Tzu Chi Med J 200618No. 3 NTV
SEN Virus Infection in Patients on Maintenance Hemodialysis in
Eastern Taiwan
Tso-Tsai Liu1, Hans-Hsienhong Lin1,2,7, Li-Yu Wang2,5, Shih-Yen Lo2,6, Teng-Yi Lin3, Chi-Tan Hu1,2,7, Chih-Hsien Wang4, Te-Chao Fang4,7, Bang-Gee Hsu2,4,7
Department of Gastroenterology1, Research Center for Hepatology2, Laboratory Medicine3, Nephrology4, Buddhist Tzu Chi General Hospital, Hualien, Taiwan; Graduate Institute of Aboriginal Health5, Medical Technology6, Medicine7, Tzu Chi University, Hualien, Taiwan
ABSTRACT
Objective: Patients on maintenance hemodialysis have a higher risk of viral hepatitis infection and this study investigates the
prevalence of SENV-D and SENV-H viremia among hemodialysis patients in eastern Taiwan. Patients and Methods: Serum samples were obtained from 119 patients attending a hemodialysis center in eastern Taiwan, and from 43 patients undergoing health examinations. These were tested for SENV-D and SENV-H viremia using polymerase chain reaction. Results: The occurrence of blood transfusion (p<0.0001), the frequency of hepatitis C virus (HCV) infection (p=0.001), and serum AST level (p=0.049) were significantly higher among hemodialysis patients. The prevalence rates of SENV (D and/or H) viremia, D viremia, SENV-H viremia were lower in hemodialysis patients than in control group (10.9% vs 32.6%; 4.2% vs 16.3%; 7.6% vs 23.4%, respectively). Only one SENV-D (+) hemodialysis patient was coinfected with HCV. No SENV-H (+) patients was positive for anti-HCV among the hemodialysis patients (p=0.047). There was no statistically significant association between SENV infection and age of recruitment, gender, transfusion history, AST level, ALT level or hepatitis B surface antigenemia among the hemodialysis patients. Conclusion: These results indicate that SENV infection is a common viral infection in healthy individuals, but, unexpectedly, the frequency of infection among hemodialysis patients in eastern Taiwan was significantly lower than the frequency among healthy individuals. Further studies are therefore needed to resolve this dichotomy. (Tzu Chi Med J 2006; 18:179-183)
Key words: SEN virus, hemodialysis, prevalence
Received: March 15, 2006, Revised: May 30, 2006, Accepted: June 20, 2006
Address reprint requests and correspondence to: Dr. Bang-Gee Hsu, Department of Nephrology, Buddhist Tzu Chi General Hospital, 707, Section 3, Chung Yang Road, Hualien, Taiwan
INTRODUCTION
Patients with chronic renal failure on maintenance hemodialysis have a high risk of viral hepatitis infec-tion [1] by hepatitis B virus (HBV) [2], hepatitis C virus (HCV) [3], hepatitis G virus (HGV) [4] and transfusion-transmitted virus (TTV) [5].
A new virus, designated as SEN virus (SENV; fam-ily Circoviridae, genus Cyrovirus), was identified to be a non-A to G hepatitis virus. This virus contains single
stranded DNA, is distantly related to TTV and can be classified into eight genotypes (A-H) based on the nu-cleotide sequence [6]. However, only two of them (SENV-D and SENV-H) have been described as related to transfusion-associated hepatitis [7].
Patients on hemodialysis are considered to be at risk of infection by blood-borne viruses [8]. SENV-D/H in-fection has been found to be common among patients on hemodialysis [8,9]. The aim of this study was to in-vestigate the prevalence of SENV-D and SENV-H viremia among hemodialysis patients in eastern Taiwan.
