Survival analyses included the Kaplan–Meier method, log-rank test and Cox proportional hazards model.PCR-detection of (hr)HPV-DNA and immunohistochemical staining for p16ink4a-overexpression and (hr)HPV-DNA were detected in 35 and 37 of the 85 tumors, respectively. Among the 35 p16ink4a-positive tumors, 10 lacked (hr)HPV-DNA (29 %). Among the 50 p16ink4a-negative tumors, (hr)HPV-DNA was detected in 12 cases (24 %). The median follow-up was 89.20 months (range 1.7–189.5 months). P16ink4a-overexpression, but not (hr)HPV-DNA positivity of the primary tumor, was correlated with prolonged overall survival (OS) (p = 0.009). P16ink4a-overexpression predicted a better response to radiotherapy (p < 0.001). Univariate analysis has demonstrated that age (p = 0.025), tumor grade (p = 0.001), lymph node metastasis (p < 0.001), FIGO stage (p < 0.001), p16ink4a-overexpression (p = 0.022), and adjuvant RTX (p < 0.001) were prognostic factors for OS. Multivariate analysis has demonstrated that lymph node metastasis , tumor grade  and p16ink4a-overexpression  are independent prognostic factors.p16ink4a in combination with HPV-DNA detection as an ancillary test for future research and clinical studies in vSCC. The prognostic and predictive value of p16ink4a-overexpression should be tested in larger cohort studies.The discovered overlap suggests the use of p16The online version of this article (doi:10.1186/s12885-016-2503-y) contains supplementary material, which is available to authorized users. The most common type of vulvar cancer is vulvar squamous cell carcinoma (vSCC) [HPV-dependent and HPV-independent vSCCs were suggested to be separate entities ; howeverink4aoverexpression within vSCC tumors. The secondary aim was to analyze the prognostic significance of p16ink4a and (hr)HPV-DNA status in vSCC patients.The aim of this study was to assess the prevalence of HPV genotypes and the concordance between the presence of (hr)HPV-DNA and p16This retrospective study was approved by the Polish Ministry of Science and Higher Education review board . The board determined that further informed consent was not required, as all patients provided informed consent for tissue sampling prior to surgical treatment, including written consent for the storage of their information in the hospital database and the use of their information for research.We studied 85 patients with primary vSCC who underwent surgical treatment at the Department of Gynaecological Oncology at The Medical University of Gdańsk between January 2002 and December 2007. All patients underwent standard surgical treatment, which was not modified by the results of the sentinel node procedure. A wide local excision was performed if the tumor diameter did not exceed 2 cm and the depth of invasion was less than 1 mm. In cases of lateral tumors with an invasion greater than 1 mm, a wide local excision or tailored radical vulvectomy with a bilateral inguinofemoral lymphadenectomy was performed. Lymphadenectomies were mostly performed with separate incisions. Postoperative radiotherapy was administered to all patients with positive inguinal lymph nodes, with the exception of those patients who had both a well-differentiated histology of the primary tumor and also only one lymph node metastasis. Overall, 33 (39 %) patients received adjuvant radiotherapy. Clinical data were obtained from the medical records and questionnaires designed specifically for our previous studies conducted in the same cohort [Histopathological data on 76 tumors were obtained from two previous studies . Nine neTumor type (pT), depth of invasion , tumor grade according to the GOG  and lymph nodes status (pN) were verified by the same two independent pathologists (without knowledge of the disease outcome). All of these patients were staged according to the new 2009 FIGO system for vulvar cancer [ink4a and PCR detection of HPV-DNA were performed on 85 paraffin-embedded tissue samples from the primary tumors.Finally, immunohistochemical (IHC) staining for p16ink4a monoclonal (sc-56330) antibody was obtained from Santa Cruz Biotechnology (USA).Mouse anti-human p16For immunohistochemical staining, four-micron-thick serial sections were cut, placed onto slides, and deparaffinized. For epitope retrieval, slides were immersed in Target Retrieval Solution  and heated in a pressure cooker. The slides were incubated for 90 min with primary antibodies. The reaction was visualized using the Novolink Polymer Detection System (Novocastra Laboratories). Appropriate positive and negative controls were included for each case. As a positive control, a case of HPV-related cervical cancer was used. For the negative control, the primary antibody was replaced with normal mouse IgG at an appropriate dilution. Immunohistochemistry results were evaluated by two independent pathologists, who were blind to the clinical data. The concordance rate between their observations was over 96 %.ink4a immunostaining was performed on 3 different staining patterns: negative, focal, and diffuse staining. For statistical purposes, p16 immunostaining was classified as positive or negative. Staining for p16ink4a was considered positive only in cases with a strong diffuse and continuous nuclear/cytoplasmic expression of p16ink4a within the cancer nests .The evaluation of the p16Genomic DNA was prepared from two to three 4 mm sections from each case using standard methods. DNA was obtained from the samples by incubating them with 250 μl proteinase K solution (1 mg/ml) for 16 h at 70 °C. Following heat inactivation at 95 °C for 10 min, 10 μl of the supernatant was used for PCR. Appropriate positive and negative controls were incorporated during the DNA preparation and subsequent testing to monitor test performance.Specimen extracts were also tested by real-time PCR for the human RNase P gene to monitor specimen quality.Broad-spectrum HPV DNA amplification and mucosal HPV genotyping was performed using the SPF10–LiPA25 system , as described previously . All tesNo aberrant results were observed in the sectioning, DNA isolation or PCR controls.ink4a and HPV-status.The impact of the following variables on overall survival was assessed: type of the tumor (pT), lymph node status (pN), tumor grade, depth of invasion, FIGO stage, age and recurrence, as well as p16p value <0.05 was considered significant. All analyses were performed using Statistica 10 software (Stat Soft Inc. USA).To determine statistically significant differences between the variables, the Mann-Whitney U test was used. Overall survival (OS) curves were estimated using the Kaplan-Meier method and compared using a two-sided log-rank test. Independent variables were first analyzed with univariate analysis. Variables shown by univariate analysis to be significantly associated with survival were entered into a Cox proportional hazards regression model for multivariate analysis. A The clinicopathological features of the patients with primary vSCC and their relationships to the course of the disease are summarized in Additional file p = 0.000743).The depth of invasion in pN-positive (median 8.2 mm) and pN-negative cases (median 6.0 mm) was significantly different    Fig  and p16i35) Fig. .Fig. 1MiHPV-DNA was detected in 38 of the 85 primary vSCC tumors (45 %).n = 48) and positive (n = 37).The HPV genotype distribution is provided in Additional file ink4a-overexpression, 10 cases lacked (hr)HPV-DNA (28.6 %). Among the 50 tumors without p16-overexpression, (hr)HPV-DNA was detected in 12 cases (24.0 %). The combined presence of (hr)HPV-DNA and p16-overexpression was detected in 25 of the 85 cases (29.4 %). P16ink4a status in 85 vSCC cases in relation to (HR)HPV genotypes is summarized in Table Among the 35 vSCC tissue samples with p16ink4a-positive and p16ink4a-negative vSCC was 98.97 and 23.33, respectively (p = 0.009). A positivity for p16ink4a in the primary tumor correlated with a prolonged survival of vSCC patients (p = 0.010) . The median OS (months) of radiated patients with p16ink4a-positive and p16ink4a-negative vSCC was 36.93 and 7.47, respectively. A positivity for p16ink4a predicted longer OS among vSCC patients who received adjuvant radiotherapy (p = 0.0006)  of patients with p1610) Fig. . There w06) Fig. .Fig. 2Prp = 0.41). A positivity for DNA of (hr) HPV in the primary tumor was not correlated with a prolonged survival of vSCC patients (p = 0.411) . The OS (in months) of radiated vSCC patients with tumors positive and negative for (hr)HPV-DNA was 18.92 and 15.77, respectively. A positivity for (hr)HPV-DNA did not predict longer OS among vSCC patients who received adjuvant radiotherapy (p = 0.201)  of patients with (hr)HPV-DNA-positive and (hr)HPV-DNA-negative vSCC was 69.31 and 41.02, respectively (11) Fig. . There w01) Fig. .p = 0.0411)  Fig. .Fig. 3Prn = 76), T2 (n = 8), T3 (n = 1), T4 (n = 0)) has not revealed any influence on overall survival (p = 0.5027).Most of the cases were staged as T1 . Tumor type (pT: T1 (p = 0.00006)  has a significant impact on overall survival (06) Fig. .p = 0.00055) .We found significant differences in the overall survival between patients with different histological tumor grades (divided in accordance with the three-tier grading scheme: G1/G2/G3) (55) Fig. , as wellp = 0.057).We did not manage to find any borderline depth of invasion with a significant impact on overall survival (for 7 mm (median) n = 52) without irradiation (p = 0.00001)  Fig. .p = 0.00379) , stage II: 2 (2.35 %), stage III: 35  and stage IV 4 . The cumulative 5-year survival was as follows: stage I: 67 %, stage II: no data , stage III: 27 % and stage IV: 25 % (79) Fig. .p = 0.004)  Fig. .p = 0.0254), lymph node status (p = 0.0005), tumor grade (G1 vs. G2 + G3) (p = 0.001), adjuvant radiotherapy (p = 0.000005) and p16-overexpression (p = 0.0216) were prognostic factors for OS (Table p = 0.001), nodal status , and tumor grade  were independent prognostic factors for OS  . An overIndeed, all of our cases come from Poland (Europe) while only 49.8 % of women with IVC included in de Sanjosé’s study were European. The prevalence of HPV-DNA was found to be higher among European women than women living in other geographical regions .Additionally, the number of well differentiated tumors was higher in our group than in IVC cohort described by Sanjosé S et al. (76 % vs. 71 %).Indeed, the proportion of histological types is crucial for cohort prevalence, as it was shown that HPV-positivity among warty-basaloid and keratinizing vSCC tumors varies, and it was described as 69 and 11.5 % of cases, respectively [Our study confirmed the predominant contribution of HPV-16 to the etiology of HPV-related vulvar cancer and suggested that other HPV types, such as HPV-33, HPV-18 and HPV-56, which are common in cervical cancer, are also important to vulvar carcinogenesis, although to a lesser degree.ink4a-positive tumors, a lack of (hr)HPV-DNA was observed, and in 24.0 % of p16ink4a-negative tumors, (hr)HPV-DNA was detected. The substantial mismatch between p16ink4a-overexpression and HPV-status reported here was also observed in the largest cohort of 1709 vSCC cases [ink4a lack (hr)HPV-DNA, and 9.4 % of tumors lack p16ink4a-overexpression despite the presence of (hr)HPV-DNA [In 28.6 % of p16CC cases . Sevente)HPV-DNA .ink4a expression [ink4a expression in cancer [RB1 mutation, deletion or methylation. The viral oncogene expression represents just one potential form of multiple possible ways of RB inactivation [A lack of (hr)HPV-DNA in p16-positive tumors could be explained by the fact that the HPV oncoprotein, E7, which functionally inactivates RB, is not the only thing responsible for the increases in p16n cancer , 3, aberink4 expression [ink4 expression in our older age cohort of vSCC patients should also be considered.Several findings have proven the strong association between age-promoting, ‘gerontogenic’ signals and p16There remains the possibility that a certain fraction of HPV-negative samples were false negatives. However, during the testing, we checked all the samples for amplifiable human genomic DNA. All samples showed the presence of human DNA by PCR (RNAseP gene). The size of the PCR fragment in this test is also 65 base pairs, and therefore, it is most sensitive PCR for formalin-fixed paraffin-embedded/FFPE/tissue samples.Ink4a-inactivation, ranging from 25 to 70 % [(hr)HPV-DNA-positive cancer cases without marked p16-overexpression could be explained by the fact that close to half of all human cancers show