Despite being preventable, cervical cancer remains a major health concern among women. Persistent Human Papillomavirus (HPV) and other viral co-infections may influence cervical dysplasia. We determined and compared the prevalence and risk factors of cervical viral infections among the tribal and general population of southern coastal Karnataka, India.A population-based cross-sectional survey was conducted among 1140 and 1100 women from tribal and general population, respectively. Cervical infections with HPV, Epstein-Barr Virus (EBV), Cytomegalovirus (CMV) and Herpes-Simplex Virus (HSV) were examined using polymerase chain reactions (PCR) and DNA sequencing.HPV prevalence was higher among tribal women (40.6%) than general population (14.3%) while the prevalence of EBV (55.1%) and CMV (49.4%) were lower among tribal women than general population . HSV infection was observed in tribal women only (1.8%). Among HR-HPV strains, HPV-18 was predominant among tribal population (28.3%) while, HPV-16 was predominant among the general population (9.1%). Infections were associated with age, educational status, unemployment and personal hygiene of tribal women. Phylogenetic analysis revealed that HPV-16 variants of tribal participants were closely related to non-European sublineages indicating greater risk of HPV persistence and carcinogenesis.The study provides a comparative estimate for DNA virus infections of the cervix among women from general as well as tribal population in this region and also reveals a different type-specific pattern of viral infection. Further research is required to delineate the role of specific interactions between multiple virus infections and their role in carcinogenesis. Cervical cancer is the leading cancer among women aged 15–44 years, especially in the developing countries, with human papillomavirus (HPV) infection being the key etiological factor . In spitThe ethnic tribes form an integral part of India’s social diversity constituting 8.6% of the total population . High prPermission was obtained from the State Tribal Welfare Department (STWD), Karnataka, Integrated Tribal Development Project (ITDP) and District Health Authority to carry out the study in the district. The study proposal was approved by the Institutional Ethics Committee, Kasturba Hospital, Kasturba Medical College, Manipal, prior to initiation of the study . Married women aged 20–65 years were included after obtaining a written consent from the eligible participants willing to take part in the study.A population-based cross-sectional survey was carried out among women from the tribal communities  Act, 1976 and as inserted by Act 39 of 1991) and from the general population of Udupi district situated in south-coastal Karnataka, of southern India. About 41,613 tribal people inhabit Udupi district . Among the three communities, Koraga is primitive and original inhabitants of coastal Karnataka. Traditional occupation of this community is basket weaving and rope making with overall low socio-economic status as illiteracy, unemployment and alcohol and tobacco use prevail in these communities.Considering the HPV prevalence to be 16.9% among rural women reported earlier [Socio-demographic, gynaecologic and reproductive data were collected using pre-designed questionnaire, after obtaining written informed consent. Socio-economic status was assessed using modified Udai Pareek scale . ParticiOn the day of camp, exfoliated cervical samples were obtained by a trained medical doctor /a trained nurse with the help of a cytobrush and transported to the laboratory in sterile 15ml tubes containing Phosphate Buffered Saline (PBS) solution on ice. Samples were centrifuged and pellets were stored at -20°C till further processing.The exfoliated cervical cells were digested with RNaseA (10mg/ml) and proteinase K (10mg/ml), treated with 1M Tris (pH 7.4), 0.5M EDTA and 20% SDS and incubated overnight at 37°C. DNA was extracted by standard phenol-chloroform method followed by precipitation with ethanol, dissolved in sterile Milli-Q water and stored at -20°C until further use.Viral type-specific PCRs were performed to detect the presence of HPV, EBV, CMV and HSV, as described in the literature –14. HPV Data was analysed using Statistical Package for Social Sciences (SPSS) version 16.0. Categorical data was summarized as mean ±standard deviation (SD). Univariable and multivariable logistic regressions were performed to estimate the crude and adjusted odds ratios (OR) with corresponding 95% confidence intervals (CI). The factors showing an association with viral infections at a p <0.2 on univariable analysis were included in multivariable analysis.Software R and online tool HighCharts were used for construction of clusters, heatmaps and graphs.DNA sequences of HPV-16 L1 locus from our results were used for construction of phylogenetic tree by maximum parsimony approach using MEGA package 7.0 with 1000 bootstrap replicates. The sequences were aligned against references collected from GenBank which included NC_001526.3 and 10 sublineages of HPV-16 viz., A1 (K02718.1), A2 (AF536179.1), A3 (HQ 644236.1), A4 (AF534061.1), B1 (AF536180.1), B2 (HQ64429.1), C (AF472509.1), D1 (HQ644257.1), D2 (HQ644270.1) and D3 (AF402678.1).A total of 1140 tribal women and 1100 from general population were screened through 80 camps . DemograA distinct pattern of viral infections was observed in the cervical samples of both populations. The prevalence of overall HPV infection was 40.6% and 14.3% among tribal women and general population, respectively, i.e. higher among the tribal women . In contrast, prevalence of EBV (55.1% vs. 74.2%) and CMV (49.5% vs. 77.5%) infections were lower among tribal population . Prevalence of HSV infection among tribal women was 1.8% and absent in women from general population. Infections with at least one virus was observed in approximately 82% of the tribal women and 91.5% of the women from the general population. Interestingly, infection with HPV, independent of viral co-infection, was detected in only 8.8% and 0.5% of tribal and general population, respectively.Among HPV negative tribal women, the prevalence of EBV, CMV and HSV infection was 42.4%, 56.0% and 1.9%, respectively, whereas the prevalence of EBV and CMV infections in HPV negative women from general population, was 74.3% and 76.1%, respectively. The prevalence of these viral infections was compared among HPV positive and HPV negative women from both populations . PrevaleA total of 14 high-risk (HR) and 15 low-risk (LR) HPV subtypes were detected from both the groups . InteresInfection with single HPV subtype was observed in 14.4% and 5.1% while, infections with multiple HPV subtypes was observed in 24.7% and 8.6% of tribal and general population, respectively. The prevalent single HPV infection among tribal women was HPV-18 (8.1%), followed by HPV-16 (4.9%) and HPV-6 (<1%) while that among general population was HPV-16 (4.1%) followed by HPV-18 (<1%).Among the HPV positive women, about 60% were infected with multiple HPV subtypes in both populations. Pattern of infections with multiple HPV subtypes was evaluated among tribal and general population  and 31–45 years , having <5 years of education , positive history of smokeless tobacco consumption , and using home-made sanitary napkins .Similarly, associations of variables and presence of EBV and CMV infections in all the women  and suchMaximum parsimony trees of HPV-16 L1 were constructed from molecular phylogenetic analysis of 11 reference sequences aligned with 113 tribal and 99 general population sample variants with 1000 bootstrap replicates to test the robustness of the tree . PhylogeIn this study, higher prevalence of HPV (40.6% vs. 14.3%) and HSV (1.8% vs. none) and lower prevalence of EBV (55.1% vs. 74.2%) and CMV (49.5% vs. 77.5%) were observed in tribal population than general population. Prevalence of HPV among general population was in concordance with that observed in asymptomatic women from British Columbia (12.3%), Iran (13%), Taiwan (13.8%) and rural Mexico (14.7%) –18. StudEBV and CMV infections were lower among tribal women than women from general population. A few studies have suggested the prevalence of EBV and CMV in the cervical secretions from healthy women and it ranges from 10–30% . This isHigh prevalence of cervical viral co-infections was observed among tribal (49.8%) and general 65.3%) population with EBV and CMV co-infection being predominant, irrespective of HPV status. Studies have reported co-infection of HPV and EBV in non-malignant and malignant cervical tissues and its significant association with cervical carcinogenesis . EBV has.3% populCervical HPV infections are reported to be acquired around adolescence, peak at middle-age and declining after 45 years, across the globe . In concHPV-16 L1 variants exhibit diverse geographical distribution and pathogenicity. The non-European sublineages, especially Asian (A4) sublineage, has been reported to be involved with persistent HPV infection, development of cervical lesions and progression towards high grade squamous intraepithelial lesions (HSIL) ,51. In oThis community-based study is first of its kind in this region assessing prevalence of cervical HPV and other DNA viral infections, co-infections and their risk factors among tribal and general populations. Higher prevalence of HPV and other viral single and co-infections was observed in both the populations. Study also highlights some of the socio-demographic and reproductive characteristics as potential risk factors of cervical HR-HPV and multiple viral infections among tribal women. The prevalent infections with herpesviruses, including EBV and CMV among non-malignant samples irrespective of HPV infection status may suggest a synergistic infection. We, therefore, recommend molecular testing of DNA viruses together with Pap smear tests for identification of latent viral infections at early stages and follow-up cohort studies to identify their role in causing cervical abnormalities. This study provides baseline data on HPV and mixed viral infections among the women from two socio-culturally diverse groups. Longitudinal studies to explore the role of multiple virus infections in cervical carcinogenesis is the need of the hour as it would help in policy making and introduction of population-specific interventions against HR-HPV and multiple viral infections.S1 FigComparison of prevalence of EBV, CMV and HSV infections among HPV positive and HPV negative women from tribal and general population . *: p-Value <0.05; **: p-Value <0.01; ***: p-value <0.001.(TIF)Click here for additional data file.S2 FigPattern of co-infection of different viruses among the samples infected with more than one virus, i.e. samples with multiple viral infections .