This study aimed to determine the feasibility of vaginal/cervical nurse-assisted self-sampling (NASS) and the agreement between human papilloma virus (HPV) tests on self-samples versus clinician-taken (CT) specimens.P < .001). Lifetime partnered with >2 men were associated with HR HPV positivity (P < .001). There was a strong statistical association between HR HPV positivity and visual inspection with acetic acid- positive (P < .001). The NASS for HPV testing could be seen as an alternative option and might be acceptable to Ethiopian women. The overall HR HPV prevalence was comparable with Sub-Saharan countries in the general population.Women participated voluntarily for cervical cancer screening at St. Aklesia Memorial Hospital. Eighty-three women provided a total of 166 coupled self-taken and CT specimens collected. Specimens were stored at room temperature for a maximum of 10 months and analyzed using validated the RIATOL qPCR HPV genotyping test, a quantitative polymerase chain reaction (qPCR) high-throughput HPV E6, E7 assay. The average age of the participating women was 32 years. Seventy-three women (87.9%) felt that NASS was easy to use. An overall HPV, high-risk (HR) HPV, and low-risk HPV prevalence was 22.7% (15/66), 18.2% (12/66), and 6.1% (4/66), respectively. The overall HR HPV prevalence was 17.2% (NASS) and 15.5% (CT). The most prevalent HPV type was HPV51; HPV 16 was only detected in 1 woman (CT+NASS) and HPV18 only in 1 woman (CT). The overall measurement agreement between self-taken and CT samples was moderate with a kappa value of 0.576 ( Ethiopia has 31.5 million women aged 15 years and older and 7.095 women were diagnosed yearly with ICC of whom 4.732 died from the disease (estimates for 2012). Currently, there are only sparse data on the human papilloma virus (HPV) burden in the general population of Ethiopia.Invasive cervical cancer (ICC) is the fourth most frequent malignancy and cause of death in women suffering from cancer worldwide. Another study in Sub-Saharan Africa indicated a comparable HPV prevalence for self- (14.6%) and physician (12.7%) samples, so similar accuracy of the test on both sampling methods. A study in Madagascar showed absolute acceptance (100%) of SS (with a flocked swab) followed by HPV testing as cervical cancer screening method.A study in Nigeria, for example, found 93% participation in the self-sampling (SS) arm, compared to only 56% in the hospital-collection arm.Available data indicate that the HPV prevalence in Ethiopia among women with normal cervical cytology varies between 15.9% and 17.5%, and 96.6% of the ICCs are attributed to HPV16 (78.4%) and 18 (18.2%). Currently in Ethiopia, 200 health facilities are providing visual inspection with acetic acid (VIA) screening followed by cryotherapy (ablative treatment technique),and >52,000 women were screened in 2016/17. Of the 20 million women eligible for screening, only 0.3% of them screened. In addition, Loop electrosurgical excision procedure service was scaled up from 5 to 15 hospitals and the Federal Ministry of Health (FMoH) is working to expand VIA screening and cryotherapy into 823 districts. However, more efforts or other screening techniques are urgently necessary to scale up the cervical cancer screening coverage in Ethiopia.Cervical cancer develops over a long period of time through precursor lesions. These lesions can be detected by  screening, and progression toward cancer can then be stopped by treatment (ablation or excision) in an early phase.This study aimed to determine the feasibility and acceptability of vaginal/cervical nurse-assisted self-sampling (NASS) and the agreement between HPV test on self-samples versus clinician-taken (CT) specimens in the Ethiopian population.2The study aimed to determine the feasibility of vaginal/cervical NASS and the agreement between HPV testing on self-samples versus CT specimens.The study was conducted in Adama Town, Oromia region, having a total population of 1,356,342 people of whom 659,992 are females. The St. Aklesia Memorial Hospital (SAMH), located in Adama Town, is a private hospital with a long-time history and expertise in cervical cancer screening.To reach in an efficient way to a lot of women for recruitment in the study, radio calls and face–to-face interactions were organized. Through these channels, women were encouraged to schedule an appointment for cervical cancer screening approximately 2 weeks (10–18 days) after the first day of their last menstrual period. Also, women visiting the hospital for reproductive health-related issues were called for participation in the study.Women were eligible if they were 20 years or older, had an intact uterus, had no history of cervical cancer, were mentally competent, and able and willing to provide informed consent. Based on the upset of this study, a cross-sectional and probability sampling technique was used. The minimum calculated sample size of this study was 73.Women who were interested in participating in the study were given following instructions: no douche 48 hours before the test; no use of tampons, birth control foams, jellies, or other vaginal creams or vaginal medications for 48 hours before the test and also advised to refrain from intercourse 48 hours before the test.After signing an informed consent document, women were subjected to 2 ways of sample collection, both performed within the clinic: NASS with supervision; CT specimens, that is, a physician collected the samples according to the standard procedure of the clinic. Women were also asked to fill in a questionnaire.2.1Women were invited to the private area of the clinic and were given verbal and printed diagrammatic instructions by the trained nurse for collecting the vaginal specimen. When the women confirmed that all instructions were clear, the nurse opened the collection kit and handed over the collection devices (in sequence order of spatula followed by cytobrush) to the womanThe vaginal fornix and ectocervix were sampled before the endocervix. To start the NASS, women were instructed to take a sample of the ectocervix using a plastic spatula, without speculum. The women were asked to insert the spatula, laying on the bed, into their vagina and to rotate 3 times at 360 degree, to remove and to handover the collection device to the nurse. The nurse then rinsed the spatula into a labeled vial with ThinPrep PreservCyt solution.In the next step, the nurse provided the cytobrush to the woman to sample the endocervix. It was inserted by the woman herself until it met with resistance, rotated 45 to 90 degree, removed, and handed over to the nurse. The nurse inserted the cytobrush sample into the same ThinPrep PreservCyt labeled vial. This procedure was not involving any invasive steps rather noninvasive simple and easy collection techniques. Collected samples were kept at 22°C (room temperature) for about 10 months, until shipment and processing.2.2The clinicians collected cervical samples according to standard protocols, that is, both ectocervix and endocervix samples were collected with a cytobrush and rinsed in a labeled vial with ThinPrep PreservCyt solution. Collected samples were kept at 22°C (room temperature) for about 10 months, until shipment and processing.2.3After the NASS and the CT sample, all women underwent VIA. A woman was classified as VIA-positive when acetowhite lesions were visualized by the clinician. All VIA-positive women were eligible for cryotherapy and were treated.2.4The ethical committee of the College of Natural Sciences, Addis Ababa University, has examined the project and approved it. The SAMH Hospital also approved the project. All women signed informed consent before enrolment in the study.2.5,9 Samples <10 cell/μL are considered as invalid and reported as samples of poor quality.Both CT and NASS specimens were tested for presence of HPV with the RIOTOL quantitative polymerase chain reaction (qPCR) HPV genotyping test . This method is validated and ISO-certified laboratory-developed (LDT) high-throughput HPV test detects 14 high-risk (HR) HPV types, that is, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 57, 58, 59, 66, and 68, four intermediate/low-risk HPV (LR HPV) types, that is, 6, 11, 53, and 67; 68 and a cell control.2.6χ2 test and 95% confidence interval (CI) were used and statistically significant if the P value was ≤0.05.Quantitative data were collected and for some of the demographic variables were decoded accordingly. Any missed variable identified during the collection of data, the supervisor was responsible to follow-up the patients and correct it accordingly. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) version 20 software. The overall measurement agreement between self- and clinician-collected samples was calculated with a kappa value. The dependent variable was HPV outcome and independent variables are sociodemographic. Pearson 3A total of 83 eligible women were enrolled between October 2015 and July 2016 at SAMH hospital, Adama, Oromia region, Ethiopia. The study had no missed data or variables.3.1The average age of eligible women was 32 years and the youngest was 20 and the oldest 65 years. Forty-seven women (56.6%) had an education level below grade 10 (high school); 33.7% (28/83) and 27.7% (23/83) of the study population were laborers and housewives, respectively. Seventy-one women (85.5%) were married at the time of the study and 69.9% had 1 lifetime partner. A total of 80.7% (67/83) had gravidity ≥1 and 7.5% (5/67) of these women had a spontaneous abortion before. Women who used birth control and smoking cigarettes were 39.8% (33/83) and 18.1% (15/83), respectively. Four (4.8%) women reported being infected with HIV at the time of the study  (Table Of 166 samples (2 specimens per women), 44 (26.6%) had not enough cells (>10 cells/μL) and were considered as samples with poor quality. According to Fisher exact test, there was no statistically significant difference in the number of samples with poor quality between the 2 sample groups (NASS: 19/83 and CT: 25/83) . A total of 47/66 (71.2%) CT and NASS samples were in agreement in terms of HPV test results. From the 15 positive HPV samples, only 33.3% (5/15) were positive in both the NASS and CT sample, whereas for the HPV-negative results, there was 82.4% agreement (42/51).The most prevalent HPV type was HR HPV51 , followed by HR HPV31, 58 and 68 and LR HPV6 and 67 which were all found twice . HPV16 was detected in 1 woman, in both the CT and the NASS sample  and HPV18 in 1 woman, and only in