Both the 3p14.2 locus copy number and FHIT protein expression levels showed significant decreases when CIN transitioned to cervical cancer. HPV copy number was also evaluated in these clinical samples, and the copy number of HPV increased significantly between CIN and cervical cancer samples. Finally, we employed receiver operating characteristic curve (ROC curve) analysis to evaluate the potential of all these indexes in distinguishing CIN and cervical cancer, and the HPV copy number, FHIT copy number and FHIT protein expression levels have good diagnostic efficiencies.Cervical cancer is the second most common cancer and the third leading cause of cancer death in females worldwide, especially in developing countries. High risk human papillomavirus (HR-HPV) infection causes cervical cancer and precancerous cervical intraepithelial neoplasia (CIN). Integration of the HR-HPV genome into the host chromatin is an important step in cervical carcinogenesis. The detection of integrated papillomavirus sequences-PCR (DIPS-PCR) allowed us to explore HPV integration in the human genome and to determine the pattern of this integration. We performed DIPS-PCR for 4 cell lines including 3 cervical cancer cell lines and 40 tissue samples. Overall, 32 HR-HPV integration loci were detected in the clinical samples and the HeLa and SiHa cell lines. Among all the integration loci, we identified three recurrent integration loci: 3p14.2 (3 samples), 13q22.1 (2 samples and a SiHa cell line) and 8q24 (1 sample and a HeLa cell line). To further explore the effect of HR-HPV integration in the 3p14.2 locus, we used fluorescence  In vitro models established by transfection of primary human keratinocytes with HPV 16 and HPV 18 have shown that cells can be immortalized by the action of the E6 and E7 genes [FHIT), may play a role in cervical tumorigenesis. The FHIT gene is encoded by 10 exons in a 1.1-Kb transcript distributed over 1 Mb of genomic DNA, and aberrant FHIT transcripts were reported in cervical cancer. These aberrant transcripts were determined to correlate with cervical cancer outcomes [Persistent infection with High risk human papillomavirus (HR-HPV) is the main risk factor for the development of cervical intraepithelial neoplasia (CIN) and cervical carcinoma, but only 0.8% of HPV infections develop into cervical cancer. In China, HPV 16 and HPV 18 are the most prevalent types . The E6 E7 genes . There aE7 genes . Integraoutcomes .FHIT gene locus had the highest frequency of HPV integration in cervical cancer tissues. Then, with fluorescence in situ hybridization (FISH), we found that the FHIT copy number was lower in cervical cancer samples compared to CIN samples. Protein levels of FHIT were also reduced during CIN transition to cervical cancer as measured by an IHC assay.In this study, we used DIPS-PCR and determined that the 37 CIN patients and 93 cervical cancer patients were recruited at Tongji Hospital between 2014 and 2016. As normal control, 9 patients in the gynecological department who were performed with hysterectomy and confirmed with normal cervix were also recruited. Samples were collected by surgery and biopsy at the same time. All pathology diagnoses were evaluated and confirmed by two different pathologists from the Pathology Department of Tongji Hospital. Among the patients with cervical lesions, 23 case of para-cancerous tissue were successfully collected. All patients signed the sample collection informed consent form. We had access to information that could identify individual participants after data collection. Tissue DNA extractions were performed for 40 cervical cancer samples and all CIN tissue samples, and 53 cervical cancer samples were formaldehyde-fixed and paraffin-embedded. Clinical data were obtained from individual patient charts. In this study, we used International Federation of Gynecology and Obstetrics (FIGO) staging. The tissue sample collection and experiments were supervised and approved by the Ethics Committee of Tongji Hospital.Genomic DNA from clinical samples was extracted with a QIAGEN Blood & Tissue Kit . The genomic DNA was dissolved in elution buffer, and its concentration was determined using a Nanodrop 2000 ; all the DNA samples were stored at -80°C.Luft et al [ The second round exponential PCR products were analyzed on 2% agarose gels stained with Cyber Green and visualized using UV light in a ChemiImager 5500 . These PCR products were sequenced by Tianyi Biotechnology Ltd.The detection of integrated papillomavirus sequences by ligation mediated PCR (DIPS-PCR) was performed to determine the HPV integration loci at the DNA level. The assay was carried out following the method of ft et al . After dhttp://genome.ucsc.edu) and NCBI nucleotide BLAST (http://blast.ncbi.nlm.nih.gov) with assembly hg19.Sequencing results were analyzed using the UCSC database  was placed on a recipient TMA block, 4 μm thick slides were obtained from the TMA block, and TMA slides were re-examined for the original lesion and used for the subsequent experiment.FHIT (RP11-960D5) was purchased from Invitrogen. The whole genomic plasmid of the most common high-risk HPV type 16 (HPV 16) was a gift from Harald Zur Hausen. FISH probes were labeled by standard nick translation with digoxigenin-dUTPs. The TMA slides were dewaxed, dehydrated, and pre-treated with hydrogen peroxide, followed by rehydration, digestion with pepsin (1:3000) in HCl at 37°C, and dehydration. Probe and target DNA were denatured simultaneously prior to hybridization overnight. After hybridization, the preparations were washed stringently in formamide, 2×SSC. The probe was detected by peroxidase-conjugated sheep anti-digoxigenin Fab fragments  and then Cy3 or FITC labeled tyramide  and was mounted in a Vectashield (Vector Laboratories) containing 4’,6-diamidino-2-phenylindole (DAPI). The results were analyzed using a fluorescence microscope (Olympus BX53) with cellSens fluorescence imaging system software. A minimum of 100 nuclei were scored for FISH copy number evaluation. The copy numbers were evaluated using punctate signal per nucleus. In order to gain precise calculation of copy numbers, the average signals per nucleus of FHIT were normalized to the average signal per nucleus found in para-cancerous biopsies and copy numbers in those samples without para-cancerous tissue were normalized to the average copy number of the 9 normal biopsies.A bacterial artificial chromosome (BAC) plasmid for IHC reactions were performed on TMA slides following the standard protocol for 3,3’-Diaminobenzidine (DAB) staining. Briefly, slides were dewaxed, rehydrated, washed with water and then pre-treated in 3% hydrogen peroxide. Then, the slides were boiled with antigen retrieval solution and incubated with 1% BSA. The TMA slides were incubated overnight in a humidified chamber at 4°C with the primary antibody, a monoclonal anti-FHIT antibody . A biotinylated antibody was applied, followed by streptavidin-peroxidase incubation. The expression staining was developed in a solution of 3,3’-Diaminobenzidine. Then, slides were counterstained with hematoxylin, dehydrated, cleared with xylene and mounted. Image pro plus (IPP) software was used to analyze the staining. IHC evaluation was based on the integral optical density (IOD) of staining intensity. The optical density means the absorbance of light in a unit area of the IHC figure, and the summation of the absorbance of an IHC figure makes integral optical density (IOD).t-test and analysis of variance, and the Chi-square test was used to compare the expression level results. Receiver operating characteristic (ROC) curve analysis was used to evaluate the diagnostic capabilities of HPV copy number and FHIT copy number and protein expression levels. Maximum value of the Youden’s index may be used as a criterion for selecting the optimum cut-off point of numeric diagnostic biomarker.Copy numbers and protein expression levels were compared with Student’s KLF5 and KLF12 genes. In the HeLa cell line, the breakpoint was identified as chr8-128230632, and this position lies within CCAT1, upstream of c-MYC. However, we did not detect any breakpoints in C33a and HEK293 cells. We further performed DIPS-PCR to identify the integration loci in 40 cervical cancer tissue samples and determined 30 integration positions in 27 cervical cancer samples, 13 of which were HPV negative. Among all the 30 integration loci of cervical cancer samples, most of the integration sites were located in introns or intergenic positions. Most integrated HR-HPV sequences were determined to be HPV 16 and only 1 were HPV 18 integration. The distribution of the integration breakpoints in the human genome and HPV genome are shown in Fig 1and Genomic integration sites of HPV were detected by DIPS-PCR assay . This prFHIT gene.Notably, HPV integration into the 3p14.2 locus was detected in 3 samples, and these breakpoints are located in intron 4 of the FHIT aroused our interest, further studies on FHIT are needed. To further evaluate the copy number and protein expression levels of the FHIT gene, we performed FISH and IHC. In addition, we assessed the potential of FHIT as a biomarker for the transition of CIN to cervical cancer.As it was the most frequent integration locus, We included 9 patients with normal cervical biopsy, 37 patients with CIN and 53 patients with cervical cancer in our study. The average age was 46.3, 47.1 and 43.3 years, respectively, the differences on age between these three groups were not significant. We evaluated cervical cancers using International Federation of Gynecology and Obstetrics (FIGO) staging.FHIT and HPV in normal cervix, CIN and cervical cancer by FISH. Typical FISH signal patterns are shown in  We did not detect any HPV signals in normal cervical tissues.We analyzed the copy number variation of Fig 3A, 1.04 to 1.92, P < 0.001). However, there was no significant increase from CINI to CINII, CINII to CINIII, CINIII to cervical cancer stage I, or cervical cancer stage I to stage II . The differentiation of cervical cancer had no effect on the HPV copy number variation .For HPV copy number, in the normal cervical biopsies, we didn’t detect any HPV signals. An elevation was observed from CIN to cervical cancer , but not from normal cervical samples to CIN . However, between different stages of CINs and different stages of cervical cancers there was no particular aberrance . The most significant decline in average copy number was in the transition of CIN III to cancer stage I . We determined that cervical cancer differentiation was not an influential factor for FHIT copy number Generally, the average copy number of Fig 2. Average IOD decreased significantly from CIN to cervical cancer , but from normal cervical tissue to CIN, the change was not significant . The most significant decrease was from CIN III to cancer stage I , suggesting that FHIT is an important factor in the transition of CIN to cervical cancer. Transitions from CINI to CINII, from CINII to CINIII and from CaI to CaII do not have significant decreases . Average IOD showed no significant differences between different grades of differentiation. .IHC was used to analyze the protein expression levels of FHIT. Representative FHIT staining patterns are shown in FHIT copy number and protein expression level as biomarkers