Clinical Note
Prevalence of hepatitis E virus in Chinese blood donors
Journal of International Medical Research 2015, Vol. 43(2) 257–262 ! The Author(s) 2015 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0300060514562054 imr.sagepub.com
Li Ma1, Pan Sun1, Fangzhao Lin1, Hongjie Wang2, Xia Rong3, Yudong Dai4, Jianqiang Liu5, Liqiong Qian6, Min Fang7, Na Su1, Wei Xiao1, Shengliang Ye1 and Changqing Li1
Abstract Objective: To investigate the prevalence of hepatitis E virus (HEV) antibodies and RNA in voluntary blood donors, in China. Methods: Serum samples from blood donors were collected sequentially at six blood donation centres in five geographical regions of China. Donors were stratified between two groups according to their serum alanine aminotransferase (ALT) concentration (40 U/l and >40 U/l. Enzyme-linked immunosorbent assay and reverse transcription–polymerase chain reaction were used to quantify anti-HEV antibodies and RNA, respectively. Results: In total, 21.1% (172/816) of donors were positive for anti-HEV immunoglobulin (Ig) G (elevated ALT samples group 18.9% [85/450]; normal ALT group 23.8% [87/366]), and 0.5% (four of 816) donors were positive for anti-HEV IgM (elevated ALT group 0.9% [four of 450]; normal ALT group 0% [none of 366]). The rate of anti-HEV IgG seropositivity was significantly higher in male than in female donors. No serum sample tested positive for HEV RNA. Conclusion: The prevalence of anti-HEV antibodies in blood donors in China is high, regardless of ALT concentration.
1 Department of Blood Biochemistry, Institute of Blood Transfusion, Chinese Academy of Medical Sciences, Chengdu, China 2 Department of Blood Components, Blood Centre of the Beijing Red Cross, Beijing, China 3 Department of Blood Components, Guangzhou Blood Centre, Guangzhou, China 4 Department of Blood Components, Blood Centre of the Nanjing Red Cross, Nanjing, China 5 Department of Blood Components, Shanxi Blood Centre, Xian, China
6
Department of Blood Components, Deyang Central Blood Station, Deyang, China 7 Department of Blood Components, Suzhou Central Blood Station, Suzhou, China Corresponding author: Fangzhao Lin, Institute of Blood Transfusion, Chinese Academy of Medical Sciences, Hua Cai Road 26 Hao, Dong San Huan Road Er Duan, Chengdu, China. Email: [email protected]
Creative Commons CC-BY-NC: This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 3.0 License (http://www.creativecommons.org/licenses/by-nc/3.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the originalDevelopment work is attributed as specified on the15, SAGE Downloaded from imr.sagepub.com at Inter-American Bank (KNL/FHL) on November 2015 and Open Access page (http://www.uk.sagepub.com/aboutus/openaccess.htm).
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Keywords Hepatitis E virus, anti-HEV antibodies, alanine aminotransferase, blood donors Date received: 23 May 2014; accepted: 7 November 2014
Introduction Hepatitis E is an important public health problem that is caused by hepatitis E virus (HEV), a member of the Hepeviridae family. Epidemiological data suggest that HEV may pose a threat to the safe supply of blood for transfusions.1–4 In China, the mandatory screening test panel for voluntary blood donors includes quantification of alanine aminotransferase (ALT),5 and elevated ALT is the main reason for discarding donated blood. However, ALT concentrations can be affected by factors other than liver disease (including drug or alcohol use, inadequate sleep and being overweight),6 with the result that blood donations may be discarded unnecessarily. Many countries either do not have compulsory ALT testing for blood donors or use a high ALT cut-off value.7,8 The aim of the current study was to investigate the prevalence of HEV antibodies and RNA in blood donors with elevated or normal ALT concentrations, and to determine whether there is a relationship between ALT concentration and HEV infection.