p16 to 70 % . Such an to 70 % , it is pink4a-overexpression as a surrogate marker for (hr)HPV infection in vSCC. The correlation between p16ink4a and (hr)HPV-DNA varies in squamous cell carcinomas. In cervical cancer, p16ink4a overexpression and (hr)HPV status are quite well correlated [Taking these facts together, we postulate not to treat p16rrelated , while irrelated .ink4a-overexpression was detected in 25 of the 85 cases (29.4 %). This result is in the range of the series reported by de Sanjosé S et al., who have reported 22.4 % HPV-driven cases out of 1709 vSCCs [The combined presence of (hr)HPV-DNA and p16In the following analyses, we assessed the prognostic significance of (hr)HPV-DNA status and p16 overexpression separately. The (hr)HPV-DNA status of the primary tumor has no impact on the survival of vSCC patients. P16-overexpression was found to be prognostic, and also predicted a better response to radiochemotherapy.Several reports investigating the relationship between HPV DNA and vSCC prognosis have produced conflicting results , 21–24. We identified only two studies that utilized p16 expression for the survival analysis of vSCC patients, and they reported contradictory results . Our resThe conducted univariate and multivariate analyses revealed that p16-overexpression is an independent prognostic factor with respect to survival.Ink4a from its negative feedback control, causing a paradoxical increase in the levels of this protein, which attempts to inhibit uncontrolled cellular replication [ink4a-overexpression itself (not the HPV virus) has a protective role in HPV-related malignancies.RB inactivation releases p16lication . Thus, iInk4a-overexpression are very sensitive to radiotherapy, and they have a better prognosis [Ink4a-overexpression predicts a better clinical outcome among patients requiring adjuvant radiotherapy. The two compared groups (irradiated p16-positive and p16-negative) were similar in terms of the number of positive nodes and the presence of extracapsular spread, which strongly supports this conclusion.It was shown that cancers that present p16rognosis . A betterognosis –32. IndeThis study has the traditional weaknesses of a retrospective design, and the results obviously represent a small cohort. Lack of information on smoking and cause of death potentially limit the prognostic analysis. The strengths of the study include the treatment of patients according to uniform standards and a sufficient follow-up duration to reveal recurrences and to allow for the reliable assessment of the prognostic significance of all analyzed biomarkers. Data on (hr)HPV-DNA prevalence were provided by highly experienced sources in the HPV-DNA detection laboratory.ink4a is not a surrogate marker for a transforming infection with HPV high-risk genotypes in vSCC. This suggests the use of p16ink4a in combination with HPV DNA-detection as an ancillary test for research and clinical studies when HPV is not a necessary cause. The prognostic and predictive significance of p16Ink4a-overexpression within cancer tissue requires further investigation in future prospective studies.The overexpression of p16Ink4a, protein, cyclin-dependent kinase inhibitor 2A, multiple tumor suppressor 1; PCR, polymerase chain reaction; vSCC, vulvar squamous cell carcinomaDFS, disease free survival; DNA, deoxyribonucleic acid; FIGO, fr. Fédération internationale de gynécologie et d’obstétrique; GOG, gynecologic oncology group; HPV, human papilloma virus; HR, hazard ratio; hr, high risk; IHC, immunohistochemistry; IVC, invasive vulvar cancer; OS, overall survival; p16


HPV is the major etiological factor in the causal pathway for cervical cancer, which is the leading cancer among women in sub-Saharan Africa. HIV is associated with a higher prevalence and a broader range of high-risk HPV genotypes. Studies have shown a positive association between Bacterial vaginosis (BV) and HPV and HIV. Also, in African women, BV was found to be significantly associated with vaginal inflammation. The high prevalence of BV, HIV and HPV infections in the African continent makes elucidation of the interactions with one another of utmost public health interest. The aims of the current study are to examine the frequency of HPV genotypes and BV as well as their respective risk factors within an HIV infected population with abnormal cytology in the resource-constrained setting of Mombasa, Kenya and, secondly, highlight issues to consider for triple co-infection clinical management.Cross-sectional analysis with a sample drawn from an ongoing cohort study. All consenting, non-pregnant HIV infected women, between 18 and 50 years of age, without a history of cervical cancer or hysterectomy, between November 2005 and April 2006 were screened for HR HPV DNA in Mombasa, Kenya. 1 out of 4 HIV positive women fulfilled the criteria by having SIL (24.9 %). 600 HIV infected women were tested to reach a cohort of 74 HIV women with abnormal cytology. To assess which factors were associated with HR HPV, crude statistical analysis was performed through logistic regression.n = 11), of which 8 had BV. The most prevalent HPV genotypes were HPV 16 (33.8), HPV 53 (24.3) and HPV 18 (17.6 %), while 65 % of the participants had multiple genotype infection.Bacterial vaginosis (BV) was found in 46 women out of 74 (62.2 %). Cervicitis was diagnosed in 15 % of women (p = 0.03) and HPV53 . A statistically significant association was found between CD4 count ≥ 350 μl and HPV 16 adjusted for age . A borderline statistically significant association was observed between BV and HPV58 .Statistically significant associations between CD4 counts <200 cells/μl and multiple HPV prevalence, adjusted for age were also noted (OR = 3.7; 95 CI: 1.2–12.1; The most prevalent HPV genotypes observed were HPV 16, HPV 53, and HPV 18, which have a combined prevalence of 76 %. Our results show that a triage based on CD4 count should start at CD4 count ≥ 350 μl as our study suggests that HPV 16 are more prevalent when women are moderately immunosuppressed. Given the high prevalence of HPV 53 in a HIV infected population with abnormal cytology, its cervical carcinoma genesis potential as a stand-alone genotype and as well as its synergism with multiple infections should be investigated. The new WHO guideline in resource-poor settings to rescreen women for HPV within ten years may be more effective if BV and cervicitis management become a major component for HIV-HPV management. Cervical carcinoma is the fourth most prevalent cancer in the world and the most common female cancer in sub-Saharan Africa . It is tInfection with a high risk (HR) Human Papillomavirus (HPV), a sexually transmitted DNA virus, is the central etiological agent in the development of cervical cancer and include HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68 [Cervical cancer is the result of a change in the cell cycle control caused by HP. Cervical intraepithelial neoplasia (CIN) can be histologically graded into mild dysplasia (CIN 1), moderate dysplasia (CIN 2), and both severe dysplasia and carcinoma in situ belonging to CIN 3 .Apart from a higher prevalence and broader range of HR HPV, HIV immunosuppression has been linked to multiple HPV infection , 9, part202-producing lactobacilli in African women is among the highest worldwide [Moreover, concurrent genital infections are common in HIV-1–infected women. The prevalence of Bacterial Vaginosis (BV), characterized by an overgrowth of vaginal anaerobic flora and reduction of Horldwide , which iorldwide , 17. A rorldwide . A recenorldwide . These fChlamydia trachomatis, Neisseria gonorrhoea, HSV-1 and 2 [202-producing lactobacilli, reduced levels of protective vaginal mucins, and increased pro-inflammatory enzymes and cytokines, which in turn may decrease the cervical mucus barrier [In previous studies, BV was associated with many sexually transmitted infections (STIs), including infection with -1 and 2 –22. Also-1 and 2 . There i-1 and 2 . BV coul barrier .New guidelines from WHO in December 2014 advocates for a “screen and treat” approach in resource poor settings, in which treatment of detected precancerous lesions ensues Visual Inspection with Acetic acid (VIAC) or HPV testing for screening soon or immediately . The useThis analysis of HIV-positive women with abnormal cytology purports to test our a priori hypotheses that HPV 53, being more prevalent in HIV-infected women is associated with immunosuppression and that BV is associated with genotypes pertaining to the alpha-group. The objectives are threefold: 1) to describe the distribution of HPV genotypes and multiple HPV infections, cervicitis, BV and STIs within our population; 2) to explore whether low CD4 count is a predictor for multiple HR HPV genotypes and the most prevalent pHR/HR HPV genotypes and BV a predictor for alpha 9 related genotypes as well as the most prevalent pHR/HR HPV genotypes 3) discuss the potential effectiveness of a screening protocol triaged by CD4 count and age and the public heath management of HIV-HPV-BV.This cross-sectional analysis reports the findings of a sample of women enrolled in a prospective cohort study, whose aim was to define the best way to manage low grade squamous intraepithelial lesions in HIV-1 infected women, aged between 18 and 50 years in Mombasa, Kenya, between November 2005 and April 2006. As all women were to receive the same intervention depending on their squamous intraepithelial lesion, no formal sample size calculation was needed. 600 women were screened within this period. Non-pregnant women of reproductive age and attending the Comprehensive Care Centre for management of HIV infection at Coast Province General Hospital in Mombasa, Kenya, were invited to participate. Within the 600 HIV positive women undergoing cytological examination, we expected to find 10 % prevalence of Squamous Intraepithelial Lesions (SIL) for the HIV positive patients not on HAART and 15 % for those under HAART period, resulting in 74 HIV women with abnormal cytology being recruited from the initial cohort.Cytological abnormalities, a prerequisite for participating in the study, were all histologically confirmed. Exclusion criteria included pregnancy, less than 6 weeks post-partum, history of cervical cancer or hysterectomy. Self-reported behavioral risk factor assessment included the presence of an STI, number of sexual partners, the age of first sexual intercourse and regular use of a condom. Blood plasma samples for measuring CD4 count were taken, and a gynaecological examination was performed with speculum insertion, and collection of endocervical and high vaginal swabs.Cervical samples were collected using a cervix brush , and cervical cytology was assessed with conventional Papanicolaou (Pap) smears. Slides were read by a cytologist with master level training, supervised by a pathologist. An external cytopathologist provided quality control. The Bethesda Reporting System was used for cytological classification .The cervix brush tips were preserved in a liquid-based cytology collection medium  and stored at 4 °C until further processing.HPV testing was done as described by Depuydt et al. (2006) Micalessi IM et al. (2012) in an accredited laboratory (ISO certification: ISO15189) , 30. BriThe national recommendation was followed to diagnose HIV and a parallel testing algorithm was used for HIV diagnosis using rapid immunoassays: Uni-Gold™ Recombigen® HIV  and Determine® HIV-1/2 . In the case of indeterminate results, an enzyme-linked immunosorbent assay was used to confirm HIV status.Trichomonas vaginalis was determined by wet mount and/or PAP smear slides. To test for Neisseria gonorrhoeae infection, culture plates  were inoculated with endocervical swabs Presence of HIV was confirmed in the patient’s serum using a parallel algorithm comprising Uni-Gold® HIV  and Determine® HIV-1/2 . Elisa was used when parallel test results were discordant.A gynecological examination was performed and blood, urine, PAP smear, and high vaginal and endocervical swabs collected. PAP smears were read using conventional cytology and classified using the Bethesda classification. Infection with Cervicitis was diagnosed from inflammatory cells by means of a microscopic test.Analysis was done using STATA version 12 .Age was dichotomized into > 30 years and ≤ 30 years; this categorization was used to reflect the WHO 2014 guideline concerning cervical screening. Condom use was dichotomized as always or irregular, according