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JCM Accepts, published online ahead <strong>of</strong> print on 23 March 2011<br />

J. Clin. Microbiol. doi:10.1128/JCM.00012-11<br />

Copyright © 2011, American Society for Microbiology <strong>and</strong>/or <strong>the</strong> Listed Authors/Institutions. All Rights Reserved.<br />

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<strong>Comparison</strong> <strong>of</strong> <strong>clinical</strong> <strong>and</strong> <strong>analytical</strong> <strong>performance</strong> <strong>of</strong> <strong>the</strong> <strong>Abbott</strong><br />

RealTime High Risk HPV test <strong>and</strong> Hybrid Capture 2 in<br />

population-based cervical cancer screening<br />

Running title: <strong>Abbott</strong> RealTime High Risk HPV in primary screening<br />

Mario Poljak, 1 * Anja Oštrbenk, 1 Katja Seme, 1 Veronika Učakar, 2 Peter Hillemanns, 3<br />

Eda Vrtačnik Bokal, 4 Nina Jančar 4 <strong>and</strong> Irena Klavs 2<br />

Institute <strong>of</strong> Microbiology <strong>and</strong> Immunology, Faculty <strong>of</strong> Medicine, University <strong>of</strong> Ljubljana,<br />

Slovenia 1 ,<br />

National Institute <strong>of</strong> Public Health <strong>of</strong> Slovenia, Ljubljana, Slovenia 2 ,<br />

Clinic <strong>of</strong> Obstetrics <strong>and</strong> Gynecology, Hannover Medical School, Hannover, Germany 3 ,<br />

Department <strong>of</strong> Obstetrics <strong>and</strong> Gynecology, University Medical Centre Ljubljana, Ljubljana,<br />

Slovenia 4<br />

*Corresponding author. Pr<strong>of</strong>. Mario Poljak, MD, PhD. Mailing address: Institute <strong>of</strong><br />

Microbiology <strong>and</strong> Immunology, Faculty <strong>of</strong> Medicine, University <strong>of</strong> Ljubljana Zaloška 4, 1000<br />

Ljubljana, Slovenia. Phone: +386 1 543 7453. Fax: +386 1 543 7418. E-mail:<br />

mario.poljak@mf.uni-lj.si<br />

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Abstract<br />

The <strong>clinical</strong> <strong>performance</strong> <strong>of</strong> <strong>the</strong> <strong>Abbott</strong> RealTime High Risk HPV test (RealTime) <strong>and</strong> Hybrid<br />

Capture 2 HPV DNA Test (hc2) was prospectively compared in <strong>the</strong> population-based cervical<br />

cancer screening setting. In women above 30 years (N=3,129), <strong>the</strong> <strong>clinical</strong> sensitivity for<br />

detection <strong>of</strong> cervical intraepi<strong>the</strong>lial neoplasia (CIN) grade 2 or worse (38 cases) <strong>and</strong> <strong>clinical</strong><br />

specificity for lesions less than CIN2 (3,091 controls) <strong>of</strong> RealTime were 100% <strong>and</strong> 93.3%,<br />

respectively, <strong>and</strong> <strong>of</strong> hc2 97.4% <strong>and</strong> 91.8%, respectively. A noninferiority score test showed<br />

that <strong>the</strong> <strong>clinical</strong> specificity (P


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High-risk genotypes <strong>of</strong> alpha human papillomaviruses (hrHPV) are etiologically linked to<br />

virtually all cervical carcinomas <strong>and</strong> <strong>the</strong>ir immediate precursors - high-grade cervical<br />

intraepi<strong>the</strong>lial neoplasia (CIN) lesions (49). HPV DNA testing has <strong>the</strong>refore become an<br />

important part <strong>of</strong> cervical carcinoma screening <strong>and</strong> management algorithms in several<br />

countries (reviewed in 10, 41). The four main <strong>clinical</strong> applications <strong>of</strong> HPV DNA testing at<br />

present are: (i) triage <strong>of</strong> women with equivocal screening cytology results, in order to<br />

determine which patients should be referred to colposcopy; (ii) follow-up <strong>of</strong> women with<br />

abnormal screening cytology results who are negative at initial colposcopy/biopsy; (iii)<br />

prediction <strong>of</strong> <strong>the</strong> <strong>the</strong>rapeutic outcome after treatment <strong>of</strong> high-grade CIN <strong>and</strong> (iv) primary<br />

screening <strong>of</strong> women aged 30 years <strong>and</strong> more in combination with Pap smear to detect cervical<br />

cancer precursors (1, 3, 10-11, 13, 15).<br />

Several in-house <strong>and</strong> more than 35 commercial assays for <strong>the</strong> detection <strong>of</strong> hrHPV are<br />

currently available (reviewed in 11, 40, 44). These assays have significantly different <strong>clinical</strong><br />

<strong>performance</strong> for high-grade CIN detection <strong>and</strong> are not necessarily useful for primary<br />

screening purposes (21, 40). The Hybrid Capture 2 HPV DNA Test (hc2) (Qiagen, Hilden,<br />

Germany) is <strong>the</strong> most frequently used diagnostic HPV assay worldwide, which has been used<br />

in <strong>the</strong> majority <strong>of</strong> key trials that have proved <strong>the</strong> <strong>clinical</strong> value <strong>of</strong> hrHPV testing (summarized<br />

in 10, 12-13, 32). In order to assess suitability <strong>and</strong> facilitate <strong>the</strong> acceptance <strong>of</strong> novel hrHPV<br />

assays for primary cervical cancer screening, it has been recently recommended that c<strong>and</strong>idate<br />

HPV assays should show similar <strong>clinical</strong> characteristics as hc2 i.e. <strong>clinical</strong> sensitivity, <strong>clinical</strong><br />

specificity <strong>and</strong> reproducibility, before <strong>the</strong>y can be used for cervical cancer screening purposes<br />

(32). Based on reported <strong>clinical</strong> characteristics <strong>of</strong> hc2, <strong>the</strong> requirement has been set that <strong>the</strong><br />

c<strong>and</strong>idate assay should have a <strong>clinical</strong> sensitivity <strong>and</strong> <strong>clinical</strong> specificity for high-grade CIN<br />

or worse (CIN2+) not less than 90% <strong>and</strong> 98% <strong>of</strong> <strong>the</strong> hc2 test, respectively, in a population <strong>of</strong><br />

women above 30 years (32). A c<strong>and</strong>idate assay should be robust <strong>and</strong> display high intra-<br />

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laboratory reproducibility <strong>and</strong> inter-laboratory agreement with a lower confidence boundary<br />

not less than 87% (32). Stoler at al. recently proposed that any novel HPV assay intended for<br />

use for cervical screening should have a <strong>clinical</strong> sensitivity <strong>and</strong> <strong>clinical</strong> specificity for CIN3+<br />

<strong>of</strong> 92% ± 3% <strong>and</strong> at least 85%, respectively (46). The common idea behind all currently<br />

proposed recommendations is that a <strong>clinical</strong>ly useful HPV assay should achieve an optimal<br />

balance between <strong>clinical</strong> sensitivity <strong>and</strong> <strong>clinical</strong> specificity for detection <strong>of</strong> CIN2+, in order to<br />

detect virtually all women with cervical cancer or immediate precursors <strong>and</strong>, at <strong>the</strong> same time,<br />

minimize redundant or excessive follow-up procedures for hrHPV positive women with<br />

transient hrHPV infections <strong>and</strong>/or without cervical lesions (19, 27, 32, 46).<br />

Several recent studies have clearly shown that a negative hrHPV result provides more<br />

reassurance against cervical precancerous lesions <strong>and</strong> cancer than a negative cervical cytology<br />

result, <strong>and</strong> <strong>the</strong>refore, safely permits longer intervals between screens (4, 8, 12-13, 16, 31, 33).<br />

However, among hrHPV positive women, only a small proportion will have a concurrent<br />

<strong>clinical</strong>ly-relevant disease, creating a dilemma <strong>of</strong> how to identify <strong>the</strong> subset <strong>of</strong> women that<br />

require fur<strong>the</strong>r immediate <strong>clinical</strong> attention, such as colposcopy (2, 8). One approach to<br />

improving <strong>the</strong> positive predictive value for disease among hrHPV positive women is <strong>the</strong> use<br />

<strong>of</strong> limited HPV genotyping for identification <strong>of</strong> <strong>the</strong> two most oncogenic HPV types: HPV16<br />

<strong>and</strong> HPV18 (9, 26). A new generation <strong>of</strong> commercial assays that test for <strong>the</strong> most important<br />

hrHPV types <strong>and</strong>, in addition have <strong>the</strong> potential to separate cytologically negative/hrHPV<br />

positive women at highest risk for CIN3+ (HPV16 or HPV18 positive) from those at lower<br />

risk (HPV16 <strong>and</strong> HPV18 negative), has been recently introduced (reviewed in 40).<br />

One <strong>of</strong> <strong>the</strong> next-generation tests, <strong>the</strong> <strong>Abbott</strong> RealTime High Risk HPV test (RealTime)<br />

(<strong>Abbott</strong>, Wiesbaden, Germany) detects a pool <strong>of</strong> 12 carcinogenic HPV genotypes in<br />

aggregate, with concurrent, separate detection <strong>of</strong> HPV16 <strong>and</strong> HPV18. The assay was<br />

launched in Europe in January 2009. In previous evaluations, RealTime demonstrated<br />

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superior <strong>analytical</strong> specificity <strong>and</strong> similar <strong>analytical</strong> sensitivity as hc2 (23, 39). The <strong>clinical</strong><br />

sensitivity <strong>of</strong> RealTime for CIN2+ lesions in six published studies performed on different<br />

study populations was at least comparable with hc2 (14, 20, 22, 25, 39, 47). The <strong>clinical</strong><br />

specificity <strong>and</strong> positive predictive value for ei<strong>the</strong>r CIN2+ or CIN3+ in a triage <strong>of</strong> women with<br />

abnormal cervical cytology smears were comparable between RealTime <strong>and</strong> hc2 in one study<br />

(22), while RealTime performed slightly better in ano<strong>the</strong>r study (14).<br />

We present here <strong>the</strong> results <strong>of</strong> <strong>the</strong> first comparative evaluation <strong>of</strong> <strong>the</strong> <strong>clinical</strong> <strong>performance</strong><br />

<strong>of</strong> RealTime <strong>and</strong> hc2 in <strong>the</strong> population-based cervical cancer screening setting. The study was<br />

primarily designed <strong>and</strong> statistically powered to determine <strong>the</strong> <strong>clinical</strong> specificity <strong>of</strong> RealTime<br />

for <strong>the</strong> detection <strong>of</strong> lesions less than CIN2 in women above 30 years, although applicable data<br />

were also obtained related to <strong>the</strong> <strong>clinical</strong> sensitivity <strong>of</strong> RealTime for <strong>the</strong> detection <strong>of</strong> CIN2+<br />

lesions. In addition, <strong>the</strong> <strong>analytical</strong> <strong>performance</strong> <strong>of</strong> <strong>the</strong> two HPV assays were compared on <strong>the</strong><br />

largest sample collection to date (4,479 samples) <strong>and</strong> <strong>the</strong> first data on intra-laboratory<br />

reproducibility <strong>and</strong> inter-laboratory agreement <strong>of</strong> RealTime are provided.<br />

MATERIALS AND METHODS<br />

Study population <strong>and</strong> sample collection. During <strong>the</strong> period from December 2009 to<br />

August 2010, we prospectively enrolled all women attending <strong>the</strong> routine organized national<br />

cervical screening program within a network <strong>of</strong> 16 outpatient gynecology services with a<br />

nationally wide geographical coverage. In <strong>the</strong> Slovenian National Cervical Cancer Screening<br />

Program, which started in 2003, each woman between ages 20 <strong>and</strong> 64 is invited to have a<br />

preventive gynecological examination, toge<strong>the</strong>r with a PAP smear, once every 3 years (after<br />

two consecutive negative smears taken one year apart) (35). All smear <strong>and</strong> histology reports<br />

are ga<strong>the</strong>red in <strong>the</strong> central database, which is linked to <strong>the</strong> Central Population Registry. The<br />

present study (Slovenian HPV Prevalence Study) was conducted in accordance with <strong>the</strong><br />

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Helsinki Declaration <strong>and</strong> was approved by <strong>the</strong> National Medical Ethics Committee at <strong>the</strong><br />

