This study aimed to evaluate the clinical and economic implications of integrating human papilloma virus (HPV) load testing into the follow-up and management protocol of women postconisation for high-grade cervical intraepithelial neoplasia (CIN2–3). We evaluated 130 suitable women: 63 were screened biannually by Pap smears  and 67 also had HPV-load testing (‘HPV approach’). More stringent criteria for undergoing colposcopy or reconisation were observed by the former group compared to the latter. Both approaches were analysed for cost effectiveness. There were 33 out of 67 (49.2%) colposcopic referrals and 24 out of 67 (35.8%) reconisation/hysterectomies with the ‘conventional approach’ compared to 9 out of 63 (14.2%) and 7 out of 63 (11.1%) with the ‘HPV approach’. Cervical intraepithelial neoplasia 2–3 residual disease was detected in 7 out of 67 (10.5%) and 7 out of 63 (11.1%) women. The ‘conventional approach’ had more negative colposcopic biopsies and more negative reconisation/hysterectomy histologies than the ‘HPV approach’. The respective cost per detection of one case of residual disease was US$3573 and US$3485. The ‘HPV approach’ required fewer colposcopic and reconisation procedures to detect one case of residual CIN2–3. Its higher positive predictive value than that of cytology provided a significant decrease in false positive rates and a reduction of US$88 per detected case. The diagnosis of high-grade cervical intraepithelial neoplasia (CIN2–3) bears a significant risk of developing invasive carcinoma if not treated . The recCervical cytology has significant inherent potential drawbacks. The first and most significant of them is a false negative result which precludes women with undiagnosed lesions from being referred for a proper evaluation, with the consequent obvious risk of developing invasive cervical disease. In addition, cervical cytology is associated with a substantial false positive rate that may create an overload in the work schedule of the limited available colposcopic units, causing a delay in the referral of true positive cases for colposcopic evaluation and leading to over-treatment, which is inevitably accompanied by anxiety, morbidity and monetary costs  will spontaneously remit (Human papilloma virus (HPV), which is the most common viral sexually transmitted disease in the United States . Adding Previous studies on the relation between the quantitative level of HPV DNA and histologic severity of cervical lesions have yielded controversial results. Several studies using the semiquantitative polymerase chain reaction analysis (PCR) have shown a correlation between HSIL and a large amount of HPV-16 DNA in cervical samples and a small amount in samples of a low-grade squamous intraepithelial lesion (LSIL) (vs the ‘HPV (load) approach’.We performed this prospective cohort study in order to test our hypothesis that adding assessment of the HPV load of high-risk types as a triaging tool would enable the reduction of the number of colposcopic and reconisation procedures for the detection of one case of residual CIN2–3. To this end, we compared the clinical and economic implications of two different approaches to the follow-up and management of women postconisation due to CIN2–3 lesions, namely the ‘conventional (cytology) approach’ This prospective study was carried out on a cohort of 139 consecutive patients with CIN2–3 lesions who underwent cone biopsies at the Cervical Pathology Unit of the Tel Aviv Medical Center between January 1994 and December 1998. A Pap smear had been used as the screening method for the first 72 patients  until October 1996, when HPV load assessment was added to the screening protocol of the remaining 67 patients . The mean follow-up was 53 months (range 25–77). Five of the 72 patients in Group 1 were excluded: three failed to attend all the follow-up visits and the other two were lost to follow-up. Four of the 67 patients in Group 2 were excluded: the HPV test results were incomplete in two and the other two were lost to follow-up.The mean age was 38.8 years (range 17–54) for the Group 1 patients and 37.4 years (range 16–51) for the Group 2 patients. The following characteristics were similar between the two study groups: contraception use, education level, marital status and positive surgical margins .The follow-up protocols as well as the indications for colposcopic and reconisation procedures are summarised in We used the type 1 Hybrid Capture Test System . Samples for the presence of a cocktail limited to the following high-risk HPV types  were collected from the external cervical os and the exocervix by a special brush applicator and transferred to the lab in a container with transfer media provided in a specific kit by Digene Corp. In the type 1 Hybrid Capture assay, light is emitted during the cleavage of the tested substrate. The intensity of the emitted light is proportional to the amount of DNA present in the examined specimen. A relative light unit (RLU) measurement greater than or equal to the cutoff value indicates the presence of HPV DNA, whereas an RLU measurement less than the cutoff value indicates the absence of an HPV DNA sequence. Based on the above principles, the following scale of HPV load was used: an RLU <0.3 units=a negative HPV DNA test, an RLU between 0.4 and 3 units=a borderline test, an RLU between 4 and 9 units=a low HPV DNA load test and an RLU >10=a high-load HPV DNA test.A standard cone biopsy was performed by loop excision of the transformation zone (LETZ) followed by laser vaporisation of the crater base and side walls, as previously described , Hybrid Capture test=$40 ($70 including doctor's visit) and cone biopsy=$80. These are the government estimates for the cost of the procedures that were issued in order to define tariffs in the public health system . The cost per detected case was calculated accordingly.Significant differences in proportions were assessed by Fisher's exact test. All the statistical analyses were performed using SPSS for Windows version 8.0 .In all, 67 women who were treated by the ‘conventional approach’ (Group 1) and 63 women who were treated by the ‘HPV approach’ (Group 2) completed the follow-up period according to the study protocol . Table 2vs 14.2%, P<0.001) and reconisation referrals  in Group 1 compared to Group 2, respectively. There was a trend of more negative colposcopic biopsies (27.2 vs 11.1%) as well as negative reconisation/hysterectomy histologies (37.5 vs 0%) in Group 1 compared to Group 2, respectively.There were significantly more colposcopic referrals  and 7 (11.1%) for Groups 1 and 2, respectively.Table 3The cost of detecting one case of a CIN2–3 lesion according to the ‘conventional approach’ (Group 1) was US$2964. If we had applied the ‘conventional approach’ to the whole study group, the cost-effectiveness ratio would be US$3573. The cost of detecting one case of a CIN2–3 lesion according to the ‘HPV approach’ was US$3485. Thus, adding an HPV load assessment to the standard Pap protocol for this selected group of women would yield a decrease in cost of US$88 per detection of one case of a residual CIN2–3 lesion.In spite of the relatively high rate of clearance , but they were to be expanded whenever only cytology had been used for triaging subjects .A comparison between the two study groups shows that HPV-load evaluation identifies the presence of CIN2–3 at a detection rate equal to the standard cytological approach, but with significantly fewer colposcopies and reconisation procedures. The main contribution of an HPV-load evaluation is its ability to identify normal or low-grade lesions that might progress to high-grade lesions. Three such patients from Group 2 with normal cytologic results were further evaluated only due to the discovery of a high HPV load and they were eventually proved to be CIN2–3. Accordingly, the PPV of a high-load HPV test was maximal  according to these findings.When an HPV-load evaluation is unavailable for the follow-up of such a high-risk group and initial triaging is performed by repeated cytologies, however, one must seriously consider further and complete histological evaluation of all ASCUS, LSIL and CIN1 results. The case of LSIL that was additionally evaluated by colposcopy and subsequently reclassified as CIN2–3 in Group 1 supports this contention. Such a policy, however, results in a high rate of colposcopic and reconisation referrals and, consequently, a high number of low-grade colposcopic and reconisation histologic results.Previous studies on the relation between the HPV load and the histologic grade of cervical lesions have yielded controversial results. Several investigations in which a semiquantitative PCR was used showed a significant correlation between cytological grade and the amount of HPV-16 DNA in cervical samples (We have shown a reduced cost per detection of one case of residual CIN2–3 lesion if HPV-load assessment were to be included in addition to the standard Pap smear. Several authors have independently reported that inclusion of HPV assessment in screening for cervical cancer may be more clinically effective than Pap tests alone at higher but still ‘reasonable costs’ (


The relationships between risk of cervical dysplasia and dietary and serum carotenoids and vitamins were investigated in a case–control study. Cases were 156 women who attended Papanicolaou test screening in nine institutes affiliated with Japan Study Group of Human Papillomavirus (HPV) and Cervical Cancer and had cervical dysplasia newly histologically confirmed. Age-matched controls were selected from women with normal cervical cytology attending the same clinic. Blood sample and cervical exfoliated cells were obtained for measuring serum retinol, α-carotene, β-carotene, zeaxanthin/lutein, cryptoxanthin, lycopene and α-tocopherol and for HPV detection. Higher serum level of α-carotene was significantly associated with decreased risk of cervical dysplasia after controlling for HPV infection and smoking status (odds ratio (OR) = 0.16, 95% confidence interval (CI) 0.04–0.62 for the highest as compared with the lowest tertile). Decreased risk for the highest tertile of serum lycopene (OR = 0.28) was marginally significant. Decreased risks observed for the highest tertiles of β-carotene (OR = 0.65) and zeaxanthin/lutein (OR = 0.53), were not statistically significant. © 1999 Cancer Research Campaign