Subjects and methods Study population Samples for this study were collected sequentially for this study, which took place between January and December 2012. All potential blood donors were required to pass a routine predonation screening process, including health-history questionnaire screening, predonation rapid testing (for screening hepatitis B virus surface antigen, ABO blood type, hemoglobin, ALT, HIV, and Treponema pallidum), and a brief physical examination. After a donor passed the
predonation screening phase, a unit of blood was collected. In addition, 5 ml of venous blood was collected in two vacuum tubes containing ethylenediaminetetra-acetate-K2 (with separator gel). Plasma was separated from red-blood cells by centrifugation (at 4 C, 2500 g for 15 min). Samples were then divided between three tubes, frozen at 30 C and shipped on dry ice to the Institute of Blood Transfusion, Chinese Academy of Medical Sciences, Chengdu, China. Donor samples came from six blood donation centres in five regions of China: Blood Centre of the Nanjing Red Cross and Suzhou Central Blood Station (both located in East China); Beijing blood Centre(located in South China); Blood Centre of the Beijing Red Cross (located in North China); Shanxi Blood Centre (Northwest China); Deyang Central Blood Station (located in Southwest China). Exclusion criteria were positive tests for hepatitis B surface antigen, hepatitis C virus (HCV) antibodies, Treponema pallidum or HIV. The study protocol was approved by the ethics committee of the Institute of Blood Transfusion, Chinese Academy of Medical Sciences, Chengdu, China, and all donors provided written informed consent.
ALT quantification The ALT level was quantified via the rate method:9 ALT concentrations 40 U/l were defined as normal; ALT concentrations >40 U/l were defined as elevated.
Anti-HEV serology Anti-HEV immunoglobulin (Ig)G and IgM antibodies were detected by enzyme-linked
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immunosorbent assay (ELISA) using HEV IgG and IgM ELISA kits (Wantai Pharmaceutical Co., Beijing, China), according to the manufacturer’s instructions. ELISAs were performed on an automated microplate reader (Molecular Devices LLC, Sunnyvale, CA, USA). According to the package insert, the assay had a specificity of 99.9% when testing 10587 healthy Asian subjects. It is to be noted that confirmatory assays for HEV IgM and IgG antibodies were not available.
HEV RNA detection All serum samples were tested for HEV RNA by reverse transcription quantitative polymerase chain reaction (RT–qPCR). Nucleic acid isolation was performed using a viral RNA extraction kit (Daan Gene Co., Guangzhou, China) according to the manufacturer’s instructions. Primer sequences for PCR were: forward 50 -ATCCCGCATG ACATCGAC-30 , reverse 50 -GGTCGGT CCTGCTCATGTT-30 , and probe FAM50 -CGGGGAATCTCGTGTAGTTATTC AG-30 -TAMRA. PCR cycling conditions were 40 cycles of denaturation at 94 C for 15 s, annealing at 55 C for 30 s, and extension at 72 C for 40 s. A positive HEV RNA control (1000 copies/ml) and negative HEV RNA control were included in each assay. Amplification, data acquisition and analysis were performed on an ABI StepOne PlusTM Detection System (Life Technologies, Carlsbad, CA, USA).
Statistical analyses Data were presented as n (%) or mean SD. Between-group comparisons were made using one-way analysis of variance followed by Student’s t-test with Tukey’s correction. All statistical analyses were performed using SPSSÕ version 12.0 (SPSS Inc., Chicago, IL, USA) for WindowsÕ .
P-values < 0.05 were considered statistically significant.
