human papillomaviruses ( hpvs ) are small double - stranded dna tumor viruses infecting oral mucosa and genital tracts ; many hpv genotypes are found in both these localizations . hpvs responsible for benign epithelial hyperproliferation are named  low - risk  types ( hpv - lr ) , while hpvs associated with premalignant lesions and invasive squamous cell carcinoma are named  high - risk  types ( hpv - hr ) [ 5 , 23 ] . in the head and neck , hpv infections may result in benign lesions of the oral cavity and larynx or in squamous cell carcinoma in the oropharynx . hpvs are not only found in cancer tissues but also detected in normal oral mucosa of healthy individuals . although hpv infection is transmitted mainly by sexual contacts , it may also be transmitted by skin - to - skin contacts . it has been proposed that hpv infection of normal skin is acquired very early in infancy . several modes of hpv transmission during birth were proposed by birth canal , transplacental infection or amniotic fluid . in the oral cavity , 24 hpv genotypes ( hpv-1 , 2 , 3 , 4 , 6 , 7 , 10 , 11 , 13 , 16 , 18 , 30 , 31 , 32 , 33 , 35 , 45 , 52 , 55 , 57 , 59 , 69 , 72 and 73 ) have been shown to be present in benign lesions and 12 hpv genotypes ( hpv-2 , 3 , 6 , 11 , 13 , 16 , 18 , 31 , 33 , 35 , 52 and 57 ) in malignant lesions . according to syrjanen the list of hpv genotypes found in the oral cavity is even longer and includes also hpv-58 genotype . many reports about oral hpv prevalence in the oropharynx in geographically distant human populations are available from cross - sectional surveys [ 3 , 10 , 17 , 19 , 20 , 22 ] . however , different methodological approaches make difficult to compare hpv frequencies and patterns of hpv genotypes in normal oral mucosa among studied groups . it should be noted that there are very few population - based screening programs restricted to small areas in poland . the purpose of the present study was to investigate the prevalence and genotypes of hpv in the oral cavity of children and adolescents in the wielkopolska region , poland . it is the first epidomiological cross - sectional survey conducted in children and adolescents in poland . our data provide novel information , still missing in poland , about hpv occurrence in the oral cavity / oropharynx in young polish population , aged 1018 . data were collected in years  20082009 as part of a large ongoing cross - sectional population - based survey of adolescents in the wielkopolska region named adopolnor ( www.adopolnor.eu ) . this region , with 3.4 million population , is situated in the central west of poland and covers 29,826.51  km . the study population consisted of 2,028 boys and 2,122 girls , aged 1018  years , pupils of all types of schools , residing in rural and urban areas of the wielkopolska region . the examination included body height and weight measurements and an extensive questionnaire where adolescents reported their state of health , health - risk behaviours , such as smoking cigarettes , drinking alcohol , taking drugs , and practicing risky sexual activities and socioeconomic life conditions of their households . oral squamous cells from all the studied subjects ( n  =  4150 ) were collected as an oral rinse with a saline solution ( 5  ml ) and processed to cellular dna extraction , as previously described . dna concentration obtained from buccal swabs ranged from 10 to 30  ng / ul . all these steps were performed as described in previous studies [ 6 , 13 ] . for statistical analysis , all subjects were grouped by age ( 1012 ; 1315 , and 1618  years ) , type of school , and residence . the urbanization factor was categorized as follows : place of residence  village ( up to 20,000 residents ) , town ( up to 500,000 residents ) , and city ( over 500,000 residents ) . the pearson 's chi - square test was used to estimate relation between hpv prevalence and variables of interest . data were collected in years  20082009 as part of a large ongoing cross - sectional population - based survey of adolescents in the wielkopolska region named adopolnor ( www.adopolnor.eu ) . this region , with 3.4 million population , is situated in the central west of poland and covers 29,826.51  km . the study population consisted of 2,028 boys and 2,122 girls , aged 1018  years , pupils of all types of schools , residing in rural and urban areas of the wielkopolska region . the examination included body height and weight measurements and an extensive questionnaire where adolescents reported their state of health , health - risk behaviours , such as smoking cigarettes , drinking alcohol , taking drugs , and practicing risky sexual activities and socioeconomic life conditions of their households . oral squamous cells from all the studied subjects ( n  =  4150 ) were collected as an oral rinse with a saline solution ( 5  ml ) and processed to cellular dna extraction , as previously described . dna concentration obtained from buccal swabs ranged from 10 to 30  ng / ul . all these steps were performed as described in previous studies [ 6 , 13 ] . for statistical analysis , all subjects were grouped by age ( 1012 ; 1315 , and 1618  years ) , type of school , and residence . the urbanization factor was categorized as follows : place of residence  village ( up to 20,000 residents ) , town ( up to 500,000 residents ) , and city ( over 500,000 residents ) . the pearson 's chi - square test was used to estimate relation between hpv prevalence and variables of interest . there were slightly more girls than boys in the study sample ( 51.13% versus 