FAM19A4/miR124-2 methylation in invasive cervical cancer: A retrospective cross-sectional worldwide study.

Widespread adoption of primary human papillomavirus (HPV)-based screening has encouraged the search for a triage test which retains high sensitivity for the detection of cervical cancer and precancer, but increases specificity to avoid overtreatment. Methylation analysis of FAM19A4 and miR124-2 genes has shown promise for the triage of high-risk (hr) HPV-positive women. In our study, we assessed the consistency of FAM19A4/miR124-2 methylation analysis in the detection of cervical cancer in a series of 519 invasive cervical carcinomas (n = 314 cervical scrapes, n = 205 tissue specimens) from over 25 countries, using a quantitative methylation-specific PCR (qMSP)-based assay (QIAsure Methylation Test®). Positivity rates stratified per histotype, FIGO stage, hrHPV status, hrHPV genotype, sample type and geographical region were calculated. In total, 510 of the 519 cervical carcinomas (98.3%; 95% CI: 96.7-99.2) tested FAM19A4/miR124-2 methylation-positive. Test positivity was consistent across the different subgroups based on cervical cancer histotype, FIGO stage, hrHPV status, hrHPV genotype, sample type and geographical region. In conclusion, FAM19A4/miR124-2 methylation analysis detects nearly all cervical carcinomas, including rare histotypes and hrHPV-negative carcinomas. These results indicate that a negative FAM19A4/miR124-2 methylation assay result is likely to rule out the presence of cervical cancer.

Intra- and inter-laboratory agreement of the FAM19A4/mir124-2 methylation test: Results from an international study.

BACKGROUND: HPV-based cervical screening detects women at an increased risk of cervical cancer and precancer. To differentiate among HPV-positive women those with (pre)cancer, triage testing is necessary. The detection of cancer-associated host-cell DNA methylation (FAM19A4 and hsa-mir124-2) in cervical samples has shown valuable as triage test. This multicenter study from 6 collaborating European laboratories and one reference laboratory was set out to determine the intra- and inter-laboratory agreement of FAM19A4/mir124-2 DNA methylation analysis utilizing the QIAsure Methylation Test. METHODS: Agreement analysis for the QIAsure Methylation Test was assessed on high-risk HPV-positive cervical specimens (n = 1680) both at the level of the assay and at the full workflow, including bisulfite conversion. RESULTS: Intra- and inter-laboratory assay agreement were 91.4% (534/584; 95% CI 88.9-93.5; κ = 0.82) and 92.5% (369/399; 95% CI 90.0-94.7; κ = 0.83), respectively. The inter-laboratory workflow (bisulfite conversion and assay combined) agreement was 90.0% (627/697; 95% CI 87.5%-92.0%; κ = 0.76). CONCLUSION: These data show that the QIAsure Methylation Test performs robust and reproducible in different laboratory contexts. These results support the use of the QIAsure Methylation Test for full molecular screening for cervical cancer, including primary HPV testing and triage testing by methylation analysis.

DNA methylation markers as a triage test for identification of cervical lesions in a high risk human papillomavirus positive screening cohort.

Objective triage strategies are required to prevent unnecessary referrals for colposcopy in population-based screening programs using primary high-risk human papillomavirus (hrHPV) testing. We have identified several DNA methylation markers with high sensitivity and specificity for detection of high-grade cervical intraepithelial neoplasia or worse (CIN2+) in women referred for colposcopy. Our study assessed diagnostic potential of these methylation markers in a hrHPV-positive screening cohort. All six markers (JAM3, EPB41L3, C13orf18, ANKRD18CP, ZSCAN1 and SOX1) showed similar association across histology in the hrHPV-positive cohort when compared to the Dutch cohort (each p > 0.15). Sensitivity for CIN2+ was higher using methylation panel C13orf18/EPB41L3/JAM3 compared to the other 2 panels (80% vs. 60% (ANKRD18CP/C13orf18/JAM3) and 63% (SOX1/ZSCAN1), p = 0.01). For CIN3+ all three methylation panels showed comparable sensitivity ranging from 68% (13/19) to 95% (18/19). Specificity of SOX1/ZSCAN1 panel (84%, 167/200) was considerably higher compared to ANKRD18CP/C13orf18/JAM3 (68%, 136/200, p = 2 × 10-5 ) and C13orf18/EPB41L3/JAM3 (66%, 132/200, p = 2 × 10-7 ). High negative predictive value (NPV) (91-95% and 96-99%) was observed for CIN2+ and CIN3+, for all three methylation panels, while positive predictive value (PPV) varied from 25 to 40% for CIN2+ and 15-27% for CIN3+. Interestingly, 118/235 samples were negative for all six markers (including 106 controls (89.8%), 6 CIN1 (5.1%), 5 CIN2 (4.2%) and 1 CIN3 (0.8%)). Methylation results from both independent cohorts were comparable as well as high sensitivity for detection of cervical cancer and its high-grade precursors in hrHPV-positive population. Our study therefore validates these methylation marker panels as triage test either in hrHPV-based or abnormal cytology-based screening programs.

