Prevalence of Human Papillomavirus Infection in Unselected SurePath Samples Using the APTIMA HPV mRNA Assay
2013; Elsevier BV; Volume: 15; Issue: 5 Linguagem: Inglês
10.1016/j.jmoldx.2013.04.002
ISSN1943-7811
AutoresMatejka Rebolj, Sarah Preisler, Ditte Møller Ejegod, Jesper Bonde, Carsten Rygaard, Elsebeth Lynge,
Tópico(s)Hepatitis B Virus Studies
ResumoThe APTIMA Human Papillomavirus (HPV) Assay detects E6/E7 mRNA from 14 human papillomavirus genotypes. Horizon was a population-based split-sample study among well-screened women, with an aim to compare APTIMA, Hybrid Capture 2 (HC2), and liquid-based cytology (LBC) using SurePath samples. APTIMA testing on the PANTHER platform, and HC2 testing on the Rapid Capture System were performed in accordance with protocols agreed on with the manufacturers before the study, on 5070 consecutive, routine, cervical cytology samples from Copenhagen, Denmark. In this high-risk population, 17% of all samples tested positive on APTIMA, 20% of samples tested positive on HC2, and 7% of samples had abnormal cytology. Among the 4411 samples without recent abnormalities, 15% tested positive on APTIMA, 19% tested positive on HC2, and 5% had abnormal cytology. The κ coefficient of 0.75 suggested substantial agreement between APTIMA and HC2. This is the first APTIMA study using SurePath samples on the PANTHER platform. The trends in positivity rates on SurePath samples for APTIMA, HC2, and LBC were consistent with studies based on PreservCyt samples, and the agreement between the two HPV assays was substantial. The high proportions of women testing positive suggest that in countries with a high HPV prevalence, caution will be needed if HPV tests, including mRNA-based tests, are to replace LBC. The APTIMA Human Papillomavirus (HPV) Assay detects E6/E7 mRNA from 14 human papillomavirus genotypes. Horizon was a population-based split-sample study among well-screened women, with an aim to compare APTIMA, Hybrid Capture 2 (HC2), and liquid-based cytology (LBC) using SurePath samples. APTIMA testing on the PANTHER platform, and HC2 testing on the Rapid Capture System were performed in accordance with protocols agreed on with the manufacturers before the study, on 5070 consecutive, routine, cervical cytology samples from Copenhagen, Denmark. In this high-risk population, 17% of all samples tested positive on APTIMA, 20% of samples tested positive on HC2, and 7% of samples had abnormal cytology. Among the 4411 samples without recent abnormalities, 15% tested positive on APTIMA, 19% tested positive on HC2, and 5% had abnormal cytology. The κ coefficient of 0.75 suggested substantial agreement between APTIMA and HC2. This is the first APTIMA study using SurePath samples on the PANTHER platform. The trends in positivity rates on SurePath samples for APTIMA, HC2, and LBC were consistent with studies based on PreservCyt samples, and the agreement between the two HPV assays was substantial. The high proportions of women testing positive suggest that in countries with a high HPV prevalence, caution will be needed if HPV tests, including mRNA-based tests, are to replace LBC. Screening with human papillomavirus (HPV) DNA tests offers excellent protection from cervical cancer,1Ronco G. Giorgi-Rossi P. Carozzi F. Confortini M. Dalla Palma P. Del Mistro A. Ghiringhello B. Girlando S. Gillio-Tos A. De Marco L. Naldoni C. Pierotti P. Rizzolo R. Schincaglia P. Zorzi M. Zappa M. Segnan N. Cuzick J. Efficacy of human papillomavirus testing for the detection of invasive cervical cancers and cervical intraepithelial neoplasia: a randomised controlled trial.Lancet Oncol. 