A total of 6112 consecutive serum samples were tested for antiHEV IgG or anti-HEV IgM at Department of Infectious Diseases, Ruijin Hospital between August 2018 and July 2019. Samples with missing information of age or gender were excluded from further analysis. For the patient who was tested repeatedly and showed no change of result category (positivity/negativity/gray zone result), the earliest result was kept and the following repeated result was excluded. Finally, 4956 results of anti-HEV IgG test and 5289 results of anti-HEV IgM test were analyzed, respectively(Fig. 1).Most of the samples were tested for anti-HEV IgG and anti-HEV IgM simultaneously (n = 4838). The median age of subjects tested for anti-HEV IgG and anti-HEV IgM was both 45 years, and around 53% of them were males (Table 1).
Anti-HEV IgG (n = 4956) Anti-HEV IgM (n = 5289) Age, median (range) 45 (0–99) 45 (0–99) Male, n (%) 2666 (53.8) 2788 (52.7) COI, median (range) 0.28 (0–24.92) 0.1 0.01–68.49)
Table 1. Characteristics of study population tested for anti-HEV IgG or anti-HEV IgM.
Among the samples tested for anti-HEV IgG, 40.2% (1994/4956) was positive, 57.6% (2856/4956) was negative, and the remaining 2.1% (106/4956) had gray zone results. The sero-positivity rate of anti-HEV IgG increased with age (Fig. 2A), from 0% in age group [0-10 years] to 61.4% in subjects older than 80 years. Males had higher sero-positivity rate than females throughout the age groups. Males over the age of 20 had higher mean level of anti-HEV IgG (represented by COI) than females and the differences gradually expanded with age (Fig. 2B).
Figure 2. The seroprevalence and distribution of HEV antibodies in study population. The prevalence and level of anti-HEV IgG (A, B) or anti-HEV IgM (C, D) by age and gender. M, male. F, female. Subjects were divided into 10-year age groups. E The overall level of anti-HEV IgG or anti-HEV IgM by gender. M, male. F, female. ****P < 0.0001. ***P < 0.001. F The prevalence of anti-HEV IgG and anti-HEV IgM across the year. The prevalence was calculated by dividing the number of seropositive subjects by the total number of subjects tested in each age or time group. The level of HEV antibodies was shown as mean ± SEM.
Among the samples tested for anti-HEV IgM, 6.0% (319/5289) was positive, 93.2% (4931/5289) was negative, and the remaining 0.7% (39/5289) had gray zone results. The prevalence of anti-HEV IgM increased until age of 70 years and dropped subsequently, which peaked in the age group of [61-70] among males and [71-80] among females, respectively (Fig. 2C). The mean level of anti-HEV IgM in males increased with age and peaked in the age group of [61-70 years]. Females showed a parallel pattern to males except that women older than 70 years had higher mean level of anti-HEV IgM than counterparts (Fig. 2D). Both antiHEV IgG and anti-HEV IgM levels were significantly higher in males than in females (Fig. 2E).
The prevalence of anti-HEV IgG maintained stable around 40% perennially, while potential heterogeneity was observed in anti-HEV IgM. The sero-positivity rate of anti-HEV IgM bottomed in November (3.9%) and June (3.6%), but peaked in February (8.6%) (Fig. 2F).
HEV RNA presence was tested in patients with evidence of active or recent HEV infection (defined by positivity of anti-HEV IgM) and patients who could not be ruled out of HEV infection (with unexplained elevated LFT results). A total of 632 samples were tested, including 243 available anti-HEV IgM-positive samples, 375 anti-HEV IgM-negative samples, 9 samples with gray zone results and 5 samples that were not tested for anti-HEV IgM (Fig. 3A). These anti-HEV IgM non-positive samples came from patients with unknown reason of hepatitis, reflected by their abnormal ALT levels (Table 2). Totally, one antiHEV IgM-negative sample and 126 anti-HEV IgM-positive samples were positive for HEV RNA.
Figure 3. The flowchart of HEV RNA detection and genotyping of HEV sequences. A The composition of samples tested for HEV RNA. N/A: not available. B The subtype composition of HEV sequences determined from the study. C The phylogenetic tree inferred using the Maximum likelihood method. The analysis involved 146 nucleotide sequences, including 21 genotype references, 42 regional references and 83 unique sequences determined from this study. The vertical scale bar represents 0.05 nucleotide substitutions per site. Available information of isolation host, year and region were provided beside the 42 regional references. ''hu'' means the strain was isolated from human, while ''sw'' means swine. For the strains isolated from China, the information of isolating province or municipality was provided if available. The tree with the highest log likelihood (-17, 368.42) was shown. Initial tree(s) for the heuristic search were obtained automatically by applying NeighborJoin and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The tree was drawn to scale, with branch lengths measured in the number of substitutions per site. All positions containing gaps and missing data were eliminated. There were a total of 630 positions in the final dataset. The subtype reference strains (3b_AP003430, 4a_AB197673, 4b_DQ279091, 4d_AJ272108, 4h_GU119961 and 4i_DQ450072) were labeled with red triangular symbols next to the isolate, and the clusters of 3b, 4a, 4b, 4d, 4 h and 4i were circled for easy identification.
