Multiple HPV Infections Research Articles

Type-Specific Detection of 30 Oncogenic Human Papillomaviruses by Genotyping both E6 and L1 Genes

Human papillomavirus (HPV) is the principal cause of invasive cervical cancer and benign genital lesions. There are currently 30 HPV types linked to cervical cancer. HPV infection also leads to other types of cancer. We developed a 61-plex analysis of these 30 HPV types by examining two genes, E6 and L1, using MassARRAY matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (PCR-MS). Two hundred samples from homosexual males (HM) were screened by PCR-MS and MY09/MY11 primer set-mediated PCR (MY-PCR) followed by sequencing. One hundred thirty-five formalin-fixed, paraffin-embedded (FFPE) cervical cancer samples were also analyzed by PCR-MS, and results were compared to those of the commercially available GenoArray (GA) assay. One or more HPV types were identified in 64.5% (129/200) of the samples from HM. Comprising all 30 HPV types, PCR-MS detected 51.9% (67/129) of samples with multiple HPV types, whereas MY-PCR detected only one single HPV type in these samples. All PCR-MS results were confirmed by MY-PCR. In the cervical cancer samples, PCR-MS and GA detected 97% (131/135) and 90.4% (122/135) of HPV-positive samples, respectively. PCR-MS and GA results were fully concordant for 122 positive and 4 negative samples. The sequencing results for the 9 samples that tested negative by GA were completely concordant with the positive PCR-MS results. Multiple HPV types were identified in 25.2% (34/135) and 55.6% (75/135) of the cervical cancer samples by GA and PCR-MS, respectively, and results were confirmed by sequencing. The new assay allows the genotyping of >1,000 samples per day. It provides a good alternative to current methods, especially for large-scale investigations of multiple HPV infections and degraded FFPE samples.

HIV/HPV co-infection during pregnancy in southeastern Brazil: Prevalence, HPV types, cytological abnormalities and risk factors

ObjectiveHIV+ pregnant women are at a higher risk of HPV infection and development of cervical cancer. Our objectives were to assess the prevalence and HPV types in HIV+ pregnant women and to identify risk factors for HPV infection and cytological abnormalities. MethodsCervicovaginal smears were collected during pregnancy from 140 women. Partial HPV L1 gene and the exon 4 of the human TP53 gene (containing codon 72) were PCR-amplified and sequenced. Amplified products indicating multiple HPV infection were further cloned and sequenced. The association of demographic, obstetric and HIV-related clinical variables with HPV infection and cervical lesions was tested by univariate analyses, and significant factors were subsequently tested by logistic regression multivariate analysis. ResultsHPV DNA tested positive for 118 patients and HPV types were identified in 104 samples. Twenty-eight different types were found, HPV-16 and HPV-58 being the most prevalent. High-risk types were present in 79.8% of samples and multiple infections in 16.3%. Abnormal cervical smears were found in 44 patients (31.4%). Absolute CD4+ T-cell counts below 350 were associated with HPV infection. Younger age was associated with cervical abnormalities and higher CD4+ T-cell count was an apparent protective factor. ConclusionsWe found a high prevalence of HPV infection and high-risk types in this cohort. Our results highlighted the relevance of immune system integrity rather than TP53 variants for protecting this highly vulnerable population to HPV infection and carcinogenesis.

HPV Infection and Intraepithelial Lesions: Comparison Between HIVPositive and Negative Women

Human Papillomavirus infections have been shown to be crucial for the development of cervical intraepithelial neoplasia and subsequent cervical cancer. The aim of this study is to describe the prevalence of different genotypes of HPV, in a population of HIV-positive women, compared to the negative ones, and their oncogenic risk. A case-control study comparing HPV genotype distribution between 93 HIV-seropositive and 186 HIV-seronegative women, matched for age and severity of cervical lesions, who attending colposcopic service of our departments for periodical Pap smear and HPV DNA full genotyping by SPF-10 LiPA assay. No significant difference was found in genotype distribution between HIV positive and HIV negative women. Only the prevalence of HPV56 was higher in HIV positive women (p=0,046). The rates of HPV 6, 11, 16 and 18 were similar in both groups. The likelihood of the detection of three or more HPV genotypes was significantly associated with CIN (OR=2.0; 95% CI=1.1-3.8; p= 0.026) but only marginally to HIV-positive serostatus (OR=1.68; 95% CI=0.89-3.16; p= 0.1). High grade cervical lesions are associated with high risk viruses like HPV 16 and 18 and with multiple cervical HPV infections. The tendency to treat HIV disease with high active antiretroviral therapy may reduce the impact of immunosuppression and make the course of such HPV infections more similar to that among women who are not HIVinfected. As in immunocompetent women, high oncogenic risk viral type and multiple infections are associated with a histologically proven cervical intraepithelial lesions.