Squamous cell carcinoma of the conjunctiva (SCCC) is associated with HIV-related immunosuppression, but human papillomavirus virus (HPV) is also suspected to have a role. We carried out a case–control study to assess the role of cutaneous and mucosal HPV types in SCCC, conjunctival dysplasia, and their combination (SCCC/dysplasia) in Uganda.We compared HPV prevalence in frozen biopsies from 94 SCCC cases (79 of whom were found to be HIV-positive), 39 dysplasia cases (34 HIV-positive), and 285 hospital controls (128 HIV-positive) having other eye conditions that required surgery. Highly sensitive PCR assays that detect 75 HPV types were used. Odds ratios (ORs) and 95% confidence intervals (CIs) were computed, adjusting for, or stratifying by age, sex, and HIV status.Cutaneous HPV types were detected in 45% of SCCC cases, 41% of dysplasia cases and 11% of controls. Human papillomavirus virus 5 and 8 were the most common types in SCCC, and most often occurred in combination with other types. Associations were observed between SCCC/dysplasia and detection of both single  and multiple  cutaneous HPV types, and were chiefly based on findings in HIV-positive patients. Cutaneous HPV infections were rarely observed among HIV-negative patients and the association with SCCC/dysplasia was not significant  among them. Squamous cell carcinoma of the conjunctiva/dysplasia risk and mucosal HPV types were not associated in either HIV-positive or HIV-negative patients.We detected cutaneous HPV types in nearly half of SCCC/dysplasia cases and often multiple types (HPV5 and 8 being most common). The role of HIV (confounder or strong enhancer of cutaneous HPV carcinogenicity) is still uncertain. Epidermodysplasia Verruciformis HPV types; Since 1980s there has been a substantial increase in the number of reported cases of squamous cell carcinoma of the conjunctiva (SCCC), mainly in sub-Saharan Africa . HoweverThis case–control study of SCCC and conjunctival dysplasia was carried out in Uganda to further explore the role of HPV and HIV infection in SCCC risk.From January 2004 to June 2007, 125 and 17 patients admitted to the Departments of Ophthalmology of Mulago Hospital, Kampala, and Jinja Hospitals, Jinja, Uganda, respectively, for eye lesions suspected to be SCCC were invited to participate in this study as cases. None had ever been included in previous scientific studies. Conjunctival biopsies from these patients were divided: one part was frozen and one part was fixed in 10% formalin and embedded in paraffin. Three histological slides were stained with haematoxylin/eosin and initially read by a pathologist (EO) at the Department of Pathology, Makerere University, Kampala, Uganda, and then by two independent pathologists from DDL Diagnostic Laboratory, Voorburg, the Netherlands. In case of disagreement on the presence of SCCC or dysplasia, a fourth expert pathologist in Kampala reviewed the slides. A consensus was achieved on the presence of SCCC in 100 patients. A diagnosis of conjunctival dysplasia was made in the remaining 42 patients.Eligible controls were patients who had been admitted to the same hospitals as cases for eye conditions other than SCCC or dysplasia, and required surgical intervention. This allowed for the collection of conjunctival biopsies that were subsequently frozen. Controls were frequency-matched by hospital and sex, and had to fall into the same expected age range as cases . The most frequent diagnoses were cataract (37%), chalazia (12%), corneal tears (10%), and eye trauma (9%). Patients presenting with pterygium or pingueculum were not included because it was previously suspected that these diseases were associated with HPV infection , one targeting mucosal HPV types, and one targeting cutaneous HPV types. β-globin-negative biopsy samples  were also excluded.Isolated DNA was tested using three different PCR-based assays, one targeting 10-line probe assay (LiPA)25-DNA enzyme immunoassay (DEIA) system,  as described previously . The LiPA25 SPF10 DEIA-positive samples, which were negative on the LiPA25 strip, were tested on an additional strip containing probes for 17 additional mucosal HPV types .Testing for mucosal HPV types was carried out using the short PCR fragment (SPF)eviously . Brieflyβ-HPV genus and targets a fragment of 117 bp from the E1 region of the HPV genome. Combined with the reverse hybridisation assay, the method allows the detection of 25 cutaneous HPV types . As no DEIA had been developed for this assay, all amplimers were directly analysed by reverse hybridisation assay.Testing for cutaneous HPV types was performed using a PCR reverse hybridisation assay method  that is reported to have 99.9% specificity and 100% sensitivity. The HIV-positive findings were confirmed using western blot analysis and all HIV-positive patients received post-test counselling. The diagnosis of AIDS was based on the presence of cytomegalovirus retinitis, cryptococcal meningitis, or skin rash with weight loss.The association of SCCC, dysplasia or (when findings were similar) their combination (SCCC/dysplasia) with various characteristics, and the positivity for mucosal and cutaneous HPV types was evaluated using unconditional multiple logistic regression equations. Odds ratios (ORs) and corresponding 95% confidence intervals (CIs) were calculated after adjustment for age (as a continuous variable) and sex. All analyses were also adjusted for, or stratified by, HIV status.A total of 94 SCCC cases , 39 dysplasia cases , and 285 controls  with HPV results were included in this study. Education, occupation, and cigarette smoking were not related to the risk of SCCC/dysplasia . An asson=15), 8 (n=16), and 24 (n=9). Human papillomavirus 5, 14, and 17 were each observed in at least five dysplasia cases. Human papillomavirus 5, 15, and 24 were each observed in at least five controls and only four controls had multiple infections. The few mucosal or cutaneous HPV types detected among HIV-negative cases and controls are shown in parentheses . This tendency derived entirely from findings among HIV-positive patients. Among HIV-nagative patients HPV8 was dectected in only one SCCC case and no controls, which raises, for the first time, the possibility that a cutaneous type, HPV8, might be a ‘high-risk’ cutaneous type. The classification of HPV types into high- and low-risk types, based on the change in their prevalence from less to more severe neoplastic lesions, has been essential in understanding the role of mucosal HPV infection in cervical cancer . A simil10 PCR we used for mucosal HPV types includes broad-spectrum primers that preferentially amplify mucosal types, but also allows for the amplification of some cutaneous HPV types. It is, therefore, possible that the uncharacterised HPV types detected by assays targeting mucosal types were actually low-viral-copy cutaneous HPV infections (Lack of association of SCCC risk with mucosal HPV types either in HIV-positive or HIV-negative patients in our study is in agreement with most recent studies (for review, see The conjunctiva is the only site in equatorial Africans that is not protected from ultraviolet light by heavy pigmentation (The important strengths of our study include the larger number of cases and controls compared with previous studies , so falsThe weaknesses of this study include lack of information on HPV viral load and HPV subtypes and variants. However, there is at present no evidence that cutaneous HPV subtypes and variants have any clinical relevance. Information on CD4+ count was not available either, but a relationship between SCCC/dysplasia risk and presence of AIDS was observed.Finally, many more studies exist on cutaneous HPV types and non-melanomatous skin cancer than on SCCC . NotwithIn conclusion, to determine whether any individual cutaneous HPV type is aetiologically important, or simply a correlate of immunosuppression, will require molecular studies of their presence in tumour cells (


Correction to:British Journal of Cancer (2002) 87, 61–64. doi:10.1038/sj.bjc.6600350Due to an error, the database used in the above study had interchanged information on sex with those of one of the participating cohorts. Consequently, some of the results in the published article were incorrect. The authors have performed new analyses and the correct data are given below:In a joint study of future risks for anal and perianal skin cancers in relation to baseline human papillomavirus (HPV) seropositivity nested in Norwegian and Finnish cohorts, it turns out that the information on sex had been interchanged in one of the Norwegian cohorts.With correct information on sex, HPV 16 seropositivity conferred an increased risk of anal/perianal cancer of 3.6 (95% CI=1.3–10). Overall, 27% of the female and 31% of the male cases were seropositive for HPV 16. Similar figures for HPV 18 were 27% and 8%, respectively. By dichotomisation on age and lag in the Norwegian cohort, we found that HPV 16 seropositive men above 45 years of age had an increased risk  as did subjects with less than 10 years lag .The corrected data are given below (tone.bjorge@oslo.online.noThe authors apologise for any inconvenience this may have caused. For a complete list of corrections to the text of the article, please contact


A series of 176 archival cervical intraepithelial neoplasia (CIN) was analysed for the presence, viral load and integration status of ‘high-risk’ types of human papillomavirus (HR-HPV). The samples were assayed using newly developed methods based on real-time PCR. Two methods for the extraction of DNA from the paraffin-embedded biopsies were compared: a protocol based on the MagNA pure system (Roche) and a Qiagen spin column kit (Qiagen). It was possible to amplify 94% (166) of the samples. Of these, 36, 63 and 80% of the CIN I, II and III cases contained HR-HPV. HPV 16 was the most prevalent, and was found in 20, 28 and 46% of the CIN I, II and III cases, respectively. The second most frequent HR-HPV was type 33 group, and in CIN II it was as prevalent as HPV 16. The median number of copies of HR-HPV per cell was not significantly different in the CIN I, II and III cases, but there was a wide range of viral load values over several magnitudes, regardless of the grade of CIN. All samples were found to contain integrated forms of HPV 16, frequently mixed with an episomal form. Intensive research has provided evidence for a central, causal role of certain types of HPV in the development of invasive cervical carcinoma . The prein situ, and even cervical squamous carcinoma .Protocol A is based on binding the isolated DNA from tissue samples to the surface of specific MGPs. This procedure is automated and performed by using the μl absolute ethanol were added and mixed gently. After centrifugation at 14 000 rpm for 2 min, the supernatant was discarded and 1 ml absolute ethanol was added. After a new 2-min centrifugation at 14 000 rpm, the pellet was dried for 10–15 min using a Speed Vac vacuum drier.The paraffin-embedded  tissue sections were deparaffinised with xylene and absolute ethanol. To melt the paraffin, the paraffin-embedded tissue sections were incubated at 65°C for 10 min, and, subsequently, 1 ml xylene and 500 μl tissue lysis buffer and 20 μl proteinase K, and was subsequently incubated at 56°C overnight. The cell suspension was mixed with a 350 μl cell lysis buffer in a sample cartridge, and then loaded into the MagNa Pure instrument. The other reagents, that is, MGPs, which bind the total DNA from the lysed cell suspension, proteinase K, and a wash and elution buffer, were also loaded into the instrument. The extracted DNA was eluted with a 50 μl elution buffer.The dried pellet was re-suspended in a 130 Protocol B is a manual DNA extraction method, which is based on the QIAamp Spin Column kit (Qiagen).