This work was supported by the State Committee for Scientific Research (KBN Poland) grant No. N401 197 32/4212.CHEK2 and CYP1B1 genes predispose to neoplasm via multiorgan mechanisms, while the human papilloma virus (HPV) may actively participate in the neoplastic transformation towards urinary bladder carcinoma as an environmental factor.Urinary bladder carcinoma ranks the fourth position in malignancy incidence rates in men (6.1%) and the 17th position in women (1.6%). In general, neoplastic diseases, should be approached from two perspectives: prevention with implementation of prophylactic measures and early diagnostics. Prophylactics is possible in the preclinical phase of neoplasm, being both justified and plausible in patients from high-risk groups. Thus, it is particularly important to select such groups, not only by referring to environmental carcinogenic factors  but also from genetic predisposition, which may be conductive for neoplasm formation. The mutations / polymorphisms of CHEK2 gene mutations, CYP1B1 polymorphism and of the oncogenic types of HPV in a group of patients with urinary bladder carcinoma and in control groups. The secondary goals included a comparison of the incidence of the above-mentioned mutation/polymorphism and of persistent infection with HPV in the studied group of patients and in the control group, with a subsequent determination of the effects, which those factors could have exerted on the neoplastic process development. In general, the studies were undertaken to determine whether there is any genetic and environmental predisposition to urothelial carcinoma and to evaluate possible advantages of the used methods for efficient prognosing of genetic predisposition to urinary bladder carcinoma development.The primary goal of the study was an evaluation of the incidence of CHEK2  mutation and for polymorphism of CYP1B1 (355T/T) gene.The studied group comprised 131 patients with urinary bladder carcinoma, diagnosed for the first time and demonstrating various clinical stages  and histological grades of malignancy . DNA from tumour cells and DNA, isolated from peripheral blood, were study materials. The obtained DNA was then submitted to an intensive search for In order to find out, whether the searched mutations occurred somatically (being not limited to neoplastic cells) or were constitutional in character, the detection was carried out in the DNA from tumour tissue and from peripheral blood. The assumed presence of oncogenic types of HPV was searched in the DNA isolated from tumour tissue.CHEK2  mutation and of CYP1B1 (355T/T) gene polymorphism and oncogenic types of HPV in DNA isolated from epithelial cells in urinary sediment. Seventy-four subjects from control group III were tested for the presence of oncogenic HPV in DNA isolated from epithelial cells in urinary sediment.The control group included 131 patients , in whom tests were run for identification of CHEK2 gene mutations were identified, while 355T/T polymorphism of CYP1B1 gene was found in 18 cases of the study group (12.9%). In 36 cases (29.3%), out of 123 examined subjects, the presence of an oncogenic HPV type was found. In the control groups (I and II), one I157T missense mutation of CHEK2 gene was detected. In both control groups, 355T/T polymorphism of CYP1B1 gene was found in 7 cases.In the study group, a total of 11 mutations of A study was carried out for the presence of oncogenic types of the virusin 72 subjects of control group II with indication to HPV infection diagnostics, demonstrating the presence of oncogenic HPV in 32 (44.4%) cases. Seventy-four subjects from control group III with no indications to tests for the presence of oncogenic HPV, constituted a reference group. The presence of the virus was identified in 8 (10.81%) cases.CHEK2 gene mutations, 355T/T polymorphism of CYP1B1 gene and the presence of oncogenic HPV types.The performed studies demonstrated a statistically significant difference between the study group and the control group in the incidence of Taking into account the obtained results, the following conclusions have been drawn:CHEK2 gene mutations, 355T/T polymorphism of CYP1B1 gene and the presence of oncogenic HPV types are observed with a higher, statistically significant prevalence in neoplastic tissue of urinary bladder carcinoma.1. CHEK2 gene mutations or 355T/T polymorphism of CYP1B1 gene and of the presence o oncogenic HPV types statistically significantly correlates with histological malignancy grades of urinary bladder carcinoma.2. The concomitance of CHEK2 gene, of polymorphism of CYP1B1 gene and of the oncogenic HPV types can be added to the list of genetic and environmental factors, predisposing to urinary bladder carcinoma development and modifying the course of the disease.3. It seems that occurrence of the mutation of 4. Carrying on the above described studies on larger patient populations can allow in the future on the implementation conditions for effective prophylactics: at the carriers of the mutation/polymorphism the elimination/limitation of the influence of the environmental risk factors , and early implementation of appropriate antiphlogistic treatment in this antiviral.


Samples that tested positive but could not be genotyped were designated HPVX. Odds ratios (OR) and 95% confidence intervals (CI) were calculated by logistic regression, adjusting for possible confounding factors. While the association of human papillomavirus (HPV) with cervical cancer is well established, the influence of HIV on the risk of this disease in sub-Saharan Africa remains unclear. To assess the risk of invasive cervical carcinoma (ICC) associated with HIV and HPV types, a hospital-based case-control study was performed between September 2004 and December 2006 in Kampala, Uganda. Incident cases of histologically-confirmed ICC (N=316) and control women (N=314), who were visitors or care-takers of ICC cases in the hospital, were recruited. Blood samples were obtained for HIV serology and CD4 count, as well as cervical samples for HPV testing. HPV DNA detection and genotyping was performed using the SPFFor both squamous cell carcinoma (SCC) and adenocarcinoma of the cervix, statistically significantly increased ORs were found among women infected with HPV, in particular single HPV infections, infections with HPV16-related types and high-risk HPV types, in particular HPV16, 18 and 45. For other HPV types the ORs for both SCC and adenocarcinoma were not statistically significantly elevated. HIV infection and CD4 count were not associated with SCC or adenocarcinoma risk in our study population. Among women infected with high-risk HPV types, no association between HIV and SCC emerged. However, an inverse association with adenocarcinoma was observed, while decrease in CD4 count was not associated with ICC risk.The ORs for SCC and adenocarcinoma were increased in women infected with HPV, in particular single HPV infections, infections with HPV16- and 18-related types, and high-risk HPV types, specifically HPV16, 18 and 45. HIV infection and CD4 count were not associated with SCC or adenocarcinoma risk, but among women infected with high-risk HPV types there was an inverse association between HIV infection and adenocarcinoma risk. These results suggest that HIV and CD4 count may have no role in the progression of cervical cancer. Human papillomavirus (HPV) infection is a necessary cause for the development of cervical cancer, and the risk of cervical cancer differs  accordinWe present here results from a study of invasive cervical carcinoma (ICC) in relation to HPV status, HIV status and CD4 count in Uganda, where the estimated world-standardised incidence rates of cervical cancer is rising and is now 52.4 per 100 000 women .We conducted a hospital-based case-control study in Mulago Hospital in Kampala, Uganda, which is the national referral and teaching hospital for Makerere University. The hospital admits about 30 ICC cases each month.Patients attending the gynaecological clinics or emergency section are mostly residents of Kampala City and the surrounding areas, with a population of about 1 000 000, although some come directly from areas outside Kampala. Like most urban areas of Uganda, HIV prevalence is relatively high in Kampala . RecruitPatients eligible for the study were women aged 18 to 74 years, residents of Uganda for at least 2 years and consecutively diagnosed with incident ICC during the period September 2004 to September 2006. ICC cases recruited had not yet undergone primary treatment, signed a written informed consent form to participate in the study, and were able to provide biological samples. ICC cases were excluded if tissue and blood samples could not be collected because they were in a terminal stage, or for any other reason that might interfere with established patterns of patient care.At the Mulago Hospital, all hospitalised patients have one or more accompanying persons, who are responsible for preparing food and taking care of basic hygiene needs and other patient requirements. These accompanying persons are in general female members of the same family or clan. Control women were recruited among those accompanying or visiting ICC cases. The motivation to enrol relatives of the cancer patients as controls was to avoid bias by including control women who would most likely live in the same areas of the country as the ICC cases and would have a similar social background. In addition, control women would probably have used the same hospital as the ICC cases if they had had a cervical cancer diagnosis. Since the evaluation of family history of cervical cancer was not part of our study hypothesis, the inclusion of blood relatives of ICC cases did not constitute a problem.Control women were frequency matched to ICC cases by 5-year age groups. Inclusion criteria for control women were the same as for ICC cases , save the cervical cancer diagnosis.Control women were offered a pelvic examination with visual inspection of the cervix uteri and Pap smear screening test. Test results were made available to control women before they left the hospital . Women in whom pre-malignant and malignant cervical abnormalities were suspected or detected were referred for standard follow-up diagnosis and treatment at Mulago Hospital. A symptomatic diagnosis of cervical and vaginal infections was done by the gynaecologists examining control women, and appropriate antibiotic treatment was offered free of charge whenever indicated.Information about reproductive history, lifestyle practices and sexual behaviour was assessed using a standardised questionnaire administered by the nurses or midwives recruiting ICC cases and control women. Blood was obtained for a full blood count, HIV serological testing, and CD4 count. All subjects underwent pelvic and abdominal examinations, and cervical samples were obtained for diagnosis and HPV detection.For ICC cases, biopsies of the cervical lesions were collected under general anaesthesia. Samples were immediately stored in sterile specimen tubes and transported in a flask containing ice to the pathology laboratory. There the specimen was divided into two parts: one part was fixed in 10% buffered formalin (for 12 to 24 hours) for histological processing and diagnosis, and the other half was kept in a Nunc tube, labelled and stored at -70°C until shipped for HPV testing. Biopsy samples were processed using the automatic tissue processor; they were paraffin