Next-generation sequencing of cervical DNA detects human papillomavirus types not detected by commercial kits

BackgroundHuman papillomavirus (HPV) is the aetiological agent for cervical cancer and genital warts. Concurrent HPV and HIV infection in the South African population is high. HIV positive (+) women are often infected with multiple, rare and undetermined HPV types. Data on HPV incidence and genotype distribution are based on commercial HPV detection kits, but these kits may not detect all HPV types in HIV + women. The objectives of this study were to (i) identify the HPV types not detected by commercial genotyping kits present in a cervical specimen from an HIV positive South African woman using next generation sequencing, and (ii) determine if these types were prevalent in a cohort of HIV-infected South African women.MethodsTotal DNA was isolated from 109 cervical specimens from South African HIV + women. A specimen within this cohort representing a complex multiple HPV infection, with 12 HPV genotypes detected by the Roche Linear Array HPV genotyping (LA) kit, was selected for next generation sequencing analysis. All HPV types present in this cervical specimen were identified by Illumina sequencing of the extracted DNA following rolling circle amplification. The prevalence of the HPV types identified by sequencing, but not included in the Roche LA, was then determined in the 109 HIV positive South African women by type-specific PCR.ResultsIllumina sequencing identified a total of 16 HPV genotypes in the selected specimen, with four genotypes (HPV-30, 74, 86 and 90) not included in the commercial kit. The prevalence’s of HPV-30, 74, 86 and 90 in 109 HIV positive South African women were found to be 14.6%, 12.8%, 4.6% and 8.3% respectively.ConclusionsOur results indicate that there are HPV types, with substantial prevalence, in HIV positive women not being detected in molecular epidemiology studies using commercial kits. The significance of these types in relation to cervical disease remains to be investigated.

Population-based study on the prevalence of and risk factors for human papillomavirus infection in Qujing of Yunnan province, Southwest China

BackgroundHuman papillomavirus (HPV) infection causes cervical cancer and premalignant lesions of the cervix. Prevalence of HPV infection and HPV genotypes vary among different regions. However there is no data on the prevalence of HPV infection and HPV genotypes from southwest China. This study was undertaken to determine the prevalence of and risk factors for HR-HPV infection in Qujing of Yunnan province, southwest China to provide comprehensive baseline data for future screening strategies.MethodsA sample of 5936 women was chosen by the multi-stage stratified cluster sampling method with selection probabilities proportional to size (PPS). An epidemiological questionnaire was conducted via a face-to-face interview and cervical specimens were taken for HPV DNA testing by Digene Hybrid Capture 2 (HC2) test. HPV Genotyping Reverse Hybridization Test was used for HPV genotyping. Proportions were compared by Chi-squared tests, and logistic regression was utilized to evaluate risk factors.ResultsThe median age was 38 years and the inter-quartile range was from 31 years to 47 years. 97.3% of the study population was Han nationality. Overall prevalence of HR-HPV infection was 8.3% (494/5936) and bimodal age distribution of HPV infection was observed. The five most prevalent HR-HPV genotypes were HPV-16(3.4%), HPV-56(1.7%), HPV-58(1.4%), HPV-33(1.2%) and HPV-52(0.88%). Multiple HPV infections were identified in 50.5% (208/412) of the positive genotyping specimens. Multivariate logistic regression model indicated that parity (OR = 1.35, 95% CI: 1.18-1.53, p < 0.0001) was a risk factor for HR-HPV infection, and age of 50–65 years (OR = 0.60, 95% CI: 0.45-0.80, p = 0.0005), being married or in stable relationship (OR = 0.55, 95% CI: 0.31-0.96, p = 0.035) were protective factors.ConclusionsThis study provided baseline data on HR-HPV prevalence in the general female population in Qujing of Yunnan province, southwest China. The finding of multiple HPV infections and bimodal age distribution revealed that HPV screening is necessary for perimenopausal women in future.