μl tissue lysis buffer and 20 μl proteinase K. After incubation of the solution at 56°C overnight, a 200 μl cell lysis buffer was added. A new incubation at 70°C for 10 min was carried out before the addition of 200 μl 95% ethanol. The mixture was then applied to the QIAamp spin column. After centrifugation of the column at 8000 rpm for 1 min, the column was washed twice with a 500 μl wash buffer AW1 followed by AW2. The purified DNA was eluted with 50 μl distilled water.The paraffin-embedded tissue sections were deparaffinised by the procedure described above. After addition of 1 ml xylene, the tube was incubated at room temperature for 30 min and subsequently centrifuged at 14 000 rpm for 5 min. This step was repeated once, after which 1 ml absolute ethanol was added to the tube, followed by centrifugation at 14 000 rpm for 5 min. The pellet was dried in the Speed Vac for 10–15 min and was then resuspended in a 180 Extracted DNA from the MagNA pure was subsequently quantified, and HR-HPV typed, by using the ‘QUANTOVIR HPV’ detection and quantification system as described by μl (22 μl Master Mix and 3 μl DNA template), and with the use of optical 96-well micro-plates were used. The Master Mix contained 1 × PCR buffer – 200 μM each of dATP, dCTP and dGTP and 400 μM dUTP – 3.5 mM MgCl2, 10 × G-carboxy-X-rhodamine,6-Rox, and 0.625 U Taq DNA polymerase final concentrations in sterile water. The PCR amplification program consisted of an initial holding step of 10 min at 95°C, followed by a two-step cycle of 15 s at 95°C and 1 min at 57°C for 40 cycles. Three parallel reactions per sample were performed: no. 1 quantified HPV 16, 31, 18 and 45 (the last two together); no. 2 quantified HPV 33, 35, 39, 52, 58 and 67 ; no. 3 quantified the single copy human gene for hydroxymethylbilane synthase. A total of six nontemplate control reactions, which consisted only of PCR components without template DNA, were used as negative controls to ensure that the reagent mix was free of contamination. The assay was performed by using the ABI PRISM 7700 Sequence Detection System, running the 1.6.3 version of the SDS program (Applied Biosystems).The ‘QUANTOVIR HPV’ detection and quantification kit was used for preparation of the Master Mix. The real-time PCR reaction was performed in a total volume of 25 Since the ABI 7700 software could not quantify three different fluorophores in the same reaction, a separate QUANTOVIR algorithm was developed, using the MATLAB program vers. 5.2.1, directly coupled to the data collection program of the ABI 7700 apparatus. This was used for calculation of the threshold cycle, and conversion of the data into HPV copy numbers per cell. The number of cells was calculated from the copy numbers of a single copy human gene (hydroxymethylbilane synthase), and this and other calculations were performed automatically by the specially developed software.2–107 copies of HR-HPV per genomic DNA equivalent . The 96-well optical plate was used to run reactions. In each experiment, samples were loaded in duplicate and three negative controls were included.The recently developed real-time PCR-based integration method of DNA from the SiHa cell line, containing one copy of fully integrated HPV 16, was also used as a control for full integration in these experiments. No E2 signal should be produced, since the assayed region of E2 is deleted in SiHa cells.The study comprises 166 cases: among these 50 (30%) CIN I, 60 (36%) CIN II and 56 (34%) CIN III. The mean age of women with CIN I was 39.6, in women with CIN II was 33.3 and in women with CIN III was 34.7.MagNA pure system has further advantages in its semi-automation and quickness. It has been used throughout this study.A comparison between the Qiagene and Magna Pure kits showed that Magna Pure provided higher DNA yields than Qiagene for the extraction of paraffin-embedded tissue sections. The Of the 176 samples analysed with the ‘Quantovir HPV’ system, 10 were totally negative, that is, no signal was obtained from the human gene. The frequencies of the different types of HR-HPV in the remaining 166 samples, from different stages of CIN, are summarised in There was a steady increase in the frequency of HR-HPV from CIN I to III. Of the CIN III samples, 45 (80%) were positive for HR-HPV. As shown in However, the high proportion (23%) of the HPV 33 group in the biopsies from CIN II seemed almost as high as that for HPV 16 . No HPV The viral loads obtained for HR-HPV in the different grades of CIN are summarised in Since HPV 16 is the most predominant type in this material, as well as in cervical squamous carcinomas, viral load values were examined separately for this type. As for HR-HPV in general, there was an equally broad distribution of viral loads . The meaE2 gene, indicating complete integration  in our material. As could be expected, the oncogenic HPV 16 predominated at all stages of CIN: 20, 28 and 46% at CIN I, II and III, respectively. The most obvious findings were that HPV types 31 and 39 did not occur more frequently in higher degrees of CIN, and that the frequency (23%) of the HPV 33 group in the biopsies from patients with CIN II seemed almost as high as that for HPV 16. In previous studies, in which the sensitive PCR technique for detection and typing of HPV DNA was used, it was reported that HPV 16 is the most common type, followed by types 31 and 18. Even in a general population of 32–38-year-old healthy women, HPV 16 is the most prevalent type (2.2%)  were found to contain integrated virus genome. However, there was a higher proportion of cases with complete integration at the CIN III grade (nine cases) than at CIN I and II (2 and 1 cases); the average ratio of (copy numbers of E2 gen/copy numbers of E6 gen) was not significantly different in the three CIN grades. The reason could be, for instance, the limited number of samples (48 samples), or the episomal signal could arise from border areas of the biopsy that are not yet malignant. If a microdissection could be performed, to select only the parts of the biopsy with clear malignant properties, it is possible that the difference in the integrated/episomal ratio would be more significant.As we expected, there was a significantly higher number of CIN III samples showing very low levels of episomal compared to integrated form (<1/100), than for the other CIN grades.in situ already several years before diagnosis, compared to women infected with a low viral dose. Viral load mainly seems to have relevance for HPV 16-related tumours. By using quantitative methods to measure HPV viral load, we probably have a sensitive tool to identify women of over-risk for cervical carcinoma. This would have important applications for the cervical screening. Prospective randomised studies including quantitative HPV testing are needed on fresh material.A combination of organised and opportunistic screening has reduced the incidence of squamous carcinoma substantially during the last decades in Sweden . AgainstVaccines emerging today are directed mainly against HPV 16 and 18. Even if vaccines were expanded to cover HPV 31 and 45, they would still only prevent 80% of cervical cancers worldwide. HPV vaccination is likely to significantly reduce HPV infection and cervical cancer burden, but the need for screening of some form is likely to remain in the foreseeable future.


Knowledge of the prevalence of type-specific human papillomavirus (HPV) infections is necessary to predict the expected, and to monitor the actual, impact of HPV immunisation and to design effective screening strategies for vaccinated populations.N=4719), CIN3/CGIN and cervical cancer biopsies (N=1515) were obtained from sites throughout England, anonymised and tested for HPV DNA using the Linear Array typing system (Roche).Residual specimens of cervical cytology  type) was 76% in squamous cell carcinomas, 82% in adeno/adenosquamous carcinomas and 63% and 91% in CIN3 and CGIN, respectively. Of all HR HPV-infected women undergoing cytology, non-vaccine HPV types only were found in over 60% of those with mild dyskaryosis or below, and in <20% of those with cancer. In women of all ages undergoing screening, HR HPV prevalence was 16% and HPV 16 and/or 18 prevalence was 5%.Pre-immunisation, high-grade cervical disease in England was predominantly associated with HPV 16 and/or 18, which promises a high impact from HPV immunisation in due course. Second-generation vaccines and screening strategies need to consider the best ways to detect and prevent disease due to the remaining HR HPV types. A wide range of international studies have found approximately 70% of cervical cancers to be associated with human papillomavirus (HPV) types 16 or 18, and therefore preventable by currently available HPV vaccines  and for each disease phase , prior to any impact of the National HPV Immunisation Programme.Two sample types were included in the study: (i) tissue sections from routinely obtained diagnostic biopsies of cervical cancers and high-grade precancerous lesions archived in NHS pathology laboratories, and (ii) residual liquid-based cytology (LBC) samples from women attending for cervical screening. Samples were obtained from eight participating NHS pathology laboratories in England. Three laboratories ) provided both tissue sections and LBC samples, three laboratories  provided tissue sections only and two laboratories  provided LBC samples only.Cancer and CIN3/CGIN biopsies Sections from archived blocks of cervical intraepithelial neoplasia 3 (CIN3), cervical glandular intraepithelial neoplasia (CGIN), squamous cell carcinoma (SCC) and adenocarcinoma (including adeno-squamous carcinoma) (ADC) tissue were collected from the six participating sites between June 2006 and July 2008. Cases were deemed suitable for inclusion in the study after confirmation of the pathological diagnosis by an experienced subspecialist gynaecological histopathologist at each of the six participating centres. The biopsies of cancer had been originally embedded between 1986 and 2008; 96% had been embedded since 2000. The CIN3 and CGIN biopsies had been collected between 2000 and 2008. Approximately six 10 μ thick sections from each tissue block were placed in a 1.6 ml Eppendorf tube. A further section was stained and re-examined by the pathologist to confirm the presence of diseased tissue. To prevent cross-contamination from residual wax from the previous block, the microtome blade was cleaned with ethanol between blocks. Tissue sections were sent to the Department of Clinical Virology, Central Manchester University Hospitals NHS Foundation Trust for HPV genotyping. The following data were collected on a study record for each sample and sent to Manchester: month and year of birth, date of sample collection, type of lesion  and histology result.