embedded, sectioned at 4 μm thickness and stained with Haematoxylin and Eosin.For control women, exfoliated cervical cells were collected with two Cervex brushes before any treatment for cervical or vaginal infection was administered. The first brush was rinsed in PreservCyt solution  according to the manufacturer's instructions and kept at room temperature until shipped to the laboratory for HPV testing. The second brush was used to make a smear and stained with Pap stain; smears were classified as normal or abnormal. All histological and cytological diagnoses were done by a pathologist at the Department of Pathology, Makerere University.HIV was measured using a rapid test , which wThe frozen ICC samples and the exfoliated cervical cell samples (in ThinPrep) from control women were shipped to DDL Diagnostic Laboratory (DDL), Voorburg, the Netherlands, for HPV analysis. Frozen samples were shipped in dry ice, while ThinPrep samples were shipped at room temperature. DNA was extracted from the frozen biopsy sections using proteinase K. From the suspension of cervical cells, DNA was isolated by use of a MagNA Pure LC instrument  using the total nucleic acid isolation kit (Roche Diagnostics).10 PCR was performed using 10 μl isolated DNA in a final reaction volume of 50 μl. Ten μl of the amplified PCR product was tested with a cocktail of general probes recognising at least 54 mucosal HPV types in a microtitre plate format for the detection of HPV DNA by use of DNA enzyme immunoassay (DEIA). Optical densities (OD450) were read on a microtitre plate reader. Subsequently, for HPV DNA-positive samples, 10 μl of the same SPF10 amplimer was genotyped by the SPF10-LiPA25 test . By reverse hybridisation of the SPF10 amplimer with 28 different probes on the LiPA strip, 25 HPV genotypes can be identified: HPV6, 11, 16, 18, 31, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 68/73, 70, 74. The sequence variation within the SPF10 primers allows the recognition of these different HPV genotypes, except for HPV68 and 73, as their innerprimer regions are identical and cannot be distinguished in this test. The positive hybridisation on the strips is visualised as a purple band by means of a precipitating colour substrate on the probe site. Samples that were HPV DNA-positive by SPF10 PCR-DEIA, but for which no type was identified by the LiPA25 strip, were designated as HPVX.SPFFor HPV-negative ICC cases, a beta-globin PCR was performed to assess the quality of DNA isolated from the biopsy.The study protocol was approved by the Higher Degree's Research Committee of the Faculty of Medicine, Makerere University, and the Uganda National Council of Science and Technology. The study also complied with the ethical norms of Mulago Hospital and those of the Ministry of Health of Uganda. All the study participants signed a written informed consent form before inclusion in the study.Our analytical strategy was based on the assumption that normal cervical cells persistently infected with high-risk HPV types may undergo several mutations, which can eventually lead to the development of ICC. In our study cervical samples for HPV testing were collected at the time of cancer diagnosis . Therefore HPV infections are only to be considered as indicators of persistent HPV infection(s).per se is associated with cervical cancer risk (as this is a question mark in the literature), and moreover whether the lowered immunity caused by HIV infection or by other causes (as reflected by decreased CD4 count) is associated with this risk. HIV infection and decline in CD4 count may increase the probability of acquiring an HPV infection, or decrease the probability of clearing an HPV infection.We also wanted to test the hypothesis that HIV infection To evaluate the risk of ICC, SCC and adenocarcinoma were analysed separately, and odds ratios (OR) and corresponding two-sided 95% confidence intervals (CI) were calculated using logistic regression models. To address the problem of small sample sizes, we calculated the logistic regression using Firth's penalised likelihood approach . As a coa) Each HPV type or group of HPV types,b) HIV status (positive or negative),c) CD4 count per 100 cell decrease, as a linear continuous variable(s),d) Each HPV type and group of HPV types in subgroups of women classified according to HIV status,e) HIV status and CD4 count (with groups of >300 or ≤300 and >500 or ≤500 cells) among women who were infected with high-risk HPV types only.The groups of HPV types analysed were "any HPV infection"; "single HPV infections" for any HPV infection except HPVX; "multiple HPV infections", i.e. concomitant infection with more than one HPV type, except for HPVX; "high-risk HPV types" for infections with any of the HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68 or 73; "low-risk HPV types" for infections with any of the HPV types 6, 11, 34, 40, 42, 43, 44, 53, 54, 66, 70 or 74; "HPV16-related types" for any of the HPV types 16, 31, 33, 35, 52 or 58; "HPV18-related types" for any of the types 18, 39, 45, 59 or 68; and HPVX.In the analysis of ICC risk according to HIV status, adjustment for CD4 count was done to rule out the possibility that HIV had an influence on risk before affecting immune status (reflected by a decrease in CD4 count).The statistical software SAS version 9.2 procedure logistic was used for all analyses. All tests of statistical hypotheses were made on the two-sided 5% level of significance.During the study period, 484 women with a diagnosis of preliminary incident ICC and 385 control women were approached and invited to participate in the study. Sixty-six (66) potential ICC cases and 22 control women declined to participate, and 41 ICC cases and 44 control women were excluded due to incomplete questionnaires or inability to provide biological samples. Further, 61 potential ICC cases were excluded because ICC diagnosis was not confirmed by histology, as well as five control women who had cervical lesions compatible with cervical intraepithelial neoplasia (CIN). Thus, a total of 316 ICC cases  and 314 control women were recruited into the study. Of these, cervical samples for HPV testing were obtained from 255 ICC cases  and 309 controls. Following HPV testing, 16 of the 255 ICC cases had to be excluded as their samples were beta-globin-negative, leaving 239 ICC cases in the final analysis  and 95 (30.7% of 309) women, respectively. Seventeen ICC cases (7.1%) were HPV-negative.There were no other statistically significant differences between ICC cases and control women regarding characteristics, including HIV positivity. The HIV test was positive in 55 (18.8%) ICC cases and 54 (17.5%) control women Table .For SCC, the age-adjusted ORs were statistically significantly increased for any HPV infection , single HPV infections , HPV16-related types , HPV18-related types , and high-risk HPV types , in particular HPV16 , HPV18 , and HPV45 .Multiple HPV infections and infections with low-risk HPV types were not associated with an increased OR for SCC. Adjustment for HIV as well as decline in CD4 count (-100 cells) did not substantially change the OR estimates among the different groups of HPV types Table .The association between infection with different HPV types and SCC was rather similar in HIV-negative and -positive women. However, the number of HIV-positive SCC in our study N = 42) was limited, and therefore the CIs of our OR estimates were rather broad , single HPV infections , infection with HPV16-related types , HPV18-related types , and high-risk HPV types, in particular HPV 16 , 18  and 45 . Adjustment for HIV status as well as decline in CD4 count (-100 cells) did not substantially change the OR for the different groups of HPV types (Table Given the very low number of HIV-positive adenocarcinomas (N = 4) in our study, our risk estimates are not sufficiently precise to reach any conclusions about differences between HIV-negative and -positive women, but there was no evidence of a difference in risk Table .Table In statistical models adjusting for age and decline in CD4 count (-100 cells), HIV-positive women had an increased OR for SCC  of borderline significance. However, the OR for SCC among HIV-positive women was no longer statistically significant after further adjustment for different groups of HPV infections , except when we adjusted for infection with low-risk HPV types alone  and multiple HPV infections , when the lower CIs were of borderline statistical significance Table . There wDecline in CD4 count (-100 cells) was not statistically significantly associated with SCC  or adenocarcinoma  in age-adjusted models including all women with available information in the study, nor in analyses adjusting for HIV and individual HPV types, or groups of HPV types (data not shown).Table The HPV types associated with both SCC and adenocarcinoma risk in our study have also been found to be associated with most ICC in other regions of the world ,18.Our study shows that SCC in Uganda is strongly associated with any HPV infection and infection with high-risk HPV types, in particular HPV16 (and related types), 18 (and related types) and 45. We found no evidence that adjustment for HIV infection or CD4 decline affect the OR for HPV infections significantly.As in our present study, several earlier studies found that HPV16 predominated in SCC . Our stuFor adenocarcinoma, we found an association between any HPV infection, in particular single infections, infections with high-risk HPV types, and most importantly HPV16 (and related types), 18 (and related types) and 45. Adjustment for HIV infection and decline in CD4 count did not alter the associations meaningfully, suggesting that there is only a small confounding effect of HIV and its associated immune deficiency on the risk of HPV in cervical adenocarcinoma.HPV18 has been reported as the most frequent HPV type found in adenocarcinoma . In a poTrichomonas vaginalis, but no associated increased risk for adenocarcinoma. Another factor which could in theory explain the differences in SCC and adenocarcinoma risk is smoking [These differences in findings across different studies may indicate that regional factors, such as differences in the prevalence of different HPV types, may affect ICC risk . These d smoking ; however smoking  and 5.3% smoking . The rel smoking , includi smoking  which fo smoking .10-LiPA25 HPV test used in our study  is highly sensitive for the HPV types tested. We tested HPV-negative ICC samples for beta-globin, and excluded from all analyses those women whose samples were beta-globin-negative. Thus, the samples included in our analysis were of sufficient quality to allow HPV detection. However, since cases were in general presenting at advanced stages (3 and 4), it is possible that HPV could no longer be detected in the cervical cells of some patients, which may explain why a small proportion of our cases were HPV-negative. This could have somewhat biased the OR for the associations between HPV and ICC towards unity.As possible caveats, our study was limited by the number of probes for HPV, which only allowed us to detect 25 HPV genotypes. These are, however, the most important HPV types described in the literature as being associated with cervical lesions including ICC. Moreover, the SPFOf particular interest was the lack of clear association between HIV infection and decline in CD4 count on the risk for SCC in our entire study population, and in the subset of women infected with