The predictive model has a high statistical significance , as well as sensitivity (Se = 0.74) and specificity (Sp = 0.75). Thus, simultaneous accounting for the clinical and molecular genetic predictors  makes it possible to effectively differentiate patients with prognostically favorable and unfavorable outcome of the disease.This study is aimed at searching for an informative predictor of the clinical outcome of cervical cancer (CC) patients. The study included 135 patients with locally advanced cervical cancer (FIGO stage II–III) associated with human papillomavirus (HPV) 16/18 types or negative status of HPV infection. Using logistic regression, we analyzed the influence of the treatment method, clinical and morphological characteristics, and the molecular genetic parameters of HPV on the disease free survival (DFS) of patients treated with radiotherapy or chemoradiotherapy. Multivariate analysis revealed three factors that have prognostic significance for DFS, i.e., HPV-related biomarker (HPV-negativity or HPV DNA integration into the cell genome) (OR = 9.67,  Cervical cancer (CC) continues to occupy one of the leading places in the morbidity and mortality of young women . DespiteThe clinical studies revealed the main factors influencing the effectiveness of CC treatment: degree of spread, form of growth, morphological structure of tumor, patient’s age, etc. . At the The individual characteristics of human papillomaviruses (HPV) of high carcinogenic risk are among the most attractive prognostic markers because of the proven ability of the virus to modulate the sensitivity of tumor cells to various impacts ,6. It isThe integration of HPV DNA into the cell genome is predominantly associated with disruption of the E2 gene open reading frame, which leads to the loss of E2 functional activity as a negative regulator of the transcription of viral E6/E7 oncogenes and, as a result, to an increase in the expression of E6 and E7 oncoproteins, although the number of viral DNA copies decreases ,16. In tAnother important mechanism of HPV influence on the chemo- and radiosensitivity of tumor cells is associated with the effect of HPV on the pool of cancer stem cells (CSC), which are resistant to many anticancer agents in comparison with the rest of the tumor cells ,18. The Taken together, these data, obtained mainly in vitro, indicate the participation of HPV in the modulation of radio- and chemosensitivity of tumor cells. At the same time, the role of HPV in vivo may be somewhat different, taking into account the influence of numerous microenvironmental factors on the sensitivity of malignant neoplasms to anticancer agents. These factors include physicochemical characteristics  and many signaling molecules  secreted by not only tumor cells but also various stromal cells, including endothelial, immune cells, tumor-associated macrophages, fibroblasts, and normal stem cells. Indeed, the results of studying the relationship between various HPV parameters and clinical outcome in patients with CC after radiation and/or chemoradiation therapy are controversial. The inconsistency in the assessment of the prognostic value refers to such HPV parameters as genotype ,26,27,28The purpose of this study was to clarify the prognostic value of such parameters of HPV infection as presence/absence of high risk HPV DNA (HPV status), its genotype, VL, and the integration of HPV DNA into the cell genome in combination with traditional clinical and morphological indicators .The HPV status and genotype were determined in tumor material of 173 patients with CC stages II–III according to the classification developed by the International Federation of Gynecology and Obstetrics (FIGO). Patients were treated at the Department of Radiation and Combined Methods for the Treatment of Gynecological Diseases, A. Tsyb Medical Radiological Research Center . This study was approved by the Ethics Committee of MRRC, informed consent was obtained from all patients for the study. One hundred thirty-five (out of 173) patients with HPV-negative or HPV16/18-positive tumors were selected for follow-up study of prognostic value of the HPV status, parameters of HPV infection, clinical and morphological indicators. The diagnosis was morphologically verified in all patients. The histological type of tumor was identified in accordance with the WHO classification . PatientThe effectiveness of CC patient treatment was assessed by disease-free survival (DFS) according to the criteria for the occurrence of loco-regional relapses and distant metastases based on the annual clinical and radiological examination using rectovaginal examination, ultrasound, computed and/or magnetic resonance imaging in accordance with the RECIST v. 1.1  . An unfaThe presence of HPV DNA of 14 high risk genotypes  was determined in united scrapings from endo- and exocervix of 173 CC patients before treatment. Real-time PCR on “Rotor-Gene”  was performed using the test-systems “AmpliSens HPV HCR screen-titer-FL” and “AmpliSens HPV HCR genotype-titer FL” .The physical state  of the viral DNA was assessed in HPV16- and HPV18-positive patients using kits containing primers and probes designed to specifically amplify the E2 and E7 genes of these viruses . This limitation is due to the fact that the set of reagents at our disposal allowed us to assess the degree of integration of HPV DNA 16 and 18 types only. Sites of HPV16 or HPV18 E2 and E7 genes and human β-globin gene were amplified in one tube in triplicate for each clinical sample. Standard samples with known concentration of HPV16 or HPV18 DNA were amplified in each experiment. The number of genomic equivalents of E2 and E7 was calculated from the calibration curves obtained on these standard samples. The quantitative load of HPV DNA was expressed in logarithms of E7 genomic equivalents, normalized to 200,000 genomic equivalents of β-globin or 100 thousand cells. The degree of HPV DNA integration was assessed by the ratio of E2 and E7 genomic equivalents, based on the fact that the E7 gene remains intact during the integration of viral DNA into the cell genome; therefore, the number of its copies in both forms of viral DNA  is the same. On the contrary, the E2 gene, as a rule, is destroyed and the number of its copies decreases. The degree of HPV DNA integration was calculated by the formula (1 − E2/E7) × 100%, where E2 and E7 are the number of genomic equivalents of the corresponding genes. The absence of amplification signal for E2 gene in the presence of such signal for E7 gene corresponds to 100% integration of viral DNA into the cell genome.-3 per 100 thousand cells (lgE7 < 3)—low viral load;The number of E7 gene copies is less than 10-3, but less than 105 per 100 thousand cells (3 ≤ lgE7 < 5)—moderate viral load;The number of E7 gene copies is equal to or more than 10-5 per 100 thousand cells (lgE7 ≥ 5)—high viral load.The number of E7 gene copies is equal to or more than 10Data on VL in HPV 16/18 positive patients were interpreted in accordance with the following criteria:Patients underwent radical courses of radiation therapy (RT) in conventional regimen, concurrent chemoradiation therapy (CCRT) or neoadjuvant chemotherapy followed by radiotherapy (NACT + RT) .60Co high activity at a single dose of 5.0 Gy two times per week to a TD of 50.0 Gy. TDs for the full course of RT were: primary focus 75.0–82.0 Gy, areas of regional metastasis 58.0–62.0 Gy, bladder 45.0–50.0 Gy, rectum 45.0–50.0 Gy. RT included external beam irradiation of primary tumor focus and areas of regional metastasis on linear electron accelerator SL-75-5  with photon radiation energy of 6 MeV at a single dose of 2.0 Gy daily on working days up to a total dose (TD) of 30.0 Gy. Then, intracavitary irradiation was performed on brachytherapy apparatus with sources of 2 and 5-fluorouracil 200 mg/m2 on days 1–5) which was started simultaneously with external beam irradiation and performed in 2–3 cycles with an interval of 21 days.CCRT included RT during polychemotherapy  and a radical course of RT, which was started 7–10 days after first cycle of polychemotherapy.NACT + RT included preradiation polychemotherapy , SPSS Statistics 23.0 , MedCalc 13.3.3.0 . For descriptive statistics, means and standard error (SE) were used. We calculated odds ratio (OR) of unfavorable outcome of the disease with 95% confidence interval (CI), sensitivity, specificity and accuracy (area under curve—AUC) of the prognostic test with Receiver Operating Characteristic (ROC) analysis.p values < 0.05 were considered statistically significant.The significance of differences for bivariate predictors was assessed using Fisher’s exact test. If the number of possible predictor values was more than two, a univariate logistic regression was constructed; significance was assessed by Wald test. Life table and Kaplan–Meier methods were used to assess DFS for various periods of observation. Log rank test was used to compare survival between groups and determine level of significance. Multivariate analysis was performed using multiple logistic regression. Hazard ratio (HR) was assessed with Cox proportional hazard model. A High risk HPV DNA was not detected in 20 out of 173 (11.5%) patients. One hundred and fifteen (66.5%) persons turned out to be HPV16/18-infected, including 95 HPV16-positive and 20 HPV18-positive cases. One patient was found to have simultaneously HPV 16 and 18 types; however, the case was assigned to the group of patients with HPV 16 due to the higher VL of type 16 (6.61) compared to type 18 (1.17). Other HPV genotypes  were found in the remaining 38 (22.0%) patients. Samples from the last 38 patients were excluded from further research due to methodological limitations in determining the degree of integration of viral DNA into the cell genome.Thus, the study group consisted of 135 patients with HPV-negative or HPV 16/18 positive tumors. The clinical and morphological characteristics of the patients are presented in Molecular genetic parameters of HPV in tumors of 115 HPV16/18-positive patients are shown in High VL  was observed most often, namely in 88 (76.5%) patients. VL was moderate  in 23 (20.0%) patients, and low VL  was found only in 4 (3.5%) patients.The presence of HPV 16/18 DNA integration of various degrees into the cell genome (integrated form) was found in the majority of patients—in 66 cases (57.4%), of which 26 cases had complete (100%) integration of viral DNA. Absence of integration  was registered in 49 (42.6%) patients.p > 0.05 according to Fisher’s test).VL and physical state of viral DNA  were not significantly different in patients with stages II and III of the disease   .p = 0.018; OR = 3.31). Interestingly, the probability of unfavorable outcome in HPV-negative patients was comparable to that observed for cases of HPV DNA integration . On this basis, cases with integrated form of HPV 16/18 DNA and HPV-negative cases were pooled in a multivariate analysis.In univariate analysis, the clinical outcome of the disease depended on the HPV status and physical state of HPV16/18 DNA  . Thus, tp = 4.7 × 10−5; OR = 3.66).The presence of HPV 16/18 in episomal form significantly increased the probability of favorable outcome , histological type  and parametrial infiltration  were associated with the clinical outcome of the disease.Of the clinical and morphological indicators, only stage (p = 0.0016) for a period of 5 years.Taking into account the data on the association of HPV DNA integration into the cell genome with unfavorable clinical outcome, it was interesting to find the optimal discriminatory level of integration degree, dividing patients into groups with a favorable and unfavorable clinical outcome. It was found by the ROC analysis that such a discriminator is the presence/absence of HPV16/18 DNA integration into the host cell genome regardless of