(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Among different types of human papillomavirus (HPV), types 16 and 18 were known to be high-risk agents causing mainly cervical cancer. Up to now, the potential of HPV E7 protein has been proved as a diagnostic marker of cervical cancer. Moreover, the levels of anti-heat shock protein (Hsp) and anti-high mobility group box-1 (HMGB1) antibodies in cancer patients have been useful in tumor diagnosis. The goal of the present study was to determine the efficiency of the potential serologic markers including HPV E7, Hsp20, Hsp27 proteins and Hp91 peptide in Iranian HPV-exposed women, for the first time.E. coli system, and purified by affinity chromatography under native conditions. Then, antibody responses were detected against the recombinant proteins as well as Hp91 peptide as potential markers in 49 Iranian women who were seropositive for HPV-16 and 18 L1 capsids  and 49 controls using indirect ELISA.At first, the recombinant HPV E7, Hsp20 and Hsp27 proteins were expressed in p < 0.05 for HPV16 or HPV18; p < 0.01 for both of them versus all markers). HPV-exposed women with high antibody responses to HPV-16 and 18 L1 capsids as a commercial biomarker had significant seroreactivity to HPV-16 and 18 E7 and Hsp27 (p < 0.05). The recombinant E7 and Hsp27 proteins showed higher efficiency than Hsp20 and Hp91 for detection of individuals exposed to HPV infections (p < 0.05).Our data indicated that the seroreactivities of women exposed to HPV16, HPV18 and both of them against the recombinant E7, Hsp20, Hsp27 proteins and Hp91 peptide were significantly higher than those in control group  contains supplementary material, which is available to authorized users. The studies indicated that most high-risk HPV infections in young subjects are transient and have little long-term significance. Only a small proportion of infected individuals (~ 5–10%) fail to clear HPV and suffer from persistent infections. Thus, serological assays are important for detecting anti-HPV antibodies and determining the subjects with previous exposure to HPV, who may be protected against subsequent HPV infection. In the current study, we showed that HPV E7 and Hsp27 can be considered as promising biomarkers similar to HPV L1 for determination of HPV-16 and 18-exposed women.Human papillomavirus (HPV) infection has been recognized as the main etiologic factor in the development of different cancers . The HPV, Hsp27 and Hsp20) [As known, heat shock proteins (HSPs) are present in all prokaryotic and eukaryotic organisms. These proteins are divided into various groups based on their molecular weight such as Hsp60, Hsp70, Hsp90, Hsp110 and small Hsps e.g.d Hsp20) . Heat sh . Heat sd Hsp20) . Among hd Hsp20) , 23. Ford Hsp20) . A studyd Hsp20) . Anotherd Hsp20) . These rOn the other hand, the high-mobility group box-1 (HMGB1) protein known as amphoterin, is a highly conserved, non-histone nuclear protein expressed in almost all eukaryotic cells. Recent clinical studies have shown that HMGB1 is a potential diagnostic or prognostic biomarker in a variety of inflammatory disorders and cancers , 28. TheEscherichia coli (E. coli) as a His-tag protein and purified using affinity chromatography. E. coli is the most popular expression system for production of the recombinant proteins. This system has some advantages compared to other systems such as low cost, high yield, easy purification, a large number of expression plasmids and strains, and many cultivation strategies [In this study, at first, the recombinant (r) HPV E7, Hsp20 and Hsp27 proteins were expressed in rategies .After production of the recombinant proteins, the seroreactivities of Iranian women who were seropositive for HPV-16 and 18 L1 capsids as mono- and co-infection  were evaluated against the rE7, rHsp20, rHsp27 proteins as well as Hp91 peptide as diagnostic markers. Indeed, we compared the efficiency of HPV-16 and 18 L1 capsids with HPV-16 and 18 E7, Hsp27, Hsp20 and Hp91 for detection of anti-HPV IgG antibodies using indirect ELISA in Iranian women exposed to HPV infections not diagnosed with cervical cancer., E7) along with a non-specific marker  possess high efficiency for diagnosis of HPV-exposed women. On the other hand, this study confirms whether Iranian women who were seropositive for HPV-16 and 18 L1 capsids are really HPV-infected women at present. However, it will be required to design new kits with suitable efficacy and low cost for serological tests.Briefly, our goal was to determine novel and effective markers instead of L1 capsid-based commercial kit for diagnosis of HPV-exposed subjects. We used the samples recognized by L1 kits whether a specific HPV marker  and one commercial standard sera  were enrolled as control group for evaluation of anti-HPV antibody in sera against the E7, Hsp20, Hsp27 proteins and Hp91 peptide  who were seropositive for HPV-16 , HPV-18  and both HPV-16 and 18  L1 capsids as observed in our previous study , the NheI/ SalI cloning site. Next, the E. coli Rosetta strains were transformed with the recombinant pET-Hsp27 plasmid and the clones were selected on LB-agar plate. These bacterial clones were grown to an optical density of 0.6–0.7 at 600 nm in Ty2x medium and protein expression was induced by 1 mM IPTG. The cell pellets were harvested at 4 h after induction and analyzed by 12% SDS-PAGE. The recombinant Hsp27 protein was purified by affinity chromatography under native conditions using imidazole buffer according to the manufacturer’s instructions (Qiagen), and identified by Western Blot analysis using anti-His tag antibody. Then, the purified protein was dialyzed against PBS1X and detected by LAL assay for the LPS contamination. This contamination was lower than 0.5 EU/mg which was suitable for the next use in ELISA. Finally, the protein concentration was assessed by NanoDrop Spectrophotometer and stored at − 70 °C.At first, the full length of human Hsp27 gene (Accession No: NM_001540) was synthesized in a prokaryotic expression vector . To generate pET-Hsp27, the Hsp27 fragment was subcloned from pQE30-Hsp27 into the pET-23a in NheI/ HindIII cloning site. All expression and purification protocols were performed similar to the production of Hsp27 as mentioned above. Herein, the endotoxin contamination was less than 0.4 EU/mg for Hsp20 protein as detected by LAL assay which was suitable for the next use in ELISA.The full length of human Hsp20 gene (Accession No: NM_144617) was synthesized in a prokaryotic expression vector . To generate pET-Hsp20, the Hsp20 fragment was subcloned from pQE30-Hsp27 into the pET-23a in The Hp91 peptide (DPNAPKRPPSAFFLFCSE) with an N-terminal biotin was synthesized and purified by BioMatik Co. . The pev/v) Tween 20 for 2 h at 37 °C. The bound human antibodies were detected by goat anti-human IgG conjugated to horseradish peroxidase  and then addition of 3,3′,5,5’-Tetramethylbenzidine  as the substrate. All washing steps were done with PBS1X containing 0.05% Tween 20. The enzyme reaction was stopped by adding 50 μl of 1 M sulphuric acid and the absorbance was determined at 450 nm.For detection of IgG antibodies against the recombinant proteins, 96-microwell plates (Greiner) were coated overnight at 4 °C with 100 μl of the recombinant HPV E7 , Hsp27 , Hsp20  proteins as well as Hp91 peptide (10 μg/ml). Then, the plates were blocked with 1% BSA in PBS for 2 h at 37 °C, and incubated with 100 μl of human sera diluted 1:100 in blocking buffer containing 0.05% . One-way ANOVA was used to analyze the differences in the levels of antibody production. For all analyses, Iranian subjects who were seropositive for HPV-16 and 18 L1 capsids were divided into three groups: seropositive for HPV-16 , seropositive for HPV-18 , and seropositive for both HPV-16 and HPV-18 infections . Indeed, seroprevalence was 45% for HPV16 L1, 24% for HPV18 L1 and 31% for both HPV16 and HPV18 L1 in 49 Iranian subjects.E. coli strains. The rE7, rHsp20 and rHsp27 proteins migrated as the 23, 20 and 27 kDa bands in SDS-PAGE, respectively. Their purification was performed under native conditions using affinity chromatography . The results showed that the levels of antibodies against rE7, rHsp20, rHsp27 proteins and Hp91 peptide in HPV-16 exposed women did not show any statistically differences with HPV-18 exposed women (p > 0.05). In addition, the levels of antibodies against rHsp20 and Hp91 peptide in HPV-18-exposed women showed no statistically differences with women exposed to both HPV-16 and HPV-18 (p > 0.05). In contrast, the levels of antibodies against the rE7 and rHsp27 in women exposed to both HPV-16 and HPV-18 were higher than women exposed to HPV-16 or HPV-18 (p < 0.05). Figure The frequency of antibodies to HPV E7, Hsp20, Hsp27 proteins and Hp91 peptide was determined in 49 serum samples from Iranian women who were seropositive for HPV-16 and 18 L1 capsids  as well E. coli were used as antigens in ELISA for screening 49 serum samples obtained from individuals who were seropositive for HPV-16 (22 cases), HPV-18 (12 cases) and both HPV-16 and HPV-18 (15 cases) L1 capsids as well as control group . SDS-PAGE analysis of IPTG-induced cell lysates showed the presence of a prominent protein band of 23, 20 and 27 kDa for E7, Hsp20 and Hsp27, respectively that was not detectable in non-induced cell lysates. Then, the small heat shock proteins and Hp91 peptide besides viral oncogenic protein (E7) could be used to screen the seropositive individuals for HPV L1 capsids. Our ELISA findings indicated that HPV-exposed women with high antibody response to HPV-16 and 18 L1 had significant seroreactivity to HPV-16 and 18 E7 and Hsp27.In the current study, the presence of human antibodies to HPV E7 protein, small heat shock proteins 20 and 27, and Hp91 peptide was indicated by ELISA in HPV-exposed Iranian women. The recombinant proteins expressed in , exposure rate to high-risk HPV types), a variety of methods was developed for these purposes [Considering the importance of detecting HPV infection  compared to un-infected women  against the recombinant HPV E7 protein. In general, the detection of HPV-specific IgG in serum is a useful assay to determine previous and current HPV infection status [As known, E6 and E7 transforming proteins are consistently expressed in HPV life cycle –41. Seven status .The studies showed that HSP70 is a promising tumor marker for both diagnostic and therapeutic applications [. Moreover, several reports showed high levels of Hsp27 in sera of patients with different types of cancers by ELISA [On the other hand, heat shock proteins are highly conserved, but their expression levels between individuals were reported to differ widely for HSP60, HSP70 and HSP90 . The stuications , 47. Morby ELISA , 48–53. by ELISA , 54, 55.Recent clinical studies have shown that HMGB1 is a potential diagnostic or prognostic biomarker in a variety of inflammatory disorders and cancers. For instance, the serum level of HMGB1 was significantly elevated in benign and malignant asbestos-related diseases (ARDs) . Other r, Hsps or Hp91) with specific markers  could enhance significantly the overall diagnostic sensitivity as compared to each marker, alone. However, it is required to evaluate these markers in larger populations from different regions.Generally, the use of commercial HPV L1 kit is efficient to determine HPV-exposed subjects, but there is a major limitation including cost and HPV type. This kit can effectively detect HPV types 16 and 18, thus the development of other markers will be necessary for this purpose especially in resource-poor countries. Our report demonstrated that oncological/viral biomarker (E7) associated with Hsp27 may be beneficial in the detection of individuals exposed to high-risk HPV types. It was interesting that individuals with strong anti-L1 antibody level showed high anti-E7 and anti-Hsp27 antibodies suggesting similar efficiency of these proteins as HPV biomarkers. Regarding the results, the combination of non-specific markers Additional file 2:Identification of the purified proteins by Western Blot analysis using anti-His antibody: lane 1: Hsp27, lane 2, Hsp20; lane 3, E7; MW is the molecular weight marker . (JPG 78 kb)


