Vitaly Smelov

Long term duration of protective effect for HPV negative women: follow-up of primary HPV screening randomised controlled trial

Objectives To assess whether the increased sensitivity of screening for human papillomavirus (HPV) may represent overdiagnosis and to compare the long term duration of protective effect against cervical intraepithelial neoplasia grade 2 or worse (CIN2+) in HPV based and cytology based screening. Design 13 year follow-up of the Swedescreen randomised controlled trial of primary HPV screening. Setting Organised cervical screening programme in Sweden. Participants 12 527 women aged 32-38 attending organised screening were enrolled and randomised to HPV and cytology double testing (intervention arm, n=6257) or to cytology only, with samples frozen for future HPV testing (control arm, n=6270). Main outcome measures Cumulative incidence of CIN2+ and CIN3+ (Kaplan Meier curves). Longitudinal test characteristics were calculated for cytology only, HPV testing only, and cytology and HPV testing combined, adjusting for censoring. Results The increased detection of CIN2+ in the intervention arm decreased over time. After six years, the cumulative incidence of CIN3+ was similar in both trial arms, and after 11 years the cumulative incidence of CIN2+ became similar in both arms. The longitudinal sensitivity of cytology for CIN2+ in the control arm at three years was similar to the sensitivity of HPV testing in the intervention arm at five years of follow-up: 85.94% (95% confidence interval 76.85% to 91.84%) v 86.40% (79.21% to 91.37%). The sensitivity of HPV screening for CIN3+after five years was 89.34% (80.10% to 94.58%) and for cytology after three years was 92.02% (80.59% to 96.97%). Conclusions Over long term follow-up, the cumulative incidence of CIN2+ was the same for HPV screening and for cytology, implying that the increased sensitivity of HPV screening for CIN2+ reflects earlier detection rather than overdiagnosis. The low long term risks of CIN3+ among women who tested negative in HPV screening, support screening intervals of five years for such women. Trial registration Clinicaltrials.gov NCT00479375.

Anal Lymphogranuloma Venereum Infection Screening With IgA Anti-Chlamydia trachomatis-Specific Major Outer Membrane Protein Serology

Background: Anal lymphogranuloma venereum (LGV) infections, caused by Chlamydia trachomatis biovar L (Ct+/LGV+), are endemic among men who have sex with men (MSM). Anal non-LGV biovar Ct infections (Ct+/LGV−) can be eradicated with 1 week doxycycline, whereas Ct+/LGV+ infections require 3-week doxycycline. To differentiate Ct+/LGV+ from Ct+/LGV− infections, biovar-specific Nucleic Acid Amplification Test (NAAT) are standard, but also expensive and laborious. A chlamydia-specific serological assay could serve as an alternative test. Methods: MSM were screened for anal Ct+/LGV+ and Ct+/LGV− infections with a commercial nonspecific NAAT and an in house biovar L-specific NAAT. Serum samples were evaluated with chlamydia-specific anti-Major Outer Membrane Protein (MOMP) and antilipopolysaccharide assays of IgA and IgG classes. Asymptomatic patients were identified as: (1) no anal complaints or (2) no microscopic inflammation (i.e., <10 leucocytes per high power field in anal smears). The best differentiating assay was subsequently evaluated in 100 Ct+/LGV+ and 100 Ct+/LGV− MSM using different cut-off points. Results: The anti-MOMP IgA assay was the most accurate to differentiate Ct+/LGV+ (n = 42) from Ct+/LGV− (n = 19) with 85.7% sensitivity (95% confidence interval [CI], 72.2–93.3) and 84.2% specificity (95% CI, 62.4–94.5), even among asymptomatic patients. In a population comprising 98 Ct+/LGV+ and 105 Ct+/LGV− patients, the anti-MOMP IgA assay scored most accurate when the cut-off point was set to 2.0 with 75.5% (95% CI, 65.8–83.6) sensitivity and 74.3% (95% CI, 64.8–82.3) specificity. Conclusions: The IgA anti-MOMP assay can identify a considerable proportion of the (asymptomatic) anal LGV infections correctly. Yet, biovar L-specific NAAT are still the preferred diagnostic tests in clinical settings.