for diagnosis of CIN evolution to cervical cancer, receiver operating characteristic curve (ROC curve) analysis was employed. Cutoff points were made according to the maximum of Youden’s index. For HPV copy number, the area under the curve (AUC) was 0.769 (95% CI: 0.659–0.880), and the cutoff point was set to 1.54. Under such conditions, the sensitivity is 59.46%, and the specificity is 87.50%. The AUC of the FHIT copy number ROC was 0.820 (95% CI: 0.717–0.924). According to a cutoff point of 1.21, the sensitivity is 87.50%, and the specificity is 80.00%. For FHIT expression level, the AUC of the ROC curve was 0.683 (95% CI: 0.558–0.807), and the cutoff point was 161383.45. Under such conditions, the sensitivity is 85.11%, and the specificity is 55.89%.To evaluate the potential of HPV copy number and Although there has been a large amount of research concentrating on HPV integration and cervical cancer, the mechanism of cervical cancer tumorigenesis still needs to be elucidated. Previous studies revealed that HPV tends to integrate near tumor associated genes, causing gene function alterations . These aFHIT is one of the highest frequency loci for HPV integration. FHIT, or fragile histidine triad, is located at 3p14.2. FHIT is a target of tyrosine phosphorylation by the Src protein kinase, and the regulation of communication by Src may be important in the control of embryonic development and cellular growth [cyclin D1, AXIN2, MMP14, and survivin. Dysfunction of FHIT protein can cause overexpression of cyclin D1 and other cell cycle genes. This can cause cell cycle disorder and carcinogenesis [FHIT works as a tumor suppressor genes. Studies showed reductions in the expression levels of FHIT have occurred in many kinds of malignancies, such as lung cancer [et al have reveal that loss of heterozygosity was an indicator of high-grade tumor, greater tumor depth and lymph node involvement [et al have identified a novel missense mutation of FHIT gene, this mutation can cause protein inactivation with loss of tumor suppressor activity [FHIT gene inactivation in cervical cancer was found to be strongly correlated with 5'-CpG island hypermethylation have been reported in previous study [FHIT in cervical carcinogenesis.In our study, DIPS-PCR was used to detect the location of HPV integration in the human genome. In the SiHa and HeLa cell lines, we detected the genomic chr13-74087562 and chr8-128230632 breakpoints. This is consistent with the next generation sequencing results in our previous study [g cancer , renal cg cancer  and colog cancer . Terry Golvement . Neyaz Mactivity . Also, Fus study . These fFHIT as a biomarker for cervical lesions. We assessed the copy number variation of the FHIT locus with FISH and the expression levels of the FHIT protein by IHC.Therefore, we investigated the potential of FHIT was absent in CIN but was frequent in invasive tumor. Also, allelic losses, homozygous deletions, and aberrant FHIT transcripts are common in cervical cancers but occur at much lower frequencies in precancerous lesions [FHIT may play an important role in CIN evolution to cervical cancer. In our study, as CIN progressed to cervical cancer, FHIT copy number and protein expression levels both showed significant differences. The most significant reductions of FHIT copy numbers and FHIT expression were from CIN III to cancer stage I , This finding indicated that there was obvious deletion in the FHIT gene locus when CIN progress into cervical cancer. And this deletion may cause protein expression decrease, suggesting that FHIT is an important factor in the transition of CIN to cervical cancer. We then explored the potential of them of being diagnostic biomarkers with ROC curve as well as HPV copy numbers. According to the ROC curve analysis, FHIT copy number has the most potential for being diagnostic biomarker compare to the HPV copy number and FHIT protein expression. The AUC of the ROC was 0.820 (95% CI: 0.717–0.924). According to a cutoff point of 1.21, the sensitivity is 87.50%, and the specificity is 80.00%.Previous report showed that methylation of  lesions . This imFHIT may be a latent target of cervical cancer treatment, but in our study, integration of HPV into the FHIT gene was only detected in 3 samples, and FHIT copy number and expression levels decreased in a large proportion of the cervical cancer samples. HPV integration only explains part of this reduction. The reasons why the remainder of the samples also decreased in copy number and protein expression levels are still unknown. Further study is needed to determine the latent mechanism.All of the results suggested that S1 TableDIPS-PCR primers for HPV 16 and HPV 18 and the formation of Sau3AI-specific adapters.(PDF)Click here for additional data file.S2 TableDIPS-PCR results of 40 cervical cancer samples. 30 integration positions in 27 cervical cancer samples were found. Of all the breakpoints, FHIT was found to be a recurrent breakpoint.(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


Head and neck (H&N) cancer is an aggressive disease and the incidence has increased in younger population worldwide. Tumour TNM staging is the main basis for treatment decision despite significant variation in clinical outcome. Survival time of these patients has marginally improved during the last 30 years. Various biomarkers with cumbersome analysis, high cost, time consumption and requirement of special laboratory facilities have been investigated. However, none of these biomarkers have been shown to be suitable to use for individual H&N cancer patient treatment selection in the clinic. For practical use in clinical settings, the given biomarkers must be simple to analyse, rapid, cost effective and available in routine laboratories. With this intension, we suggested the combination of standard TNM staging and biomarkers associated with inflammation such as neutrophils, neutrophil to lymphocyte ratio, plasma C-reactive protein or plasma tumour necrosis factor alpha (TNFa) and single-nucleotide polymorphism in TNFa rs1800629 using blood-based analysis. The optimal treatment outcome of H&N cancer by using combination of TNM stage and these blood-based biomarkers for individual patient selection need further investigation. Head and neck (H&N) cancer is an aggressive disease and the incidence is increasing, especially in younger population worldwide , 2. RelaTumour TNM staging is the primary guide for treatment modality despite significant heterogeneity in clinical outcome. Independent from TNM stage, number of circulating tumour cells, lymphocytes expressing cytolyses granules, neutrophils, neutrophil to lymphocyte ratio (NLR), plasma C-reactive protein (CRP), tumour necrosis factor alpha (TNFa) or downregulated T cell receptor Zeta chain was associated with the clinical outcome of cancer patients –19.Prognostic biomarkers for individual patient treatment selection in the clinical practice need to be established for optimal clinical outcome of H&N cancer patients. We emphasize the cost-effective, rapid and automatic blood-based biomarkers in combination with TNM staging for individual treatment selection in the clinical setting of this patient group.Epidemiological data have established that cigarette smoking, alcohol consumption or HPV infection are the main risk factors for H&N cancer –23. MoreSNP is the most common source of human genetic variation in the individual germline DNA. SNP sequence variation may lead to altered effects of the gene and causes inter-individual susceptibility to oncogenesis. One SNP, TNFa rs1800629 is associated with increased risk of H&N cancer, tumour recurrence and survival time of the patients .HPV is a common sexually transmitted infection and most of the sexually active women and men worldwide are infected by low-risk or high-risk HPV in their life time , 28, 29.High-risk HPV type might be necessary for tumorigenesis but it is not sufficient on its own . IndividThe presence of high-risk HPV-DNA is insufficient to classify accurately tumours as an HPV associated since it might be present as the passenger, inactive virus and not the cause of malignancy , 38. TheTwo techniques, PCR and in situ hybridization (ISH), are generally used for diagnosis of high-risk HPV status in biopsy material. The presence of high-risk HPV-DNA shown by PCR or ISH is, however, insufficient to accurately classify tumours as HPV associated. Fluorescence in situ hybridization (FISH) could identify whether the high-risk HPV is oncogenic with integrated HPV-DNA or inactive with episomal HPV-DNA in the infected cells . This anCigarette smoking and alcohol consumption are independent risk factors but when combined a synergistic effects is observed . In vitrSystemic inflammation from smoking induces massive cell death in the body might indicate by increasing circulating neutrophil, NLR, perforin expression in T cells and plasma CRP or TNFa levels were detected in healthy smokers . HPV infection and alcohol consumption could also increase normal cell death , 42, 49.Human solid tumours are generally infiltrated by inflammatory cells and considered to be a host attempt at the detection of emerging tumour cells and their elimination . H&N tumElevated frequency and enhanced suppression capacity of regulatory T cells (Treg) was detected in blood circulation of H&N cancer patients , 54. IncPeripheral blood contains immune response cells, plasma proteins, cytokines and variable mediators with wide-ranging biologic effects. Circulating blood are in direct contact with all organs, non-invasive to obtain and suitable for transportation. Analysis of SNPs and inflammatory biomarkers could be simply done using this peripheral blood sample.Using only TNM stage as treatment selection might be practical but not optimal to predict clinical outcome of H&N cancer patients. Independent from tumour etiological or TNM stage, SNP in TNFa and elevating number of inflammatory biomarkers obtained from pre-treatment peripheral blood sample is associated with the clinical outcome of H&N cancer patients –17, 27. With intension to be used as biomarkers in clinical practice, such analysis must be rapid, available in routine laboratory facility and cost effective. For optimal personalized H&N cancer treatment, we propose the pre-treatment blood-based biomarkers with focus on TNFa rs1800629, neutrophil counts, NLR, plasma CRP and TNFa in combination with standard pathological diagnosis and TNM classification for individual H&N cancer patient treatment selection. The prognostic value of this combination warrants future clinical investigation.The impact of genetic variation, smoking, alcohol consumption, HPV infection and HPV integration significantly influence host immune response , 42, 52.The analysis of TNFa rs1800629 and systemic inflammatory biomarkers such as plasma CRP, TNFa or neutrophil and NLR using blood sample are rapid, cost effective and require only common facility in the routine laboratory. Thus, blood-based biomarkers offer an attractive possibility to use in combination with standard tumour TNM stage for individual H&N cancer patient treatment selection in the clinical setting. The prognostic value of TNM stage combined with these blood-based biomarkers for treatment selection of H&N cancer patients warrants further investigation.


