Incidence and risk factors of de novo Hepatitis E virus infection after receiving liver transplantation

doi:10.21203/rs.3.rs-4023335/v1

Download PDF

Research Article

Incidence and risk factors of de novo Hepatitis E virus infection after receiving liver transplantation

https://doi.org/10.21203/rs.3.rs-4023335/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Background/Aims:

Organ transplant recipients with hepatitis E virus (HEV) infection bears high risk to develop chronic hepatitis, which is generally associated with immunosuppressive therapies. This study aimed to identify the incidence and predictors of de novo HEV infection in patients after receiving transplantation.

Methods

We performed a large retrospective study to investigate the prevalence of anti-HEV at baseline, incidence of de novo HEV infection after transplantation, and the risk factors of HEV infection among patients with liver transplant in China. A total of 407 liver transplant recipients were examined for the presence of anti-HEV immunoglobulin G (IgG), IgM antibodies, and HEV RNA in serum. Basal indexes in individuals with evidence of post-transplant HEV infection were compared with those without evidence of that, and risk factors associated with HEV infection were assessed.

Results

The prevalence of anti-HEV at pre-transplant in liver transplant recipients was 25.8% (105/407). Serum-negative conversion occurred in 34 (32.38%) of 105 liver transplant patients. Sixty-five out of 302 patients had de novo HEV infection after transplantation, with a cumulative incidence of 42.74% during follow-up. After transplantation, HEV infection was associated with liver failure (P = 0.012), hypoproteinemia (P = 0.030) and higher level of r-glutamyl transferase (GGT) (P = 0.022) before transplantation. Graft rejection (OR 0.075; P = 0.045) was negatively associated with serum-negative conversion in patients who had positive anti-HEV antibody before transplantation.

Conclusions

The incidence of de novo HEV infection after transplantation were higher in China. Liver failure, hypoproteinemia, and GGT elevation may be associated with HEV infection after liver transplantation. This study suggests that prevention and control of HEV infection after liver transplantation should be paid attention in patients bearing these risk factors.

hepatitis E virus

liver transplantation

incidence

solid-organ transplant recipients

risk factors

liver failure

hypoproteinemia

r-glutamyl transferase

graft rejection

infection

The study investigated the burden and risk factors of HEV infection in liver transplant recipients with a large and long-term follow-up retrospective cohort. We found that the incidence of de novo HEV infection after transplantation were higher in China. Liver failure, hypoproteinemia, and GGT elevation may be associated with HEV infection after liver transplantation. These findings suggest that prevention and control of HEV infection after liver transplantation should be paid attention in patients bearing these risk factors.

Hepatitis E virus (HEV) is a leading global cause of acute viral hepatitis, with 20 million estimated infections and 70,000 deaths per year.¹ HEV belongs to the Herpesviridae family, and HEV isolates that infect humans are currently categorized into four genotypes (HEV1 to HEV4).² Genotype (GT) 1 and GT2 HEV are routinely encountered in developing countries and generally transmitted through the fecal-oral route. GT3 and GT4 HEV are associated with sporadic autochthonous infection among western countries and predominantly transmitted through animal reservoirs and ingestion of uncooked meat.³ Blood products and solid organ transplants are also considered to be the transmission modalities.⁴

HEV infection was initially considered to be a self-limited disease and generally did not cause chronic infection. However, in recent years, severe complications and chronic hepatitis due to HEV infection have been widely reported in immunosuppressed patients, including solid organ transplantation (SOT) recipients, human immunodeficiency virus (HIV) patients, and a few hematological-disordered patients receiving chemotherapy.^5–7 Epidemiological studies from Europe, America, and Asia estimate that HEV seroprevalence among SOT recipients differs widely, ranging from 2.5% up to 55.6%, and seems to be higher in middle-income countries compared to high-income countries.^8–10 Most of these studies were performed on liver, kidney, heart, and lung transplant recipients. The prevalence of HEV infection in liver transplant recipients is considerably more common than in other organ transplants, and lung transplant recipients are less susceptible to HEV infection.¹⁰ Most cases of chronic HEV infection in SOT recipients are attributable to GT3.¹¹ A retrospective study performed in 17 centers in Europe and the United States indicated that 65.9% of solid organ transplant recipients infected with GT3 HEV progressed to chronic hepatitis. Moreover, nearly 10% of these chronic HEV-infected patients developed cirrhosis within 3–5 years after infection, indicating HEV infection presents great potential harm to transplant recipients. Liver fibrosis can evolve rapidly in solid organ transplant recipients, leading to hepatic decompensation and death.¹² In addition to liver damage, acute and chronic HEV infections can cause neurological impairments, renal impairment, and gastrointestinal diseases.¹³

To date, various studies of HEV infection in SOT recipients have been reported in European and American countries but not yet in China. Although several investigations about the prevalence and clinical courses of HEV infection in SOT recipients were performed, few studies provide data on de novo HEV infection and accumulated de novo HEV infection rate after transplantation with long-term follow-up. In this study, we conducted a large retrospective study to investigate the seroprevalence of HEV in liver transformed recipients, the frequency of de novo HEV infection after transplantation, and associated risk factors of HEV infection.

2.1 Study Subjects

From 2006 to 2018, patients who received liver transplantation in the Fifth Medical Center of PLA General Hospital were enrolled in the retrospective study. Those with incomplete data and unavailable serum were excluded. Serum samples at baseline and post-transplant were collected and routinely tested for anti-HEV IgM, IgG, and HEV RNA. The demographic features, clinical manifestations and laboratory tests of enrolled patients were recorded.

2.2 Case Definitions

HEV seropositivity was defined by positive serum anti-HEV IgM and/or anti-HEV IgG antibodies. Patients who were positive for HEV RNA were defined as HEV infection, regardless of anti-HEV antibodies. Patients who were negative for anti-HEV antibodies and HEV RNA at transplantation but showed positive any of anti-HEV IgG, anti-HEV IgM, or HEV RNA after transplantation were considered to have a de novo HEV infection. In contrast, patients who have positive any of anti-HEV IgG, IgM, or HEV RNA before transplantation but present negative for all HEV indicators during the follow-up after transplantation were considered to have a serum-negative conversion.

