the association of hepatitis b virus ( hbv ) and hematologic malignancies , including hodgkin lymphoma and non - hodgkin lymphoma ( nhl ) has been previously studied ( 1 ) . hepatitis b virus causes a wide range of liver diseases , including subclinical infection , acute , chronic and fulminant hepatitis . chronic hbv may lead to cirrhosis or hepatocellular carcinoma ( hcc ) ( 2 ) . hepatitis b virus is a dna virus , which belongs to the hepadnaviridae family ( 3 ) . the genome of hbv is a partially double stranded circular dna with molecular weight of 3.2 kbp . the viral genome encodes four open reading frames ( orfs : s , c , p and x ) . the orf s encodes hbsag , which consists of pre - s1 , pre - s2 and s regions . the orf x encodes the x protein ( hbx ) and is a transactivator that influences the transcription of hbv genes by regulating the activity of transcriptional promoters and orf p , which encodes dna polymerase and reverse transcriptase ( 4 ) . genotypes a and d are prevalent in europe , united states , and africa , whereas genotypes b and c are prevalent in asia . genotype e is the dominant genotype in west africa whereas f and h have been isolated in the america ( 5 ) . based on amino acid substitutions in positions 122 and 160 of the s region of hbv dna , four major subtypes : adw , adr , ayw and ayr , and nine minor subtypes have been described ( 6 ) . hepatitis b virus is a hepatotropic virus but recently hbv dna has been detected in peripheral blood mononuclear cell ( pbmc ) of patients with acute and chronic infection ( 7 - 9 ) . some studies have suggested that the hbv dna could replicate in pbmc and proved this suggestion by detection of hbv mrnas and expression of hbsag and hbcag in pbmcs ( 10 - 15 ) . these observations show that the lymphoid system could be an important reservoir for hepatitis b virus ( 16 ) . the hbv dna integration in pbmcs has been demonstrated previously ( 7 , 17 - 19 ) . hodgkin lymphoma and non - hodgkin lymphoma are lymphoid malignant tumors , and are prevalent worldwide . recent studies have revealed the association between hbv infection and hodgkin and non - hodgkin lymphoma ( 20 - 22 ) . the aim of this study was to determine the prevalence of hbv dna and s region mutations among block samples of patients with hodgkin and non - hodgkin lymphoma in ahvaz city , iran . paraffin embedded tissues of 41 block samples including 12 ( 29/26% ) hodgkin and 29 ( 70.73% ) non - hodgkin patients were collected from imam khomeini hospital located in ahvaz , iran , during 2002 to 2011 . the following steps were then carried out for the detection of hbv dna in the patient samples . statistical analysis was done with the chi square test using the spss software , version 20 . the dna extraction of all the tissue samples was carried out using the high pure pcr template kit ( roche , germany ) , according to the manufacturer s instructions . the nested pcr for detection of hbv dna was performed for all the tissue samples . the primers for partial sequencing of the  s  region are presented in table 1 ( 23 ) . for the first round , 5 l of the extracted dna from each sample was added to a pcr reaction mixture containing 0.5 l dntp ( 10 mm ) , 2.5 l pcr buffer ( 10x ) , 0.15 l 5u taq dna polymerase ( roch , germany ) , 50 pmol/l of the fhbs1 and rhbs1 primers , 0.5 l dntp ( 10 mm ) and 15.85 l distilled water . the samples were placed in the thermal cycler ( techne company , uk ) and the first round amplification was carried out with initial denaturation at 94c for five minutes , followed by denaturation at 94c for 30 seconds , annealing at 56c for 30 seconds and extension at 72c for 30 seconds , for a total of 30 cycles . for the second round , 5 l of the pcr product was added to 25 l of reaction mixture containing the same components mentioned in the first run including dntp , pcr buffer and taq dna polymerase with 50 pmol/l of each of fhbs2 and rhbs2 primers . amplification was carried out in the thermal cycler with the same program as the first round . next , 8 l of the nested pcr product ( 417 bp ) was analyzed by 2% agarose gel electrophoresis . amongst the ten samples positive for hbv dna , six samples were sequenced ( applied bi benier company , south