Results The study included 450 blood donors with elevated ALT (396 male/54 female; mean age 30 8 years; age range 18–55 years; Guangzhou Blood Centre n ¼ 33; Xian Blood Centre n ¼ 100; Nanjing Blood Centre n ¼ 50; Suzhou Central Blood Station n ¼ 90; Deyang Central Blood Station n ¼ 127; Beijing Blood Centre n ¼ 50), and 366 blood donors with normal ALT concentrations (183 male/183 female; mean age 24 8 years; age range 19–53 years; Guangzhou Blood Centre n ¼ 20; Beijing Blood Centre n ¼ 20; Nanjing Blood Centre n ¼ 20; Xian Blood Centre n ¼ 30; Suzhou Central Blood Station n ¼ 90; and Deyang Central Blood Station n ¼ 186). All serum samples were negative for HEV RNA. Data regarding the prevalence of HEV IgG and IgM in donors stratified by ALT concentration are given in Table 1. In the total donor population, 21.1% (172/816) were positive for anti-HEV IgG and 0.5% (four of 816) were positive for anti-HEV IgM. A single donor was positive for both IgM and IgG anti-HEV antibodies. There was no significant difference in the prevalence of anti-HEV IgG between donors with normal ALT or those with elevated ALT (23.8% and 18.9%, respectively). In donors with elevated ALT, anti-HEV IgG seropositivity was significantly more common in those aged 36–45 years compared with other age groups (18–35 or 46–55 years; P < 0.05, Table 1). In contrast, in donors with normal ALT levels, anti-HEV IgG seropositivity was significantly more common with increasing age, being higher among older donors (aged 46–55 years) than those aged 18–35 or 36–45 years (P < 0.05, Table 1). In the elevated ALT group, antiHEV IgG seropositivity was significantly
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more common in male than in female donors (P < 0.05; Table 1).
3 (0.4) 1 (0.7) 0 (0.0)
3 (0.5) 1 (0.4)
4 (0.5)
Anti-HEV IgMþ
Journal of International Medical Research 43(2)
91 (14.2)b 68 (48.2) 13 (39.4) Data presented as n (%). a P < 0.05 versus female sex; one-way analysis of variance followed by Student’s t-test with Tukey’s correction. b P < 0.05 versus all other age groups; one-way analysis of variance followed by Student’s t-test with Tukey’s correction.
642 141 33 – – – 51 (17.2) 30 (49.2) 6 (66.7)b 296 61 9 40 (11.6) 38 (47.5)b 7 (29.2) 346 80 24
3 (0.9) 1 (1.3) 0 (0)
133 (23.0) 39 (16.5) 579 237 – – 50 (27.3) 37 (20.2) 183 183 3 (0.8) 1 (1.9) 83 (21.0)a 2 (3.7) 396 54
172 (21.1) 816 0 (0) 87 (23.8) 366 4 (0.9) 450
Total Sex Male Female Age range, years 18–35 36–45 46–55
Anti-HEV IgMþ Total Characteristic
85 (18.9)
Anti-HEV IgGþ Total Anti-HEV IgGþ Anti-HEV IgGþ
Total
ALT40 U/l n ¼ 366
Anti-HEV IgMþ
All donors n ¼ 816
Discussion
ALT >40 U/l n ¼ 450
Table 1. Prevalence of anti-hepatitis E virus (HEV) immunoglobulin (Ig)G and IgM in volunteer blood donors from Chinese blood centres, stratified according to serum alanine aminotransferase (ALT) concentration.
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Transfusion-transmitted HEV infections due to contaminated blood products have been reported in Europe (including the UK) and Japan.1–4,8 Since the majority of HEV infections are subclinical,10 the risk of asymptomatic infection in blood donors is a major concern. High rates of anti-HEV IgG seropositivity have been demonstrated among blood donors in many countries, with considerable variance between populations (China 0.07%;11 USA 18.8%;12 Germany 5.94–15.5%;13 Switzerland 4.1–5.4%;14 Brazil 2.3%;15 France 3.2–16.6%;16 Japan 3.4–6.6%;17 Scotland 4.7%).18 The rate of anti-HEV IgG seropositivity in the present study (21.1%) was considerably higher than the rates described above,11–18 irrespective of ALT concentrations. The anti-HEV IgM seropositivity rates in the present study were 0.9% and 0.0% for donors with and without elevated ALT, respectively. Others reported rates of 0.94–1.8% in China,11 0.5– 1.6% in Japan,17 and 0.5% (normal ALT) and 8.08% (elevated ALT) in Germany.13 These differences may be explained by demographic differences, and variations in ALT reference ranges. In China, the majority of voluntary blood donors are young men. The prevalence of anti-HEV IgG seropositivity was significantly higher in male than in female donors in the present study, but the reasons for this pattern of infection remain unclear and require further investigation. It may be due to more frequent exposure to potential sources of contamination among donors, perhaps due to greater activity in the labour market (such as involvement in high-risk work in livestock farming, cleaning services or the sewage disposal system) in those who were anti-HEV IgG seropositive.