high-risk HPV types. Although there was a non-significant increase in SCC risk associated with HIV infection, it is not probable that this effect was due to immune depression on disease progression, since the OR was not decreased when adjusting for CD4 count.For adenocarcinomas, there was no evidence of association with HIV infection in the entire study population, regardless of adjustment for different HPV types or groups of types and CD4 count. However, there was an inverse association between HIV infection and adenocarcinoma risk when restricting the analysis to women who were infected with high-risk HPV types. There was no association between CD4 count and adenocarcinoma risk when analysing all women in our study, or when restricting the analyses to the subset of women infected with high-risk HPV types. These finding must be interpreted with caution, as the number of HIV-positive women in our study, in particular among adenocarcinoma cases, was small, and therefore we cannot rule out the possibility of a chance finding.The overall HPV prevalence among HIV-positive control women was higher than among HIV-negative control women. This may be explained by common sexual transmission routes, or it may be a reflection of the HIV-associated decrease in immune response, which increases the probability of being infected with HPV, and of clearing the infection less effectively than women who are not infected with HIV. As a consequence, the association between HPV infection and ICC among HIV-positive women was in general weaker than among HIV-negative women.Several previous studies have shown varying risk of ICC in association with HIV infection ,35-41. EIn our case-control study design, cases were recruited when ICC was already present, and exposures were measured at the time subjects were recruited into the study. Thus, we do not know if HIV infection, or if decrease in CD4 count, occurred before or after the onset of HPV infection or cervical lesions. It is possible that some HIV infections occurred before the onset of HPV infections and before the development cervical pre-invasive or invasive lesions. HIV infection could also have occurred while women were infected with HPV, thus eventually decreasing their capacity to clear the infection. It is also plausible that HIV infections may have occurred too late in life to have any effect on the occurrence or persistence of HPV infection, as suggested by a study from Nairobi, Kenya .The lack of association between HIV infection, CD4 count and SCC risk in our study could also be explained by the fact that progression of CIN to ICC is multifactorial, and not dependent on immune status only , by the Our study had a number of advantages compared to previous studies. We enrolled all consecutive patients suspected to have ICC, aiming to reduce bias that could occur if any criteria associated with the exposure were used to select ICC cases, for example stage, duration of symptoms, tribe, etc. While other studies have used women with other disease conditions as controls ,12, we wper se and the HPV risk associated with HIV status on ICC.Another strength was that we carried out the study in an area where both HIV prevalence  and cervOur study had a relatively small number of HIV-positive SCC and, in particular, adenocarcinoma. In addition, assessment of immunity was based only on CD4 count, which may reflect HIV infection on the systemic immune system. Although CD4 count has been used before , it is dIn conclusion, the OR for SCC and adenocarcinoma of the cervix in Uganda were increased in women infected with HPV, in particular single HPV infections, infections with HPV16- and 18-related types and infections with high-risk HPV types, specifically with HPV16, 18 and 45. HIV infection and CD4 count were not clearly associated with SCC or adenocarcinoma in our entire study population. Among women infected with high-risk HPV types, there was no association between SCC, HIV and CD4 count, while there was an inverse association between HIV infection - but not CD4 count - and adenocarcinoma.CD4 gene; CI: confidence intervals; CIN: cervical intraepithelial neoplasia; DDL: DDL Diagnostic Laboratory; DEIA: DNA enzyme immunoassay; DNA: deoxyribonucleic acid; HIV: human immunodeficiency virus; HPV: human papillomavirus; ICC: invasive cervical carcinoma; OR: odds ratio; PCR: polymerase chain reaction; SCC: squamous cell carcinoma. SPF10-LiPA25: system for HPV genotypingAIDS: acquired immune deficiency syndrome; BD FACSCount: automated instrument for monitoring of HIV/AIDS; CD4: cluster of differentiation 4, glycoprotein, in humans encoded by the The authors declare that they have no competing interests.MO, FM and EW were responsible for developing the concept, full proposal development and obtaining ethical approvals. MO carried out the field work and was responsible for data collection. BK and WQ made all laboratory analyses and contributed to the interpretation of laboratory results. SS provided statistical analyses and interpretation of the results. MO and EW wrote the manuscript while all other authors; FM, BK, WK and SS gave their critical comments upon the writing process and revised the final manuscript. All authors approved the version to be published.


The development of cervical cancer is associated with high-risk Human papilloma viruses (HPV-HR). In sub-Saharan Africa cervical cancer is the most common cancer among women and the leading cause of death attributed to malignant tumors. This study aims to identify HPV genotypes within the 30′S and 50′S HPV families found in two previous studies from our laboratory, and to determine the prevalence of twelve HPV-HR genotypes in a population of women in Ouagadougou. The twelve HPV-HR genotypes were determined by real-time multiplex PCR, in 180 samples from the general population and among a group of HIV-1 infected women.The most common genotypes found were HPV-35 (29.4%) and HPV-31 (26.1%) of the 30′S family, and HPV-52 (29.4%) and HPV-58 (20.6%) of the 50′S family. Multiple infections of HPV-HR were observed in 78.03% of infected women.The frequencies of HPV genotypes from the 30′S and 50′S families were higher, while the genotypes HPV-16 and18 were lower among the women in our study. Human papilloma viruses (HPV) are responsible for cervical cancer (CC), genital warts and verruca.One hundred and eighty (180) women divided into three (3) groups were included in this study.Cohort 1 consisted of 63 women of unknown HPV status, recruited from November 2012 to January 2013. Cohorts 2 and 3 were formed by 34 women positive for 30′S and 50′S HPVAll women who could write signed a consent forms to participate to the study.Samples were collected from the endocervical region with a sterile cotton swab. The collected samples were stored in transport medium (Sacace biotechnologies) and frozen at − 20° C.DNA extraction was performed using the DNA-Sorb-A kit  following the manufacturer’s instructions. HPV-HR genotyping was performed by real time PCR using the HPV High Risk Typing Real-TM kit (Sacace Biotechnologies) in the SaCycler-96 Real Time PCR machine (Sacace Biotechnologies).The HPV High Risk Typing Real-TM kit is based on two major processes: isolation of DNA from specimens, and multiplex Real Time amplification of 4 tubes for each sample. Each tube contains primers directed against regions of three HPV types and the β-globin gene used as Internal Control.PCR conditions were as follows : 1 cycle of 95°C for 15 minutes ; 5 cycles of 95°C for 05s, 60°C for 20s and 72°C for 15s; and 40 cycles of 95°C for 05s, 60°C for 30s and 72°C for 15s.Data were analyzed using SPSS 17 .0 and Epi Info 3.5.1 softwares. The Chi-square test was used for comparisons. A difference was significant for p <0.05. The confidence interval (CI) and odd ratios (OR) were calculated with Epi Info 6.The average age of women in cohorts 1 and 2 were respectively 33.2 ± 8.3 years (21 – 51) and 31.5 ± 10.3 years (19 – 60). Among the HIV-1 seropositive women, the mean age was 33.7 ± 6.2 years (20–53). The majority of the HPV-HR infected women was married and had a low level of education. Almost all of the women in the study had only one sexual partner. Among all the 180 HPV-HR genotypes found, the most frequent were HPV-35 (29.4%) and HPV-52 (29.4%) followed by HPV-31 (26.1%), HPV-18 (25. 0%), HPV-58 (20.6%), HPV-56 (18.9%), HPV-51 (14.4%), HPV-59 (13.9%), HPV-45 (12.8%) and HPV-33 (11.1%). The least frequent genotypes were HPV-16 (7.2%) and HPV-39 (6.7%). Among the HIV-1 seropositive women, the most represented genotype was HPV-35 with a prevalence of 49.4% while HPV-39 (9.6%) was the least common genotype. In women from cohort 1, 30.20% (19/63) were infected with at least one HPV-HR. HPV-52 (15. 9%) was more prevalent while HPV-16 with a frequency of 1.6% was less frequent . The cumThe number of HPV-HR genotypes in women ranged from 1 to 7 in HIV-1 seropositive women, and 1 to 5 in women from cohorts 1 and 2 . The average number of genotype HPV-HR was significantly higher in HIV-1 positive women (3.5 ± 1.52) compared to women of the general population (2.1 ± 1.17) (p <0.001). In addition the majority of HIV-1 positive women (74.1%) had at least 3 HPV-HR genotypes, while the majority of women of the general population (66.7%) had no more than 2 HPV-HR genotypes .HPV-HR multiple infections were observed in 78.03% of the women of the study; but were 90.1% in HIV-1 infected women.This study first aim was to diagnose individually by real-time PCR genotypes HPV-HR 30′S and 50′S detected in two previous studies from our laboratory and the second aim was to genotype 12 HPV-HR in a cohort of women in Ouagadougou.The prevalence of 30.2% of HPV-HR infection observed in this study (cohort 1) was comparable to that of 27.9% reported in Colombia.12In cohort 1 of this study (cohort 1) the cumulative prevalence of HPV-30′S and HPV-50′S families was 43.0%. We also found that the HPV-52 genotype was the most frequent both in our study population as well as in cohort 2 the . However, HPV-16 was the least common genotype observed in women. Our findings support those of Chen et al. who observed a high prevalence of HPV-52 (33.4%) and HPV-58 15.93%) and a relatively low prevalence of HPV-16 (20.95%) and HPV-18 (8.36%) among women in China..93% and Indeed, HIV increases the risk of developing cervical intra-epithelial neoplasia (CIN) and cervical cancer of the uterus.The major limitation of this report is the sample size which is not representative of the female population of Ouagadougou. However, this pilot study has an epidemiological and clinical importance because it shows the prevalence of HR-HPV genotypes in Burkina Faso is different from other regions of the world with a preponderance of HPV-35, HPV-52, HPV-31 and HPV-18 genotypes. This study can have a real impact on the choice of immunization policy of the country as the existing HPV vaccines only cover HPV-16, HPV-18, HPV-6 and HPV-11 genotypes. The development of HPV vaccines covering the of HPV genotypes 30′S and 50′S families should be considered to protect all women from getting infected with these viruses.We showed in this study a predominance of genotypes HPV-35, HPV-52, HPV-31 and HPV-18, respectively in HIV-1 seropositive women and in those of the two cohorts studied from the general population. A low prevalence of HPV-16 genotypes was also found. A large-scale study is needed to determine the epidemiology of HPV in the population in Burkina Faso.