48.87% ) . for more detailed description of sociodemographic data of the participants evaluated in the investigation , refer to table  1 . table  1general characteristics of the study samplevariablesnumber%genderboys2,02848.87girls2,12251.13age ( years)1012 ( primary school)1,31331.641315 ( secondary school)1,55137.371618 ( high school)1,28630.99place of residencevillage1,60338.63town : 21,000500,000 inhabitants1,67540.36city over 500,000 inhabitants80719.45socio - economic status ( ses)on the basis of complex indicator)low1,33832.24average1,30131.35high1,40133.76sexual intercourseno2,35686.52yes , once1033.78yes , more than once2649.6965 individuals did not state their place of residence ( blank space in the questionnaire)one hundred and ten individuals did not state their ses ( blank space in the questionnaire)only adolescents ( 1315 and 1618 age categories ) were asked with question about sexual intercourse ( n  =  2,837 ) . 114 individuals did not want to answer this question ( blank space in the questionnaire ) , thus n  =  2,723 in this category general characteristics of the study sample 65 individuals did not state their place of residence ( blank space in the questionnaire ) one hundred and ten individuals did not state their ses ( blank space in the questionnaire ) only adolescents ( 1315 and 1618 age categories ) were asked with question about sexual intercourse ( n  =  2,837 ) . 114 individuals did not want to answer this question ( blank space in the questionnaire ) , thus n  =  2,723 in this category the total prevalence of hpv in the oral cavity was 1.08% and four different hpv genotypes were detected . no correlation was found between hpv prevalence and diseases of upper respiratory tract diagnosed by clinicians in the study group ( including chronic diseases , such as asthma , tuberculosis , mucoviscidosis ; these variables were not included in table  2 ) . the distribution of positive cases for infections with hpv was almost even as far as the different sizes of places of residence in the wielkopolska region is concerned . for details , see table  2 . correlations between hpv prevalence in the oral cavity and age , gender , socioeconomical status , as well as several risk factors enhancing hpv - related cell transformation , as described in literature ( smoking , alcohol consumption , and sexual intercourse history ) , were sought , but none was observed . table  2hpv prevalence and selected features of the study samplevariablescategorynumber of hpv - infected individualschi - squaredfp valuegenderboys210.08610.7690girls24age101293.5620.1685131522161814place of residencevillage : up to 20,000 inhabitants135.59520.0610town : 21,000500,000 inhabitants17city : over 500,000 inhabitants15seslow130.4120.8150average16high16sexual intercourse*no342.2920.3180yes , once0yes , more than once2*only adolescents ( 1315 and 1618 age categories ) were asked with question about sexual intercourse ( n  =  2,837 ) . 114 individuals did not want to answer this question ( blank space in the questionnaire ) , thus n  =  2,723 in this category hpv prevalence and selected features of the study sample * only adolescents ( 1315 and 1618 age categories ) were asked with question about sexual intercourse ( n  =  2,837 ) . 114 individuals did not want to answer this question ( blank space in the questionnaire ) , thus n  =  2,723 in this category after the initial my - pcr amplification , over 53 samples could be classified as positive for hpv dna presence . in order to confirm these results , a second round of my - pcr was performed ; eight probes were negative for the presence of 450-bp hpv amplicon and were excluded from further analysis to avoid overestimation of positive results . it might have been due to the lack of viral dna stability and/or too low viral dna copy numbers very close to the detection threshold using my - pcr method . in any case , the 450-bp hpv amplicon was redetected in 45 probes . the presence of hpv dna was additionally confirmed and checked for hpv genotypes by direct dna sequencing from pcr reaction tube ( without purification from my09 and my11 oligonucleotides ) , even though the ethidium bromide signal from the 450-bp hpv amplicon had been very weak ( hardly visible ) in agarose gel after electrophoresis of several probes . in general ,  seq files  contained sequences of perfect readability covering over 300400 nucleotides ; in three cases , only 50100 nucleotides could have beed read because of numerous n  nucleotides ( background noise ) . with the help of the corresponding chromatogram file in our study group , none of hpv - hr types was detected , while among hpv - lr types , hpv-11 ( 38/45 hpv - positive cases ) , hpv-6 ( 3/45 hpv - positive cases ) , hpv-57 ( 3/45 hpv - positive cases ) , and hpv-12 ( 1/45 hpv - positive cases ) were found ( table  3 ) . table  3prevalence of hpv genotypes in the study samplehpv genotypen ( hpv - positive cases)% ( out of n  =  4,150)hpv genotype / all hpv caseshpv11380.9238/45hpv630.073/45hpv5730.073/45hpv1210.021/45all hpv cases451.08 prevalence of hpv genotypes in the study sample a great variation in hpv rates detected in the oral mucosa of healthy individuals was reported , and it depends on the population studied and the choice of method . it is also known that hpv occurrence in the normal oral mucosa comprises sublinical and/or latent infections ; moreover , hpv infections of low viral load are frequent in the oral cavity . given the abundance of literature describing results concerning hpv prevalence in the oral cavity and oropharynx