MAJOR ARTICLE
Endemic Hepatitis E in the Czech Republic Pavel Chalupa,1 Petra Vasickova,2 Ivo Pavlik,2 and Michal Holub1 1
Department of Infectious and Tropical Diseases, First Faculty of Medicine, Charles University in Prague and Na Bulovce Hospital, Prague, and Department of Food and Feed Safety, Veterinary Research Institute, Brno, Czech Republic
2
Keywords.
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Background. An increasing incidence of endemic hepatitis E (HE) has been reported in developed countries. Thus, an evaluation of the clinical characteristics of the disease and the utility of the current diagnostic methods is warranted. Methods. Fifty-one adult acute patients with HE hospitalized in a single center between the years 2009 and 2012 were evaluated. Serological and molecular techniques (detection of hepatitis E virus [HEV] RNA from stool and serum samples by quantitative reverse transcription polymerase chain reaction) with sequencing and phylogenetic analysis were used for diagnosis, and the clinical, laboratory, and epidemiological parameters of the patients were evaluated. Results. Forty-nine (96.1%) patients had acute endemic HE and 2 (3.9%) had an imported infection. In the cohort of patients with acute symptomatic HE (n = 47), men outnumbered women (40:7), the patients were in older middle age (mean, 60.57 years), and they had elevated median values of total bilirubin (6.67 mg/dL), alanine aminotransferase (2288.82 U/L), aspartate aminotransferase (1251.76 U/L), gamma-glutamyl transferase (360.53 U/L), and alkaline phosphatase (197.06 U/L). Serology was positive in 50 (98%) of the patients, and 1 case was diagnosed by polymerase chain reaction only. HEV RNA was detected in at least 1 specimen from 84.3% of the patients, and 28 of 29 tested isolates belonged to genotype 3. The eating of meat, innards, other home-prepared pork products, or the tasting of raw meat before cooking were the most frequently reported data (reported by 25 patients [49.0%]). Conclusions. Large numbers of the endemic cases of HE were caused by HEV genotype 3, and the clinical characteristics of endemic HE were demonstrated. hepatitis E; endemic cases; clinical findings; zoonosis.
Hepatitis E (HE) is one of the most common forms of hepatitis transmitted via the fecal-oral route worldwide. The causative agent of the disease, the hepatitis E virus (HEV), is classified in the genus Hepevirus, family Hepeviridae [1]. The Hepevirus genus also includes HEV strains that are capable of infecting other animal species such as avian hosts (chicken) [2]. Although only 1 serotype was described within the genus Hepevirus, 4 major genotypes with different modes of transmission, pathogenicity, reservoirs, and capacity for interspecies transmission were recognized in this genus [3].
Received and accepted 17 November 2013; electronically published 26 November 2013. Correspondence: Pavel Chalupa, MD, PhD, Department of Infectious and Tropical Diseases, Na Bulovce Hospital, Budinova 2, CZ-180 81 Praha 8, Czech Republic ( [email protected]). Clinical Infectious Diseases 2014;58(4):509–16 © The Author 2013. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: [email protected]. DOI: 10.1093/cid/cit782
Although each HEV genotype is believed to have a specific geographic distribution, exceptions have been observed. Genotype 1 is mostly associated with epidemic or sporadic cases in the tropical and subtropical countries of Asia and Africa, whereas genotype 2 has been detected in Mexico and some African countries. The relative conservation of these 2 genotypes corresponds to their primary circulation among humans. Conversely, the diversity of the HEV genotypes 3 and 4 is related to their zoonotic potential. Genotype 3 has been isolated from patients with acute HE and from different animal species (in particular, domestic pigs, wild boar, and deer are the ones most likely to be involved in human transmission) worldwide. Genotype 4 contains human and animal strains originating particularly from Asian countries [4, 5]. Recently, genotype 4 was isolated from patients with endemic HE and domestic pigs in Europe [6, 7]. HEV infection has at least 2 epidemiological profiles: large waterborne outbreaks and sporadic cases. The
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Figure 1. Number of imported and endemic (autochthonous) cases of acute hepatitis E reported to the central Czech database EPIDAT in the National Institute of Public Health in Prague (http://www.szu.cz/publikace/ data/vybrane-infekcni-nemoci-v-cr-v-letech-2003-2012-absolutne). Abbreviation: HE, hepatitis E.
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significant increase in the number of endemic HE cases has been observed (Figure 1) [14]. The disease is frequently recognized only by serological testing. Routine laboratory diagnostics for HE may be one reason for the observed increasing incidence. Almost all of the endemic HE cases are associated with genotype 3 or 4, and HEV isolates from patients show a high sequence homology to those found in domestic pigs and other animals [5, 15]. Despite an increasing incidence, data regarding epidemiology and clinical manifestations of HEV infections in Central Europe are very limited. Therefore, the aim of the present study was characterization of acute hepatitis E cases hospitalized in a large referral center for infectious patients in Prague (Czech Republic), with a special attention to endemic cases of the disease. PATIENTS AND METHODS Fifty-one patients (43 men, mean age, 60 years [range, 37–74 years]; 8 women, mean age, 56 years [range, 42–77 years]) with acute HE were enrolled between 2009 and 2012 (Table 1). Informed consent was obtained from all patients after obtaining approval from the local ethics committee (IRB00002721, Eticka komise Nemocnice Na Bulovce, IRB No. 1, Biomedical). The diagnosis of acute HE was established by the detection of antiHEV antibodies, and positivity on enzyme-linked immunosorbent assay was confirmed by Western blotting. For patients 1– 7, the EIAgen HEV IgM Kit, EIAgen HEV IgG Kit (both Adaltis, Italy), and Recomblot HEV IgM/IgG (Microgen GmbH, Germany) were used; for patients 8–51, the recomWell HEV IgM, recomWell HEV IgG, and recomLine HEV IgG/IgM (Microgen GmbH) were used. In addition, all patients tested negative for hepatitis A, B, and C virus infections, and the use of hepatotoxic drugs was ruled out. We also evaluated the clinical, biochemical, hematological, and epidemiological parameters of the enrolled patients. Correlations between the biochemical and hemocoagulation examinations were tested using Spearman and Pearson correlations (Sigma Stat; Jandel Scientific, San Rafael, California). The serum and stool samples originating from the enrolled patients were tested for the presence of the HEV genome (RNA) through the use of molecular methods. If possible, samples were collected at the beginning (stool and serum) and at the end (stool) of a patient’s hospitalization. Detection of HEV RNA was performed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) [16, 17]. Subsequently, the partial parts of the open reading frame (ORF) 1 and the overlapping region of ORFs 2 and 3 (ORF2/3) of the acquired HEV isolates were subjected to sequencing (Eurofins MWG Operon, Germany); sequence and phylogenetic analyses were performed using MEGA 3.1 software. The neighbor-joining method with 1000 replications in the bootstrap test was used
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outbreaks are usually caused by genotype 1 or 2, and they result in high morbidity and mortality among pregnant women and young children in developing countries. The most common clinical presentation is acute icteric hepatitis, which is indistinguishable from other forms of viral hepatitis. HE attack rates peak among young to middle-aged adults (15–30 years). Rates of infection are usually higher in men than in women; however, no sex difference exists in exposure to HEV. The sporadic cases of HE, which present clinically as icteric hepatitis or anicteric illnesses with nonspecific symptoms, are associated with genotypes 3 and 4 in both developing and industrialized countries. Endemic (autochthonous) HEV infections in developed countries, which are not related to travel in epidemic regions, have certain clinical features. Most patients are older (middle-aged and elderly people) and predominantly are men with an increased frequency of underlying liver disease or alcohol use. HEV can also cause neurological manifestations and chronic infection in immunocompromised persons, including those infected with the human immunodeficiency virus (HIV) [8, 9]. In contrast to epidemic HE, pregnant women do not show a severe course of endemic HEV infection. Nevertheless, sporadic HEV infections are associated with morbidity and mortality [10–12]. Reports of HEV infections without travel association have become more frequent in nonhyperendemic regions (industrialized countries) in recent years [13]. According to the periodic reports of the National Institute of Public Health in Prague (Czech Central Database EPIDAT, http://www.szu.cz/publikace/ data/vybrane-infekcni-nemoci-v-cr-v-letech-2003-2012-absolutne), the numbers of imported cases of HE in the Czech Republic have remained stable during the last 2 decades, whereas a
Table 1.
Characterization of Patients and Detection of Hepatitis E Virus RNA by Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction Hematological Parameters
Biochemical Examinations
Patient No.