Slovenian Ministry <strong>of</strong> Health. Women were eligible for inclusion if <strong>the</strong>y were attending<br />

routine organized Slovenian national cervical cancer screening program. The exclusion<br />

criteria were: attendance for a gynecological examination after an atypical/abnormal cytology<br />

result, history <strong>of</strong> CIN <strong>of</strong> any grade or treatment for cervical disease in <strong>the</strong> preceding year,<br />

hysterectomy, menstruating or pregnancy at presentation (36). A total <strong>of</strong> 34 gynecologists<br />

were responsible for patient recruitment <strong>and</strong> management. Written informed consent was<br />

obtained from all women by <strong>the</strong> participating gynecologists. Patient identity was blinded to all<br />

study participants except <strong>the</strong> participating gynecologist. During <strong>the</strong> gynaecological<br />

examination, <strong>the</strong> cervix was visually inspected <strong>and</strong> a sample was taken for routine cervical<br />

cytology, following <strong>the</strong> procedures normally used in each gynaecological practice. Samples<br />

were most <strong>of</strong>ten taken with a wooden or plastic spatula or with an endocervical brush,<br />

smeared onto a microscope slide <strong>and</strong> spray fixed. In addition to this st<strong>and</strong>ard procedure at<br />

gynecology examination for cervical cancer screening purposes, a second sample was<br />

obtained for HPV DNA testing using ei<strong>the</strong>r a Cervex-Brush (Rovers Medical Devices, Oss,<br />

<strong>the</strong> Ne<strong>the</strong>rl<strong>and</strong>s) (in 87.6% <strong>of</strong> cases) or a Pap Perfect Plastic Spatula <strong>and</strong> Cytobrash Plus GT<br />

Gentle Touch (Medsc<strong>and</strong> Sample Collection Kit, Medsc<strong>and</strong> Medical, Berlin, Germany) (in<br />

12.4% <strong>of</strong> cases) <strong>and</strong> placed into ThinPrep PreservCyt Solution (Hologic, Marlborough, MA).<br />

Coded ThinPrep vials <strong>and</strong> all accompanying data collection forms were transported to <strong>the</strong><br />

laboratory on a weekly basis. Immediately on arrival at <strong>the</strong> laboratory, <strong>the</strong> specimens were<br />

split into several aliquots. The first two aliquots were used alternately for hc2 <strong>and</strong> RealTime<br />

testing <strong>and</strong> remainder aliquots were stored at -70 o C.<br />

RealTime HPV testing. RealTime assay was performed on <strong>the</strong> fully automated nucleic<br />

acid preparation instrument m2000sp <strong>and</strong> <strong>the</strong> real-time PCR instrument m2000rt (<strong>Abbott</strong>),<br />

following <strong>the</strong> manufacturer’s instructions, as previously described (20, 23). The assay uses<br />

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four channels for <strong>the</strong> detection <strong>of</strong> fluorescent signals; one for <strong>the</strong> detection <strong>of</strong> an internal<br />

process control (136-bp region <strong>of</strong> human beta-globin) for sample adequacy, DNA extraction<br />

<strong>and</strong> amplification, a second one for <strong>the</strong> detection <strong>of</strong> HPV16, a third for <strong>the</strong> detection <strong>of</strong><br />

HPV18 <strong>and</strong> a fourth for <strong>the</strong> aggregate detection <strong>of</strong> <strong>the</strong> 12 HPV types: HPV31, HPV33,<br />

HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66 <strong>and</strong> HPV68<br />

(23). The PCR amplification <strong>of</strong> HPV targets was achieved using a modified GP5+/6+ primer<br />

mix consisting <strong>of</strong> three forward <strong>and</strong> two reverse primers (24). The assay cut<strong>of</strong>f is set at a fixed<br />

cycle threshold (Ct) value <strong>of</strong> 32. According to <strong>the</strong> manufacturer’s instructions, <strong>the</strong> assay was<br />

repeated on samples that showed initial invalid results for internal control <strong>and</strong>, additionally, at<br />

our discretion, on samples that showed some degree <strong>of</strong> HPV-specific positive amplification<br />

signal(s) but Ct values were above <strong>the</strong> manufacturer’s fixed assay cut<strong>of</strong>f cycle <strong>and</strong> on samples<br />

that showed an initial HPV-negative result but had been defined as cases (CIN2+) during<br />

evaluation <strong>of</strong> <strong>clinical</strong> <strong>performance</strong>.<br />

hc2 HPV testing. hc2 is a hybridization assay designed for aggregate detection <strong>of</strong> 13 HPV<br />

types: HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56,<br />

HPV58, HPV59 <strong>and</strong> HPV68, using a mixture <strong>of</strong> unlabeled single-str<strong>and</strong>ed full-genomic-<br />

length RNA probes (30, 37). Testing was performed following <strong>the</strong> manufacturer’s<br />

instructions. Samples with a relative light unit per cut-<strong>of</strong>f (RLU/CO) ratio higher than 2.50<br />

were considered positive <strong>and</strong> samples with a RLU/CO value <strong>of</strong> less than 1.00 were considered<br />

negative. According to <strong>the</strong> manufacturer’s instructions, all samples with a RLU/CO ratio<br />

between 1.00 <strong>and</strong> 2.50 were retested <strong>and</strong> <strong>the</strong> results interpreted according to <strong>the</strong><br />

manufacturer’s instructions. Additionally, hc2 was repeated at our discretion on samples that<br />

showed an initial hc2 borderline HPV-negative result (RLU/CO ratio from 0.80-0.99) <strong>and</strong> on<br />

samples that showed an initial hc2 negative result but had been defined as cases (CIN2+)<br />

during evaluation <strong>of</strong> <strong>clinical</strong> <strong>performance</strong>.<br />

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HPV genotyping <strong>and</strong> discordant analysis. To detect HPV type(s) present in <strong>the</strong> sample,<br />

all samples with RealTime/hc2 concordant positive results <strong>and</strong> all samples with discordant<br />

results were additionally tested using <strong>the</strong> Linear Array HPV Genotyping Test (Linear Array)<br />

(Roche Molecular Diagnostics, Branchburg, NJ), which is capable <strong>of</strong> recognizing 36 different<br />

HPV types <strong>and</strong> one HPV subtype (including all 13 assay-common HPV types, i.e., types<br />

targeted by both RealTime <strong>and</strong> hc2), following <strong>the</strong> manufacturer’s instructions (45). Samples<br />

with a positive Linear Array HPV52 cross-reactive probe signal were additionally tested with<br />

an HPV52 type-specific real-time PCR assay, as previously described (29). All Linear Array<br />

HPV-negative samples <strong>and</strong> all samples in which no assay-common HPV types were identified<br />

by Linear Array, were additionally tested with <strong>the</strong> INNO-LiPA HPV Genotyping Extra Test<br />

(INNO-LiPA) (Innogenetics, Gent, Belgium), which is capable <strong>of</strong> recognizing 28 different<br />

alpha-HPV types (including all 13 assay-common types), following <strong>the</strong> manufacturer’s<br />

instructions. Finally, all INNO-LiPA HPV-negative samples <strong>and</strong> all samples in which no<br />

assay-common types were identified by INNO-LiPA, were tested using an in-house<br />

GP5+/GP6+ PCR assay targeting a 150-bp fragment in HPV L1 gene with additional HPV68<br />

specific primers, as previously described (18, 34). Direct sequencing <strong>of</strong> <strong>the</strong> GP5+/GP6+ PCR<br />

products with <strong>the</strong> same primers was used for genotyping, as previously described (28). All<br />

HPV types identified by any <strong>of</strong> <strong>the</strong> genotyping tests were considered when interpreting <strong>the</strong><br />

RealTime/hc2 discordant results. The <strong>analytical</strong> reliability <strong>of</strong> <strong>the</strong> applied three-step<br />

genotyping strategy was verified using <strong>the</strong> HPV DNA Pr<strong>of</strong>iciency 2010 Panel prepared by <strong>the</strong><br />

World Health Organization HPV Laboratory Network – LabNet (17). The panel consisted <strong>of</strong><br />

46 coded samples with a titration series <strong>of</strong> purified plasmids <strong>of</strong> 16 different HPV types<br />

(including all 13 assay-common types), in concentrations ranging from 5-500 IU <strong>of</strong> HPV16 or<br />

HPV18 DNA <strong>and</strong> 5-500 genome equivalents <strong>of</strong> <strong>the</strong> o<strong>the</strong>r 14 HPV types. The genotyping<br />

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strategy used in this study showed 100% specificity <strong>and</strong> 100% sensitivity for a total <strong>of</strong> 72<br />

HPV types present in 46 pr<strong>of</strong>iciency panel specimens.<br />

Cytological examination. All cervical smears were examined under routine screening<br />

conditions by certified cytologists normally used by each participating gynecology practice<br />

who were blinded to HPV results.<br />

Colposcopic referral. According to <strong>the</strong> criteria <strong>of</strong> <strong>the</strong> Slovenian National Cervical Cancer<br />

Screening Program, women were called for immediate colposcopy using a cytology threshold<br />

<strong>of</strong> atypical squamous cells - cannot exclude high-grade lesion (ASC-H)/atypical gl<strong>and</strong>ular<br />

cells (AGC) or worse. In addition, irrespective <strong>of</strong> cytology result, according to our study<br />

protocol, women were also invited for colposcopy if <strong>the</strong>y were positive for HPV16 or HPV18.<br />

In women positive for hrHPV o<strong>the</strong>r than HPV16 <strong>and</strong> HPV18, an immediate colposcopy was<br />

performed at <strong>the</strong> physician’s discretion; o<strong>the</strong>rwise <strong>the</strong> woman was invited to a control<br />

gynaecological examination after six months to one year. Colposcopy was performed by<br />

certified colposcopists according to st<strong>and</strong>ard operating procedures, using <strong>the</strong> international<br />

nomenclature (48). During colposcopy, punch biopsy specimens were taken from any regions<br />

suspicious for CIN. No biopsy was taken from women with normal colposcopy, since this is<br />

considered unethical in Slovenia. The three-tier CIN nomenclature was used for biopsy<br />

classification <strong>and</strong> <strong>the</strong> most severe abnormality was selected for final histopathological<br />

diagnosis. An expert histopathology system was used for histopathological assessment: all<br />

biopsies were first examined by a certified pathologist with more than 20 years <strong>of</strong> experience<br />

in gynecological pathology, followed by independent, blinded histopathological review. In<br />

discrepant cases, <strong>the</strong> final diagnosis was <strong>the</strong> consensus reached by a panel <strong>of</strong> three<br />

pathologists. Pathologists performing histopathological assessments were blinded to <strong>the</strong> HPV<br />

status but did have access to concurrent cytology results.<br />

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Intra-laboratory reproducibility <strong>and</strong> inter-laboratory agreement <strong>of</strong> RealTime. To<br />

assess RealTime intra-laboratory reproducibility in time, a total <strong>of</strong> 500 samples (167<br />

r<strong>and</strong>omly selected HPV positive <strong>and</strong> 333 r<strong>and</strong>omly selected HPV negative samples) were<br />

retested after 61 to 226 days (median 73 days) from initial testing, as recently recommended<br />

(32). In addition, two sets <strong>of</strong> coded 0.7 ml ThinPrep aliquots (2 x 500 samples) were prepared<br />

from <strong>the</strong> same 167 HPV positive <strong>and</strong> 333 HPV negative samples <strong>and</strong> shipped on dry ice to a<br />

collaborative laboratory in Hannover (Germany), where two additional HPV testing rounds<br />

were performed. The obtained results were used to calculate intra-laboratory reproducibility<br />

in time in <strong>the</strong> two participating laboratories, as well as inter-laboratory agreement between <strong>the</strong><br />

Ljubljana <strong>and</strong> Hannover laboratories. Reproducibility testing was performed using coded<br />

samples <strong>and</strong> <strong>the</strong> technicians performing <strong>the</strong> assay in <strong>the</strong> two laboratories were completely<br />

blinded to <strong>the</strong> HPV status <strong>of</strong> <strong>the</strong> samples.<br />

Statistical analysis. For assessment <strong>of</strong> <strong>the</strong> <strong>clinical</strong> <strong>performance</strong> <strong>of</strong> RealTime <strong>and</strong> hc2,<br />

cases were defined as women with high-grade cervical disease (CIN2+) <strong>and</strong> controls as<br />

women without high-grade cervical disease (less than CIN2). The <strong>clinical</strong> <strong>performance</strong> <strong>of</strong><br />

both assays was compared using a noninferiority score test, as recently recommended (32).<br />

The thresholds used for noninferiority were specificity for <strong>the</strong> detection <strong>of</strong> lesions less than<br />

