV-3 could be higher than might have been assumed to be the case. The strengths of our study are that it was performed in a single centre which employed well established and validated diagnostics for HEV using a relatively stable diagnostic algorithm for
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HEV testing. Data retrieval was good, and causation of mortality was approached in meaningful ‘real-life’ context. In common with any retrospective study, our study has several weaknesses, including incomplete data capture and possible case-selection bias. Case selection bias probably accounts for the very small number of chronic and neurological cases we observed in our cohort. For example, we documented only two (2.5%) of cases of HEVassociated neurological injury. Previous studies have shown a much higher incidence, with neurological injury observed in 5.5%–7.5% in cohorts from France and England [36,39], and a recent French study which observed neurological injury in 22% of cases of hepatitis E in the immunocompetent [40]. The reason why we found so few cases of neurological damage may relate to the fact that such cases often have only minor liver function test derangement and frequently present to neurologists who may be less likely to request HEV testing. However, the main weakness of the study was the relatively limited number of cases available. This could well have a resulted in a type II error in the statistical analysis of risk factors. For example, underlying CLD was a risk factor for death on univariant but not multivariant analysis: this discrepancy might simply be due to insufficient number of observations. A multi-centre international study of outcomes in locally acquired hepatitis E infection is currently in progress, which will address this issue. In conclusion, hepatitis E in Marche Italy is mostly locally acquired and caused by HEV-3. Eight patients (9.9%) with acute hepatitis E infection died all of whom had significant and often multiple co-morbidity; six cases (7.4%) were found to have HEV as a causal or contributory role in the patients’ death. The presence of diabetes was an independent risk factor for death. 55% of patients required hospitalisation, consuming 785 days of in-patient hospital care. The burden of disease caused by HEV-3 may be higher than previously thought, but this requires confirmation with larger multicentre studies. Conflict of interest None declared. Acknowledgements We must dedicate a special acknowledgement to Dr. Harry R. Dalton, for his decisive role in drafting this work, as he has encouraged us to write it, for his critical re-analysis of data, and his intellectual and linguistic support. References [1] Dalton HR, Bendall R, Ijaz S, Banks M. Hepatitis E: an emerging infection in developed countries. Lancet Infect Dis 2008;8:698–709. [2] Kamar N, Bendall R, Legrand-Abravanel F, Xia NS, Ijaz S, Izopet J, et al. Hepatitis E. Lancet 2012;379:2477–88. [3] Kamar N, Dalton HR, Abravanel F, Izopet J. Hepatitis E virus infection. Clin Microbiol Rev 2014;27:116–38. [4] Kamar N, Izopet J, Pavio N, Aggarwal R, Labrique A, Wedemeyer H, et al. Hepatitis E virus infection. Nat Rev Dis Primers 2017;3(November):17086. [5] Kamar N, Selves J, Mansuy JM, Ouezzani L, Peron JM, Guitard J, et al. Hepatitis E virus and chronic hepatitis in organ-transplant recipients. N Engl J Med 2008;358:811–7. [6] Versluis J, Pas SD, Agteresch HJ, de Man RA, Maaskant J, Schipper ME, et al. Hepatitis E virus: an underestimated opportunistic pathogen in recipients of allogeneic hematopoietic stem cell transplantation. Blood 2013;122(August (6)):1079–86. [7] Dalton HR, Bendall R, Keane F, Tedder R, Ijaz S. Persistent carriage of hepatitis E virus in patients with HIV infection. N Engl J Med 2009;361:1025–7. [8] von Felden J, Alric L, Pischke S, Aitken C, Schlabe S, Spengler U, et al. The burden of hepatitis E among patients with hematological malignancies: a retrospective European cohort study. J Hepatol 2019;(May). [9] Dalton HR, Izopet J. Transmission and epidemiology of hepatitis E virus genotype 3 and 4 infections. Cold Spring Harb Perspect Med 2018;8(November (11)). [10] Lee GH, Tan BH, Teo EC, Lim SG, Dan YY, Wee A, et al. Chronic infection with camelid hepatitis E virus in a liver transplant recipient who regularly consumes camel meat and milk. Gastroenterology 2016;150(February (2)):355–7.
Please cite this article in press as: Tarantino G, et al. Locally acquired hepatitis E virus in Marche Italy: Clinical/laboratory features and outcome. Dig Liver Dis (2019), https://doi.org/10.1016/j.dld.2019.11.015
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Please cite this article in press as: Tarantino G, et al. Locally acquired hepatitis E virus in Marche Italy: Clinical/laboratory features and outcome. Dig Liver Dis (2019), https://doi.org/10.1016/j.dld.2019.11.015