2.3 Study Design

Demographic, clinical, and routine biochemistry data at inclusion were recorded. Two comparisons were performed. Firstly, we compared the baseline characteristics between de novo HEV infection and non-infected patients to determine the risk factors of de novo HEV infection after liver transplantation. Patients who were negative for anti-HEV antibodies and HEV RNA at transplantation but showed positive any of anti-HEV IgG, anti-HEV IgM, or HEV RNA during the three follow-up years after transplantation were included in the de novo HEV infection group. Conversely, patients who were negative for anti-HEV antibodies and HEV RNA both at transplantation and within the three follow-up years were considered to be the non-infected patients. Secondly, we determined the associated factors of serum-negative conversion after liver transplantation by comparing serum-negative and persistent positive patients. Patients who have positive any of anti-HEV IgG, IgM, or HEV RNA before transplantation but present negative for all HEV indicators during the three follow-up years after transplantation were included in the serum-negative conversion group. In contrast, patients showed positive any of anti-HEV IgG, anti-HEV IgM, or HEV RNA both at transplantation and within the three follow-up years were considered to be the persistent positive patients. Besides, only patients who have follow-up data more than three years after liver transplantation were eligible for the above two comparisons, in which the serum samples collected at less than four weeks post-transplantation were excluded.

2.4 HEV serological test and HEV molecular test

Anti-HEV IgM and IgG antibodies were tested using ELISA Kit (Wantai, Beijing, China) according to the manufacturer's instructions. Samples with signal-to-noise ratio (S/N ratio) N 1.0 were considered positive. Serum HEV RNA detection and sequence analysis were performed as before.¹⁴ Chloroform-free RNA extraction kit (Bioteke Corporation) was used to extract RNA from serum samples, followed by reverse transcription with RT-PCR kit (MCE). Nested-PCR and agarose gel electrophoresis were carried out to detect HEV using the specific primers as described.¹⁵ The samples with positive band of target gene fragment were further confirmed by sequencing.

2.5 Statistical Analyses

All data were analyzed using the software GraphPad Prism 9 and SPSS statistics 26.0. Categorical variables were compared using the chi-square test as appropriate. Student t test was used to compare continuous variables. Poisson regression was used to analyze the association between categorical variables and HEV seropositivity for each factor separately. Normally distributed continuous variables were expressed in mean ± standard deviation (SD), whereas other continuous variables were expressed in median (interquartile range [IQR]). Categorical variables were presented as counts (percentage). P < 0.05 was considered statistically significant.

2.6 Ethics

Sample collection and research were in accordance with regulations issued by the National Health Commission of China and the ethical standards formulated in the Helsinki Declaration. The permission for retrospective study was obtained from the institutional review board of The Fifth Medical Center of Chinese PLA General Hospital.

3.1 Patient Characteristics

A total of 780 patients were received organ transplantation in our center from 2006 to 2018, of which 373 patients were ruled out either due to data missing (N = 159), serum unavailability (N = 202), or non-liver transplantation (N = 12), leading to 407 patients eligible in this study (Fig. 1). A total of 969 serum samples were collected, with two to 18 samples per persons.

Clinical characteristics of all enrolled patients were summarized in Table 1. The median age was 50 years with a male predominance (N = 330, 81.08%). More than ninety percent patients had liver cirrhosis at transplantation, among which viral hepatitis (73.22%) is the most common etiology followed by alcoholic hepatitis (18.67%). There were 194 patients suffering from hepatocellular carcinoma and 61 patients having liver failure. The common complications included anemia (n = 146, 42.82%), ascites (n = 154, 45.16%), electrolyte disturbance (n = 98, 28.74%), and Inflammation (n = 91, 26.69%). In regard to biochemical parameters, the median levels of total bilirubin (TBiL) and international normalized ratio (INR) at pre-transplantation, as well as direct bilirubin (DBiL), TBiL, alanine aminotransferase (ALT), and 𝛾-glutamyl transferase (GGT) at post-transplantation, were beyond normal ranges. Aspartate aminotransferase (AST), aspartate aminotransferase (ALP) and creatinine tended to be frequently abnormal in these patients, despite the median values being in normal ranges. The median values of the blood routine tests were within normal ranges except for lymphocyte percentage (LY%/WSCR), which was lower than normal value. 341 patients received immunosuppressive therapy. All of them received tacrolimus, among which 340 received steroid as well, 306 received additional mycophenolate. The follow-up time after transplantation varied from 7 to 4352 days, and the median was 383 days. 27.03% of patients received re-hospitalization for graft rejection during the follow-up.

