D O N O R - R E L AT E D I N F E C T I O N R I S K Hepatitis E virus seroprevalence and molecular study among blood donors in China Furong Ren,1 Chenyan Zhao,2,3 Ling Wang,4 Zhuoyan Wang,1 Xiaoyan Gong,1 Meilan Song,1 Hui Zhuang,4 Yi Huang,5 Hua Shan,6 Jingxing Wang,5 Qiang Liu,2 Paul Ness,6 Kenrad E. Nelson,7 and Youchun Wang2
BACKGROUND: The risk of hepatitis E virus (HEV) infection from blood transfusion has aroused increasing concern in many countries. The aim of this study was to analyze the potential risk of HEV infection through blood transfusion in China. STUDY DESIGN AND METHODS: Qualified blood donations and donations with isolated alanine aminotransferase (ALT) elevations from five geographically diverse Chinese regions were tested for anti-HEV immunoglobulin (Ig)M and IgG and HEV antigen. The positive samples for anti-HEV IgM and HEV antigen were tested for HEV RNA. HEV open reading frame (ORF)2 partial sequences were analyzed from HEV RNA–positive samples. RESULTS: The seroprevalence rates of HEV antigen and anti-HEV IgM and IgG among qualified donations were 0.06% (6/10,741), 1.02% (109/10,741), and 27.42% (2945/10,741), respectively. Samples with isolated ALT elevations had higher prevalence of HEV markers, namely, HEV antigen of 0.25% (2/797), antiHEV IgM of 2.76% (22/797), and anti-HEV IgG of 40.02% (319/797). The HEV antibody prevalence varied significantly by age, sex, and geographic region. All 131 samples that were anti-HEV IgM positive were negative for HEV RNA, whereas four of eight (50%) samples positive for HEV antigen were HEV RNA positive. HEV ORF2 sequences from three of four HEV RNA– positive samples were determined and grouped with Genotype 4. CONCLUSION: Qualified donations after routine blood donor screening still carry potential risk for transmitting HEV. HEV antigen screening could be one measure to reduce the risk of HEV transmission by blood transfusion.
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TRANSFUSION Volume 54, March 2014
A
s the causative agent of hepatitis E, hepatitis E virus (HEV) is a nonenveloped RNA virus with a positive sense single-stranded genome of approximately 7.2 kb containing three open reading frames (ORF1, 2, and 3).1 Circulating HEV strains among humans are divided into four genotypes and several subgenotypes.2,3 Genotypes 1 and 2 infect only
ABBREVIATIONS: HEV = hepatitis E virus; ORF = open reading frame. From the 1Beijing Red Cross Blood Center, Beijing, China; the 2 Division of HIV and Sexual Transmitted Virus Vaccine, National Institutes for Food and Drug Control, Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products, Beijing, China; the 3College of Life Science, Jilin University, Changchun, China; the 4Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China; the 5Institute of Blood Transfusion, Chinese Academy of Medical Sciences, Chengdu, China; and the 6 Division of Blood Transfusion, HIV Specialty Testing Laboratory, Department of Pathology, Johns Hopkins Medical Institutions, and the 7Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland. Address reprint requests to: Youchun Wang, MD, PhD, Division of HIV and Sexual Transmitted Virus Vaccine, National Institutes for Food and Drug Control, Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products, Beijing 100050, China; e-mail: [email protected]. This study was supported by 863 projects from National Science and Technology (No. 2006AA02Z453). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Received for publication August 11, 2013; revision received November 2, 2013, and accepted November 3, 2013. doi: 10.1111/trf.12530 © 2013 AABB TRANSFUSION 2014;54:910-917.
