Low grade squamous intra-epithelial lesions could be considered as a manifestation of human papillomavirus exposition, however the discrepancy between rates of infection with human papillomavirus and development of low grade squamous intra-epithelial lesions is notable. Here we report a cross-sectional three-armed case–control study in the Colombian population, to compare the risk factors of women with low grade squamous intra-epithelial lesions with that of human papillomavirus DNA-negative and positive women with normal cytology.British Journal of Cancer (2002) 87, 1417–1421. doi:10.1038/sj.bjc.6600650www.bjcancer.com© 2002 Cancer Research UK Cervical cancer, the main cause of death from cancer among Colombian women, is known to be preceded by a series of squamous intra-epithelial lesions (SIL). Recently it has been established that human papillomavirus (HPV) is not only the central cause of cervical cancer, but also a necessary cause  and cancer. The category HPV infection could include a range of diagnoses from HPV DNA detectable only by PCR without cytological abnormalities (latent HPV infection) to CIN-1 lesions indistinguishable from koilocytic atypia, called low-grade squamous intra-epithelial lesions (LSIL) or productive HPV infection. LSIL should then be considered as a sign of exposure to HPV.It is currently unclear if the determinants of HPV DNA detection in women with normal cytology are the same or similar to those of LSIL. A recent follow-up study in young women suggests that risk factors for HPV infection are different from those of LSIL , 14 did not fill in the epidemiological questionnaire, 29 did not provide cervical scrapes and 1010 had inadequate cervical specimens for amplification, as measured by failure to amplify a human β-globin fragment. Thus 1995 (90.7%) women with a cytological diagnosis and with adequate cervical samples for HPV DNA and The smears taken at recruitment were read by two expert cytopathologists as follows: normal cytology in 1845 women (92.5%), LSIL in 70 (3.5%) women, atypical squamous cells of undetermined significance (ASCUS) in 30 (1.5%), atypical glandular cells of undetermined significance (AGUS) in 14 (0.7%), HSIL in 8 (0.4%), invasive cervical cancer in 2 (0.1%) and 26 (1.3%) had inadequate Pap smears.in situ at histology and were excluded). Controls were selected from the pool of 1845 women with normal cytology. Eight controls per case with LSIL diagnosis were selected, matched for age (±2 years) and date of recruitment (±6 months), giving a total of 557 control women. Two subgroups of control women were compared to the cases: one of 459 HPV DNA-negative and another of 98 HPV DNA-positive women.Cases for the case–control analysis were those with a diagnosis of LSIL  and 95% confidence intervals (CIs) were estimated using unconditional logistic regression. Two separate analyses were performed. The first, analysing risk factors for the development of LSIL, compared LSIL  to a matched sample of cytology controls. The second compared LSIL-positive for HPV DNA with two subgroups of normal cytology controls, one including only HPV DNA-negative controls, the other including only HPV DNA-positive controls.The ORs estimates were adjusted for age .The estimated overall HPV DNA prevalence in the total study population from which we selected our three study groups was 16.5%. Table 1Table 2C. trachomatis was 7.1% in the overall LSIL group and 5.2% in the control group (Table 3The age-adjusted OR for LSIL associated with single HPV infections was 6.1 (95% CI=3.4–10.9) while that for multiple infections was 7.6 (95% CI=3.4–16.9). The median age of women with LSIL was 30.8. The prevalence of this diagnosis was higher in women younger than 25 years where it was 4.4% and then decreased to 1.7% in women over 55–64 years old and 0.0 in women over 65. The prevalence of C. trachomatis and non-significant decreased risks with number of partners, age at first sexual intercourse and smoking were observed  were compared with all controls (HPV-positive and negative) and ORs adjusted for age and HPV were estimated, a significantly reduced risk appeared with ever use of OCs. Non-significant increased risk associated with the detection of observed . When woobserved , a signiWe report here the prevalence of cervical neoplasia and of HPV types in a Colombian high-risk population.The estimated overall prevalence of HPV was 16.5%. Among women with normal cytology it was 14.9%, while in LSIL it was 55.7%. Under the premise that HPV is a necessary cause of cervical cancer, we should expect that all true pre-neoplastic lesions contain HPV DNA. Thus, the 44.3% of HPV-negative LSIL could represent errors of diagnosis , low sensitivity of the HPV assay or both. Considering that the PCR-based assays used in this study have shown excellent performance in previous investigations, the possibility of a misdiagnosis of LSIL is more likely. In line with this possibility is the fact that the prevalence of LSIL in this Colombian population (3.5%) was higher than the one reported in another high-risk population in Costa Rica (2.2%) using more strict diagnostic criteria  were compared with a control group of normal cytology, independently of HPV status. Then LSIL HPV-positive cases were compared with two control groups: one composed by HPV-negative women with normal cytology and a second one of HPV-positive women with normal cytology.Surprisingly, no association with the number of sexual partners was observed when LSIL cases were compared with HPV negative controls and a reduced risk of LSIL was associated with oral contraceptive (OC) use. These findings are in contrast to the risk factors for HPV DNA detection identified in women with normal cytology from the same study population. In relation to OC use, our results are in line with those reported by In contrast to our results, tobacco smoking has been identified as a risk factor for LSIL in other populations. For example, Although our study was limited, for the small number of cases, it indicates that with the possible exception of OC, we were unable to identify any other important co-factor for the development of LSIL. An additional major limitation of our study, as well as of the vast majority of the studies of LSIL is the inaccuracy of the cytological diagnosis of LSIL. We have attempted to circumvent this limitation by restricting the analysis to HPV-positive-LSIL, but this considerably limited the power of our study.On the other hand, studies aimed to assess cofactors for HSIL and cervical cancer have identified parity, smoking and long-term OC use as important cofactors that modulate the risk of progression from HPV infection or LSIL to HSIL and cervical cancer (


Quality control included reviews of cytology, histology, colposcopy images and retesting of samples with polymerase chain reaction. Test diagnostic performances were based on 7908 women who had complete baseline and follow-up results. Routine histology identified 86 women with high-grade cervical intraepithelial neoplasia (CIN2+), which was confirmed by review histology in only 46 cases. Sensitivity of routine cytology for the detection of CIN2+ was 43.5%, with a specificity, positive predictive value (PPV), negative predictive value (NPV) of 98.0, 11.4 and 99.7%, respectively. Sensitivity of the HC2 test for the detection of CIN2+ was 97.8%, with a specificity, PPV and NPV, of 95.3, 10.9 and 100%, respectively. No high-grade neoplasia was detected in the randomly selected control group. A negative HPV-test result, even in combination with a positive Papanicolaou (Pap) result, virtually excluded any risk of underlying high-grade disease, but this was not the case for a negative Pap result. These data show that HPV testing is of value for the detection or exclusion of prevalent CIN in a routine cervical cancer-screening setting and could be used for further risk classification of women for follow-up management.In a prospective cohort study 8466 women attending routine cervical cancer screening were recruited. Colposcopy was performed on women with any degree of atypia on cytology and/or a positive high-risk human papillomavirus (HPV)-DNA test (HC2; Hybrid Capture 2 Despite widespread cervical cancer-screening programmes, 30 000 of the 190 million women living in Europe still die every year from this disease  is the major risk factor for the development of cervical cancer . To avoid confounding factors, we implemented different levels of quality controls  were excluded, leaving 4333. Of the 3900 women enrolled from Hannover, 132 women (3.4%) were excluded leaving 3768. Reasons for exclusion (365 women) were: 43 women with genital warts; 13 women with a history of conisation or hysterectomy; 11 women who were pregnant; eight women with abnormal cytology within 1 year of study entry; 167 women who were under the age of 30 years and 123 women who had not given written consent.Written informed consent was obtained from the patients by the participating gynaecologists. The study was approved by the local ethics committees at the Universities of Hannover and Tuebingen.At the first gynaecological examination, the cervix was visualised and a sample was taken for routine cervical cytology following the procedures normally used in each gynaecological practice . This practice of sample taking is the standard recommended procedure in Germany . A seconAll cervical smears were analysed at one of the cytology laboratories normally used by each participating office-based gynaecology practice and reported in accordance with the Second Munich Cytological Classification . All samples with RLU values in the range of 0.7–2.0 RLU were retested in duplicate, although subsequent statistical analysis was based only on the primary measurement.Samples for HPV testing were stored at 4°C for a maximum of 4 weeks prior to testing. All primary HPV testing was undertaken using the Hybrid Capture 2 test  at the clinical diagnostic laboratory of the Medizinische Hochschule Hannover or at the Department of Experimental Virology at the University of Tuebingen. All samples were analysed for the presence of 13 HR-HPV types  following the manufacturer's instructions. Therefore, a positive HPV result refers to a patient who is positive for one or more of the 13 HR-HPV types included in the high-risk probe mix. Infection with low-risk HPV types was not evaluated. Samples were considered positive if they attained or exceeded the FDA-approved threshold of 1.0 pg HPV DNA mlβ-globin gene. Three different primer combinations were used  producing amplicons ranging in size from 220 to 450 bp, all located in the highly conserved helicase region of the E1 gene. The sensitivity of this PCR system for different HPV types was established by testing serial dilutions of HPV plasmids for the following types: HPV1–8, 10–19, 21–26, 30–38, 40, 45–47 and 60, both in the presence and absence of human placenta DNA (1 μg). Primers PPF1/PPR2, which are not degenerate, reach the highest sensiti-vity of 10 genome copies per sample and result in a single 220 bp amplification product when applied to DNA extracted from clinical samples. PPF1/CP5 and CP4/CP5 have a detection limit of 100 copies, which is by far sufficient for the testing of clinical samples of the cervix. Primers PPF1/CP5, where the downstream primer is degenerate in three positions, detect at