Whilst the imputed role of High Risk (HR) HPV infection in the development of cervical lesions and cancer has been established, the high number of HPV genotypes that Female Sex workers (FSW) harbour warrants that the synergistic effects of potential HR (pHR) and HR HPV genotypes be elucidated to assess the potential impact of prophylactic vaccines. This population in Kenya also harbours a number of other vaginal infections and STIs, including bacterial vaginosis (BV), trichomonas vaginalis (TV) and The aims of this cross-sectional analysis in Kenya are to explore the epidemiology of abnormal cytology and the pairing of pHR/HPV genotypes in HIV-negative and HIV-infected FSW.A cross-sectional study design of 616 FSW from Western Kenya aged between 18 and 61 years during 2009–2015 using a peer recruitment sampling strategy.Of the 599 FSW who underwent cytological examination, 87 had abnormal cytology . A combined prevalence of HPV16 and 18  was observed in abnormal cytology. HPV 53 and 51 were the most observed pairing in FSW with abnormal cytology. Significant adjusted associations were found between abnormal cytology and TV , multiple HR HPV , HPV 51  and HPV 52 .HPV 51 and 52 were independently associated with abnormal cervical cytology in both HIV negative/positive FSW. The strong association between TV and cervical dysplasia and the high percentage of FSW harbouring more than one STI underscore the need for enhanced STI management within the framework of cervical cancer prevention. Human Papilloma Viruses (HPV) are double-stranded DNA viruses which are now deemed to be the chief etiological agents in cervical intraepithelial neoplasias and cancers . High riCervical intraepithelial neoplasia (CIN) can be histologically graded into mild dysplasia (CIN 1), moderate dysplasia (CIN 2), and severe dysplasia to carcinoma in situ CIN 3). Several studies have reported a robust association between HIV and HPV co-infection and therefore the development of CIN and genital cancer, . Several along wiIt is well recognized that among the 14 HR HPV genotypes, HPV 16 and 18 are associated with approximately two thirds of all invasive cervical carcinomas , 8.Prophylactic vaccines against HPV 16 and 18 are currently being rolled out across the globe for the prevention of cervical cancer, which is likely to yield a significant impact on the future burden of cervical cancer, particularly in sub Saharan Africa, where screening is scarce. Although the bivalent/quadrivalent vaccines, including the LR HPV 6 and 11 constitute a crucial milestone in cervical cancer prevention in HIV-negative women, epidemiological data available suggest that in HIV positive populations, HPV 16 has shown to be frequent, but not as predominant as seen in most HIV negative populations , 10. MorAlso, epidemiological knowledge of pHR HPV types is highly limited, mainly because commercial molecular assays focus on HR HPV genotypes. There is a paucity of data on these genotypes in HIV positive women with abnormal cytology, notwithstanding their potential enhanced role in cervical dysplasia development in HIV positive patients , 16.In Kenya, the Ministry of Public Health and Sanitation has developed a comprehensive cervical cancer prevention strategy, entailing plans for administrating quadrivalent vaccine to preteen girls in the near future. Currently they are running the pilot programme Kituwi in Eastern Kenya and awaiting approval for nationwide rollout and successful global funding . The notTrichomonas vaginalis (TV), have been associated with an increased risk of squamous intraepithelial lesions and/or CIN based on biopsy results [In Kenya, as in many parts of sub-Saharan Africa, where the penal code specifically penalizes prostitution , FSW bea results , 22.As early as 1985, a study reported that HIV prevalence was as high as 61% among a group of FSW in Nairobi . A recenThe objectives of this analysis were primarily to assess genotype-specific distribution of pHR/HR HPVs in FSW with abnormal cytology, as well as the pairing prevalence of certain pHR/HR HPV genotypes found in HIV negative and HIV infected women with abnormal cytology; secondly, to investigate which HPV genotypes and other variables were associated with abnormal cytology.A cross-sectional design was used to explore associations between abnormal cytology and pHR/HR HPV genotypes. This cross-sectional study based on record reviews adhered to the methodological guidelines recommended in the STROBE document on observational studies .Women were excluded if they were pregnant, <18 years of age, had a history of cervical dysplasia or cancer, had current abnormal bleeding or bloody discharge, and/or had a hysterectomy. Snowball sampling was undertaken instead of randomized sampling, which is an often used strategy for locating difficult-to-reach and stigmatized populations . This inThe sample size was calculated to allow for a prevalence of at least 15% for abnormal cytology,  with a cA structured paper questionnaire was privately administered by trained interviewers covering socio-demographic characteristics, and sexual behavior. Participants were offered testing for HIV and HPV. HIV results were disclosed to participants and women infected with HIV received counseling and treatment.Trichomonas vaginalis was diagnosed by PCR, using a validated method which was part of the HPV genotyping essay.A gynaecological examination was performed using a swab. Candida colonization was diagnosed by Gram stain; bacterial vaginosis was scored according to Nugent’s criteria. Infection with A HIV diagnosis was performed using rapid immunoassays: Uni-Gold™ Recombigen® HIV  and Determine® HIV-1/2 . In the case of indeterminate results, an enzyme-linked immunosorbent assay was used to confirm HIV status.Cervical samples were collected using a cervix brush , and cervical cytology was assessed with conventional Papanicolaou (Pap) smears. Slides were read by a cytologist with master level training, supervised by a pathologist. An external cytopathologist provided quality control. The Bethesda Reporting System was used for cytological classification .The cervix brush tips were preserved in a liquid-based cytology collection medium  and stored at 4 °C until further processing.th, 2011.Ethical approval for the study was obtained from the Institutional Research and Ethics Committee at the MOI University in Kenya (No 000187) on August 11Data analysis was done using STATA version 12 . Due to incomplete information about the study samples, we checked whether the missing data (10%) were randomly distributed by performing the Little’s MCAR test. Continuous variables were then converted into categorical. Age was dichotomized into ≥30 years and <30 years; this categorization was used to reflect the WHO 2014 guideline concerning cervical screening. The number of pHR/HR HPV co-infections was also dichotomized as a categorical variable with 1 and ≥2 genotypes.We first described the distribution of pHR/HR HPV types observed among women with both normal cytology and abnormal cytology, for which the overall prevalence and 95% confidence intervals (95% CI) based upon normal distributions were calculated.To examine patterns of clustering of high-risk HPV types, the prevalence of pHR/HR HPV genotypes in presence of another pHR/HR HPV genotypes by abnormal cytology was calculated, which was defined as the proportion of women with abnormal cytology who were positive for the pHR/HR HPV genotypes. The prevalence of pairings detected in women with HSIL was also calculated.χ2 test was used to assess whether there was an association between CIN 2+ and various risk factors. In building the regression models, age, STIs and multiple pHR/HR HPV genotypes tested were entered. A multivariable logistic regression analysis was performed to assess the association between pHR/HR HPV genotypes and abnormal cytology, ASC-US or higher, the main outcome of interest and to simultaneously control for potential confounders. The Likelihood Ratio Test (LRT) was used to measure the association of each variable with the outcome.The variables were explored by means of tabulation and cross-tabulation. The p ≤ 0.05.To assess for a possible interaction due to age, logistic regression models were fitted with and without the interaction term; significance for interaction was then checked through visual inspection of the OR and LRT. Statistical significance was considered at candida spp (7). The Little’s MCAR test revealed that the missing values were randomly distributed among the participants (p = 0.4), therefore unlikely to have introduced information bias.Out of the 616 participants, data from only 599 participants could be analysed because of the quality of the specimen. Missing values were observed for BV (61), TV (7) and The mean age of study participants was 28 years and median parity was 2 (range: 0–7 children). Regular condom use, which was defined as always or almost always was reported in 21.7% of 369 women. (95% CI = 17.6–26.2%). The median number of partners in the past week was 4 (IQR: 2–7). Table N = 616).Of the 616 FSW who underwent cytological examination, 512 (85.5%: 95% CI: 82.4–88.2) had normal cytology, 87 had abnormal cytology  and 17 were excluded due to poor quality of the sample, leaving 599 participants on whom we could perform the analysis. Of the FSW population, 192 FSW were HIV positive, of which 27.1% (95% CI: 20.9–34.0%) had abnormal cytology. Table p < 0.001.The prevalence of pHR/HR HPV and multiple pHR/HR co-infections in the 616 FSW was 57.7 and 32.8% respectively. In HPV infected FSW, HIV-infected FSW had a significantly higher number of co-infections (2.0) than HIV negative FSW (0.9), candida spp 19.9%. FSW with BV had the highest prevalence of multiple pHR/HR co-infections with 53.4%, followed by TV, and candida spp, 38.8and 23.9% respectively. Table The prevalence of BV in this population was 48.3%, followed by TV 31.4%, and The combined prevalence of HPV 16 and HPV 18 was 27.1%, and of the two pHR HPV genotypes tested, HPV 53 and 66, 20.8% (95% CI: 17.6–24.2%). In women with abnormal cytology, we observed a multiple pHR/HR HPV genotype prevalence of 65.5%. See Table HPV 53 and HPV 66 were found as a stand-alone pHR HPV genotype in two LSIL cases.n = 3) occurrences, and HPV 31 and 52 (n = 2), involving genotypes phylogenetically related to the HPV 16.A higher prevalence of pairings in abnormal cytology were observed in HIV infected FSW than HIV negative FSW. The most frequently observed pairings in HIV- negative FSW were HPV 18 and 31  and HPV 51  figure prominently. See Table p = 0.6) of having abnormal cytology compared to older women.In the univariate analysis, age did not appear to be associated with abnormal cytology: women under 30 years of age have a crude OR 1.1  adjusting for age and pHR/HR HPV genotypes, BV and p < 0.001; 95%CI: 1.9–7.8) when adjusted for HIV.A statistically significant OR, adjusted for age was found for multiple HPV and abnormal cytology compared to single HPV genotype infection. This association decreased but remained significant  for cervical dysplasia and for a wider encompassing cervical cancer prevention framework.


