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Comparison of the Roche Cobas® 4800 HPV assay to Digene Hybrid Capture 2, Roche Linear Array and Roche Amplicor for Detection of High-Risk Human Papillomavirus Genotypes in Women undergoing treatment for cervical dysplasia
Journal of Clinical Virology 62 (2015) 63–65
Contents lists available at ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Short Communication
Comparison of the Roche Cobas® 4800 HPV assay to Digene Hybrid Capture 2, Roche Linear Array and Roche Amplicor for Detection of High-Risk Human Papillomavirus Genotypes in Women undergoing treatment for cervical dysplasia Samuel Phillips a,c , Suzanne M. Garland a,b,c , Jeffery H. Tan b,d , Michael A. Quinn b,d , Sepehr N. Tabrizi a,b,c,∗ a Regional HPV Lab Net Reference Laboratory, Department of Microbiology and Infectious Diseases, The Royal Women’s Hospital, Parkville, Victoria, Australia b Department of Obstetrics and Gynecology, University of Melbourne, Victoria, Australia c Murdoch Childrens Research Institute, Parkville, Victoria 3052, Australia d Oncology and Dysplasia Unit, Royal Women’s Hospital, Parkville, Victoria, Australia
a r t i c l e
i n f o
Article history: Received 5 July 2014 Received in revised form 6 November 2014 Accepted 9 November 2014 Keywords: Human papillomavirus Roche Cobas Hybrid capture Linear array Amplicor Diagnostic
a b s t r a c t Background: The recently FDA (U.S. food and drug administration) approved Roche Cobas® 4800 (Cobas) human papillomavirus (HPV) has limited performance data compared to current HPV detection methods for test of cure in women undergoing treatment for high grade lesions. Objective: Evaluation of Cobas HPV assay using historical samples from women undergoing treatment for cervical dysplasia. Study design: A selection of 407 samples was tested on the Cobas assay and compared to previous results from Hybrid Capture 2, HPV Amplicor and Roche Linear Array. Results: Overall, a correlation between high-risk HPV positivity and high grade histological diagnosis was 90.6% by the Cobas, 86.1% by Hybrid Capture 2, 92.9% by HPV Amplicor and 91.8% by Roche Linear Array. Conclusion: The Cobas HPV assay is comparative to both the HPV Amplicor and Roche Linear Array assays and better than Hybrid capture 2 assay in the detection of High-Risk HPV in women undergoing treatment for cervical dysplasia. © 2014 Elsevier B.V. All rights reserved.
1. Background The Roche Cobas 4800 HPV assay (Cobas) is currently accredited by Food and Drug Administration (FDA) for reflex testing of patients with atypical squamous cells of undetermined significance (ASC-US) cytology and in conjunction with routine Pap testing of women over age 30. This assay was also recently approved for use in primary screening of women over 25 years of age [1]. The clinical evaluation was primarily provided through a multicenter trial designed to demonstrate validation of the Cobas HPV test in these scenarios [2]. Another potential clinical utility of this HPV assay
∗ Corresponding author at: Department of Microbiology and Infectious Diseases, The Royal Women’s Hospital, Bio 21 Institute, Parkville 3052, Australia. Tel.: +61 3 8345 3672; fax: +61 3 9348 0796. E-mail address: [email protected] (S.N. Tabrizi). http://dx.doi.org/10.1016/j.jcv.2014.11.017 1386-6532/© 2014 Elsevier B.V. All rights reserved.
which this study did not evaluate is post-treatment follow-up of women who have been treated for histologically confirmed high grade cervical intraepithelial lesions (CIN 2/3), to ensure adequacy of treatment [3].
2. Objectives The Objectives of the present study was to evaluate the performance of the Roche Cobas 4800 (Cobas) compared to the Qiagen Hybrid Capture 2(1201 Clopper Road Gaithersburg: MD 20878, USA) (HC2) Roche HPV Amplicor (Branchburg, NJ 08876, USA) (AMP), and Roche Linear Array (Branchburg, NJ 08876, USA) (LA) assays in the detection of High-Risk HPV from liquid based cytology samples collected from women undergoing treatment for cervical dysplasia.