least 64 different HPV types and produce a 280 bp amplicon, while primers CP4/CP5 with five degenerate nucleotide positions generate a fragment of 450 bp that has been described elsewhere (PPF15′-(nt 2082)-AAC-AAT-GTG-TAG-ACA-TTA-TAA-ACG-AGC-(nt 2108)-3′PPR25′-(nt2336)-ATT-AAA-CTC-ATT-CCA-AAA-TAT-GA-(nt2314)-3′CP45′-(nt1942)-ATG-GTA-CAR-TGG-GCA-TWT-GA-(nt1961)-3′CP55′-(nt 2400)-GAG-GYT-GCA-ACC-AAA-AMT-GRC-T-(nt 2378)-3′DNA extraction, sample preparation and sample analysis procedures were carried out in separate rooms. All samples were tested for DNA integrity by PCR using primers for the human Numbers are according to the sequence of HPV16W12E (Genbank IDNr. AF125673).Direct automated sequencing of the PCR products was carried out using the Big Dye Terminator Cycle Sequencing Ready Reaction Kit . Sequencing was performed in 47 cm capillaries with the use of an ABI 310 sequencer (PE Biosystems). Sequences with <5% unidentifiable bases were processed and compared with those of known HPV types available through the GenBank database  using the Blast program. A nucleotide sequence was regarded as a distinct HPV type if it demonstrated <90% homology with a known type.Women were called for colposcopy at either of the two university gynaecology clinics if they demonstrated any degree of cytological abnormality  and/or if they were positive for HR-HPV. In addition, a random sample of 5% of women who were negative on both screening tests were invited to attend for colposcopy in order to assess the prevalence of high-grade disease in this population. The median recall interval  was 79 days with a range from 12 to 128 days. At colposcopy, punch biopsy specimens were taken from any regions suspicious for CIN and final diagnoses were based on histological results expressed using the Bethesda terminology. Cases were defined as women with high-grade cervical disease (⩾CIN2) using expert histology based on the primary histological result and two independent, blinded reviews.All histology diagnoses were classified into cases (⩾CIN2) and noncases .If routine histology and the review differed between those categories, a third histology diagnosis (second review) was included. A patient was then defined as case if the quotient of case/noncase was 2.0 and as noncase if the quotient was 0.5.An extensive series of quality control procedures was implemented . All abnAll independent experts were completely blinded to previous findings. Colposcopy was quality controlled using a computer-assisted digital imaging system  initially developed for the ASCUS-LSIL Triage Study (ALTS) conducted by the NCI  and vulvar intraepithelial neoplasia (VIN) were not considered as positive results for this analysis. The test sensitivity, specificity, PPV and negative predictive value (NPV) were calculated by contingency table analysis with 95% confidence intervals assessed using logistic regression, using a weighting factor. The women were divided into four groups according to their cytology and HPV-test results: both cytology and HPV−; cytology+, HPV−; cytology negative, HPV+; both cytology and HPV+. In women with both cytology and HPV− test results, only a small percentage has had a colposcopic examination and histology results were available. The assumption was made that this selected group of women who underwent colposcopy are representative of all the women in the double-negative group. By multiplying the cases  of the double-negative group by the weight factor  the total number of cases could be estimated as if every woman would have had histology. The confidence intervals were calculated with logistic regression taking into account this weight factor. In some subgroups, test properties were 100%. In these instances, the log likelihood for the underlimit of the confidence intervals was calculated by the following formula 1−eln(0.05)/n, with n being the number of observations in women who had a colposcopy result.All statistical analyses were undertaken by an independent group that did not participate in the screening trial or in the collection of the data. All test results were stored in a Microsoft Access database in Hannover and Tuebingen and later combined into one file which was imported into SPSS 9.0 for analysis. The cytology results were categorised into positive  or negative results. An HPV-HC2 result was considered positive when it attained or exceeded the FDA-approved threshold of 1.0 pg HPV DNA mlThe mean age of the 8101 women included in the study was 42. 7 years, with 94.6% of the women between 30 and 60 years of age. Of the 8101 women included in the study, 18 women did not have a cytology result, leaving 8083 women with both a cytology result and an HPV-HC2 result at baseline. Of the 8083 women with complete baseline results, 251 women (3.1%) had cytological abnormalities⩾PapIIw (≈borderline/ASCUS), which included one woman with PapIVb (⩾CIN3), eight women with PapIVa (CIN3), 61 women with PapIIID (CIN1–CIN2), 14 women with PapIII (ASC-H/AGUS) and 167 women with PapIIw (≈borderline/ASCUS). In all, 521 (6.4%) women were positive for HR-HPV  responded to this invitation. The results for these women were considered representative for all double-negative women. Based on extrapolation of the primary histology results of the double-negative control group (five cases of CIN2+ in 250 women), we calculated that 247 out of the total cohort of 7908 women would have had the routine diagnosis of high-grade cervical disease (⩾CIN2). However, independent blinded reviews by two pathologists of all histology slides stated that none of these controls showed any degree of neoplasia. Further, review of the digital colposcopic images confirmed the absence of disease in this group. There was complete agreement between primary and review colposcopy in terms of normal colposcopy results for all those women with normal Pap smears and negative HPV tests who had no cervical biopsy.Overall, 86 cases were found on primary histology among the 536 women attending colposcopy, from which only 46 cases were confirmed on expert review histology. Of the 46 women with high-grade cervical disease on expert histology, 20 would have been detected on the basis of cytology alone , one had PapIII (ASC-H/AGUS), nine had PapIIID (CIN1-CIN2), four had PapIva (CIN3) and one had PapIvb (⩾CIN3). The one woman with invasive carcinoma had a Pap smear classified as PapIII (ASC-H/AGUS). Human papillomavirus testing of these 37 women identified 36 women who were positive for HR-HPV, with the one case missed having a borderline negative result (0.83) that stayed negative after three-fold repeated retesting (average=0.993) with HC2 and was subsequently found to be positive for HPV 18 upon testing by PCR and sequence analysis. Of the 17 women who would have been detected by cytology, 16 were also positive for HR-HPV with the one discrepant case being the woman with CIN3 who was positive for HPV 18 on PCR/sequencing analysis. Using a case definition of CIN3+, the sensitivity, specificity, PPV and NPV of cytology were 46, 98.0, 9.7 and 99.7%, respectively, while the corresponding parameters for HPV testing were 97.3, 95.2, 8.7 and 100.0% .The sensitivity of cytology given above is based upon a Pap-smear result of ⩾PapIIw (≈borderline/ASCUS), which is lower than the usual threshold used in Germany. Using the usual threshold of ⩾PapIII (⩾ASC-H/AGUS), as the basis for referral to colposcopy, the sensitivity of cytology would have decreased to 37.0%, while the positive predictive value would have increased to 27.0% . It has A total of 925 of the routine cytology samples were reviewed by an independent expert, who noted at least minor deficiencies of sample fixation, staining or mounting in 19.8% of all slides. The primary cytology result and the review result were in agreement for 719 out of 925 (77.7%) Pap smears. In total, 87 out of 206 (42%) of the different cases involved a discrepancy of two or more grades . Using review cytology with PapIIw (≈borderline/ASCUS) or higher as the basis for referral to colposcopy would have missed two cases identified on primary cytology, and would have identified an additional five cases.QA testing of HC2-positive samples demonstrated a very high correlation between the two laboratories involved in this study. On the whole, 300 samples from the clinical laboratory at the Medizinische Hochschule Hannover were retested at the Department of Virology University of Tuebingen, and 300 samples from Tuebingen were retested in Hannover. In all, 96.6% of the samples yielded the same result on repeat HC2 testing for a kappa value of 0.75 (95% CI: 0.64–0.85).This trial is one of the first large-scale evaluations of HPV testing within an established nationwide cervical cancer-screening programme under routine screening conditions. Owing to the nonselected study population and the inclusion of rural and urban regions from both Northern and Southern Germany, the data are highly representative of Western Germany as a whole.Ideally, routine screening for cervical cancer based on Pap smears should detect all high-grade neoplasia in a given population, while testing for HPV, as the causative agent of cervical cancer, should identify all individuals either having prevalent cervical neoplasia or of being at risk for developing incident cancer within the next decade. Combining both methods should enable a reliable risk stratification of the screened population and allow for the development of a risk-adapted screening programme with increased intervals for women at greatly reduced risk. Important aspects of this hypothesis are indeed supported by the results of this study. As expected, the group of Cyto+/HPV+ women contained the highest proportion of high-grade cervical neoplasia (19 out of 54), while no cases were found on colposcopic examination of 250 randomly selected women who were negative on both cytology and HPV testing. It is a limitation of this study that we were not able to examine 10% of double negatives as claimed by others to be adequate to assess verification bias, although the results from our colposcopy review process and preliminary follow-up data make it unlikely that cases were missed. This verifies the widespread belief that Cyto−/HPV− women are at significantly reduced risk of having high-grade CIN or cancer at the time they are tested. In addition, they should be protected for an extended period against the development of cervical cancer as suggested by other investigators , as in Germany, holds great promise. Given the low prevalence of HPV infection in the female population aged 30 or above, combined with the high sensitivity of the HC2 test, ≈94% of women could have been returned safely to routine screening, while detecting virtually all of clinically relevant diseases. Theoretically, in a country like Germany, a significant benefit could be expected even from a twice in a lifetime HPV test. The timing of the first lifetime test should be sufficiently late in life, such that most HPV exposure has already occurred, but, on the other hand, sufficiently early to keep the risk of missing prevalent invasive cervical cancer to a minimum. It is expected that the cost efficiency of HPV testing will be poor in age groups under 30 years of age, because transient HPV infections are common (


