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Cited by 61Cervical Cancer and HPV ResearchWomen's cancer prevention and managementReproductive tract infections research

Clinical validation of Anyplex™ II HPV HR Detection according to the guidelines for HPV test requirements for cervical cancer screening

Open access
Hesselink, A.T., Sahli, R., Berkhof, J., Snijders, P.J.F., van der Salm, M.L., Agard, D., Bleeker, M.C.G., Heideman, D.A.M.·2016·Journal of Clinical Virology
DOI 10.1016/j.jcv.2016.01.009
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Abstract

BACKGROUND: Anyplex™ II HPV HR Detection (Seegene, Seoul, Korea) is a multiplex real-time PCR using tagging oligonucleotide cleavage and extension (TOCE) technology for simultaneous detection and genotyping of 14 high-risk (HR) HPV types, including HPV16 and HPV18. OBJECTIVES: To evaluate whether the clinical performance and reproducibility of Anyplex™ II HPV HR Detection meet the international consensus guidelines for HPV test requirements for cervical cancer screening [1]. STUDY DESIGN: The clinical performance of Anyplex™ II HPV HR Detection for detecting cervical intraepithelial neoplasia grade 2 or worse (CIN2+) was determined relative to that of the reference assay, i.e., HR HPV GP5+/6+-PCR-EIA, by analysis of a total of 879 cervical liquid based cytology (LBC) specimens from a screening population, of which 60 were from women with CIN2+. The intra-laboratory reproducibility and inter-laboratory agreement were determined on 509 LBC samples, of which 172 were positive by the reference assay. RESULTS: Anyplex™ II HPV HR Detection showed a clinical sensitivity for CIN2+ of 98.3% (59/60; 95% CI: 89.1-99.8) and a clinical specificity for CIN2+ of 93.6% (764/816; 95% CI: 89.8-96.1). The clinical sensitivity and specificity were non-inferior to those of HR HPV GP5+/6+-PCR-EIA (non-inferiority score test: P=0.005 and P=0.023, respectively). Both intra-laboratory reproducibility (96.8%; 95% CI: 95.3-98.1; kappa value of 0.93) and inter-laboratory agreement (96.0%; 95% CI: 94.3-97.4; kappa value of 0.91) were high. CONCLUSIONS: Anyplex™ II HPV HR Detection performs clinically non-inferior to HR HPV GP5+/6+-PCR-EIA. Anyplex™ II HPV HR Detection complies with international consensus validation metrics for HPV DNA tests for cervical cancer screening [1].

AI snapshot

Written from the paper’s full text.

TL;DR

Anyplex™ II HPV HR Detection showed clinical sensitivity and specificity for CIN2+ non-inferior to HR HPV GP5+/6+-PCR-EIA in cervical liquid-based cytology samples, with high intra- and inter-laboratory reproducibility. The authors conclude the assay meets international consensus validation metrics for HPV DNA tests in cervical cancer screening.

Key findings

  • Clinical sensitivity for CIN2+ was 98.3% (59/60; 95% CI: 89.1–99.8) and clinical specificity was 93.6% (764/816; 95% CI: 89.8–96.1).
  • Sensitivity and specificity were non-inferior to GP5+/6+-PCR-EIA (non-inferiority score test: P = 0.005 and P = 0.023, respectively); relative clinical sensitivity was 1.00 and relative specificity 0.99.
  • In the Results, intra-laboratory reproducibility was 96.0% (485/505; 95% CI: 94.3–97.4; kappa 0.91) and inter-laboratory agreement was 96.8% (489/505; 95% CI: 95.3–98.1; kappa 0.93). The abstract reports these two values with the percentages/kappas swapped.
  • Genotype agreement was moderate to perfect: overall kappa 0.87 for intra-laboratory and 0.89 for inter-laboratory genotyping. Most discrepant samples had low signal intensity (18/20 intra-lab and 13/16 inter-lab; P = 0.0008 and P = 0.007, respectively).

Methods

  • Design: clinical validation study comparing Anyplex™ II HPV HR Detection with reference HR HPV GP5+/6+-PCR-EIA, with testing blinded to prior reference results and cyto-/histopathology outcomes; non-inferiority thresholds were relative sensitivity 90% and relative specificity 98%, and reproducibility required lower confidence bound ≥87% with kappa ≥0.5.
  • Samples: 879 cervical LBC specimens for clinical performance, including 60 histologically confirmed CIN2+ cases (25 CIN2, 31 CIN3, 4 squamous cell carcinomas) and 819 controls with normal cytology and no CIN2+ within 2 years; 509 LBC samples for reproducibility, of which 172/509 (33.8%) were reference-assay positive.
  • Analysis: clinical sensitivity/specificity for CIN2+ compared using a non-inferiority score test; intra- and inter-laboratory agreement and kappa values; genotype agreement by kappa; association between semi-quantitative viral load and discordance by chi-square test for trend.