2010; 11: 249-257Abstract Full Text Full Text PDF PubMed Scopus (763) Google Scholar, 2Rijkaart D.C. Berkhof J. Rozendaal L. van Kemenade F.J. Bulkmans N.W. Heideman D.A. Kenter G.G. Cuzick J. Snijders P.J. Meijer C.J. Human papillomavirus testing for the detection of high-grade cervical intraepithelial neoplasia and cancer: final results of the POBASCAM randomised controlled trial.Lancet Oncol. 2012; 13: 78-88Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar but many women with transient HPV infections test positive.3Rebolj M. Pribac I. Lynge E. False-positive human papillomavirus DNA tests in cervical screening: it is all in a definition.Eur J Cancer. 2011; 47: 255-261Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar Integration of HPV into the host cell genome leads to expression of E6 and E7 oncogenes, causing disruption in p53 and pRb cell-cycle control pathways.4Doorbar J. Molecular biology of human papillomavirus infection and cervical cancer.Clin Sci (Lond). 2006; 110: 525-541Crossref PubMed Scopus (759) Google Scholar Therefore, detection of E6/E7 mRNA (mRNA) from HPV may be more specific for the detection of high-grade cervical intraepithelial neoplasia (CIN) than HPV DNA testing.5Cuschieri K. Wentzensen N. Human papillomavirus mRNA and p16 detection as biomarkers for the improved diagnosis of cervical neoplasia.Cancer Epidemiol Biomarkers Prev. 2008; 17: 2536-2545Crossref PubMed Scopus (214) Google Scholar The Food and Drug Administration–approved APTIMA HPV Assay (Hologic/Gen-Probe, San Diego, CA) is an in vitro nucleic acid amplification test for qualitative detection of E6/E7 viral mRNA from 14 HPV genotypes (genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68; Hologic/Gen-Probe, San Diego, CA) detected in combination. Of these, 13 are known to be carcinogenic in humans, and 1 (genotype 66) is possibly carcinogenic in humans.7Szarewski A. Ambroisine L. Cadman L. Austin J. Ho L. Terry G. Liddle S. Dina R. McCarthy J. Buckley H. Bergeron C. Soutter P. Lyons D. Cuzick J. Comparison of predictors for high-grade cervical intraepithelial neoplasia in women with abnormal smears.Cancer Epidemiol Biomarkers Prev. 2008; 17: 3033-3042Crossref PubMed Scopus (206) Google Scholar The assay has an internal control spiked in each reaction, and these controls monitor each step of the process. APTIMA's sensitivity for high-grade CIN appears to be similar to that of HPV DNA tests, and much better than that of cytology, which historically has been the standard cervical screening test. At the same time, studies indicate that APTIMA has better clinical specificity than HPV DNA tests.7Szarewski A. Ambroisine L. Cadman L. Austin J. Ho L. Terry G. Liddle S. Dina R. McCarthy J. Buckley H. Bergeron C. Soutter P. Lyons D. Cuzick J. Comparison of predictors for high-grade cervical intraepithelial neoplasia in women with abnormal smears.Cancer Epidemiol Biomarkers Prev. 2008; 17: 3033-3042Crossref PubMed Scopus (206) Google Scholar, 8Waldstrom M. Ornskov D. Comparison of the clinical performance of an HPV mRNA test and an HPV DNA test in triage of atypical squamous cells of undetermined significance (ASC-US).Cytopathology. 2012; 23: 389-395Crossref PubMed Scopus (22) Google Scholar, 9Ovestad I.T. Vennestrom U. Andersen L. Gudlaugsson E. Munk A.C. Malpica A. Feng W. Voorhorst F. Janssen E.A. Baak J.P. Comparison of different commercial methods for HPV detection in follow-up cytology after ASCUS/LSIL, prediction of CIN2-3 in follow up biopsies and spontaneous regression of CIN2-3.Gynecol Oncol. 2011; 123: 278-283Crossref PubMed Scopus (46) Google Scholar, 10Dockter J. Schroder A. Hill C. Guzenski L. Monsonego J. Giachetti C. Clinical performance of the APTIMA HPV Assay for the detection of high-risk HPV and high-grade cervical lesions.J Clin Virol. 