CXCL12 gene including rs1801157 in the 3′-untranslated region, which is characterized by a substitution of a guanine for an adenine.Human papillomavirus (HPV) is the most common sexually transmitted virus in women worldwide. The persistence of the virus may cause warts that are considered benign lesions and low or high grade intraepithelial lesions (LSIL/HSIL). Immunological system plays an important role in the resolution of infections. In this context, we highlight the chemokines, which are important regulators in the development of viral infections and inflammation. Among which CXCL12 stands out, due to its pro-inflammatory features, acting as chemoattractant recruiting immune cells. Several polymorphisms were identified in In this study, 195 women were classified as HPV non-infected and 169 as HPV-infected. HPV-DNA was detected by polymerase chain reaction (PCR) and the polymorphism was assessed in blood cells through restriction fragment length polymorphism analysis.p = 0.007), among those who presented lower number of pregnancies (p = 0.017). HPV was more prevalent among allele A carriers confirmed by logistic regression analysis adjusted for several confounding factors . An association between allele A carriers and HSIL development (p = 0.003) was also observed.HPV infection was more incident in women who had more than 4 sexual partners during lifetime (CXCL12 rs1801157 is independently associated with HPV infection and exerts influence in HSIL development, suggesting it as a promising susceptibility biomarker for HPV infection and lesions development.In the present study, we demonstrated that  Human papillomavirus (HPV) is the most common sexually transmitted infection in women worldwide. Infection may resist asymptomatic and is, usually, transient. Most of the women eliminate the virus from the body with the immune system effective action within 5–15 months . SeveralMoreover, immunological system plays an important role in the infection resolution. HPV-HR presence may not be elucidated and persist through several years, inducing an inflammatory microenvironment leading to pre-cancerous lesions development –6. It isCXCL12 gene is located on long arm of chromosome 10 and was first cloned from a bone marrow-derived stromal cell line and then, identified as pre-B cell growth stimulating factor /2 N, where h represents the heterozygous genotype, H is the homozygous genotype, and N is the sample size for each population. Hardy-Weinberg equilibrium in infected and non-infected women was tested using x2 test. Differences in the distribution of genotypes were assessed by x2 test between non-infected and infected women, and among women with or without low- and high-grade squamous intraepithelial lesions. Adjusted Odds Ratio with 95% confidence interval was calculated to estimate the association between HPV presence, sociodemographic, reproductive and sexual behavior features, as well to analyze association of CXCL12 polymorphism with HPV presence and lesions development. Binary logistic regression model adjusted for confounding factors was performed to establish the association between HPV presence and CXCL12 polymorphism. All statistical analysis were performed in SPSS Statistics 22.0 . A p value < 0.05 was considered statistically significant.Differences in sociodemographic and sexual behavioral data between infected and non-infected women were examined using contingency tables and Pearson’s xIn the present study, 364 women were included and categorized as HPV non-infected patients (195/53.6%) and HPV infected patients (169/46.4%) according to the molecular detection of HPV-DNA. Non-infected women mean age was 42 ± 12 years (median = 42), while HPV infected patients mean age was 36 ± 13 years (median = 33).p = 0.024), were younger than 24 years old (p = 0.001), single (p = 0.002), smokers (p < 0.001) and received less than 1 minimum wage (p = 0.040).Sociodemographic characteristics of both groups, HPV infected and HPV non-infected women are presented in Table p = 0.007), among those who presented lower number of pregnancy (p = 0.017).Sexual and reproductive characteristics data are presented in Table CXCL12 rs1801157 polymorphism genotypes distribution among HPV non-infected and infected patients were in Hardy-Weinberg equilibrium (p ≥ 0.05). A higher frequency of allele A was observed in HPV infected women (p < 0.001) which was confirmed by codominant, dominant and recessive models , p < 0.001].In order to confirm whether on Table . A signip = 0.476) compared to women without lesion. However, it was significantly associated to HSIL (p = 0.003) development.Considering the polymorphism influence in lesions development, the dominant model was adopted in order to make a better distribution among genotype groups Table . We obseCXCL12 rs1801157 polymorphism HPV infection and HSIL.To the best of our knowledge, this is the first study that demonstrated an independent association between According to sociodemographic data, HPV was more frequent within patients who had no knowledge about the virus which may indicated lack of information about HPV and also, the ways to avoid virus exposure; among women younger than 24 years old, single and who had more than 1 sexual partner during lifetime.Young age has been associated in an independent way to HPV infection  and it ip < 0.001), this could be explained by the fact that tobacco smoking may cause immunosuppression [In this study, smoking status was associated to HPV infection  was also associated to HPV presence. During pregnancy, elevated estrogen and progesterone may lead to the squamous-columnar junction exposure and metaplasia. Parity might increase the risk of cervical cancer because it maintains the cervix transformation zone for many years, facilitating exposure to HPV infection and others cofactors [Lower pregnancies number . Increased gradient of CXCL12 concentration was observed from LSIL to HSIL in women with HR-HPV [Precursor lesions can occur as consequence of persistent infection. In the present study, allele A influence in cervical lesion development was also evaluated and a significant association was observed for allele A carriers with HSIL (h HR-HPV . Hence, + T cells in progressing cervical cancer [CXCL12 has been considered as a standard proinflammatory molecule for a long time, since it attracts leukocytes to inflammatory sites contributing to their activation , 29. Datl cancer .CXCL12 rs1801157 polymorphism influence in its expression, plasmatic or cervical levels in HPV infection or SIL development, but it has been widely studied in others diseases and tumors, showing conflicting results. De Oliveira et al. [CXCL12 mRNA in the peripheral blood samples when compared to GG patients. Controversially, Hirata et al. [Until present there are no studies about the a et al.  observedImmunohistochemistry profile of CXCL12 in colorectal cancer showed weak or negative in normal mucosa and strongly increased in cancer tissues especially in well-differentiated tumors, 73.5% of patients that expressed a strong CXCL12 immunostaining in the membrane and cytomembrane presented AA or GA genotype. By the other hand, 88.6% of those with negative immunoreactivity presented GG genotype . HoweverCXCL12 rs1801157 SNP impact on chemokine levels [The 3′ untranslated region of genes contains several regulatory motifs that is target of posttranscriptional regulation through interaction with microRNAs, RNA-binding proteins, and long non-coding RNAs, which influence on mRNA turnover, stability and localization. SNPs located in these motifs may prevent miRNA binding and cause mRNA transcript stabilization and increased protein expression . Althouge levels –39, evide levels . AvailabEvidence for the involvement of the CXCL12 in the HPV life cycle arose from the abnormal and specific expression of CXCL12 observed in keratinocytes of HPV-productive skin or mucosal lesions .CXCL12 proximal promoter in its 5′-flanking and 5′-untranslated region contain six Sp1 binding sites, and Sp1 transcription factor seems to be the major positive regulator of CXCL12 expression [CXCL12 gene expression [CXCL12 expression levels may increase in keratinocytes as a consequence of HPV genome expression, generating an autocrine signaling loop essential for keratinocyte proliferation and migration . A reasopression . Additiopression .CXCL12 rs1801157 polymorphism to HPV infection and HSIL, suggesting it as a promising susceptibility biomarker for HPV infection and the development of cervical lesions.More studies are necessary to determine the rs1801157 polymorphism influence in CXCL12 expression and in its cervical levels, in order to establish its influence in HPV infection and in cervical lesion development. However, our work is pioneer in demonstrating the association of


Background: People living with human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) and, men who have sex with men (MSM) are disproportionately affected by genital warts and cancers caused by human papillomavirus (HPV). We assessed community-based HIV/AIDS service organizations' (ASOs) staff awareness, knowledge, attitudes, and beliefs about HPV and effective cancer prevention tools, namely HPV vaccination, Pap, and HPV tests. The potential engagement of ASO staff in future efforts to reduce the disproportionate burden of genital warts and HPV-related cancers among HIV-positive women and MSM was explored.Methods: In May-June 2016, staff were recruited from three ASOs located in the South United States Census region—a geographical area disproportionately affected by HIV/AIDS. Participants completed a 30-min self-administered, 118-item paper and pencil survey about HPV and cancer. Data analysis was conducted using Stata/SE 14.2.Results: ASO staff (n = 30) were 83% non-Hispanic Black, 40% lesbian/gay, and worked with people living with HIV for an average of 11.4 ± 7.7 years. All reported hearing of HPV and 77% had heard of the HPV vaccine (n = 23). While all knew HPV can cause cervical cancer, only 67% knew HPV can cause anal cancer. Most (61%) thought the HPV vaccine could prevent cervical cancer. Fewer (39–48%) thought the HPV vaccine could prevent anal, oral, penile, vaginal, and vulvar cancers. All were willing to encourage MSM and female clients to talk to a healthcare provider about HPV vaccination. Almost all were willing to promote HPV vaccination to clients (91–95%) and navigate clients to adult safety net HPV vaccine providers (86–95%). More than half (59–67%) thought they could positively influence their MSM and female clients' HPV vaccine decision-making.Conclusion: HPV vaccination and the Pap and HPV tests are effective cancer prevention tools that can reduce the disproportionate burden of genital warts and HPV-related cancers among HIV-positive women and MSM. Engaging ASO staff in cancer prevention efforts may increase HPV vaccination rates and early detection of HPV-related cancers among HIV-positive women and MSM. Exploring ASOs as community-based settings for promoting effective cancer prevention tools may foster opportunities to reduce the disproportionate burden of genital warts and HPV-related cancers among HIV-positive women and MSM. Human papillomavirus (HPV) is the most common sexually transmitted infection worldwide , 2. An HBecause of their increased risk for developing cervical cancer, HIV-positive women are screened more regularly than HIV-negative women  . Even wiData from HPV vaccine clinical trials supports the benefits of HPV vaccination among at-risk populations disproportionately affected by HPV-related diseases and cancers, such as HIV-positive women and MSM, in light of minimal risks  and decreased effectiveness for those already exposed to some HPV types. Yet, HPV vaccine coverage remains suboptimal among MSM , 12, andHealthy People 2020 goals for the general population (USDHHS). In this study, we examined HPV-related awareness, knowledge, attitudes, and beliefs among staff at ASOs located in the South