Concerns have been raised that the proportion of cervical cancer preventable by human papillomavirus (HPV) 16/18 vaccines might be lower in sub-Saharan Africa than elsewhere.In order to study the relative carcinogenicity of HPV types in Nigeria, as well as to estimate the vaccine-preventable proportion of invasive cervical cancer (ICC) in the country, we compared HPV type prevalence among 932 women from the general population of Ibadan, Nigeria, with that among a series of 75 ICC cases diagnosed in the same city. For all samples, a GP5+/6+ PCR based assay was used for the detection of 44 genital HPV types.versus the general population . Other high-risk HPV types, as well as low-risk and multiple HPV infections were less common in HPV-positive women with ICC than from the general population.In the general population, 245  23.5% - 29.2%) women were HPV-positive, among whom the prevalence of HPV35 and HPV16 were equally frequent . In ICC, however, HPV16 predominated strongly (67.6% of 68 HPV-positive cases), with the next most common types being 18 , 35, 45 and 56 . Comparing among HPV-positive women only, HPV16 and 18 were over-represented in ICC Our study confirms that in Nigeria, as elsewhere, women infected with HPV16 and 18 are at higher risk of developing ICC than those infected with other high-risk types, and that current HPV16/18 vaccines have enormous potential to reduce cervical cancer in the region. Cervical cancer incidence in sub-Saharan Africa is among the highest in the world , as is tHPV16 prevalence was also the highest in Nigeria. However, in relative terms, HPV-positive women in Nigeria were less likely to be infected with HPV16 and more likely to be infected with other high-risk HPV types, in particular HPV35, but also HPV45, 52, 56 and 58 [In order to study the relative carcinogenicity of HPV types in Nigeria, as well as to obtain a more robust measure of the vaccine-preventable proportion of ICC in the country, we compared HPV type-specific prevalence in the previously published IARC prevalence study with that among a newly collected series of ICC cases diagnosed in the same city and tested for HPV using the same protocol.Between April 1999 and April 2000, a previously published study of HPV prevalence was carried out among a representative sample of women living in Ibadan, the third largest city in Nigeria, according to the standardized protocol of the IARC HPV Prevalence Surveys , which wTumour biopsies, formalin fixed and embedded in paraffin, were retrieved from the pathology archives for all women diagnosed with histologically confirmed ICC (n = 102) at the College of Medicine, University of Ibadan, Nigeria, between 2004 and 2006.2 of tissue were predigested with Proteinase K after which DNA was extracted using magnetic beads . For all samples, a general primer GP5+/6+-mediated PCR was used for the detection of 44 genital HPV types, after screening for adequate DNA by PCR with beta-globin gene-specific primers. Subsequent HPV typing was performed by reverse-line blot hybridization of PCR products.HPV testing of cervical samples from both the general population and ICC cases were performed at the VU University, Amsterdam, the Netherlands. Tumour biopsies were sectioned using a 'sandwich' approach: inner sections were destined for HPV testing and outer sections for histological confirmation. One or more five μM sections representing approximately 1 cmPrevalence ratios and corresponding 95% confidence intervals (CI) were used to compare the relative frequency of HPV types in HPV-positive women with ICC with that among HPV-positive women from the general population.in situ (HPV18/42/52/81/83) or invasive (HPV82) cervical carcinoma.For the general population sample, type-specific HPV prevalence was evaluated among 932 women with valid HPV results, among whom 844 and 68 had normal and abnormal (positive on PAP and/or VIA) cervical findings, respectively. Overall HPV prevalence was 26.3% (95% CI 23.5 - 29.2) (n = 245). HPV16 and 35 were equally common (n = 30), followed by HPV31 (n = 27), 56 (n = 22) and 58 (n = 21). Two women with abnormal cervical findings were subsequently histologically confirmed with For ICC, after exclusion of biopsies without any histological evidence of tumour (n = 4) or inadequate DNA for HPV testing (n = 23), type-specific HPV prevalence was evaluated among 75 histologically confirmed ICC cases. They included 62 squamous-cell, 11 adeno-, and 2 small cell carcinomas. The median age of women with ICC was 55 years (range: 31-80 years). Overall HPV prevalence was 90.7% (95% CI 81.7 - 96.2). In seven ICC, including 3 squamous-cell and four adenocarcinoma (ADC), no HPV DNA could be detected. The five most commonly detected types in ICC were HPV16 (n = 46), 18 (n = 7), 35, 45 and 56 (n = 4 each). The HPV types among the 7 HPV-positive ADC were four HPV16 and one each of HPV18, 35 and 56. One ICC was positive for a low-risk type only (HPV30).The comparison of type-specific HPV prevalence among the 68 women with HPV-positive ICC and 245 HPV-positive women from the general population is shown in Table We previously reported that HPV-positive women from the general population in Nigeria were less likely to be infected with HPV16 and more likely to be infected with other high-risk HPV types, in particular HPV35 . Indeed,Reassuringly, however, we confirmed that HPV16 and 18 are the two most frequently detected types among ICC in Nigeria, and that they account for 78% of HPV-positive ICC, which is very similar to the proportion estimated in other world regions by recently published large meta-analyses  and caseDespite being as common as HPV16 in the general population, HPV35 accounted for only 5.9% of ICC. This figure is compatible with estimates of 5.0% (95% CI: 3.3% - 7.1%) and 4.1% (95% CI: 2.3% - 7.4%) among two recently published studies including 544 and 1,668 African ICC cases, respectively, assessed for this type , but is A relatively high importance of HPV45 (5.3%) and of HPV51 (4.0%) in Nigerian ICC is also consistent with that observed in other sub-Saharan Africa ICC in comparison to other world regions ,15. On tIn summary, our study confirms that current HPV vaccines have enormous potential to reduce cervical cancer in Nigeria, where annual ICC incidence is estimated to be 19.3 cases/100,000 women , then thADC: adenocarcinoma; CI: confidence interval; HPV: human papillomavirus; IARC: International Agency for Research on Cancer; ICC: invasive cervical cancer; OR: odds ratio.The authors declare that they have no competing interests.GC conceived the study, performed statistical analysis, and drafted the manuscript. MF, IA and SF participated in the design and coordination of the study and helped to draft the manuscript. CO and JT were responsible for sample and data acquisition. PS and CM were responsible for all laboratory analyses. All authors contributed to data interpretation and read and approved the final manuscript.