Limitations

  • The paper does not include a dedicated limitations section.
  • Text-evident limitation: the CIN2+ sensitivity cases were selected from women with histologically confirmed CIN2+ detected by abnormal cytology and/or positive GP5+/6+-PCR, so they are not an unselected screening cohort; controls had normal cytology and no CIN2+ within 2 years.
  • Text-evident limitation: the abstract and Results report the intra- and inter-laboratory reproducibility percentages and kappa values in opposite order, which creates ambiguity in the reported reproducibility figures.
  • Text-evident limitation: the study was funded by Seegene, and several authors disclose employment, minority stake, speaker bureau, consultancy, or advisory relationships with relevant companies; the paper states the funder had no influence on design or analysis.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1016/j.jcv.2016.01.009

Cites

6 of 6 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

4 of 10 cited sources checked · 3 supported · 0 flagged · accuracy not yet measured

  • Study design and conductcitation

    Clinical sensitivity and specificity values for CIN2+ were compared using a non-inferiority score test with relative sensitivity threshold 90% and relative specificity threshold 98% as proposed in consensus guidelines.

    “Clinical sensitivity and specificity values for CIN2+ of Anyplex TM II HPV HR Detection were compared to those of GP5+6+-PCR using a non-inferiority score test, as described by Tang et al. [6], with a relative sensitivity threshold for CIN2+ of 90% and a relative specificity threshold for CIN2+ of 98% as proposed in the consensus guidelines [1].”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [6] source
    • cites [1] source
  • Interpretationown data

    Anyplex II HPV HR Detection complies with international consensus validation metrics for HPV DNA tests for cervical cancer screening.

    “Anyplex TM II HPV HR Detection complies with international consensus validation metrics for HPV DNA tests for cervical cancer screening [1].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [1] source

      The ‘Validation guidelines for candidate HPV assays’ section sets criteria for HPV tests used in cervical screening, including reproducibility and inter-laboratory agreement. The paper identifies hc2 and GP5+/6+-PCR EIA as meeting its requirements, but it does not assess Anyplex II HPV HR Detection or establish that it complies with them. Page numbers are unavailable in our copy. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
  • Findingown data

    Genotyping data of Anyplex II HPV HR Detection turned out to be highly reproducible in this study, with moderate to perfect agreement.

    “Genotyping data turned out to be highly reproducible in this study as well, with moderate to perfect agreement.”
    Quote in the paperSubstantial claim
  • Findingown data

    Inter-laboratory agreement of Anyplex II HPV HR Detection was 96.0% (95% CI: 94.3-97.4) with kappa 0.91.

    “and inter-laboratory agreement (96.0%; 95% CI: 94.3-97.4;kappa value of 0.91) were high.”
    Quote in the paperSubstantial claim
  • Findingown data

    Intra-laboratory reproducibility of Anyplex II HPV HR Detection was 96.8% (95% CI: 95.3-98.1) with kappa 0.93.

    “Both intra-laboratory reproducibility (96.8%; 95% CI: 95.3-98.1;kappa value of 0.93)”
    Quote in the paperSubstantial claim
  • Classified as probably not a substantial claim. 6
  • May combine two claims. 10

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    The relative sensitivity threshold for CIN2+ of 90% and relative specificity threshold for CIN2+ of 98% are proposed in the consensus guidelines.

    “with a relative sensitivity threshold for CIN2+ of 90% and a relative specificity threshold for CIN2+ of 98% as proposed in the consensus guidelines [1].”
    Quote in the paperSubstantial claim1 of 1 sources checked

  • May combine two claims.

    Interpretationown data

    Both intra-laboratory reproducibility and inter-laboratory agreement fulfilled the validation metrics, with lower confidence bound of percentage of agreement >87% and kappa >0.5.

    “Both the intra-laboratory reproducibility over time and the inter-laboratory agreement fulfilled the validation metrics given a lower confidence bound of percentage of agreement that was higher than 87%, and a corresponding kappa value that was higher than 0.5 [1].”
    Quote in the paperSubstantial claim1 of 1 sources checked

  • May combine two claims.

    Findingown data

    The clinical sensitivity and specificity of Anyplex II HPV HR Detection for CIN2+ were non-inferior to those of HR HPV GP5+/6+-PCR-EIA (non-inferiority score test P=0.005 and P=0.023, respectively).