the CT sample . Two women were coinfected with at least 2 HPV types (multiple infections: 2/66 = 3.0%). One woman of these 2 was coinfected with 5 HPV subtypes: HPV6, 16, 51, 67, and 68 (according to the NASS HPV DNA result). A total of 12 different HPV types were identified in this study, of the 18 HPV types that were tested for  women, acetowhite lesions were visualized. When excluding the CT samples with poor sample quality, 5 of the 9 women with a positive VIA result were HPV-positive (sensitivity of 55.5% [95% CI: 26.6%–81.1%]) and 84.5% (49/58). However, 45 of the 49 women with no acetowhite lesions were also HPV-negative (specificity of 91.8% [95% CI: 80.8%–96.8%]). The overall agreement between HPV and VIA result from CT sample was 86.2%  and 52 women of the 56 with no visual lesions were HPV-negative (sensitivity 92.8% [95% CI: 83.0%–97.2%]). The overall agreement between HPV and VIA result from NASS sample was 92.2% (Table 3.6χ2 test using the HR HPV test result (combined NASS and CT results) and all collected variables with HR HPV-negative status as the reference group. Having >2 lifetime sexual partners (P = .000447) and being VIA-positive were causally associated with an HR HPV-positive test result and not a difference by chance. Spontaneous abortion (P = .021) and being a housewife (P = .016) were also associated with HR HPV-positive results. Younger age groups (<40 years) showed a trend toward a correlation with a positive HPV test result (P = 0.058); there were about 19.6% (11/56) HR HPV-positive women in the age groups under 40 years, whereas only 10% (1/10) in the combined age groups above 40. Housewife and laborer were statistically associated with HR HPV (χ2 = 13.880 and P = .0016). No statistical association was found between HR HPV positivity and all other collected variables.Table 44.1,11Self-sampling devices are not commercially available in Ethiopia and not used for routine sample collection system on cervical cancer screening program. Thus, this study may be considered as first in kind to used SS collection device in Ethiopia. The acceptability of a SS device were very high and women felt SS device was easy to use, insert and collect, and user-friendly. Women were willing to perform the SS because of its private nature. Ghanaian women reported that 76.3% self-collected (SC) was very easy technique and easy to obtain; 57.7% preferred SS over CT sample and felt SC would increase their likelihood to access cervical cancer screening which was comparable percentage of women who felt same in our study too. Furthermore, a number of studies report that HPV SS was found to be highly acceptable and feasible among hard-to-reach women.Our study was further supported by data from Bolivia where SS was generally preferred over CT for a screening program based on HPV detection. Other studies from American-Indian and Hopi women have also supported our findings wherein samplings HPV testing was feasible and acceptable that may contribute to an increase of uptake.A study in El Salvador reported that SS revealed acceptability of 68%, although lower than reported in our study.Most women showed a willingness to pay for SS services and believed their results which could be seen as a driving force for screening among hard-to-reach women.Almost the same percentage of women between our study and Japanese were reported; they would use SS again and found instructions easy to follow and reported no issues with the usability of the SS device. However, women in our study reported that they had confidence in the results of self-taken sampling unlike women who lacked confidence in the test. Furthermore, in German, SS considered being easy by 89.0% as well as user-friendly by 96.0% of the women. Therefore, Ethiopian women might use NASS service as an alternative option for fighting cervical cancer prevention.Similar studies supported our findings from Latinas and Haitian populations where women agreed HPV SS was faster, more private, easy to use, and would prefer to use it again.4.2 Two studies elsewhere in Ethiopia reported that the HR HPV prevalence was 17.3% and 15.8%.,21 Thus, our study revealed HR HPV prevalence was consistent with sub-Saharan Africa report where ours is slightly higher. The overall HPV prevalence of SS and CT samples were 10.8% and 13.2% respectively. The authors could not find similar report on the prevalence of HR HPV among SS and doctor sampling which were 14.1% and 12.1%, respectively, in the general population of Ethiopia.The authors reported that an overall HPV prevalence was 22.7% and a prevalence of HR HPV and LR HPV were 18.2% and 6.1%, respectively. HPV prevalence in Africa varied within a range of 12% to 46%. The prevalence of HR HPV in Dakar was 17.4% as compared to ours that is 18.2%, for which geographical areas and population difference could be a reason. The HR HPV prevalence in Cameroon was 18.5% that was comparable to our findings.In Rwanda, the HR HPV prevalence was 19.0% which was slightly higher than our result. However, a study done by Traore et al in Burkina Faso revealed that HR HPV prevalence was 38.3%, which was twice of our result. Therefore, HPV prevalence was varied based on geographical areas and population segment as indicated in entire previous studies.A study from Northern Africa, a Muslim community, HPV infection was 6.3% (4.0% of them were HR types), with no significant variation by age. the estimated prevalence of HPV in Sub-Saharan Africa and global prevalence was 24.4% and 11.7%, respectively, which was almost comparable to our study. Further studies from 11 countries  without cytological abnormalities were included and age-standardized HPV prevalence varied nearly 20 times between populations, from 1.4% in Spain to 25.6% in Nigeria where 22.5% HPV prevalence was presented in our study.In a study done by Bruni et al,4.3 Thus, even within the same country, it possible to observe difference among segments of population.From our study, the most prevalent HPV type was HPV51 and followed by HPV31, 58 and 68 (HR types) and HPV6 and 67 (LR types). Women were coinfected with at least 2 HPV types and the higher were coinfected with 5 HPV subtypes: HPV6, 16, 51, 67, and 68. A total of 12 HPV types were identified in this study of 19 HPV types tested. HPV16 was the most frequent genotype identified in samples from previous Ethiopia studies and HPV52, 58, and 18 were the second, third, and fourth common genotypes identified respectively, whereas in our study, HPV51 and 31 were the common genotypes identified. In Mozambique, the most frequent were HPV51, HPV35, HPV18, HPV31, and HPV52. Also multiple infection were detected in normal cytology of types HPV51 and HPVs 16/18. And HPVs 51 and 35 were the two most common types.In a study from South Africa, HPV16, 35, and 58 were the most common high-risk HPV types with no major differences in the type distribution by HIV status.,31 In a study from Burkina Faso, HPV52, 33, and 59 were most identified genotypes, whereas HPV51, 31, and 58 were the most prevalent in our study.HPV-positive women in Europe were significantly more likely to be infected with HPV16 than those in sub-Saharan Africa. Heterogeneity between areas of Asia was significant were that supported by previous Ethiopian studies. In a study from Nigeria, the prevalence of HPV35 and HPV16 was equally frequent. HPV16 was the most common type among the general population of Guinea (7.3%). Thus, these indicated that different genotypes were identified in different geographical areas and population.HPV52 (3.2%) was the most prevalent HPV type, followed by HPV31 (3.0%) and HPV16, 45, and 53 .4.4In this study, the overall agreement between SS HPV and VIA results was higher than CT results. The sensitivity between HPV and VIA test results was relatively higher on SS over CT samples. There was an almost equal specificity value found between SS and CT samples. A study from Cameroon indicated that the sensitivity and specificity of VIA/VILI among HPV-positive women were 80.0% and 44.0%, respectively, which were less compared to our study.A combination of HPV-based and VIA screen-and-treat approach may be feasible in a low-resource context and may contribute to improving the effectiveness of CC prevention programs. The combination of HPV-testing and VIA/VILI for CC screening might reduce over treatment.4.5 A study from Sub-Saharan Africa revealed that the overall HPV positivity agreement between self- and doctor was κ value of 0.52, respectively which had similar agreement with our study.The overall agreement of HPV test results between NASS and CT samples was moderate, with a kappa value of 0.58. A study from Bolivia showed good agreement between self- and physician-collected samplesin where HR HPV detection (κ = 0.71) was higher as compared to our study.5There was a moderate agreement between NASS and CT sample for HPV detection. NASS might be used alternatively as sample collection strategy for HPV testing in cervical cancer screening program in Ethiopia; however, the quality sample should need improvement. NASS HPV testing is a valuable tool for the follow-up of women in low-resource settings. SC sampling may be used as alternative strategy to increase cervical cancer screening coverage in Ethiopia.Although our study revealed that HPV51, 31, 16, 45, 52, and 58 were mostly identified, a large-scale study is required to study circulated genotypes in Ethiopia and select an appropriate HPV vaccine accordingly. Genotyping information could be important to guide vaccine policy. Our study was the first to report on HPV detection on SS using ThinPrep PreservCyt solution and may be used as a platform for similar studies in the future.6Although this research was carefully prepared, we concluded that the sample size was small and not able to generalize.The authors acknowledged SAMH‘s staff for the support of sample collection activities and Hologic Inc Company, USA for donating ThinPrep PreservCyt solution, cytobrush, spatula through IPRH.Conceptualization: Eshetu Lemma Haile.Data curation: Eshetu Lemma Haile.Formal analysis: Eshetu Lemma Haile, Benoy Ina.Investigation: Ransom Sharon.Methodology: Eshetu Lemma Haile, Benoy Ina, Gurja Belay.Resources: Van geertruyden Jean Pierre, Ransom Sharon.Supervision: Eshetu Lemma Haile, Van geertruyden Jean Pierre, Bogers Johannes Paul.Writing – original draft: Eshetu Lemma Haile.Writing – review & editing: Eshetu Lemma Haile, Simoens Cindy, Benoy Ina, Van geertruyden Jean Pierre, Lebelo Ramokone Lisbeth, Bogers Johannes Paul.