the degree of integration . The AUCp = 0.0001) for period of 5 years. Separately for stage III, OR was 5.8 . OR for stage II was not calculated, since all patients under observation with episomal form of HPV 16/18 DNA were alive for 5 years without disease progression.The study results served as the basis for combining patients with integrated HPV16/18 DNA (regardless of the degree of integration) and HPV-negative patients into one prognostic group. As a result of this combining, a single biomarker including HPV-negativity or presence of HPV16/18 DNA integration into the cell genome was created. In the general group of CC patients with stages II–III, OR of unfavorable outcome was several times higher in patients with presence of the biomarker than in other patients, and reached 8.9 ; with stage III–50.0 ± 35.3%, 36.4 ± 20.2% and 47.3 ± 9.4%, respectively (p = 0.864).In the general group of CC patients with stages II–III, DFS did not differ significantly between patients with different levels of VL at all periods of observation. The same data were obtained as a result of separate analysis for stages II or III of the disease. Thus, 5-year DFS of patients with stage II was 100% at low VL, 65.0 ± 14.9% at moderate VL, and 81.7 ± 7.4% at high VL (p = 0.0002 according to Log Rank test). At stage II of the disease, the 5-year DFS of patients with episomal form was 100%, with integrated form—68.8 ± 9.4% (p = 0.012); at stage III of the disease—76.1 ± 11.0% and 29.1 ± 10.6%, respectively (p = 0.004) (The significant decrease in DFS was found in patients with an integrated form of HPV 16/18 DNA (regardless of the integration degree) compared with an episomal form: 48.8 ± 7.6% versus 88.5 ± 5.6% for all periods of observation (= 0.004) .p = 0.58 and p = 0.96, respectively).Patients with HPV-negative CC showed low 5-year DFS , similar to that in HPV 16/18—positive patients with integrated form (p = 4.55 × 10−5 according to LogRank test), while with stage II-100% and 60.9 ± 10.4%, respectively (p = 0.006), with stage III-76.1 ± 11.0% and 33.4 ± 8.7%, respectively (p = 0.005)  was significantly higher than in the prognostically unfavorable group (presence of the biomarker): 88.8 ± 5.6% versus 44.8 ± 6.8% (= 0.005) .p = 1.2 × 10−4; OR = 9.67), stage of the disease , age   and molecular genetic parameters of HPV infection . As a result of multivariate analysis, three independent predictors of clinical outcome of locally advanced CC were identified: presence/absence of the biomarker (HPV-negative status or HPV16/18 DNA integration) ( = 0.61) .p = 0.33. The predictive model has a high statistical significance , as well as sensitivity (Se = 0.74) and specificity (Sp = 0.75). Positive predicted value (PPV) = 0.57; negative predicted value (NPV) = 0.86.Thus, the results of multivariate analysis showed that within certain stages of the disease and the age category, the unfavorable prognosis of CC is not influenced by the HPV genotype, number of genotypes, VL, such clinical and morphological characteristics of CC as form of tumor growth, parametrial infiltration, histological type and grade. Along with stage of the disease and age of the patients, HPV-negative status or the presence of HPV 16/18 DNA integration had high prognostic significance regardless of the treatment method. p = 0.001), FIGO stage , and age  remained significant prognostic factors for DFS in all patients.The forward stepwise multivariate Cox proportional hazards model showed that the presence of HPV-related biomarker  of locally advanced cervical cancer according to the following criteria: HPV DNA status (negative/positive), physical state of HPV 16/18 DNA , stage of the disease, age. The application of our model in clinical practice makes it possible to identify CC patients with a high risk of disease progression in order to personalize treatment approaches in the future.This approach can be used to predict the treatment effectiveness in 78.0% of all patients with locally advanced CC since we were unable to analyze the molecular genetic characteristics and prognostic significance of other (rarer) HPV genotypes of high carcinogenic risk .HPV status was determined in scrapings from the cervix of 173 CC patients before treatment. The tumors of 20 patients (11.5%) were HPV-negative for 14 high risk genotypes. HPV types 16 or 18 were detected in 115 patients (66.5%). A small proportion of HPV-negative tumors were found in almost all studies using highly sensitive HPV tests. Until now, the question of the existence of true HPV negative CC remains controversial. A number of authors believe that detection of HPV-negative samples can be, at least partially, due to inadequate sampling, limitations in the testing of cytology specimens, deletion or rearrangement of the HPV gene being deleted, low viral load in some cancers, the presence of other HPV genotypes not detected with the current PCR primers and erroneous diagnoses ,46,47,48True HPV-negative CC appears to represent a biologically distinct subset of tumors that develop through an HPV-independent pathway and have different sensitivity to treatment as compared with HPV-positive CC. Numerous publications report lower DFS of HPV-negative patients with CC after radiation or chemoradiation therapy regardless of other prognostic factors  ,49,50,51Our study revealed significant decrease in 5-year DFS in CC patients with integrated HPV 16/18 DNA compared to episomal form regardless of the FIGO stage (II or III) of the disease. A number of authors also report a reduction in DFS of CC patients with stage Ib-IV in cases of HPV DNA integration regardless of the disease stage ,52,53. TContradictory data on the prognostic value of HPV load was observed ,30,55,56p = 0.0001) in the presence of this biomarker before the treatment. The mathematical algorithm allowing us to predict the clinical outcome of CC patients with high accuracy was developed on the basis of multivariate analysis according to the following criteria: high risk HPV status (negative/positive), physical state of HPV 16/18 DNA , stage of the disease, and age. As shown in numerous studies, FIGO stage is the well-known and widely used predictor of clinical outcome in CC patients, taking into account the size of the primary tumor, the state of the lymph nodes, infiltration of the parametrium, and the depth of stromal invasion [A single HPV-related biomarker of unfavorable outcome was developed in our study, as DFS of HPV-negative patients was similar to that of HPV 16/18-positive patients with an integrated form of viral DNA. In univariate analysis for patients with stages II-III, OR of unfavorable outcome was 8.9  of patients with locally advanced CC. The rest of the patients were found to have more rare genotypes of high risk HPV. Further research is needed to elucidate the prognostic value of HPV DNA integration and other molecular parameters of such HPV genotypes. The relevance of this issue will, apparently, increase over time during large-scale HPV vaccination, which is expected to reduce the prevalence of the more aggressive HPV genotypes 16 and 18 in cervical cancer.


Performance characteristics were calculated for three screening models: primary HRHPV with p16/Ki67 triage, primary cytology with reflex HPV and primary cytology alone. Primary HRHPV with p16/Ki67 triage was significantly more sensitive in high-grade squamous intraepithelial lesion quantified with cervical intraepithelial neoplasia grade 2 or worse [HSIL(CIN2+)] detection than cytology with reflex HRHPV and cytology alone (83.3% vs. 70.8%/45.8%) and had significantly higher diagnostic predictive values . The number of colposcopies per HSIL(CIN2+) detection indices was 3.4, 4.7 and 4.4, respectively. Primary HPV testing in women <30 years with p16/Ki67 triage of HPV-positive cases might be an effective cervical cancer screening strategy for HSIL(CIN2+) detection with superior diagnostic performance when compared with primary cytology-based models. Women <25 years might also benefit from an introduction to a more sensitive screening approach.Recently, cervical cancer rates elevation has been noted in women aged 20–39 years in regions with a very high human development index (HDI). The onset of cancer elevation rates is observed in the age range of 25–29 years, which should necessitate effective precancer screening in younger age groups, including those <25 years. From 30.066 liquid-based screening tests results ( The incidence and mortality of cervical cancer vary globally depending on the geographic region and the level of the HDI . In counHuman papillomavirus DNA testing is the globally recommended primary screening strategy for cervical cancer prevention for women 25–65 years old in all resource settings ,8. The rDual immunocytochemical staining in cervical cytology specimens with the simultaneous use of anti-proliferative p16 and Ki67 proliferative proteins is a morphologic-independent cell cycle dysregulation biomarker. A diagnostic value profile of p16/Ki67 testing is characterized by high sensitivity and high specificity for histologic high-grade intraepithelial lesions with evidenced effectiveness in HPV-positive women and/or with abnormal cytology results ,18,19,20Data on the use of p16/Ki67 dual-stained cytology in women under 25 years are limited, especially when used as a secondary test in HPV-positive young women undergoing primary HPV screening. Due to increasing cervical cancer rates in women aged 25–29 years, we investigated in our study whether primary HPV screening with triage HPV-positive cases with the p16/Ki67 biomarker can be an alternative screening strategy to cytology-based screening in young women under age 30 years. For this purpose, we conducted a retrospective analysis of cytologic–virologic–immunocytochemical reporting rates with histologic correlation at this age range and an evaluation of the diagnostic value of three screening strategies for the HSIL (CIN2+) detection.We conducted a retrospective analysis of cervical cancer liquid-based screening (LBS) tests results, including liquid-based cytology (LBC), 14 types of high-risk human papillomavirus testing (HRHPV14) and p16/Ki67 dual-stained cytology, undertaken at a private-based opportunistic cervical cancer screening at one of the largest Lower-Silesian private outpatient gynecological clinics  from August 2015 to July 2020. There were three cervical cancer screening modalities used in the center over the period considered: cytology with reflex HRHPV14, co-testing (HRHPV14 simultaneously performed with LBC) and co-testing plus (co-testing simultaneously performed with p16/Ki67). The baseline analysis included a total of 30,066 screening tests results, including 20,605 LBC, 8331 HRHPV14 and 1130 p16/Ki67 tests sampled in women aged 15 to 92; mean age of 40.9. From the baseline group, all screening test results in women under age 30 were selected. Following the selection, 3849 LBC results, 1321 HRHPV14 and 316 p16/Ki67 tests results were obtained. The final study group consisted of 121 patients <30 years with available LBC, HRHPV14 and p16/Ki67 tests results who had colposcopy with biopsy and cytologic–virologic–immunocytochemical–histologic correlations could be assessed. Including all the LBS and histology results, in the final group, a retrospective analysis was conducted, and the diagnostic value was evaluated together with assessment numbers of colposcopies needed per detection of HSIL (CIN2+). This analysis comprised 3 screening models (the first screening round with unknown screening history), as follows: (1) model No 1 (M1)—primary HPV with reflex p16/Ki67 for all HPV-positive results; (2) model No 2 (M2)—primary cytology with reflex HRHPV, based on the risk-based management of ASCCP 2019 using a mobile application; (3) model No 3 (M3)—primary cytology alone without any reflex