HEV AMONG BLOOD DONORS IN CHINA
humans or nonhuman primates and have been associated with large epidemics of hepatitis E. In contrast, Genotypes 3 and 4 have a zoonotic reservoir in pigs and other animals and cause sporadic infections in humans.4 Recently, the prevalence of anti-HEV immunoglobulin (Ig)M has been studied among blood donors in several countries, including Japan, the United States, Denmark, southwest England, southwest France, southwest Switzerland, and China. The HEV seroprevalence in these studies ranged from 3.4% to 52.5%.5-11 In addition to these seroprevalence studies, several transfusion-transmitted cases have been described.12-14 According to the annual reports by National Health and Family Planning Commission of the People’s Republic of China, the number of new reported cases of clinical hepatitis E has been increasing from 16,444 cases in 2004 to 27,271 cases in 2012 (http://www.chinacdc.cn/ tjsj/fdcrbbg/200506/t20050614_25233.htm, http://www .china.com.cn/guoqing/2013-01/16/content_27700969 .htm). Epidemiologic studies from China indicate that the seroprevalence rates of anti-HEV and HEV RNA in the general population as well as special populations including blood donors, pig farmers, and slaughtermen are higher compared with developed countries.15-17 Additionally, HEV infection is widespread in domestic animals, particularly pigs in China, which increases the risk of human infection.18 China is an endemic area for hepatitis E. However, data on the HEV seroprevalence and RNA prevalence among blood donors are limited.19,20 The aim of this study was to investigate the prevalence of HEV antibodies, antigen, and RNA among qualified blood donors and those who were deferred because they had isolated elevations of alanine aminotransferase (ALT) from blood collection centers in five cities in China. In addition, we analyzed partial nucleotide sequences from the ORF2 region of several HEV isolates.
MATERIALS AND METHODS Blood donor samples Donor samples were obtained from six blood centers in five geographically diverse cities in China in 2005.19 All donor samples were tested for ALT, hepatitis B surface antigen (HBsAg), anti-hepatitis C virus (anti-HCV), antihuman immunodeficiency virus (anti-HIV) type 1 and 2, and syphilis during routine donor screening. Our study included 10,741 samples that were negative for all routinely screened markers and 797 samples from donations that were deferred due to isolated elevated ALT (≥40 IU/L by the speed rate method). Whole blood samples were collected in two ethylenediaminetetraacetate-K2 (with separator gel) vacuum tubes (Greiner, Kremsmünster, Austria) at the blood collection sites. Plasma was sepa-
rated from the red blood cells by centrifugation. Samples were then frozen and shipped on dry ice to the Beijing Red Cross Blood Center, where they were stored at −80°C.
Detection of anti-HEV and HEV antigen Serum samples were tested for anti-HEV IgM and IgG antibodies using commercial enzyme-linked immunosorbent assay (ELISA) kits (Wantai Biopharmaceutical, Beijing, China). HEV antigen in the samples was detected by a monoclonal antibody-based ELISA kit (Wantai Biopharmaceutical Co., Beijing, China). All assay procedures were carried out according to the manufacturer’s instructions. Fifty microliters of each sample was used for antibody detection, and 100 μL, for antigen detection.21 The mean OD value of negative controls plus 0.26, plus 0.16, and plus 0.12 were used as the cutoff values for antiHEV IgM, IgG, and HEV antigen, respectively. The limitation of detection for HEV antigen assay is 0.05 to 0.10 ng/mL recombinant ORF2 polypeptide as manufacture described.
Detection of HEV RNA by real-time fluorescence reverse transcription polymerase chain reaction Detection of HEV RNA was performed using a real-time fluorescent reverse transcription polymerase chain reaction (RT-PCR) diagnostic kit after extracting nucleic acid from 200 μL of each plasma sample and eluting RNA from a spin column in 50 μL of elution buffer and amplifying RNA using RT-PCR (Kinghawk Pharmaceutical Co., Beijing, China) in a 50-μL reaction under the following conditions: 10 μL of RNA, 1 × RT-PCR buffer including MgCl2, dNTPs, primers, and probe as described previously.22 The RT-PCR mixture was incubated as follows: 50°C for 30 minutes, one cycle of denaturation at 95°C for 3 minutes, five preliminary amplification cycles of 95°C for 10 seconds, 50°C for 20 seconds, 72°C for 30 seconds, 40 cycles of 95°C for 10 seconds, and 55°C for 40 seconds utilizing a sequence detection system (ABI Prism 7000, Applied Biosystems, Foster City, CA). One negative and two positive (weak and high) controls were used for detecting RNA with each run and the results should meet the requirements. The results with the Ct values less than 32.0 were reported positive for HEV RNA. Its sensitivity was approximately 103 copies/mL22 and was validated by participating in the WHO international standard to harmonize assays for detection of HEV RNA.23
Detection of HEV RNA by nested RT-PCR Nested RT-PCR was carried out on plasma samples to identify HEV sequences using degenerate oligonucleotide primers that corresponded to a region of ORF2, as described previously.24 The external primer pair was 5′-CCCTTATCCTGCTGAGCATTCTC-3′ and 5′-AAYTAT GCWCAGTACCGGGTTG-3′; the internal primer pair was Volume 54, March 2014 TRANSFUSION
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REN ET AL.
