Evaluation of the Roche AMPLICOR® Human

Papillomavirus (HPV) Test*

Rita Sun, Janet Kornegay, Anona Ahmad, Robert Current, Karen Ding, Snehal Joshi,
Dmitriy Kosarikov, Rashmi Kotadia, Namneet Rudra, Theresa Young, Karen Yu
Roche Molecular Systems Inc., 4300 Hacienda Drive, Pleasanton, CA 94588, USA

Introduction Results
● No cross-reactivity with the non-HPV microorganisms and HPV ‘low-risk’ genotypes was observed
● The Roche AMPLICOR® Human Papillomavirus (HPV) Test* is a PCR-based qualitative assay for the (A450 ≥0.2) using the AMPLICOR HPV Test (Table 3)
detection of high-risk human papillomavirus types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68
● The test permits simultaneous PCR amplification of HPV target DNA within the L1 region of the HPV Table 3. Non-HPV microorganisms and HPV ‘low-risk’ genotypes tested
genome, and a human DNA marker (β-globin) as a cellular and inhibition control Achromobacter xerosis Derxia gummosa Mycobacterium avium
● The amplified products (amplicon) are detected by hybridization to a pool of HPV probes coated on a Acinetobacter sp. genospecies 3 Eikenella corrodens Mycoplasma hominis
single microwell plate well, followed by an enzymatic reaction; detection of the β-globin control is Actinomyces israelii Enterobacter cloacae Neisseria gonorrhea
performed in a separate well Aerococcus viridans Enterococcus faecalis Pasteurella maltocida
Aeromonas hydrophila Erysipelothrix rhusiopathiae Pediococcus acidilactica
Objective Agrobacterium radiobacter Escherichia coli Peptostreptococcus productus
Alcaligenes faecalis Ewingella americana Prevotella corporis
● To evaluate the non-clinical performance characteristics of the AMPLICOR HPV Test Bacillus thuringiensis Flavobacterium meningosepticum Propionibacterium acnes
Bacteroides fragilis Gamella morbillorum Proteus mirabilis
Limit of detection and genotype inclusivity Bifidobacillus longum Gardnerella vaginalis Providencia stuartii
Bifidobacterium adolescentis Haemophilus ducreyi Pseudomonas aeruginosa
Materials and methods Branhamella catarrhalis Herpes simplex virus 1 Rahnella aquatilis
● Each of the 13 high-risk type HPV plasmids was tested at concentrations ranging from 10 to Brevibacterium linens Herpes simplex virus 2 Salmonella minnesota
1920 copies/mL Candida albicans Kingella kingae Staphylococcus aureus
– A minimum of 24 replicates were tested for each plasmid level Chlamydia trachomatis ser E Klebsiella pneumoniae ss ozaenae Streptococcus anginosus
Chlamydia trachomatis ser A Lactobacillus vaginalis Streptococcus epidermidis
● The positive hit rate was calculated, to determine the limit of detection (LOD; positivity hit rate >95%)
Chromobacter violaceum Lactococcus lactis cremoris Trichomonas vaginalis
for each high-risk HPV genotype
Citrobacter braakii Legionella pneumophila Treponema phagedenis
Results Clostridium perfringens Leuconostoc paramesenteroides Vibrio parahaemolyticus
● The positivity rate for the AMPLICOR HPV Test was >95% for all 13 HPV high-risk types at a Corynebacterium genitalium Micrococcus luteus Yersinia enterocolitica
concentration of 240 HPV DNA copies/mL; at a concentration of 100 HPV DNA copies/mL, the Cryptococcus neoformans Moraxella osloensis
positivity rate was >95% for all types except 31, 52, 58 and 59 (Table 1) Deinococcus radiopugnans Morganella morganii
Human papillomavirus 6 Human papillomavirus 43S Human papillomavirus 57
● All genotypes were detected with a positivity rate of 100% at 480 copies/mL
Human papillomavirus 11 Human papillomavirus 44 Human papillomavirus 64
Table 1. Limit of detection for the AMPLICOR HPV Test Human papillomavirus 26 Human papillomavirus 53 Human papillomavirus 66
Human papillomavirus 40 Human papillomavirus 54 Human papillomavirus 67
HPV genotype Limit of detection (copies/mL)
Human papillomavirus 42 Human papillomavirus 55B Human papillomavirus 70
16 100
18 100
31 240 Clinical specificity and correlation
33 100
35 100
Materials and methods
39 100 ● A total of 762 clinical specimens that were previously tested for ‘high-risk’ HPV by the Digene Hybrid
45 100 Capture® 2 (hc2) test were tested in this study:
51 100 – 478 of these cervical specimens were collected in PreservCyt® preservative solution and 284 in
52 240 SurePath™ preservative fluid
56 100 ● Discrepant results between the AMPLICOR HPV Test and the hc2 test were resolved by a combination of
58 240 modified E7 type-specific PCR1 and the Roche LINEAR ARRAY® HPV Test*2
59 240 ● Sensitivity and specificity for the presence of high-risk HPV DNA in cervical specimens collected in
68 100 both PreservCyt® preservative solution and SurePath™ preservative fluid were determined for both the
AMPLICOR HPV Test and the hc2 test (Table 4)
Reproducibility Results
Materials and methods ● From a total of 762 valid results, the AMPLICOR HPV Test showed a specificity of 95.6% and a sensitivity
● The high-risk type 16 HPV plasmid was tested at three concentrations (480, 960 and of 96.1%, while the hc2 test showed a specificity of 93.4% and a sensitivity of 86.1% (Table 4)
1440 copies/mL) in PreservCyt® preservative solution by two operators for 15 days Table 4. Sensitivity and specificity of the AMPLICOR HPV Test and the hc2 test for the presence of
● Data for a total of 90 replicates for each concentration were generated, as well as for 90 replicates of an high-risk HPV DNA in cervical specimens collected in liquid-based cytology media
HPV-negative control [HPV(-)C] (PreservCyt® and SurePath™)
● The positive hit rate for the samples tested and the negativity rate for HPV(-)C were calculated, to Test Parameter Estimate 95% CIa
determine the reproducibility
AMPLICOR HPV Sensitivity 96.1% 93–99%
Results AMPLICOR HPV Specificity 95.6% 93–97%
● The reproducibility results showed a positivity rate of 100% at all concentrations tested; a negativity hc2 Sensitivity 86.1% 81–90%
rate of 100% was observed for HPV(-)C (Table 2) hc2 Specificity 93.4% 91–95%
a95% Wald confidence interval
Table 2. Reproducibility of the AMPLICOR HPV Test