testing, based on the risk-based management of ASCCP 2019 using a mobile application. All analyzed data came from the center’s registry.HPV detection and genotyping were performed by the qualitative in vitro PCR Abbott RealTime High Risk HPV as recommended by the manufacturer, according to a report from the laboratory performing the test. This assay phenotypes 14 types of high-risk HPV DNA, including as follows  and specifically genotypes HPV 16 and HPV 18. If type 16 and/or 18 HPV is detected, the result was categorized as HPV16 or 18 or both positive. If one or more of the other 12 HRHPV14 types were detected, the result was categorized as N16/N18 positive.Liquid-based SurePath slides were obtained using the automatic laboratory PrepStain Slide processor  by a laboratory external to the center, and it was ensured that a laboratory procedure strictly adhered to the manufacturer’s protocol. All cytology samples were reported by a gynecological cytopathologist and classified according to the Bethesda 2014 guidelines. A cytopathologist was aware of the HRHPV14 status of the cervical samples. All residual cervical samples were stored for 6 months in the laboratory under the conditions specified by the manufacturer. This strategy enabled to perform additional tests also ordered later (including the p16/Ki67 triage) and with the patient’s comfort, who did not have to be called for another gynecological visit, and all cervical cancer diagnostics was performed from one cytological sample collection.p16/Ki67 testing was performed in HRHPV14-positive cases and in women who underwent co-testing plus. Dual immunocytochemical staining of cervical samples was carried out using p16 and Ki67 proteins in CINtec PLUS detection kit  processed in an automatic BenchMark XT laboratory system , according to the manufacturer’s protocol, and with one control specimen present in each run. The p16/Ki67 testing was performed from the same LBC sample with residual material stored in a laboratory in the original SurePath vials . An immuno-expression status was assessed by a gynecological cytopathologist specially trained and certified in p16/Ki67 review and classified as positive, negative, or unsatisfactory. A positive result of the p16/Ki67 test was the presence of at least one cell in the preparation meeting the following evaluation criteria: simultaneous red nuclear stain for Ki67 and brown cytoplasmatic stain for p16 in the same epithelial cell. For a morphological evaluation of the cell groups, the positive diagnosis was determined by a strong diffuse p16 stain within the group and the presence of at least one cell with a Ki67 stain in the nucleus and a p16 stain in the cytoplasm at the edge of the assessed group and/or sheet of cells. The negative p16/Ki67 test result was defined when no staining or single staining of p16 or Ki67 was present in the epithelial cell. An unsatisfactory p16/Ki67 test result was determined by features included in the manufacturer’s kit and/or by cellularity criteria, which are consistent with the criteria for a liquid-based cytological preparation.Women with positive p16/Ki67 test result, ASC-US or LSIL HRHP14 positive, with cytologic ASC-H or HSIL regardless of HRHPV14 status, or with ASC-H or HSIL without HRHPV14  were referred for a colposcopy. Indications for the colposcopy were according to Polish guidelines with the extension of the 2012 ASCCP guidelines and 2015 ASCCP interim guidelines. The colposcopy protocol included endocervical sampling as a minimum sampling approach and additional direct or random biopsies if required. Histologic diagnoses of cervical biopsies and endocervical specimens (obtained using curettage with brushing) were reviewed by a gynecological pathologist, according to the LAST 2012/WHO 2014 terminology ,23. Colpp < 0.05 was considered significant.Statistical analysis was conducted using licensed PQStat Software. A diagnostic value of tests, measured with sensitivity, specificity, positive (PPV) and negative (NPV) predictive values, and positive (PLR) and negative (NLR) likelihood ratios, was calculated according to standard definitions. Differences in diagnostic value between the analyzed screening models were evaluated, and the colposcopy number needed for each model was also compared. A In the analyzed period, from August 2015 to July 2020, a total of 5.486 LBS tests were performed in patients aged 15–29 years, with a mean age of 25.3 years. A total of 3.849 LBC results were analyzed, with 1.321 HRHPV14 and 316 p16/Ki67 tests results. The following reporting rates were obtained for cytological diagnoses compared to the group of women over 30 years of age, NILM, ASC-US, LSIL and ASC-H+ (% <30 years of age/% ≥30 years of age) 91.3/94.7, 3.9/3.2, 4.2/1.5 and 0.6/0.57, respectively. The biggest difference in the baseline group <30 years of age was for diagnosis of LSIL, which in women <30 years of age was almost three times more frequent than in the 30+ group. In the final study group of 121 women, detected HSIL (CIN2+) cases were 19.83% of all histologic diagnoses. No screening in this group of women would lead to remain these HSIL (CIN2+) lesions undetected, with all the potential consequences. The highest level of the diagnostic value, including sensitivity, PPV, NPV, PLR and NLR, was in screening model 1 of 83.3, 29.4, 91.7, 1.60 and 0.35, respectively. Only in specificity, the highest value of 61.9% was in screening model 3, but it should be remembered that this would be at the expense of not detecting 13 cases of HSIL (CIN2+). In M3, the lowest absolute number of colposcopies needed to perform would also be obtained, but the detection index (number of colposcopies needed per HSIL (CIN2+) detection) is significantly higher in M3 than in M1. The results of the diagnostic value for the analyzed models, a number of HSIL (CIN2+) missing cases and a number of colposcopies performed in each model along with detection of HSIL (CIN2+) vs. colposcopies needed are presented in In this study, we compared the diagnostic value of primary HPV screening with p16/Ki67 triage and cytology-based screening strategies  in women under 30 years of age. The sensitivity of screening model 1 (primary HPV with p16/Ki67 triage) was significantly higher than the sensitivity of cytology with reflex HPV and cytology alone screening models for detection histologic HSIL (CIN2+) in this age range . Moreover, the diagnostic predictive values were significantly higher for primary HPV with p16/Ki67 triage screening model (29.4% for PPV) compared to cytology with reflex HPV and cytology alone . The negative predictive value of primary HPV with p16/Ki67 triage for HPV-positive cases was very high (91.7% at CIN2+ threshold), which suggests the high safety of HPV-positive and p16/Ki67 negative women. The specificity for the detection of HSIL (CIN2+) of primary HPV triaging with p16/Ki67 was statistically significantly higher compared to cytology alone (47.8% vs. 35.1%) but was lower than for cytological screening with reflex HPV testing (61.9%). Our results also indicate a higher level of true screening test results in primary HPV with p16/Ki67 triage compared to cytology with reflex HPV and cytology alone screening models, due to the highest PLR  and lowest NLR (0.35 vs. 0.83 and 0.88) in this screening strategy.Cytology is well known and easily available triaging option for HPV-positive women; however, it is suboptimal due to the lower sensitivity. This requires the use of a secondary screening tool that is more focused on the detection of cervical cancer precursors in HPV-positive patients. This approach was represented in our study by screening model no. 1, in which a highly specific and highly sensitive p16/Ki67 biomarker was incorporated as a triage test in women who had undergone primary HPV screening and had positive HPV status. Our study results are essentially consistent with the data from other reports on the evaluation of the diagnostic performance of p16/Ki67 dual-stained cytology for triaging HPV-positive cases ,20,21,24Similarities with several studies in the sensitivity levels for detection HSIL (CIN2+) obtained in our screening model 1 were noted, including the study of the New Technologies for Cervical Cancer Screening group  (81.8%),High or very high levels of negative predictive value for detecting cervical cancer precursors, regardless of the threshold taken (HSIL(CIN2+) or HSIL(CIN3+)), in most other studies and in our observed at a level higher than 90% or higher than 95% ,21,25, sAs part of our retrospective assessment of diagnostic performance in analyzed screening models was the determination of the HSIL (CIN2+) detection versus colposcopies need index. In screening model 3 with primary cytology alone, the lowest absolute number of colposcopies needed to perform for HSIL (CIN2+) detection was identified. However, the histologic HSIL detection index (CIN2+) per colposcopy rate in this model was higher than in the screening model based on primary HPV testing with p16/Ki67 triaging and based on cytology with reflex HPV. The best rate in the number of colposcopies needed to perform for detection of histologic HSIL was observed in screening model 1 with biomarker triage used. In both analyzed cytology-based screening models, 7 (in M2) and 13 (in M3) cases of HSIL (CIN2+) HRHPV14-positive and p16/Ki67-positive would not be detected.We believe that promoting an effective cervical cancer screening with primary HPV in younger age groups of women, including women below 25, may be associated with building appropriate habits, which may result in a positive change in recently recorded increases in cancers rate among women 25–29 years. This may be particularly important in countries with very high HDI and with still too low HPV vaccination coverage and may be a factor making it independent of changes in sexual behavior . FurtherStrengths of this study: (1) One of the few studies evaluating the diagnostic value of p16/Ki67 triage in HPV-positive women <30 years who underwent primary HPV screening, including women below 25 years. (2) A thorough cervical disease ascertainment due to the extended colposcopic protocol used. (3) Our study gives an insight into screening tests results obtained in a private-based opportunistic cervical cancer screening. (4) All diagnostic tests were performed in the same specimen. (5) Immediate histologic correlation results due to the interval between colposcopy and abnormal screening test results not exceeding 3 months. (6) All p16/Ki67 tests were evaluated by a qualified gynecologic cytopathologist. Limitations of the study: (1) This study shows a post-hoc analysis. (2) We currently do not have a large enough group of patients to evaluate a diagnostic value at the CIN3+ threshold in the presented screening approaches. (3) Not all patients with indications for colposcopy decided to do it at the center. Due to different colposcopic protocols used, different histologic terminology and/or lack of p16 stain in cervical histologic specimens, the results of colposcopic biopsies performed outside the center were not included in the study.In conclusion, our study showed that in women under 30 years, cervical cancer screening model based on primary HPV testing with p16/Ki67 dual-staining triage of HPV-positive women can be an effective screening approach in detecting cervical precancers and provide superior diagnostic value when compared with primary cytology with reflex HPV or primary cytology alone models. This investigation also demonstrated that young women below 25 years screened by primary HPV and triage with p16/Ki67 histologic HSIL biomarker might benefit from introducing this more sensitive screening algorithm, especially in the light of new data on the cervical cancer rates elevation in younger groups of women in countries with very high HDI.