Association of High-risk Human Papillomavirus (HR-HPV) with oral cancer has been established recently. Detecting these viruses in oral cavity is important to prevent oral lesions related to them. The purpose of this study was to evaluate the prevalence of HR-HPV in the oral cavity of women with cervical cancer, and their children. A total of 70 women, previously diagnosed with cervical cancer, and 46 children of these women, born by vaginal delivery only, were selected for this study. Buccal swabs were collected from their oral cavity and HPV detection was carried out using Hybrid Capture 2 high-risk HPV (HC2 HR-HPV) detection system. Out of 70 women with cervical cancer, four (5.71%) were found to be positive for HR-HPV in their oral cavity. No association of HR-HPV was found with sociodemographic profile, marital status, reproductive history, tobacco and alcohol usage, contraceptive pills usage, and presence of oral lesions (p>0.05). Among children, HR-HPV in the oral cavity was detected in only 1 of the 46 subjects examined (2.17%). Clinically healthy oral mucosa, without any oral lesions, was observed in all the HR-HPV positive subjects.The result of this study showed that there is low, if any, risk of HR-HPV infection in the oral cavity of women with cervical cancer. Further, our study suggests that there is very low risk for children of women with cervical cancer, to acquire and sustain HR-HPV in their oral cavity until childhood or adolescence. Human Papillomavirus (HPV) is an epitheliotropic, double stranded, circular DNA virus from Papovavirus family , which iA recent meta-analysis has established HPV as an independent risk factor for oral carcinomas as well . Our recDigene HC2® assay is United States Food and Drug Administration (USFDA) approved commercially available kit. HC2® assay is a nonradioactive, immuno-chemiluminescence method that is based on the hybridization of genotype-specific RNA probes to HPV genomic sequence. Compared with PCR, HC2® has excellent clinical sensitivity, almost up to 100%, as it shows only positive result when risk of disease progression exists. Studies have shown that HC2® is comparable to PCR and thus, it can be used as an adjunct or stand-alone test in HPV detection [Different methods have been used to detect HPV in concurrent infections giving varied results. PCR is used in many studies and it is known to be very sensitive in detection of HPV . The Digetection .While the HPV related genital lesions are more frequently seen in adults, lesions like skin warts, oral and laryngeal papillomas are more frequently seen in children . FurtherTherefore, the purpose of this study was to detect the prevalence of HR-HPV in the oral cavity of women with cervical cancer, and to evaluate the risk factors which contribute to its occurrence. Also, this study aimed to detect the existence of HR-HPV in children of the women with cervical cancer, for evaluation of vertical transmission of this virus, and to assess any associated risk factors.In total, 70 women diagnosed with cervical cancer and 46 children of these women, were recruited for this study. The demographic profile of the women and their children is given in Table ® detection system, with RLU/CO value being >1. Among the children, only 1 (2.17%) sample was found to be positive for HR-HPV in the oral cavity. All the positive subjects showed clinically healthy oral mucosa without any lesions. Association of HPV in cervical cancer subjects was evaluated with age range, ethnicity, marital status, age when got married, number of children, pregnancy status, contraceptive pills usage, tobacco and alcohol habits, and any oral lesions. Chi square/Fisher's Exact test showed no significant association between HR-HPV infection with any of these variables (p > 0.05) (Table Four samples (5.71%) from the oral cavity of women with cervical cancer showed positivity for HR-HPV using HC25) Table . As the Various studies have been conducted to study the role of HPV in oral lesions and malignancies. However, the association of HPV between cervical and oral cavity remains unclear. Further, recent studies on mother-to-child transmission by perinatal infection with HPV have been inconclusive . Consideet.al [Our results showed the prevalence of HR-HPV in oral cavity of women with cervical cancer to be quite low, with only 4 out of 70 subjects (5.71%) testing positive for HR-HPV. The results found in this study were in concordance with the study done by Kellokoski et.al , which eet.al .While recent studies have shown the presence of HR-HPV in a faction of oral pre-malignant and malignant lesions, this may suggest a role of HPV in only a portion of oral malignancies, contrary to cervical region, where its association is noted in almost all the cervical malignancies . This diet.al [et.al studied concurrent HPV infection in oral & genital mucosa by using PCR based assay. Sixteen subjects positive for HPV (31.25%) showed simultaneous genital & oral cavity infections, while HPV type-specific concordance was detected in only 3 patients [®, on the other hand, detects clinically relevant viral levels, that is 5000 copies and above [®. Moreover, HC2® detects only 13 types of HR-HPVs, while PCR based assays detect all types of HPVs.In contrast to our findings, a higher percentage (15.4% and 29.4%) of concurrent HPV infection between clinically normal oral mucosa and genital region, by using Southern blot and PCR procedures respectively, was obtained by Kellokoski patients . These dnd above . At thesOur study did not find any association of HPV in cervical cancer subjects with age range, ethnicity, marital status, number of children, tobacco or alcohol usage, contraceptive pills usage, or any oral lesions. This could be due to small percentage of positivity seen in our results and smaller sample size. Studies have shown that smoking has potential to alter oral epithelium, thus it has an influence on HPV expression in oral cavity . HoweverThis study did not assess the presence of HPV in the genital regions of women with cervical cancers at the time of examining their oral cavity for HR-HPV. It is because this study was more focused on detecting HR-HPV in the oral cavity, which might cause benign or malignant HPV related oral lesions in their future, rather than detecting concurrent infections in genital and oral regions, as reported in previously mentioned studies.et al tested the presence of HPV in the anal region and the oral cavity of Danish children, aged 0 to 17 years, by PCR targeting the L1 region of the HPV genome. Only four of the 249 anal samples and one of 392 oral samples were found to be HPV positive. The authors concluded that ano-genital types of HPV are not transmitted by non-sexual routes, and that HPV infection mainly occurs later in life [This study also aimed to detect the presence of HPV in oral cavity of the children of these women. Only 1 of the 46 children analysed was found to be positive for oral HR-HPV (2.17%). Further questioning from this HR-HPV positive subject revealed that he was sexually active. Thus, the positivity could be, in part, due to the sexual activities rather than transmission from the mother. Similar to our results, Koch  in life .Children of older age groups were chosen for analysis in our study. This is because the infants were shown to have a higher percentage of oral HPV, which could be due to the result of surface contamination that might occur in these infants during childbirth rather than HPV infection per se . This fiOur study was cross-sectional as compared to several other studies which were longitudinal, starting from the HPV detection since the mother became pregnant, until the delivery or a few months after birth ,21,44,45This study shows that there is low, if any, risk of HR-HPV infection in oral cavity of women with cervical cancer. There were no relevant risk factors that contributed to the development of this infection. Further, our study suggests that there is very low risk for children of women with cervical cancer, to acquire and sustain HR-HPV in their oral cavity until childhood or adolescence. More studies with bigger sample size are recommended to determine the relationship of HPV infection in these two areas. For more accurate evaluation of vertical transmission, long term follow-up studies should be conducted.This was a cross-sectional study that involved 70 women who were diagnosed to have cervical cancer, undergoing active treatment and routine follow up in Obstetrics and Gynaecology department or Oncology department of Hospital Universiti Sains Malaysia. A total of 46 children of these women, born only with vaginal delivery, were also examined. Only those children, who were born after the women were suspected or diagnosed of cervical cancer, were recruited in this study. Ethical approval was obtained from the Human Research Ethics Committee of Universiti Sains Malaysia. After obtaining the consent and explaining the procedure to each participant, questions about sociodemographics, marital status, reproductive history, tobacco or alcohol usage, contraceptive pills usage, oral and medical health status were asked using questionnaire provided to women at the time of enrollment. The questionnaire given to children was to inquire about their sociodemographic profile and habits. The children, who were older than 18 years of age, were married, or in any immunocompromised state like diabetes, were excluded from this study. Oral cavity of the subjects was also checked for any oral lesions, including HPV related oral lesions like papillomas, condylomas and focal epithelial hyperplasia, by a single oral medicine specialist to avoid inter-examiner variability.® (HC2) high-risk (HR) HPV DNA detection system located in the Department of Pathology, Universiti Sains Malaysia, and the procedure followed was according to the instruction of manufacturer. Digene HC2® detects HR- HPV  by using RNA probe cocktails to detect 13 HPV-HR which are HPV type-16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. HC2® technology is a nucleic acid hybridization assay for detection of HPV with signal amplification using microplate chemiluminescent detection. Light emitted is measured in terms of relative light units (RLUs) using Luminometer. RLU value is converted into ratio by the Cutoff value (5000 copies/ml). Any specimen with RLU/CO ≥1 was considered positive.The buccal swabs were collected using DNAPap Cervical Sampler™, from right and left buccal mucosa by moving the brush in circular motions, and then kept in the transport medium. All the sample tubes were stored immediately in -20°C until testing for HPV. All collected specimens were tested using Hybrid Capture 2The prevalence of HPV in the oral cavity of cervical cancer subjects was analysed using estimation method. The association of HPV infection with risk factors  was analyzed using Chi square test/Fisher's exact test with software SPSS version 16.0.The authors declare that they have no competing interests.RS designed the research project and drafted the manuscript. TPK collected and processed the samples from the women with cervical cancer. SBR collected and processed the samples from the children of the women with cervical cancer. RS and TTH guided the bench work of the procedure. MI did the bench work for detecting HR-HPV in samples. RS and TTH critically reviewed the final manuscript. All authors read and approved the final manuscript.


