the human papillomavirus ( hpv ) belongs to the papillomaviridae family and persistent infection of high - risk hpv is the direct cause of cervical carcinoma , which is the second most common malignancy among women worldwide .
hpv detection and genotyping is the most effective and accurate approach in screening of the early cervical lesions and cervical cancer . with hpv genotyping becoming more prevalent , over 120 types of hpv
have been identified , of which at least 40 types are indicated to infect the genital epithelium .
its genotypes are generally classified into high - risk ( hr- ) and low - risk ( lr- ) groups based on their carcinogenic potential .
hr - hpvs include hpv 16 , 18 , 31 , 33 , 35 , 39 , 45 , 51 , 52 , 56 , 58 , 59 , 68 , 73 , 82 , and so on , and the lr - hpvs include 6 , 11 , 40 , 42 , 43 , 44 , 54 , 61 , 70 , 72 , 81 , cp6108 , and so on . it should be noted that it is of particular interest to know the difference in the clinical properties of cervical neoplasia according to hpv types , which will help us in estimating the gravity of the disease and evaluating the prognosis after therapy according to different hpv types . although there are geographical differences in the distribution of hpv types among populations , globally , it has been shown that hpv 16 , 18 , 45 , 33 , and 31 are the most prevalent hpv types associated with cervical cancer .
it is of indicated that a vaccine that included the 7 most common hpv types can prevent 87.4% of cervical cancer worldwide .
therefore , it is of considerable clinical value to establish a reliable and convenient method to detect and genotype hpv .
food and drug administration ) commercially available method for the detection of hpv dna , the well - established hybrid capture ii system ( hc - ii ) has been proven to be a sensitive and reliable assay , which can detect 13 types of carcinogenic - hpv types or 5 types of low - risk hpv in a single test [ 6 , 7 ] . however , its main undeniable limitation is that hc - ii can not distinguish between different hpv genotypes definitely [ 5 , 8 ] .
recently , a new hpv - genotyping method combining two advanced techniques , the flow - through hybridization and gene chip ( hybrimax ) has been used to detect and genotype hpv , which can distinguish 21 different types of hpv dna in a single test and diagnose multiple infections [ 911 ] . in this study , we have evaluated the efficacy of hybrimax on cervical hpv genotyping through comparison of the results with hybrid capture ii ( hc - ii ) and in situ hybridization ( ish ) .
we showed that the most common hpv types tested by hybrimax in different grades of cervical disease could be determined , suggesting that hybrimax is an efficient method for hpv genotyping and is more suitable for clinical use .
591 out of 7520 women who accepted liquid - based cytology examination in china - japan friendship hospital from august 2004 to may 2005 were randomly selected for detection of the 21 hpv genotypes by hybrimax , and their mean age was 35.4  7.7 ( ranging from 20 to 64 ) . among them , 138 women ( mean age was 35.8  7.8 ) , who diagnosed within normal limits with cervical cytology for at least two years without any cervical disease or operation , were described as 
a total of 453 women were diagnosed with abnormal cytology , and the mean age was 34.1  6.9 .
cytological diagnosis ( according to the 2001 bethesda system ) of those 453 patients were as follows : 161 cases with atypical squamous cells ( asc ) , 187 cases with low - grade squamous intraepithelial lesion ( lsil ) , 105 cases with high - grade squamous intraepithelial lesion ( hsil ) , or squamous cell carcinoma ( scc ) .
patients were classified into 6 groups according to their histopathology diagnosis from specimens of olcposcopic biopsy , loop electrosurgical excision procedure ( leep ) , or cold - knife conization .
these 6 groups include 152 cases of chronic cervicitis , 101 cases of cervical intraepithelial neoplasia ( cin , which indicates that dysplasia is seen on a biopsy of the cervix ) i ( mild dysplasia ) , 77 cases of cin ii ( moderate to marked dysplasia ) , 76 cases of cin iii ( severe dysplasia to carcinoma in situ ) , 27 cases of scc , and 20 cases of condyloma acuminata . with liquid - based cytology
, samples were taken with the cervical brush at gynecological examination for hpv dna testing .
it is prohibitive to apply vaginal douching three days prior to the collection of samples or to have sexual intercourse within one day .