Table 1

Patient Characteristics
Characteristic	Patients (n = 407)
Age at transplantation, median (range)	50 (9–72)
Male gender, n (%)	330 (81.0)
Follow-up weeks, median (range)	54.71 (1-621.71)
Re-hospitalization due to graft rejection, n (%)	110 (27.03)
Hepatobiliary disease at transplantation	407(100)
Liver failure, n (%)	61 (14.99)
Cirrhosis, n (%)	367 (90.17)
Viral hepatitis and related cirrhosis, n (%)	298 (73.22)
HBV, n (%)	271 (66.58)
HCV, n (%)	42 (10.32)
HEV, n (%)	1 (0.2)
Biliary cirrhosis, n (%)	27 (6.63)
Alcoholic cirrhosis, n (%)	76 (18.67)
Autoimmune cirrhosis, n (%)	19 (4.67)
Hepatocellular carcinoma, n (%)	194 (47.67)
Cholangiocarcinoma and/or Gallbladder carcinoma, n (%)	3 (0.74)
DILI, n (%)	2 (0.49)
Others, n (%)(Parasite, Bujia, hepatolenticular degeneration, congestion cirrhosis, liver metastasis from other cancers, unexplained cirrhosis)	15 (3.69)
Complications at transplantation (n = 341)
Inflammation, n (%)	91 (26.69)
Hypoproteinemia, n (%)	48 (14.08)
Electrolyte disturbance, n (%)	98 (28.74)
Kidney disease, n (%)	34 (9.97)
Hepatic encephalopathy, n (%)	42 (12.32)
Portal hypertension, n (%)	57 (16.72)
Anemia, n (%)	146 (42.82)
Ascites, n (%)	154 (45.16)
Hypertension, n (%)	24 (7.04)
Diabetes, n (%)	56 (16.42)
Liver function at pre-transplantation (n = 367)
MELD score	13 (3–40)
TBiL 3.4–20.5 (µmol/L)	42.4 (6.8-725.5)
Creatinine 62–115 (umol/L)	75 (32–541)
INR 0.8–1.2	1.36 (0.89–5.05)
Liver function at post-transplantation (n = 339)
DBiL, 0–6.8 (umol/L)	24.2 (0.4-335.8)
TBiL 3.4–20.5 (umol/L)	37.2 (5.4-444.4)
DBiL/TBiL	0.67 (0.04–0.90)
AST 5–40 (U/L)	32 (9-7576)
ALT 5–40 (U/L)	82 (9-4155)
AST/ALT	0.41 (0.15–1.82)
ALP 40–150 (U/L)	98 (34–884)
GGT 11–50 (U/L)	109 (22–934)
Creatinine 62–115 (umol/L)	69 (21–631)
Hematological examination
WBC 3.97–9.15 (10⁹/L)	7.11 (1.51–21.9)
LY%/WSCR 20–40 (%)	11.7 (2.2–31.3)
LY%/WSCC 0.8-4.0 (10⁹/L)	0.8 (0.1–9.01)
Platelets count 85–303 (10⁹/L)	92 (8.4–666)
Immunosuppressive therapy (n = 341)
Steroid, n (%)	340 (99.71)
Tacrolimus, n (%)	341 (100.00)
Mycophenolate, n (%)	306 (89.74)
Double regime, n (%)	36 (10.56)
Triple regime, n (%)	305 (89.44)
immune-modulating therapy (n = 341)
Thymosin, n (%)	124 (36.36)
Interleulin, n (%)	18 (5.28)
Human Immunoglobulin, n (%)	13 (3.81)
Abbreviations: HBV, hepatitis B virus; HCV, hepatitis C virus; HEV, hepatitis E virus; DILI, drug-induced liver injury; TBiL, total bilirubin; INR, international normalized ratio; DBiL, direct bilirubin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; GGT, 𝛾-glutamyl transferase; WBC, white blood cell; LY, lymphocyte. Steroid, including prednisone acetate/ methylprednisolone/ prednisolone/ dexamethasone
The continuous variables are expressed in median (interquartile range [IQR]). Categorical variables are presented as counts (percentage).

3.2 HEV Seroprevalence at transplantation

Overall, 302 patients (74.20%) were negative for anti-HEV IgG, anti-HEV IgM and HEV RNA before transplantation. 105 (25.8%) cases presented anti-HEV IgG positivity, of which five patients were also positive for anti-HEV IgM, and one patient had detectable HEV RNA with negative anti-HEV IgM (Fig. 1).

3.3 Post-transplant HEV Seroprevalence

Among 302 patients with negative serum before transplantation, de novo HEV infection were found in 65 individuals after transplantation, with a cumulative incidence of 42.74% during follow-up (Fig. 1–3). Of note, the seroprevalence rates dramatically increased in the first follow-up year after transplantation with a ratio of 19.35%, but kept at low infection risk between the second to fourth follow-up year. Subsequently, a gradual increase in incidence was observed from the fifth to the ninth follow-up year, with the exception of the sixth year, during which no instances of HEV infection were identified (Fig. 3). Of these 65 patients, five patients were positive for both anti-HEV IgM and HEV IgG and two showed only positive anti-HEV IgM, indicating they were experiencing HEV infection. The remaining 58 individuals were only positive for anti-HEV IgG. None of the patients had detectable HEV RNA (Fig. 1, 2).

3.4 Risk factors of de novo HEV infection after liver transplantation

Clinical characteristics were compared between 51 de novo HEV infection cases and 73 non-infected cases. A total of 124 patients were eligible for the analysis of the risk factors of de novo HEV infection after liver transplantation. The mean age of the patients with de novo HEV infection was same to that of the non-infected patients. The percentage of males in the de novo HEV infection group was 88%, higher than that in non-infected patients with a percentage of 73.97%, but not significantly (P = 0.051). Since graft rejection is frequently encountered post liver transplantation, especially in humans, we subsequently investigated whether graft rejection influenced de novo HEV infection. The rate of graft rejection was significantly lower in HEV infected patients (19.61%) compared with non-infected patients (39.73%) after transplantation (P = 0.018). The prevalence of cirrhosis was similar in both groups, but patients with liver failure were more likely to have de novo HEV infection (P < 0.001). Extra-hepatic complications seemed not associated with HEV infection, except hypoproteinemia that was found to frequently present in patients developing HEV infection after transplantation (P < 0.001). In terms of the usage of immunosuppressive drugs, patients received triple drug more likely to have HEV infection than those received double drug, despite the difference was not significant (P = 0.076) (Table 2).