Seropositivity to Multiple Anogenital Human Papillomavirus (HPV) Types Is Associated With Current Anogenital HPV Infection, Abnormal Cytology, and Seropositivity for Nongenital HPVs.

Background: Antibodies against human papillomaviruses (HPVs) are biomarkers for current or past infections. We assessed whether antibodies against multiple HPV types were determinants of current multiple anogenital HPV infections, abnormal cytology, and seropositivity for cutaneous HPVs. Methods: A total of 1848 Slovenian women attended 2 rounds of cervical cancer screening 3 years apart and provided data on HPV antibodies and HPV DNA at both visits. Antibodies against 15 anogenital HPV types and 6 cutaneous HPVs were determined using pseudovirion-Luminex serology and anogenital HPV DNA using Linear Array. Antibodies to polyomaviruses were evaluated as a control. Women were grouped as either HPV seronegative or having antibodies to 1-2 HPV types or to ≥3 HPV types. Results: Presence of antibodies to multiple anogenital HPV types at baseline was associated strongly with (i) presence of HPV DNA at the cervix (χ2 = 68.8; P < .0001), (ii) multiple types of HPV DNA at baseline (χ2 = 58.6; P < .0001), (iii) HPV DNA at follow-up (χ2 = 22.9; P < .0001), (iv) abnormal cytology (χ2 = 9.8; P = .0017), and (v) concomitant presence of antibodies to any of 6 nongenital HPV types (χ2 = 40.1; P < .0001). Presence of antibodies to ≥3 anogenital HPV types tended to persist over time. Conclusions: Seropositivity against at least 3 anogenital HPV types is associated with current multiple anogenital HPV infections, abnormal cytology, and seropositivity to nongenital HPVs.

Impact of the human papillomavirus status on the development of high-grade cervical intraepithelial neoplasia in women negative for intraepithelial lesions or malignancy at the baseline: A 9-year Swedish nested case-control follow-up study.

BACKGROUND: The causal relation between high-risk human papillomavirus (HPV) and cervical cancer and its precursor lesions has led to the use of sensitive HPV molecular tests for screening. This study examined the impact of the baseline HPV status on the future risk of cervical intraepithelial neoplasia grade 2 or worse (CIN2+) among women with cytology negative for intraepithelial lesions or malignancy (NILM). METHODS: This was a nested case-control study including women with NILM baseline cytology participating in the Swedish cervical screening program in 2005-2007. Ninety-six cases of CIN2+ and 5 age-matched controls per case were identified through the National Cervical Screening Registry by follow-up through 2014. Baseline liquid-based cytology samples were tested for HPV. Conditional logistic regression analysis was used to calculate odds ratios (ORs) with confidence intervals (CIs). RESULTS: The risk of future high-grade cervical intraepithelial neoplasia (CIN) was strongly associated with the baseline HPV status. For women younger than 30 years, HPV-16/18 showed a significant association with future risk for CIN2+ (OR, 9.44; 95% CI, 3.37-26.4). Other HPV types were not significantly associated with future CIN2+ in these younger women. For women 30 years old or older, both HPV-16/18 and other HPV subtypes conferred a significant risk. CONCLUSIONS: The presence of HPV-16/18 among women with NILM cytology is associated with an elevated future risk of high-grade CIN. HPV types other than HPV-16/18 seem to have a greater impact on women 30 years old or older than younger women. Women with NILM cytology and HPV-16/18 need specific follow-up management within screening.

Validation of EUROArray HPV test using the VALGENT framework.