Detection of multiple human papillomavirus (HPV) types in the genital tract is common. Associations among HPV types may impact HPV vaccination modeling and type replacement. The objectives were to determine the distribution of concurrent HPV type infections in cervicovaginal samples and examine type-specific associations. We analyzed HPV genotyping results from 32,245 cervicovaginal specimens collected from women aged 11 to 83 years in the United States from 2001 through 2011. Statistical power was enhanced by combining 6 separate studies. Expected concurrent infection frequencies from a series of permutation models, each with increasing fidelity to the real data, were compared with the observed data. Statistics were computed based on the distributional properties of the randomized data. Concurrent detection occurred more than expected with 0 or ≥3 HPV types and less than expected with 1 and 2 types. Some women bear a disproportionate burden of the HPV type prevalence. Type associations were observed that exceeded multiple hypothesis corrected significance. Multiple HPV types were detected more frequently than expected by chance and associations among particular HPV types were detected. However vaccine-targeted types were not specifically affected, supporting the expectation that current bivalent/quadrivalent HPV vaccination will not result in type replacement with other high-risk types. Genital HPV is the most common sexually transmitted infection The current HPV vaccines target the two HR-HPV types (HPVs 16 and 18) associated with 70% of cervical cancers. If, however, types display positive associations to inflate infection rates, broad HPV vaccination coverage may lead to reduction of HPV types not targeted by the vaccine, i.e. “cross-protection” not based on cross-reaction immunity but as a result of reduced fitness of positively associated types. Alternatively, negative associations among types may lead to type replacement of non-vaccine types as competing types targeted by vaccines are reduced Associations among multiple HPV types have been examined in prior studies, but the conclusions are contradictory The aim of the present study is to address overall and type-specific HPV associations by taking advantage of a large laboratory database of HPV results obtained using the same validated HPV typing assay. Aggregating multiple study datasets provides greater statistical power in analyzing potential HPV type combinations. We employed a permutation methodology to test first a complete null model of random type association, and then gradually less naïve models with preserved higher orders of data structure The dataset includes anonymized HPV typing results from 32,245 cervicovaginal samples from six studies of women aged 11 to 83 years conducted between 2001 through 2011 . BecauseAll samples were extracted to yield DNA or total nucleic acids (DNA and RNA), and 0.5–1% of the total sample extract was tested for HPV. HPV typing was performed using the Linear Array HPV Genotyping Test  according to the manufacturer's protocol. The LA detects 37 HPV types , 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73, 81, 82, 83, 84, 89, IS39). HPV types 33, 35, and 58 have type-specific probes, but HPV 52's probe (XR) also reacts with HPV 33, 35, and 58. Therefore when both a HPV 33, 35, or 58 probe and the XR probe is positive, the LA result is equivocal as to whether HPV 52 is being detected or the probe is just reacting with HPV 33, 35, or 58. Samples with equivocal results for HPV 52 were tested with a quantitative type-specific assay for HPV 52 In order to test the type specificity of the LA for HPVs 56 and 66, high copy numbers of their L1 genes were amplified from plasmids. The primers were designed to flank the LA target region . Three dbase package version 2.14.1, preserving the count of each HPV type (fixed column sums) while allowing the number of types per subject to vary (variable row sums).To determine the expected number of concurrent infections, the matrix of observed results was randomized in five different ways, depending on the characteristics of the observed data being controlled, using R  [31]. Ob7 for the ‘burnin’ parameter as this value maximized Bray-Curtis dissimilarity values, indicating effective matrix randomization For the subsequent matrix randomizations, the ‘permatswap’ function created a series of matrix permutation models, each with increasing fidelity to the real data, where some of the higher order data structure is preserved during the randomization Counting occurrences of type combinations, whether in the observed data or in the randomized matrices for a given model, was done in Perl . A key feature of the counting, whether in observed or permuted data, was that specific type combinations were counted whether or not additional types were present. The results for the randomized matrices were then matched to type combinations observed in the real data.−4, 10−6, 10−8, etc. were calculated for both the right (observed > expected) and left (observed < expected) tails of the Poisson distribution. Observed counts were then plotted against expected counts for a given permutation model so that type combinations falling outside the boundary value lines could be easily seen. Besides the hypothetical p-value boundaries, actual p-values and Z-scores were computed for each specific type combination using the Poisson fit mean values from the permutation runs. Because the type combination frequencies observed in the 1,000 randomized matrices precisely fit a known and well characterized distribution, p-values <0.001 can be reliably estimated.Results for the Perl scripts above were then read into a Mathematica program to assess statistical significance . First, the expected counts of any given type combination in the permutation models were fit to a Poisson probability density function pdf; . The PoiDue to the number of HPV type combinations analyzed, the Benjamini and Hochberg false-discovery rate (fdr) was calculated using ‘p.adjust’ in R to control for spurious results Further details on calculating the statistics are in All code  is available upon request.Of 32,245 subjects, 13,729 were positive for ≥1 HPV type, and 7,358 were positive for multiple HPV types . Allowin8 (3.22×104 people/iteration × 1×104 iterations) are expected to have this many concurrent HPV types detected, whereas the observed data contains a continuous distribution of subjects with up to 14 HPV types detected at the same time. Furthermore, infection with 0 and 3 through 14 HPV types occurred more than expected by the null model. In contrast, infection with 1 and 2 HPV types occurred less than expected.Because such differences in general trends between the observed data and null model would bias the analysis of specific HPV type associations, these trends were preserved in subsequent randomizations to create continuously less naïve models. This randomization resulted in the distribution of each of the 10,000 permutation matrices exactly matching the observed results  of a Poisson probability density function fit to the observed counts for a given type combination in 100 iterations of a permutation model Monte Carlo randomization. The plot (B) shows the number of observations needed to meet p = 0.0001 significance (y-axis) vs. number expected by a given permutation model (x-axis). The x-axis corresponds to the mean number of counts observed in the 1000 Monte Carlo runs from any of the models. The equation embedded shows the power law fit used to create the boundary value lines on the left side of the plot (i.e. over observed type combinations) in 8 = 256 counts are expected in the given permutation model, then roughly 340 observations in the real data are needed to satisfy p = 0.0001. If only 16 counts are expected, the needed number observed counts increases (as a ratio relative to expected) to about 40 for the same level of significance. Rarely observed type combinations with very rare expected values in the permutation models  were excluded from the analysis of significance. This is because, for example, it is difficult to determine the true significance or impact of a 4 type combination that is seen in the observed data only once, even if it was expected <0.001 times (for a very large observed/expected ratio) in a database of 30,000 specimens.(TIF)Click here for additional data file.Figure S2Difference between the observed data and the 10,000 non-strata model segregated by high-risk and low-risk HPV types: all subjects (A), general population (B), colposcopy population (C).(TIF)Click here for additional data file.Figure S3Testing specificity of HPV DNA genotyping test on PCR amplified plasmid DNA for (A) HPV 56 and (B) 66.(TIF)Click here for additional data file.Table S1Primer sequences used for HPV 56 and 66 PCR amplification.(DOC)Click here for additional data file.Table S2Key parameters and assumptions of the different randomization models.(DOC)Click here for additional data file.Table S3Significant 3 HPV type combinations.(DOC)Click here for additional data file.Table S4Significant 4 HPV type combinations.(DOC)Click here for additional data file.Table S5Number of times HPV types appeared in significant combinations.(DOC)Click here for additional data file.Methods S1Supplementary methods for statistical calculations.(DOC)Click here for additional data file.Data S1Supplementary data.(TXT)Click here for additional data file.