μl of methanol. Tubes were vortexed and incubated at 56 °C for 30 min followed by centrifugation at 13 000 r.p.m. for 1 min after which the octane layer was removed using a fine-tipped Pasteur pipette. The tissue pellet was then washed with 1.0 ml of ethanol, centrifuged as before and the ethanol removed. The tubes were then left at 56 °C for 30–45 min to evaporate off residual ethanol. The tissue was digested in 600–1000 μl (depending on size of pellet) of proteinase K lysis buffer containing 10 mM Tris/HCl buffer pH 8.3; 1 mM EDTA, 0.5% Triton X-100, 0.002% SDS and 250 μg ml–1 of proteinase K. Tissue digestion was carried out at 56 °C for 72 h with constant agitation. DNA was then extracted from a 200 μl aliquot of digested tissue using the Roche MagNA Pure  automated extraction system with an elution volume of 100 μl.Tissue sections were de-waxed by adding a 1 ml of octane to each Eppendorf tube followed by 75 LBC samples Residual LBC samples were collected prospectively from women undergoing cervical screening between October 2007 and January 2009 at five participating sites. Stratified sampling was used to obtain sufficient high-risk (HR) HPV-positive samples to analyse type distribution within each combination of age-band and cytology grade, thus a disproportionately higher number of older women and those with more severe cytology outcomes were sampled. Before inclusion in the study, samples were collected and handled according to local protocols: Thinprep and Surepath LBC systems were both in use, with samples stored at ambient temperatures while awaiting cytological examination. After completion of cytology, residual LBC samples that matched an unmet age-group and grade (according to the site’s stratified sampling frame) were anonymised and sent to the Health Protection Agency (HPA) Virus Reference Department for HPV genotyping. For Surepath samples, the tubes (enriched sample), not the vials, were used as the vials were found to have an inadequate amount of cellular material. The tube approximated to levels found using Thinprep, although with more variability. The following data were collected on a study record for each sample and sent to the HPA: month and year of birth, cytology result, date of sample, outward postcode , biopsy taken (and histology result if applicable).μl of cold sterile phosphate-buffered saline. After a lysis step (addition of 40 μl Qiagen Protease and 265 μl Qiagen Buffer AL containing guanidine hydrochloride) samples were stored at −25 °C before nucleic acid extraction. Nucleic acid extraction was conducted on the BioRobot Universal platform  using the QIAamp DNA Blood BioRobot MDx kit and the extraction protocol QIAamp ‘One for All UNIV rcV23’.1 ml of each LBC sample was centrifuged for 5 min at 13 000 r.p.m. and the cellular pellet suspended in 300 μl aliquot of the extracted DNA from the biopsy samples and a 10 μl aliquot of the extracted DNA from the LBC samples  test ) was used for the PCR reaction stage of the Roche LA test. The Roche LA test was used according to manufacturer’s instructions, with the exception of the sample extraction methods described above. Individual oligonucleotide capture probes enabled identification of 37 HR and low-risk HPV genotypes and the β-globin amplicon acted as a control for cell adequacy, extraction and amplification. Identification of HPV 52 in this system was possible by the use of a cross-reactive oligonucleotide probe (52 M) that hybridises with HPV genotypes 33, 35, 52 and 58. The presence of HPV 52 was therefore inferred by reactivity to the 52 M probe with no reactivity to the individual oligonucleotide probes representing HPV 33, 35 and 58. The presence of a co-infection of 52 with 33, 35 or 58 would not have identified HPV 52. The HPV-positive and -negative controls provided within the Roche LA test kit were included for every 46 tissue section samples and every 22 LBC samples tested. The time from LBC sample collection to receipt at HPA  was not negatively associated with detection of β-globin or HPV DNA: this variable was not considered further.A 50 β-globin low control band were recorded.Following an assessment of type-specific assay reproducibility (details in β-globin negative or in which no HR HPV DNA was detected using Roche LA test (β-globin control), however, it amplifies a much smaller fragment of the DNA than the Roche LA assay making it less likely to be affected by DNA fragmentation, which may occur during tissue fixation. A 5 μl aliquot of extracted DNA was amplified and detected according to manufacturer’s instructions.For tissue section samples only, additional analysis using the Inno-Lipa Extra  genotyping assay was performed on those samples that were  LA test . The Innβ-globin result by either Roche LA or Inno-Lipa Extra. Only samples with a valid HPV typing result , while the LBC samples were from 2452 women without cervical disease, 1051 with borderline, 697 with mild, 276 with moderate and 243 with severe dyskaryosis. The mean (s.d.) age of the women at biopsy was 31.9 (8.1), 49.7 (16.1), 35.4 (7.8) and 45.5 (14.1) years for CIN3, SCC, CGIN and ADC, respectively. The numbers of samples included in the study, by participating site, are available in The prevalence of each of our defined categories of HR HPV, by age-group and disease-grade, from normal to cancer, are shown in In biopsies from high-grade cervical abnormalities, HR HPV were detected in 94.2% (95% CI 87.7–97.3%) and 98.1% (95% CI 86.4–99.8%) of CIN3 and CGIN, respectively. HPV 16 and/or 18 were detected in 63.2% (95% CI 57.0–69.1%) and 90.7% (95% CI 85.0–94.4%) of CIN3 and CGIN, respectively, and were the only HR types detected in 53.9% (95% CI 48.7–59.0%) and 83.3% (95% CI 61.1–94.1%), of CIN3 and CGIN, respectively .n=226) and 62.6% (n=149), respectively. Women of all ages with moderate dyskaryosis also had high prevalence of HR HPV and of HPV 16 and/or 18 infection   and 5.1% (95% CI 3.3–8.0%), respectively . The prectively) . With inctively) .χ2P>0.05). Further exploration of HR HPV prevalence in young women (25–29 years) with normal cytology results also found no significant differences between regions (data not shown).Prevalence of HR HPV by region ranged from 13.3% in Gloucestershire to 20.1% in London, while prevalence of HPV 16 and/or 18 ranged from 4.2% in Gateshead to 6.5% in London . DiffereHPV 16 was the most commonly detected type in all cervical grades apart from CGIN wherein HPV 18 was the most commonly detected type. The four next most frequently identified HPV types in the SCC biopsy samples (in single or multiple infections) were, in descending order, HPV 33, 45, 52 and 31. The prevalence of these six types in cancer, precancerous lesions and normal cytology samples is shown in The four HR types found most commonly in SCC biopsy samples, after HPV 16 and 18, showed a general pattern of increasing prevalence with increasing grade of disease although all, except HPV 45, had higher prevalence in severe grade lesions and CIN3 than in cervical cancers . The ageThe prevalence, in the absence of HPV 16 or 18, in SCC and ADC of selected HPV types from the A9 and A7 species, closely related to HPV 16 and 18 respectively, is shown in n=46, 0.8% weighted prevalence). Of these samples, 34.8% (n=16) had normal cytology, 30.4% (n=14) had borderline and 34.8% (n=16) had mild dyskaryosis. No women with moderate or severe dyskaryosis were found to have HPV 6 and/or 11 without a HR HPV type. Overall (irrespective of other types), the age-weighted (to NHS CSP population) prevalence of HPV 6 and/or 11 was 0.9% (95% CI 0.6–1.5%) in normal samples, 0.1% (95% CI 0.08–0.14%) in borderline samples, 0.1% (95% CI 0.05–0.3%) in mild dyskaryotic samples, 0.02%  in moderate dyskaryotic and 0.01%  in severe dyskaryotic samples. In women with normal cytology, the weighted prevalence of HPV 6 was 0.8% and HPV 11 was 0.2%  samples and biopsies of cervical cancers and high-grade lesions, collected from eight sites, to describe type-specific HPV prevalence in women in England, across the full spectrum of cervical pathology, from normal to cancer.Assuming the relative frequency of SCC and ADC in our study are representative of all cervical cancers, our results suggest that at least 73% of cervical cancers in England are potentially preventable by type-specific protection from HPV 16/18 immunisation , rising to around 77% if co-infection with other HR types is never the causative HPV infection. Similarly, at least 56% and up to 65% of CIN3/CGIN are potentially preventable by HPV 16/18 immunisation. The difference between the percentage of cases with HPV 16 and/or 18 only (minimum attributable fraction), and the percentage with HPV 16 and/or 18 and another HR HPV (probable attributable fraction), indicates the potential for unmasking of disease caused by other HR HPV types if HPV 16 and 18 infections are prevented. By the same reckoning, between 32 and 49% of the moderate and 40 and 64% of the severe dyskaryotic cervical abnormalities identified by cervical screening may be preventable by HPV 16/18 immunisation.HPV 31 and 45 were each associated  with an additional 3% of SCC and 2% of ADC. As there is some evidence of cross-protection from current HPV 16/18 vaccines against infection and disease due to these types and HPV 33, 52 and 58, which are closely related to HPV 16 or HPV 18 (N=222) and 91% of ADC (N=47) diagnosed between 2000 and 2006. Three other studies have looked at fewer than 50 cases each . Our prevalence estimates do not provide data on the 20% of the population who do not regularly attend for screening , and whoThis is the first study to describe comprehensively type-specific HPV prevalence across England, including samples from women with normal cytology and all stages of cervical disease in order to be able to estimate the proportion of disease potentially preventable, nationally, by immunisation. These data will be used in models to assess the likely benefit from HPV immunisation (


The polymerase chain reaction has been used to determine the presence of human papillomavirus (HPV) 16 and HPV 18 DNA sequences in archival histological material removed from a cohort of untreated women with cervical epithelial abnormalities. The detection of HPV 16 or HPV 18 DNA sequences in the initial biopsy specimen was associated with a significantly increased risk of subsequent disease progression.