BACKGROUND: Robust clinical and analytical validation of human papillomavirus (HPV) tests is a pre-requisite for their use in cervical cancer screening given the transience of most high-risk HPV infections. OBJECTIVES: To evaluate the EUROArray HPV test (PCR-based full HPV genotyping test) using the international validation of the VALGENT framework, which offers an opportunity to determine analytical and clinical performance according to internationally accepted performance metrics. STUDY DESIGN: A total of 1300 consecutive and 300 abnormal cervical samples derived from the Slovenian screening programme were tested with the EUROArray HPV test. Clinical performance for the detection of cervical intraepithelial neoplasia grade 2 and above (CIN2+) was performed and compared to a standard comparator test (Hybrid Capture 2). Intra- and inter-laboratory reproducibility of the assay was performed in a subset of 500 samples. RESULTS: The relative clinical sensitivity and specificity of EUROArray HPV vs HC2 was 0.93 (95% Confidence Interval (CI), 0.88-0.99; P non-inferiority(ni) = 0.1413) and 1.01 (95% CI, 0.99-1.02; Pni = 0.0001), respectively. Application of an a-posteriori cut-off for HPV16 led to relative values of 0.98 (95% CI, 0.92-1.03; Pni = 0.0076) and 1.00 (95% CI, 0.97-1.03; Pni = 0.007), respectively. The assay showed excellent intra- and inter-laboratory reproducibility (concordance ≥ 94%, Kappas ≥0.85). CONCLUSION: At the predefined cut-off, EUROArray HPV was less sensitive than HC2 for the detection of CIN2+. However, when an optimised cut-off was applied, EUROArray HPV fulfilled international criteria for its use in cervical cancer screening.

Clinical Evaluation of INNO-LiPA HPV Genotyping EXTRA II Assay Using the VALGENT Framework.

In this diagnostic test validation study, we assessed the clinical accuracy and HPV genotyping performance of the INNO-LiPA HPV Genotyping Extra II (INNO-LiPA) within the VALGENT-3 framework. VALGENT is designed to assess the analytical and clinical performance of HPV tests with genotyping capacity. The VALGENT-3 panel comprised 1300 consecutive cervical cell specimens enriched with 300 samples with abnormal cytology obtained from women attending the Slovenian cervical cancer screening programme. The INNO-LiPA allows type-specific detection of 32 HPV types; however, for the clinical accuracy assessment, we considered it as high-risk (hr)HPV positive when at least one of the following HPV types was present: HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, and HPV68. Clinical accuracy for detection of cervical intraepithelial neoplasia grade 2 or worse (CIN2+) was compared between INNO-LiPA and Hybrid Capture 2 (HC2), which is a standard comparator test for HPV tests used in cervical cancer screening. In addition, hrHPV and type-specific detection HPV types were compared between INNO-LiPA and Linear Array HPV Genotyping Test (Linear Array). The prevalence of hrHPV determined by INNO-LiPA was 17.1% (95% CI, 15.0⁻19.2%) in the screening population. HrHPV testing with INNO-LiPA had a sensitivity for CIN2+ of 96.9% (95% CI, 92.1⁻99.1%) which was non-inferior to HC2 (relative sensitivity of 1.01; 95% CI, 0.97⁻1.04; pn.inf = 0.0002) and a specificity for ≤CIN1 of 85.3% (95% CI, 83.2⁻87.3%) which was inferior to HC2 (relative specificity of 0.95; 95% CI, 0.93⁻0.97; pn.inf = 0.9998). Genotyping agreement between INNO-LiPA and Linear Array was excellent for hrHPV, HPV16, HPV18, HPV35, HPV45, HPV58 and HPV59, but good or fair for other HPV types. To conclude, INNO-LiPA demonstrated non-inferior clinical sensitivity but lower specificity compared to HC2 in addition to excellent concordance compared to Linear Array for hrHPV and some genotypes.

Clinical and Analytical Evaluation of the Anyplex II HPV HR Detection Assay within the VALGENT-3 Framework.

In 2012, VALidation of human papillomavirus (HPV) GENotyping Tests (VALGENT) was initiated to provide a formalized and uniform study framework for comparison and validation of HPV assays with genotyping capability. In VALGENT-3, the clinical and analytical performance of Anyplex II HPV HR detection (Anyplex) was compared to that of the Hybrid Capture 2 HPV DNA test (hc2) and the cobas 4800 HPV test (cobas). The panel comprises 1,300 stored samples that were obtained from women 25 to 64 years old who participated in the Slovenian cancer screening program, enriched with 300 samples from women with abnormal cervical cytology. The sensitivity and specificity of Anyplex were noninferior to those of hc2, with a relative sensitivity of 1.01 (95% confidence interval [CI], 0.97 to 1.04) for cervical intraepithelial neoplasia grade 2 or worse (CIN2+) and 1.01 (95% CI, 0.97 to 1.06) for CIN3+ and relative specificity of 1.02 (95% CI, 1.00 to 1.03) for a CIN grade of ≤1. The clinical sensitivity of Anyplex for CIN2+ and CIN3+ was comparable to that of hc2 (P values for McNemar test [pMcN] of 0.655 and 0.564, respectively), but its specificity was significantly higher (pMcN = 0.008). The sensitivity and specificity of Anyplex were also noninferior to those of cobas, with relative sensitivity of 1.01 (95% CI, 0.98 to 1.04) for CIN2+ and 1.01 (95% CI, 0.99 to 1.04) for CIN3+ and relative specificity of 1.00 (95% CI, 0.99 to 1.01) (pMcN value of >0.05 in all cases). Regardless of the clinical outcome (CIN2+ or CIN3+), age restriction (women ≥30 years old), or comparator test used, Anyplex consistently showed excellent clinical performance and can be considered validated for primary cervical cancer screening.

