Evaluation of a Novel Isothermal Amplification Assay

for Rapid HPV Detection and Genotyping

Michelle Kleeman ,
1 Xin Chen ,
2 Caroline Reuter ,
1 Youxiang Wang ,
2 A;la Lorincz1 and Belinda Nedjai 1

1. Wolfson InsFtute of PrevenFve Medicine, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ
2. AFla BioSystems, Inc. 740 Sierra Vista Ave, Unit E, Mountain View, CA 94043

BACKGROUND
Early human papillomavirus (HPV) screening and treatment of precancerous
lesions is known to be highly effecFve in reducing the cervical cancer death
rate. A large number of assays designed for HPV genotyping have been
developed in recent decades. They have variable analyFcal sensiFvity and
specificity for different HPV genotypes and may be used for rouFne clinical
diagnosis, epidemiological studies and evaluaFon of vaccine efficacy and
monitoring. However, HPV infecFon is sFll a global issue, especially in poor
resource areas. Despite there being many HPV screening technologies
available, they oben require expensive equipment, are complicated and can
be Fme-consuming.
RESULTS
• AmpFire assay has almost idenFcal results when compared to Abbog (n=60,
96.7%) and Genera (n=58, 97%). See table 1.
• Table 2 shows the compared performance between AmpFire and Genera
PapType genotyping results in 45 mulFple infecFon samples. 44.4% showed
idenFcal genotyping results and 33.3% showed parFal agreement. Overall, the
two assays largely agree with each other.
• AmpFire assay showed excellent consistency between two batches of samples
(n=8) compared to PapType genotype results, which indicates AmpFire assay
has great reproducibility. See table 3.

AmpFire HPV assay by AFla Biosystems is an isothermal amplificaFon assay, • 17 samples out of 20 archived samples tested by AmpFire assay showed
that has a simple protocol with no need for DNA extracFon. The test detects idenFcal results to all the other methods (Abbog, Cobas, BD and Genera).
fibeen high-risk HPV types in a single tube reacFon and can also genotype • AmpFire assay demonstrated an excellent reproducibility and great sensiFvity
HPV16 and HPV18 using real-Fme fluorescent detecFon. Raw samples are based on 8 diluted samples and HPV 16 plasmid diluFon test. See figure 2.
heated and lysed prior to isothermal amplificaFon. The test can be completed
within an hour, including hands-on Fme. The simplicity and speed of AmpFire AmpFire Results No. of %
Assay Summary Category
+ - Total Samples
would be a great fit for poor resources areas for HPV screening.
+ 48 1 49
PA= 98.0% IdenFcal Results 20 44.4%
Extra Types for AmpFire 11
OBJECTIVE Abbo:
-
Tota
1 10 NA= 90.9%
11
OA= 96.7% Extra Types for Genera 2 33.3%
Assess the performance of the AmpFire HPV assay, comparing results to those 49 11 60 k=0.89
l Extra Types for both assays 2
previously obtained using other HPV tests on the market (including Abbog + 39 3 42 PA= 79.6% AmpFire +, Genera - 8
RealTime High Risk HPV assay and Genera Biosystems PapType high-risk HPV - 10 8 18 NA= 72.7% 22.2%
Genera AmpFire -, Genera + 2
DetecFon and Genotyping Assay), as well as the reproducibility and analyFcal Tota OA= 78.3%
49 11 60 Total 45 100.0%
l k=0.42
sensiFvity. Table 2. HPV Genotyping results comparison-
Table 1. Agreements between AmpFire HPV
mulFplex assay and Abbog RealTime HPV assay AmpFire Vs Genera.
MATERIALS & METHODS or Genera PapType assay.

• 60 fresh samples stored in ThinPrep PreservCyt soluFon were tested with Sample AmpFire HPV MulFplex Assay Results Genera Abbo:
the AmpFire HPV MulFplex assay. 45 samples with mulFple infecFons Undiluted 1:5 DiluFon 1:50 DiluFon (Undiluted) (Undiluted)
(besides HPV16 and HPV18) were further analysed with AmpFire HPV 1 HPV16 HPV16 HPV16 HPV16 HPV16
Genotyping assay. 8 samples were chosen for repeat tests as well as tesFng 2 HR HPV Neg Neg Neg HR HPV
at 1:5 and 1:50 template diluFons. 3 HR HPV HR HPV HR HPV HPV59 HR HPV
4 HR HPV HR HPV HR HPV HPV66 HR HPV
• An addiFonal 20 archived samples (stored in PreservCyt soluFon at -20 °C for
5 HR HPV HR HPV HR HPV HPV31 HR HPV
over 10 years) were tested to evaluate the performance on old samples.
HPV16, HR HPV16, HPV16, HPV16, 56 & HPV16,
6
• HPV16 plasmids were diluted to assess the analyFcal sensiFvity. HPV HR HPV HR HPV 68 HR HPV
7 Neg Neg Neg Neg Neg
8 HR HPV HR HPV HR HPV HPV58 HR HPV
Table 3 Sample diluFon tolerance test for AmpFire HPV assay

Figure 2. AnalyFcal sensiFvity test using HPV16 plasmid DNA by

Figure 1. AFla Biosystems Ampfire HPV Assay Workflow Ampfire HPV mulFplex Assay.

CONCLUSION
AmpFire HPV assays showed high reproducibility and high sensiFvity on clinical samples. The assay is promising as a new rouFne HPV DNA detecFon and genotyping
test. The main advantages of AmpFire are the simple procedure and use of minimal instrumentaFon. AmpFire HPV assay demonstrates the shortest sample-to-result
Fme among many of the HPV assays on the market.

Topic: HPV TesFng www.Wolfson.qmul.ac.uk/centres/CCP