Increasing numbers of human immunodeficiency virus (HIV)-infected women are now accessing life-prolonging highly active antiretroviral therapy (HAART) in developing countries. There is a need for better understanding of interactions of human papillomavirus (HPV) and HIV, especially in the context of increasing life expectancy due to HAART. The data regarding the impact of HAART on reducing the incidence and progression and facilitating the regression of HPV infection and cervical abnormalities is largely inconsistent. Published studies differ in their study designs (prospective or retrospective cohorts or record linkage studies), screening and diagnostic protocols, duration and type of HAART use, recruitment and referral strategies, and definitions of screening test and disease positivity. Due to the ethical and resource limitations in conducting randomized trials of the impact of HAART on incidence of HPV, CIN, and cervical cancer among HIV-infected women, it is important to consider innovative study designs, including quasi-experimental trials and operations research in sentinel populations to answer the critical research questions in this area. Invasive cervical cancer (ICC), although entirely preventable by early detection and treatment, remains one of the most common malignancies among women worldwide. ICC andThe developing world carries the largest burden over 80%) of ICC, largely as a result of the expense and logistical challenges in establishing and sustaining population-level screening and treatment programs. Figure  In many 0% of ICCThe introduction of highly active antiretroviral therapy (HAART*) in the late nineties resulted in dramatic improvement of clinical outcomes and life expectancies for people living with HIV/AIDS. It also gave hope that improved immunological status would result in better clearance of HPV infection in HIV-infected women, much like other opportunistic and AIDS-associated infections, and result in a gradual decrease in the incidence and progression of cervical neoplasia. However, data from the industrialized world does not point to a clear reduction in the burden and severity of cervical disease with introduction of HAART, in contrast to other AIDS-related malignancies, most notably Kaposi's sarcoma and non-Hodgkin lymphoma -12.The need for better understanding of the interactions between HIV and HPV in the context of HAART is therefore even more pressing as increasing numbers of HIV-infected women are living longer with a persistent risk of ICC. Guidelines for prevention and treatment strategies for cervical cancer among HIV-infected women are largely based on limited evidence, or in the case of resource limited settings, are completely lacking. In this review, we summarize the available epidemiological evidence in this area, nearly all of which originates from industrialized nations. We discuss the priorities for further research in relation to resource-limited settings, home to over 90% of women living with HIV/AIDS.A systematic review of literature documenting the impact of HAART on development of HPV-induced cervical intraepithelial neoplasia (CIN) in HIV-infected women was conducted using PubMed. Article selection criteria included any clinic-based observational or population-based linkage studies documenting both HAART status and HPV/CIN/ICC rates. PubMed was searched with an end date of January 2009 using Medical Subject Headings (MeSH) "Uterine Cervical Neoplasms" OR "AlphaPapillomavirus" AND "Antiretroviral Therapy, Highly Active" limiting to English language literature. A total of 59 articles retrieved through this search were reviewed manually and additional articles were retrieved by cross referencing. The final analysis included 22 papers that are tabulated in Table The published literature on this subject is striking in lack of standardization in study designs and methods of screening as well as the dearth of studies among populations from resource-limited settings. The differences in clinical versus population-based study designs, use of prevalent versus incident lesions as endpoints, different thresholds of diagnostic criteria for CIN, differing standards of lengths of follow-up evaluation periods, and nonuniform use of diagnostic assessments by colposcopy and/or histopathology make it difficult to draw overall conclusions about the impact of HAART on cervical disease. We discuss these differences by stage of HPV infection/cervical disease and highlight trends and similarities, as well as differences, in conclusions.The definition of HAART also varies across studies, reflecting the evolution in terminology as well as the time horizon of adoption of newer classes of drugs over the past decade and a half. Several authors have defined HAART conventionally, i.e. as consisting of 2 nucleoside reverse transcriptase inhibitors (NRTIs) + 1 protease inhibitor (PI) or 1 non-nucleoside reverse transcriptase inhibitor (NNRTI) -15. SomeThere are significant differences between studies in the utilization of the screening/diagnostic methods for cervical pathology. Most investigators have used cytological (Pap smear) changes with reporting based on the revised Bethesda classification, relying on low and high-grade squamous intraepithelial lesions (SIL) as endpoints. However, cervical cytology has only moderate clinical sensitivity (55%-65%) for detection of histopathologically confirmed 'true disease status' , and witOur review covers studies that evaluate the effects of HAART on incidence, progression, and regression of HPV infection and CIN lesions/ICC. We only include studies among HIV-infected women which have controlled for cervical treatment to tease out the independent effect of HAART on recurrent lesions. We present salient features of studies grouped by impact of HAART on HPV infection, on CIN, and on ICC, in Tables In the sections below, we discuss the salient study findings that highlight the heterogeneity in studies and the current dearth of evidence to definitively demonstrate the impact of HAART on HPV-associated cervical neoplastic disease in HIV-infected women.Studies that evaluate HPV infection have been limited, primarily because of limited availability of high quality HPV polymerase chain reaction (PCR) typing on cervical samples, while simultaneously evaluating the outcomes of cytological/colposcopic abnormalities over time among HIV-infected women seeking HAART. Table  In an ItIn summary, the evidence about the impact of HAART on incidence, progression and clearance of HPV infection and lesions remains inconsistent and inconclusive. While these cohort studies are limited by their modest sample sizes in general, this limitation is especially significant when establishing impact on individual HPV types or phylogenetically similar types of HPV. It is expected that with improved techniques, expanded availability, and standardization of HPV primers that is being attempted at an international level, , future 3 and with no previous SIL, there was no significant difference in SIL incidence between groups receiving versus not receiving HAART[Since the goal of early detection (screening) for cervical cancer is to target precancerous lesions, studies that measure the impact of HAART on these lesions have significant implications for informing clinical practice as well as public health guidelines. Results from a pre-protease inhibitor-era cohort of HIV-infected women from the United States did not report a protective effect of antiretroviral treatment on SIL, even after controlling for CD4+ status and HPV-DNA status. In a weing HAART.Similar findings are also reported from another Spanish cohort that indirectly assessed the impact of HAART on incidence of SIL as outcome on its effect on CD4+ T-cell counts or its effect on HIV-1 viral loads. The HERThus, most published literature ,25,28,323) was associated with significantly higher odds of progression[Studies evaluating the impact of HAART on progression of preexisting SIL have reported mixed results. Table  Studies ogression. A more ogression. The twoogression,15. Howeogression in a 6-mThus, data on impact of HAART on progression from low to higher grade SIL/CIN remains inconclusive with conflicting study results. While it is biologically possible that immune competence restored due to HAART may in fact prevent the progression, it appears more likely that progressive lesions are reflective of slow oncogenic changes due to persistent HPV infection that are possibly unaffected to the relatively short duration and typically moderate immunocompetence induced due to HAART. Comparison across studies is difficult since they differ in how they adjust for markers of immunological status of the patient  as well as differences in HAART .10 increase in HIV viral load, but this decline was not independently related to CD4+ counts or HAART status[A 2001 report from the WIHS study concluded that women on ART who were infected with at least 1 high-risk HPV genotype were 40% more likely to experience regression of SIL than those not receiving ART, after adjustment for CD4+ cell counts and baseline cytology status. The folRT status. Also, aRT status. Other sRT status,35. HoweThus, the evidence that HAART causes regression of lesions is also mixed just as the evidence about HAART on progression of lesions. In addition to the challenges in controlling for the markers of immune suppression and the type and nature of HAART, these cohort studies also differ by varying definitions of clinical endpoints of SIL/CIN, possible misclassification of results, and the small sample sizes. Moreover, in the context of regression, the immunological and virological interplay between HIV, HPV, and HAART is not particularly well-understood. The pathways by which HIV may interact with HPV are still under investigation. The HIVIn 2000, the International Collaboration on HIV and Cancer pooled incidence estimates of ICC among all till-date prospective cohort studies in North America, Europe, and Australia in the pre-HAART and post-HAART eras. The study reports no difference in ICC incidence rates pre- or post HAART, although the pooled cases of ICC in the study were too few (36) to make definitive comparisons with the impact of HAART on other AIDS-associated cancers such d non-Hodgkin lymphoma. The ItaIt has been suggested, however, that some record linkage methodologies may have underestimated the risks of some cancers among people withAIDS. Thus, iEvident in this review is that most research on the impact of HAART on cervical cancer has been conducted and reported from high-income nations while the developing world unquestionably shoulders a disproportionate burden of the morbidity and mortality associated with cervical cancer. The samClearly, a trial randomizing women to HAART versus no HAART to study impact of HAART on incidence, progression or regression of HPV infection or CIN would be unethical. Yet, opportunities for answering these questions abound. It is quite feasible to nest outcomes research studies in HIV/AIDS care and treatment settings that may allow for accumulation of data in developing evidence-based guidelines for cervical cancer prevention in HIV-infected women. Some possible approaches to develop this evidence in the context of public health implementation programs and clinical, epidemiological, or implementation research studies could include:Dovetailing observational studies within pre-existing cohorts of HIV-infected women followed-up primarily for other HIV/AIDS related  outcomes could be valuable. In many such cohort studies, especially those in middle incomes countries, data of varying degrees of completeness exists for cervical disease status  and HAART status for HIV-infected women. These data can be gainfully exploited for evaluating associations between the HAART and cervical neoplasia that may have context specific significance.In settings where HAART programs are being newly instituted, it may be possible to conduct innovative quasi-experimental studies to answer some important research questions. In these settings, a large proportion of HIV-infected women have never been screened for cervical cancer. By providing cervical cancer screening services through cost-effective and locally appropriate methods, it is possible to compare CIN disease status (baseline and follow-up) of cervical screening-naïve women who are initiating HAART newly and those who are HAART experienced for longer periods (e.g. >6 months). This design may provide valuable research opportunities without additional investments or formal randomization procedures for HAART exposure.Multiple studies are underway evaluating the utility of early versus deferred initiation of HAART among HIV-infected persons,52. SuchIn settings with large numbers of women being treated with HAART, sentinel surveys can be conducted by documenting cervical disease status at predetermined frequencies and pre-determined sample sizes to assess changes in CIN outcomes over time. With careful data collection approaches and quality assurance measures, such sentinel sites will serve as important resources for monitoring disease trends over longer periods of time.There remain significant challenges for elucidating the impact of ART on CIN disease outcomes. As evidenced in the review of studies above, the interactions between HIV, HPV, HAART and the development of cervical neoplasia are not clearly understood. Evidence of the impact of HAART on incidence, progression and regression of CIN is largely mixed. Given the small absolute numbers of invasive cervical cancer cases in HIV-infected women in industrialized nations, the role of HAART on ICC outcomes is difficult to determine with certainty. The reasons for the wide variation in the medical literature likely reflect complex immunological or viral-immune interactions yet to be elucidated. The heterogeneity of HPV and its ubiquity in populations, the difficulty in diagnosing CIN, and its long natural history may also play a role. Certainly, the biological interactions between HIV, HPV, and HAART appear different from other cancers of viral origins common in HIV-infected persons.Innovative approaches are needed to elucidate the impact of HAART in HIV-infected women in resource-limited settings. Given that the nature of this research is intertwined with clinical care, conduct of such epidemiology studies dovetailed within existing public health care and treatment programs for HIV-infected women can have significant patient benefits as well as provide insights to the interplay between two preventable diseases: AIDS and cervical cancer.* Significant differences exist while describing the drug treatments for HIV. In this article, we use the term highly active antiretroviral therapy (HAART) broadly in place of antiretroviral therapy (ART), although but most published literature only uses the terminology HAART to describe the more effective combination regimens introduced after 1996 and those currently recommended by the World Health Organization.The authors declare that they have no competing interests.VS conceived the manuscript idea and developed it along with LB. LB conducted the data abstraction. The manuscript was drafted by both LB and VS and both authors read and approved the final manuscript.