Age, y/Sex
Presence of HEV RNAa
Hospital Stay, d
Anti-HEV IgM/IgG
Bilirubin Total, mg/dL
Bilirubin Direct, mg/ dL
ALT, U/L
AST, U/L
GGT, U/L
ALP, U/L
INR
AT III, %
PLT, 109/L
2.69
4639.41
3045.88
543.49
407.06
1.17
59
263
6.00 × 100 3.36 × 102
na
Pork meat
Serum
Stool 1
Stool 2
Possible Risk Factor of HEV Infection
Genotype
10
+/+
4.50
2
74/M
15
+/+
11.88
na
1310.00
1910.59
160.77
197.06
1.04
61
193
1.30 × 101 na
na
nd
3f
3
62/M
41
+/+
35.92
na
1872.94
2658.82
298.74
339.41
1.25
51
143
6.00 × 100 1.40 × 101
nd
nd
3f
4
61/M
13
+/−
8.89
na
2258.82
1135.29
335.93
284.12
1.17
82
180
nd
2.88 × 103
nd
nd
3e
5
45/M
15b
−/−
25.80
na
2788.82
1251.76
136.17
157.65
1.31
73
219
na
4.00 × 102
na
Butcher
nd
6
58/M
10
+/−
17.37
9.13
5202.35
2680.59
271.15
147.06
1.56
76
165
na
nd
na
nd
nd
7
65/M
11
+/+
6.44
3.80
2398.82
512.35
923.82
278.24
0.98
110
269
nd
4.00 × 100
nd
Home pig slaughter
3g
8
51/M
10
+/−
3.33
1.87
2980.59
1321.76
571.69
179.41
1.07
88
154
1.59 × 102 1.00 × 101
na
nd
3i
9
60/M
45
+/+
2.11
na
3096.47
1665.29
485.90
107.06
1.06
na
130
nd
7.00 × 100 Excessive pork intake
3c
10
60/M
7
+/+
11.41
na
2240.00
1625.88
247.75
150.00
1.12
94
173
6.20 × 101 2.00 × 102
3.00 × 101 Mincemeat, liver sausage
3e
11
61/M
16
+/+
7.55
na
4521.76
3075.88
363.53
159.41
1.00
70
178
3.70 × 101 na
na
Brawn
nd
12
69/M
14
+/+
6.67
na
2869.41
2293.53
368.93
461.76
1.02
108
261
nd
8.00 × 100
nd
Liver sausage
nd
13
48/F
15
+/+
17.08
na
2910.59
1941.18
172.77
123.53
1.25
55
248
nd
nd
na
Raw pork meat
nd
14
53/M
18
+/+
14.89
13.06
1144.12
313.53
207.56
154.71
0.96
82
247
nd
1.00 × 100
6.00 × 100 nd
3
15
56/M
14
+/+
1.40
na
2357.06
882.94
458.31
135.29
0.98
94
168
nd
4.70 × 105
na
Grilled pork meat
3f
16
73/M
4
±/−
1.93
na
32.94
25.29
24.00
65.29
1.00
92
216
nd
nd
na
Contact with HEV
nd
17
49/M
11
+/+
3.51
1.93
2560.59
1687.65
887.82
382.35
0.94
157
316
1.22 × 102 9.22 × 102
na
nd
3c
18
62/M
11
+/+
6.20
2.75
867.06
213.53
779.84
217.65
0.97
80
247
nd
1.56 × 102
na
Home pig slaughter
3e
19
69/M
9
+/+
12.17
5.91
770.59
15.88
194.36
191.76
0.97
84
247
nd
na
na
Liver sausage
nd
20
37/M
0
−/+
0.59
63.53
38.82
56.99
55.19
0.87
na
191
9.70 × 101 nd
nd
Contact with HEV patient
nd
21
65/M
12
+/+
2.40
0.88
1448.23
280.59
199.16
227.06
1.10
91
110
nd
nd
Home pig slaughter
nd
22
43/M
7
+/+
1.64
0.59
374.71
70.59
354.53
155.88
0.94
na
232
1.00 × 101 5.00 × 100
na
nd
nd
23
69/M
45
+/+
19.31
8.95
1420.00
438.24
1019.80
201.18
1.23
36
124
nd
nd
Mincemeat
nd
24
59/M
11
+/−
2.75
1.52
1714.12
422.94
314.94
126.47
1.11
59
102
2.18 × 102 7.27 × 102
na
nd
3e
25
74/M
15
+/+
6.67
4.27
510.59
226.47
482.90
340.59
1.06
na
160
3.40 × 101 nd
na
nd
nd
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26
60/M
18
+/±
7.78
5.21
4684.12
3584.70
507.5
87.65
1.65
49
124
9.90 × 101 na
4.81 × 104 nd
3g
27
42/F
12
+/+
17.96
8.72
2739.41
3390.59
125.88
2.44
31
195
8.00 × 100 3.74 × 105
1.49 × 103 Travel - India
1a
Endemic Hepatitis E
77/F
•
3f
1
na
43.19
1.18 × 102
nd nd
1
2
28
55/M
10
+/+
4.62
1.76
5776.47
3535.29
400.72
345.29
1.26
81
138
3.10 × 10
29
71/M
8
+/+
2.11
0.94
1993.53
885.88
211.76
166.47
1.05
84
150
1.00 × 100 7.60 × 101
6.00 × 100 nd
nd
30
54/M
41
+/+
25.27
na
3640.00
2953.53
257.35
155.88
1.37
30
97
7.00 × 100 1,38 × 102
na
Greaves
3e
31
46/F
8
+/−
1.76
na
994.12
323.53
364.73
134.12
0.89
na
223
nd
1.21 × 102
na
Mincemeat
nd
32
60/M
20
+/+
31.47
4655.88
2725.88
128.97
109.41
2.11
24
146
1.90 × 101 9.00 × 100
na
Precooked sausage meat
3e
•
33
38/M
10
+/±
1.93
34
50/F
11
+/+
11.06
18.31 0.76 na
2.96 × 10
na
Home pig slaughter
3
1980.00
772.94
392.32
432.94
1.07
108
170
6.00 × 100 2.86 × 102
na
Home pig slaughter
3f
1453.53
1187.06
365.33
225.88
0.81
112
273
8.10 × 103 1.98 × 107
na
nd
3e
511
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Table 1 continued.
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Hematological Parameters
Biochemical Examinations
Age, y/Sex
Hospital Stay, d
Anti-HEV IgM/IgG
Bilirubin Total, mg/dL
Bilirubin Direct, mg/ dL
ALT, U/L
AST, U/L
GGT, U/L
ALP, U/L
INR
AT III, %
PLT, 109/L
Serum
Stool 1
35
61/M
9
+/+
3.39
0.76
2100.00
1530.59
420.52
160.00
0.94
106
165
8.09 × 104 1.26 × 104
36
69/M
20
+/+
15.74
8.54
867.06
1087.06
431.91
124.71
1.83
46
196
nd
37
68/M
11
+/+
4.10
1.70
2288.82
1685.29
587.28
427.65
5.00c
61
173
38
60/M
9
+/+
4.80
2.75
6176.47
4352.94
365.93
228.82
1.12
112
39
71/M
13
+/+
14.22
7.66
1533.53
1056.47
743.85
295.29
1.13
40
66/M
8
+/−
7.84
4.45
2590.59
1063.53
234.55
355.88
41
65/F
11
+/±
1.11
0.47
2070.00
2093.53
241.15
42
65/F
12
+/+
2.93
1.70
2641.18
2000.00
430.71
43
63/M
15
+/−
25.10
14.63
196.47
194.71
44
56/F
14
+/+
8.60
4.15
3061.76
45
34/M
8
+/+
6.61
3.80
3647.06
46
60/M
13
+/+
5.91
3.10
2823.53
2705.88
221.96
47
65/M
8
+/+
5.32
3.16
3259.41
1170.00
291.54
48
61/M
14
+/+
8.34
5.24
5294.12
4941.17
623.88
49
59/M
9
+/+
3.86
2.11
1823.53
1235.29
50
70/M
10
+/+
31.94
19.19
4147.06
6531.17
51
64/M
12
+/+
12.64
6.44
321.76
150.00
Stool 2
Possible Risk Factor of HEV Infection
Genotype
6.27 × 103 nd
3e
1.15 × 103 Liver sausage
nd
4.00 × 101 6.38 × 105
5.78 × 104 Pork meat
3f
162
3.82 × 103 1.03 × 105
na
Pork meat
3f
70
113
8.60 × 101 1.30 × 104
na
nd
nd
1.07
99
222
7.90 × 101 9.50 × 101
nd
Homemade sausage
nd
181.76
1.24
92
122
nd
2.23 × 103
na
Liver sausage
3a
284.71
1.07
93
180
na
1.14 × 106
4.32 × 104
nd
3f
1199.76
481.18
1.14
79
399
nd
1.18 × 102
nd
nd
nd
960.59
520.10
270.59
1.28
60
197
nd
2.10 × 101
nd
nd
3
1705.88
394.12
456.47
0.96
114
265
nd
nd
nd
Travel to Thailand
nd
394.12
0.96
105
241
4.70 × 101 9.24 × 102
na
Wild boar goulash
nd
122.35
1.01
81
225
2.24 × 103 2.22 × 103
na
Sausage
3
294.12
1.17
50
235
nd
2.70 × 101
nd
nd
nd
419.92
129.41
1.02
81
148
5.66 × 102 1.25 × 104
na
nd
3a
221.96
122.94
2.78
12
146
1.00 × 102 2.13 × 102
na
nd
3
713.86
505.29
0.91
na
193
nd
na
nd
nd
1.33 × 103
nd
Abbreviations: ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; AT III, antithrombin III; GGT, gamma-glutamyl transferase; HEV, hepatitis E virus; IgG, immunoglobulin G; IgM, immunoglobulin M; INR, international normalized ratio; na, not analyzed; nd, not detected; PLT, platelets. a Numbers of detected copies of HEV RNA are recalculated per 1 µL of isolated total RNA. b Following unknown time of hospitalization. c Patient treated with warfarin.