Table 2

Baseline characteristics, complications and biochemical indexes between HEV infection and non-infection patients at post-transplantation
	HEV infection (N = 51)	No infection (N = 73)	P
Age	48.8 ± 8.5	48.3 ± 9.4	0.743
Male gender (%)	45 (88.24)	54 (73.97)	0.051
Graft rejection (%)	10 (19.61)	29 (39.73)	0.018
Cirrhosis (%)	48 (94.12)	72 (98.63)	0.305
Autoimmune cirrhosis (%)	2 (3.92)	5 (6.85)	0.699
Viral hepatitis (%)	42 (82.35)	61 (83.56)	0.860
HBV (%)	42 (82.35)	53 (72.60)	0.207
HCV (%)	1 (1.96)	10 (13.70)	0.052
Alcoholic cirrhosis (%)	11 (21.57)	8 (10.96)	0.107
Biliary cirrhosis (%)	4 (7.84)	5 (6.85)	1.000
Hepatocellular carcinoma (Including post-treatment) (%)	25 (49.02)	33 (45.21)	0.675
Liver failure (%)	13 (25.49)	2 (2.74)	< 0.001
Inflammation (%)	13 (25.49)	12 (16.44)	0.216
Hypoproteinemia (%)	12 (23.53)	1 (1.37)	< 0.001
Electrolyte disturbance (%)	18 (35.29)	17 (23.29)	0.144
hepatitis-related kidney disease/renal (%)	6 (11.76)	5 (6.85)	0.531
Hepatic encephalopathy (%)	7 (13.73)	6 (8.22)	0.325
portal hypertension (%)	9 (17.65)	14 (19.18)	0.829
Anemia (%)	25 (49.02)	24 (32.88)	0.070
Ascites (%)	24 (47.06)	34 (46.58)	0.958
Hypertension (%)	6 (11.76)	2 (2.74)	0.064
Diabetes (%)	7 (13.73)	9 (12.33)	0.819
Steroid (%)	51 (100.00)	73 (100.00)	-
Tacrolimus	51 (100.00)	73 (100.00)	-
Mycophenolate (%)	48 (94.12)	61 (83.56)	0.076
Thymosin (%)	21 (41.18)	33 (45.21)	0.656
Interleulin (%)	1 (1.96)	8 (10.96)	0.122
Double regime (%)	3 (5.88)	12 (16.44)	0.076
Triple regime (%)	48 (94.12)	61 (83.56)	0.076
Human Immunoglobulin(pH4) (%)	1 (1.96)	4 (5.48)	0.648
Others (%)	0 (-)	4 (5.48)	0.143
Liver function
MELD score	14.29 (8.93–21.26)	11.99 (9.26–18.13)	0.318
TBiL 3.4–20.5 (umol/L)	47.10 (23.70-207.30)	35.15 (21.05–62.15)	0.061
Cr 62–115 (umol/L)	75 (64–89)	77 (66–87)	0.657
INR 0.8–1.2	1.42 (1.16–1.69)	1.33 (1.15–1.70)	0.384
DBiL 0-6.8 (umol/L)	23.10 (12.40–47.60)	25.50 (11.50–60.10)	0.851
D/T	0.69 (0.54–0.76)	0.66 (0.54–0.75)	0.718
AST 5–40 (U/L)	30 (21–44)	36 (22–56)	0.111
ALT 5–40 (U/L)	73 (55–105)	107 (70–149)	0.013
AST/ALT	0.39 (0.30–0.52)	0.36 (0.30–0.50)	0.153
ALP 40–150 (U/L)	114 (87–172)	90 (71–118)	< 0.001
GGT 11–50 (U/L)	123 (81–176)	89 (67–153)	0.043
Hematological examination
WBC 3.97–9.15 (10⁹/L)	7.11 (5.36–8.40)	7.88 (5.14–10.90)	0.238
LY%/WSCR 20–40%	12 (8–17)	10 (8–14)	0.154
LY#/WSCC 0.8-4.0 (10⁹/L)	0.77 (0.52–1.33)	0.79 (0.45–1.30)	0.728
Platelets count 85–303 (10⁹/L)	97 (50–128)	88 (48–161)	0.911
Abbreviations: HBV, hepatitis B virus; HCV, hepatitis C virus; Steroid, including prednisone acetate/ methylprednisolone/ prednisolone/ dexamethasone; MELD, Model for end-stage liver disease; TBiL, total bilirubin; Cr, creatinine; INR, international normalized ratio; DBiL, direct bilirubin; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; GGT, 𝛾-glutamyl transferase; WBC, white blood cell; LY, lymphocyte.
Normally distributed continuous variables are expressed in mean ± standard deviation (SD), whereas other continuous variables are expressed in median (interquartile range [IQR]). Categorical variables are presented as counts (percentage).

Serum liver function and other laboratory tests were summarized in Table 2. The incidence of HEV de novo infection was found not related to baseline model for end-stage liver disease (MELD) score and the level of TBiL, DBiL, INR and creatinine. However, the transaminase showed distinct trends when compared between HEV infection and non-infection groups. ALT level was lower while GGT and ALP levels were higher significantly in HEV infected patients than in those without HEV infection. The blood routine tests such as WBC, LY%/WSCR, lymphocyte absolute value (LY#/WSCC), and platelets count (PLT) were comparable between the two groups (Table 2).

In order to identify risk factors of de novo HEV infection after liver transplantation, we selected significantly different parameters to perform further multivariable analysis. We found liver failure (OR 11.906; P = 0.012), hypoproteinemia (OR 14.466; P = 0.030), and higher GGT (OR 10.088; P = 0.022) at transplantation were independent factors associated with de novo HEV infection after liver transplantation (Table 3).

Table 3

Risk factors of *de novo* HEV infection at post-transplantation (multivariate analysis)
	OR (95% CI)	P
Graft rejection	0.446 (0.173–1.147)	0.094
Liver failure	11.906 (1.734–81.751)	0.012
Hypoproteinemia	14.466 (1.293-161.895)	0.030
GGT > 50 U/L	10.088 (1.390-73.233)	0.022
Anemia	1.345 (0.552–3.278)	0.515
HEV infection (N = 51) and no infection group (N = 73). GGT, 𝛾-glutamyl transferase.

3.5 Post-transplant serum-negative conversion

Among the 105 patients with positive serum before transplantation, serum-negative conversion was found in 34 (32.38%) individuals after transplantation (Fig. 1). For 99 patients with solely positive anti-HEV IgG before transplantation, 66 patients remained positive after transplantation, while only 33 patients converted to negativity. In the setting of five patients with both positive antibodies at pre-transplantation, we found one cases achieved completed serum-negative conversion, three presented positive IgG but negative IgM, and one kept both antibodies positivity during follow-up. The only one patient with positive HEV RNA before transplantation successfully cleared viral genome after transplantation (Fig. 1, 2).