Human immunodeficiency virus (HIV)-infected patients are at an increased risk of co-infection with human papilloma virus (HPV), and subsequent malignancies such as oral cancer. To determine the role of HIV-associated immune suppression on HPV persistence and pathogenesis, and to investigate the mechanisms underlying the modulation of HPV infection and oral cancer by HIV, we developed a mathematical model of HIV/HPV co-infection. Our model captures known immunological and molecular features such as impaired HPV-specific effector T helper 1 (Th1) cell responses, and enhanced HPV infection due to HIV. We used the model to determine HPV prognosis in the presence of HIV infection, and identified conditions under which HIV infection alters HPV persistence in the oral mucosa system. The model predicts that conditions leading to HPV persistence during HIV/HPV co-infection are the permissive immune environment created by HIV and molecular interactions between the two viruses. The model also determines when HPV infection continues to persist in the short run in a co-infected patient undergoing antiretroviral therapy. Lastly, the model predicts that, under efficacious antiretroviral treatment, HPV infections will decrease in the long run due to the restoration of CD4+ T cell numbers and protective immune responses. Infection with the human immunodeficiency virus (HIV) afflicts over 35 million people worldwide and results in impaired immune responses which may affect defenses against other pathogens. In the absence of protective vaccines, current management of HIV consists of administration of combination antiretroviral therapy (cART)—which suppresses viral replication and, consequently, drastically reduces morbidity and mortality , 2. cARTHelicobacter pylori, Salmonella typhimurium, Chlamydophila pneumonia . The. TheY1), surface , 30. Theed to Y1 . For siml layer) . The rate, as in , both ty rate c2 . The c2 Y2, effector cells specific to HPV, E, expand with a maximum per capita rate ω and carrying capacity K. This carrying capacity is an addition to the original work  com comY2, eE kills both classes of infected cells at the same rate a, since both infected cells populations express oncogenes E6 and E7 . Add. AddE kiPV types , are alsY1(0) = Y10,Y2(0) = Y20,W(0) = W0 and E(0) = E0.The following system of differential equations represents these dynamics:tat, secreted from HIV-infected intraepithelial immune cells, is known to play an important role in the disruption of epithelial tight junctions, thereby facilitating the entry of HPV into the mucosal epithelium  [15 [1548] . Indeed,To address the possible interactions leading to HPV persistence, we highlighted specific scenarios presenting an increased persistence of HPV due to the permissive immune environment created in an HIV-infected individual. Our model predicted that among the HIV infected individuals, those who had CD4+ T cells corresponding to average chronic HIV CD4+ T cell levels were more likely to clear HPV than those who had the CD4+ T cell levels reported in the co-infection clinical study , or thosWe used the model to study the impact of cART on HPV persistence in HIV/HPV co-infected individuals and compared the findings with those from the clinical study , which sεRT = 0.2 and εPI = 0 with intermediate CD4+ T cell levels such that 5 cells. These results provide further evidence to support the findings from other studies that show immunosuppression plays a role prior to cancer diagnosis [tat, which are removed when HIV is removed, and therefore do not influence HPV clearance. Finally, our results show that when HPV infection occurs at the same time as the start of cART, HPV levels increase and stay elevated throughout the 24 weeks of study. The findings support previous reports that predicted an increased risk of oral HPV infection among immunosuppressed individuals, which can be explained by the reactivation or reacquisition of a previously acquired infection [tat and CD4+ T cell rebound. A finding from our model highlights the need of a higher carrying capacity in a co-infected individual for HPV clearance to occur. This suggests a potentially clinically testable hypothesis that—if a HIV/HPV co-infected individual is immunosuppressed, then they should be treated for immunosuppression first. Once the CD4+ T cell levels are restored, the individual can be treated against HPV. Moreover, if a person treated for HIV has restored CD4+ T cell levels, HPV treatment should be tailored towards type specific HPV. In particular, our simulation result show that individuals with high CD4+ T cell levels post cART, produce more HPV when their oncogenic expression is high, compared with a patient with a weaker CD4+ T cell restoration but a lower oncogenic expression. Hence, the treatment (vaccine) against the type specific HPV, may aid the viral clearance.Our study predicts that the timing of viral clearance is determined by the timing of cART compared to the timing of HPV co-infection, as well as the CD4 T cell level. When cART is started shortly after HPV infection, HPV will expand and will not be controlled in the 24 weeks of cART as described for the 28% cases in . This caiagnosis  and faciiagnosis ,30. The nfection . We furtThis work is one of the first HIV/HPV co-infection models that investigates the dynamics of HPV in HIV-infected individuals. The only other published HIV/HPV co-infection model is a transitional probability-based model , which wCM) are the major cellular reservoirs for HIV in the peripheral blood [To understand the mechanisms responsible for progression to AIDS and for CD4+ T cell rebound following cART, it is necessary to understand the impact of HIV in the dynamics of each functional CD4+ T cell subsets –58. Th17al blood . Furtheral blood , 65 in tThe present modeling study should be evaluated in the context of several limitations. First, it does not take into account the spatial structure of the epithelial tissue. A generalized model that takes in to account the infectivity and layer transition terms described by probabilities or one which would consider spatial structures for epithelial cells in different layers, requires extensive knowledge of numerous parameters, that are currently unknown. The second limitation concerns the immune clearance of lesions caused by HPV infections, which can lead to asymptomatic or latent infections with possibility of increased virion production upon immunosuppression –69. ThisThe next step would be investigating the T cell subsets involved in the HIV/HPV co-infection in our model. Additionally, the findings of the modeling study and associated limitations guarantees and necessitates inclusion of latent T cell reservoirs which are involved in the activation of residual cART induced immune reconstitution. Furthermore, incorporating the immune activation in the T cells under the effect of cART during a HIV/HPV co-infection would corroborate the findings regarding the presence of new detectable HPV DNA.In summary, we developed a novel mathematical and computational model of HIV/HPV co-infection and used it to present hypotheses for the mechanisms underlying HPV persistence in HIV/HPV co-infected individuals. Our model can be applied to studying interactions between HIV and other widespread microbes to gain a better mechanistic understanding, guide the rational for the design of clinical trials, and accelerate the path to safer and more effective vaccines and therapeutics. We use this study as an alternative approach to determining how overall CD4+ T cell levels influence HPV prognosis in an HIV-infected individual. Overall, a better understanding of the cell specificity of HIV infection integrated with the cellular environment in HPV infection would facilitate the development of more effective therapeutic strategies in HIV/HPV co-infections.The model was deposited in BioModels  and assiS1 FigT), productively infected CD4+ T cells (I) and HIV (V) are shown. The figure also includes the effect of reverse transcriptase (RT) and protein inhibitor (PI) (shown by red line—inhibition). The right side of the figure represents the HPV dynamics wherein the interaction between infected basal cells (Y1), suprabasal transit-amplifying cells (Y2), HPV specific (E) cells and HPV (W) are shown.The left side of the figure represents the HIV dynamics wherein the interaction between target CD4+ T cells ((PDF)Click here for additional data file.S2 Fig(A) HIV/HPV co-infection model comparison. (a) HPV W and (b) CTL E as given by model (5), solid blue lines and model (26), dashed red lines, for ε = 0.5 per day, parameters are listed in 6 cells per ml (first row); 5 cells per ml (second row); 5 cells per ml (third row); and 5 cells per ml (fourth row). (B) HIV/HPV dynamics when cART and HPV infection coincide. (a) HPV W; (b) CD4+ T cells (T) as given by model (5) solid blue lines and model (26) dashed red lines under cART. Here, ε = 0.5, εRT = 0.95, εPI = 0.5, and all other parameters are listed in t = 0 is the start of cART. Over the first 24 weeks HPV persists (panel a), and in the long term HPV is cleared (zoomed out panel a).(PDF)Click here for additional data file.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.