hpv genotyping by hybrimax was performed using an hpv genoarray test kit ( hybribio ltd . ,
this assay can determine 21 hpv types , including 14 high - risk hpv types ( 16 , 18 , 31 , 33 , 35 , 39 , 45 , 51 , 52 , 56 , 58 , 59 , 66 , and 68 ) , five low - risk hpv types ( 6 , 11 , 42 , 43 , and 44 ) , and two unknown - risk types ( 53 and cp8304 ) , by the flow - through hybridization technique using hpv dna amplified by pcr .
in brief , 0.5  ml specimen was centrifuged at 13,000  g for 15  min with kubota 6930 . then
, the supernatant was removed and the pellet was resuspended in 200  l pbs buffer .
high - quality dna was yielded from lysis of cells by isolation of dna , precipitation , and purification .
the instrument used for pcr amplification was pe 9600 thermal cycler ( applied biosystems , usa ) .
we prepared the pcr master mix by mixing 19.25  l pcr - mix solution and 0.75  l dna taq polymerase for each reaction , adding 5  l dna template in each tube , and then running the amplification program .
the amplification program was denaturing at 95c for 9  min , 40 cycles at 95c for 20 seconds , 55c for 30 seconds , and 72c for 30 seconds , and finally extension at 72c for 5  min .
the flow - through hybridization was made on a prewarmed instrument at 45c , and the hybrimem hpv-21 dna microarray membrane was placed , which is marked with 21 hpv - genotype probes including hpv 6 , 11 , 42 , 43 , 44 , 16 , 18 , 31 , 33 , 35 , 39 , 45 , 51 , 52 , 53 , 56 , 58 , 59 , 66 , 68 , and cp8304 .
the number of samples tested in a batch could be adjusted from 1 to 15 as required .
the pcr products were denatured at 95c for 5  min just before hybridization and then was chilled on ice for at least 2  min .
we mixed the pcr products with hybridization solutions and then added the mixture into sample wells to proceed with flow - through hybridization for about 510  min .
the membrane was washed with hybridization solution , and the empty space was blocked without reaction . adding nbt / bcip solution to display the results , a positive result was indicated by a clearly visible indigo dot .
the hpv - genotype result was determined according to the position of the hpv - genotype probes on the microarray chip .
413 samples were detected by the commercially available hc - ii assay ( digene co. , gaitherburg , md , usa ) .
the probes used were designed to detect 13 types of high - risk hpv , including 16 , 18 , 31 , 33 , 35 , 39 , 45 , 51 , 52 , 56 , 58 , 59 , and 68 .
this enzyme - linked immunosorbent assay is based on a sandwich hybridization followed by a nonradioactive alkaline phosphatase reaction with chemiluminescence in the microplate .
samples were classified as positive for hpv dna if their chemiluminescence was more than 1.0  pg / ml of control .
it was performed with rembrandt universal dish and ap detection kit ( panpath , the netherlands ) . after being dewaxed and hydrated , paraffin sections ( 5 - 6  m )
specimens were denatured at 95c for 5  min by applying the probe solution to them and hybridized at 37c for 2 hours .
then , they were incubated with pan wash at 37c for 15  min except the positive control .
we dropped the conjugate by heating for 30  min and the nbt / bcip substrate by heating for 10  min at 37c in the dark and finally counterstaining and mounting .
the statistical analysis was performed using the spss software ( version 10.0 ) for chi - square test and kappa index was calculated to evaluate the accordance of the results .
the positive rate of hybrimax for 13 types of hr - hpv ( detected by hc - ii ) was 73.7% ( 303/413 ) and the positive rate of hc - ii was 70.7% ( 292/413 ) ( table 1 )
. in general , hpv - detection rates by hybrimax agreed well with those of hc - ii with a total accordance rate of 92.5% .
it is shown that the accordance rates were excellent in general ( kappa index ( ki ) = 0.814 ) in the group with normal cytological diagnosis ( ki = 0.750 ) , chronic cervicitis ( ki = 0.781 ) , condyloma acuminata ( ki = 0.80 ) , group cini ( ki = 0.755 ) , cin ii ( ki = 0.723 ) , and good at group cin iii ( ki = 0.547 ) ( 0.75 > ki > 0.4 , good ; ki  0.75 , excellent ) .
in addition , we found that there were 21 cases with hc - ii - negative and hybrimax - positive diagnoses , including 8 cases of hpv18 ( or multiple infections that include hpv18 ) , 5 cases of hpv 68 ( or that include hpv 68 ) , 4 cases of hpv 31 ( or that include hpv 31 ) , 2 cases of hpv 33 ( or that include hpv 33 ) , and 2 cases of hpv 51 ( or that include hpv 51 ) .
another two cases with genotyping results of hpv 66 , 2 cases with hpv 6 , one case with hpv 6/11 , and one case with hpv 44 by hybrimax were hc - ii positive , the results of which may be out of the hc - ii testing limits . 