3.6 Associated factors of serum-negative conversion after liver transplantation

thirty-four subjects were eligible for the study to determine the associated factors of serum-negative conversion after liver transplantation according to the excluding criteria, including 19 serum-negative conversion subjects and 15 persistent seropositive subjects. Forty (88.24%) patients were male with a similar sex ratio in both groups (89.47% and 86.67%, P = 1.000). No significant differences were found with regard to etiologies for liver transplantation, including cirrhosis, hepatocellular carcinoma (HCC), alcohol liver disease (ALD), and liver failure between the two groups. The incidence of common complications before transplantation appeared not corrected with HEV antibody negative conversion. Intriguingly, graft rejection occurred in around 46.67% patients that kept positive HEV antibody versus 15.79% patients achieved negative conversion (P = 0.068), reflecting a trend towards short antibody response in immunotolerant patients. In parallel, patients received triple immunosuppressants were more likely to have negative conversion than those received double immunosuppressants. However, there were no significant difference in laboratory parameters and hematological examination between the two groups (Table 4).

Table 4

**Baseline characteristics, complications and biochemical indexes between persistent seropositive and serum-negative conversion patients at post-transplantation**
	Positive N = 15	negative N = 19	P
Age (year)	50 (43–57)	48 (41–58)	0.622
Gender (male) (%)	13 (86.67)	17 (89.47)	1.000
Cirrhosis (%)	14(93.33)	17(89.47)	1.000
Virus hepatitis (%)	13 (86.67)	14 (73.68)	0.426
HBV (%)	9 (60.00)	14 (73.68)	0.475
HCV (%)	4 (26.67)	1 (5.26)	0.146
Biliary cirrhosis (%)	1 (6.67)	0 (-)	0.441
HCC (%)	5 (33.33)	9 (47.37)	0.495
ALD (%)	4 (26.67)	5 (26.32)	1.000
Liver failure (%)	1 (6.67)	7 (36.84)	0.053
Graft rejection (%)	7 (46.67)	3 (15.79)	0.068
Inflammation (%)	4 (26.67)	9 (47.37)	0.296
Hypoproteinemia (%)	1 (6.67)	1 (5.26)	1.000
Electrolyte disturbance (%)	1 (6.67)	3 (15.79)	0.613
Renal disease (%)	0 (-)	2 (10.53)	0.492
HE (%)	2 (13.33)	3 (15.79)	1.000
PH (%)	3 (20.00)	1 (5.26)	0.299
Anemia (%)	2 (13.33)	8 (42.11)	0.129
Ascites (%)	6 (40.00)	5 (26.32)	0.475
Hypertension (%)	2 (13.33)	1 (5.26)	0.571
Diabetes (%)	3 (20.00)	2 (10.53)	0.634
Steroid (%)	15 (100.00)	19 (100.00)	-
Tacrolimus (%)	15 (100.00)	19 (100.00)	-
Matikao (%)	12 (80.00)	17 (89.47)	0.634
2 drug (%)	3 (20.00)	2 (10.53)	0.634
3 drug (%)	12 (80.00)	17 (89.47)	0.634
Thymosin (%)	7 (46.67)	7 (36.84)	0.728
IL (%)	3 (20.00)	0 (-)	0.076
PH4 (%)	0 (-)	1 (5.26)	1.000
Laboratory parameters
MELD (pre)	15.04 (9.28–22.14)	16.67 (11.07–23.31)	0.842
TBiL (pre) 3.4–20.5 (umol/L)	50.5 (23.4-129.2)	42.8 (27.1–289.0)	0.925
Creatinine (pre) 62–115 (umol/L)	67 (59–80)	82 (67–88)	0.171
INR (pre)	1.35 (1.24–1.73)	1.46 (1.26–1.96)	0.779
DBiL 0-6.8 (umol/L)	17.6 (13.0-60.3)	29.9 (10.3–90.2)	0.315
TBiL 3.4–20.5 (umol/L)	32.0 (20.4–74.8)	48.0 (26.5-123.3)	0.228
D/T	0.63 (0.54–0.73)	0.64 (0.58–0.79)	0.656
AST 5–40 (U/L)	27 (20–39)	30 (18–57)	0.817
ALT 5–40 (U/L)	80 (51–94)	58 (32–101)	0.298
S/L	0.44 (0.33–0.53)	0.53 (0.44–0.69)	0.120
ALP 40–150 (U/L)	85 (68–106)	97 (58–175)	0.509
GGT 11–50 (U/L)	87 (43–97)	185 (51–305)	0.067
Creatinine (post) 62–115 (umol/L)	65 (58–97)	75 (56–120)	0.509
Hematological examination
WBC 3.97–9.15 (× 10⁹)	6.85 (5.90–9.40)	6.39 (4.20–7.34)	0.095
LY%/WSCR (0.20–0.40)	0.12 (0.08–0.16)	0.11 (0.09–0.12)	0.804
LY#/WSCC (0.8-4.0× 10⁹)	0.90 (0.50–1.34)	0.62 (0.40–0.86)	0.126
PLT (× 10⁹)	81 (59–103)	101 (53–128)	0.638
Abbreviations: HBV, hepatitis B virus; HCV, hepatitis C virus; HCC, hepatocellular carcinoma; ALD, alcohol liver disease; HE, hepatic encephalopathy; PH, portal hypertension; IL, interleukin; MELD, model for end stage liver disease; TBiL, total bilirubin; INR, international normalized ratio; DBiL, direct bilirubin; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; GGT, 𝛾-glutamyl transferase; WBC, white blood cell; LY, lymphocyte; PLT, platelets count. Steroid, prednisone acetate/ methylprednisolone/ prednisolone/ dexamethasone.
The continuous variables are expressed in median (interquartile range [IQR]). Categorical variables are presented as counts (percentage).

According to the multivariate analysis, no occurrence of graft rejection (OR 0.075; P = 0.045) was identified independent factor associated with serum-negative conversion in patients after transplantation (Table 5).

Table 5

Associated factors with serum-negative conversion in patients at post-transplantation (multivariate analysis)
	OR (95% CI)	P
Liver failure	4.071 (0.300-55.228)	0.291
Graft rejection	0.075 (0.006–0.946)	0.045
GGT 11–50 (U/L)	1.009 (0.999–1.018)	0.074
IL	< 0.001(< 0.001- >999.999 )	0.965
WBC	0.749 (0.487–1.153)	0.189
Abbreviations: GGT, 𝛾-glutamyl transferase; WBC, white blood cell.