CIN2 <strong>of</strong> at least 98% <strong>and</strong> sensitivity for <strong>the</strong> detection <strong>of</strong> CIN2+ lesions <strong>of</strong> at least 90%<br />

relative to <strong>the</strong> results <strong>of</strong> hc2, as previously described (32). Since we enrolled more than 2,500<br />

women above 30 years, for <strong>clinical</strong> specificity <strong>the</strong> power <strong>of</strong> <strong>the</strong> study was more than 99%<br />

(32). The <strong>analytical</strong> <strong>performance</strong> <strong>of</strong> <strong>the</strong> two HPV assays was determined against hrHPV<br />

status, which was defined by <strong>the</strong> concordance between RealTime <strong>and</strong> hc2 <strong>and</strong>, for discordant<br />

specimens, by genotyping results. Genotyping results were designated as hrHPV positive<br />

when at least one <strong>of</strong> <strong>the</strong> 13 assay-common HPV types was detected: HPV16, HPV18, HPV31,<br />

HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59 <strong>and</strong> HPV68.<br />

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Sensitivity, specificity, negative predictive value <strong>and</strong> positive predictive value were calculated<br />

using <strong>the</strong> conventional contingency tables <strong>and</strong> 95% confidence intervals (95% CI) were<br />

computed using exact binomial methods. The level <strong>of</strong> agreement between tests was assessed<br />

by <strong>the</strong> Kappa statistics. The Chi-square test was used for intercomparison <strong>of</strong> proportions. All<br />

statistical analyses were performed using R s<strong>of</strong>tware vs. 2.12.0 (Free S<strong>of</strong>tware Foundation,<br />

Boston, MA). The level <strong>of</strong> statistical significance was set at a value <strong>of</strong> 0.05.<br />

RESULTS<br />

Between December 2009 <strong>and</strong> August 2010, 4,602 eligible women were invited to<br />

participate in <strong>the</strong> study, <strong>of</strong> which 88 (1.9%) declined to participate for various reasons. Seven<br />

women were excluded from <strong>the</strong> study due to ThinPrep sample spill out during transport (3<br />

women) or missing ThinPrep samples (4 women). HPV testing was thus finally performed<br />

using RealTime <strong>and</strong> hc2 on a total <strong>of</strong> 4,507 women.<br />

RealTime was repeated on a total <strong>of</strong> 41 samples. The assay was repeated according to <strong>the</strong><br />

manufacturer’s instructions on 10 samples due to initial invalid results for internal control; all<br />

10 samples also had repeated invalid results <strong>and</strong> were excluded from <strong>the</strong> study. All excluded<br />

samples were repeatedly hc2 negative <strong>and</strong> were obtained from women who had normal<br />

cytology. Of 30 samples that initially showed RealTime HPV-specific positive amplification<br />

but Ct values were above <strong>the</strong> manufacturer’s fixed assay cut<strong>of</strong>f cycle <strong>and</strong> were repeated at our<br />

discretion, eight samples turned out to be HPV-positive after repeat testing <strong>and</strong> 22 samples<br />

again showed HPV-specific Ct values above <strong>the</strong> manufacturer’s cut<strong>of</strong>f. A single sample that<br />

showed an initial RealTime HPV-negative result but was defined as a case (CIN2+) during<br />

evaluation <strong>of</strong> <strong>clinical</strong> <strong>performance</strong> (repeated at our discretion), was again tested RealTime<br />

HPV-negative on repeat testing.<br />

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Hc2 was repeated on a total <strong>of</strong> 137 samples. The test was repeated according to <strong>the</strong><br />

manufacturer’s instructions on 101 samples due to an initial RLU/CO ratio between 1.00 <strong>and</strong><br />

2.50 (borderline HPV-positive results), among which 43 samples had to be retested twice. Of<br />

<strong>the</strong>se 101 samples, after discordant analysis <strong>and</strong> HPV genotyping, 41 samples were finally<br />

considered to be hc2 <strong>analytical</strong>ly true positive (<strong>the</strong> sample contained at least one targeted<br />

HPV type) <strong>and</strong> 60 samples to be hc2 <strong>analytical</strong>ly false positive (no targeted HPV types were<br />

detected). All 101 women with hc2 initially borderline HPV-positive samples were defined as<br />

controls (less than CIN2+) in <strong>clinical</strong> <strong>performance</strong> assessment. Of 32 samples that showed an<br />

initial hc2 borderline HPV-negative result (RLU/CO ratio between 0.80-0.99) <strong>and</strong> were<br />

repeated at our discretion, seven samples turned out to be hc2 positive after repeat testing <strong>and</strong><br />

25 samples again had a RLU/CO ratio below 1.00. Of <strong>the</strong> four samples that showed an initial<br />

hc2 negative result but were defined as cases (CIN2+) during evaluation <strong>of</strong> <strong>clinical</strong><br />

<strong>performance</strong>, <strong>and</strong> were repeated at our discretion, three samples again tested hc2 negative on<br />

repeat testing (initial RLU/CO ratios 0.29, 0.42, 0.55; repeat RLU/CO ratios 0.43, 0.74, 0.73,<br />

respectively) <strong>and</strong> one sample turned out to be hc2 positive after repeat testing (initial<br />

RLU/CO ratio 0.84; repeat RLU/CO ratio 1.44).<br />

Clinical <strong>performance</strong> <strong>of</strong> RealTime <strong>and</strong> hc2. Of 4,497 women who had valid HPV results<br />

in both assays, 14 women were excluded from assessment <strong>of</strong> <strong>clinical</strong> <strong>performance</strong> <strong>of</strong><br />

RealTime <strong>and</strong> hc2 due to missing cytology results <strong>and</strong> 51 women eligible for colposcopy<br />

(according to <strong>the</strong> protocol criteria) were excluded because <strong>the</strong>y did not respond in time to<br />

repeated invitations for colposcopy or <strong>the</strong>y refused colposcopy. The <strong>clinical</strong> <strong>performance</strong> <strong>of</strong><br />

<strong>the</strong> HPV assays was finally assessed on a total <strong>of</strong> 4,432 women using two study groups:<br />

women above 30 years <strong>and</strong> all participating women (<strong>the</strong> number <strong>of</strong> women in age groups ≤29,<br />

30-39, 40-49, 50-59 <strong>and</strong> ≥60 years were 1,304, 1,528, 976, 542 <strong>and</strong> 82).<br />

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Clinical <strong>performance</strong> <strong>of</strong> RealTime <strong>and</strong> hc2 in women above 30 years. Women above<br />

30 years represented our primary study group for evaluation <strong>of</strong> <strong>the</strong> <strong>clinical</strong> <strong>performance</strong> <strong>of</strong><br />

RealTime <strong>and</strong> hc2 (N=3,129 women; mean age 41.5 years; median age 40 years). The overall<br />

prevalence <strong>of</strong> HPV infection in women above 30 years assessed by RealTime <strong>and</strong> hc2 was<br />

7.8% (243/3,128; 95% CI, 6.9 to 8.8%) <strong>and</strong> 9.3% (290/3,128; 95% CI, 8.3 to 10.4%),<br />

respectively, <strong>and</strong> <strong>the</strong> HPV prevalence in women with a cytology result negative for<br />

intraepi<strong>the</strong>lial lesion <strong>and</strong> malignancy (NILM) was 6.1% (180/2,974; 95% CI, 5.2 to 7.0%) <strong>and</strong><br />

7.4% (219/2,974; 95% CI, 6.5 to 8.4%), respectively. Table 1 shows <strong>the</strong> RealTime results in<br />

women above 30 years, stratified for cases <strong>and</strong> controls, in comparison to <strong>the</strong> hc2 findings. A<br />

total <strong>of</strong> 38 cases were identified in women above 30 years: 18 CIN2 lesions, 16 CIN3 lesions,<br />

one carcinoma in situ <strong>and</strong> three invasive carcinomas <strong>and</strong> 3,091 women were classified as<br />

controls. The <strong>clinical</strong> sensitivity for <strong>the</strong> detection <strong>of</strong> CIN2+, <strong>clinical</strong> specificity for <strong>the</strong><br />

detection <strong>of</strong> lesions less than CIN2, PPV <strong>and</strong> NPV <strong>of</strong> RealTime <strong>and</strong> hc2 at cut<strong>of</strong>f values <strong>of</strong><br />

1.00 <strong>and</strong> 2.50 RLU/CO are shown in Table 2. The single CIN2+ case missed by hc2 (at a<br />

cut<strong>of</strong>f value <strong>of</strong> 1.00 RLU/CO) had RLU/CO values <strong>of</strong> 0.42 <strong>and</strong> 0.74 in initial <strong>and</strong> repeat hc2<br />

testing, respectively. A noninferiority score test performed to determine whe<strong>the</strong>r <strong>the</strong> <strong>clinical</strong><br />

specificity <strong>of</strong> RealTime for <strong>the</strong> detection <strong>of</strong> lesions less than CIN2 <strong>and</strong> <strong>the</strong> <strong>clinical</strong> sensitivity<br />

<strong>of</strong> RealTime for <strong>the</strong> detection <strong>of</strong> CIN2+ lesions were noninferior to those <strong>of</strong> <strong>the</strong> hc2 (cut<strong>of</strong>f<br />

1.00 RLU/CO) at recommended thresholds <strong>of</strong> 98% <strong>and</strong> 90% (32), respectively, showed that<br />

both <strong>clinical</strong> specificity (P < 0.0001) <strong>and</strong> <strong>clinical</strong> sensitivity (P = 0.011) <strong>of</strong> RealTime were<br />

noninferior to that <strong>of</strong> hc2.<br />

Clinical <strong>performance</strong> <strong>of</strong> RealTime <strong>and</strong> hc2 in <strong>the</strong> total study population. The total<br />

study population represented our secondary study group for evaluation <strong>of</strong> <strong>the</strong> <strong>clinical</strong><br />

<strong>performance</strong> <strong>of</strong> RealTime <strong>and</strong> hc2 (N=4,432 women; mean age 36.6 years; median age 35<br />

years). The overall prevalence <strong>of</strong> HPV infection assessed by RealTime <strong>and</strong> hc2 were 11.6%<br />

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(515/4,431; 95% CI, 10.7 to 12.6) <strong>and</strong> 13.3% (589/4,431; 95% CI, 12.3 to 14.3%),<br />

respectively. The prevalence <strong>of</strong> HPV infection in women with NILM cytology assessed by<br />

RealTime <strong>and</strong> hc2 were 9.7% (411/4,217; 95% CI, 8.9 to 10.7%) <strong>and</strong> 11.2% (474/4,217; 95%<br />

CI, 10.3 to 12.2%), respectively. As shown in Table 1, a total <strong>of</strong> 57 cases were found in <strong>the</strong><br />

total study population: 31 CIN2 lesions, 22 CIN3 lesions, one carcinoma in situ <strong>and</strong> three<br />

invasive carcinomas <strong>and</strong> 4,375 women were classified as controls. The <strong>clinical</strong> sensitivity,<br />

<strong>clinical</strong> specificity, PPV <strong>and</strong> NPV <strong>of</strong> RealTime <strong>and</strong> hc2 at cut<strong>of</strong>f values <strong>of</strong> 1.00 <strong>and</strong> 2.50<br />

RLU/CO are shown in Table 2. A noninferiority score test showed that both <strong>clinical</strong><br />

specificity (P < 0.0001) <strong>and</strong> <strong>clinical</strong> sensitivity (P = 0.015) <strong>of</strong> RealTime were also noninferior<br />

to that <strong>of</strong> hc2 in <strong>the</strong> total study population.<br />

Analytical <strong>performance</strong> <strong>of</strong> RealTime <strong>and</strong> hc2. Of 4,497 women who had valid HPV<br />

results in both assays, 18 women were excluded from an assessment <strong>of</strong> <strong>analytical</strong><br />

<strong>performance</strong> because HPV genotyping showed <strong>the</strong> presence <strong>of</strong> HPV66 alone or in<br />

combination with o<strong>the</strong>r non-targeted HPV genotypes. All excluded HPV66-positive samples<br />

tested RealTime positive <strong>and</strong> 16 out <strong>of</strong> 18 samples tested hc2 positive (although HPV66 is not<br />

targeted by hc2). The <strong>analytical</strong> <strong>performance</strong> <strong>of</strong> <strong>the</strong> HPV assays was finally assessed on a<br />

total <strong>of</strong> 4,479 specimens. As shown in Table 3, after hc2 repeat testing <strong>of</strong> 101 samples<br />

according to <strong>the</strong> manufacturers’ instructions (initial results), excellent agreement between<br />