Human papillomaviruses are the most important causative agents for invasive cervical cancer development. HPV type-specific vaccination and HPV cervical cancer screening methods are being widely recommended to control the disease but the epidemiology of the circulating HPV types may vary locally. The circulating HPV-strains have never been assessed in Burundi. This study determined the prevalence and genotype-specific distribution of HPV in four different strata in Burundi: HIV-infected or non-infected and women living in rural or urban areas. Implications for HPV diagnosis and vaccine implementation was discussed.Four cross-sectional surveys were conducted in Burundi  among HIV-infected and uninfected women living in rural and urban areas. Liquid-Based Cytology (LBC) and HPV genotyping were performed and risk factors for HPV infection and cervical pre-cancer lesions were determined using logistic regression model.HPV prevalence was very high in urban area with significant differences between HIV-positive and negative women (p<0.0001). In fact, 45.7% of HIV-positive participants were infected with any HPV type and all were infected with at least one HR/pHR-HPV type. Among the HIV-negative participants, 13.4% were HPV-infected, of whom, only four women (2.7%) were infected with HR/pHR-HPV types. In rural area, HPV infection did not significantly differ between HIV-positive and negative women . In urban area, multiple infections with HR/pHR-HPV types were detected in 13.9% and 2.7% among HIV-positive and negative women respectively (p<0.0001), whereas in rural area, multiple infections with HR/pHR-HPV types were detected in 4.7% and 3.3% of HIV-positive and negative women respectively (p = 0.56). The most prevalent HR/pHR-HPV types in HIV-positive women living in urban area were HPV 52, 51, 56, 18 and 16 types. In HIV-negative women living in urban area, the most prevalent HR/pHR-HPV types were HPV 66, 67, 18, 45 and 39 types. In HIV-positive women living in rural area, the most prevalent HR/pHR-HPV types were HPV 66, 16, 18 and 33 types. In HIV-negative women living in rural area, the most prevalent HR/pHR-HPV types were HPV 16, 66, 18, 35 and 45 types. Independent risk factors associated with cervical lesions were HPV and HIV infections.There is a high burden of HR and pHR-HPV infections, in particular among HIV-infected women living in urban area. The study points out the need to introduce a comprehensive cervical cancer control programme adapted to the context. This study shows that the nonavalent vaccine covers most of the HR/pHR-HPV infections in rural and urban areas among HIV-infected and uninfected women. Since about three decades, human papillomaviruses (HPVs) have been firmly proven to be the most important etiologic agents for the development of invasive cervical cancer (ICC) –3. WorldHPVs are small double-stranded DNA viruses, with a large epithelial tropism. Basal epithelial cells are infected with HPVs, causing benign and malignant lesions of the skin and the ano-genital mucosae and the upper aero-digestive tract . StudiesRisk factors for HPV infection have been documented and include infection with other STIs (including HIV), high number of lifetime sexual partners, early sexual debut and host susceptibility , 10.Both HIV and HPV are STIs and infection with either one of the two viruses may facilitate transmission of the other , 12. FurCurrently, there are three prophylactic HPV vaccines licensed: (1) Cervarix ; a bivalent vaccine targeting HPV16/18; (2) Gardasil , a quadrivalent vaccine targeting HPV6/11/16/18 and (3) Gardasil-9 , a nonavalent vaccine targeting HPV 6/11/16/18/31/33/45/52/58 .In Burundi, ICC represents the most common female cancer, accounting for approximately 39% of all female cancers . It is rSince end of 2016, a demonstration project on HPV vaccination started and was limited to two districts where girls aged 9–13 years old are being vaccinated with Cervarix. A conclusive evaluation of this project could lead to its large scale up at national level in a second phase . HoweverTo date, information about the epidemiology of the circulating HPV strains in Burundi has not yet been ever documented. In the current context of type-specific HPV vaccination and of HPV-based cervical cancer screening, information about the prevalence and the circulating HPV types and their relative contribution to ICC is of great importance to assist in planning for vaccine, cervical cancer screening implementation as well as to monitor the potential impact on circulating HPV types after vaccination.Hence, this study aimed to document the prevalence and genotype-specific distribution of cervical HPV types in both HIV-infected and uninfected Burundian women living in urban (Bujumbura) or in rural settings (Kirundo).Four cross-sectional surveys were conducted: two were conducted from May to July 2013 in Kirundo, a rural health district in the northern part of Burundi. Another two were conducted from March to May 2016 in Bujumbura, the capital city of Burundi.In the rural area, participants were women attending Kirundo District Hospital and ANSS -Kirundo antenna. The province of Kirundo is divided into 4 health district zones but has only 2 district hospitals (Kirundo and Mukenke). Kirundo district hospital is located at the provincial town of Kirundo and is the biggest hospital in the province. Patients are referred from health centres in Kirundo district and the neighbouring Busoni and Vumbi health districts. Patients can also come directly to the hospital to consult a General Practitioner (GP). ANSS-Kirundo is a local NGO, champion in HIV-care in Kirundo and has the longest active list of HIV-patients coming from the 4 health districts and few from the neighbouring Ngozi province. It is located near Kirundo district hospital.In the urban area, participants were women attending an HIV clinic, located in the University Teaching Hospital of Bujumbura, which follows up around 3500 HIV-positive patients. Participants were also women attending a reputed family planning centre, ABUBEF , in Bujumbura with clients from all neighbourhoods and all social classes.The study was approved by the Burundian National Ethics Committee. A written informed consent to participate in the survey  was obtained and signed by all participants before being enrolled in the study. Girls aged below 18 years were asked to bring a signed parent’ consent before being enrolled. Information collected was kept confidential by the use of codes.General information about the study objectives were given to all participants every morning in the waiting room. During the consultation in the outpatient department, a GP gave clear and detailed information on the study objective and procedures, and proposed the women to participate in the study. Women who signed an informed consent were assisted by the GP to complete a short risk factor questionnaire and blood was collected for HIV testing. Women who declared being HIV-negative or with an unknown HIV status received a pre-test counselling before HIV-testing. A post-test counselling was done before giving back the result. Those who tested HIV-positive were referred to any HIV clinic of their choice for follow up. Women known to be HIV-positive, followed up at the University HIV clinic or ANSS were not retested for HIV.After filling the questionnaire, a gynaecologic examination was conducted. Cervical samples were collected using a cytobrush  and placed into liquid-based cytology (LBC) medium . Vials were stored at room temperature until shipment. Administrative clearances for sample shipment were obtained before the onset of the study. In rural or urban areas, specimen collection took on average 6 weeks and samples were shipped in the following 5 days. Specimens were sent to the Department of Virology at Sefako Makgatho Health Science University (SMU) in Pretoria, South Africa and to the Laboratory of Molecular Pathology, AML, Sonic Healthcare, Antwerp, Belgium for HPV genotyping and cytology reading. Specimens were sent via a specialised transport company, DHL, and transportation lasted around 3 days in both rural and urban settings. The genotyping process was started upon arrival of the specimens and results were available 4 weeks later.Any woman (or girl) aged between 17–65 years, declaring having had vaginal sexual intercourse and agreeing to participate could be included in the study. Exclusion criteria were pregnancy, menstrual period, vaginal discharge and hysterectomy.We estimated HPV prevalence in HIV-negative and positive women to be 21% and 36.3% respectively , 22, 27.We initially planned that all samples would be tested at SMU, South Africa. At the time of sample collection of urban specimens, there was a discontinuation of HPV genotyping and we therefore, decided to send urban samples at AML, Antwerp in Belgium. For the 300 samples from the urban area, thin-layer slides were prepared with the ThinPrep 5000 Processor with Autoloader System  and stained with the Papanicolaou stain on the Tissue-Tek Prisma and Film Automated Slide Stainer and Coverslipper . After scanning of the slides with the ThinPrep Imaging System, cytology reading was performed by image-guided screening, with prior knowledge of HPV infection status. Cytological diagnoses were reported according to the Bethesda 2001 terminology system as 1) normal, 2) atypical squamous cells of undetermined significance; atypical squamous cells cannot exclude high-grade lesion; atypical glandular cells; low-grade squamous intraepithelial lesions (ASCUS/ASC-H/AGC/LSIL), 3) high-grade squamous intraepithelial lesions (HSIL), or 4) invasive cancer. After the LBC preparations are made, 800μL of the remaining cell suspension was used for DNA extraction. Cytology reading was performed only on the 300 urban samples due to budget constraints.For the urban samples, DNA isolation from liquid-based cytology was performed on the Medium Throughput Automation (MTA) (Hologic Inc) with the Genfind DNA extraction kit. Subsequently, the DNA is amplified using a series of real-time qPCR reactions in the LightCycler 480 (Roche) as previously described by Micalessi et al . BrieflyThe 300 specimens from rural area were tested at the Department of Virology, SMU, Pretoria, RSA. HPV DNA was extracted using AmpliLute liquid media Extraction kit following manufacturer’s instructions . HPV DNA was detected following conventional nested HPV. PCR was performed with MY09/MY11 (450 bp) and GP5+/GP6+ (150 bp). First round PCR was carried out in a final reaction of 50 μl containing 1 X buffer II , 1.5 mM MgCl2 , 200 μM of each deoxynucleoside triphosphate (dNTP), 0.2 μM of MY09/MY11 primers and 0.2 μM PCO3 and PCO4 primers (110 bp) , 1 unit of Bio Taq DNA polymerase , and 10 μl of DNA template. The cycling conditions included an initial denaturation step for 2 minutes at 94°C followed by 40 cycles at 94°C for denaturation, 30 seconds at 55°C for annealing, 1 minute at 72°C for elongation and a final elongation for 5 minutes at 72°C. The PCR product detected using gel electrophoresis (2% w/v agarose in Tris-acetate EDTA) and visualised using ethidium bromide. Bands of the appropriate size were identified by comparison with a DNA molecular weight marker and the gel was viewed using a Gel DOC system . For HPV genotyping the PCR products were used and genotyping was performed using Linear array genotyping test that identifies 37 HPV genotypes  as previously described by Coutlee et al. . BrieflyProportions were compared using Pearson’s Chi-square test (or Fisher’s exact test when appropriate). T-test was used to compare means of normally distributed variables . Bivariate analysis was performed to generate odds ratios (ORs) with their 95% confidence interval (CI) to analyse the relationship between each sociodemographic variable and cervical cancer lesions or with HPV infection status. Variables with a bivariate p-value <0.20 were entered in a multivariate logistic regression model to determine adjusted odds ratios (AORs) with their 95% CIs. Analysis was conducted using EpiInfo 3.5.4 software and α-error margin of 5% was considered significant.In rural area, we enrolled 150 HIV-negative and 150 HIV-positive women. The mean age of the participants was 39.9 years (SD = 8.3) and 36.4 years (SD = 7.9) for HIV-positive and HIV-negative women respectively (p = 0.0002). Profession categories and marital status differed between HIV-positive and negative women (p<0.0001 for both). Farmers represented 71.3% and 50% in HIV-positive and negative women respectively. The other HIV-negative women were ‘‘employees/shopkeepers” (47.3%) or ‘‘housewife/Non-working/student/other” (2.7%). 