P<0.001), whereas the prevalence of HPV 6 and 11 infection was similar . HPV 52, 58, 16, 39 and 18 were the five most prevalent types in both sexes. Sexual and hygienic behaviors were associated with HPV infection in both women and men. We found that oncogenic HPV DNA detection is more prevalent in women than in men in China, whereas the prevalence of HPV 6 and 11 is similar in both sexes. The data indicate that the interaction of host and virus might be different among high- and low-risk HPV types.Oncogenic human papillomavirus (HPV) infection is a cause of many anogenital cancers in women and men; however, there is little research on HPV prevalence and risk factors that includes both women and men from the same population. A total of 4687 participants, including 2378 women and 2309 men aged 18–55 years old from the same community, were enrolled in the study in Liuzhou, China. Exfoliated cells were collected from the participants from different anatomic sites and were tested for 13 oncogenic and 3 non-oncogenic HPV types. The prevalence of any oncogenic HPV type was higher in women than in men (18.7% vs 9.4%,  HPV is assumed to be responsible for 100% of cervical cancers, 90% of cancers of the anus, 50% of cancers of the penis, 40% of cancers of the vulva (VU), 70% of cancers of the vagina (VA) and 20%–60% of cancers of the oropharynx.1 Licensed HPV vaccines are used mainly in girls aged ≥nine years and in young women. HPV is mainly transmitted through sexual activity, and case–control studies have shown that the sexual behavior of male partners affects a woman's risk of cervical neoplasia.4 Recently, a cohort study conducted in heterosexual couples illustrated that the male-to-female and female-to-male HPV transmission rates were 7.11 and 5.56 per 1000 person months, respectively.5 Therefore, the epidemiological characterization of HPV infection in both sexes from the same population is important to understand the whole picture of HPV transmission and natural history in humans.The first generation of preventive HPV vaccines are bivalent (HPV 16 and 18) or quadrivalent  and can prevent ~70% of cervical cancer cases. The second-generation HPV vaccine is nine-valent, adding five more HPV types  to the quadrivalent HPV vaccine and is expected to prevent 20% more cervical cancer cases.10 A direct comparison of the prevalence data between sexes in different studies has been hampered by the differences in the population sampling strategy, the sensitivity of the HPV genotyping assay and the number of genotypes detected. Thus, less is known about the sex-dependent differences of anogenital HPV infection characteristics. Only one study reported that HPV prevalence was lower in the cervical samples of women than in the penile samples of men  in an HIV-negative population in South Africa, but the sample size was relatively small.11Much has been learned about HPV prevalence in women and men, but few studies have included both sexes.The aim of this study was to define the prevalence and type distribution of HPV in the general population of women and men in southern China using the same protocol for HPV genotyping. The whole picture of HPV distribution and the risk factors in both sexes would be helpful for developing more effective preventive policies for HPV infection.A population-based study among women and men was conducted in a rural site and an urban site of Liuzhou city, Guangxi, China, from March to July 2014. Participants were eligible if they were 18–55 years old, had ever been involved in sexual activity at enrollment, and were willing to refrain from sexual activity  and to avoid washing the genitals for 48 h before sample collection. Exclusion criteria consisted of having received an HPV vaccine, having reported serious illnesses or current pregnancy for women. Most of the volunteers were recruited from the general population through media advertising, including local newspapers and TV news, and educational presentations in communities. In addition, to increase the enrollment of younger people, students from Guangxi University of Science and Technology were recruited through flyers and posters.Before study initiation, the Ethics Committee of Liuzhou Centers for Disease Controls approved all of the study procedures. At the enrollment visit, all of the participants gave written informed consent and underwent a clinical examination. Each participant was interviewed individually by a trained interviewer to complete a questionnaire. Then, genital exfoliated cell samples were obtained from each participant.For women, three iCleanhcy flocked swabs  were used to collect specimens from the VU, VA and perianal/anal (PA) canal. Physicians used a swab to swipe the region from the clitoris to the labia minora and the labia majora in a zigzag pattern to ensure acquisition of the exfoliated cells. A second swab was used to collect a sample from the lower 1/3 of the VA by separating the labia of the VU. Then, a single-use speculum was inserted into the VA, and exfoliated cells from the upper 1/3 of the VA were obtained. A third swab was used to sweep 360° around the perianal area and was then inserted into the anal canal to obtain exfoliated cells.For men, two pre-wetted saline iCleanhcy flocked swabs  were used to collect specimens from the penis/glans penis/coronary sulcus (PGC) and PA canal. Physicians used a swab to rub up and down the entire skin surface of each of the quadrants of the penis shaft. Then, the same swab was swept 360° around the glans penis and coronary sulcus. For uncircumcised men, exfoliated cells of the foreskin were collected using the same swab. The cells of the perianal and anal canal were obtained using the second swab in the same way as for women.The swab samples were placed into separate vials containing 1 ml of PreservCyt solution , and the tubes containing the specimens were stored at room temperature until they were transported to the laboratory of Zeesan Biotech Corporation, Xiamen, China, within two weeks for detection and typing of HPV DNA.14 DNA extraction was performed using the Lad-Aid 824 system  according to the instructions of the manufacturer. In brief, 200 μL aliquots of the clinical material were digested with 3 M guanidine hydrochloride solution for 300 s. The DNA was eluted in 150 μL of 10 mmol/L Tris-HCl buffer (pH 7.5) at room temperature. The DNA was stored at −80 °C until use.HPV testing of the swab specimens was conducted using a multicolor real-time PCR and melting curve analysis. GP5+/6+ was used as the general primer rather than the MY09/MY11 or SPF10 primers, which amplified longer or shorter target L1 fragments, respectively.2SO4, 4 mmol/L MgCl2, 0.01% (vol/vol) Tween 20, 1 unit of Taq HS DNA polymerase, 200 μmol/L of each deoxynucleoside triphosphate, 100 nmol/L primer GP5+, 1 μmol/L primer GP6+, 100 nmol/L primer IPC-F, 1 μmol/L primer IPC-R, and 250 nmol/L of each of the 16 HPV and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probes, which were used to determine the adequacy of specimens. For every PCR plate, a negative control (H2O) and a positive control  were run to control for possible contamination and accuracy. The samples were amplified using an SLAN-96p real-time PCR instrument. The following amplification profile was used: 50 °C 5 min→95 °C 3 min→(95 °C 15 s→50 °C 20 s (−1 °C per cycle)→78 °C 20 s) for 10 cycles→(95 °C 15 s→56 °C 16 s→78 °C 20 s) for 50 cycles. HPV genotyping was conducted based on the specific melting temperature of the product produced by hybridization of the PCR products and fluorescent probes. The procedure was set up as follows: 95 °C 1 min→35 °C 3 min→40 °C ~85 °C (at a heating rate of 0.04 °C /s).The specimens were tested for the presence of HPV by amplifying 5 μL of the DNA extracts with the GP5+/6+ L1 consensus primer system and Taq HS polymerase . Each 25 μL amplification reaction contained 75 mmol/L Tris-HCl (pH 8.0), 20 mmol/L (NH4)15 A specimen was deemed adequate if the GAPDH or HPV genotyping was positive. The subject having at least one adequate specimen was included in the analysis. The presence of any HPV DNA in the VA, VU or PA for women or in the PGC or PA for men was defined as a positive result. The absence of any HPV DNA from the VA, VU and PA for women or in the PGC and PA for men was defined as a negative result. Subjects without adequate samples were excluded from the analysis. The classification of any HPV or oncogenic HPV was defined as a positive test result for at least one of the 16 tested HPV types or at least one of 13 oncogenic HPV types.Thirteen HPV types are labeled oncogenic types, including 12 types  that are carcinogenic to humans and HPV 68, which is probably carcinogenic to humans. In addition, HPV types 6, 11 and 66 were also detected in our study. HPV 66 is possibly carcinogenic to humans, and HPV 6 and 11 are not carcinogenic to humans.P<0.05 was considered statistically significant. The HPV prevalence in women and men was estimated for any, oncogenic and specific HPV type. The HPV prevalence was compared using Pearson's χ2-test, correction for the χ2-test, or the Fisher exact test. The 95% confidence intervals (CIs) for prevalence were calculated by the binomial exact test.The data were analyzed using SAS version 9.4 , and P-value<0.20 in the bivariate logistic analysis were included in the multivariate logistics model using stepwise selection, with a 0.05 significance level for entry into the model and 0.05 for removal.Bivariate analysis and multivariate logistic regression were conducted to assess the odds ratios (ORs) and 95% CIs, respectively, for oncogenic HPV and HPV 6/11 infection factors. Variables with a P<0.0001). Overall, the majority of the participants reported their age at first sexual intercourse was 18–25 years. At enrollment, most (56.2%) of the participants had one lifetime sex partner (LSP), 30.4% had two to three LSPs and 13.3% had four or more LSPs. Women were more conservative than men, having fewer LSPs and sexual partners in the year before the study (P<0.0001). The percent of women reporting a history of sexually transmitted diseases (STDs) was more than that of men .A total of 4687 participants, including 2378 women and 2309 men, aged 18–55 years were enrolled. After removing the participants without adequate samples, 2344 (98.6%) women and 1937 (83.9%) men were included in the analysis. P<0.001), any oncogenic type , HPV 16/18 , HPV 6/11/16/18  and HPV 6/11/16/18/31/33/45/52/58 .The overall prevalence of any HPV infection in the study population was 15.4% (95% CI: 14.4–16.6). Statistically significant differences were observed by sex for the prevalence of any tested HPV , HPV 58 (3.5%) and HPV 16 (2.2%) were the most commonly detected infections, followed by HPV 39 (2.0%) and HPV18 (1.2%). The prevalence of any other HPV type was <1.0%. For women, the three most common oncogenic HPV types were HPV 52 (6.6%), HPV 58 (3.5%) and HPV 16 (3.4%), whereas in men, HPV 58 (3.5%), HPV 52 (1.5%) and HPV 39 (0.9%) were the most common .P>0.05).To our knowledge, this is the first study to compare the prevalence of anogenital HPV and associated risk factors between sexes in the same community in China. The standardized clinical, sampling and laboratory methods allow a direct comparison of HPV DNA prevalence and risk factor data across both sexes. The data indicate that the prevalence of any oncogenic HPV type in women is higher than in men, whereas the prevalence of HPV 6/11 is comparable between the sexes. HPV infection peaks in women aged 18–25 years and then decreases slowly, whereas the prevalence remains stable among all age groups in men. In addition to sexual behavior, health habits, such as using a towel supplied by a hotel, are significantly associated with HPV infection in both women and men.16 In the current study, we assessed only 16 HPV types, including 13 high- and 3 low-risk types. For carcinogenic HPV types, the estimated prevalence is 9.4%, which is higher than in the Henan study (6.4%). The discrepancy might be explained by the different anatomic sampling sites and the different HPV