There are limited data on the epidemiology of HPV in different anatomical sites of female sex workers (FSW). We investigated the prevalence and concordance of cervical, vulval, oral and anal HPV among FSW in Ibadan, Nigeria.FSWs aged 18–45 years were enrolled in a cross-sectional survey. After interview and clinical examination, samples were collected from mouth, cervix, vulva and anus. HPV genotyping was done with Anyplex II 28HPV assay. Multivariable analyses were performed to explore associated risk factors and concordance of HPV infections across sites.In total, 315 FSWs participated in the study with a mean age of 30–6.5 years. The prevalence of any HPV infection was 88% in the vulva, 84% in the cervix, 75% in the anus and 24% in the oral cavity. HPV 35 was the most prevalent and concordant high-risk type in the four sites. The risk factors for HPV infection by anatomic site varied.This large study showed a high prevalence and concordance of HPV infections of cervical, vulval, oral and anal HPV among FSWs in Nigeria. The potential to acquire and transmit HPV is high in this population, and we highlighted the urgency to protect young women through HPV vaccination. Human papillomavirus (HPV) infection occurs in nearly 80% of sexually active people at least once in a lifetime , 2. MostFSWs have more than two-times the risk of acquiring genital HPV infections and increased prevalence of abnormal pap smears than women in the general population . In 2020In 2019, a systematic review showed that sexually active women including FSWs engage in heterosexual oral and anal sex in SSA . HoweverThis cross-sectional survey was conducted among FSWs aged 18–45 years in brothels as part of the Sexual behaviour and HPV Infection in Nigerians in Ibadan (SHINI) study . BrothelBefore enrolment, trained field workers mapped and updated the existing list of brothels in the six urban LGAs in Ibadan. Eligible participants were listed and assigned a unique number by female research assistants by questioning the manager/chairlady (leader of FSWs). For brothels that had 10 FSWs or fewer, we invited all eligible FSWs while 90% of the FSWs were selected by a simple random sampling in brothels that had 11 or more eligible participants. Female research assistants visited each brothel to explain the study objectives to potential participants collectively and then individually in their rooms, and a copy of an information leaflet was also given to each of them. After, a signed consent for was obtained which also covered storage of samples for future use.Interview and sample collection were done inside the bedroom of each participant. A face-to-face interview with paper questionnaires was conducted in English or pidgin English for participants that could not understand English by female research assistants. The interview covered socio-demographics, sexual behaviours, condom use and hygiene practices, intravaginal practices, alcohol consumption, smoking, stimulant use, previous STI and awareness about HPV vaccine. After the interview, a female nurse collected blood and performed rapid diagnostic HIV test (RDT using Nigerian HIV test protocol ). Partic©). The nurse demonstrated the rinse and gargle procedure to individual participant. The participant sample was then collected into a 10 ml labelled sample bottle and placed immediately into a cold box (2–4°C). For the vulvar sample, the tip of a Dacron swab was used to rub the introitus on either side of the vaginal orifice without touching the urethral orifice. The cervical sample was then collected by inserting a sterile Cusco speculum into the vagina to expose the cervix. The tip of a new Dacron swab was inserted into the cervical os and gently rotated 360 degrees to avoid trauma to the cervix and potential bleeding before removing it. An anal sample was collected with the participant in a left lateral position. A Dacron swab was inserted into the anal canal  and rotated 360 degrees with gentle pressure around the anal verge before removing it. Swab were placed in separate 2 ml cryotubes that were labelled and barcoded prior to being placed into a cold box (2–4°C).Biological samples were collected by a trained nurse from the cervix, vulva and oral and anal cavities in separate sample bottles. Briefly, an oral sample was collected using a 30 second oral rinse and gargle method with 10mls of Scope mouth wash , while cervical, vulvar and anal dry swabs were performed using the Maxwell 16 Buccal swab LEV DNA Purification kit, following the procedures previously described [DNA extraction from the mouthwash was performed using the Maxwellescribed .™ II HPV28  assay [™ II HPV28 detection test was performed according to the manufacturer’s instructions using 10 μl of sample DNA [HPV genotyping of samples were performed at the Catalan Institute of Oncology, Spain using the Anyplexa) assay . The Anymple DNA . Human BVanderbilt University, Nashville Tennessee, USA). The CSV data format were imported into STATA 16.0 software for analysis. Descriptive statistics were performed and presented. The primary outcome was prevalence of any HPV infection. The prevalence of HR-HPV and LR-HPV infection and by 2009 IARC epidemiological oncogenic classification for each of the anatomical sites were calculated. The trends of association between each classification of HPV infection and the age group of participants were calculated with ANOVA. The association between any HPV infection and explanatory variables were tested using hierarchical logistic model as shown in the conceptual framework (a priori. Explanatory variables were grouped into Level 1 (sociodemographic factors), Level 2  and Level 3  (The interview data were double entered into REDCap software (ramework . Any HPVfactors) .Each variable in Level 1 (sociodemographic factors) was added one by one to the model that included age group and study site . P-valueEthical approvals were obtained from the ethical committees of the London School of Hygiene and Tropical Medicine, London (LSHTM 9736–3); the University of Ibadan/the University College Hospital, Ibadan (UI/EC/16/005); and the Oyo State Government (AD13/479/712) in Nigeria. Participants gave a written consent for participation and for their data to be published in a scientific journal.Out of 31 brothels in the 6 LGAs, two had closed and the management of one brothel declined participation. Three hundred and forty-four FSWs were listed from the remaining 28 brothels; 319 FSWs were randomly selected proportionate to brothel size. Of these, 315 FSWs consented and 4(1%) declined to partook in the study .The mean age of FSWs was 30.4 years (SD = 6.5); 55% were 25 to 34 years old. The median age of vaginal sexual debut of participants and their partners was 18.0 years (inter quartile range = 4.0) and 23.0 years (Inter quartile range = 7.0), respectively. About a quarter had passed their sexual debut by 15 years. Most (94%) FSWs reported using condoms during their last vaginal sex act and 160 (51%) reported more than 50 vaginal sex partners in the past 3 months.Sixty-one (19%) FSWs reported ever giving a male partner oral sex while 135 FSWs (43%) had ever received oral sex from a male partner. Only eight (3%) had received anal sex. A third (32%) were initiated into sex work by the age of 24 years. Fourteen (4%) FSWs had heard of HPV vaccine. Forty-two (13%) participants were HIV positive .Of the 1260 samples collected from the 4 anatomic sites, two (0.6%) cervical, four (1.3%) anal swabs, and 33 (10.5%) mouthwash samples were declared as invalid in the laboratory due to the lack of amplification of HPV and the internal control. These invalid samples were treated as missing variables. In total, 303 (96%) FSWs were positive for at least one HPV genotypes, 274 (87%) FSWs had one HR-HPV and 257 (82%) FSWs had one LR-HPV.Overall, the prevalence of any HPV among the FSWs was 87.9%  in the vulva, 84.0%  in the cervix, 74.6%  in the anus and 24.1%  in the mouth .There was an inverse relationship between the age of participants and any HPV, HR-HPV, and multiple HPV at all anatomic sites. Specifically, there was a significant inverse association between any HPV and age of participants in the vulvar and cervical samples. There was no association with age and HPV detection in oral samples by any HPV classification . HPV-35 The results of crude and adjusted models for the risk factor analyses associated with each of the four anatomic sites were presented in Tables Only age of participants and presence of any cervical and anal HPV were independently associated with detection of vulvar HPV. The odds of detecting any vulvar HPV was lower among women aged 25–34 years  and 35–45 years  compared to those aged 18–24 years. Detection of vulvar HPV was associated with higher odds of having any anal  and cervical  HPV.Factors associated with detection of any anal HPV include age, income and detection of any vulvar HPV. FSWs aged 25–34 years and 36–45 years had 0.39  and 0.37  odds of having any anal HPV, respectively, relative to those aged 18–24 years. The odds of having anal HPV was higher among those that earned 20,001 to 40,000 Naira  and more than 40,000 Naira  relative to those that earned less than 20,000 Naira a month. Reported anal sex was not considered as an explanatory factor for anal HPV due to small number of observations. FSWs that had any vulvar HPV had higher odds  of having any anal HPV than those without vulvar HPV. There was higher odds of oral HPV among FSWs that had ever heard of HPV  compared to those with no information about HPV. HIV positive FSWs had 2.40  times higher odds of having any oral HPV compared to those that were HIV negative.Results of concordance of specific HPV genotypes were presented in In the six possible two-sites comparisons, concordance of HR and LR-HPV was highest between cervix and vulva, and least between oral and anal cavity. HPV-68 (17/83) and HPV-35 (13/86) were the mostly frequently detected HR genotypes between cervix and vulvar sites . Of the In this study, HPV infection was highly prevalent with 96% of FSWs having at least 1 genotype-specific infection in one of the 4 sites. More than 70% of FSW had HPV in the vulva, cervix, and anus, and 24% had an oral HPV infection. Multiple HPV infections were also common, especially in the vulva. HPV-35 was the most common HR-HPV infections in the 4 anatomic sites. To our knowledge this is the largest data on the prevalence of, risk factor and concordance of HPV infections in the oral, genital and anal sites among FSWs.The prevalence of any HPV, HR-and LR-HPV, and multiple HPV infections in the oral, cervical, vulvar and anal sites in this study was higher than the similar prevalences by anatomical sites reported among women in the general population in Ibadan, Nigeria . This isMost studies reported on cervical HPV and fewer studies were on anal, vulvar, anal and oral HPV infections in FSWs –23. The To date, this study presents the first data on oral HPV among FSWs in SSA. Oral HPV infection was higher in our study than two previous studies that reported oral HPV in FSW; 7.6% among 185 FSWs aged 18–26 years in Peru and 6.1% among 196 FSWs aged18-45 years in Japan . The obsContrary to previous studies that showed that HPV-16, HPV-31, HPV-51 or HPV-58 as the most frequent genotypes in the cervix and anus of FSWs in Europe and SSA , 20, 21,The low level of awareness of HPV vaccination among our participants despite having high prevalence of HPV infections in multiple anatomic sites is worrisome. Previous Nigerian studies have also reported low level of awareness and knowledge of HPV vaccination in sexually active women , 31. ThoSimilar to other studies, presence of HPV infection in a genital or anal sites as a common risk factor for detection of HPV infection in another anatomical site , 28. ConHIV infection is another risk factor found to be associated with cervical and oral HPV infections in our study, as in previous studies , 18. TheWe observed a reduced risk of cervical HPV infection among FSWs with clinical evidence of FGM. It is plausible that the observed reduced risk might be due to less sexual activity from lack of sexual pleasure or orgasm and or increased sexual dysfunctions among people with FGM , 38. HowThe major strength of this study was the collection of multiple HPV data at the same time, and this is the first study to date with such diverse anatomic sampling among FSWs. We used probability sampling to select participants, and also limited recruitment to FSWs in the brothels. Previous studies that combined different sub-groups of FSWs from brothels, entertainment venues and streets, while others provided no information on the sub-group of FSWs that were recruited , 17, 27.In conclusion, we found a high prevalence of HPV in FSW in Nigeria, and this is higher compared to similar studies among FSW in different settings. Similar to our previous study, HPV-35 was the most common HR-HPV in all sites. The study also showed evidence of high infection among HIV positive participants especially in the cervical and oral sites. Future studies should include other categories of FSWs such as those working on the streets and entertainment venues who might be even more vulnerable to HPV infection. Longitudinal studies will help to answer other critical issues such as the incidence, clearance, and persistence of HPV in this group. Over 60% of FSW had evidence of HR-HPV in the cervical, vulvar and anal sites and in over 60% of women aged 35 years and around 15% had HR-HPV in the oral cavity, highlighting the need for prophylactic HPV vaccination and screening in Nigeria.S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