64
S. Phillips et al. / Journal of Clinical Virology 62 (2015) 63–65
Table 1 Concordance between HC2, AMP, LA and Cobas tests in detecting HR-HPV genotypes among 406 PreserveCyt specimens. HR-HPV
HC2 result Negative Positive AMP result Negative Positive
Cobas test result Negative
Positive
69 9
44 284
65 13
3 325
%Concordance
Table 3 Summary of discordant HPV results. HR-HPV result
Cohen’s kappa statistic hc2+/Cobas− hc2−/Cobas+ AMP+/Cobas− AMP−/Cobas+ LA+/Cobas− LA−/Cobas+
0.70 (95% CI: 0.63–0.77)
P ≤ 0.0001
0.87 (95% CI: 0.80–0.93)
P ≤ 0.0001
LA
AMP
HC2
Positive
Negative
Positive
Negative
1a 40 10e 0
8 4b 3 3f
2c 41
7 3d
i
10 5
0 0
Positive
Negative
2g 9 1j 1
11 3h 9 4k
a
HPV 16 LA. 2 × neg, 1 × hpv 61 and 62, 1 × hpv 54, 73, 83. c 1 × LA pos, 1 × LA neg. d 3 × LA neg. e 1 × 56: 1 × 16: 1 × 53, 59: 1 × 51, 58: 1 × 16, 84: 1 × 51: 1 × 52, 54, 59, 61: 1 × 58, 73: 1 × 6, 52: 1 × 18. f 1 × HPV 16, 2 × HPV Other (1 × HPV 54, 73, 83, 1 × Neg). g AMP OD = 0.923 and 0.211, HC OD = 5 and 6, LA = 16 and 82. h Same as (f). i Same as (e). j 1 × HPV 16. k 1 × 61 and 62, 1 × 54, 73 and 83, 2 × LA Neg. b
LA result Negative Positive
68 10
5 323
0.88 (95% CI: 0.826–0.94)
P ≤ 0.0001
3. Study design Overall, 339 women with histologically confirmed high grade abnormalities and 68 women with histological confirmed low grade abnormality were randomly selected and their liquid based cytology samples evaluated [4]. The samples were randomly selected to include approximately 2/3 of the original pool of all high grade abnormality samples available and 1/5 of all low grade and normal samples. Stored frozen PreservCyt samples were tested by the Cobas according to the manufacturing instructions. 4. Methods Briefly, 400 l of sample was extracted and eluted in final volume of 100 l, 25 l of which was then used in a qPCR. This assay is able to detect HPV high-risk types 16 and 18 individually, as well as a group of 12 other known HPV High-Risk types (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) with a single probe different to the type 16 and 18 probes. HC2, Amp, and LA HPV tests results were available from previously conducted studies [4,5]. The Cobas results were compared to HC2, Amp, and LA HPV using 2-by-2 contingency tables, with two-sided P values calculated using Fisher’s exact test. Agreement between tests was assessed by Cohen’s kappa statistic. Confidence intervals (95% CI) for proportions were calculated using a two-tailed test and assuming a random sample of the population [6] (Table 1). The relationship between the histological and HPV results was analysed for sensitivity and specificity and also positive or negative predictive values (95% CI), using ≥CIN 2 as a measure of clinically significant disease (Table 2). 5. Results From 407 cytology Thinprep samples tested on the Cobas, one specimen was negative for both the internal control and HPV, and
hence was removed from further analyses. Among the 406 assessable specimens, HR HPV positivity was 80.8% (Cobas), 72.2% (HC2), and 83.3% (Amp), and 82.0% (LA). Agreement between the Cobas and other HPV tests for the presence of HR HPV types was good (HC2, = 0.70) or very good (Amp, = 0.87 and LA, = 0.88) (Table 1) [6]. 