Steep decline of HPV 6 and genital warts despite low HPV vaccination coverage in young women in Germany: a long-term, population-based cohort study

Background: The introduction of human papilloma virus (HPV) vaccination has resulted in a remarkable decline of genital warts in women and men, but in Germany historical rates of vaccination are relatively low. We report long-term surveillance data on changes in HPV 6 and 11 infection and the prevalence of genital warts in young women in the Wolfsburg HPV epidemiological study (WOLVES). Methods: Women born in 1983/84, 1988/89, and 1993/94 participated in four cohorts between 2009/10 and 2014/15. Vaccination coverage and prevalence of HPV 6/11 and genital warts are reported for participants aged 19−22 years and 24−27 years at the time of analysis. Statistical analyses were done to compare similarly aged participants using 2 x 2 contingency tables (Röhmel-Mansmann unconditional exact test; two-side alpha of 0.05). Results: A total of 2,456 women were recruited. In 2010, vaccination rates were 40/659 (6.1%) in women aged 24−27 years and 142/600 (23.7%) in those aged 19−22 years; 5 years later, vaccination rates had increased to 135/733 (18.4%) and 177/368 (48.1%), respectively. Between 2010 and 2015, there was a statistically significant decrease in the prevalence of HPV 6 among women aged 24−27 years (2.12% versus 0%; P<0.0001) and women aged 19−22 years (2.0% versus 0%; P=0.0056). In total, 52 of 2341 participants were diagnosed with genital warts. There was a statistically significant decrease in the life-time risk of developing genital warts in women aged 24−27 years between 2010 and 2015 (4.7% versus 1.68%, respectively; P=0.0018). The overall life-time risk of developing genital warts in women aged 19−27 years decreased from 3.1% in 2010 to 1.2% in 2015 (P=0.0022). Conclusions: An increase in vaccination coverage was associated with a decreased prevalence of genital

3 immunity alone was seen despite low vaccination rates. Quadrivalent vaccine had a protective effect on genital HPV 6 positivity and a fully protective effect on the development of genital warts in the youngest population.

