Cervical human papillomavirus (HPV) infection is a dangerous disease, whose subtypes exhibit different distribution patterns in particular countries, regions, and races. In this study, the HPV distribution in patients with cervical precancerous lesions in Western China was investigated to assess a probability for prevention of cervical cancer and the clinical application of an HPV vaccine in China. The retrospective study of patients with different HPV subtypes and cervical precancerous lesions, who have undergone loop eelectrosurgical excision procedure, cold knife conization, or a total hysterectomy in our hospital from January 2016 to August 2016, was performed. All patients were tested for 27 HPV subtypes via the liquid suspension chip technology (Luminex 200). A total of 3393 cases of cervical intraepithelial neoplasia (CIN) were investigated, including 1098 cases of CIN I, 762 cases of CIN II, and 1533 cases of CIN III. The overall HPV infection rate was 82.58%. The high-risk HPV infection rate was 76.61%, and the low-risk rate was 9.88%. The most common 5 subtypes were HPV16, HPV52, HPV58, HPV33, and HPV18. The patients were grouped into 6 age groups: ≤20, 21 to 30, 31 to 40, 41 to 50, 50 to 64, and ≥65. The HPV subtypes’ distribution varied across different age groups. In patients with cervical precancerous lesions in Western China, the top 5 HPV subtypes with the highest infection rates were HPV16, HPV52, HPV58, HPV33, and HPV18. The rate of cervical precancerous lesions unrelated to HPV was 17.42%. Thus, HPV screening with no cytology may leave unobserved about 20% of cervical precancerous lesions, which is worth of significant clinical attention. It is the fourth most common cancer in women worldwide.Cervical cancer is a serious threat to female health. In 2015, there were 98,900 new cases of cervical cancer and 30,500 cervical cancer-related deaths in China. In general, it takes approximately 10 to 20 years of precancerous lesions before cervical cancer develops. Therefore, standard and reasonable early screening and early treatment may effectively reduce the incidence of cervical cancer and improve survival and life quality for thousands of female patients.Cervical intraepithelial neoplasia (CIN) is a one-way pathophysiological process with 2 distinct outcomes. The first one is a spontaneous regression of the cervical lesions after evolution, which, in general, does not develop into cervical invasive carcinoma. The second outcome is the potential occurrence of cancer during the development of cervical lesions: if not timely screened or treated properly at early stages, it may develop into invasive carcinoma. Studies have shown that the probability for CIN I, CIN II, and CIN III to develop into cervical cancer are15%, 30%, and 45%, respectively, and occasionally, CIN I and CIN II may directly develop into cervical invasive carcinoma without turning into CIN III first.–6 As HPV infection rates and distribution of different HPV types are known to vary significantly in different countries and regions,–12 their investigation in specific areas is expedient for providing a scientific basis for the measures and methods aimed at distribution minimization and more effective treatment of cervical cancer, including the development of an HPV vaccine. Thus, the results obtained would be instrumental for developing more effective prevention and control strategies for the regional cervical cancer treatment. Cervical cytology and HPV tests are widely used for cervical cancer screening and can effectively reduce its incidence. However, the incidence of cervical cancer in China exhibits an increase and shifts to younger age groups. This can be attributed to the fact that China is a developing country, whose citizens are not fully aware of the importance of screening for cervical cancer, and many areas do not yet have universal cervical cancer screening. In this study, the HPV subtypes in patients with cervical precancerous lesions, who were treated in the West China Second University Hospital of Sichuan University  from January 2016 to August 2016, were retrospectively analyzed.The main prerequisite for development of cervical cancer and precancerous cervical lesions is persistent infection with high-risk human papillomavirus (HPV).22.1The study subjects were patients with cervical precancerous lesions, who were treated in our hospital from January 2016 to August 2016, regardless of age. These patients have undergone loop eelectrosurgical excision procedure, cold knife conization, or a total hysterectomy. Any patients with a medication treatment history for HPV infection, with cervical surgery and/or hysterectomy history, with liver, kidney, or systemic diseases, and any patients treated with vaginal medication within a week were excluded from the analysis.2.2The retrospective studies were conducted, where each patient was placed in the lithotomy position and sterile cotton swabs were used to clean mucus. A dry, sterile cotton swab was inserted into the cervix and rotated clockwise for 5 to 8 circles, and the exfoliated cervical cells were stored in a cell preservation solution. A kit  for purifying DNA from it produced by Qiagen was used to extract DNA from the specimens. Afterward, the extracted DNA was dissolved in 50 μL of elution buffer and stored at −20 °C for use. The purified DNA was placed in the genotyping kit, namely, the HPV nucleic acid typing detection kit (flow fluorescence hybridization) produced by Qiagen, including 17 high-risk types  and 10 low-risk types . The hybridization products were tested by using a Luminex 200 , which is a multifunctional suspension array system.+), CIN II, CIN II or worse (CIN II+), CIN III, or CIN III or worse (CIN III+). Any patients with negative or invasive cervical cancer were excluded from the study. As this study is retrospective, no ethical committee approval was required for its conduction, which is in compliance with the institutional and national policies concerning research approvals.All patients underwent a colposcopy, and a cervical biopsy was performed. Two independent pathology specialists reviewed the paraffin-embedded tissue sections of cervical precancerous lesions. Cervical histopathological examination results were grouped into the following grades: CIN I, CIN I or worse  was used for the data analysis. The measurement data were represented as x ± s (mean ± standard deviation) and analyzed with the 33.1The basic characteristics of the research subjects are shown in Table 3.2The distributions of HPV subtypes in patients with CIN are tabulated in Table 3.3The distribution of HPV subtypes among patients from different age groups is presented in Tables Among the CIN II+ patients, the HPV infection was highest among patients of age ≥65, with a value of 100%; the most common 5 subtypes were HPV33, HPV58, HPV16, HPV51, and HPV52, a indication which holds for all patients. The lowest infection rate was for patients of age ≤20, with a value of 75%, and the most common 5 subtypes were HPV51, HPV33, HPV55, HPV59, and HPV66, which was also relevant for all patients. The distribution of HPV subtypes exhibited a variation among age groups.3.4The multiple infections of HPV subtypes are tabulated in Table 4Persistent infection with high-risk HPV is a prerequisite for the occurrence and development of cervical cancer and precancerous lesions in women. The distribution of HPV subtypes varies across different countries and even across different regions of a country, and the cervical precancerous lesions and cervical cancer related subtypes are different as well. Therefore, an analysis of the HPV subtypes of different ethnic groups in a country or a region is critical for the prognosis evaluation, as well as for making review plans for outpatients, clinical treatment plans for inpatients, and developing preventive and therapeutic HPV vaccines. A study conducted by Xiao et al on HPV infections in patients with cervical precancerous lesions in the Beijing area demonstrated that the high-risk HPV infection rates are 38.1% and 32.4%, respectively, for CIN I and CIN II+ patients, and the most prevalent HPV subtypes for CIN I patients were HPV16, HPV58, HPV52, and HPV18. In contrast, the most prevalent ones for CIN II+ patients were HPV16, HPV58, HPV33, and HPV52. The reports on HPV infection in patients with CIN in the Western China are scarce, and the sample sizes of these studies are nonrepresentative.–17 Thus, to develop reasonable screening and treatment plans for patients with HPV infections in the Western China, as well as provide evidence for the development of preventive and therapeutic HPV vaccines, we treated patients with cervical precancerous lesions admitted in our hospital from January 2016 to August 2016 as our study subjects and analyzed their HPV infection subtypes. The study demonstrated the following results: the overall HPV infection rate was 82.58%, and the high-risk HPV infection rate was 76.61%. The most common 5 subtypes were HPV16, HPV52, HPV58, HPV33, and HPV18, and 17.42% of the patients were HPV negative. Among the CIN II+ patients, the HPV infection rate was 84.97%, and the most common 5 subtypes were the same as for all patients, and 15.03% of the patients were HPV-negative. Except for the fact that HPV16 had the highest infection rate, the infection rates of other subtypes were quite different.Studies have shown that the overall HPV infection rate is 38.1% in the Henan area, and HPV16 was the most common 1 among all subtypes, with a value of 8.9%.–20 Our study showed higher HPV infection rates among women over 40 years of age, and lower one for younger ones. This result is consistent with the high incidence of cervical cancer in women aged 45 to 60 in China. Therefore, it has become particularly important to disseminate information about cervical diseases in elder women, as well as perform HPV infection screening for this age group.The HPV infections were different for patients of different age groups. In addition, the human immune system may eventually clear the virus, and clinically, the HPV test result would be negative, even if cervical lesions have formed by that time. HPV has proven to be the primary cause of cervical lesions, but it is not the only one. The occurrence and development of cervical lesions is the result of multiple factors. In this study, 17.42% of patients were HPV-negative, and 15.03% of patients with CIN II+ were HPV-negative.Previous studies have revealed that 80% of women have been infected with HPV throughout their lives; nevertheless, most of them were transient infections. The virus can be cleared by autoimmune mechanisms, usually in 8 to 24 months after infection. Only a small number of patients suffer from a persistent viral infection that further develops into lesions.In summary, the top 5 HPV subtypes with the highest infection rates in cervical precancerous lesions patients in the Western China were HPV16, HPV52, HPV58, HPV33, and HPV18. The HPV infection and its distribution varied across different age groups. Some patients with cervical precancerous lesions could not be detected through HPV screening. Therefore, solo HPV screening without cytology may miss approximately 20% of patients with cervical precancerous lesions, which requires additional clinical attention. This particular research effort being a single-center and retrospective study with a small sample size, its results should be confirmed and generalized by further large-sample, multicenter, prospective studies.