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Chalupa et al
Patient No.
Presence of HEV RNAa
for the phylogenetic analysis, and bootstrap values >70% were considered as significant. The sequences of the Czech human HEV isolates were compared with prototype sequences representing different genotype clusters (1, 2, 3, and 4), and the 2 sequences with a homology of 95% were supplemented from GenBank according to the BLAST algorithm (http://blast.ncbi. nlm.nih.gov/Blast.cgi) [16, 17]. RESULTS
Laboratory Test
Normal Value
Median
Maximum Value
Minimum Value
Bilirubin, mg/dL Bilirubin direct, mg/dL
0–1.17 0–0.41
6.67 3.13
35.92 18.31
1.11 0.47
AST, U/L ALT, U/L
0–38.24 0–47.06
1251.76 2288.82
6531.17 6176.47
70.59 196.47
ALP, U/L
29.41–117.65
197.06
505.29
87.65
GGT, U/L INR
0–35.99 0.8–1.20
360.53 1.07
1198.56 2.78a
128.97 0.81
AT III, %
80–120
81
157
12
PLT, 109/L
140–440
178
399
97
Abbreviations: ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; AT III, antithrombin III; GGT, gammaglutamyltransferase; INR, international normalized ratio; PLT, platelets. a
Without patient number 37, who was treated with warfarin.
before the cooking; and eating raw or inadequately cooked venison or boar meat (eg, wild boar goulash). Only 1 patient (number 50, Table 1) with endemic HE progressed to acute liver failure. This 70-year-old man was admitted for acute hepatitis with underlying liver disease and alcohol abuse in his personal history. HEV infection was confirmed serologically (the anti-HEV IgM and IgG results were positive) and also by qRT-PCR. His liver and coagulation disorders gradually progressed, which necessitated a substitution of coagulation factors. A treatment with ribavirin (1200 mg/day) was introduced because liver transplant was contraindicated due to the patient’s poor clinical condition and comorbidities. The patient died 10 days after admission due to bleeding in the gastrointestinal tract. HEV RNA was detected in at least 1 sample of serum or stool originating from 43 (84.3%) patients; HEV RNA was not detected in the clinical samples originating from the 8 (15.7%) patients with acute HE. Overall, HEV RNA was detected in the stool samples from 39 of 51 (76.5%) patients, in the serum samples from 28 of 48 (58.3%) patients, and in both the stool and serum samples from 24 of 51 (47.1%) patients. In addition, the second stool samples collected prior to the end of hospitalization from 22 (43.1%) patients were tested for HEV RNA presence, and HEV excretion was found in 10 (45.5%) of these samples (Table 1). Subsequently, the sequences originating from HEV ORF1 and HEV ORF2/3 from 24 and 5 HEV isolates, respectively, were analyzed. The sequence and phylogenetic analyses demonstrated that 28 HEV isolates belonged to genotype 3; 1 HEV isolate belonging to genotype 1 was detected in 1 patient who had recently returned from India (Table 1, Figure 2). Overall, genotype determination was possible in 29 of 43 (67.4%) patients with a positive HEV RNA detection.
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HEV infection was serologically confirmed in 50 (98.0%) patients, and serum from 1 (2.0%) patient (number 5) was found to be anti-HEV immunoglobulin M (IgM) and immunoglobulin G (IgG) negative (Table 1). Forty-eight patients with confirmed anti-HEV IgM and IgG positivity had acute symptomatic HE. Two of these patients (numbers 27 and 45) reported traveling to endemic regions (India and Thailand). Two patients (numbers 16 and 20) had asymptomatic courses of acute HE and were diagnosed by repeated serological testing because they had contact with a person with acute HE. In both patients, specific antibodies were detected. The first patient had mild hyperbilirubinemia, and the second patient had HEV RNA viremia and mildly elevated alanine aminotransferase (ALT). The patient (number 5) with acute endemic HE and negative serological examination was diagnosed by HEV RNA detection only; notably, this patient had a significant professional exposure (butcher). Demographic and clinical characteristics were evaluated in the group of 47 patients with symptomatic endemic HE: the ratio of men to women was 40:7; higher age was noted in both sexes (median, 61 years [range, 38–77 years]); the median duration of hospitalization was 14.69 days (SD, 9.21), with a range of 7–38 days. The laboratory parameters demonstrated a high level of total bilirubin; markedly elevated median levels of ALT, aspartate aminotransferase (AST), alkaline phosphatase (ALP), and gamma-glutamyl transferase (GGT); and no changes in median values of platelets, international normalized ratio (INR), or antithrombin III (AT III) (Table 2). Significant correlations (P < .001) were found between total bilirubin and the following parameters: INR, AT III, and length of hospitalization (r = 0.472; r = −0.557; r = 0.469, respectively); the criteria for discharge from the hospital were total bilirubin 90% of patients infected with HEV have detectable anti-HEV IgM antibodies during the first 2 weeks after the onset of illness [19]. The serological appearance of anti-HEV IgM is followed shortly by anti-HEV IgG, and it seems that both antibodies appear almost simultaneously during the acute phase of the infection. Notably, anti-HEV IgM antibodies usually disappear during convalescence, whereas antiHEV IgG antibodies may be detectable for up to 14 years [20].
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Figure 2. Phylogenetic tree constructed by neighbor-joining method with 1000 replications in bootstrap test based on partial nucleotide sequences (242 nt) of hepatitis E virus (HEV) open reading frame 1 (ORF1; nucleotides 125–366); only bootstrap values ( percentages) >70 are indicated on the tree. Present Czech human isolates (•) as well as Czech animal isolates of swine (Δ) and wild boar (□) origin are marked. Sequences of Czech human HEV isolates were compared with prototype sequences representing different genotype 1, 2, 3, and 4 clusters. The 2 sequences with homology of 95% were supplemented from GenBank using BLAST utility. Avian hepatitis E virus was used as an outgroup for the analysis. GenBank accession numbers of chosen sequences are IND-HEV-FHF2-2004 (JF443723), Nep1 (AF051830), B1 (M73218), C1 (D11092), P1 (M80581), I1 (X9822), Cs13 (AF082092), T3 (AY204877), Morocco (AY230202), M1 (M74506), swCH25 (AY594199), T1 (AJ272108), HE-JVN1 (AB168095), V091ORF1 (AB075967), V228ORF1 (AB075970), JAK-Sai (AB074915), S15 (AF082093), CCC220 (AB108537), VH1 (AF195064), Gr1 (AF110388), HEV ORF1/swine/Ger/06 (JN415695), HEV ORF1/swine/Ger/03 (JN415693), Gr2 (AF110389), UK1 (AJ315768), Osh 205 (AF455784), HEV/SW/NL/2005–1048 (EF372554), NLSW22 (AF336002), US2 (AF060669), swArkell (AY115488), swMexico (AF521654), swJL97 (AB108662), JDEER-Hyo03 (AB189071), It1 (AF110387), swNZ (AF215661), Au1 (AF279122), swAr (AY258006), 3039 2 (JQ863408), NLSW105 (AF336013), NLSW68 (AF336007), and avian hepatitis E virus (JN597006).