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To obtain the true rate of HEV infection in Chinese blood donors we recruited subjects sequentially, with the result that the mean ALT concentration of samples in this study was not very high. In addition, HEV infection was asymptomatic and all donors were negative for HEV RNA. Since the main factors influencing ALT levels are nonpathological, a large number of healthy blood donors are unnecessarily excluded in China. This can result in serious economic loss and also undue psychological stress to these excluded donors. It would therefore be useful to redefine testing methods and ALT cut-off levels, such has been undertaken in other countries. China has a high risk of hepatitis, so redefining ALT cut-off values or cancelling ALT testing altogether should be considered only after in-depth studies. If ALT testing is no longer recommended, anti-HEV IgM and IgG testing should be initiated, instead. The current study had some limitations. We demonstrate that HEV circulates in five geographical regions of China, but data regarding HEV prevalence in other Chinese regions are lacking and merit exploration. Further studies are necessary to define the clinical and epidemiological importance of HEV infection in China, to identify sources and routes of transmission, and assess the possible risk of transfusion-transmitted HEV. Factors other than liver disease, including tobacco and alcohol use, can influence ALT concentrations. In China, these lifestyle choices are more common among men than women, and men account for the majority of blood donors. This compounds the risk of blood donations being discarded due to high ALT concentrations. A great deal of time was required to recruit the 54 female donors with elevated ALT in the present study, and it was not possible to sex match the two study groups. In conclusion, this cross-sectional study shows a high prevalence of anti-HEV
antibodies in blood donors in China, regardless of ALT concentration, and indicates that HEV is highly prevalent in this population. It remains unclear whether cancelling ALT screening or redefining ALT cut-off values might be required. We plan to undertake a large-scale study, in collaboration with Chinese Blood Centres, that includes other hepatitis viruses (hepatitis A and hepatitis G), to examine this issue further.
Declaration of conflicting interest The authors declare that there are no conflicts of interest.
Funding This work was supported by the research special fund for public welfare industry of health (Grant Nos. 200902008 and 201002005).
References 1. Boxall E, Herborn A, Kochethu G, et al. Transfusion-transmitted hepatitis E in a ‘nonhyperendemic’ country. Transfus Med 2006; 16: 79–83. 2. Colson P, Coze C, Gallian P, et al. Transfusion-associated hepatitis E, France. Emerg Infect Dis 2007; 13: 648–649. 3. Matsubayashi K, Kang JH, Sakata H, et al. A case of transfusion-transmitted hepatitis E caused by blood from a donor infected with hepatitis E virus via zoonotic food-borne route. Transfusion 2008; 48: 1368–1375. 4. Matsubayashi K, Nagaoka Y, Sakata H, et al. Transfusion-transmitted hepatitis E caused by apparently indigenous hepatitis E virus strain in Hokkaido, Japan. Transfusion 2004; 44: 934–940. 5. Ren FR, Wang JX, Huang Y, et al. Hepatitis B virus nucleic acid testing in Chinese blood donors with normal and elevated alanine aminotransferase. Transfusion 2011; 51: 2588–2595.
