HPV-DNA was identified using two methodologies: multiplex PCR (PGMY09 and MY11 primers) and nested-PCR . Perinatal transmission was considered when concordance was found in type-specific HPV between mother/newborn or mother/child. HPV-DNA genital was detected in 49 pregnant women submitted to delivery. Eleven newborns  were HPV-DNA positive. In 8 cases  there was type specific HPV concordance between mother/newborn samples. At the end of the first month of life three children  became HPV-DNA positive, while two remained positive from birth. In 3 cases  there was type specific HPV concordance between mother/newborn samples. In the 6th month, a child  had become HPV-DNA positive between the 1st and 6th month of life, and there was type specific HPV concordance of mother/newborn samples. All the HPV-DNA positive children  at birth and at the end first month of life  became HPV-DNA negative at the age of 6 months. The HPV-DNA positive child  from 1st to the 6th month of life became HPV-DNA negative between the 6th and 12th month of life and one child had anogenital warts. In the twelfth month all  the children studied were HPV-DNA negative. A positive and significant correlation was observed between perinatal transmission of HPV-DNA and the immunodepression of maternal variables . Finally, the study suggests that perinatal transmission of HPV-DNA occurred in 24.5% (n = 12/49) of the cases studied.The purpose was to study the perinatal transmission of human papillomavirus DNA (HPV-DNA) in 63 mother-newborn pairs, besides looking at the epidemiological factors involved in the viral DNA transmission. The following sampling methods were used: (1) in the pregnant woman, when was recruited, in cervix and clinical lesions of the vagina, vulva and perineal region; (2) in the newborn, (a) buccal, axillary and inguinal regions; (b) nasopharyngeal aspirate, and (c) cord blood; (3) in the children, buccal was repeated in the 4 Sexual transmission of human papillomaviruses (HPV) is widely recognized as a cause of anogenital warts and cervical cancer,2. TodayHPV is a sexually transmitted disease common in adolescents and adults. It is bTherefore a prospective study was proposed in pregnant women with molecular diagnosis of genital HPV-DNA, as well as their newborns, in order to seek a better understanding of the risks of perinatal transmission of DNA virus, and also to study a few maternal variables that might be correlated with DNA virus transmission.Between April 2005 and December 2008, a cross-sectional, prospective study was performed on pregnant women with a prior history of HPV infection, or who had abnormal Papanicolaou smear or genital warts, due to the high probability that they could have HPV infection. The women were referred from the Obstetrical Service of the University of Caxias do Sul and by the Basic Health Units of the Single Health System of Caxias do Sul. This study was performed with the approval of the Ethics Committee in Research at the University of Caxias do Sul, and of the Editorial and Scientific Board of the General Hospital of Caxias do Sul, and did not present a conflict of interest. The Letter of Free and Informed Consent and the epidemiological evaluation tool were obtained from all the women by individual interviews during the obstetrical examinations. At the end of the study 63 pairs of mothers/newborns were included in the research.The epidemiological study was performed taking the following variables into account: age, race, level of education, smoking, marital status, marital stability, history of immunodepression (HIV – acquired immunodeficiency syndrome), type of HPV lesion , site of HPV lesion , numbers of types of HPV-DNA in maternal genital , gestational age at the time HPV infection was diagnosed (weeks), time of labor (minutes), time of amniotic membrane rupture (minutes), type of delivery  and HPV lesion at delivery .The maternal genital samples were obtained during pregnancy, at the first visit, when the pregnant woman was recruited. The sample was obtained using a special brush for cytopathological sampling of the cervix, which was used for genital brushing in the following order: cervix and possible clinical and subclinical lesions of the vagina, vulva and perineal region. The brush was placed in a TE solution , and the material collected was kept frozen at -20°C, until the desoxyribonucleic acid (DNA) was extracted.In newborns, in the first minutes of life, buccal, body, nasopharyngeal aspirates and arterial blood were obtained from the umbilical cord samples.The swabs were collected in the first minutes of life, using the special brush for cytopathological sampling of the cervix, with which brushing was performed in the following order: buccal cavities, axillary and inguinal regions of the newborn. The brush was placed in a TE solution  and kept frozen at -20°C, until DNA was extracted.th week and 6th and 12th month of life.Collection with a brush from the buccal of the newborn was repeated in the 4® Linha Sondas Descartáveis Mercosul®, Empresa CPL Medical's Produtos Médicos LTDA), used to aspirate the upper airways  of the newborn immediately after birth, was removed. The distal extremity of the catheter (about 4 cm long) was cut and placed in TE solution , keeping it frozen at -20°C, until DNA was extracted.The distal extremity of the tracheal aspiration catheter  to obtain about 1 ml of fetal blood. The collection was performed after clamping the cord and complete delivery of the placenta and fetal membranes. The fetal blood was placed in a KMA type tube with EDTA and frozen at -20°C, until DNA was extracted.Wizard Genomic DNA Purification Kit (Promega), according to the manufacturer's specifications. In the brush samples, DNA was extracted using 600 μl of NaOH 50 mM, stirred in a vortex for 5–10 seconds and later incubated at 95°C for 5 minutes. The solution was then neutralized with 60 μl of Tris HCl pH 8.0 and kept in a freezer at -20°C, until it was submitted to the next stages.DNA was extracted in the blood and tissue samples using the After the DNA extraction methodology, the products were submitted to two different PCR methods for HPV-DNA identification and typing: PCR multiplex and type specific nested multiplex PCR.Human Papillomavirus[φ X 174RF HaeIII molecular weight marker were used. The presence or absence of HPV-DNA fragments and β-globin amplified from the oligonucleotides was analyzed in 1.5% agarose gel, in buffer TBE 0.5× with 0.3% ethidium bromide (0.1 mg/μL solution), under ultraviolet light.The DNA samples obtained from the extraction methodology were amplified in multiplex PCR, and this was composed by the PCO4 oligonucleotides (CAA CTT CAT CCA CGT TCA CC) and GH20 (GAA GAG CCA AGG ACA GGT AC), which amplified the segment of 268 base pairs (pb) of the human β-globin gene, ensuring the qualification and quantification of DNA for HPV analysis, and by the PGM09 and PGMY11 oligonucleotides, which amplify a segment of 450 pb of a preserved region of gene L1 of lomavirus. The theThe HPV positive samples were submitted to a new type of PCR-specific for viral type identification. For this purpose the RFLP (Restriction Fragment Length Polymorphism) technique was used, according to the methodology described by Bernard et al. (1994). The ampThe nested multiplex PCR (NMPCR) assay combines degenerate E6/E7 consensus primers and type-specific primers for the detection and typing of HPV genotypes 6/11, 16, 18, 31, 33, 35, 39, 42, 43, 44, 45, 51, 52, 56, 58, 59, 66 and 68. As to sensitivity and performance with clinical samples, the novel NMPCR assay is a potentially useful tool for HPV-DNA detection in epidemiologic and clinical follow-up studies, especially when accurate HPV typing and the detection of multiple HPV infections are required.The samples were amplified in the first PCR reaction using the degenerated primers GP-E6-3F (GGG WGK KAC TGA AAT CGG T), GP-E6-5B (CTG AGC TGT CAR NTA ATT GCT CA) and GP-E6-6B (TCC TCT GAG TYG YCT AAT TGC TC), W being A/T; K, G/T; R, A/G; Y, C/T and N, A/C/G/T. These primers amplify a region of 630 pb of the E6/E7 region of the 38 most common types of HPV. The nested-PCR reaction is specific and was performed for types 6/11, 16, 18, 31, 33, 42, 52 and 58, which represent the viral types that are most prevalent in the region. The priIn the study, perinatal transmission of HPV-DNA was considered when HPV type-specific agreement was observed between the samples mother/newborn or mother/child.Statistical analyses were performed with the SPSS computer software package (version 12.0 for Windows). Frequency tables were analyzed by using the chi-square test, with Pearson and likelihood ratio tests for the significance of differences between the categorical variables. The 95% confidence interval (95% CI) was calculated where appropriate. Differences in the means of continuous variables between the groups were analyzed by using nonparametric tests. In all analyses, probability values of < 0.05 were regarded as significant.The genital HPV-DNA was detected in 49 women of the 63 who underwent delivery . The distribution of the viral types identified in the maternal genital samples are shown in table When analyzing the samples obtained from buccal and body scrapings, nasopharyngeal aspirate and arterial cord blood obtained during the first minutes of life, it was observed that 11 newborns (NB)  were positive for the research of HPV-DNA Table . Of the Studying the buccal samples of infant, obtained at the end of the first month of life, it was observed that five children  had HPV-DNA+ Table . Three oth month of life, it was observed that one child  had become HPV-DNA+ between the 1st and 6th month of life  who were HPV-DNA+ from birth to the 6th month of life became HPV-DNA negative between the 6th and 12th month of life  child had anogenital warts in the 12Analyzing the demographic and behavioral factors Table  a positiThe statistical analysis did not show any significant association between the presence of HPV-DNA in the NB or child and the other variables studied Tables  and 5.Evaluating the HPV-DNA detection and typing methods it was observed that the multiplex PCR methodology identified HPV-DNA in 41 pregnant . In 31 pregnant women  only a single type of HPV-DNA was identified, and in 10 pregnant women  two or more types of HPV-DNA. The nested multiplex PCR method  identified HPV-DNA in 43 pregnant women . In 28 pregnant women  only one type of HPV-DNA was identified and in 15 pregnant women  two or more types of HPV-DNA. Together the multiplex PCR and nested multiplex PCR methods identified HPV-DNA in 49 pregnant women . In 19 pregnant women  only one type of HPV-DNA was identified and in 30 pregnant women  two or more types of HPV.The multiplex PCR method identified HPV-DNA only in two newborns or children , while the nested multiplex PCR method identified it in 13 newborns or children .HPV infection is considered a sexually transmitted disease common in sexually active young women, with an estimated prevalence between 20% and 46%,23. In pThe presence of HPV-DNA in the maternal genital area may be considered a risk factor for fetal exposure to the virus. In this study, among the types of HPV identified in the maternal genital samples, 54.9% were considered a high carcinogenic risk. HPV infection has been identified in 1% to 20%,29 of baSedlacek et al. (1989) showed tIn the study discussed here, on analyzing the samples of buccal and body scrapings, nasopharyngeal aspirate and arterial cord blood obtained in the first minutes of life, it was observed that 11 NB  were positive for HPV-DNA research, 54.9% of which were considered of high carcinogenic risk. Concordance of the type specific HPV was also observed between mother/NB in 16.3% (n = 8/49) of the pairs, characterizing the possibility of transmission of HPV-DNA intrauterine or during the delivery. The different types of HPV identified among the mother/newborn pairs  can be explained by contamination of the sample or of the PCR technique , or by infection from multiple types of HPV, or by viral subtypes and/or variants.This study also showed the efficacy of the multiple sample methodology in the newborn, eliminating false negative results for HPV-DNA research. The results obtained corroborate Mazzatenta et al. (1996) who in tth month of life, all of them became HPV-DNA negative, even if a new case of a HPV-DNA+ appeared for buccal samples. This "regression" of the presence