The Expanded Program on Immunization currently considers offering Human Papilomavirus vaccine on a routine basis in Vietnam. However, as the current available vaccine can prevent only two types HPV 16 and 18, before implementing a large-scale vaccine campaign we need information about the prevalence of infection with only HPV 16 and 18 in Viet Nam. This study was done in 5 large cities in Vietnam to estimate the prevalence of HPV 16 and/or 18 infections and to explore the distribution of other high risk types of HPV among married women in these provinces.The study employed a cross-sectional design with multistage sampling. The sample size included 4500 married women in two rounds . Participant were randomly selected, interviewed and given gynaecological examinations. HPV infection status  and HPV genotyping test (by Reverse dot blot) were done for all participants.The prevalence of cervical infection with HPV type 16 and/or 18 among married women in this study ranged from 3.1% to 7.4%. Many positive HPV cases (ranged from 24.5% to 56.8%) were infected with other type of high risk HPV which can lead to cervical cancer and cannot prevented by currently available vaccines. In addition to HPV 16 and/or 18, most common types of high risk HPV were types 58, 52, 35 and 45. Awareness about HPV and HPV vaccines was still low in the study samples.While it is relevant to implement an HPV vaccine campaign in Viet Nam, it is important to note that one can be infected with multiple types of HPV. Vaccination does not protected against all type of high risk HPV types. Future vaccine campaigns should openly disclose this information to women receiving vaccines.High prevalence of infection with HPV high risk types was observed in this study. As HPV infection has a high correlation with cervical cancer, this study emphasizes the need for both primary prevention of cervical cancer with HPV vaccines as well as secondary prevention with screening. Human papillomavirus (HPV) is a very common sexually transmitted virus but the infection are often gone without any treatment . HoweverCurrently, two efficacious prophylatic HPV vaccines are available (Cervarix made by GlaxoSmithKline) protects against only HPV 16 and 18, Gardasil made by Merck protects against HPV 16,18, 6 and 11 ,5. In deIn the year 2010 and 2011, a survey was conducted in 5 large cities in Vietnam  to collect information on prevalence of HPV cervical infection and risk factors of HPV cervical infection among married women. As reported elsewhere, the prevalence of HPV cervical infection in those cities ranged from 6.1% to 10.2% and the prevalence of high risk HPV infection was from 5.6% to 9.3% . The preThe first round of the study was done in 2010 in two cities, Hanoi and HCM. The estimated sample size for each city was 850 women. Using formula for sample size for a proportion estimate with relative precision, sample size was calculated with the following parameters: anticipated prevalence of HPV as 10%, [In each city, eligible women were randomly selected from the list of married women aged from 18–69 year old provided by the Women Union. These selected women were visited by staffs of Women Union at their home to explain about the objectives of the study as well as to obtain a written consent form for their participation in this study. The response rates in both rounds were very high due to the benefits of attending this study , however due to limited funding for testing, we were able to select only 750 women/each city in round 1 and 1000 women/each city in round 2.Information and specimen collection used the following steps. First, a personal interview was done to collect information on socio-demographic variables, obstetric/gynecologic history, and sexual lifestyle. After interviewing, each participant was scheduled for a pelvic examination carried out by a gynaecologist. For Pap sample, a wooden spatula was gently used to collect cells from the ectocervix, the cells then were place on a glass slide preserved with a fixative, stained. Another wooden spatula was used to collect HPV sample. Both HPV and Pap samples were sent to the laboratory of Vietnam Institute of Dermatology on the same day of collection.The HPV genotyping protocol includes 4 steps. The first step is to receive and treat samples using cotton swab and storage solution. The second step is to extract DNA HPV using DNA extraction kit based on phenol/chloroform. The third step is to use the real-time PCR kit to detect HPV DNA using TaqMan probe. In step 4, genotype by reverse dot blot (RDB), was applied for all the HPV postive samples. RDB was a simple and fast typing procedure for detecting 37 mucosotropic HPV types with high sensitivity/specificity .The study protocol was submitted to the Hanoi school of Public Health IRB, registered with U.S. Dept. of Health and Human Services - IORG number 0003239, FWA number FWA00009326. The protocol was reviewed and cleared by this ethical committee .Prevalence of cervical infection with HPV type 16 and/or 18.Table Figure Figure Figure This study detected a total of 24 HPV types; of these, 8 types were low risk  and 16 types were high risk . Table In addition to HPV high risk type 16 and 18, there were other 14 HPV high risk types. The 5 most common other HPV high risk types among the whole sample were HPV58 (1.47%), HPV52 (0.49%), HPV35 (0.44%), HPV45 (0.38%) and HPV59 (0.38%).Of 4500 women participated in this study, 177 women 3.9%) had abnormal Pap smear results. Specifically, 104 women had ASCUS , 39 had AGUS , 26 had LSIL , and 8 had HSIL . Table .9% had aFigure The prevalence of cervical infection with HPV type 16 and/or 18 among married women in this study ranged from 3.1% to 7.4%. This prevalence was highest in Can Tho, this figure was also consistent with the fact that among 5 cities, Can Tho had highest rate of cervical cancer . Most ofResults also indicated that among cases with abnormal Pap results, 16.9% of them infected with HPV 16 and/or 18; however, the sample size of women with abnormal Pap smear was not sufficient enough to examine the distribution of HPV 16 and/or 18 among abnormal Pap cases. Data about the composition of HPVs present in cervical cancer in Vietnam was also not available from previous research. It is important to note that the distribution of HPV type 16/and or 18 among general population would be different to that among cancer cases. So the distribution of HPV 16/and or 18 among general married women in this study cannot be considered as a proxy for that in cervical cancer and it was a limitation of this study.In addition to HPV 16 and 18, this study also reported 14 other types of high risk HPV, most notably was HPV type 58, 52, 35 and 45. Other studies also reported that HPV 58 was among the most common types found in cervical cancer specimens in China, Thailand and The Philippines . A recenVietnam still applies the opportunistic cytology-based screening model and this model has failed to have an impact on the overall incidence of cervical cancer in Vietnam. A new potential model “screen and treat” using either HPV DNA testing or VIA  followed by treatment with cryotherapy (freezing) proposed by the Alliance for Cervical Cancer Prevention may be applied to the Vietnam context .Results showed that about 37.4 women ever heard about HPV and 31.2% knew about HPV vaccine. A previous study among women in the United states reported that the level of awareness of HPV and of HPV vaccine were 84.3% and 78.9% . More coStrict protocols to avoid biases were followed in this study: women were randomly chosen, all clinical examination and specimen collections were done by qualified gynaecologists and all the samples were examined by a nationally qualified laboratory. The detection of HPV positivity using real time PCR methods and the genotyping of HPV type using reverse dot blot method in this study also provided more precise results compared to the Hybrid Capture Tube Method applied in previous studies . One alsThis is a limitation of this study because we were not able to invite unmarried women to attend study with pelvic examinations and cervical sampling collect due to the strict cultural/ethical norms in Vietnam so the results did not cover a subgroup a the young population already sexually active but not yet married. It can also be argued that probably HPV detected in older women would have more likely to be present for a longer period, at higher risk of persistent infection and higher risk to develop lesions and cancer so the data presented in this study would be very useful for the cervical screening programming in Vietnam.In conclusion, a high prevalence of HPV infection, especially high risk types, was observed in this study. As HPV infection has a high correlation with cervical cancer, this study emphasizes the need for both primary prevention of cervical cancer with HPV vaccines as well as secondary prevention with screening. Currently, in Vietnam HPV vaccines is not yet offered in routine EPI program and the cervical screening program still applies the opportunistic cytology-based screening model. Policy-makers in Viet Nam should consider making HPV vaccines and screening become organized routine practices for cervical cancer prevention.The authors declare that they have no competing interests.LV designed, carried out the survey, coordinated the sequence alignment and drafted the manuscript. HL and DB participated in the design of the study and performed the statistical analysis. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/13/53/prepub


Human papillomavirus (HPV) represents an exciting subject of study because it is currently established as an essential etiological factor of uterine cervical cancer and strongly implicated in the development of other genital cancers as well, in addition to benign genital warts. Additionally, substantial amount of new data have been elaborated linking HPV with head and neck cancer and, more tentatively, also with esophageal, breast, prostate, and lung cancers. Despite the existing controversies, the possible link of HPV infection with these nongenital carcinomas opens a new fascinating era of HPV research.Concomitantly, the HPV vaccination has emerged as a new paradigm to cancer prevention programs worldwide. As much as 10% of all cancers can be related to certain HPV types, and therefore we can anticipate a substantial reduction in cancers worldwide with the implementation of HPV prophylactic vaccines. HPV vaccination provides a realistic option to reduce cervical cancer incidence and mortality in poor and developing countries, where the secondary prevention options  are not easily implemented due to lacking infrastructure, low human resources, lack of population adherence, and lack of political commitment. We can anticipate substantial reduction of HPV-related diseases.During the past decades, HPV research has been pursued along different lines, which has resulted in an ever-increasing number of publications dissecting the multifaceted mechanisms of HPV infections and the complexity of biological cascade related to HPV-associated human carcinogenesis.We are pleased to introduce this special issue dedicated to Papillomavirus, from the bench to the clinics, providing us an opportunity of entering this fascinating world that integrates a plethora of specialties studying the relation between this virus and human cancer.Adhemar Longatto FilhoAdhemar Longatto FilhoLuisa Lina VillaLuisa Lina VillaKari SyrjKari Syrj


