clonal proliferation of histiocytes was reported in langerhans cell histiocytosis ( lch ) by willman et al . in 1994 ( 1 ) . langerhans cells are a type of nonlymphoid mononuclear cells involved in inflammatory responses and also in neoplastic processes . they are immature dendritic cells which express lysosomal enzymes cd1a and s-100 protein and ultrastructurally contain racket shaped organelles of birbeck granules ( 2 ) . the etiology is unknown ; however , environmental agents and viruses , especially epstein - barr virus ( ebv ) , or vaccination , have been proposed to play a role in the pathogenesis of lch ( 3 , 4 ) . the role of ebv as the etiologic agent of several malignancies is known and herpes viruses are shown to be responsible for persistent infection ( 5 , 6 ) . in addition , ebv and cytomegalovirus ( cmv ) are considered to be responsible for hemophagocytic syndromes in human with several inherited immunodeficiencies ( 7 , 8) . one study showed 30% positivity for cmv in lch ( 9 ) ; however , others have failed to show any association with adenovirus , cytomegalovirus , epstein - barr virus , herpes simplex virus , human herpes virus type 6 , human immunodeficiency virus , human t - cell leukemia virus types i and ii , and parvovirus using pcr ( 10 - 12 ) . in this study , we have investigated the presence of cmv in lch by qualitative pcr method . formalin - fixed , paraffin - embedded ( ffpe ) tissue samples of 30 patients with pathologic diagnosis of lch were extracted from archive of pathology department of mofid children s hospital in tehran ( one of the referral centers from all over the country ) for a ten year period ( 2002 to 2012 ) . the diagnosis of lch was made by a pediatric pathologist , using the histological criteria mentioned in the pathology textbooks , i.e. granulomas composed of langerhans cells with typical grooved nuclei mixed with eosinophils and other inflammatory cells . the diagnoses were confirmed using immunohistochemical technique for cd1a , s-100 protein and cd68 when available . thirty tissue samples with non lch diagnoses were also selected ( between the years 2002 and 2012 ) as age and site - matched controls to lch cases . paraffin was removed from the ffpe samples after undergoing xylene treatment , followed by two washes with pure ethanol . total dna was extracted from the air - dried pellet tissues according to the rtp dna / rna virus mini kit procedure ( stratec molecular , berlin , germany ) . the extracted nucleic acid was stored at -20c until it underwent pcr . to assess the quality of the extracted genome , as well as inhibition of pcr test , all extracted and stored nucleic acid underwent beta - globin pcr , using the pco3/pco4 primer set , as described previously with modifications in method ( 13 ) . the beta - globin pcr was performed in syber green real - time pcr - melting curve format ( figure 1 ) . to detect cmv , real - time pcr was performed using envelope glycoprotein b ( gpul55 ) gene primer sets amplifying 116 bp gene region of the virus genome ( cmv - r ; 5-aagtacccctatcgcgtgtg-3 ) , cmv - f ; 5-atgatgccctcrtccargtc -3 , with an internal probe , cmv - p ; 5-fam - tggcccagggtacggatcttattcg - bhq1 - 3 ) ( 14 ) . amplification of cmv gpul55 genes was performed in reaction volumes of 20 l under the following conditions : first the samples underwent denaturation at 94c for 10 minutes , followed by denaturation at 94c for 10 seconds , followed by annealing and extension at 60c for one minute , 50 cycles ( figure 2 ) . real - time pcr system cfx-96 ( bio - rad , usa ) with hs prime taq premix taqman reagent ( genetbio , korea ) was used in real - time pcr assay . the limit detection of 5 genome copies of cmv per reaction was determined by the real - time assay , using the serial dilutions of amplirun cytomegalovirus dna control ( vircell , spain ) . the graph shows that the melting temperature of beta - globin pcr product is around 82.5c . the achieved data were compared by chi - square ( or fisher s exact test if appropriate ) . p value of less than 0.05 was considered to indicate statistical significance . in this study , no ethical issues were involved ; only the pathology reports were reviewed retrospectively , and the patients were anonymous . formalin - fixed , paraffin - embedded ( ffpe ) tissue samples of 30 patients with pathologic diagnosis of lch were extracted from archive of pathology department of mofid children s hospital in tehran ( one of the referral centers from all over the country ) for a ten year period ( 2002 to 2012 ) . the diagnosis of lch was made by a pediatric pathologist , using the histological criteria mentioned in the pathology textbooks , i.e. granulomas composed of langerhans cells with typical grooved nuclei mixed with eosinophils and other inflammatory cells . the diagnoses were confirmed using immunohistochemical technique for cd1a , s-100 protein and cd68 when available . thirty tissue samples with non lch diagnoses were also selected ( between the years 2002 and 2012 ) as age and site - matched controls to lch cases . paraffin was removed from the ffpe samples after undergoing xylene treatment , followed by two washes with pure ethanol . total dna was extracted from the air - dried pellet tissues according to the rtp dna / rna virus mini kit procedure ( stratec molecular , berlin , germany ) . to assess the quality of the extracted genome , as well as inhibition of pcr test , all extracted and stored nucleic acid underwent beta - globin pcr , using the pco3/pco4 primer set , as described previously with modifications in method ( 13 ) . the beta - globin pcr was performed in syber green real - time pcr - melting curve format ( figure 1 ) . to detect cmv , real - time pcr was performed using envelope glycoprotein b ( gpul55 ) gene primer sets amplifying 116 bp gene region of the virus genome ( cmv - r ; 5-aagtacccctatcgcgtgtg-3 ) , cmv - f ; 5-atgatgccctcrtccargtc -3 , with an internal probe , cmv - p ; 5-fam - tggcccagggtacggatcttattcg - bhq1 - 3 ) ( 14 ) . amplification of cmv gpul55 genes was performed in reaction volumes of 