U.S. Census region—a geographical area disproportionately affected by HIV/AIDS  emailed a description of the research study to leaders at four ASOs located in South Carolina (SC). The three ASO leaders that responded were asked to invite eligible staff to participate. Eligibility criteria included being an employee, volunteer, or community partner who provided HIV/sexually transmitted disease (STD) risk-reduction counseling to PLWH or high-risk HIV-negative men (including MSM) or women.The HINTS is a population-based mail survey that was used to obtain a subsample of respondents that resided in the same U.S. Census Region (South) and Division (South Atlantic) as our study participants . The HINParticipants in our ASO study completed a 30-min, self-administered, 118-item paper and pencil survey that included questions about HPV and HPV vaccine awareness, knowledge, and attitudes/beliefs drawn heavily from the HINTs survey. For this analysis, we are only focusing on these domains . The principal investigator (LTW) conducted the survey in a group setting and was able to provide oversight to ensure that the respondents did not get help answering the knowledge questions from either from the Internet or their colleagues.Both samples were asked if they had ever heard of HPV and the HPV vaccine. We used an adapted version of the HINTS HPV vaccine awareness question to ask our ASO sample if they had heard of specific HPV vaccines  and cancer screening tests . Response options were yes/no.Both samples were asked about knowledge that HPV can cause anal, cervical, oral, and penile cancers; is a sexually transmitted disease; and will usually clear . We used an adapted version of the HPV knowledge question to assess the ASO sample's knowledge that HPV causes more health problems for PLWH and other types of cancers . Questions from older HINTS iterations and existing surveys were adapted to assess the ASO sample's HPV vaccine knowledge about the following: main purpose is to prevent HPV infection, 3-doses recommended for females and males (including PLWH) through 26 years old, not recommended for persons older than 26 years old, cervical cancer screening is still needed , and safIn both samples, beliefs about the HPV vaccine's cervical cancer prevention effectiveness was assessed. We adapted the HINTS HPV vaccine effectiveness question to include other HPV-related cancers . A similar question from an older HINTS iteration was used to assess ASO staff's beliefs that the Pap test was effective at detecting cervical cancer in its earliest stages when treatment is more effective. Response options were: not at all successful, a little successful, pretty successful, very successful, and don't know. We dichotomized this variable as successful or not at all successful/don't know.We also adapted the HINTS question about cervical cancer being preventable to include other HPV-related cancers . ASO staff who worked with female clients were asked how much they agreed/disagreed that anal, cervical, oral, vaginal, and vulvar cancers were preventable. Similarly, ASO staff who worked with MSM clients were asked if anal, oral, and penile cancers were preventable. Response options were: strongly agree, agree, disagree, strongly disagree: We dichotomized these questions as agree or disagree.We used a single item to assess ASO staff's perceptions of barriers that their clients might encounter when trying to access the HPV vaccine, cervical cancer screening tests , and/or follow-up treatment or diagnostic tests if their Pap test result was unclear or abnormal. Response options included patient , provider , and system-level  barriers. An open-ended response option was used to assess other barriers.n = 365/820 South Atlantic division residents; H5C1: n = 323/754 South Atlantic division residents). Frequencies and percentages were calculated independently for each of the three study samples. The variability between our research study design and the HINTS sample design and weighting procedures limited our ability to merge datasets. Although we were not able to perform chi-square tests between our sample and each of the HINTS samples, we were able to use proportion tests to determine whether or not the observed differences in HPV-related awareness, knowledge, and beliefs between our sample and the HINTS samples were statistically significant.Stata/SE 14.2  was used to perform all data analyses. We restricted our HINTS data analyses to the subpopulation of US adults who lived in the South Atlantic division of the South US Census division and had no missing observations for any of our covariates , 83% non-Hispanic Black or African American , and 77% had completed a college degree or higher  compared to US adults residing in the South Atlantic division of the South U.S. Census region. (Table n = 22) or part-time (n = 1) at their ASOs, and almost all worked with HIV-positive (or high-risk HIV-negative) women (93%) or MSM (97%) clients . A larger percentage of ASO staff knew that HPV can cause anal (67%), penile (59%), and oral (38%) cancers compared to US adults residing in the South Atlantic division: anal ; oral ; and penile , and cancers. Eighty-three percent of ASO staff knew that HPV can cause vaginal cancer, and almost half (48%) knew HPV can cause vulvar cancer. The proportions of ASO staff who knew that HPV is an STD (80%) that will usually go away on its own without medical treatment  were higher compared to US adults residing in the South Atlantic region: HPV is an STD , …that will usually clear  had heard of the HPV vaccine (Table n = 18/23) who had heard of the nonavalent HPV vaccine  and 86% (H5C1: p = 0.0001) of US adults residing in the South Atlantic division  of ASO staff . While 3–7% of ASO staff thought that anal, cervical, oral, and vulvar cancers were not preventable, almost half thought that penile (45%) and vaginal (43%) cancers were not preventable  knew that vaccinated females still needed to be screened for cervical cancer. The HINTS 3 2008) iteration is the last time that this survey question was assessed. Comparatively 94% of US adults at that time knew that vaccinated females still needed to be screened for cervical cancer (008 iterap = 0.0100) in 2014. However, compared to US adults residing in the South Atlantic region in 2017, the proportion of ASO staff (47%) that thought the HPV vaccine can prevent cervical cancer was not statistically higher . Fewer than half of ASO staff thought the HPV vaccine could prevent anal (33%), oral (48%), penile (30%), vaginal (48%), and vulvar (48%) cancers , 77% of ASO staff had heard of the HPV vaccine. Almost half (47%) of ASO staff thought that the HPV vaccine can prevent cervical cancer, which was a statistically significant higher than the proportion of US adults residing in the South Atlantic region . In contrast, a smaller proportion of ASO staff who worked with MSM clients thought their opinion could influence these male's HPV vaccine decision-making (59%) than ASO staff who worked with female clients . Similarly, a smaller proportion of ASO staff who worked with MSM clients were willing to help navigate these males to adult safety net HPV vaccine providers (86%) where they could get the HPV vaccine for free or at a reduced cost than those who worked with female clients  were willing to encourage these males to get the HPV vaccine, compared to ASO staff who worked with female clients , and were also willing to help these females better understand their Pap test results  were willing to encourage females aged 30+ years to talk to their healthcare provider about getting an HPV DNA test. Almost all ASO staff  that worked with female clients were willing to help these females find free or low-cost cervical cancer screening tests  and screening  tools. This may be attributed to the disproportionate burden of HPV-related diseases and cancers among their clients. That said, the lower level of awareness about the anal Pap test raised some concern given the high proportion of ASO staff that participated in our research study who work with MSM clients and the high burden of anal diseases and cancers among MSM (which is further exacerbated by HIV infection). ASO staff's lack of knowledge about anal Pap tests could be attributed (at least in part) to the fact that in contrast to cervical cancer, there are no recommended screening guidelines for anal cancer. However, the general sense is that for high-risk populations such as MSM , the potential for improving HPV-related cancer outcomes outweigh the potential harm. In this regard, the potential for ASO staff to help their clients overcome barriers and enable them to make informed (or at a minimum shared) decisions about cancer screening with their health care providers becomes increasingly important .Although some ASO staff may not share the same positive attitudes and beliefs about HPV vaccination and anal and/or cervical cancer screening, almost all of the ASO staff in our research study were willing to promote patient-provider communication and provide patient navigation support to their clients. Studies have shown that ASO clients' health outcomes are positively associated with how effectively ASOs can integrate various services . FurtherThis study is one of only a few that have compared the US population-based HINTS sample to a special subpopulation of US adults , 22. OurThe main limitation of our study is its small sample size. However, it should be noted that our study was a sample of provider-level participants, and not a population-level sample. Further, our sample of provider-level participants served a vulnerable subgroup of the population . Although we were able to ask more specific questions, this limited our ability to make additional comparisons between our sample and the HINTS sample. Our study findings may not be generalizable to other ASO staff in other locales. Another limitation is that we did not assess gay stigma as a potential healthcare access barrier.HPV vaccination can reduce the burden of HPV vaccine preventable diseases and cancers. Future research to assess what ASO staff's beliefs are about the HPV vaccine specifically for special populations such as PLWH and MSM is warranted, especially since most knew that the HPV vaccine was recommended for PLWH, but only about half (or fewer) thought that the HPV vaccine was safe and effective. Similarly, early detection of precancerous cells can improve health outcomes. Because ASO play an important role in improving health outcomes for their clients , it seemData collection activities for this research study were carried out in accordance with the recommendations of the University of South Carolina's Office of Research Compliance with verbal informed consent from all subjects that participated in our needs assessment survey. All participants gave verbal informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the institutional review board of the University of South Carolina's Office of Research Compliance.LW conceptualized the research project and HB . LW conducted the literature review that informed the development of the survey, which LW developed and HB and SB contributed to expert review and content validity. LW conducted the research study . LW lead data analysis with assistance from NS. All authors  have all contributed to the interpretation of the findings. LW provided the initial draft of the findings, and all authors  assisted with revising subsequent drafts for important intellectual content. All authors  read and provided approval for publication of the final draft of the manuscript that was submitted. LW along with her K01 mentors (HB and MO) agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work were appropriately investigated and resolved.HB was and still is a member of the United States HPV Advisory Board of Merck. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