However, TIS may also influence the interpretation of Pap tests, leading to changes in the frequency of various interpretive categories. The effect of the TIS on rates of TPPT interpretation as atypical squamous cells of undetermined significance (ASC-US) is of concern because any shift in the frequency of ASC-US will alter the sensitivity and specificity of the Pap test. We have sought to determine whether automated screening of TPPT has altered ASC-US rates in our institution when compared with manual screening (MS) of TPPT.Automated screening of Papanicolaou tests (Pap tests) improves the productivity of cytopathology laboratories. The ThinPrepA computerized search for all ASC-US with reflex Human Papillomavirus (HPV) testing over a one-year-period (7/1/06 to 6/30/07) was conducted. Cases included both TPPT screened utilizing TIS and screened manually. HPV test results for both groups were recorded. Pertinent follow-up cervical cytology and histology results were retrieved for the period extending to 11/30/07. Automated screening was in clinical use for 10 months prior to the start of the study.Automated screening was performed on 23,103 TPPT, of which 977 (4.23%) were interpreted as ASC-US. Over the same period, MS was performed on 45,789 TPPT, of which 1924 (4.20%) were interpreted as ASC-US. Reflex HPV testing was positive for high risk (HR) types in 47.4% of the TIS cases and 50.2% of MS cases. Follow-up cervical dysplasia found by colposcopy was also distributed proportionally between the two groups. Cervical intraepithelial neoplasia (CIN) was found on follow-up biopsy of 20.1% of the TIS cases (5.2% CIN 2/3) and 21.2% of MS cases (5.1% CIN 2/3). None of these differences were statistically significant.® Imaging System did not appreciably change ASC-US rates or follow-up reflex HPV test results in our laboratory. This demonstrates that the benefits of automated screening may be obtained without increasing the rate of referral to colposcopy for ASC-US follow-up.Use of the ThinPrep The ThinPrep® Imaging System (TIS)  is the most widely used and studied automated system currently available. The operational details of the TIS have previously been published [Automated screening of ThinPrepublished . Brieflyublished -8. This ublished .The effect of the TIS on rates of atypical squamous cells of undetermined significance (ASC-US), however, is less clear. Two studies reported an increase in the ASC-US rate ,8, whileASC-US is the most common abnormal interpretation of Pap tests. Consequently, this category accounts for the highest proportion of the cervical intraepithelial neoplasia (CIN) 2 and 3 detected by screening (38.8% of cases in the study by Kinney and coworkers ). ASC-USIf the ASC-US rate were to increase as a result of TIS use, the sensitivity of the Pap tests would almost certainly rise. However, the cost of cervical cancer screening programs would also rise. At the very least, the number of reflex HPV tests would increase. Referrals to colposcopy could also increase if the number of reflex HPV tests positive for HR-types went up as a result of more frequent testing. Depending on the size of these increases, the rise in costs could become substantial.A decrease in the ASC-US rate as a result of using the TIS would lead to a regrettable loss of Pap test sensitivity. If the frequency of LSIL and HSIL interpretations increased simultaneously, however, this could at least partially offset the sensitivity losses. The costs of Pap test screening programs would reflect the overall sensitivity of the test, with declining ASC-US rates probably resulting in a less costly system.The introduction of reflex HPV testing for ASC-US into clinical practice has created a new metric for the evaluation of ASC-US rates: the frequency of results positive for high-risk viral types. The ALTS trial, which established the efficacy of HPV triage, also set a benchmark for HR-HPV positive rates in ASC-US of 50% . At presThis study evaluates the effect of the TIS on ASC-US rates on TPPT and subsequent follow-up in a screening population arbitrarily divided between manual and TIS-assisted screening, over a single time period, with reflex HPV test result feedback.® liquid-based preparations . This study focuses on TPPT. We receive specimens from a mix of clinical settings which include both low-risk populations (privately insured women) and high-risk populations . Specimens were examined by a team of 12 CTs and 4 cytopathologists during the study period. The ASC:SIL ratio for the laboratory was 1.3 in 2006 and 2007.Our laboratory processes approximately 70,000 Pap tests per year, of which more than 95% are TPPT with the remainder consisting of conventional smears and SurePathThe TIS has been in clinical use in our laboratory since September of 2005. Although all TPPT were processed through the imager during the study period, many were manually screened because of an insufficient number of special automated microscopes. Selection for MS was made essentially randomly by the CT with no systematic bias we are aware of. Cases screened with the assistance of the TIS are identified by a standardized comment included in the reports of all such cases.Reflex HPV testing is performed by the Hybrid Capture 2 method  using a mixed probe targeting 13 cancer-associated HR-HPV types. The test is performed by the microbiology section of our department based on the manufacturer's protocol. Specimens without sufficient material remaining in the vial, approximately 4 mL, were excluded from this study.The cytopathologists in our laboratory were aware of the results of reflex HPV testing for their ASC-US cases. The goal of the laboratory was to maintain a HR-HPV positivity rate of close to 50% during this period.Cervical biopsy specimens are reviewed by the surgical pathology department which includes 19 pathologists.The computerized laboratory records for our institution were reviewed for the one-year period from July 1, 2006 to June 30, 2007 to find all TPPT interpreted as ASC-US. Follow-up reflex HPV test and biopsy results, as well as additional Pap test results for the time period extending from July 1, 2006 to November 30, 2007, were also reviewed. The institutional review board authorized this study.The statistical calculations employed the Yates correction of the chi-square test using an online resource . We consDuring the study period, 68,898 Pap tests were performed in our laboratory. Of these, 2,901 tests from 2,716 women , were interpreted as ASC-US and had a reflex HPV test.One third (33.5%) of the cases were screened with the assistance of the TIS. The comparative rates of ASC-US interpretations for the two groups as well as the reflex HPV test results are displayed in Table Among HR-HPV positive cases, follow-up biopsy results were available for almost half of the women as shown in Table Among women negative for HR-HPV, 80 had histological follow-up, including 25 women screened by the TIS and 55 by MS. Among these there were 10 cases of dysplasia  in the TIS group and 11 cases  in the MS group.In the subset of HR-HPV positive women who did not have histological sampling, 387 had follow-up Pap tests available, including 132 women screened by the TIS and 255 by MS. Of these a few had an interpretation of dysplasia in both the TIS group  and the MS group .Since reflex HPV testing in adolescent women is now deemed "unacceptable" according to the recently revised ASCCP guidelines  we have Previous studies of the clinical performance of the TIS have yielded conflicting results about the effect on the ASC-US rate. Most studies have used a non-synchronous design, with the Pap test interpretations from the year before the introduction of the TIS compared with the interpretations from the first year of TIS use. Using this method, Lozano found an increase in the ASC-US rate from 4.09% to 6.52% (p < 0.001)  and DziuThese studies, in contrast to ours, have the strength of allowing for and measuring changes in clinical practice following the introduction of the TIS. If the TIS somehow revolutionized the interpretation of Pap tests, these studies would be more likely to detect the difference. However, this design of comparing consecutive years also has serious problems. Any change in clinical practice from year to year will affect the results, confounding the analysis of the influence of the TIS alone.Lozano demonstrated an impressive "learning curve" effect in the early months of TIS use which corresponded to a sizeable, sudden change in both the ASC-US rate and the follow-up HPV results . This "lAnother factor to consider is the influence of changing interpretive thresholds. Although it is not feasible to speculate on the cause of such changes, the consistent observation that the HR-HPV positivity rate changed in accord with the ASC-US rate in all three studies makes this mechanism a likely explanation for the findings. Lowered thresholds for ASC-US will increase the ASC-US rate and decrease the HR-HPV frequency in reflex testing, with raised ASC-US thresholds having the opposite effect. These scenarios correspond to the findings of the three studies that reported follow-up HPV results -8. Of coOur study, in comparison with these, has the merit of synchronous analysis of the TIS and MS groups. This should minimize interpretive threshold differences and other potentially confounding factors such as changes in cytology staffing or differing specimen sources over time. We also designed our study to begin well after the introduction of the TIS to our laboratory (10 months) to eliminate any potential "learning curve" effect.Only a few studies have analyzed the effects of the TIS on ASC-US rates using single-patient comparisons ,4. UnforAnother significant difference between our study and those performed previously was our use of reflex HPV test results as a quality metric in our laboratory. We were consciously trying to remain near the benchmark of 50% HR-HPV positive ASC-US interpretations established by the ALTS trial . This prSome criticisms of the ALTS trial have been put forward. The patients used in the trial were younger and at higher risk for HPV exposure than the population at large , raisingIdeally any new technology would improve both the sensitivity and the specificity of the Pap test. Although studies agree that the TIS improves sensitivity, the effect on specificity is far less clear. In the current environment, with discussions beginning as to whether HPV testing should replace cytology as the preferred mode of screening , it is iOur results indicate that use of the TIS has had no appreciable effect on the ASC-US rate in our laboratory. The percentage of TPPT interpreted as ASC-US was essentially the same in cases screened by the TIS as in TPPT screened by MS. This indicates that the use of the TIS, with its productivity benefits, appears not to be having a significant negative impact on the performance characteristics of the Pap test. We believe that the use of the HR-HPV positivity rate in ASC-US as a performance metric contributed to our ability to maintain a stable ASC-US rate while using the TIS.® Imaging System; TPPT – ThinPrep® Papanicolaou test.ASC-US – atypical squamous cells of undetermined significance; CIN – cervical intraepithelial neoplasia; CT – cytotechnologist; FOV – fields of view; HPV – Human Papillomavirus; HR – high-risk; HSIL – high-grade squamous intraepithelial lesion; LSIL – low-grade squamous intraepithelial lesion; MS – manual screening; Pap test – Papanicolaou test; SIL – squamous intraepithelial lesion; TIS – ThinPrepThe authors declare that they have no competing interests.MT collected and compiled the study data, performed the statistical analyses, and drafted the manuscript. DR assisted in the design of the study and contributed to data collection. TB assisted in the design of the study and helped to revise the manuscript. RH supervised the study and helped to revise the manuscript. All authors read and approved the final manuscript.


Infection with certain types of human papillomavirus (HPV) presents a high risk for the subsequent development of cervical intraepithelial neoplasia (CIN) and cervical carcinoma. Immunological mechanisms are likely to play a role in control of cervical HPV lesions. The HPV E2 protein has roles in virus replication and transcription, and loss of E2 functions may be associated with progression of cervical neoplasia. Accordingly, it is of interest to monitor immune responses to the E2 protein, and previous studies have reported associations between serological reactivity to E2 peptide antigens and cervical neoplasia. In order to investigate serological responses to native, full-length E2 protein, we expressed HPV-16 E2 proteins with and without an N-terminal polyhistidine tag using the baculovirus system. Purified HPV-16 E2 protein was used to develop enzyme-linked immunosorbent assays to detect serological IgG and IgA responses in cervical neoplasia patients and controls. We found that serum IgA levels against the E2 protein were elevated in CIN patients relative to normal control subjects but were not elevated in cervical cancer patients. Moreover, there appeared to be a gradient of response within cervical neoplasia such that the highest antibody levels were seen in lower grades of neoplasia up to CIN 2, whereas lower levels were observed in CIN 3 and still lower levels in cervical carcinoma. These findings suggest that the IgA antibody response to E2 may associate with stage and progression in cervical neoplasia.