of HPV-DNA in buccal could be explained by the silent neutralization of antibodies that have migrated transplacentally, from the mother to the fetus, and that are functionally active in neonatal circulation, or contamination by maternal infected cells in NB delivered vaginally and that tend to disappear during the first months of life. This diminished number of HPV-DNA+ children could be explained by the results shown by Kawana et al. (2003)[The behavior of the presence of HPV-DNA in newborns can be understood by prospective follow up studies and obtaining repeated samples. The present study suggests that the presence of the HPV-DNA in children of mothers HPV-DNA+ in genital sample fluctuates during the first six months of life. Out of the 11 NB who presented HPV-DNA+, only two children continued HPV-DNA+ in samples obtained from the buccal after the first month of life. Three children  became HPV-DNA+ during the first month of life. When children were evaluated in the 6l. (2003). These al. (2003), Cason el. (2003) Tenti etl. (2003) demonstrl. (2003),38.The new cases of children who were HPV-DNA+ in the buccal at the end of the first (n = 3) and sixth (n = 1) month of life, could be explained by the agreement (100%) of the type specific HPV between mother/NB. This agreement of HPV-DNA suggests that these children may have been exposed to HPV-DNA: during the intrauterine period or during the delivery and that the HPV-DNA was only identified after birth; or durith month of life. The incidence of anogenital warts in children has increased dramatically since 1990 [One  child had anogenital warts (HPV-DNA type 6/11) in the 12nce 1990 -43. Befonce 1990 ,43. The nce 1990  classifince 1990 ,43. For nce 1990 .In three cases no concordance of the type specific HPV between mother/NB were observed. The different types of HPV-DNA identified among the mother/NB pairs can be explained by infection from multiple types of HPV, or by viral subtypes and/or variants.The positive and significant correlation between presence HPV-DNA+ in the newborn or child and the maternal variables "history of immunodepression"  may be related to the special characteristics of the pregnancy, especially to the changes in the hormonal and immunological balance prevailing during this period, which could favor vertical transmission of the virus. In adults, the risk factors for HPV-DNA transmission have been well established. The chances of perinatal transmission and the differences in the known rates of HPV-DNA transmission, are probably more closely related to the viral load of the infected cells than to the risk factors established for HPV infection.Although the nested multiplex PCR methodology is used to identify only 9 types of HPV represented as the most prevalent in the city of Caxias do Sul, it had an excellent performance to identify maternal HPV-DNA, and also considerably increased the total number of pregnant women with infections caused by multiple viruses. In the samples of newborns, the nested multiplex PCR showed its great sensitivity and specificity to identify HPV. The use of that method was also essential to evaluate the agreement of type specific HPV-DNA between the maternal/newborn samples, thus defining the perinatal transmission rates.th month of life. In conclusion, perinatal transmission of HPV-DNA was suggested in 24.5% (n = 12/49) of the newborns of mothers with genital warts or intraepithelial lesions of the uterine cervix. Thus, a different management can be adopted for each of the different stages , both from the diagnostic and therapeutic perspective. Clinical observation of the mother and the newborn must be maintained, and preventive educational measures established for forms of HPV-DNA transmission, besides effective strategies for specific immunization.The perinatal transmission of HPV-DNA was suggested when concordance of the type specific HPV was observed between mother/NB and mother/child: eight newborns, three children at the end first month of life and a child in the 6The authors declare that they have no competing interests.RLR participated in the design of the study, sampling methods and performed the statistical analysis. RLR made substantial contributions to conception and design, acquisition of data and analysis and interpretation of data. EPS has been involved in drafting the manuscript or revising it critically for important intellectual content.EPS and JM conceived of the study, and participated in its design and coordination. RLR, EPS, JM, KPL and EZ participated in the sequence alignment and drafted the manuscript. JM, KPL and EZ carried out the molecular studies.RLR, EPS, JM, KPL and EZ have given final approval of the version to be published.


A prophylactic quadrivalent vaccine can cost-effectively reduce the incidence of cervical cancer, cervical intraepithelial neoplasia, and genital warts. We present a transmission dynamic model that can assess the epidemiologic consequences and cost-effectiveness of alternative strategies of administering a prophylactic quadrivalent (types 6/11/16/18) human papillomavirus (HPV) vaccine in a setting of organized cervical cancer screening in the United States. Compared with current practice, vaccinating girls before the age of 12 years would reduce the incidence of genital warts (83%) and cervical cancer (78%) due to HPV 6/11/16/18. The incremental cost-effectiveness ratio (ICER) of augmenting this strategy with a temporary catch-up program for 12- to 24-year olds was US $4,666 per quality-adjusted life year (QALY) gained. Relative to other commonly accepted healthcare programs, vaccinating girls and women appears cost-effective. Including men and boys in the program was the most effective strategy, reducing the incidence of genital warts, cervical intraepithelial neoplasia, and cervical cancer by 97%, 91%, and 91%, respectively. The ICER of this strategy was $45,056 per QALY. Human papillomavirus (HPV) causes cervical intraepithelial neoplasia (CIN); cervical, anal, penile, vaginal, vulvar, and head/neck cancers; anogenital warts; and recurrent respiratory papillomatoses, resulting in disease and death in both women and men , to prevent cervical cancer, precancerous and low-grade lesions, and genital warts caused by HPV types 6, 11, 16, or 18. Policymakers will need information on the epidemiologic and economic impact of HPV vaccination to formulate guidelines  vaccination strategies. A We assumed that the vaccine will be combined with current screening and HPV disease treatment practices. We defined the reference vaccination strategy to be routine HPV vaccination of girls by age 12 (F12-only)  against incident infection (HPV 6/11 or 16/18) was 90%; against associated disease the degree of protection was 100% (We assumed the cost of the HPV vaccine for 3 doses and administration would be US $360 (range $300–$500), consistent with previous analyses , quality-of-life weights, discounting, and duration of natural immunity. We also conducted a multivariate sensitivity analysis that examined a pessimistic scenario . We also examined the role of herd immunity.Model predictions generally fell within the range of values reported in the literature. Overall, HPV 6/11 steady-state prevalence among females was 0.7%, which is similar to that reported by Giuliano et al. . Without>12 years of age (2.4%) was higher than that for boys or men (1.7%) and increased with level of sexual activity (data not shown). For both sexes, prevalence increased with age, reached a peak in the 20- to 24-year age group and continuously declined thereafter.Steady-state HPV prevalence rates were higher for boys or men than for girls or women across all age groups . OverallAcross all strategies, the effect of the vaccine was to steadily reduce CIN 2/3 incidence until the system approached a steady state . The larFor genital warts, the reduction occurred sooner  and 5B. F&M12+CUF&M had the most effect on the number of cases of genital warts, CIN, and cervical cancer. Compared with screening only, this strategy substantially reduced the long-run, overall number of genital warts (97%), CIN 2/3 (91%), and cervical cancer cases (91%) among adolescent girls and women.F&M12 was less effective and more costly (dominated) than F12-only+CUF-only . The incWith 10 years’ duration of protection, vaccination reduced disease incidence steadily until ≈10–15 years after vaccination, when the loss of immunity among vaccinated persons and increased numbers of unvaccinated persons reversed these trends and caused the incidence to rise  and 6B. The long-term cervical cancer incidence and ICER were not very sensitive to changes in the degree of vaccine protection against infection and disease. However, the results were sensitive to varying vaccination coverage. For example, the impact of vaccination on cervical cancer was lower when coverage was 50% compared with 90%  and 7B. Lower discount rates resulted in higher costs and QALY for each vaccination strategy. Discounting both costs and QALY at 1% decreased ICERs of the nondominated strategies: F12-only+CUF-only had an ICER of $448/QALY, whereas the ICER of F&M12+CUF&M was $28,614 /QALY. With a 5% discount rate, ICERs of these 2 strategies increased to $10,138/QALY and $64,413/QALY, respectively. HPV prevalence and burden of HPV-related diseases increased with shorter duration of natural immunity. A higher background rate of disease made the impact of vaccination look more favorable. For example, with 10-year duration of natural immunity, F12-only+CUF-only was cost-saving, whereas the ICER of F&M12+CUF&M was $11,567/QALY.When the effects of herd immunity and benefits of prevention of HPV 6/11 were removed, the ICER of F12-only increased to $21,404. If 1 assumes a pessimistic scenario, the ICER of the F12-only+CUF-only strategy increased from $4,446/QALY to $29,053/QALY and the ICER of the F&M12+CUF&M increased from $45,056/QALY to $124,063/QALY.Because vaccination coverage rates are expected to be lower among older age groups, we assumed a rate of 50% among 15- and 18-year-olds. With these rates, F12-only+CUF-only had an ICER of $8,357/QALY compared with delaying age of vaccination to 18 years . ICERs oFinally, to estimate the additional value of preventing HPV 6/11 infection, we conducted an analysis in which we assumed that persons had no protection against HPV 6/11 infection and related disease. The results of this analysis showed that ICERs of F12-only+CUF-only and F&M12+CUF&M increased to $11,254/QALY and $74,151/QALY, respectively.We developed an integrated transmission dynamic model and economic evaluation to inform HPV vaccine policy recommendations and decisions. We gained valuable insights by comparing various vaccination strategies. In general, the results suggest that a quadrivalent HPV vaccine program that targets female adolescents and women, ages 12–24 years, can be cost-effective  when compared with other commonly accepted medical interventions  are needed. Also, given the influence utility weights have on ICERs, more studies are needed to collect health utilities data on HPV disease states.Second, we modeled only 4 HPV types and their associated diseases and assumed that HPV types have independent natural histories with no interaction among them. If cross-immunity exists between HPV types, a vaccine that reduces the prevalence of 1 type may promote the prevalence of other types through a process of competitive release. If, however, current or prior infection with 1 HPV type facilitates concurrent or subsequent infection with another HPV type, or if the vaccine provides cross-protection against other types, HPV vaccination could have the additional benefit of reducing the prevalence of HPV infection of types not covered by the vaccine , as was done in other analyses  vaccine can 1) substantially reduce genital warts, CIN, and cervical cancer, 2) improve quality of life and survival, 3) be cost-effective (across a reasonably wide range of assumptions) when administered to girls before age 12 years (with or without a catch-up program), and 4) have a cost-effectiveness ratio near or below (depending on the underlying assumptions of the model) that of several other recommended vaccines, when implemented as a strategy that combines vaccination of both girls and boys before age 12 with a 12–24 years of age catch-up program.Demographic ModelA Technical Report Accompanying Manuscript