P16 immunohistochemistry, FISH, and genetic analyses of the HPV gene from histopathological and liquid biopsy specimens have been employed. Based on the results of the differential diagnosis, various treatments employing EGFR TKI and low-dose radiation have been employed. Here, we review the involvement of the HPV virus in HNSCCs as well as the molecular mechanism of carcinogenesis, classification, prognosis, diagnostic procedures, and therapy of the disease.Head and neck squamous cell carcinomas (HNSCCs) arise in the mucosal lining of the upper aerodigestive tract. Tobacco and alcohol use have been reported to be associated with HNSCC. Infection with high-risk human papillomaviruses (HPVs) has recently been implicated in the pathogenesis of HNSCCs. It is now widely accepted that high-risk HPV is a cause of almost all cervical cancers as well as some forms of HNSCCs. HPV-related HNSCCs are increasing. HPV-related HNSCCs and HPV-unrelated HNSCCs differ with respect to the molecular mechanisms underlying their oncogenic processes. HPV-related HNSCCs are known to have a better prognosis response to treatment as compared with HPV-unrelated HNSCCs. Therefore, in recent years, it has been required to accurately discriminate between HPV-related and HPV-unrelated HNSCCs. To diagnose the HPV-related HNSCCs, various methods including  The role of human papillomavirus (HPV) in carcinogenicity was confirmed in 1983 following the cloning of HPV 16 type from cervical carcinoma tissue by Durst and colleagues . As reviVarious studies have reported that oral and tonsillar epithelial cells can be immortalized by full-length HPV 16 or its E6/E7 oncogenes ,12,13,14A higher frequency of oral sex and a greater number of sex partners are thought to increase the risk of HPV-related cancer in the oropharynx ,20. http://senseaboutscience.org/activities/2017-john-maddox-prize/). This review summarizes the involvement of HPV virus, molecular pathological implications, classification and prognosis, and prospects for future treatment in head and neck cancers.The development of a vaccine for the primary prevention of HPV infection subsequently became an urgent worldwide priority. In 2017, Muranaka was awarded the John Maddox Prize for raising public awareness of the efficacy of the HPV vaccine for the prevention of cervical and other cancers , inhibits phosphorylation of Rb, and inhibits transcription factor E2F-related cell rotation (pRB pathway) ..P16 is lThus, the phenotype at the molecular level is completely different between HPV-positive and HPV-negative cancer of the head and neck. Various methods have therefore been employed for the detection of HPV in head and neck cancer such as consensus primer or type-specific PCR, real-time PCR, in situ hybridization, and serum antibody assays. For cervical cancer screening, accepted international guidelines recommend using hybrid capture II (QIAGEN) and PCR (GP 5/GP 6). P16 immunohistochemistry is also useful as a surrogate maker for HPV infection detection, especially in head and neck cancers. P16 immunohistochemistry has 100% sensitivity and 79% specificity; it is the gold standard for HPV detection, based on HPV 16 E6 and E7 mRNA, in head and neck cancer specimens and is useful as a surrogate maker for clinical HPV detection. P16 is also a useful molecular marker for judging prognosis and is a component of the WHO classification scheme, described below. P16-positive and P16-negative HNSCCs could clearly be distinguished in our specimens , CCND 1 , and PIK 3 CA . Changes in TPV, CDKN2A, and CCND1 genes occurred more frequently in HPV-negative tumors, but the total amount of mutations was similar between HPV-negative and HPV-positive tumors. HPV-positive tumors were significantly associated with immune-related genes compared to HPV-negative tumors. Mutations in NOTCH1 (p = 0.027), CDKN2A (p < 0.001), and TP53 (p = 0.038) were significantly associated with decreased overall survival. FAT1 mutation was highly enriched in cisplatin responders and targetable alterations such as PIK3CA E545K and CDKN2A R58X were found in 14 patients (15%) [Studies using next-generation sequencing of HNSCCs have also been reported in recent years. Matthias Lechner did 20 HPV+ and 20 HPV-laser capture microdissection pharyngeal carcinoma studies. HPV-positive and HPV-negative oropharyngeal cancers are divided into two different subgroups. A TP53 mutation was detected in 100% of HPV-negative cases and invalidation of G1/S checkpoint by CDKN2A/B deficiency and/or CCND1 amplification was shown to occur in the majority of HPV tumors . In a stAs an emerging technology, liquid biopsies, involving the use of a small amount of DNA and mRNA collected from blood samples, have been used in recent cancer studies. Allen et al.  reportedP16. For HPV-positive and HPV-negative cancers the respective response rates were 84% versus 57%, the 2-year progression-free survival rates were 86% vs. 53%, the overall 2-year survival rates were 95% vs. 62%, and the prognosis for HPV-positive cancer was significantly better [P16, and the outcomes of oropharyngeal cancer in relation to tobacco exposure. In a phase III study involving 400 oropharyngeal carcinomas, the 2-year progression-free survival rates for HPV-positive and HPV-negative cancer were 72% vs. 51% and the 2-year overall survival rates were 88% vs. 67%. Due to differences in prognosis, squamous cell carcinoma of the head and neck was classified in the new WHO scheme as HPV-negative or HPV-positive. Classification by P16 immunostaining or HPV testing is recommended. In addition, some recently proposed classification schemes are based on the EGFR status according to 2 categories: HPV-positive/P16 positive squamous cell carcinoma and HPV-negative/P16 negative squamous cell carcinoma [In general, HPV-positive oropharyngeal carcinoma is highly susceptible to radiation and anticancer drugs and has a better prognosis compared with HPV-negative cancer. HPV-negative oropharyngeal carcinoma is caused by the disappearance of function due to p53 gene mutation ,57,58,59y better . Previouarcinoma . Cetuximarcinoma ,62 and harcinoma ,64, and P16 immunostaining, Furthermore, HPV vaccination may also be useful for preventing HPV-related cancers other than cervical cancer. For example, most cases of oropharyngeal carcinoma are caused by HPV 16 (about 90%) and HPV 18, and HPV vaccination for this condition can be expected to have a greater disease-suppressing effect than in cervical cancer. Randomized trials have provided strong evidence for high efficacy of the 2 FDA-approved VLP vaccines: the bivalent HPV16/18 vaccine  and the quadrivalent HPV 6/11/16/18 vaccine  against cervical, vaginal, and vulvar HPV16/18 infections and related diseases, and against anal HPV16/18 infections in women [For the reasons outlined herein, HPV virus is expected to be a therapeutic target in the treatment of human cancer  We beliein women ,68,69,70in women . Also, Reduction surgery or minimally invasive treatment should be considered in cases of HPV virus-related oropharyngeal carcinoma. Although limited to the T1 and T2 stages of oropharynx cancer, transoral robotic surgery approved by the FDA since 2009. In addition, many reduction surgeries and post-operative adjuvant therapies based on pathologic staging are being studied ,73,74. P16-positive OPCs treated 2 cycles of IC (paclitaxel and carboplatin was for 21 days), followed by radiation and paclitaxel. The radiation dose was also reduced in complete or partial responders , and even in patients with partial or no response . Except for 1patient, all patients completed the IC. Two years PFS and local region control were 92% (95% CI 77–97) and 95% (95% CI 80–99), respectively. The two-year degree of freedom for grade 3 or adverse events of worsening mucosa and esophagus was 85% (95% CI 80–90) in patients treated with 54 Gy. These results were achieved with doses reduced by 15–20% compared with these in the ECOG 2399 trial using the same protocol, except that a dose of 70 Gy, dose was used as a standard chemoradiotherapy regimen. Besides that, Removal of chemotherapy and Alternative to the “conventional” photon beam therapy are considered as new treatment methods [As mentioned above, it is known that EGFR is expressed in HNSCCs, and combination use of cetuximab and radiation is, therefore, being studied for treatment instead of standard cisplatin therapy ,76,77,78% 95% CI –99, respDifferences in the prognosis and etiologic mechanisms of HPV-related head and neck cancer from conventional head and neck cancers (mostly HPV-negative) suggest that the detection of HPV may significantly change the future diagnosis, treatment, and management . HPV not


Incidence and mortality rates of cervical cancer in Bolivia are the highest in Latin America. Vaginal cell self-sampling can improve screening coverage. Information on common reasons for low screening coverage and preferences for future screening are essential to reduce cervical cancer incidence. We aimed to evaluate the knowledge about human papillomavirus (HPV) and cervical cancer of Bolivian women from urban, peri-urban and rural areas of Cochabamba and to determine their degree of acceptability and confidence towards vaginal HPV self-sampling. In addition, we assessed the impact of self-sampling on cervical cancer screening coverage in a selected peri-urban area.We gathered information from women living in urban, peri-urban and rural areas of Cochabamba province in Bolivia using two different structured questionnaires. In Survey1, we collected information from 222 women about their knowledge on HPV and cervical cancer. In Survey 2, the acceptance and confidence towards vaginal HPV self-sampling compared to the physician-sampling was assessed in 221 women. A non-probabilistic stratified sampling by areas was carried out for the two questionnaires. In the third phase of the study, we determined the impact of HPV self-sampling collection on screening coverage in a peri-urban area of Cochabamba.Bolivian women knew little or nothing about cervical cancer and HPV infection in all areas. They all found self-sampling collection easier to perform (86.9 to 93.2%) and more comfortable (79.4 to 83.3%) compared to physician sampling. Sampling accuracy to detect cervical cancer was probably higher in their point of view when it was taken by physician (35.1 to 63.5%). However in rural areas women preferred self-sampling. Accordingly, the campaign of vaginal HPV self-sampling in this peri-urban area increased screening coverage, reaching in three months the annual rate average.The knowledge about cervical cancer and HPV infection is poor in Bolivia. Despite greater acceptance of the vaginal HPV self-sampling in all areas, women kept greater confidence in the screening performed by the gynecologist although HPV self-sampling improved coverage rate.The online version of this article (10.1186/s12889-019-6401-5) contains supplementary material, which is available to authorized users. The development of cervical cancer depends on high-risk human papillomavirus (HR-HPV) persistent infection in the uterine cervix . The 2–1Cervical cancer-related deaths have nevertheless declined significantly in developed countries because of extensive cytology screening. However, similar initiatives in developing countries have not been equally successful because of the complexity of the required elements, such as laboratory expertise, depending