131 HIV-positive women (87.3%) was married compared to 78 (52%) in the HIV-positive group. In the HIV-positive group, 38% were “widowed or divorced” compared to 10% in HIV-negatives. HIV-positive women started earlier sexual intercourse, got married earlier, got pregnant earlier and had a higher median number of sexual partners compared to their HIV-negatives counterparts  .In urban area, a total of 151 HIV-positive and 149 HIV-negative participants were enrolled. The mean age of our participants was 41.1 years (SD = 9.7) and 39.7 years (SD = 8.7) for HIV-positive and negative women respectively (p = 0.19). Profession categories and marital status differed between HIV-positive and negative women (p<0.0001 for both). In the HIV-positive group, majority (42.4%) was in the category ‘‘housewife/Non-working/student/other”. In the HIV-negative group, 77.2% of participants were employees (in public or private) or shopkeepers. In the HIV-negative group, 91.9% were married versus 53% in the HIV-positive group. A high proportion of HIV-positive women were widowed/divorced (36.4%) whereas only 4% who belonged to this category were in the HIV-negative group. HIV-positive women started earlier sexual intercourse, got married earlier, got pregnant earlier and had a higher median number of sexual partners compared to their HIV-negative counterparts  .In rural area, all participants had valid data on HPV genotyping. The HPV prevalence (any) was 30% (45/150) and 31.3% (47/150) among the HIV-positive and negative women respectively (p = 0.80). Multiple HPV infection was 11.3% and 14% among the HIV-positive and negative women respectively (p = 0.49). The HR/pHR-HPV prevalence was 18.7% and 17.3% in HIV-positive and negative respectively (p = 0.76). Multiple HR/pHR-HPV infections was 4.7% versus 3.3% among HIV-positive and negative women respectively (p = 0.56).In the HIV-positive group, infection with any HR-HPV, pHR-HPV and LR-HPV type was 14.7%, 7.3% and 19.3% respectively. In HIV-negative women, infection with any HR-HPV, pHR-HPV and LR-HPV type was 12%, 6.7% and 24.7% respectively . InfectiIn the HIV-negative group, the most frequent HR-HPV types isolated were HPV 16 (4.7%), followed by HPV 18 (2%). The most frequent pHR-HPV types were HPV 66 (4%) and HPV 67 (1.3%).In the HIV-negative group, HR/pHR-HPV prevalence was 8.4% (12/142), 100% (5/5) and 100% (2/2) in women with normal cytology, ASCUS/LSIL and HSIL/ASC-H/ICC respectively.Most prevalent HR/pHR-HPV types, in HIV-positive women, were HPV 52, 18, 51 and 58 among women with normal cytology; HPV 56, 51 and 52 in women with ASCUS/LSIL and HPV16 and 18 in women with HSIL/ASC H/ICC. In the HIV-negative women, the most prevalent HR/pHR-HPV types were HPV 67, 39, 45 and 66 in women with normal cytology; HPV 58 and 66 in women with ASCUS/LSIL and HPV 16 and 18 in women with HSIL/ASC-H/ICC.Overall, among these HPV infections, HR-HPV types are the most predominant. In urban, HR/pHR-HPV infections decreased with increasing age, with a second peak in the age group ≥46 years, both in HIV-positive and negative women. In rural, the decrease in HR/pHR-HPV prevalence was slow compared to urban across all age groups.Cytology results were available for all urban women except one HIV-positive woman with an undetermined cytological result. Cytological abnormalities were more frequent in HIV-positive women compared to HIV-negative women (p = 0.0005).In the HIV-positive group, among 150 women with a valid result on LBC, 23.3% (35/150) had an abnormal cytological result, including 8.7% (13/150) with ASCUS, 10.6% (16/150) with LSIL, 1.3% (2 patients) with ASC-H, 2% with HSIL and 1 patient with ICC. In the HIV-negative group, among 149 women with valid results on LBC, 4.7% (7/149) had abnormal cytological results including 2 patients (1.3%) with ASCUS, 3 patients (2%) with LSIL and 2 patients (1.3%) with HSIL.In the bivariate analysis, abnormal cytology was significantly associated with HR/pHR-HPV infection , age (older women i.e. age groups 36–45 years and ≥46 years being protected compared to younger women aged 17–25 years), number of lifetime sexual partners , HIV infection status  and profession  . After hIn the HIV-positive women living in rural area, infection with HPV 16 or 18 represented 32.1% of all HR/pHR-HPV infections. In the HIV-negative women living in rural, infection with HPV 16 or 18 represented 30.8% of all HR/pHR-HPV infections. Infections with HR-HPV included in the Nonavalent vaccine (i.e. HPV 16/18/31/33/45/52/58) represented 60.7% and 53.9% of the circulating HR/pHR-HPV infections among HIV-positive and negative women living in rural area respectively. In urban area, HPV 16/18 infections represented 8.7% and 15.8% of all HR/pHR-HPV infections in HIV-positive and negative women respectively. Infections with the HR-HPV types included in the Nonavalent vaccine represented 31.9% and 42.1% of all HR/pHR-HPV infections in HIV-positive and negative urban women respectively .This study describes, to our knowledge, for the first time in Burundi the HPV prevalence and type-distribution according to HIV status in rural and urban areas. This may put in perspective diagnostic screening and vaccination programme using type-specific tools. Moreover, it presents HPV prevalence and type-distribution according to cytological findings in the urban area.These data are of young adult women, never screened for cervical lesions and before any national screening and/or vaccination programme is rolled out. According to Bruni L. et al. based on an extensive meta-analysis , the ageOur results show a high overall HPV prevalence ranging from of 13.4% to 45.7% in urban HIV-negative and positive women respectively (p<0.0001). Our data show significant differences in HR/pHR-HPV prevalence between HIV-positive and negative women in urban . This pattern has been described in other studies , 35. StuThe overall HPV prevalence was respectively 30% and 31.3% in HIV-positive and negative women living in rural area with lack of significant difference by HIV status (p = 0.80). This high HPV prevalence is in line with other studies conducted in East Africa . This waMoreover, a selection bias cannot be ruled out since the participants were not randomly selected; all women were enrolled from the health facilities and thus may not represent the rural population. Therefore, more information in robust studies is needed to fully understand the pattern in this area. In the literature, lack of significant differences in HPV prevalence among HIV-positive and negative women has also been reported in a study done in a rural community in Zimbabwe .Stronger independent predictors for abnormal cytology results were, as expected, a positive HR/pHR-HPV infection and HIV infection . Indeed,Stratified by cytological results, our findings show a HPV prevalence of 28.7% and 9.2% in HIV-positive and negative women respectively with normal cytology, which is in line with data from East Africa showing HPV prevalence in women with normal cytology ranging from 3.2% to 41.4% , 40, 41.HR/pHR-HPV prevalence in HIV-positive women with ASCUS/LSIL was 100% in our results and is higher than the prevalence found in a meta-analysis by Clifford M. et al ; which wOur findings on the most prevalent HPV strains among HIV-positive urban women with normal cytology, are in agreement with results found in two robust meta-analyses in Africa despite some differences in ranking , 41. ConIn the meta-analysis by Guan et al. , AfricanOur data from rural area cannot be disaggregated by cytological results but results are roughly similar to those found in Rwanda and Kenya , 37. StrIn rural area, that the Cervarix or Gardasil vaccines would target 32.1% and 30.8% of the HR/pHR-HPV infections among HIV-positive and negative women respectively. The nonavalent vaccine, Gardasil-9, would target 60.7% and 53.9% of the circulating HR/pHR-HPV infections among HIV-positive and negative women respectively.In urban women, Gardasil or Cervarix vaccine would target 8.7% and 15.8% of all HR/pHR-HPV infections in HIV-positive and negative women respectively and Gardasil-9 would prevent 31.9% and 42.1% of all HR/pHR-HPV infections in HIV-positive and negative women respectively. Concerning the HPV genotype distribution, almost 7 on 8 and 100% of all HSIL/ASC-H/ICC among HIV-positive and negative women respectively were targeted by the prophylactic Gardasil-9 HPV vaccine, constituting the basis of the rational of introducing primary prevention against HPV-related cancer in young women by prophylactic vaccination. We acknowledge however, a reported cross-protection of the HPV-16/18 vaccine against non-vaccine oncogenic HPV types, namely HPV-31, HPV33, HPV45, and HPV51 . This isOur study participants were recruited at health facilities on a voluntary basis and may not be representative of the rural or urban Burundian women population. The participants were chosen in 4 similar strata according to HIV status and urban/rural areas. This was a deliberate choice as we wanted to avoid that urban women and HIV-infected women living in either rural or urban setting would be underrepresented. In fact, 91% of Burundian women live in rural areas where HIV prevalence is estimated at 0.8% in women aged 15–49 years old, whereas in urban area, HIV prevalence in women aged 15–49 years old is estimated at 3.5% . Given tAnother limitation is related to the use of two different HPV tests for rural and urban data which might have different sensitivity. We believe that this has not much affected our results since the 2 test platforms used in rural and urban samples both fulfil the VALGENT criteria , 50. TheAlso, cytology results were only available for urban population with relatively few numbers across different cytological strata. Cytology could not be done on rural samples due to budget constraints. Complementary studies with more reliable data are being prepared to overcome these limitations.In Burundi there is a high burden of HPV infection, in particular HR/pHR-HPV infections which mimics other similar settings. We observed that HIV-infected women in Bujumbura are a specific risk group to be targeted in contrast to the rural area where all women seem to have similar distributions of HPV circulating strains. This study strengthens the urgent need to introduce a comprehensive cervical cancer control programme adapted to the context. This most probably may include a HPV-vaccination programme, although it cannot be the sole component due to the actual burden, the coverage, efficacy and the lag time of the impact. Complementary studies in invasive cervical cancer cases are needed to further elucidate the most appropriate control tools.S1 FigAge-specific prevalence of HPV (A) and of abnormal cytological results (B) stratified by HIV status in rural and urban women, Burundi.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.


