Anna Söderlund-Strand

Modified General Primer PCR System for Sensitive Detection of Multiple Types of Oncogenic Human Papillomavirus

Human papillomavirus (HPV) infection is a necessary cause of cervical cancer and cervical dysplasia. Accurate and sensitive genotyping of multiple oncogenic HPVs is essential for a multitude of both clinical and research uses. We developed a modified general primer (MGP) PCR system with five forward and five reverse consensus primers. The MGP system was compared to the classical HPV general primer system GP5+/6+ using a proficiency panel with HPV plasmid dilutions as well as cervical samples from 592 women with low-grade cytological abnormalities. The reference method (GP5+/6+) had the desirable high sensitivity (five copies/PCR) for five oncogenic HPV types (HPV type 16 [HPV-16], HPV-18, HPV-56, HPV-59, and HPV-66). The MGP system was able to detect all 14 oncogenic HPV types at five copies/PCR. In the clinical samples, the MGP system detected a significantly higher proportion of women with more than two concomitant HPV infections than did the GP5+/6+ system (102/592 women compared to 42/592 women). MGP detected a significantly greater number of infections with HPV-16, -18, -31, -33, -35, -39, -42, -43, -45, -51, -52, -56, -58, and -70 than did GP5+/6+. In summary, the MGP system primers allow a more sensitive amplification of most of the HPV types that are established as oncogenic and had an improved ability to detect multiple concomitant HPV infections.

Comparison between the Hybrid Capture II Test and a PCR-Based Human Papillomavirus Detection Method for Diagnosis and Posttreatment Follow-Up of Cervical Intraepithelial Neoplasia

ABSTRACT Human papillomavirus (HPV) infection is the major cause of cervical cancer and its precursor, cervical intraepithelial neoplasia (CIN), and HPV testing has therefore been proposed for improved triaging and follow-up of women treated for CIN. We compared two common HPV DNA detection tests (Hybrid Capture II [HCII] and PCR-enzyme immunosorbent assay (EIA) using the primers GP5+/GP6+ followed by HPV typing with reverse dot blot hybridization) for sensitivity and specificity for detection of CIN and of CIN recurrence after treatment. Two hundred and thirty-nine women referred to the Department of Obstetrics and Gynaecology in Västerås, Sweden, were enrolled because of atypical Pap smears; 177 of these were later treated for dysplasia by conization or loop diathermy. Samples for HPV DNA testing were taken before and 4 to 6 months after treatment. There was substantial agreement between the HCII and PCR-EIA (kappa, 0.70 before treatment and 0.72 after treatment). The sensitivity for histopathologically confirmed CIN III was 100.0% for PCR-EIA and 95.6% for HCII. For patients with CIN II or worse (CIN II+), the sensitivities were 92.9% (PCR-EIA) and 91.8% (HCII). The specificities for CIN II+ in the pretreatment setting were 30.4% for PCR-EIA and 24.1% for HCII. After treatment, the sensitivities for CIN III in cytology were 100.0% by both methods, and for CIN II+, sensitivities were 80.0% by both methods. The specificities for CIN II+ in the posttreatment setting were 83.5% for PCR and 85.4% for HCII. In conclusion, the sensitivities of both PCR-EIA and HCII are high and almost equal, suggesting that both methods are suitable as tools for detection and posttreatment follow-up of CIN II-III.

Change in Population Prevalences of Human Papillomavirus after Initiation of Vaccination: The High-Throughput HPV Monitoring Study

Background: Organized human papillomavirus (HPV) vaccination was introduced in Sweden in 2012. On-demand vaccination was in effect from 2006 to 2011. We followed the HPV prevalences in Southern Sweden from 2008 to 2013. Methods: Consecutive, anonymized samples from the Chlamydia trachomatis screening were analyzed for HPV DNA for two low-risk types and 14 high-risk types using PCR with genotyping using mass spectrometry. We analyzed 44,146 samples in 2008, 5,224 in 2012, and 5,815 in 2013. Results: Registry-determined HPV vaccination coverages of the population in Southern Sweden increased mainly among 13- to 22-year-old women. Most analyzed samples contained genital swabs from women and the HPV6 prevalence in these samples decreased from 7.0% in 2008 to 4.2% in 2013 [−40.0%; P < 0.0005 (χ2 test)]. HPV16 decreased from 14.9% to 8.7% (−41.6%; P < 0.0005) and HPV18 decreased from 7.9% to 4.3% (−45.6%; P < 0.0005) among 13- to 22-year-old women. There were only small changes in vaccination coverage among 23- to 40-year-old women. In this age group, HPV18 decreased marginally (−19.6%; P = 0.04) and there were no significant changes for HPV6 or HPV16. Two nonvaccine HPV types (HPV52 and HPV56) were increased among 13- to 22-year-old women, both in 2012 and 2013. Conclusions: A major reduction of HPV6, 16, and 18 prevalences is seen in the age groups with a concomitant increase in HPV vaccination coverage. The minor changes seen for nonvaccine types will require further investigation. Impact: Monitoring of type-specific HPV prevalences may detect early effects of HPV vaccination. Cancer Epidemiol Biomarkers Prev; 23(12); 2757–64. ©2014 AACR.