The diagnosis of acute HE can also be established in patients after contact with HE using repeated serological examinations. Only 2 such patients were detected in our group of 51 patients (Table 1). This number indicates a low contagiousness of the patients with acute HE caused by genotype 3 even with their close contacts. In developed countries, anti-HEV antibody prevalence rates ranging from 1% to >20% have been reported; this rate may reflect exposure to HEV-infected animals or their excrement, asymptomatic HEV infection, serologic cross-reactivity with other causal agents, and/or false-positive serologic testing. It was also demonstrated that veterinarians and farmers coming in close contact with pigs have higher anti-HEV seroprevalence rates than the general population [33]. Moreover, the data from the Czech Central Database EPIDAT show a significantly increased number of endemic cases compared to imported HE cases in the Czech Republic in the last 2 decades (Figure 1) [14]. This trend is partly due to increased awareness of HE among physicians, resulting in more extensive serologic testing of patients with a suspicion of hepatitis. However, negative epidemiological trends with an increased incidence of transmission of HEV from animals to humans cannot be ruled out. The source of HEV infection remains unidentified in the majority of HE cases, but the gathering evidence suggests zoonotic transmission of HEV infection: Consumption of raw or not properly heat-treated meat, porcine offal during the pig home slaughter, tasting of raw meat products before cooking, and cross-contamination with blood or meat juice were the most frequent epidemiological data obtained from our patients (Table 1). The possible zoonotic origin of HEV infection was also suggested by sequence and phylogenetic analyses of human and animal HEV strains in the Czech Republic. The zoonotic genotype 3 was detected in 28 (96.6%) of the sequenced HEV isolates; in addition, high phylogenetic relatedness was observed between the present human and animal HEV isolates originating from Czech domestic pigs and wild boars (Figure 2). It is worth noting that although the main route of HEV genotype 3 transmission is most likely zoonotic transmission, rare cases of transfusion-associated infections have also been reported [34, 35]. Our study has certain limitations. First, the patients with acute HE were enrolled at a single center. However, this center has a catchment area of 2 million inhabitants, which represents one-fifth of the entire Czech population. Second, the laboratory diagnostics of HEV infection were not standardized and there is a risk of false-negative or false-positive results. Thus, the positive results of serology testing should be confirmed by Western blotting, which has been a standard approach introduced in our institution since the beginning of the study period. Third, sequencing and following the sequence and phylogenetic analyses of the detected HEV genome was not possible in all of the
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As was already mentioned, the diagnosis of acute HEV infection can also be based on molecular methods, which allow direct detection of the causative agent (precisely, its genome). HEV RNA can be detected in serum and/or in stool samples of infected patients during the acute phase of the infection by RT-PCR using conventional and quantitative formats [21]. The detection of HEV RNA has the advantage of providing additional information about the viral load in the tested sample, and the additional sequencing of a specific portion of the viral genome allows for molecular epidemiological studies. On the other hand, the detection of HEV RNA has a limited value for the diagnosis of acute HEV infection due to usually brief periods of viremia (up to 2 weeks) and fecal shedding (up to 4 weeks) [22]. Nevertheless, our results indicate the frequent presence of fecal shedding of HEV and relatively frequent viremia during acute HE infection. The clinical features of HE are similar to those of acute viral hepatitis caused by other hepatotropic viruses, but some differences exist. In our group of 47 patients with clinically symptomatic endemic acute HE, the patients were significantly older, with a clear prevalence of men. The patients’ liver function tests revealed the prevalence of icteric forms of hepatitis, sometimes with very high levels of total bilirubin (the highest value was 35.92 mg/dL), very often with markedly elevated serum levels of ALT, AST, ALP, and GGT and without any dilation of bile ducts on ultrasonography (data not shown). The process of the normalization of patients’ hyperbilirubinemia and liver function tests was frequently protracted, which led to relatively long hospital stays. Although clinically expressed hemocoagulation disorders were very rare in our cohort, some patients had significantly prolonged INR and decreased AT III levels. Prolonged INR and high bilirubin levels are well-known predictors of the severity of viral hepatitis; however, less is known about AT III levels in patients with acute hepatitis. The decreased AT III levels have already been observed in severe shock and some other serious infections, but their importance in viral hepatitis has not yet been established [23, 24]. Thus, it is of interest that the low AT III levels were associated with a longer hospital stay and that the lowest AT III level was found in the patient (number 50) who progressed to acute liver failure with a fatal outcome (Table 1). Several factors have been reported as being potentially associated with acute liver failure or a fatal outcome of acute endemic HE, including presenting or underlying chronic liver disease or being infected with HEV of genotype 4 or 1 [25–27]. Substantially fewer cases of fatal fulminant hepatitis have been described following genotype 3 HEV infections [28, 29]. We assume that the fatal outcome of our patient was due to an underlying chronic alcoholic liver disease. Unfortunately, the fatal outcome of the patient was not reversible, even after treatment with ribavirin, which was previously reported to be highly effective in treating chronic and acute HEV infections [30–32].
patients with HEV infection enrolled in the study. Based on previous studies, sequencing is successful only when the viral load in the tested sample is >102 of genomic equivalents, which allowed for the determination of the HEV genotype in more than half of enrolled patients. In conclusion, our results demonstrated specific clinical, laboratory, and epidemiologic characteristics of patients with acute endemic HE. The knowledge of these findings, together with the routine use of a combination of serologic and molecular methods, may improve the diagnostic process of HE. Moreover, the use of molecular methods may help in our understanding of the zoonotic potential of HEV and process of its transmission to humans. Notes
References 1. Carstens EB. Ratification vote on taxonomic proposals to the International Committee on Taxonomy of Viruses (2009). Arch Virol 2010; 155:133–46. 2. Drexler JF, Seelen A, Corman VM, et al. Bats worldwide carry hepatitis E virus-related viruses that form a putative novel genus within the family Hepeviridae. J Virol 2012; 86:9134–47. 3. Khuroo MS. Discovery of hepatitis E: the epidemic non-A, non-B hepatitis 30 years down the memory lane. Virus Res 2011; 161:3–14. 4. Lu L, Li CH, Hagedorn CH. Phylogenetic analysis of global hepatitis E virus sequences: genetic diversity, subtypes and zoonosis. Rev Med Virol 2006; 16:5–36. 5. Vasickova P, Psikal I, Kralik P, Widen F, Hubalek Z, Pavlik I. Hepatitis E virus: a review. Vet Med (Praha) 2007; 52:365–84. 6. Hakze-van der Honing RW, van Coillie E, Antonis AF, van der Poel WH. First isolation of hepatitis E virus genotype 4 in Europe through swine surveillance in the Netherlands and Belgium. PLoS One 2011; 6: e22673. 7. Tesse S, Lioure B, Fornecker L, et al. Circulation of genotype 4 hepatitis E virus in Europe: first autochthonous hepatitis E infection in France. J Clin Virol 2012; 54:197–200. 8. Chalupa P, Holub M. Jaundice complicated by an atypical form of Guillain-Barré syndrome. J Clin Virol 2010; 49:229–30. 9. Hoofnagle JH, Nelson KE, Purcell RH. Hepatitis E. N Engl J Med 2012; 367:1237–44. 10. Aggarwal R, Jameel S. Hepatitis E. Hepatology 2011; 54:2218–26. 11. Borgen K, Herremans T, Duizer E, et al. Non-travel related hepatitis E virus genotype 3 infections in Netherlands; a case series 2004–2006. BMC Infect Dis 2008; 8:61. 12. Mansuy JM, Peron JM, Abravanel F, et al. Hepatitis E in the south west of France in individuals who have never visited an endemic area. J Med Virol 2004; 74:419–24.
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Financial support. The study was supported by the Ministry of Health (grant number NT13884-4/2012), Ministry of Agriculture (grant number MZE0002716202), and Ministry of Education, Youth and Sports of the Czech Republic (grant number AdmireVet CZ 1.05/2.1.00/01.0006-ED 0006/01/01); and by Charles University in Prague (grant number PRVOUK/P24/LF1/3). Potential conflicts of interest. P. C. has received funding for travel to meetings from Roche and Gilead. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.