to self-report. CD4 cell count was analyzed both as a continuous variable and as two categorical variable, using a cut off of CD4 count <200 cells/μl (vs ≥ 200 cells/μl) and CD4 count <350 cells/μl (vs ≥350 cells/μl), when HIV infected women are severely immunosuppressed and moderately immunosuppressed, respectively. The number of sexual partners was also dichotomized as per other studies (up to 5 versus 6 or more), this dichotomization was used to make our studies more comparable to others and to explore the risk from a public health point of view.The method employed for the first objective was to calculate the proportion of the specific HPV genotype divided by 74 (the sample size), the number of co-infected women with abnormal cytology, as well as the proportion of STI, BV and cervicitis, along with a 95 % CI. For the second objective, logistic regression analysis was performed to identify risk factors for HR-HPV. This included the associations between low CD4 count and the most prevalent pHR/HR HPV genotypes  and absence of HPV genotypes versus HR-HPV genotypes (see above). The resulting odds ratio was used to measure the strength of the association between HPV infection and each risk factor in turn. A multivariable logistic regression analysis was performed to simultaneously control for potential confounders and to assess the adjusted association for various risk factors. To assess the impact of CD4 count on the effects of the parameters, interaction parameters were assessed in the models by doing a Wald test. As no interaction terms were significant, no model reflecting the differential impact for that particular outcome was included.The same method was also used to explore the simultaneous effect of age and number of sexual partners on the HPV genotype and to explore whether there was an association between the two or more HR HPV co-infections and CD4 count, while adjusting for age and the number of sexual partners.p-value, derived from the Wald Test, is 0.05 or less.Statistical tests were considered significant when the Staff obtained written informed consent from patients, collected demographic and behavioural data using structured questionnaires. All human subject protocols were approved by the Ethics Committee at the Kenyatta National Hospital, which also gave overall ethical approval for this study (Ref: KNH-ERC/01/3618).The study included 74 non-pregnant HIV-infected women with an abnormal cytology. The mean age was 34.2 years of age (SD = 6.5). The median age at first sexual intercourse was 18.0 (IQR: 15.5–20.0). The median number of sex partners was 2 (IQR: 1–4). The median CD4 count was 236 cells μl/(IQR: 158–374). The mean number of HR HPV genotypes per participant was 2.3 (SD = 1.6). The majority of patients (82 %) were under treatment with highly active antiretroviral therapy (HAART)  were detected in 4.0 %, atypical squamous cells of undetermined significance (ASC-US) in 16.2, LSIL 58.1 and HSIL in 20.3 % of the patients.Histology results found the following abnormalities: Cervical intraepithelial neoplasia (CIN I) in 58.3 %, CIN 2+ 43.2 %, of which 1 had ICC. Cervicitis was recorded in 15 and normal biopsy in 4.0 % of the participants. In 5 women with CIN 1, HPV was absent and in the two normal biopsies, HPV was present.The most prevalent HPV genotypes found in this HIV-infected cohort of women are displayed in Table A large proportion, 64.9 %, of the women in this cohort has multiple HPV genotypes Table . Most muAt least one of the HR HPV genotypes was observed in 86.5 of the women, 5.4 with LR-HPV types and 8.1 % without any contemporaneous HPV infection.trichomonas vaginalis and 10.8 % had genital warts.STIs were observed in 68.9 of all women, and 15.7 % had more than 1 STI co-infection , adjusted for ≥6 sexual partners ; HPV 53:  adjusted for ≥6 partners  and HPV 18 : adjusted for ≥6 partners . No significant association was found for multiple HPV genotypes and age  and adjusted for sexual partners   but a non-significant association between CD4 count ≥350 cells/μl and multiple HPV infections . Low CD4 counts <200 cells/μl was found to be a significant predictor of only HPV 53  and HPV 16   compared to women with CD4 count >200 (mean of pHR HPV genotypes coinfections = 2.0) : (p = 0.006 for the difference in mean). However, there was no significant difference (p = 0.5) between women with a CD4 count <350 cells/μl (mean = 2.4) in and women with CD4 count ≥350 cells/μl (mean = 2.1).A p = 0.07), no significant association was found between BV and HPV 16 and its phylogenetically-related genotypes, HPV 31, HPV 33, HPV 58, HPV 52 . However, a South African study suggested that the prevalence of HPV 18 is inversely correlated with the level of immunosuppression, (10.6 %) in women with CIN 2/3 and 5 % with women with normal cytology .A high prevalence of HPV53 had also been observed in other studies from Kenya. A study in HIV positive women (median CD4 count 281/μl) from Nairobi found high numbers of HPV53 in normal smears, whilst rare in HIV negative women . In a Nap = 0.01; 95 % CI: 1.4–13.6) and the prevalence of multiple infection. Cervical HPV infection with multiple genotypes in HIV infected women has already been reported in regional studies [Although, intuitively, it would seem reasonable that all HPV genotypes should increase in frequency in HIV positive individuals, strong statistically significant associations were only detected between CD4 count <200 cells/μl and HPV53, the second most prevalent potentially oncogenic HPV genotype in our study (aOR = 4.4;  studies –40.Whilst younger age has been linked with higher prevalence of HR-HPV genotypes in HIV-negative women , we did Estimates in BV range from 20 % to 50 % in African populations , 44. A rA major strength of our study is that our analysis is limited to histological, considered to be the gold standard instead of cytological endpoints. However, the total sample size was rather limited, which may have occulted some plausible association of risk factors which may be evident with a larger sample size; for this reason, adjustment for various factors was limited. In addition, another limitation relates to the cross-sectional design, where the simultaneous data collection of BV and acquisition of HIV infection, as well as all other variables, the temporal criterion of causality is not fulfilled, thereby reversal causality cannot be excluded. These limitations may result in a sub-optimal internal validity of our study.Our results can be generalized to other women receiving HIV care management. Our lack of association found between age and the most common HPV genotypes and multiple HPV genotypes do not appear to support the recommendation for a screen and treat protocol for women starting from thirty years of age. Also, a CD4 based triage may not be effective as HR HPV genotypes are seen at different levels of immunosuppression, at both CD4 < 200 cells/μl and CD4 count ≥350 cells/μl.The new WHO 2014 HPV screen and treat recommendation comes with close timing with the WHO 2013 recommendation to initiate HAART when asymptomatic at CD4 count ≥350 cells/μl, which may offer opportunities to prevent HPV 53 and multiple HPV infections, thereby potential cervical dysplasia although it may not prevent HPV 16 and HPV 18. However, given the limitation of this study, including small sample size and its cross-sectional design, a prospective cohort or RCT with a larger size is warranted to elucidate potential relevance to plan screening according to the level of immunosuppression.Biologic susceptibility to HPV acquisition and immune competence for clearance of an HPV infection could be affected by BV underscores the importance of prevention and successful treatment of BV. In light of this finding, the new WHO guideline to rescreen within ten years may be more effective if it is implemented in conjunction with a BV screening programme. Given the high prevalence of cervicitis in this population and its potential public health impact, it is important that BV clinical management becomes a major component for HIV-HPV management. To this effect, more health care personnel, including front line personnel, including nurses who make up the majority of the work force in sub Saharan Africa should be trained in microscope, Nugent score or Amsel reading of BV in HIV positive women. Furthermore, symptomatic management algorithm be established for cervicitis in this study population and its aetiology elucidated.Effective treatment of cervicitis resulting in significant decreases in shedding of HIV-1 virus and infected cells in cervical secretions depends on the aetiology of BV and other pathogens involved implicated in cervicitis. In this study, cervicitis was common and predominantly non-gonococcal, although it is not possible to say that it was non-chlamydial in etiology as CT was not diagnosed and was found predominantly in women with BV.Although high prevalence cervicitis has been reported in Africa, local epidemiological data regarding the prevalence, etiologies, and risk factors for cervicitis in HIV-HPV co-infected women in Kenya is lacking for guiding syndromic management of cervicitis in this group. The high prevalence in this study group may also affect sexual health at a population level as inflammation in the genital tract results in shedding of HIV in HIV-positive women may increase HIV to the general population. A recent meta analysis including HIV viral load observations in blood plasma, showed the average effect of a STI co-infection on HIV viral load in individuals on HAART was unlikely to decrease the effectiveness of treatment as prevention, although there is evidence of HIV compartmentalization in some treated patients where viral loads as measured in genital secretions are congruent with a non-negligible risk of transmission despite very low blood plasma viral loads –48.Our marginal association between HPV 16 genetically related HPV 58 as well as the high prevalence of BV in HIV-HPV co-infected women along with a high prevalence of STIs underscores the need to elucidate its synergistic effects in increasing risk of HIV and HPV acquisition. Finally, the high level of co-infection with STIs underscores the value of screening for the simultaneous presence of different genital infections in HPV positive patients. This study also evidenced the lack of protective precautions against STIs, notwithstanding the lack of effectiveness of condoms reported in reducing HPV transmission , 50.Epidemiological considerations will be needed to determine the best screening approach for different geographical settings within Kenya. A screening interval for HPV 16, HPV 53, and HPV 18, the most prevalent HPV genotypes observed, triaged by age may not be optimal in this study population of HIV- infected women as these genotypes are often found in presence of other pHR/HR genotypes. The concomitant WHO recommendation to initiate HAART when asymptomatic at CD4 count ≥350 cells/μl, instead of at CD4 count ≥200 cells/μl, may offer women the possibility to ensure continuous cervical screening during regular follow up, thereby preventing HPV 16 and HPV 18 induced cervical dysplasia.Given the high prevalence of HPV 53 in a HIV infected population with abnormal cytology, its cervical carcinoma genesis potential as a stand alone genotype and as well as its synergism with multiple infections should be investigated.The new WHO guideline to rescreen within ten years may be more effective if it is interspersed with BV management. Given the high prevalence of cervicitis in this population and its potential public health impact, it is important that bacterial ecology monitoring becomes an integral component of HIV-HPV management. To this effect, there is a need for training of health care personnel in validated point of care diagnostics for diagnosing BV in HIV infected women. In addition, it is pivotal that the aetiology of cervicitis be elucidated within this study population and a symptomatic management algorithm for cervicitis be established.Furthermore, the association between BV and HPV 16 and its related genotypes must be further explored. A high BV prevalence, together with high concomitant STIs detected in our study underscores the need that the possible synergistic effects of co-infections be elucidated for public health interest. In addition, both high concomitant STIs prevalence and very irregular use of condoms make the case for strengthening STD counseling within HIV care.