all over the world , data presented in this study are to be compared to similar surveys , which were conducted on adolescents aged 1018  years old using the same my - pcr method . in the present study , hpv-11 genotype was detected at surprisingly high rate ( 38/45 hpv - positive cases ) , while hpv-6 type , frequently reported to be present in the oropharynx , was detected only in three cases ( 3/45 hpv - positive cases ) . in our concurrent study , hpv-6 genotype has been identified in 30% cases of juvenile - onset recurrent respiratory papillomatosis among 47 patients investigated ( 312  year old ) . concordant results concerning high hpv-11 prevalence in normal oral mucosa were not reported in literature . one should keep in mind that in some cases , localized hpv-11 infection may lead to severe clinical symptoms , such as recurrent respiratory papillomatosis mentioned above , or genital warts if infection is transmitted . another important finding of our study was the hpv-57 type present in three hpv - positive samples ( 3/45 hpv - positive cases ) . this hpv type had been previously found in numerous locations ( inverted papillomas of the maxillary sinus , an oral verruca , and a genital condyloma ) . in rare cases , hpv-11 is known , especially for transformation of cells of the larynx and lungs , while hpv-57 of the nasal cavity . it was also surprising to detect hpv-12 genotype in one specimen , because in the majority of studies , it was not mentioned to infect buccal mucosa [ 9 , 21 ] and is rather considered to be etiologically significant in cutaneous epidermodysplasia verruciformis ( ev)-associated hpv ( ev - hpv ) . however , hpv-12 tends also to persistently infect within oral mucosa as shown in the study on hpv prevalence in japan . in the present study hpv - hr genotypes were not detected , whereas their presence was reported at relatively high rate in similar studies by other researchers [ 19 , 20 ] . it may be due to the my - pcr method , solely employed in our experimental procedure . it has been reported that type - specific nested pcr greatly improves detection limit of hpv-16 in buccal samples , setting it below ten viral dna copies . however , in one study performed with the sole usage of my09 and my11 oligonucleotides in pcr reaction , hpv-16 dna was detected in one sample ( among 662 screened ) . these results could support ours , in which case , indeed hpv - hr types were not present in the study sample of polish adolescents or hpv - hr dna load was significantly lower ( several viral copies ) than the viral load of detected hpv - lr genotypes . the overall hpv prevalence in the study group was 1.08% , and its distribution was even among different strata of age . this observation remains in opposition to those from other studies establishing that hpv prevalence depends strongly upon age . whereas the hpv prevalence reported herein is low and consistent with the results mentioned above ; where the mean hpv prevalence in the 1215  year - old group of american adolescents was 1.5% . in the present study correlations between hpv , prevalence and other variables concordant results were described in a number of studies [ 19 , 20 ] ; where sociodemographic gender , sexual intercourse ( only adolescents over 13  years old were asked the question about their sexual activity ) , smoking cigarettes , and drinking were taken into account . in our study correlation of hpv , prevalence with upper respiratory chronic diseases ( such as asthma , tuberculosis , mucoviscidosis ) was also sought but was not observed . however , it is noteworthy that in several schools , localized in small towns around pozna and in pozna itself , the number of hpv - positive children / adolescents was elevated ( not included in table  2 ) . a non - invasive method for oral squamous cells collection , dna extraction , and hpv dna detection described herein are suitable for large cross - sectional studies of children and adolescents .
human papillomaviruses ( hpvs ) are a very complex group of pathogenic viruses , with more than 80 types , causing human infection . 
 given the prevalence of hpv infection and its relationship with the development of cervical and many other cancers , hpv vaccine development has been a major public health initiative worldwide in the last decade . 
 the aim of the presented study was to identify hpv dna by my - pcr in 4,150 school children and adolescents , aged 1018  years in the wielkopolska region , poland . 
 all individuals were asked to fill in extensive questionnaires ; further normal , oral squamous cells were collected from each pupil . 
 cellular dna was isolated and used as a my - pcr template to estimate the incidence of hpv - active infection . 
 forty five subjects ( 1.08% of the sample ) were carriers of oropharyngeal hpvs . 
 hpv status and variables of interest , such as age , gender , socioeconomical status , and risk factors ( smoking and sexual intercourse history , alcohol consumption ) were not correlated . 
 the presence of hpvs in the oral cavity was cumulated in several schools of the region . 
 dna sequencing of my - pcr products revealed only four hpv genotypes . 
 the most frequent genotype was hpv11 ( 38/45 hpv - positive cases ) , while other more rare genotypes were hpv6 ( 3/45 ) , hpv12 ( 3/45 ) , and hpv57 ( 1/45 ) . 
 conclusion : our findings presented herein , reveal a relatively low prevalance of oropharyngeal hpvs in polish adolescents and fill an important gap in the knowledge of oral hpv infections of children above 10  years and adolescents .