Population-Level Effects of Human Papillomavirus Vaccination Programs on Infections with Nonvaccine Genotypes

After introduction of vaccination, some prevalences of nonvaccine types changed, without clear evidence for type replacement.

Human papillomavirus type-specific persistence and recurrence after treatment for cervical dysplasia.

Human papillomavirus (HPV) infection is a necessary factor in the cervical cancer development. Also after treatment for cervical dysplasia, HPV can be present and promote the recurrence of cervical disease. In the present study, the aim was to perform a long‐term follow‐up on the ability of HPV testing with genotyping, as compared with cytology, to predict recurrence of high‐grade cervical intraepithelial neoplasia and to evaluate the effectiveness of treatment with loop electrosurgical excision procedure (LEEP) conization. Cervical samples for HPV DNA testing and cytological analysis were obtained from 178 women with abnormal smears referred for treatment with LEEP conization. These women were scheduled for HPV DNA testing and Pap smears before and 3, 6, 12, 24, and 36 months after treatment. Three years after treatment 3.1% (N = 4) of women were still persistently HPV‐positive with the same type as had been detected at treatment. Recurrent or residual cervical intraepithelial neoplasia II+ in histopathology was found among 9 (5.1%) women during follow‐up. All of these women had type‐specific HPV‐persistence (sensitivity 100% [95% CI 63–100%] and specificity 94.7% [89.8–97.4%]), but only 7/9 had abnormal cytology (sensitivity 77.8% [40.2–96.1%] and specificity 94.7% [89.8–97.4%]). No recurrent or residual disease was found among women with any other patterns of HPV positivity (e.g., type change or fluctuating positivity) (sensitivity 0% [95% CI 0–37.1%] and specificity 80.5% [73.5–86.0%]). In conclusion, only type‐specific HPV persistence predicted recurrent or residual disease, and HPV genotyping appears useful to improve the specificity when using HPV testing in post‐treatment follow‐up. J. Med. Virol. 86:634–641, 2014.

Diversity of human papillomaviruses in skin lesions

Pools of frozen biopsies from patients with squamous cell carcinoma (SCC) (n=29) actinic keratosis (AK) (n=31), keratoacanthoma (n=91) and swab samples from 84 SCCs and 91 AKs were analysed with an extended HPV general primer PCR and high-throughput sequencing of amplimers. We found 273 different HPV isolates (87 known HPV types, 139 previously known HPV sequences (putative types) and 47 sequences from novel putative HPV types). Among the new sequences, five clustered in genus Betapapillomavirus and 42 in genus Gammapapillomavirus. Resequencing of the three pools between 21 to 70 times resulted in the detection of 283 different known or putative HPV types, with 156 different sequences found in only one of the pools. Type-specific PCRs for 37 putative types from an additional 296 patients found only two of these putative types. In conclusion, skin lesions contain a large diversity of HPV types, but most appeared to be rare infections.

Genotyping of human papillomavirus in triaging of low-grade cervical cytology.

OBJECTIVE The objective of the study was to evaluate whether typing of human papillomavirus (HPV) among women with low-grade cervical cytology can improve the ability to identify women with cervical cancer or cervical intraepithelial neoplasia grade III (CIN III or worse). STUDY DESIGN A total of 1595 women with low-grade cervical cytology participating in a randomized implementation trial of HPV triaging using Hybrid Capture II were also HPV genotyped and CIN III or worse predictive values evaluated. RESULTS HPV 16 was detected in 57% of cases with CIN III or worse but only among 24% of all tested women. Testing for the 3 HPV types with highest risk (HPV16/31/33) detected 77% of CIN III or worse, with 36% of women testing positive. Positivity for the other high-risk HPV types had a decreased risk for CIN III or worse. CONCLUSION Different high-risk HPV types confer different risks for the presence of CIN III or worse, implying that HPV genotyping could be useful for the optimization of triaging strategies.

Patterns of human papillomavirus types in multiple infections: an analysis in women and men of the high throughput human papillomavirus monitoring study.