typing techniques. On the other hand, the sexual behavior characteristics differ between north and south China. Most (87.6%) of the participants in Henan reported having zero to one lifetime sexual partner, whereas the proportion with zero to one partner in our study was only 45.0%. The number of lifetime sexual partners is significantly associated with increased HPV prevalence in men.19 A higher rate of any tested HPV (21.2%) was reported among men on five continents, although a similar number of HPV types was assessed.17 Among the five continents, the participants in Asia/Pacific had the lowest HPV DNA prevalence. The reported prevalence of total and high-risk HPV, respectively, was much higher in men from the United States , Mexico  and Brazil , likely because of sexual behavior—the average reported LSPs of the participating men were much higher than in the current study.9There is a paucity of studies on the prevalence of HPV in males in China. We observed a lower prevalence of HPV infection (10.5%) in men than in the only other study we are aware of, which reported the HPV prevalence of 40 types (17.8%) in males in Henan province, north China.20For the women in our study, the prevalence of oncogenic HPV infection (18.7%) was similar to the rate reported by a pooled study that included a total of 30 207 women (17.7%) in 17 studies throughout China, and the young women aged 18–25 years (15.3%) in a large phase III clinical trial in Jiangsu, China; however, these studies collected samples from the cervix, in contrast with the three anatomic  sites used in our study.11 In the current study, samples from more anatomic sites were assessed, and the data showed that the estimated prevalence of carcinogenic HPV was higher in women than in men. A potential explanation for this phenomenon might be the different physiological structures, and therefore compared with men, women may have a higher incidence rate or lower clearance rate.21 A Dutch study found that persistent infection was observed more frequently in women than in men (OR=3.88).22 Further prospective studies should be conducted to illustrate the differences in HPV natural history in both sexes.Few studies enroll both women and men from the same population; therefore, a direct comparison of the prevalence of HPV in women and men is difficult due to the methodological differences among studies. A study from South Africa reported that the prevalence of carcinogenic HPV was similar in HIV-negative men (22.4%) and HIV-negative women (22.7%), but was higher for non-carcinogenic HPV in men (33.2%) than in women (14.0%).27 In men, the three most common types were HPV 58, 52 and 39, which was different from other studies in Brazil , Mexico , the United States , Anyang, China , and on five continents .17 In both women and men, HPV 52 and 58 were the most prevalent types in our study, which further implies the necessity to vaccinate against HPV 52 and 58 in China.The five most dominant circulating high-risk HPV types were HPV 52, 58, 16, 39 and 18, which is concordant in the women and men in the current study, although the order is slightly different between the sexes. Epidemiological studies have found that type-specific HPV infection distribution is different among world regions. Our finding that HPV 52, 58 and 16 were the most frequent oncogenic HPV types in women was concordant with the findings of previous studies in China and different from an American study  and a meta-analysis of one million women on five continents .9 For women, the age trend of HPV infection differed by region.28 In general, the rate peaks in the years after sexual debut (<25 years old), is then relatively steady across age, or declines in older ages, or is characterized by a U shape with a second peak during older age . In China, Zhao et al. reported that age-stratified high-risk HPV infection in women presented a U shape, which was different from our finding of a downward trend as age increased. Whether this difference is due to different anatomic sampling sites is not clear.7In agreement with most studies, we found that age does not influence genital HPV prevalence in men.30 We also found an association of ever having used a towel supplied by a hotel with oncogenic HPV infection in both sexes, which may provide new information for the secondary prevention of oncogenic HPV infection among women and men. No other questionnaire-based information, including age at first sexual intercourse, condom use or STDs, had a significant association with HPV infection.For non-oncogenic HPV infection types 6 and 11, sexual behavior, such as LSPs and/or the number of sex partners in the past year, played a statistically significant role among men and women. This finding mirrors those observed in other studies.32 Our study did not collect exfoliated cells from the scrotum, which may have resulted in a lower detected prevalence in men. Third, we tested for only 16 HPV types, which excluded data on the prevalence of additional non-oncogenic HPV types.Limitations need to be considered for this study. First, compared with women, the lower proportion of GAPDH in men, which may be partially caused by the sampling method and the difference in genital structure, may result in an underestimate of HPV prevalence in men; however, we believe this possibility is quite low because there was no significant difference when we analyzed a subset of men who had adequate specimens in all the anatomic sites (data not shown). Second, some studies indicated that HPV detection at the scrotum (20%–34%) was higher than the perianal area (14%–20%) and anal canal (11%–17%).In conclusion, this preliminary study highlights a higher burden of oncogenic HPV infection in generally healthy women compared with men in China, whereas the prevalence of HPV 6 and 11 is similar between the sexes. The data indicate that the interaction of host and virus might be different among high- and low-risk HPV types.


High-risk human papillomaviruses (hrHPV) are responsible for anogenital and oropharyngeal cancers, which together account for at least 5% of cancers worldwide. Industrialised nations have benefitted from highly effective screening for the prevention of cervical cancer in recent decades, yet this vital intervention remains inaccessible to millions of women in low- and middle-income countries (LMICs), who bear the greatest burden of HPV disease. While there is an urgent need to increase investment in basic health infrastructure and rollout of prophylactic vaccination, there are now unprecedented opportunities to exploit recent scientific and technological advances in screening and treatment of pre-invasive hrHPV lesions and to adapt them for delivery at scale in resource-limited settings. In addition, non-surgical approaches to the treatment of cervical intraepithelial neoplasia and other hrHPV lesions are showing encouraging results in clinical trials of therapeutic vaccines and antiviral agents. Finally, the use of next-generation sequencing to characterise the vaginal microbial environment is beginning to shed light on host factors that may influence the natural history of HPV infections. In this article, we focus on recent advances in these areas and discuss their potential for impact on HPV disease. The overall burden of HPV-related disease is difficult to estimate, but it is believed that approximately 600,000 annual cases of cervical, anal, penile, vulvar, and vaginal cancers combined are attributable to hrHPV4 (Human papillomavirus (HPV) is the most common viral infection of the reproductive tract. Even though most HPV infections are asymptomatic and clear spontaneously, persistent infections with “high-risk” (oncogenic) mucosal HPV (hrHPV) cause approximately 5% of all cancers worldwide. These include almost all cases of cervical cancer––with annually over 500,000 newly diagnosed cases and over 260,000 cervical cancer deaths worldwide––as well as a large proportion of other anogenital carcinomas and oropharyngeal tumours in both women and menhrHPV4 .5. An additional concern is that the HIV epidemic has disproportionately affected the same regions and is an important contributing factor to the prevalence and persistence of HPV infections6. Closing the gap between wealthy and low-income countries in prophylactic HPV vaccine rollout is an international priority. However, even if this were to be achieved in the near future, hundreds of millions of women who have already been exposed to hrHPV will remain at risk of developing cervical cancer without access to affordable tools for screening and treatment of pre-invasive disease. In this article, we review innovations in testing and treatment for hrHPV infection and intraepithelial neoplasias from molecular, clinical, and operational perspectives alongside new insights into pathogenesis  of the global burden occurs in the less-developed regions with little or no access to screening or prophylactic vaccine programmes7. HrHPV-16 and -18 DNA prevalence was consistently lower than antibody prevalence; however, rates varied widely according to age. In a study assessing the impact of prophylactic vaccination in England, prevalence of hrHPV DNA (any of 13 types) in women aged 16–24 during 2012–2013 ranged from 35–44%8. Although rare, the presence of antibodies against hrHPV in prepubertal children may suggest non-sexual  transmission routes10. HPV-16 and -18 cause nearly 70% of cervical cancers. HPV-16 also contributes largely to hrHPV-associated vulvar, vaginal, and penile pre-cancer11.HPV prevalence varies largely by geographic region, age, gender, and sexual behaviour. HPV-16 antibody prevalence in females aged 9–26 ranged from 0–33% in Europe and North America to 13–43% in Africa and Central and South America12. However, in the absence of hrHPV testing, high false-positive detection rates can lead to unnecessary referrals to colposcopy. This triggered the recent use of hrHPV DNA testing to triage women with abnormal cytology at screening. Although this is standard of preventive care in high-income countries, access to screening in LMICs is still limited or unavailable. Population-based models have calculated a projected reduction in cervical cancer incidence of 50–60% by 2040 if effective screening and/or treatment methods were to be implemented13. Fortunately, the growing importance of reducing the burden of HPV-induced cancers has been recently acknowledged by the World Health Organisation and led to the listing of screening and treatment of precancerous lesions to prevent cervical cancer as a “best buy” intervention14.For the past five decades, the introduction of screening with the Papanicolaou (Pap) smear test to detect cervical disease early and the implementation of national screening programs have reduced the incidence and mortality of a disease that once caused more deaths amongst women than any other cancer. Liquid-based cytology has widely replaced the conventional Pap testing in recent years, owing to its greater effectiveness, i.e. comparable sensitivity coupled with reduced risk of inadequate sampling15. Large randomised controlled trials have shown that hrHPV testing, when implemented over several rounds of screening, provided greater protection against CIN2+ or invasive cervical cancer than did cytological screening. Of note, protection was greater with hrHPV screening in women at 30+ years of age and with a screening interval of 5 years than cytological screening every 3 years19. Even a single round of HPV testing can have a significant impact on the rate of advanced cervical cancers and cancer deaths in rural India20. Currently, the implementation of molecular testing in LMICs is limited because of cost and infrastructure requirements15. However, this might be addressed by using rapid low-cost point-of-care tests such as Xpert HPV, which has shown promise in a field trial in Papua New Guinea21.HrHPV testing detects over 90% of all high-grade cervical intraepithelial neoplasia (CIN) and is therefore an attractive screening tool, especially in countries with established screening infrastructure. Several countries