Acute GT3 HEV infections are increasingly reported in developed countries and may establish chronic infection in immuno-compromised patients, leading to rapidly progressive liver fibrosis and cirrhosis, as demonstrated in cohorts of organ transplant adult recipients. The burden of HEV infection in liver transplant recipients in China, which GT4 HEV is predominant, remains uncertain because they are not routinely tested in last decade. Unless HEV screening is included in systematic analyses, the diagnosis can be easily missed because clinical features are often unremarkable.⁶ The majority of acute HEV infections will clear uneventfully either spontaneously or following a reduction in the levels of immunosuppression.

The current study investigated the burden and risk factors of HEV infection in liver transplant recipients with a large and long-term follow-up retrospective cohort. The cumulative incidence of de novo HEV infection after transplantation was 42.74%, although no viremia was detected. Liver failure, hypoproteinemia, and GGT at pre-transplantation were found to be independently associated with HEV de novo infection after transplantation during follow-up. For patients with HEV seropositivity before transplantation, graft rejection was negatively associated with serum-negative conversion at post-transplantation.

The seropositivity of HEV is 25.8% in patients prior to liver transplantation, slightly lower than the prevalence of 30% among blood donors reported in Chinese cohort.¹⁶ Likewise, French studies showed that the HEV seropositivity was similar between solid-organ transplant recipients (14.1%) and blood donor (16.6%).^{17, 18} Variations in HEV seroprevalence between countries may reflect not only the differences in viral circulation within a geographical region but also the impact of assays used in particular studies. Therefore, the data should be cautiously interpreted.

Largely different form our results indicating a cumulative incidence of 42.74% for de novo HEV infection following transplantation, reports of anti-HEV-IgG seroprevalence after solid organ transplantation in Western countries vary from 1% in Netherlands to 4% in North American.^{19, 20} This discrepancy with our data reinforces the need for studies on factors associated with HEV infection in different regions worldwide. Meanwhile, the difference may in part be blamed for different research designs, rather than true differences in prevalence of de novo HEV infection after transplantation. Netherlands and North American data are derived from cross-sectional study, whereas our study is estimated a cumulative incidence for a twelve follow-up year, which certainly much higher than that in cross-sectional studies. Furthermore, we noted a strikingly elevated rate of de novo HEV infection during the initial year post-transplantation than the following years. Transplant recipients typically receive quite low immunosuppression therapy after the first year post-LT and this may account for the high prevalence of HEV infection in the first-year post-transplantation. It is of utmost importance to maintain heightened vigilance in monitoring patients during the first year following transplantation.

Liver failure at baseline was identified as an independent risk factor for de novo HEV infection post-transplantation. It is well-documented that patients with liver failure often exhibit a severely dysregulated immune system, encompassing various aspects from antigen processing to effector cell functions and cytokine release. In this context, the activated immune cells seem to be dysfunctional, paralyzed and energy-depleted, rendering individuals more susceptible to infections.²¹ Specifically, MER receptor tyrosine kinase (MERTK) expressed by monocytes and macrophages was reported as a critical protein greatly increased in patients with liver failure and contributed to down-regulation of innate immune response to microbes.²² Accordingly, the reduced cellular immune function in subjects with liver failure might contribute to the increased infectious morbidity of these patients and provide a rational basis for prevention strategies.

Hypoproteinemia is recognized as a significant marker of liver dysfunction, protein-calorie malnutrition, and an active acute-phase response. It has been consistently linked to surgical site infections in patients undergoing general surgery procedures.²³ Notably, a prior study specifically highlighted hypoproteinemia as an independent factor associated with the occurrence of surgical site infections following hepatectomy.²⁴ Furthermore, hypoalbuminemia is established as an independent risk factor for pulmonary infection, contributing to a decrease in the lung tissue's anti-infection and anti-inflammatory capabilities.²⁵ In our current investigation, we identified hypoproteinemia as an independent factor linked to de novo HEV infection after liver transplantation. Collectively, hypoproteinemia appears to heighten patients' susceptibility to infections. However, it remains imperative to explore the underlying mechanisms how hypoproteinemia associated with the incidence of de novo HEV infection in post-liver transplant patients.

32.38% HEV seropositive patients at pre-transplantation experienced serum-negative conversions after liver transplantation. In line with our result, a previous study from France documented the loss of anti-HEV IgG in 32 of 89 patients (35.9%) following solid-organ transplantation. ¹⁷ The decline in anti-HEV antibodies is likely to be attributed to immunosuppressive therapy. Notably, rapamycin has been shown to inhibit IgG production by pure B cells when stimulated with IL2 and Staphylococcus aureus Cowan I in vitro.²⁶ Additionally, previous reports have indicated reduced vaccine efficacy against various diseases, including influenza, tetanus, diphtheria, hepatitis A, and hepatitis B, as well as diminished antibody titers in transplant patients.²⁷ For instance, Severson et al. found that after two years’ immunization, seroprotection rates (defined as an antibody titer of ≥ 1:40) against influenza A strains ranged from 65–74% in lung transplant recipients compared to 77–100% in healthy controls.²⁸ This suggests that organ transplant recipients are prone to loss vaccine-induced antibodies than healthy individuals. In our present study, it appeared that patients with liver failure, no graft rejection, a taking triple immunosuppressive drug regimen, were more likely to experience negative conversion, indicating a tendency toward a shorter duration of antibody response in immunotolerant patients. Particularly, graft rejection emerged as an independent factor associated with sustained seropositivity. After transplantation of liver or other organs, antibody-mediated, hyperacute vasculitic rejection can occur in individuals with preformed antibodies against the donor’s major histocompatibility complex (MHC) class I– encoded antigens. Cells of the innate immune system, such as natural killer (NK) cells, are also present in allografts during rejection.²⁹ Therefore, graft rejection may maintain the anti-HEV antibody levels through both innate and adaptive immune cells which produce antibodies. However, another study found that biopsy-proven acute rejection was independent risk factor for loss of anti-HBV antibodies in kidney transplant recipients.³⁰ Further studies are need to clarify the causality of anti-virus antibodies disappearance in SOT recipients with graft rejection.

