Clinical Validation of Anyplex II HPV HR Detection Test for Cervical Cancer Screening in Korea.
Molecular HPV testing has been considered as an adjunct/ follow-up to the Papanicolaou test for cervical cancer in women 30 years and older. (3) Recently, the US Food and Drug Administration approved molecular HPV testing as a first-line, primary screening test for cervical cancer in women 25 years and older. (3)
Several molecular test kits for the detection of high-risk human papillomavirus (hrHPV) are currently available. Evaluation of their clinical performance is critical before they are routinely used in clinical practice. According to international guidelines (4) for validation of hrHPV DNA tests, candidate assays should demonstrate clinical noninferiority to an established and clinically validated reference assay, such as Hybrid Capture 2 (HC2; Qiagen, Valencia, California) or [GP5.sup.+]/[6.sup.+] polymerase chain reaction (PCR). The candidate assays should have demonstrable sensitivity for cervical intraepithelial neoplasia (CIN) grade 2 or 3 and treatable cancer of 0.90 or greater and specificity for [CIN2.sup.+] of 0.98 or greater. In addition, candidate assays should display intralaboratory reproducibility and interlaboratory agreement, with a lower confidence bound of not less than 87% ([kappa] [less than or equal to] 0.5).
The recently developed Anyplex II HPV HR detection kit (Seegene Inc, Seoul, Korea) is a new multiplex, real-time PCR assay designed to detect and individually distinguish 14 hrHPV types * using tagging oligonucleotide cleavage and extension (TOCE) technology. (5) The TOCE technology uses artificial, template-based melting temperature instead of the current, probe-based melting temperature and distinguishes multiple targets in a single channel in a real-time PCR reaction. (6) In addition, the cyclic-catcher melting temperature analysis with TOCE enables a semiquantitative estimation of viral load through repeated melting temperature analysis expression during the TOCE reaction. (7)
Currently, the prevalence of HPV among Korean women with normal cytology results is higher than in more-developed countries. (8,9) Human papillomavirus cytology examination is carried out using a program incorporating a high level of cytopathology control.
We present the results of the first comparative evaluation of HPV HR relative to HC2 according to international guidelines (4) in the setting of Korean cervical cancer screening.
MATERIALS AND METHODS
Cervical Sample Collection
Specimens (n = 1137) from Korean women were randomly selected from the liquid-based cervical specimens submitted to the Seegene Medical Foundation (Seoul, Korea) between January and June 2014. All specimens were collected with a cytobrush, and were placed in a Huro Path liquid-based cytology sampling device (CelltraZone, Seoul, Korea). They were stored at -70[degrees]C until HPV testing. Each specimen was divided into 2 aliquots and then used for HPV detection with HC2 (4 mL) and HPV HR (1 mL) tests. The cervical cytologic examination was performed by experienced cytopathologists. To examine the clinical performance of HPV assays for histology of [CIN2.sup.+], the medical records were reviewed to gather information about colposcopic examination and histologic diagnosis. The study protocol was approved by the institutional review board of the Seegene Medical Foundation (Seoul, Korea; No. ND-IRB-2013-06).
The HC2 testing was performed according to the manufacturer's instructions. The RNA cocktail probes in the HC2 assay detect 13 hrHPV genotypes (HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68). Briefly, the specimens were denatured at 65[degrees]C for 45 minutes and hybridized with a mixture of RNA probes. The DNA-RNA hybrid was captured by an anti-DNA-RNA antibody on the surface of the microtiter plate wells and reacted with an alkaline phosphatase-conjugated antihybrid monoclonal antibody. Chemiluminescence substrate was added and the light emitted by the bound enzyme-antibody conjugate was measured with a luminometer. All specimens with a relative light unit:cutoff ratio of 1.0 or greater were considered positive. An HC2 sample conversion kit (Qiagen) was used with cervical specimens collected in Huro Path solution. The validity of placing specimens in Huro Path solution for the HC2 test, instead of PreservCyt solution (ThinPrep, Cytyc, Boxborough, Massachusetts), was confirmed by comparison of results from duplicate serial 10-fold dilutions from [10.sup.5] to [10.sup.-1] copies of HPV DNA of HeLa (HPV-18) and SiHa (HPV-16) cell lines spiked into Huro Path and PreservCyt.