    “The clinical sensitivity and specificity were non-inferior to those of HR HPV GP5+/6+-PCR-EIA (non-inferiority score test: P = 0.005 and P = 0.023, respectively).”
    • Also stated in: § 4.1. Clinical sensitivity and clinical specificity · plain
      “Both clinical sensitivity and specificity for CIN2+ of Anyplex TM II HPV HR Detection were non-inferior to that of GP5+6+-PCR, i.e., relative clinical sensitivity for CIN2+ of 1.00 (P = 0.005) and relative clinical specificity for CIN2+ of 0.99 (P = 0.023).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Discrepant results at the type-specific level were most commonly found for HPV39 and HPV45, and mostly concerned multiple infections and low signal intensities for respective types.

    “Discrepant results at the type-specific level, that were most commonly found for HPV39 and HPV45, mostly concerned multiple infections and low signal intensities for respective types.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Inter-laboratory genotyping agreement was moderate to perfect, with overall kappa 0.89 (range 0.54-1.00).

    “At the genotype level, moderate to perfect agreement was observed, with overall kappa of 0.87 (range: 0.50-1.00)and 0.89 (range: 0.54-1.00)for intra-and inter-laboratory genotyping agreement, respectively (Tables 3A and 3B ).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Most samples with discrepant findings (18/20 intra-lab and 13/16 inter-lab) had low signal intensity (score +), suggesting association of discrepant results with low viral loads (P=0.0008 and P=0.007, respectively).

    “Most samples with discrepant findings (18/20 in intralab and 13/16 in inter-lab analysis) had low signal intensity (score +), suggesting association of discrepant results with low viral loads (P = 0.0008 and P = 0.007, respectively).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductown data

    Of the 509 samples, 506 (99.4%) had a valid test result in run 1 at lab A, 507 (99.6%) in run 2 at lab A, and one sample had insufficient material left for testing at lab B, leaving 505 samples with valid data for both intra- and inter-laboratory analyses.

    “Of the 509 samples, 506 (99.4%) had a valid test result in run 1 at lab A, 507 (99.6%) in run 2 at lab A, and one sample had insufficient material left for testing at lab B, leaving 505 samples with valid data for both intra-and inter-laboratory analyses.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductown data

    The reproducibility testing was performed in a blinded manner: two portions tested within Lab A by different technicians at different time points, and the third set analyzed blinded for Lab A results at Lab B.

    “Two portions were tested in a blinded manner within the same laboratory (Lab A; VU University Medical Center, Amsterdam, The Netherlands) by different technicians at different time points.The third set was analyzed blinded for the results of lab A, at another laboratory (Lab B; Institute of Microbiology, CHUV, University of Lausanne, Lausanne, Switzerland).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Several HPV assays have partially or completely been clinically validated using these criteria.

    “Several HPV assays have partially or completely been clinically validated using these criteria [3].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [3] source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    This study followed the local ethical guidelines of VU University Medical Center.

    “This study followed the local ethical guidelines of VU University Medical Center.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Anyplex II HPV HR Detection is a multiplex real-time PCR using tagging oligonucleotide cleavage and extension (TOCE) technology for simultaneous detection and genotyping of 14 high-risk HPV types, including HPV16 and HPV18.

    “Anyplex TM II HPV HR Detection (Seegene, Seoul, Korea) is a multiplex real-time PCR using tagging oligonucleotide cleavage and extension (TOCE) technology for simultaneous detection and genotyping of 14 high-risk (HR) HPV types, including HPV16 and HPV18.”
    • Also stated in: § 1. Background · plain
      “The multiplex real-time PCR design using tagging oligonucleotide cleavage and extension (TOCE) technology allows for simultaneous detection and genotyping of 14 high-risk (HR) HPV types, including HPV16,-18,-31,-33,-35,-39,-45,-51,-52,-56,-58,-59,-66, and -68 (L1 gene), and an internal control (human beta-globin) in a single reaction [4].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [4] source

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductcitation

    Anyplex II HPV HR Detection was performed blinded for earlier obtained GP5+/6+-PCR-EIA results and cyto-/histopathology outcomes, and data were correlated afterwards.

    “Anyplex TM II HPV HR Detection was performed blinded for earlier obtained GP5+/6+-PCR-EIA results [5] and cyto-/histopathology outcomes, and data were correlated afterwards.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [5] source
12 of 12

  • The paper relies on this without support.

    Anyplex II HPV HR Detection had not yet been evaluated according to international consensus validation metrics.

    “An assay not yet evaluated according to these international consensus validation metrics is Anyplex TM II HPV HR Detection (Seegene, Seoul, Korea).”

  • The paper relies on this without support.

    The 60 cervical LBC samples from women with CIN2+ used for clinical sensitivity analysis are representative of women with CIN2+ in the screening population.