To evaluate the effectiveness of human papillomavirus (HPV) testing in primary cervical screening. This was a cross-sectional study from the recruitment phase of a prospective randomised trial. Women were screened for HPV in addition to routine cervical cytology testing. Greater Manchester, attendees at routine NHS Cervical Screening Programme. In all, 24 510 women aged 20–64 screened with liquid-based cytology (LBC) and HPV testing at entry. HPV testing in primary cervical screening. Type-specific HPV prevalence rates are presented in relation to age as well as cytological and histological findings at entry. In all, 24 510 women had adequate cytology and HPV results. Cytology results at entry were: 87% normal, 11% borderline or mild, 1.1% moderate and 0.6% severe dyskaryosis or worse. Prevalence of HPV decreased sharply with age, from 40% at age 20–24 to 12% at 35–39 and 7% or less above age 50. It increased with cytological grade, from 10% of normal cytology and 31% of borderline to 70% mild, 86% moderate, and 96% of severe dyskaryosis or worse. HPV 16 or HPV 18 accounted for 64% of infections in women with severe or worse cytology, and one or both were found in 61% of women with severe dyskaryosis but in only 2.2% of those with normal cytology. The majority of young women in Greater Manchester have been infected with a high-risk HPV by the age of 30. HPV testing is practicable as a primary routine screening test, but in women aged under 30 years, this would lead to a substantial increase in retesting and referral rates. HPV 16 and HPV 18 are more predictive of underlying disease, but other HPV types account for 30% of high-grade disease. Cervical cancer is the second most common cancer among women worldwide, and ranks first in many developing countries . Human pIn order to evaluate the effectiveness of HPV testing in primary cervical screening, the Trial, A Randomised Trial in Screening to Improve Cytology (ARTISTIC) Trial is being conducted within the routine NHS Cervical Screening Programme in Greater Manchester. This randomised trial will compare outcomes in women whose HPV test result is concealed with those in whom it is revealed and acted upon. In this paper, we report prevalence rates of HPV 16, HPV 18 and other high-risk HPVs in relation to age, cytology and histology at entry to the trial.Between July 2001 and October 2003, women aged 20–64 years attending for routine cervical screening in four health authorities in the Manchester area  were invited to participate. The trial, which was approved by the North West Multicentre Research Ethics Committee, was explained in a letter enclosed with the invitation to attend for a routine smear. Women willing to participate were asked to sign informed consent before having a smear collected for LBC and HPV testing. This is a population-based study and is therefore representative of women in Greater Manchester, a heavily populated British conurbation.The cervical sample was collected using the broom-like device of the Thin-Prep™ (Cytyc) kit and rinsed into a vial containing PreservCyt® transport medium. The sample and consent form were sent to the Manchester Cytology Centre laboratory in Manchester Royal Infirmary if they were collected in Manchester, Salford & Trafford, or Wigan & Leigh, and to Stepping Hill Hospital cytology laboratory if they were from the Stockport area. Samples accompanied by a consent form were flagged on receipt on the laboratory database. Consent forms were checked and forwarded to the ARTISTIC trial office, which sent a letter notifying participants of their random allocation .The data reported in this study are derived from the cytology and HPV testing performed during the recruitment phase. All HPV results at entry and subsequently are concealed from the women and from clinical staff including the HPV results at 36 months. All women in the concealed arm, and those in the revealed arm who remained HPV-negative, were referred to colposcopy according to national guidelines at that time, that is (i) after three consecutive inadequate samples; (ii) after three consecutive borderline smears; (iii) after an initial borderline or mildly dyskaryotic smear followed by another showing mild dyskaryosis or worse; or (iv) following a single smear showing moderate dyskaryosis or worse.Women in the revealed arm with normal cytology at entry who tested HPV positive were invited for a second HPV test 12 months after entry. If this second test was also positive, they were invited to choose between immediate colposcopy or a repeat HPV test after a further 12 months. If this third test was again positive, women were referred to colposcopy.Primary outcome measures will include the proportion of additional CIN3 lesions diagnosed in the revealed arm in cytologically normal women who were HPV positive at entry and any resulting reduction in their CIN3 detection rate at the 3 year second screening round . Other relevant outcomes are (a) the sensitivity and specificity of persistently detectable HPV for diagnosis of CIN3 or cancer; (b) the cost effectiveness of HPV testing; (c) the psychological effects of HPV testing; (d) attendance rates among HPV positive women when recalled after 1 year for retesting and (e) their preference between immediate colposcopy and further HPV testing if they are still HPV positive. Some HPV positive women fail to return for a second HPV test after 12 months, and the trial will also provide substantial evidence on the effects of retesting HPV positive women after 3 years, which may prove as sensitive but more specific for CIN3 diagnosis compared with retesting after 1 year.ThinPrep® vials were collected from the participating practices or clinics and sent either to Manchester Cytology Centre or to Stepping Hill Hospital Cytology Laboratory. Vials not accompanied by a consent form were not processed until the consent form was received at the laboratory. Laboratory numbers were allocated to the samples before automatic staining and routine processing in cytology.Most were processed using the multi sample ThinPrep® 3000 processor (Cytyc). The single sample ThinPrep® 2000 (Cytyc) was used if an extra slide needed to be made from a sample because the initial slide appeared unsatisfactory or if only a few samples needed to be processed.Vials containing the residual sample were sent to the Virology Laboratory in Manchester Royal Infirmary for HPV testing by the Digene Hybrid Capture® 2 (hc2) assay. Laboratory numbers were allocated to the samples before automatic staining and routine processing. Procedures are in place to ensure that cross contamination by HPV does not occur in the T3000 processor and swabs are taken to ensure no contamination is present.−1 of HPV DNA. The high-risk HPV types detected by the assay are 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. We classified samples as HPV positive according to the manufacturer's instructions at the outset of the study, which was to use a positive cut off as 1 RLU/control.Cervical samples collected into Cytyc LBC medium were tested for high-risk HPV DNA by hc2 according to the manufacturer's instructions. Briefly, after treatment with conversion buffer, single-stranded DNA (ssDNA) was produced by adding denaturing fluid and heating at 65°C for 45 min. Following hybridisation of ssDNA to high-risk HPV specific RNA probes the DNA/RNA hybrids were captured onto anti-DNA/RNA antibody coated micro-titre plates prior to detection of the hybrids using an alkaline phosphatase conjugated anti-DNA/RNA antibody in conjunction with a light emitting substrate. Positive results were expressed in relative light units (RLUs) compared with a positive control containing 1 pg mlGenotyping of hc2 positive samples was carried out using the prototype reverse line blot assay supplied by Roche.μl of the pelleted sample stored at −70°C using the automated Roche MagNAPure LC system. Extracted DNA was amplified using the PGMY primer reagents provided by Roche. Biotinylated PCR product was then denatured and captured onto nylon strips coated with HPV type specific oligonucleotides. Immobilised product was visualised using streptavidin-horseradish peroxidase-mediated colour precipitation.DNA was extracted from 50 Trial participants were flagged on the routine cytology and histology records of both cytology laboratories. In order to avoid the possibility that any histology results have been obtained from the second screening round, we have censored histology records at 2 years. Name, address and date of birth are routinely used by cytology laboratories to match new smears against a woman's previous screening record. A separate database was maintained by the virology laboratory. Relevant results were sent to the trial office in Manchester regularly, where they were matched and appended to participants' records on an Access 2000 database. Stata version 8 statistical software  was usedA total of 25 020 women were enrolled of whom 397 (1.6%) had an inadequate first smear at entry including 71 (0.3%) who did not provide an adequate repeat smear. These 71 women were referred for colposcopy according to national guidelines and excluded from further analysis. The cytology results presented thus correspond to the first adequate smear. In all, 85 women had a conventional smear and no HPV test at entry, 137 had samples that were insufficient for HPV testing, 84 were younger than 20 and 133 were older than 64 years of age. There were thus 24 510  eligible women with satisfactory cytology and HPV results at entry.The lower part of The ARTISTIC trial cohort represents the largest population of women in the UK to have undergone routine cervical screening with both LBC and HPV testing. The study population spanned the 20–64 age range of screened women when the trial opened, although the lower age threshold for routine cervical screening in England has since been increased from 20 to 25 years.Our age-specific HPV positive rates in different grades of cytological abnormality were similar to those in the HART study in which over 10 000 women were screened with conventional cytology and hc2 testing, but our overall HPV prevalence was slightly higher at each age. HPV prevalence in the HART study declined from 14.5% in women aged 30–34 years to 3.8% in women aged 55–59 years  of women aged under 30 years with mild dyskaryosis are HPV positive, this proportion falls to 58% (233/398) at age 30–49 years and to only 28% (18/65) at age 50–64 years suggesting a role for HPV triage. The prevalence of borderline abnormalities in HPV positive women is about twice as high as in HPV-negative women at each age, and although there may be some overcalling by LBC, our results indicate that many borderline abnormalities are not caused by HPV. The prevalence of moderate or severe dyskaryosis in HPV-positive women is 11.6% throughout the premenopausal years suggesting that the natural history of HPV infection may be much the same in premenopausal women irrespective of age, although CIN3 is rarer in HPV-infected women aged 50 years or over. In women aged 30 years or over, our data suggest that the main effect of replacing cytology by HPV testing in primary screening would be the replacement of HPV negative abnormal smears, most of which would be borderline, by a similar number of HPV positive normal smears among women referred for follow-up. For those aged 20–29 years, however, the number who were HR HPV positive was 52% greater than the number with abnormal cytology, suggesting the need for a secondary test prior to colposcopy.Primary screening with HPV testing in combination with cytology triage has been recommended only in women aged over 30 years (The ARTISTIC trial has also provided the largest collection of HPV typed primary screening cervical samples from the UK. The HPV type may be clinically important as the proportion of hc2 positive women who were infected with HPV16 increased with cytological abnormality, from 14% in those with normal cytology to 55% in those with severe dyskaryosis . The HPVWomen are still undergoing repeat cytology and colposcopy as necessary. It is therefore not yet possible to compare the sensitivity and specificity of cytology and HPV testing for detecting CIN2 and CIN3 at entry to the trial, particularly in relation to specific HPV types. As with any quantitative test, the performance of hc2 will depend on the choice of cutoff.The second round of screening at 3 years began in July 2004 and we anticipate reporting final results at the end of 2007.