13. Wichmann O, Schimanski S, Koch J, et al. Phylogenetic and casecontrol study on hepatitis E virus infection in Germany. J Infect Dis 2008; 198: 1732–41. 14. The National Institute of Public Health in Prague. Incidence of chosen infectious diseases in the Czech Republic in the years 2003–2012. Available at: http://www.szu.cz/publikace/data/vybrane-infekcni-nemoci-vcr-v-letech-2003-2012-absolutne. Accessed 12 December 2013. 15. Vasickova P, Slany M, Chalupa P, Holub M, Svoboda R, Pavlik I. Detection and phylogenetic characterization of human hepatitis E virus strains, Czech Republic. Emerg Infect Dis 2011; 17:917–19. 16. Vasickova P, Kralik P, Slana I, Pavlik I. Optimisation of a triplex RTPCR for detection of hepatitis E virus RNA and validation on biological samples. J Virol Meth 2012; 180:38–42. 17. Vasickova P, Psikal I, Widen F, et al. Detection and genetic characterisation of hepatitis E virus in Czech pig production herds. Res Vet Sci 2009; 87:143–48. 18. Khudyakov Y, Kamili S. Serological diagnostics of hepatitis E virus infection. Vir Res 2011; 161:84–92. 19. Favorov MO, Fields HA, Purdy MA, et al. Serologic identification of hepatitis E virus infections in epidemic and endemic settings. J Med Virol 1992; 36:246–25. 20. Bryan JP, Tsarev SA, Iqbal M, et al. Epidemic hepatitis E in Pakistan: patterns of serologic response and evidence that antibody to hepatitis E virus protects against disease. J Infect Dis 1994; 170:517–21. 21. Mushahwar IK. Hepatitis E virus: molecular virology, clinical features, diagnosis, transmission, epidemiology and prevention. J Med Virol 2008; 80:646–58. 22. Krawczynski K, Meng XJ, Rybczynska J. Pathogenetic elements of hepatitis E and animal models of HEV infection. Vir Res 2011; 161:78–83. 23. Warren BL, Eid A, Singer P, et al. Caring for the critically ill patient. High-dose antithrombin III in severe sepsis: a randomized controlled trial. JAMA 2001; 286:1869–78. 24. Chalupa P, Holub M. Favorable outcome of severe acute hepatitis B in a patient treated with antithrombin III and antiviral therapy. Clin Infect Dis 2009; 49:481. 25. Dalton HR, Hazeldine S, Banks M, Liaz S, Bendall R. Locally acquired hepatitis E in chronic liver disease. Lancet 2007; 369:1260. 26. Ramachandran J, Eapen CE, Kang G, et al. Hepatitis E superinfection produces severe decompensation in patients with chronic liver disease. J Gastroenterol Hepatol 2004; 19:134–8. 27. Inoue J, Nishizawa T, Takahashi M, et al. Analysis of the full-length geonome of genotype 4 hepatitis E virus isolates from patients with fulminant or acute self-limited hepatitis E. J Med Virol 2006; 78:476–84. 28. Péron JM, Bureau C, Poirson H, et al. Fulminant liver failure from acute autochthonous hepatitis E in France: description of seven patients with acute hepatitis E and encephalopathy. J Viral Hepat 2007; 14:298–303. 29. Dalton HR, Stableforth W, Thurairajah P, et al. Autochthonous hepatitis E in southwest England: natural history, complications and seasonal variation, and hepatitis E virus IgG seroprevalence in blood donors, the elderly and patients with chronic liver disease. Eur J Gastroenterol Hepatol 2008; 20:784–90. 30. Kamar N, Rostaing L, Abravanel F, et al. Ribavirin therapy inhibits viral replication on patients with chronic hepatitis E virus infection. Gastroenterology 2010; 139:1612–18. 31. Mallet V, Nicand E, Sultanik P, et al. Brief communication: case report sof ribavirin treatment for chronic hepatitis E. Ann Intern Med 2010; 153:85–9. 32. Gerolami R, Borentain P, Raissouni F, Motte A, Solas C, Colson P. Treatment of severe acute hepatitis E by ribavirin. J Clin Virol 2011; 52:60–2. 33. Aggarwal R. Hepatitis E: historical, contemporary and future perspectives. J Gastroent Hepatol 2011; 26(suppl 1):72–82. 34. Boxall E, Herborn A, Kochethu G, et al. Transfusion-transmitted hepatitis E in a ‘nonhyperendemic’ country. Transfus Med 2006; 16:79–83. 35. Colson P, Coze C, Gallian P, Henry M, De Micco P, Tamalet C. Transfusion-associated hepatitis E. Emerg Infect Dis 2007; 13:648–9.
Endemic Hepatitis E in the Czech Republic Pavel Chalupa,1 Petra Vasickova,2 Ivo Pavlik,2 and Michal Holub1 1
Department of Infectious and Tropical Diseases, First Faculty of Medicine, Charles University in Prague and Na Bulovce Hospital, Prague, and Department of Food and Feed Safety, Veterinary Research Institute, Brno, Czech Republic
2
Keywords.
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Background. An increasing incidence of endemic hepatitis E (HE) has been reported in developed countries. Thus, an evaluation of the clinical characteristics of the disease and the utility of the current diagnostic methods is warranted. Methods. Fifty-one adult acute patients with HE hospitalized in a single center between the years 2009 and 2012 were evaluated. Serological and molecular techniques (detection of hepatitis E virus [HEV] RNA from stool and serum samples by quantitative reverse transcription polymerase chain reaction) with sequencing and phylogenetic analysis were used for diagnosis, and the clinical, laboratory, and epidemiological parameters of the patients were evaluated. Results. Forty-nine (96.1%) patients had acute endemic HE and 2 (3.9%) had an imported infection. In the cohort of patients with acute symptomatic HE (n = 47), men outnumbered women (40:7), the patients were in older middle age (mean, 60.57 years), and they had elevated median values of total bilirubin (6.67 mg/dL), alanine aminotransferase (2288.82 U/L), aspartate aminotransferase (1251.76 U/L), gamma-glutamyl transferase (360.53 U/L), and alkaline phosphatase (197.06 U/L). Serology was positive in 50 (98%) of the patients, and 1 case was diagnosed by polymerase chain reaction only. HEV RNA was detected in at least 1 specimen from 84.3% of the patients, and 28 of 29 tested isolates belonged to genotype 3. The eating of meat, innards, other home-prepared pork products, or the tasting of raw meat before cooking were the most frequently reported data (reported by 25 patients [49.0%]). Conclusions. Large numbers of the endemic cases of HE were caused by HEV genotype 3, and the clinical characteristics of endemic HE were demonstrated. hepatitis E; endemic cases; clinical findings; zoonosis.
Hepatitis E (HE) is one of the most common forms of hepatitis transmitted via the fecal-oral route worldwide. The causative agent of the disease, the hepatitis E virus (HEV), is classified in the genus Hepevirus, family Hepeviridae [1]. The Hepevirus genus also includes HEV strains that are capable of infecting other animal species such as avian hosts (chicken) [2]. Although only 1 serotype was described within the genus Hepevirus, 4 major genotypes with different modes of transmission, pathogenicity, reservoirs, and capacity for interspecies transmission were recognized in this genus [3].
Received and accepted 17 November 2013; electronically published 26 November 2013. Correspondence: Pavel Chalupa, MD, PhD, Department of Infectious and Tropical Diseases, Na Bulovce Hospital, Budinova 2, CZ-180 81 Praha 8, Czech Republic ( [email protected]). Clinical Infectious Diseases 2014;58(4):509–16 © The Author 2013. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: [email protected]. DOI: 10.1093/cid/cit782
Although each HEV genotype is believed to have a specific geographic distribution, exceptions have been observed. Genotype 1 is mostly associated with epidemic or sporadic cases in the tropical and subtropical countries of Asia and Africa, whereas genotype 2 has been detected in Mexico and some African countries. The relative conservation of these 2 genotypes corresponds to their primary circulation among humans. Conversely, the diversity of the HEV genotypes 3 and 4 is related to their zoonotic potential. Genotype 3 has been isolated from patients with acute HE and from different animal species (in particular, domestic pigs, wild boar, and deer are the ones most likely to be involved in human transmission) worldwide. Genotype 4 contains human and animal strains originating particularly from Asian countries [4, 5]. Recently, genotype 4 was isolated from patients with endemic HE and domestic pigs in Europe [6, 7]. HEV infection has at least 2 epidemiological profiles: large waterborne outbreaks and sporadic cases. The
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Figure 1. Number of imported and endemic (autochthonous) cases of acute hepatitis E reported to the central Czech database EPIDAT in the National Institute of Public Health in Prague (http://www.szu.cz/publikace/ data/vybrane-infekcni-nemoci-v-cr-v-letech-2003-2012-absolutne). Abbreviation: HE, hepatitis E.