HPV HR Test
Liquid-based cytology samples (1 mL) were centrifuged at 15 000g for 15 minutes. Each cell pellet was resuspended in 450 [micro]L of phosphate-buffered saline and added to the Microlab STARiet automated purification system (Hamilton, Reno, Nevada). For each sample, HPV detection and genotyping were performed according to the manufacturer's instructions for the HPV HR test using a CFX96 real-time thermocycler (Bio-Rad, Hercules, California). The TOCE technology of the HPV HR is initiated with hybridization of the dual priming oligonucleotide primers and the "Pitcher" to the target sequence. Taq polymerase, with 5'-nuclease activity, encounters the target-bound Pitcher and releases the tagging portion of the Pitcher. The sequence of the released tagging portion is complementary to the capturing portion of the "Catcher" as an artificial template. As the tagging portion is fully extended on the Catcher to create the "Duplex Catcher," quenching is diminished, and the fluorescent signal can be detected. Briefly, each PCR reaction was performed in a 20-[micro]L reaction consisting of 5 [micro]L extracted DNA, 4X HPV HR TOCE oligo mix, and Anyplex PCR mix containing uracil-DNA glycosylase. The cycling conditions were initial incubation at 50[degrees]C for 4 minutes for activation of the uracil-DNA glycosylase system to prevent contamination, denaturation at 95[degrees]C for 15 minutes, followed by 50 cycles of denaturation (30 seconds at 95[degrees]C), annealing (1 minute at 60[degrees]C), and elongation (30 seconds at 72[degrees]C). Cyclic-catcher melting temperature analysis was performed after PCR cycles 30, 40, and 50. Catcher melting temperature analysis conditions were as follows: cooling the reaction mixture to 55[degrees]C, holding at 55[degrees]C for 30 seconds, and heating from 55[degrees]C to 85[degrees]C (5 s/0.5[degrees]C). The L1 gene of HPV DNA and the human housekeeping gene (human [beta]-globin) were coamplified simultaneously, and the human housekeeping gene was used as an internal control to monitor DNA purification efficiency, PCR inhibition, and cell adequacy. Data interpretation was automated with the Anyplex software (Seegene) according to the manufacturer's instructions.
HPV HR Reproducibility Test
To assess the reproducibility of the HPV HR, 504 samples, including 152 [HC2.sup.+] samples, were used. Intrareproducibility tests were performed by retesting HPV HR 4 to 6 weeks after the initial test. Interlaboratory reproducibility studies were conducted at 2 additional laboratories of Seegene Medical Foundation and Seegene Institute of Life Science.
All statistical analyses were carried out using GraphPad Prism (GraphPad Software, La Jolla, California) and Excel 2007 (Microsoft, Seattle, Washington). Agreement between the HC2 test and HPV HR was assessed with the Cohen's [kappa] statistic, with values of 0.00 to 0.20 indicating poor agreement, 0.21 to 0.40 fair agreement, 0.41 to 0.60 moderate agreement, 0.61 to 0.80 substantial agreement, and 0.81 to 1.00 almost perfect agreement. Clinical sensitivity, clinical specificity, positive predictive value, and negative predictive value were calculated with the conventional contingency tables, and 95% confidence intervals (95% CI) were computed using the exact binomial method. The clinical sensitivity and specificity of the HPV HR for high-grade squamous intraepithelial lesion (HSIL) were compared with those of HC2 with a noninferiority score test as previously described. (4) In addition, differences in the clinical sensitivity and specificity between the two methods were assessed using the McNemar exact [chi square] test. A P value less than .05 was considered significant.
Our study included 1137 cervical cell specimens collected in Huro Path medium from patients whose mean age was 41.8 years (range, 18-96 years; median age, 41 years). The cytologic grades of specimens were classified as normal (n = 857; 75.4%), atypical squamous cell of undetermined significance (n = 85; 7.5%), low-grade squamous intraepithelial lesion (n = 123, 10.8%), and HSIL (n = 72, 6.3%). Cervical biopsies were performed for 75 women, and 40 samples from women with a biopsy of [CIN2.sup.+] (CIN2, 12; CIN3, 24; and 4 squamous cell carcinomas) within 12 months were included to evaluate clinical sensitivity for [CIN2.sup.+].
