Twenty-four years ago, it was clear to us that reaching hundreds of millions of women in medically underserved regions with traditional Pap screening, was impossible. Upon the arrival of HPV screening, the potential for self-collection became the focus of our research team. It required 8 more years to discover how a self-collected specimen could be equal in sensitivity to a direct endocervical sample. Up to that point we had incorrectly assumed the collection device was the key. However, in women with high-grade disease, we realized the viral load in self-collected vaginal specimens is significantly lower than in direct endocervical samples, thereby requiring PCR to amplify the specimen.^1,2 Since that time, we have moved from slow cumbersome expensive technologies to today, where we have a choice of excellent PCR-based assays for everyday clinical use; and self-collection is being explored in all parts of the world. For this work we are always on the lookout for technologies that have the potential to synergize with self-collection especially for LMICs.

Remember, systems make things work, not isolated technologies. However, sometimes a technology comes along that “encourages” a system to be built around it, since it solves many unique problems.

LMIC: low-middle income countries.

The novel platform of AmpFire HPV assays we believe is such a technology. Developed by the California-based Atila Biosystems, there are two platforms: a screening assay for 16, 18, with a pool of thirteen high-risk HPV types; and a genotyping assay for 15 specific types. AmpFire has regulatory approval from CE-IVD, China, Indonesia, Israel and Belgium. It is in use in eight African countries, plus regions of several other LMICs.

The AmpFire assay uses isothermal amplification to amplify the specimen and this methodology results in a constellation of assays addressing not only HPV, but all common STDs as well as COVID-19 in a simple, fast, versatile platform.

More specifically the assay detects HPV from raw samples. Therefore, no DNA extraction is needed. Best is a simple dry brush sample, or a non-alcohol containing liquid transport media. Virtually all common media types will work; but by eliminating the need to remove alcohol from the sample (as with PreservCyt), or the technology required to punch-process solid media cards, sample prep and analysis is reduced to less than 1hour.³ Also, the assay has been successfully used for stored FFPE samples which require only 1.5 hours sample preparation time.⁴ Therefore, no special labs or air flow management are needed, just a simple room. The AmpFire assay requires only 1 tube for its screening assay and four tubes are used for the full genotyping assay, which also takes less than 1hour and can be run individually, sequentially or simultaneously. Samples do not require batch processing, and the equipment is inexpensive and portable (Figure 1). Under consideration is removing HPV53 from the pool and possibly HPV66 to further improve the specificity with negligible effect on sensitivity.

We designed a pilot study to test the AmpFire HPV screening assay using CIN2 or worse as the endpoint. The results are shown in Table 1.⁵ For self and direct testing the Kappas ranged from 0.86 – 0.90 and there was only 1 discordant case >CIN3 (a non-16/18 AmpFire self-test positive, Cobas self-test negative). The AmpFire assay demonstrated equal sensitivity and significantly better specificity than the Cobas assay.

Finally, returning to what started this discussion, the amplification works beautifully for self-collected samples, and the technologies’ acceptance of dry-brush transport and resistance to enzyme inhibition makes the “system” simple, inexpensive and adaptable to large population-based screening programs. This is an assay that stands out in a crowd and should be explored by multiple investigators and tailored to their special applications.

DISCLOSURE
The authors declare no conflicts of interest to disclose.

References

1. Belinson JL, Hu S, Niyaxi M et al. Prevalence of type-specific human papillomavirus in endocervical, upper and lower vaginal, perineal, and vaginal self-collected specimens; implications for vaginal self-collection. Int. J Cancer 2010;127(5):1511-7. Available from: https://pubmed.ncbi.nlm.nih.gov/20039323/

2. Belinson JL, Yang B, Wu R et al. Improved sensitivity of vaginal self-collection and high-risk human papillomavirus testing. Int J Cancer 2012;130(8):1855-60. Available from: https://pubmed.ncbi.nlm.nih.gov/21630255/

3. Jang D, Shah A, Arias M et al. Performance of AmpFire HPV assay on Neck Cervical Lymph Node Aspirate and Oropharyngeal Samples. J Virol Methods. 2020 Feb 24:113840. Available from: https://pubmed.ncbi.nlm.nih.gov/32105752/

4. Tang YW, Lozano L, Chen X et al. An Isothermal, Multiplex Amplification Assay for Detection and Genotyping of Human Papillomaviruses in Formalin-Fixed, Paraffin-Embedded Tissues. J Mol Diagn. 2020 Mar;22(3):419-428. Available from: https://pubmed.ncbi.nlm.nih.gov/31978559/

5. Wei Zhang, Hui Du, Chun Wang, Xia Huang, Jerome L Belinson, and Ruifang Wu. Evaluation of an isothermal amplification HPV detection assay (AmpFire) for primary cervical cancer screening. Abstract/Poster IPVC 2020.