We analyzed the evolutionary dynamics of deletion-containing mitochondrial DNA (ΔmtDNA) molecules, previously characterized as selfish elements, in six different natural strains of the nematode Caenorhabditis briggsae allowed to undergo experimental evolution in a range of population sizes  for a maximum of 50 generations. Mitochondrial DNA (mtDNA) was analyzed for replicate lineages at each five-generation time point. Ten different ΔmtDNA molecule types were observed and characterized across generations in the experimental populations. Consistent with predictions from evolutionary theory, lab lines evolved in small-population sizes  were more susceptible to accumulation of high levels of preexisting ΔmtDNA compared with those evolved in larger populations. New ΔmtDNA elements were observed to increase in frequency and persist across time points, but almost exclusively at small population sizes. In some cases, ΔmtDNA levels decreased across generations when population size was large . Different natural strains of C. briggsae varied in their susceptibilities to ΔmtDNA accumulation, owing in part to preexisting compensatory mtDNA alleles in some strains that prevent deletion formation. This analysis directly demonstrates that the evolutionary trajectories of ΔmtDNA elements depend upon the population-genetic environments and molecular-genetic features of their hosts.Evolutionary interactions across levels of biological organization contribute to a variety of fundamental processes including genome evolution, reproductive mode transitions, species diversification, and extinction. Evolutionary theory predicts that so-called “selfish” genetic elements will proliferate when the host effective population size ( Drosophila species infected with Wolbachia support this hypothesis (Selfish genetic elements (SGEs) occur throughout the eukaryotic phylogeny and influence diverse evolutionary processes . Two feapothesis , as do opothesis . SGEs capothesis  and the pothesis .Organelle genomes generally undergo uniparental inheritance  in most eukaryotic species studied. Although maternal inheritance is thought to facilitate coevolution of organelle and nuclear genomes, it also renders cytoplasmic DNA susceptible to Muller’s ratchet dynamics and associated accumulation of deleterious variants, including SGEs . OrganelSaccharomyces cerevisiae experimental evolution study demonstrated that levels of selfish mutant mitochondrial DNA (mtDNA) molecules, with associated respiration defects, remained high only when the yeast population size was small (What factors influence the evolutionary success (or demise) of cytoplasmic SGEs? This question has received theoretical attention  and a sias small  and seleCaenorhabditis briggsae nematodes offer an effective system for investigating diverse evolutionary questions including reproductive mode evolution  because new ΔmtDNA types will be reported later in the Results. Our initial study  of sequence, including part of a pseudogene element named ψnad5-2  occur in some C. briggsae strains from North America and Europe that are part of the “temperate” intraspecific clade  and DNA sequencing analyses to identify and characterize the state of mtDNA across multiple generational time points. This approach identified many new ΔmtDNA variants, some originating during the course of the evolution experiment, and provided new insights into the evolution of cytoplasmic SGEs in different host genetic backgrounds and at varying host population sizes.Here, we investigate the transgenerational dynamics of C. briggsae strains . Replicates were maintained by transferring either one or ten L4-stage hermaphrodites to a new 35 mm Petri plate every 48 ± 2 h. If a hermaphrodite in the N = 1 treatment laid no eggs within this timeframe, an additional 2 days was allowed for reproduction. If it failed to reproduce after this time, another of its kin was selected for propagation . A line was considered extinct after five consecutive failures of the GTB procedure.Replicate populations of N = 100 and N = 1,000 were propagated by harvesting all eggs from each replicate and transferring the desired number to a new plate every 48 ± 2 h. Populations were cultured on 60 and 100 mm Petri plates, respectively. Specifically, nematodes from replicates were collected into 5 ml M9 buffer and 2 ml of 2:1 (v:v) 6% bleach:5 M NaOH solution was added to dissolve the cuticles. Eggs were rinsed twice and resuspended in M9 buffer. Pellets of eggs were transferred with 1 ml fresh M9 to a 1.7 ml microcentrifuge tube to achieve uniform dispersion within a column. After vortexing briefly, egg density was calculated for each replicate from eight 1 ul samples and the volume appropriate to achieve the desired population bottleneck size was transferred to a fresh Petri plate. Accuracy of calculated egg densities was determined by counting the surviving worms of one replicate per strain immediately prior to transfer.Populations of N = 1,000, we followed the approach of a previous study . Plates were prepared by transferring a volume of eggs expected to yield approximately 200 individuals.After observing an increased prevalence of males within the AF16 lines evolving at us study  to estimA). The presence of any bands which differed in expected size from wild-type or ΔmtDNA-C were recorded. To determine the location of deletions, PCR products smaller than intact mtDNA from at least one generation of each line in which they appeared were sequenced using BigDye (Applied Biosystems) with standard reaction conditions. If only a band of the smaller, deletion-bearing mtDNA was produced, that PCR product was purified using magnetic beads before sequencing. When PCR amplification produced amplicons from both intact mtDNA and deletion containing mtDNA, the smaller, deletion-bearing band was isolated using a QIAquick gel extraction kit in preparation for sequencing. In cases where sequencing from gel extracted products failed, the PCR products were cloned into the pCR8/GW/TOPO vector (Life Technologies). The resulting clones were screened to find those with plasmids of the predicted size for containing the deletion PCR product. Plasmid DNA was purified with the QIAprep spin miniprep kit (Qiagen) for sequencing. To determine deletion boundaries, sequences were aligned with the AF16 reference mtDNA sequence in GenBank (AC186293) if the deletion occurred in AF16 or with sequences from DNA was extracted from pools of approximately 50–500 worms (with a few exceptions when only approximately 10 worms were available) using a standard proteinase K-based lysis buffer method. In order to detect mtDNA size variants, the whole mtDNA genome was amplified via four overlapping PCR fragments using previously described primers , involving strain AF16 and a derivative line evolved at N = 1 for 50 generations, for the purpose of further corroborating the presence of ΔmtDNA in this species using a complimentary experimental method. The specific bottlenecked AF16 line was from a different lab evolution experiment relative to those that were the focus of this paper. For this analysis, thousands of L1-stage nematodes were collected subsequent to standard hypochlorite treatment of large mixed-stage populations  for each of AF16 (unevolved) and its derivative bottlenecked line. DNA was extracted using a Qiagen DNeasy kit. This total DNA was then analyzed on an Illumina MiSeq system available at the Oregon State University Center for Genome Research and Biocomputing. Sequencing was carried out as paired-end 150 bp reads. After sequencing, reads were mapped to the reference C. briggsae AF16 genome sequence  and under strict mapping parameters (98% length identity and 98% sequence identity). The Illumina data were archived with the NCBI Short Read Archive under accession numbers SRX1014084 and SRX1014093.We also provided a high-throughput DNA sequencing-based analysis of ΔmtDNA occurrence . Odds ratios reported in N = 1,000 used as the reference group. All observations from HK105 were excluded from this analysis due to high prevalence of missing data at population size of 1.An ordered logistic regression model  was usedfigs. 4N = 1, 10, 100, and 1,000) and six C. briggsae natural strains  was observed for only one C. briggsae strain (HK105), in three-fifth replicate lines of this strain. Extinction in these three cases occurred at 14, 25, and 36 generations, respectively. All replicates of all strains at the three larger population sizes  achieved Gmax without extinction. The frequency of transfers successfully establishing new generations for each replicate was quantified as the ratio of the number of transfers to Gmax , following . The resulting mortality patterns mirrored those of failed transfers reported above. HK105 performed worst with 16% mortality per transfer (one-quarter of which resulted from premature hatching of eggs within the parent). Replicates of HK104 showed 7% mortality, followed by AF16 (3%), EG4181 (0.004%), and PB800 and VT847 (both 0%).We analyzed among-strain variation in extinction, successful transfer frequencies, developmental delay, and mortality for the nematodes evolved at ollowing . This raN = 100 and 1,000 as early as G3 and G14, respectively. An analysis of male frequency conducted at G41 for all N = 1,000 lineages revealed average male frequency in AF16 lines to be 22.1% as compared with approximately 0% in all other strains . These AF16 males were in several instances observed to be copulating with hermaphrodites. Thus, outcrossing may have been a significant contributor to evolution in the AF16 lineages but not to that in the other strains. Furthermore, unlike the N = 1 and 10 treatments where hermaphrodites were directly transferred each generation, the N = 100 and 1,000 lines were maintained by bulk population transfers  that may have included male offspring. Our observations and analysis suggest that this was a rare occurrence for strains other than AF16. To the extent that male offspring were included in the AF16 population transfers, the actual eN of AF16 lineages could differ compared with other strains.During lab evolution, we noted an increased presence of males within replicate lineages of AF16 evolving at N = 1 for 50 generations, using Illumina MiSeq technology . Read coverage at the nad5 sequences deleted in ΔmtDNA-C molecules were much lower in the