Anti-HEV IgM category (n) HEV RNA detected, n (%) ALT, 9 ULN1 Positive (243) 126 (51.9) 11.2±1.1 Negative (375) 1 (0.3) 8.4±0.5 Gray zone (9) 0 (0.0) 1.0±0.5 Not available (5) 0 (0.0) 5.3±2.2 1 Results are presented as mean ± SEM.
Table 2. Characteristics of samples tested for HEV RNA by real time RT-PCR.
The 127 HEV RNA-positive samples came from 111 patients, 15 of whom had sera collected at sequential time points. Partial ORF2 region was successfully amplified from 83 unique patients. Sequence analysis showed 82 (98.8%) patients were infected with HEV genotype 4 and one (1.2%) patient was infected with genotype 3. Subtypes were determined by creating a Maximum likelihood phylogenetic tree from all patient sequences and reference sequences obtained from GenBank (Fig. 3B). Subtype 4d was the most frequently detected (67.5%), followed by subtypes 4b (24.1%). Subtypes of 4a, 4 h, 4i and 3b were rarely detected (Fig. 3C).
There existed close phylogenetic relation between HEV sequences isolated from distant areas. For example, genotype 4b sequences identified from the current study were clustered with sequences isolated from Jiangsu (JX997647), Guangxi (EU676172) and Guangdong (JX855794) provinces, the latter two of which are more than 1200 km from Shanghai. We only detected one patient of genotype 3b infection (E122), who had travel history to Japan ten days before illness onset, but denied consumption of undercooked meat or seafood there. The identification of homologous swine sequence (FJ527832) to E122 argues the indigenous circulation of this subtype in Shanghai.
Subjects with positive HEV RNA were defined as laboratory-confirmed cases of HEV infection. Distinguishing features were addressed between confirmed HEV-infected subjects and subjects with positive anti-HEV IgM but undetected HEV RNA. The latter group may consist of subjects who recovered from recent infection or had viral load below detection limit. Overall, subjects with low level of anti-HEV IgM were often PCR-negative. Anti-HEV IgM, ALT and AST levels (P < 0.0001), as well as ALP, GGT and TBIL levels (P < 0.05), were significantly higher in double positive group (Table 3). This suggests that clearance or lowering of HEV viremia was accompanied by alleviation of symptoms and recovery of liver function.
HEV RNA (+) (n = 126) HEV RNA (-) (n = 117) P value Anti-HEV IgG COI) 10.3±0.4 11.7±0.6 0.0674 Anti-HEV IgM (COI) 31.9±1.5 11.1±1.3 < 0.0001 ALT, × ULN 18.2±1.8 2.1±0.4 < 0.0001 AST, × ULN 18.4±2.7 2.4±0.3 < 0.0001 ALP, × ULN 1.5±0.1 1.1±0.1 0.0133 GGT, × ULN 3.5±0.2 2.2±0.5 0.0052 TBIL, × ULN 5.6±0.5 3.8±0.7 0.0243 1Results are presented as mean ± SEM; 2ULV: Upper limit of normal value.
Table 3. Comparison of anti-HEV IgM (+) patients with or without HEV RNA detected 1.
Understanding demographic and clinical features of cases with HEV infection may help us identify patients at high risk in the future. Confirmed cases of HEV infection included 8 outpatients and 103 inpatients. The majority of them were males (62.2%). None of the females were pregnant at infection. About 77% were aged > 40 years. Thirty-nine cases (35%) occurred in January and February. Among patients who had available LFT results at presentation, 100 out of 106 had elevated ALT, AST or GGT above normal range; 78 out of 106 had elevated ALP; 90 out of 105 had elevated TBIL. The median level and range of LFT were listed in Table 4. As for outcome, 45 patients had normal ALT level at discharge, 33 patients had ALT level restored during follow up, 10 patients had improvement of ALT level at discharge, 15 patients had negative HEV RNA detected during follow up, 1 patient died during treatment and 7 outpatients were lost to follow-up.