Proliferative verrucous leukoplakia (PVL) is an oral potentially malignant disorder, characterized by multifocal expression, progressive clinical evolution, and a high rate of malignant transformation. Evidence-based information regarding optimal PVL management is lacking, due to the paucity of data. The present report describes a case of PVL associated with HPV-16 infection and epithelial dysplasia treated by diode laser surgery, and the outcome of disease clinical remission over a 2-year follow-up period.A 61-year-old Caucasian male with oral verrucous hyperkeratosis presented for diagnosis. The lesions were localized on the maxillary gingiva and palatal alveolar ridge. Multiple biopsy specimens have been taken by mapping the keratotic lesion area. Microscopic examination was compatible with a diagnosis of PVL with focal mild dysplasia, localized in the right maxillary gingiva. Polymerase chain reaction (PCR) was done for human papillomavirus (HPV) detection which revealed presence of HPV DNA, and the genotype revealed HPV 16 in the sample. The PVL in the right gingival area was treated on an outpatient basis by excision with a diode laser. This approach resulted in good clinical response and decreased morbidity over a 2-year follow-up period.This case illustrates the benefit of a conservative approach by diode laser treatment than wide surgical excision for management of the PVL lesions associated with mild dysplasia and HPV-16 infection. Tobacco use is not a common factor associated with PVL, being present in about a third of the cases , 5. The As regard the clinical management, there is lack of high-quality data to support the routine surgical excision of the non-invasive phase of PVL (Hansen’s grades 3–5) in an attempt to prevent future malignant transformation .The role of the human papillomavirus (HPV) infection in the pathogenesis of the PVL is never been defined, and current evidences are contradictory , 9–11.However, it should be considered that the infection of PVL lesions with high-risk HPV subtypes—such as HPV 16 and 18—could increase the transforming potential as a result of the expression of E6 and E7 viral oncoproteins, which functionally inactivate two human tumor suppressor proteins, p53 and pRb, respectively , 13. BelIn January 2015, a 61-year-old Caucasian male was referred to our Oral Pathology and Medicine Unit by his own dentist for white oral lesions, since it was initially thought to be a fungal infection and he had received treatment based on topical antifungals, without a significant clinical improvement.Past medical history included appendectomy, cholecystectomy, and tonsillectomy. He had never smoked or used drugs recreationally and did not drink alcohol. At the time of the first visit, he was not taking any medication. No symptoms were referred by the patient, but he had noted a progressive increase of the lesion size in recent months. Clinical examination showed a thickened hyperkeratosis, which was confluent in widely exophytic papillary plaques. These lesions appeared homogeneously whitish-gray with verrucous surface and localized on the maxillary gingiva and palatal and alveolar ridge mucosa Fig. . IncisioThe histopathological analysis revealed exophytic, hyperkeratotic lesions with prominent verruciform or papillary surface and acanthosis forming droplet-shaped epithelial projections into the lamina propria Fig. . A samplCurrently, no specific treatment modality has proven to be effective in the management of the PVL, either to slow down its progression toward malignant transformation and limit the high frequency of recurrence , 14. TheThe conventional treatment of PVL is surgical excision, although complete removal of the lesions may be challenging because of the multifocal manifestation of the oral lesions . This apAlthough recurrence and progression are very common in the PVL lesions, whether the microscopic evaluation of the tissue sample confirms the HPV infection associated with mild dysplasia, the laser excision can be considered as surgical option. Biopsy mapping and complete laser excision of PVL tissue associated with epithelial dysplasia appear to be reliable, reproducible procedures associated with low complications and morbidity rates in terms of preventing malignant transformation. Strict follow-up is recommended for patients with PVL associated with HPV-16 infection and low grade dysplasia to detect oral cancer at an early stage.Nonsurgical therapeutic approaches for PVL have been considered such as external beam radiation therapy, cryotherapy, topical vitamin, and topical chemotherapy, but with little success. Cryosurgery, photodynamic therapy, and antiviral methisoprinol therapy have also been suggested with significant benefit. In the future, anti-HPV, anti-TGF, and pro-apoptotic management strategies may be assessed , 4, 14.


The infection with human papillomavirus (HPV) is the major cause of genital disease. This study assessed the prevalence and genotype of HPV among outpatient women and healthy women in Jiangsu, East China.A total of 65,613 women aged 16–85 years were recruited from Nanjing Kingmed Diagnostics, including 45,736 outpatients and 19,877 healthy women. The cervico-vaginal cells were collected and then HPV types were detected using the Tellgenplex™ HPV DNA Test.The overall HPV prevalence was 17.7% for outpatients and 10.6% for healthy women. 13.7% outpatients were infected with a single HPV type and 4.0% were infected with multiple HPV types. Regarding healthy women, 8.5 and 2.1% were infected with single and multiple HPV types, respectively. The two most commonly detected HPV types were HPV 16 and 58 regardless of single- or multiple-type infection or source of the participants. HPV16 + 58 was the most commonly identified multiple genotype in outpatients, while HPV16 + 52 was frequently detected in healthy women. Highest prevalence rate was found in outpatients aged < 20 years and ≥ 60 years.This study revealed the prevalence characteristics of HPV in both outpatient women and healthy women in Jiangsu province.The online version of this article (10.1186/s12889-019-6487-9) contains supplementary material, which is available to authorized users. The infection with human papillomavirus (HPV) is strongly associated with genital disease. Type-specific persistent infection with high-risk (HR) HPV is the major reason of cervical squamous intraepithelial lesions as well as cervical cancer –3. AccorThere are many reports on the prevalence of HPV in different populations. The prevalence of HPV infection varies greatly by country, region within country, and population , 9. TherThe women who received HPV screening at Nanjing KingMed Diagnostics between January of 2016 and May of 2017 were recruited. We excluded the women who did not have valid testing results  or who were not the residents of Jiangsu Province. Finally, we enrolled 65,613 women aged 16–85 years, including 45,736 outpatients and 19,877 healthy women who received routine examination. All the data were retrospectively collected from Laboratory Information Management System of Nanjing KingMed Diagnostics.All the participants were asked to do not douche or use tampons or vaginal creams, deodorants, or medications for 2 days before the test. The participants were not in menstruation period and refrained from sex for 48 h before the test. The cervico-vaginal cells were collected from the transformation zone of the uterine cervix by a gynecologist or a trained physician using a standard cytobrush, then suspended in a standard transport medium (STM) for the Tellgenplex™ HPV DNA Test  and stored at 4 °C until analyzed (24 h–72 h) .HPV types were detected using the Tellgenplex™ HPV DNA Test . It could identify the 27 most common HPV genotypes including 14 HR-HPV genotypes  and 13 LR-HPV/undetermined risk (UR-) HPV genotypes . Briefly, DNA was extracted, then 100 ng HPV DNA was used as template and added into 20 μl reaction mixture that includes 10 μl PCR premixed solution, 0.8 μl Taq DNA Polymerase and 5 μl primer mixture.x2) was used to test for departure from homogeneity in prevalence across all the 6 age groups. The prevalence was estimated separately for outpatients and healthy women. We used data of healthy women to estimate percentage of women who will be protected by the vaccines targeting different HPV types. p < 0.05 was considered statistically significant.The SPSS version 19.0  was used to perform all statistical analyses. HPV prevalence rate was estimated by a proportion and summarized as a percentage, with an exact binomial 95% confidence interval (95% CI). Prevalence estimates are presented overall and by age . The chi-squared (P < 0.001). The HR-HPV prevalence rate  was higher than the LR/UR-HPV rate   were infected with at least one HPV type, including 6253 participants  infected with a single HPV type and 1828 participants  infected with multiple HPV types (P < 0.001). HR-HPV and LR/UR-HPV infection was detected in 2002  and 611  healthy women, respectively  were infected with single HPV subtype, whereas 2.1% (95% CI: 1.9–2.3%) were infected with multiple HPV subtypes . In the healthy women, people aged 50–59 years had the highest prevalence rate  and the 30–39 age group had the lowest prevalence rate  (P < 0.001) , followed by the over 60 age group , while the 30–39 age group had the lowest prevalence rate  in the outpatient women  . FortunaMost of the previous researches on HPV epidemiology were only based on outpatient samples. Since the outpatients usually come to the hospital for some medical problems, the HPV prevalence is different from that of healthy women who participate in normal medical examinations. In our study, both outpatients and healthy participants were recruited from Jiangsu, China. In the 45,736 outpatient women, the prevalence of HPV was 17.7% and the HR-HPV prevalence was 15.7%. Compared with similar studies in China, the prevalence in our study was higher than that in Beijing (9.9%)  and YunnThe analysis of HPV prevalence showed that the most commonly identified HR-HPV genotypes were HPV 16, 58, 52, 39 and 18 among the outpatient women. It was different from the healthy women, where HPV 58, 16, 52, 39 and 56 were the five most commonly detected HR-HPV genotypes. The present study showed that HPV16, 58 and 52 were the top three detected HR-HPV genotypes in the two populations, which was similar to the other studies conducted in China, despite the prevalence varied widely , 16, 17.In the outpatient women with multiple HPV infection, the most commonly identified combinations of HPV types were HPV16 + 58, HPV52 + 58 and HPV39 + 52. While the top three combinations of types were HPV16 + 52, HPV52 + 58 and HPV39 + 58 for the healthy women. It was easy to note that these top three multiple-type infections were the different combinations of HPV 16, 39, 52 and 58. We also found that the vaccines targeting HPV 16/39/52/58 will protect more women in East China, when four HPV types were included in vaccines Additional file 2:Table S2. The all multiple HPV infection mode in participants. (XLSX 29 kb)