We investigated the role of infection with genital and cutaneous human papillomavirus types (HPV) in the aetiology of ocular surface squamous neoplasia  and carcinoma) using data and biological material collected as part of a case-control study in Uganda.Among 81 cases, the prevalence of genital and cutaneous HPV types in tumour tissue did not differ significantly by histological grade of the lesion. The prevalence of genital HPV types did not differ significantly between cases and controls . The prevalence of cutaneous HPV types was 22% (18/81) among cases and 3% (1/29) among controls .We find no evidence of an association between genital HPV types and ocular surface squamous neoplasia. The prevalence of cutaneous HPV was significantly higher among cases as compared to controls. Although consistent with results from two other case-control studies, the relatively low prevalence of cutaneous HPV types among cases  indicates that there remains considerable uncertainty about a role for cutaneous HPV in the aetiology of this tumour. In the years before the HIV epidemic, corneo-conjunctival intraepithelial neoplasia (CIN) and carcinoma ) were reported to be more frequent in African countries than in Europe and the USA -3. UsingSince the 1980s there has been a marked increase in cases of conjunctival neoplasia, mostly in sub-Saharan Africa -19. In Ubetapapillomavirus genus. A variety of HPV types has already been identified in some, but not in all, tumour specimens from several small case series and results from case-control studies have, to date, been inconclusive ). The most frequently detected genital type was HPV 11, which was found in 22 cases and in 10 controls. The prevalence of cutaneous HPV types was 22% (18/81) among cases and 3% (1/29) among controls  and did not differ significantly between HIV infected and uninfected cases and controls  inveThere is substantial variation in HPV prevalence rates between different studies, which may have arisen, in part, because of differences in patient selection, sample taking, preparation and storage and detection method. Even for PCR as a detection system, there are many variables that influence the sensitivity and specificity and so could impact on the reported prevalence. These include PCR design , the size of the amplified product and the choice of the polymerase used. This review was not done to draw attention to these differences, but rather to show that there is no consistent evidence for a causal association between HPV and OSSN. In addition, however, it should be noted that the total number of cases and controls studied in this and in other studies, remains relatively small.Results reported here are also broadly similar to those from case-control studies investigating the role of HPV in the aetiology of cutaneous squamous cell carcinoma (SCC) ,72. ModeThe possibility that the presence of a tumour facilitates detection of antibodies against HPV is supported by the findings of Favre et al (2000), who reported a higher seroprevalence of HPV-5 among patients with burns or with proliferative cutaneous autoimmune diseases than among controls . Patient10 PCR – DEIA (see Methods section) more than 50 HPV types can be detected. It cannot, therefore, be excluded that the two cases with an indeterminate genital HPV result actually represent a cutaneous HPV type. The SPF10-LiPA25 system amplifies a small fragment from 65 base pairs and is therefore very suitable for the testing of paraffin-embedded, formalin-fixed samples. Although the conjunctiva represent mucosal tissue, the detection of genital HPV types in 40% of the HIV seronegative cases and in 32% of the HIV seronegative controls was unexpected. This finding indicates that the natural history of HPV and their tissue tropism is not fully understood.It should be noted that the relatively high percentage of samples with unclassified cutaneous HPV types could represent infections with novel types of which only subgenomic amplicons have been sequenced . HoweverWe find no evidence of an association between genital HPV types and ocular surface squamous neoplasia. The prevalence of cutaneous HPV was significantly higher among cases as compared to controls. Although consistent with results from two other case-control studies, the relatively low prevalence of cutaneous HPV types among cases  indicates that there remains considerable uncertainty about a role for cutaneous HPV in the aetiology of this tumour.From November 1995 to May 2001 in country-wide clinics, anyone with a suspect corneo-conjunctival lesion was offered removal and histology, and enrolment in a follow-up study with home visits. HIV serology was also offered after pre-test counselling. Lesions were photographed and details of the eyes and general health were recorded and analysed in EPI INFO version 6. Those who subsequently turned out to have lesions other than ocular surface squamous neoplasia were used as a control group in the analyses of HPV.Information about the disease, its treatment and HIV testing was given in private in vernacular by counsellors, and consent confirmed by signature or thumbprint. The study was approved by the Science and Ethics Committee of the Uganda Virus Research Institute, and by the Uganda National Council for Science and Technology.Venous blood was taken and screening tests for HIV antibodies done, with confirmation at the Uganda Virus Research Institute . Biopsies went to St Thomas' Hospital London for histopathology. CIN was classified (by SBL) into 3 stages according to one, two or three thirds thickness being dysplastic; invasive tumours were diagnosed when the epithelial basement membrane was breached.10-LiPA25 system  as described previously , supplemented by searches of references in identified papers, by hand searches of relevant journals and by direct contact with authors. No restriction was placed on language of publication. No attempt was made to identify unpublished studies or to obtain unpublished data from published studies. There were no prospective studies. The odds ratios used here are either those presented in the paper or, where none were provided, they were estimated for each study by the authors, using published figures.The authors declare that they have no competing interests.KW, JM, RD and RN conducted the original study and collected all the biological material used for work described here. MK and WQ developed the HPV assays, which MK used in this study, with assistance from KP, CP and CH. SL conducted the histopathology. RN conducted the statistical analyses. The manuscript was drafted by RN and MK. All authors read, contributed to and approved the manuscript.