5′-GTYATGYTYTGCATACATGGCT-3′ and 5′-AGCCGACG AAATYAATTCTGTC-3′. Nested RT-PCR was carried out in 50-μL reactions mixtures with 7 μL of RNA, 20 pmol of each primer, 5 U of AMV reverse transcriptase, and 5 U of Taq polymerase. The RT and amplification were performed in the same tube. The RT was performed at 50°C for 30 minutes, followed by 35 cycles of amplification at 94°C for 1 minute, 50°C for 45 seconds, and 72°C for 1 minute, with a final extension of 72°C for 10 minutes. Second-round reactions were carried out in a volume of 50 μL with 2 μL of first-round product, 20 pmol of each primer, and 5 U of Taq polymerase. The amplification variables were the same as for the first round. A final amplification product of 345 bp was then analyzed by electrophoresis through 1.0% agarose gel.
DNA sequencing and sequence analyses After purification using a column kit (Quick Spin, Qiagen, Hilden, Germany), the PCR products were sequenced directly on both strands with the specific PCR primers synthesized by Invitrogen (Beijing, China). An DNA sequencer by Sanger’s dideoxy terminator method (ABI Model 3730, Applied Biosystems) and an ABI sequencing ready reaction kit (Perkin Elmer, Downers Grove, IL) were used for sequencing in this study according to the manufacturer’s instructions. Sequences were analyzed using computer software (MEGA4.0, http:// www.megasoftware.net/). The HEV reference sequences used in the phylogenetic and sequence analyses were as follows: Genotype 1, AKL-90 (AF124407), Chi (AF141652) and Moro (AF065061); Genotype 2, M1 (M74506); Genotype 3, 01-9913 (AF466676), HE-JA5 (AB082561), and NLSW20 (AF336290); Subtype 4a, Ch87 (AJ344171), HF-030 (AF134916), Ch266 (AJ344193), and Ch-T21 (AF151962); Subtype 4b, Ch181 (AJ344188), Ch254 (AJ344186), Ch120 (AJ344192), and LZ105 (AF103940); Subtype 4c, HE-JA1 (AB097812), HE-JK4 (AB099347), HE-JI4 (AB080575), JYWSap02 (AB161719), and JSMSap95 (AB161717); Subtype 4d, T1 (AJ272108), swCH25 (AY594199), Ch108 (AJ344181), and Ch202 (AJ344184); Subtype 4e, IND-SW1 (AF324501) and IND-SW2 (AF324502); Subtype 4f, HE-JA2 (AB082558); and Subtype 4g, CCC220 (AB108537). Sequence alignments were made using computer software (MEGA 4.0, Version 4.0) to generate a phylogenetic tree with 1000 bootstrap replicates. Genetic distances were calculated by using the Kimura two-parameter method. The three sequences isolated in this study have been deposited in GenBank and the accession numbers are KF957726, KF957727, and KF957728.
Statistical analysis Statistical analysis was performed by the chi-square test using computer software (SPSS 17.0, SPSS, Chicago, IL). 912
TRANSFUSION Volume 54, March 2014
The differences within the subgroups of qualified or ALT elevated alone by region, age, and sex were analyzed with Fisher’s exact test (HEV RNA and antigen) or chi-square test (IgM and IgG). The effect of age or sex on the geographic difference was analyzed by a multivariable analysis using logistic regression. A difference with a p value of less than 0.05 was considered significant.
RESULTS Prevalence of HEV antigen and anti-HEV IgM and IgG among qualified donors in five cities Among 10,741 qualified samples from blood donors in five cities (Urumqi, Beijing, Hangzhou, Guangzhou, Kunming), six (0.06%) were HEV antigen positive, 109 (1.02%) were anti-HEV IgM positive, and 2945 (27.4%) were anti-HEV IgG positive (Table 1). The highest prevalence of HEV antigen (0.20%) and anti-HEV IgM (1.36%) were found in the samples from Hangzhou. Samples from Guangzhou had the lowest prevalence of anti-HEV IgM (0.63%). The highest prevalence of anti-HEV IgG (38.35%) occurred in Kunming. The mean prevalence of anti-HEV IgG was higher in the northern cities (Urumqi and Beijing) with 33.26% than the southern cities (Hangzhou, Guangzhou, and Kunming) with 18.63% (799/4288; chi-square test, p < 0.01; Table 1). After controlling for age and sex, the significant difference was not found within the subgroup of qualified donation by region (logistic regression test, p = 0.12). If Beijing was a control, only Guangzhou had the significantly lower prevalence of anti-HEV IgM (logistic regression test, p = 0.04). As the prevalence of anti-HEV IgG was concerned, there were significant differences in various cities, compared with Beijing (logistic regression test, p < 0.01).