Of the 1137 specimens, HPV HR detected 263 (23.1%) cases of hrHPV and HC2 detected 261 (23.0%) cases, with no statistically different detection rate evident between them (P = .91) (Table 1). According to the cytologic grades and histology, the [hrHPV.sup.+] rates by the HPV HR and the HC2 were 9.9% (85 of 857) and 9.8% (84 of 857) in normal cases, 29.4% (25 of 85) (identical) in cases of atypical squamous cell of undetermined significance, 69.1% (85 of 123) (identical) in cases of low-grade squamous intraepithelial lesion, 94.4% (68 of 72) and 93.1% (67 of 72) in cases of HSIL, and 92.5% (37 of 40) and 87.5% (35 of 40) in [CIN2.sup.+] cases, respectively. Overall agreement between the HPV HR and HC2 was 92.4% (1051 of 1137), with a [kappa] value of 0.787 (95% CI, 0.744-0.830). Agreement for cases with less than an HSIL diagnosis was 92.4% (984 of 1065), with a [kappa] value of 0.745 (95% CI, 0.693-0.798), whereas the agreement for cases with an HSIL diagnosis was 93.1% (67 of 72), with a [kappa] value of 0.408 (95% CI, 0.000-0.829). Agreement for the [CIN2.sup.+] was 95.0%, with a [kappa] value of 0.724 (95% CI, 0.366-1.000). The HC2 and HPV HR test cytologic findings and histology are shown in Table 1.
Of the 280 specimens with abnormal cytologic findings, 251 (89.6%) specimens had concordant results in both assays. Of these, 46.2% (116 of 251) had a single genotype (including HPV-16, n = 37; HPV-18, n = 2), whereas 18.7% (47 of 251) had multiple genotypes (including HPV-16, n = 23; HPV-18, n = 4; HPV-16 and HPV-18, n = 3) ranging between 2 and 3 different genotypes by the HPV HR. Also, 35.1% (88 of 251) of specimens were not detected by both methods. Of the 29 specimens showing discordant results and having abnormal cytologic findings, 15 specimens (51.7%) were positive in only the HPV HR and 14 specimens (48.3%) were positive in only the HC2 (Table 2).
The clinical sensitivity, clinical specificity, positive predictive value, and negative predictive value of the HPV HR and HC2 are shown in Tables 3 and 4. The clinical sensitivity of the HPV HR and HC2 for the detection of HSIL was 94.4% (95% CI, 89.2-99.7) and 93.1% (95% CI, 87.2-98.9), respectively, and clinical specificity was 81.7% (95% CI, 79.4-84.0) and 81.8% (95% CI, 79.5-84.1), respectively. Using a noninferiority score test, the clinical sensitivity and specificity of the HPV HR were not inferior to those of the HC2 assay (P = .005 and P = .04, respectively) (Table 3).
With [CIN2.sup.+] as reference standard, clinical sensitivity and specificity was analyzed in 40 samples with [CIN2.sup.+] and 892 samples with normal cytology and without evidence of a [CIN2.sup.+] diagnosis within a 12-month period. The clinical sensitivity of the HPV HR and HC2 for the detection of [CIN2.sup.+] was 92.5% (95% CI, 84.3-100.0) and 87.5% (95% CI, 77.3-99.7), and clinical specificity was 88.0% (95% CI, 85.9 90.1) and 87.9% (95% CI, 85.8-90.0), respectively. Using a noninferiority score test, the clinical sensitivity and specificity of the HPV HR were not inferior to those of the HC2 assay (P = .007 and P = .02, respectively) (Table 4).
The HPV HR exhibited excellent intralaboratory and interlaboratory reproducibility, showing 98.0% (494 of 504; [kappa] = 0.953; 95% CI, 0.925-0.982) and 97.4% (491 of 504; [kappa] = 0.940; 95% CI, 0.908-0.972), respectively (Tables 5 and 6). For both the intralaboratory and interlaboratory reproducibility, the lower confidence bounds were less than 87%, with [kappa] values less than 0.5. The results complied with the international guidelines. (4) For each genotyping agreement in the intralaboratory and interlaboratory reproducibility test of HPV HR, the lower confidence bounds were greater than 87%, with [kappa] values greater than 0.5. The results complied with the international guidelines (data not shown). (4)
The purpose of this study was to evaluate the clinical performance of the novel, real-time, multiplex HPV HR using TOCE technology for the detection of 14 hrHPV genotypes. We compared HPV HR to HC2 as an established reference method in the setting of Korean cervical cancer screening based on international guidelines. To use routinely requested liquid-based cervical cytology samples in Huro Path medium, we validated the HC2 test using Huro Path medium versus PreservCyt medium. There was an excellent agreement between HC2 results using the 2 different media, demonstrating Huro Path medium to be an appropriate alternative to PreservCyt medium.