Cervical smears prepared around the time of menses have been linked to unsatisfactory specimens and false negative results; however, it is unclear whether liquid-based cytology is similarly affected and data relating date of last menstrual period (LMP) to human papillomavirus (HPV) DNA testing are conflicting. Accordingly, we evaluated liquid-based cytology and HPV test results using Hybrid Capture 2 and PCR by LMP . We studied 5060 participants in ALTS, the Atypical Squamous Cells of Undetermined Significance (ASCUS) Low Grade Squamous Intraepithelial Lesion (LSIL) Triage Study. On average, women had 3.4 examinations  during a 2-year period of observation permitting an examination of intra-individual variation in cytology and HPV by LMP. Although uncommon, unsatisfactory cytology specimens were most likely on days 0–10. For satisfactory specimens, the frequency with which cytologic categories were reported varied by time since LMP, although differences were modest and did not affect the chance of abnormal cytology or its severity among women diagnosed with CIN2+. The frequency of positive HC2 tests did not vary with date of LMP. Among HPV infected women, independent of eventual diagnosis and the number of viral genotypes present, mid-cycle specimens yielded the highest frequency of LSIL cytologic interpretations and the highest HPV load; however, the magnitude of these effects were small. Intraindividual correlations of cytology or HPV by LMP were generally weak. We conclude that mid-cycle specimens yield slightly higher HPV DNA loads and slightly increased LSIL interpretations, but the clinical impact is marginal. Standardizing collection times would slightly improve interpretation of trends in HPV load. Finally, these data are consistent with the view that the biological properties of the HPV-infected cervix vary with the date of the LMP. Important goals of cervical cancer research include improving detection of precancerous lesions and reducing equivocal results by employing better collection, preparation, and testing methods  DNA and cytologic testing in some settings highlights the need to clarify inconsistencies in reported analyses assessing the performance of HPV testing in relation to LMP (n=3488) or LSIL (n=1572) at four clinical centres in the US , was placed in PreservCyt  and used to prepare a ThinPrep (Cytyc) cytology slide and to perform the Hybrid Capture 2  test, which targets 13 oncogenic HPV types. The second sample, collected with a Dacron swab, was placed in Specimen Transport Medium (Digene), frozen, and later used for HPV typing by a polymerase chain reaction (PCR)-based assay.Subjects were randomized to one of three management arms: (1) Conservative Management consisting of colposcopy referral for repeat cytology of a high-grade squamous intraepithelial lesion (HSIL); (2) HPV triage, with colposcopy referral for a positive HC2 test for oncogenic types ; and (3) Immediate Colposcopy. Subjects who received histologic diagnoses at the Clinical Centers of cervical intraepithelial neoplasia 2 or worse (CIN2+) were treated with loop electrical excision procedure (LEEP). The follow-up protocol was identical for all women: repeat cytology every 6 months for 2 years, with colposcopy referral for Clinical Center cytology of HSIL. At the 24-month (exit) visit, all data were unmasked and reviewed for every subject, colposcopy was performed, and women with CIN2+ or persistent ASCUS or LSIL diagnosed by the Clinical Center were offered treatment with LEEP, permitting detection of histologic CIN2+ that was undetected by cytology and colposcopy. All referral community smears and enrollment ThinPreps, most follow-up ThinPreps, and all histology specimens were reviewed by a Pathology Quality Control (QC) Group to provide standardized interpretations and added subject safety .−1 of HPV 16 DNA (approximately 5000 copies) were considered positive. The content of HPV DNA in specimens that tested positive (‘HPV load’) was determined as relative light units (RLU), calculated as the ratio of the signal of the specimen to that of the standard, which is linearly related to load  using a PCR-based assay employing L1 consensus primers and PGMY09/11 amplification followed by reverse line blot hybridization as described elsewhere  and Pathology QC histology results (the reference standard for outcomes) were used for all analyses. For most analyses, we stratified these data in three periods, reflecting serum hormone fluctuations during a normal 28-day cycle: (1) days 1–10 ; (2) days 11–21 ; and (3) days 22–28 . For ease of interpretation of odds ratios (ORs), we selected the late period as the referent group, which generally yielded ORs ⩾1.0 for most comparisons.n=4098), failure to collect cytology specimens (n=16) and LMPs greater than day 28 day (n=5797) accounted for the exclusions. HC2 data were available for 14 490 of these visits.Of 25 300 potential cytologic examinations that could have been performed in ALTS (five examinations of 5060 women), cytology results with associated LMP data in the range of 0–28 days were available for 15 389 interpretations. Missed visits  to take into account possible intra-individual ‘dependencies’ or ‘auto-correlation’  because many women contributed multiple data points to the analysis . This approach provides an estimate of whether data from individual women are correlated; strong auto-correlation reduces the precision gains achieved for larger sample sizes, resulting in reduced statistical power. We present ORs with 95% confidence intervals (CI) using GEE models, although auto-correlation was generally weak and adjustment widened CI only slightly.We assessed specimen quality by LMP by tabulating the frequency of reports of Bethesda System categories of ‘satisfactory for interpretation’ or ‘unsatisfactory’ . Next, fTo consider how cytologic interpretations related to the most severe histologic diagnosis per woman during the entire trial, we stratified the cytologic results by whether or not a woman ever received a histologic diagnosis of CIN2+. Previous work within ALTS showed that subsequent risk of CIN2+ or CIN3+ was similar for women with a colposcopically-directed biopsy result of CIN1, a negative biopsy, or a normal colposcopic appearance, which did not prompt a biopsy . We only−1  for which the corresponding PCR test demonstrated only one of the 13 oncogenic types included in the HC2 kit. The specimens could contain other types as well, not targeted by the assay. Of 3739 specimens that tested positive for only one of the 13 types by PCR and which were associated with suitable LMP dates, there were 3004 specimens that tested positive by HC2 for which we assessed viral load. To confirm the conclusions from this approach, we analyzed associations between LMP data and viral load <1.0 , and also analyzed LMP associations for specimens containing any single HPV type, including types that are not targeted by the HC2 kit. These analyses were confirmatory and are not presented.Subsequent analyses regarding HPV results were restricted to specimens with positive HC2 tests  specimens collected early in the cycle, five of 6792 (0.07%) collected during mid-cycle, and eight of 3213 (0.25%) collected late in the cycle. Considering specimens collected late as the referent, the association with unsatisfactory specimens yielded an OR=1.87 (95% CI=0.88–4.43) for the early period and OR=0.30 (95% CI=0.09–0.89) for mid-cycle collections. Unsatisfactory reports were most common on days 0–4 (data not shown). Intra-individual correlation was minimal (−0.0019).Among the 15 351 satisfactory specimens that comprised the rest of the analysis, the reported frequency of cytologic interpretations in ALTS varied significantly by LMP, although the magnitudes of absolute differences were slight . For exaTo explore the possible clinical relevance of the variation in cytology by time since LMP, we restricted the study population to women who received a histologic diagnosis of CIN2+ in ALTS . Among tWe compared the frequency of HPV DNA positive test results by HC2 for the three intervals of the menstrual cycle, without restrictions. The auto-correlation estimate was 0.39, showing a slight tendency of women that were HPV positive once to be positive repeatedly. This strength of correlation was typical of all HPV analyses, whether assessed as negative vs positive or as viral load.n=6468 associated with one or multiple types by PCR), we obtained slightly significant associations. Compared to specimens collected late in the cycle, specimens obtained early in the cycle had viral loads that were only 83% (95% CI=0.71–0.98) as high, whereas mid-cycle specimens yielded average loads that were 22% higher (95% CI=1.05–1.43).Overall, 44.6% of specimens were HC2 positive. Frequencies of HC2 positive results were unrelated to the date of LMP. When we assessed viral load among all HC2-positive tests .To study the associations between HPV viral load per type and time of LMP, independent of number of HPV types present, we assessed the frequency of positive HC2 results among specimens associated with PCR detection of one of the 13 carcinogenic types targeted by HC2. Positive HC 2 results were obtained for 1094 of 1377 (79.4%) specimens collected early in the cycle, 1315 of 1614 (81.5%) obtained at mid-cycle, and 595 of 748 (79.6%) late cycle specimens . Compare−1=403.1, median 93.6) peaked at mid-cycle  containing only one of the 13 types, HPV load  and HPV18 (n=192) separately. Among specimens collected early in the cycle that were associated with concurrent single infections with HPV 16 (determined by PCR), 77.7% of HC2 tests were positive, which was less sensitive than HC2 tests performed on specimens collected late in the menstrual cycle . Viral load was nonsignificantly lower (21.9%) for the specimens collected early as opposed to late in the cycle. For mid-cycle specimens associated with HPV 16, the frequency of HC2 positive results and load determinations did not differ significantly from those obtained for specimens late in the cycle. Among specimens associated with concurrent detection of single infections with HPV 18 (detected by PCR), positive HC2 results were more frequent at mid-cycle  and load was 55% higher (though non-significantly) compared to specimens collected late in the cycle.Finally, we analysed data for single infections with HPV16  are probably optimal for detection of HPV DNA by HC2 testing and for identification of LSIL by thin-layer cytology. However, the effects of the date of LMP on the performance of cytology and HC2 testing among women diagnosed with histologic CIN2+ was minor, suggesting that it is unnecessary to defer testing for women who present outside this interval.Ideally, optimizing the timing of specimen collection would minimize unsatisfactory specimens and cytologic reporting of ASCUS  overall and maximize clear-cut results of SIL among women with underlying CIN2+. Similar to results obtained with conventional smears, specimens obtained early in the menstrual cycle were associated with the highest frequency of unsatisfactory samples  as compared to mid-cycle (4.4%) and late in the cycle (2.9%), although these differences were not significantly significant based on limited data. To account for these findings, we speculate that misclassification of endometrial cells as HSIL contributes to higher reporting of HSIL during the early part of the cycle. In support of this hypothesis: (1) endometrial cells are generally identified only during the first 10 days of the cycle; (2) endometrial cells may mimic HSIL because of their similar small size and high nuclear to cytoplasmic ratio, especially in liquid-based cytology; and (3) the inconspicuousness of blood in most thin-layer slides disguises the occurrence of menses. Previous studies have emphasized that interpretation of HSIL in thin-layer cytology may pose difficulties, especially with regard to false negative results  rather than analytical sensitivity (detection of the lowest number of copies possible). Finally, some collections that yielded positive PCR and negative HC2 tests could reflect errors in HPV typing, differences in HPV content between samples tested by HC2 and PCR or other factors.Although this analysis was based on a clinical trial of over 5000 women who were followed with repeated examinations for 2 years, we recognize some limitations. The majority of women in this study were young and eligibility criteria included a recent cytology report of ASCUS or LSIL, therefore, these results may not apply to all women. In addition, the measurement of HPV load in cervical cellular collections using HC2 does not necessarily reflect the concentration of the virus in cervical tissue. In fact, it is recognized that HC2 pg mlIn conclusion, the LMP date is not a critical clinical consideration if women with cytology of ASCUS+ are closely monitored and HPV test results are categorized as negative or positive for management. However, it is prudent when possible to schedule screening tests at mid-cycle to optimize sensitivity and limit the occurrence of unsatisfactory cytology. Standardized timing of specimen collections may also minimize the inherent variability of test results for individual patients, thereby facilitating interpretation of serial results. Improved understanding of the interactions between hormones and HPV may eventually provide clues that are useful for cancer prevention.