To estimate the prevalence, incidence and persistence of anal HPV infection and squamous intra-epithelial lesions (SILs) among men living with HIV (MLHIV), and determine their risk factors.We enrolled MLHIV ≥18 years, who attended 6-monthly visits for 18 months. Socio-behavioural data were collected by questionnaire. Clinicians collected blood sample , anal swabs (HPV DNA testing) and anal smears (Bethesda classification) at each visit. HPV DNA testing and classification of smears were done at enrolment and last follow-up visit (two time points). Factors associated with persistent anal HPV infection and SILs were evaluated with generalized estimating equations logistic regression and standard logistic regression respectively.Mean age of 304 participants was 38  years; 25% reported >1 sexual partner in the past 3 months. Only 5% reported ever having sex with other men. Most (65%) participants were taking antiretroviral treatment (ART), with a median CD4+ count of 445 cells/μL . Prevalence of any-HPV infection at enrolment was 39% (88/227). In total, 226 men had anal HPV DNA results at both enrolment and final visits. Persistence of any-anal HPV infection among 80 men who had infection at enrolment was 26% (21/80). Any persistent anal HPV infection was more frequent among MLHIV with low CD4+ count . Prevalence of anal SILs at enrolment was 49% (118/242) while incidence of SILs among MLHIV who had no anal dysplasia at enrolment was 27% (34/124). Of the 118 men who had anal dysplasia at enrolment, 15% had regressed and 38% persisted by month 18. Persistent anal HPV infection was associated with persistent SILs . ART status or duration at enrolment were not associated with persistent anal HPV infection or persistent SILs during follow-up.In spite of a high prevalence of anal HPV, HIV-positive heterosexual men have a low burden of anal HPV related disease. HPV vaccine and effective ART with immunological reconstitution could reduce this burden of infection. Anogenital human papillomavirus (HPV) is the most common sexually transmitted infection worldwide and responsible for a large burden of disease. High-risMen living with HIV (MLHIV) have a high prevalence of HPV infection and are more likely to be infected with multiple high-risk types compared to HIV-negative men. HIV co-iThe natural history of cervical cancer and progression from HPV infection to development of invasive cervical disease has been well documented. The slowTo help inform the selection of future HPV prevention interventions among MLHIV in South Africa, we established a cohort study of HIV-positive men. We estimated the prevalence, persistence and incidence of anal HPV infections and SILs. We further evaluated the effect of factors such as ART status, duration on ART, CD4+ cell count and HIV plasma viral load (PVL) at baseline on these outcomes.Full details of the cohort study and prevalence of HPV and SIL at enrolment have been published elsewhere. Briefly,Anogenital examination, including digital anorectal examination, was performed by a trained clinician at each visit. Two intra-anal specimens were collected by blindly (i.e. without anoscopy) inserting Dacron swabs three centimetres into the anal canal and removing them whilst rotating and applying pressure on the walls of the canal. One swab was used for HPV DNA testing, whilst the other was used to prepare a conventional anal smear for cytological analysis. Swabs we™ BD Biosciences, United States) and HIV-1 plasma viral load . The standard of care at the time was men could only start ART at CD4+ count of 350 cells/μL or less. [Venous blood was taken at each visit to test for CD4+ cell count  visit. Testing was only done at two time points due to funding limitations. The MagNA Pure LC DNA Isolation Kit I  was used to extract HPV DNA from the swabs. HPV genotyping was determined by the Roche Linear Array assay . HPV 16 and HPV 18 VL in copies per million human cells were quantified at enrolment on samples that were positive for these types using a quantitative duplex real-time PCR method.  This metConventional smears were performed as liquid based cytology was not available at the time that the study was undertaken. Smears were graded as either unsatisfactory for analysis, negative for intraepithelial lesion or malignancy (NILM), atypical squamous cells of undetermined significance (ASCUS), atypical squamous cells-high grade lesions cannot be ruled out (ASC-H), and low- (LSIL) or high-grade squamous intraepithelial lesions (HSIL) in accordance with the Bethesda System. Smears wpersistence, i.e. detection of the same HPV DNA type that was detected at enrolment, and (ii) clearance, i.e. absence of an HPV DNA type that was detected at enrolment, and (iii) incident infection, i.e. detection of any HPV DNA type that was not detected at enrolment. Groups of HPV genotypes were categorised as follows: (i) any HPV infection, i.e. detection of at least one of the 37 HPV genotypes that can be identified by the Roche LA; (ii) any HR-HPV infection, i.e. detection of at least one of the following HPV genotypes: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 or 68; (iii) any LR-HPV infection, i.e. detection of at least one of the following HPV genotypes: 6, 11, 26, 40, 42, 53, 54, 55, 61, 62, 66, 69, 70, 71, 72, 73, 81, 83, 84, IS39 or CP6108; (iv) any alpha 7 infection, i.e. detection of at least one of the following HR-HPV genotypes: 18, 45; 39 or 59; and any alpha 9 infection, i.e. detection of at least one of the following HR-HPV genotypes: 16, 31, 33, 35, 52 or 58. The HPV 16 or 18 viral loads were log transformed to base 10 to normalise distributions, as done previously.[HPV DNA infection outcomes at final visit were defined as (i) eviously., 21Abnormal anal cytology was defined as a diagnosis of ASCUS or higher. Anal cytology at final visit was defined as (i) regression to a lower grade from a higher grade OR to normal cytology from ASCUS/ASC-H at enrolment, (ii) persistent, i.e. abnormal anal cytology at both enrolment and final visits or transition from ASCUS/ASC-H to LSIL, and (iii) incident, i.e. any abnormality not detected at enrolment. High stable CD4+ count was defined as a CD4+ count >500 cells/μL at all follow-up visits. Sustained HIV virological control was defined HIV-1 PVL <40 copies/mL at all follow-up visits. Controlled HIV disease was defined as being on ART for at least 6 months, with CD4+ count of 350 cells/μL or higher and undetectable HIV-1 PVL (i.e. <40 copies/ml).Descriptive statistics were used to summarize the prevalence of the anal HPV infection and SILs. Chi-square tests were run to explore differences between proportions. Persistent anal HPV infection and anal SILs were categorised as a binary outcome and reported as proportions since data were available at enrolment and final visits only. The incidence rate and 95% confidence interval (95% CI) of anal HPV infection was estimated by the Kaplan-Meier method. Person time was calculated as the time from the date of sample collection at enrolment to the date of sample collection at the final follow-up visit. Multivariate models were constructed to identify socio-demographic, sexual behaviour and HIV-related factors associated with these outcomes.Associations between persistent anal HPV infection and exposure variables were evaluated with generalized estimating equations (GEE) logistic regression with robust standard errors (vce) to account for multiple HPV genotypes and multiple infection states (persistence and clearance) that could occur within each participant. The exchAssociations between anal cytological abnormalities and exposure variables were assessed using logistic regression. Adjusted standard logistic regression models were constructed to explore independent associations between these cytological abnormalities and risk factors using the same model-building approach as described above. Analysis was performed using Stata version 13 .The study was approved by the Wits Human Research Ethics Committee (Reference numbers: M111191 and M160859). Written, informed consent was obtained from all study participants after full explanation of the study objectives and testing procedures.A full description of the study population has been published elsewhere. Briefly,The data on prevalence of anal HPV infection and cytology has been published elsewhere . In brieA total of 226 men  had valid anal HPV DNA results at both enrolment and final visits. The median (IQR) follow-up time was 1.49 (1.34–1.50) years. Although 74% (59/80) of the men who had anal HPV infections at enrolment cleared them during follow-up, a substantial proportion, 26% (21/80) had persistent infection. The proportion of MLHIV with persistent infection when this limited to heterosexual men was 23% . Levels of persistence were 17% with alpha-7 types, 13% with alpha-9 types and 17% with HPV 16 . There wAmong 250 anal smears that were collected at enrolment, 8 (3.2%) were of poor quality and could not be analysed. Of the remaining 242 smears, 124 (50.8%) were graded as NILM, 48 (19.7%) were ASCUS and 70 (28.99%) were LSIL. Of the 169 smears collected at the final visit, 12 (7.1%) were excluded due to poor quality. Among the remaining 157 smears, 62 (39.5%) were graded NILM, 33 (21.0%) were ASCUS and 62 (39.5%) were LSIL. There were no cases of HSIL diagnosed at either enrolment or final visits. The proportion of smears with abnormal anal cytology was significantly higher at month 18 compared to the enrolment visit  .Regression of anal dysplasia at final follow-up visit among the 118 men who had abnormal cytology at enrolment was 15% (n = 18). Most of the cases changed from LSIL to NILM (n = 10), whilst eight regressed from ASCUS to NILM. Thirty eight percent (n = 45) had persistent anal dysplasia at final follow-up visit with the majority being LSIL cases (n = 28), whilst eight cases were ASCUS and 9 cases transited from ASCUS to LSIL . At finaThere was some evidence of inverse relationship between persistent anal cytological abnormalities and enrolment CD4+ cell count. MLHIV who had a shorter duration of ART were more likely to have persistent anal cytological abnormalities. This was significant among men who had been on ART for 12 months or less  when compared those who had been on ART for more than 12 months at enrolment.At enrolment, HR-HPV  or any alpha-9 type infections  were strongly associated with persistent anal cytological abnormalities. Similarly, any persistent anal HPV  and any persistent HR-HPV  infections were associated with persistent anal cytological abnormalities.ART status, HIV-1 PVL and HPV VL at enrolment were not independently associated with persistent anal cytological abnormalities .In this study of predominantly heterosexual men, we found that the prevalence of anal HPV infection was 39% and 26% of men with at least one anal HPV infection at enrolment had persistence 18 months later. The incidence of anal HPV was low at 0.79 per 100 person-years. By comparison, a study in China, also among heterosexual men, reported an incidence of 4.1 per 100 person-years, but the confidence interval of that study overlapped with ours.  Another A third of participants in this cohort had persistent anal dysplasia, and a further quarter (27%) of MLHIV who had normal cytology at enrolment also developed dysplasia during follow-up. Despite this, there were no high-grade SIL cases at baseline or follow-up. The reason for this is unclear but could be related to the relatively short (18 months) follow-up period as progression of anal lesions may take a number of years. However,per se that is important but other factors such early ART initiation, virological control and HIV disease status also influence the natural history of anal HPV infection and related disease.[Lower CD4+ count had a strong inverse association with persistent anal HPV infection and persistent anal dysplasia. Quite likely, as in women living with HIV for cervical dysplasia, this is related to the role of cell-mediated immunity in the clearance of HPV infection or cells with pre-neoplastic changes. ART stat disease. This is The strong association between anal HPV infection  and persistent anal dysplasia suggests that HPV vaccination could be used as an effective way of managing the future burden of anal dysplasia among MLHIV in high HIV prevalence settings such as South Africa. HPV vaccines can reduce the burden of HPV-related diseases, however the girls-only vaccination programme in South Africa might need to be extended to include boys if men are to be effectively protected. This is based on emerging evidence which suggest that the impact of a girls-only programme and herd immunity to men may be reduced by the high HIV prevalence in the country. MoreoverOur study had a number of limitations. Conventional anal smears were used, which could have misclassified some cytological outcomes. Anal cytology screening is a relatively insensitive and nonspecific means of diagnosing anal HSIL., 38 We dIn spite of a high prevalence of anal HPV, HIV-positive men have a low burden of anal HPV related disease. Men living with HIV face a significant burden of anal HPV infection and anal lesions. Effective, and possibly early, use of ART with immunological reconstitution and HIV virological control may contribute to the control of the HPV infection. HPV vaccination, if extended to boys, could also reduce this burden among both homosexual and heterosexual men in the future.S1 Table*Values could not be calculated as all participants in that category had HPV VL above the cut-off point.(DOC)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 File(XLS)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(PDF)Click here for additional data file.S4 File(PDF)Click here for additional data file.