RealTime <strong>and</strong> hc2, with a kappa value <strong>of</strong> 0.83 (95% CI, 0.80 to 0.85) <strong>and</strong> a percentage <strong>of</strong><br />

total agreement <strong>of</strong> 96.0% (4,304/4,479; 95% CI 95.5 to 96.6%), was obtained. After repeat<br />

testing <strong>of</strong> 62 samples (30 samples by RealTime <strong>and</strong> 32 samples by hc2) at our discretion<br />

(resolved results; see reasons for repeated testing in Materials <strong>and</strong> Methods), excellent<br />

agreement with a kappa value <strong>of</strong> 0.84 (95% CI, 0.82 to 0.87) <strong>and</strong> a percentage <strong>of</strong> total<br />

agreement <strong>of</strong> 96.4% (4,319/4,479; 95% CI 95.8 to 96.9%) was obtained.<br />

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HPV types determined in 160 samples with <strong>analytical</strong>ly discordant results between<br />

RealTime <strong>and</strong> hc2 are presented in Table 4. Of <strong>the</strong> 44 samples detected by RealTime <strong>and</strong> not<br />

by hc2 (resolved results), 38 samples were positive for at least one assay-common HPV type<br />

<strong>and</strong> were considered to be <strong>analytical</strong>ly RealTime true positive/hc2 false negative. The hc2<br />

most frequently missed HPV51 (in nine samples), followed by HPV16 <strong>and</strong> HPV18 (in six<br />

samples each) (Table 4). Five RealTime positive/hc2 negative samples tested HPV DNA<br />

negative using all three broad-range PCR-based genotyping methods applied <strong>and</strong> only low-<br />

risk HPV6 was found in one RealTime positive/hc2 negative sample. These six samples were<br />

considered to be <strong>analytical</strong>ly RealTime false positive/hc2 true negative samples.<br />

Of <strong>the</strong> 116 samples detected by hc2 <strong>and</strong> not by RealTime (resolved results), only non-<br />

targeted HPV types were identified in 62 samples <strong>and</strong> 24 samples tested HPV DNA negative<br />

using all three broad-range PCR-based genotyping methods applied (Table 4). These 86<br />

samples were considered to be <strong>analytical</strong>ly hc2 false positive/RealTime true negative. The<br />

most frequently identified non-targeted HPV type causing an hc2 false positive result was<br />

HPV53 (in at least 15 samples), followed by HPV67 <strong>and</strong> HPV70 (in at least five samples<br />

each). In 30 RealTime negative/hc2 positive samples, genotyping showed <strong>the</strong> presence <strong>of</strong> at<br />

least one assay-common HPV type <strong>and</strong> <strong>the</strong>se samples were considered to be <strong>analytical</strong>ly hc2<br />

true positive/RealTime false negative. Of <strong>the</strong>se 30 samples, 16 samples repeatedly showed<br />

some degree <strong>of</strong> RealTime HPV-specific amplification but Ct values were above <strong>the</strong><br />

manufacturer’s fixed assay cut<strong>of</strong>f (cycle <strong>of</strong> 32 nd ) <strong>and</strong> 14 samples were RealTime HPV non-<br />

reactive. Among <strong>the</strong>se RealTime negative/hc2 positive samples, <strong>the</strong> RealTime most<br />

frequently missed HPV68 (in ten samples).<br />

Comparing RealTime <strong>and</strong> hc2 results against resolved HPV status, <strong>the</strong> <strong>analytical</strong><br />

sensitivity <strong>of</strong> RealTime was 94.8% (543/573; 95% CI, 92.6 to 96.4%) <strong>and</strong> <strong>analytical</strong><br />

specificity 99.8% (3,900/3,906; 95% CI, 99.7 to 99.9%). By comparison, <strong>the</strong>se <strong>analytical</strong><br />

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values for hc2 at a cut<strong>of</strong>f value <strong>of</strong> 1.00 RLU/CO were 93.4% (535/573; 95% CI, 91.0 to<br />

95.3%) <strong>and</strong> 97.8% (3,820/3,906; 95% CI 97.3 to 98.2%), respectively. The <strong>analytical</strong><br />

accuracy <strong>of</strong> RealTime <strong>and</strong> hc2 in detecting 13 HPV types targeted by <strong>the</strong> two assays was<br />

99.2% (4,443/4,479; 95% CI, 98.9 to 99.4%) <strong>and</strong> 97.2% (4,355/4,479; 95% CI, 96.7 to<br />

97.7%), respectively; <strong>the</strong> values were significantly different (P < 0.0001). The <strong>analytical</strong><br />

accuracy <strong>of</strong> RealTime in detecting HPV16 <strong>and</strong> HPV18 at manufacturer’s fixed assay cut<strong>of</strong>f<br />

(cycle <strong>of</strong> 32 nd ) was 99.8% (4,471/4,479; 95% CI, 99.6 to 99.9%) <strong>and</strong> 99.8% (4,473/4,479;<br />

95% CI, 99.6 to 99.9%), respectively.<br />

Intra-laboratory reproducibility <strong>of</strong> RealTime. In Ljubljana, <strong>the</strong>re was excellent<br />

agreement <strong>of</strong> overall RealTime HPV result between <strong>the</strong> two rounds <strong>of</strong> testing, with a kappa<br />

value <strong>of</strong> 1.0 (95% CI, 0.98-1.0), percentage <strong>of</strong> agreement <strong>of</strong> 100% (500/500; 95% CI, 99.0 to<br />

100.0%) <strong>and</strong> percentage <strong>of</strong> positive agreement <strong>of</strong> 100% (167/167; 95% CI, 97.1 to 100.0%).<br />

After stratification <strong>of</strong> RealTime HPV-positive results into three categories (HPV16 positive,<br />

HPV18 positive <strong>and</strong> positive for <strong>the</strong> o<strong>the</strong>r 12 HPVs), <strong>the</strong> percentage <strong>of</strong> agreement was 99.0%<br />

(495/500; 95% CI, 97.5 to 99.6%), <strong>the</strong> percentage <strong>of</strong> positive agreement was 97.0% (162/167;<br />

95% CI, 92.7 to 98.8%), with <strong>the</strong> kappa value being 0.98 (95% CI, 0.96-0.99). All HPV type-<br />

specific discordant results were obtained in samples containing several HPV types (mixed<br />

HPV infection): additional HPV type(s) were detected in four samples in <strong>the</strong> second testing<br />

round (3x HPV18, 1x o<strong>the</strong>r hrHPVs) <strong>and</strong> in one sample in <strong>the</strong> first testing round (o<strong>the</strong>r<br />

hrHPVs).<br />

In Hannover, <strong>the</strong>re was also excellent agreement <strong>of</strong> overall RealTime HPV result between<br />

<strong>the</strong> two rounds <strong>of</strong> testing, with a kappa value <strong>of</strong> 0.99 (95% CI, 0.98-1.0), percentage <strong>of</strong><br />

agreement <strong>of</strong> 99.8% (499/500; 95% CI, 98.7 to 99.9%) <strong>and</strong> percentage <strong>of</strong> positive agreement<br />

<strong>of</strong> 99.4% (166/167; 95% CI, 96.2 to 99.9%). One sample was positive for HPV16 in <strong>the</strong> first<br />

testing round (Ct = 30.21) <strong>and</strong> HPV negative in <strong>the</strong> second, but with HPV16-specific<br />

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amplification near cut<strong>of</strong>f (Ct = 32.28). After stratification <strong>of</strong> RealTime HPV-positive results<br />

into three categories, <strong>the</strong> percentage <strong>of</strong> agreement was 99.0% (495/500; 95% CI, 97.5 to<br />

99.6%), <strong>the</strong> percentage <strong>of</strong> positive agreement was 97.0% (162/167; 95% CI, 92.7 to 98.8%),<br />

with <strong>the</strong> kappa value being 0.98 (95% CI, 0.96-0.99). HPV type-specific discordant results<br />

were obtained in a total <strong>of</strong> five samples: in a previously described sample with an HPV16<br />

discordant result <strong>and</strong> in four samples with mixed HPV infection: additional HPV type(s) were<br />

detected in two samples in <strong>the</strong> first testing round (1x HPV18, 1x o<strong>the</strong>r hrHPVs) <strong>and</strong> in two<br />

samples in <strong>the</strong> second testing round (2x HPV18).<br />

Inter-laboratory agreement <strong>of</strong> RealTime. There was excellent agreement <strong>of</strong> overall HPV<br />

result between Ljubljana <strong>and</strong> Hannover laboratories in <strong>the</strong> first <strong>and</strong> second testing rounds,<br />

with kappa values <strong>of</strong> 1.0 (95% CI, 0.98-1.0) <strong>and</strong> 0.99 (95% CI, 0.98-1.0), respectively,<br />

percentage <strong>of</strong> agreement <strong>of</strong> 100.0% (500/500; 95% CI, 99.0%-100.0%) <strong>and</strong> 99.8% (499/500;<br />

95% CI, 98.7%-99.9%), respectively, <strong>and</strong> percentage <strong>of</strong> positive agreement <strong>of</strong> 100.0%<br />

(167/167; 95% CI, 97.1%-100.0%) <strong>and</strong> 99.4% (166/167; 95% CI, 96.2%-99.9%),<br />

respectively. In <strong>the</strong> second testing round, one sample was positive for HPV16 in Ljubljana (Ct<br />

= 30.15) <strong>and</strong> HPV16 negative when tested in Hannover but with HPV16-specific<br />

amplification near <strong>the</strong> cut<strong>of</strong>f (Ct = 32.28). After stratification <strong>of</strong> RealTime HPV-positive<br />

results into three categories, in <strong>the</strong> first <strong>and</strong> second testing rounds <strong>the</strong> percentage <strong>of</strong> agreement<br />

was 99.2% (496/500; 95% CI, 97.8%-99.7%) <strong>and</strong> 98.2% (491/500; 95% CI, 96.4%-99.1%),<br />

respectively, percentage <strong>of</strong> positive agreement 97.6% (163/167; 95% CI, 93.5%-99.2%) <strong>and</strong><br />

94.6% (158/167; 95% CI, 89.7%-97.3%), respectively, with kappa values <strong>of</strong> 0.98 (95% CI,<br />

0.96-0.99) <strong>and</strong> 0.96 (95% CI, 0.94-0.98), respectively. In <strong>the</strong> first testing round, all HPV type-<br />

specific discordant results were obtained in samples with mixed HPV infection: additional<br />

HPV type(s) were detected in three samples in Ljubljana (1x HPV16, 2x HPV18) <strong>and</strong> in one<br />

sample in Hannover (1x o<strong>the</strong>r hrHPVs). In <strong>the</strong> second testing round, HPV type-specific<br />

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discordant results were obtained in a total <strong>of</strong> nine samples: in a previously described single<br />

sample with HPV16 discordant result; in one sample in which different HPV types were<br />

detected (in Ljubljana HPV16, in Hannover o<strong>the</strong>r hrHPVs); <strong>and</strong> in seven samples with mixed<br />

HPV infection: additional HPV type(s) were detected in six samples in Ljubljana (5x HPV18,<br />

1x o<strong>the</strong>r hrHPVs) <strong>and</strong> in one sample in Hannover (1x HPV18).<br />

DISCUSSION<br />

The most important consideration when evaluating an assay for routine detection <strong>of</strong><br />

hrHPVs in cervical specimens is <strong>the</strong> <strong>clinical</strong> accuracy for <strong>the</strong> detection <strong>of</strong> cervical high-grade<br />

lesions (10, 27, 32, 46). A <strong>clinical</strong>ly useful hrHPV assay should have balanced <strong>clinical</strong><br />

sensitivity <strong>and</strong> <strong>clinical</strong> specificity for CIN2+ lesions to ensure reliable detection <strong>of</strong> women<br />

with high-grade disease <strong>and</strong> to minimize HPV positive results in those with minimal risk <strong>of</strong><br />

disease (19, 32, 46). In recent years, it has become clear that many currently available<br />

commercial hrHPV assays are not very useful for primary cervical cancer screening, mainly<br />

as a result <strong>of</strong> misguided attempts to achieve perfect <strong>clinical</strong> sensitivity via increasing analytic<br />

sensitivity (27, 32, 40). Such <strong>analytical</strong>ly highly sensitive HPV assays, although capable <strong>of</strong><br />

recognizing almost all women with underlying high-grade disease, usually yield a large<br />

number <strong>of</strong> <strong>clinical</strong>ly insignificant positive results, which cause unnecessary <strong>clinical</strong> follow-<br />

up, unnecessary diagnostics procedures <strong>and</strong> unnecessary treatment <strong>of</strong> healthy women (27). In<br />

order to facilitate <strong>the</strong> evaluation <strong>and</strong> acceptance <strong>of</strong> novel hrHPV assays, guidelines describing<br />

requirements for <strong>the</strong> use <strong>of</strong> HPV assay for primary cervical cancer screening have recently<br />

been provided with European-North American collaboration (32). These guidelines<br />

recommend <strong>the</strong> use <strong>of</strong> a so-called <strong>clinical</strong> validation strategy, based on analysis <strong>of</strong> <strong>the</strong><br />

equivalence <strong>of</strong> <strong>the</strong> result <strong>of</strong> <strong>the</strong> c<strong>and</strong>idate hrHPV assay relative to that <strong>of</strong> an already <strong>clinical</strong>ly<br />

validated reference HPV assay such as hc2, with <strong>clinical</strong> samples that originate from a<br />