Prevalence of sexually transmitted diseases, HIV, and hepatitis among male sex workers in Ho Chi Minh City, Vietnam

Background: This study determined the prevalence of HPV infection inmen andwomen in north-east Croatia and the association betweenhigh-riskHPVgenotypeswith age, sex andbehavioral risk factors. Methods: From December 2007 to December 2011, 2521 clinical specimens (436 urethral swabs from men and 2085 cervical swabs) collected in the Osijek-Baranya County Institute of Public Health and gynecologist’s offices from symptomatic patients were tested for high-risk HPV infection using the AMPLICOR HPV test (Roche Diagnostics). Subsequently, 301 HR HPV-positive samples were genotyped by the Linear Array HPV Genotyping Test (Roche Diagnostics). Results: High-risk HPV infection was significantly more prevalent in women than in men of all age groups (52% vs. 31%); its highest prevalence was found in 21-25 year-old women (62%) and 25–30 year-oldmen (26%). Themost common high-risk HPV genotype was HPV 16 (18.8%) followed by HPVs 51, 31, 52, 39, 66, 6, 16, 62, 33, 58, 45 and 56. Multiple HPV infection was detected in 46.2% of all high-risk HPV-positive subjects (139 out of 301). Unmarried status, >1 sexual partner in the past year, active smoking and cervical neoplasia positively correlatedwith HPV infection. When compared to neighbouring European countries, HPV subtype 52 was more prevalent in our population (6.1% vs 1.4–5%) while HPV 18 was significantly lower (3.8% vs 5–30%). Conclusion: In conclusion, this study showed unexpectedly high HPV prevalence in the young population of north-east Croatia. For this reason,mass vaccination against HPVs of youngwomen and men in north-east Croatia is highly recommended.

Frequency and multiplicity of human papillomavirus infection in HIV-1 positive women in Italy

BackgroundNatural history of HPV infection is altered in HIV positive women, with increased risk of cervical dysplasia. Limited data are available about the influence of HPV genotypes and HPV multiple infections on cervical disease in HIV positive women. ObjectivesWe determined HPV genotype prevalence in cervical samples from 553 HIV-infected women attending the gynaecological service at “L. Spallanzani” Hospital. Association of HPV multiple infections with cervical abnormalities was investigated. Study designMY09/MY11 consensus primers were used to detect HPV-DNA; HPV typing was performed by RFLP. A commercial array based kit was used to define unresolved RFLP patterns. ResultsHPV was detected in 244/553 (44.1%) patients, correlating with low CD4 counts (p<0.001) and age (p=0.001). Multiple HPV types were observed in 44.4% of cases, more frequently involving HR than LR HPV (OR=12.8, p<0.00001). Multiple HPV infections were associated with low CD4 counts (OR=3.8 in CD4<200 vs CD4≥500cells/mm3). Dyskaryosis was associated with decreased CD4 counts (≥500 vs 200–499 vs <200cells/mm3, χ2 for trend, p=0.001) and with HPV types multiplicity (1 vs 2–3 vs ≥4, χ2 for trend, p<0.00001). Notably, in 3 H-SIL cases only LR types were detected (HPV62, n=2; HPV81, n=1). ConclusionsMultiple HPV infections, often involving HR types, are frequent in HIV-infected women. Association between multiple HPV infection, low CD4 count and cytological abnormalities supports the interplay of virological and immunological factors in cervical cancer pathogenesis. Assessment of multiple HPV infections might gain importance in cervical cancer screening, particularly in patients with predisposing factors like immuno-suppression.