The local immune response is considered a key determinant in cervical carcinogenesis after persistent infection with oncogenic, high-risk human papillomavirus (HPV) infections. Genetic variation in various immune response genes has been shown to influence risk of developing cervical cancer, as well as progression and survival among cervical cancer patients. We reviewed the literature on associations of immunogenetic single nucleotide polymorphism, allele, genotype, and haplotype distributions with risk and progression of cervical cancer. Studies on HLA and KIR gene polymorphisms were excluded due to the abundance on literature on that subject. We show that multiple genes and loci are associated with variation in risk of cervical cancer. Rather than one single gene being responsible for cervical carcinogenesis, we postulate that variations in the different immune response genes lead to subtle differences in the effectiveness of the antiviral and antitumour immune responses, ultimately leading to differences in risk of developing cervical cancer and progressive disease after HPV infection. Infection with human papillomavirus (HPV) is highly common across human populations. Worldwide, prevalence estimates of HPV infection among women range from 2% to 44% . InvasivApproximately 200 HPV types have been identified to date, with new types constantly being discovered. Types may differ in tissue tropism and may preferentially infect skin or mucosa. Certain HPV types are pathogenic, leading to a variety of benign conditions  as well as malignant disease  , 4. HPV Although infection and colonisation of the cervical epithelium by oncogenic, high-risk human papilloma viruses (hr-HPVs) are prerequisites for the development of cervical cancer, the local immune response is thought to be an important determinant of progression and disease outcome . The higGenetic variation in various immune mediators has been shown to be an important determinant in susceptibility to a wide variety of autoimmune disorders and neoplasms, as well as in progression and disease outcome –10. ThisA systematic search in the NCBI PubMed bibliographic database and HuGE navigator was conducted . Since mInformation on allele or genotype frequencies and, if available, odds or hazard ratios with associated 95% confidence intervals were extracted from the studies. If unavailable in the original studies, these ratios were calculated along with the population attributable faction (PAF), according to Miettinen's formula .Cytokines play a crucial role in mounting and maintaining immune responses against a host of pathogens, including viral infections and tumours . Though  IL1B gene (c.-511C>T) encoding interleukin-1 beta , which has been demonstrated to be associated with cervical carcinoma risk in Korean, North Indian, Chinese Han, and Egyptian populations [Although several polymorphisms in the genes encoding various interleukins have been described in relation to cervical carcinoma and its precursor lesions –63, the ulations –15. Two ulations , 64. TNF gene, encoding tumour necrosis factor (TNF), have been described in relation to HPV infection and cervical neoplasia. TNF is a key proapoptotic cytokine, involved in cell proliferation and differentiation [ TNF gene polymorphisms, including microsatellite polymorphisms, is available but conflicting, probably due to small sample sizes [Several polymorphisms in thentiation . Variatintiation . Howeverntiation , 18, 66.ntiation . Data rele sizes –70.γ) plays an important role in antiviral immunity [ IFNG gene, has been shown to be associated with altered levels of IFNγ production in response to immunogenic stimuli, with minor allele carriers having decreased IFNγ production [Interferon-gamma , which regulates the biological activity of the two potent proinflammatory cytokines interleukin-1α and interleukin-1β [ IL1B gene. In contrast, a study of Austrian patients with high-grade CIN lesions demonstrated no significant association with the IL1RN VNTR polymorphic site [Theeukin-1β . Most ofeukin-1β , 77, bothic site . Unfortu IL10 c.-1082A>G polymorphism has been shown to influence levels of IL-10 production, with major allele homozygosity being associated with low IL-10 levels, heterozygosity with intermediate levels, and minor allele homozygosity with high levels [ IL10 promotor SNP (c.-592C>A), with occurrence of the minor allele associated with increased cervical cancer risk, especially among Asian patients [ IL10 polymorphisms in HPV and cervical cancer might be tied to the stage of lesions.Interleukin-10 (IL-10) has both immunosuppressive and antiangiogenic effects and may therefore exert both tumour-promoting and antitumour effects , 80. Assh levels , 83–86. h levels . Howeverpatients . This aspatients , 26–28. patients . It is t CXCL12, have been reported in relation to cervical carcinoma. Chemokine CXCL12, also known as stromal-cell derived factor 1 (SDF-1), directs leucocyte migration and, through interactions with its receptor CXCR4 [ CXCL12 SNPs in cervical cancer and identified one intronic SNP (rs266085), where the minor allele was associated with a decreased risk of cervical cancer. Using haplotype interaction analysis, this group identified a combination of three SNPs , which was associated with cervical cancer risk [ CXCL12 gene is consistent with several possible aetiologic mechanisms for the identified association with cervical carcinoma, including alternative splicing and regulation of CXCL12 induction [ CXCR4 gene, which lead to increased cellular responsiveness to CXCL12, yielding a rare syndrome characterised by immune deficits and extensive HPV-induced lesions [To date, polymorphisms in only one chemokine gene,or CXCR4 , it is aor CXCR4 , 90. Onecer risk . The locnduction –93. In cnduction . Stronge lesions , 95.Receptors bind a wide variety of ligands, including cytokines, costimulation transmembrane proteins, and pathogen-associated molecular patterns (PAMPs). Due to the different ligands and functions associated with them, many different receptors have been found to be associated with antitumour immunity. Genetic variation in the corresponding genes has been found to be strongly associated with HPV infection and subsequent cervical carcinoma. FAS gene have been identified: c.-1377G>A and c.-670G>A  . Two SNP.-670G>A . These Subgroups . The c.-ubgroups  but has ubgroups . FASL gene has been investigated for association with cervical cancer ( FASL c.-844T>C lies within a putative binding motif for CAAT/enhancer-binding protein (C/EBPβ) and the two resulting alleles have different affinities for C/EBPβ. This SNP has been demonstrated to have functional consequences: minor allele homozygosity has been linked to increased FASL expression and alteration of FASL-mediated signalling in lymphocytes [ FASL c.-844T>C SNP was not associated with cervical carcinoma risk [One SNP in thel cancer . FASL c.phocytes . Consequphocytes . Howeveroma risk .Toll-like receptors (TLRs) are transmembrane proteins that recognise pathogen-associated molecular patterns (PAMPs), the conserved structural motifs in pathogenic organisms . TLRs ar TLR2 c.+613T>C SNP showed association with cervical cancer in a Costa Rican population [Toll-like receptor 2 (TLR2) is a pattern recognition receptor that senses the presence of bacterial lipoproteins and other components of bacteria and fungi . Howeverpulation . TLR4 c.+936C>T polymorphism was associated with stage II cervical cancer in a North Indian population [TLR4 plays an important role in recognising lipopolysaccharide molecules present in Gram-negative bacteria; however, as in the case of TLR2, recognition of viruses has also been implicated . Heterozpulation . TLR9 polymorphisms may therefore influence HPV persistence and cervical carcinogenesis. The TLR9 c.+2848G>A SNP was shown to be associated with risk of cervical carcinoma, with the minor allele associated with an increased cancer risk among Chinese Han women [ TLR9 c.-1486T>C SNP has been associated with cervical cancer risk in various studies [Toll-like receptor 9 (TLR9) recognises nonmethylated CpG DNA sequences, ubiquitous in bacterial and viral genomes, for instance, HPV16. Viral oncoproteins E6 and E7 block the expression of this receptor, thereby contributing to HPV immune evasion . TLR9 poan women . Similaran women . Similar studies , 40. LMP7, TAP2, and ERAP1 have been found to be associated with cervical carcinoma risk, lymph node metastases, and overall survival, both individually and in specific haplotype configurations [The antigen-processing machinery (APM) is responsible for the generation, trimming, transport, and loading of peptides derived from intracellular proteins to be presented by molecules of the human leucocyte antigen (HLA) family to cytotoxic T lymphocytes . The maiurations , 45. Thourations –44, 107.There is a great diversity of immunogenetic associations with HPV infection, persistence, and cervical neoplastic transformation, based on the overwhelming body of continuously growing data. Contradicting results indicated by different studies attest to the complexity of the topic. Moreover, the wide variation in sample size amongst the various studies can lead to underestimation of the actual strength of association of particular loci with disease . MultiplAn emerging field in genetic association studies is that of haplotype interaction analysis, which investigates the association of specific SNP combinations (often spanning multiple chromosomes) with particular diseases . This is MAL/CADM1 methylation pattern, p16INK-4a/Ki-67 dual immunostaining, and viral integration [The increasingly popular genome-wide association study (GWAS) approach for the purpose of identifying polymorphisms of interest appears promising for the purpose of elucidating the susceptibility to persistent HPV infection as well as progression of cervical neoplasia , 110. Alegration . These megration . ElucidaUltimately, by combining data regarding HPV infection and distribution with host genomic data, it may be possible to make individual “predictions” not just of the risk of developing cervical cancer and its progression but also of the efficacy of therapies and, equally important, the efficacy of anti-HPV vaccination programmes, all of which will ultimately facilitate the development of tailor-made, personalised interventions.