6. Discussion Correlation of HR HPV to histological diagnosis (using ≥CIN2 as a measure of clinically significant disease), demonstrated clinical sensitivities of 90.5% (95% CI of 86.9–93.2) and specificity of 67.6% (95% CI of 55.8–77.6) for the Cobas, 92.9% (95% CI of 89.7–95.2) for the Amplicor HPV test, 80.8% (95% CI of 76.2–84.6) for HC2, and 91.7% (95% CI of 88.3–94.2) for the LA HPV test (Table 2). The Cobas, LA, and AMP HPV tests correctly predicted 33, 37 and 41 respectively more histologically confirmed cervical disease (CIN2) than the HC2 HPV test. Overall, 53 (13%), 16 (3.9%), 15 (3.6%) of results were discordant between the Cobas and HC, AMP and LA tests. The majority (40/44) of HC negative/Cobas positive were also positive by LA and AMP (Table 3). Among the HC negative/Cobas positives, there were 22 samples from women with histological diagnosis of CIN3. This lower sensitivity of HC is due to the higher sensitivity in detection of low concentrations of viral DNA with “the PCR based” target amplification assays. Of the 9 samples positive by HC and negative by the Cobas (Table 1), only 1 of these was positive by LA for HPV 16, a type detected by all methods (Table 3). The other 8 HC2/Cobas
Table 2 Relationship between histological diagnosis and HR-HPV testing. Normal
CIN 1
CIN 2
CIN 3
>CIN 3
Total
≥CIN 2
≤CIN 1
Specificity or sensitivity (95% CI)a
HC2− HC2+ AMP− AMP+ LA− LA+ Cobas− Cobas+
23 5 22 6 22 6 24 4
25 15 22 18 23 17 22 18
31 66 16 81 16 81 19 78
33 197 8 222 12 218 13 217
1 10
11
113 293 68 338 73 333 78 328
65 273 24 314 28 310 32 306
48 20 44 24 45 23 46 22
70.6 (58.9–80.1) 80.8 (76.2–84.6) 64.7 (52.8–75.0) 92.9 (89.7–95.2) 66.2 (54.3–76.3) 91.7 (88.3–94.2) 67.6 (55.8–77.6) 90.5 (86.9–93.2)
Total
28
40
97
230
11
406
338
68
11 11
Negative and positive predictive values (95% CI) 42.5 (33.8–51.7) 93.2 (89.7–95.5) 64.7 (52.8–75.0) 92.9 (89.7–95.2) 61.6 (50.2–71.9) 93.1 (89.8–95.4) 59.0 (47.9–69.2) 93.3 (90.1–95.5)
HC2 – Hybrid Capture 2; AMP – Roche Amplicor; LA – Roche Linear Array; Cobas – Roche Cobas 4800; 95% CI – 95% confidence intervals; CIN – cervical intraepithelial neoplasia. a The Specificity and Sensitivity were determined using ≥CIN 2 and ≤ CIN 1.
S. Phillips et al. / Journal of Clinical Virology 62 (2015) 63–65
discrepant samples may be a result of DNA degradation due to the historical nature of the samples used. In respect to HPV types that are not detected across all PCR-based assays, 10 samples (Table 3) were Cobas negative and AMP and LA positive, most likely due to low HPV copy numbers and differential sensitivities across these assays. In addition, the extraction method for the Cobas is only representative of 400 l of a sample (100 l equivalent is tested in each reaction), whereas LA uses an extraction method that represents 1 ml of clinical sample (500 l equivalent was tested in each reaction). Overall 5 samples were positive by the Cobas and negative by LA. This may be due to competition of assessable types at low copy numbers, with other type’s present, but not analysed or possibly due to sub-types not detectable on LA, such as 68a. This study focused on a population of women being treated for cervical dysplasia, representing a cohort with an inflated disease prevalence and as such this clinical performance should not be extrapolated to the general screening population. However, this cohort of women with heightened levels of disease provides a valuable population for sensitivity testing to assess the Cobas assay system [7]. The performance of the Cobas was equivalent to the AMP and LA HPV tests for HR HPV detection. The Cobas predicted more underlying histologically-confirmed high-grade lesions than the HC2 HPV test, with the added advantage of identifying HPV 16 and 18 genotypes present. The follow up algorithm of women with PCR-based detection of HR HPV as marker of residual disease would need to be confirmed with addition of cytology/histology to ensure no residual disease remain in patients after treatment, in particular with younger women who may have higher frequency of transient HPV infection. Funding Roche Molecular Systems supplied the detection kits utilised for this study.