bottlenecked line compared with that in the ancestral line . This pattern is consistent with the presence of ΔmtDNA-C molecules in C. briggsae, as previously reported using PCR methodologies . When size variants were detected on agarose gels, amplicon sequencing was conducted to reveal the DNA sequence characteristics of the variant. New PCR primers were developed as needed to more specifically characterize newly discovered mtDNA variants. This approach was used to comprehensively search for new ΔmtDNA molecules across all mtDNA regions. We also implemented a conventional PCR assay developed  in all experimental populations of eveloped  to coarsC. briggsae lineages. Nine different specific ΔmtDNA sequence types were identified among these 19 lab lineages  were detected in 19 different experimental lineages , with dee region . Similarae lines , in thisN = 1 and 10).After initial detection of ΔmtDNA molecules by long PCR, conventional PCR was used to more finely characterize deletion size differences on agarose gels  and for nad5 region of the mitochondrial genome where ΔmtDNA-C was originally discovered  while the boundaries of six newly discovered deletions occurred entirely within the nad5 gene. All of these deletions likely disrupt nad5 protein-coding function. Like ΔmtDNA-C, all but one new deletion were flanked by directly repeated DNA sequence . The proportion of intact banding type among lines increased across generations in the N = 1,000 populations derived from strains EG4181 and PB800 .The vast majority of experimental lines (115/120) began with the intermediate-category ΔmtDNA-C gel banding pattern at G0. Likewise, the majority (86.4%) of the 1,271 total DNA samples analyzed  produced the intermediate banding type . The ovefig. 1nd PB800 . For exaThe PCR approaches implemented in this study revealed the presence of previously known (ΔmtDNA-C) and novel ΔmtDNA variants. Although this approach offered a reproducible and sensitive method for identifying and characterizing new variants, the variably biased nature of PCR  precluded efforts to quantify relative abundances of the many different ΔmtDNA circle types discovered across the greater than 1,000 nematode samples analyzed here. Thus, we based our analysis on the categorical gel banding patterns observed across generations and selected an ordered logistic regression statistical model where the outcome variable—deletion score—is ordered, but the quantitative distances between deletion categories are not assumed to be equal.Five categorical deletion scores (1–5) were possible in our statistical analysis, with increasing scores reflecting increasing severity of ΔmtDNA state . Explanatory variables examined in the statistical analysis were population size, strain, and generation number. A random effect was included for line to account for correlation of observations across generations for a given experimental line.supplementary table S2, Supplementary Material online). Positive coefficients for population sizes of 1 and 10 indicated that evolution in small populations is strongly associated with increased ΔmtDNA prevalence , ΔmtDNA scores were also more likely to increase with each generation, reflected by significant interaction effects between population size and generation number. However, in large populations, ΔmtDNA scores decreased over time, indicated by the negative coefficient of generation number. Note that ΔmtDNA scores were not significantly different in population sizes of 100 and 1,000, after accounting for other factors (P = 0.21).Likelihood ratio tests indicated that population size, strain, and generation number are significant predictors of ΔmtDNA score; the effect of generation number depended on population size . The estimates in ΔmtDNA scores were strongly associated with strain, with VT847, AF16, and HK104 having significantly greater odds of higher ΔmtDNA scores compared with EG4181  as generation number increased. By contrast, HK104 was overall much more likely to receive band scores of 2 or greater as compared with EG4181; for population sizes of 1 or 10, the probability of a receiving a score above 2 increased dramatically as generation increased.The overall statistical trends were illustrated by the probabilities predicted for each ΔmtDNA score based on the chosen model for a given generation number, strain, and population size . For insC. briggsae, but only when population size is small (N = 1 in HK104), but can be subject to selective elimination in larger nematode populations —see N = 1 we observed that it was possible for new ΔmtDNAs of smaller genome size to arise and quickly outcompete previous ΔmtDNAs of larger size across generations . In PB800, for example, levels of ΔmtDNA-C were observed to be intermediate . A similar pattern was observed for HK104. At generations 0, 5, and 10, the deletion ΔmtDNA-C band category was prevalent in the N = 1,000 experimental lines. At generation 15 and beyond, however, the intermediate category predominated, again indicating that ΔmtDNA-C levels decreased across generations. For the other three C. briggsae strains , more static patterns were observed across generations at N = 1,000. This observation is particularly noteworthy for strain AF16 where high male frequencies were observed in the N = 100 and N = 1,000 lines . The similarity between AF16 and other strains lacking compensatory alleles, rather than EG4181 and PB800, suggests that the prevalence of males had a minimal impact on ΔmtDNA-C evolution in AF16. Our combined results implicate host population size as a key factor that shapes the evolutionary trajectories of naturally occurring ΔmtDNA elements.We observed that preexisting ΔmtDNA levels sometimes decreased across generations, but only when nematode population size was large  in HK105, and to a lesser extent in VT847, the strains previously determined to harbor the highest natural levels of ΔmtDNA-C ; instead, static ΔmtDNA-C patterns were observed. As previously discussed, EG4181 harbors putative compensatory mtDNA mutations that are associated with lower overall ΔmtDNA-C levels as compared with strains lacking those mutations. The compensatory mutations also occur in PB800 where similar static patterns were observed in smaller populations. The differential occurrence of compensatory mtDNA mutations among strains might explain the relative stability of ΔmtDNA-C levels in EG4181 and PB800 compared with the other four strains. However, this factor cannot explain the occurrence of new ΔmtDNA types (involving different direct repeats compared with those associated with ΔmtDNA-C) in AF16, HK104, HK105, and VT847 strains but not in EG4181 and PB800. We searched for potential compensatory mutations in the direct repeats associated with new ΔmtDNAs discovered here but discovered none in EG4181, PB800, or the other four strains. This suggests that the low occurrence of all ΔmtDNA types in EG4181 and PB800 might result from superior mitochondrial quality control  and/or lower baseline mtDNA deletion formation rates as compared with the other four strains. These observations also suggest that ΔmtDNA might be more deleterious in EG4181 and PB800 genetic backgrounds as compared with the other four strains.Although population size was a major factor in determining patterns of variation in ΔmtDNA accumulation in our study, we also observed variable patterns of accumulation among nematode strains. For example, in HK104, new ΔmtDNA molecules arose and persisted at  = 1,000  and 6. SΔmtDNA-C . By contN = 100 and 1,000 lines compared with those of the other five progenitor strains . Why elevated male frequency was observed for AF16 alone remains a mystery, but the fact may have impacted our observations in at least two ways. First, if the ΔmtDNA elements studied here have any direct selective effects on males (unknown) or if their transmission was biased toward male offspring, ΔmtDNA elements in these lines would have been exposed to an additional form of selection compared with those in all other lines where males were essentially absent  sizes. If true, increased selection efficiency afforded by sexual reproduction may have helped to limit ΔmtDNA accumulation—or in some cases to eliminate ΔmtDNA elements—in this strain , however, were found in more than one experimental line, and also from two or more different progenitor strains.Ten different ΔmtDNA types were detected in the present study . The ΔmtN = 1 lines and 2/30 N = 10 lines. ΔmtDNA-5 was found in 5/6 progenitor strain backgrounds . Is it possible that these patterns might be entirely explained by de novo deletion mutation formation (rather than segregation of preexisting heteroplasmic variants) during the experiment? A mutation rate of 3.3 × 10−3 would be required for de novo mutation to explain the observed occurrences of ΔmtDNA-5 at N = 1 in our study. This value is approximately 3 times higher than the rate for new deletion formation in C. briggsae mtDNA reported in ΔmtDNA-5 was detected in seven different experimental lines . This elN = 10 lineage of HK104, and one N = 10 lineage of VT847. In both cases, the first evidence of ΔmtDNA-6 occurred midway through the experiment (G25 in HK104 and G30 in VT847). The observation of this element at N = 10 only is peculiar when compared with patterns at other ΔmtDNA elements which predominated at N = 1. These observations suggest that ΔmtDNA-6 probably arose through de novo mutation and accumulated to detectable levels two independent times during the experiment. Our combined observations suggest that C. briggsae ΔmtDNA elements likely evolve through a complex combination of high baseline mutation rates of de novo formation  in male sperm-fertilized progeny  selfish DNA system  as technical error.Why are such heritable ΔmtDNA molecules found in erations . Thus, n progeny . Naturaloduction , are notA system . AlthougC. briggsae are differentially susceptible to the accumulation of deleterious ΔmtDNA. Knowledge on the (presumably) nuclear genome-encoded determinants of this variation will require future studies involving recombinant inbred line approaches  population size is small, and decline when host population size is large. It was also revealed that different natural strains  of proaches . We alsoSupplementary figure S1 and tables S1 and S2 are available at Genome Biology and Evolution online .