Several countries are implementing a transition to HPV testing for cervical screening in response to the introduction of HPV vaccination and evidence indicating that HPV screening is more effective than cytology. In Australia, a 2017 transition from 2-yearly conventional cytology in 18–20 to 69 years to 5-yearly primary HPV screening in 25 to 74 years will involve partial genotyping for HPV 16/18 with direct referral to colposcopy for this higher risk group. The objective of this study was to determine the optimal management of women positive for other high-risk HPV types (not 16/18) ('OHR HPV').We used a dynamic model of HPV transmission, vaccination, natural history and cervical screening to determine the optimal management of women positive for OHR HPV. We assumed cytology triage testing was used to inform management in this group and that those with high-grade cytology would be referred to colposcopy and those with negative cytology would receive 12-month surveillance. For those with OHR HPV and low-grade cytology , we evaluated (1) the 20-year risk of invasive cervical cancer assuming this group are referred for 12-month follow-up vs. colposcopy, and compared this to the risk in women with low-grade cytology under the current program ; (2) the population-level impact of the whole program, assuming this group are referred to 12-month surveillance vs. colposcopy; and (3) the cost-effectiveness of immediate colposcopy compared to 12-month follow-up. Evaluation was performed both for HPV-unvaccinated cohorts and cohorts offered vaccination (coverage ~72%).The estimated 20-year risk of cervical cancer is ≤1.0% at all ages if this group are referred to colposcopy vs. ≤1.2% if followed-up in 12 months, both of which are lower than the ≤2.6% benchmark risk in women with low-grade cytology in the current program (who are returned for 12-month follow-up). At the population level, immediate colposcopy referral provides an incremental 1–3% reduction in cervical cancer incidence and mortality compared with 12-month follow-up, but this is in the context of a predicted 24–36% reduction associated with the new HPV screening program compared to the current cytology-based program. Furthermore, immediate colposcopy substantially increases the predicted number of colposcopies, with >650 additional colposcopies required to avert each additional case of cervical cancer compared to 12-month follow-up. Compared to 12-month follow-up, immediate colposcopy has an incremental cost-effectiveness ratio (ICER) of A$104,600/LYS  in unvaccinated women and A$117,100/LYS  in cohorts offered vaccination .In primary HPV screening programs, partial genotyping for HPV16/18 or high-grade triage cytology in OHR HPV positive women can be used to refer the highest risk group to colposcopy, but 12-month follow-up for women with OHR HPV and low-grade cytology is associated with a low risk of developing cervical cancer. Direct referral to colposcopy for this group would be associated with a substantial increase in colposcopy referrals and the associated harms, and is also cost-ineffective; thus, 12-month surveillance for women with OHR HPV and low-grade cytology provides the best balance between benefits, harms and cost-effectiveness. Several countries are currently evaluating or implementing a transition from cytology to primary HPV testing for cervical screening,2,3, basAustralia was the first country to initiate a national publicly-funded vaccination program in 2007. Three dose vaccination uptake is 72–73% in 12–13 year old females; catch-up in 18–26 year old females (conducted from 2007–9) achieved coverage rates of the order of 30–50%. After thRenewal. This commenced in 2011, and led to an evaluation of the evidence in 2013, including modelled evidence of the impact of the renewed program in both unvaccinated cohorts and in cohorts offered vaccination, which was performed on behalf of the Australian Government’s Medical Services Advisory Committee (MSAC)., is expected to provide direct evidence in the future on the outcomes in women with OHR HPV and ASC-US/LSIL cytology within a primary HPV screening program. Compass is a large scale randomised controlled trial of 5-yearly HPV versus 2.5 yearly image read LBC cytology screening in women aged 25–69 years, and it is being conducted in the state of Victoria, Australia. HPV screening in the trial incorporates the use of partial genotyping and 12-month follow-up for women testing positive for high-risk HPV (not 16/18) and cytology ASC-US/LSIL, as considered in the current evaluation. Recruitment is stratified according to whether women are in age cohorts that were offered vaccination. In HPV-screened women, a secondary randomisation process for women testing positive for other high-risk HPV types (i.e. not HPV16/18) is implemented, and these women are randomised to be triaged either with LBC or with dual-stained p16/Ki67 cytology . In conjunction with the implementation of the renewed NCSP, Compass will provide emergent evidence both on the performance of LBC triage of women with OHR HPV infections, but also data on new options for management in this group. In the future, this is expected to provide the basis for further review and, if warranted, consideration of the role of other options for management.In our evaluation, we assumed that the HPV test technologies would provide information separately for HPV types 16/18, but that other high-risk HPV types would be managed in the same way as each other. However, there is evidence to suggest that there is heterogeneity in the risk in women positive for types other than 16/18 –for instance, the risk of high-grade CIN in women positive for HPV types 33 and 31 is significantly higher than some of the other high-risk HPV types, and moreTo our knowledge, this is the first detailed evaluation of the management of women testing positive for high-risk HPV (not 16/18) in a primary HPV screening program. These findings are currently informing the development of new clinical management guidelines in Australia and willWe conclude that 12-month surveillance of women who test positive for other high-risk HPV (not 16/18) and cytology ASC-US/LSIL, appears to provide the best balance of benefits, harms and cost-effectiveness in the context of partial genotyping for HPV 16/18 within the new Australian primary-HPV based screening program.


CC) is the leading cause of cancer‐related death among women in sub‐Saharan Africa, primarily because of limited access to effective screening and preventive treatment. Our aim was to assess the feasibility of a human papillomavirus (HPV)‐based CC screen‐and‐treat approach in a low‐resource context. We recruited 1012 women aged 30–49 years through a CC screening campaign conducted in the District Hospital of Dschang, Cameroon. Participants performed HPV self‐sampling, which was tested for high‐risk HPV (HR‐HPV) DNA using the point‐of‐care Xpert HPV assay. All HPV‐positive women were invited for visual inspection with acetic acid and Lugol's iodine (VIA/VILI) to exclude CC or enable triage. A cervical sample for histological analysis was also collected. Women positive for HPV 16/18/45 and for other HR‐HPV with pathological VIA/VILI were selected to undergo treatment with thermocoagulation. The HPV prevalence in the study population was 18.5% (n = 187); of these cases, 20 (10.6%), 42 (22.3%) and 140 (74.9%) were positive for HPV16, HPV18/45 and other HR‐HPV types, respectively. Overall, 107/185 (57.8%) VIA/VILI examinations were classified as pathological and 78 (42.2%) as normal. Women positive for HPV16/18/45 were 4.2 times more likely to harbor cervical intraepithelial neoplasia grade 2 or worse (CIN2+) than those with other HPV types. The specificity of HPV 16/18/45 genotypes for detection of high‐grade lesions among HR‐HPV positive women was higher than that of VIA/VILI in all age groups. The sensitivity and specificity of VIA/VILI in detecting CIN2+ among HPV positive women were 80% and 44%, respectively. Overall, 110/121 screen‐positive women (90.9%) were eligible for, and were treated with, thermocoagulation. An HPV‐based screen‐and‐treat approach is feasible in a low‐resource context and may contribute to improving the effectiveness of CC prevention programs. Immediate thermocoagulation treatment for women who are HPV16‐ and/or HPV18/45‐positive is a practical approach for the treatment of CIN2+. The combination of HPV‐testing and VIA/VILI for CC screening might reduce overtreatment.Cervical cancer ( Cervical cancer (CC) is the second most common cancer in women worldwide, and is a leading cause of cancer death in women in low‐resource countries such as Cameroon, despite being largely avoidable. Although cytology‐based CC screening programs have successfully reduced CC incidence in high‐income countries, such dramatic reduction in incidence has not been observed in low‐ and medium‐income countries (LMIC) Recent studies have shown that HPV‐based screening has a greater sensitivity than conventional cytology‐based screening programs for the detection of precancerous lesions and CC ®, hereafter referred as “Xpert”) has become available. This method uses real‐time PCR to detect HR‐HPV DNA and identify genotype . Xpert is a quick (1‐hour), non‐batch HPV assay that could facilitate same‐day screening and management strategies Recently, a point‐of‐care assay . Women were included if they were aged 30–49 years, understood the study procedures and voluntarily agreed to participate by signing an informed consent form. Exclusion criteria were pregnancySocio‐demographic data and gynecological and obstetrical history were collected to evaluate the association of risk factors with HPV infection. After being informed about HPV infection and CC, the participants were invited to perform HPV self‐sampling. Dry cotton swabs were used for cervicovaginal sampling, and samples were immediately prepared for on‐site analysis. The swab was introduced in a tube containing 5 mL of NaCl medium and vortexed for 10 sec. After vortexing, 1 mL of each sample was transferred to the GeneXpert cartridge and run on a four‐module GeneXpert machine.We used an Xpert HPV assay consisting of real time PCR that uses detection