Cervical cancer is one of the most common malignant diseases of women; it is diagnosed in almost half a million women every year and half as many die from it annually. In Canada and other industrialized countries, its incidence has decreased due to cytology screening. However, invasive cases still occur, particularly among immigrant groups and native Canadian women. Although incidence of squamous cell carcinomas has decreased, the proportion of adenocarcinomas has increased because Pap cytology is ineffective to detect these lesions.In Canada, cervical cancer will cause an estimated 11,000 person-years of life lost. In most Canadian provinces, early detection is dependent on opportunistic screening. Primary prevention can be achieved through health education  and vaccination to prevent infection from Human Papillomavirus (HPV). The initial results from vaccination trials are encouraging but wide scale use is more than a decade away.Most cases of cervical cancer occur because the Pap smear was either false negative, was not done or not done often enough. Appropriate recommendations and guidelines exist on implementation of cytology-based programs. However, most Canadian women do not have access to organized screening. Further research is needed to 1) evaluate automated cytology systems; 2) define appropriate management of precursor lesions and 3) deliver definitive evidence of HPV testing efficacy in long-term follow-up studies with invasive cancer as an outcome and 4) provide Canadian data to justify augmenting or modifying current programs to use HPV testing in secondary triage of equivocal Pap smears. Cervical cancer is a malignant neoplastic disease that tends to begin slowly when there is a disruption of the cervical epithelium, near the squamocolumnar junction of the uterine cervix. Initially, this pre-invasive process is limited to the cervical epithelium and is known variably as cervical intraepithelial neoplasia (CIN), according to the classification scheme mostly used in histopathology, or as squamous intraepithelial lesion (SIL), as per the classification system favoured for cytopathological diagnosis. Low-grade SIL (LSIL)  and high-grade SIL  are invariably asymptomatic and can be detected through cytological examination using the Papanicolaou technique (the Pap test). Their presence is confirmed by magnification during colposcopic examination and by biopsy. If left untreated, LSIL may become HSIL, and the latter may eventually extend to the full thickness of the cervical epithelium, a condition that is recognized as cervical carcinoma in situ (CIS).Subsequently, the disease may become invasive. This process may take a decade or longer. There are two main histological types of invasive cancer: squamous cell carcinomas and adenocarcinomas. The invasive lesion may metastasize to nearby pelvic and distant lymph nodes and other body sites. The symptoms and signs in most women with invasive cervical cancer include post-coital bleeding, recurrent bladder infections and ulcers on the cervix. Pressure against nerve trunks and the sacral plexus produces persistent pain. As soon as lymph node metastasis occurs the disease worsens considerably.The women most at risk of this disease in North America are Native populations, Black women, the Hispanic population and recent immigrant groups. Screening programs may not be reaching these women, and when cervical cancer is eventually diagnosed there may be barriers in the way of speedy access to the most recent and effective treatment methods. Women of low socio-economic status also face a less favourable outcome.Data on incidence rates and rates of mortality from cervical cancer in Canada were obtained from Statistics Canada and the Ministère de la santé et des services sociaux du Québec, and in the United States from the Surveillance, Epidemiology, and End Results (SEER) Program. Other epidemiologic and risk factor information was found in published surveillance and research reports.Canada has been a pioneer among Western countries in adopting wide-scale cervical cancer screening with the Pap test, and this has greatly contributed to its current status as a country with one of the lowest incidence rates of the disease worldwide. Cervical cancer is expected to be newly diagnosed in an estimated 1,400 Canadian women in 2003 and will claim approximately 420 lives in that period.[Figure As a general rule, incidence and mortality rates of cervical cancer have declined in North America during the last 50 years in consequence of the increased availability of Pap smear screening programs and probably also the decline in fertility rates during the last half-century. Figure The decline in incidence has varied by province, as shown in Figure The decline in cervical cancer incidence in Canada can be further examined by comparing age-specific rates by successive five-year periods Figure . The redMost cervical cancers are squamous cell carcinomas. While the decline of cervical cancer incidence applies to this histological type, adenocarcinomas have actually increased in incidence over the last 30 years in most Western countries. The latter used to account for about 5% of all invasive cervical cancers in the early 1970s and now constitute approximately 20% of all such neoplasms.Cervical cancer is one of the most common malignant diseases of women. An estimated 471,000 new cases of invasive cervical carcinoma are diagnosed annually worldwide, with a disproportionately heavy burden of the disease  occurring in developing countries. In 2000 there were an estimated 233,000 deaths from cervical cancer worldwide.[Figure Cervical cancer takes a particularly heavy toll in North American Native populations, Black women, and Hispanic minorities. Black women in the United States have about a 50% greater risk of acquiring cervical cancer than white women but more than twice the risk of dying from the disease. Ethnic dAn average 26 years of life are lost per woman dying of cervical cancer in Canada. On the basis of this average loss of life and of the number of deaths each year in Canada, it is estimated that cervical cancer caused an estimated 11,000 person-years of life lost in 1997.[Figure Three main factors influence the magnitude of the survival rates: (i) the relative proportions of patients with advanced versus early disease stage, (ii) the age distribution of the cohort of patients, and (iii) access to the main treatment modalities for cervical cancer  and their combination integrated into specific treatment protocols. These thAbility to pay for health care could be a determinant of the substantially different survival rates between white and Black women in the United States and, in specific situations, between the United States and Canada. A recent study comparing cancer survival in Detroit and Toronto found that socio-economic status was associated with cervical cancer survival in Detroit but not in Toronto. Furthermore, the survival rate among the poorest patients in Toronto was significantly better than that among the poorest ones in Detroit for most types of cancer, including cervical cancer. NevertheHPVs are small, double-stranded DNA viruses. As infectious agents, they are highly specific to their respective hosts. More than 120 different HPV types, derived from DNA sequence homology, have been catalogued so far.Clinical, sub-clinical and latent HPV infections are the most common sexually transmitted viral diseases today, with a pIn epidemiologic studies conducted during the past 10 years, the relative risks (RRs) for the association between HPV infection  and risk of cervical cancer are high – in some studies greater than 100.,19 No otToday, it is well established that infection with the HPV types associated with high oncogenic risk  is the central causal factor in cervical cancer.,21 It maSee "The Role of HPV." Other prominent risk factors are the role of two measures of sexual activity, namely, number of sexual partners and age at first intercourse, and the sexual behaviour of the woman's male partner(s).,26,27Tobacco smoking has consistently emerged as a risk factor for cervical cancer. A directThe number of live births per woman is a consistent risk factor for cervical cancer. There is a linear trend in the parity-risk association, as seen in large studies in North America and in Latin America.,35 It isAn increased risk of cervical cancer among oral contraceptive (OC) users is found mainly among long-term users. The plausibility of the association rests on the potential for hormonal effects on HPV-containing cervical cells, as it has been shown that steroid stimulation may trigger viral oncogene-related events that could culminate in integration of the virus into the host's genome. ConfoundHigh intake of foods (fruits and vegetables) containing carotenoids and vitamin C and, to a lesser extent, intake of vitamins A and E seem to reduce the cervical cancer risk. The resuPatients infected with HIV are prone to develop a variety of infections attributed to their debilitated immune system. HIV infection impairs cell-mediated immunity, thus increasing the risk of HPV-associated diseases, such as genital warts and malignancies. Latent HPV infection and SIL are much more common among HIV-infected women than HIV-negative women from the same populations.,44 HPV aSquamous and adenocarcinomas present many characteristics in common; however, there is also evidence to show that these types may actually have distinct causes despite being so close together anatomically. Most risk factors are indeed common to both types. Four main features differentiate their epidemiologic and prevention characteristics:1. unlike squamous carcinomas, the incidence of adenocarcinomas has been increasing in recent years, particularly among younger women in Canada and othe2. HPV 16 is the HPV type most frequently found in squamous carcinomas, whereas HPV 18 is found in more than half of adenocarcinomas;3. increased parity is associated with an increased risk of squamous but not adenocarcinomas;4. Pap cytology (mentioned below) is not as efficacious in detecting adenocarcinomas.The implications of such discrepancies are yet to have an impact on policy recommendations because current screening recommendations are based on Pap cytology. New screening technologies need to take into account the need to incorporate more sensitive methods for detecting adenocarcinomas.Primary prevention of cervical cancer can be achieved through prevention and control of genital HPV infection. Health promotion strategies geared to a change in sexual behaviour and targeting all sexually transmitted infections of public health significance can be effective in preventing HPV infection.,51 AlthoTwo main types of HPV vaccine are currently being developed: prophylactic vaccines to prevent HPV infection and associated diseases, and therapeutic vaccines to induce regression of precancerous lesions or remission of advanced cervical cancer. Such vaccines are already under evaluation in phase I and II trials in different populations. ImmunizaCervical cancer screening is currently one of the most active areas of research in cancer prevention (see "Other Cytology Methods" below). Several new technologies are under evaluation, and professional and government groups are considering their contribution in a reassessment of practice guidelines currently being undertaken.Efficacy of Pap Cytology Screening: There have been no controlled trials of Pap screening efficacy, either randomized or not. The evidence for the efficacy of Pap smear screening in cervical cancer comes mainly from three sources: (i) epidemiologic studies reporting a risk of invasive cervical cancer 2–10 times greater among women who have not been screened and an increased risk with time since last normal smear or with lower frequency of screening; (ii) cervical cancer incidence and mortality rates, which decreased sharply following the introduction of cytology screening in Scandinavian countries, Canada and the United States, and did so in proportion to the intensity of the screening efforts; and (iii) multiple national and international consensus panels worldwide.In spite of its success, cytology has important limitations, false negative results being the most significant. One recent meta-analysis indicated that the average sensitivity of a single Pap test to detect HSIL or cervical cancer was 51%, whereas its specificity was 98%. The soluThe Canadian Task Force on Preventive Health Care and a seIn essence, the prevailing guidelines recommend that Pap screening is to begin at age 18 or at initiation of sexual activity and to continue annually. After two negative consecutive smears, one year apart, screening is to proceed every three years to age 69. The national workshop recommendations also state that this screening schedule be implemented in combination with an efficient information system allowing rapid case notification and recall. The empiThere are several automated systems being tested and marketed. In one of these, liquid-based cytology, the sample recovered from the cervix is suspended in a cell-preserving solution rather than placed on a glass slide. Excess blood and inflammatory cells are lysed, and approximately 50,000 diagnostic cells are randomly transferred by the equipment as a thin layer onto a glass slide by a robotic cell processor. The slides are stained and then read by cytotechnologists. Results from clinical studies have shown that automated thin-layer slides can improve detection of atypical cells, precursor lesions and cancer by producing uniformly cleaner slides free of blood, debris and cell clumps that interfere with microscopic reading.,61 A recComputer-assisted scanners map the smear in order to detect abnormal cells, thereby separating any slides that contain suspect images for subsequent reading by a cytotechnologist. A key advantage is the potential to alleviate the shortage of qualified workers in cytopathology. Comparative trials, mostly funded by the private sector, are taking place in many laboratories in North America and Europe to answer questions related to screening efficacy and the cost-effectiveness of automated devices.Since the mid-1990s there has been substantial interest in the use of standardized HPV DNA testing as a cervical cancer screening tool under the premise that it will provide acceptable diagnostic performance while being more reproducible and more easily adapted for clinical practice than conventional Pap cytology. Several studies have assessed the test's diagnostic performance (for high-risk types) in a variety of populations, using cross-sectional or short-term follow-up rather than more extended review of incidence or mortality rates. -69 LesioHPV DNA testing has been shown to have, on average, a 25% greater sensitivity than Pap cytology but somewhat lower specificity  for detecting CIN 2/3 or cancer. Screening of women aged 30 or older tended to improve the specificity considerably, because viral infections in this age group are less likely to be of a transient nature than those in younger women. An important finding of most studies was that the combination of cytology and HPV testing attained very high negative predictive values (approaching 100%), which, at least in theory, could safely permit longer screening intervals, thus lowering costs. Definitive evidence of efficacy is still needed from long-term follow-up studies with invasive cancer as an outcome and from randomized controlled trials.One other screening application of HPV testing is in the secondary triage of equivocal Pap smears. Results from two large-scale studies have indicated that HPV testing has greater sensitivity than a repeat Pap test for detecting hidden HSIL or cancer among women referred because of an equivocal Pap smear. At the same time it results in reduced costs in terms of colposcopy referrals.,71There is a lack of Canadian data on cervical cancer in women from different ethnic backgrounds, and this precludes preventive efforts targeted at vulnerable groups.Most cases of cervical cancer occur because of a false-negative result of the Pap test, or because the woman did not receive a Pap test at all or did not receive it often enough. Well-developed recommendations and guidelines exist as part of a succession of national consensus reports concerning the implementation and maintenance of cytology-based screening programs. However, most Canadian women do not yet have access to organized, centralized cervical cancer screening.Despite the availability of some excellent Canadian surveys of Pap test utilization among women who developed the invasive form of the disease, we still lack critical information concerning the appropriate management of precursor lesions from audit studies. Various automated systems aimed at improving the performance of the cytology test are currently being tested. Large-scale prospective studies are still needed to evaluate these systems.There have been several studies assessing the relative utility of HPV testing in addition to or compared with the Pap test as a cervical cancer screening tool; these have been cross-sectional or short-term follow-up investigations, and no randomized controlled trials have yet been published. HPV testing seems to be a promising screening approach, but definitive evidence of efficacy is still needed from long-term follow-up studies with invasive cancer as an outcome and from randomized controlled trials. One other screening application for HPV testing is in the secondary triage of equivocal Pap smears. Results from large-scale studies have indicated that HPV testing has greater sensitivity than a repeat Pap test for detecting hidden precursor lesions or cancer among women referred because of an equivocal Pap smear, while resulting also in reduced costs for colposcopy referrals. However, taking into account the totality of the evidence, we still lack Canadian data to justify augmenting or modifying current screening programs.The views expressed in this report do not necessarily represent the views of the Canadian Population Health Initiative, the Canadian Institute for Health Information or Health Canada.