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population-based cervical cancer screening, as assessed by <strong>the</strong> use <strong>of</strong> a noninferiority score<br />

test (21, 32). According to <strong>the</strong> guidelines, three characteristics <strong>of</strong> <strong>the</strong> c<strong>and</strong>idate hrHPV assay<br />

should be assessed during <strong>the</strong> <strong>clinical</strong> validation process, using predetermined thresholds:<br />

<strong>clinical</strong> sensitivity, <strong>clinical</strong> specificity <strong>and</strong> reproducibility (32).<br />

RealTime is a recently launched next-generation HPV assay designed to detect a pool <strong>of</strong> 12<br />

carcinogenic HPV genotypes in aggregate, with concurrent, separate detection <strong>of</strong> HPV16 <strong>and</strong><br />

HPV18. In <strong>the</strong> past, <strong>the</strong> <strong>clinical</strong> <strong>performance</strong> <strong>of</strong> RealTime has been assessed on preselected<br />

archived cervical samples obtained from women with histologically confirmed cervical high-<br />

grade lesions (25, 39, 47) or in triage settings on cervical samples obtained from women with<br />

abnormal cytology referred for colposcopy (14, 20, 22). The <strong>clinical</strong> sensitivity <strong>of</strong> RealTime<br />

for cervical high-grade lesions in <strong>the</strong>se studies performed on a total <strong>of</strong> 1,481 histologically<br />

confirmed CIN2+ lesions was above 96% in all six studies <strong>and</strong> noninferior to hc2 (14, 20, 22,<br />

25, 39, 47). In <strong>the</strong> present study, we assessed for <strong>the</strong> first time <strong>the</strong> two remaining requirements<br />

for <strong>the</strong> use <strong>of</strong> RealTime for primary cervical cancer screening: <strong>clinical</strong> specificity for <strong>the</strong><br />

detection <strong>of</strong> lesions less than CIN2 in women above 30 years in <strong>the</strong> population-based primary<br />

cervical cancer screening setting <strong>and</strong> intra-laboratory reproducibility/inter-laboratory<br />

agreement. Although our study was primarily designed <strong>and</strong> statistically powered to determine<br />

<strong>the</strong> <strong>clinical</strong> specificity <strong>of</strong> RealTime (with power greater than 99%), applicable data were also<br />

obtained relevant to <strong>clinical</strong> sensitivity <strong>of</strong> RealTime. In our primary study group for<br />

evaluation <strong>of</strong> <strong>the</strong> <strong>clinical</strong> <strong>performance</strong> <strong>of</strong> RealTime (3,128 women above 30 years; 38 cases<br />

<strong>and</strong> 3,091 controls), both <strong>the</strong> <strong>clinical</strong> specificity <strong>of</strong> RealTime for <strong>the</strong> detection <strong>of</strong> lesions less<br />

than CIN2 <strong>and</strong> <strong>the</strong> <strong>clinical</strong> sensitivity <strong>of</strong> RealTime for <strong>the</strong> detection <strong>of</strong> CIN2+ lesion were<br />

noninferior to those <strong>of</strong> <strong>the</strong> <strong>clinical</strong>ly validated reference HPV assay (hc2) with <strong>the</strong> use <strong>of</strong><br />

predetermined thresholds <strong>of</strong> 98% <strong>and</strong> 90% (relative specificity <strong>and</strong> relative sensitivity <strong>of</strong><br />

RealTime versus hc2), respectively. The favorable <strong>clinical</strong> <strong>performance</strong> <strong>of</strong> RealTime was also<br />

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confirmed in our secondary study group - total study population - which comprised 4,432<br />

women 20-64 years old (57 cases <strong>and</strong> 4,375 controls). Some previous studies have shown that<br />

hc2 might benefit from adjusting <strong>the</strong> cut<strong>of</strong>f value to 2.0-2.5 RLU/CO, which improved hc2<br />

<strong>clinical</strong> specificity <strong>and</strong> PPV, while <strong>the</strong> effect on <strong>clinical</strong> sensitivity was minimal (42-43). This<br />

was not confirmed in our study, since adjustment <strong>of</strong> <strong>the</strong> hc2 cut<strong>of</strong>f value from 1.00 to 2.5<br />

RLU/CO resulted in a marginal improvement <strong>of</strong> hc2 <strong>clinical</strong> specificity but a substantial<br />

reduction <strong>of</strong> its <strong>clinical</strong> sensitivity. This was probably <strong>the</strong> result <strong>of</strong> an specific distribution <strong>of</strong><br />

hc2 RLU/CO values among cases, i.e., three women with CIN2+ had RLU/CO values<br />

between 1.00 <strong>and</strong> 2.00: 1.18, 1.44 <strong>and</strong> 1.67.<br />

The <strong>clinical</strong> specificity <strong>of</strong> <strong>the</strong> hc2 assessed in our study is in agreement with results<br />

obtained in similar previous studies. Recent meta-analysis <strong>of</strong> HPV primary screening trials<br />

thus showed a pooled <strong>clinical</strong> specificity <strong>of</strong> hc2 in North American <strong>and</strong> European trials <strong>of</strong><br />

91.3% (95% CI, 89.5 to 93.1%; range: 85 to 95%)(1,32). In European HPV primary screening<br />

studies, hc2 <strong>and</strong> GP5+/6+ PCR pooled <strong>clinical</strong> specificity was 93.3% (95% CI, 92.9 to<br />

93.6%) for women 35–49 years <strong>of</strong> age <strong>and</strong> 90.7% (95% CI, 90.4 to 91.1%) for all women<br />

(12). In recent trials, hc2 <strong>clinical</strong> specificities were 93.2% (95% CI, 92.8 to 93.6%) for<br />

women 35–60 years <strong>of</strong> age (41) <strong>and</strong> 94.1% (95% CI, 93.4 to 94.8%) for women 30–69 years<br />

<strong>of</strong> age (31). Due to <strong>the</strong> fact that <strong>the</strong> hrHPV prevalence among Slovenian women is still<br />

relatively high in <strong>the</strong> age group 30-34 years (12.8% by <strong>Abbott</strong>; 14.4% by hc2), based on our<br />

results HPV primary screening in our country would be feasible only if it starts at <strong>the</strong> age <strong>of</strong><br />

35 years. Similar findings have been also recently been described in o<strong>the</strong>r European countries<br />

(1, 12, 16, 41). The <strong>clinical</strong> specificity <strong>of</strong> <strong>the</strong> two HPV assays in our study improved<br />

substantially in women above 35 years: RealTime had a <strong>clinical</strong> specificity <strong>of</strong> 94.4%<br />

(2,188/2,317; 95% CI, 93.4 to 95.3%) <strong>and</strong> hc2 had a <strong>clinical</strong> specificity at a cut<strong>of</strong>f value <strong>of</strong><br />

1.00 RLU/CO <strong>of</strong> 93.0% (2,154/2,317; 95% CI, 91.9 to 94.0%).<br />

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In addition to <strong>the</strong> assessment <strong>of</strong> <strong>clinical</strong> <strong>performance</strong>, six previously published RealTime<br />

evaluations have also assessed some <strong>analytical</strong> characteristics <strong>of</strong> <strong>the</strong> novel assay, mainly in<br />

comparison to hc2 (14, 20, 22, 25, 39, 47) <strong>and</strong> one study examined RealTime <strong>analytical</strong><br />

<strong>performance</strong> in details (23). In <strong>the</strong>se seven studies, RealTime showed comparable <strong>analytical</strong><br />

sensitivity to hc2 but superior <strong>analytical</strong> specificity. These findings were also confirmed in <strong>the</strong><br />

present study, in which <strong>the</strong> <strong>analytical</strong> <strong>performance</strong> <strong>of</strong> <strong>the</strong> two HPV assays were compared on<br />

<strong>the</strong> largest sample collection to date (4,479 samples). Excellent <strong>analytical</strong> agreement between<br />

RealTime <strong>and</strong> hc2, with a kappa value <strong>of</strong> 0.84 <strong>and</strong> a total agreement <strong>of</strong> 96.4% was obtained in<br />

our study but <strong>the</strong> <strong>analytical</strong> accuracy <strong>of</strong> RealTime in detecting 13 assay-common HPV types<br />

was significantly higher than that <strong>of</strong> hc2. The vast majority <strong>of</strong> 116 RealTime negative/hc2<br />

positive samples were considered to be <strong>analytical</strong>ly hc2 false positive <strong>and</strong> were probably <strong>the</strong><br />

consequence <strong>of</strong> previously described hc2 probe cocktail cross-reactivity with untargeted HPV<br />

types (7, 38). Similarly to previous findings, <strong>the</strong> four most frequently identified non-targeted<br />

HPV types causing hc2 false positive results in our study were HPV66, HPV53, HPV67 <strong>and</strong><br />

HPV70. One quarter <strong>of</strong> <strong>the</strong> RealTime negative/hc2 positive samples were considered to be<br />

<strong>analytical</strong>ly RealTime false negative results: approximately half <strong>of</strong> <strong>the</strong>se samples repeatedly<br />

showed some degree <strong>of</strong> RealTime HPV-specific amplification but with a cycle number<br />

beyond <strong>the</strong> assay cut<strong>of</strong>f <strong>and</strong> approximately half <strong>of</strong> samples were RealTime HPV non-reactive.<br />

RealTime most <strong>of</strong>ten missed HPV68, probably due to <strong>the</strong> lower ability <strong>of</strong> <strong>the</strong> GP5+/6+ primer<br />

mix to detect <strong>the</strong> HPV68 prototype. This problem is not limited only to GP5+/6+ based assays<br />

such as RealTime. PGMY-primer based assays such as Linear Array, designed to detect<br />

HPV68 subtype b, also cannot detect <strong>the</strong> HPV68 prototype because <strong>of</strong> several mismatches<br />

(34). HPV68 was <strong>the</strong> least commonly detected hrHPV type in <strong>the</strong> recent 2009 <strong>and</strong> 2010 HPV<br />

DNA Pr<strong>of</strong>iciency Panels prepared by <strong>the</strong> World Health Organization HPV Laboratory<br />

Network – LabNet; this HPV type was correctly identified by fewer than 38% <strong>of</strong> participating<br />

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laboratories (17). Of <strong>the</strong> 44 samples detected in our study by RealTime <strong>and</strong> not by hc2, 38<br />

(86.3%) were considered to be <strong>analytical</strong>ly RealTime true positive. hc2 most <strong>of</strong>ten missed<br />

HPV51, HPV16 <strong>and</strong> HPV18, probably due to <strong>the</strong> presence <strong>of</strong> low levels <strong>of</strong> <strong>the</strong> HPV target,<br />

which may not be reliably detected by hc2; i.e., approximately half <strong>of</strong> <strong>the</strong>se samples had<br />

repeated hc2 RLU/CO values between 0.60-0.99. Five RealTime positive/hc2 negative<br />

samples tested HPV DNA negative using all three broad-range PCR-based genotyping<br />

methods applied <strong>and</strong> only low-risk HPV6 was found in one RealTime positive/hc2 negative<br />

sample. These six samples were considered to be <strong>analytical</strong>ly RealTime false positive; <strong>the</strong><br />

most probable reason for false positivity was amplicon contamination (all samples had late<br />

Ct).<br />

In <strong>the</strong> present study, intra-laboratory reproducibility <strong>and</strong> inter-laboratory agreement <strong>of</strong><br />

RealTime were assessed for <strong>the</strong> first time in two laboratories (Ljubljana <strong>and</strong> Hannover),<br />

following <strong>the</strong> requirements set in guidelines for <strong>the</strong> evaluation <strong>of</strong> c<strong>and</strong>idate HPV assays for<br />

cervical cancer screening purposes (32). According to this document, intra-laboratory<br />

reproducibility in time <strong>and</strong> inter-laboratory agreement should be determined by evaluation <strong>of</strong><br />

at least 500 samples, 30% <strong>of</strong> which tested positive in a reference laboratory using a <strong>clinical</strong>ly<br />

validated assay. This should result in an agreement with a lower confidence boundary not less<br />

than 87% (kappa value <strong>of</strong> at least 0.5 in this series <strong>of</strong> samples including 30% positives) <strong>and</strong><br />