are due to implement primary hrHPV testing with triage by cytology or, in some settings, partial genotyping22. However, the effectiveness of screening by self-sampling is determined in part by the performance of the HPV PCR test when applied to self-samples23. To bridge the prevention gap in low-/middle- income settings, approaches such as hrHPV self-testing are viable and enable invitation of communities who test positive to attend further triaging25. Visual inspection of the cervix with acetic acid (VIA) or Lugol’s iodine (VILI) is a particularly simple and low-cost screening tool that enables trained healthcare workers to detect abnormalities with a speculum examination. Both methods are especially attractive tools for establishing screening in LMICs, where the infrastructure to provide screening at a national level is not yet in place despite increasing awareness of its importance28. HPV urine testing is a non-invasive alternative to conventional cytology sampling that could improve screening uptake in individuals for whom cervical or vaginal sampling may be difficult. A large meta-analysis confirmed that detection HPV test results in urine generally correlate with HPV testing in clinician-taken samples30. However, data to support urinary HPV testing for the detection of CIN are currently lacking.HPV self-testing to collect cervical/vaginal cells offers the potential to improve screening coverage: meta-analyses have concluded that self-testing increased the participation of non-attenders in screening programmes when sampling kits were provided directly to all participants31. Whether a productive life cycle is completed or not depends on the infected epithelial site and the hormonal environment32. Nevertheless, hrHPV can persist––often for many years––and can drive cell cycle entry and cell proliferation in the basal and parabasal cell layers33, which distinguishes them from low-risk HPVs. Progression to cancer is a consequence of persistent hrHPV infection that, following integration of the viral genome into host DNA, drives dysregulated viral gene expression. The hrHPV early proteins E6 and E7 inactivate the host tumour suppressors p53 and retinoblastoma protein, resulting in neoplastic transformation. The distinct susceptibility of the transformation zone to neoplastic transformation and progression may also be linked to the increased accessibility and proliferation of the basal cell layers at this metaplastic epithelium33.HrHPV genotypes are maintained in the general population owing to productive infections rather than inadvertent cancers. Low-grade lesions (CIN1), where infectious particles are produced and shed, tend to regress spontaneously within 18–36 months in immunocompetent hosts36 and low viral load, or a >100-fold decline over time, is associated with clearance39. Higher viral load was also associated with decreased HPV clearance rates in uncircumcised males and homosexual men who smoke41. Methylation of CpG sites in HPV DNA is a marker of persistent infection, particularly for HPV-16, that may have utility in triage: in a large randomised controlled trial, methylation triage on cervicovaginal self samples was non-inferior to cytology triage on clinician-taken samples for the detection of high-grade CIN42.Recently, the predictive value of HPV viral load as a measure of persistence has been investigated: several studies have shown that high baseline HPV-16 and -18 viral load or viral load increase over time are associated with persistent infections43. Recently, more conservative approaches in the management of moderate (CIN2) lesions have been adopted in the light of large population-based data indicating high regression rates in young, non-smoking, immunocompetent women in whom minimising the risk of future adverse pregnancy outcomes (second-trimester miscarriage and spontaneous pre-term delivery) is a priority47.Precancerous cervical lesions can be treated by loop excision of the transformation zone or ablative techniques such as cryotherapy or laser therapy. The use of novel, cheap ablation techniques that do not require external gas supply (such as CryoPen) could provide affordable treatment of precancerous lesions and prevent progression to cancer in LMICs49. Multiple small molecule inhibitors targeting hrHPV DNA-binding activities or the apoptotic sequences of E6/E7 or exhibiting synthetic lethal interactions are still in preclinical development50. Adoptive T cell therapy comprising infusion of E6- and E7-specific tumour-infiltrating T lymphocytes to facilitate tumour shrinkage is a novel approach to the treatment of metastatic cervical cancer that has shown promise in an exploratory clinical study51. However, such individualised therapies are challenging to deliver at scale.A non-surgical therapy for pre-invasive hrHPV lesions is highly desirable given the disadvantages outlined above and the infrastructure required to deliver and monitor the efficacy of ablative treatment, which is a major challenge for LMICs. In addition, there is a need for better therapeutic options for hrHPV-driven lesions at anogenital sites other than the cervix. Tackling chronic and recurrent HPV-induced lesions and the possibility to scale up patient-applied use in LMICs have led to the development of the topical immune response modulators such as imiquimod and Yallaferon®, a recombinant interferon alpha-2b gel, but recurrence rates remain high. Lopinavir and cidofovir, both antiviral drugs, are currently being trialled in precancerous lesions as well as HPV-associated cancers of varying sites54. Regression of CIN is associated with infiltration of the lesion by CD8+ T cells55. CD4+ T cells play a crucial role in orchestrating this effector response: immunosuppressive states  are associated with reduced HPV clearance rates and increased risk of progression to invasive cancer59. T cells recognise viral antigens in the form of peptides that are generated from proteolytically cleaved internal and external proteins and presented on the cell surface in association with HLA class I (CD8+ T cells) and class II (CD4+ T cells) molecules. It is hypothesised that boosting naturally induced hrHPV-specific T cell responses to early viral antigens by vaccination should accelerate the regression of CIN and clearance of infection. Licensed prophylactic vaccines prevent the acquisition of infection through the induction of antibodies to late (capsid) proteins presented in the form of virus-like particles. They have no impact on disease once infection has occurred; therefore, a different vaccine strategy is required to achieve a therapeutic effect. HPV  presents non-self antigens; the development of T cell tolerance is therefore far less likely than is the case for cancers expressing self-antigens. The oncoproteins E6 and E7 have been the antigens of choice for most therapeutic vaccine candidates to date, as they are expressed throughout the virus life cycle and on transformed cells. The development of therapeutic vaccines for hrHPV has encompassed proteins and peptides, viral, bacterial, and DNA vectors, RNA replicons, and dendritic cell-based approaches. As there have been several recent comprehensive reviews61, we focus on the most clinically advanced strategies.Several aspects of hrHPV biology and pathogenesis make it an attractive candidate for targeting with a therapeutic vaccine. The development of a high-grade cervical lesion is the result of failure of host T cell responses to control or indeed clear hrHPV infection, an uncommon event that typically takes 10–15 years and is reversible in up to 30% of cases62. This was an important finding given the very low rate of spontaneous regression of vulval intraepithelial neoplasia and high recurrence rate. A randomised placebo-controlled phase II trial was subsequently initiated to assess the same vaccine strategy in women with HPV-16-positive high-grade CIN. Unfortunately, the study was terminated prematurely because of the reluctance of enrolled patients to defer excisional treatment; therefore, the results were inconclusive63. A recombinant protein vaccine, GTL001, comprising HPV-16 and -18 E7 fused to catalytically inactiveBordetella pertussis adenylate cyclase, was tested alone and with imiquimod in a phase I study of women with HPV-16 or HPV-18 and normal cytology64. Sustained viral clearance was observed in the majority of women receiving the higher vaccine dose together with imiquimod.Peptide- and protein-based vaccines offer safety, stability, and ease of manufacture. However, they are poorly immunogenic unless administered with adjuvants, which may cause unwanted reactogenicity, and efforts to apply this approach to immunotherapy of hrHPV have generally been unsuccessful. A mix of adjuvanted synthetic long peptides from HPV-16 E6 and E7 was found to induce complete regression in 9 out of 19 women with high-grade vulval intraepithelial neoplasia65. In the modified intention-to-treat analysis, histopathological regression to CIN1 or normal was significantly more frequent in VGX-3100 recipients than in placebos (48.2% vs. 30%). In a post-hoc analysis, the magnitude of E6-specific T cell responses was associated with regression of CIN and viral clearance65. A similar DNA vaccine expressing E6/E7, GX-188E, was tested in a small uncontrolled study: complete regression and virus clearance was observed in seven out of nine women with CIN3, in association with polyfunctional CD8+ T cell responses66.DNA vaccines are safe and easy to manufacture, do not require a cold chain, and can be administered repeatedly, as they do not elicit vector-specific immunity. However, despite eliciting potent immune responses in small animal models, immunogenicity in humans has been disappointing. Electroporator delivery and adjuvants can overcome this, albeit at the cost of increasing complexity and reactogenicity. The leading candidate is the VGX-3100 vaccine , which comprises synthetic consensus HPV-16 and HPV-18 E6 and E7 genes. It has shown the most encouraging results of any therapeutic HPV vaccine candidate to date in a randomised controlled phase IIb trial in 154 women with CIN2/367. The first trial of a replication-competent vaccinia virus vectored HPV vaccine, TA-HPV, was conducted 20 years ago68. A modified vaccinia Ankara (MVA) vectored vaccine encoding E2, a transcriptional regulator of E6 and E7, has been tested in a phase III trial that included 1,176 female subjects with HPV-driven oncogenic and non-oncogenic intraepithelial lesions and 180 males with condylomata only69. The vaccine was delivered directly into the affected anogenital tissues. The investigators reported elimination rates of 89% in females and 100% in males. When considering only the participants with high-grade lesions , the cure rate was 73%69. Although this is higher than the expected spontaneous regression rate, the results should be interpreted with caution, as a control group was lacking.Viral vector vaccines elicit potent cell-mediated immune responses owing to their capacity for high levels of transgene expression and natural adjuvant properties. Heterologous prime-boost viral vector regimens targeting diverse human pathogens have been shown to induce high frequencies of T cells in clinical trials while avoiding vector-specific immunity71. Vaginal dysbiosis , a state characterised by paucity or absence of lactobacilli, overgrowth of anaerobes, and high pH, has been implicated in the acquisition and outcome of HPV infections. A systematic review and meta-analysis of 19 studies confirmed an association between BV and CIN72. However, causality remains uncertain, since the majority of studies were cross-sectional. A reduction in Lactobacilli, increasing vaginal microbial diversity, and dominance of Sneathia species were observed in association with progression of cervical histology from normal through to high-grade CIN74. In a longitudinal study in which women were sampled twice weekly for 16 weeks, community state types  were observed to influence both the incidence and the clearance rates of prevalent HPV infection75. A low lactobacillus