Sexually transmitted infections (STIs) are prevalent throughout the world and impose a significant burden on individual health and public health systems. Missed diagnosis and late treatment of STIs can lead to serious complications such as infertility and cervical cancer. Although sexually transmitted co-infections are common, most commercial assays target one or a few STIs. The HPV-STI ChapterDx Next Generation Sequencing (NGS) assay detects and quantifies 29 HPVs and 14 other STIs in a single-tube and single-step PCR reaction and can be applied to tens to thousands of samples in a single sequencing run.A cohort of 274 samples, previously analyzed by conventional cytology/histology and Roche cobas HPV Test, were analyzed by ChapterDx HPV-STI NGS assay for detection of 43 HPV and STI. A set of 43 synthetic control DNA fragments for 43 HPV and STI were developed to evaluate the limit of detection, specificity, and sensitivity of ChapterDx HPV-STI NGS assay.C. trachomatis, T. vaginalis, M. genitalium, and HSV2 were identified.The assay was evaluated in this study, and the limit of detection was 100% at 50 copies for all targets, and 100%, 96%, 88% at 20 copies for 34, 8, and 1 target, respectively. The performance of this assay has been compared to Roche cobas HPV test, showing an overall agreement of 97.5% for hr-HPV, and 98.5% for both, HPV16 and HPV18. The assay also detected all HPV-infected CIN2/3 with 100% agreement with Roche cobas HPV results. Moreover, several co-infections with non-HPV STIs, such as The ChapterDx HPV-STI NGS assay is a user-friendly, easy to automate and cost-efficient assay, which provides accurate and comprehensive results for a wide spectrum of HPVs and STIs.The online version contains supplementary material available at 10.1186/s13027-022-00420-8. Chlamydia trachomatis, Neisseria gonorrhoeae, Treponema pallidum (syphilis), and Trichomonas vaginalis. Prevalence of several viral STIs is similarly high with an estimated 500 million people infected with herpes simplex type 2 (HSV2), and approximately 291 million women harboring human papillomaviruses (HPV) [Sexually transmitted infections (STIs) are prevalent globally and World Health Organization (WHO) estimates that annually there are 376 million new cases of four curable STIs: HPVs are a large group of viruses with more than 200 completely characterized types, and new HPV types being continuously found . Among tHPV viral load quantification has been shown to be a predictor of infection persistence or clearance . It has Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, Ureaplasma urealyticum, and HSV2 have been reported to be associated with HPV persistence, cervical dysplastic and neoplastic lesion [Besides HPVs, several bacteria and viruses infect human anogenital area. Earlier studies indicated that non-HPV STIs could facilitate the entry of HPV virions into host cells, change in the immunological response pathways, and decrease the host’s ability to clear HPV infection –18. Co-ic lesion –26. Co-iThis work presents the evaluation of the ChapterDx HPV-STI NGS assay (NGS assay in short) that amplifies 29 HPVs and 14 STIs  was selected for this study. Within this cohort, 102, 101 and 57 samples were negative for intraepithelial lesions or malignancy (NILM), low-grade squamous intraepithelial lesion (LSIL) and high-grade squamous intraepithelial lesion (HSIL), respectively; and five were invasive carcinoma and nine samples had no cytology results. The available histological results for this cohort were: cervical intraepithelial neoplasia grade 1 (CIN1) (N = 4), CIN2 (N = 1) and CIN3 (N = 58), 3 squamous cell carcinoma (SCC) and 2 adenocarcinoma (AdC). HPV samples were shipped from Oncogenic Viruses Service, National and Regional HPV Reference Laboratory, National Institute of Infectious Diseases-ANLIS “Dr. Malbrán”, Buenos Aires, Argentina to Chapter Diagnostics in Menlo Park, California, USA, for comprehensive HPV-STI analysis.The 274-sample cohort were analyzed by Roche cobas HPV automated platform . The ampChapterDx HPV-STI NGS kit targets 19 hr-HPVs, 10 lr-HPVs, 14 non-HPV STIs, and GAPDH as internal control. Table Chlamydia trachomatis (serovars A to K) (CP018052.1), Chlamydia trachomatis serovar L1 (JN795429.1), Chlamydia trachomatis serovar L2 (JN795428.1), HSV1 (KX946970.1), HSV2 (KU886303.1), VZV (KX262866.1), Haemophilus ducreyi (CP015434.1), Mycoplasma genitalium (CP003773.1), Mycoplasma hominis (CP011538.1), Neisseria gonorrhoeae (LT592163.1), Treponema pallidum (CP016048.1), Trichomonas vaginalis (XM_001582357.1), Ureaplasma parvum (AP014584.1) and Ureaplasma urealyticum (CP041200.1). The DNA controls were pooled and diluted into 100, 50 and 20 copies per vial in a final volume of 5 µl. Each pool was spiked with 10 ng human genomic DNA. The pools were amplified and sequenced in 23 replicates for 100 copies and 24 replicates for 50 and 20 copies to assess the limit of detection (LoD) of the NGS assay. The acceptable consensus for an assay’s LoD at the lowest concentration is ≥ 95% for at which 19/20 replicates are positive [A set of 43 synthetic control DNA fragments for all targets were synthesized by Integrated DNA Technologies . The controls were designed based on following references derived from PapillomaVirus Episteme (PaVE) database: HPV6 KU298876.1), HPV11 (KU298879.1), HPV16 (LC193821.1), HPV18 (KU707825.1), HPV26 (KF444058.1), HPV31 (KU163582.1), HPV33 (KU050111.1), HPV35 (JX129488.1), HPV39 (KC470231.1), HPV40 (KU298895.1), HPV42 (JQ902118.1), HPV43 (HE962401.1), HPV44 (HE963128.1), HPV45 (KU049754.1), HPV51 (KU050115.1), HPV52 (KU298909.1), HPV53 (KU951265.1), HPV55 (KU298899.1), HPV56 (KU050122.1), HPV58 (KU550636.1), HPV59 (KC470266.1), HPV61 (KU298923.1), HPV66 (KU298928.1), HPV68a (KC470268.1), HPV68b (KU298932.1), HPV73 (KU298935.1), HPV81 (KU298939.1), HPV82 (AB027021.1), HPV83 (AF151983.1), 8876.1, HGenomic DNA extraction for 274-sample cohort were performed by Roche cobas HPV Test (cobas for short) platform. All the specimens were measured by Qubit 3  to monitor the presence and concentration of DNA prior to PCR. One-step multiplex PCR was performed in 15 µl final volume in a 96-well plate on a Veriti thermocycler . The PCR reaction consisted of target-specific primers , barcoded universal primers, sample DNA, DNA polymerase, dNTPs and PCR buffer. The PCR conditions were according the ChapterDx HPV-STI NGS kit instructions.After PCR, the amplified products were pooled into a 1.5 ml tube (or a 15 ml tube depending on the sample size). A portion of the pooled amplicons was purified with SPRIbeads  according to the manufacturer’s instructions. The purified pooled amplicons concentration was measured on a Qubit fluorometer, and the concentration was adjusted for sequencing according to Illumina library preparation instructions. The library was sequenced on an Illumina MiniSeq platform using an Illumina Mid-Output sequencing kit . The workflow is depicted in Fig. (1) Decoding: sequencing reads (FASTQ format) for forward and reverse reads and forward and reverse indexing reads are input in ChapterDx Analysis Software. The software assigns sequencing reads for a sample based on the sequence of both forward and reverse index reads with no mismatch; (2) Mapping: sequencing reads are mapped onto reference sequences using the Smith-Waterman algorithm with options as nucleotide match reward is 1, nucleic mismatch penalty is -3, cost to open a gap is 5, and cost to extend a gap is 2. Only the alignment of best match is kept for each sequencing read if the alignment score is beyond 60. Alignments of paired reads with identical reference sequences are kept. Reads are labeled to the reference based on their corresponding alignments; (3) Genotyping: a sample is called positive for a species/type when there are more reads than the preset cutoff reads labelled with the corresponding reference.For determining the cutoff detection threshold, samples from HPV LabNet international proficiency panel  as well The HPV and STI species/types frequencies, cytology and histology were calculated by Microsoft excel. Overall agreement for 14 hr-HPV, HPV16, 18 and other 12 HPVs between ChapterDx HPV-STI NGS and Roche cobas HPV assays were calculated by Cohen’s Kappa coefficients with 95% confidence intervals (CIs).Mycoplasma genitalium .To evaluate the analytical performance and sensitivity of the assay, a set of 43 synthetic control DNA fragment pools of 100 copies (23 replicates), 50 copies (24 replicates), 20 copies (24 replicates) and 0 copies (blank) spiked with 10 ng human genomic DNA were analyzed to determine the LoD. 100 copies and 50 copies control DNA were detected in all replicates. For the 20 copies pool replicates, the detection rate was 100% (24/24) for 34 targets, 95.8% (23/24) for HPV6, 11, 39, 42, 43, 45, 56 and 83 (8 targets) and 87.5% (21/24) for To evaluate the agreement with the Roche cobas HPV test, 274 clinical samples were analyzed by ChapterDx HPV-STI NGS assay. Out of 274 samples, the results for 267 samples agreed with those by cobas HPV test for 14 hr-HPVs. Among those 267 samples, 241 were positive and 26 were negative for 14 hr-HPVs. The overall agreement for 14 hr-HPVs between both methods was 97.5% with kappa 0.867. The CIN1 (n = 4), CIN2/3 (n = 59) were positive for hr-HPV by both cobas and the NGS assays  with a kappa value of 0.967 (Table U. parvum (32.1%) and M. hominis (25.2%) comprised the majority of the non-HPV STIs , existing in both healthy and symptomatic individuals [Earlier studies have shown evidence of association between hr-HPVs and non-HPV STIs  with cervical carcinogenesis –26, 33. ividuals . Such coHPV genotyping has been mostly focused on HPV16 and HPV18 due to their high prevalence in cervical cancer, although, other hr-types indicate high positive predictive value (PPV) . EarlierIt has been reported that non-transient hr-HPV infections and high viral loads is a risk marker for dysplasia and carcinoma in situ in normal cytology –16. SuchThere are several NGS-based HPV assays –40 that ChapterDx HPV-STI NGS Assay is cost-effective as it detects a much broader spectrum of HPV/STI than other commercial kits, at a similar or lower cost per sample, with proven analytical performance; therefore, it is a valuable tool for large epidemiology and surveillance studies. Once evaluated with larger, well-characterized cohorts for clinical validation, this assay could also be applied to cervical cancer screening programs.Additional file 1. Comparison of Illumina sequencing platforms and sequencing kits for HPV-STI analysis.