Tzu Chi Med J 200618No. 3 NUM
PATIENTS AND METHODS Patients
Serum samples were obtained from 43 patients un-dergoing health examinations and from 119 patients on maintenance hemodialysis at a medical center in Hualien, eastern Taiwan, in December 2004. The Pro-tection of Human Subjects Institutional Review Board Tzu Chi University and Hospital approved this study. Maintenance hemodialysis was being performed two or three times a week as clinically indicated using dispos-able dialysers with standard bicarbonate dialysate. Hypochloride sodium (6%) was used to disinfect the hemodialysis machines between patients. Patients with HCV infection were isolated and treated on separate hemodialysis machines limited to these patients. Blood samples were taken before hemodialysis from each sub-ject and were immediately centrifuged for biochemical study and identification of hepatitis markers. About 2.5 mL of serum was stored at -70˚C in liquid nitrogen for later screening by polymerase chain reaction.
Laboratory tests
Blood samples were immediately centrifuged at 3,000 g for 10 minutes. Serum aminotransferases (AST and ALT) were measured using an autoanalyzer (Hitachi 747, Tokyo, Japan). The serum hepatitis B surface anti-gen (HBsAg) and the antibody to hepatitis C virus (anti-HCV) were detected using commercially available en-zyme immunoassays (Abbott Laboratories, North Chicago, IL). All samples were processed at room tem-perature and prepared according to the manufacturer's directions.
Nucleic acid extraction and Polymerase Chain Re-action (PCR)
The presence of SENV-D and SENV-H DNA was determined by semi-nested PCR. The viral nucleic acid was extracted from 200 µL serum using a QIAamp DNA Blood Mini Kit (Qiagen Ltd, Crawley, UK). The viral nucleic acid was then dissolved in 50 µL RNase-free water and used directly for PCR amplification of SENV-D and -H SENV-DNA. The oligonucleotide primers used for the detections of SENV-D and SENV-H DNA were the same as those reported previously [10]. The first-round PCRs were performed with the sense primer AI-1F (5'-TWCYCMAACGA CCAGCTAGACCT-3') [W=A or T, Y=C or T, M= C or A]) and the antisense primer AI-1R (5'-GTTTGTGGTGAGCAGAACGGA-3') for all genotypes of SENV. These primers were used in a 10
µL PCR mixture containing PCR reaction buffer, 0.25
mM dNTPs, 2.5 mM magnesium chloride, 2.0 U PowerTaq DNA Polymerase (GeneTek BioScience Inc., Taipei), and 1.25 µL of extracted DNA. PCR involved 35 cycles (94 ˚C for 30 seconds, 58 ˚C for 30 seconds, and 72˚C for 45 seconds for each cycle) followed by the extension reaction at 72˚C for 7 minutes in a Perkin Elmer 9700 thermal cycler. An aliquot (1.25 µL) of the products of first round PCR was be used for the second round PCR amplification.
The second round PCR were performed with the sense primer D-1148F(5'-CTAAGCAGCCCTAACA-CTCATCCAG-3') and the antisense primer D-1341R (5'-GCAGTTGACCGCAAAGTTACAAGAG-3') for SENV-D detection, and with the sense primer H-1020F (5'-TTTGGCTGCACCTTCTGGTT-3') and the antisense primer H-1138R (5'-AGAAATGATGGG-TGAGTGTTAGGG-3') for SENV-H detection. The sec-ond round PCR involved 25 cycles (94 ˚C for 30 secsec-onds, 58 ˚C for 30 seconds, and 72 ˚C for 30 seconds for each cycle) followed by the extension reaction at 72˚C for 7 minutes. The amplification products (193 bp fragments for SENV-D and 118 bp fragments for SENV-H) were separated by electrophoresis on a 2% agarose gel, stained with ethidium bromide and visualized using an ultra-violet transilluminator.
We had evaluated the reliability of the PCR meth-ods for the detection of serum SENV DNA. Our results showed that the kappa value of the two tests was 0.88 for SENV-D DNA and 0.80 for SENV-H DNA.
The amplified products of second-round PCR were also purified using a Gel/PCR DNA fragments extrac-tion kit (Geneaid, Taoyuan, Taiwan) and then sequenced by a fluorescent dye terminator cycle system with a com-mercial DNA sequencing kit (Applied Biosystems, Fos-ter City, CA, USA) and ABI PRISM 3730 Genetic Ana-lyzer (Applied Biosystems) according to the manufacturer's instructions.
