Year : 2009 | Volume
: 20 | Issue : 3 | Page : 398--401
HBV-DNA in hemodialysis patients infected by HCV
Mohammad Kazemi Arababadi, Gholamhossein Hassanshahi, Hassan Yousefi
Department of Microbiology and Immunology, Faculty of Medicine, Rafsanjan University of Medical Science, Rafsanjan, Iran
Mohammad Kazemi Arababadi
Department of Microbiology and Immunology, School of Medicine, Rafsanjan University of Medical Sciences, Rafsanjan
End-stage renal disease patients on chronic hemodialysis (HD) patients are at risk for both hepatitis B virus (HBV) and hepatitis C virus (HCV) infection, and they may coexist. To determine the prevalence and clinical impact of HBV and HCV infection, we studied poly chain reaction (PCR) and reverse transcription (RT)-PCR on the blood samples of 90 HD patients in Kerman, Iran. ELISA test was used to detect anti-HBc, anti-HBs and HBsAg. We found that 30 out of 90 (33.3%) patients were PCR-RT-PCR positive for HCV-RNA. No HBV-DNA (0%) was detected through the PCR study in both positive and negative HCV-RNA patient groups. Though none of the samples was HBsAg positive, 10 (33.3%) HCV-RNA positive patients were anti-HBc positive, and 12 (40.7%) were anti-HBs positive. We conclude that prevalence of hepatitis C infection is high in HD patients in our region, but not associated with active HBV infection.
|How to cite this article:|
Arababadi MK, Hassanshahi G, Yousefi H. HBV-DNA in hemodialysis patients infected by HCV.Saudi J Kidney Dis Transpl 2009;20:398-401
|How to cite this URL:|
Arababadi MK, Hassanshahi G, Yousefi H. HBV-DNA in hemodialysis patients infected by HCV. Saudi J Kidney Dis Transpl [serial online] 2009 [cited 2021 Mar 1 ];20:398-401
Available from: https://www.sjkdt.org/text.asp?2009/20/3/398/50769
Hepatitis C virus (HCV) and hepatitis B virus (HBV) infections are the most common causes of liver disease in hemodialysis (HD). ,, HBV infection in patients with undetectable HBsAg is called occult HBV infection. , It is possible that host immune mechanisms and viral interactions can maintain HBV infection in a latent state until more profound immunosuppression supervenes. , Occult HBV infection carries its own risks of disease transmission, acute exacerbations and development of hepatocellular carcinoma (HCC). ,,,
Although prevalence and clinical significance is unknown in HD patients with chronic hepatitis C, occult HBV infection is frequently reported in patients with chronic hepatitis C liver disease. , Viral reactivation in patients undergoing immunosuppressive therapy is a well-known complication of occult HBV infection.  High prevalence of occult HBV and chronic hepatitis C may coexist in patients with HCC, HD, cryptogenic liver disease, illicit drug contaminated injections, HIV patients, and frequent blood transfusion (i.g., hemophilia, thalassemia disease, etc.) be sides blood donors. ,,,,,,
The prevalence of occult HBV infection in dialysis patients ranges between 0% and 58%. 
The aim of our study is to determine the prevalence of coexistence HBV and HCV infection in chronic HD patients in our region.
Patients and Methods
We studied in a cross-sectional pattern the blood samples of 90 chronic hemodialysis patients in Kerman, Iran to detect HCV and HBV infection in them. There were 38 (42.2%) males, and the average age of the patients was 50-70 years.
0.1 mL of samples were added to phenol and after vortexing, centrifuged for 5 min at 12000g. The supernatants were transferred to new tubes and 0.2 ml chloroform was added and this was mixed vigorously for 15 seconds with each sample, which were then incubated at room temperature for 5 minutes and centrifuged at 12000g for 15 minutes at 4°C. The supernatants (containing RNA) were transferred to a fresh tubes. 0.5 mL isopropanol was added to each isolated supernatant and the mixture was incubated at room temperature for 10 minutes. The RNA was precipitated by centrifugation at 12,000g for 10 minutes at 4°C. Each pellet was washed with 1 mL 75% (v/v) ethanol and centrifuged at 7500g for 5 minutes at 4°C. The final pellets were airdried for 10-15 minutes and dissolved afterwards in 25-50 µL DEPC-treated water (volume added was dependent on the pellet size) at 60°C for 10 minutes.