65
Competing interests No competing interests are declared.
Ethics approval Ethical approval was received for the study by the Royal Women’s Hospital research and ethics committees under reference number 00/30.
References [1] Stoler MH, Wright Jr TC, Sharma A, Zhang G, Apple R, Wright TL, et al. The interplay of age stratification and HPV testing on the predictive value of ASC-US cytology. Results from the ATHENA HPV study. Am J Clin Pathol 2012;137:295–303. [2] Wright Jr TC, Stoler MH, Behrens CM, Apple R, Derion T, Wright TL. The ATHENA human papillomavirus study: design, methods, and baseline results. Am J Obstet Gynecol 2012;206, 46.e1–e11. [3] Moss SM, Bailey A, Cubie H, Denton K, Sargent A, Muir P, et al. Comparison of the performance of HPV tests in women with abnormal cytology: results of a study within the NHS cervical screening programme. Cytopathology 2014, http://dx.doi.org/10.1111/cyt.12210. [4] Stevens MP, Garland SM, Rudland E, Tan J, Quinn MA, Tabrizi SN. Comparison of the Digene Hybrid Capture 2 assay and Roche AMPLICOR and LINEAR ARRAY human papillomavirus (HPV) tests in detecting high-risk HPV genotypes in specimens from women with previous abnormal Pap smear results. J Clin Microbiol 2007;45:2130–7. [5] Stevens MP, Garland SM, Tan JH, Quinn MA, Petersen RW, Tabrizi SN. HPV genotype prevalence in women with abnormal pap smears in Melbourne, Australia. J Med Virol 2009;81:1283–91. [6] Mackinnon A. A spreadsheet for the calculation of comprehensive statistics for the assessment of diagnostic tests and inter-rater agreement. Comput Biol Med 2000;30:127–34. [7] Tabrizi SN, Stevens MP, Khan ZA, Chow C, Devitt MA, Garland SM. Comparison of PapType to Digene Hybrid Capture 2, Roche linear array, and Amplicor for detection of high-risk human papillomavirus genotypes in women with previous abnormal pap smears. J Clin Microbiol 2012;50:2796–8.
Contents lists available at ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Short Communication
Comparison of the Roche Cobas® 4800 HPV assay to Digene Hybrid Capture 2, Roche Linear Array and Roche Amplicor for Detection of High-Risk Human Papillomavirus Genotypes in Women undergoing treatment for cervical dysplasia Samuel Phillips a,c , Suzanne M. Garland a,b,c , Jeffery H. Tan b,d , Michael A. Quinn b,d , Sepehr N. Tabrizi a,b,c,∗ a Regional HPV Lab Net Reference Laboratory, Department of Microbiology and Infectious Diseases, The Royal Women’s Hospital, Parkville, Victoria, Australia b Department of Obstetrics and Gynecology, University of Melbourne, Victoria, Australia c Murdoch Childrens Research Institute, Parkville, Victoria 3052, Australia d Oncology and Dysplasia Unit, Royal Women’s Hospital, Parkville, Victoria, Australia
a r t i c l e
i n f o
Article history: Received 5 July 2014 Received in revised form 6 November 2014 Accepted 9 November 2014 Keywords: Human papillomavirus Roche Cobas Hybrid capture Linear array Amplicor Diagnostic
a b s t r a c t Background: The recently FDA (U.S. food and drug administration) approved Roche Cobas® 4800 (Cobas) human papillomavirus (HPV) has limited performance data compared to current HPV detection methods for test of cure in women undergoing treatment for high grade lesions. Objective: Evaluation of Cobas HPV assay using historical samples from women undergoing treatment for cervical dysplasia. Study design: A selection of 407 samples was tested on the Cobas assay and compared to previous results from Hybrid Capture 2, HPV Amplicor and Roche Linear Array. Results: Overall, a correlation between high-risk HPV positivity and high grade histological diagnosis was 90.6% by the Cobas, 86.1% by Hybrid Capture 2, 92.9% by HPV Amplicor and 91.8% by Roche Linear Array. Conclusion: The Cobas HPV assay is comparative to both the HPV Amplicor and Roche Linear Array assays and better than Hybrid capture 2 assay in the detection of High-Risk HPV in women undergoing treatment for cervical dysplasia. © 2014 Elsevier B.V. All rights reserved.