Prevalence of HEV antigen and anti-HEV IgM and IgG by age and sex Among 9719 qualified donations and 747 samples with isolated ALT elevation with age and sex information from the donors the anti-HEV IgG prevalence increased significantly with age and was higher among male qualified donors (chi-square test, p < 0.01). However, the prevalence of HEV antigen and anti-HEV IgM did not differ by age or sex (Tables 2 and 3)
Relationship between ALT and HEV serologic markers Donors with isolated ALT elevation had higher prevalence of both anti-HEV IgM and IgG than qualified donors. The prevalence of anti-HEV IgM was 2.76% in those with ALT elevation and 1.02% among qualified donors (p < 0.01) and the anti-HEV IgG was 40.02% in those with elevated ALT and 27.42% among qualified donors (p < 0.01;
HEV AMONG BLOOD DONORS IN CHINA
TABLE 1. Prevalence of HEV antigen and anti-HEV IgM and IgG by region in qualified donation and elevated ALT alone donation Group Region Qualified* Urumqi Beijing Hangzhou Guangzhou Kunming Total p value† ALT elevated alone Urumqi Beijing Hangzhou Guangzhou Kunming Total p value Total Urumqi Beijing Hangzhou Guangzhou Kunming Total p value
Number
Positive for antigen
Positive rate for antigen (%)
Positive rate for anti-HEV IgM
Positive for anti-HEV IgM (%)
Positive for anti-HEV IgG
Positive rate for anti-HEV IgG (%)
1,910 2,378 1,980 2,520 1,953 10,741
1 0 4 1 0 6
0.05 0.00 0.20 0.04 0.00 0.06 0.04
14 30 27 16 22 109
0.73 1.26 1.36 0.63 1.13 1.02 0.06
341 458 651 746 749 2,945
17.85 19.26 32.88 29.60 38.35 27.42
BACKGROUND: The risk of hepatitis E virus (HEV) infection from blood transfusion has aroused increasing concern in many countries. The aim of this study was to analyze the potential risk of HEV infection through blood transfusion in China. STUDY DESIGN AND METHODS: Qualified blood donations and donations with isolated alanine aminotransferase (ALT) elevations from five geographically diverse Chinese regions were tested for anti-HEV immunoglobulin (Ig)M and IgG and HEV antigen. The positive samples for anti-HEV IgM and HEV antigen were tested for HEV RNA. HEV open reading frame (ORF)2 partial sequences were analyzed from HEV RNA–positive samples. RESULTS: The seroprevalence rates of HEV antigen and anti-HEV IgM and IgG among qualified donations were 0.06% (6/10,741), 1.02% (109/10,741), and 27.42% (2945/10,741), respectively. Samples with isolated ALT elevations had higher prevalence of HEV markers, namely, HEV antigen of 0.25% (2/797), antiHEV IgM of 2.76% (22/797), and anti-HEV IgG of 40.02% (319/797). The HEV antibody prevalence varied significantly by age, sex, and geographic region. All 131 samples that were anti-HEV IgM positive were negative for HEV RNA, whereas four of eight (50%) samples positive for HEV antigen were HEV RNA positive. HEV ORF2 sequences from three of four HEV RNA– positive samples were determined and grouped with Genotype 4. CONCLUSION: Qualified donations after routine blood donor screening still carry potential risk for transmitting HEV. HEV antigen screening could be one measure to reduce the risk of HEV transmission by blood transfusion.