2009; 45: S55-S61Abstract Full Text PDF PubMed Scopus (101) Google Scholar, 11Monsonego J. Hudgens M.G. Zerat L. Zerat J.C. Syrjanen K. Halfon P. Ruiz F. Smith J.S. Evaluation of oncogenic human papillomavirus RNA and DNA tests with liquid-based cytology in primary cervical cancer screening: the FASE study.Int J Cancer. 2010; 129: 691-701Crossref PubMed Scopus (124) Google Scholar, 12Ratnam S. Coutlee F. Fontaine D. Bentley J. Escott N. Ghatage P. Gadag V. Holloway G. Bartellas E. Kum N. Giede C. Lear A. Aptima HPV E6/E7 mRNA test is as sensitive as Hybrid Capture 2 Assay but more specific at detecting cervical precancer and cancer.J Clin Microbiol. 2011; 49: 557-564Crossref PubMed Scopus (104) Google Scholar, 13Clad A. Reuschenbach M. Weinschenk J. Grote R. Rahmsdorf J. Freudenberg N. Performance of the APTIMA high-risk HPV mRNA assay in a referral population in comparison with Hybrid Capture 2 and cytology.J Clin Microbiol. 2011; 49: 1071-1076Crossref PubMed Scopus (49) Google Scholar, 14Wu R. Belinson S.E. Du H. Na W. Qu X. Wu R. Liu Y. Wang C. Zhou Y. Zhang L. Belinson J.L. Human papillomavirus messenger RNA assay for cervical cancer screening: the Shenzhen Cervical Cancer Screening Trial I.Int J Gynecol Cancer. 2010; 20: 1411-1414Crossref PubMed Scopus (38) Google Scholar, 15Reuschenbach M. Clad A. von Knebel D.C. Wentzensen N. Rahmsdorf J. Schaffrath F. Griesser H. Freudenberg N. von Knebel D.M. Performance of p16INK4a-cytology. HPV mRNA, and HPV DNA testing to identify high grade cervical dysplasia in women with abnormal screening results.Gynecol Oncol. 2010; 119: 98-105Crossref PubMed Scopus (58) Google Scholar, 16Arbyn M. Roelens J. Cuschieri K. Cuzick J. Szarewski A. Ratnam S. Reuschenbach M. Belinson S. Belinson J.L. Monsonego J. The APTIMA HPV assay versus the hybrid capture 2 test in triage of women with ASC-USor LSIL cervical cytology: a meta-analysisof the diagnostic accuracy.Int J Cancer. 2013; 132: 101-108Crossref PubMed Scopus (108) Google Scholar APTIMA has been validated for use only on samples stored in PreservCyt (Hologic, Boxborough, MA). Arbyn et al16Arbyn M. Roelens J. Cuschieri K. Cuzick J. Szarewski A. Ratnam S. Reuschenbach M. Belinson S. Belinson J.L. Monsonego J. The APTIMA HPV assay versus the hybrid capture 2 test in triage of women with ASC-USor LSIL cervical cytology: a meta-analysisof the diagnostic accuracy.Int J Cancer. 2013; 132: 101-108Crossref PubMed Scopus (108) Google Scholar have called for evaluation of APTIMA using other storage media. SurePath (BD Diagnostics–TriPath, Burlington, NC) is the other widely used medium; however, the formaldehyde-based fixative in SurePath represents a special challenge for molecular-based testing because it leads to extensive cross-linking of cellular proteins and nucleic acids, rendering the genomic material less easily accessible for extraction and subsequent analysis.17Horvath C.A. Boulet G. Sahebali S. Depuydt C. Vermeulen T. van den Broeck D. Vereecken A. Bogers J. Effects of fixation on RNA integrity in a liquid-based cervical cytology setting.J Clin Pathol. 2008; 61: 132-137Crossref PubMed Scopus (19) Google Scholar, 18Powell N. Smith K. Fiander A. Recovery of human papillomavirus nucleic acids from liquid-based cytology media.J Virol Methods. 2006; 137: 58-62Crossref PubMed Scopus (45) Google Scholar To some extent, this cross-linking can be reversed, and the amount of amplifiable RNA increased, by proteinase K treatment,18Powell N. Smith K. Fiander A. Recovery of human papillomavirus nucleic acids from liquid-based cytology media.J Virol Methods. 2006; 137: 58-62Crossref PubMed Scopus (45) Google Scholar, 19Murphy P.G. Henderson D.T. Adams M.D. Horlick E.A. Dixon E.P. King L.M. Avissar P.L. Brown C.A. Fischer T.J. Malinowski D.P. Isolation of RNA from cell lines and cervical cytology specimens stored in BD SurePath preservative fluid and downstream detection of housekeeping gene and HPV E6 expression using real time RT-PCR.J Virol Methods. 