In addition to the absence of a definitive confirmation of the HEV genotype for all patients, several other limitations in our study merit acknowledgment. The variable durations of follow-up and irregular timing of sample collection have made it challenging to determine the rate of chronic infection in cases of de novo HEV infection. Furthermore, it is important to note that our study was limited to a single center. Larger and multicenter studies with a greater number of cases are required to provide more comprehensive understanding on HEV infection among liver transplant recipients in China. Nonetheless, this study, with its long-term follow-up and multiple samples for liver transplant patients, represented the first large cohort investigating the HEV infection at pre- and post-transplantation in China. Most importantly, we explored the frequency of de novo HEV infection and serum-negative conversion after transplantation, and did a thorough analysis of their associated factors. We firmly believe that our noteworthy findings have provided crucial insights into HEV infection among liver transplant recipients, serving as a valuable foundation for further research in this field.

In summary, the seroprevalence of anti-HEV in liver transplant recipients was high in China. It is recommended that liver transplant recipients should avoid eating uncooked meat and avoid contact with possibly HEV-infected animals. Liver transplant recipients with graft hepatitis should be carefully monitored by testing HEV-RNA.

HEV

hepatitis E virus

IgG

immunoglobulin G

IgM

immunoglobulin M

GGT

r-glutamyl transferase

Genotype

SOT

solid organ transplantation

HIV

human immunodeficiency virus

TBiL

total bilirubin

INR

international normalized ratio

DBiL

direct bilirubin

ALT

alanine aminotransferase

AST

aspartate aminotransferase

ALP

aspartate aminotransferase

LY%/WSCR

lymphocyte percentage

MELD

model for end-stage liver disease

LY#/WSCC

lymphocyte absolute value

PLT

platelets count

HCC

hepatocellular carcinoma

ALD

alcohol liver disease

MHC

major histocompatibility complex.

Conflict of interest declaration: The authors declare that they have no conflict of interest. All authors read and approved the final manuscript.

Funding: This work was supported by Natural Science Foundation of ShenZhen (JCYJ20210324103808023); International science and technology cooperation of ShenZhen (GJHZ20220913142608016); International science and technology cooperation of Guangdong (2023A0505050115); National Natural Science Foundation of China (82370610) (to Y. Wang).

Author contributions：PH and JL contributed to acquisition and analysis of data, prepared the tables and drafted the manuscript; PH, JL, JZ and CW contributed to lab experiments; PH, CW and YJ contributed to sample collection. JL and HL contributed to critical revision of the manuscript; YG contributed to the study concept and critical revision of the manuscript; YW contributed to study concept and design, obtained funding, supervision of the study, literature search, writing of the manuscript and revised the final version of manuscript. All the authors read the article and approved the submission.

Ethics: Sample collection and research were in accordance with regulations issued by the National Health Commission of China and the ethical standards formulated in the Helsinki Declaration. The permission for retrospective study was obtained from the institutional review board of The Fifth Medical Center of Chinese PLA General Hospital.

Data availability statement: Aggregated data will be available on reasonable request to the corresponding author. Approvals must be obtained from all collaborators, with a signed data access agreement. No date restrictions apply to data availability.