Analysis of cervical HPV infection in HIV positive Chinese women

To investigate prevalence HPV infections in cervix among HIV-infected Chinese women. From September 2009 to May 2011, 293 women with positive HIV underwent cervical cancer screening as study group matched with 200 women with negative HIV as control group. Questionnaires including demographic information and HIV associated information were collected, Pap smear and 23 subtype of HPV were performed in those women. The women with positive HPV were followed up per 6 months, and the period of following up were more than 12 months. Binary logistic analysis was used for high risk factors of HPV persistent infection. Prevalent HPV infection was 44.4% (130/293) in study group and 20.0% (40/200) in control group, respectively, which reached statistical difference (P < 0.05). The most common genotype of HPV was HPV 16, which prevalence was 13.7% (40/293) in study group and 7.0% (14/200) in control group. The other HPV subtype prevalence was HPV-58, HPV-52, HPV-43 and HPV-18, which was 9.2% (27/293), 8.2% (24/293), 8.2% (24/293), 6.8% (20/293) in study group and 3.0% (6/200), 2.5% (5/200), 1.5% (3/200), 2.5% (5/200) in control group. At time point of 12 months following up, the persistent prevalence of HPV was 47.5% (48/101) in study group and 21.1% (8/38) in control group, which reached statistical difference (P < 0.05). Multiple HPV infections (OR = 6.4, 95%CI: 1.6 - 25.6), abnormal cytology (OR = 18.1, 95%CI: 4.5 - 76.9) and lower CD(4) T cell count (compared with CD(4) > 3.5 × 10(8)/L, if 2.0 × 10(8) ≤ CD(4) ≤ 3.5 × 10(8), OR = 8.1, 95%CI: 1.3 - 56.3; if CD(4) < 2.0 × 10(8)/L, OR = 9.1, 95%CI: 1.8 - 46.9) were independently associated with HPV persistence among HIV-positive subjects. Prevalence and persistence of HPV infections were more common among HIV-positive Chinese women than those in HIV-negative Chinese women. Improving immune function, decreasing multiple HPV infections, treating abnormal cervical cytology could decrease prevalence of HPV infection.

One virus, one lesion—individual components of CIN lesions contain a specific HPV type

In 20-40% of cervical intra-epithelial neoplasia (CIN) and in 4-8% of cervical carcinoma tissue specimens, multiple HPV genotypes have been detected. Whole tissue section (WTS) PCR does not determine how the individual types relate causally to complex and multiple CIN. Our objective was to determine whether laser capture micro-dissection (LCM) with HPV PCR genotyping (LCM-PCR) could accurately recover type-specific HPV DNA from epithelial cells in individual areas of CIN and normal epithelium, and whether one or more viruses are present in one lesion. For that, histologically selected samples of CIN and normal epithelium were isolated by LCM and analysed by the SPF(10) PCR/LiPA(25) (version 1) HPV genotyping system for 25 HPV genotypes. HPV genotypes detected in 756 areas of CIN (grade 1, 2 or 3) by LCM-PCR were compared with results obtained by WTS-PCR in 60 cases (74 biopsies). We showed that when a single HPV type is detected by WTS-PCR, that type was almost always (94%; 29/31) recovered by LCM-PCR from CIN. When multiple HPV types were present by WTS-PCR, their distribution within histological sections could be mapped by LCM-PCR. Association of a single HPV type with a discrete area of CIN was found for 93% (372/399) of LCM fragments analysed by PCR. We found colliding CIN lesions associated with separate HPV types and only 62% (61/99) of HPV types detected by WTS-PCR were found in CIN by LCM-PCR. Therefore, the LCM-PCR technique was found very accurate for high-resolution HPV genotyping and for assigning an individual HPV type to an area of CIN. At LCM level, in cervical biopsy sections with multiple HPV infections, the relation between HPV types and CIN lesions is often complex. Almost every HPV type found in CIN by LCM-PCR is associated with a biological separate independent CIN lesion-one virus, one lesion.

Type-dependent E6/E7 mRNA expression of single and multiple high-risk human papillomavirus infections in cervical neoplasia

BackgroundCoinfection with multiple HPV types is common in cervical lesions, but the biological significance of the individual infections is difficult to establish. Expression of oncogenic E6/E7 HPV mRNA is correlated to risk of malignant progression, commercial assays for genotyping E6/E7 mRNA of all HR-HPV are lacking. ObjectivesTo characterize the tendency of 12 HR-HPV to express mRNA, correlated to the severity of the cervical lesion. Furthermore, we wanted to analyse mRNA expression in multiple infections, in order to establish which genotype may be responsible for cellular transformation. Study design245 samples from women with normal histology, various grades of dysplasia (cervical intraepithelial neoplasia grade 1–3), and cancer, were analysed for presence and genotyping of HPV DNA and mRNA using an in house real-time PCR test. ResultsPresence of mRNA was detected for 64% of the in total 422 HPV infections present in the samples, and more commonly in high-grade lesions. In 88% of DNA-positive samples from CIN2+ lesions, mRNA could be detected, compared to 33% of DNA-positive samples from women in screening with normal cytology. The genotype most prone to express mRNA in high-grade lesions was HPV45, followed by HPV16 and HPV31, less prone was HPV59. Expression of mRNA was significantly enhanced in CIN2+ lesions, an association also found for HPV16. In 52% of multiple infections (in which mRNA expression was generally more common), more than one genotype expressed mRNA, a phenomenon increasing with severity of lesion. Presence of mRNA could more often be detected in samples with multiple infections than in samples with single infections. ConclusionsThe frequent expression of E6/E7 by HPV45 may promote oncogenicity and could be of clinical importance. Since presence of E6/E7 mRNA was common in multiple infections regardless of histology, multiple infection could be a clinically important finding. In multiple HPV infections, mRNA testing may identify the genotype that causes transformation. However, since mRNA expression of several genotypes in one sample is common, further and larger studies using complementary techniques are required.