Epidermodysplasia verruciformis (EV)-related human papillomaviruses (HPVs) were searched by broad-spectrum PCR in 86 conjunctival neoplasia biopsies and 63 conjunctival non-neoplastic control tissue from Ugandan subjects. Seven different EV-related HPV types, including a putative new HPV, and two mucosal HPVs were detected in 25% (14 out of 56) of HIV-positive, in 10% (three out of 30) of HIV-negative conjunctival neoplasia samples, and rarely (0–1.6%) in control subjects. The absence of high-risk HPVs and the low detection frequency of EV-related HPV types in more advanced tumour stages (10%) raise doubts about their role in conjunctival carcinomas.Mucosal, cutaneous and  The 125 cancer-free controls, frequency matched to the cases by sex and age (±10 years), were consecutively randomly enrolled from a pool of patients treated for benign eye lesions  or eye injuries, within the same period of time, in the same eye clinics. All enrolled subjects, representing more than 95% of those approached, agreed to provide informed consent to participate. Blood serum samples were prospectively obtained from all cases (patients) as well as control subjects and analysed for HIV serology (ELISA screening followed by Western blot confirmation). The histological evaluation confirmed a total of 118 case biopsies as conjunctival neoplasias, the remaining seven were not confirmed as neoplastic tissue and excluded from the analysis.μg ml−1 at 60°C for 30 min) in lysis buffer , followed by DNA purification, phenol and phenol–chloroform–isoamyl alcohol (25 : 24 : 1) extraction and ethanol precipitation in 0.3 M sodium acetate (pH 4.6). DNA quality test, performed by amplification with specific oligoprimers targeting a fragment of the exon 7 within the TP53 gene, and DNA quantity analysis, evaluated by spectrophotometric measurements, rendered 86 cases and 63 controls suitable for the HPV detection. Testing for HPV was carried out with general-primer-mediated PCR using the following primer pairs: (a) MY09/MY11 . Differences were considered to be statistically significant when P-values were less than 0.05.The data were analysed using P=0.0095) than that observed in controls, confirming that HIV infection is a relevant risk factor in the pathogenesis of conjunctival carcinoma in this population . A total of 14 samples (25.0%), among the 56 conjunctival neoplasia from HIV-positive subjects, tested positive for HPV sequences (P=0.0312). Only 10% (three out of 30) of the neoplastic samples, from HIV-negative subjects, were HPV positive.The HPV-positive samples comprise 17 (19.8%) conjunctival neoplasia and one (1.6%) control biopsy  lesions as ICSCC, 23 (26.7%) as CIN3, 18 (20.9%) as CIN2 and 16 (18.6%) as CIN1. The HPV distribution among cases, grouped by histological types, also stratified by HIV serology, are summarised in Characterisation of the amplified HPV sequences allowed the identification of two mucosal HPVs (6 and 18 types) and six EV-related HPVs . A putative new HPV sequence (CJ198) was identified in one ICSCC and in one CIN1 sample.Overall, this study suggests that mucosal and EV HPVs do infect the conjunctival epithelium, but does not confirm a role for specific HPVs in the development of conjunctival carcinoma. Furthermore, the low frequency of EV HPV types in ICSCC compared to that found in preneoplastic lesions (CIN1-2) suggests that these HPV types are not involved in tumour progression.These results are concordant with several others epidemiological studies, performed by broad consensus PCRs, clearly indicating that EV HPV types are commonly found in skin lesions (up to 90%) from immunosuppressed renal transplant recipients.The high detection rates of HPV DNA both in healthy skin biopsies, plucked hairs or skin swab samples in immunocompetent (25–65% HPV-positivity) and in immunosuppressed (30–90% positivity) individuals may suggest an ubiquity nature of these viruses. In conclusion, we report that in our study on conjunctival neoplasia from sub-Sahara region, cutaneous HPVs were not detected while mucosotropic HPV types have been rarely found. Moreover, EV-related HPVs were frequently identified in low-grade conjunctival neoplasia of HIV-positive patients but less commonly in invasive conjunctival carcinomas. Thus, HPV DNA found in conjunctival lesions might merely be a passenger.


Human papillomavirus (HPV) and HIV are each responsible for a considerable burden of disease. Interactions between these infections pose substantial public health challenges, especially where HIV prevalence is high and HPV vaccine coverage low.Between July 2005 and January 2006, a cross-sectional community-based survey in Mombasa, Kenya, enrolled female sex workers using snowball sampling. After interview and a gynaecological examination, blood and cervical cytology samples were taken. Quantitative real-time PCR detected HPV types and viral load measures. Prevalence of high-risk HPV was compared between HIV-infected and -uninfected women, and in women with abnormal cervical cytology, measured using conventional Pap smears.P < 0.001). Of HIV-infected women, 73.3% had high-risk HPV (200/273) and 35.5% had HPV 16 and/or 18 (97/273). Corresponding figures for HIV-negative women were 45.5% (229/503) and 15.7% (79/503). After adjusting for age, number of children and condom use, high-risk HPV was 3.6 fold more common in HIV-infected women (95%CI = 2.6-5.1). Prevalence of all 15 of the high-risk HPV types measured was higher among HIV-infected women, between 1.4 and 5.5 fold. Median total HPV viral load was 881 copies/cell in HIV-infected women  and 48 copies/cell in HIV-uninfected women . HPV 16 and/or HPV 18 were identified in 42.7% of LSIL (32/75) and 42.3% of HSIL (11/26) lesions (P = 0.98). High-risk HPV types other than 16 and 18 were common in LSIL  and HSIL ; even higher among HIV-infected women.Median age of the 820 participants was 28 years (inter-quartile range [IQR] = 24-36 years). One third of women were HIV infected  and these women were y more likely to have abnormal cervical cytology than HIV-negative women (27%, 73/269, versus 8%, 42/503; HIV-infected sex workers had almost four-fold higher prevalence of high-risk HPV, raised viral load and more precancerous lesions. HPV 16 and HPV 18, preventable with current vaccines, were associated with cervical disease, though other high-risk types were commoner. HIV-infected sex workers likely contribute disproportionately to HPV transmission dynamics in the general population. Current efforts to prevent HIV and HPV are inadequate. New interventions are required and improved implementation of existing strategies. Human papillomavirus (HPV) is the most common sexually transmitted infection (STI) worldwide. It is the pre-eminent etiologic agent of cervical cancer, the second most common cancer among women . Over 10Women with HIV infection commonly have a broader range of HPV genotypes, often with multiple concurrent HPV infections . HIV disNeisseria gonorrhoea.Besides the effects of HIV on the natural history of HPV infection, it is possible that HIV affects the transmissibility of HPV, and even possibly vice versa -19. CausThe HPV vaccine, including high-risk HPV types 16 and 18, is effective in reducing HPV infection and HPV-related disease in women, and potentially in men . These vmatatu (minibus taxi) touts or drivers.Mombasa, in Kenya's Coast province, is a major economic centre in the region, with important tourism, port, rail and industrial enterprises, as well as a large FSW population. Sex workers in Mombasa are working either full- or part-time from bars, hotels, streets or from their homes ,23. AbouBetween July 2005 and January 2006, 820 FSW from Chaani and Kisauni divisions of Mombasa were enrolled in a community-based, cross-sectional survey. FSW were recruited using snowball sampling, a method appropriate for locating difficult-to-reach populations, particularly those with stigmatized behaviours or professions . Full deIn the two study areas, an existing drop-in centre for FSW and a municipality run health centre were modified to include clinical examination areas and related research facilities. Here staff obtained written informed consent, collected demographic and behavioural data using structured questionnaires, and offered HIV counselling and same-day testing. Blood plasma samples were taken and a gynaecological examination was done with speculum insertion, and collection of endocervical and high vaginal swabs.®, Rovers®, Oss, The Netherlands) and cervical cytology was assessed with conventional Papanicolaou (Pap) smears. Slides were read by a cytologist with masters level training, supervised by a pathologist. An external cytopathologist provided quality control. The Bethesda Reporting System was used for cytologic classification [®, Tripath Imaging Inc., Burlington, North Carolina, USA) and stored at 4°C until further processing.Cervical samples were collected using a cervix brush  probes for high-risk HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66 and 68 [HPV testing was done as described by Depuydt et al  in an ac® HIV  and Determine® HIV-1/2 . In the case of indeterminate results, an enzyme-linked immunosorbent assay was used to confirm HIV status.A parallel testing algorithm was used for HIV diagnosis using rapid immunoassays: Uni-Gold™ RecombigenWhere indicated, FSW received STI treatment free of charge. Women with a positive HIV test were referred to a nearby HIV clinic where medical care and antiretroviral treatment are provided, with no user fees. Women with abnormal cervical cytology were referred for further management and follow-up at the provincial teaching and referral hospital  or a local municipal clinic supported by the International Centre for Reproductive Health.U test was used to compare the HPV viral load of HIV infected and uninfected women who had ≥ 1 HPV copy/cell. Total HPV viral load was also calculated for each woman . To account for differences in age distribution between HIV infected and uninfected women, age-adjusted risk ratios are presented comparing the risk of HPV infection in these two groups. A multivariable logistic regression model was constructed, in which the presence of any high-risk HPV infection was the dependent variable. Fitting of the regression model was done using stepwise backward elimination, with variables retained if their removal markedly altered model parameters [P < 0.1) were included in the initial model. Associations between high-risk HPV infection and abnormal cervical cytology were also assessed.Data were double entered by separate data clerks. Following data checking and cleaning, analysis was done using Intercooled Stata, version 10.0 . The presence of any detectable viral load for specific HPV types was considered diagnostic for infection with that specific type. Chi-square tests were used to detect differences in HPV prevalence in women with and without HIV infection. For each HPV type, the Mann-Whitney rameters . VariablThe median age of the 820 enrolled FSW was 28 years , though about a fifth worked mainly from home (163/820). The median income from sex work per week was 14.3 US$ , obtained from a mean of 2.3 sex partners per week (standard deviation (sd) = 0.7). About two thirds of women reported consistently using a condom with clients , while consistent condom use with boyfriends was reported by only 92 of 437 women who currently had a boyfriend (21.1%).P < 0.001). Prevalence of HIV was lower in Muslim  than in other women  and women with a shorter duration of sex work . HIV prevalence was also higher in widowed women  and in those with a recent genital sore .A third of participants were HIV infected ; HIV status was unknown in 17 women. Compared to HIV-negative women, infected women were a median 3 years older. With each increase in decade of age, the odds of HIV infection increased 1.6 fold . Among these women, over half had at least two different high-risk HPV types . HPV types 16 and/or 18 were detected in 22.8% of women (180/789), but more than twice as many were infected with high-risk HPV types other than 16 and 18 . A quarter of women were infected with low-risk HPV type 6 and/or 67 (194/789).P < 0.001). Co-infection with two or more high-risk HPV types was more frequent among HIV positive than negative women . Fifteen percent of HIV-infected FSW had four or more high-risk HPV types.Co-infection with both HIV and high-risk HPV occurred in 25.8% (200/776) of FSW . Similarly, a linear decrease in the prevalence of HPV 16 and/or HPV 18 was noted in HIV-negative women, from a prevalence of 40.5% (32/169) in women below 25 years to 7.6% (6/57) in women above 40 years . Among women with HIV infection, the prevalence of HPV 16 and/or HPV 18 did not vary across age groups.Among the whole study population, age was not associated with prevalence of high-risk HPV. However, in HIV-negative women, age effects were noted. Here, HPV prevalence decreased with increasing age, for example, 52.1% of HIV-negative women