20 l under the following conditions : first the samples underwent denaturation at 94c for 10 minutes , followed by denaturation at 94c for 10 seconds , followed by annealing and extension at 60c for one minute , 50 cycles ( figure 2 ) . real - time pcr system cfx-96 ( bio - rad , usa ) with hs prime taq premix taqman reagent ( genetbio , korea ) was used in real - time pcr assay . the limit detection of 5 genome copies of cmv per reaction was determined by the real - time assay , using the serial dilutions of amplirun cytomegalovirus dna control ( vircell , spain ) . the graph shows that the melting temperature of beta - globin pcr product is around 82.5c . the achieved data were compared by chi - square ( or fisher s exact test if appropriate ) . p value of less than 0.05 was considered to indicate statistical significance . in this study , no ethical issues were involved ; only the pathology reports were reviewed retrospectively , and the patients were anonymous . all patients were iranians ( 16 males and 14 females ) and the age ranged 2 months to 10 years . thirty tissue samples with non lch diagnoses were also selected as controls which were age and site - matched to lch cases . cmv dna was detected in only 2 ( 6.66% ) lch patients out of 30 cases ( positive results in 6.66% and negative results in 93.34% ) . in control group , we detected cmv virus in 1(3.3% ) sample out of a total of 30 cases ( positive results in 3.3% and negative results in 96.7% of controls ) with a p value of 1.00 ( 1.00 > 0.05 ) and or : 2.07 ; 95% ci of or : 0.18 - 24.15 which shows no significant difference in cmv prevalence results between lch patients and controls . the lch patients with positive result for cmv expression were a 2 year old boy with frontal bone lesion and a 1 year old girl with chest skin lesion ; the positive control was in a 6 year - old boy with skin biopsy diagnosed as pilonidal fistula . lch is a rare disease involving clonal proliferation of bone marrow derived langerhans cells which mostly affects children , particularly children younger than 5 years with an incidence of about 5 per 1,000,000 ( 2 ) . clinically , there are three overlapping clinical syndromes including multifocal multisystemic lch ( letterer - siwe syndrome ) , multifocal unisystemic lch ( hand - schuller - christian syndrome ) and unifocal lch ( solitary eosinophilic granuloma ) ( 2 ) . histologically , the findings are the same in various tissues , characterized by granulomas composed of a mixture of langerhans cells , macrophages , eosinophils , multinucleated giant cells and lymphocytes ( 15 , 16 ) . most adult patients with lung involvement usually have an indolent course of regress , whereas children need to be treated ( 17 - 20 ) . ( 9 ) detected cmv in lesional langerhans cells in 30% of 29 patients by immunochemistry , in situ hybridization , and pcr , indicating that release of cytokines stimulates langerhans cell growth and induction of lch . ( 10 ) , however , did not support the hypothesis of the role of ebv , cmv , or hhv-6 , in the pathogenesis of lch in their study . ( 11 ) also failed to find evidence of genomes for adenovirus , cmv , ebv , hsv , hhv-6 , human immunodeficiency virus , human t - cell leukemia virus types i and ii , and parvovirus in 56 cases of lch employing in situ hybridization and pcr technique . we detected cmv dna in only 2 patients with lch ( positive results in 6.66% and negative results in 93.34% ) . only one in our control group had cmv virus ( positive results in 3.3% and negative results in 96.7% of controls ) , which could be attributed to a coincidence . the p value of 1.00 ( 1.00 > 0.05 ) and or : 2.07 ; 95% ci of or : 0.18 - 24.15 indicates no significant difference in cmv prevalence results between lch patients and controls . this is in keeping with some other results reported in the literature ( 10 , 12 ) . in previous studies ( 9 - 12 ) authors declared no limitations and the methods used included serology , immunochemistry , in situ hybridization , and pcr . however , immunochemistry and in situ hybridization for cmv were not available to be employed in our study and the patients were not checked for the evidence of cmv infection serologically ( limitations in our study ) . our study is the first study performed in iran on this subject and does not support the hypothesis of a role for cmv in the pathogenesis of lch , in concordance with some other studies in the literature ( 10 - 12 ) . our study is the first study performed in iran on this subject and does not support the hypothesis of a role for cmv in the pathogenesis of lch , in concordance with some other studies in the literature ( 10 - 12 ) .
background : langerhans cell histiocytosis is a rare proliferative histiocytic disease of unknown etiology . 
 histologically , it is characterized by granuloma - like proliferation of langerhans - type dendritic cells derived from bone marrow . 
 many investigators have suggested the possible role of viruses such as epstein - barr virus , human herpesvirus-6 ( hhv-6 ) , herpes simplex virus ( hsv ) types 1 and 2 , and cytomegalovirus in the pathogenesis of langerhans cell histiocytosis.objectives:in this study , we have investigated the presence of cytomegalovirus in langerhans cell histiocytosis in iranian children.patients and methods : in this retrospective study , we have investigated the presence of cytomegalovirus dna expression , using paraffin - embedded tissue samples of 30 patients with langerhans cell histiocytosis and 30 age and site - matched controls by qualitative polymerase chain reaction ( pcr ) method.results:no significant difference in prevalence of cytomegalovirus presence between patients and controls was found . 
 cytomegalovirus was found by qualitative pcr in only 2 ( 6.66% ) out of 30 patients and in 1 ( 3.3% ) of 30 control samples with a p value of 1 ( 1.00 > 0.05 ) using chi - square test with or : 2.07 ; 95% ci of or : 0.18 - 24.15.conclusions:our findings do not support the hypothesis of a possible role for cytomegalovirus in the pathogenesis of langerhans cell histiocytosis .