of a human reference gene  and an internal Probe Check Control (PCC) as an internal assay control for specimen adequacy. The PCC was used to verify reagent rehydration, PCR tube filling in the cartridge, probe integrity and dye stability. The Xpert test included reagents for the simultaneous detection of 14 HR‐HPV genotypes . The assay used multiple fluorescent channels for the detection of individual types of HPV, groups of HPV, and the human reference gene. Each fluorescent channel had specific cutoff parameters for target detection and validity. If a sufficient amount signal was detected for the human reference gene, the assay results were reported as an overall positive. Additionally HPV16, pooled HPV18/45 and pooled other HR‐HPV types detected by the assay were reported separately as positive or negative.Each HPV test was analyzed within 1 hour of sample collection and the results were immediately communicated to the participants. Depending on the women's HPV status, management was as follows: HPV‐negative women were reassured and advised to repeat the test in 5 years. HPV‐positive women  with a normal VIA/VILI test underwent cervical 6 o'clock biopsy and endocervical curettage (ECC) to exclude CC, and for quality control. HPV‐positive women  with an abnormal VIA/VILI test underwent biopsy of the abnormal area followed by thermo‐coagulation treatment. Additionally, women positive for HPV16 and/or HPV18/45 were immediately treated with thermo‐coagulation, regardless of the VIA/VILI outcome. A control visit at 1 month was arranged for all women treated with thermocoagulation in order to exclude the presence of posttreatment side effects, such as bleeding and infection. Follow‐up visits at 6 and 12 months were arranged for all HPV‐positive women in order to assess viral clearance. HPV‐positive women had three pictures of the cervix  taken using a Smartphone  for quality control. The study design is illustrated in the flowchart in Figure VIA and VILI tests were performed by three trained local gynecologists. VIA consisted of application of a 3–5% acetic acid solution to the cervix. Appearance of acetowhite areas touching the squamocolumnar junction (SCJ) was used to help identify pathological areas of the cervix. VILI consisted of application of Lugol's iodine to the cervix. Appearance of a well‐defined, bright yellow area touching the SCJ indicated a suspicious lesion. VIA and VILI were conducted and the results interpreted according to the World Health Organization's recommendations We used biopsy forceps to perform cervical biopsy and an endocervical brush to perform ECC. Material collected was conserved in formalin‐based solution. An experienced pathologist, blind to the HPV and the VIA/VILI test results, conducted the histological analyses in Geneva. Histological analyses were conducted in accordance with Swiss standards and international recommendations.Thermo‐coagulation involved application of a probe heated to 100°C to the cervix for 50 sec, to achieve an expected tissue destruction of 5–7 mm depth. An appropriately sized probe was selected based on the area of the pathological cervical tissue. Several applications were performed, if necessary, to cover the entire pathological area.P < 0.05 was considered statistically significant. Analyses were performed for the whole study population and separately for women of different age groups. Relative risk of CIN2+ was calculated for the different HPV genotypes. Data analysis was conducted using Stata Statistical software Release 13 .A personalized electronic medical chart including sociodemographic and medical information was created using the SecuTrial online database to collect and manage data. Sensitivity, specificity, positive predictive value and negative predictive value were calculated for the VIA/VILI, HPV16, HPV18/45 and HPV16/18/45 assays, using histopathology as the gold standard. A bivariate logistic regression was performed to identify factors associated with HPV prevalence. The socio‐demographic characteristics of the participants are shown in Table n = 187/1012). Eighteen (9.6%) samples were positive for HPV16, 29 (15.5%) were positive for HPV18/45, 125 (66.8%) were positive for other HR‐HPV types, 2 (1.1%) were co‐positive for HPV16 and other HR‐HPV types and 13 (7.0%) were co‐positive for HPV18/45 and other HR‐HPV types  HPV‐positive women underwent VIA/VILI examination. Overall, 107 (57.8%) VIA examinations were classified as pathological. A total of 121 (65.4%) women had a positive screen, of whom 110 (90.9%) were eligible to be treated with thermo‐coagulation. Histological analyses revealed 10 cases with (5.3% of women with an HPV positive screen) CIN1, 3 (1.6%) with CIN2, 15 (8.0%) with CIN3 and 1 (0.5%) with CC. Four histological samples were lost between the transportation and the laboratory analysis processes.n = 1), presence of a lesion extending to more than 2 mm inside the cervix (n = 5), presence of a lesion suspicious for cancer (n = 3), failure of probe heating (n = 1) and undeclared pregnancy at the time of enrollment (n = 1). Women presenting with large pathological areas or with lesions suspicious for cancer were referred to LEEP or hysterectomy. Pregnant women were invited for a follow‐up visit after childbirth.Among 62 women positive for HPV 16/18/45, 61 (98.4%) underwent screening and treatment in the same 1‐day session. Among 126 women positive for other HR‐HPV types, 125 (99.2%) underwent VIA/VILI triage. Two women (1.1%) were lost between HPV testing and VIA/VILI triage. In total, 110 of the 121 women requiring treatment were treated in the same‐day session. Reasons for not being able to treat women with a positive screen included: no visualization of the SCJ , and decreased in women aged 40 years and older. HPV 18/45 prevalence was highest among women aged 35–39 years (5.5%). HPV 16 prevalence increased with age, reaching a peak among women aged 40–45 (2.5%). CIN1 prevalence was higher among younger women. Most CIN3 lesions were observed in women older than 35 years.P = 0.015). Women who had had more than six sexual partners had 2.12 times the odds of presenting with a HPV infection . Number of children was a protective factor for presenting with a HPV infection : women with more than two children had 0.66 times the odds to have an HR‐HPV infection as women with one or no children .The likelihood of HR‐HPV infection decreased with age . The VIA/VILI positivity rate was 73.2% when women were positive for HPV 18/45, and 77.1% when women were positive for either HPV 16 or HPV 18/45 or both. The VIA/VILI positivity rate was lower (50.4%) in women positive for other HR‐HPV types. Performance of VIA/VILI and HPV genotyping for detection of high‐grade cervical lesions is presented in Table P = 0.005). Among women of all ages, those who were positive for HPV 16 and/or HPV 18/45 had 4.2 times greater risk of having a CIN2+ than those who were positive for other HR‐HPV types (P = 0.004)  for CIN2+ in women positive for HPV 16 and/or HPV 18/45 compared with in women positive for other HR‐HPV genotypes was 4.2 (95% CI: 1.6–11.5). Furthermore, HPV 16‐positive women had 6.5 times the risk of CIN2+ compared with women positive for other HR‐HPV genotypes (95% CI: 2.21–19.64). Such results highlight the importance of genotyping as a valuable tool to identify higher‐risk groups among HPV‐positive women and to increase the specificity of HPV testing. Our findings are consistent with those of previous studies that detected HPV 16, 18 and/or 45 in 71.1% of patients with CC Viral genotyping allowed us to detect a 2% prevalence of HPV 16, a 4.2% prevalence of HPV 18/45, and a 13.9% prevalence of other HR‐HPV types in the study population. The HPV prevalence in our study population was similar to that observed in other studies in Sub‐Saharan Africa According to our results, the sensitivity and specificity of VIA/VILI in detecting CIN2+ among HPV positive women were 80% and 44%, respectively. This finding means that although most HPV positive women were VIA/VILI positive, more than half of them did not actually have precancerous lesions and were, therefore, un‐necessarily treated. Indeed, one of the issues of a screen‐and‐treat approach is the risk of overtreatment One strength of our study was the low number of patients lost to follow‐up (1.1%). We observed a drastic reduction in loss to follow‐up, compared with the 25% rate in a previous study of HPV‐positive women in Cameroon In this study, we did not follow the manufacturer's protocol for sample collection for the Xpert HPV assay, as we used a 0.9% NaCl solution for sample collection instead of the manufacturer's proprietary solution, PreservCyt. This choice reflects a real‐life situation that can be frequently encountered in low‐income settings, where the availability of resources is not always guaranteed. Because NaCl solution is cheaper and readily available in resource‐constrained settings, its use in future screening programs is recommended. The low number of invalid results in our study supports this recommendation. However, equivalence of the two methods has yet to be demonstrated through appropriately designed studies. Our study was limited by the small number of CIN2+ cases in the study population. Estimates based on a larger number of cases would provide more accurate, reliable results. Another limitation of this study is that, although our results allow us to make optimistic assumptions about the management of HPV‐positive women, this pilot study does allow us to draw a conclusion on the subject. Such delicate matter requires careful consideration and should be supported by further, prospective studies.In conclusion, we have shown that rapid HPV testing is technically accessible and appropriate in low‐resource settings. Our results show that a single‐day, screen‐and‐treat approach can drastically increase compliance with a screening program in a developing country. The success of this type of CC prevention program, however, must further be assessed based on its capacity to ensure sustainability and appropriate scale‐up to the general population of the country and other low‐resource settings.The authors declare no conflict of interest.