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significant increase in the number of endemic HE cases has been observed (Figure 1) [14]. The disease is frequently recognized only by serological testing. Routine laboratory diagnostics for HE may be one reason for the observed increasing incidence. Almost all of the endemic HE cases are associated with genotype 3 or 4, and HEV isolates from patients show a high sequence homology to those found in domestic pigs and other animals [5, 15]. Despite an increasing incidence, data regarding epidemiology and clinical manifestations of HEV infections in Central Europe are very limited. Therefore, the aim of the present study was characterization of acute hepatitis E cases hospitalized in a large referral center for infectious patients in Prague (Czech Republic), with a special attention to endemic cases of the disease. PATIENTS AND METHODS Fifty-one patients (43 men, mean age, 60 years [range, 37–74 years]; 8 women, mean age, 56 years [range, 42–77 years]) with acute HE were enrolled between 2009 and 2012 (Table 1). Informed consent was obtained from all patients after obtaining approval from the local ethics committee (IRB00002721, Eticka komise Nemocnice Na Bulovce, IRB No. 1, Biomedical). The diagnosis of acute HE was established by the detection of antiHEV antibodies, and positivity on enzyme-linked immunosorbent assay was confirmed by Western blotting. For patients 1– 7, the EIAgen HEV IgM Kit, EIAgen HEV IgG Kit (both Adaltis, Italy), and Recomblot HEV IgM/IgG (Microgen GmbH, Germany) were used; for patients 8–51, the recomWell HEV IgM, recomWell HEV IgG, and recomLine HEV IgG/IgM (Microgen GmbH) were used. In addition, all patients tested negative for hepatitis A, B, and C virus infections, and the use of hepatotoxic drugs was ruled out. We also evaluated the clinical, biochemical, hematological, and epidemiological parameters of the enrolled patients. Correlations between the biochemical and hemocoagulation examinations were tested using Spearman and Pearson correlations (Sigma Stat; Jandel Scientific, San Rafael, California). The serum and stool samples originating from the enrolled patients were tested for the presence of the HEV genome (RNA) through the use of molecular methods. If possible, samples were collected at the beginning (stool and serum) and at the end (stool) of a patient’s hospitalization. Detection of HEV RNA was performed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) [16, 17]. Subsequently, the partial parts of the open reading frame (ORF) 1 and the overlapping region of ORFs 2 and 3 (ORF2/3) of the acquired HEV isolates were subjected to sequencing (Eurofins MWG Operon, Germany); sequence and phylogenetic analyses were performed using MEGA 3.1 software. The neighbor-joining method with 1000 replications in the bootstrap test was used
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outbreaks are usually caused by genotype 1 or 2, and they result in high morbidity and mortality among pregnant women and young children in developing countries. The most common clinical presentation is acute icteric hepatitis, which is indistinguishable from other forms of viral hepatitis. HE attack rates peak among young to middle-aged adults (15–30 years). Rates of infection are usually higher in men than in women; however, no sex difference exists in exposure to HEV. The sporadic cases of HE, which present clinically as icteric hepatitis or anicteric illnesses with nonspecific symptoms, are associated with genotypes 3 and 4 in both developing and industrialized countries. Endemic (autochthonous) HEV infections in developed countries, which are not related to travel in epidemic regions, have certain clinical features. Most patients are older (middle-aged and elderly people) and predominantly are men with an increased frequency of underlying liver disease or alcohol use. HEV can also cause neurological manifestations and chronic infection in immunocompromised persons, including those infected with the human immunodeficiency virus (HIV) [8, 9]. In contrast to epidemic HE, pregnant women do not show a severe course of endemic HEV infection. Nevertheless, sporadic HEV infections are associated with morbidity and mortality [10–12]. Reports of HEV infections without travel association have become more frequent in nonhyperendemic regions (industrialized countries) in recent years [13]. According to the periodic reports of the National Institute of Public Health in Prague (Czech Central Database EPIDAT, http://www.szu.cz/publikace/ data/vybrane-infekcni-nemoci-v-cr-v-letech-2003-2012-absolutne), the numbers of imported cases of HE in the Czech Republic have remained stable during the last 2 decades, whereas a
Table 1.
Characterization of Patients and Detection of Hepatitis E Virus RNA by Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction Hematological Parameters
Biochemical Examinations
Patient No.
Age, y/Sex
Presence of HEV RNAa
Hospital Stay, d
Anti-HEV IgM/IgG
Bilirubin Total, mg/dL
Bilirubin Direct, mg/ dL
ALT, U/L
AST, U/L
GGT, U/L
ALP, U/L
INR
AT III, %
PLT, 109/L
2.69
4639.41
3045.88
543.49
407.06
1.17
59
263
6.00 × 100 3.36 × 102
na
Pork meat
Serum
Stool 1
Stool 2
Possible Risk Factor of HEV Infection
Genotype
10
+/+
4.50
2
74/M
15
+/+
11.88
na
1310.00
1910.59
160.77
197.06
1.04
61
193
1.30 × 101 na
na
nd
3f
3
62/M
41
+/+
35.92
na
1872.94
2658.82
298.74
339.41
1.25
51
143
6.00 × 100 1.40 × 101
nd
nd
3f
4
61/M
13
+/−
8.89
na
2258.82
1135.29
335.93
284.12
1.17
82
180
nd
2.88 × 103
nd
nd
3e
5
45/M
15b
−/−
25.80
na
2788.82
1251.76
136.17
157.65
1.31
73
219
na
4.00 × 102
na
Butcher
nd
6
58/M
10
+/−
17.37
9.13
5202.35
2680.59
271.15
147.06
1.56
76
165
na
nd
na
nd
nd
7
65/M
11
+/+
6.44
3.80
2398.82
512.35
923.82
278.24
0.98
110
269
nd
4.00 × 100
nd
Home pig slaughter
3g
8
51/M
10
+/−
3.33
1.87
2980.59
1321.76
571.69
179.41
1.07
88
154
1.59 × 102 1.00 × 101
na
nd
3i
9
60/M
45
+/+
2.11
na
3096.47
1665.29
485.90
107.06
1.06
na
130
nd
7.00 × 100 Excessive pork intake
3c
10
60/M
7
+/+
11.41
na
2240.00
1625.88
247.75
150.00
1.12
94
173
6.20 × 101 2.00 × 102
3.00 × 101 Mincemeat, liver sausage
3e
11
61/M
16
+/+
7.55
na
4521.76
3075.88
363.53
159.41
1.00
70
178
3.70 × 101 na
na
Brawn
nd
12
69/M
14
+/+
6.67
na
2869.41
2293.53
368.93
461.76
1.02
108
261
nd
8.00 × 100
nd
Liver sausage
nd
13
48/F
15
+/+
17.08
na
2910.59
1941.18
172.77
123.53
1.25
55
248
nd
nd
na
Raw pork meat
nd
14
53/M
18
+/+
14.89
13.06
1144.12
313.53
207.56
154.71
0.96
82
247
nd
1.00 × 100
6.00 × 100 nd
3
15
56/M
14
+/+
1.40
na
2357.06
882.94
458.31
135.29
0.98
94
168
nd
4.70 × 105
na
Grilled pork meat
3f
16
73/M
4
±/−
1.93
na
32.94
25.29
24.00
65.29
1.00
92
216
nd
nd
na
Contact with HEV
nd
17
49/M
11
+/+
3.51
1.93
2560.59
1687.65
887.82
382.35
0.94
157
316
1.22 × 102 9.22 × 102
na
nd
3c
18
62/M
11
+/+
6.20
2.75
867.06
213.53
779.84
217.65
0.97
80
247
nd
1.56 × 102
na
Home pig slaughter
3e
19
69/M
9
+/+
12.17
5.91
770.59
15.88
194.36
191.76
0.97
84
247
nd
na
na
Liver sausage
nd
20
37/M
0
−/+
0.59
63.53
38.82
56.99
55.19
0.87
na
191
9.70 × 101 nd
nd
Contact with HEV patient
nd
21
65/M
12
+/+
2.40
0.88
1448.23
280.59
199.16
227.06
1.10
91
110
nd
nd
Home pig slaughter
nd
22
43/M
7
+/+
1.64
0.59
374.71
70.59
354.53
155.88
0.94
na
232
1.00 × 101 5.00 × 100
na
nd
nd
23
69/M
45
+/+
19.31
8.95
1420.00
438.24
1019.80
201.18
1.23
36
124
nd
nd
Mincemeat
nd
24
59/M
11
+/−
2.75
1.52
1714.12
422.94
314.94
126.47
1.11
59
102
2.18 × 102 7.27 × 102
na
nd
3e
25
74/M
15
+/+
6.67
4.27
510.59
226.47
482.90
340.59
1.06
na
160
3.40 × 101 nd
na
nd
nd
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26
60/M
18
+/±
7.78
5.21
4684.12
3584.70
507.5
87.65
1.65
49
124
9.90 × 101 na
4.81 × 104 nd
3g
27
42/F
12
+/+
17.96
8.72
2739.41
3390.59
125.88
2.44
31
195
8.00 × 100 3.74 × 105
1.49 × 103 Travel - India
1a
Endemic Hepatitis E
77/F
•
3f
1
na
43.19
1.18 × 102
nd nd
1
2
28
55/M
10
+/+
4.62
1.76
5776.47
3535.29
400.72
345.29
1.26
81
138
3.10 × 10
29
71/M
8
+/+
2.11
0.94
1993.53
885.88
211.76
166.47
1.05
84
150
1.00 × 100 7.60 × 101
6.00 × 100 nd
nd
30
54/M
41
+/+
25.27
na
3640.00
2953.53
257.35
155.88
1.37
30
97
7.00 × 100 1,38 × 102
na
Greaves
3e
31
46/F
8
+/−
1.76
na
994.12
323.53
364.73
134.12
0.89
na
223
nd
1.21 × 102
na
Mincemeat
nd
32
60/M
20
+/+
31.47
4655.88
2725.88
128.97
109.41
2.11
24
146
1.90 × 101 9.00 × 100
na
Precooked sausage meat
3e
•
33
38/M
10
+/±
1.93
34
50/F
11
+/+
11.06
18.31 0.76 na
2.96 × 10
na
Home pig slaughter
3
1980.00
772.94
392.32
432.94
1.07
108
170
6.00 × 100 2.86 × 102
na
Home pig slaughter
3f
1453.53
1187.06
365.33
225.88
0.81
112
273
8.10 × 103 1.98 × 107
na
nd
3e
511
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• CID 2014:58 (15 February)
Table 1 continued.