Randomised controlled trials showed human papillomavirus (HPV)-based screening leads to a significant reduction in cervical cancer incidence compared with cytology-based screening only.Non-hysterectomised participants ≥30 years underwent co-testing with Papanicolaou (Pap) smear and HR-HPV testing . Women with normal findings had their next screening round after 5 years, and HC2+ and Pap abnormal cases were immediately referred for colposcopy, while cases with discordant findings had repeat testing after 12 months with referral to colposcopy in cases with persistent positive findings.p < 0.0001) and cervical cancer  between the first and subsequent rounds. Approximately 90% (246/274) of CIN3+ cases were diagnosed at first colposcopy.Twenty-six thousand six hundred and twenty-four women were recruited between February 2006 and December 2016. Two hundred and seventy-four CIN3+ cases were diagnosed , including 31 invasive cervical cancers . No CIN3+ was detected in HPV− women with abnormal cytology. We observed a significant decline in the 5-year incidence of CIN3+ (from 0.96% [95% CI 0.85–1.09%] to 0.16% [95% CI 0.10–0.25%]; The decline in disease rates with 5-yearly co-testing seems mainly attributable to HPV testing since no CIN3+ occurred in HPV−/Pap+ women. Findings from controlled clinical trials may differ from observations in the real-world for several reasons related to selection of subjects and participating healthcare professionals, differences between study protocols and everyday clinical pathways and the lack of a control arm for comparison and mitigation of bias. Here we report 11-year follow-up data from a local screening programme based on HPV and cytology co-testing in the Wolfsburg region of Germany. The main focus of this analysis is to determine the incidence of cervical cancer diagnosed in the first screening round compared with subsequent rounds.An important consequence of the higher sensitivity of HPV testing for CIN2+Wolfsburg pilot project for better prevention of cervical cancer with primary HPV screening (WOLPHSCREEN) has been described in detail previously.15 In brief, WOLPHSCREEN was a locally organised HPV screening programme for non-hysterectomised women aged 30–70 years, covered by ‘Deutsche BKK’ and ‘Audi BKK’ health insurance companies. The upper age limit of 70 years, recommended at the start of the WOLPHSCREEN programme, became mandatory from 2012. Every woman coming for screening could freely choose to do either yearly Pap-smear or Pap-smear and HPV-test with a 5-year interval. Although both possible screening modalities were reimbursed by the health insurance companies, women opted almost entirely for co-testing using Pap smear and HPV (Hybrid Capture 2 (HC2)) tests. The included population was, therefore, unselected and non-randomised. Data were collected in an observational fashion, with all patients co-tested with HC2 and cytology (no control arm) and followed according to regular care using a rigorous, individualised, risk-based follow-up.The 15The same testing protocols were used for all screening rounds. Women with positive HC2 test results and abnormal Pap smears were referred immediately for colposcopy; those with positive HC2 tests and normal cytology were retested annually and referred for colposcopy if HPV infection persisted, while those with negative HC2 tests and normal Pap smears returned for their next screening round 5 years later. Opportunistic testing did not play a role in the WOLPHSCREEN protocol.Screening round 1 included all participants recruited between 1 February 2006 and 31 December 2016. Round 1 started with the first primary screening and ended with the second screening round, loss to follow-up or the end of 2017. Screening rounds 2 and 3 covered procedures among eligible women who had completed a planned 5-year screening interval after rounds 1 and 2, respectively. Follow-up data were included up to 31 December 2017. As there was no formal notification that participants had left WOLPHSCREEN or the Wolfsburg area, follow-up data for participants who left the programme because they changed their health insurance provider, reached the study exit age of 70 years, underwent hysterectomy, were diagnosed with other gynaecologic malignancies or left the area were obtained if possible from the tumour registry of Lower Saxony, insurance databases or our internal hospital database to identify cases of invasive cervical cancer occurring outside of WOLPHSCREEN. We also compared all cervical cancer cases registered within the Cancer Centre Wolfsburg 2006–2017 with cases registered by the cancer registry of Lower Saxony for the Wolfsburg region. For the very few cases that were not treated at our institution, we contacted the health insurer, responsible gynaecologist and/or the treating institution to clarify if these cases occurred in former or active WOLPHSCREEN participants. The aim was to identify incident cancer cases occurring up to 6 years after screening to mitigate the risks of underestimation (due to missing diagnosed cancer cases or loss to follow-up) or overestimation . To ensure a follow-up period of at least 1 year for each woman included in a screening round, we did not include screening performed after 1 January 2017.16 which are equivalent to, or worse than, atypical cells of undetermined significance (ASC-US) in the Bethesda system. After February 2011, optional repeat Pap smear after 6 months for women with initial positive result was replaced with p16/Ki-67 dual-stained cytology test , as described previously.18Testing procedures: HC2 tests, done according to the manufacturer’s protocol , detect high-risk HPV subtypes . Abnormal smear test results were classified as Pap IIw or IIp or worse, using the Munich Cytological Classification,Rates of invasive cervical cancer, CIN3+ and CIN2+ detected in screening round 1 and subsequent screening rounds among the overall population.Proportion of participants in defined risk groups  according to test findings at screening round 1 and subsequent outcomes  in these defined risk groups.The time between primary screening and diagnosis of CIN2+, CIN3+ or cervical cancer.The main objective of this analysis was to determine the rates of invasive cervical cancer and CIN3 or cancer (CIN3+) during the first and subsequent screening rounds. The predefined study endpoints for this analysis were:Outcomes data for cases of invasive cervical cancer, CIN3+ and CIN2+ over time were reported descriptively. Cases occurring >6 years after the last screening round were excluded from the analysis. In women with multiple outcomes in the same round (e.g. CIN2 and CIN3), the highest (most severe) clinical finding was always used for the analysis. The time to diagnosis was counted as the time to the first diagnosis of any clinical outcome . Confidence intervals (CIs) were calculated using R (Clopper Pearson formula for proportion).A total of 26,624 women, recruited between 1 February 2006 and 31 December 2016, had primary screening tests in round 1. Figure n = 48) or ovarian cancer (n = 14), 32 of whom were aged <70 years at the time of diagnosis. All 28 participants diagnosed with cervical cancer in round 1 were excluded from subsequent screening rounds, as were 15 women who had a hysterectomy because of CIN3 or adenocarcinoma in situ of the cervix. Overall, 707 participants aged <70 years with normal cytology and a negative HPV test underwent hysterectomy for benign indications; no cases of CIN2+ were diagnosed on the final histological cervical specimen in this cohort. There were 347 withdrawals from WOLPHSCREEN in screening round 1 and none afterwards. The status of a further 2618 women is uncertain. Because of a merger between Deutsche BKK and BARMER health insurance, we were unable to identify whether these women joined Audi BKK or other health insurance providers, left the project for other reasons or are delayed in attending the next screening round.We registered 46 deaths not related to cervical cancer in WOLPHSCREEN participants aged <70 years. Overall, 62 participants were treated for endometrial .Overall, 274 CIN3+ cases were diagnosed, including 31 invasive cervical cancers among women followed per protocol. Among 28 cases of cervical cancer diagnosed in round 1, 14 were detected within 6 months and 23 within 2 years of recruitment, while 2 patients had a delayed diagnosis because of lack of compliance with colposcopy and/or proposed treatment of CIN3 and 1 patient had a delayed diagnosis because of colposcopy failure. In round 2, three cases of cervical cancer were diagnosed, one within 2 years of entry to round 2. One patient who had HPV persistency after assessment in round 1 had a severely delayed diagnosis in round 2 because of colposcopy failure.19 The two other cases were microinvasive cancers; the first was diagnosed at our institution after 8 years, while another occurred in a patient who underwent CIN3 treatment within WOLPHSCREEN, left the Wolfsburg region and did not attend follow-up examinations as advised and was finally diagnosed with microinvasive cervical cancer.Follow-up of women excluded from WOLPHSCREEN in 2012 or later, using searches of the hospital registry, the cancer registry of Lower Saxony and the health insurance providers’ databases, showed no further cases of invasive cervical cancer among women who reached the age of 70+ years or those who had hysterectomy for benign indications. We identified three cancer cases who were excluded because these occurred >6 years after recruitment to round 1 and never attended for round 2. One of these excluded cases of cervical cancer was diagnosed after 10 years at another institution as a stage IV adenocarcinoma of the cervix and could be a misclassification because there was no histology review or surgical staging performed; misclassifying cancers of other origin as cervical cancer is common without surgical staging.n = 28 cases) in round 1 to 0.025%  in subsequent rounds. The difference in cervical cancer incidence between the first and the subsequent screening rounds was significant (Fisher exact test p = 0.01). The incidence of CIN3+ was 0.96%  in round 1 and dropped to 0.16%  in subsequent screening rounds (p < 0.0001).Figure p = 0.007), CIN3+  and CIN2+ .As the CIN3 incidence declines with age in a population aged 30–70 years, we did an additional sensitivity analysis excluding participants aged 30−34 years, which confirmed declines between round 1 and subsequent rounds for cervical cancer  to 0% (0/37) in the HC2+/Pap-abnormal