Long‐term HPV type‐specific risks of high‐grade cervical intraepithelial lesions: A 14‐year follow‐up of a randomized primary HPV screening trial

Quantitative knowledge of the long‐term human papillomavirus (HPV) type‐specific risks for high‐grade cervical intraepithelial neoplasias Grades 2 and 3 (CIN2 and CIN3) is useful for estimating the effect of elimination of specific HPV types and clinical benefits of screening for specific HPV types. We estimated HPV type‐specific risks for CIN2 and CIN3 using a randomized primary HPV screening trial followed up for 14.6 years using comprehensive, nationwide registers. Poisson regression estimated cumulative incidences, population attributable proportions (PAR) and incidence rate ratios (IRRs) of high‐grade lesions by baseline HPV type, with censoring at date of first CIN2/3 or last registered cytology. Multivariate analysis adjusted for coinfections. IRRs were highest during the first screening round, but continued to be high throughout follow‐up (IRRs for CIN3 associated with high‐risk (HR) HPV positivity were 226.9, 49.3, 17.7 and 10.3 during the first, second and third screening round and for >9 years of follow‐up, respectively). Increased long‐term risks were found particularly for HPV Types 16, 18 and 31 and for CIN3+ risks. HPV16/18/31/33 had 14‐year cumulative incidences for CIN3+ above 28%, HPV35/45/52/58 had 14 year risks between 14% and 18% and HPV39/51/56/59/66/68 had risks <10%. HPV16 contributed to the greatest proportion of CIN2+ (first round PAR 36%), followed by Types 31, 52, 45 and 58 (7–11%). HPV16/18/31/33/45/52/58 together contributed 73.9% of CIN2+ lesions and all HR types contributed 86.9%. In summary, we found substantial differences in risks for CIN2 and CIN3 between different oncogenic HPV types. These differences may be relevant for both clinical management and design of preventive strategies.

Next generation sequencing for human papillomavirus genotyping

BACKGROUND Human papillomavirus (HPV) genotyping using next generation sequencing (NGS) could be useful to study the HPV variant-specific epidemiology, including monitoring for possible emergence of new HPV variants after introduction of HPV vaccination programs. OBJECTIVES We wished to design and validate a method for rapid HPV detection, typing and sequencing in clinical samples. STUDY DESIGN Plasmids of 15 different HPV types were mixed and serially diluted in human DNA in concentrations ranging from 1 to 100 copies per sample, amplified using the HPV general PCR primer pair PGMY and sequenced using 454 technology. Sixty cervical samples were tested both with the NGS-based method and with a comparison method based on genotyping using type-specific probes bound to fluorescent beads (Luminex). Thirty-three clinical samples were repeat tested using NGS to evaluate reproducibility. RESULTS The NGS-based method correctly identified all 15 mixed HPV types when present in 100 copies/sample and 13/15 types when present in 10 copies/sample. For 36/60 cervical samples genotyping results using NGS and Luminex were identical. For 12/60 samples the NGS method was more sensitive than the Luminex test and most of the remaining discrepancies could be explained by the different type coverage of the assays. Reproducibility testing found complete or partial concordance in 30/33 samples. CONCLUSIONS NGS provides a sensitive and accurate method for genotyping of HPV. The fact that also the amplimer sequence is obtained could be important for studying the epidemiology of viral variants and monitoring of HPV vaccination.

Detection of DNA viruses in prostate cancer

We tested prostatic secretions from men with and without prostate cancer (13 cases and 13 matched controls) or prostatitis (18 cases and 18 matched controls) with metagenomic sequencing. A large number (>200) of viral reads was only detected among four prostate cancer cases (1 patient each positive for Merkel cell polyomavirus, JC polyomavirus and Human Papillomavirus types 89 or 40, respectively). Lower numbers of reads from a large variety of viruses were detected in all patient groups. Our knowledge of the biology of the prostate may be furthered by the fact that DNA viruses are commonly shed from the prostate and can be readily detected by metagenomic sequencing of expressed prostate secretions.