it is shown that the accordance rate of the two methods was 89.1% , and their kappa index was 0.776 . 

the total hpv - positive rate of patients with abnormal cytological diagnosis was 89.6% ( 406/453 ) , with 80.9% in group a , 90.1% in group b , 92.2% in group c , 97.4% in group d , and 100% in groups e and f. in the group of patients with normal cytological diagnosis , the positive rate was 30.4% .
the 10 most common genotypes and their infection rates with abnormal cytological diagnosis in turn ( descending ) were hpv 16 ( 28.9% ) , 52/58 ( 19.0% ) , 18 ( 16.8% ) , 33 ( 9.9% ) , 31 ( 9.7% ) , 81 ( 8.4% ) , 53 ( 8.6% ) , 68 ( 8.4% ) , 66 ( 5.1% ) , and 43 ( 0% ) .
the 10 most common genotypes in normal groups were hpv 16 ( 8.0% ) , 68 ( 7.2% ) , 18 ( 6.5% ) , 52/58 ( 3.6% ) , 11 ( 2.9% ) , 53 ( 2.2% ) , 31/39 ( 1.4% ) , and 33 ( 0.7% ) , while hpv 35 , 45 , 59 , 66 , 42 , 43 , and 44 were not detected .
the 10 most common genotypes in different groups ( in descending order ) were as follows : hpv 16 , 18/58 , 52 , 31 , 53 , 68 , 81 , 33 , and 39 in the group of chronic cervicitis ; hpv 58 , 16/52 , 18 , 33 , 68 , 53 , 56/81 , and 31 in the group of cin ; hpv 16 , 52 , 58 , 18 , 33/81 , 31/51 , 53 , and 68 in cin ; hpv 16 , 58 , 52 , 18 , 31 , 33 , 81 , 53/68 , and 66 in cin iii ; hpv 16 , 18 , 52 , 58 , 33 , 66 , 68 , and 31/51/53 in group of scc .
we come to the conclusion that hpv 16 , 18 , 52 , 58 , 33 , and 31 were the 6 most common hpv types that can infect the patients with cervical lesions .
the most common hpv types causing condyloma acuminata were hpv 11 ( with a total positive rate of 55.0% ) and hpv 6 ( with a total positive rate of 30.0% ) .
the positive rates of the 6 most common genotypes in different groups were shown in figure 1 .
hpv 16 was the most frequent type in almost all the groups ( except for cin i , less than hpv 58 ) .
hpv 18 was the second most frequent type in scc , but the fourth most frequent type in all groups of cin .
similarly , the positive rates of hpv 18 increase with the development of disease . hpv 52 and hpv 58 were the third and fourth most common types in the group of scc , but the second and third most frequent types in all groups of cin .
hpv 33 was the fifth most frequent type in the group of scc , and the positive rates increase with the progress of disease in general .
previous studies have documented that hpv plays a central role in the etiology of cervical cancer [ 12 , 13 ] . it is indicated that women positive for hpv dna have a risk of developing cervical cancer 1550 times higher than those without hpv dna .
therefore , it is a preferred approach to combine the liquid - based cytology diagnosis with hpv dna testing in cervical - cancer screening .
traditional hpv - genotyping methods , such as southern - blot hybridization , direct sequencing , and restriction fragment length polymorphisms ( rflp )   based on polymerase chain reaction ( pcr ) are unsuitable for clinical use due to various reasons , such as low sensitivity , difficultly of handling , and time consumption .
ish is an easy to handle , reliable method for hpv detection and typing , working on pap smears and paraffin - embedded sections [ 17 , 18 ] ; however , its sensitivity and genotype detection are limited .
hc - ii , as the most wildly accepted hpv - dna - testing method for clinical use , is considered to be reliable , sensitive and easy to handle [ 6 , 7 ] .
however it has a limitation in discriminating hpv genotype and multiple infections , for hpv infection can only be attributed to a  low - risk  or  high - risk  group [ 5 , 8 ] .