Seroprevalences of Antibodies to 11 Human Papillomavirus (HPV) Types Mark Cumulative HPV Exposure.

Background: The antibody responses against human papillomavirus type 16 (HPV-16) and HPV-18 are well known, but many genital HPV types are oncogenic. We assessed the correlation between detection of type-specific HPV DNA and antibodies for 11 HPV types. Methods: A total of 2024 women attending the organized national cervical cancer screening program in Slovenia were tested for cervical high-risk HPV DNA (HPV-16, -18, -31, -33, -35, -39, -45, -52, -56, -58, -59, and -68) and serum anti-HPV antibodies. Of these, 1848 women were tested with the same methods 3 years earlier. Results: Type-specific antibodies against 10 of 11 HPV types (HPV-16, -18, -31, -33, -35, -39, -45, -52, -56, and -58) were associated with concomitant presence of type-specific DNA (median odds ratio [OR], 7.5; 95% confidence interval [CI], 2.2-26.1). When the concomitant presence of type-specific HPV DNA at the 3-year visit was combined with the presence of the same HPV DNA type 3 years earlier, the statistical precision was greatly improved, and antibodies against all 11 types (HPV-16, -18, -31, -33, -35, -39, -45, -52, -56, -58, and -59) were associated with the presence of DNA of the same HPV type (median OR, 7.4; 95% CI, 4.2-12.8). Sensitivity had a slight tendency to increase (from 47% to 52%) when DNA positivity at the earlier time point was included, whereas specificity was the same (88%). Seroconversion was associated with previous HPV DNA positivity. Seropositivity mostly remained stable during the observation period. Conclusions: For 11 HPV types, type-specific seropositivity was associated with the presence of DNA of the same HPV type (either concomitantly or previously). Antibodies to these HPV types mark cumulative HPV exposure.

Assessment of the Roche Linear Array HPV Genotyping Test within the VALGENT framework.

BACKGROUND: Cervical cancer screening programs are switching from cytology-based screening to high-risk (hr) HPV testing. Only clinically validated tests should be used in clinical practice. OBJECTIVES: To assess the clinical performance of the Roche Linear Array HPV genotyping test (Linear Array) within the VALGENT-3 framework. STUDY DESIGN: The VALGENT framework is designed for comprehensive comparison and clinical validation of HPV tests that have limited to extended genotyping capacity. The Linear Array enables type-specific detection of 37 HPV types. For the purpose of this study, Linear Array results were designated as positive only if one of the 13 hrHPV types also included in the Hybrid Capture 2 (HC2) was detected. The VALGENT-3 framework comprised 1600 samples obtained from Slovenian women (1300 sequential cases from routine cervical cancer screening enriched with 300 cytological abnormal samples). Sensitivity for cervical intraepithelial neoplasia of grade 2 or worse (CIN2+) (n=127) and specificity for

Molecular characterization, tissue tropism, and genetic variability of the novel Mupapillomavirus type HPV204 and phylogenetically related types HPV1 and HPV63.

HPV204 is the only newly identified Mupapillomavirus (Mu-PV) type in more than a decade. To comprehensively characterize HPV204, we performed a detailed molecular analysis of the viral genome and evaluated its clinical relevance in comparison to the other Mu-PVs, HPV1 and HPV63. The 7,227-bp long genome of HPV204 exhibits typical genomic organization of Mu-PVs with eight open reading frames (ORFs) (E6, E7, E1, E2, E8, E4, L2, and L1). We developed three type-specific quantitative real-time PCRs and used them to test a representative collection (n = 1,006) of various HPV-associated benign and malignant neoplasms, as well as samples of clinically normal cutaneous, mucosal, and mucocutaneous origins. HPV204, HPV1, and HPV63 were detected in 1.1%, 2.7%, and 1.9% of samples tested, respectively, and were present in skin and mucosa, suggesting dual tissue tropism of all Mu-PVs. To evaluate the etiological role of Mu-PVs in the development of HPV-associated neoplasms, Mu-PV viral loads per single cell were estimated. HPV1 and HPV63 were present in high viral copy numbers in 3/43 and 1/43 cutaneous warts, respectively, and were identified as the most likely causative agents of these warts. HPV204 viral load was extremely low in a single HPV204-positive cutaneous wart (7.4 × 10-7 viral copies/cell). Hence, etiological association between HPV204 and the development of cutaneous warts could not be established. To the best of our knowledge, this is the first study to evaluate the genetic variability of Mu-PVs by sequencing complete LCR genomic regions of HPV204, HPV1, and HPV63. We detected several nucleotide substitutions and deletions within the LCR genomic regions of Mu-PVs and identified two genetic variants of HPV204 and HPV63 and five genetic variants of HPV1.