HPV is important in a subset of HNSCC. Our meta-analysis determined the clinical characteristics of HPV-related HNSCC.Pubmed search terms "HPV" and "HNSCC" were used to identify 34 studies since 1980. We obtained pooled adjusted odds ratio (OR) or hazard ratio (HR) using random or fixed-effects model and compared OS depicted in forest plot.A total of 5681 patients were included. The prevalence of HPV+ tumors was 22%, with 86.7% of HPV16+ genotype. The OR for HNSCC in HPV16+ patients was 4.44 (95% CI = 2.87-6.02). HPV status was associated with p16 expression , and HPV+ tumors were less likely to harbor p53 mutations . The HR for death in HPV+ patients was 0.42 (0.27-0.57). HPV+ HNSCC also had a better response to therapy.HPV+ HNSCC are established as a separate biologic entity. Prospective trials are needed to establish the optimal therapy for HPV+ HNSCC. Squamous cell carcinoma of the head and neck (HNSCC) has an estimated incidence of 35,310 new cases in 2008 in the United States, with an expected 7590 deaths due to these cancers. The male to female ratio is approximately 2:1. Recent Recent emerging data indicates that HPV related HNSCC define a unique population of patients with distinct biology that likely should be treated separately from non-HPV related HNSCC. The aimTo identify articles, we searched the Pubmed database, combining permutations of the terms ["human papillomavirus" or "HPV] and ["squamous cell carcinoma" or "cancer"] and ["oropharyngeal" or "oropharynx" or "head and neck" or "tonsil"]. Additional articles from reference lists of retrieved articles were reviewed and included as deemed appropriate. Only articles which solely or separately reported on oropharyngeal cancers, were published from 1980-2008 in English language, and reported HPV prevalence were considered for inclusion. A total of 34 articles met our inclusion criteria for this meta-analysis.The presence of study heterogeneity was evaluated using the Chi-squared based Q test. In order to quantify the extent (estimated as a percent ranging from 0% to 100%) of any existing heterogeneity in the meta-analyses, the I2 index was used. Pooled adjusted odds ratios (ORs) for risk or hazard ratios (HRs) for overall survival were obtained using a fixed-effects model (or random-effects model if needed due to among-study heterogeneity). If adjusted ORs or HRs were not available, then we used the same methods as above to pool unadjusted point estimates of risk and survival. Potential sources of publication bias were detected using a funnel plot which is simple scatter plots of the effect of interest versus study size. An asymmetrical funnel plot is indicative of potential publication bias. If necessary, we omitted studies prior to the final meta-analytic estimate that were identified as influential  in the funnel plot. A forest plot was constructed to visually compare all included studies with the final pooled statistic. When possible, we also performed the above meta-analyses for the subset of oropharyngeal cancers. All analyses were completed using STATA 10.0.A total of 5681 patients in 34 studies were included. To detect HPV DNA in tumor biopsies, 33 studies used PCR, and one study used fluorescence in situ hybridization (FISH). We established a prevalence of HPV among all head and neck squamous cell carcinoma (HNSCC) patients of 21.95% (95% CI = 0.21-0.23). The most prevalent HPV genotype was HPV-16, representing 86.69% (95% CI = 0.85-0.89) of all HPV positive tumors  conferred an increased risk for HNSCC  as seen in Figure Survival was improved in HPV positive patients compared to HPV negative patients  Figure . The surA total of ten publications included treatment information, but only two reported statistical information on treatment response to radiotherapy and two reported efficacy results to chemotherapy/radiotherapy Table . HPV posSeveral studies reported p16 immunohistochemical expression in HPV positive HNSCC-29. We fWe have conducted a literature review and large meta-analysis in HPV related HNSCC to determine the prevalence of the disease and assess the ramifications on survival and associated molecular biomarkers. Given prior published reports of a higher prevalence of HPV in carcinomas of the oropharynx, we concentrated this study on oropharyngeal carcinomas to enrich and focus the meta-analysis. Therefore, the number of studies included in our review is smaller than other previously reported meta-analyses with different inclusion criteria,31. SeveIn the current study we confirmed the role of p16 immunohistochemical expression as a surrogate marker for HPV infection in HNSCC, which is not only positively associated with HPV infection but also predicts for an improved survival outcome,28,29. TAs HPV-related oropharyngeal cancers are reported to be rising in incidence,3, it isThere are several limitations inherent to our methodology, which was based on literature review, not individual patient data. While the total number of patients included in our meta-analysis is large, for many specific associations the number of the studies that actually could be combined to provide information is relatively small. This is a limitation which unfortunately cannot be overcome without access to primary data from all included publications. Also, there is overlap of the same patient series included in multiple reports. We tried to account for this fact as much as possible by reviewing the author list and institution of each paper and including only the largest study if several were apparently from the same group. Finally, given the heterogeneity of the kinds of studies our meta-analysis was based on, it is difficult to combine conclusions drawn, e.g. assessing HPV as risk factor for HNSCC from case series, retrospective and prospective trials. There is no valid formulation to give adequate weight to the results of each kind of the studies mentioned. In spite of the limitations inherent to this type of a meta-analysis, our results are in keeping with published literature and our current understanding of the natural biology of HPV-related HNSCC. We also performed as a validation control a meta-analysis for the established HNSCC risk factors smoking and alcohol consumption. Our results confirmed prior published results ,21.For the future, there is a need for randomized prospective data to identify the optimal treatment for patients based on the tumor's HPV status and to also develop potential prevention programs. First, given the apparent improved response to current treatment modalities, patients with HPV-related HNSCC may be able to receive adequate curative intent therapy from a reduction in radiation or chemotherapy dose. These issues will require future clinical trial designs to either prospectively stratify patients by HPV status or to conduct trials which are dedicated to HPV positive HNSCC. In addition, it is necessary to determine the best method of detecting the HPV status and validate whether p16 overexpression should be utilized as a surrogate or in conjunction with direct HPV testing. Given the etiologic involvement of the HPV oncoproteins E6 and E7 during carcinogenesis of these tumors, it is aThe issue of preventing HPV associated HNSCC from occurring requires investigation as well. Two recently approved vaccines to prevent HPV-related cervical cancers,46 coverHPV-related HNSCC comprise about 25% of all HNSCC. They are predominantly tumors of the oropharynx, and exhibit a separate biologic behavior including improved response to (chemo)-radiation and survival compared to HPV-negative HNSCC. The optimal therapy for this subset of patients remains unclear, but the growing data supports the theory that this subset of patients should be treated differently than HPV negative patients. In addition to this avenue of clinical research, identifying peripheral surrogate biomarkers and possible screening and prevention methods is an important field of future investigation.The authors declare that they have no competing interests.FD and AST designed the study, acquired and interpreted the data, and wrote the manuscript. CJE, ML, and CHH acquired, analyzed, and interpreted the data, and contributed to manuscript writing. SML contributed to study design, data interpretation and manuscript writing. All authors have given final approval of the version to be published.