Statistical analysis
An unpaired t-test was used to quantify the signifi-cance of continuous variables and the Fisher's exact test was used for categorical data. All test statistics were two-sided. A p-value less than 0.05 was considered statisti-cally significant.
RESULTS
Table 1 shows a comparison of clinical backgrounds of hemodialysis patients and the control group. There is higher prevalence of blood transfusion (p<0.0001), hepa-titis C virus (HCV) infection (p=0.001), and serum AST
Tzu Chi Med J 200618No. 3 NUN
level (p=0.049) among the hemodialysis patients. The prevalence rate of SENV (D and/or H) viremia, SENV-D viremia, SENV-H viremia is lower in hemodialysis patients than in the control group (Table 2). Only one SENV-D (+) patient was coinfected with HCV in he-modialysis group. None of the SENV-H (+) hemodialy-sis patients were also positive for anti-HCV (p=0.047). There were no statistically significant differences in mean age, gender distribution, transfusion history, mean AST level, mean ALT level and HBsAg status between the various the groups of hemodialysis patients (Table 3).
DISCUSSION
SENV has a worldwide distribution, having been detected in Japan [11,12], Germany [13], Canada [14], Thailand [15], United States [16] and the Taiwan [9, 17]. The prevalence varies from 5% to 28.6% across these countries. In our study, the prevalence is about 32.6% in healthy adults and this is similar to Dai et al, whose data was from southern Taiwan [9], but is higher than data available for northern Taiwan (15%) [17].
Patients on hemodialysis are at risk of infection by blood-borne viruses. The prevalence of SENV infection has been reported to be high among hemodialysis patients. Kobayashi et al reported a prevalence of 37.6% among hemodialysis patients in Japan [8], Dai et al. reported a prevalence of 61.6% among hemodialysis patients in southern Taiwan [9] and Kao et al reported a prevalence of 68% among hemodialysis patients in
north-Table 3. Comparisons of Clinical and Virological Backgrounds among Hemodialysis Patients with and without SENV Infection Background characteristics SENV (-)(n=106) SENV (+) (n=13) SENV-D (+) (n=5) SENV-H (+) (n=9) Both D and H (n=1)
Age (years)a 59.1±13.8 57.9±19.0 50.4±18.3 59.3±20.3 33
Gender (female %)b 50 46.2 40 44.4 0
Duration of hemodialysis (months)a 52.8±40.6 35.8±28.6 31.2±27.8 35.4±30.2 10
Transfusion history (%)b 83 92.3 100 88.9 100 HBsAg (%)b 14.2 23.1 40 11.1 0 Anti-HCV (%)b 30.2* 7.7 20 0* 0 AST (IU/L)a 18.6±13.9 21.3±14.3 26.0±14.1 17.8±13.7 13 ALT (IU/L)a 17.3±20.8 19.6±16.1 24.8±25.9 16.3±4.8 16 a
Data are expressed as means ± SD; bData are expressed as positive % (number); *p< 0.05; p-value of t test for SENV (-) vs SENV-H (+); Anti-HCV: antibodies against hepatitis C virus; HBsAg: hepatitis B surface antigen
Table 1. Comparisons of Clinical Backgrounds between Hemodialysis and Control Patients
Background characteristics Age (years)a Gender (F, %)b Transfusion (%)b HBsAg (%)b Anti-HCV (%)b AST (IU/L)a ALT (IU/L)a
Control group (n=43) 63.5±14.3 32.6 10.3 14.0 4.7 15.0±9.9 15.8±12.9
Hemodialysis (n=119) 58.9±14.4 49.6 84 15.1 27.7 18.9±13.9 17.5±20.3
p-value* 0.082 0.073 <0.0001 0.85 0.001 0.049 0.52
aData are expressed as means ± SD; bData are expressed as positive % (number); *two-sided test; Anti-HCV; antibodies against hepatitis C virus; HBsAg: hepatitis B surface antigen
Table 2. Prevalence of SENV Infection among Hemodialysis and Control Patients
SENV-D (+) SENV-H (+) SENV (+) Both D and H
N
N (%) N (%) N (%) N (%)
Control group 43 7 (16.3) 10 (23.4) 14 (32.6) 3 (7.0)
Hemodialysis group 119 5 (4.2) 9 (7.6) 13 (10.9) 1 (0.8)
Tzu Chi Med J 200618No. 3 NUO
ern Taiwan [17]. In contrast, our results showed a low prevalence of 10.9%, which is similar to that reported among hemodialysis patients by Schroter et al [18]. Interestingly, the prevalence among hemodialysis pa-tients is much lower than the rate for the general popu-lation in our study. This is indirectly supported by a pre-vious study among hemodialysis patients by ourselves that showed a significantly lower infection rate of TTV, a virus with closely similarity to SENV [19]. Two obvi-ous reasons for the lower SENV infection rate in our hemodialysis unit are the use of different nested PCR primers across the different studies or population isola-tion caused by the mountains of eastern Taiwan. Other possible reasons include the possibility that SENV vire-mia may disappear as time passes as has been found in a longitudinal study [20] or the use of hypochloride so-dium (6%) to disinfect hemodialysis machines between different patient sessions, which may help to prevent parenteral transmission between patients. It is clear that further studies are needed to resolve this anomaly.