•
Hematological Parameters
Biochemical Examinations
Age, y/Sex
Hospital Stay, d
Anti-HEV IgM/IgG
Bilirubin Total, mg/dL
Bilirubin Direct, mg/ dL
ALT, U/L
AST, U/L
GGT, U/L
ALP, U/L
INR
AT III, %
PLT, 109/L
Serum
Stool 1
35
61/M
9
+/+
3.39
0.76
2100.00
1530.59
420.52
160.00
0.94
106
165
8.09 × 104 1.26 × 104
36
69/M
20
+/+
15.74
8.54
867.06
1087.06
431.91
124.71
1.83
46
196
nd
37
68/M
11
+/+
4.10
1.70
2288.82
1685.29
587.28
427.65
5.00c
61
173
38
60/M
9
+/+
4.80
2.75
6176.47
4352.94
365.93
228.82
1.12
112
39
71/M
13
+/+
14.22
7.66
1533.53
1056.47
743.85
295.29
1.13
40
66/M
8
+/−
7.84
4.45
2590.59
1063.53
234.55
355.88
41
65/F
11
+/±
1.11
0.47
2070.00
2093.53
241.15
42
65/F
12
+/+
2.93
1.70
2641.18
2000.00
430.71
43
63/M
15
+/−
25.10
14.63
196.47
194.71
44
56/F
14
+/+
8.60
4.15
3061.76
45
34/M
8
+/+
6.61
3.80
3647.06
46
60/M
13
+/+
5.91
3.10
2823.53
2705.88
221.96
47
65/M
8
+/+
5.32
3.16
3259.41
1170.00
291.54
48
61/M
14
+/+
8.34
5.24
5294.12
4941.17
623.88
49
59/M
9
+/+
3.86
2.11
1823.53
1235.29
50
70/M
10
+/+
31.94
19.19
4147.06
6531.17
51
64/M
12
+/+
12.64
6.44
321.76
150.00
Stool 2
Possible Risk Factor of HEV Infection
Genotype
6.27 × 103 nd
3e
1.15 × 103 Liver sausage
nd
4.00 × 101 6.38 × 105
5.78 × 104 Pork meat
3f
162
3.82 × 103 1.03 × 105
na
Pork meat
3f
70
113
8.60 × 101 1.30 × 104
na
nd
nd
1.07
99
222
7.90 × 101 9.50 × 101
nd
Homemade sausage
nd
181.76
1.24
92
122
nd
2.23 × 103
na
Liver sausage
3a
284.71
1.07
93
180
na
1.14 × 106
4.32 × 104
nd
3f
1199.76
481.18
1.14
79
399
nd
1.18 × 102
nd
nd
nd
960.59
520.10
270.59
1.28
60
197
nd
2.10 × 101
nd
nd
3
1705.88
394.12
456.47
0.96
114
265
nd
nd
nd
Travel to Thailand
nd
394.12
0.96
105
241
4.70 × 101 9.24 × 102
na
Wild boar goulash
nd
122.35
1.01
81
225
2.24 × 103 2.22 × 103
na
Sausage
3
294.12
1.17
50
235
nd
2.70 × 101
nd
nd
nd
419.92
129.41
1.02
81
148
5.66 × 102 1.25 × 104
na
nd
3a
221.96
122.94
2.78
12
146
1.00 × 102 2.13 × 102
na
nd
3
713.86
505.29
0.91
na
193
nd
na
nd
nd
1.33 × 103
nd
Abbreviations: ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; AT III, antithrombin III; GGT, gamma-glutamyl transferase; HEV, hepatitis E virus; IgG, immunoglobulin G; IgM, immunoglobulin M; INR, international normalized ratio; na, not analyzed; nd, not detected; PLT, platelets. a Numbers of detected copies of HEV RNA are recalculated per 1 µL of isolated total RNA. b Following unknown time of hospitalization. c Patient treated with warfarin.
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Chalupa et al
Patient No.
Presence of HEV RNAa
for the phylogenetic analysis, and bootstrap values >70% were considered as significant. The sequences of the Czech human HEV isolates were compared with prototype sequences representing different genotype clusters (1, 2, 3, and 4), and the 2 sequences with a homology of 95% were supplemented from GenBank according to the BLAST algorithm (http://blast.ncbi. nlm.nih.gov/Blast.cgi) [16, 17]. RESULTS
Laboratory Test
Normal Value
Median
Maximum Value
Minimum Value
Bilirubin, mg/dL Bilirubin direct, mg/dL
0–1.17 0–0.41
6.67 3.13
35.92 18.31
1.11 0.47
AST, U/L ALT, U/L
0–38.24 0–47.06
1251.76 2288.82
6531.17 6176.47
70.59 196.47
ALP, U/L
29.41–117.65
197.06
505.29
87.65
GGT, U/L INR
0–35.99 0.8–1.20
360.53 1.07
1198.56 2.78a
128.97 0.81
AT III, %
80–120
81
157
12
PLT, 109/L
140–440
178
399
97
Abbreviations: ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; AT III, antithrombin III; GGT, gammaglutamyltransferase; INR, international normalized ratio; PLT, platelets. a
Without patient number 37, who was treated with warfarin.
before the cooking; and eating raw or inadequately cooked venison or boar meat (eg, wild boar goulash). Only 1 patient (number 50, Table 1) with endemic HE progressed to acute liver failure. This 70-year-old man was admitted for acute hepatitis with underlying liver disease and alcohol abuse in his personal history. HEV infection was confirmed serologically (the anti-HEV IgM and IgG results were positive) and also by qRT-PCR. His liver and coagulation disorders gradually progressed, which necessitated a substitution of coagulation factors. A treatment with ribavirin (1200 mg/day) was introduced because liver transplant was contraindicated due to the patient’s poor clinical condition and comorbidities. The patient died 10 days after admission due to bleeding in the gastrointestinal tract. HEV RNA was detected in at least 1 sample of serum or stool originating from 43 (84.3%) patients; HEV RNA was not detected in the clinical samples originating from the 8 (15.7%) patients with acute HE. Overall, HEV RNA was detected in the stool samples from 39 of 51 (76.5%) patients, in the serum samples from 28 of 48 (58.3%) patients, and in both the stool and serum samples from 24 of 51 (47.1%) patients. In addition, the second stool samples collected prior to the end of hospitalization from 22 (43.1%) patients were tested for HEV RNA presence, and HEV excretion was found in 10 (45.5%) of these samples (Table 1). Subsequently, the sequences originating from HEV ORF1 and HEV ORF2/3 from 24 and 5 HEV isolates, respectively, were analyzed. The sequence and phylogenetic analyses demonstrated that 28 HEV isolates belonged to genotype 3; 1 HEV isolate belonging to genotype 1 was detected in 1 patient who had recently returned from India (Table 1, Figure 2). Overall, genotype determination was possible in 29 of 43 (67.4%) patients with a positive HEV RNA detection.
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HEV infection was serologically confirmed in 50 (98.0%) patients, and serum from 1 (2.0%) patient (number 5) was found to be anti-HEV immunoglobulin M (IgM) and immunoglobulin G (IgG) negative (Table 1). Forty-eight patients with confirmed anti-HEV IgM and IgG positivity had acute symptomatic HE. Two of these patients (numbers 27 and 45) reported traveling to endemic regions (India and Thailand). Two patients (numbers 16 and 20) had asymptomatic courses of acute HE and were diagnosed by repeated serological testing because they had contact with a person with acute HE. In both patients, specific antibodies were detected. The first patient had mild hyperbilirubinemia, and the second patient had HEV RNA viremia and mildly elevated alanine aminotransferase (ALT). The patient (number 5) with acute endemic HE and negative serological examination was diagnosed by HEV RNA detection only; notably, this patient had a significant professional exposure (butcher). Demographic and clinical characteristics were evaluated in the group of 47 patients with symptomatic endemic HE: the ratio of men to women was 40:7; higher age was noted in both sexes (median, 61 years [range, 38–77 years]); the median duration of hospitalization was 14.69 days (SD, 9.21), with a range of 7–38 days. The laboratory parameters demonstrated a high level of total bilirubin; markedly elevated median levels of ALT, aspartate aminotransferase (AST), alkaline phosphatase (ALP), and gamma-glutamyl transferase (GGT); and no changes in median values of platelets, international normalized ratio (INR), or antithrombin III (AT III) (Table 2). Significant correlations (P < .001) were found between total bilirubin and the following parameters: INR, AT III, and length of hospitalization (r = 0.472; r = −0.557; r = 0.469, respectively); the criteria for discharge from the hospital were total bilirubin 90% of patients infected with HEV have detectable anti-HEV IgM antibodies during the first 2 weeks after the onset of illness [19]. The serological appearance of anti-HEV IgM is followed shortly by anti-HEV IgG, and it seems that both antibodies appear almost simultaneously during the acute phase of the infection. Notably, anti-HEV IgM antibodies usually disappear during convalescence, whereas antiHEV IgG antibodies may be detectable for up to 14 years [20].