The raw incidence of cancer of the uterine cervix is Spain is 7,8 per 100.000 inhabitants (adjusted incidence is 5.6). The incidence of this tumor is still low, but a steady increase has been seen, probably related to increasing risk factors.To determine the frequency of infection by different types of human papillomavirus (HPV) in Papanicolau smears from women with and without cancer of the uterine cervix in Spain.A case-control study was performed in women with and without cervical cancer from Zaragoza, Spain. Pap smears from 600 cases  and 60 with invasive cancer) and 1200 controls (women without those lesions) were tested by polymerase chain reaction (PCR) and typed by oligonucleotide microarray-based detection.HPV was detected in 93.3% of all samples with invasive cancer versus 17.5% of controls. OR for invasive cancer was 55 . Statistically significant associations were also found for different grades of cervical dysplasia.The strong association found between HPV infection, specifically types 16 and 18 and cancer of the uterine cervix in Zaragoza, Spain, stresses the importance of ongoing efforts to institute a vaccine program with recently approved HPV vaccines in order to prevent cervical cancer in this population. Cancer of the uterine cervix is more frequent in developing countries than in the Western world. Spain, a country where screening through Papanicolau smears has been performed for more than 30 years now, has a low raw incidence of 7.8 in 2000 (age-adjusted rate is 5.6) .In the last decade, an increasing incidence of cervical cancer has been noted, most likely related to increasing numbers of young women with risk factors, namely sexual promiscuity and prostitution.The fact that these high-risk women, some of whom are foreign immigrants, are not adhering to screening programs, stresses the importance of developing alternative strategies for prevention.While there are many genotypes of human papillomavirus and most of them are frequently found in Pap smears of healthy women, two genotypes, 16 and 18, of human papillomavirus have been consistently associated to intraepithelial neoplasia and invasive cancer of the uterine cervix in Western populations  and haveWe have performed a case-control study to assess the incidence of infection by different HPV types in women with and without cervical dysplasia and carcinoma. Following PCR-based detection of HPV, oligonucleotide microarray-based detection was used for genotyping, given its reliability and ease of use.Following approval by the Institutional review Board of the University of Zaragoza, the study included six hundred consecutive women with some degree of cervical dysplasia or neoplasia  excluding atypical squamous cells of uncertain significance (ASCUS) who had Pap smears at the University Hospital of Zaragoza, Spain from 1999 to 2005. Cytological diagnoses were required to have been subsequently confirmed by histology for study entry.Controls were 1200 age-matched women who had no cervical dysplasia, neoplasia or ASCUS in the Pap smear, for a case:control ratio of 1:2.In addition to standard cytologic examination, all Pap smears from cases and controls underwent DNA extraction and were tested for HPV by polymerase chain reaction (PCR) and typed by oligonucleotide microarray-based detection  with posStatistical analysis included a logistic regression analysis adjusted for age, calculating odds ratio (OR) for each type of cervical lesion  and 95% confidence intervals in women with and without HPV positivity in the Pap smear.The study population included 1800 women. Median age was 36 years. There were 3.3% foreign-born individuals. The number of samples in each diagnostic group was: 120 patients (20%) with CIN I, 210 (35%) with CIN II, 210 (35%) with CIN III and 60 (10%) with invasive cervical carcinoma. All these cytological diagnoses had been subsequently confirmed by histology. The frequency of HPV positivity according to type of cervical lesion is shown in Table As for HPV types Table , the mosIt is remarkable that HPV 16 and HPV 18 were infrequent among controls, comprising only 1% of all HPV infections.Table In the present study, the correlation between HPV and cervical neoplasia has been different for early lesions  than for higher-risk lesions .Overall, the odds ratio found in our study is well within values reported in prior articles, ranging from a lowest value of 2  in a series of women with cancer of the uterine cervix from the United Kingdom to a high odds ratio of 813  seen in another study . Other aSuch large differences in studies that are otherwise concordant in showing a significant association between HPV infection and cervical cancer may well be due to differences in the type of pathological lesion, the viral load and the duration of infection.In a study from Mexico , the oddIn the present study, a higher odds ratio was found for more severe cervical lesions, reaching a maximum of 55 for invasive carcinoma, a figure that is remarkably similar to that found in prior studies.The fact that HPV detection was performed just after cytological examination, without the long storing periods seen in some prior studies  supportsBoth HPV-16 and HPV-18 were significantly more frequent in cases than in controls, as seen in prior reports -15. TherAn interesting finding in our study is the high prevalence of HPV-54 in controls (12%) and patients with CIN I (27%). The fact that the prevalence of HPV-54 in patients with CIN III and invasive carcinoma was much lower (0% and 2%) suggests that this type has a low potential to induce invasive cancer.Prior studies have found other HPV genotypes different from HPV-16 and HPV-18 to be related to cervical cancer in specific geographic areas  and ChinAs in other studies performed in Spain , HPV 16 It is remarkable that a significant percentage of healthy women (controls) harbour HPV , even if the types were different from women with cervical lesions and the prevalence of types associated with cancer (namely HPV-16 and HPV-18) was very low.The high frequency of HPV found in controls suggests that most were transient infections. Only women infected with high-risk HPV types and with ineffective immune response would develop neoplasia -21.The distribution of HPV types associated to cervical cancer in each area of the world needs to be taken into account when designing HPV vaccines. This is an exciting time for prevention of cancer of the uterine cervix, when two HPV vaccines have already been proven to be effective in preventing cervical cancer, and may soon improve public health strategies against this disease, currently based solely on early detection by Pap smears.The strong association found in this report between HPV infection, specifically types 16 and 18 and cancer of the uterine cervix in Zaragoza, Spain, stresses the importance of ongoing efforts to institute a vaccine program with two recently approved HPV vaccines in order to prevent cervical cancer in this population.MB and IB designed the study, MM, RB and JG carried out the molecular genetic studies, JIM drafted the manuscript. All authors read and approved the final manuscript.


TP53 mutations, 11q13 amplification (amp) and CDKN2A/p16 mutations or promoter methlyation. However, in HPV+ HNSCC it is frequent to observe wild-type TP53 and expression of p16. The relationship of this unusual pattern with 11q13 amp has not been tested. In a retrospective study on 125 HNSCC patients, only 17% (five out of 30) of HPV+ vs 44% (39 out of 89) of HPV − tumours expressed 11q13 amp (adjusted odds ratio (OR)=0.2, 95% confidence interval (CI)=0.1–0.6). A subpopulation of tumours (n=69) were classified according to the three molecular markers, TP53, p16 and 11q13 amp. In addition to wild-type TP53, and p16 expression, HPV+ tumours were more likely not to be amplified at 11q13 . As HPV+ HNSCC lack the genetic alterations which are common in other tumours, we hypothesise that HPV infection may represent an early event in the HNSCC carcinogenic process, thus suggesting a distinct molecular pathway.Two distinct etiologies of head and neck squamous cell carcinoma (HNSCC) have been proposed, DNA damage owing to tobacco and alcohol exposure and human papillomavirus (HPV) oncogene-mediated transformation. Common genetic alterations in HNSCC include  Although the majority of cases of head and neck squamous cell carcinoma (HNSCC) are attributed to tobacco and alcohol consumption  is thought to play a role in tumorigenesis. Cyclin D1 is an important cell cycle protein that binds cyclin-dependent kinase (CDK) 4 or 6 to phosphorylate and inactivate the RB1 protein thus driving the cell from G1 into S-phase. Therefore, overexpression of cyclin D1 may lead to deregulate cell cycling from G1 to S-phase and may result in a selective growth advantage for the affected cell.Head and neck squamous cell carcinoma frequently carries numerous genetic alterations, such as mutations, chromosomal losses, gains and amplifications (amp) . Common  cancers . A numbeCDKN2A/p16 opposes cyclin D1 function by negatively regulating RB1 phosphorylartion and a genetic (loss of heterozygosity (LOH)) or epigenetic (promoter methylation) alteration in this gene is frequently observed, resulting in a lack of p16 expression in most HNSCC  in patients with first primary head and neck tumours.In contrast to most HNSCC, HPV-positive tumours are more likely to have wild-type N=185) included patients undergoing surgical resection of their tumours between September 1992 and February 1997, following informed consent under a protocol approved by the University of Pittsburgh Biomedical Institutional Review Board. Patients with recurrent tumours, second primaries and metastatic tumours (lymph node and distant) at the time of enrollment were excluded from this analysis (n=58). The remaining subjects (n=127), with first primary tumours were enrolled. Human papillomavirus could be tested in only 125 of 127 tumours; therefore, the number of patients included in this analysis was 125.The study population  to form the head and neck database. This database contains clinical, treatment and follow-up information for the first surgical resection at the time of enrollment and all subsequent resections for all patients enrolled in the study.The head and neck database was established by CCRR, JSW and JLW in June 2004. Information, including demographics, smoking and alcohol use, family history, tumour site, clinical characteristics of the initial primary tumour and subsequent tumours, follow-up data (such as disease outcome and time to next disease occurrence) through May 2004 were obtained from the University of Pittsburgh Tumor Registry. Patients' clinical and demographic information from the Tumor Registry was re-verified from each patient's original deidentified and coded questionnaires, pathology and surgical reports. All data from the Tumor Registry was combined with the corresponding patient's laboratory data , p16 immunohistochemistry (unpublished data) and 11q13 amp . Cells were considered p16-positive if more than 5% of cells in a tumour island or mucosal surface stained visibly stronger than the negative control. When more than 5% of the cells were stained weakly, the result was considered to be weakly positive for p16. Amplification of 11q13 was assessed using fluorescence in situ hybridisation (FISH) probes for FGF3/INT2, FGF4/HST1 or CCND1 (Smoking status was defined as follows: never smoker (never smoked), current smoker (recorded as an ever smoker who had not quit at the time of enrollment), former smoker (quit smoking at least 1 month before enrollment in the study). Alcohol use