on high operating costs , 4. It iPrevention of cervical cancer in Bolivia is primarily based on Papanicolaou smear cytology test (Pap) and more recently on visual inspection under acetic acid (VIA) , 8. ScreThe health personnel estimated that 50 to 80% of Pap screened women were lost to follow-up, mainly because of delays in result delivery .Bolivia’s Ministry of health has introduced the HPV vaccine in 2016 as an alternative to reduce the incidence of cervical cancer. In 2017, 80% of the target population (girls between 10 and 12 year old) were vaccinated with the first dose .Bolivia is characterized by significant disparities between rural and urban areas, including education and access to basic health services as some of the variables contributing to this difference , 11. AltThe screening coverage rate is a key component in cervical cancer prevention programs. Vaginal self-testing of HR-HPV could reduce screening barriers for those women and therefore increase their screening coverage –18. IndeThe objectives of this study were to obtain information among the Bolivian women about their level of knowledge on HPV and cervical cancer and to evaluate the degree of the vaginal self-sampling test acceptability and confidence in comparison to gynecologist sampling at urban, peri-urban and rural areas, in order to evaluate the need for an adapted strategy within each three geographical areas, potentially corresponding to a characteristic population. Furthermore, this study assessed the impact of the HR-HPV self-sampling test on screening coverage in a restricted Bolivian peri-urban area.This cross sectional study is based on the analysis of two surveys with a non-probabilistic stratified sampling by geographical areas . The evaluation of coverage impact through self-sampling was performed in a peri-urban area, with a mixed population . The Bio-ethical committee of “Universidad Mayor de San Simon” approved the study protocol . The signed informed consent was an indispensable requirement for the inclusion of patients. Pregnant women over 20 weeks, calculated on the basis of last menstrual period, and women with hysterectomy were excluded from this study. This study was divided in three phases. For surveys, a person speaking Quechua and Spanish was available to read and help to fill the survey files in case of analphabetism.n = 96), the peri-urban (surrounding area n = 66) and the rural  areas with a range of age between 20 and 64 years. One structured questionnaire was conducted with four multiple selection questions in the three geographic areas mentioned above to determine the degree of knowledge about cervical cancer and HPV, , we obtained information from 222 women about their knowledge on HPV and cervical cancer and their prevention. The target population was women from the urban , the peri-urban (surrounding area n = 63) and the rural  areas, with a range of age between 25 to 64 years.The second phase of the study (survey 2) assessed 221 women through eight closed-ended questions after self-sampling and physician-sampling to determine their acceptance and confidence towards vaginal HPV self-sampling. The target population was women from the urban . This was carried out through campaigns with the participation of volunteers . All of them were trained for three days to give correct information on cervical cancer, HPV and on the self-sampling technique and procedure. Two strategies to promote and collect self-samplings were performed: 1) direct contact with the population through visits at home, informing and offering the self-sampling device, 2) installation of tents with isolated cubicles in populated areas, generating a favorable environment for self-sampling. This was carried out during 9 one-day campaigns over a three month period. Cervical cancer screening was also promoted for 8 months in the primary Health Centers of District 9 Cochabamba, starting four months before the beginning of the self-sampling campaigns. The device for the vaginal and cervical cell self-collection was the same as in phase 2. The results in the coverage achieved through this intervention were estimated for twelve months and compared to the cytology coverage obtained in the previous year.All cervico-vaginal samples in this study were analyzed for HR-HPV DNA within two weeks after they were collected. The procedure for DNA extraction from samples transported and conserved on glass slides has been described previously . Only DNP value greater than 0.05), assuming that differences in education between the geographic residences of women are not influencing the survey responses. This null hypothesis is justified to detect any difference between areas on the survey responses.Statistical analyses were performed using hypothesis test for proportion (Chi-square). The null hypothesis was that there are no differences between the responses of the urban, peri-urban and rural areas, .In the first and second phase of the study, most of the population (83% for the survey 1 and 95% for the survey 2) was in the range of 20 to 49 years (data not shown). As expected, the level of education of the survey 1 population decreased in relationship with the distance to the urban region .As summarized in Table P value> 0.05). Compared to self-sampling, samples taken by physicians with vaginal speculum seemed less comfortable (from 40.5 to 71.6%), and more painful, especially in rural areas , in peripheral areas of Cochabamba city and in the central area of Cochabamba city survey 2). Three questions were asked to evaluate the easiness, the comfort and the experienced sensation of pain using the cotton swab for the self-sampling . However, in regards to the question: what test would you recommend? women from the rural areas tended to recommend the self-sampling with a significant statistical difference in the three areas .In Survey 2, women were also asked for their long term screening strategy preference: physician-sampling versus self-sampling procedure Table . There was Table . SimilarIn 2015, starting in January, we promoted cervical cancer screening in District 9 health centers. Health personnel from 8 public health primary care centers in the intervention area were informed and trained about the relationship between HR-HPV and cervical cancer, prevention and molecular detection of HPV. Women attending those primary health care centers were encouraged to visit gynecologists to request the physician-collected HPV and Pap smear test. The purpose of this activity was to be able to respond to the demand for information and / or follow-up of any woman in the area requesting it.Four months later, we started campaigns (9 days distributed in a three month period), to collect self-sampling from the same population. Campaigns were performed informing and offering the self-sampling device either by visiting women from house to house or by meeting them in populated areas under a tent with isolated cubicles, providing favorable environment for self-sampling. This 9 days campaign allowed collecting 902 HPV self-sampling tests from the same population. From 902 100%) women who underwent self-sampling, 792 (87%) samples were analyzed, 95 (12%) were HR-HPV positive, and only 13 (13.7% of women with HR-HPV) came back for follow-up Three women had a low-grade intraepithelial lesion and two had a high-grade intraepithelial lesion. The coverage calculation in District 9 was performed taking into account the population assigned to the eight health centers . The Pap smear test coverage achieved in the year 2014 was 3.9%, and coverage obtained in 2015 in three months by the HPV self-sampling test was 4.6%, making an estimated annual coverage that could achieve 18.6%, namely 4.7 fold higher with respect to the Pap coverage of the previous year  were always provided 2–3 weeks after collection (by physician or self-sampling), while cytology results, when available, were mostly provided after 3 months.In order to reduce the cervical cancer incidence and mortality in the department of Cochabamba in Bolivia, we aimed to develop and assess a low cost HPV self-sampling screening. We believe that this strategy could improve the poor screening programs developed in our country. Not only the HPV DNA detection rate in self-sampling has been reported to be in good agreement with physician-sampling, but also the self-sampling method seems better accepted among women , 25. It In our study, in the Survey 1, we confirmed that the level of knowledge about cervical cancer and HPV, in the three geographical areas, was low, diminishing in function of the distance between the area of residence and the capital city (urban area), and of the education level (the null hypothesis is rejected). A study in Cameroon mentioned that education about HPV and cervical cancer is associated to a greater acceptability towards the HPV self-sampling test. The provided information had a positive impact on the patient’s concerns, about her ability to perform the test . TherefoAccording to the results obtained in the survey 2, most of the women found, in the three geographic areas, self-sampling easy, comfortable and without pain. The level of the self-sampling acceptance was thus good, allowing to consider it as a promising screening tool to break sociocultural barriers. A similar result was observed in a study performed on three different populations  as 75% women found that self-sampling was easy and 90% found it very comfortable . HoweverA meta-analysis in which six studies were reviewed about self-sampling acceptability revealed that the majority of women interviewed preferred the self-sampling test which did not require a gynecological examination . The vagIn our third study, assessing self-sampling on cervical cancer screening coverage in a peri-urban area (9th District), we observed up to a 4.7 fold higher screening coverage compared to the previous year Pap coverage. Similarly, another study in South America mentioned a 4 fold increase in screening coverage using self-sampling  and simiIf self-sampling would be routinely used in Bolivia, additional infrastructures and guidelines will be needed, especially to provide clear information, among others to prevent insecurity feelings during samplings, to increase appropriated patient engagement, to improve the follow-up rate, but also to reduce the risk of patient anxiety after a positive result diagnosis transfer .In conclusion, the self-sampling is well accepted by the Bolivian surveyed women, regardless of demographic differences. This suggested that in Bolivia also, cervical cancer detection coverage can be expanded using this form of cervical sampling (self-sampling). Knowledge about cervical cancer and HPV should be optimized to improve cervical cancer prevention programs. This aspect is fundamental to improve confidence and motivation of women performing self-sampling Studies to assess follow-up efficacy in regards to screening strategy should be further performed.Additional file 1:Survey 1: Level of knowledge about HPV and cervical cancer. Multiple choice questionnaire in the three geographical areas of study to determine the degree of knowledge about cervical cancer and HPV. (PDF 414 kb)Additional file 2:HPV self-sampling kit. Self-sampling kit consisting of a cotton swab and a glass slide for cervical / vaginal sampling (TIF 222 kb)