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6. Piton A, Poynard T, Imbert-Bismut F, et al. Factors associated with serum alanine transaminase activity in healthy subjects: consequences for the definition of normal values, for selection of blood donors, and for patients with chronic hepatitis C. Hepatology 1998; 27: 1213–1219. 7. Cable R, Badon S, Pray C, et al. Limited utility of alanine aminotransferase screening of hepatitis C antibody screened blood donors. Transfusion 1997; 37: 206–210. 8. Notari EP 4th, Orton SL, Cable RG, et al. Seroprevalence of known and putative hepatitis markers in United States blood donors with ALT levels at least 120 IU per L. Transfusion 2001; 41: 751–755. 9. Karmen A, Wroblewski F and Ladue JS. Transaminase activity in human blood. J Clin Invest 1955; 34: 126–133. 10. Mitsui T, Tsukamoto Y, Yamazaki C, et al. Prevalence of hepatitis E virus infection among hemodialysis patients in Japan: evidence for infection with a genotype 3 HEV by blood transfusion. J Med Virol 2004; 74: 563–572. 11. Guo QS, Yan Q, Xiong JH, et al. Prevalence of hepatitis E virus in Chinese blood donors. J Clin Microbiol 2010; 48: 317–318. 12. Xu C, Wang RY, Schechterly CA, et al. An assessment of hepatitis E virus (HEV) in US blood donors and recipients: no detectable
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HEV RNA in 1939 donors tested and no evidence for HEV transmission to 362 prospectively followed recipients. Transfusion 2013; 53: 2505–2511. Vollmer T, Diekmann J, Johne R, et al. Novel approach for detection of hepatitis E virus infection in German blood donors. J Clin Microbiol 2012; 50: 2708–2713. Kaufmann A, Kenfak-Foguena A, Andre´ C, et al. Hepatitis E virus seroprevalence among blood donors in southwest Switzerland. PLoS One 2011; 6: e21150. Bortoliero AL, Bonametti AM, Morimoto HK, et al. Seroprevalence for hepatitis E virus (HEV) infection among volunteer blood donors of the Regional Blood Bank of Londrina, State of Parana´, Brazil. Rev Inst Med Trop Sao Paulo 2006; 48: 87–92. Boutrouille A, Bakkali-Kassimi L, Crucie`re C, et al. Prevalence of anti-hepatitis E virus antibodies in French blood donors. J Clin Microbiol 2007; 45: 2009–2010. Fukuda S, Ishikawa M, Ochiai N, et al. Unchanged high prevalence of antibodies to hepatitis E virus (HEV) and HEV RNA among blood donors with an elevated alanine aminotransferase lever in Japan during 1991–2006. Arch Virol 2007; 152: 1623–1635. Cleland A, Smith L, Crossan C, et al. Hepatitis E virus in Scottish blood donors. Vox Sang 2013; 105: 283–289.
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Prevalence of hepatitis E virus in Chinese blood donors
Journal of International Medical Research 2015, Vol. 43(2) 257–262 ! The Author(s) 2015 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0300060514562054 imr.sagepub.com
Li Ma1, Pan Sun1, Fangzhao Lin1, Hongjie Wang2, Xia Rong3, Yudong Dai4, Jianqiang Liu5, Liqiong Qian6, Min Fang7, Na Su1, Wei Xiao1, Shengliang Ye1 and Changqing Li1
Abstract Objective: To investigate the prevalence of hepatitis E virus (HEV) antibodies and RNA in voluntary blood donors, in China. Methods: Serum samples from blood donors were collected sequentially at six blood donation centres in five geographical regions of China. Donors were stratified between two groups according to their serum alanine aminotransferase (ALT) concentration (40 U/l and >40 U/l. Enzyme-linked immunosorbent assay and reverse transcription–polymerase chain reaction were used to quantify anti-HEV antibodies and RNA, respectively. Results: In total, 21.1% (172/816) of donors were positive for anti-HEV immunoglobulin (Ig) G (elevated ALT samples group 18.9% [85/450]; normal ALT group 23.8% [87/366]), and 0.5% (four of 816) donors were positive for anti-HEV IgM (elevated ALT group 0.9% [four of 450]; normal ALT group 0% [none of 366]). The rate of anti-HEV IgG seropositivity was significantly higher in male than in female donors. No serum sample tested positive for HEV RNA. Conclusion: The prevalence of anti-HEV antibodies in blood donors in China is high, regardless of ALT concentration.