2009; 156: 138-144Crossref PubMed Scopus (19) Google Scholar which is part of the pre-analytical treatment for APTIMA (Schröder AI, Guzenski LI, Dockter J: Poster handout. Eurogin, February 17–20, 2010, Monte Carlo). Previous studies performed the testing using either the TIGRIS platform8Waldstrom M. Ornskov D. Comparison of the clinical performance of an HPV mRNA test and an HPV DNA test in triage of atypical squamous cells of undetermined significance (ASC-US).Cytopathology. 2012; 23: 389-395Crossref PubMed Scopus (22) Google Scholar, 10Dockter J. Schroder A. Hill C. Guzenski L. Monsonego J. Giachetti C. Clinical performance of the APTIMA HPV Assay for the detection of high-risk HPV and high-grade cervical lesions.J Clin Virol. 2009; 45: S55-S61Abstract Full Text PDF PubMed Scopus (101) Google Scholar, 20Waldstrom M. Ornskov D. Clinical performance of a human papillomavirus messenger RNA test (Aptima HPV Assay) on residual material from archived 3-year-old PreservCyt samples with low-grade squamous intraepithelial lesion.Arch Pathol Lab Med. 2011; 135: 1052-1056Crossref PubMed Scopus (24) Google Scholar or a semiautomated Direct Tube Sampling system.9Ovestad I.T. Vennestrom U. Andersen L. Gudlaugsson E. Munk A.C. Malpica A. Feng W. Voorhorst F. Janssen E.A. Baak J.P. Comparison of different commercial methods for HPV detection in follow-up cytology after ASCUS/LSIL, prediction of CIN2-3 in follow up biopsies and spontaneous regression of CIN2-3.Gynecol Oncol. 2011; 123: 278-283Crossref PubMed Scopus (46) Google Scholar, 10Dockter J. Schroder A. Hill C. Guzenski L. Monsonego J. Giachetti C. Clinical performance of the APTIMA HPV Assay for the detection of high-risk HPV and high-grade cervical lesions.J Clin Virol. 2009; 45: S55-S61Abstract Full Text PDF PubMed Scopus (101) Google Scholar, 12Ratnam S. Coutlee F. Fontaine D. Bentley J. Escott N. Ghatage P. Gadag V. Holloway G. Bartellas E. Kum N. Giede C. Lear A. Aptima HPV E6/E7 mRNA test is as sensitive as Hybrid Capture 2 Assay but more specific at detecting cervical precancer and cancer.J Clin Microbiol. 2011; 49: 557-564Crossref PubMed Scopus (104) Google Scholar A new, smaller, fully automated and Food and Drug Administration–approved platform PANTHER now also is available (all Hologic/Gen-Probe). An aim of Horizon, a population-based split-sample study, was to compare APTIMA with the Hybrid Capture 2 High-Risk HPV assay (HC2; Qiagen, Hilden, Germany) and liquid-based cytology (LBC) on unselected samples from Copenhagen, Denmark. Danish women are well screened but have a high prevalence of HPV infection and a high background risk of cervical cancer.21Clemmesen J. Nielsen A. The social distribution of cancer in Copenhagen, 1943 to 1947.Br J Cancer. 1951; 5: 159-171Crossref PubMed Scopus (62) Google Scholar, 22Kjaer S.K. Breugelmans G. Munk C. Junge J. Watson M. Iftner T. Population-based prevalence, type- and age-specific distribution of HPV in women before introduction of an HPV-vaccination program in Denmark.Int J Cancer. 2008; 123: 1864-1870Crossref PubMed Scopus (149) Google Scholar In Denmark, SurePath has an 80% market share, and gradual implementation of HPV-based screening therefore has to be based on SurePath samples. Unlike previous studies evaluating the APTIMA assay, the Horizon study was performed on samples stored in SurePath, and testing was performed on the PANTHER platform. The Department of Pathology at Hvidovre University Hospital in Copenhagen is accredited with Joint Commission International, and is the largest cervical cytology laboratory in Denmark. While the present study was ongoing, the Department of Pathology at Hvidovre University Hospital in