Background
Human papillomavirus (HPV) is one of the most frequently sexually transmitted viral infections in the world (1). Persistent infections with high-risk HPV types (or class I and IIa carcinogenic types as defined by the International Agency for Research on Cancer) is a necessary risk factor for the development of cervical cancer, other anogenital malignancies, oropharyngeal cancer, and possibly nonmelanoma skin tumors, while lowrisk and other HPV types can lead to benign tumors of the skin and mucosa, for example, genital warts. The term 'genital wart' is not well-defined and covers a wide spectrum of diverse skin and mucosal lesions including the typical condylomata acuminata (2), associated with low-risk HPV genotypes 6 and 11 (3). Risk factors include acquisition of new sex partners, a higher number of sex partners, and concurrent infection with high-risk HPV types (4). Condylomata acuminata are highly contagious and will develop in approximately 65% of individuals with an infected partner (5).
Prophylactic HPV vaccines, either the quadrivalent vaccine against HPV types 6, 11 and 16, 18 or the nonavalent vaccine against types 6,11,16,18,31,33,45,52, and 58 can minimize the overall burden of HPV and genital warts (6). An Australian vaccination program using the quadrivalent HPV vaccine quickly led to a decline and almost complete disappearance of genital warts in both women and men (7). The steep decrease in the prevalence of genital warts occurred before widespread HPV immunization started in boys in 2013 suggesting a herd immunity in the nonvaccinated population of the same age group as the vaccinated girls (7,8).
In contrast to countries with school-based HPV vaccination programs, such as Australia, 4 the UK, and Austria, Germany has a relatively low vaccine coverage. Although HPV vaccination for the prevention of cervical cancer was recommended in March 2007 for females aged 12-17 years, in 2008-2009 the vaccine uptake rate was about 40% in females aged [16][17][18] years and remained at a low 42% vaccination rate in 2017. Genital warts and HPV infections are not reportable diseases and reliable surveillance data are not available in Germany to estimate the incidence of genital warts and the impact of HPV vaccination (9). Additional data from surveillance studies are necessary to understand the impact of HPV vaccination on the prevalence of HPV infection and associated genital warts. Here we report long-term surveillance data on changes in the prevalence of All participants were asked to complete a standardized medical questionnaire that included questions on education, birth country, marital status, pregnancies, parity, contraception, smoking, number of sex partners, age at first intercourse, history of abnormal smears, vaccination status, sexually transmitted infections, and genital warts.
During each medical examination, women had a pelvic examination with visualization of the uterine cervix. Pap smears were taken using a spatula and endocervical brush. A second sample was then obtained with a Qiagen Cervical Sampler (Medscan, Uppsala, Sweden) and suspended in 1 ml of specimen transport medium (STM/ Qiagen Inc., Hilden, Germany) for HPV DNA testing. From 2011, samples were taken for liquid cytology using a Cervex brush and ThinPrep (Hologic) transport medium. The gynecologist collected data on HPV vaccination status by checking the certificate of vaccination.
Women were referred to colposcopy if they had genital warts or an abnormal high-grade Pap smear, or they had an abnormal Pap smear classified as borderline or low-grade and tested positive for high-risk HPV. Colposcopy examinations were done at the Klinikum Wolfsburg.
Genital warts were classified as: (i) typical condylomata acuminata for lesions with typical acuminate morphology, which persist on mucosal epithelium or cornifying (cutaneous) skin and have highly vascularized dermal cores producing typical punctuated or cauliflower-like patterns; typical condylomata acuminata tend to be pigmentless and are mostly seen on 6 the pigmented skin (10); (ii) flat condylomata for genital papillomas with a more hyperkeratotic and pigmented surface and flat condylomata with a smooth surface and non-pigmented papules; or (iii) seborrheic wart-like lesions of the cutaneous skin of the external anogenital area. Patients presenting with vulvar intraepithelial neoplasia, Bowenoid papulosis, Naevi or Mollusca contagiosa were excluded (2). HPV genotyping using SPF-10-PCR, followed by Reverse Line Probe Assay LiPA Extra (SPF-10-PCR) (11) was done on all HC2-positive samples and 10% of HC2-negative samples.
Total DNA was isolated from the cervical samples using a QiaSymphony device (Qiagen, Hilden, Germany) and analyzed by the INNO-LiPA Extra HPV prototype assay (FujiReBioGent, Belgium) according to the manufacturer´s instructions. All PCR reactions were done with 10 µl input DNA in a final of 50 µl using reagents provided by Innogenetics according to the following sequence: 10 min 37°C, 9 min 94°C, and 40 cycles of 30 sec of denaturation at 94°C, followed by 45 sec at 52°C unchanging temperature and 45 sec of extension at 72°C run on a MJ Thermocycler PCT 200. The PCR product was then denaturated and a 10 µl aliquot hybridized to one strip at 49°C for 60 min, followed by multiple washing steps. The INNO-LiPA Extra test allowed identification of 13 established high-risk HPV types (16,18,31,33,35,39,45, 51, 52, 56, 58, 59, and 68), five known or 7 potential high-risk types (26, 53, 66, 73, and 82), seven low-risk HPV types (6,11,40,43,44, 54, and 70), additional non-differentiated HPV types, and types with undefined risk (69, 71, and 74). The strips were analyzed on a flatbed scanner using LiRAS prototype software (Innogenetics, Inc), which displays the patterns and relative intensity of positive bands as arbitrary grey-tone values between 0.1 and 1.0 and allows direct data transfer to Excel spreadsheets. All HC2-low-risk positive samples, which were negative in the LiPA Extra, were retested with the CP4/5 PCR (12). The positive PCR products were purified with the Qiagen Gel Extraction Test QIAquick and then directly sequenced using the CP4 primer.

Study endpoints
The predefined endpoints were the rates of full HPV vaccination coverage (three doses), the prevalence of HPV 6 and 11, and of genital warts (condylomata acuminata). We reported changes in these prevalence rates according to vaccination status and lifestyle factors (sexual history and smoking history) among participants in the four cohorts.

Statistical analysis
Vaccination coverage and prevalence rates of HPV 6/11 and genital warts are reported for participants aged 19−22 years (cohorts 2 and 4) and 24−27 years (cohorts 1 and 3) at the time of analysis. Statistical analyses were done to compare similarly aged participants (cohort 1 versus 3 and cohort 2 versus 4) using 2 x 2 contingency tables for the prevalence of HPV 6/11 infection (positive versus negative) and genital warts (no versus yes). The Röhmel-Mansmann unconditional exact test was used to test for difference, with a two-side alpha of 0.05. Statistical analyses were performed using Testimate V.6.5.14.

Results
Between October 2009 and January 2018, 2,456 women were included. Data are available 8 from 2,360 women for vaccination status and from 2,341 for prevalence of HPV infections and genital warts.
Vaccination coverage Table 1  Prevalence of HPV 6 and 11 HPV 6 was the most prevalent low-risk HPV type (Table 2) Table 2 shows that the prevalence of HPV 11 was low. Among participants aged 24−27 years, the prevalence of HPV 11 was 0.15% in cohort 1 and 0.14% in cohort 3, whereas in those aged 19−22 years the rates were 0.67% in cohort 2 and 0% in cohort 4. The observed decline in HPV 11 prevalence was not significant.