CST was associated with a nearly two-fold higher rate ratio of incident HPV, and HPV clearance was fastest in women withLactobacillus gasseri-dominated CSTs.HrHPV infections are highly prevalent in sexually active women, yet only a small minority persist and progress to cancer. Reported risk factors for carcinogenesis include smoking, other sexually transmitted pathogens, oral contraceptive use, and socio-economic status. However, few studies have established which of these associations have a mechanistic basis and which are confounders reflecting high-risk sexual behaviour. Recent studies using next-generation sequencing to characterise the microbial communities inhabiting the vagina and cervix  have suggested a possible role of altered vaginal microbiota in the development of CIN and cervical cancer. The cervico-vaginal microbiome is typically of low diversity and dominated by Lactobacillus species, which are assumed to protect against pathogens by maintaining a low pH76. Metatranscriptomics approaches can yield more precise information regarding function and thus identify potential targets, such as the metabolically active bacteria in dysbiotic states77. A possible interaction between cervical microbiota diversity, histopathological diagnosis of cervical HPV lesion, and cervical cytokine expression was explored in a recent cross-sectional study of women across a spectrum of HPV disease. Expression of IL-4, IL-10, and TGF-β was increased in women with cervical cancer compared with those with low-grade CIN74. It was hypothesised that hrHPV E2, E6, and E7 drive the production of immunosuppressive cytokines, which, in turn, increase cervical microbial diversity and proliferation of Sneathia and Fusobacterium species. Collectively, these studies highlight the need for more longitudinal data and for a combined metagenomics/metatranscriptomics approach in order to better understand the complex interplay between the cervico-vaginal microbiota, sexually transmitted pathogens, and host immune system in determining the outcome of hrHPV infections.To date, the microbial composition in the vagina and cervix of healthy women has been studied largely through parallel sequencing of the highly conserved 16s ribosomal RNA subunit genes, which indicates the abundance and diversity of bacteria but does not provide information on the functional activity of the human vaginal microbiota or the diversity of other microbes present. Direct DNA sequencing (metagenomics) has revealed not only a very high prevalence of HPV in healthy women but also a far greater diversity of HPV types than can be detected by conventional typing80. The use of a low-cost ultra-portable tampon-based digital colposcope has been proposed as a novel approach to increase access to screening at the community level and also as a tool for virtual training81. Collectively, these efforts could have significant health and socio-economic benefits.Implementation of effective cervical screening is a major medical advance of the late twentieth century, and it will remain an essential tool for cancer prevention for several decades while prophylactic HPV vaccination programmes take effect. The transition from cytology to hrHPV detection as a screening tool has the potential to improve the effectiveness of screening at longer intervals and to increase coverage by enabling self-testing in the community or even at home. Stratification according to age and HR genotype, with fast-tracking of HPV-16-positive cases to colposcopy and treatment could reduce unnecessary referrals and healthcare costs in high-income countries and also facilitate the allocation of limited resources in LMICs. The opportunities available to LMICs through technological innovations in screening and preventive treatment will be missed unless innovative approaches to implementation are adopted. These include “screen and treat” protocols that embed screening by VIA or HPV tests in existing primary healthcare systems, scaling up training of healthcare workers to enable provision of treatment in a greater range of settings than specialised cancer services, and use of smartphone technology for tracking, recalls, and staff trainingAlthough the development of specific antiviral agents is slow, alternatives to surgical excision or ablation of CIN such as therapeutic DNA vaccines are on the horizon. With the application of potent viral vector vaccine technologies, we anticipate improvements in the clinical efficacy of these approaches. As our understanding of the microbial and mucosal immune states that lead to persistent hrHPV infection grows, we may identify new agents that can be used to manipulate the local microenvironment for therapeutic benefit.


However, screening with human papillomavirus (HPV) molecular tests holds promise for reducing cervical cancer incidence and mortality in low- and middle-income countries. The performance of the Abbott RealTiThe AbRT assay detected at least one HPV genotype in 44.57% of women regardless of the grade of cervical abnormalities. Except for one case, good concordance was observed for the genotypes detected with the AbRT assay in the high-risk HPV category determined with HTS of the amplicon generated by conventional nested PCR.The AbRT test is an easy and reliable molecular tool and was as sensitive as conventional nested PCR in cervical smear specimens for detection HPVs associated with high-grade lesions. Moreover, sequencing amplicons using an HTS approach effectively identified the genotype of the hrHPV identified with the AbRT test. Infections with human papillomavirus (HPV) are the most common sexually transmitted infections worldwide . PersistBased on the clear causal link between hrHPVs and cervical cancer, the guidelines of the American Society for Colposcopy and Cervical Pathology recommend HPV DNA testing instead of cytology to carry out triage in a screened population. Molecular tests for HPV detection may be a good alternative to cytology for early screening because an HPV-negative result is sufficient to declare an extremely low risk of developing CIN3 or worse (CIN3+) for 5 years in contrast to a negative cytology result . Other rme High-Risk HPV (AbRT) assay for the detection of HPV in cervical cell samples of women with or without cervical abnormalities. The performance of the test was evaluated in regard to the detection of two main HPV genotypes, HPV16 and 18, and a panel of 12 hrHPVs, all constituting one single hrHPV category. HPV genotypes identified after sequencing amplicons generated by conventional nested PCR allowed us to determine the non-HPV16/non-HPV18 genotypes detected by the AbRT assay.This study was based on the use of the Abbott RealTin = 50), atypical squamous cells of undetermined significance , low-grade squamous intraepithelial lesions , high-grade squamous intraepithelial lesions  and carcinoma . Women infected by a low-risk HPV genotype — which cannot be detected by the AbRT assay — were excluded.Participating women were selected from a 960 patient cohort previously constituted from March 2013 to January 2014 during a cervical lesion screening program as part of a multi-center cross-sectional study carried out in two hospitals located in Libreville, Gabon . A totalDNA was extracted from exfoliated cells obtained from liquid-based cytology (LBC) using the DNeasy Blood and Tissue kit  according to the manufacturer’s instructions. DNA was quantified using the Qubit® dsDNA BR Assay kit with the Qubit 2.0 fluorimeter  primarily to confirm the previous DNA purification step.m2000rt automated analyzer according to the manufacturer’s instructions. Detected HPVs were validated by genotyping a 150 bp fragment of the L1 HPV gene and discriminating between 14 hrHPVs: HPV16, HPV18 and a pool of 12 other hrHPVs . PCR amplification included 5 μL of the extracted DNA sample along with the GP5+/6+ primers designed for use with a high-throughput sequencing approach (HTS): HTS-GP5+: 5′ tcg-tcg-gca-gcg-tca-gat-gtg-tat-aag-aga-cag-TTG-TTA-CTG-TGG-TAG-ATA-CTA-C 3′; HTS-GP6+: 5′ gtc-tcg-tgg-gct-cgg-aga-tgt-gta-taa-gag-aca-gGA-AAA-ATA-AAC-TGT-AAA-TCA-TAT-TC 3′) and corresponding HPV probes [The AbRT assay  was performed with the V probes   and theStatistical analyses were performed using R software version 3.4.0. Cohen’s Kappa value, a measure of agreement, was used to evaluate the concordance between the two HPV detection methods used here. The Kappa statistic varies from 0 to 1, with unity indicating perfect agreement.p value˂0.001; odds ratio, OR = 8.89) and increased with the severity of the cervical lesions and the age of the patient (p < 0.05). Although 10.81% (4/37) of the women were infected by at least two different categories of HPV (16 and/or 18 and/or other hrHPVs), HPV16 was the most frequently detected genotype, followed by other hrHPVs and HPV18 with detection rates of 52.63%, 39.47% and 21.05%, respectively.The AbRT assay detected at least one HPV genotype in 44.57% of women (37/83). The mean age of the women who were positive for HPV DNA was 50 years. HPVs (genotypes 16 and/or 18 and/or other targeted hrHPVs) were identified in 26% (13/50) and 72.72% (24/33) of cases among women presenting normal and abnormal cytology, respectively. Moreover, the association between abnormal cytology and HPV infection was significant  among 100% (14/14) of the HSIL+ cases tested  and 43.5% (30/69) of cases by the AbRT assay and the nested PCR method, respectively Table . HPV18 wGenotypes detected by the AbRT assay in the hrHPV category were compared with the results obtained from amplicons generated by the nested PCR method. The hrHPV category was detected in women presenting cytological abnormalities, with 14.28% (1/7), in 28.57% (2/7) and in 57.14% (4/7) of cases in carcinoma, HSIL and LSIL, respectively. Furthermore, the hrHPV category was also detected in 43.8% (7/10) of cases for women with normal cytology. Except for case no. 9, all HPV33 and HPV45 genotypes identified by PCR were also detected by the AbRT assay  were identified by both methods. Two cases (no. 21 and 22) were not identified by nested PCR, whereas for case no. 20, the AbRT assay failed to detect HPV33, which was identified by nested PCR. Among samples with normal cytology, at least one hrHPV genotype was detected in 94.7% (18/19) of cases by nested PCR  can detect a total of 14 different high-risk HPV genotypes whereas the conventional nested PCR that uses the primers MY09/11 and GP5+/6+ can amplify around 30 different low- and high-risk HPV genotypes [m200sp, Abbott Molecular) with this assay for HPV detection, extractions were performed manually as described in literature as a practicable alternative. In a recent study, Kocjan et al. (2012) showed that similar results are obtained for HPV detection from biological tissues of head-neck squamous cell cancers regardless of the extraction method used with the AbRT assay [This study describes the first use of the Abbott RealTienotypes , 14. TheRT assay , 16. MorIn this study, the AbRT generally gave consistent genotyping results for the hrHPV screened in HSIL+ cases in comparison with the conventional nested PCR. These data are similar to other studies that compared the AbRT kit with this nested PCR method or with other molecular methods of HPV genotyping , 16. HowSeveral studies have shown that the sensitivity of the AbRT assay is similar to that of the hc2 test and that the specificity of detection of HPVs associated with the development and/or spread of CIN2+ can reach 92% , 24, 25.This study on the performance of the AbRT assay shows that it is an easy and reliable molecular tool for HPV detection in HSIL+ LBC samples. However, as expected, the AbRT assay is less sensitive than conventional nested PCR for the detection of HPV in HSIL-. Moreover, the sequencing of amplicons using a HTS approach effectively identified the genotype of hrHPV detected with the AbRT assay. Finally, our data showed that the use of AbRT in co-testing with VIA/VILI in low-income countries holds promise as an interesting alternative to other combinations, such as VIA and cytology.