HPV-16 long control region (LCR) has been shown to be the most variable region of the HPV-16 genome and may play important roles in viral persistence and the development of cervical cancer. This study aimed to assess the risk of HPV-16 LCR variants for cervical cancer in women of Southwest China. 2146 cervical scrapings of volunteer outpatients and 74 cervical cancer tissues were screened.14 entire HPV-16 LCRs from asymptomatic carriers and 34 entire HPV-16 LCRs from cervical cancer patients were successfully amplified and sequenced to align to others described. 58 different point mutations were detected in 54 nucleotide sites of HPV-16 LCR. G7193T and G7521A variants, accounting for 100% of the infections, were predicted to locate at the binding site for FOXA1 and SOX9, respectively. A7730C variant which showed a high mutation frequency in cervical cancer was predicted to be a binding site for the cellular transcription factor PHOX2A. In addition, phylogenetic analysis displayed a high prevalence of A lineage in HPV-16 LCR in this Southwest China population. This study may help understanding of the intrinsic geographical relatedness and the correlations between LCR mutations and the development of carcinogenic lesions in Southwest China population. And it provides useful data for the further study of the biological function of HPV-16 LCR variants. Human papillomavirus type 16 (HPV-16) is prevalent worldwide and is the main etiological agent of cervical cancer .HPV-16 is a capsid-enclosed circular double-stranded DNA virus from the papillomavirus family with a genome approximately 7.9 kb in length, consisting of an early region , a late region , a small non-coding region (NCR) localized between E5 and L2, and a long control region (LCR) , which iThe LCR region contains the early promoter and various transcriptional regulatory sites for both viral and cellular proteins –6, such In this study, samples from asymptomatic carriers and cervical cancer patients were obtained from women in Southwest China, and polymorphic sites within the HPV-16 LCR were detected and compared to investigate the association of specific LCR mutations with the development of cervical cancer. A phylogenetic study and functional prediction of HPV-16 LCR in asymptomatic and cervical cancer specimens were also performed to determine the circulating lineages and investigate the polymorphisms of transcription factor binding sites of the HPV-16 LCR variants in Southwest China.All participants were informed of the study aims, and a written informed consent was received from each patient before sample collection. The study was conducted with the approval of the Institutional Ethics Committee of Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College and was performed in accordance with the Declaration of Helsinki for Human Research of 1974 (last modified in 2000).Cervical scrapings of 2,146 volunteer outpatients were collected from the First Affiliated Hospital of Kunming Medical University in 2015. 74 cervical cancer samples were drawn from Yunnan Provincial Tumor Hospital of Kunming Medical University. After routine cytology and HC2 testing, each sample was transferred to a 1.5 ml Eppendorf tube.The extraction of specimen DNA of frozen scraped cells was performed as previously described . BrieflyCGTCCAAAAGGAAACTGAGC, MY11: GCACAGGGACATAACAATGG) followed by GP5+/GP6+ primer pair  according to the previous publications [2O with the following parameters for the reactions: an initial heating at 94°C for 5 min, followed by 38 cycles of 94°C for 1 min, 55°C for 1 min, 72°C for 1 min, and a final elongation at 72°C for 10 min. In the second-round PCR, 5 μl of the first-round products along with 25 μl 2× Power Taq PCR MasterMix (BioTeke), 2 μl 20mM Primer GP5+, 2 μl 20mM Primer GP6+ and 5 μl H2O were brought to a volume of 50 μl and subjected to thermocycling as follows: 95°C for 5 min, followed by 38 cycles of 95°C for 1 min, 50°C for 1 min, 72°C for 50 s, and then 72°C for 10 min. β-globin  was used as a positive control [™ nucleic acid stain. Positive samples with a single 150 bp band were then subjected to DNA sequencing and aligning on NCBI. Then the score, query cover, E value and identity of each alignment were taken into overall consideration for HPV genotyping.HPV genotyping was performed using a nested PCR assay. The extracted DNA was first amplified by the MY09/MY11 primer pair  under the accession number: KX912865 to KX912878 for the sequences from asymptomatic carriers, and KX912879 to KX912912 for the sequences from cervical cancer patients.According to the HPV reference strain (NCBI Reference Sequence: NC_001526.3), two pairs of primers that were used in the amplification of the two partially overlapping fragments were designed to cover the entire HPV-16 LCR as listed in Molecular characterization was performed by a sequence analysis of HPV-16 LCR amplicons. To identify mutational sites in LCR, all of the studied LCR sequences were aligned to the HPV-16 reference sequence NC_001526.3) by MEGA 7.0 package [26.3 by MThe phylogenetic tree of HPV-16 LCR was constructed by the Neighbor-Joining method and the Kimura 2-Parameter model by MEGA package 7.0. The number of bootstrap replications was 1,000. The reference HPV-16 LCR sequences that were used to construct the phylogenetic branches were collected from the GenBank sequence database and included NC_001526.3, K02718 (A1), AF536179 (A2), HQ644236 (A3), AF534061 (A4), AF536180 (B1), HQ644298 (B2), AF472509 (C), HQ644257 (D1), HQ644270 (D2) and AF402678 (D3).http://jaspar.genereg.net/) [The high-quality transcription factor binding profile database JASPAR (eg.net/) ,17 was uTo examine the distributions of the HPV 16 LCR mutations with respect to tumorigenicity of cervical cancer, a Fisher’s exact test was performed. A P-value less than 0.05 was considered statistically significant. The entire database was analyzed with SPSS 12.0.Of the 2146 collected outpatient samples, 248 samples were HPV-positive. By genotyping, 21 HPV types were identified, including HPV-6, 11, 16, 18, 33, 39, 43, 52, 53, 54, 58, 59, 61, 62, 66, 67, 81, 83, 84, 90 and 91. Of the types observed, HPV-16, 81, 11, 58, and 6 were the five most common types, accounting for 72.58% (180/248). The most common high-risk and low-risk types were HPV-16  and HPV-81 , respectively .Of the 74 cervical cancer samples, 61 samples were identified HPV positive and belonging to high-risk types. They were HPV-16, 18, 58, 56 and 59, accounting for 73.77% (45/61), 16.39% (10/61), 6.56% (4/61), 1.64% (1/61), 1.64% (1/61), respectively.Among the 47 HPV-16-positive samples from the outpatients (asymptomatic carriers), only 14 entire LCR sequences were obtained. A possible explanation is the small copy number of HPV in some women. Meanwhile, 34 entire LCR sequences were obtained from the cervical cancer samples. When aligned to the reference LCR, nucleotide variations of HPV-16 LCR were found in all of the studied HPV-16 positive asymptomatic carrier samples (14/14) and cervical cancer samples (34/34). In total, 58 different point mutations were detected in 54 nucleotide sites within the LCR segment with 832 bp in length, and mutations occurred over a span of nucleotides 7157 to 82 . The mutWhen compared with the asymptomatic carriers, A7175C, T7177C, T7201C, C7270T, A7287C, A7289C, A7730C, G7842A and C24T got higher mutation frequencies in cervical cancer. Additionally, the variation sites such as A7233C and C7395T were only detected in cervical cancer . These mWhen considering the entire LCR sequence, combinations of the single nucleotide polymorphisms (SNPs) resulted in 30 unique sequences (variant IDs: 1 to 30) . VariantTo assess the effects of the variations in HPV-16 LCR on binding sites of cellular transcription factors, the JASPAR database was used. The results showed that several variations in the HPV-16 LCR spanning nucleotides 7157 to 7906 altered the binding sites of transcription factors , which pG7193T and G7521A variants, which accounted for 100% of the infections, were located at the binding site for Forkhead box protein A1 (FOXA1) and sex-determining region Y-box 9 (SOX9), respectively. The variations of A7730C contributed to influence the binding of paired-like homeobox 2a (PHOX2A). Furthermore, the variations of C7310T, C7395T and C7886G were predicted to locate at the binding site for CCAAT/enhancer binding protein beta (CEBPB), ETS proto-oncogene 1 (ETS1) and signal transducer and activator of transcription 3 (STAT3), respectively. While, the mutations C24T and C31T within 1 to 82 nucleotides of the HPV-16 LCR were not predicted to affect the binding sites of transcription factors.More than 170 different types have been characterized in the HPV family . HPV preHPV-16 variants have been found to show different geographic distributions  and exhiThe LCR has been shown to be the most variable region of the HPV-16 genome in different populations  and contCompared to the control group of HPV16-positive women without HSIL or cervical cancer, those polymorphic sites only found within the HPV-16 LCR region of isolates from cervical cancer patients, should receive attentions. The variations of C7394T and C7395T were predicted within the binding site for Ets transcription factor 1, which has been demonstrated to play roles in the complex biological control of tumor progression , 33. CEBIn this study, 58 different point mutations were detected in 54 nucleotide sites of HPV-16 LCR. Phylogenetic analysis displayed a high prevalence of A lineage in HPV-16 LCR in this Southwest China population. G7193T and G7521A detected in all the infections, were predicted to locate at the binding site for FOXA1 and SOX9, respectively. In addition, A7730C variant, which showed a high mutation frequency in cervical cancer, was predicted to be a binding site for the cellular transcription factor PHOX2A. Our study provided a helpful experimental basis for understanding the intrinsic geographical relatedness of HPV-16 variants, the interaction of HPV-16 LCR mutation variants and transcription factors, and the correlation between mutations of the LCR region and carcinogenesis. It also helps performing further study to demonstrate the biological function of HPV-16 LCR variants.S1 Table(DOC)Click here for additional data file.