Clinical and Pathological Heterogeneity of Cervical Intraepithelial Neoplasia Grade 3

ObjectiveCervical intraepithelial neoplasia grade 3 (CIN3), the immediate cervical cancer precursor, is a target of cervical cancer prevention. However, less than half of CIN3s will progress to cancer. Routine treatment of all CIN3s and the majority of CIN2s may lead to overtreatment of many lesions that would not progress. To improve our understanding of CIN3 natural history, we performed a detailed characterization of CIN3 heterogeneity in a large referral population in the US.MethodsWe examined 309 CIN3 cases in the SUCCEED, a large population-based study of women with abnormal cervical cancer screening results. Histology information for 12 individual loop electrosurgical excision procedure (LEEP) segments was evaluated for each woman. We performed case-case comparisons of CIN3s to analyze determinants of heterogeneity and screening test performance.ResultsCIN3 cases varied substantially by size (1–10 LEEP segments) and by presentation with concomitant CIN2 and CIN1. All grades of CINs were equally distributed over the cervical surface. In half of the women, CIN3 lesions were found as multiple distinct lesions on the cervix. Women with large and solitary CIN3 lesions were more likely to be older, have longer sexual activity span, and have fewer multiple high risk HPV infections. Screening frequency, but not HPV16 positivity, was an important predictor of CIN3 size. Large CIN3 lesions were also characterized by high-grade clinical test results.ConclusionsWe demonstrate substantial heterogeneity in clinical and pathological presentation of CIN3 in a US population. Time since sexual debut and participation in screening were predictors of CIN3 size. We did not observe a preferential site of CIN3 on the cervical surface that could serve as a target for cervical biopsy. Cervical cancer screening procedures were more likely to detect larger CIN3s, suggesting that CIN3s detected by multiple independent diagnostic tests may represent cases with increased risk of invasion.

Human Papillomavirus Genotype Spectrum in Studied Group of Montenegrin Women

The risk of high-grade lesions and carcinoma is known to correlate with specific high-risk HPV genotypes. The distribution of HPV types varies between countries and little is known about HPV distribution in our country. Therefore, the purpose of this study was to determine the range and frequency of HPV genotypes in studied group of women in Montenegro. HPV genotypes were determined using the method of enzyme restriction of PCR products amplified with group-specific primers MY09/MY11 and restricted with seven different restriction endonucleases. Out of the total number of women HPV infection was found in 1/5 of participants (20%). Genotyping performed in HPV DNA positive women shows that the HPV genotype 16 is dominant and present in more than 1/3 of the participants (36.8%). The second most frequent HPV infection is with HPV genotype 58 and it is found in 10.5% of participants. HPV 31 and HPV 6 infections are present in 7.9% of women, while infections with other genotypes were demonstrated individually by 2.6%. Multiple HPV infection was demonstrated in 18.4% and they are dominant in younger women (aged 25 to 30 years).