aged 17-24 years had high-risk HPV (88/169) while this figure was 38.6% in women older than 40 years  than women with no children. High levels of reported condom use were not protective against high-risk HPV infection, nor were other measures of sexual behaviour or sex work duration associated with HPV prevalence.When adjusted for age, having a child and condom use, HIV status remained a predictor for high-risk HPV infection .Compared with HIV uninfected women, prevalence of all HPV types was higher in HIV-infected women, with the age-adjusted risk ratio a median 2.7 fold higher . HPV 16 and/or HPV 18 infection was found in 35.5% (97/273) of HIV-positive women and in 15.7% (79/503) of HIV-negative women (P < 0.001).HIV infection was significantly associated with an elevated HPV viral load for types 16, 31, 39, 45, 51, 52, 59 and 66. For 9 of the 15 high-risk HPV types measured, the median viral load for high-risk HPV was 5 or more times higher in HIV-positive than HIV-negative women. The total HPV viral load was 16.9 times higher in women with HIV infection compared to uninfected women  and 82.7% with LSIL (62/75). HPV type 16 and/or 18 was detected in 42.7% of women with LSIL (32/75) and 42.3% with HSIL (11/26). However, high-risk HPV types other than 16 and 18 were more frequent, occurring in 74.7% of LSIL (56/75) and 84.6% of HSIL (22/26), even more so among HIV-infected women (12 of 13 women with HSIL). High-risk HPV type 52 (907 copies/cell) and low-risk type 67 were detected in the woman who had invasive carcinoma on cytology.Half of women with a normal Pap smear had high-risk HPV (327/648), though high-risk HPV was more common among HIV-infected women with normal cytology . Abnormal Pap smears were also markedly more common in HIV-infected women, of whom about 5% had HSIL (13/269) and 20% LSIL (54/269). The corresponding figures for HIV-negative women were 2.4% (12/503) and 4.2% (21/503), with one woman having squamous cell carcinoma. The 13 HIV-infected women with HSIL had a median of 3 high-risk HPV infections (IQR = 2-3).4 copies/cell, 24 (47.1%) had LSIL and 6 had HSIL lesions (11.8%). Only 18 HIV-negative women had a total HPV viral load = 104 copies/cell, of whom 4 had LSIL and 1 HSIL.Of the 51 HIV-infected women whose total HPV viral load was ≥10In this study, about one-third of female sex workers were infected with HIV and more than half with high-risk HPV. In multivariable analysis, high-risk HPV was more than three times as common among HIV-positive than negative women. Prevalence of high-risk HPV in the study population is notably higher than in a meta-analysis of HIV-infected women including 20 studies, mostly from North America . Though Simultaneous infection with multiple HPV types was more frequent in HIV-positive women. This finding is consistent with previous reports , and accPersistent HPV infection is the main cause of cervical cancer, and since this is more common in HIV-infected women, it makes the presence of cervical lesions in a quarter of HIV-positive women in this study even more concerning . Many prBroadly, the distribution of high-risk HPV types were similar in HIV-infected and uninfected women, albeit that the most common HPV types occurred in slightly differing order of prevalence. However, the most common HPV types in our study differ from those in other settings ,40. ThesAn association between parity and high-risk HPV has been noted previously, though might be accounted for by confounding due to sexual behaviour, such as unprotected sex or age at coital debut ,35,42. AAge, as previously shown ,30,43,44Results presented here are consistent with a meta-analysis by De Vuyst et al. describing higher median viral loads of high-risk HPV in HIV-positive women, regardless of HPV type . HPV virEven though current HPV vaccines offer some cross-protection against several HPV types , type-spIt is difficult to ascertain to what degree the sample obtained through snowballing methods is representative of the FSW population in Mombasa. Though snowball sampling is appropriate for locating difficult-to-reach populations, as it is a non-random sampling method, selection bias may occur. The investigators elected to have a large sample size, in an attempt to mitigate such effects. As a further limitation, information was missing on both HIV and high-risk HPV types, which could introduce bias. Such bias, however, is likely minimal as the missing information can be assumed to be non-differential in nature .Patterns of HPV prevalence will likely alter the impact of prophylactic HPV vaccination . The extGiven the common occurrence of high-grade cervical lesions, especially in HIV-infected women, regular HPV screening and follow-up is essential for prevention of cervical cancer. Even though much is known about these diseases and their optimal control, current measures to prevent HIV and HPV in this population are clearly inadequate. The lack of control of these infections means that sex workers likely play a disproportionate role in transmission dynamics of both infections in the general population. Additional prevention measures, especially higher population-level coverage with HPV vaccine, are required.The authors declare that they have no competing interests.SL participated in the design of the study, supervised the study and contributed to interpretation of findings and drafting of the final manuscript. DVB and MFC assisted in drafting the manuscript. MFC and WD performed the data analysis. AN critically reviewed the manuscript and assisted in interpretation of the study findings. KM participated in the study coordination, collection of data and helped draft the manuscript. CED analysed the samples and assisted in interpretation of the results. PC, JPB and MT made a substantial contribution to the study design and interpretation of findings. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/10/18/prepub


Studies on HPV infection in pregnant women and HPV transmission to the child have yielded inconsistent results.To estimate mother-to-child HPV transmission we carried out a prospective cohort study that included 66 HPV-positive and 77 HPV-negative pregnant women and their offspring attending a maternity hospital in Barcelona. To estimate HPV prevalence and genotype distribution in pregnancy we also carried out a related screening survey of cervical HPV-DNA detection among 828 pregnant women. Cervical cells from the mother were collected at pregnancy (mean of 31 weeks) and at the 6-week post-partum visit. Exfoliated cells from the mouth and external genitalia of the infants were collected around birth, at the 6-week post-partum visit, and around 3, 6, 12, and 24 months of age. All samples were tested for HPV using PCR. Associations between potential determinants of HPV infection in pregnant women and of HPV positivity in infants were also explored by logistic regression modelling.Overall cervical HPV-DNA detection in pregnant women recruited in the HPV screening survey was 6.5% (54/828). Sexual behavior-related variables, previous histories of genital warts or sexually transmitted infections, and presence of cytological abnormalities were statistically significantly and positively associated with HPV DNA detection in pregnant women recruited in the cohort. At 418 infant visits and a mean follow-up time of 14 months, 19.7% of infants born to HPV-positive mothers and 16.9% of those born to HPV-negative mothers tested HPV positive at some point during infants' follow-up. The most frequently detected genotype both in infants and mothers was HPV-16, after excluding untyped HPV infections. We found a strong and statistically significant association between mother's and child's HPV status at the 6-week post-partum visit. Thus, children of mothers' who were HPV-positive at the post-partum visit were about 5 times more likely to test HPV-positive than children of corresponding HPV-negative mothers (p = 0.02).This study confirms that the risk of vertical transmission of HPV genotypes is relatively low. HPV persistence in infants is a rare event. These data also indicate that vertical transmission may not be the sole source of HPV infections in infants and provides partial evidence for horizontal mother-to-child HPV transmission. Despite the overwhelming evidence for a sexual transmission of high-risk HPVs, other routes of transmission have been proposed. Several studies have explored whether HPVs can be vertically transmitted from mother to child by direct contact during labor, or horizontally through manipulation of the child with infected hands, bathing, towels and fomites . StudTo assess in a low-risk country the prevalence and determinants of HPV infection in pregnant women as well as perinatal transmission and persistence of HPV types, we carried out an HPV screening survey in pregnant women and a prospective study of HPV-positive and HPV-negative mothers and their infants.The project was initiated in 1995 by carrying out a prospective cohort study of pregnant women attending a public maternity hospital for prenatal care in urban Barcelona, Spain. The hospital covers mostly general, non-selected population in its catchment area. Pregnant women were selected according to their potential risk of HPV exposure as based on their history of high-risk sexual behavior, genital warts, abnormal colposcopy, and HPV-related cervical lesions by cytology or histology. Emphasis was placed in recruiting high-risk women to procure a high yield of HPV-positive mothers and thus increase statistical power to properly study HPV transmission. A total of 115 consenting pregnant women were included and classified according to their risk of HPV exposure into high- (n = 73) and low-risk (n = 42) groups. At their first or second obstetric visit , consenting women were tested for cervical HPV-DNA status using a screening consensus primer PCR (see below). Since the yield of HPV-positive pregnant women was too low  to adequately assess mother-to-child HPV transmission, we extended the study by carrying out an HPV screening survey among unselected pregnant women to identify and recruit additional HPV-positive subjects for the prospective cohort study.Subjects for this HPV screening survey included 858 pregnant women consecutively attending the prenatal care clinic of the maternity hospital between 1997 and 2000. Consenting women were tested for cervical HPV-DNA status at their obstetric visit  using the same consensus primer PCR (see below) as that used in the initial cohort study. Women were included in the prospective cohort study only when a positive result was found (i.e. HPV-negative pregnant women from this HPV screening survey were not included in the prospective cohort study). Participation rates were not estimated and refusals were replaced by the next consenting women. This limitation should not bias our results as the purpose of the HPV screening survey was mainly the recruitment of additional HPV positive pregnant women to the prospective cohort study. Thirty women were excluded because the cervical samples collected were invalid for PCR testing leaving a total number of 828 women for the analysis of HPV prevalence among unselected pregnant women. Women from this survey that were not recruited into the prospective cohort study (those testing HPV-negative) did not receive the risk-factor questionnaire (see below).Women in the final prospective cohort study included those from the initial cohort (n = 115) and all HPV-positive women from the HPV screening survey (n = 54) along with their offspring. At the enrollment visit, performed between 2 and 12 weeks after the HPV screening visit, all 169 women signed a written informed consent and were administered a full standardised questionnaire by personal interview that included socioeconomic, reproductive, sexual behavior, and risk factor variables. A standardised clinical form was also used to systematically collect all relevant clinical parameters during pregnancy, labor, and follow up visits. The questionnaires and clinical forms were only collected for the women participating in the prospective cohort study but not for the group of women that tested HPV-negative in the HPV screening survey.