External anogenital warts (EGW) are primarily associated with the low-risk human papillomavirus (HPV) genotypes 6 and 11, though coinfection with other low-risk and oncogenic high-risk HPV genotypes also occurs. Although there have been many studies on HPV-associated disease, the prevalence of HPV genotypes associated with EGW is not well characterized. The objective of our retrospective study was to determine the prevalence of HPV genotypes among patients diagnosed with EGW in the south-west of Hungary. Archived formalin-fixed paraffin-embedded tissues from 94 patients were processed in our study. HPV genotypes were determined, applying HPV Direct Flow CHIP test. The overall prevalence of HPV DNA in the EGW samples was 100%, yielding 131 infections among the 94 samples. Of these cases, 72.3% were mono while 27.6% were multi-infections. Out of the 131 infections, the cumulative prevalence of HPV 6 and 11 was 71%. A total of 98.9% of the samples were carrying at least one of these genotypes, while 19.1% of the cases occurred with at least one high-risk genotype. Data from our study could provide invaluable information concerning the prevalence of HPV types among patients with EGW, enabling improved assessment of the actual and future efficacy of vaccination programs, vaccine development, and forecast changes in infection patterns. Human papillomavirus (HPV) infection is responsible for the development of external anogenital warts (EGW), often referred to as external condyloma acuminata . Externan = 66 females; n = 28 males), earlier diagnosed with EGW, were processed in our retrospective study. The patients mostly resided in the south-west part of Hungary. The study was performed based on females and males that attended the Departments of Obstetrics and Gynecology, Urology and Department of Dermatology, Venereology, and Oncodermatology at the University of Pécs, between 2004 and 2020, due to routine gynecological and urological screening, or because of symptoms of external anogenital warts, such as pain, bleeding or aesthetic reasons. Patient’s data  were extracted from medical records.Archived, formalin-fixed paraffin-embedded (FFPE) samples from 94 patients . The technology is based on the amplification of a short DNA fragment of HPV L1 ORF, enabling the use of FFPE samples. The test allows the qualitative detection of thirty-five different genotypes of HPV  based on direct PCR from crude-cell extracts, automatic flow-through hybridization and colorimetric detection. All the virus types are determined separately except for the low-risk 44/55 and 62/81 genotypes, which are identified in clusters.The test was performed according to the manufacturer’s instructions. In brief: 3 pieces of 10 µM thick sections were treated with 400 µL of mineral oil then incubated at 95 °C for 2 min. After removing the mineral oil, 60 µL lysis buffer with 1.5 µL DNA release  was added to the samples and incubated at 60 °C for 30 min, followed by inactivation at 98 °C for 10 min. To lyophilized PCR mix, 3 µL of crude cell extract and 27 µL DNase/RNase free water were added. The lyophilized PCR mix contained PCR buffer, dNTP(U/T), DNase/RNase free water, biotinylated primers, DNA polymerase, and UNG. The primers included are specific for the amplification of a fragment of the L1 region of the HPV genome. Besides, primers for the amplification of the human beta-globin gene are included and used as an internal control for the PCR reaction. The amplification cycling conditions in IANLONG PCR Thermal Cycler (Genesy 96T) were the following: pre-incubation at 25 °C for 10 min then 94 °C for 3 min; 15 cycles of denaturation at 94 °C for 30 s, annealing at 47 °C for 30 s and elongation at 72 °C for 30 s; 35 cycles of denaturation at 94 °C for 30 s, annealing at 65 °C for 30 s and elongation at 72 °C for 30 s and final elongation at 72 °C for 5 min; followed by cooling at 8 °C. Amplicons were denatured at 95 °C for 10 min then cooled on ice for 5 min.The full hybridization process was performed semi-automatically in hybriSpot (HS12) following the instructions provided by the system. The management of the samples, the capture of images, and the analysis and report of the results were performed by hybriSoft software .p values < 0.05 were considered to be statistically significant.The overall prevalence of HPV genotypes was calculated. The distribution of specific HPV genotypes was expressed as the proportion of HPV DNA-positive specimens among all cases of EGW. Qualitative variables were studied using the two-sided Chi test or Fisher’s exact test as appropriate. Quantitative data are expressed as mean (±SD) and range. p = 0.035, at p < 0.05).Samples from sixty-six females  and twenty-eight males  were processed in the study; all of them provided positive β-globin results, suggesting that the DNA amount were suitable for PCR analysis. All patients were confirmed to be HPV positive and all the genotypes had been identified. The patient characteristics are presented in p = 0.276; at p < 0.05). The presence of ≥1 high-risk genotype was as common among males as females  and occurred as frequently in the new cases as in the clinically recurrent cases . Our results also indicated that ≥1 high-risk genotype was mostly found at ages under thirty years .HPV genotype classification was based on the HPV Direct Flow CHIP test’s  description. HPV DNA was detected in all of the ninety-four EGW cases and genotypes were also determined in every case. From the ninety-four cases, sixty-eight (72.3%) showed one HPV genotype, while in twenty-six cases (27.6%), multi-infections were found. According to our results, none of the cases occurred with high-risk genotype without a low-risk genotype, whereas a total of eighteen (19.1%) samples demonstrated coinfection with low-risk and high-risk genotypes, and 4.3% of the samples were infected with more than one high-risk HPV . Comparip = 0.46 at p < 0.05). In the case of the low-risk genotypes, 60% of the detected HPV types shared homology  in female and male EGW cases, while 40% of the genotypes (HPV 44/55 and 62/81) were only found in females  HPV genotypes, ten low-risk, and eleven different high-risk HPV genotypes were detected, yielding a total number of one hundred thirty-one infections among the ninety-four samples . Out of  females .p = 0.035). According to our results, among the examined Hungarian patients the most common HPV genotypes were HPV 6 and 11. The vast majority of the samples, 72.3%, were diagnosed with mono-infection of HPV 6 and 11 genotypes, while these two genotypes either alone or in combination with other HPV genotypes, occurred in 98.9% of the cases. Data regarding the prevalence of HPV 6 and 11 genotypes are comparable to the data reported from other European countries [It has been proposed that low-risk HPV genotypes do not integrate their DNA into the chromosomes of the infected cells, hence such low-grade lesions have a low risk of progression to malignancy. However, around 19–33% of EGW cases are coinfected with oncogenic, high-risk HPV genotypes ,22. Whilountries ,22. By aountries ,30,31,32We found that oncogenic, high-risk HPV genotypes never occurred as mono-infection. Our study revealed, that 19.1% of the cases occurred with ≥1 high-risk HPV genotype. Although data were not significant, by analyzing the prevalence of high-risk genotypes according to different age distributions, in line with other studies, we found that ≥1 high-risk genotype was more represented in the age group of under thirty years of age . This coBased on the analysis of Mayenaux and Chaturwedi, the incidence of HPV infection could be kept in check; although the continuously increasing number of risk factors, such as the growing number of lifetime sexual partners, earlier sexual debut (<16 ages), homosexual relationships, history of other STDs, smoking and human immunodeficiency virus (HIV), can only be counteracted by screening programs and vaccination ,33,34.Currently, three licensed HPV vaccines are available using L1 capsid antigens of two, four, or nine HPV genotypes. All three vaccines include HPV 16 and 18, which cause the majority of HPV-related cancers; the quadrivalent vaccine (Gardasil 4) also targets low-risk types HPV 6 and 11, which are primarily associated with external anogenital warts, while the nonavalent (Gardasil 9) vaccine also includes five other high-risk HPV types such as HPV 31, 33, 45, 52, and 58. Preliminary data also indicate that vaccination against one HPV strain may, in turn, confer protection against heterologous HPVs ,28,35. HThe published data on type-specific HPV-associated diseases in Central and East European countries are scarce. Our retrospective study is the first to provide data on the prevalence of different HPV genotypes among Hungarian patients diagnosed with EGW. While most of the studies have analyzed only small numbers of genotypes, our wide spectrum analysis of HPV genotypes will contribute to understanding the HPV prevalence in EGW and may have an impact on vaccine development and the HPV vaccination program in Hungary.Although our results show high accordance with those of other studies on HPV and EGW, the relatively small sample size and limited clinical data for male patients made the statistical comparison difficult.