This study was undertaken to determine the distribution of type-specific human papillomavirus (HPV) in external anogenital warts, and the correlation with clinical presentation of warts and demographic data of patients.Genital warts specimens were obtained from 129 men and 27 women attending a dermatology clinic, who had been advised surgical excision. The tissues were fixed and screened for HPV DNA by using real-time PCR. HPV genotype was determined by PCR-based sequencing.Sixteen different HPV genotypes were detected, comprising 4 oncogenic HPV genotypes , 2 low-risk HPV types (LR) , HPV 9, and other types associated with common warts . Oncogenic HPV types were found in 34.62% of patients, LR HPV types in 14.4%, HPV 9 in 0.64%, and common warts type in 50.6%. The prevalence of HPV infection with a single type was 88.4, 9.0% for two types, and 2.6% for three types. Multiple logistic regression model showed that age, gender, nationality, number of warts, size of each wart, and positive history of wart in sexual partner, were not predictors of HPV type. However, patients with anogenital warts of one to six months duration were three times more likely to have oncogenic HPV infection compared to those with less than one month.This study shows that oncogenic HPV types are detected in around 35% of patients with genital warts, and are prevalent in warts of one to six months duration. Anogenital condyloma acuminata are warts around the genital and anal areas. More than 90% of anogenital warts are caused by HPV type 6 and 11 , 28. In Limited reports are available on HPV infection and anogenital warts locally and worldwide. In the Kuwaiti context’, one report published in 2004 by Al-Fouzan and Al-Mutairi  presenteWe have previously conducted a study to determine the prevalence and type specific distribution of HPV in women with normal cervical cytology samples in Kuwait. HPV DNA was detected in 2.4% of women with cytologically normal cervices; low-risk (LR) HPV types were detected in 71.8% of the infected samples, high-risk (HR) HPV types in 32.3%, and intermediate-risk (IR) types in 7% [Three different prophylactic HPV vaccines are currently approved. Gardasil/Silgard which targets HPV6, 11, 16, and 18; Cervarix which targets HPV 16 and 18; and Gardasil 9 which targets HPV6, 11, 16, 18, 31, 33, 45, 52, and 58, reviewed in Gupta et al., 2018 .In order to investigate whether the HPV types associated with genital warts are covered by the current licensed HPV vaccines, the HPV types detected in the genital warts were identified by direct sequencing. Demographic data and warts characteristics were used in a regression model to predict the HPV type.A cross sectional study that uses a non-probabilistic sample was conducted. Patients attending dermatology clinics at Farwania Health District Area were recruited in the study consecutively from November 2016 to May 2017. Immunocompetent patients with previous clinical diagnosis of external anogenital warts scheduled for cryotherapy or laser treatment were asked to sign the consent form after a verbal explanation from the dermatologist soliciting their participation. At the time of sampling, wart specimens were collected in universal transporting medium , and stored at − 80 °C. Ethical approval was obtained from the Health Sciences Center Ethical Committee (Number: VDR/EC/2310) and Ministry of Health.Demographic and clinical information were taken from each patient, including: age (years), gender , nationality , number of warts , duration of the presence of warts , size of each wart (in centimeter) , partners has warts , and if warts were subject to prior treatment . None of the participants received HPV vaccination, because HPV vaccination has not yet been implemented in Kuwait, although its implementation is currently being discussed at the Ministry of Health.Received tissue samples were stored at − 80 °C. Due to the large number of samples received, all warts per patient were minced together and were subjected to one DNA isolation. Tissue was minced with sterile scissor and forceps on a Petri dish and about 25-mg of tissue was weighed. The tissue was washed in phosphate buffered saline (PBS) and transferred into 1.5-ml Eppendorf tubes. Genomic DNA was extracted from tissue samples using a QIAmp DNA Mini kit  according to the manufacturer’s instructions.HPV type 2, HPV type 1 and HPV type 16 vectors  were used as controls in PCR experiments.The real-time PCR assay was carried out to screen for HPV DNA in the genital warts. Five microlitres (5-μl) of the extracted DNA was combined with 12.5-μl of 2X Syber Green master mix  containing ROX as a passive reference, and 10 pmol of forward and reverse primers . All sets of the primers were custom synthesised by Thermo Fisher Scientific, Waltham, Massachusetts, USA. The mixture was made up to 25-μl volume with nuclease free water . In order to reduce the number of false positive or negative results, samples were analyzed in duplicate on a 96 optical well reaction plate . Positive and negative controls were included in each amplification batch. In order to quantify the HPV in the samples, a five to 10 fold serial dilution of the known positive control DNA was performed alongside the samples. Real-time PCR amplification was carried out in ABI 7500 real-time PCR .The real-time PCR assay was carried out on three different plates. The first plate was used to determine the integrity of the target DNA by β-globin PCR assay, amplifying a target of 268- bp fragment, as described previously by Lum and Le Marchand in 1998 . The nucThe second plate was used to screen for the presence of HPV infection using MY11/GP6 primers from the HPV L1 ORF [The third plate was used to screen for the presence of HPV infection using HVP2/B5 primers from the HPV L1 ORF . The nucFluorescence spectra were recorded during the elongation phase of each PCR cycle. Sequence Detection Software (SDS v1.7) of ABI 7500 real-time PCR was used to generate the amplification curve for each reaction. A dissociation curve was generated after each reaction to differentiate between specific and non-specific amplicons. On the basis of the amplification curve, all samples with HPV amplification starting at any cycle and up to cycle number 40 (with a cut-off line of 0.2) were selected for the analysis. Only samples with a dissociation curve between 70 °C and 80 °C and with a derivative value between 0.100–0.500 were considered HPV DNA positive.Samples showing positive amplification for the presence of HPV by MY11/GP6 were subjected to conventional PCR assay. The expected HPV genotype spectrum to be detected from mucosal warts using MY11/GP6 includes, HR HPV genotypes: 9, 16, 18, 26, 31, 33, 35, 38, 39, 45, 51, 52, 55, 56, 58, 59 and 68, LR HPV genotypes: 6, 11, 40, 42, 53, 54, 57, 66 and 81 [HPV DNA in warts was amplified by nested PCR prior to sequencing, using the AmpliTaq Gold Master Mix  HVP2/B5 primers were used in the first PCR, and CN1F/CN1R, CN2F/CN2R, CN3F/CN3R and C4F/C4R primers  in the sThe PCR products were purified using a PCR purification kit  as per the manufacturer’s instructions. They were then subjected to Sanger sequencing reaction using BigDye terminator v3.1 cycle sequencing mix , and the nested PCR primers described above. Post sequencing PCR purifications were performed to remove unbound fluorescent dye deoxy terminators using BigDye XTerminator™ Purification kit . The samples were denatured for 2 min at 95 °C and immediately chilled on ice and loaded on an ABI 3100 Genetic Analyzer . Samples were electrophoresed on a 50-cm capillary array using POP 6 polymer  as a separation medium.https://blast.ncbi.nlm.nih.gov/) and the Los Alamos Data National Laboratory Theoretical Biology and Biophysics HPV database (https://pave.niaid.nih.gov/).The samples were analyzed using Sequencing Analysis software v 3.7 . The HPV sequence alignment was performed with sequences presented in the GenBank database using BLASTn software  following purification with the Wizard SV Gel and PCR Clean-Up System kit (Promega), and the sequencing of inserts was performed using M13 forward and reverse primers.P value less than 5% was considered statistically significant.Data of the clinical diagnosis, demographic data and virological analysis were tabulated and analyzed using SPSS statistical software ‘Statistical Package for Social Sciences, SPSS version 25.0’ . Descriptive statistics: frequencies/percentages, measures of center and dispersion were calculated. For testing equality of means, the two-sample t-test was used for all continuous variables when normality was held, otherwise Mann Whitney U test was used. For comparing means of three groups, either analysis of variance or Kruskal-Wallis test was used depending on the assumptions being satisfied. Pearson chi square test for independence or Fisher exact test were used to test associations between two categorical variables when the assumptions were satisfied. To model a binary outcome with a set of covariates, logistic regression was implemented, and crude and adjusted odds ratios and their 95% confidence intervals were estimated. All tests were two tailed and a External anogenital wart samples obtained from 156 patients attending outpatient dermatology clinics were included in the study. Anogenital warts included warts taken from penis, pubic area, anus, perianal area and vulva. The mean of their ages was 35.36 years, Standard Deviation (SD) was 9.59, median was 33 years, and range was 16–56 years. The mean of male patient ages was 34 and the mean of female ages was 42.Data revealed that the majority of patients 98.1%) had more than one wart for a duration of more than one month, and of size between one and two centimeters. Of note, 75.6% of the patient partners had no anogenital warts  with demographic data and warts characteristics is shown in Table This study aimed to investigate the relationship between the type specific distribution of HPV genotypes and clinical characteristics of patients with HPV-infected anogenital warts. It is among few that correlate clinical presentation of warts and demographic data of patients with HPV infection. Unexpectedly, for single type infection, HPV 16 was the most common HPV genotype detected in anogenital warts (28.2%), while HPV 6 prevalence was only 10.3%. As commonly known, LR HPV 6 and HPV 11 are mostly encountered for anogenital warts and HR HPV 16 is always encountered in lower prevalence , 7–9.The authors were surprised to find that HPV 11 was not detected in this cohort. This could be due to the low prevalence of this HPV genotype in anogenital warts. This study is the first of its kind in the Arabian Gulf region further studies are needed in Kuwait and in the region to confirm the outcome of this study. Around 83% of patients in this study were men, due to the fact that, in Kuwait, most women with external genital warts are referred to a gyneacology clinic. The findings of this study show that the mean age of patients infected with HPV (35.3 years) or with HR HPV genotypes (36.2 years) was higher than what is documented worldwide .HPV vaccines were first developed to reduce the number of HPV related cancers and precancerous changes in women. For long-term benefit, it was shown that HPV vaccination reduced the number of HPV related diseases in women, including condyloma acuminata of the vagina, vulva and, of course, perianal areas and cervical intraepithelial neoplasia; and, in men, including anogenital condyloma acuminata and penile intraepithelial neoplasia , 24. GarThis study shows that the risk of having a genital wart infected with HPV types 16, 18, 33 or 38 increases in a wart of one to six months duration. This could be due to the fact that HR HPV genotypes, especially HPV 16, have longer time to clearance than other genotypes . Other sThis study shows that oncogenic HPV types are detected in around 35% of patients with genital warts, and are prevalent in warts of one to six months duration.