korea ) . the sequences of all the six samples were submitted to ncbi and registered in gen bank ( table 2 and figure 1 ) . hepatitis b virus sequences were aligned with the hbv reference sequence from gene bank using the hbv data - base and online blast . a phylogenetic tree was constructed using the mega 5 software . to evaluate the hbs subtypes and s region mutations of the positive samples , ( figure 1 ) the amino acid substitutions in positions 122 and 160 of the s region of hbv dna were compared with the hbv reference sequence . interestingly , the presence of an unpredictable amino acid substitution ( l88p ) was observed , which indicated a new mutation in the s  region of hbv dna in patients with hodgkin and non - hodgkin lymphoma , ( figure 2 ) . the consensus amino acid sequence of the reference d genotype is shown on the top . the partial sequences of all the six hbs genes in position 88 compared with the consensus sequence amino acid indicated a proline substitution with leucine . so far this type of mutation has not been reported in any hbv d genotype . the dots show conserved amino acid and the amino acid code indicates differences with the consensus amino acid . statistical analysis was done with the chi square test using the spss software , version 20 . the dna extraction of all the tissue samples was carried out using the high pure pcr template kit ( roche , germany ) , according to the manufacturer s instructions . the nested pcr for detection of hbv dna was performed for all the tissue samples . the primers for partial sequencing of the  s  region are presented in table 1 ( 23 ) . for the first round , 5 l of the extracted dna from each sample was added to a pcr reaction mixture containing 0.5 l dntp ( 10 mm ) , 2.5 l pcr buffer ( 10x ) , 0.15 l 5u taq dna polymerase ( roch , germany ) , 50 pmol/l of the fhbs1 and rhbs1 primers , 0.5 l dntp ( 10 mm ) and 15.85 l distilled water . the samples were placed in the thermal cycler ( techne company , uk ) and the first round amplification was carried out with initial denaturation at 94c for five minutes , followed by denaturation at 94c for 30 seconds , annealing at 56c for 30 seconds and extension at 72c for 30 seconds , for a total of 30 cycles . for the second round , 5 l of the pcr product was added to 25 l of reaction mixture containing the same components mentioned in the first run including dntp , pcr buffer and taq dna polymerase with 50 pmol/l of each of fhbs2 and rhbs2 primers . amplification was carried out in the thermal cycler with the same program as the first round . next , 8 l of the nested pcr product ( 417 bp ) was analyzed by 2% agarose gel electrophoresis . amongst the ten samples positive for hbv dna , six samples were sequenced ( applied bi benier company , south korea ) . the sequences of all the six samples were submitted to ncbi and registered in gen bank ( table 2 and figure 1 ) . hepatitis b virus sequences were aligned with the hbv reference sequence from gene bank using the hbv data - base and online blast . a phylogenetic tree was constructed using the mega 5 software . to evaluate the hbs subtypes and s region mutations of the positive samples , ( figure 1 ) the amino acid substitutions in positions 122 and 160 of the s region of hbv dna were compared with the hbv reference sequence . interestingly , the presence of an unpredictable amino acid substitution ( l88p ) was observed , which indicated a new mutation in the s  region of hbv dna in patients with hodgkin and non - hodgkin lymphoma , ( figure 2 ) . the consensus amino acid sequence of the reference d genotype is shown on the top . the partial sequences of all the six hbs genes in position 88 compared with the consensus sequence amino acid indicated a proline substitution with leucine . so far this type of mutation has not been reported in any hbv d genotype . the dots show conserved amino acid and the amino acid code indicates differences with the consensus amino acid . out of 12 hodgkin samples , seven ( 58.33% ) and five ( 41.66% ) were males and females , respectively . the age of the patients with hodgkin lymphoma was between 5 and 34 with a mean age of 20  7 years . of the 29 non - hodgkin samples , 17 ( 58.62% ) and 12 ( 41.37% ) were