was defined as follows: never drinker , current drinker  and former drinker . For the duration of alcohol use, each patient was categorised as never , short-term , or long-term  users. Tumour site, histology, stage and grade were classified according to the American Joint Committee on Cancer (AJCC) ICD9 codes, ICD-morphology, stage and grade classification, respectively. Sites of the oral cavity included cheek, retromolar area, alveolar ridge, oral tongue, palate, floor of mouth and overlapping lesions of other and unspecified parts of the mouth. The oropharynx included sites involving the base of tongue, soft palate, tonsil and overlapping lesions of the oral cavity and pharynx. Analyses of β-globin gene fragment was performed using the PCO4 and GH20 primers (β-globin amplicon (n=125). Human papillomavirus status was determined by a nested real-time PCR assay using consensus HPV primers PGMY09/11/HMB01 . All DNA amplification reactions were set up according to the manufacturer's protocol (using 3 mM MgCl2 and 0.3 μM each PGMY09/11/HMB01 primers (for the primary PCR reaction) or 1.0 μM each GP5+/GP6+ primers (for the nested PCR reaction)) and were performed in a separate room from that used for the DNA amplification steps. For all HPV-positive samples from the previously described PCR protocol, confirmation of the appropriately sized amplicon was carried out by running an aliquot of each sample on a 10% PAGEr® polyacrylamide gel , at 250 volts for 55 min. The gel was stained with the highly sensitive GelStar® nucleic acid stain  and the 150 bp GP5+/6+ amplicon was visualised using a Clare Chemical Dark Reader transilluminator .Extracted DNA was available from 125 tumours. Polymerase chain reaction (PCR) amplification of a β-globin gene. The PCR products were chemically denatured and hybridised to linear array strips which contained specific and one cross-reactive oligonucleotide probes for the HPV genotypes listed above as well as a high and low concentration of a β-globin probe. The specific genotypes were visualised using streptavidin-horseradish peroxidase conjugate and a substrate solution containing hydrogen peroxide and 3,3′,5,5′-tetramethylbenzidine which yielded a blue precipitate at the probe positions where hybridisation occurred.HPV genotyping was performed using the Linear Array HPV Genotyping kit . Briefly, the assay involved amplification of samples by PCR using a master mix which contained biotin-labelled primers for 33 HPV genotypes , 81, 82(MM4), 83(MM7), 84(MM8), IS39 and CP6108) as well as the human Ten samples, which were positive by nested-PCR, were not able to be genotyped using the Linear Array kit because they were below the detectable limit of the assay. Human papillomavirus genotyping of these samples was accomplished by pyrosequencing  performed according to the manufacturer's instructions. Briefly, the nested PCR was performed as described above. The GP6+ primer was biotinylated at the 3′-end. The PCR product was denatured to obtain single stranded DNA, followed by pyrosequencing using the GP5+ as the sequencing primer. The following dispensation order was used GCACTACGCTAGCTACTACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTG. To identify the HPV genotype, sequencing results were aligned with the expected sequences for the 37 most common HPV genotypes or blasted against HPV sequences in the NCBI database.χ2 test or Fisher's exact test, where appropriate. Logistic regression was used to perform the analyses of associations between demographic, clinical and pathologic variables with HPV status. Crude odds ratio (OR) were first calculated. Univariate analyses for the association of selected variables with 11q13 amp status were performed in the subset of HPV-negative subjects to identify possible confounding factors of association. Potential confounders were identified based on P-values ⩽0.1. Logistic regression was used to calculate the relative odds of HPV status with 11q13 amp after adjusting for the potential confounders .Clinical and laboratory risk factors for the study population were extracted from our head and neck database and imported to a statistical software package for analysis. Statistical analyses were performed using the Intercooled STATA (version 8.2) software . Factors associated with HPV status were selected based on cross-tabulations and univariate logistic regression analyses. Cross-tabulations were analysed using the n=76, 61%). The majority of the study population (65%) was dead by the last follow-up (May 2004). Our study population ranged in age from 18 to 92 years; the median age at diagnosis was 63 years, and the median age at death was 67 years.Twenty-four percent (30 out of 125) of the tumours were positive for HPV. All HPV-positive tumours contained HPV16 whereas two out of 30 (7%) were coinfected with other HPV types. One had an additional low-risk HPV type 11 and the other had an additional high-risk HPV type 33 and possibly 52  .The majority of tumours occurred in the oral cavity . Human papillomavirus-positive tumours occurred at various sites throughout the oral cavity and pharynx (data not shown). Approximately 20% (18 out of 91) of tumours from the oral cavity were found to be HPV-positive, whereas a slightly higher proportion of HPV-positive tumours were observed in the oropharynx (35% (12 out of 34). When stratified by smoking status, seven HPV-positive tumours from the oropharynx of current smokers occurred in the base of tongue (3), soft palate (2), tonsil (1) and an overlapping lesion of the oropharynx (1). In contrast, the five HPV-positive tumours from the oropharynx of nonsmokers and former smokers occurred in the tonsil (3) and base of tongue (2) only.vs ⩾55 years, 19% were HPV-positive: adjusted OR=0.3, 95% confidence interval (CI)=0.1–0.8), and were less likely to carry amplified genes in chromosomal band 11q13. After adjusting for age, tumour site and stage, 11q13 amp status was still statistically associated with HPV status, with only 17% (five out of 30) of HPV-positive tumours vs 44% (39 out of 89) of HPV-negative tumours having 11q13 amp .Analyses of factors associated with a positive HPV status are summarised in We evaluated the pattern of tobacco and alcohol risk behaviours by HPV status. Although the majority of patients from both the HPV-positive and negative groups were ever smokers and ever drinkers , these patients were no more likely to be HPV-positive than those who never smoked or consumed alcohol . However, no statistical interaction between alcohol and drinking habits with HPV-positivity was observed.Individuals with HPV-positive tumours were less likely to have subsequent tumours, either recurrences, metastases or new primaries . Subsequent tumours that developed in HPV-positive patients  were confined to head and neck sites. In contrast, among patients with HPV-negative tumours, 57% (54 out of 95) developed recurrences, metastases or new primaries at head and neck sites, as well as other sites throughout the body.A higher proportion of HPV-positive tumours occurred in the oropharynx. Forty percent (12 out of 30) of HPV-positive tumours compared to 23% (22 out of 95) of HPV-negative tumours occurred in the oropharynx . A high tumour stage  and family history of cancer  were also more likely to be associated with HPV-positive tumours, but none of these associations were statistically significant.vs 44%, respectively, Fisher's exact P=0.009). In the oral cavity, 22% of the HPV-positive tumours were amplified at 11q13, whereas 39% of the HPV-negative tumours had 11q13 amp (Fisher's exact P=0.270). In the oropharynx, a significantly smaller proportion of HPV-positive tumours had 11q13 amp when compared with HPV-negative tumours .Amplification status at chromosomal band 11q13 was available for 119 tumours from our study population. Approximately 37% (44 out of 119) were amplified at 11q13. n=69) for which data on all three molecular markers were available , their associations with HPV status was assessed . As expected, HPV-positive tumours were more likely to express p16 . Sixty percent (nine out of 15) of the HPV-positive tumours exhibited high expression of p16 whereas 33% (18 out of 54) of HPV-negative tumours expressed high levels of p16.In a subset of tumours . The tumours that contained wild-type TP53 overexpressed p16, but those with 11q13 amp were less likely to be HPV-positive than those without 11q13 amp and were no different from all other tumours .We classified the subpopulation according to combined p16 expression, TP53 mutation status, 11q13 amp and p16 protein expression in a subpopulation of 69 unselected tumours. Our analysis shows that along with wild-type TP53 and p16 overexpression, HPV-positive tumours were less likely to carry gene amplification of chromosomal band 11q13, which further defines the characteristic of HPV-positive head and neck tumours and suggests a distinct molecular pathway for HNSCC development.This retrospective study of 125 patients with first primary head and neck tumours evaluated the association between tumour HPV status and patient demographics, clinical risk factors and amplification status at chromosomal band 11q13. We further evaluated the relationship between genetic alterations: TP53, 11q13 amp and HPV has been shown in one study including only 37 HNSCC  of their samples and p16 overexpression was not evaluated. The study reported a lower frequency of 11q13 amp in HPV-positive tumours (HPV+ with 11q13 amp=2 out of 4 (50%); HPV− with 11q13 amp=5 out of 7 (71%) but their HPV-positive tumours were more likely to have LOH at TP53 (three out of four (75%)). Earlier studies utilising immunohistochemistry have shown that the overexpression of p53 and p16 and reduced expression of cyclin D1 were associated with HPV-positive tumours but 11q13 amp was not evaluated and these studies only included tumours arising in the tonsils  and 11q13 amp  were evaluated to further characterise the role of HPV in head and neck tumourigenesis. Tumours that were HPV-positive were more likely to carry all three markers . We included in our analyses tumours that were first primaries from various sites within the oral cavity and pharynx and demonstrate that HPV-positive tumours with this distinct molecular phenotype can arise not only in the oropharynx  but also in the oral cavity .The relationship between LOH at et al ). Our results show that HPV-positive tumours are less likely to be amplified at 11q13 and at the same time p16 is overexpressed, providing indirect evidence that the infection may have occurred at an earlier time point in the carcinogenic process.Our results shed further light on the multiple genetic alterations that are commonly found in of head and neck tumours . A genetet al  and descTP53 by mutation seems not to be necessary in HPV-positive HNSCC, as p53 protein loss results from increased degradation in these tumours  . ApproxiTP53 and in addition, a low frequency of amplification at 11q13. Further analyses are warranted to determine whether this lack of 11q13 amp also contributes to the improved prognosis observed in patients with HPV-positive HNSCC.Several studies report that patients with HPV-positive head and neck tumours have an improved prognosis , whereas