Copenhagen evaluated approximately 66,000 cervical SurePath samples per year. It handles all cervical cytology from women living in the municipalities of Copenhagen and Frederiksberg (denoted as Copenhagen in the remainder of the article), regardless of the reason for sample collection. Since the 1960s, Copenhagen has been covered by an organized cervical screening program. At present, women aged 23 to 49 years are targeted for screening every 3 years, and women aged 50 to 65 years are targeted for screening every 5 years. In 2011, 76% of women in the Capital Region, which includes Copenhagen, were screened at least once within the recommended interval.23Styregruppen for DKLS: Årsrapport DKLS 2011: Dansk Kvalitetsdatabase for Livmoderhalskræftscreening [annual report 2011: Danish Quality Assurance database for cervical cancer screening]. Hvidovre, DKLS, 2012, pp 1–122Google Scholar Horizon was nested into routine laboratory practice. Samples were identified by the internal laboratory specimen identifier and a barcode before they were divided sequentially into racks of 48 samples. For this study, samples were collected from June 10, 2011, to August 25, 2011, equally from Monday to Friday. Based on capacity and processing considerations at the molecular pathology laboratory, the target number of samples was set to 5000. After completion of routine LBC evaluation [which includes HC2 triage of women aged 30 years and older with atypical squamous cells of undetermined significance (ASCUS)], approximately 2 mL of residual cellular suspension of SurePath per sample was collected for Horizon from each of the tubes in, at most, the first four racks processed on the sampling days. This method mimicked collection of unselected consecutive LBC samples, assuming that the time of day that the sample arrived in the laboratory was not associated with any specific characteristics. In accordance with the protocol agreed on with the assay manufacturers before the study, collected samples were diluted with 2 mL of SurePath (dilution factor, approximately 1:1) to obtain enough volume for additional testing, which will be reported separately (unpublished data). All testing was performed in the same laboratory and by the same staff. Cytologic evaluation of SurePath samples was performed by cytoscreeners, in collaboration with a pathologist in cases of abnormal findings. Reading of the specimens was assisted by the FocalPoint GS Imaging System (SlideWizard; BD, Burlington, NC). Cytologic findings were reported using the Bethesda 2001 system, and abnormalities were classified as ASCUS, low-grade squamous intraepithelial lesions, or high-grade squamous intraepithelial lesions or worse. The last category includes atypical squamous cells, which cannot exclude high-grade squamous intraepithelial lesions, atypical glandular cells, adenocarcinoma in situ, and carcinoma. Samples with ASCUS or worse (≥ASCUS) were considered to have abnormal cytology. Cytoscreeners and pathologists were blinded to the results of HPV testing in Horizon. For handling of SurePath samples, we followed a protocol provided for this study by Hologic/Gen-Probe. Diluted sample (1 mL) was aliquoted into an APTIMA Specimen Transfer Tube containing 2.9 mL of buffered solution (Hologic/Gen-Probe). Before APTIMA testing, proteinase K treatment was performed using the Pace 2 Fast Expression Kit containing 1 mL diluent and lyophilized reagent (all from Hologic/Gen-Probe). Reconstituted proteinase K (100 μL) was added to each Specimen Transfer Tube and incubated at 65°C for 2 hours. The treated specimen tube was stored at 2°C to 8°C until testing. Testing was performed on the PANTHER platform. After lysis of the cells, mRNA was captured using oligonucleotide probes attached to magnetic