males and females , respectively , table 3 . out of the 41 samples , three out of twelve ( 25% ) hodgkin samples and seven of twenty - nine ( 24.13% ) all the positive hbv patients were within the normal range for alanine aminotransferase ( 20 - 32 iu / dl ) and aspartate aminotransferase ( 18- 27 iu / dl ) . the youngest hodgkin patient was a female with 17 years of age and the oldest was a male with 29 years of age . the youngest non - hodgkin patient with hbv infection was a male with two years of age and the oldest patient was a female with 75 years of age . the sequences of six positive samples ( two hodgkin and four non - hodgkin lymphoma ) were registered in genbank with the following accession numbers , kj398939.1 to kj398944.1 . the results of the alignment showed that the d genotype was the predominant genotype among the patients with hepatitis b infection . mutation analysis indicated amino acid substitutions in positions 88 , 122 and 160 of the  s  region of hbv dna . all of the six sequences were recognized as the  awy  subtype because of substitution in positions 122 and 160 . the sequences of six positive samples ( two hodgkin and four non - hodgkin lymphoma ) were registered in genbank with the following accession numbers , kj398939.1 to kj398944.1 . the results of the alignment showed that the d genotype was the predominant genotype among the patients with hepatitis b infection . mutation analysis indicated amino acid substitutions in positions 88 , 122 and 160 of the  s  region of hbv dna . several epidemiological studies have revealed the persistence of hepatitis b virus ( hbv ) infection amongst patients with hodgkin ( hl ) and non - hodgkin lymphoma ( nhl ) . in our study , 25% of hodgkin and 24.13% of non - hodgkin patients had positive hbv dna , which indicates the high rate of hbv infection among these patients . the youngest hodgkin patient with hbv infection was a female with 17 years of age and the oldest was a male with 29 years of age . the youngest non - hodgkin patient with hbv infection was a male with two years of age and the oldest was a female with 75 years of age . the frequency of hbv infection among the male and female patients was not significantly different ( p > 0.05 ) . in our study fernndez et al . in spain reported the ad and ay hbs subtypes amongst 40 chronic hbv patients ( 24 ) . de souza et al . from brazil reported hbsag subtypes adw2 , adw4 , and ayw amongst patients with mental problems ( 25 ) . in our study genes , showed mutation at position 88 ( l88p ) . so far this type of mutation has not been reported in any hbv d genotype . in the study conducted by okamoto et al . a point mutation was found in the s gene of hepatitis b virus for two blood donors carrying a surface antigen with subtype adyr or adwr ( 26 ) . the patient s history revealed that they were not tested for hbv and hepatitis c virus and it is not clear whether these patients had chronic hbv or occult hbv infection . however , there are some reports about the association of occult hbv infection and non - hodgkin lymphoma ( 27 , 28 ) . the presence of unpredictable amino acid proline at position 88 of hbs gene indicates a new mutation in the s  region of hbv dna in patients with hodgkin and non - hodgkin lymphoma . in our survey the d1 genotype was dominant in all the hbv patients , which is similar to the report of marcucci et al . the role of other viruses including hepatitis c virus in hodgkin and non - hodgkin lymphoma was not studied however this requires further investigations ( 30 , 31 ) . there have been reports about the reactivation of hepatitis b and hepatitis c virus ( hcv ) infections during nhl treatment ; in these studies it was revealed that the patients who were under chemotherapy showed hbv and hcv reactivation , yet other patients who received lamivudine before chemotherapy did nt show any reactivation for hbv or hcv infection ( 32 - 35 ) . in conclusion , a high prevalence of hbv infection ( 25% and 24.13% , respectively ) was found in patients with hodgkin and non - hodgkin lymphoma , indicating the importance of hbv and hcv screening by sensitive tests such as pcr before chemotherapy to prevent hbv or hcv reactivation in patients with hl and nhl .