The majority of cervical cancers are associated with infection by one or more Human Papillomavirus (HPV) types from just two distinct Alpha-Papillomavirus species groups, A7 and A9. The extent to which the current HPV16/18 vaccines will protect against other genetically related HPV types is of interest to inform vaccine implementation, cervical disease surveillance and the development of second generation HPV vaccines. The aim of this study was to determine the frequency and titer of neutralizing antibodies against a range of A7  and A9  HPV types using sera from individuals immunized with the bivalent HPV vaccine within the school-based, UK national HPV immunization programme. Serum samples were collected from 69 girls aged 13–14 years, a median 5.9 months  after their third vaccine dose. Cross-neutralizing antibodies against HPV31, HPV33, HPV35 and HPV45 were common and strongly associated with the titer for the related vaccine-type, but were considerably lower (<1%) than their related vaccine type-specific response. The low prevalence of these HPV types in the population and the ages within the study cohort suggest these responses are due to vaccination. It is unclear whether such low levels of neutralizing antibodies would be sufficient to protect at the site of infection in the absence of other immune effectors but the coincidence with HPV types reported from efficacy studies is intriguing. The utility of neutralizing antibodies as surrogate markers of protection remains to be determined. The vaccines were delivered through the UK's school-based national HPV Immunization Programme within the recommended dosing intervals Anonymized serum samples were collected, following informed consent, from 13 to 14 years old girls approximately six months after completion of a three-dose vaccination schedule with the bivalent HPV vaccine, CervarixAnonymized serum samples from infants  participating in a clinical trial where consent had been given for anonymous testing for other vaccine-related antibodies were used to gauge the potential for non-specific assay interference. Environmentally acquired HPV infection was expected to be rare in this group and, for the present purposes, we considered these individuals to represent a true HPV negative population; herein referred to as HPV-naïve.2.2http://home.ccr.cancer.gov/lco/default.asp). The consensus sequences were derived from NCBI database sequences  and contemporary sequences from anonymous, HPV-infected cytology samples .Bicistronic vectors  representing the HPV types 16, 18, 31, 45, 52, 58 and Bovine Papillomavirus (BPV) were obtained from J.T. Schiller, National Cancer Institute, Bethesda, MD, USA, while p33sheLL and p68sheLL were obtained from H. Faust and J. Dillner, Malmö University Hospital, Malmö, Sweden. Constructs representing HPV35 (p35sheLL), HPV39 (p39sheLL) and HPV59 (p59sheLL) were generated by the insertion of codon optimized genes  based upon consensus L1 and L2 amino acid sequences into p5shell (http://home.ccr.cancer.gov/lco/ripcord.htm), developed to reduce the inclusion of excess non-reporter-containing ‘cold capsids’, and by using luciferase  as the encapsidated reporter. Briefly, 293TT cells were transfected with equal amounts of pXsheLL and pGL4.51 [luc2/CMV/Neo] plasmids  and the encoded proteins expressed for 48 h before the cells were lysed, the capsids matured overnight in the presence of ribonucleases  and the double-clarified supernatant subjected to iodixanol gradient fractionation.The production of L1L2 pseudovirus stocks was performed as described elsewhere 50) was estimated using the Spearman–Karber equation. The average of three such estimations was made for each pseudovirus stock used in this study. Pseudovirus-mediated reporter gene transduction of target cells in both the infectivity and neutralization assays was measured using the Steady-Glo Luciferase Assay Reagent (Promega) and the Glomax Multi Detection System (Promega) according to manufacturer's instructions.Purified pseudovirus stocks were titrated on 293TT cells in quadruplicate, five-fold serial dilutions and the equivalent of a Tissue Culture Infectious Dose 50%  demonstrated that 300 TCID50 was within the linear range of the assay for all A7, A9 and BPV pseudoviruses and a median 3.35  Log10 fold over the background level of the assay; linear regression, r2 = 0.908  50, spanning ca. 4 Log10 range of L1 protein and ca. 2 Log10 difference in particle to infectivity ratios between the standard and alternative protocol-produced stocks were not significantly different . Thus, 300 TCID50 was deemed an appropriate pseudovirus input and used for all subsequent neutralization assays.It was important to ascertain the appropriate input level of pseudovirus for the neutralization assays. A plot of input TCIDn) neutralization titers were as follows: HPV16 65,564 ; HPV31 449 ; HPV33 62 ; HPV35 21 ; HPV52 43 ; HPV58 413 ; HPV18 17,632 ; HPV39 <20 ; HPV45 70 ; HPV59 <20 ; HPV68 <20 ; BPV <20 . As HPV39, 59, 68 and BPV were not neutralized by this control serum pool, neutralization tests using these pseudoviruses were repeated against all study sera to confirm the lack of activity and included Heparin  as a positive inhibitor control. All A7, A9 and BPV pseudoviruses were sensitive to heparin with a median 80% inhibition concentration of 14.3  μg/mL Inter-assay reproducibility of neutralizing antibody titers was demonstrated by including in every experiment a vaccinee serum pool control, comprising study sera selected following an initial neutralization screen against HPV16, HPV18, HPV31, HPV45, HPV52 and HPV58. Median .A small panel of nine sera samples was also retested at the end of the study against six pseudoviruses HPV16, 31, 33, 35, 52 and 58 (2.32-tailed Fisher's exact test and two sample Wilcoxon rank-sum (Mann–Whitney) test were used to compare proportions of individuals with positive neutralizing antibody and antibody titers of vaccinees versus HPV-naïve individuals, respectively. Spearman's and Kendall's rank correlations and Pearson's product-moment correlation were used to compare the neutralizing antibody titers against non-vaccine types and the corresponding vaccine type within a species group. Wilcoxon paired signed rank test was used to determine whether the range of neutralizing antibody titers against two HPV types were different. Kruskal–Wallis equality-of-populations rank test and the test for trend across ordered groups  were used to assess the difference between non-vaccine type neutralization data ordered into tertiles based upon neutralizing antibody titers against the vaccine type. All tests were performed using the statistical package, Stata 10.1 .3® vaccine.Sixty-nine serum samples were collected a median 5.9 (IQR 5.7–6.0) months after receiving a third dose of the Cervarixn = 69, 100%) individuals generated high titer neutralizing antibodies against HPV16 and HPV18 following vaccination (p < 0.001). Sera capable of neutralizing non-vaccine A9 HPV types were commonly found among this group of vaccinees  with neutralization detected most frequently for HPV31, followed by (in order) 33, 52, 35, and 58. Sera capable of neutralizing non-vaccine HPV types within the A7 species group were fewer and almost completely restricted to reactivity against HPV45. No inhibition of the control BPV pseudovirus was seen using these vaccine sera.As expected, all . However, using the more stringent Pearson's product-moment correlation coefficient only HPV31 , HPV33 , HPV35  and HPV45  gave significant associations with their respective type-specific titers. As expected rho = 0.673; p < 0.001; Pearson's r = 0.657; p < 0.001).Significant associations were found between the neutralizing antibody titers observed against HPV31, 33, 35, 45, 52 and 58 and the titers observed against their related vaccine-type . For HPV types 31, 33, 35, 45 and 58 the percentage of individuals with a positive, non-vaccine type neutralization titer increased with each tertile of vaccine-type titer . For exap < 0.001).Neutralizing antibody titers against non-vaccine types HPV31, 33, 35, 45, 52 and 58 increased in association with increasing vaccine-type tertiles . For exa3.3p = 0.018).Cross-neutralization titers were overall very low, being <1% of the respective type-specific, HPV16 or HPV18 titer: for example, HPV31 (median 0.49% [IQR 0.24–1.02%]), HPV33 (0.13% [0.09–0.24%]) and HPV45 (0.50% [0.18–1.02%]). In contrast to the increase across the vaccine-type tertiles of the percentage of individuals with, and levels of, cross-neutralizing titers , the rel4In this study we have attempted to estimate the propensity for serum taken from 13 to 14 year old girls recently vaccinated with the bivalent HPV vaccine to neutralize pseudoviruses representing genetically related, non-vaccine HPV types within the A9 and A7 species groups. Neutralizing antibodies against non-vaccine A9 HPV types were commonly detected within this study group, with antibodies against HPV31 and HPV33 being the most frequently detected and of the highest titer. The only A7 non-vaccine HPV type for which a significant neutralizing antibody response was found was HPV45.Neutralizing antibody titers against HPV31, 33, 35, 45 (and to a lesser extent HPV52 and 58) were significantly associated with their related vaccine-type antibody titers, suggesting that the generation of cross-neutralizing antibodies is at least coincident with the host immune response to vaccination. No significant neutralization was seen against the A7 HPV types 39, 59 and 68 or the control BPV. It is noteworthy perhaps that the individuals with a positive neutralizing antibody score against either HPV59 or HPV68 were also in the highest tertile of vaccine-type HPV18 neutralizing antibody titers, suggesting that responses against these types, although not significant overall and a rare occurrence (<5% of vaccinees), may indeed be vaccine-related. The fewer number of samples positive for neutralizing antibodies against non-vaccine HPV types from the A7 species group, being almost exclusively directed against HPV45, than from the A9 species group, is likely to be related to the lower (3.5 fold) titers generated against the vaccine-type HPV18 compared to HPV16, which appears to be a common finding for the HPV vaccines ®A recent study We have expanded the currently available panel of HPV L1L2 pseudoviruses to represent all those HPV types within the vaccine-related A9  and A7  species groups that have been considered by the International Agency for Research on Cancer to be at least ‘probably carcinogenic to humans’ We did not have pre-vaccine sera, or sera from unvaccinated 13–14 year old girls, with which to gauge background levels of naturally induced HPV antibodies and non-specific assay interference. However, the sera included in our study were from young girls close to the target age (12 years) for routine HPV immunization in the UK and within an age-group where the level of naturally induced antibodies was expected to be very low As the sera used for this study were collected within six months of the third vaccine dose and given the apparent improved immunogenicity within this age group per se does not, of course, provide sufficient evidence for antibodies being mechanistically associated with cross-protection. Furthermore, type-specific antibody titers in genital secretions are orders of magnitude lower than those found in the periphery The detection of cross-neutralizing antibodies in vaccine sera These data may be helpful to parameterize epidemiological models to predict the impact of the current HPV vaccines on the population and to inform the development of second generation HPV vaccines.This study was given a favorable ethical opinion by the Tameside & Glossop Local Research Ethics Committee, Manchester, UK (REC reference number 09/H1013/33).