Human Papillomavirus Persistence in Young Unscreened Women, a Prospective Cohort Study

ObjectiveTo evaluate hr-HPV persistence and associated risk factors in a prospective cohort of young unscreened women. Additionally, the relation between hr-HPV status and cytology/histology results is examined.Methods and Principal FindingsTwo year follow-up of 235 out of 2065 young women (18–29 years), participating in a large, one year epidemiological study, with questionnaires, self-collected cervico-vaginal samples (Vibabrush), and SPF10LiPA for HPV detection. Only women hr-HPV positive at sample month 12 were invited for a second year of follow-up. After study follow-up, available cytology/histology data were requested from PALGA (the national network and registry of histo- and cytopathology in The Netherlands). These data were compared with available cytology/histology data of the month 12 hr-HPV negative women from the same cohort. 44.1% of the hr-HPV types detected at study month 12, persisted during follow-up. HPV types 45, 31, 16 and 18 were most likely to persist with percentages of 60.0%, 56.8%, 54.4%,and 50.0%, respectively. Compared to newly detected infections at month 12, infections present since 6 months or baseline had an increased risk to persist (OR 3.09 [95% CI: 1.74–5.51] and OR 4.99 [95% CI: 2.67–9.32], respectively). Other co-factors influencing persistence were, multiple HPV infections, smoking and multiple lifetime sexual partners. The percentage of women with a HSIL/CIN2+ (12.1%) in the persistent HPV group, was not significantly different (p = 0.107) from the 5.3% of the women who cleared the hr-HPV infection, but was significantly (p 0.000) higher than to the 1.6% of women in the hr-HPV negative control group.ConclusionWe showed that HPV genotype, multiple infections, smoking, and multiple lifetime sexual partners are co-factors that increase the risk of hr-HPV persistency. Most importantly, we showed that hr-HPV infections are more likely to persist the longer they have been present and that women with a persistent hr-HPV infection have a high risk of HSIL/CIN2+ development.

HPV16 is associated with younger age in women with cervical intraepithelial neoplasia grade 2 and 3

ObjectiveTo evaluate if women with HPV16 positive CIN2 and CIN3 are diagnosed at a younger age. MethodsWe conducted a population-based cohort study including more than 40,000 women having a liquid based cervical cytology sample taken as part of routine screening. HPV analysis was performed using Hybrid Capture 2 and LiPAv2. The study population was linked to the Danish Pathology Data Bank to retrieve information on subsequent cervical histology. We included HR HPV positive CIN2/3 samples, comprising 173 CIN2 and 467 CIN3 lesions. Due to a high number of multiple concurrent HPV infections, the causative HPV type was assigned to a hierarchically group. ResultsIn CIN3, the estimated proportion of lesions positive for HPV16 was 68.1% among women aged 20years and decreased to 38.9% among women aged 50years. A decrease in HPV16 positivity with increasing age was also observed in CIN2. In a multinomial logistic regression analysis, young age was strongly associated with HPV16 positivity in CIN3 lesions (OR=0.46 per 10year increase in age, 95% CI: 0.32–0.65). The proportion of HPV16 and/or 18 positive lesions among women diagnosed with CIN2 and CIN3 below 30years of age was 44% and 75%, respectively. ConclusionsHPV16 positivity was significantly associated with younger age at diagnosis of CIN3. In a population vaccinated against HPV16 and 18, we will experience a shift to older ages in cervical precancerous lesions. These findings may imply that cervical cancer screening programs could start at an older age in HPV vaccinated populations.

O1-S02.03 Multiple sex partners and lack of condom use for anal sex are associated with multiple anal HPV infections among men having sex with men: the HIM study

BackgroundHuman Papillomavirus (HPV) infection is the primary cause of anal cancer. While multiple HPV infections in the anal canal may accelerate disease progression, there are no reports of behavioural factors...

Prediction of residual/recurrent disease by HPV Genotype after loop excision procedure for high-grade cervical intraepithelial neoplasia with negative margins

This study aims to evaluate whether the preconisation of high-risk human papillomavirus (HR-HPV) genotype and multiple HPV infection is predictive for residual/recurrent disease during the follow-up of high-grade cervical intraepithelial neoplasia (CIN) treated by loop electrosurgical excision procedure (LEEP) with negative margins. Two hundred and thirty-six women (mean age 37; range 20-61) with CIN2/3 treated by LEEP conisation with negative margins confirmed by pathology examination of the surgical specimen were included. The cervical cells for HPV genotype testing by the polymerase chain reaction (PCR) were collected before, and 6, 12, 24 months after treatment, respectively. HPV genotype and multiple HPV infection were evaluated as possible predictors of residual/recurrent disease. Residual/recurrent disease was demonstrated by colposcopy-guided biopsy in 62 patients (26.3%) who underwent loop conisation with negative margins. Preoperative infection with either HPV16 (P = 0.007), HPV18 (P = 0.000), HPV33 (P = 0.001) or HPV45 (P = 0.019) was associated with higher rates of residual/recurrent lesions after conisation with negative margins. Preoperative infection with multiple HPV types was associated with the highest rate of residual/recurrent lesions compared with infection with single HPV type and HPV-negative cases (χ2 = 16.599, P < 0.001). Results demonstrate that the presence of HPV-16, 18, 33 and 45, as well as multiple HPV types pre-LEEP, is associated with higher rates of residual/recurrent disease after LEEP.