    “For clinical sensitivity analysis, a representative set of 60 cervical liquid based cytology (LBC) samples (PreservCyt) from women participating in population-based screening in the Netherlands, who were diagnosed with histologically confirmed CIN2+, were used.”

  • The paper relies on this without support.

    The 819 consecutively collected LBC samples from women with normal cytology and without evidence of CIN2+ within 2 years of follow-up are representative of women without CIN2+ in the screening population.

    “For clinical specificity analysis, we used 819 consecutively collected LBC samples (PreservCyt) from the screening population of women (median age of 41 years, range 31-60 years) with normal cytology and without evidence of CIN2+ within 2 years of follow-up.”

  • The paper relies on this without support.

    The 2-year follow-up without evidence of CIN2+ is sufficient to classify samples as controls without CIN2+.

    “For clinical specificity analysis, we used 819 consecutively collected LBC samples (PreservCyt) from the screening population of women (median age of 41 years, range 31-60 years) with normal cytology and without evidence of CIN2+ within 2 years of follow-up.”
4 of 4

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • The Anyplex assay has reported sensitivities ranging from 92.5 to 98.3% and specificities from 81.7 to 94.1% for detecting CIN2+.

    “For example, the Anyplex assay has been reported with sensitivities ranging from 92.5 to 98.3% and specificities from 81.7 to 94.1% for detecting cervical intraepithelial neoplasia grade 2 or worse (CIN2+) [16, 17, 18].”

    From Comparative analysis of SD biosensor standard™ M10 HPV and seegene anyplex™ II HPV HR for detecting high-risk human papillomavirus: a concordance study (2025) · cites this paper as [17]

    The Results passage in the indexed copy (Abstract; page not provided) reports 98.3% sensitivity and 93.6% specificity for detecting CIN2+, both within the cited ranges. It does not report the 92.5% sensitivity or the 81.7% and 94.1% specificity endpoints, so this source alone does not establish the full ranges attributed to the three citations. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Abstract swaps intra- and inter-laboratory results

    Minor

    The abstract assigns 96.8% agreement and kappa 0.93 to intra-laboratory reproducibility, and 96.0% and kappa 0.91 to inter-laboratory agreement. The Results section assigns those values the other way around. Both sets of values still exceed the stated validation thresholds.

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Authors
Hesselink, A. T., Sahli, R., Berkhof, J., Snijders, P. J. F., van der Salm, M. L., Agard, D., Bleeker, M. C. G., & Heideman, D. A. M.
Affiliations
  • Self-screen BV (Netherlands) (NL)
  • Centre Hospitalier Universitaire Vaudois (CH)
  • Amsterdam UMC Location Vrije Universiteit Amsterdam (NL)
  • Vrije Universiteit Amsterdam (NL)
Corresponding author
Heideman, D.A.M.
ORCID
Venue
Journal of Clinical Virology
Publisher
Elsevier BV
Published
2016-03
Volume
76
Pages
36-39
ISSN
1386-6532 (ISSN-L)
DOI
10.1016/j.jcv.2016.01.009
OpenAlex
W2260212312
Semantic Scholar
62100de09d6fc1ffc3c3ec71a387f691b5a74888
Cited by
61
References
5
Citations by year
2026: 5 · 2025: 3 · 2024: 7 · 2023: 3 · 2022: 8 · 2021: 8 · 2020: 4 · 2019: 8 · 2018: 8 · 2017: 4 · 2016: 3
FWCI
6.27
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Clinical Virology (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • Amsterdam UMC (VU Amsterdam) - Institutional Repository (publishedVersion)
  • IRIS (submittedVersion)
  • Pure Amsterdam UMC (submittedVersion)
  • SERVAL (Université de Lausanne) (submittedVersion, other-oa)
  • Data Archiving and Networked Services (DANS) (submittedVersion)
Topics
  • Health Sciences › Medicine › Epidemiology › Cervical Cancer and HPV Research
  • Health Sciences › Medicine › Public Health, Environmental and Occupational Health › Women's cancer prevention and management
  • Life Sciences › Immunology and Microbiology › Microbiology › Reproductive tract infections research
Keywords
high-risk HPV genotyping, cervical cancer screening, cervical intraepithelial neoplasia, multiplex real-time PCR, HPV16, HPV 18, test validation, non-inferiority, liquid-based cytology, interlaboratory agreement
MeSH terms
Adult, Uterine Cervical Dysplasia, Uterine Cervical Neoplasms, Colposcopy, Female, Genotype, Humans, Mass Screening, Middle Aged, Pregnancy, Reagent Kits, Diagnostic, Sensitivity and Specificity, Reproducibility of Results, Papillomavirus Infections, Alphapapillomavirus, Early Detection of Cancer, Republic of Korea, Real-Time Polymerase Chain Reaction

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