Effect of naturally acquired type-specific serum antibodies against human papillomavirus type 16 infection.

BACKGROUND: While vaccine-induced antibodies are known to confer protection against incident human papillomavirus (HPV) infection, there is inconsistent data regarding the protective effect of naturally acquired anti-HPV antibodies. OBJECTIVES: To estimate the protective effect of naturally acquired anti-HPV16 serum antibodies against incident anogenital infection with HPV16 in females aged 20-64 years and to assess whether antibodies influence the persistence/clearance of anogenital HPV16 infection. STUDY DESIGN: 4432 women attending the organized national cervical cancer screening program in Slovenia were initially enrolled. 2199 and 1848 women had valid HPV DNA results obtained using PCR-based assays and HPV antibody serotyping results obtained using pseudovirion-based serological assay, at baseline and at three-year follow-up, respectively. RESULTS: Baseline HPV16 seroprevalence was 2.4-fold higher among HPV16 DNA-positive women (55.7% vs. 23.2%; p<0.01). Baseline HPV16 DNA-positive/seronegative women frequently acquired anti-HPV16 antibodies during follow-up (OR=8.2; 95% CI: 3.8-17.8). Baseline anti-HPV16 antibodies persisted at follow-up, irrespective of baseline HPV16 DNA status (OR=40.6; 95% CI: 30.3-54.5). Baseline HPV16 DNA-negative/seropositive women were less likely to acquire HPV16 infection at follow-up (unadjusted OR=0.2; 0.1-0.9). However, the age-adjusted association was non-significant (adjusted OR=0.3; 0.1-1.2). The tendency for protective effect was stronger among women older than 25 years (OR=0.2; 0.03-1.8). Baseline anti-HPV16 antibodies were not associated with persistence/clearance of HPV16 infection at follow-up (OR=0.8; 0.3-1.9). CONCLUSIONS: Naturally acquired anti-HPV16 serum antibodies appeared to protect against anogenital HPV16 infection, but this association was at least partially confounded by age. Baseline anti-HPV16 serum antibodies did not influence persistence/clearance of HPV16 infection at follow-up.

Global Genomic Diversity of Human Papillomavirus 11 Based on 433 Isolates and 78 Complete Genome Sequences.

UNLABELLED: Human papillomavirus 11 (HPV11) is an etiological agent of anogenital warts and laryngeal papillomas and is included in the 4-valent and 9-valent prophylactic HPV vaccines. We established the largest collection of globally circulating HPV11 isolates to date and examined the genomic diversity of 433 isolates and 78 complete genomes (CGs) from six continents. The genomic variation within the 2,800-bp E5a-E5b-L1-upstream regulatory region was initially studied in 181/207 (87.4%) HPV11 isolates collected for this study. Of these, the CGs of 30 HPV11 variants containing unique single nucleotide polymorphisms (SNPs), indels (insertions or deletions), or amino acid changes were fully sequenced. A maximum likelihood tree based on the global alignment of 78 HPV11 CGs (30 CGs from our study and 48 CGs from GenBank) revealed two HPV11 lineages (lineages A and B) and four sublineages (sublineages A1, A2, A3, and A4). HPV11 (sub)lineage-specific SNPs within the CG were identified, as well as the 208-bp representative region for CG-based phylogenetic clustering within the partial E2 open reading frame and noncoding region 2. Globally, sublineage A2 was the most prevalent, followed by sublineages A1, A3, and A4 and lineage B. IMPORTANCE: This collaborative international study defined the global heterogeneity of HPV11 and established the largest collection of globally circulating HPV11 genomic variants to date. Thirty novel complete HPV11 genomes were determined and submitted to the available sequence repositories. Global phylogenetic analysis revealed two HPV11 variant lineages and four sublineages. The HPV11 (sub)lineage-specific SNPs and the representative region identified within the partial genomic region E2/noncoding region 2 (NCR2) will enable the simpler identification and comparison of HPV11 variants worldwide. This study provides an important knowledge base for HPV11 for future studies in HPV epidemiology, evolution, pathogenicity, prevention, and molecular assay development.

Commercially available molecular tests for human papillomaviruses (HPV): 2015 update.