Reverse Transcription Polymerase Chain Reaction (RT-PCR)
To make complementary DNA (cDNA), reverse transcription reactions were performed using the following protocol: 4 µL 5x strand buffer (125 mM tris-HCl pH 8.3, 188 mM KCl, 7.5 mM MgCl2 25 mM DTT); 1 µL of each dNTP [dATP, dCTP, dGTP, dTTP (stock concentration of 10 mM in DEPC-treated water)]; 4 µL oligo-dT (stock concentration of 125 µg/mL); 1 µL RNA (1µg/ µL); 4 µl DEPC-treated water; 1.5 µL M-MLV reverse transcriptase enzyme. After addition of M-MLV-reverse transcriptase and mixing, the samples were incubated for 1 hour at 37°C. To amplify cDNA species, PCR reaction mixture was prepared by addition of the following reagents to a 0.2 mL microcentrifuge tube in ice: 5 µL Taq polymerase buffer (10x); 1.5 µL MgCl2 (stock concentration 1.5 mM); 1 µL of dNTPs [(dATP, dCTP, dGTP, dTTP) stock concentration of 10 mM]; 2 µL of each primer pair [(forward and reverse) stock concentration of 25 ng/µL]; 4 µL cDNA; and sterile double distilled water to a final volume of 50 µL. The sequence of forward primer was 5'-TGGTGGAGTTTACTTGTT-3' and the sequence of reverse primer was 5'TCGTCGGCGCCCCTCTTG-3'. The PCR thermocycler was adjusted accordingly: 94°C for 5 minutes, 94°C for 40 second, 40 second at 63°C annealing temperature, and 72°C for 45 second. Denaturation, annealing and elongation procedures were repeated for 30 cycles. During the last 45 seconds of first stage 1 mL Taq polymerase was added to the mixture for each sample. The presence of a 354 bp fragment indicated positive result. All the HCV-RNA positive patients were followed up for at least 6 months and accepted for this study.
Enzyme Linked Immunosurbent Assay (ELISA)
HBsAg screening test were performed using HBsAg ELISA kit (RADIM, Italy). Anti-HBc and anti-HBs tests were also performed by a manual microplate enzyme immunoassay using anti-HBc and anti-HBs kits provided by RADIM (Italy).
Viral DNA was purified from 100 µL of plasma samples. Briefly, each serum sample was incubated at 72°C for 10 minutes and then cooled at 4°C for 5 minutes in 100 µL proteinase K (200 µg/mL). After phenol/chloroform extraction (1:1), the viral DNA was precipitated with ethanol and the pellet was resolved in DNase free, deionized water and stored at -20°C for future use.
PCR and Gel Electrophoresis
PCR was carried out in a 50 µL mixture containing 10 mM tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.01 % gelatin, 5 units recombinant Taq DNA polymerase, 200 µM of each dNTPs, 0.6 µM of each primer, and 5 µL of the DNA extracted from 100 µL of plasma. The sequence of forward primer was 5'TCGTGG-TGGACTTCTCTC-3' and the sequence of reverse primer was 5'-ACAGTGGGGGAAAGCCC-3'. These primers amplify a 500bp of the HBV genome. A fast temperature cycling was performed. PCR amplification was performed by including one cycle of 93°C for 180 sec, 63°C for 30 sec and 72°C for 40 sec, then 35 cycles of 93°C for 40 sec, 63°C for 40 sec and 72°C for 40 sec. HBV genome provided by Sinagen company was used as positive control. For the analysis of PCR amplification, 10 µL of the amplified DNA were run on a 2% agarose gel after adding 4 µL loading dye. The presence of a 500 bp fragment indicated a positive result. Ladder was also run in parallel with samples on the gels to estimate the molecular weights of DNA fragments in the gel.
All data are expressed as mean ± SEM. Comparisons of variables were performed using an unpaired Student's "t" test. Differences were considered significant when P (26.7%). Furthermore, there was no significant difference in the duration of receiving blood and their components between the positive and negative patients as shown in [Table 1].
Our results showed that no HBV-DNA (0%) was detected through the PCR study in both patient groups with positive and negative HCV-RNA. In addition, none of the samples was HBsAg positive, but 10 out of 30 (33.3%) HCV-RNA positive were anti-HBc positive, and 12 out of 30 (40%) patients were positive for anti-HBs.
As they are similar transmission models, HBV and HCV co-infection is prevalent. In our study, HBV-DNAand HBsAg were not detected in hemodialysis patients with chronic HCV hepatitis, but 33% were anti-HBc positive and 40% were positive for anti-HBs. Besisik et al found chronic hepatitis C in 33 HD patients who were and positive for HCV-RNA and negative for HBsAg, but serum HBV-DNA PCR study yielded positive results in 12 (36.4%) patients. 
Furthermore, the reported anti-HBc prevalence in patients with chronic HCV infection is 50%-55%; ,, this percentage is higher than the 33% in our study and may reflect less contact with HBV in our population. A study from Loyola University at Chicago-USA showed that the prevalence of anti-HBc was high among anti-HCV positive individuals;  our results are in agreement with theirs results and much higher than the 5.18% in blood donors reported previously from our region. 
Most studies disclosed the existence HBV DNA genome in 22%-87%, , of patients with negative HBsAg and positive HCV-RNA. HBVDNA is observed in 46% of anti-HBc positives, and in 20% of anti-HBc negative patients.  In contrast to these studies, , we found no in creased incidence of occult HBV infection in hemodialysis patients with chronic hepatitis C in our region. However, our results are comparable with those of Vedat et al  from Turkey. They were unable to detect HBV-DNA in hemodia lysis HCV infected patients. It is probably due to several factors such as the intermediate prevalence of HBV in our region  and safety of blood and its components. However, fluctuations of HBV viremia associated with occult HBV infection may also explain the variability of the prevalence HBV-DNA. 
We conclude that prevalence of hepatitis C in fection is high in HD patients in our region, but not associated with active HBV infection.
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