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TRANSFUSION Volume 54, March 2014
A
s the causative agent of hepatitis E, hepatitis E virus (HEV) is a nonenveloped RNA virus with a positive sense single-stranded genome of approximately 7.2 kb containing three open reading frames (ORF1, 2, and 3).1 Circulating HEV strains among humans are divided into four genotypes and several subgenotypes.2,3 Genotypes 1 and 2 infect only
ABBREVIATIONS: HEV = hepatitis E virus; ORF = open reading frame. From the 1Beijing Red Cross Blood Center, Beijing, China; the 2 Division of HIV and Sexual Transmitted Virus Vaccine, National Institutes for Food and Drug Control, Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products, Beijing, China; the 3College of Life Science, Jilin University, Changchun, China; the 4Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China; the 5Institute of Blood Transfusion, Chinese Academy of Medical Sciences, Chengdu, China; and the 6 Division of Blood Transfusion, HIV Specialty Testing Laboratory, Department of Pathology, Johns Hopkins Medical Institutions, and the 7Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland. Address reprint requests to: Youchun Wang, MD, PhD, Division of HIV and Sexual Transmitted Virus Vaccine, National Institutes for Food and Drug Control, Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products, Beijing 100050, China; e-mail: [email protected]. This study was supported by 863 projects from National Science and Technology (No. 2006AA02Z453). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Received for publication August 11, 2013; revision received November 2, 2013, and accepted November 3, 2013. doi: 10.1111/trf.12530 © 2013 AABB TRANSFUSION 2014;54:910-917.
HEV AMONG BLOOD DONORS IN CHINA
humans or nonhuman primates and have been associated with large epidemics of hepatitis E. In contrast, Genotypes 3 and 4 have a zoonotic reservoir in pigs and other animals and cause sporadic infections in humans.4 Recently, the prevalence of anti-HEV immunoglobulin (Ig)M has been studied among blood donors in several countries, including Japan, the United States, Denmark, southwest England, southwest France, southwest Switzerland, and China. The HEV seroprevalence in these studies ranged from 3.4% to 52.5%.5-11 In addition to these seroprevalence studies, several transfusion-transmitted cases have been described.12-14 According to the annual reports by National Health and Family Planning Commission of the People’s Republic of China, the number of new reported cases of clinical hepatitis E has been increasing from 16,444 cases in 2004 to 27,271 cases in 2012 (http://www.chinacdc.cn/ tjsj/fdcrbbg/200506/t20050614_25233.htm, http://www .china.com.cn/guoqing/2013-01/16/content_27700969 .htm). Epidemiologic studies from China indicate that the seroprevalence rates of anti-HEV and HEV RNA in the general population as well as special populations including blood donors, pig farmers, and slaughtermen are higher compared with developed countries.15-17 Additionally, HEV infection is widespread in domestic animals, particularly pigs in China, which increases the risk of human infection.18 China is an endemic area for hepatitis E. However, data on the HEV seroprevalence and RNA prevalence among blood donors are limited.19,20 The aim of this study was to investigate the prevalence of HEV antibodies, antigen, and RNA among qualified blood donors and those who were deferred because they had isolated elevations of alanine aminotransferase (ALT) from blood collection centers in five cities in China. In addition, we analyzed partial nucleotide sequences from the ORF2 region of several HEV isolates.
MATERIALS AND METHODS Blood donor samples Donor samples were obtained from six blood centers in five geographically diverse cities in China in 2005.19 All donor samples were tested for ALT, hepatitis B surface antigen (HBsAg), anti-hepatitis C virus (anti-HCV), antihuman immunodeficiency virus (anti-HIV) type 1 and 2, and syphilis during routine donor screening. Our study included 10,741 samples that were negative for all routinely screened markers and 797 samples from donations that were deferred due to isolated elevated ALT (≥40 IU/L by the speed rate method). Whole blood samples were collected in two ethylenediaminetetraacetate-K2 (with separator gel) vacuum tubes (Greiner, Kremsmünster, Austria) at the blood collection sites. Plasma was sepa-
rated from the red blood cells by centrifugation. Samples were then frozen and shipped on dry ice to the Beijing Red Cross Blood Center, where they were stored at −80°C.
Detection of anti-HEV and HEV antigen Serum samples were tested for anti-HEV IgM and IgG antibodies using commercial enzyme-linked immunosorbent assay (ELISA) kits (Wantai Biopharmaceutical, Beijing, China). HEV antigen in the samples was detected by a monoclonal antibody-based ELISA kit (Wantai Biopharmaceutical Co., Beijing, China). All assay procedures were carried out according to the manufacturer’s instructions. Fifty microliters of each sample was used for antibody detection, and 100 μL, for antigen detection.21 The mean OD value of negative controls plus 0.26, plus 0.16, and plus 0.12 were used as the cutoff values for antiHEV IgM, IgG, and HEV antigen, respectively. The limitation of detection for HEV antigen assay is 0.05 to 0.10 ng/mL recombinant ORF2 polypeptide as manufacture described.