1. Background The Roche Cobas 4800 HPV assay (Cobas) is currently accredited by Food and Drug Administration (FDA) for reflex testing of patients with atypical squamous cells of undetermined significance (ASC-US) cytology and in conjunction with routine Pap testing of women over age 30. This assay was also recently approved for use in primary screening of women over 25 years of age [1]. The clinical evaluation was primarily provided through a multicenter trial designed to demonstrate validation of the Cobas HPV test in these scenarios [2]. Another potential clinical utility of this HPV assay
∗ Corresponding author at: Department of Microbiology and Infectious Diseases, The Royal Women’s Hospital, Bio 21 Institute, Parkville 3052, Australia. Tel.: +61 3 8345 3672; fax: +61 3 9348 0796. E-mail address: [email protected] (S.N. Tabrizi). http://dx.doi.org/10.1016/j.jcv.2014.11.017 1386-6532/© 2014 Elsevier B.V. All rights reserved.
which this study did not evaluate is post-treatment follow-up of women who have been treated for histologically confirmed high grade cervical intraepithelial lesions (CIN 2/3), to ensure adequacy of treatment [3].
2. Objectives The Objectives of the present study was to evaluate the performance of the Roche Cobas 4800 (Cobas) compared to the Qiagen Hybrid Capture 2(1201 Clopper Road Gaithersburg: MD 20878, USA) (HC2) Roche HPV Amplicor (Branchburg, NJ 08876, USA) (AMP), and Roche Linear Array (Branchburg, NJ 08876, USA) (LA) assays in the detection of High-Risk HPV from liquid based cytology samples collected from women undergoing treatment for cervical dysplasia.
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S. Phillips et al. / Journal of Clinical Virology 62 (2015) 63–65
Table 1 Concordance between HC2, AMP, LA and Cobas tests in detecting HR-HPV genotypes among 406 PreserveCyt specimens. HR-HPV
HC2 result Negative Positive AMP result Negative Positive
Cobas test result Negative
Positive
69 9
44 284
65 13
3 325
%Concordance
Table 3 Summary of discordant HPV results. HR-HPV result
Cohen’s kappa statistic hc2+/Cobas− hc2−/Cobas+ AMP+/Cobas− AMP−/Cobas+ LA+/Cobas− LA−/Cobas+
0.70 (95% CI: 0.63–0.77)
P ≤ 0.0001
0.87 (95% CI: 0.80–0.93)
P ≤ 0.0001
LA
AMP
HC2
Positive
Negative
Positive
Negative
1a 40 10e 0
8 4b 3 3f
2c 41
7 3d
i
10 5
0 0
Positive
Negative
2g 9 1j 1
11 3h 9 4k
a
HPV 16 LA. 2 × neg, 1 × hpv 61 and 62, 1 × hpv 54, 73, 83. c 1 × LA pos, 1 × LA neg. d 3 × LA neg. e 1 × 56: 1 × 16: 1 × 53, 59: 1 × 51, 58: 1 × 16, 84: 1 × 51: 1 × 52, 54, 59, 61: 1 × 58, 73: 1 × 6, 52: 1 × 18. f 1 × HPV 16, 2 × HPV Other (1 × HPV 54, 73, 83, 1 × Neg). g AMP OD = 0.923 and 0.211, HC OD = 5 and 6, LA = 16 and 82. h Same as (f). i Same as (e). j 1 × HPV 16. k 1 × 61 and 62, 1 × 54, 73 and 83, 2 × LA Neg. b
LA result Negative Positive
68 10
5 323
0.88 (95% CI: 0.826–0.94)
P ≤ 0.0001
3. Study design Overall, 339 women with histologically confirmed high grade abnormalities and 68 women with histological confirmed low grade abnormality were randomly selected and their liquid based cytology samples evaluated [4]. The samples were randomly selected to include approximately 2/3 of the original pool of all high grade abnormality samples available and 1/5 of all low grade and normal samples. Stored frozen PreservCyt samples were tested by the Cobas according to the manufacturing instructions. 