<strong>the</strong> same intra-laboratory reproducibility should be achieved after testing <strong>the</strong> same set <strong>of</strong><br />

samples several weeks later. Our evaluation showed that RealTime can be considered to be a<br />

reliable <strong>and</strong> robust HPV assay, since intra-laboratory <strong>and</strong> inter-laboratory kappa <strong>and</strong><br />

agreement values exceeded those set in <strong>the</strong> recommendations <strong>and</strong> those obtained in similar<br />

previous evaluations <strong>of</strong> hc2 (5-6). As expected, <strong>the</strong> vast majority <strong>of</strong> o<strong>the</strong>rwise infrequent HPV<br />

type-specific discordant RealTime results were obtained in samples containing several HPV<br />

types (mixed HPV infection), probably due to amplification competition. However, HPV<br />

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type-specific discordant results did not influence RealTime overall HPV positivity with <strong>the</strong><br />

exception <strong>of</strong> a single sample in <strong>the</strong> Hannover laboratory, in which presumably <strong>the</strong> low<br />

quantity <strong>of</strong> HPV16 present in <strong>the</strong> sample produced a discordant borderline positive <strong>and</strong><br />

borderline negative result in <strong>the</strong> first <strong>and</strong> second testing rounds, respectively.<br />

In summary, <strong>the</strong> evaluation <strong>of</strong> RealTime in <strong>the</strong> population-based cervical cancer screening<br />

setting showed that <strong>the</strong> <strong>clinical</strong> <strong>performance</strong> <strong>of</strong> RealTime is not inferior to that <strong>of</strong> <strong>the</strong><br />

<strong>clinical</strong>ly validated reference HPV assay hc2, in women above 30 years <strong>and</strong> in women 20-64<br />

years. Excellent <strong>analytical</strong> agreement between RealTime <strong>and</strong> hc2 results was obtained while<br />

<strong>the</strong> <strong>analytical</strong> accuracy <strong>of</strong> RealTime was significantly higher than that <strong>of</strong> hc2. The typing<br />

information for HPV16 <strong>and</strong> HPV18 provided by RealTime could serve as a valuable<br />

additional tool in patient risk stratification <strong>and</strong> management. RealTime displayed high intra-<br />

laboratory reproducibility <strong>and</strong> inter-laboratory agreement. According to our results <strong>and</strong> <strong>the</strong><br />

results <strong>of</strong> previous studies, RealTime can be considered to be a reliable <strong>and</strong> robust HPV assay<br />

<strong>clinical</strong>ly comparable with hc2 for <strong>the</strong> detection <strong>of</strong> CIN2+ lesions in population-based<br />

cervical cancer screening setting.<br />

ACKNOWLEDGEMENT<br />

<strong>Abbott</strong> Molecular, <strong>the</strong> National Institute <strong>of</strong> Public Health <strong>of</strong> Slovenia <strong>and</strong> <strong>the</strong> Institute <strong>of</strong><br />

Microbiology <strong>and</strong> Immunology, Faculty <strong>of</strong> Medicine, University <strong>of</strong> Ljubljana provided<br />

financial support for <strong>the</strong> assays <strong>and</strong> logistical conduct <strong>of</strong> <strong>the</strong> study. <strong>Abbott</strong> Molecular was not<br />

involved in <strong>the</strong> study design, data collection, data analysis <strong>and</strong> interpretation, or writing <strong>the</strong><br />

manuscript. The authors would like to thank <strong>clinical</strong> colleagues: Petra Bavčar, Irena Begič,<br />

Lara Beseničar Pregelj, Martina Bučar, Simona Čopi, Petra Eržen Vrlič, Andreja Gornjec,<br />

Mojca Grebenc, Mojca Jemec, Jožefa Kežar, Tatjana Kodrič, Zdravka Koman, Jasna<br />

Kostanjšek, Jasna Kuhelj Recer, Zlatko Lazić, Sonja Lepoša, Mili Lomšek, Sladjana Malić,<br />

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Petra Meglič, Maja Merkun, Aleks<strong>and</strong>er Merlo, Anamarija Petek, Suzana Peternelj Marinšek,<br />

Igor Pirc, Uršula Reš Muravec, Filip Simoniti, Lucija Sorč, Tina Steinbacher Kokalj, Mateja<br />

Darija Strah, Vesna Šalamun, Ksenija Šelih Martinec <strong>and</strong> Andrej Zore for patient recruitment<br />

<strong>and</strong> management, Petra Markočič for study management, Petra Čuk, Robert Krošelj, Boštjan<br />

J. Kocjan <strong>and</strong> Mateja Jelen for excellent laboratory assistance, Jasna Šinkovec, Marja Lenart,<br />

<strong>and</strong> Boštjan Luzar for cytology <strong>and</strong> histology review, Matthias Jentschke for reproducibility<br />

testing, Johannes Berkh<strong>of</strong> for help with noninferiority test calculations <strong>and</strong> Miha Pirc for<br />

sample transportation.<br />

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REFERENCES<br />

1. Arbyn, M., P. Sasieni, C. J. Meijer, C. Clavel, G. Koliopoulos, <strong>and</strong> J. Dillner. 2006.<br />

Chapter 9: Clinical applications <strong>of</strong> HPV testing: a summary <strong>of</strong> meta-analyses. Vaccine.<br />

24(Suppl. 3):78-89.<br />

2. Arbyn, M., G. Ronco, J. Cuzick, N. Wentzensen, <strong>and</strong> P. E. Castle. 2009. How to<br />

evaluate emerging technologies in cervical cancer screening? Int. J. Cancer. 125:2489-2496.<br />

3. Barzon, L., C. Giorgi, F. M. Buonaguro, G. Palù, <strong>and</strong> Italian Society for Virology.<br />

2008. Guidelines <strong>of</strong> <strong>the</strong> Italian Society for Virology on HPV testing <strong>and</strong> vaccination for<br />

cervical cancer prevention. Infect. Agent Cancer. 3:14.<br />

4. Bulkmans, N. W., J. Berkh<strong>of</strong>, L. Rozendaal, F. J. van Kemenade, A. J. Boeke, S. Bulk,<br />

F. J. Voorhorst, R. H. Verheijen, K. van Groningen, M. E. Boon, W. Ruitinga, M. van<br />

Ballegooijen, P. J. Snijders, <strong>and</strong> C. J. Meijer. 2007. Human papillomavirus DNA testing<br />

for <strong>the</strong> detection <strong>of</strong> cervical intraepi<strong>the</strong>lial neoplasia grade 3 <strong>and</strong> cancer: 5-Year follow-up <strong>of</strong><br />

a r<strong>and</strong>omised controlled implementation trial. Lancet. 370:1764–1772.<br />

5. Carozzi, F. M., A. Del Mistro, M. Confortini, C. Sani, D. Puliti, R. Trevisan, L. De<br />

Marco, A. G. Tos, S. Girl<strong>and</strong>o, P. D. Palma, A. Pellegrini, M. L. Schiboni, P. Crucitti, P.<br />

Pierotti, A. Vignato, <strong>and</strong> G. Ronco. 2005. Reproducibility <strong>of</strong> HPV DNA Testing by Hybrid<br />

Capture 2 in a Screening Setting. Am. J. Clin. Pathol. 124:716–721.<br />

6. Castle, P. E., C. M. Wheeler, D. Solomon, M. Schiffman, C. L. Peyton, <strong>and</strong> ALTS<br />

Group. 2004. Interlaboratory reliability <strong>of</strong> Hybrid Capture 2. Am. J. Clin. Pathol. 122:238–<br />

245.<br />

7. Castle, P. E., D. Solomon, C. M. Wheeler, P. E. Gravitt, S. Wacholder, <strong>and</strong> M.<br />

Schiffman. 2008. Human papillomavirus genotype specificity <strong>of</strong> hybrid capture 2. J. Clin.<br />

Microbiol. 46:2595-2604.<br />

8. Castle, P. E., A. C. Rodríguez, R. D. Burk, R. Herrero, S. Wacholder, M. Alfaro, J.<br />

Morales, D. Guillen, M. E. Sherman, D. Solomon, M. Schiffman, <strong>and</strong> Proyecto<br />

Epidemiológico Guanacaste (PEG) Group. 2009. Short term persistence <strong>of</strong> human<br />

papillomavirus <strong>and</strong> risk <strong>of</strong> cervical precancer <strong>and</strong> cancer: population based cohort study. Br.<br />

Med. J. 339:b2569.<br />

9. Castle, P. E., M. Sadorra, T. Lau, C. Aldrich, F. A. Garcia, <strong>and</strong> J. Kornegay. 2009.<br />

Evaluation <strong>of</strong> a prototype real-time PCR assay for carcinogenic human papillomavirus (HPV)<br />

detection <strong>and</strong> simultaneous HPV genotype 16 (HPV16) <strong>and</strong> HPV18 genotyping. J. Clin.<br />

Microbiol. 47:3344-3347.<br />

10. Cox, J. T. 2009. History <strong>of</strong> <strong>the</strong> use <strong>of</strong> HPV testing in cervical screening <strong>and</strong> in <strong>the</strong><br />

management <strong>of</strong> abnormal cervical screening results. J. Clin. Virol. 45(Suppl. 1):S3-S12.<br />

11. Cuschieri, K. S., H. A. Cubie. 2005. The role <strong>of</strong> human papillomavirus testing in cervical<br />

screening. J. Clin. Virol. 32(Suppl. 1):S34-42.<br />

25


1<br />

2<br />

3<br />

4<br />

5<br />

6<br />

7<br />

8<br />

9<br />

10<br />

11<br />

12<br />

13<br />

14<br />

15<br />

16<br />

17<br />

18<br />

19<br />

20<br />

21<br />

22<br />

23<br />

24<br />

25<br />

26<br />

27<br />

28<br />

29<br />

30<br />

31<br />

32<br />

33<br />

34<br />

35<br />

36<br />

37<br />

38<br />

39<br />

40<br />

41<br />

42<br />

43<br />

44<br />

45<br />

46<br />

47<br />

48<br />

49<br />

12. Cuzick, J., C. Clavel, K. U. Petry, C. J. Meijer, H. Hoyer, S. Ratnam, A. Szarewski,<br />

P. Birembaut, S. Kulasingam, P. Sasieni, <strong>and</strong> T. Iftner. 2006. Overview <strong>of</strong> <strong>the</strong> European<br />

<strong>and</strong> North American studies on HPV testing in primary cervical cancer screening. Int. J.<br />

Cancer. 119:1095-1101.<br />

13. Cuzick, J., M. Arbyn, R. Sankaranarayanan, V. Tsu, G. Ronco, M. H. Mayr<strong>and</strong>, J.<br />

Dillner, <strong>and</strong> C. J. Meijer. 2008. Overview <strong>of</strong> human papillomavirus-based <strong>and</strong> o<strong>the</strong>r novel<br />

options for cervical cancer screening in developed <strong>and</strong> developing countries. Vaccine.<br />

26(Suppl. 10):K29-41.<br />

14. Cuzick, J., L. Ambroisine, L. Cadman, J. Austin, L. Ho, G. Terry, S. Liddle, R. Dina,<br />

J. McCarthy, H. Buckley, C. Bergeron, W. P. Soutter, D. Lyons, <strong>and</strong> A. Szarewski. 2010.<br />

Performance <strong>of</strong> <strong>the</strong> RealTime RealTime high-risk HPV test in women with abnormal cervical<br />

cytology smears. J. Med. Virol. 82:1186-1191.<br />

15. Desai, M.S., <strong>and</strong> H. A. Cubie. 2005. The HPV test in cervical screening: a brave new<br />

world? Cytopathology. 16:3-6.<br />

16. Dillner, J., M. Rebolj, P. Birembaut, K. U. Petry, A. Szarewski, C. Munk, S. de<br />

Sanjose, P. Naucler, B. Lloveras, S. Kjaer, J. Cuzick, M. van Ballegooijen, C. Clavel, T.<br />

Iftner, <strong>and</strong> Joint European Cohort Study. 2008. Long term predictive values <strong>of</strong> cytology<br />

<strong>and</strong> human papillomavirus testing in cervical cancer screening: Joint European cohort study.<br />