Detection of HEV RNA by real-time fluorescence reverse transcription polymerase chain reaction Detection of HEV RNA was performed using a real-time fluorescent reverse transcription polymerase chain reaction (RT-PCR) diagnostic kit after extracting nucleic acid from 200 μL of each plasma sample and eluting RNA from a spin column in 50 μL of elution buffer and amplifying RNA using RT-PCR (Kinghawk Pharmaceutical Co., Beijing, China) in a 50-μL reaction under the following conditions: 10 μL of RNA, 1 × RT-PCR buffer including MgCl2, dNTPs, primers, and probe as described previously.22 The RT-PCR mixture was incubated as follows: 50°C for 30 minutes, one cycle of denaturation at 95°C for 3 minutes, five preliminary amplification cycles of 95°C for 10 seconds, 50°C for 20 seconds, 72°C for 30 seconds, 40 cycles of 95°C for 10 seconds, and 55°C for 40 seconds utilizing a sequence detection system (ABI Prism 7000, Applied Biosystems, Foster City, CA). One negative and two positive (weak and high) controls were used for detecting RNA with each run and the results should meet the requirements. The results with the Ct values less than 32.0 were reported positive for HEV RNA. Its sensitivity was approximately 103 copies/mL22 and was validated by participating in the WHO international standard to harmonize assays for detection of HEV RNA.23
Detection of HEV RNA by nested RT-PCR Nested RT-PCR was carried out on plasma samples to identify HEV sequences using degenerate oligonucleotide primers that corresponded to a region of ORF2, as described previously.24 The external primer pair was 5′-CCCTTATCCTGCTGAGCATTCTC-3′ and 5′-AAYTAT GCWCAGTACCGGGTTG-3′; the internal primer pair was Volume 54, March 2014 TRANSFUSION
911
REN ET AL.
5′-GTYATGYTYTGCATACATGGCT-3′ and 5′-AGCCGACG AAATYAATTCTGTC-3′. Nested RT-PCR was carried out in 50-μL reactions mixtures with 7 μL of RNA, 20 pmol of each primer, 5 U of AMV reverse transcriptase, and 5 U of Taq polymerase. The RT and amplification were performed in the same tube. The RT was performed at 50°C for 30 minutes, followed by 35 cycles of amplification at 94°C for 1 minute, 50°C for 45 seconds, and 72°C for 1 minute, with a final extension of 72°C for 10 minutes. Second-round reactions were carried out in a volume of 50 μL with 2 μL of first-round product, 20 pmol of each primer, and 5 U of Taq polymerase. The amplification variables were the same as for the first round. A final amplification product of 345 bp was then analyzed by electrophoresis through 1.0% agarose gel.
DNA sequencing and sequence analyses After purification using a column kit (Quick Spin, Qiagen, Hilden, Germany), the PCR products were sequenced directly on both strands with the specific PCR primers synthesized by Invitrogen (Beijing, China). An DNA sequencer by Sanger’s dideoxy terminator method (ABI Model 3730, Applied Biosystems) and an ABI sequencing ready reaction kit (Perkin Elmer, Downers Grove, IL) were used for sequencing in this study according to the manufacturer’s instructions. Sequences were analyzed using computer software (MEGA4.0, http:// www.megasoftware.net/). The HEV reference sequences used in the phylogenetic and sequence analyses were as follows: Genotype 1, AKL-90 (AF124407), Chi (AF141652) and Moro (AF065061); Genotype 2, M1 (M74506); Genotype 3, 01-9913 (AF466676), HE-JA5 (AB082561), and NLSW20 (AF336290); Subtype 4a, Ch87 (AJ344171), HF-030 (AF134916), Ch266 (AJ344193), and Ch-T21 (AF151962); Subtype 4b, Ch181 (AJ344188), Ch254 (AJ344186), Ch120 (AJ344192), and LZ105 (AF103940); Subtype 4c, HE-JA1 (AB097812), HE-JK4 (AB099347), HE-JI4 (AB080575), JYWSap02 (AB161719), and JSMSap95 (AB161717); Subtype 4d, T1 (AJ272108), swCH25 (AY594199), Ch108 (AJ344181), and Ch202 (AJ344184); Subtype 4e, IND-SW1 (AF324501) and IND-SW2 (AF324502); Subtype 4f, HE-JA2 (AB082558); and Subtype 4g, CCC220 (AB108537). Sequence alignments were made using computer software (MEGA 4.0, Version 4.0) to generate a phylogenetic tree with 1000 bootstrap replicates. Genetic distances were calculated by using the Kimura two-parameter method. The three sequences isolated in this study have been deposited in GenBank and the accession numbers are KF957726, KF957727, and KF957728.