However, the HPV vaccine has, to the best of our knowledge, not been tried in head and neck carcinoma. The role of HPV in etiopathogenesis, prevalence in benign and malignant lesions of this area and vaccination strategies are briefly reviewed here.Head and neck malignancies are characterized by a multiphasic and multifactorial etiopathogenesis. Tobacco and alcohol consumption are the most common risk factors for head and neck malignancy. Other factors, including DNA viruses, especially human papilloma virus (HPV), may also play a role in the initiation or development of these lesions. The pathways of HPV transmission in the head and neck mucosal lesions include oral-genital contact, more than one sexual partner and perinatal transmission of HPV to the neonatal child. The increase in prevalence of HPV infection in these lesions may be due to wider acceptance of oral sex among teenagers and adults as this is perceived to be a form of safe sex. The prevalence of HPV in benign lesions as well as malignancies has been assessed by many techniques. Among these, the polymerase chain reaction is the most sensitive method. Review of literature reveals that HPV may be a risk factor for malignancies, but not in all cases. For confirmation of the role of HPV in head and neck squamous cell carcinoma, large population studies are necessary in an assortment of clinical settings. Prophylactic vaccination against high-risk HPV types eventually may prevent a significant number of cervical carcinomas. Of the two vaccines currently available, Gardasil Head and neck squamous cell carcinoma (HNSCC) is a significant cause of cancer worldwide . IncidenTobacco and alcohol consumption are the most common risk factors for HNSCC . In addiThe participation of HPV in oral and oropharyngeal carcinogenesis was first proposed by Syrjanen et al in 1983  and thenMany detection methods have been used to study the role of HPV virus, like in-situ hybridization (ISH), Immunohistochemistry (IHC), Southern Blotting, Polymerase Chain Reaction (PCR), DNA micro-arrays and Hybrid Capture-II (HC-II) high risk and low risk detection methods. In the PubMed database, the following keywords were used for the literature survey: HPV detection methods, HPV in head and neck cancer, HPV in benign lesion, premalignant and malignant lesion of head and neck, HPV in oral pre-cancer, HPV in oral cancer, HPV in larynx, HPV in Pharynx, Expression of HPV in OSMF, detection of high risk HPV by PCR etc. The objective of this review is to summarize the various techniques of detection and the role of HPV in the etiology of benign, potentially malignant and malignant lesions of head and neck.6 D. On the basis of DNA base pair (bp) distribution, the viral DNA is divided into three parts: first a 4,000 bp region that responsible for viral DNA replication and cell transformation, second 3,000 bp region that encodes the structural proteins of the virus particles and last 1,000 bp non-coding region (NCR) that contains the origin of viral DNA replication.Human Papilloma virus is about 55 nm in diameter. It has a single circular double stranded DNA molecule and belongs to the family papillomaviridae. Its genome is made up of 7,200 – 8,000 base pairs with a molecular weight of 5.2 × 10Zur et al suggested that the genomic HPV DNA has nine open-reading frame sequences (ORFS) -present on single strand of DNA and are divided into seven early (E) and two late-phase genes (L). The transcription of viral DNA is regulated by early phase gene, while the capsid proteins  are regulated by late phase gene . The earHPV must adhere to a specific receptor protein on the keratinocytes membrane. Once the virus entered into the cell, it transforms itself of its protein coat and the viral DNA may then utilize host cell themselves. These viruses elaborate early gene proteins (E) that are able to regulate the host cell cycle, or mitotic capabilities. The E6 and E7 proteins are most important in this respect; they bind two host proteins that are regulators of the keratinocytes at the time of cell division. E6 binds to a protein designated p53, a molecule that arrests cell division. However, once bound, it is degraded and this inhibition of keratinocytes mitosis is abrogated. Likewise, E7 binds a protein termed Rb; and, similarly, cell cycle regulation is troubled.On the basis of their genotype, more than 120 types of HPV viral DNA genotypes have been fully sequenced ,27. It cAlthough HNSCC is closely linked to tobacco and alcohol use, there is an increasing incidence HPV16-related HNSCC arising in the oropharynx ,22,31. BPintos et al reported that HPV 16 was identified in tonsil-related cancers. . HPV hasDifferent methods are used to detect specific types of HPV DNA in lesions and shows varying sensitivity and specificity . The detIn situ hybridization (ISH) techniques employ the use of type-specific radioactively labeled DNA probes, which are complementary to HPV DNA sequences used for detection of viruses in the premalignant and malignant lesions of the head and neck. ISH and immunohistochemistry have low sensitivity because these tests only detect the virus when it is present in more than 10 copies of the viral DNA per cell. Lee et al studied the formalin-fixed, paraffin-embedded tissue sections through in situ hybridization using newly available probe sets and concluded that the new probe sets for HPV can be used very efficiently in clinical pathology material of HNSCC .Southern Blotting is an assay that has long been one of the standard techniques for the detection of HPV DNA. This blotting has the ability to differentiate between episomal and integrated DNA and can detect up to 0.1 copy of viral DNA per cell. This method has some technical variability and requires a significant amount of DNA. Frazer and Kay et al suggested that Southern blot may boast a theoretically higher specificity but is clearly less sensitive than PCR ,43. SimiPCR is highly sensitive detection method for specific subtypes of HPV because it detects the virus in less than 1 copy of the viral DNA per cell . UniversQuantitative PCR utilizes a fluorescent probe which helps to measure the degree of fluorescence in the reaction mixture. Ha et al reported the advantages of quantitative PCR in premalignant and malignant lesions of the oral cavity and analyzed the samples previously found to be HPV positive by other molecular methods . TermineThe HC II technique is a nucleic acid hybridization assay with signal amplification that utilizes microplate chemiluminescent detection. First, double stranded DNA is denatured by using a strong alkaline denaturation solution that converts it into single stranded DNA (ssDNA). This ssDNA is then hybridized in-solution to a cocktail of specific 13 high risks HPV RNA probes. The resultant DNA-RNA hybrids are captured onto the surface of a micro-well plate coated with particular antibodies for DNA-RNA hybrids. The immobilized hybrids are reacted with alkaline phosphate conjugated antibody and detected by cleavage of the chemiluminescent substrate. The emitted light is measured as relative light unit (RLU) in a luminometer. The intensity of the light is proportional to the amount of target DNA in the sample. Specimens with RLU greater than or equal to the mean of the three positive control values are considered HPV positive.® is approved by Food and Drug Administration (FDA) USA for routine clinical purposes. Detection of HPV DNA is carried out by using probes of 13 high risks HPV genotypes .Estimation of HPV Viral Load, a ratio of relative light unit (RLU) of the sample to mean of RLU of the three positive controls (PC), has been taken as estimate of approximate viral load, which relates to the index of intensity of HPV infection. This ratio of any specimen represents empirically a relative measure of its viral load. Cut off value (RLU of specimen/mean RLU of PC) is 1.0 pg/ml of sample. The hybridization method of HC II DigeneDNA microarray is a compilation of microscopic DNA spots on a solid surface by covalent attachment to a chemical matrix. Each gene on the solid supports referred to as spot or probe is usually less than 200 μm in diameter. Each spot has a unique sequence different from the others in the array and will hybridize only to its complimentary strand. This technique uses a DNA probe labeled with either a radioisotope or a fluorescent tag. The probe is applied to the fragment of DNA or RNA to be studied and by the rules of base pairing  "sticks" to its complementary sequence. This technology has made it possible to miniaturize methods of probe detection for DNA and allow detection of several thousand DNA or RNA sequences in one experiment.DNA microarrays have been successfully used to identify global patterns of gene expression in different human neoplasia, including head and neck cancers. . Many in® and suggested the specific expression of gene patterns in HPV-positive and HPV-negative oropharyngeal squamous carcinomas that may serve as potential biomarkers for the development of HNSCC [Ivan et al compared the cellular gene expression profiles of HPV-positive and negative in normal epithelium and oropharyngeal carcinomas with the help of Affymetrix Human U133A Gene Chipof HNSCC . DNA micDetection methods of HPV in benign lesions indicates that not all types of viral infections can cause disease and it would be essential to recognize the factors that determine its capability to induce malignant transformation in head and neck lesions. Respiratory papillomatosis associated with human papilloma virus (HPV) infection is the most common benign laryngeal neoplasm . LaryngeSeveral investigators have studied the prevalence of HPV in potentially malignant lesions, hoping to find a similar HPV prevalence as in malignant diseases. An increasing prevalence of HPV in premalignant lesions suggests that it may play a role in malignant transformation. Ha et al reported 1% prevalence of HPV 16 and 18 in premalignant oral lesions by using the quantitative PCR. . On the Furrer et al reported 41% HPV DNA detection by PCR-Southern blot analysis in premalignant and malignant lesions . OstwaldRecently, a study was conducted by the author's group with the aim to analyze the high-risk HPV DNA in oral submucous fibrosis (OSMF) and to determine the role of HPV infections. 105 cases, 33(31.4%) patients were positive for high risk HPV, while 72 68.6%) were negative. On correlating HPV with histopathological grading, 7(29.1%) patients of grade I, 11(28.9%) of grade II and 15(34.8%) of grade III OSMF were positive. While correlating addiction habits with HPV, 18 (34.6%) areca nut chewers were positive and showed a significant p value (<0.05). The results showed that the level of agreement between the HPV and koilocytosis was moderate, with a Cohen kappa value of 0.524 . The sensitivity of the hybrid capture test vis-à-vis presence of koilocytosis was 59% (95% CI: 0.4427 – 0.7363) and specificity was 91% (95% CI: 0.8102 – 0.9714) while the positive predictive value was 84% (95% CI: 0.6810 – 0.9489) and negative predictive value was 73% (95% CI: 0.6190 – 0.8330 (Unpublished data). Chen et al reported the positivity of HPV16 as 52.6% and HPV 18 as 25% in OSMF by the PCR method . Vidal e.6% were Human papillomavirus especially the high-risk (HR) types 16 and 18), are widely recognized in the promotion of tumorigenesis in the uterine cervical squamous cell carcinoma , are wid.D'Costa et al suggested that HPV 16 plays a direct role in a certain populations of oral cancers whereas in a subpopulation, HPV16 infection may be vital in the early events associated with development of potentially oral lesion to malignancy . Mishra Torrente et al reported that HPV DNA was found in 32% of laryngeal carcinoma biopsies. The genotypes identified were high-risk types, the most frequent being HPV 16. Viral DNA was integrated into the host genome (genotype HPV 16), providing supporting evidence for a role of HPV in the carcinogenic pathway of laryngeal squamous cell carcinoma . Montald®' developed by Merck which protects against HPV types 6, 11, 16 and 18 and another is bivalent Cervarix® vaccine by Glaxo Smith Kline (GSK), protects against HPV types 16 and 18. Prophylactic vaccinations against high-risk HPV types eventually prevent a significant number of carcinomas. Both vaccines are Virus like particle (VLP) vaccines has been developed for primary HPV vaccination and targets HPV 16 and 18, while Gardasil, includes a standard alum adjuvant, also targets HPV 6 and HPV 11. These vaccines are recommended for vaccinating young adolescent girls at or before onset of puberty. Gardasil®  has gained regulatory approval from U.S.-Food and Drug Administration while Cervarix®  has been approved in Australia and pending approval from regulatory agencies in the USA. (Table Currently, two prophylactic HPV vaccines, one is quadrivalent 'GardasilA. Table Giorgi Rossi et al reported that the identification of the HPV as the necessary cause of cervical cancer introduced two new tools for prevention: HPV DNA test and vaccine and suggested that vaccine and screening interventions must be integrated in a unique public health program and screening should use less aggressive protocols, to be less expensive and more sustainable and efficient . Marra eIn 2007, New South Wales, Australia implemented the National human papillomavirus vaccination program, which provides quadrivalent HPV vaccine free to all women aged 12–26 years. In Australia, favorable estimates of cost effectiveness have supported funding of a 'catch-up' programme to females up to 26 years of age. . BrotherThe Rural Southern areas of North Carolina, USA have accepted the HPV vaccine . GoncalvHPV may play a role in the carcinogenesis of head and neck malignancies. Molecular mechanisms have been able to recognize its ability to disrupt key cellular elements responsible for the regulation of cell division and apoptosis. Review of literature reveals that HPV may be a risk factor for some head and neck malignancies, but not in all cases. The prevalence of HPV in virus-related benign lesions as well as malignancies has been confirmed by many molecular techniques, among those, PCR is the most sensitive method. For the confirmation of the role of HPV in head and neck malignancies, large population studies are necessary in an assortment of clinical settings.The authors declare that they have no competing interests.All authors read and approved the final manuscript.