4. Methods Briefly, 400 l of sample was extracted and eluted in final volume of 100 l, 25 l of which was then used in a qPCR. This assay is able to detect HPV high-risk types 16 and 18 individually, as well as a group of 12 other known HPV High-Risk types (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) with a single probe different to the type 16 and 18 probes. HC2, Amp, and LA HPV tests results were available from previously conducted studies [4,5]. The Cobas results were compared to HC2, Amp, and LA HPV using 2-by-2 contingency tables, with two-sided P values calculated using Fisher’s exact test. Agreement between tests was assessed by Cohen’s kappa statistic. Confidence intervals (95% CI) for proportions were calculated using a two-tailed test and assuming a random sample of the population [6] (Table 1). The relationship between the histological and HPV results was analysed for sensitivity and specificity and also positive or negative predictive values (95% CI), using ≥CIN 2 as a measure of clinically significant disease (Table 2). 5. Results From 407 cytology Thinprep samples tested on the Cobas, one specimen was negative for both the internal control and HPV, and
hence was removed from further analyses. Among the 406 assessable specimens, HR HPV positivity was 80.8% (Cobas), 72.2% (HC2), and 83.3% (Amp), and 82.0% (LA). Agreement between the Cobas and other HPV tests for the presence of HR HPV types was good (HC2, = 0.70) or very good (Amp, = 0.87 and LA, = 0.88) (Table 1) [6]. 6. Discussion Correlation of HR HPV to histological diagnosis (using ≥CIN2 as a measure of clinically significant disease), demonstrated clinical sensitivities of 90.5% (95% CI of 86.9–93.2) and specificity of 67.6% (95% CI of 55.8–77.6) for the Cobas, 92.9% (95% CI of 89.7–95.2) for the Amplicor HPV test, 80.8% (95% CI of 76.2–84.6) for HC2, and 91.7% (95% CI of 88.3–94.2) for the LA HPV test (Table 2). The Cobas, LA, and AMP HPV tests correctly predicted 33, 37 and 41 respectively more histologically confirmed cervical disease (CIN2) than the HC2 HPV test. Overall, 53 (13%), 16 (3.9%), 15 (3.6%) of results were discordant between the Cobas and HC, AMP and LA tests. The majority (40/44) of HC negative/Cobas positive were also positive by LA and AMP (Table 3). Among the HC negative/Cobas positives, there were 22 samples from women with histological diagnosis of CIN3. This lower sensitivity of HC is due to the higher sensitivity in detection of low concentrations of viral DNA with “the PCR based” target amplification assays. Of the 9 samples positive by HC and negative by the Cobas (Table 1), only 1 of these was positive by LA for HPV 16, a type detected by all methods (Table 3). The other 8 HC2/Cobas
Table 2 Relationship between histological diagnosis and HR-HPV testing. Normal
CIN 1
CIN 2
CIN 3
>CIN 3
Total
≥CIN 2
≤CIN 1
Specificity or sensitivity (95% CI)a
HC2− HC2+ AMP− AMP+ LA− LA+ Cobas− Cobas+
23 5 22 6 22 6 24 4
25 15 22 18 23 17 22 18
31 66 16 81 16 81 19 78
33 197 8 222 12 218 13 217
1 10
11
113 293 68 338 73 333 78 328
65 273 24 314 28 310 32 306
48 20 44 24 45 23 46 22
70.6 (58.9–80.1) 80.8 (76.2–84.6) 64.7 (52.8–75.0) 92.9 (89.7–95.2) 66.2 (54.3–76.3) 91.7 (88.3–94.2) 67.6 (55.8–77.6) 90.5 (86.9–93.2)
Total
28
40
97
230
11
406
338
68
11 11
Negative and positive predictive values (95% CI) 42.5 (33.8–51.7) 93.2 (89.7–95.5) 64.7 (52.8–75.0) 92.9 (89.7–95.2) 61.6 (50.2–71.9) 93.1 (89.8–95.4) 59.0 (47.9–69.2) 93.3 (90.1–95.5)
HC2 – Hybrid Capture 2; AMP – Roche Amplicor; LA – Roche Linear Array; Cobas – Roche Cobas 4800; 95% CI – 95% confidence intervals; CIN – cervical intraepithelial neoplasia. a The Specificity and Sensitivity were determined using ≥CIN 2 and ≤ CIN 1.