1 Department of Blood Biochemistry, Institute of Blood Transfusion, Chinese Academy of Medical Sciences, Chengdu, China 2 Department of Blood Components, Blood Centre of the Beijing Red Cross, Beijing, China 3 Department of Blood Components, Guangzhou Blood Centre, Guangzhou, China 4 Department of Blood Components, Blood Centre of the Nanjing Red Cross, Nanjing, China 5 Department of Blood Components, Shanxi Blood Centre, Xian, China
6
Department of Blood Components, Deyang Central Blood Station, Deyang, China 7 Department of Blood Components, Suzhou Central Blood Station, Suzhou, China Corresponding author: Fangzhao Lin, Institute of Blood Transfusion, Chinese Academy of Medical Sciences, Hua Cai Road 26 Hao, Dong San Huan Road Er Duan, Chengdu, China. Email: [email protected]
Creative Commons CC-BY-NC: This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 3.0 License (http://www.creativecommons.org/licenses/by-nc/3.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the originalDevelopment work is attributed as specified on the15, SAGE Downloaded from imr.sagepub.com at Inter-American Bank (KNL/FHL) on November 2015 and Open Access page (http://www.uk.sagepub.com/aboutus/openaccess.htm).
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Keywords Hepatitis E virus, anti-HEV antibodies, alanine aminotransferase, blood donors Date received: 23 May 2014; accepted: 7 November 2014
Introduction Hepatitis E is an important public health problem that is caused by hepatitis E virus (HEV), a member of the Hepeviridae family. Epidemiological data suggest that HEV may pose a threat to the safe supply of blood for transfusions.1–4 In China, the mandatory screening test panel for voluntary blood donors includes quantification of alanine aminotransferase (ALT),5 and elevated ALT is the main reason for discarding donated blood. However, ALT concentrations can be affected by factors other than liver disease (including drug or alcohol use, inadequate sleep and being overweight),6 with the result that blood donations may be discarded unnecessarily. Many countries either do not have compulsory ALT testing for blood donors or use a high ALT cut-off value.7,8 The aim of the current study was to investigate the prevalence of HEV antibodies and RNA in blood donors with elevated or normal ALT concentrations, and to determine whether there is a relationship between ALT concentration and HEV infection.
Subjects and methods Study population Samples for this study were collected sequentially for this study, which took place between January and December 2012. All potential blood donors were required to pass a routine predonation screening process, including health-history questionnaire screening, predonation rapid testing (for screening hepatitis B virus surface antigen, ABO blood type, hemoglobin, ALT, HIV, and Treponema pallidum), and a brief physical examination. After a donor passed the
predonation screening phase, a unit of blood was collected. In addition, 5 ml of venous blood was collected in two vacuum tubes containing ethylenediaminetetra-acetate-K2 (with separator gel). Plasma was separated from red-blood cells by centrifugation (at 4 C, 2500 g for 15 min). Samples were then divided between three tubes, frozen at 30 C and shipped on dry ice to the Institute of Blood Transfusion, Chinese Academy of Medical Sciences, Chengdu, China. Donor samples came from six blood donation centres in five regions of China: Blood Centre of the Nanjing Red Cross and Suzhou Central Blood Station (both located in East China); Beijing blood Centre(located in South China); Blood Centre of the Beijing Red Cross (located in North China); Shanxi Blood Centre (Northwest China); Deyang Central Blood Station (located in Southwest China). Exclusion criteria were positive tests for hepatitis B surface antigen, hepatitis C virus (HCV) antibodies, Treponema pallidum or HIV. The study protocol was approved by the ethics committee of the Institute of Blood Transfusion, Chinese Academy of Medical Sciences, Chengdu, China, and all donors provided written informed consent.
ALT quantification The ALT level was quantified via the rate method:9 ALT concentrations 40 U/l were defined as normal; ALT concentrations >40 U/l were defined as elevated.
Anti-HEV serology Anti-HEV immunoglobulin (Ig)G and IgM antibodies were detected by enzyme-linked
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immunosorbent assay (ELISA) using HEV IgG and IgM ELISA kits (Wantai Pharmaceutical Co., Beijing, China), according to the manufacturer’s instructions. ELISAs were performed on an automated microplate reader (Molecular Devices LLC, Sunnyvale, CA, USA). According to the package insert, the assay had a specificity of 99.9% when testing 10587 healthy Asian subjects. It is to be noted that confirmatory assays for HEV IgM and IgG antibodies were not available.