Commercial molecular tests for human papillomaviruses (HPV) are invaluable diagnostic tools in cervical carcinoma screening and management of women with cervical precancerous lesions as well as important research tools for epidemiological studies, vaccine development, and implementation and monitoring of vaccination programs. In this third inventory of commercial HPV tests, we identified 193 distinct commercial HPV tests and at least 127 test variants available on the market in 2015, which represents a 54% and 79% increase in the number of distinct HPV tests and variants, respectively, in comparison to our last inventory performed in 2012. Identified HPV tests were provisionally divided into eight main groups and several subgroups. Among the 193 commercial HPV tests, all but two target alpha-HPV types only. Although the number of commercial HPV tests with at least one published study in peer-reviewed literature has increased significantly in the last three years, several published performance evaluations are still not in line with agreed-upon standards in the HPV community. Manufacturers should invest greater effort into evaluating their products and publishing validation/evaluation results in peer-reviewed journals. To achieve this, more clinically oriented external quality-control panels and initiatives are required. For evaluating the analytical performance of the entire range of HPV tests currently on the market, more diverse and reliable external quality-control programs based on international standards for all important HPV types are indispensable. The performance of a wider range of HPV tests must be promptly evaluated on a variety of alternative clinical specimens. In addition, more complete HPV assays containing validated sample-extraction protocols and appropriate internal controls are urgently needed. Provision of a broader range of automated systems allowing large-scale HPV testing as well as the development of reliable, rapid, and affordable molecular point-of-care tests are priorities for the further improvement of HPV tests.

Three-year longitudinal data on the clinical performance of the Abbott RealTime High Risk HPV test in a cervical cancer screening setting.

BACKGROUND: Testing cervical smears for the presence of high-risk human papillomaviruses (hrHPV) increases the sensitivity for detecting women with underlying high-grade cervical intraepithelial neoplasia (CIN) and provides better and longer protection against invasive cervical cancer compared to cytology testing alone. The Abbott RealTime High Risk HPV test (RealTime) is a hrHPV DNA test with concurrent partial genotyping for HPV16 and HPV18 and aggregate detection of 12 other hrHPV types that have been extensively analytically and clinically evaluated over the last 6 years. OBJECTIVES AND STUDY DESIGN: To provide the first 3-year longitudinal data regarding the clinical performance of RealTime, the risk of CIN2+ according to various negative baseline characteristics, and baseline and future risk for CIN2+ at 3 years for women with baseline infection with various hrHPV types were assessed in a cohort of 3,920 Slovenian women that had hrHPV DNA and/or cytology in 36- to 48-month follow-up results after a baseline screening round in 2009/2010. RESULTS: A total of 36 CIN2+ cases were identified in the second screening round. Of these, 17 CIN2+ cases were identified passively through questionnaires/data registries and 19 cases actively as the result of actions triggered by second-round cytology and/or HPV test results. Accumulation of CIN2+ cases during follow-up occurred predominantly in woman with normal cytology at baseline. Among women >30 years old, significantly better protection against CIN2+ at 3 years was associated with a negative hrHPV DNA result at baseline (risk for CIN2+ 0.04% [95 CI, 0.00-0.22%]) than by normal cytology at baseline (risk for CIN2+ 0.68% [95 CI, 0.40-1.08%]). Women with baseline HPV16 infection had a significantly higher risk of CIN2+ at baseline (21.9% [95 CI, 15.2-30.4%]) and baseline plus future risk at 3 years for CIN2+ (33.3% [95 CI, 24.7-44.0%]) in comparison to women with baseline non-HPV16/18 hrHPV infection (7.0% [95 CI, 4.6-10.2%]) or those that were hrHPV-positive (11.7% [95 CI, 9.1-14.9%]). CONCLUSIONS: 3-year longitudinal data reinforce evidence from previous studies that RealTime can be safely used in primary HPV-based cervical cancer screening. Concurrent partial genotyping for HPV16/18 should be strongly considered as a triage method for HPV screen-positive women.

Identification of a Novel Human Papillomavirus, Type HPV199, Isolated from a Nasopharynx and Anal Canal, and Complete Genomic Characterization of Papillomavirus Species Gamma-12.