beads. The released target mRNA molecules were amplified using transcription-mediated amplification, in which two different primers, nucleotides, the T7 RNA polymerase and the Moloney murine leukemia virus reverse transcriptase with RNase H activity produced mRNA amplicons in an isothermal reaction. The amplicons were hybridized to DNA probes labeled with an acridinium ester detector molecule, leading to photon emission. The light signal (relative light units) was detected on a luminometer and a signal to cut-off ratio (S/CO) was calculated from the positive and negative calibrators loaded onto the machine. An internal control was included in each sample as supplied in the kit.6Bouvard V. Baan R. Straif K. Grosse Y. Secretan B. El Ghissassi F. Ibrahim-Tallaa L. Guha N. Freeman C. Galichet L. Cogliano V. A review of human carcinogens–part B: biological agents.Lancet Oncol. 2009; 10: 321-322Abstract Full Text Full Text PDF PubMed Google Scholar Positive APTIMA samples were defined in accordance with the manufacturer's specifications as an S/CO of 0.5 or greater. We also evaluated APTIMA's intralaboratory reproducibility in a routine setting, based on a selection of 361 negative and 375 positive APTIMA samples from different Horizon batches. These samples were retested once within an average of 8 days (range, 1 to 50 days). For samples with nonreproducible APTIMA results (disagreement between initial and retest outcomes), viral DNA was extracted from separate 1-mL aliquots using a LC Total Nucleic Assay kit on the automated LC96 MagnaPure platform (Roche Diagnostics, Rotkreuz, Switzerland) and subsequently analyzed using the CLART HPV2 genotyping assay (Genomica, Madrid, Spain) according to the manufacturer's specifications. CLART targets L1 genomic sequences of the DNA from 35 HPV genotypes. HC2 is widely considered the standard HPV DNA assay.24Meijer C.J. Berkhof J. Castle P.E. Hesselink A.T. Franco E.L. Ronco G. Arbyn M. Bosch F.X. Cuzick J. Dillner J. Heideman D.A. Snijders P.J. Guidelines for human papillomavirus DNA test requirements for primary cervical cancer screening in women 30 years and older.Int J Cancer. 2009; 124: 516-520Crossref PubMed Scopus (488) Google Scholar It is a hybridization assay detecting 13 high-risk genotypes (genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) in combination. The assay has no internal control for sufficiency of test material. Sample DNA was either denatured before testing by pretreating manually according to the manufacturer's protocol (n = 5043), or DNA was isolated and purified using the DSP AXpH DNA kit on the QIASymphony SP platform (Qiagen, Hilden, Germany) (n = 1086). Testing of these samples was performed on the automated Rapid Capture System (Qiagen, Gaithersburg, MD) using different scripts depending on pretreatment. A minority of samples used for routine triage of women aged 30 years and older with ASCUS were denatured and tested manually (n = 106). The remaining 23 samples were not tested with HC2. Positive HC2 samples were defined as those having a relative light unit/cut-off value of 1 or greater. The screening history of women from January 1, 2000, onward was retrieved from the Danish Pathology Data Bank.25Bjerregaard B. Larsen O.B. The Danish Pathology Register.Scand J Public Health. 