Expressed prostate secretions in the study of human papillomavirus epidemiology in the male.

Introduction Exploring different sampling sites and methods is of interest for studies of the epidemiology of HPV infections in the male. Expressed prostate secretions (EPS) are obtained during digital rectal examination (DRE), a daily routine urological diagnostic procedure, following massage of the prostate. Materials and Methods Urethral swabs and EPS samples were obtained from a consecutive sample of 752 men (mean age 32.4 years; median life-time sex partners 34) visiting urology outpatient clinics in St. Petersburg, Russia and tested for HPV DNA by general primer PCR, followed by genotyping using Luminex. Results Overall, 47.9% (360/752) of men were HPV-positive, with 42.0% (316/752) being positive for high-risk (HR-) HPV and 12.6% (95/752) for multiple HPV types. HPV-positivity in the EPS samples was 32.6% (27.7% HR-HPV) and in the urethral samples 25.9% (24.5% HR-HPV). 10.6% were HPV positive in both EPS and urethral samples. 6.4% had the same HPV-type in both EPS and urethral samples. 10.6% were HPV positive in both EPS and urethral samples. 6.4% had the same HPV-type in both EPS and urethral samples. The concordance between the urethral samples and EPS was 62.5% (470/752), with 80 cases double positive and 390 cases double negative in both sites. The sensitivity of urethral samples for overall HPV detection was 54.2% (195/360). Compared to analysis of urethral samples only, the analysis of EPS increased the HPV prevalence in this population with 26.2%. Conclusion EPS represent informative sampling material for the study of HPV epidemiology in the male.

Metagenomic sequencing of expressed prostate secretions.

To investigate which microorganisms may be present in expressed prostate secretions (EPS) metagenomic sequencing (MGS) was applied to prostate secretion samples from five men with prostatitis and five matched control men as well as to combined expressed prostate secretion and urine from six patients with prostate cancer and six matched control men. The prostate secretion samples contained a variety of bacterial sequences, mostly belonging to the Proteobacteria phylum. The combined prostate secretion and urine samples were dominated by abundant presence of the JC polyomavirus, representing >20% of all detected metagenomic sequence reads. There were also other viruses detected, for example, human papillomavirus type 81. All combined prostate secretion and urine samples were also positive for Proteobacteria. In summary, MGS of expressed prostate secretion is informative for detecting a variety of bacteria and viruses, suggesting that a more large‐scale use of MGS of prostate secretions may be useful in medical and epidemiological studies of prostate infections. J. Med. Virol. 86:2042–2048, 2014.

Are human papillomavirus DNA prevalences providing high-flying estimates of infection? An international survey of HPV detection on environmental surfaces

Most epidemiological studies of human papillomavirus (HPV) infections rely on HPV DNA detection. Recent studies have reported very high prevalence and acquisition rates in men.1 However, presence of HPV DNA is not proof of infection, as it might represent environmental contamination. Studies of HPV DNA on environmental surfaces could provide insights of the possible magnitude of this problem. We studied surfaces that frequently contact anogenital skin: toilet seats in airport restrooms. Apparently clean seats in 23 airports located in 13 countries (Belgium, Denmark, Finland, France, Germany, Italy, Jordan, the Netherlands, Russia, …

Long‐term HPV type‐specific risks for ASCUS and LSIL: A 14‐year follow‐up of a randomized primary HPV screening trial