In comparative analysis of HPV HR, high agreement between the results of HPV HR and HC2 was observed. For samples diagnosed as less than HSIL, HSIL, and [CIN2.sup.+], the agreement rates between the 2 tests was similar at 92.4% ([kappa] = 0.408), 93.1% ([kappa] = 0.745), and 95.0% ([kappa] = 0.724), respectively. A recent study (10) compared the HPV 28 test (Seegene) with the HC2 test for the detection of 13 hrHPV genotypes (HPV HR provides 14 hrHPV genotypes, whereas the HPV 28 test provides 19 hrHPV genotypes, including 14 hrHPV genotypes of the HPV HR and 9 low-risk genotypes). The overall agreement between 2 assays was 91.4% with [kappa] = 0.50, which was similar to our results.
Regarding the genotype detection results, of 163 abnormal cytologic cases that were positive in both assays (Table 2), 5 cases (3.1%) showed only a single HPV-66 genotype in HPV HR (data not shown). This genotype is not covered by HC2, but the HC2 yielded a positive result. Even though we did not evaluate this result with an additional reference test, such as a sequencing analysis, because of inadequate sample volume, this result supports a previous report (11) of cross-reactivity of high-risk probes of HC2 with low-risk HPV types in the HC2 assay. The HPV HR using the type-specific primer amplification technique might report the more-accurate results without cross-reactivity. Further studies are needed to confirm the current outcome.
The clinical sensitivity and specificity of the HPV HR were noninferior to those of HC2 using the predetermined thresholds of 90% and 98%, respectively. (4) The HPV HR showed good intralaboratory and interlaboratory reproducibility because the lower confidence bound of the percentage of agreement was higher than 87% and the corresponding [kappa] value was higher than 0.5, as suggested by guidelines. (4) Poljak et al (12) have reported that the multiplex, real-time PCR showed discordant results on mixed HPV infections, probably because of competition in amplification. However, the level of intralaboratory reproducibility of the HPV HR in this study was comparable with that of previous assays. (13-15)
The HPV HR has some advantages for hrHPV detection and extended genotyping. It can detect both single infections and multiple infections for 14 hrHPV genotypes using the TOCE technology. Other commercial kits (13-17) can report 3 kinds of results, such as HPV-16 and HPV-18, and a pool of 12 carcinogenic HPV genotypes, whereas the HPV HR can detect and distinguish all 14 hrHPV types individually. Presently, of the 251 specimens with positive results by both HPV HR and HC2 tests, 47 specimens (18.7%) had multiple infections for the hrHPV genotypes. Considering the strong relationship between multiple infections and cervical cancer, (18) the hrHPV genotype information provided by the HPV HR can serve as an additional tool in treating patients infected with HPV. The exact identification of the HPV genotypes could help assess whether an HPV infection is transient or persistent by comparing the results of the primary cervical screening and follow-up. In addition, because a high viral load is closely associated with persistent infection, the results of HPV genotyping, including semiquantitative information (ie, viral load) using the cyclic-catcher melting temperature analysis, by the HPV HR could provide more accurate diagnosis of cervical cancer. (19,20)
This is the first study, to our knowledge, comparing the HPV HR and the HC2, based on the Meijer guidelines, (4) in the Korean population. There are several potential limitations. For the samples showing discordant results between HPV HR and HC2, further genotypes were not confirmed. Histologic diagnosis was possible only in women who were referred to colposcopy and was not performed in women with [HPV.sup.+] but cytology-negative results. Nevertheless, considering that the overall concordance rate between the cytology and histologic diagnoses was 93.0% with a [kappa] value of 0.77 in a Korean population study (21) and a high cytology-histology correlation in a Croatian population study, (22) the comparative analysis data for HSIL detection might also be useful for evaluation of the clinical performance of the HPV HR.