Prevalence study of HPV mixed infections in Italian HIV positive women

Introduction: HIV positive women, show a higher frequency of multiple HPV infections than HIV negative.The immune response seems to be genotype-specific, but evidence on different genotypes distribution and involvement of coinfections in the development of invasive cervix cancer (ICC) remains limited. The aim of our study was to assess the prevalence of multiple infections in a group of Italian HIV positive women, the distribution of High risk (HR) strains and Low Risk (LR) strains in multiple and single infections, and their correlation with immune status and cervical lesions. Methods: 553 women were considered in the study. HPV search was performed with MY09-MY11 primers. HPV positive samples were typed with the Clinical Genomic array (HPV) test (Genomica, Spain). Results: 244 samples were HPV positive (44.1%).129/244 (52.9%) had a single infection and 103/244 (42.2%) multiple infections.Among the 412 performed typing, 223 (54.1%) were HR strains, while 189 (45.9%) were LR strains.The HPV61 (40 times) was more frequent among the LR strains.Among HR strains, the most frequently observed was the HPV16 (30 times). In 92% of multiple infections, at least one HR strain was found. 36% of LR strains was presented in single infections compared to 27% of HR strains (p = 0.06). The clades A3 (n = 124, 65.3% multiple infections) and A10 (n = 37, 56.8% multiple infections) were the most represented in LR;A9 (n = 95, 67.4% multiple infections) and A6 (n = 57, 70.2%) clades were the most representative among HR strains. Differences in age between women with single infection and those with multiple infection were not observed (p = 0.33) .Women with the best immune status (CD4 cell count of &gt;500 cell/ mm3) showed a higher prevalence of single infection. HPV was positive in 75% of ASCUS/LSIL lesion and 77.3% of H-SIL. Conclusions: HPV-16 is the most frequent in both single and multiple infections as reported in a recent study about HIV negative women. Follow-up studies are necessary to assess the impact of multiple infections on the progression of different cervical lesion degrees.

HPV genotype distribution according to severity of cervical neoplasia

Objective To analyse the HPV genotype profile and the presence of multiple HPV infections according to severity of cervical intraepithelial neoplasia. Methods From a population of 424,143 women in Norway, we included all women ( n = 643) with histologically confirmed cervical intraepithelial neoplasia grade 2 or higher (CIN2+) and evaluable HPV test during 2005 and 2006. Histology revealed CIN2 in 135 women, CIN3/ACIS in 495, and invasive carcinoma in 13 women. HPV genotyping was performed on cell suspensions from cervix by linear array which differentiates 37 HPV genotypes. Results HPV was detected in 98.4% (633/643) of the women, of whom 52.5% (338/643) were infected with more than one HPV genotype. HPV16 was most common, being detected in 51.2% (329/643) of all cases, followed by HPV31, 33, 52, 18, and 51. Overall, HPV 16 or 18 were detected in 58.0% (373/643), with 34.7% (223/643) without concurrence of other high-risk genotypes. HPV16 and HPV33 as single infections were more common in women with CIN3+ as compared to CIN2 (age-adjusted odds ratio = 5.93, 95% CI = 2.73–12.87, and age-adjusted odds ratio = 4.53, 95% CI = 1.42–14.46, respectively). Concurrent infection with other HPV genotypes did not significantly alter the associations to CIN3+ for HPV16 or HPV33. A single HPV infection, other than HPV16, 18, 31, or 33, was used as the reference. HPV18 or multiple HPV infections not including HPV16 or HPV33 were not associated with the severity of cervical neoplasia. Conclusion HPV16 and HPV33 appear to have a higher oncogenic potential than other HPV genotypes.