Urine sampling may offer a less invasive solution than cervical sampling to test for human papillomavirus (HPV) for HPV vaccine impact monitoring.Paired samples of urine and exfoliated cervical cells were obtained for 89 women with history of high-risk (HR) HPV-positive normal cytology in Bhutan. Urine sampling protocol included self-collection of first-void urine immediately into a conservation medium and procedures to optimize DNA yield. Colposcopical abnormalities were biopsied. Two HPV assays were used: a multiplex type-specific PCR (E7-MPG) and a less analytically sensitive GP5+/6+ PCR followed by reverse line blot.p = 0.32) but lower in urine by GP5+/6+ . HPV6/11/16/18 positivity did not significantly differ between urine and cells by either assay. Sensitivity of urine (using cells as gold standard) to detect 21 HPV types was 80% and 58% for E7-MPG and GP5+/6+, respectively, with specificity 61% and 89%. HPV type distribution in urine and cells was similar, regardless of assay. The 5 detected CIN3+ were HR-HPV positive in cells by both assays, compared to 4 and 3 by E7-MPG and GP5+/6+, respectively, in urine samples.HPV positivity for 21 types common to both assays was similar in urine and cells by E7-MPG (62.9% and 57.3%, respectively, For the monitoring of vaccine impact, we demonstrate validity of a urine sampling protocol to obtain HPV prevalence data that are broadly comparable to that from cervical cells. However, detection of HPV in urine varies according to assay sensitivity, presumably because low level infections are frequent. Confidence in the use of urine samples for the detection of human papillomavirus (HPV) has been increasing in recent years. Systematic reviews have shown a reasonable concordance with cervical cells for HPV positivity in women –3, with Using a device for self-collection of first-void urine and implementation of optimized procedures for urine sample management, we have recently reported good acceptability and performance of a protocol of HPV testing from urine to monitor HPV vaccine impact in young women in Bhutan and Rwanda . Indeed,However, the choice of assay for HPV testing in urine can impact estimates of epidemiological associations and HPV vaccine effectiveness , so thatWe aimed to estimate the performance of the above mentioned urine sampling protocol directly against paired cervical cytological samples, and using two HPV assays of differing sensitivity, namely GP5+/6+RLB, an assay developed for clinical specificity in HPV-based cervical screening and E7-MPG, which has greater analytical sensitivity . This waDuring a population-based study in 2012, 2505 women aged 18–69 years were invited for the collection of exfoliated cervical cells for a PAP smear and HPV testing by GP5+/6+RLB . Women wImmediately prior to colposcopy, participants self-collected a urine sample using a device  designed to collect the first 14 ml of first-void urine immediately into 7 ml of a urine-conservation medium to avoid DNA degradation [After the urine collection, during colposcopy, a cytobrush  was used for the collection of exfoliated cervical cells. After preparation of a conventional Pap smear, the brush containing cellular material was placed in a vial containing 20 ml PreservCyt medium. DNA was extracted from the PreservCyt sample in the Department of Pathology at the VU University Medical Centre, Amsterdam, using magnetic beads (Macherey Nagel) on a robotic system (Hamilton Star).Two methods of different analytical sensitivity were used for HPV DNA testing.A type specific E7 PCR bead-based multiplex genotyping assay (E7-MPG) was performed at IARC, Lyon using a Luminex bead-based platform . The assIn the Department of Pathology at the VU University Medical Centre, Amsterdam, β-globin polymerase chain reaction (PCR) analysis was conducted to confirm the presence of human DNA in all specimens  and a geHPV-positivity refers only to positivity for the 21 HPV types in common to both HPV genotyping methods . Other HPV types detected by GP5+/6+RLB were ignored.P-values from McNemar’s test for paired nominal data was used to evaluate the homogeneity of HPV findings between urine and cells by each assay. Graphs include two lines: a dotted line represents a theoretical scenario where types are detected equally in both samples, and that corresponds to a urine:cell PR of one; a solid line represents the slope of a linear regression passing through the origin and the 21 type-specific points, hence representing the average urine:cell PR. The strength of the association of this linear regression was assessed by using the coefficient of determination R2 or explained variation. Finally, sensitivity and specificity was computed for HPV detection in urine compared to cervical cells, i.e., the gold standard in cervical HPV-based screening.Type-specific positivity is compared between urine and cells by urine:cells prevalence ratios (PRs), as well as graphically. Of 95 HR-HPV-positive women with normal cytology recalled for colposcopy, six had β-globin-negative urine samples invalid for HPV testing, leaving 89 women with paired urine and cervical samples. Median age was 39 years (5–95 percentile = 30–54 years), 83% reported only 1 lifetime sexual partner, and none were vaccinated against HPV.p = 0.32), whereas it was lower in urine (30.3%) than in cells (40.4%) by the GP5+/6+ test . HPV positivity for vaccine types 6/11/16/18 was similar in urine and cells by both E7-MPG , and by GP5+/6+  .Figure Eleven cervical intraepithelial neoplasia (CIN) grade 1, 6 CIN2, 4 CIN3 and 1 invasive cancer were histologically diagnosed among these 89 patients. Table We confirmed a >70% concordance of HPV testing between urine and paired cervical cell samples among women in Bhutan using two differently sensitive assays. Furthermore, whereas previous studies have almost all reported a moderate under detection of HPV in urine compared to paired cervical cell samples , we repoHowever, urine:cell PR can also vary according to the HPV assay used. When these same samples were tested by GP5+/6+, an assay developed for clinical specificity in HPV-based cervical screening, HPV prevalence was lower than by E7-MPG, as expected. However, the difference in HPV prevalence between the two tests was greater in urine than in cells. This resulted in significantly lower detection of HPV in urine compared to cervical cells when relying on GP5+/6+, which is similar to findings of a meta-analysis of previous studies , althougWe have recently shown that E7-MPG infections non-detected by GP5+/6+RLB are associated with low viral copy number  , a findiThis study was neither designed nor powered to evaluate the utility of urine testing for detection of precancerous lesions in cervical cancer screening. Indeed, to obtain more information of the performance of cells and urine samples, we chose a group of women especially likely to be HPV-positive, and numbers were small. Nevertheless, we notice anecdotally that all 5 cervical cell samples from CIN3+ cases were HR-HPV-positive, irrespective of the test used and, of these, 4 out of 5 were positive by E7-MPG and 3 out of 5 by GP5+/6+RLB when tested from urine. Some previous studies have shown similar results of CIN3 sensitivity (74–100%) , 19, 20,In conclusion, we demonstrate the validity of a protocol of urine self-collection and HPV testing that we are already using for the long-term monitoring of vaccine impact in Bhutan and Rwanda . In youn


Background. Human papillomavirus virus type 16 (HPV-16) and HPV-18 cause a large proportion of oropharyngeal cancers, which are increasing in incidence among males, and vaccine efficacy against oral HPV infections in men has not been previously evaluated.Methods. Sera and saliva collected in mouthwash and Merocel sponges at day 1 and month 7 were obtained from 150 men aged 27–45 years from Tampa, Florida, and Cuernavaca, Mexico, who received Gardasil at day 1 and months 2 and 6. Specimens were tested for anti–HPV-16 and anti–HPV-18 immunoglobulin G (IgG) levels by an L1 virus-like particle–based enzyme-linked immunosorbent assay.Results. All participants developed detectable serum anti–HPV-16 and anti–HPV-18 antibodies, and most had detectable antibodies in both oral specimen types at month 7 . Antibody concentrations in saliva were approximately 3 logs lower than in serum. HPV-16– and HPV-18–specific antibody levels, normalized to total IgG levels, in both oral specimen types at month 7 were significantly correlated with serum levels .Conclusions. This is the first study demonstrating that vaccination of males with Gardasil induces HPV antibody levels at the oral cavity that correlate with circulating levels. Human papillomaviruses (HPVs) cause approximately 70% of oropharyngeal cancers (OPCs), with the majority due to HPV-16 . US inciThe quadrivalent HPV vaccine (Gardasil) is highly efficacious in reducing anogenital HPV-6, -11, -16, and -18 infections, subsequent development of HPV-related external genital lesions, and anal diseases in young males (ages 16–26 years) . These fPost hoc vaccine trial evidence suggests that the bivalent HPV vaccine protects against 1-time-detected prevalent oral infections in females . HoweverHence, we assessed the levels of HPV-16– and HPV-18–specific immunoglobulin G (IgG) antibodies at the oral cavity following a phase 2 vaccine trial of mid-adult-aged men ages 27–45) from the United States and Mexico who received 3 doses of Gardasil /[specimen weight (in grams) − mean dry sponge weight (in grams)]; “0.6 g” refers to the weight of the extraction buffer added to each specimen.Oral fluid was extracted from Merocel sponges according to a previously published protocol . A totalAnti-HPV IgG antibodies were detected by an ELISA, as previously described –12. ThisTotal human IgG levels were measured in duplicate in each specimen type , using an ELISA according to the manufacturer's protocol  . Total IP values from the demographic parameters were calculated using Pearson χ2 analysis (Monte Carlo), for categorical variables, and the Wilcoxon rank sum test (Monte Carlo), for continuous variables. The proportions of men who had measurable serum or oral HPV-16/18 IgG, as well as associated 95% confidence intervals (CIs), were estimated. Geometric mean titers (GMTs) and 95% CIs for HPV-16 and HPV-18 antibody levels were calculated and reported by categories of the covariates examined. GMTs were compared across groups, using the Wilcoxon rank sum test or the Kruskal–Wallis test. Correlations between serum-specific and oral-specific IgG levels were determined by Spearman correlation coefficients. Among trial participants with detectable antibodies in both serum and oral