We reportthe role of visual inspection with acetic acid (VIA) as a triage test forwomen testing positive with the careHPV test onself-collected vaginal samples.Human papillomavirus (HPV) DNA screening reduces cervical cancer incidenceand mortality in low-resource settings. Self-collected vaginal samplestested with affordable HPV tests such as careHPV test on self-collected vaginal samples,provider-collected cervical samples, the Papanicolaou test, and VIA. Allwomen who tested positive for any of the screening tests were evaluated withcolposcopy and guided biopsies, followed by treatment if any cervicallesions were detected. The data from the 377 women who tested positive forHPV in the self-collected vaginal samples were also analyzed to assess theperformance of VIA, conventional cytology, and colposcopy, as triage testsin the detection of cervical cancer and precancerous lesions.As part of a multicountry demonstration study, 5,207 women 30 to 49 years ofage were recruited from urban slums to undergo four cervical screening testsusing the Nineteen percent of women who tested positive for vaginal HPV (V-HPV) alsotested positive with the VIA test; cervical intraepithelial neoplasia 2+lesions were detected in 58% of these women. In the 30 % of the women whotested positive for V-HPV with cytology triage, cervical intraepithelialneoplasia 2+ lesions were detected in 80% of these women. The colposcopyreferrals for women who tested positive for V-HPV were reduced from 7.6% to1.5% by VIA triage, and to 2.3% by cytology triage. Although the sensitivitywas reduced, the positive predictive value improved after triage with VIAand cytology.This study reflects the optimal role of VIA triaging for treatment selectionof lesions among those who test positive for V-HPV in screen and treatscreening programs that use an HPV test in low-resource settings. Visual inspection with acetic acid (VIA)—a simple test thatcan be performed easily by trained mid-level health care providers—is anoptimal screening method for a single-visit screen-and-treat approach forresource-constrained countries. In a recent meta-analysis, the sensitivity of VIAfor the detection of high-grade cervical lesions ranged from 41% to 92% because ofthe test's subjectivity.3Similarly, specificity ranged from 49% to 98%.3 The human papillomavirus (HPV) DNA test is a more objectiveand sensitive test than VIA and cytology, and it is suitable for population-basedscreening in India.4 A single roundof HPV DNA screening is associated with a reduction in cervical cancer incidence andmortality in low-resource settings.5Screening services are inadequate in remote and rural areas, and cytology-basedscreening test performance is suboptimal. Cervical cytology (the Papanicolaou [Pap]test) has been implemented widely in developed countries, but it is not suitable forareas with limited resources because of the test's complexity, the lack ofcytotechnologists to review the slides, and the test’s suboptimalsensitivity.careHPV test.6Self-collection of vaginal samples for HPV DNA testing is acceptable towomen7 and is effective forthe detection of high-grade cervical lesions.4 A strategy that is based on self-collected vaginal samplesfor HPV testing could increase access to cervical screening and improve populationcoverage, especially in rural and remote areas. Because women who test negative forHPV have a negligible risk of developing cervical cancer over a 5- to 10-yearperiod, screening intervals may be increased significantly in women with a negativeHPV screening result.8 A positiveHPV test indicates the presence of infection with any of the 14 types of high-riskoncogenic HPV types. However, the specificity of the test is not optimal because ofthe occurrence of transient infections without any cervical lesions. Therefore, themanagement of women who screen HPV-positive requires a triage test to identify anddiagnose the precancerous or cancerous lesions requiring treatment. The inclusion ofa second test for triaging women whose primary screen is HPV-positive in screeningprotocols may also reduce unnecessary referrals for diagnosis and treatment, whichcould decrease the burden on the weak health care systems in areas with limitedresources.One affordable, simple, and rapid HPV DNA test on the market is the9 WHOguidelines recommend several options for primary screening, including HPVgenotyping, cytology, or VIA triage for follow-up of women who test positive for HPVinfection.10 However,using VIA as a triage option for primary HPV testing has not been investigatedextensively in field studies in developing countries. The careHPVtest has been evaluated in comparison with other screening tests in a real-lifefield setting in the urban slums of Hyderabad (the Screening Technologies to AdvanceRapid Testing–Utility and Program Planning [START-UP] project) in SouthIndia.4 Using data fromthis community-based cervical screening program, we report on the performance of VIAand cytology as triage tests in the detection of cancer and precancers in women whotest positive with the careHPV DNA test using self-collectedvaginal samples.Screening protocols from wealthier countries recommend using HPV testing as theprimary screening test and cytology as the triage test to improvespecificity.careHPV test with VIA and Pap smears to detect cervical cancerand precancerous lesions was conducted in Hyderabad, India, from January 2010 toDecember 2013. This study was approved by institutional ethics committees of the MNJInstitute of Oncology and the Program for Appropriate Technologies in Health (PATH),in the United States. The comparative results of all four screening tests in thedetection of cervical intraepithelial neoplasia (CIN) 2+ lesions have beenpublished.4 The data from377 women who tested positive for vaginal HPV (V-HPV) in this study were alsoanalyzed to evaluate the performance of VIA, cytology, and colposcopy as triagetests in the detection of CIN 2+ lesions.As a part of the international START-UP project, a comparative evaluation of thecareHPVtesting in privacy using a soft-bristled brush. The samples were transported inDigene collection media . They then underwentspeculum examination to collect cervical samples for the careHPVtest. A Pap test and VIA were performed sequentially on all the women by trainedhealth workers. Women with positive results on any of the screening tests underwentcolposcopy and guided biopsies for histopathologic confirmation by a trained medicalofficer. Women who tested VIA-positive underwent colposcopy and biopsy in the samevisit, but women who were HPV- and cytology-positive were informed and laterexamined after the results became available, which ranged from 1 to 4 weeks. Biopsyspecimens were taken from colposcopically abnormal areas or at the 12 o'clockposition if there was no visible acetowhite lesion. Women with confirmed CIN grade2+ lesions were either treated with cryotherapy at the local clinic or referred to ahospital for additional evaluation and treatment.Five thousand two hundred seven women, from 30 to 49 years of age, were recruitedfrom the general population residing in urban slums to undergo cervical cancerscreening. The participants were married, non-pregnant women with an intact uterus.They had not been diagnosed previously with cervical cancer or precancer, werewilling to undergo screening, and were able to give informed consent. Localcommunity motivators conducted health education sessions and group discussionsbefore the cervical screening took place. Women were invited for screening either inthe outreach community screening clinic or in the makeshift mobile screening campsnear their localities. All eligible women who consented were offered four screeningtests during the same screening visit. First, they were instructed by the healthworker on how to take a self-collected vaginal sample for careHPV testing were transported tothe base hospital and stored at room temperature (15°C to 30°C) for amaximum of 14 days, at 2°C to 8°C for a maximum of 30 days, or at−20°C for a maximum of 60 days. The careHPV testresults were quantified as a ratio of viral load (expressed in relative light units)to the mean relative light units from a positive control set at a 1 pg/mL cutoff.The equipment had been set by the manufacturer to call a sample positive if theratio was ≥ 1.0 pg/mL. A local pathologist who was aware that the sampleswere part of a study to compare multiple screening options examined the Pap testsamples, maintaining quality assurance. The samples were evaluated according to theBethesda classification system; any smear with atypical squamous cells ofundetermined significance or more severe changes was considered positive. Women withthe appearance of acetowhite areas in the transformation zone with 4% acetic acidwere classified as VIA positive by the trained health workers.Vaginal and cervical samples for Data entry and analysis were performed using Stata software, version 12.0 . Sensitivity, specificity, predictive values, and 95% CIs forthe screening tests were calculated using two-by-two tables and standardformulas.careHPV testing were collected from 4,947 women. Samples wereself-collected by 85% of these women. Twenty-four participants with inadequatesamples and eight with missing samples were excluded from the analysis.Out of the 5,207 women recruited for screening, self-collected or vaginal samples forcareHPV test for V-HPV was positive in 377 women. The mean ageof the women who were V-HPV positive was 36.3 years , and 15% were postmenopausal  among the women who were V-HPVpositive were detected by both VIA and cytology triage, but 13% were missed by bothtests. VIA triage alone detected 58% of CIN 2+ and 73% of CIN 3+ lesions, whereascytology detected 80% and 88%, respectively. VIA detected all 13 invasive cancers,but cytology missed one 7.7%; . Only72careHPV, is available for cervical cancerprevention in low- and middle-income countries. Our study reflects the data from areal-life limited resource setting. If careHPV testing onself-collected vaginal samples is used as a primary screening test in a populationsuch as that in the low socioeconomic slums of Hyderabad where it is readilyacceptable, only a limited number of women would be referred for additionalevaluation, and most CIN2+ lesions would be detected. Self-sampling has theadvantage of being better accepted because it does not require a pelvic examinationand thus improves screening coverage.10 Although approximately 5,000 women were screened in our study,only 377 women (7.5%) who were V-HPV positive would be referred to the healthfacility for a pelvic evaluation, which is a more manageable number when resourcesare limited. In resource-constrained settings, a primary HPV screening method withlow PPV would lead to excessive referrals of women with transient or insignificantHPV infections, and to the treatment of nonprogressive cervical lesions, resultingin overtreatment if used as a screen-and-treat method. Because triaging of thesewomen with quality-assured cytology or genotyping may not be possible because ofcost implications, alternatives such as VIA should be the choice. The WHO expertpanel also recommends VIA triage of women who test positive for HPV as ascreen-and-treat strategy in low- and middle-income countries, especially whereresources are limited or quality assurance is not maintained for cytologyprograms.11With the emergence of HPV DNA testing as a primary screening modality, a promisingand affordable new test, 12 in a study in India,observed that detection rates of CIN 2+ lesions were similar when women who testedpositive for HPV were triaged with either cytology or VIA triage tests. VIA triagesignificantly improved the PPV, which was comparable to that of cytology triaging,and it also reduced the colposcopy referral rate by 59%; however, it missed 18% ofCIN 2+ lesions.12In our study, the detection rate of CIN 2+ lesions was similar with both cytology andcolposcopy triage and was higher than that with VIA triaging. The colposcopyreferral rate would be reduced to six times in VIA triage and five times in cytologytriage for these women at risk. A limitation of our study is that all the women whotested positive for V-HPV did not undergo diagnostic confirmation, and it was alsoobserved that 80% and 95% of these women were cytology and VIA negative,respectively. In addition, approximately 17% of women who were cytology-positive andwho required colposcopy did not undergo diagnostic confirmation because there was awaiting period of 3 to 4 weeks for cytology results. In this study, VIA triaging ofwomen who tested positive for V-HPV were detected by primary HPV testing alone witha sensitivity of 51.47% . Muwonge13 The loss of sensitivity after triaging was high in thisstudy as well, and the VIA triage missed 31.6% of the CIN 3+ lesions detectedoriginally by the HPV test alone.13 Similar observations were also found in a randomized controlledtrial from sub-Saharan Africa that used self-collected samples.15It is crucial that the triaging strategy not reduce the sensitivity of the primaryscreening test. The sensitivity of detection of CIN 3+ lesions with VIA triage inour study was also reduced significantly because of its questionable usefulness.Moreover, VIA or cytology triage improved the PPV. In another large HPV-basedscreening program conducted in West Bengal, India, VIA triaging of women who testedpositive for HPV using provider-collected cervical samples improved the PPV fordetection of CIN 3+ lesions by almost 30%.17 VIA triage should be preferred inlow-resource areas because the necessary consumables like acetic acid are readilyavailable, trained nurses can perform the test, and the results are availableimmediately, which would allow triage and treatment in a single visit.Although VIA triage in our study failed to detect one half of the high-grade lesions,it effectively detected three quarters of the CIN 3+ lesions and all invasivecancers that required immediate treatment. If VIA were to be used to triage womenwho are HPV positive and to determine who should be referred for additionaltreatment and for treatment selection, as in our study, 72 women would have beenselected for additional diagnostic evaluation and treatment. Overall, 40% of thewomen with CIN2+ lesions that were detected originally by primary HPV screeningwould be missed for treatment by VIA triage. In contrast, a high-quality Pap testtriage such as that used in our study would miss 21% of the CIN 2+ cases detected byHPV testing alone. The lesions missed by VIA triage included only 27% of lesions atthe CIN 3+ threshold and 74% of CIN grade 2 lesions. Therefore, VIA triageeffectively detects most CIN 3 lesions and all invasive cancers but fails to detectthe majority of CIN 2 lesions. Most of these CIN grade 2 lesions undetected by VIAtriage could be either nonprogressing or regressive lesions requiring closefollow-up because V-HPV positivity indicates a higher risk of developingneoplasia.18Large prospective studies using this strategy with longitudinal follow-up will berequired to establish its effectiveness in low- and middle-income countries.Hence, if VIA were to be used to triage women who test positive for HPV, it coulddetermine who should be referred for additional treatment and for treatmentselection of high-grade cervical lesions. Therefore, VIA triage could be consideredan optimal component of a screen-and-treat strategy in limited settings where womenwho are VIA positive can receive immediate treatment, and in which self-sampling,combined with an affordable and field-friendly HPV test, is used as the primarycervical screening method. Adopting this strategy would not only improve screeningcoverage, but also benefit women at risk by allowing treatment in the same visit,minimizing the loss of these women at risk to follow-up and treatment delays becauseof the need for multiple visits.