Human Papillomavirus (HPV) is considered as a necessary, but not sufficient, cause of cervical cancer. In this study, we aimed to assess the prevalence of HPV in a series of pre-malignant and malignant cervical lesion cases, to identify the virus genotypes, and to assess their distribution pattern according to lesion type, age range, and other considered variables. The samples were submitted to histopathological revision examination and analysed by polymerase chain reaction (PCR) for the presence of HPV DNA, followed by HPV typing by dot blot hybridisation.Of the analysed samples, 53.7% showed pre-malignant cervical lesions, and 46.3% presented with cervical cancer. Most cancer samples (84.1%) were classified as invasive carcinoma. The mean age of these cancer patients was 47.3 years. The overall HPV prevalence was 82.4% in patients with pre-malignant lesions and 92.0% in the cancer patients. HPV 16 was the most prevalent type, followed by HPV 18 and 58, including both single and double infections. Double infection was detected in 11.6% of the samples, and the most common combination was HPV 16+18.Cervical cancer appears to occur in women in a lower age range in the studied area, compared to the situation in other Brazilian regions. Furthermore, among the patients with CIN 3 and those with cancer, we observed a higher proportion of married women, women with more than one sexual partner, smokers, and individuals with less than an elementary education, relative to their counterparts.The overall HPV prevalence was 82.4% in patients with pre-malignant lesions and 92.0% in the cervical cancer patients from Northeast Brazil. HPV 16 was the most prevalent type, followed by HPV 18 and 58. The most common double infection was HPV 16+18. Cervical cancer appears to occur in women in a lower age range in the Northeast Brazil. Among the patients with CIN 3 and those with cancer, we observed a higher proportion of married women, women with more than one sexual partner, smokers, and individuals with less than an elementary education, relative to their counterparts. Squamous cell cervical carcinoma is the third most common malignancy among women around the world, especially in underdeveloped countries, and it is the second cause of cancer death in women worldwide -3. It isIn Brazil, the occurrence of cervical cancer continues to be a serious public health problem, showing remarkable differences in incidence among different regions of the country ,10. ArchWe have analysed a large series of cervical specimens of archival formaldehyde-fixed, paraffin-embedded tissues from women in a high-risk area for cervical cancer in northeastern Brazil. The samples were re-examined, and the lesions were histologically classified and analysed by polymerase chain reaction (PCR) for the presence of HPV, followed by HPV typing by dot blot hybridisation.A series of pre-malignant and malignant samples obtained from archival paraffin-embedded tissues were analysed. Initially, we selected specimens that had been collected between January 1996 and December 2000 from patients with a previous diagnosis of cervical lesions, treated at Luis Antonio Hospital, a cancer referral centre in the city of Natal, State of Rio Grande do Norte (RN), Brazil. Six serial 5-μm sections from each block were prepared: one was used for histopathological revision examination, while the remaining samples were processed for HPV DNA detection. After histopathological revision, we selected 215 samples diagnosed with severe cervical intraepithelial neoplasia (CIN 3) or cervical cancer, among which 9 cases were excluded due to the lack of representative neoplastic tissue in the block after histopathological evaluation, and 16 more were excluded due to the lack of amplifiable DNA, leaving a total of 190 samples for this study.This study was approved by the Ethical Committee in Research at the Federal University of Rio Grande do Norte.DNA was extracted as previously described by Banerjee et al. (1995) , with soDNA samples were submitted to PCR analysis with the β-globin primers, PCO3/PC04 . The res2 test and the proportion comparison test were performed using the Statistica 7.0 software. Values of p ≤ 0.05 were considered as statistically significant.To verify the association between HPV prevalence and the considered variables, the χThe presence of cervical lesions was confirmed in 206 of the 215 samples analysed by histopathological revision exam; these samples were then submitted to DNA extraction for molecular analysis. Among all of the samples, 190 were positive for the amplification of a human β-globin gene segment. Among them, 53.7% showed pre-malignant lesions classified as severe CIN 3; 38.9% presented with invasive squamous cell carcinoma (SCC); and 7.4% were classified as adenosquamous carcinoma (ADSQ).Among the samples with only malignant lesions, most 84.1%) were histologically classified as SCC, whereas only 15.9% were categorised as ADSQ. We verified that the majority of the samples (60.0%) were obtained from non-Caucasian women (African-mixed-descendant) considered as non-white in this study, whereas 40.0% of the samples were from Caucasians considered as white. The age varied from 19 to 69 years, with a mean age of 44.3 ± 11.25 years for patients with CIN 3 and 47.3 ± 10.9 years for those with cervical cancer. Most of the CIN 3 samples (61.7%) were obtained from women between 19 and 47 years of age, while most of the cervical cancer samples (86.4%) were obtained from patients between 38 and 57 years of age , women with more than one sexual partner in their lifetime , smokers , and individuals with less than an elementary education , relative to their counterparts. Moreover, we observed a higher incidence of cancer among the non-white women (p = 0.002) . Similar differences were found between the samples with SCC and those with ADSQ   were histologically classified as SCC, while ADSQ was found in the remaining 15.9% of cases, a ratio similar to that described by the National Institute of Cancer, Brazil . ConsideThe average age of the cervical cancer patients in this study was 47.3 years, which is lower than that of Brazilian women from other regions of the country, such as central 49 years) [ years 9, and souWe observed a high incidence of cervical cancer in the non-white women. However, due to the high degree of interbreeding in the population of Rio Grande do Norte , the impWe also found a high incidence of cervical cancer in married women, women with a low degree of education, women with more than one sexual partner in their lifetime, and smokers.The overall HPV prevalence in cancer samples in this study was 92.0%, higher than that described for women from Goiânia, in the central region of Brazil (80.3%) ,22,23.Comparing the distribution of HPV types by prevalence order with that in other regions of Brazil, as described by Rabelo-Santos et al. (2003), the overall HPV 16 prevalence rate found in the current study (59.1%) was similar to that described for the central region (57.1%), almost equal to that described for the northeastern region (59.3%), but higher than that in the southern (52.2%), southeastern (53.8%), and northern regions (43.5%). The HPV 18 prevalence was higher than that reported for all other regions of the country, including the northeastern region. With regard to HPV 31, the prevalence rate observed in the present study was higher than that described for the central region, similar to that in the southern and southeastern regions, and lower than that described for the northeastern region. The HPV 33 prevalence rate found in this study was lower than that reported for the central, southern, and southeastern regions but similar to that for the northeastern region. However, these results of genotype distribution might also be influenced by the HPV genotyping techniques used in the different studies.When only considering cancer cases and by comparing the prevalence rates of HPV types with a consistent worldwide study conducted by Bosch et al., 1995 .Geographical differences in HPV distribution of cervical cancer have recently been compiled ,8 and seCervical cancer appears to occur in a lower age-range in women in this study, when compared with studies conducted in other Brazilian regions. Among the patients with CIN 3 and cancer, we observed a higher proportion of married women, women with more than one sexual partner, smokers, and individuals with less than an elementary education, relative to their counterparts.The authors declare that they have no competing interests.JVF conceived and coordinated the study, participated in its design, carried out the molecular analysis, and drafted the manuscript; RVM participated in the molecular analysis; MGFC carried out the histological analysis; TAAMF participated in the molecular analysis and helped to draft the manuscript; PRMA performed the statistical analysis; JSM and JCMP carried out the HPV typing; and LLV participated in the design and coordination of the study. All authors read and approved the final manuscript.