Parameter Value Male, n (%) 69 (62.2) Age (years), median (range) 58 (25–86) Baseline laboratory tests1, median (range) ALT, × ULN 13.6 (0.6–96.8) AST, × ULN 8.3 (0.6–185.7) ALP, × ULN 1.4 (0.5–4.2) GGT, × ULN 3 (0.5–12.5) TBIL, × ULN 4.2 (0.3–26.8) Symptoms2, n (%) Abdominal pain 21 (20.4) Anorexia 70 (68.0) Fever 29 (28.2) Jaundice 78 (75.7) Malaise 49 (47.6) Nausea 44 (42.7) Vomiting 29 (28.2) Concomitant liver disease2, n (%) Alcoholic fatty liver disease 5 (4.9) Non-alcoholic fatty liver disease 17 (16.5) HBV carriage 13 (12.6) 1106 patients had available ALT, AST, ALP and GGT test results and 105 patients had available TBIL result at presentation to our hospital
2Information of symptoms and concomitant liver disease was available for 103 inpatients.
Table 4. Characteristics of laboratory-confirmed cases of HEV infection.
Comprehensive analyses based on detailed medical records of the 103 inpatients were carried out (Table 4). Patients presented within one day to one month after symptom onset. Ten patients recalled consumption of aquatic products, six patients reported eating takeout or dining out, one patient had definite history of consuming pork liver, one patient had close contact with swine, one patient traveled outside China within a month, and two patients' spouses were infected by HEV recently. Jaundice was the most reported clinical symptom, followed by anorexia, malaise, nausea, fever, vomiting, and abdominal pain. No case of extrahepatic manifestation was recorded. Pre-existing liver diseases were recorded in 13 patients with HBV carriage (represented by HBsAg presence), five patients with alcoholic fatty liver disease (AFLD) and 17 patients with non-alcoholic fatty liver disease (NAFLD). Eight patients had liver cirrhosis and three of them had decompensated cirrhosis due to HBV infection.
Next, we want to address the major factors correlated to patients' severity of illness, indicated by the length of hospital stays. All patients were discharged from hospital after improvement in LFT except that a 62-year-old male patient with late stage lung cancer died from renal failure 29 days after admission. The median hospital stay of the remaining 102 inpatients was 14 days, with a range between 5 and 128 days. Univariate analysis showed that the length of hospital stay was significantly correlated with age (P = 0.0384), anti-HEV IgG (P = 0.0408), ALP/ULN (P = 0.0166) and TBIL/ULN (P < 0.0001) level at admission. After adjusting the confounding factors, the length of hospital stay was significantly correlated with anti-HEV IgM (P = 0.0062) and TBIL/ULN (P < 0.0001) level, but not with age, HBsAg status, anti-HEV IgG, ALT, AST, ALP or GGT level by multivariate linear regression analysis. Remarkably, the association between hospitalization duration and TBIL/ULN level at admission was strongly positive (Table 5). No significant differences were found for age or any of the LFT results among patients with or without pre-existing HBV carriage (Table 6). Notably, patients co-infected with HBV had longer hospital stay (P = 0.0318) and higher level of TBIL, although not reaching statistical significance. No significant difference was found among patients with or without fatty liver diseases (data not shown).
Variables Length of hospital stay Univariate Multivariate β Coefficient P value β Coefficient P value Age 4.40 0.0384 Anti-HEV IgG 4.30 0.0408 Anti-HEV IgM 0.22 0.6389 7.83 0.0062 HBsAg (±) 1.41 0.2383 ALT/ULN 0.01 0.9391 AST/ULN 0.00 0.9628 ALP/ULN 5.94 0.0166 GGT/ULN 2.16 0.1447 TBIL/ULN 74.80 < 0.0001 87.33 < 0.0001
Table 5. Univariate and multivariate linear regression analysis for variables associating with the length of hospital stay.
Parameter HBsAg (+) (n = 13) HBsAg (-)(n = 89) P value Age (years) 54.4 ± 3. 5 55.5 ± 1.5 0.7526 Discharge days 24.2 ± 4.0 18.4 ± 1.8 0.0318 ALT, × ULN 16.6 ± 5.6 21.3 ± 2.1 0.3099 AST, × ULN 25.1 ± 9.5 20.8 ± 3.3 0.7191 ALP, × ULN 1.7 ± 0.2 1.5 ± 0.1 0.5041 GGT, × ULN 3.2 ± 0.5 3.8 ± 0.3 0.6391 TBIL, × ULN 8.0 ± 2.0 5.4 ± 0.4 0.2403 1Results are presented as mean ± SEM
Table 6. Demographic and baseline clinical parameters comparison of inpatients with or without HBV carriage1.