fluid (mouthwash gargle and Merocel sponge), HPV-specific antibody levels were normalized to the total IgG level in the serum or oral specimen, respectively, and are reported as ratios of the HPV-specific IgG concentrations to the total concentrations of IgG. In Supplementary Figure 1, P values were calculated by the Wilcoxon rank sum test, and Bonferroni correction was used to set significance at a P value of < .0167.We conducted an intent-to-treat analysis that included all men in the trial regardless of HPV DNA and antibody status at enrollment. The A total of 150 men were enrolled in this study, but only a subset of men with specimens available at both baseline (day 1) and month 7 (1 month after the third dose of vaccine) visits were tested and included in this analysis . Table P < .001).The level of HPV-16 and HPV-18 antibodies induced by vaccination in serum and oral fluid collected in gargles and in sponges are shown in Table At day 1, <9% of the participants had detectable anti–HPV-16 or anti–HPV-18 levels in any of the 2 oral fluid specimens tested. Following vaccination, most participants had measurable HPV-16– and HPV-18–specific antibodies in oral fluids . Strong correlations were observed between the levels of anti–HPV-16 and anti–HPV-18 antibodies found in the 2 oral sample types . In addition, HPV-16– and HPV-18–specific antibody levels at month 7 in both oral fluid specimens were significantly correlated with serum levels .HPV-specific antibody levels after vaccination in Merocel sponge specimens were approximately 262–495-fold lower than in serum . Serum HPV-18 antibody levels were inversely associated with alcohol intake but were not affected by any other demographic factors. HPV-16 antibody levels in oral fluids differed by country, tobacco use, and frequency of tooth brushing and gum bleeding. HPV-18 antibody levels in oral fluids differed by country in both specimen types and by gum bleeding in mouthwash gargles. These effects were no longer observed when antibody levels in oral fluids were normalized to total oral IgG levels (Supplementary Table 2).Serum anti–HPV-16 antibody levels were not influenced by any of the demographic factors examined (To date, no vaccine trials have evaluated quadrivalent HPV vaccine efficacy against persistent oral HPV infections, the presumed obligate precursor to OPC. Gardasil has demonstrated robust efficacy against HPV infection at the cervix, vulva, and vagina in females and at the external genital skin and anal canal in men , 14, 15.Using 2 independent oral sampling methods, HPV-16– and HPV-18–specific antibodies were detectable at the oral cavity in the majority of HPV vaccine recipients. A strong correlation was observed between antibody levels measured in mouthwash gargle specimens and in Merocel sponge extracts. These findings suggest that both methods could be used to monitor oral antibody levels in HPV vaccine studies. For some studies, mouthwash gargle specimen collection will be more convenient, as this collection method is commonly used for HPV genotyping , 17, 18.Data presented in this study demonstrate for the first time that HPV-16– and HPV-18–specific antibodies induced by vaccination can be detected at the oral mucosa in 2 different oral specimen types. It is still uncertain whether the antibody levels measured in this study at the oral cavity are sufficient to provide long-term protection against acquisition of oral HPV-16 and HPV-18 infections. Future efficacy trials addressing the longevity of the immune responses in serum and at the oral cavity, as well as other aspects of B-cell immunity in relationship to HPV vaccine efficacy among men, are needed to inform vaccine protection against oral HPV infection and cancer in men.Supplementary materials are available at http://jid.oxfordjournals.org. Consisting of data provided by the author to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the author, so questions or comments should be addressed to the author.


Plasmodium. Reliable diagnosis is crucial since it allows the early detection of the disease. The development of rapid, sensitive and low-cost diagnosis tools is an important research area. Different studies focused on the detection of hemozoin, a major by-product of hemoglobin detoxification by the parasite. Hemozoin and its synthetic analog, β-hematin, form paramagnetic crystals. A new detection method of malaria takes advantage of the paramagnetism of hemozoin through the effect that such magnetic crystals have on Nuclear Magnetic Resonance (NMR) relaxation of water protons. Indeed, magnetic microparticles cause a shortening of the relaxation times. In this work, the magnetic properties of two types of β-hematin are assessed at different temperatures and magnetic fields. The pure paramagnetism of β-hematin is confirmed. The NMR relaxation of β–hematin suspensions is also studied at different magnetic fields and for different echo-times. Our results help to identify the best conditions for β–hematin detection by NMR: T2 must be selected, at large magnetic fields and for long echo-times. However, the effect of β-hematin on relaxation does not seem large enough to achieve accurate detection of malaria without any preliminary sample preparation, as microcentrifugation.200 million patients suffer from malaria, a parasitic disease caused by protozoans of the genus  The protozoan parasites of the genus Plasmodium causing the disease are transferred to humans by Anopheles mosquito bites. In the fight against malaria, reliable diagnosis tools are crucial since they allow the early detection of the disease, which increases the efficiency of the treatments, but also because they prevent overprescription of antimalarial drugs which may lead to drug resistance. The standard diagnosis method consists in the examination of Giemsa stained blood smears by light microscopy, which requires efficient microscopes and trained microscopists. Polymerase Chain Reaction (PCR) analysis of DNA extracted from the patient’s blood is also very sensitive, but rather expensive and difficult to use on the field. As the disease is mainly present in developing countries, the diagnosis tests must be rapid, cheap and easy to implement. Therefore, rapid diagnosis tests (RDTs) – based on lateral flow immunochromatographic detection of malaria antigens – were created and are now widely used3 even if their sensitivity is clearly lower than that of microscopy and PCR5. In this context, the development of new, rapid and sensitive diagnosis tools remains an important research area. Some groups focused on the detection of a major by-product of malaria infection, hemozoin. Indeed, during their proliferation in red cells, the parasites feed on haemoglobin, which results in the release of free heme. This redox agent being toxic, the parasite transforms it into an insoluble polymer, hemozoin, the so-called “malaria pigment”. Hemozoin and its synthetic analog, β-hematin, have comparable structural properties but their immunogenic properties may differ because of different crystal sizes and/or shapes9. They are both paramagnetic because they contain Fe3+ ions in high-spin configuration13. Values of the magnetic susceptibility of hemozoin and β-hematin were published by Brémard et al.10, Bohle et al.11, Hackett et al.14 and Butykai et al.15. In 2016, a study16 surprisingly proposed a superparamagnetic model for β-hematin, which however remains to be confirmed. The magnetic properties of hemozoin are at the origin of two detection methods which were recently proposed. The first method17 uses the magnetic moment of the pigment crystals to align them in a specific direction which makes them magnetically driven optical polarizers. The second method, evaluated by two different groups19, tries to take advantage of the paramagnetism of hemozoin through the effect that such magnetic crystals can have on Nuclear Magnetic Resonance (NMR) properties of neighbouring water protons in whole blood. Indeed, magnetic nano- and micro-particles cause a shortening of the relaxation times T1 and T2 of water protons, a phenomenon which is at the origin of the use of iron oxide particles as Magnetic Resonance Imaging (MRI) contrast agents. Karl et al.19 were the first to show the influence of hemozoin on water transverse relaxation time T2. Even if the effect was noticeable, they concluded that it was unlikely that this technique could achieve the requested sensitivity to become an efficient diagnosis tool on the field, since they were only able to detect parasitemia levels higher than 10,000 parasites per µl of blood, corresponding to 0.2% infected Red Blood Cells (RBC). A few years later, Peng et al.18 and Kong et al.20 reported a new technique for the rapid and sensitive detection of Plasmodium infected RBC using the measurement of T2. Their micromagnetic resonance relaxometry system uses the transverse relaxation of RBC pellets – obtained after microcentrifugation of a patient’s blood sample – to detect parasitemia levels of less than 10 parasites per µl, which is comparable to the detection limit of light microscopy, the gold standard. The marked discording conclusions of the two studies led to a reaction of Karl et al.21 who questioned the sensitivity reported by Peng et al.In 2015, 200 million patients were suffering from the tropical disease malaria, and half a million died from the infection the same yearIn this work, the magnetic properties of two types of β-hematin will be assessed, to discriminate between paramagnetic and superparamagnetic behaviours, and the NMR relaxation of β-hematin-containing suspensions will be studied at different magnetic fields, in order to determine the best experimental conditions for the hemozoin and malaria detection by NMR.22, the crystals present a rod shape. An estimation of the mean size and standard deviation of the crystals long axis was obtained by measuring 60 particles. For the commercial sample, L = 1.01 ± 0.34 µm while for the Mons sample L = 0.67 ± 0.28 µm. This is slightly over the size of Plasmodium falciparum, P. vivax, P. ovale and P. malariae hemozoin crystals 23.Figure 15. Our data are in agreement with previously published data obtained at 2 K15. A good agreement is also obtained when looking at the evolution of the magnetisation at 0.5 T with the inverse of temperature (1/T)  Fig. . FinallyT1 NMRD profiles – the curve showing the evolution of the longitudinal relaxation rate with the magnetic field – of β-hematin suspensions. 