Statistical analysis Statistical analysis was performed by the chi-square test using computer software (SPSS 17.0, SPSS, Chicago, IL). 912
TRANSFUSION Volume 54, March 2014
The differences within the subgroups of qualified or ALT elevated alone by region, age, and sex were analyzed with Fisher’s exact test (HEV RNA and antigen) or chi-square test (IgM and IgG). The effect of age or sex on the geographic difference was analyzed by a multivariable analysis using logistic regression. A difference with a p value of less than 0.05 was considered significant.
RESULTS Prevalence of HEV antigen and anti-HEV IgM and IgG among qualified donors in five cities Among 10,741 qualified samples from blood donors in five cities (Urumqi, Beijing, Hangzhou, Guangzhou, Kunming), six (0.06%) were HEV antigen positive, 109 (1.02%) were anti-HEV IgM positive, and 2945 (27.4%) were anti-HEV IgG positive (Table 1). The highest prevalence of HEV antigen (0.20%) and anti-HEV IgM (1.36%) were found in the samples from Hangzhou. Samples from Guangzhou had the lowest prevalence of anti-HEV IgM (0.63%). The highest prevalence of anti-HEV IgG (38.35%) occurred in Kunming. The mean prevalence of anti-HEV IgG was higher in the northern cities (Urumqi and Beijing) with 33.26% than the southern cities (Hangzhou, Guangzhou, and Kunming) with 18.63% (799/4288; chi-square test, p < 0.01; Table 1). After controlling for age and sex, the significant difference was not found within the subgroup of qualified donation by region (logistic regression test, p = 0.12). If Beijing was a control, only Guangzhou had the significantly lower prevalence of anti-HEV IgM (logistic regression test, p = 0.04). As the prevalence of anti-HEV IgG was concerned, there were significant differences in various cities, compared with Beijing (logistic regression test, p < 0.01).
Prevalence of HEV antigen and anti-HEV IgM and IgG by age and sex Among 9719 qualified donations and 747 samples with isolated ALT elevation with age and sex information from the donors the anti-HEV IgG prevalence increased significantly with age and was higher among male qualified donors (chi-square test, p < 0.01). However, the prevalence of HEV antigen and anti-HEV IgM did not differ by age or sex (Tables 2 and 3)
Relationship between ALT and HEV serologic markers Donors with isolated ALT elevation had higher prevalence of both anti-HEV IgM and IgG than qualified donors. The prevalence of anti-HEV IgM was 2.76% in those with ALT elevation and 1.02% among qualified donors (p < 0.01) and the anti-HEV IgG was 40.02% in those with elevated ALT and 27.42% among qualified donors (p < 0.01;
HEV AMONG BLOOD DONORS IN CHINA
TABLE 1. Prevalence of HEV antigen and anti-HEV IgM and IgG by region in qualified donation and elevated ALT alone donation Group Region Qualified* Urumqi Beijing Hangzhou Guangzhou Kunming Total p value† ALT elevated alone Urumqi Beijing Hangzhou Guangzhou Kunming Total p value Total Urumqi Beijing Hangzhou Guangzhou Kunming Total p value
Number
Positive for antigen
Positive rate for antigen (%)
Positive rate for anti-HEV IgM
Positive for anti-HEV IgM (%)
Positive for anti-HEV IgG
Positive rate for anti-HEV IgG (%)
1,910 2,378 1,980 2,520 1,953 10,741
1 0 4 1 0 6
0.05 0.00 0.20 0.04 0.00 0.06 0.04
14 30 27 16 22 109
0.73 1.26 1.36 0.63 1.13 1.02 0.06
341 458 651 746 749 2,945
17.85 19.26 32.88 29.60 38.35 27.42