In conclusion, our data indicate that the HPV HR is highly comparable to HC2 for clinical equivalence and reproducibility based on international guidelines. The individual typing information for 14 hrHPV genotypes, including HPV-16 and HPV-18, provided by the HPV HR can be an important tool in patient management.
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(11.) Castle PE, Solomon D, Wheeler CM, Gravitt PE, Wacholder S, Schiffman M. Human papillomavirus genotype specificity of Hybrid Capture 2. J Clin Microbiol. 2008; 46(8):2595-2604.
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(17.) Depuydt CE, Benoy IH, Beert JF, Criel AM, Bogers JJ, Arbyn M. Clinical validation of a type-specific real-time quantitative human papillomavirus PCR against the performance of Hybrid Capture 2 for the purpose of cervical cancer screening. J Clin Microbiol. 2012; 50(12):4073-4077.
(18.) Nobbenhuis MA, Walboomers JM, Helmerhorst TJ, et al. Relation of human papillomavirus status to cervical lesions and consequences for cervical-cancer screening: a prospective study. Lancet. 1999; 354(9172):20-25.
(19.) Kim YJ, Kwon MJ, Woo HY, Paik SY. Prevalence of human papillomavirus infection and genotype distribution determined by the cyclic-catcher melting temperature analysis in Korean medical checkup population. J Microbiol. 2013; 51(5):665-670.
(20.) Dalstein V, Riethmuller D, Pretet JL, et al. Persistence and load of high-risk HPV are predictors for development of high-grade cervical lesions: a longitudinal French cohort study. Int J Cancer. 2003; 106(3):396-403.
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Sunkyung Jung, MD; Byungdoo Lee, MD; Kap No Lee, MD, PhD; Yonggoo Kim, MD, PhD; Eun-Jee Oh, MD, PhD
Accepted for publication June 23, 2015.
From the Molecular Diagnostics Testing Center (Drs Jung and K. N. Lee) and the Department of Pathology (Dr B. Lee), Seegene Medical Foundation, Seocho-gu, Seoul, Republic of Korea; and the Department of Laboratory Medicine, College of Medicine, Seoul St. Mary's Hospital, Catholic University of Korea (Drs Kim and Oh).
Drs Jung, B. Lee, and K. N. Lee are employees of Seegene Medical Foundation. The study was supported by a grant from Seegene Inc (Seoul, Korea). The source of funding did not have any influence on the design and analysis of the results.
Reprints: Eun-Jee Oh, MD, PhD, Department of Laboratory Medicine, College of Medicine, Seoul St Mary's Hospital, Catholic University of Korea, Banpo-daero 222, Seocho-gu, Seoul 137-701, Republic of Korea (email: firstname.lastname@example.org).
* References 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68.
Table 1. Comparison of Anyplex II HPV HR Detection Kit (HPV HR) (a) and Hybrid Capture 2 (HC2) (b) Results by Cytology and Histology (c) Cytology/ HPV HR HC2 Result, No. Histology Result Positive Negative Total Less than Positive 154 41 195 HSIL (d) Negative 40 830 870 Total 194 871 1065 HSIL (e) Positive 65 3 68 Negative 2 2 4 Total 67 5 72 [CIN2.sup.+] (f) Positive 35 2 37 Negative 0 3 3 Total 35 5 40 Abbreviations: [CIN2.sup.+], cervical intraepitherial neoplasia grade 2 or 3 and treatable cancer; 95% CI, 95% confidence interval; HSIL, high-grade squamous intraepithelial lesion. (a) Seegene Inc, Seoul, Korea. (b) Qiagen, Valencia, California. (c) Overall agreement, 92.4%; [kappa] = 0.787; 95% CI, 0.744-0.830. (d) Agreement on less than HSIL, 92.4%; [kappa] = 0.745; 95% CI, 0.693-0.798. (e) Agreement on HSIL, 93.1%; [kappa] = 0.408; 95% CI, 0.000-0.829. (f) Agreement on [CIN2.sup.