Br. Med. J. 337:a1754.<br />

17. Eklund, C., T. Zhou, J. Dillner, <strong>and</strong> WHO Human Papillomavirus Laboratory<br />

Network. 2010. Global pr<strong>of</strong>iciency study <strong>of</strong> human papillomavirus genotyping. J. Clin.<br />

Microbiol. 48:4147-4155.<br />

18. Evans, M. F., C. S. Adamson, L. Simmons-Arnold, <strong>and</strong> K. Cooper. 2005. Touchdown<br />

General Primer (GP5+/GP6+) PCR <strong>and</strong> optimized sample DNA concentration support <strong>the</strong><br />

sensitive detection <strong>of</strong> human papillomavirus. BMC Clin. Pathol. 5:10.<br />

19. Gravitt, P. E., F. Coutlée, T. Iftner, J. W. Sellors, W. G. Quint, <strong>and</strong> C. M. Wheeler.<br />

2008. New technologies in cervical cancer screening. Vaccine. 26(Suppl. 10):K42-52.<br />

20. Halfon, P., D. Benmoura, A. Agostini, H. Khiri, G. Penar<strong>and</strong>a, A. Martineau, <strong>and</strong> B.<br />

Blanc. 2010. Evaluation <strong>of</strong> <strong>the</strong> <strong>clinical</strong> <strong>performance</strong> <strong>of</strong> <strong>the</strong> RealTime RealTime High-Risk<br />

HPV for carcinogenic HPV detection. J. Clin. Virol. 48:246-250.<br />

21. Hesselink, A. T., D. A. Heideman, J. Berkh<strong>of</strong>, F. Topal, R. P. Pol, C. J. Meijer, <strong>and</strong> P.<br />

J. Snijders. 2010. <strong>Comparison</strong> <strong>of</strong> <strong>the</strong> <strong>clinical</strong> <strong>performance</strong> <strong>of</strong> PapilloCheck human<br />

papillomavirus detection with that <strong>of</strong> <strong>the</strong> GP5+/6+-PCR-enzyme immunoassay in populationbased<br />

cervical screening. J. Clin. Microbiol. 48:797-801.<br />

22. Huang, S., B. Erickson, N. Tang, W. B. Mak, J. Salituro, J. Robinson, <strong>and</strong> K.<br />

Abravaya. 2009. Clinical <strong>performance</strong> <strong>of</strong> RealTime RealTime High Risk HPV test for<br />

detection <strong>of</strong> high-grade cervical intraepi<strong>the</strong>lial neoplasia in women with abnormal cytology. J.<br />

Clin. Virol. 45(Suppl. 1):S19–23.<br />

26


1<br />

2<br />

3<br />

4<br />

5<br />

6<br />

7<br />

8<br />

9<br />

10<br />

11<br />

12<br />

13<br />

14<br />

15<br />

16<br />

17<br />

18<br />

19<br />

20<br />

21<br />

22<br />

23<br />

24<br />

25<br />

26<br />

27<br />

28<br />

29<br />

30<br />

31<br />

32<br />

33<br />

34<br />

35<br />

36<br />

37<br />

38<br />

39<br />

40<br />

41<br />

42<br />

43<br />

44<br />

45<br />

46<br />

47<br />

48<br />

49<br />

50<br />

23. Huang, S., N. Tang, W. B. Mak, B. Erickson, J. Salituro, Y. Li, E. Krumpe, G.<br />

Schneider, H. Yu, J. Robinson, <strong>and</strong> K. Abravaya. 2009. Principles <strong>and</strong> <strong>analytical</strong><br />

<strong>performance</strong> <strong>of</strong> RealTime RealTime HR HPV test. J. Clin. Virol. 45(Suppl. 1):S13–17.<br />

24. Jacobs, M. V., P. J. F. Snijders, A. J. C. van den Brule, T. J. M. Helmerhorst, C. J. L.<br />

M. Meijer, <strong>and</strong> J. M. M. Walboomers. 1997. A general primer GP5+/GP6+-mediated PCRenzyme<br />

immunoassay method for rapid detection <strong>of</strong> 14 high-risk <strong>and</strong> 6 low-risk human<br />

papillomavirus genotypes in cervical scrapings. J. Clin. Microbiol. 35:791-795.<br />

25. Kaliterna, V., S. Z. Lepej, <strong>and</strong> A. Vince. 2009. <strong>Comparison</strong> between <strong>the</strong> RealTime<br />

RealTime High Risk HPV assay <strong>and</strong> <strong>the</strong> Hybrid Capture 2 assay for detecting high-risk<br />

human papillomavirus DNA in cervical specimens. J. Med. Microbiol. 58:1662-1663.<br />

26. Khan, M. J., P. E. Castle, A. T. Lorincz, S. Wacholder, M. Sherman, D. R. Scott, B. B.<br />

Rush, A. G. Glass, <strong>and</strong> M. Schiffman. 2005. The elevated 10-year risk <strong>of</strong> cervical precancer<br />

<strong>and</strong> cancer in women with human papillomavirus (HPV) type 16 or 18 <strong>and</strong> <strong>the</strong> possible utility<br />

<strong>of</strong> type-specific HPV testing in <strong>clinical</strong> practice. J. Natl. Cancer Inst. 97:1072-1079.<br />

27. Kinney, W., M. H. Stoler, <strong>and</strong> P. E. Castle. 2010. Special commentary: Patient safety<br />

<strong>and</strong> <strong>the</strong> next generation <strong>of</strong> HPV DNA tests. Am. J. Clin. Pathol. 134:193-199.<br />

28. Kocjan, B. J., M. Poljak, K. Seme, M. Potocnik, K. Fujs, <strong>and</strong> D. Z. Babic. 2005.<br />

Distribution <strong>of</strong> human papillomavirus genotypes in plucked eyebrow hairs from Slovenian<br />

males with genital warts. Infect. Genet. Evol. 5:255-259.<br />

29. Kocjan, B. J., M. Poljak, <strong>and</strong> K. Seme. 2010. Universal ProbeLibrary based real-time<br />

PCR assay for detection <strong>and</strong> confirmation <strong>of</strong> human papillomavirus genotype 52 infections. J.<br />

Virol. Methods. 163:492-494.<br />

30. Lörincz, A. T. 1996. Hybrid Capture method for detection <strong>of</strong> human papillomavirus DNA<br />

in <strong>clinical</strong> specimens: a tool for <strong>clinical</strong> management <strong>of</strong> equivocal Pap smears <strong>and</strong> for<br />

population screening. J. Obstet. Gynaecol. Res. 22:629-636.<br />

31. Mayr<strong>and</strong>, M. H., E. Duarte-Franco, I. Rodrigues, S. D. Walter, J. Hanley, A.<br />

Ferenczy, S. Ratnam, F. Coutle´e, E. L. Franco, <strong>and</strong> Canadian Cervical Cancer<br />

Screening Trial Study Group. 2007. Human papillomavirus DNA versus Papanicolaou<br />

screening tests for cervical cancer. N. Engl. J. Med. 357:1579–1588.<br />

32. Meijer, C. J., J. Berkh<strong>of</strong>, P. E. Castle, A. T. Hesselink, E. L. Franco, G. Ronco, M.<br />

Arbyn, F. X. Bosch, J. Cuzick, J. Dillner, D. A. Heideman, <strong>and</strong> P. J. Snijders. 2009.<br />

Guidelines for human papillomavirus DNA test requirements for primary cervical cancer<br />

screening in women 30 years <strong>and</strong> older. Int. J. Cancer. 124:516-520.<br />

33. Naucler, P., W. Ryd, S. Törnberg, A. Str<strong>and</strong>, G. Wadell, K. Elfgren, T. Radberg, B.<br />

Str<strong>and</strong>er, O. Forslund, B. G. Hansson, E. Ryl<strong>and</strong>er, <strong>and</strong> J. Dillner. 2007. Human<br />

papillomavirus <strong>and</strong> Papanicolaou tests to screen for cervical cancer. N. Engl. J. Med.<br />

357:1589–1597.<br />

34. Nazarenko, I., L. Kobayashi, J. Giles, C. Fishman, G. Chen, <strong>and</strong> A. Lorincz. 2008. A<br />

novel method <strong>of</strong> HPV genotyping using Hybrid Capture sample preparation method combined<br />

27


1<br />

2<br />

3<br />

4<br />

5<br />

6<br />

7<br />

8<br />

9<br />

10<br />

11<br />

12<br />

13<br />

14<br />

15<br />

16<br />

17<br />

18<br />

19<br />

20<br />

21<br />

22<br />

23<br />

24<br />

25<br />

26<br />

27<br />

28<br />

29<br />

30<br />

31<br />

32<br />

33<br />

34<br />

35<br />

36<br />

37<br />

38<br />

39<br />

40<br />

41<br />

42<br />

43<br />

44<br />

45<br />

46<br />

47<br />

48<br />

49<br />

50<br />

with GP5+/6+ PCR <strong>and</strong> multiplex detection on Luminex XMAP. J. Virol. Methods. 154:76-<br />

81.<br />

35. Nicula, F. A., A. Anttila, L. Neamtiu, M. P. Zakelj, R. Tachezy, A. Chil, M. Grce, <strong>and</strong><br />

V. Kesić. 2009. Challenges in starting organised screening programmes for cervical cancer in<br />

<strong>the</strong> new member states <strong>of</strong> <strong>the</strong> European Union. Eur. J. Cancer. 45:2679-2684.<br />

36. Petry, K. U., S. Menton, M. Menton, F. van Loenen-Frosch, H. de Carvalho Gomes,<br />

B. Holz, B. Schopp, S. Garbrecht-Buettner, P. Davies, G. Boehmer, E. van den Akker, T.<br />

Iftner. 2003. Inclusion <strong>of</strong> HPV testing in routine cervical cancer screening for women above<br />

29 years in Germany: results for 8466 patients. Br. J. Cancer. 88:1570-1577.<br />

37. Poljak, M., A. Brenčič, K. Seme, A. Vince, <strong>and</strong> I. J. Marin. 1999. Comparative<br />

evaluation <strong>of</strong> first- <strong>and</strong> second-generation Digene Hybrid Capture assays for detection <strong>of</strong><br />

human papillomaviruses associated with high or intermediate risk for cervical cancer. J. Clin.<br />

Microbiol. 37:796-797.<br />

38. Poljak, M., I. J. Marin, K. Seme, <strong>and</strong> A. Vince. 2002. Hybrid Capture II HPV test<br />

detects at least 15 human papillomavirus genotypes not included in its current high risk<br />

cocktail. J. Clin. Virol. 25(Suppl. 3):S89-S97.<br />

39. Poljak, M., A. Kov<strong>and</strong>a, B. J. Kocjan, K. Seme, N. Jancar, <strong>and</strong> E. Vrtacnik-Bokal.<br />

2009. The RealTime RealTime High Risk HPV test: comparative evaluation <strong>of</strong> <strong>analytical</strong><br />

specificity <strong>and</strong> <strong>clinical</strong> sensitivity for cervical carcinoma <strong>and</strong> CIN 3 lesions with <strong>the</strong> Hybrid<br />

Capture 2 HPV DNA test. Acta Dermatovenerol. Alp. Panonica Adriat. 18:94-103.<br />

40. Poljak, M., <strong>and</strong> B. J. Kocjan. 2010. Commercially available assays for multiplex<br />

detection <strong>of</strong> alpha human papillomaviruses. Exp. Rev. Anti. Infect. Ther. 8:1139-1162.<br />

41. Ronco, G., M. van Ballegooijen, N. Becker, A. Chil, M. Fender, P. Giubilato, J.<br />

Kurtinaitis, L. Lancucki, E. Lynge, A. Morais, M. O'Reilly, P. Sparen, O. Suteu, M.<br />

Rebolj, P. Veerus, M. P. Zakelj, <strong>and</strong> A. Anttila. 2009. Process <strong>performance</strong> <strong>of</strong> cervical<br />

screening programmes in Europe. Eur. J. Cancer. 45:2659-2670.<br />

42. Sargent, A., A. Bailey, A. Turner, M. Almonte, C. Gilham, H. Baysson, J. Peto, C.<br />

Roberts, C. Thomson, M. Desai, J. Ma<strong>the</strong>r, <strong>and</strong> H. Kitchener. 2010. Optimal threshold for<br />

a positive hybrid capture 2 test for detection <strong>of</strong> human papillomavirus: data from <strong>the</strong><br />