The novel human papillomavirus type 199 (HPV199) was initially identified in a nasopharyngeal swab sample obtained from a 25 year-old immunocompetent male. The complete genome of HPV199 is 7,184 bp in length with a GC content of 36.5%. Comparative genomic characterization of HPV199 and its closest relatives showed the classical genomic organization of Gammapapillomaviruses (Gamma-PVs). HPV199 has seven major open reading frames (ORFs), encoding five early (E1, E2, E4, E6, and E7) and two late (L1 and L2) proteins, while lacking the E5 ORF. The long control region (LCR) of 513 bp is located between the L1 and E6 ORFs. Phylogenetic analysis additionally confirmed that HPV-199 clusters into the Gamma-PV genus, species Gamma-12, additionally containing HPV127, HV132, HPV148, HPV165, and three putative HPV types: KC5, CG2 and CG3. HPV199 is most closely related to HPV127 (nucleotide identity 77%). The complete viral genome sequence of additional HPV199 isolate was determined from anal canal swab sample. Two HPV199 complete viral sequences exhibit 99.4% nucleotide identity. To the best of our knowledge, this is the first member of Gamma-PV with complete nucleotide sequences determined from two independent clinical samples. To evaluate the tissue tropism of the novel HPV type, 916 clinical samples were tested using HPV199 type-specific real-time PCR: HPV199 was detected in 2/76 tissue samples of histologically confirmed common warts, 2/108 samples of eyebrow hair follicles, 2/137 anal canal swabs obtained from individuals with clinically evident anal pathology, 4/184 nasopharyngeal swabs and 3/411 cervical swabs obtained from women with normal cervical cytology. Although HPV199 was found in 1.4% of cutaneous and mucosal samples only, it exhibits dual tissue tropism. According to the results of our study and literature data, dual tropism of all Gamma-12 members is highly possible.

Specificity of the Linear Array HPV Genotyping Test for detecting human papillomavirus genotype 52 (HPV-52).

INTRODUCTION: HPV-52 is one of the most frequent human papillomavirus (HPV) genotypes causing significant cervical pathology. The most widely used HPV genotyping assay, the Roche Linear Array HPV Genotyping Test (Linear Array), is unable to identify HPV-52 status in samples containing HPV-33, HPV-35, and/or HPV-58. METHODS: Linear Array HPV-52 analytical specificity was established by testing 100 specimens reactive with the Linear Array HPV-33/35/52/58 cross-reactive probe, but not with the HPV-33, HPV-35, and/or HPV-58 individual probes, using an HPV-52-specific real-time PCR assay with a detection level of 3.9 DNA copies/reaction. In addition, we established the prevalence of HPV-52 in 49 samples reactive with the Linear Array HPV-33/35/52/58 cross-reactive probe, but containing either HPV-33, HPV-35, and/or HPV-58 using the same approach. RESULTS: The HPV-52-specific assay detected HPV-52 in all 100 samples reactive with the Linear Array HPV-33/35/52/58 cross-reactive probe, but not with the HPV-33, HPV-35, and/or HPV-58 probes. The presence of up to six other HPV genotypes did not influence the HPV-52 analytical performance of Linear Array. Only 6/49 (12.2%) samples reactive with the Linear Array HPV-33/35/52/58 cross-reactive probe, but containing either HPV-33, HPV-35 or/and HPV-58, were HPV-52 positive. CONCLUSIONS: Linear Array is a highly reliable test for detecting HPV-52 in the absence of genotypes HPV-33, HPV-35, and HPV-58. Additional testing using an HPV-52-specific test is necessary when HPV-33, HPV-35, and/or HPV-58 are present in the sample.

Pre-vaccination prevalence of infections with 25 non-high-risk human papillomavirus types among 1,000 Slovenian women in cervical cancer screening.

Cervical infections with non-high-risk human papillomavirus (non-HR-HPV) types have been associated with genital warts and a fraction of atypical squamous cells of undetermined significance and low-grade squamous intraepithelial lesions. The pre-vaccination prevalence of cervical infections with 25 non-HR-HPV types has been estimated, regardless of and without the coexistence of infection with HR-HPV types among Slovenian women 20-64 years old in cervical cancer screening, overall and according to age and cytology result. One thousand cervical specimens selected randomly from 4,455 specimens collected in 2010 in the Slovenian HPV prevalence survey were tested with Linear Array HPV Genotyping Test. Prevalence of cervical infections with any of the 25 non-HR-HPV types was 10.0% (95% CI: 8.1-11.9%) and with exclusively non-HR-HPV types 4.5% (95% CI: 3.2-5.8%). Prevalence of infections with any non-HR-HPV types among women with normal cytology was 8.8%, with atypical squamous cells of undetermined significance 30.4%, with low-grade squamous intraepithelial lesions 60.0%, and with high-grade squamous intraepithelial lesions 7.7%. Non-HR-HPV types without coexisting HR-HPV types were found in 4.0% of women with normal cytology, 26.1% with atypical squamous cells of undetermined significance, 6.7% with low-grade squamous intraepithelial lesions, and none with high-grade squamous intraepithelial lesion. Non-HR-HPV type cervical infections without coexisting HR-HPV infections were common among Slovenian women in cervical cancer screening with atypical squamous cells of undetermined significance, while rare in those with low-grade squamous intraepithelial lesions or worse. J. Med. Virol. 86: 1772-1779, 2014. © 2014 Wiley Periodicals, Inc.