The role of human papillomavirus (HPV) in the development of esophageal cancer remains controversial. Our study aims to test the association between HPV 16 infection and esophageal cancer in China, providing useful information on this unclear association in Chinese population.Studies on HPV infection and esophageal cancer were identified. A random-effects model was used to calculate the odds ratios (ORs) and corresponding 95% confidence intervals (CIs) comparing cases with controls.CI: 2.17, 6.13) to 15.44 . The pooled estimates for OR was 6.36 . In sensitivity analysis, the estimates for OR ranged from 5.92  to 6.97 .A total of 1442 esophageal cancer cases and 1602 controls from 10 included studies were evaluated to estimate the association between HPV 16 infection and esophageal cancer risk. The ORs for each case–control studies ranged from 3.65 (95% This study indicates that HPV-16 infection may be a risk factor for esophageal cancer among Chinese population, supporting an etiological role of HPV16 in this malignancy. Results in this study may have important implications for esophageal cancer prevention and treatment in China. Esophageal cancer is the eighth most common malignancy worldwide and a majority of cases show poor prognosis in clinical practice . Based oIn the past few decades, many risk factors for esophageal cancer have been investigated, including tobacco smoking, alcohol drinking, dietary and micronutrient deficiency, high temperature of beverage and food consumption, poverty and history of head and neck cancer . HoweverHuman papillomavirus (HPV) has been suggested as a distinct possible cause of esophageal cancer. To date, more than 140 HPV genotypes have been recognized and subdivided into cutaneous and mucosal HPV types. Based on the oncogenicity, HPV are classified into high-risk and low-risk types. High-risk HPV 16 infection is more prevalent than any other high-risk HPV type in most regions of the world . As the The role of HPV in the development of esophageal cancer remains controversial. The prevalence of HPV infection in esophageal lesion or carcinomas varied largely in different studies . SyrjaneA systematic search was conducted to identify relevant articles, using MEDLINE (via PubMed), Excerpta Medica database (EMBASE) for English language, and using Chinese National Knowledge Infrastructure and Wanfang Data Knowledge Service Platform for Chinese language. Date of the literature was specified between 1 Jan 2005 and 1 July 2014. The search strategy was verified by a medical reference librarian and research articles were selected using the following keywords: human papillomavirus, papillomavirus infections, (o) esophageal neoplasms, (o) esophageal cancer, and (o) esophageal carcinoma. The search strategy was adapted for each database in order to maximize the ability to identify eligible studies. Reference lists of the included studies and published meta-analyses on related topics were also screened for additional studies.Two authors independently reviewed the identified relevant articles and judged whether they met the inclusion criteria for meta-analysis. Uncertainties and discrepancies were resolved by consensus after discussion with a senior author. The meta-analysis included studies in adults meeting the following criteria: (1) case–control studies or cohort studies; (2) studies detected HPV DNA in the tissues of subjects; (3) samples of control group must be taken from normal population without esophageal cancer; (4) studies explicitly provided the information on HPV DNA detection method. HPV DNA must be tested either by polymerase chain reaction (PCR)-based methods, including broad-spectrum PCR primers, type-specific PCR primers, or a combination of both kinds of primers; (5) necessary data could be directly extracted or calculated from the original article; (6) studies were peer-reviewed publications with HPV prevalence data from a minimum of 30 cases of esophageal cancer; and (7) studies conducted in the Chinese population. If the study was reported in duplication, the one published earlier or provided more detailed information was included. Review articles and editorials were included if they contained original data. Abstracts were excluded.Two authors performed the data extraction from each article and discrepancies were resolved by consensus. For studies meeting the inclusion criteria, a standard data extraction form was used to extract the following data: general information, including name of first author, year of publication, geographical areas of the study origin; numbers of cases/controls and HPV positive cases/controls; HPV detection method; and types of specimen (paraffin-embedded fixed biopsies (PE), fresh or frozen biopsies (FF)).In this meta-analysis, the association between HPV infection and cancer risk was estimated by means of odds ratios (ORs) and corresponding 95% confidence intervals (CIs) comparing cases with controls through the method of DerSimonian and Laird using the assumptions of a random-effects model . For subI2  and Cochrane Q test  [Heterogeneity between eligible studies were assess by geneity) . Stratifgeneity) ,16. In aIn this study, meta-analyses were performed using STATA version 12 for Windows . A two-tailed P < 0.05 was considered statistically significant.Figure Individual characteristics of the included 10 studies were summarized in Table Q test Pheterogeneity < 0.001, I2 = 55.1%). Therefore, the random-effects model was chosen to evaluate the pooled ORs. Individual and pooled OR estimates derived from a random effect model analysis were illustrated in the Forest plot. As shown in Figure CI: 3.42, 69.70). The pooled estimates for OR was 6.36 , indicating a significant association between HPV 16 infection and esophageal cancer.Based on the heterogeneity test, there was a moderate heterogeneity between included studies , compared to the latest study in 2014 with a cumulative estimate of 6.36 . The confidence interval for the summary estimate of the cumulative studies decreased with time of studies  to 6.97 . Results did not show significant differences when any study was omitted, which indicated that each single study did not influence the stability of the association between HPV 16 and esophageal cancer.To address the potential bias due to the quality of the included studies, we performed the sensitivity analysis by calculating pooled OR again when omitting one study each time. Figure P = 0.721, Egger P = 0.878).The funnel plot did not show evidence of asymmetry (Not shown). According to Begg’s and Egger’s test, there was no evidence of publication bias  in our meta-analysis is relatively higher than other studies. This is not surprising if we consider the inclusion criteria of this meta-analysis. We only included studies provided the information of HPV DNA tested by PCR-based assay which is more sensitive than other methods, such as in situ hybridization histochemistry. Even more importantly, samples of control group were taken from normal population without esophageal cancer. Studies using other normal participants had a higher risk than studies with adjacent normal mucosa as the normal control [Results in our meta-analysis are consistent with other studies. Yong et al. reported an OR of 3.55  between HPV 16 infection and esophageal cancer. Li et al. reported a similar OR of 3.52  . Hardefe control . This waSome factors might contribute to the variability of results on evaluating the association of HPV 16 infection and esophageal cancer. In our study, stratified analyses were performed according to geographical areas of the study origin, publication years, HPV detection method and types of specimen. Based on the results, we found that samples from FF tissues had a higher point estimate of OR than samples from PE tissues. This is mainly due to the DNA degradation in PE tissue . We alsoOur results may have important implications for esophageal cancer prevention and treatment. To date, two vaccines have been developed and approved for use against HPV. Gardasil is a quadrivalent vaccine that targets HPV-6, −11, −16, and −18. The bivalent vaccine Cervarix targets HPV-16 and −18. Recently, the success of the prophylactic immunization campaign for cervical cancer has attracted a lot of interest in preventable HPV related cancers, including esophageal cancer. However, the association between HPV and esophageal cancer is controversial since it was first reported by Syrjänen . TherefoThis meta-analysis shows minimal heterogeneity or publication bias, and that no single study affects the summary effect significantly. The pooled result is further validated by the stratified analysis. All these strengthen the finding of the association between HPV 16 and esophageal cancer. However, there are still some limitations that should be addressed. First of all, due to lack of detailed information on confounders such as age, gender, smoking, assumption of alcohol, which were also risk factors of esophageal cancer, we could not adjust for these confounders which may affect the association between HPV 16 and esophageal cancer. Further studies should focus on this issue and provide more information on type-specific HPV risk on esophageal cancer. Secondly, we cannot exclude the effect of contamination of samples in this study which can directly affect the detection of HPV 16. Roden et al. reported that dehydrated HPV could maintain 100% infectivity for one day . FerenczThis study indicates that HPV-16 infection may be a risk factor for esophageal cancer among Chinese population, supporting an etiological role of HPV16 in this malignancy. We believe that this is a major step forward for this controversial issue and our results may have significant implications for esophageal cancer prevention in China. Further studies are needed to elucidate the role of HPV in esophagus carcinogenesis with careful consideration of study design and laboratory detection method, providing more accurate assessment of type specific HPV risk on esophageal cancer.


Human Papillomavirus (HPV) genotype distribution varies according to the method of assessment and population groups. This study analyzed type-specific HPV infections among women ranging from 14–95 years old, displaying normal and abnormal cytology, from São Paulo and Barretos cities, Brazil.n = 431) plus a random sample of 223 negative by both assays and 11 samples with indeterminate results, totalizing 665 samples, were submitted to HPV detection by the PapilloCheck test. Cytological distribution included 499 women with a cytological result of Negative for Intraepithelial Lesion or Malignancy and 166 with some abnormality as follows: 54 Atypical Squamous Cells of Undetermined Significance; 66 Low-Grade Squamous Intraepithelial Lesion; 43 High-Grade Squamous Intraepithelial Lesion and 3 (0.5 %) Invasive Cervical Cancer.Women found positive for High Risk-HPVs DNA by either the Hybrid Capture 2 (HC2) or Cobas HPV Test (From the 323 samples (48.6 %) that had detectable HPV-DNA by the PapilloCheck assay, 31 were HPV negative by the cobas HPV and HC2 assays. Out of these 31 samples, 14 were associated with HR-HPVs types while the remaining 17 harbored exclusively low-risk HPVs. In contrast, 49 samples positive by cobas HPV and HC 2 methods tested negative by the PapilloCheck assay (19.8 %). Overall, the most frequent HR-HPV type was HPV 16 (23.2 %), followed by 56 (21.0 %), 52 (8.7 %) and 31 (7.7 %) and the most frequent LR-HPV type was HPV 42 (12.1 %) followed by 6 (6.2 %). Among the HR-HPV types, HPV 56 and 16 were the most frequent types in NILM, found in 19.1 and 17.7 % of the patients respectively while in HSIL and ICC cases, HPV 16 was the predominant type, detected in 37.2 and 66.7 % of these samples.In the population studied, HPV 16 and 56 were the most frequently detected HR-HPV types. HPV 56 was found mainly in LSIL and NILM suggesting a low oncogenic potential. HPV 16 continues to be the most prevalent type in high-grade lesions whereas HPV 18 was found in a low frequency both in NILM and abnormal smears. Surveillance of HPV infections by molecular methods is an important tool for the development and improvement of prevention strategies. Two hundred different HPV types, classified into 49 species, are currently recognized