S. Phillips et al. / Journal of Clinical Virology 62 (2015) 63–65
discrepant samples may be a result of DNA degradation due to the historical nature of the samples used. In respect to HPV types that are not detected across all PCR-based assays, 10 samples (Table 3) were Cobas negative and AMP and LA positive, most likely due to low HPV copy numbers and differential sensitivities across these assays. In addition, the extraction method for the Cobas is only representative of 400 l of a sample (100 l equivalent is tested in each reaction), whereas LA uses an extraction method that represents 1 ml of clinical sample (500 l equivalent was tested in each reaction). Overall 5 samples were positive by the Cobas and negative by LA. This may be due to competition of assessable types at low copy numbers, with other type’s present, but not analysed or possibly due to sub-types not detectable on LA, such as 68a. This study focused on a population of women being treated for cervical dysplasia, representing a cohort with an inflated disease prevalence and as such this clinical performance should not be extrapolated to the general screening population. However, this cohort of women with heightened levels of disease provides a valuable population for sensitivity testing to assess the Cobas assay system [7]. The performance of the Cobas was equivalent to the AMP and LA HPV tests for HR HPV detection. The Cobas predicted more underlying histologically-confirmed high-grade lesions than the HC2 HPV test, with the added advantage of identifying HPV 16 and 18 genotypes present. The follow up algorithm of women with PCR-based detection of HR HPV as marker of residual disease would need to be confirmed with addition of cytology/histology to ensure no residual disease remain in patients after treatment, in particular with younger women who may have higher frequency of transient HPV infection. Funding Roche Molecular Systems supplied the detection kits utilised for this study.
65
Competing interests No competing interests are declared.
Ethics approval Ethical approval was received for the study by the Royal Women’s Hospital research and ethics committees under reference number 00/30.
References [1] Stoler MH, Wright Jr TC, Sharma A, Zhang G, Apple R, Wright TL, et al. The interplay of age stratification and HPV testing on the predictive value of ASC-US cytology. Results from the ATHENA HPV study. Am J Clin Pathol 2012;137:295–303. [2] Wright Jr TC, Stoler MH, Behrens CM, Apple R, Derion T, Wright TL. The ATHENA human papillomavirus study: design, methods, and baseline results. Am J Obstet Gynecol 2012;206, 46.e1–e11. [3] Moss SM, Bailey A, Cubie H, Denton K, Sargent A, Muir P, et al. Comparison of the performance of HPV tests in women with abnormal cytology: results of a study within the NHS cervical screening programme. Cytopathology 2014, http://dx.doi.org/10.1111/cyt.12210. [4] Stevens MP, Garland SM, Rudland E, Tan J, Quinn MA, Tabrizi SN. Comparison of the Digene Hybrid Capture 2 assay and Roche AMPLICOR and LINEAR ARRAY human papillomavirus (HPV) tests in detecting high-risk HPV genotypes in specimens from women with previous abnormal Pap smear results. J Clin Microbiol 2007;45:2130–7. [5] Stevens MP, Garland SM, Tan JH, Quinn MA, Petersen RW, Tabrizi SN. HPV genotype prevalence in women with abnormal pap smears in Melbourne, Australia. J Med Virol 2009;81:1283–91. [6] Mackinnon A. A spreadsheet for the calculation of comprehensive statistics for the assessment of diagnostic tests and inter-rater agreement. Comput Biol Med 2000;30:127–34. [7] Tabrizi SN, Stevens MP, Khan ZA, Chow C, Devitt MA, Garland SM. Comparison of PapType to Digene Hybrid Capture 2, Roche linear array, and Amplicor for detection of high-risk human papillomavirus genotypes in women with previous abnormal pap smears. J Clin Microbiol 2012;50:2796–8.