High-risk HPV types , in particular HPV-16+18 , and multiple HPV infections  were more prevalent in HIV-1-infected women. High-grade SIL (HSIL) was identified in 3.8% of the women. Human immunodeficiency virus type 1 infection was strongly associated with presence of HSIL  after controlling for high-risk HPV infection and other risk factors for HSIL. Nine of 14 (63%) HSIL cases were associated with HPV-16 or HPV-18 infection, and might have been prevented by an effective HPV-16/18 vaccine.Human papillomavirus (HPV) infection and cervical squamous intraepithelial lesions (SILs) were studied in 379 high-risk women. Human papillomavirus DNA was detected in 238 of 360 (66.1%) of the beta-globin-positive cervical samples, and 467 HPV isolates belonging to 35 types were identified. Multiple (2–7 types) HPV infections were observed in 52.9% of HPV-infected women. The most prevalent HPV types were HPV-52 (14.7%), HPV-35 (9.4%), HPV-58 (9.4%), HPV-51 (8.6%), HPV-16 (7.8%), HPV-31 (7.5%), HPV-53 (6.7%), and HPV-18 (6.4%). Human immunodeficiency virus type 1 (HIV-1) seroprevalence was 36.0%. Human papillomavirus prevalence was significantly higher in HIV-1-infected women (87  Cervical cancer is the most frequent cancer of women in developing countries, particularly in sub-Saharan Africa (Human papillomavirus prophylactic vaccines are now being developed and promising results have been obtained with recombinant L1 capsid protein virus-like particles (VLPs). However, the current HPV vaccines target the most prevalent high-risk HPV (HR-HPV) types worldwide, namely HPV-16 and HPV-18 , virtually nothing is known of the epidemiology of HPV and HPV-related malignancies, and the impact of HIV on these conditions. The aim of the present study was to determine the prevalence of HPV infection, the distribution of HPV genotypes, the frequency of SIL, and their relationships with HIV-1, in a group of high-risk women in Bobo Dioulasso, the second largest city of Burkina Faso.In Burkina Faso, a country in West Africa with an estimated HIV-1 prevalence among the general adult population of 4.2%  among female sex workers (FSWs). The cohort inclusion process has been described elsewhere . The brush was stirred into a vial of PreservCyt transport solution (Cytyc) and kept at room temperature until shipment to the Pathology Laboratory at the University Hospital, Montpellier, France. The Yerelon Cohort Research Programme has been approved by the Ethical Committee of the Centre Muraz, Bobo Dioulasso, the National Ethical Committee of the Ministry of Health, Burkina Faso, and the Ethics Committee of the London School of Hygiene and Tropical Medicine.Thin-layer cytological smears were prepared from the Cytic cell suspension vial by controlled membrane transfer technology using the ThinPrep 2000 processor (Cytyc). After Papanicolaou (Pap) staining, slides were independently examined by two senior pathologists who were blinded to all other study results. The 2001 Bethesda classification  was usedM (MY09/MY11 primers), or 4 mM (GP5+/GP6+ primers) MgCl2, 200 μmol (each) dNTPs, 1.5 U of AmpliTaq gold DNA polymerase , and 5 μl of DNA extract in a volume of 50 μl. The PCR conditions were 95°C for 9 min, followed by 40 cycles of 95°C for 1 min, 55°C (MY09/MY11 primers) or 38°C (GP5+/GP6+ primers) for 1 min and 72°C for 1 min, and a final extension at 72°C for 5 min. For the nested PCR, 2 μl of the first-round product were used in the second-round PCR.DNA was extracted from the residual cell suspension as described previously . This test is based on the amplification of a part of the L1 region of the HPV genome using the broad-spectrum SPFhttp://www.ncbi.nlm.nih.gov/) by use of the CLUSTAL W multiple sequence alignment program. Phylogenetic trees were constructed by the neighbour-joining method and reliability of the branching orders was assessed by the bootstrap approach with CLUSTAL W. A nucleotide sequence was assigned to a HPV type if it displayed greater than 90% homology with this type  on the ABI PRISM 3100 genetic analyser (Applied Biosystems); GP5+ and GP6+ primers served as forward and reverse primers, respectively. The nucleotide sequences were aligned and compared with those of known HPV types available through the GenBank database  in this population ; and HSV-2 serology, using a specific gG2-ELISA test  proved to have a good sensitivity and specificity on African sera  and corresponding 95% CI were calculated by multiple logistic regression for factors significantly associated with HSIL in univariate analysis , or for known risk factors for HSIL reported in the literature . Associations of prevalence of HPV types with cervical lesions categories were tested by χ2 for trend.Data were analysed using STATA 8.1 . Associations between HIV and HPV were assessed with prevalence ratios (PR) and their 95% confidence intervals (95% CI). Associations between HSIL and exposures were assessed with odds ratios (OR) and 95% CI, as HSIL was a rare event, and statistically tested by Fisher's exact test and C. albicans, and T. vaginalis were 42.4, 6.8, and 9.6% respectively, and no diagnosis of N. gonorrhoeae was made. Only two cases of serological syphilis (RPR- and TPHA-positive sera) were detected. Human immunodeficiency virus type 1 and HSV-2 seroprevalence were 36.6 and 69.8%, respectively.A total of 379 women were seen during the study period and all agreed to participate. Their median age was 28.0 years (range 16–54 years) and the mean age at first sexual intercourse was 16.7 years. Participants had been involved in commercial sex for a median of 3.2 years, with a median number of 3.3 weekly sexual partners. Condoms were reportedly used in all sexual encounters with first-time clients by 77.1% of women, but were seldom used with their regular partner. Only 12% of participants used hormonal contraception and 7% were current smokers. Prevalence of bacterial vaginosis, Cervical samples were obtained from all 379 women, and 360 (95.0%) were positive for beta-globin DNA. The prevalence of cervical HPV was 66.1% (238 of 360): 114 samples were positive with the MY09/MY11 primer pair, 50 were positive with the GP5+/GP6+ primer pair, and 74 were positive by nested PCR. Human papillomavirus types could be identified by LiPA in 211 samples and by sequencing in the remaining 27 samples. A total of 467 HPV infections belonging to 35 types were identified . The mosInterpretable cervical smears were obtained from 366 (97%) women. Squamous intraepithelial lesion were detected in 88 (24.0%) women, with 74 (20.2%) being classified as LSIL and 14 (3.8%) as HSIL. Thirteen (3.5%) samples were classified as atypical squamous cells of undetermined significance (ASCUS).μl−1. One woman, infected with HIV-2 alone (and infected with HPV-70) was excluded from further analyses. Subsequent analyses are based on the 349 remaining women.A total of 350 women had a complete set of data available, including an interpretable Pap smear, a positive beta-globin DNA PCR and known behavioural and demographic characteristics. Among these women, 126 (36.0%) were infected with HIV-1 and 30 (23.8%) of them had a CD4 cell count <200 cells P=0.03). In contrast, there was no statistically significant association of HPV with age among HIV-1-seropositive women (P=0.9).As shown in P<0.001), and among women with multiple HPV infections compared to those infected with a single HPV type (54 of 123 (43.9%) vs 24 of 109 (22.0%), P<0.001). Among HPV-infected women, prevalence of SIL was significantly higher among those co-infected with HIV-1 (P<0.001). HSIL was observed exclusively among HPV-infected women, and all but one HSIL cases were observed among HIV-1-infected women (P<0.001) (μl−1) had an association of borderline significance. None of the classical cofactors of HSIL  reported in the literature were associated with HSIL in this study, in part because of the small number of HSIL cases and/or the low prevalence of some of these variables. In multivariate analysis, only HIV-1 infection remained strongly associated with the presence of HSIL , while there was suggestion for a nonstatistically significant association between HR-HPV and HSIL .Despite the participants' young age, a high prevalence (24.9%) of cervical cytological abnormalities was observed . As expeP<0.001) . Only HIP<0.001) , while lTo explore the potential impact of a bivalent HPV-16/18 vaccine, we have examined the prevalence of HPV-16- and HPV-18-related infections according to HIV-1 serostatus and cervical lesions categories . Human pThis is the first report of an epidemiological study of cervical HPV and SIL in Burkina-Faso. We did not conduct a general population-based study but decided to target a population of highly sexually exposed women. Such women are likely to concentrate the majority of HPV types circulating in the general population of the region, thereby providing some of the information required to assess the usefulness of an HPV vaccine. Moreover, our choice of study population allowed, with an economical sample size, exploration of the relationships between HPV subtypes, presence of SIL, and the role played by HIV in promoting persistence of HPV and progression to HSIL. This is critical information since, ultimately, an HPV vaccine's effectiveness will depend upon the proportion of HSIL and ICC cases attributable to the subtypes included in the vaccine, and possibly the background HIV prevalence in the population, which might act as a catalyst in the upregulation of certain HPV types. Moreover, in some sub-Saharan settings, it is conceivable that HSIL and ICC may disproportionately be found among young HIV-positive women. Thus, although the epidemiological picture found in our study population might not be indicative of infection levels in the general population of Burkina Faso, the diversity of HPV types found and their association with cervical lesions would constitute valid and important data to inform HPV vaccine strategies in the country. Finally, our study represents one of the most detailed and exhaustive molecular studies of HPV in sub-Saharan Africa, which is an important prerequisite in describing HPV epidemiology. Indeed, we detected HPV DNA in cervical samples using the two most commonly used consensus PCR primer sets for genital HPV, namely MY09/MY11 and GP5+/GP6+  and older (>40 years) HIV-1-seronegative women compared to the middle age groups is consistent with high rates of HPV acquisition following entry into sexually active life, and high rates of recurrence or persistence associated with older ages, as described elsewhere (A high prevalence of HIV-1 infection was observed in our study population, and HIV-1 infection was associated with an increased rate of HPV infection, mainly restricted to HR-HPV types. This finding may result from the high level of sexual exposure to both viruses as well as, as mentioned above, from persistence or recurrence of oncogenic HPV following HIV-1 induced immunosuppression. We also found that HIV-1 infection was independently associated with an increase in SIL among HPV-positive women and that HIV-1 represented an important risk factor for the presence of HSIL. These findings are in agreement with other African series (More than half of the HPV-infected FSWs presented multiple HPV infection, which was associated with higher HIV-1 and SIL prevalence. Previous studies have reported a high prevalence of multiple HPV infections among HIV-infected women (As was found in other African studies (In conclusion, our study confirms the high diversity of HPV genotypes, the burden of HPV-related cervical lesions, the role of HIV-1 as a cofactor of HPV persistent infection, multiple-type infection, and SIL in highly sexually exposed young African women. The potentially preventable nature of prevalent HPV types constitutes a compelling argument to propose HPV-16/18 vaccination to such highly vulnerable populations. However, the safety, immunogenicity and impact of these vaccines in populations with high background HIV-1 prevalence, remain to be determined.