by the International HPV Reference Center . From thThe majority of HPV infected women will never develop cancer since most HPV infections are transient and asymptomatic. As expected, HPV is more prevalent in young sexually active women (18 to 30 years) , decayinInvasive Cervical Cancer (ICC) is worldwide the third most frequent cancer among women . In BrazThe distribution and prevalence of the HPV genotypes vary with the grade of cervical disease, age and the geographical location of the patients , 13–15. As a result of accumulated knowledge over the past three decades on the causal relationship between HPV infection and ICC, detection of the viral DNA became an attractive approach to identify women at risk for developing ICC , 28. EviSince June 2013, the Brazilian Ministry of Health has announced the decision to introduce the quadrivalent HPV vaccine in the national immunization program, the vaccination schedule (0 and 6 months) was updated in January 2016. From 2014, this vaccine was offered to girls and adolescents 11–13 years old and from 2015 for adolescents between 9 and 11 years . In thisPapilloCheck assay is a robust method for HPV genotyping, which is based on PCR amplification of a fragment of approximately 350 bp from the E1 region of HPV genomes, using broad-spectrum consensus primers, allowing the simultaneous detection and genotyping of 24 different HPV types by DNA chip technology including low-risk HPVs .We aimed to uncover the HPV type-distribution hiding under an HPV-HR result in the HC2 test or the 12 HR-HPVs cocktail in the Cobas HPV test to analyze the HPVs genotype distribution in women undergoing routine cervical cancer screening and women with a previous abnormal Pap test in São Paulo and Barretos cities and their correlation to cytological results and age groups.n = 156 from São Paulo and 247 from Barretos), in addition to 262 referral women with a previous abnormal Pap test in the year preceding study enrollment (163 from São Paulo and 99 from Barretos). A cervical sample was obtained and processed as described elsewhere [Specimens were collected in between October, 2009 and April, 2011, in two reference centers of São Paulo State, Brazil: Hospital das Clínicas from the São Paulo Medical School, Universidade de São Paulo and Hospital de Câncer de Barretos. Participants, with age ranging from 14 to 95 years, average 43 yo, median 43yo (SD = ±14 yo), consisted of 403 women undergoing routine screening (n = 431) were submitted to HPV genotyping by the PapilloCheck assay  [Briefly, all samples were submitted to Cobas 4800 HPV  and the Hybrid Capture 2  assays. Only the HR-HPV cocktail probe was employed on the HC2 testing. Samples from women found positive for high-risk HPVs DNA by either the HC2 or Cobas HPV (Germany)  plus a rCervical scrapes obtained by the SurePath collection kit  and preserved in liquid cytology medium were transported to the lab where DNA was extracted using the QIAamp DNA Mini Kit , according to manufacturer’s instructions. For each reaction, 5 μL of DNA eluate was used in the PapilloCheck assay. Specimens containing the target DNA are hybridized to specific oligonucleotide probes immobilized on a DNA chip and detected by the binding of a Cy5-dUTP labeled oligonucleotide probe to the tag sequence. The DNA chip is scanned by the CheckScanner apparatus at wavelengths of 532 and 635 nm. This test detects HPV genotypes 6, 11, 16, 18, 31, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53.56, 58, 59, 66, 68, 70, 73 and 82. In addition, human ADAT1 gene  is used as an internal control to assess the quality of the DNA .smears were examined by staff cytopathologists at both institutions and classified according to the Bethesda system [Six hundred sixty five a system : NILM  women with negative cytology (NILM) and 166 (25.0 %) with some cytological abnormality as follows: 54 (8.1 %) ASCUS; 66 (9.9 %) LSIL; 43 (6.5 %) HSIL and 3 (0.5 %) ICC.When stratified by age group, LSIL was less frequent in women older than 45 years old (p trend = 0.01), whereas HSIL was more frequent (p trend = 0.03). All 3 ICC cases were found in women older than 45 years: 48, 52 and 84 years old.Among the 665 samples analyzed 323 (48.6 %) were HPV positive by the PapilloCheck assay. Single type infection was observed in 65 % (210/323) of them, while 59 (18 %) had co-infection by 2 HPV types, 34 (11 %) by three, 14 (4 %) by four and 6 (2 %) by five or more types. The frequency of HPV infection decreased with aging (p trend <0.001); in women under 31 years old the prevalence of HPV DNA was 58.2 % (92/158) decreasing to 52.3 % (116/222) in women between 31 and 45 years and 40.4 % (115/285) in women older than 45 years old.The most frequent HR-HPV type among HPV positive women was 16 (23.2 %), followed by 56 (21.0 %), 52 (8.7 %), 31 (7.7 %), 53 (7.7 %), 51 (7.4 %), 39, 59 and 66 (6.5 % each), 33 (5.3 %), 58 (5 %), 18 (5 %), 82 (4 %), 45 and 70 (4 %) each, 68 (3.4 %), 73 (2.5 %) and 35 (2.5 %). The frequency of LR- HPV types was: 42 (12.1 %), 6 (6.2 %), 44 (4.3 %), 43 (4 %), 40 (2.8 %) and 11 (1.5 %). No association between LR-HPV and HR-HPV types with age group was observed.vs 8.9 %. This was also observed for HPV 56, the second most common genotype in both groups, found in 13.7 and 7.9 % respectively. Table When stratified by women undergoing routine screening and those with a previous abnormal Pap test , HPV 16 was detected more often in referral than in the screening group; 14.9 % Comparing the HPV result obtained by the PapilloCheck to the cytology diagnosis, we found 41.8 % (209/499) of HPV positive results among women with negative cytology; 55.6 % (30/54) ASCUS; 80.3 % (53/66) LSIL; 67.4 % (29/43) HSIL and 100 % (3/3) among women with ICC. Eight HSIL cases, 18.6 % (8/43) were positive either by Hybrid Capture 2 (HC2), Cobas HPV or both, while negative for the Papillocheck assay, whereas in the three ICC cases HPV was detected by all three methods.HPV 56 and HPV 16 were the most frequent in negative cytology being found in 19.1 % (40/209) and 17.7 % (37/209) of the patients, respectively. In LSIL cases, HPV 56 was the most frequent type present in 28.8 % (19/66), while in HSIL and ICC cases, HPV 16 was the predominant type, detected in 37.2 % (16/43) and 66.7 % (2/3) of these samples, respectively whilst, the third ICC case harbored HPV 33. Distribution of HPV types according to the cytological diagnosis is shown in Fig. Concerning samples that showed negative results in molecular screening by HC2 and Cobas HPV on the original study, 13.9 % (31/223) were HPV positive by the Papillocheck. Of these, 54.8 % (17/31) were positive for LR-HPV, whereas 45.2 % (14/31) were associated with HR- HPVs types 16, 33, 39, 51, 53, 56, 58, 59, 66, 68, 70 or 73. In contrast, 49 samples positive by both Cobas HPV and HC 2 methods tested negative by the PapilloCheck assay .The two HR-HPV types found more frequently in all ages from the screening group were HPV 16 and 56. HPV 16 was observed in 15.5 % (15/97) of samples from women under 31 years and in 7.7 % (14/183) of samples from women older than 45 years, followed by HPV-56 that was detected in 9.3 % (9/97) of the patients under 31 years and in 6.0 % (11/183) of the older than 45 years. In samples from women aged 31–45 years old, the most frequent HR-HPV type was HPV 56 9.8 %; 12/123), followed by HPV 16 . The most frequent HPV types according to age groups are depicted in Fig. /123, folWhen HR-HPV genotypes are clustered in HPV species according to the classification of de Villiers et al. , it may In the present study, the specimens analyzed were enriched from a group of HPV positive samples and a random subsample of specimens displaying HPV negative molecular testing results, as described above. Thus, it has to be considered that this rate of HPV infection does not represent the true prevalence in women submitted to cervical cancer screening, in whom HR-HPV DNA was detected in about 9 % of subjects under routine screening, nor the prevalence in the referral group, of 24 % in women with a previous abnormal Pap test .Differences in the prevalence and distribution of HPV types worldwide have been observed in several studies , 39, andHPV 16 was the most frequent type in the present series as found in almost all screening population studies, irrespective of the study design –44. An iIn the current study, HPV 56 was the most frequent type among HPV+ women bearing NILM and LSIL cytology, found respectively in 19.1 and 28.8 % of these samples. In contrast Oliveira-Silva et al.  studied In fact, other studies using PapilloCheck assay reported rates of HPV 56 above the expectation , 46, whin = 175) also harbored HPV 16 as the most frequent type (77.6 %), followed by HPV 18 (12.3 %), HPV 31 (8.8 %), HPV 33 (7.1 %) and HPV 35 (5.9 %) [In this study, HPV 16 was the predominant type found in 38.1 and 66.7 % of the HPV+ HSIL and ICC cases respectively. These findings corroborate the results of a systematic review and meta-analysis in Latin America and the Caribbean including 2446 cases of HSIL and 5540 of ICC that reported HPV 16 as the most frequent type, verified in 46.5 % of HSILs and 53.2 % of ICC cases . In anot (5.9 %) .Detection of of LR-HPV types in the current study is explained by the inclusion of probes for these genotypes in the PapilloCheck assay, depicting them when present in co-infections with the HR-HPV genotypes, or in single type infections in the HPV screening (Cobas-/HC2-) negative group. Regarding the LR-HPV types, HPV 42 was the most common as observed in other studies using the same method of genotyping , 49, 50.Currently, HPV DNA testing has been incorporated into cervical cancer screening algorithms to triage cases with indeterminate cytologic results (ASC-US) ; clinicaIn the current study, HPV DNA was not detected in 32.6 % of HSIL samples. This rate is higher than found by Guo et al. , whom reWhen stratified by age group, women under 31 years old had the highest HPV frequency which mostly represents transient infections . HoweverMultiple infections were detected in 35 % of samples, and were more frequent in women under 31 years old. This observation is consistent with others studies , 62, andCurrent US guidelines recommend genotyping only for HPV 16 and 18, and detection of either of these two HPV types is sufficient to warrant direct referral to colposcopy, avoiding additional triage methods. However, some studies suggested that it may be also clinically useful to identify other HPV types such as HPV 31, 33, 33/58 (combined), which showed comparable or increased risk for the development of CIN 2+ –67.The nonavalent HPV vaccine appears to be safe and effective in preventing persistent infection and precancerous lesions associated with HPV types 16/18/31/33/45/52/58, as well as genital warts related to HPV types 6 and 11 . MoreoveThe prevalence and genotype distribution of human papillomavirus (HPV) provides the basis for designing HPV prevention programs. HPV types 53, 66, 68, 70, 73 and 82 included in the Papillocheck assay and other types such as HPVs 26 and 67 have been rarely but consistently identified as single HPV infections in about 3 % of cervical cancer tissues [