2011; 39: 72-74Crossref PubMed Scopus (265) Google Scholar Horizon samples with an earlier diagnosis of cervical cancer, a CIN diagnosis up to 3 years earlier, ASCUS in the previous 15 months, or more severe cytologic abnormalities or a positive HPV test in the past 12 months were considered follow-up samples. Horizon samples with no recent abnormality were considered primary samples. Reflecting routine practice, primary samples included screening samples and a small proportion of samples taken by indication. Differences in the distributions of age, screening history, cytology, and HC2 results between the included and excluded samples were tested with the χ2 test. Trends in HPV positivity by age were tested with the Mantel-Haenszel χ2 test for trend. Agreement between the APTIMA and HC2 assays was measured by the κ coefficient. The 95% CIs for the differences in the relative proportions (RPs) of women testing positive on APTIMA or HC2 by cytology grade were calculated by assuming that the logarithms of RPs were approximately normally distributed. Horizon was designed as a quality development study, using only residual material that otherwise would have been discarded. According to Danish regulations of biomedical research, published on May 5, 2011, in the Guidelines about Notification of a Biomedical Research Project to the Committee System on Biomedical Research Ethics no. 9154 section 2.5, quality development studies do not require ethical approval. Of the 12,138 samples processed by the laboratory during the sample collection period, 6258 (52%) were collected and processed for the Horizon study. All collected samples were tested with LBC, and all but 23 samples (0.4%, for technical reasons) were tested with HC2. In total, 1188 (19.0%) of the 6258 samples were not tested with APTIMA owing to insufficient volume for the complete study (n = 1165) or because of technical reasons (n = 23), and were excluded from the analysis. There was no significant difference in the distribution of age (P = 0.08), cytology (P = 0.46), or HC2 result (P = 0.31) between the 5070 included and the 1188 excluded samples. There was a small but significant difference in women's screening history, with 87.0% of the included and 89.9% of the excluded samples being primary (P = 0.01). Among the 5070 samples, 5011 (98.8%) women had one sample collected during the course of the study, whereas 59 (1.2%) samples were from 29 women, reflecting consecutive sampling from routine practice. The mean age of the women was 37.3 years (SD, 12.3 years; range, 16 to 89 years), and 64% of the samples were obtained from women younger than age 40 years (Table 1), reflecting a predominantly young source population (Statistics Denmark, http://www.statbank.dk/folk1; last accessed, April 11, 2013). The mean number of days between the receipt of the specimen in the laboratory and storage in the APTIMA Specimen Transport Tubes was 2 (range, 1 to 5 days). The mean number of days between storing and proteinase K treatment was 81 (range, 45 to 112 days); and the mean number of days between proteinase K treatment and testing with APTIMA was 12 (range, 0 to 25 days). All of these times were in accordance with the protocol agreed on with the manufacturer before the study.Table 1APTIMA Results for 5070 Primary and Follow-Up Samples by Age, Screening History, Cytology, and HC2 ResultsCharacteristicsResults on APTIMA, N (%)PositiveNegativeInvalidTotalTotal849 (16.7%)4220 (83.2%)1 (0.0%)5070 (100%)Age, years 16–2272 (44.4%)90 (55.6%)0 (0.0%)162 (100%) 23–29427 (27.8%)1108 (72.2%)0 (0.0%)1535 (100%) 30–39225 (14.7%)1301 (85.3%)0 (0.0%)1526 (100%) 40–4982 (8.3%)911 (91.7%)0 (0.0%)993 (100%) 50–5927 (5.3%)479 (94.5%)1 (0.2%)507 (100%) 60–659 (3.8%)225 (96.2%)0 (0.0%)234 (100%) >657 (6.2%)106 (93.8%)0 (0.0%)113 (100%)Screening history Primary sample676 (15.3%)3734 (84.7%)1 (0.0%)4411 (100%) Follow-up sample173 (26.3%)486 (73.7%)0 (0.0%)659 (100%)Cytology Normal587 (12.6%)4083 (87.4%)1 (0.0%)4671 (100%) ASCUS58 (47.2%)65 (52.8%)0 (0.0%)123 (100%) LSIL110 (76.9%)33 (23.1%)0 (0.0%)143 (100%) ≥HSIL93 (86.9%)14 (13.1%)0 (0.0%)107 (100%) ≥ASCUS261 (70.0%)112 (30.0%)0 (0.0%)373 (100%) Inadequate1 (3.8%)25 (96.2%)0 (0.0%)26 (100%)HC2∗Six samples were not tested with HC2. Positive747 (72.2%)288 (27.8%)0 (0.0%)1035 (100%) Negative99 (2.5%)3929 (97.5%)1 (0.0%)4029 (100%)≥HSIL, high-grade intraepithelial lesions or worse; LSIL, low-grade squamous intraepithelial lesions.