HPV 16 DNA Number AMPLICOR HPV Test results

(copies/mL) tested HPV-positive (A450 ≥ 0.2)
Summary of results
n % 95% CIa ● The LOD of the AMPLICOR HPV Test was at least 240 HPV DNA copies/mL, and most subtypes
showed an LOD of 100 HPV DNA copies/mL
1440 90 90 100 96–100%
● The AMPLICOR HPV Test reproducibly detected samples near the LOD
960 90 90 100 96–100%
● Clinical specificity of the test was 95.6% and clinical sensitivity was 96.3%. No cross-reactivity was
480 90 90 100 96–100%
found upon testing samples containing other microorganisms or low-risk types of HPV
HPV-negative (A450 < 0.2)
0 90 90 100 96–100%
a95%
Wald confidence interval
Conclusion
● Results from this study indicate that the AMPLICOR HPV Test is suitable for the
Exclusivity detection of high-risk HPV DNA, with good sensitivity, specificity and reproducibility

Materials and methods

● A panel of 64 non-HPV microorganisms and 15 HPV ‘low-risk’ genotype plasmids were tested, to
References
evaluate the analytical specificity of the AMPLICOR HPV Test 1. Walboomers JM, Jacobs MV, Manos MM et al. J Pathol 1999;189:12–9
● The HPV ‘low-risk’ genotype plasmids were tested at 2 x 107 copies/mL, and the non-HPV micro- 2. Shepard A, Boyle S, Kosarikov D et al. 21st International Papillomavirus Conference, 2004, Mexico City, Mexico
organisms were tested at the highest levels available *Under development