European Association for the Study of the Liver. Electronic address eee, European Association for the Study of the L. EASL Clinical Practice Guidelines on hepatitis E virus infection. J Hepatol. 2018;68(6):1256–71.
Okamoto H. Genetic variability and evolution of hepatitis E virus. Virus Res. 2007;127(2):216–28.
Pavio N, Meng XJ, Doceul V. Zoonotic origin of hepatitis E. Curr Opin Virol. 2015;10:34–41.
Kamar N, Bendall R, Legrand-Abravanel F, Xia NS, Ijaz S, Izopet J, Dalton HR, Hepatitis E. Lancet. 2012;379(9835):2477–88.
Kamar N, Selves J, Mansuy JM, Ouezzani L, Peron JM, Guitard J, Cointault O, Esposito L, Abravanel F, Danjoux M, et al. Hepatitis E virus and chronic hepatitis in organ-transplant recipients. N Engl J Med. 2008;358(8):811–7.
Jagjit Singh GK, Ijaz S, Rockwood N, Farnworth SP, Devitt E, Atkins M, Tedder R, Nelson M. Chronic Hepatitis E as a cause for cryptogenic cirrhosis in HIV. J Infect. 2013;66(1):103–6.
le Coutre P, Meisel H, Hofmann J, Rocken C, Vuong GL, Neuburger S, Hemmati PG, Dorken B, Arnold R. Reactivation of hepatitis E infection in a patient with acute lymphoblastic leukaemia after allogeneic stem cell transplantation. Gut. 2009;58(5):699–702.
de Oliveira JMNS, Freitas NR, Teles SA, Bottino FO, Lemos AS, de Oliveira JM, de Paula V, Pinto MA, Martins RMB. Prevalence of hepatitis E virus RNA and antibodies in a cohort of kidney transplant recipients in Central Brazil. Int J Infect Dis. 2018;69:41–3.
Komolmit P, Oranrap V, Suksawatamnuay S, Thanapirom K, Sriphoosanaphan S, Srisoonthorn N, Posuwan N, Thongmee T, Treeprasertsuk S, Poovorawan Y. Clinical significance of post-liver transplant hepatitis E seropositivity in high prevalence area of hepatitis E genotype 3: a prospective study. Sci Rep. 2020;10(1):7352.
Wijarnpreecha K, Choudhury A, Kaewput W, Mao SA, Mao MA, Jadlowiec CC, Cheungpasitporn W. Hepatitis E in solid organ transplant recipients: A systematic review and meta-analysis. World J Gastroenterol. 2021;27(12):1240–54.
Agarwala P, Gupta E, Choudhary MC, Pamecha V. Absence of chronic hepatitis E virus infection in liver transplant recipients: Report from a hyperendemic region. Indian J Gastroenterol. 2018;37(2):160–3.
Kamar N, Garrouste C, Haagsma EB, Garrigue V, Pischke S, Chauvet C, Dumortier J, Cannesson A, Cassuto-Viguier E, Thervet E, et al. Factors associated with chronic hepatitis in patients with hepatitis E virus infection who have received solid organ transplants. Gastroenterology. 2011;140(5):1481–9.
Whitsett M, Feldman DM, Jacobson I, Hepatitis E. Virus Infection in the United States: Current Understanding of the Prevalence and Significance in the Liver Transplant Patient Population and Proposed Diagnostic and Treatment Strategies. Liver Transpl. 2020;26(5):709–17.
Wang Y, Wang S, Wu J, Jiang Y, Zhang H, Li S, Liu H, Yang C, Tang H, Guo N et al. Hepatitis E virus infection in acute non-traumatic neuropathy: A large prospective case-control study in China. EBioMedicine 2018, 36:122–130.
Huang FF, Haqshenas G, Guenette DK, Halbur PG, Schommer SK, Pierson FW, Toth TE, Meng XJ. Detection by reverse transcription-PCR and genetic characterization of field isolates of swine hepatitis E virus from pigs in different geographic regions of the United States. J Clin Microbiol. 2002;40(4):1326–32.
Zhang L, Jiao S, Yang Z, Xu L, Liu L, Feng Q, Zhang X, Hou Y, He S, Saldanha J, et al. Prevalence of hepatitis E virus infection among blood donors in mainland China: a meta-analysis. Transfusion. 2017;57(2):248–57.
Legrand-Abravanel F, Kamar N, Sandres-Saune K, Lhomme S, Mansuy JM, Muscari F, Sallusto F, Rostaing L, Izopet J. Hepatitis E virus infection without reactivation in solid-organ transplant recipients, France. Emerg Infect Dis. 2011;17(1):30–7.
Mansuy JM, Legrand-Abravanel F, Calot JP, Peron JM, Alric L, Agudo S, Rech H, Destruel F, Izopet J. High prevalence of anti-hepatitis E virus antibodies in blood donors from South West France. J Med Virol. 2008;80(2):289–93.
Haagsma EB, Niesters HG, van den Berg AP, Riezebos-Brilman A, Porte RJ, Vennema H, Reimerink JH, Koopmans MP. Prevalence of hepatitis E virus infection in liver transplant recipients. Liver Transpl. 2009;15(10):1225–8.
Sue PK, Pisanic N, Heaney CD, Forman M, Valsamakis A, Jackson AM, Ticehurst JR, Montgomery RA, Schwarz KB, Nelson KE, Karnsakul W. Hepatitis E Virus Infection Among Solid Organ Transplant Recipients at a North American Transplant Center. Open Forum Infect Dis. 2016;3(1):ofw006.
Sarin SK, Choudhury A. Acute-on-chronic liver failure: terminology, mechanisms and management. Nat Rev Gastroenterol Hepatol. 2016;13(3):131–49.
Bernsmeier C, Pop OT, Singanayagam A, Triantafyllou E, Patel VC, Weston CJ, Curbishley S, Sadiq F, Vergis N, Khamri W, et al. Patients with acute-on-chronic liver failure have increased numbers of regulatory immune cells expressing the receptor tyrosine kinase MERTK. Gastroenterology. 2015;148(3):603–e61514.
Pu S, Niu S, Zhang C, Xu X, Qin M, Huang S, Zhang L. Epidemiology, antifungal susceptibilities, and risk factors for invasive candidiasis from 2011 to 2013 in a teaching hospital in southwest China. J Microbiol Immunol Infect. 2017;50(1):97–103.
Moreno Elola-Olaso A, Davenport DL, Hundley JC, Daily MF, Gedaly R. Predictors of surgical site infection after liver resection: a multicentre analysis using National Surgical Quality Improvement Program data. HPB (Oxford). 2012;14(2):136–41.
Li F, Yuan MZ, Wang L, Wang XF, Liu GW. Characteristics and prognosis of pulmonary infection in patients with neurologic disease and hypoproteinemia. Expert Rev Anti Infect Ther. 2015;13(4):521–6.
Luo H, Chen H, Daloze P, Chang JY, St-Louis G, Wu J. Inhibition of in vitro immunoglobulin production by rapamycin. Transplantation. 1992;53(5):1071–6.
Sester M, Gärtner BC, Girndt M, Sester U. Vaccination of the solid organ transplant recipient. Transpl Rev (Orlando). 2008;22(4):274–84.
Severson JJ, Richards KR, Moran JJ, Hayney MS. Persistence of influenza vaccine-induced antibody in lung transplant patients and healthy individuals beyond the season. Hum Vaccin Immunother. 2012;8(12):1850–3.
Sánchez-Fueyo A, Strom TB. Immunologic basis of graft rejection and tolerance following transplantation of liver or other solid organs. Gastroenterology. 2011;140(1):51–64.
Meng C, Belino C, Pereira L, Pinho A, Sampaio S, Tavares I, Bustorff M, Sarmento A, Pestana M. Reactivation of Hepatitis B virus in kidney transplant recipients with previous clinically resolved infection: A single-center experience. Nefrologia (Engl Ed). 2018;38(5):545–50.

Download PDF

Version 1

posted

You are reading this latest preprint version

Incidence and risk factors of de novo Hepatitis E virus infection after receiving liver transplantation

Status:

Version 1

Abstract

Background/Aims:

Methods

Results

Conclusions

Figures

Lay Summary

1. Introduction

2. Methods

2.1 Study Subjects

2.2 Case Definitions

2.3 Study Design

2.4 HEV serological test and HEV molecular test

2.5 Statistical Analyses

2.6 Ethics

3. Results

3.1 Patient Characteristics

3.2 HEV Seroprevalence at transplantation

3.3 Post-transplant HEV Seroprevalence

3.4 Risk factors of de novo HEV infection after liver transplantation

3.5 Post-transplant serum-negative conversion

3.6 Associated factors of serum-negative conversion after liver transplantation

4. Discussion

Abbreviations

Declarations

References

Status:

Version 1