∗ Six samples were not tested with HC2. Open table in a new tab ≥HSIL, high-grade intraepithelial lesions or worse; LSIL, low-grade squamous intraepithelial lesions. Overall, APTIMA testing had to be repeated on 11 (0.2%) samples to obtain valid results. The internal control failed in seven of these, whereas for the remaining four samples there were errors in the testing process (data analysis error, failure in sample dispense verification, and/or incorrect sample volume). One sample (<0.1%) remained invalid after the testing had been repeated. APTIMA was positive in 849 (16.7%) samples. For women targeted by the screening program (age, 23 to 65 years), the proportion testing positive was 16.1%. This proportion decreased from 27.8% at ages 23 to 29 years to 3.8% at ages 60 to 65 years (P < 0.0001 for trend across age categories) (Table 1). In women ages 16 to 22 and older than age 65, the proportions testing positive on APTIMA were 44.4% and 6.2%, respectively. Women in this age range are not invited routinely for screening, but may have presented for medical conditions or may have sought screening themselves. In total, 38 (0.7%) samples tested positive on APTIMA at S/CO values of 0.50 or greater and less than 1.00. From the 5064 samples that were tested with HC2, 1035 (20.4%) tested positive. Although the proportion testing positive was higher overall than with APTIMA, it showed a similar decrease with increasing age. Among women ages 16 to 22 years, 56.8% of samples tested positive on HC2, 33.0% of samples tested positive at ages 23 to 29 years, 17.4% of samples tested positive at ages 30 to 39 years, 11.3% of samples tested positive at ages 40 to 49 years, 7.3% of samples tested positive at ages 50 to 59 years, 6.0% of samples tested positive at ages 60 to 65 years, and 6.2% of samples tested positive at age older than 65 years (data not shown). Of the 846 APTIMA-positive samples for which HC2 results were available, 747 (88.3%) were also HC2 positive, and of the 4217 APTIMA-negative samples with HC2 results, 3929 (93.2%) were also HC2 negative. The κ coefficient of 0.75 indicated substantial agreement in these samples between the APTIMA and HC2 assays. Cytology was abnormal in 373 (7.4%) of 5070 samples. The proportion of samples testing positive on APTIMA and HC2 increased with cytologic grade. Among samples with normal cytology, 12.6% tested positive on APTIMA and 15.6% tested positive on HC2 (RP, 0.81; 95% CI, 0.73 to 0.89); among samples showing ASCUS 47.2% and 64.2% were positive, respectively (RP, 0.73; 95% CI, 0.58 to 0.92); among samples showing low-grade squamous intraepithelial lesions, 76.9% and 89.5% were positive, respectively (RP, 0.86; 95% CI, 0.77 to 0.96); and among samples with high-grade squamous intraepithelial lesions or worse, 86.9% and 90.7% were positive, respectively (RP, 0.96; 95% CI, 0.87 to 1.05) (for both assays, P < 0.0001 for trend). These results were fairly similar in the subset of 4411 primary samples (Table 2). In the primary samples, 676 (15.3%) tested positive on APTIMA, 822 (18.6%) tested positive on HC2 (κ coefficient for agreement between APTIMA and HC2, 0.75), and 242 (5.5%) samples had ≥ASCUS. Among women ages 23 to 29 years, 26.8% tested positive on APTIMA, 31.7% tested positive on HC2, and 7.4% tested positive on LBC (Table 3). Among women ages 30 to 65 years, these proportions were 9.4%, 11.7%, and 4.4%, respectively. Because women from Copenhagen are on average younger than Danish women in general, the proportion
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