Infection, coinfection and type-specific human papillomavirus (HPV) distribution was evaluated in human immunodeficiency virus (HIV)-positive women from paired cervical and urine samples. Paired cervical and urine samples (n = 204) were taken from HIV-positive women for identifying HPV-DNA presence by using polymerase chain reaction (PCR) with three generic primer sets . HPV-positive samples were typed for six high-risk HPV (HR-HPV)  and two low-risk (LR-HPV) (HPV-6/11) types. Agreement between paired sample results and diagnostic performance was evaluated. HPV infection prevalence was 70.6% in cervical and 63.2% in urine samples. HPV-16 was the most prevalent HPV type in both types of sample  followed by HPV-31(47.2%) in cervical samples and HPV-58 (35.7%) in urine samples. There was 55.4% coinfection (infection by more than one type of HPV) in cervical samples and 40.2% in urine samples. Abnormal Papanicolau smears were observed in 25.3% of the women, presenting significant association with HPV-DNA being identified in urine samples. There was poor agreement of cervical and urine sample results in generic and type-specific detection of HPV. Urine samples provided the best diagnosis when taking cytological findings as reference. In conclusion including urine samples could be a good strategy for ensuring adherence to screening programs aimed at reducing the impact of cervical cancer, since this sample is easy to obtain and showed good diagnostic performance. The human immunodeficiency virus (HIV) is a sexually-transmitted infection (STD) having a great impact around the world due to the large amount of people living with such infection (34.2 million) and the frequent appearance of new cases (2.5 million in 2011) Some studies have suggested that women living with HIV/AIDS have increased frequency and incidence of single and multiple infections caused by human papillomavirus (HPV) The immunosuppression can be attenuated through using antiretroviral therapy which favors balanced counts of CD4 lymphocytes, however, this therapy has not been consistently implicated in the reduction of HPV-related diseases The CC incidence in the Colombian general population is 36.4 cases/year/100,000 women Cervical cytology is the most widely used strategy for reducing the cervical cancer burden around the world In view of the above, the use of complementary techniques to the Papanicolau test could represent a useful tool in detecting women at risk. Some of these methods are non-invasive, such as self-sampling, as when they are used in screening programs they could provide advantages related to increased acceptance regarding sample-taking, adherence and following-up women, especially those having some form of immunological compromise Specimen tampons, vaginal swabs and urine samples have been studied as self-sampling methods; such sampling methods are also used for detecting other sexually-transmitted pathogens affecting the cervical area In spite of their limitations, using urine samples as a test for detecting HPV-DNA presence could facilitate frequent sample-taking due to their practicality and greater acceptance among women. This could be useful in studies involving a large number of samples and a pelvic examination is also not required, meaning that sample-taking will not affect the natural history of HPV infection as there is no risk of micro-lesions being produced, nor will inflammatory reactions occur Despite of multiple studies available in the literature that have evaluated HPV-DNA detection from urine sample In Colombia high prevalence of HPV infection and co-infection in healthy women population have been reported, using cervical samples This study aimed at identifying the infection, coinfection (defined here as being infection by more than one type of HPV simultaneously) and type-specific distribution profile of six high-risk HPV (HR-HPV) types and two low-risk (LR-HPV) types, from paired cervical and urine samples of women diagnosed with HIV/AIDS, confirmed by Western blot. Finally, we evaluated the diagnostic performance of urine samples compared to cervical samples for detecting HPV infection.HIV-infected women (such infection having been confirmed by Western blot) participating in cervical cancer screening campaigns being offered by the Centro de Asistencia Científica de Alta Complejidad S.A.S., in Bogotá, Colombia, were included in the present study. The study was approved and supervised by the participating institutions' ethics committees: Fundación Instituto de Inmunología de Colombia's ethics committee and Centro de Asistencia Científica de Alta Complejidad S.A.S.' ethics committee.Sample size was calculated assuming an estimated 80% HPV infection rate in HIV-positive women All the women enrolled in the study were informed about the research objective; they signed an informed consent form and filled in a questionnaire to facilitate collecting socio-demographic data and information regarding their sexual habits and other risk factors related to acquiring HPV infection.Each woman's urine and cervical samples were taken on the same day; the first sample from a midstream urine specimen was self-collected, kept at 4°C and processed within 72 hours after being collected. The second sample taken from cervical cells was obtained during Papanicolau test, following Colombian obligatory health plan guidelines regarding cervical cancer detection and control programs in Colombia g for 20 minutes at 4°C for urine samples and at 15,000× g for 10 minutes at 4°C for cervical samples. DNA was extracted from cell pellets of paired samples using a QuickExtract DNA extraction kit , following the manufacturer's instructions. Two PCR amplifications were made with specific primers directed at a segment of the human β-globin constitutive gene (GH20/PC04 and PC03/PC04) for evaluating DNA integrity The cells were precipitated by spinning at 2,300× Samples yielding a positive result for the human β-globin gene were amplified for detecting HPV using three consensus primer sets (for detecting more infected women) as it has been reported that using a single set might lead to underestimating viral prevalence compared to studies using more than one generic detection system Two of the primers sets were directed to the region encoding late viral protein L1: GP5+/6+ and MY09/11 2 and 104 plasmid copies in 100 ng HPV-DNA for HPV-16 and -45, respectively, using a GP5+/6+ primer set 2 HPV copies of HPV-16, -18, -33, -45 and -58 using an MY09/11 primer set Generic primer sets' HPV-DNA detection analytical sensitivity has been reported in previous studies, the detection limit being 10Samples which proved positive for any of the three generic primer sets were amplified by PCR for identifying the six HR-HPV viral types , using type-specific primers targeting the E5, E6 and E7 regions, according to that reported in the literature for each viral type Means and standard deviations were used for describing continuous variables; categorical variables were expressed in terms of frequencies and percentages. The frequency of events of interest was reported together with their corresponding 95% confidence intervals that were calculated using the bootstrap method. The association between categorical variables was evaluated with Chi-square (χ2) tests, using a significance level of 0.05.Agreement between HPV results for urine and cervical samples was evaluated using the kappa coefficient (κ), and its corresponding 95%CI, classified as follows: negative values, as well as values from 0 to 0.2 = poor, from 0.21 to 0.4 = slight, from 0.41 to 0.6 = fair, from 0.61 to 0.8 = moderate, from 0.81 to 0.99 = substantial and 1.0 = perfect agreement Two hundred and forty five women between 20 and 73 years old, were enrolled in the study  . Two hunHPV infection frequency in cervical and urine samples was 70.6%  and 63.2% , respectively. Type-specific viral identification revealed that HPV-16 had the greatest prevalence in both samples, whilst HPV-31 had the second greatest prevalence in the cervical samples and HPV-58 in urine samples; the other viral types had a variable distribution in both samples .It was found that 55.4%  of the cervical samples had coinfection, compared to 40.2%  of the urine samples. Regarding a description of the number of types of HPV simultaneously present in each sample analyzed, urine samples revealed more uninfected women or those having just one HPV-type compared to the results obtained for cervical samples where more coinfections were detected (2 to 8 types of HPV). The presence of multiple infection per sample type had a statistically significant relationship  .The Papanicolau test gave abnormal cytological findings in 28.9% of the population , results being classified as follows: 10.3% (n = 20) had atypical squamous cells of undetermined significance (AS-CUS), 16.5% (n = 32) low-grade squamous intraepithelial lesions (L-SIL) and 2.1% (n = 4) high-grade squamous intraepithelial lesions (H-SIL).The HPV infection results obtained from the two samples were classified according the cytological results; data are shown in 2(1) = 2.69; p = 0.101). On the other hand, it was found that 19.6% (n = 11) of the samples having abnormal cytological findings had negative test for HPV-DNA in the urine sample, compared to 80.4% (n = 45) where viral DNA was detected; this trend was statistically significant (χ2(1) = 9.22; p = 0.002).The association between the presence of HPV-DNA in each sample and the cytological findings  revealed that 20.4% (n = 12) of the women having abnormal cytological findings had a negative result for HPV infection in the cervical sample while 78.6% gave a positive result (n = 44). Such difference was not statistically significant (χThe results obtained for infection (generic and type-specific) in both samples  were compared to cytological findings, categorizing the population as normal or abnormal . However, the κ values gave poor correlation in all cases .The urine sample's diagnostic performance revealed 68.8% sensitivity (60.5–76.2 95%CI), 50% specificity (36.8–63.2 95%CI), 76.7% positive predictive value (PPV) (68.5–83.7 95%CI), 40% negative predictive value (NPV) (28.9–52 95%CI) and a 0.59 ROC area (0.52–0.67 95%CI).Developing cervical cancer has been related to factors determining its progression, including the type of HPV infection, viral load and persistence of the infection However, when immune system activity becomes compromised, as in HIV-positive women, the elimination of concomitant infection is less efficient; a clear example lies in that described for women suffering simultaneous HIV-HPV infection whose natural history of infection becomes altered, thereby leading to the appearance of cervical lesions in less time. This is related to a reduction in HPV elimination rates, greater efficiency regarding the cellular transformation of all viral types and lower lesion regression rates According to the data obtained in this study, DNA integrity confirmed by amplifying two segments of the human β–globin gene (as an indirect measurement method) revealed that the percentage of samples having degraded or non-amplifiable DNA were low in both cervical and urine samples, thereby highlighting that the latter also represents a good source of DNA for amplifying specific targets using molecular biology techniques and could thus be considered as a useful cervical screening tool (in spite of 30% inhibition having been reported for such amplification) et al., who described 73.6% crude HPV infection prevalence from cervical samples taken from sexually-active HIV-positive women The frequency of HPV infection detected in the present population agreed with that reported in previous studies carried out on populations having similar characteristics, such as that reported by Ferenczy Regarding type-specific distribution, the data obtained from cervical samples agreed with published reports concerning the general Colombian population, HPV-16 being the most prevalent type, followed by HPV-31 In addition, the variations in HPV type-specific distribution profile could have been related to the presence of HIV infection as it has been described that such distribution in immunologically-compromised women could vary; moreover, it has been described that such incidence is 16 times higher in the immunologically-compromised group than that found in immunologically-competent women Coinfection was found in both urine and cervical samples in around half the study population; this could have been attributed to the low infection elimination rate allowing different viral types to settle in the cervical epithelium; multiple infection events could have been also due to the reduced systemic and local cell immunity found in HIV-positive women There was poor agreement between generic and type-specific identification results; this may have been related to the samples' different nature, as well as HPV tropism for cervical epithelium. A lower number of viral copies in urine are expected regarding cervical samples, as the latter would have been taken from the pathogen's direct localization site.Interestingly, the test involving self-collected urine samples had greater sensitivity (68.8% in this study vs. 55.3%) and more specificity (50.0% in this study vs. 44.9%) for detecting HPV-DNA compared with a previous study using the same identification protocol Developing strategies in cervical cancer control and prevention programs will be particularly determinant in contributing towards increasing coverage, sample taking, adherence and follow-up of women, mainly those presenting some type of immunosuppression. According to the results obtained here, self-sampling methods, such as urine sampling, could be taken into account as useful tools for preventing this pathology, since they offer good diagnostic performance and greater acceptability among women.