HEV RNA detection All serum samples were tested for HEV RNA by reverse transcription quantitative polymerase chain reaction (RT–qPCR). Nucleic acid isolation was performed using a viral RNA extraction kit (Daan Gene Co., Guangzhou, China) according to the manufacturer’s instructions. Primer sequences for PCR were: forward 50 -ATCCCGCATG ACATCGAC-30 , reverse 50 -GGTCGGT CCTGCTCATGTT-30 , and probe FAM50 -CGGGGAATCTCGTGTAGTTATTC AG-30 -TAMRA. PCR cycling conditions were 40 cycles of denaturation at 94 C for 15 s, annealing at 55 C for 30 s, and extension at 72 C for 40 s. A positive HEV RNA control (1000 copies/ml) and negative HEV RNA control were included in each assay. Amplification, data acquisition and analysis were performed on an ABI StepOne PlusTM Detection System (Life Technologies, Carlsbad, CA, USA).
Statistical analyses Data were presented as n (%) or mean SD. Between-group comparisons were made using one-way analysis of variance followed by Student’s t-test with Tukey’s correction. All statistical analyses were performed using SPSSÕ version 12.0 (SPSS Inc., Chicago, IL, USA) for WindowsÕ .
P-values < 0.05 were considered statistically significant.
Results The study included 450 blood donors with elevated ALT (396 male/54 female; mean age 30 8 years; age range 18–55 years; Guangzhou Blood Centre n ¼ 33; Xian Blood Centre n ¼ 100; Nanjing Blood Centre n ¼ 50; Suzhou Central Blood Station n ¼ 90; Deyang Central Blood Station n ¼ 127; Beijing Blood Centre n ¼ 50), and 366 blood donors with normal ALT concentrations (183 male/183 female; mean age 24 8 years; age range 19–53 years; Guangzhou Blood Centre n ¼ 20; Beijing Blood Centre n ¼ 20; Nanjing Blood Centre n ¼ 20; Xian Blood Centre n ¼ 30; Suzhou Central Blood Station n ¼ 90; and Deyang Central Blood Station n ¼ 186). All serum samples were negative for HEV RNA. Data regarding the prevalence of HEV IgG and IgM in donors stratified by ALT concentration are given in Table 1. In the total donor population, 21.1% (172/816) were positive for anti-HEV IgG and 0.5% (four of 816) were positive for anti-HEV IgM. A single donor was positive for both IgM and IgG anti-HEV antibodies. There was no significant difference in the prevalence of anti-HEV IgG between donors with normal ALT or those with elevated ALT (23.8% and 18.9%, respectively). In donors with elevated ALT, anti-HEV IgG seropositivity was significantly more common in those aged 36–45 years compared with other age groups (18–35 or 46–55 years; P < 0.05, Table 1). In contrast, in donors with normal ALT levels, anti-HEV IgG seropositivity was significantly more common with increasing age, being higher among older donors (aged 46–55 years) than those aged 18–35 or 36–45 years (P < 0.05, Table 1). In the elevated ALT group, antiHEV IgG seropositivity was significantly
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more common in male than in female donors (P < 0.05; Table 1).
3 (0.4) 1 (0.7) 0 (0.0)
3 (0.5) 1 (0.4)
4 (0.5)
Anti-HEV IgMþ
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91 (14.2)b 68 (48.2) 13 (39.4) Data presented as n (%). a P < 0.05 versus female sex; one-way analysis of variance followed by Student’s t-test with Tukey’s correction. b P < 0.05 versus all other age groups; one-way analysis of variance followed by Student’s t-test with Tukey’s correction.
642 141 33 – – – 51 (17.2) 30 (49.2) 6 (66.7)b 296 61 9 40 (11.6) 38 (47.5)b 7 (29.2) 346 80 24
3 (0.9) 1 (1.3) 0 (0)
133 (23.0) 39 (16.5) 579 237 – – 50 (27.3) 37 (20.2) 183 183 3 (0.8) 1 (1.9) 83 (21.0)a 2 (3.7) 396 54
172 (21.1) 816 0 (0) 87 (23.8) 366 4 (0.9) 450
Total Sex Male Female Age range, years 18–35 36–45 46–55
Anti-HEV IgMþ Total Characteristic
85 (18.9)
Anti-HEV IgGþ Total Anti-HEV IgGþ Anti-HEV IgGþ
Total
ALT40 U/l n ¼ 366
Anti-HEV IgMþ
All donors n ¼ 816
Discussion
ALT >40 U/l n ¼ 450
Table 1. Prevalence of anti-hepatitis E virus (HEV) immunoglobulin (Ig)G and IgM in volunteer blood donors from Chinese blood centres, stratified according to serum alanine aminotransferase (ALT) concentration.