Quercus robur L.) throughout most of its geographic range in Europe. We found that the damage caused by chewing insect herbivores as well as the incidence of leaf‐mining and gall‐inducing herbivores consistently decreased with increasing impervious surface around focal oaks. Herbivory by chewing herbivores increased with increasing forest cover, regardless of impervious surface. In contrast, an increase in local canopy cover buffered the negative effect of impervious surface on leaf miners and strengthened its effect on gall inducers. These results show that—just like in non‐urban areas—plant–herbivore interactions in cities are structured by a complex set of interacting factors. This highlights that local habitat characteristics within cities have the potential to attenuate or modify the effect of impervious surfaces on biotic interactions.Urbanization is an important driver of the diversity and abundance of tree‐associated insect herbivores, but its consequences for insect herbivory are poorly understood. A likely source of variability among studies is the insufficient consideration of intra‐urban variability in forest cover. With the help of citizen scientists, we investigated the independent and interactive effects of local canopy cover and percentage of impervious surface on insect herbivory in the pedunculate oak ( We investigated the independent and interactive effect of impervious surface and local canopy cover on insect herbivory on the pedunculate oak throughout most of its distribution in Europe through a citizen science project. We found that local canopy cover within cities has the capacity to mitigate the effect of impervious surface on biotic interactions, as it influences the effect of impervious surface on herbivores differently. Therefore, this study supports the importance of maintaining trees in urban areas as it may consequently contribute to the preservation of biodiversity of insect herbivores in urban areas. To this end, we quantified herbivory as the proportion of leaf area consumed or impacted by chewing and leaf‐mining herbivores as well as the incidence of leaf‐mining and gall‐inducing herbivores in leaf samples collected by professional scientists and schoolchildren in European countries between 2018 and 2020. We specifically predicted that: (a) insect herbivory decreases with impervious surface and increases with canopy cover; (b) the effects of impervious surface and canopy cover on leaf herbivory vary among the herbivore guilds; and (c) impervious surface and local canopy cover have an interactive effect on insect herbivory that vary among herbivore guilds. Overall, this work provides one of the most comprehensive studies to test for effects of impervious surface on plant–herbivore interactions and shed light on potential mechanisms underlying such effects.In this study, we investigated the independent and interactive effects of impervious surface and local canopy cover on insect herbivory on the pedunculate oak  , 2019 (n = 56), and 2020 (n = 113), three of them been sampled twice in 2019 and 2020 . Leaves collected by partner schools (n = 98 oaks) were oven‐dried when received by the project coordinators, to warrant optimal preservation prior to herbivory assessment , scientists and partner schools haphazardly selected four low hanging branches per tree facing approximately compass directions. They haphazardly collected 30 leaves per branch . Then, 60 leaves were drawn blindly to reduce unconscious bias in leaf sampling. Scientists oven‐dried leaves for at least 48 h at 45°C right after collection  in a buffer with a radius of 200 m centered on the focal oaks based on oak coordinates as retrieved from Google Maps by project partners , and were independent of latitude  and climate . Although latitude was negatively correlated with temperature  and precipitation  which could have caused collinearity issue, a previous study found that climatic variables were better predictors of variation in herbivory, and therefore we decided to only include climatic variables in the models   , Local canopy cover (%), Impervious surface ×Local canopy cover, Year (as a factor), Spring temperature (°C), and Spring precipitation (mm) were included as fixed effects; and Partner ID as a random factor to account for the fact that some partners surveyed multiple trees and/or several years .iw)—i.e., the probability that a given model is the best model within the set of candidate models—and also the relative variable importance (RVI), which reflects the importance of a particular variable in relation to all other variables, as the sum of iw of every model including this variable. When multiple models were competing with the best model , we implemented a multi‐model inference approach, constructing a consensus model that comprised the selected variables from the set of best models. We subsequently averaged their effect sizes over all models in the set of best models, utilizing iw as the weighting parameter . A certain predictor was deemed to have a statistically significant effect on the response variable if its 95% confidence interval (CI) did not bracket zero  and ranged from 0 to 70%. Local canopy cover in a buffer of 20 m radius centered on focal oaks was on average 46.51 ± 1.90% and ranged from 0 to 100% cover.Impervious surface in a buffer of 200 m radius was on average 9.72 ± 0.91% . Model selection retained models that included the percentage of impervious surface and local canopy cover, year, and spring precipitation as predictors explaining variability in leaf damage Figure . AlthougInsect galls were present on 6.34 ± 0.01% of the inspected leaves. Model selection retained the percentage of impervious surface and local canopy cover, their interaction, year, spring temperature, and spring precipitation as important predictors explaining variability in leaf‐gall incidence Table . The mosLeaf miners were present in 17.98 ± 0.01% of the sampled leaves. Model selection retained the percentage of impervious surface, local canopy cover, their interaction, year, and spring precipitation as important predictors explaining variability in leaf‐miner incidence Table . The mos4Our study revealed that impervious surfaces can consistently reduce insect herbivory on the pedunculate oak throughout its geographic range. The effect of impervious surface was partially modulated by the percentage of canopy cover around oaks, with differences among herbivore feeding guilds. Specifically, we found that impervious surface and local canopy cover had independent and opposite effects on overall leaf damage. In contrast, effects of impervious surface on gall‐inducer and leaf‐miner incidence depended on local canopy cover, with a more pronounced negative effect of impervious surface on gall‐inducer and leaf‐miner incidence when there was a greater and lower local canopy cover, respectively. These results show that—just like in non‐urban areas—plant–herbivore interactions in cities are structured by a complex set of interacting factors. This highlights that local habitat characteristics within cities have the potential to attenuate or modify the effect of impervious surfaces on biotic interactions.4.1Quercus lobata) in urban areas than in natural areas. Similarly, Dobrosavljević et al.  may not scale proportionally with the abundance and diversity of insect herbivores. Several other ecological factors that are influenced by impervious surface may determine the amount of damage caused by herbivores. They include the top‐down control of herbivore populations by their enemies ; Data curation (lead); Formal analysis (lead); Investigation (lead); Methodology (lead); Project administration (lead); Supervision ; Validation (lead); Visualization (lead); Writing – original draft (lead). Anna Popova: Methodology ; Validation . Andrea Galmán: Investigation ; Methodology ; Validation . Andreas Prinzing: Investigation ; Methodology ; Validation . Andrey V. Selikhovkin: Methodology ; Validation . Andy G. Howe: Investigation ; Methodology ; Validation . Anna Mrazova: Methodology ; Validation . Anne‐Maïmiti Dulaurent: Investigation ; Methodology ; Validation . Arndt Hampe: Investigation ; Validation . Ayco Jerome Michel Tack: Investigation ; Methodology ; Validation . Christophe Bouget: Methodology ; Validation . Daniela Lupaștean: Methodology ; Validation . Deborah Harvey: Investigation ; Methodology ; Validation . Dmitry L. Musolin: Investigation ; Methodology ; Validation . Gábor L. Lövei: Investigation ; Methodology ; Validation . Giada Centenaro: Methodology ; Validation . Inge Van Halder: Investigation ; Methodology ; Validation . Jonas Hagge: Investigation ; Methodology ; Validation . Jovan Dobrosavljević: Investigation ; Methodology ; Validation . Juha‐Matti Pitkänen: Methodology ; Validation . Julia Koricheva: Investigation ; Methodology ; Validation . Katerina Sam: Investigation ; Methodology ; Validation . Luc Barbaro: Investigation ; Methodology ; Validation . Manuela Branco: Investigation ; Methodology ; Validation . Marco Ferrante: Investigation ; Methodology ; Validation . Maria Faticov: Investigation ; Methodology ; Validation . Markéta Tahadlová: Methodology ; Validation . Martin Gossner: Investigation ; Methodology ; Validation . Maxime Cauchoix: Investigation ; Methodology ; Validation . Michał Bogdziewicz: Investigation ; Methodology ; Validation . Mihai‐Leonard Duduman: Methodology ; Validation . Mikhail V. Kozlov: Investigation ; Methodology ; Validation . Mona C. Bjoern: Investigation ; Methodology ; Validation . Nikita A. Mamaev: Methodology ; Validation . Pilar Fernandez‐Conradi: Investigation ; Methodology ; Validation . Rebecca L. Thomas: Methodology ; Validation . Ross Wetherbee: Investigation ; Methodology ; Validation . Samantha Green: Methodology ; Validation . Slobodan Milanović: Methodology ; Validation . Xoaquín Moreira: Investigation ; Methodology ; Validation . Yannick Mellerin: Methodology ; Validation . Yasmine Kadiri: Methodology ; Validation . Bastien Castagneyrol: Conceptualization (lead); Data curation ; Formal analysis (lead); Funding acquisition (lead); Investigation (lead); Methodology (lead); Project administration (lead); Supervision (lead); Validation (lead); Visualization (lead); Writing – original draft (lead).Figure S1Click here for additional data file.Figure S2Click here for additional data file.Figure S3Click here for additional data file.Table S1Click here for additional data file.Supplementary MaterialSupinfoClick here for additional data file.


Cancer is a multi-factorial disease, with various intrinsic and environmental factors contributing to its occurrence. Human papillomavirus (HPV) has been associated with the occurrence of many cancers. India severely suffers from 3 HPV-associated cancers . Hence, the present study aimed to evaluate the HPV burden in these 3 cancers among patients from the western region of India.DNA was isolated from samples from 400 cervical cancer, 127 oral cancer, and 75 oropharyngeal cancer patients. Polymerase chain reaction was performed using degenerate primers for HPV infection.Overall, HPV infection was observed in 87% of cervical cancer cases, 12.5% of oral cancer cases, and 26.7% of oropharyngeal cancer cases when analyzed with a cumulative detection method using the MY 09/11, GP 5+/6+, and CP I/II primer sets.A significant prevalence of HPV infection was detected in all 3 cancers using the degenerate primer sets. This finding implies that testing for HPV infection using multiple primer sets is crucial for determining its actual prevalence in various malignancies. Human papillomavirus (HPV) has been identified as an etiological factor for cancer that contributes to 600,000 cancer cases every year worldwide . HPV hasDegenerate primer sets, including MY 09/11 and GP 5+/6+, are specifically designed in tandem with the highly conserved L1 region of the HPV genome, whereas CP I/II is designed for the E1 region. The degenerate primers were shown to have an improved HPV detection rate in our previous study . Even thHence the present study aimed to evaluate the prevalence of HPV in cervical cancer, oral cancer, and oropharyngeal cancer patients in the western region of India. Degenerate primer sets were used for HPV detection, with the specific aim of detecting HPV infection of any strain.The present study evaluated the burden of HPV infection in the 3 most prevalent HPV-associated cancers  using degenerate primer sets.The patients were enrolled in the study after histopathological confirmation from the pathology department at the Gujarat Cancer and Research Institute, Ahmedabad. Tissue biopsy samples of 400 cervical cancer, 127 oral cancer, and 75 oropharyngeal cancer patients were collected for the study after informed consent. The patients did not undergo any prior treatment for the malignancy. Patients with a simultaneous diagnosis of other major illness or coinfections with human immunodeficiency virus, hepatitis C virus, and hepatitis B virus were excluded from the study. Immediately after collection, the tissue samples were washed with phosphate-buffered saline (pH 7.4) and promptly stored at -80°C until analysis.DNA was isolated from tissues using a commercially available DNA isolation kit . The DNA yield was checked by spectrophotometric analysis  and DNA integrity was checked by agarose gel electrophoresis using an 0.8% gel.The DNA was amplified using the degenerate primer sets MY 09/11, GP 5+/6+, and CP I/II separately on a thermal cycler . The polymerase chain reaction (PCR) was prepared using a previously standardized protocol .For the MY 09/11 primers, PCR was carried out as follows: initial denaturation at 95°C for 3 minutes, followed by 40 cycles of denaturation at 95°C for 1 minute, annealing at 49.3°C for 1 minute, and extension at 72°C for 1 minute. In the final cycle, extension was carried out for 10 minutes. For the GP 5+/6+ primers, PCR was carried out as follows: initial denaturation at 95°C for 3 minutes, followed by 40 cycles of denaturation at 95°C for 1 minute, annealing at 48°C for 1 minute, and extension at 72°C for 1 minute. In the final cycle, extension was carried out for 10 minutes. For the CP I/II primers, PCR was carried out as follows: initial denaturation at 95°C for 3 minutes, followed by 40 cycles of denaturation at 95°C for 1 minute, annealing at 56.5°C for 1 minute, and extension at 72°C for 1 minute. In the final cycle, extension was carried out for 10 minutes. The samples were checked for the presence of HPV infection using 2% agarose gel electrophoresis.The MY 09/11 and GP 5+/6+ primers are designed to detect the L1 region of a wide range of the HPV genome, while CP I/II detects the E1 region . A combiThe study was approved by the Gujarat Cancer and Research Institute/the Gujarat Cancer Society Ethics Committee . Informed consent was confirmed by the committee.The present study was carried out in 3 highly prevalent HPV-associated cancers . The clinical and clinicopathological parameters of the cervical cancer patients n= 400), oral cancer patients (n= 127), and oropharyngeal cancer patients (n= 75) are given in 00, oral All 3 cohorts were screened for comprehensive HPV infections using the degenerate primer sets MY 09/11, GP 5+/6+ and CP I/II. Representative PCR patterns are depicted in Out of the cervical cancer patients enrolled in the study, 67.5% were positive for HPV infection by the MY 09/11 primer set, 80.7% of cases were positive by the GP 5+/6+ primer set, and 13.0% of cases were positive by the CP I/II primer set . The cumIn the investigation of 127 oral cancer cases, we observed that 7.8% of cases were positive for HPV infection by MY 09/11, while 4.7% of cases were positive by GP 5+/6+ and all cases were negative for HPV infection by the CP I/II primer set . The totAmong 75 oropharyngeal cancer cases, HPV positivity was found in 10.7% of cases using the MY 09/11 primer set and in 17.3% of cases using the GP 5+/6+ primers. Similar to oral cancer, no positivity was observed using the CP I/II primer set . A totalThe present study evaluated the burden of HPV infection in the 3 most prevalent HPV-associated cancers . The study was carried out in patients from the western region of India using degenerate primer sets. Our study demonstrated a comprehensive screening module to detect HPV infections without exclusion of any HPV type.We observed that 67.5% of cervical cancer cases were positive for HPV infection using the MY 09/11 primer set. Similar results were obtained in a study conducted in Odisha, which showed a 60.3% infection rate when analyzed using the PGMY 09/11 primer sets . In thisIn the investigation of 127 oral cancer cases, we observed that 7.8% of cases were positive for HPV infection by the MY 09/11 primer set, while 4.7% of cases were positive by the GP 5+/6+ primer set. This rate is lower than that reported in another study that recorded an HPV prevalence of 35.8% in oral mucosa samples when analyzed with the GP 5+/6+ primer set, whereas a positivity rate of only 2.2% was reported using the MY 09/11 primer set . FurtherWe also investigated the presence of HPV infection in 75 oropharyngeal cancer cases and observed HPV positivity in 10.7% of cases by the MY 09/11 primer set and 17.3% of cases by the GP 5+/6+ primer set. Similar to oral cancer, no positivity was observed using the CP I/II primer set. A study conducted in Delhi reported a 22.8% HPV infection rate in oropharyngeal cancer cases when analyzed using consensus primers . A totalThe study demonstrated a high rate of HPV prevalence (87.0%) in cervical cancer patients. Moreover, we also observed a notable HPV prevalence 12.5 and 26.7%) in oral and oropharyngeal cancers, respectively. HPV-positive cancers represent a distinct molecular and clinical entity from HPV-negative cancers .7% in or,30,31. FIn conclusion, the present investigation provides vital data for the study of the burden of HPV-associated cancers. The study found a high rate of HPV infection in cervical cancer and a notable prevalence of HPV infection in oral and oropharyngeal cancers using degenerate primers. Thus, the present study demonstrates the use of a comprehensive screening module for the effective detection of HPV infection in these 3 major malignancies without bias towards any HPV type.