Global genomic diversity of human papillomavirus 6 based on 724 isolates and 190 complete genome sequences.

UNLABELLED: Human papillomavirus type 6 (HPV6) is the major etiological agent of anogenital warts and laryngeal papillomas and has been included in both the quadrivalent and nonavalent prophylactic HPV vaccines. This study investigated the global genomic diversity of HPV6, using 724 isolates and 190 complete genomes from six continents, and the association of HPV6 genomic variants with geographical location, anatomical site of infection/disease, and gender. Initially, a 2,800-bp E5a-E5b-L1-LCR fragment was sequenced from 492/530 (92.8%) HPV6-positive samples collected for this study. Among them, 130 exhibited at least one single nucleotide polymorphism (SNP), indel, or amino acid change in the E5a-E5b-L1-LCR fragment and were sequenced in full. A global alignment and maximum likelihood tree of 190 complete HPV6 genomes (130 fully sequenced in this study and 60 obtained from sequence repositories) revealed two variant lineages, A and B, and five B sublineages: B1, B2, B3, B4, and B5. HPV6 (sub)lineage-specific SNPs and a 960-bp representative region for whole-genome-based phylogenetic clustering within the L2 open reading frame were identified. Multivariate logistic regression analysis revealed that lineage B predominated globally. Sublineage B3 was more common in Africa and North and South America, and lineage A was more common in Asia. Sublineages B1 and B3 were associated with anogenital infections, indicating a potential lesion-specific predilection of some HPV6 sublineages. Females had higher odds for infection with sublineage B3 than males. In conclusion, a global HPV6 phylogenetic analysis revealed the existence of two variant lineages and five sublineages, showing some degree of ethnogeographic, gender, and/or disease predilection in their distribution. IMPORTANCE: This study established the largest database of globally circulating HPV6 genomic variants and contributed a total of 130 new, complete HPV6 genome sequences to available sequence repositories. Two HPV6 variant lineages and five sublineages were identified and showed some degree of association with geographical location, anatomical site of infection/disease, and/or gender. We additionally identified several HPV6 lineage- and sublineage-specific SNPs to facilitate the identification of HPV6 variants and determined a representative region within the L2 gene that is suitable for HPV6 whole-genome-based phylogenetic analysis. This study complements and significantly expands the current knowledge of HPV6 genetic diversity and forms a comprehensive basis for future epidemiological, evolutionary, functional, pathogenicity, vaccination, and molecular assay development studies.

The Abbott RealTime High Risk HPV test is a clinically validated human papillomavirus assay for triage in the referral population and use in primary cervical cancer screening in women 30 years and older: a review of validation studies.

INTRODUCTION: Human papillomavirus (HPV) testing has become an essential part of current clinical practice in the management of cervical cancer and precancerous lesions. We reviewed the most important validation studies of a next-generation real-time polymerase chain reaction-based assay, the RealTime High Risk HPV test (RealTime)(Abbott Molecular, Des Plaines, IL, USA), for triage in referral population settings and for use in primary cervical cancer screening in women 30 years and older published in peer-reviewed journals from 2009 to 2013. RealTime is designed to detect 14 high-risk HPV genotypes with concurrent distinction of HPV-16 and HPV-18 from 12 other HPV genotypes. The test was launched on the European market in January 2009 and is currently used in many laboratories worldwide for routine detection of HPV. METHODS: We concisely reviewed validation studies of a next-generation real-time polymerase chain reaction (PCR)-based assay: the Abbott RealTime High Risk HPV test. RESULTS: Eight validation studies of RealTime in referral settings showed its consistently high absolute clinical sensitivity for both CIN2+ (range 88.3-100%) and CIN3+ (range 93.0-100%), as well as comparative clinical sensitivity relative to the currently most widely used HPV test: the Qiagen/Digene Hybrid Capture 2 HPV DNA Test (HC2). Due to the significantly different composition of the referral populations, RealTime absolute clinical specificity for CIN2+ and CIN3+ varied greatly across studies, but was comparable relative to HC2. Four validation studies of RealTime performance in cervical cancer screening settings showed its consistently high absolute clinical sensitivity for both CIN2+ and CIN3+, as well as comparative clinical sensitivity and specificity relative to HC2 and GP5+/6+ PCR. CONCLUSION: RealTime has been extensively evaluated in the last 4 years. RealTime can be considered clinically validated for triage in referral population settings and for use in primary cervical cancer screening in women 30 years and older.