Background To evaluate the pattern of co-infection of human papillomavirus (HPV) types in both sexes in Sweden. Methods Cell samples from genital swabs, first-void urine, and genital swabs immersed in first-void urine were collected in the present cross-sectional High Throughput HPV Monitoring study. Overall, 31,717 samples from women and 9,949 from men (mean age 25) were tested for 16 HPV types using mass spectrometry. Multilevel logistic regression was used to estimate the expected number of multiple infections with specific HPV types, adjusted for age, type of sample, and accounting for correlations between HPV types due to unobserved risk factors using sample-level random effects. Bonferroni correction was used to allow for multiple comparisons (120). Results Observed-to-expected ratio for any multiple infections was slightly above unity in both sexes, but, for most 2-type combinations, there was no evidence of significant departure from expected numbers. HPV6/18 was found more often and HPV51/68 and 6/68 less often than expected. However, HPV68 tended to be generally underrepresented in co-infections, suggesting a sub-optimal performance of our testing method for this HPV type. Conclusions We found no evidence for positive or negative clustering between HPV types included in the current prophylactic vaccines and other untargeted oncogenic types, in either sex.

Impact of gender-neutral or girls-only vaccination against human papillomavirus—Results of a community-randomized clinical trial (I)

Human papillomavirus (HPV) vaccine is efficacious but the real‐life effectiveness of gender‐neutral and girls‐only vaccination strategies is unknown. We report a community‐randomized trial on the protective effectiveness [(PE) = vaccine efficacy (VE) + herd effect (HE)] of the two strategies among females in virtually HPV vaccination naïve population. We randomized 33 Finnish communities into Arm A) gender‐neutral vaccination with AS04‐adjuvanted HPV16/18 vaccine (11 communities), Arm B) HPV vaccination of girls and hepatitis B‐virus (HBV) vaccination of boys (11 communities) or Arm C) gender‐neutral HBV vaccination (11 communities). All resident 39,420 females and 40,852 males born 1992‐95 were invited in 2007–09. Virtually all (99%) 12‐ to 15‐year‐old participating males (11,662) and females (20,513) received three doses resulting in uniform 20–30% male and 50% female vaccination coverage by birth cohort. Four years later (2010–14) 11,396 cervicovaginal samples obtained from 18.5 year‐old women were tested for HPV DNA, and prevalence of cervical HPV infections by trial arm and birth cohort was the main outcome measure. VEs against HPV16/18 varied between 89.2% and 95.2% across birth cohorts in arms A and B. The VEs against non‐vaccine types consistent with cross‐protection were highest in those born 1994–95 for HPV45 (VEA 82.8%; VEB 86.1%) and for HPV31 (VEA 77.6%, VEB 84.6%). The HEs in the non HPV‐vaccinated were statistically significant in those born 1994–95 for HPV18 (HEA 51.0%; 95% CI 8.3–73.8, HEB 47.2%; 6.5–70.2) and for HPV31/33 in arm A (HEA 53.7%; 22.1–72.5). For HPV16 and 45 no significant herd effects were detected. PE estimates against HPV16/18 were similar by both strategies (PEA 58.1%; 45.1–69.4; PEB 55.7%; 42.9–66.6). PE estimates against HPV31/33 were higher by the gender‐neutral vaccination (PEA 60.5%; 43.6–73.4; PEB 44.5%; 24.9–60.6). In conclusion, while gender‐neutral strategy enhanced the effectiveness of HPV vaccination for cross‐protected HPV types with low to moderate coverage, high coverage in males appears to be key to providing a substantial public health benefit also to unvaccinated females. Trial registration www.clinicaltrials.gov.com NCT000534638

Evaluation of human papillomavirus DNA detection in samples obtained for routine Chlamydia trachomatis screening

BACKGROUND The costs and logistics involved in obtaining samples is a bottleneck in large-scale studies of the circulation of human papillomavirus (HPV), which are useful for monitoring and optimisation of HPV-vaccination programs. Residual samples obtained after screening for Chlamydia trachomatis could constitute a convenient, low-cost solution. OBJECTIVES We evaluated HPV DNA detection and typing using (i) the residual samples routinely taken for C. trachomatis screening or (ii) the sample types used in large-scale phase III HPV vaccination trials (cervical, vulvar, labial, perineal, perianal, scrotal and penile shaft samples). STUDY DESIGN Samples from 127 men and 110 women attending two sexual health clinics were analysed using PCR for HPV DNA, with typing using mass spectrometry. RESULTS The HPV DNA prevalence was 7.1% in male urine samples, but 57.3% in female urine/vaginal samples, which was even higher than the HPV prevalence found in cervical samples (54.1%). The sensitivity for HPV DNA detection in the urine/vaginal samples was 7.9% (95% CI 3.0-16.4) for men and 78.9% (95% CI 67.6-87.7) for women, using detection in any one of the reference samples as reference. With cervical samples as reference, the sensitivity was 89.3 % (95% CI 78.1-95.9). CONCLUSIONS Among men, low sensitivity of urine for HPV detection suggests limited usefulness. Among women, the high sensitivity of urine/vaginal samples for HPV detection suggests a useful low-cost solution for the study of HPV epidemiology.