group and from 0.71% (12/1699) to 0.19% (1/518) in the HC2+/Pap-normal group. No cases of CIN3+ or cervical cancer were diagnosed in the HC2−/Pap-abnormal group (0/309 in round 1 and 0/119 in subsequent rounds). In the HC2−/Pap-normal group, 1 case was diagnosed among 24,362 women within round 1 and another 1 among 11,356 women during subsequent screening rounds .Overall, 270 of the 274 CIN3+ cases were HC2+ and 4 were HC2−, while 29 of the 31 invasive cervical cancers were HC2+ and 2 were HC2−.Figure p < 0.0001) and no cases of CIN3 were detected in the HC2−/Pap-abnormal group.Figure This locally organised HPV-based screening programme with individualised, risk-based follow-up protocol was implemented to improve detection of cervical cancer in a real-world setting. The current analysis with 11 years follow-up shows that co-testing detects approximately 90% of precursor lesions and invasive cervical cancer at first screening leading to a decline in disease rates in subsequent screening rounds.20 The ‘HPV FOCAL Randomised Clinical Trial’ including 19,009 women compared the cumulative incidence of CIN3+ using primary HPV testing alone or liquid-based cytology (both groups had HPV and liquid-based cytology co-testing at the 48-month exit point). The incidence of CIN3+ at 48 months was lower in the HPV testing group .20 An analysis of another four randomised trials  comprising a total of 176,464 women aged 20–64 years followed for a median of 6.5 years showed that HPV screening significantly reduced the incidence of invasive cervical cancer beyond 2.5 years compared with cytology-based screening.9 At 5.5 years, the cumulative incidence of invasive cervical carcinoma  in women with negative tests at entry was 8.7 with HPV screening compared with 36.0 with cytology. In the WOLPHSCREEN analysis, the cumulative incidence of invasive cervical cancer was 4.1 per 100,000 in women with a HC2− test and normal Pap cytology at round 1, which was even lower than the estimate for the HPV test only from the analysis of randomised trials. Although there is an inherent risk of bias in single-arm, observational studies in unselected populations, it is reassuring that WOLPHSCREEN provided evidence to support an HPV-based screening approach. Since the WOLPHSCREEN project started, Germany has decided to switch from yearly Pap screening to Pap and HPV co-testing with a 3-year interval for 35–60-year-old women in 2020. The 3-year interval was a political decision. Data from randomised controlled trials show that the screening interval can be extended up to 5 years,9 and this recommendation is supported by the WOLPHSCREEN data.Randomised clinical trials previously demonstrated improved outcomes with HPV screening compared with Pap cytology.21There was a notable reduction in the incidence of precursor lesions between round 1 and subsequent screening rounds, particularly for women with an initially positive HPV test. The reduction in the incidence of cervical cancer in subsequent rounds can be explained by improved detection of precursor lesions during round 1 screening, allowing at-risk individuals to be referred for colposcopy and definitive treatment where indicated. Of note, 28 cases of cervical cancer were associated with HPV infection present at the time of recruitment. Moreover, other HPV tests showed that two HC2-negative cases tested positive for HPV-DNA in the tumour tissue and the initial sample. Therefore, only 1 case, a microinvasive adenocarcinoma diagnosed 7.5 years after recruitment, may have developed from a newly acquired HPV infection. Long-term follow-up data showed a very low risk of CIN3+ among women with negative HPV tests. During the 11-year follow-up, we did not find any cases of CIN3 or invasive cervical cancer in women with a negative HPV test and abnormal cytology. Our data suggest that the risk of CIN3+ and cervical cancer is determined mainly by HPV status, whereas Pap cytology is a marker of additional risk in HPV-positive women, as shown in the Kaplan–Meier plots Fig. . Our res15 and, therefore 25 of the 28 cancer cases detected during round 1 occurred in women whose previous screening had been done within the last 6 years and often annually.Although there was a notable difference between cohort sizes in round 1 and subsequent rounds, the data obtained per-protocol allow a reliable comparison over time. We identified 28 cases of cervical cancer in round 1, while another 3 cases were observed beyond round 1. The three cervical cancers detected in the second and third screening rounds could be explained by failure of screening tests or colposcopy, while the majority of CIN3 identified within round 2 and 3 were classified as new lesions in women who tested negative for HPV at recruitment. It is important to note that WOLPHSCREEN participants were regularly screened with cytology before recruitment to the pilot projectA limitation of this analysis is loss to follow-up or indeterminate status of 2965 participants, mostly because of a change of insurance provider and subsequent difficulties in keeping track of young women among a highly mobile workforce; however, external clinical databases and registries were used to mitigate this issue and allowed us to identify additional diagnoses of cervical cancer made outside the protocol. Despite this loss to follow-up, data are available from almost 12,000 women with a mean follow-up of 1401 days or 3.84 years in subsequent screening rounds. To minimise the possibility that a similar reduction of cervical cancer incidence did not occur among participants who left or were excluded from WOLPHSCREEN, we searched all possible sources and identified just three cases of cervical cancer in that cohort.An additional limitation of the analysis is that we could not directly compare CIN3 and cervical cancer data before and after the WOLPHSCREEN protocol was introduced. Although the reported incidence of cervical cancer in Wolfsburg could be used for such an analysis, CIN3 is not monitored reliably by German cancer registries making direct comparisons impossible. In addition, it is expected that a superior screening programme will lead to an increased incidence of cervical cancer and CIN3 in the first screening round because of better detection of cases, whereas the incidence will decrease in subsequent rounds, which is the effect that we observed in WOLPHSCREEN. As CIN3 is negatively associated with age, a decrease in incidence between the first and subsequent screening rounds may be anticipated. We did a sensitivity analysis, excluding women aged 30−34 years, which showed that the impact of ageing seems minor and the drop in CIN3 is not explained by 5 years' ageing of the population. Furthermore, the risk of invasive cancer does not have a linear association with age (peak incidence occurs among women aged 35–44 years) and we found that the decrease in cancer incidence (−78%) was almost as strong as the decrease in CIN3+ (−81%).22 is expected to decrease the risk of invasive cervical cancer and, in combination with HPV screening, may lead to eradication of cervical cancer.23 However, this effect is not likely to have occurred in our cohort already because vaccination started in 2007 in women aged <18 years and only exceptionally in older women. The COMPASS trial of cytology versus HPV testing in vaccinated women (aged ≤33 years) showed that HPV screening can increase detection of cervical precancerous lesions in vaccinated, as well as unvaccinated, populations.24 Nevertheless, despite the implementation of HPV vaccines, it is important to guard against complacency because cervical cancers are still occurring. Figure 13 The results from this study suggest that an adjusted colposcopy protocol may allow a few hundred cancer cases per year to be avoided in Germany.The analysis is robust and shows a reduction in the risk of invasive cervical cancer in women aged >30 years screened using HPV testing and risk-adapted follow-up. The introduction of HPV vaccination, which significantly reduces HPV-related lesions,25 found that primary high-risk HPV screening detected more CIN3+ at first screening compared with cytology while co-testing did not show initial increased CIN3+ detection in randomised controlled trials. We feel that the observed significant reduction of CIN3+ between the first and subsequent screening rounds in WOLPHSCREEN may be explained at least in part by our colposcopy protocol. Histological assessment was mandatory even in cases with minor changes and >93% of all women transferred for colposcopy received a histological diagnosis immediately, which may have avoided missing more CIN3 lesions at first colposcopy and changed the clinical course of some of the cases with HPV persistency. We observed that taking biopsies significantly increased the regression rate of persistent HPV infections compared with women who were followed without biopsies.26A recent systematic review by the US Preventive Services Task force17 Overlooked CIN3 lesions or delayed diagnoses because of false-negative colposcopy in HPV-positive women with normal cytology will directly result in underestimation of the sensitivity of HPV-based screening programmes and miss the chance to increase the number of women at risk who are protected from cervical cancer. Therefore, it seems to be a major challenge for all new HPV-based screening programmes to identify and eliminate factors that may result in a delayed or missed diagnosis of CIN3+ lesions by colposcopy.While a negative HPV test can identify individuals not at high risk of developing invasive cervical cancer, a positive HPV test signals the need for appropriate intervention. Improving outcomes depends on the effectiveness of follow-up procedures and treatments to manage women identified with persistent HPV infection, cytological abnormalities and precursor lesions. The quality of colposcopy is particularly important because all screening analyses are based on the rate of lesions detected by colposcopy and not on the true rate of lesions. We showed previously that failure of colposcopy to detect prevalent CIN3 in women with abnormal cytology is significantly lower than in HPV-positive women with normal cytology.In conclusion, the observed decline in disease rates with 5-yearly co-testing seems mainly attributable to HPV testing since no CIN3+ occurred in HPV−/Pap+ women. These findings support routine HPV screening outside the setting of randomised controlled trials, although a formal assessment of benefits and harm is still required.