Our results showed that the SENV infection is not associated with blood transfusion within hemodialysis units as was found by the Kobayashi et al [8] and Dai et al [9] and therefore the role of parenteral transmission of SENV or other possible routes need to be further investigated.
It should be noted that there are also variations in the prevalence of different SENV genotypes, and geno-types D and H are particularly associated with post-trans-fusion hepatitis [7]. The prevalence of SENV-D infec-tion in hemodialysis patients was found to be higher in Japan (Kobayashi et al, 22.7%) [8] and southern Tai-wan (Dai et al, 46.5%) [9]. In our study, the prevalence of D infection is 4.2%. The prevalence of SENV-H infection in hemodialysis patients was found to be higher in Germany (Schroter et al, 12.8%) [17] and south-ern Taiwan (Dai et al, 27.3%) [9]. Our result, which is similar to Kobayashi et al, is about 7.6%. The preva-lence of mixed SENV-D/H infection in our series is also lower (0.8%) than reported by Kobayash et al and Dai et al.
Umemura et al reported that SENV infection is present in approximately 20% of patients infected by either HBV or HCV [7]. SENV coinfection did not af-fect the severity or persistence of hepatitis C [21]. Fol-lowing treatment with interferon-α, the sustained re-sponse rate for hepatitis C clearance did not differ sig-nificantly between patients with SENV and those with-out SENV infection [21]. How SENV interacts with other hepatitis viruses is still not known. Umemura et al. re-ported SENV-D (+) and HCV coinfection to be about 9% in hemodialysis patients [7]. Our results showed that
the SENV infection is not associated with HCV infec-tion among our patients. In contrast, SENV (+) hemodi-alysis patients have a lower incidence of HCV infection. Only one SENV-D (+) patient was coinfected with HCV among the hemodialysis patients. None of the SENV-H (+) patients were positive for anti-HCV among the he-modialysis patients. The reasons for the lower SENV and HCV coinfection rate in our hemodialysis unit may be because patients with HCV infection at our hemodi-alysis unit are isolated and only use specific hemodialy-sis machines. Furthermore, we use hypochloride sodium (6%) to disinfect the hemodialysis machines between patients. The interaction between SENV and HCV in patients on hemodialysis also needs to be further investigated.
Dai et al reported that concurrent SENV-D and SENV-H infection was an independent factor associ-ated with increased ALT level [9]. However, our results showed that SENV infection is not associated with an increased serum ALT level.
In conclusion, patients on hemodialysis in eastern Taiwan have a lower prevalence of SENV infection than healthy individuals. There is no statistically significant association of SENV infection with mean age, gender distribution, transfusion history, mean AST level, mean ALT level and HBsAg status in hemodialysis patients in eastern Taiwan. SENV and HCV infection may interact, but further studies are needed to elucidate this, because, specifically, in this study, SENV (+) hemodi-alysis patients have a lower incidence of HCV infection.
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