Two transport media, PreservCyt and SurePath, are widely used for cervical cytology screening. There are concerns that they may perform differently for HPV testing.A comparison of the performance of six different HPV tests in SurePath and PreservCyt in a referral population using two samples from each woman. The primary goal was to compare the performance of each test in the two media. Comparisons between assays and viral load comparisons between media were secondary aims.digene HC2 High-Risk HPV DNA Test, Abbott Realtime, BD Onclarity and Genera PapType, an RNA based test—: Hologic Aptima and a protein test: OncoHealth.Two cervical samples were collected in random order at the same visit in women with abnormal cytology. One sample was placed in 20 ml of PreservCyt and the other in 10 ml of SurePath. Aliquots were taken for 4 DNA based tests: me and Aptima, and generally lower relative signal strengths were seen with SurePath except for Onclarity, especially when it was the second sample.630 sample pairs were included in the analyses. For all tests except the protein test sensitivities were in excess of 90% for CIN2+ and 95% for CIN3+ for both media and with no significant differences except for a lower sensitivity for CIN2+ of Aptima in SurePath . Specificity for <CIN2 was significantly better in Surepath for HC2, RealTiWe found similar sensitivity for CIN3+ in PreservCyt and SurePath for 5 nucleic acid tests in the two media in a referral population, but signal strength and positivity rates were lower in SurePath except for the Onclarity test. These results need to be replicated in a screening population. Slide preparation procedures from these media are different An advantage of LBC is that additional tests, notably HPV, can be run from a single sample, although only PreservCyt is approved by the FDA for this. Unlike PreservCyt, SurePath contains formaldehyde to preserve cell morphology and cross-linkage between protein and nucleic acid can occur which can make DNA undetectable and reduce DNA yield. This is partially reversible using proteinase K (PK) digestion and/or heat treatment prior to nucleic acid purification digene HC2 High-Risk HPV DNA Test (HC2) in PreservCyt. In a study of 972 SurePath and 1033 PreservCyt screening samples in different women Zhao et al. found no significant difference in sensitivity and specificity for the detection of CIN2+ by HC2 The majority of early studies of HPV testing in a medium also suitable for cytology have been conducted using Qiagen’s 2The objective of this study was a comparison of the performance of different HPV testing assays in SurePath and PreservCyt in a routine clinical setting. We used a colposcopy referral population and compared six HPV assays using two samples from each woman—one collected in PreservCyt and the other in SurePath. Our primary goal was to compare the performance of each test in the two media. Comparisons between assays were secondary aims.3The study was conducted in the Colposcopy Unit of St. Mary’s Hospital, London among women who had been referred with an abnormal screening result within three months and never treated for CIN. All women provided written informed consent.Two cervical samples were collected with Cervex-Brushes immediately prior to colposcopic examination in accordance with the European guidelines for quality assurance with cervical cancer screening Within one day of receipt in the laboratory, samples were warmed to room temperature, agitated for 60 s and aliquotted into a fixed order set of tubes, appropriate for six assays. This was pseudo-randomised to vary the aliquot assigned to each assay by using one of four dispensing patterns . Samples were only identifiable to laboratory staff by participant number. All pathology was reviewed by M.S. who was blinded to results and participant information.4Sample storage before testing, aliquot volumes and positivity cut-off values were all in accordance with the manufacturers’ instructions . No testManufacturers use ‘Invalid’ or ‘Indeterminate’ to denote failed results including when a whole plate or run fails. We refer to all as ‘Failed’ results in this paper.4.14.1.1•digene HC2 High-Risk HPV DNA Test: the QIAsymphony automated platform was used for nucleic acid extraction with the DSP AXpH DNA Kit . This consensus DNA test detects a panel of 13 high-risk HPV types . PreservCyt and SurePath samples were processed using different protocols: PC_AXpH_hc2_V1_DSP protocol and a modified SP2000_V1_DSP protocol including PK digestion and extended heated lysis time (provided by Qiagen for research purposes only) respectively •me High-Risk HPV assay used the m2000 processing System  for the detection of 14 high-risk HPV types, utilising Abbott reaction vessels as sample input tubes. Types 16 and 18 are individually reported. The remaining 12 high-risk types are reported together as a pool .The Abbott RealTi•The Becton Dickinson Onclarity HPV Assay using the BD Viper LT System is a real-time PCR based DNA test which detects 14 high-risk HPV types. Types 16,18,31,45,51,52 are detected individually. The remaining eight high-risk types are reported in three groups: ,  and . A 0.5 ml aliquot of thoroughly vortexed SurePath or PreservCyt was added to 1.7 ml of a proprietary HPV diluent. A heat step was employed to ensure that exfoliated cells were lysed and the sample homogenized prior to extraction of sample DNA •The Genera PapType Test is a semi-automated, bead-based multiplex full genotyping DNA assay for 14 high-risk HPV types  and two low-risk HPV types . The Sirocco platform  was used. Prior nucleic acid extraction was done using the Abbott m2000sp instrument 4.1.2•The Hologic Aptima HPV assay is based on target capture, transcription-mediated amplification and hybridization protection for the detection of E6/E7 mRNA expression of 14 high-risk HPV types . A consensus result for positivity to other high-risk types was provided. The Direct Tube Sampling platform was used. Typing for 16 and 18/45, available as a reflex test, was not done here. The SurePath sample was treated with PK at 65 °C for 2 h before being assayed manually. The cut-off was specified to be 0.5 of the ratio of the intensity to the reference standard.4.1.3•OncoHealth  protein test is a direct E6/E7 HPV Whole-Cell ELISA carried out in microtitre wells and is based on detection by non-type specific HPV E6 and E7 monoclonal antibodies me, PapType and Aptima have been described previously For all HPV tests except HC2 both samples were processed using an identical assay workflow. (To distinguish this from workflow associated with sample preparation). Test details using Preservcyt for HC2, Onclarity, RealTi5me were given a Ct value of 40 and signal strength 0 for Aptima. SAS (version 9.2) and R (version 3.2.2) were used. All statistical tests were two-sided and a p-value of 0.05 were accepted as statistically significant.The primary analyses consisted of paired comparison of the two samples from each woman. For some assays confounding was observed related to the order in which the sample was taken. Subsequently additional non-paired analyses by the Wilcoxon Ranksum test and a robust L1 based linear model with allowance for test order were also conducted 6me. For PapType one sample pair was not tested with either medium and 46 tests (44 sample pairs) failed . For Aptima there were 22 failed tests .The analysis was based on 630 sample pairs obtained from 652 participating women. Reasons for drop out are shown in Entry cytology was borderline dyskaryosis 193(30.6%), mild dyskarosis 380(60.4%), moderate dyskaryosis 37(5.9%) and severe dyskaryosis or glandular abnormality 20(3.2%). A total of 176(28.0%) histology results were CIN2 or worse, including 94(15.0%) cases of CIN3 or CGIN and 2(0.3%) cases of invasive cancer. (Supplementary Table S1).me, Aptima and OncoHealth in SurePath. Although showing some predictive ability above chance in PreservCyt, the OncoHealth test was substantially and significantly less sensitive than all other tests (≤60% for both media for both CIN2+ and CIN3+), but was more specific than the other assays. There was no significant difference however with the OncoHealth assay between media.Overall positivity, sensitivity for CIN3+ and CIN2+ and specificity for <CIN2 for the different tests and transport media are shown in me in SurePath (P < 2 × 10−5) and HC2 in PreservCyt (P = 0.009). For HC2 this probably reflects a larger sample volume for PreservCyt. Significantly higher values were seen for PreservCyt (vs SurePath) when both were used as a second sample especially for HC2—again with the exception of Onclarity. Type specific results for HPV 16 and 18 for RealTime, Onclarity and PapType gave a similar pattern  differed by transport medium and test order . Little  pattern .me and Onclarity and lower levels for the other tests. For HC2 the RLU values were much lower in the second sample for SurePath, possibly due to the smaller sample volume assayed.PreservCyt values were not statistically significantly different between the first versus second samples in all cases except for OncoHealth where they were substantially lower in the second sample . For SurThe correlation between the signal strength measurements for the two media for each test is shown in 7me and Aptima in SurePath, but this was minimal for CIN3+. The largest differences were seen for specificity which was generally better for SurePath, especially for HC2, RealTime and Aptima. This is likely to also be true for primary screening but direct verification in this setting is needed. Poor performance was seen for the OncoHealth protein test in both media. This protein-based test however is known to be less stable in alcohol and a second generation test has been developed since this study was carried out.Our results indicate that similar sensitivities and specificities can be achieved with either PreservCyt or SurePath for 5 of the 6 HPV tests, provided that the manufacturer’s recommended pre-treatments are observed. Some loss of sensitivity for CIN2 was seen for RealTiThe failure rate for PapType was relatively high . No specific reason could be identified, but this was a prototype test with the complexity of full typing, so improvements are likely in the future. The failure rate was 1.3% for Aptima, but there were no failures for any other tests.The differences between tests were greater for the second than the first sample, illustrating the differences in a true diagnostic situation where only a first sample would be used. This highlights the need for an adequate sample and may be a factor in the discordant results between assays as found by Rebolj et al. Although not of direct clinical relevance, comparison of the quantitative measures of signal strength as a surrogate measure of viral load provides additional insight into the comparative performance of the different tests in the two transport media. We recognise that there are several confounding factors to this measure including cell number and specific methods of measuring signal strength. In general lower signal strength values were obtained for SurePath. The largest differences were seen for HC2 which could partly be attributed to a smaller sample volume for SurePath.Most HPV assays have been more fully optimized for PreservCyt, which has been in use for longer. An exception is the Onclarity assay, developed by the manufacturer of SurePath. The Onclarity assay uses a heat step in sample pre-processing for both sample types and little difference between media was seen. At the time of this study no HPV test manufacturer had an approved protocol for their assay in the SurePath medium and it is possible that this will impact on performance.In summary this prospective study is the first comprehensive comparison of a range of HPV tests in the two most commonly used LBC transport media where two samples are taken from each woman. No major differences in performance were seen when the manufacturer’s protocols were used. These tests have all performed well in this referral population and although all appear suitable for screening. They need to be validated using the Arbyn criteria in a screening population Funding: funded from Cancer Research UK Programme grant C569/A16891, and supplemented by financial contributions and assay kits from Qiagen, BD, Abbott, Genera, Hologic and OncoHealth.JC has received honoraria for lectures from Abbott and Qiagen and served on advisory boards for Hologic and BD.All authors have attended meetings with manufacturers of HPV assays but none was compensated for their work on this project.All manufacturers had the right to comment on a draft version of this manuscript, but had no involvement in the final content or decision to publish.Received in August 2011 from NHS Health Research Ethics Service Committee London–Hampstead [Reference 11/LO/1147].


There is an error in the second and third to last sentences of the “Subjects and groups” subsection of the Materials and Methods. The correct sentences are: Clearance was defined for both follow-up groups as HPV positivity at baseline with HPV negativity 1 year (n = 148) or 2 years later (n = 66) regardless of intermediate results. Persistence was defined as HPV positivity at baseline with HPV positivity at 1 (n = 136) or 2 years later (n = 56), regardless of intermediate results.There are multiple errors in