® collection swab, Spain), at two time points: at the pregnancy visit , and at the 6-week postpartum visit. All samples were screened for cytological abnormalities.Cervical samples from each woman were collected using a dry swab , at or around 6 weeks, and at or around 3, 6, 12, and 24 months of age. Genital samples from boys were obtained by gently rubbing the penis glans and the inner mucosal part of the prepuce. The penile shaft was not sampled. Genital samples from girls were obtained by gently rubbing throughout the mucosal part of the vulva. Mouth samples were obtained by firmly rubbing the dors of the tongue, mucosal part of the cheeks and the hard palate, using a single swab. Particular care was taken with sample collection procedures to prevent cross-contamination between subjects and different anatomical sites by using disposable equipment and changing bed lining and gloves for each woman. All the samples from recruited infants were mostly collected by the same pediatrician (JMP) throughout the study. Since the initial anal specimens did not yield sufficient DNA for adequate HPV testing, collection of anal samples was discontinued early in the follow-up and the few results obtained for this site were not considered in our analyses. All samples were sent to the laboratory, suspended in 1 mL of 0.1 M PBS , transferred to clean sterile eppendorf tubes and kept frozen at -20°C until processed.At each visit a physical examination of the child that included also a clinical inspection of the oral cavity and the external anogenital area was performed.DNA extraction was performed using 200 μL of cervical or oral cells suspensions and 400 μL from genital cells using the Qiamp Viral DNA kit  in accordance with the manufacturer's instructions.Amplification was performed using 5 μL extracted DNA with the consensus primers MY09/MY11, which amplify a fragment from the L1 HPV gene . PCR wasAll specimens were processed blindly to child or mother HPV-risk or HPV DNA initial status. Each PCR run included DNA extraction and HPV negative controls. Particular care was taken to prevent carry-over contamination by separating pre- and post-PCR areas.HPV prevalence was estimated among women at the pregnancy visit and at the 6-week postpartum visit. HPV prevalence estimates in pregnant women did not include those in the initial cohort study as these women were selected according to their risk of HPV exposure and would yield an overestimated HPV prevalence. Determinants of both HPV positivity in pregnant women and in their offspring were explored by multivariate unconditional logistic regression models to estimate prevalence odds ratios (ORs) and 95 percent confidence limits. ORs where adjusted by age in tertiles . Even though these women were selected based on their HPV status and thus biased towards HPV positivity, this should not affect the validity of exploring potential risk factors for HPV infection in pregnant women.HPV prevalence in infants and mother to child transmission rates were estimated at the perinatal period (0 to 6 days of life), at the 6-week postpartum visit and, since few HPV positive samples were detected after the 6-week visit, all childrens' HPV results from successive visits (3 months throughout 24 months) were combined in one single prevalence estimate. Overall and type-specific HPV transmission rates were also estimated at any point during follow-up. Estimates are given according to the HPV status of the mother at pregnancy and at the 6-week postpartum visit. HPV type-specific concordance among paired mother-child samples was estimated among the HPV-positive samples that could be genotyped. Paired samples with HPV X were not counted as concordant.Two parameters were used to explore horizontal transmission: HPV positivity rates in infants born to HPV-negative mothers, and the correlation between HPV status in the mother and HPV status in the child in paired mother-child samples simultaneously collected at the 6-week visit.All women participating in the cohort study signed a written informed consent. Protocols were approved by the local ethical committees of the Maternity Hospital, where the study was carried out, and the Ciutat Sanitaria i Universitaria de Bellvitge, where the coordinating center of the study was located.Of the initial 169 recruited pregnant women , 82  were HPV positive at the pregnancy visit. The genotype distribution in these HPV-positive samples from pregnant women was in descending order of frequency: HPV 16 (32.9%), HPV X (28.0%), HPV 6/11 (25.6%), HPV 31 (11.0%), HPV 39 (9.8%), HPV 18 (7.3%), HPV 33 (3.7%). Multiple types were present in 14 women (17.1%). The most frequent combinations among these were co-infection of HPV 16 with other types (50.0%) and co-infection of HPV 31 with HPV 39 (35.7%). Cervical HPV prevalence in the unselected group of pregnant women from the HPV screening survey was 6.5% .Of the initial 169 recruited women with a known HPV-DNA status, 26 mother-infant pairs  were excluded from the prospective cohort study because no adequate sample from the child could be obtained for HPV testing neither at birth nor at any of the subsequent follow-up visits. Of the 26 excluded pairs, 16  were HPV positive. The distribution of HPV types in these 16 excluded HPV-positive women did not statistically differ from the 66 HPV-positive women finally included in the cohort (p = 0.23). Thus, the final prospective cohort study included 143 mother-infant pairs with a valid PCR result.A total of 418 study visits were performed with a mean follow-up time of 14 months. All pregnancies ended on single births.The characteristics of the 143 women and infants enrolled in the prospective cohort study along with the obstetric clinical details are detailed in Table As shown in Table The age-adjusted analyses to identify determinants of cervical HPV infection in pregnant women yielded the expected pattern of established risk factors for HPV infection Table . No assoAs expected we found a strong association between HPV positivity and presence of cervical lesions (LSIL or worse). All HSIL cases tested HPV positive being HPVs 16, 18, and HPV X the most frequently detected types among women with HSIL.At 418 infant visits and a mean follow-up time of 14 months, overall prevalence of HPV in infants at any visit was 18.2% . New infections at the post-partum visit among HPV-negative women at pregnancy occurred in only one woman . Thus, there was a strong association between HPV status at pregnancy and HPV status in the mother at the post-partum visit (p < 0.0001). Type-specific concordance among HPV-positive women between pregnancy and post-partum visit was 100% (17/17 after excluding 7 women with HPV X at either visit). The overall type-specific distribution of HPV-positive mothers at pregnancy was not statistically significantly different from that at the post-partum visit (p = 0.83). Of the 19 mothers that tested HPV X at pregnancy only 2 were HPV X persistent at the post-partum visit, 9 became HPV negative, 3 became HPV-16 positive, and in the remaining 5, no PCR result was available at the post-partum visit.Of the 26 children that tested HPV positive at some point during follow-up, 18 had at least two consecutive samples to assess HPV persistence. Three (16.7%) had two consecutive HPV-positive samples and in two of them (11.1%) there was genotype-specific persistence. In the remaining 15 infants (83.3%) their first HPV-positive sample was followed by a subsequent HPV-negative sample. There was no statistical evidence that the distribution of HPV types detected from the infants changed over time (data not shown). At the end of the study none of the infants developed oral, anogenital or cutaneous, macroscopically identifiable HPV-related lesions.Our data from both the HPV screening survey of unselected pregnant women and the prospective cohort study provide for the first time estimates for HPV prevalence, type-specific distribution, mother-to-child transmission rates as well as HPV persistence in pregnant women and in infants in Spain.Consistent with previous reports , Although HPV-DNA detection rates in samples of newborns and infants vary widely in the literature, well conducted prospective studies suggest that the risk of perinatal transmission, although existent, is relatively low. Several studies have tested infants for HPV-DNA or antibodies . Secondly, we found an association between HPV status in the mother at the 6-week postpartum visit and the HPV status in children at the same visit or thereafter. Thus, at the 6-week post-partum visit, children of mothers' who were HPV-positive at the post-partum visit were about 5 times more likely to test HPV-positive than children of corresponding HPV-negative mothers . In contrast, no association was found between mothers' HPV status at pregnancy and children's HPV status at any of the visits combined. Thus, all together, the data in Table In assessing HPV positivity in children born to HPV-negative mothers we can not rule out that these mothers were false HPV negatives at pregnancy. We need to take into account that 52% of the HPV-negative mothers came from the high-risk group of women included in the initial cohort study. Thus, increased HPV exposure may increase risk of false negative results which might somehow partially explain HPV transmission among HPV-negative mothers.Our results do not support a high prevalence of HPV during pregnancy: 6.5% of the unselected group of women was positive for HPV-DNA by consensus PCR. This relatively low HPV prevalence in pregnant women correlates with the low prevalence of HPV infection in the female general population (between 1.3% and 2.4%) ,15 and tAs expected, and consistent with the sexual mode of transmission of HPV infections, we found that an early age at first sexual intercourse, a high number of sexual partners, particularly before the age of 20, a previous history of genital warts or STDs as well as a current squamous intraepithelial lesion in the cervix as diagnosed by cytology, were the strongest determinants of HPV positivity in pregnant women Table . These eAs shown in Table In interpreting these results it should be considered that our reported HPV type-specific distribution is probably biased towards an overestimated detection of HPV X, as the samples that tested positive with the generic probe were tested with only seven type-specific probes . Thus the high percentage of samples classified as HPV X could be true rare genital HPV types, cutaneous types (unlikely because of the poor efficiency of the primer system in detecting cutaneous types) or still untyped HPVs. Furthermore the MY09/MY11 primer system has been improved by the PGMY primer system in terms of higher reproducibility of primer synthesis as well as detection of mucosal HPVs. Unfortunately, very few samples remained available for re-testing with the newer PGMY system. This limitation may also have resulted in an underestimation of the true underlying type-specific concordance.In conclusion, our study, conducted in a population at low risk for HPV and cervical cancer, confirms that high-risk HPV genotypes can be vertically transmitted to the child, although the risk of vertical transmission is relatively low. In this study, if we exclude untyped (HPV X) infections, HPV 16 has been found to be the most frequent type detected both in mothers and infants. Infants of women who tested HPV positive at six weeks after delivery are nearly five times more likely to test HPV positive than infants of HPV-negative mothers. HPV persistence in infants is a rare event. Given the substantial HPV positivity observed in children born to HPV-negative mothers, these data suggest that vertical transmission may not be the sole source of HPV infections in children and that horizontal mother-to-child transmission may play also a role. It remains to be seen whether this alternative mode of HPV transmission and acquisition may have an impact in several areas, including vaccination strategies, epidemiological studies, and the clinical management of children with HPV-associated diseases.XC: Research Grants ; Speakers Bureau ; Steering Committee . GA: Travel grants . SdS: Research Grants . FXB: Advisory Board ; Speakers Bureau (GlaxoSmithKline); Research Grants .XC, TD, FXB, SdS were the principal epidemiological investigators in the various phases of this long study, conceived the study, wrote the protocols, assured funding, identified clinical investigators and study personnel, supervised statistical analyses and performance of laboratory assays, and wrote the manuscript. MPC, TD wrote the HPV assays protocols, performed the HPV DNA detection and genotyping of the samples validating the techniques with other referent HPV laboratories, created the HPV data base, wrote part of the methods section and made substantial comments to the manuscript. AG, RR, JMP were the clinical investigators (two ObGyn and one pediatrician), trained and supervised study clinical staff  and sample collection throughout the study, and made substantial contributions to the manuscript. MJQ, JM, GA coordinated the field work in terms of study implementation and data collection, designed the study data collection forms, created clinical and laboratory databases, implemented quality assurance procedures, made the statistical analyses, produced the working and final tables and made substantial comments to the manuscript.The work was partially supported by Spanish public grants from the Instituto de Salud Carlos III , from the Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR 2005SGR 00695), and from the Marató de TV3 Foundation (051530), who had no role in the data collection, analysis or interpretation of the results.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/9/74/prepub