flow - through hybridization is the most efficient method for molecular hybridization [ 911 ] .
a newly developed biotechnology named hybrimax , combining two advanced techniques , the flow - through hybridization and gene chip , is developeded to be used in clinical practice for the detection and genotyping of 21 different types of hpvs at one test .
the hpv genotypes detected by hybrimax include not only the 5 low - risk hpvs and 13 high - risk hpvs that hc - ii can detect , but also hpv 66 , 53 , and cp8304 that hc - ii can not detect .
in addition , hybrimax provides much more information than that afforded by hc - ii . in this study
, our results indicated that hybrimax was highly comparable to hc - ii in the detection of 13 types of hr - hpv , and has good accordance with ish in the detection of hpv 16/18 .
therefore , it is suggested that hybrimax can serve as an ideal method for hpv genotyping . among the 21 cases which hc - ii diagnosed negative while hybrimax revealed positive
, there were 8 cases of hpv 18 and 5 cases of hpv 68 , which probably suggested that hc - ii was less sensitive to those hpv types .
there were some samples detected hpv 6 , 11 , 44 , and 66 positive by hybrimax , present also positive by hc - ii , which should be negative ( not included in the range of genotype which can detected by hc - ii ) .
it was indicated that there was cross - reaction between the probes of the hc - ii hpv types .
it had been reported that the probes of hc - ii can have a cross - reaction with less than 22 types of hpv dna other than 13 types of hc - ii .
a potential disadvantage of hybrimax comes from the procedure of pcr , which generally was confronted with the problem of contamination .
in addition , this study revealed that , in china , the 6 most common genotypes in cervical lesions were hpv 16 , 18 , 52 , 58 , 33 , and 31 included in cervical cancer .
the recent international prevalence surveys by the international agency of research on cancer ( iarc ) reported that the most common hpv types of invasive cancer were 16 ( 57.4% ) , 18 ( 16.6% ) , 45 ( 6.8% ) , 31 ( 4.3% ) , 33 ( 3.7% ) , 52 ( 2.5% ) , 58 ( 2.3% ) , 35 ( 2.2% ) , 59 ( 1.5% ) , and 56 ( 1.3% ) , but the study did not include the chinese population . a meta - analysis made by clifford in 2003 revealed that in cases from asia , hpv 58 ( 5.8% ) and 52 ( 4.4% ) were more common than hpv 45 , 31 , and 33 , which supports our results .
a large - scale survey on the hpv types of 809 cervical cancer cases in china showed that hpv 16 and hpv 18 were the first and second most common hpv types , and hpv 58 and 52 were the third and fourth most common genotypes , followed by 31 and 33 , which also supported our results .
this study also found that the positive rates of hr - hpvs in groups of abnormal cytological diagnoses were prominently higher than that of normal groups .
therefore , it is suggested that hpv detection is especially important for women with abnormal cytological findings .
 persistent infection of high - risk human papillomavirus ( hpv ) has been recognized as the direct cause of cervical carcinoma . therefore , detection and genotyping of hpv are important to cervical - cancer screening . in this study , we have evaluated the efficacy of flow - through hybridization and gene chip ( hybrimax ) on hpv genotyping through comparison of the results with hybrid capture ii ( hc - ii ) and in situ hybridization ( ish ) . 
 591 women were classified into 6 groups according to their histological diagnoses . 
 the overall accordance rate on 13 types of hpv genotypes between hybrimax and hc - ii were 92.5% and 100% in the cancer group . 
 the overall accordance was excellent with the kappa index ( ki ) of 0.814 . 
 the value of ki in each group was 0.750 ( normal cytological diagnosis ) , 0.781 ( chronic cervicitis ) , 0.80 ( condyloma acuminatum ) , 0.755 ( cervical intraepithelial neoplasia ( cin ) 
 i ) , 0.723 ( cin ii ) , and 0.547 ( cin iii ) ( 0.75 
 > ki > 0.4 , good ; ki  0.75 , excellent ) . the 10 most common hpv subtype detected by hybrimax 
 were 16 , 52/58 , 18 , 33 , 31 , 81 , 53 , 68 , and 66 in patients , and 16 , 68 , 18 , 52 , 58 , 11 , 53 , 31/39 , and 33 in normal controls . in conclusion 
 , hybrimax is an efficient method for hpv genotyping and more suitable for clinical use . 
1. Introduction
2. Material and Methods
3. Results
4. Discussion