Abstract 4899: Identification of epigenomics biomarkers for early detection and progression of cervical carcinogenesis

Abstract Introduction: Pap smears and HPV infection tests have been the most widely used tools to detect cytological abnormalities and women's risk of developing cervical cancer. However, they do not distinguish between lesions that will progress to an invasive carcinoma and those that will not. Epigenetic biomarkers may prove to be useful early detection and progression markers for cervical carcinoma. Objective: To identify epigenetic biomarkers for early detection and monitor progression of premalignant lesions in cervical cancer. Materials and Methods: A total of 221 samples were analyzed: 25 normal, 66 Low Grade Lesions (LSIL), 91 High Grade Lesions (HSIL) and 39 cervical cancers (CC). The Reverse Line Blot technique was used for viral detection and HPV genotyping. DNA isolated from 12 normal samples and 7 cervical cancers was enriched with Methylated DNA Immunoprecipitation (MeDIP) and hybridized to Nimblegen 385K CpG Islands plus Promoter arrays. Bioinformatics strategies were used for background correction, array normalization and data analysis of differentially methylated genomic regions between tumor and normal tissue. Methylation Specific PCR (MSP) validated promoter methylation frequency in the 221 genotyped samples. Correlation analyses, and Receiver Characteristic Operator Curves (ROC) were used to determine the association between HPV infection, DNA methylation, and cancer status. Results. Infection by single and multiple HPV subtypes was observed in normal (4%), LSIL (80.3%), HSIL (93.4%) and cervical cancer (92.3%) samples. HPV16 was the most frequent genotype across sample histology: LSIL (45.3%), HSIL (47.1%) and CC (55.6%). Multiple HPV infections were more frequent in cancer samples (31%). Five hypermethylated genes were identified as potential biomarkers after validation by MSP. The methylation frequency for all five genes was higher in tumor, Gene 1 (100%); Gene 2 (98%); Gene 3 (96%); Gene 4 (100%) and Gene 5 (98%); than in normal samples: Gene 1 (12%): Gene 2 (12%); Gene 3 (16%); Gene 4 (40%); and Gene 5 (44%). The methylation status of Gene 2 and Gene 4 showed a progression in methylation frequency from normal samples to invasive carcinoma. The correlation of cancer status with of Gene 1 (0.90) methylation was higher than with HPV infection (0.87), Gene 2 (0.87), Gene 3 (0.81), Gene 4 (0.69) and Gene 5 (0.62) methylation. All correlations were statistically significant at the 95th percentile. The Receiver Characteristic Operator Curves (ROC) for HPV and individual genes revealed Area Under the Curve values of: HPV (0.94), Gene 1 (0.94), Gene 2 (0.93), Gene 3 (0.89), Gene 4 (0.80), Gene 5 (0.77). Conclusions: A panel combining HPV infection status and methylation in one of the five candidate genes identified had a higher predictive value than HPV infection or genotype and methylation status of any one candidate gene. Validation of these markers in a case-control cohort is warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4899.

Prevalence and genotype identification of human papillomavirus in women undergoing voluntary cervical cancer screening in Molise, Central Italy

We examined the prevalence of HR- and LR-HPV by Linear Array genotyping test in 299 women aged 18–63 years who consecutively visited Molise Region main hospitals for routine Pap smear between February and August 2008. Ninety women were positive for any HPV (30.1%), and 66 for any HR-HPV (22.1%). The most prevalent HR-HPV types were HPV 16 (22.2% of all women with HPV infection), HPV 53 (14.4%), and HPV 66 (14.4%). HPV infections increased from 15.8% in the 18–20 years group to 50.0% in the 21–23 years group and then decreased to 9.1% in those aged 50 years or more ( p = 0.008). Multiple HPV infections were observed in 15.7% of the study sample (52.2% of all HPV positive). There is a significantly higher prevalence of multiple infections in 18–32 years group women (24.5%) compared with females aged 33 years or more (6.8%) ( p < 0.005). Current smokers were at increased risk of HPV infection (44.2% of HPV infections compared with 23.5% in never smokers, and 25.3% of multiple HPV infections compared with 11.3%; p = 0.001). HR-HPV infections were higher in women never been pregnant (27.1% compared with 7.7%; p = 0.001). Oral contraceptive use was completely unrelated to infection. Among the 122 women who had both cytological examination and HPV results, multiple HR-HPV types were found in 36.8% of those with abnormal cervical findings, and in 13.6% of those with normal cervical findings ( p = 0.05). The results of the present investigation provide further evidence for the notion that cervical HPV infection is more widespread than previously suggested.