In France, human papillomavirus (HPV) vaccination has been recommended in 2016 for men who have sex with men (MSM) up to age 26 years.We aimed to estimate HPV vaccine coverage in 18–28 year-old MSM and identify uptake determinants.We collected data on socio-demographic characteristics, sexual behaviour, sexually transmitted diseases (STI) screening and vaccination uptake using a voluntary cross-sectional online survey conducted in 2019 targeting MSM. We calculated coverage of at least one dose of HPV vaccine and prevalence ratios (PR) of determinants with 95% confidence intervals (CI) using Poisson regression.Of 9,469 respondents (age range: 18–28 years), 15% (95% CI: 14–16) reported being vaccinated for HPV. Coverage was significantly higher among MSM < 24 years , with education level below university degree , living in rural areas , attending sex parties , using HIV-related biomedical prevention methods , with STI diagnosis  and with hepatitis A or B vaccination .The HPV vaccination uptake among MSM in France was not satisfactory. It was higher among MSM benefitting from other vaccinations and biomedical preventive methods against HIV, suggesting a synergistic effect of the national preventive sexual health recommendations for MSM. Further efforts to improve HPV vaccination coverage targeting MSM are warranted. Human papilloma virus (HPV) is a sexually transmitted infection that can lead to anal, genital and oral cancers. Approximately 4.5% of all new cancers cases per year worldwide roughly 630,000) are attributable to HPV, with an estimated 8.6% in women and 0.8% in men . Biologi0,000 areThere are 13 types of HPV considered at high risk of causing genital and anal premalignant lesions and cancers. Most frequently responsible for cancers are HPV 16/18  [Strategy Nationale de Sante Sexuelle 2017–2030 [Among a total of 352,000 estimated new cancer cases reported in France in 2015, an estimated 6,333 were attributable to HPV, of which 28% affected men . Later o017–2030 ; however017–2030 .Since 2016, MSM and other targeted groups can access vaccination via family physicians, sexual health practitioners and primary healthcare facilities; however, the 2016 High Council for Public Health (Haute Autorité de Santé) recommendation about HPV vaccination for men emphasised the need to offer the HPV vaccine free of charge to MSM also in sexual health centres  ,18, a reTo date, information on HPV vaccination uptake in MSM population in France is limited. The CeGIDD reported the number of HPV vaccination doses administered between 2016 and 2018 ; howeverEnquête rapport au sexe' (ERAS), was performed in France to describe the trends of the adoption of different methods of HIV prevention [In 2019, a cross-sectional study targeting MSM, called the 'We used data from the ERAS cross-sectional study performed in France from 16 February to 31 March 2019. Details of the methodology used for this survey have been already published elsewhere [In brief, a questionnaire was posted online on different networks such as gay dating  or information websites , generally targeting gay communities. In addition, the survey was distributed via other platforms such as Facebook and was available via links in websites accessed when using keywords related to homosexuality and encounters between men. Participation criteria were men 18 years and older.We included in our analysis all participants that declared to be 'homosexual' or 'bisexual' or reported having at least one male sexual intercourse during their life. We selected for our analysis all MSM resident in metropolitan France and overseas French departments and all those up to the age of 28 years. This age was chosen to allow all MSM aged 26 years (maximum age for HPV vaccination eligibility) in 2016, who answered the questionnaire in 2019, to be included in the analysis.The questionnaire gathered information on self-reported vaccination status for HPV, hepatitis A and hepatitis B  and on the screening uptake for sexually transmitted infections. Information gathered on potential determinants of vaccination included: socio-demographic characteristics ; lifestyle such as circle of friends, number of sexual partners, attendance to saunas, bars, backrooms, outdoor meeting places or sex parties, use of web applications to date MSM; use of HIV prevention methods, namely condoms or biomedical HIV-related preventive measures (e.g. HIV pre-exposure prophylaxis (PrEP), treatment as prevention (TasP), treatment post-exposure (TPE)); and health determinants including perception of risk of being infected with an STI; uptake of screening for STI , and STI diagnoses.Supplement.The questionnaire did not have the option to skip any of the questions; therefore, we did not have missing data. For numeric variables, we calculated means (and standard deviations) or medians (and ranges). We calculated the HPV vaccination coverage for at least one dose or for at least two doses, using as denominator those who answered 'yes', 'no' or 'unknown' to the relevant question. In this paper, we report the results from this analysis. The results of the sensitivity analysis where we excluded the unknown vaccination status from the denominator are in the To identify factors associated with HPV vaccination (at least one dose), we calculated prevalence ratios (PR) and 95% confidence intervals (95% CI). To calculate adjusted PR, we used Poisson regression. We included in the initial regression models all factors with a p value < 0.2 in the univariable analysis and those that could have been relevant for vaccination coverage based on the literature. We removed variables one at a time using the Wald test or the Akaike information criterium.The study follows the ethical guidelines of the 1975 Helsinki Declaration. By clicking on a link or banner, the participant was directed to the survey site, which contained information about the aims and contents of the survey, terms of participation and data privacy. By clicking on a button containing the text “I have read and understood the information above”, the participant provided informed consent and was directed to the online questionnaire. The questionnaire was anonymous and self-administered and no IP addresses were collected. Santé Publique France review board approved the consent procedures on 20 March 2017 (DDPS-09).Of the 24,308 respondents, 10,553 were between 18 and 28 years-old and within this population, 9,469 (median age: 23 years) met the inclusion criteria and were included in the analysis. Participants were from various regions and 22% lived in Île-de-France . Among aAmong the MSM, 15%  reported being vaccinated against HPV, any dose, and 4.3%  reported being vaccinated with two doses or more.Of the MSM who reported being vaccinated with at least one HPV dose, 90% n = 1,274) reported having been vaccinated for hepatitis A and 94%  for hepatitis B; 67% (n = 947) reported having done at least one screening for STI including HIV; 3.4% (n = 48) reported a previous HPV infection or condyloma and 15% (n = 214) a positive diagnosis to any of the STI screenings performed in the last 12 months , among those with an education level below university degree , those living in rural areas  and those attending sex parties  . The MSMWe conducted the above analysis considering two doses of HPV vaccination. However, the number of observations was very small and not sufficiently powered for a meaningful analysis. Supplement), where we excluded the unknown vaccination status from the denominator, were consistent with the main results as presented above, namely associations between higher HPV vaccination and younger age, residence in rural area, lower education status and reporting other vaccinations such as hepatitis A and hepatitis B. Living in regions other than Île-de-France was positively associated with HPV vaccination, possibly reflecting residence in rural areas. The sensitivity analysis diverged from the presented analysis for other factors such as association with the use of biomedical HIV prevention methods and attendance at sex parties as these variables were significant in the univariable analysis, but did not remain significant in the multivariable analysis.Results from the sensitivity analysis  of 15% among all MSM aged 18–28 years, indicating an unsatisfactory level of vaccination in this high-risk group. A similar HPV coverage  wereWe also identified a positive association between attendance at sex parties and HPV vaccination, although in the sensitivity analysis, this association did not remain significant. Attendance at sex parties where group sex can occur has previously been associated with higher risk of STI and with use of PrEP in HIV-negative MSM . MSM engBased on these results it would be important to identify ways to reach adolescent males after sexual debut and ensure that HPV prevention measures are known and adopted. It should be considered that while we cannot rule out any bias caused by the classification of men with unknown status as unvaccinated in the main analysis, these discrepancies between the main and the sensitivity analyses may be due to the high number of unknown answers and the consequent elimination of more than half of the available observations in the sensitivity analysis, which may have decreased the power and therefore the possibility to obtain significant associations, apart from the strongest.The main strength of our study was that we used a large dataset with a diversified the MSM population in terms of socio-demographic characteristics.Our study had some limitations. Firstly, men assertive about their homosexuality, more interested in sexual health prevention or affected by STI may have been more likely to complete the online questionnaire, suggesting that participants might not be representative of the whole MSM population and introducing a potential selection bias . This biThe low HPV vaccination coverage among MSM raises concerns about the possibility of decreasing the burden of HPV-related lesions and cancers in this population in France. Lower uptake was associated with older age, higher education level and living in urban areas. Higher vaccine uptake was observed among MSM benefitting from other vaccinations and biomedical preventive methods against HIV. It is essential to continue the vaccination programme targeting MSM and to invest further resources to improve awareness among MSM of the risk of HPV-related diseases and the importance of vaccination. In addition, it would be useful to establish a surveillance strategy to periodically estimate vaccination uptake in this population and to assess the impact of this vaccination approach by monitoring the prevalence of HPV infections in the MSM population. Improvement of HPV vaccination coverage in MSM will eventually contribute to the national HPV vaccination objectives.