Human papillomavirus (HPV) infections result in a significant burden of low‐grade cervical lesions. Between 1997 and 2000, our randomized trial of primary HPV screening enrolled 12,527 women participating in population‐based screening. Women between 32 and 38 years of age (median: 34, interquartile range: 33–37) were randomized to HPV and cytology double testing (intervention arm, n = 6,257 enrolled, n = 5,888 followed‐up) or to cytology, with samples frozen for future HPV testing (control arm, n = 6,270 enrolled, n = 5,795 followed‐up). We estimated the HPV type‐specific, long‐term absolute risks (AR), and population attributable proportions (PAR) for cytological diagnoses of atypical squamous cells of undetermined significance (ASCUS) or low‐grade squamous intraepithelial lesion (LSIL) and for histopathologically diagnosed cervical intraepithelial neoplasia grade 1 (CIN1). The women were followed using comprehensive, nationwide register‐based follow‐up. During a mean follow‐up time of 11.07 years, 886 ASCUS and LSIL lesions were detected, 448 in the intervention arm and 438 in the control arm. Poisson regression estimated the incidence rate ratios (IRRs) of low‐grade lesions by HPV type. The IRRs were strongly dependent on follow‐up time. The IRRs for ASCUS/LSIL associated with high‐risk HPV positivity were 18.6 (95% CI: 14.9–23.4) during the first screening round, 4.1 (95% CI: 2.8–6.2) during the second, 2.6 (95% CI: 1.7–4.1) during the third, and 1.1 (95% CI: 0.7–1.8) for >9 years of follow‐up, with similar declines seen for the individual types. Type 16 contributed consistently to the greatest proportion of ASCUS, LSIL, and CIN1 risk in the population (first screening round PAR: ASCUS: 15.5% (95% CI: 9.7–21.9), LSIL: 14.7% (95% CI: 8.0–20.9), and CIN1: 13.4% (95% CI: 3.2–22.5)), followed by type 31 [8.4% (95% CI: 4.2–12.5) for ASCUS to 17.3% (95% CI: 6.8–26.6) for CIN1]. In summary, most ASCUS/LSIL lesions associated with HPV infection are caused by new HPV infections and most lesions are found during the first screening round.

No high-risk human papillomavirus infection in prostate cancer tissues

To the Editor, Although human papillomavirus (HPV) has been associated with the development of specifi c cancers, its role as an aetiological agent of prostate cancer (PCa) is still the subject of debate. Therefore, we read with great interest the article by Dr Aghakhani and co-workers in which they reported the detection of high-risk (HR)-HPV DNA in 13 of 104 (12.5%) PCa and 8 of 104 (7.7%) benign prostatic hyperplasia (BPH) formalin-fi xed paraffi n-embedded tissue specimens [1]. Prior to this article, other investigators had reported higher rates of PCa in men with a history of exposure to HPV [2] and HPV associated with at least a subset of prostate carcinomas [3], while others had found no HPV DNA in PCa tissue [4], with most studies having limited power. Recently we investigated the presence of 14 HRHPV types in prostatic biopsy specimens from 61 Russian patients with PCa and 14 men with BPH (mean age 71.4 and 74.9 y, respectively) using an in-house PCR; only men with abnormal digital rectal examination (DRE) results and serum prostate specifi c antigen (PSA) levels above 4 ng/ml were included in the study. We found no association with PCa: no viral DNA was found in the BPH cases and HPV-16 and HPV-33 DNA were detected in only 3.3% and 1.6%, respectively, of PCa cases. To avoid sampling errors resulting in the taking of no malignant tissue by biopsy, we later sought to determine the presence of 14 HR-HPV types using the validated GP5 /6 assay [5], used worldwide. A total of 80 PCa tissue specimens obtained by taking the 4 histopathologically most suspect slides out of 20 archival paraffi n tissues from Dutch men who had undergone a radical prostatectomy (mean age 61 y, preoperative PSA 13 ng/ml) were investigated (this was supported by a European Urological Scholarship Programme Clinical Fellowship 2007 to Dr Vitaly Smelov); all samples were negative. Our recent data based on HR-HPV DNA detection indeed corroborate the conclusions of Aghakhani and colleagues that this transient most common sexually transmitted viral infection has no major role in the development of PCa [1]. However, men still represent an important source for the transmission of HPV: we need to focus on lowering HPV transmission between sexual partners and the role of male transmission of HPV in the development of other cancers [6].