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Transfusion-transmitted HEV infections due to contaminated blood products have been reported in Europe (including the UK) and Japan.1–4,8 Since the majority of HEV infections are subclinical,10 the risk of asymptomatic infection in blood donors is a major concern. High rates of anti-HEV IgG seropositivity have been demonstrated among blood donors in many countries, with considerable variance between populations (China 0.07%;11 USA 18.8%;12 Germany 5.94–15.5%;13 Switzerland 4.1–5.4%;14 Brazil 2.3%;15 France 3.2–16.6%;16 Japan 3.4–6.6%;17 Scotland 4.7%).18 The rate of anti-HEV IgG seropositivity in the present study (21.1%) was considerably higher than the rates described above,11–18 irrespective of ALT concentrations. The anti-HEV IgM seropositivity rates in the present study were 0.9% and 0.0% for donors with and without elevated ALT, respectively. Others reported rates of 0.94–1.8% in China,11 0.5– 1.6% in Japan,17 and 0.5% (normal ALT) and 8.08% (elevated ALT) in Germany.13 These differences may be explained by demographic differences, and variations in ALT reference ranges. In China, the majority of voluntary blood donors are young men. The prevalence of anti-HEV IgG seropositivity was significantly higher in male than in female donors in the present study, but the reasons for this pattern of infection remain unclear and require further investigation. It may be due to more frequent exposure to potential sources of contamination among donors, perhaps due to greater activity in the labour market (such as involvement in high-risk work in livestock farming, cleaning services or the sewage disposal system) in those who were anti-HEV IgG seropositive.
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To obtain the true rate of HEV infection in Chinese blood donors we recruited subjects sequentially, with the result that the mean ALT concentration of samples in this study was not very high. In addition, HEV infection was asymptomatic and all donors were negative for HEV RNA. Since the main factors influencing ALT levels are nonpathological, a large number of healthy blood donors are unnecessarily excluded in China. This can result in serious economic loss and also undue psychological stress to these excluded donors. It would therefore be useful to redefine testing methods and ALT cut-off levels, such has been undertaken in other countries. China has a high risk of hepatitis, so redefining ALT cut-off values or cancelling ALT testing altogether should be considered only after in-depth studies. If ALT testing is no longer recommended, anti-HEV IgM and IgG testing should be initiated, instead. The current study had some limitations. We demonstrate that HEV circulates in five geographical regions of China, but data regarding HEV prevalence in other Chinese regions are lacking and merit exploration. Further studies are necessary to define the clinical and epidemiological importance of HEV infection in China, to identify sources and routes of transmission, and assess the possible risk of transfusion-transmitted HEV. Factors other than liver disease, including tobacco and alcohol use, can influence ALT concentrations. In China, these lifestyle choices are more common among men than women, and men account for the majority of blood donors. This compounds the risk of blood donations being discarded due to high ALT concentrations. A great deal of time was required to recruit the 54 female donors with elevated ALT in the present study, and it was not possible to sex match the two study groups. In conclusion, this cross-sectional study shows a high prevalence of anti-HEV
antibodies in blood donors in China, regardless of ALT concentration, and indicates that HEV is highly prevalent in this population. It remains unclear whether cancelling ALT screening or redefining ALT cut-off values might be required. We plan to undertake a large-scale study, in collaboration with Chinese Blood Centres, that includes other hepatitis viruses (hepatitis A and hepatitis G), to examine this issue further.
Declaration of conflicting interest The authors declare that there are no conflicts of interest.
Funding This work was supported by the research special fund for public welfare industry of health (Grant Nos. 200902008 and 201002005).
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