Prevalence of genital warts
Between 2010 and 2015, a total of 52 of 2341 participants were diagnosed with genital warts (Table 3) The presence of HPV 6 in cervical samples directly correlated to the genesis and development of genital warts, while co-infection with HPV 6 and 11 was found in only one case. Table 4 shows the prevalence of genital warts in the cohort with the lowest vaccine coverage (6.1% in cohort 1) compared with cohorts with greater coverage. An increase in the rate of vaccine coverage was associated with a decrease in the prevalence of genital warts.
Lifestyle factors 10 Complete sexual behavior and smoking history data in this large population of women showed some differences between cohorts. The mean age at first intercourse was 16.9 years in cohort 1, 16.5 years in cohorts 2 and 3, and 16.2 in cohort 4 but this decline was not significant. There were no remarkable differences in numbers of sexual partners Results of univariate testing (Wilcoxon-Mann-Withney-U-Test) and multivariate testing (logistic regression) showed a significant correlation between the number of sexual partners and risk of genital warts in all cohorts (p<0.0001). Young age at first intercourse and currently smoking were not associated with an increased risk for genital warts. We warts. Somewhat unexpectedly, the decrease in genital warts and HPV 6 infection was observed despite the low vaccination coverage in our population (<50%). It is possible that a single HPV dose may be protective, as suggested by recent publications (13,14). In this study, we did not observe a single case of persistent infection with HPV 6,11,16,  Previous studies have shown that high levels of HPV vaccine coverage may virtually eliminate genital warts in young women and men (reviewed in (15,16)). Australia and other countries with a high vaccine coverage already observed the near-extinction of genital warts in adolescents and young adults (17). Surveillance studies from countries with vaccination programs based on bivalent HPV vaccine and from countries with low vaccine coverage showed a less pronounced decline in the incidence of genital warts.
Recent publications showed the early benefit of vaccination such as the decrease in the incidence of anogenital warts in women aged <22 years and the possible development of herd immunity within population with high vaccination coverage (18).
The results from our study also show a clear correlation between an increase in vaccination coverage and a reduction in the life-time risk of developing genital warts. In WOLVES, it was notable that a decline of genital warts and complete disappearance of HPV 6 was observed in young women with the highest vaccination coverage, i.e. those aged 19−22 years. Nevertheless, the vaccination coverage in our study population was lower than in previously conducted studies, but seems to be high enough to confer herd immunity (19). A systematic review and meta-analysis of transmission-dynamic models concluded that strong herd effects are expected from vaccinating girls only, even with coverage rates as low as 20% (20). Other studies in Germany have also suggested the possibility of herd immunity despite low vaccine coverage. Mikolajczyk et al. reported a decrease of the incidence of genital warts in females aged 16 years (47%), 17 years (45%) and 18 years (35%) at the end of 2008, one year after the recommended introduction of HPV vaccination (21). Furthermore, Thöne et al. reported a maximum reduction of up to 60% in genital warts among 16-to 20-year-old females and no decrease for corresponding groups outside the range 14-24 years (22). We interpret our data as a proof that transmission of HPV 6 as the causal agent of condylomata acuminata and even of other genital warts is strongly inhibited even in populations with low vaccination coverage, although the exact mechanism of this protection is unknown and the necessary level of vaccination coverage remains uncertain (23).
The major limitation of this analysis is the low number of genital warts reported in our population (52 cases). An increase in the use of self-applied topical treatment of genital warts over time may have contributed to the decline in the surgical treatments seen in our analysis, however, such an effect would be expected in all cohorts and, therefore, the decline observed in our youngest cohort (1993/94) cannot be explained by changes in the topical treatment of genital warts alone. It is possible that some self-reported diagnoses of genital warts were not valid and the true prevalence could be higher than reported in our previous analysis (2). The rate of underdiagnosed women is probably not related to vaccination status and the low occurrence of genital warts did not have any impact on our analyses.
The major strengths of this study are the recruitment of a large general population and the prospective nature of the design, rather than being only a retrospective analysis of existing databases. We recruited women aged 19 to 27 years and to our knowledge, this is the first complete population-based analysis of low-risk HPV and genital wart prevalence Authors' contributions: AD drafted the manuscript, collected data, and examined patients.
KUP is the scientific leader of WOLVES and responsible for the overall study protocol and the concept of the submitted analysis, MO did the statistical analyses for this publication, AI and TI were responsible for HPV testing, AL collected data and examined patients, KT was responsible for data documentation. All authors have reviewed and approved the final version of the manuscript and consent to publication. defined as 3 doses.