1/T1 monotonously decreases with the field for both samples with a broad dispersion after 1 MHz, similar to what was observed for methemoglobin24. The effect of β-hematin on the longitudinal relaxation of water is really weak. Indeed, the relaxation rate (1/T1) normalised by the β-hematin concentration (in mg/mL) is always smaller than 0.55 s−1 mL mg−1. When expressed in terms of relaxivity – relaxation rate normalized by the iron concentration in mM – it gives less than 0.35 s−1 mM−1 which is far below the relaxivities of usual MRI contrast agents. This is also an order of magnitude smaller than the relaxivity of methemoglobin25. The transverse relaxation of water protons is more affected by the presence of β-hematin crystals as shown in Fig. T2 significantly increases with the field. This increase is more important for the commercial β-hematin compared to the Mons β-hematin. Such an increase reflects the field-dependent increase of the magnetic moment of the crystals. Their effect on transverse relaxation becomes stronger when they present larger magnetic moments. This is consistent with the predictions of the classical relaxation theories26. The transverse relaxation rate normalised by the β-hematin concentration (in mg/mL) are given in Table Figure 16 had another origin than the presence of β-hematin; this may come from a contamination of β-hematin by a superparamagnetic agent, such as hematite27. This clearly impacts the potential of conventional NMR relaxometry for the detection of malaria: paramagnetic particles are much harder to detect than superparamagnetic ones.Our results confirm the pure paramagnetism of β-hematin. It seems the superparamagnetism reported recentlyet al.19 for β-hematin suspensions. But our data bring more information: 1/T2 increases with the field , the following approximation was applied. Assuming that, in each parasitized RBC, 50% of hemoglobin has been converted into hemozoin, one can roughly estimate that an hemozoin content of 30 µg hemozoin/ml corresponds to a 1% parasitemia, as shown in Newman et al.17. This allows to normalize the 1/T2 values obtained for infected RBCs samples  was purchased from InvivoGen . This sample is referred to as “commercial β-hematin” throughout the paper.30 with slight modifications; briefly, a solution of 4.54 mM porcine hemin  in 0.04 M NaOH was adjusted to pH 4.0 with 2% propionic acid dropwise and incubated at 70°C for 18 h. The formed crystals were filtered on cellulose, washed with 1 M acetic acid, dried over phosphorus pentoxide, manually powdered and stored at 4°C. The powder was characterized by infrared spectroscopy, yielding 3 bands characteristic of hemozoin7 at 1711, 1662 and 1209 cm−1. This sample is referred to as “Mons β-hematin” throughout the paper.A second sample of β-hematin was synthesized according to published protocolsElectron microscopy images were acquired directly on the powders with a Hitachi Scanning Electron Microscope, model SU8020 using a 3 kV voltage.3 reported by Coronado et al.8. The molecular weight of β-hematin was taken as 633.5 g/mol.All the magnetic measurements were carried out on a mini high field measurement system from Cryogenics Limited  with the vibrating sample option. The maximum field is 5 T and the minimum temperature is 1.7 K. The measurements were directly performed on powders of β-hematin. When the diamagnetic contribution of the sample holder was non negligible , it was subtracted from the raw data. It was especially the case at low fields and high temperature. For the comparison of the susceptibility values with the literature data, the density of β-hematin was needed. We used the value of 1440 kg/mT1) of aqueous suspensions were measured from 0.015 to 40 MHz with a Spinmaster fast field-cycling relaxometer  using 600 µL of suspensions in a dedicated NMR tube. The T2 and T1 measurements at higher fields were carried out on a homemade variable field relaxometry system using an electromagnet and a lapNMR RF spectrometer covering a range of Larmor frequency from 10 to 90 MHz. The CPMG sequence was used, with an interecho time of 1 ms unless other mention. The temperature of all the NMR measurements was 25 °C ± 1°C.For NMR results, the magnetic field is expressed in terms of the proton Larmor frequency: a field of 1 Tesla corresponds to a Larmor frequency of 42.6 MHz. The suspensions were sonicated during 3 min before the session of NMR measurements and the tubes were vigorously shaken by hand before each NMR measurement. Low-field NMRD profiles (All data generated or analysed during this study are included in this published article.


A variety of cutaneous human papillomaviruses (HPV) are detectable in genital epithelial lesions in men and non-melanoma skin cancer patients. It remains unclear whether these viruses are associated causally with skin lesions. To date, no study has prospectively examined the association between cutaneous HPV seropositivity and development of external genital lesions (EGLs) in men.To examine the association between seropositivity to cutaneous HPV types and the risk of subsequent development of EGLs.the HPV Infection in Men (HIM) Study cohort was conducted. Cases were ascertained at each of up to 10 biannual clinical visits and verified through biopsy and pathological diagnoses. EGLs were categorized as condyloma, suggestive of condyloma, penile intraepithelial neoplasia (PeIN), and other EGLs. Archived serum specimens collected at baseline were tested for antibodies against 14 cutaneous HPV types , α type 27, γ type 4, μ type 1, and ν type 41) using a GST L1-based multiplex serology assay. Socio-demographic and sexual behavior data were collected through a questionnaire. Using logistic regression, adjusted odds ratios (AOR) and 95% confidence intervals (CI) were estimated.A nested case-control study including 163 incident EGL cases and 352 EGL-free controls in Overall, seropositivity to ≥1 cutaneous HPV type (any-HPV) and ≥1 β types (any-β) was 58.3% and 37.5% among other EGL cases, 71.6% and 46.8% among condyloma, 66.8% and 50.0% among PeIN, and 71.9% and 38.4% among controls, respectively. Type-specific seropositivity was most common for ɤ-HPV 4, μ-HPV 1, and β-HPV 8. No statistically significant association was observed between any-HPV, any-β, and type-specific HPV seropositivity and subsequent development of EGLs across all pathological diagnoses.Overall, seropositivity to cutaneous HPV was common among men; however, it appears that cutaneous HPV is not associated with the development of genital lesions in men. Over 200 types of human papillomaviruses (HPV) have been identified, and clasEmerging evidence shows that cutaneous HPV infection may increase the risk of squamous cell carcinoma (SCC) of the skin , 7. Seve we detected a lower prevalence of β-HPV DNA on the surface of EGLs compared to controls, and found that some β-HPV types were inversely associated with condyloma. To our knowledge, no study has yet investigated the association of seropositivity to cutaneous HPV and the risk of EGLs among men prospectively. The purpose of this study was to examine the association between seropositivity to cutaneous HPV types and the risk of subsequent development of EGLs among men in a case-control study nested in the HIM cohort.The detection of cutaneous HPV DNA on the surface of EGLs may suggest a role in the pathogenesis of the squamous epithelium lesions in the UV unexposed areas of the body. However, little data exist on the epidemiology and serology of cutaneous HPV and their distributions and etiologic role in the development of EGLs. Previously we detected β-HPV DNA on the surface of 61.1% of all EGLs . SubsequHIM cohort, a multinational prospective study of the natural history of HPV infection in men. Study population and methods have been described previously in details . However, a non-significant pattern of elevated risk was observed for any-β HPV and α-HPV type 27 seropositivity in all categories of EGLs compared to controls. In contrast, a non-significant pattern of reduced risk was observed for μ-HPV 1 seropositivity in all categories of EGLs.As this is the first study evaluating the role of antibodies to cutaneous HPV in the development of genital lesions located on UV-unexposed skin, no published literature exists that we can use to contextualize our findings. However, there exists a considerable amount of literature on the role of cutaneous HPV and squamous epithelial lesions of skin located on UV-exposed areas of the body that we can use to compare and contrast our findings with. The null findings in this study suggest that cutaneous HPV is not associated with EGLs on UV-unexposed skin in contrast to previous studies of other cutaneous lesions of UV-exposed skin. Reasons for these differences may include anatomic location of the lesions (i.e. UV exposed versus unexposed), immune status of the participants, and different high-risk groups of populations as previously reported .It is hypothesized that cutaneous HPV infection might modify the effect of UV-induced DNA damage and apoptosis leading to accumulation of mutations and cancer initiation . If UV eCutaneous HPV DNA is abundantly found on skin surfaces and persists in hair follicles of healthy individuals . This ubmanuscript under review), in the same HIM Study population, men with condyloma were significantly less likely to have beta-HPV 24 and 47 detected on the surface of the lesion than on the normal genital skin of the controls.In this work a non-significant pattern of reduced risk of EGLs for some HPV types was observed. Previously a few studies have also reported a protective effect for some cutaneous HPV types against skin lesions. For example, Masini et al.  reportedHigh seroprevalence estimates in both cases and controls in the current study may suggest that cutaneous HPV are ubiquitous on skin and that cutaneous HPV might be a commensal component of the microbiological flora of the skin , 45. It The current study is an extension of our previous work examining the role of cutaneous HPV in the development of external genital lesions in men. Biopsy-confirmed incident EGLs, prospective evaluation of the association, and the use of a previously validated , 42, higOur findings show that cutaneous HPV are not associated with the development of EGLs. Further research is need to understand the natural history and seroepidemiology of HPV infections and their role in the development of skin lesions and EGLs among men utilizing longitudinal study designs with repeated measures.Overall, exposure to cutaneous HPV was common among men; however, it appears that cutaneous HPV is not associated with the development of genital lesions in men. Further investigations are required to determine the role of cutaneous HPV in the development of skin tumors in prospective settings.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.