+], 95.0%; [kappa] = 0.724; 95% CI, 0.366-1.000. Table 2. Comparison of Anyplex II HPV HR Detection Kit (HPV HR) (a) and Hybrid Capture 2 (HC2) (b) for 280 Abnormal Cytologic Grades Agreement No. Cytology Grades Genotyping Results by (No.) the HPV HR (No.) Concordant 251 HPV HR/HC2 (+/+) 163 Single infection 116 HSIL (47), LSIL HPV-16 (37), HPV-18 (54), ASCUS (15) (2), other types (c) (77) Multiple infections 47 HSIL (18), LSIL HPV-16 and other types (c) (23); HPV-18 and other types (c) (4); (24), ASCUS (5) HPV-16, HPV-18 and other types (c) (3); HPV HR/HC2 (-/-) 88 other types (c) (17) Discordant 29 HPV HR/HC2 (+/-) 15 Single infection 14 HSIL (3), LSIL HPV-16 (2), HPV-18 (6), ASCUS (5) (1), other types (c) (11) Multiple infections 1 HSIL (0), LSIL HPV-18 and other HPV HR/HC2 (-/+) 14 (1), ASCUS (0) types (c) (1) Abbreviations: ASCUS, atypical squamous cell of undetermined significance; HSIL, high-grade squamous intraepithelial lesion; LSIL, low-grade squamous intraepithelial lesion. (a) Seegene Inc, Seoul, Korea. (b) Qiagen, Valencia, California. (c) Non-HPV-16 and non-HPV-18. Table 3. Clinical Sensitivity and Clinical Specificity of Anyplex II HPV HR Detection Kit (HPV HR) (a) and Hybrid Capture 2 (HC2) (b) for the Detection of High-Grade Squamous Intraepithelial Lesion Clinical Clinical Sensitivity, Specificity, Test % (95% CI) % (95% CI) HPV HR 94.4 (89.2-99.7) 81.7 (79.4-84.0) HC2 93.1 (87.2-98.9) 81.8 (79.5-84.1) PPV, NPV, % (95% CI) % (95% CI) Test HPV HR 25.9 (20.6-31.1) 99.5 (99.1-100.0) HC2 25.7 (20.4-31.0) 99.4 (98.9-99.9) Abbreviations: 95% CI, 95% confidence interval; NPV, negative predictive value; PPV, positive predictive value. (a) Seegene Inc, Seoul, Korea. (b) Qiagen, Valencia, California. Table 4. Clinical Sensitivity and Clinical Specificity of Anyplex II HPV HR Detection Kit (HPV HR) (a) and Hybrid Capture 2 (HC2) (b) for the Prediction of Cervical Intraepithelial Neoplasia Grade 2 or 3 and Treatable Cancer Clinical Clinical Sensitivity, Specificity, Test % (95% CI) % (95% CI) HPV HR 92.5 (84.3-100.0) 88.0 (85.9-90.1) HC2 87.5 (77.3-99.7) 87.9 (85.8-90.0) PPV, NPV, % (95% CI) % (95% CI) Test HPV HR 25.7 (18.6-32.8) 99.6 (99.2-100.0) HC2 24.5 (17.4-31.5) 99.4 (98.8-99.9) Abbreviations: 95% CI, 95% confidence interval; NPV, negative predictive value; PPV, positive predictive value. (a) Seegene Inc, Seoul, Korea. (b) Qiagen, Valencia, California. Table 5. Intralaboratory Reproducibility (a) Over Time for the Anyplex II HPV HR Detection Kit (b) Second Results First Positive Negative Total Results Positive 150 8 158 Negative 2 344 346 Total 152 352 504 (a) Agreement, 98.0%; [kappa] = 0.953; 95% confidence interval, 0.925-0.982. (b) Seegene Inc, Seoul, Korea. Table 6. Interlaboratory Agreement (a) Over Time for the Anyplex II HPV HR Detection Kit (b) Second Results First Positive Negative Total Results Positive 150 12 162 Negative 1 341 342 Total 151 353 504 (a) Agreement, 97.4%; [kappa] = 0.940;95% confidence interval, 0.908-0.972. (b) Seegene Inc, Seoul, Korea.
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|Author:||Jung, Sunkyung; Lee, Byungdoo; Lee, Kap No; Kim, Yonggoo; Oh, Eun-Jee|
|Publication:||Archives of Pathology & Laboratory Medicine|
|Date:||Mar 1, 2016|
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