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Saudi Journal of Kidney Diseases and Transplantation
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ORIGINAL ARTICLE Table of Contents   
Year : 2009  |  Volume : 20  |  Issue : 3  |  Page : 398-401
HBV-DNA in hemodialysis patients infected by HCV


Department of Microbiology and Immunology, Faculty of Medicine, Rafsanjan University of Medical Science, Rafsanjan, Iran

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   Abstract 

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.

Keywords: Hepatitis C, Hepatitis B, HBV infection, Hemodialysis patients

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 2020 Nov 29];20:398-401. Available from: https://www.sjkdt.org/text.asp?2009/20/3/398/50769

   Introduction Top


Hepatitis C virus (HCV) and hepatitis B virus (HBV) infections are the most common causes of liver disease in hemodialysis (HD). [1],[2],[3] HBV infection in patients with undetectable HBsAg is called occult HBV infection. [4],[5] It is possible that host immune mechanisms and viral inter­actions can maintain HBV infection in a latent state until more profound immunosuppression supervenes. [4],[5] Occult HBV infection carries its own risks of disease transmission, acute exacer­bations and development of hepatocellular car­cinoma (HCC). [4],[6],[7],[8]

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. [7],[8] Viral reactivation in patients undergoing immu­nosuppressive therapy is a well-known compli­cation of occult HBV infection. [8] 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. [4],[9],[10],[11],[12],[13],[14]

The prevalence of occult HBV infection in dia­lysis patients ranges between 0% and 58%. [15]

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 Top


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.

RNA Extraction

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).

DNA Extraction

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.

Statistical Analysis

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< 0.05.


   Results Top


HCV-RNA RT-PCR examination was posi­tive in 30 (33.3%) patients. The average age of the HCV patients was 51.16 years and 50.50 in HCV negative patients. Therefore, the significant difference was not observed prevalence of HCV infection and the age of patients as shown in [Table 1].

Our results also revealed no significant diffe­rence between the RNA-HCV positive male (38.2%) and female HD patients (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.


   Discussion Top


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. [16]

Furthermore, the reported anti-HBc preva­lence in patients with chronic HCV infection is 50%-55%; [9],[10],[17] 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; [7] our results are in agreement with theirs results and much higher than the 5.18% in blood donors repor­ted previously from our region. [4]

Most studies disclosed the existence HBV­ DNA genome in 22%-87%, [18],[19] 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. [19] In contrast to these studies, [18],[19] 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 [15] 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 [20] 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. [21]

We conclude that prevalence of hepatitis C in­ fection is high in HD patients in our region, but not associated with active HBV infection.[21]

 
   References Top

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2.Lopez-Alcorocho JM, Barril G, Ortiz-Movilla N, et al. Prevalance of hepatitis B, hepatitis C, GB virus C/hepatitis G and TT viruses in hemodialysis patients. J Med Virol 2001;63: 103-7.  Back to cited text no. 2    
3.Huang CC. Hepatitis in patients with end-stage renal disease. J Gastroenterol Hepatol 1997;12: 236-41.  Back to cited text no. 3    
4.Jafarzadeh A, Kazemi Arababadi M, Mirzaee M, Pourazar A. Occult hepatitis B virus infec-tion among blood donors with antibodies to hepatitis B core antigen. Acta Medica Iranica 2008;46(1):27-32.  Back to cited text no. 4    
5.Pourazar A, Salehi M, Jafarzadeh A, Kazemi-Arababadi M, Oreizi F, Shariatinezhad K. Detection of HBV DNA in HBsAg negative normal blood donors. Iran J Immunol 2005;2: 172-6.  Back to cited text no. 5    
6.Chan HL, Tsang SW, Leung NW, et al. Occult HBV infection in cryptogenic liver cirrhosis in an area with high prevalence of HBV infection. Am J Gastroenterol 2002;97:1211-5.  Back to cited text no. 6  [PUBMED]  [FULLTEXT]
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11.Allain JP. Occult hepatitis B virus infection: implications in transfusion. Vox Sang 2004;86: 83-91.  Back to cited text no. 11  [PUBMED]  [FULLTEXT]
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13.Toyoda H, Hayashi K, Murakami Y, et al. Prevalence and clinical implications of occult hepatitis B viral infection in hemophilia patients in Japan. J Med Virol 2004;73:195-9.  Back to cited text no. 13  [PUBMED]  [FULLTEXT]
14.Oesterreicher C, Hammer J, Koch U, et al. HBV and HCV genome in peripheral blood mono-nuclear cells in patients undergoing chronic hemodialysis. Kidney Int 1995;48:1967-71.  Back to cited text no. 14    
15.Goral V, Ozkul H, Tekes S, Sit D, Kadiroglu AK. Prevalence of occult HBV infection in haemodialysis patients with chronic HCV. World J Gastroenterol 2006;12(21):3420-4.  Back to cited text no. 15    
16.Besisik F, Karaca C, Akyuz F, et al. Occult HBV infection and YMDD variants in hemo-dialysis patients with chronic HCV infection. J Hepatol 2003;38:506-10.  Back to cited text no. 16    
17.Khan MH, Farrell GC, Byth K, et al. Which patients with hepatitis C develop liver compli-cations? Hepatology 2000;31:513-20.  Back to cited text no. 17  [PUBMED]  [FULLTEXT]
18.Fukuda R, Ishimura N, Niigaki M, et al. Sero-logically silent hepatitis B virus coinfection in patients with hepatitis C virus-associated chro-nic liver disease: clinical and virological signi-ficance. J Med Virol 1999;58:201-7.  Back to cited text no. 18  [PUBMED]  [FULLTEXT]
19.Cacciola I, Pollicino T, Squadrito G, Cerenzia G, Orlando ME, Raimondo G. Occult hepatitis B virus infection in patients with chronic hepa-titis C liver disease. N Engl J Med 1999;341: 22-6.  Back to cited text no. 19  [PUBMED]  [FULLTEXT]
20.World Health Organization. Hepatitis B: World Health Organization Fact Sheet 204. 2000. http://www.who.int/mediacentre/factsheets/fs204/en/.  Back to cited text no. 20    
21.Gaeta GB, Rapicetta M, Sardaro C, et al. Prevalence of anti- HCV antibodies in patients with chronic liver disease and its relationship to HBV and HDV infections. Infection 1990; 18:277-9.  Back to cited text no. 21  [PUBMED]  

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Correspondence Address:
Mohammad Kazemi Arababadi
Department of Microbiology and Immunology, School of Medicine, Rafsanjan University of Medical Sciences, Rafsanjan
Iran
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    Tables

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