ARTISTIC trial. J. Clin. Microbiol. 48:554-558.<br />

43. Seme, K., K. Fujs, B. J. Kocjan, <strong>and</strong> M. Poljak. 2006. Resolving repeatedly borderline<br />

results <strong>of</strong> Hybrid Capture 2 HPV DNA Test using polymerase chain reaction <strong>and</strong> genotyping.<br />

J. Virol. Methods. 134:252-256.<br />

44. Snijders, P. J., D. A. Heideman, <strong>and</strong> C. J. Meijer. 2010. Methods for HPV detection in<br />

exfoliated cell <strong>and</strong> tissue specimens. APMIS. 118:520-528.<br />

45. Stevens, M. P., S. M. Garl<strong>and</strong>, <strong>and</strong> S. N. Tabrizi. 2006. Human papillomavirus<br />

genotyping using a modified linear array detection protocol. J. Virol. Methods.135:124-<br />

126.<br />

28


1<br />

2<br />

3<br />

4<br />

5<br />

6<br />

7<br />

8<br />

9<br />

10<br />

11<br />

12<br />

13<br />

14<br />

15<br />

16<br />

17<br />

18<br />

46. Stoler, M. H., P. E. Castle, D. Solomon, <strong>and</strong> M. Schiffman. 2007. The exp<strong>and</strong>ed use <strong>of</strong><br />

HPV testing in gynecologic practice per ASCCP-guided management requires <strong>the</strong> use <strong>of</strong> wellvalidated<br />

assays. Am. J. Clin. Pathol. 127:335–337.<br />

47. Tang, N., S. Huang, B. Erickson, W. B. Mak, J. Salituro, J. Robinson, <strong>and</strong> K.<br />

Abravaya. 2009. High-risk HPV detection <strong>and</strong> concurrent HPV 16 <strong>and</strong> 18 typing with<br />

RealTime RealTime High Risk HPV test. J. Clin. Virol. 45(Suppl. 1):S25–29.<br />

48. Walker, P., S. Dexeus, G. De Palo, R. Barrasso, M. Campion, F. Girardi, C. Jakob,<br />

M. Roy, <strong>and</strong> Nomenclature Committee <strong>of</strong> <strong>the</strong> International Federation for Cervical<br />

Pathology <strong>and</strong> Colposcopy. 2003. International terminology <strong>of</strong> colposcopy: an updated<br />

report from <strong>the</strong> International Federation for Cervical Pathology <strong>and</strong> Colposcopy. Obstet.<br />

Gynecol. 101:175-177.<br />

49. zur Hausen, H. 2009. Papillomaviruses in <strong>the</strong> causation <strong>of</strong> human cancers - a brief<br />

historical account. Virology. 384:260-265.<br />

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8<br />

TABLE 1. <strong>Comparison</strong> <strong>of</strong> RealTime <strong>and</strong> hc2 resolved results stratified for cases (CIN2+<br />

lesions) <strong>and</strong> controls (less than CIN2) in women above 30 years (N=3,129) <strong>and</strong> total study<br />

population (N=4,432).<br />

Study group <strong>and</strong><br />

RealTime results<br />

Women above 30 years<br />

Controls<br />

30<br />

hc2 results<br />

No. (%) <strong>of</strong> samples<br />

- + Total<br />

- 2,816 (91.1) 69 (2.2) 2,885 (93.3)<br />

+ 21 (0.7) 185 (6.0) 206 (6.7)<br />

Total 2,837 (91.8) 254 (8.2) 3,091<br />

Cases<br />

Total study population<br />

- 0 (0.0) 0 (0.0) 0 (0)<br />

+ 1 (2.6) 37 (97.4) 38 (100.0)<br />

Total 1 (2.6) 37 (97.4) 38<br />

Controls<br />

- 3,800 (86.9) 115 (2.6) 3,915 (89.5)<br />

+ 39 (0.9) 421 (9.6) 460 (10.5)<br />

Total 3,839 (87.8) 536 (12.2) 4,375<br />

Cases<br />

- 1 (1.8) 0 (0.0) 1 (1.8)<br />

+ 2 (3.5) 54 (94.7) 56 (98.2)<br />

Total 3 (5.3) 54 (94.7) 57


1<br />

2<br />

3<br />

TABLE 2. <strong>Comparison</strong> <strong>of</strong> <strong>clinical</strong> sensitivity for <strong>the</strong> detection <strong>of</strong> CIN2+, <strong>clinical</strong> specificity for <strong>the</strong> detection <strong>of</strong> lesions less than CIN2, positive<br />

predictive value <strong>and</strong> negative predictive value <strong>of</strong> RealTime <strong>and</strong> hc2 at cut<strong>of</strong>f values <strong>of</strong> 1.00 <strong>and</strong> 2.50 RLU/CO in women above 30 years <strong>and</strong> in<br />

total study population.<br />

Women above 30 years<br />

RealTime 100.0%<br />

Clinical sensitivity Clinical specificity Positive predictive value Negative predictive value<br />

(38/38; 95% CI, 86.5-100.0%)<br />

hc2 (1.00 RLU/CO) 97.4%<br />

(37/38; 95% CI, 86.2-99.9%)<br />

hc2 (2.50 RLU/CO) 92.1%<br />

Total study population<br />

(35/38; 95% CI, 78.6-98.3%)<br />

RealTime 98.2%<br />

(56/57; 95% CI, 90.6-100.0%)<br />

hc2 (1.00 RLU/CO) 94.7%<br />

(54/57; 95% CI, 85.4-98.9%)<br />

hc2 (2.50 RLU/CO) 89.5%<br />

(51/57; 95% CI, 78.5-96.0%)<br />

93.3%<br />

(2,885/3,091; 95% CI, 92.4-94.2%)<br />

91.8 %<br />

(2,837/3,091; 95% CI, 90.8-92.7%)<br />

92.9%<br />

(2,872/3,091; 95% CI, 92.0-93.8%)<br />

89.5%<br />

(3,915/4,375; 95% CI, 88.5-90.4%)<br />

87.7 %<br />

(3,839/4,375; 95% CI, 86.7-88.7%)<br />

89.1%<br />

(3,900/4,375; 95% CI, 88.2-90.0%)<br />

31<br />

15.6%<br />

(38/244; 95% CI, 11.3-20.7%)<br />

12.7%<br />

(37/291; 95% CI, 9.1-17.1%)<br />

13.8%<br />

(35/254; 95% CI, 9.8-18.6%)<br />

10.9%<br />

(56/516; 95% CI, 8.3-13.9%)<br />

9.2%<br />

(54/590; 95% CI, 7.0-11.8%)<br />

9.7%<br />

(51/526; 95% CI, 7.3-12.6%)<br />

100.0%<br />

(2,885/2,885; 95% CI, 99.8-100.0%)<br />

99.9%<br />

(2,837/2,838; 95% CI, 99.8-100.0%)<br />

99.9%<br />

(2,872/2,875; 95% CI, 99.7-100.0%)<br />

100.0%<br />

(3,915/3,916; 95% CI, 99.9-100.0%)<br />

99.9%<br />

(3,839/3,842; 95% CI, 99.8-100.0%)<br />

99.8%<br />

(3,900/3,906; 95% CI, 99.7-99.9%)


1<br />

2<br />

3<br />

4<br />

5<br />

6<br />

7<br />

8<br />

9<br />

10<br />

TABLE 3. Analytical results <strong>of</strong> testing for 13 assay-common HPV types using RealTime <strong>and</strong><br />

hc2 on 4,479 samples after repeat testing <strong>of</strong> 101 samples by hc2 according to <strong>the</strong><br />

manufacturers’ instructions (initial results) <strong>and</strong> after repeat testing <strong>of</strong> 62 samples at our<br />

discretion (30 samples by RealTime <strong>and</strong> 32 samples by hc2) (resolved results).<br />

Testing result Initial results<br />

No. (%) <strong>of</strong> samples<br />

32<br />

Resolved results<br />

No. (%) <strong>of</strong> samples<br />

RealTime positive/hc2 positive 490 (10.9) 505 (11.2)<br />

RealTime negative/hc2 negative 3,814 (85.2) 3,814 (85.2)<br />

RealTime positive/hc2 negative 51 (1.1) 44 (1.0)*<br />

RealTime negative/hc2 positive 124 (2.8) 116 (2.6)**<br />

TOTAL 4,479 4,479<br />

*38 samples considered to be <strong>analytical</strong>ly RealTime true positive <strong>and</strong> 6 samples to be<br />

RealTime false positive.<br />

**86 samples considered to be <strong>analytical</strong>ly hc2 false positive <strong>and</strong> 30 samples to be hc2 true<br />

positive.


1<br />

2<br />

3<br />

TABLE 4. HPV types determined in 160 <strong>analytical</strong>ly discordant samples based on <strong>the</strong><br />

resolved hc2/RealTime results. Assay-common HPV types are shown in bold.<br />

Sample no hc2 RealTime HPV type(s) Interpretation Frequency<br />

result result<br />

1-6 negative positive 18 RealTime TP 6<br />

7-11 negative positive 51 RealTime TP 5<br />

12-15 negative positive 16 RealTime TP 4<br />

16-18 negative positive 45 RealTime TP 3<br />

19-21 negative positive 52 RealTime TP 3<br />

22-23 negative positive 31 RealTime TP 2<br />

24-25 negative positive 59 RealTime TP 2<br />

26 negative positive 35 RealTime TP 1<br />

27 negative positive 6, 16 RealTime TP 1<br />

28 negative positive 16, 55 RealTime TP 1<br />

29 negative positive 39, 66 RealTime TP 1<br />

30 negative positive 45, 89 RealTime TP 1<br />

31 negative positive 51, 73 RealTime TP 1<br />

32 negative positive 51, 89 RealTime TP 1<br />

33 negative positive 56, 74 RealTime TP 1<br />

34 negative positive 35, 53, 61 RealTime TP 1<br />

35 negative positive 40, 52, 89 RealTime TP 1<br />

36 negative positive 51, 53, 54 RealTime TP 1<br />

37 negative positive 59, 62, 66 RealTime TP 1<br />

38 negative positive 51, 56, 59, 89 RealTime TP 1<br />

39-43 negative positive neg RealTime FP 5<br />

44 negative positive 6 RealTime FP 1<br />

45-68 positive negative neg RealTime TN 24<br />

69-83 positive negative 53 RealTime TN 15<br />

84-88 positive negative 67 RealTime TN 5<br />

89-93 positive negative 70 RealTime TN 5<br />

94-97 positive negative 42 RealTime TN 4<br />

98-101 positive negative 69/71 RealTime TN 4<br />

102-104 positive negative 82 RealTime TN 3<br />

105-107 positive negative 89 RealTime TN 3<br />

108-110 positive negative 53, 55 RealTime TN 3<br />

33


1<br />

2<br />

111-112 positive negative 55 RealTime TN 2<br />

113-114 positive negative 61 RealTime TN 2<br />

115 positive negative 40 RealTime TN 1<br />

116 positive negative 67 RealTime TN 1<br />

117 positive negative 81 RealTime TN 1<br />

118 positive negative 84 RealTime TN 1<br />

119 positive negative 6, 81 RealTime TN 1<br />

120 positive negative 6, 84 RealTime TN 1<br />

121 positive negative 42, 82 RealTime TN 1<br />

122 positive negative 42, 89 RealTime TN 1<br />

123 positive negative 53, 62 RealTime TN 1<br />

124 positive negative 53, 70 RealTime TN 1<br />

125 positive negative 54, 62 RealTime TN 1<br />

126 positive negative 62, 67 RealTime TN 1<br />

127 positive negative 67, 89 RealTime TN 1<br />

128 positive negative 6, 53, 62 RealTime TN 1<br />

129 positive negative 54, 61, 62 RealTime TN 1<br />

130 positive negative 42, 44, 70, 83 RealTime TN 1<br />

131-140 positive negative 68 RealTime FN 10<br />

141-145 positive negative 31 RealTime FN 5<br />

146-148 positive negative 58 RealTime FN 3<br />

149-150 positive negative 52 RealTime FN 2<br />

151 positive negative 16 RealTime FN 1<br />

152 positive negative 59 RealTime FN 1<br />

153 positive negative 16, 67 RealTime FN 1<br />

154 positive negative 31, 39 RealTime FN 1<br />

155 positive negative 31, 53 RealTime FN 1<br />

156 positive negative 31, 68 RealTime FN 1<br />

157 positive negative 42, 45 RealTime FN 1<br />

158 positive negative 45, 53 RealTime FN 1<br />

159 positive negative 39, 53, 73 RealTime FN 1<br />

160 positive negative 51, 67, 82 RealTime FN 1<br />

TP, true positive; FP, false positive; TN, true negative; FN, false negative.<br />

34

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