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Figure 2. Phylogenetic tree constructed by neighbor-joining method with 1000 replications in bootstrap test based on partial nucleotide sequences (242 nt) of hepatitis E virus (HEV) open reading frame 1 (ORF1; nucleotides 125–366); only bootstrap values ( percentages) >70 are indicated on the tree. Present Czech human isolates (•) as well as Czech animal isolates of swine (Δ) and wild boar (□) origin are marked. Sequences of Czech human HEV isolates were compared with prototype sequences representing different genotype 1, 2, 3, and 4 clusters. The 2 sequences with homology of 95% were supplemented from GenBank using BLAST utility. Avian hepatitis E virus was used as an outgroup for the analysis. GenBank accession numbers of chosen sequences are IND-HEV-FHF2-2004 (JF443723), Nep1 (AF051830), B1 (M73218), C1 (D11092), P1 (M80581), I1 (X9822), Cs13 (AF082092), T3 (AY204877), Morocco (AY230202), M1 (M74506), swCH25 (AY594199), T1 (AJ272108), HE-JVN1 (AB168095), V091ORF1 (AB075967), V228ORF1 (AB075970), JAK-Sai (AB074915), S15 (AF082093), CCC220 (AB108537), VH1 (AF195064), Gr1 (AF110388), HEV ORF1/swine/Ger/06 (JN415695), HEV ORF1/swine/Ger/03 (JN415693), Gr2 (AF110389), UK1 (AJ315768), Osh 205 (AF455784), HEV/SW/NL/2005–1048 (EF372554), NLSW22 (AF336002), US2 (AF060669), swArkell (AY115488), swMexico (AF521654), swJL97 (AB108662), JDEER-Hyo03 (AB189071), It1 (AF110387), swNZ (AF215661), Au1 (AF279122), swAr (AY258006), 3039 2 (JQ863408), NLSW105 (AF336013), NLSW68 (AF336007), and avian hepatitis E virus (JN597006).
The diagnosis of acute HE can also be established in patients after contact with HE using repeated serological examinations. Only 2 such patients were detected in our group of 51 patients (Table 1). This number indicates a low contagiousness of the patients with acute HE caused by genotype 3 even with their close contacts. In developed countries, anti-HEV antibody prevalence rates ranging from 1% to >20% have been reported; this rate may reflect exposure to HEV-infected animals or their excrement, asymptomatic HEV infection, serologic cross-reactivity with other causal agents, and/or false-positive serologic testing. It was also demonstrated that veterinarians and farmers coming in close contact with pigs have higher anti-HEV seroprevalence rates than the general population [33]. Moreover, the data from the Czech Central Database EPIDAT show a significantly increased number of endemic cases compared to imported HE cases in the Czech Republic in the last 2 decades (Figure 1) [14]. This trend is partly due to increased awareness of HE among physicians, resulting in more extensive serologic testing of patients with a suspicion of hepatitis. However, negative epidemiological trends with an increased incidence of transmission of HEV from animals to humans cannot be ruled out. The source of HEV infection remains unidentified in the majority of HE cases, but the gathering evidence suggests zoonotic transmission of HEV infection: Consumption of raw or not properly heat-treated meat, porcine offal during the pig home slaughter, tasting of raw meat products before cooking, and cross-contamination with blood or meat juice were the most frequent epidemiological data obtained from our patients (Table 1). The possible zoonotic origin of HEV infection was also suggested by sequence and phylogenetic analyses of human and animal HEV strains in the Czech Republic. The zoonotic genotype 3 was detected in 28 (96.6%) of the sequenced HEV isolates; in addition, high phylogenetic relatedness was observed between the present human and animal HEV isolates originating from Czech domestic pigs and wild boars (Figure 2). It is worth noting that although the main route of HEV genotype 3 transmission is most likely zoonotic transmission, rare cases of transfusion-associated infections have also been reported [34, 35]. Our study has certain limitations. First, the patients with acute HE were enrolled at a single center. However, this center has a catchment area of 2 million inhabitants, which represents one-fifth of the entire Czech population. Second, the laboratory diagnostics of HEV infection were not standardized and there is a risk of false-negative or false-positive results. Thus, the positive results of serology testing should be confirmed by Western blotting, which has been a standard approach introduced in our institution since the beginning of the study period. Third, sequencing and following the sequence and phylogenetic analyses of the detected HEV genome was not possible in all of the
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As was already mentioned, the diagnosis of acute HEV infection can also be based on molecular methods, which allow direct detection of the causative agent (precisely, its genome). HEV RNA can be detected in serum and/or in stool samples of infected patients during the acute phase of the infection by RT-PCR using conventional and quantitative formats [21]. The detection of HEV RNA has the advantage of providing additional information about the viral load in the tested sample, and the additional sequencing of a specific portion of the viral genome allows for molecular epidemiological studies. On the other hand, the detection of HEV RNA has a limited value for the diagnosis of acute HEV infection due to usually brief periods of viremia (up to 2 weeks) and fecal shedding (up to 4 weeks) [22]. Nevertheless, our results indicate the frequent presence of fecal shedding of HEV and relatively frequent viremia during acute HE infection. The clinical features of HE are similar to those of acute viral hepatitis caused by other hepatotropic viruses, but some differences exist. In our group of 47 patients with clinically symptomatic endemic acute HE, the patients were significantly older, with a clear prevalence of men. The patients’ liver function tests revealed the prevalence of icteric forms of hepatitis, sometimes with very high levels of total bilirubin (the highest value was 35.92 mg/dL), very often with markedly elevated serum levels of ALT, AST, ALP, and GGT and without any dilation of bile ducts on ultrasonography (data not shown). The process of the normalization of patients’ hyperbilirubinemia and liver function tests was frequently protracted, which led to relatively long hospital stays. Although clinically expressed hemocoagulation disorders were very rare in our cohort, some patients had significantly prolonged INR and decreased AT III levels. Prolonged INR and high bilirubin levels are well-known predictors of the severity of viral hepatitis; however, less is known about AT III levels in patients with acute hepatitis. The decreased AT III levels have already been observed in severe shock and some other serious infections, but their importance in viral hepatitis has not yet been established [23, 24]. Thus, it is of interest that the low AT III levels were associated with a longer hospital stay and that the lowest AT III level was found in the patient (number 50) who progressed to acute liver failure with a fatal outcome (Table 1). Several factors have been reported as being potentially associated with acute liver failure or a fatal outcome of acute endemic HE, including presenting or underlying chronic liver disease or being infected with HEV of genotype 4 or 1 [25–27]. Substantially fewer cases of fatal fulminant hepatitis have been described following genotype 3 HEV infections [28, 29]. We assume that the fatal outcome of our patient was due to an underlying chronic alcoholic liver disease. Unfortunately, the fatal outcome of the patient was not reversible, even after treatment with ribavirin, which was previously reported to be highly effective in treating chronic and acute HEV infections [30–32].
patients with HEV infection enrolled in the study. Based on previous studies, sequencing is successful only when the viral load in the tested sample is >102 of genomic equivalents, which allowed for the determination of the HEV genotype in more than half of enrolled patients. In conclusion, our results demonstrated specific clinical, laboratory, and epidemiologic characteristics of patients with acute endemic HE. The knowledge of these findings, together with the routine use of a combination of serologic and molecular methods, may improve the diagnostic process of HE. Moreover, the use of molecular methods may help in our understanding of the zoonotic potential of HEV and process of its transmission to humans. Notes
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Financial support. The study was supported by the Ministry of Health (grant number NT13884-4/2012), Ministry of Agriculture (grant number MZE0002716202), and Ministry of Education, Youth and Sports of the Czech Republic (grant number AdmireVet CZ 1.05/2.1.00/01.0006-ED 0006/01/01); and by Charles University in Prague (grant number PRVOUK/P24/LF1/3). Potential conflicts of interest. P. C. has received funding for travel to meetings from Roche and Gilead. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.
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