Redesign of a rapid, low‐cost HPV typing assay to support risk‐based cervical screening and management

Abstract Accelerated cervical cancer control will require widespread human papillomavirus (HPV) vaccination and screening. For screening, sensitive HPV testing with an option of self‐collection is increasingly desirable. HPV typing predicts risk of precancer/cancer, which could be useful in management, but most current typing assays are expensive and/or complicated. An existing 15‐type isothermal amplification assay (AmpFire, Atila Biosystems, USA) was redesigned as a 13‐type assay (ScreenFire) for public health use. The redesigned assay groups HPV types into four channels with differential cervical cancer risk: (a) HPV16, (b) HPV18/45, (c) HPV31/33/35/52/58 and (d) HPV39/51/56/59/68. Since the assay will be most useful in resource‐limited settings, we chose a stratified random sample of 453 provider‐collected samples from a population‐based screening study in rural Nigeria that had been initially tested with MY09‐MY11‐based PCR with oligonucleotide hybridization genotyping. Frozen residual specimens were masked and retested at Atila Biosystems. Agreement on positivity between ScreenFire and prior PCR testing was very high for each of the channels. When we simulated intended use, that is, a hierarchical result in order of clinical importance of the type groups (HPV16 > 18/45 > 31/33/35/52/58 > 39/51/56/59/68), the weighted kappa for ScreenFire vs PCR was 0.90 (95% CI: 0.86‐0.93). The ScreenFire assay is mobile, relatively simple, rapid (results within 20‐60 minutes) and agrees well with reference testing particularly for the HPV types of greatest carcinogenic risk. If confirmed, ScreenFire or similar isothermal amplification assays could be useful as part of risk‐based screening and management.


What's new?
Due to cost and perceived complexity, most existing human papillomavirus (HPV) assays for cervical cancer screening are designed to yield a pooled result for the carcinogenic HPV types.
Here, to promote rapid, affordable and risk-based cervical screening, an existing isothermal DNA amplification test was redesigned to group the carcinogenic HPV types into four channels based on clinical importance (HPV16; HPV18/45; HPV 31/33/35/52/58 and HPV 39/51/56/59/68). In masked retesting of 453 Nigerian specimens, the new ScreenFire assay showed good-to-excellent type group agreement with prior PCR testing. When validated, the redesigned test could support risk-based screening in resource-limited settings.

| INTRODUCTION
Cervical cancer incidence and mortality are highest in lower-resource regions of the world. 1 The World Health Organization (WHO)'s global call for eliminating cervical cancer relies on human papillomavirus (HPV) vaccination, two rounds of HPV based screening in midadulthood and treatment of cervical precancers and cancers. 2 Given the necessary and causal role of HPV in cervical carcinogenesis, 3 sensitive HPV-test based screening is now recognized as the preferred screening test for cervical cancer prevention worldwide. [4][5][6] Nevertheless, HPV infections are too common and benign in many high-risk regions to treat all infected women. 7,8 Most HPV infections clear rapidly within 1 to 2 years of initial detection. 9,10 Only persistent HPV infections, in the absence of clearance, progress to precancer and cancer. 9 The risk of progression, given persistence, depends on HPV type and varies substantially even among the 13 high-risk (HR) HPV types defined as human carcinogens (ie, Group 1) or probable carcinogens (ie, Group 2A) by the International Agency on Research for Cancer (IARC). 11 Evidence to date suggests that at least four distinct risk categories exist, including: (a) HPV16 (species alpha-9) with the highest risk of cervical cancer, causes $60% of squamous cancers, (b) HPV18 and HPV45 (species alpha-7) with an intermediate risk of cervical cancer, cause $15% of squamous cancers but together with HPV16 account for >90% of adenocarcinomas, 12 (c) other HPV16-related alpha-9 types, namely, HPV31, HPV33, HPV35, HPV52 and HPV58, with intermediate risk of cancer, cause another 15% of squamous cancers and (d) other HR types, namely, HPV39, HPV59, HPV68 (species alpha-7), HPV56 (species alpha-6) and HPV51 (species alpha-5), with low risk of cervical cancer, account for only $5% of squamous cancers but represent a quarter of HR13 HPV infections. 9,13-18 Risk-based extended HPV typing, if incorporated with minimal additional cost into primary HPV screening, could provide risk stratification for triage of HPV-positive women in a single step. 19 Due to cost and perceived complexity, most existing HPV assays are designed to yield a pooled result for the carcinogenic HPV types with no or only limited genotyping (mainly of HPV16 and HPV18), obscuring the finer risk-stratifications within the other HR HPV types (eg, HPV16-related alpha-9 types pose a substantially greater risk than the "other HR" types). Moreover, many existing assays also include HPV66 and a few include HPV53 (species alpha-6). The latest version of the IARC monograph, rectifying the mistake of the previous versions, reclassifies HPV66 along with HPV53 as only possibly and rarely carcinogenic to humans (ie, Group 2B). 11 HPV66 and HPV53 are relatively common in the general population and frequently cause high-grade appearing lesions but rarely cause cancer. 20 Thus, including these marginally carcinogenic HPV types in a screening assay is likely to harm public health due to a decreased specificity and positive predictive value without appreciable gain in sensitivity and negative predictive value for screening to prevent cervical cancer. 20,21 Additionally, HPV assays need to be rapid, low-cost and practical for scale-up in cervical cancer screening programs in resource-limited settings. An isothermal amplification technology-based AmpFire HPV assay (Atila Biosystems, Inc., Mountain View, California) shows promise in this regard. 22 The assay in its two current formats offers (a) multiplex detection in a single tube of 15 HPV types (ie, including HPV53 and HPV66) with separate detection of types 16/18 or (b) full genotyping of 15 individual types in four tubes. The assays are Conformité Européenne (CE) marked 23 and previously demonstrated to have satisfactory performance in analytical 24 and clinical validation. 23 We collaborated with the scientists at Atila Biosystems to redesign the AmpFire HPV assay into a single-step 13-type assay providing risk-based, extended HPV genotyping in four channels in a single  The study methodology has been described in detail previously. 25 Briefly, $1420 eligible women (not pregnant, sexually experienced and without hysterectomy, 15+ years old and living in the house for more than 3 months) from randomly selected houses in the village of Irun in Nigeria were invited and attended the screening clinic and provided informed consent to be enrolled in the study. At the screening visit, locally trained nurses performed cervical exams and collected cervical cell sample in Preservcyt (Hologic, Marlborough, Massachusetts) using a broom-type device and endocervical brush for liquidbased cytology (LBC) and HPV testing.
One milliliter aliquots of the frozen residual cytology samples were tested in the United States (Albert Einstein Cancer Center) for HPV using a AmpliTaq Gold MY09-MY11 PCR-based test that included additional type-specific primers, and primers to amplify a cellular beta-globin fragment as an internal control (IC) for amplification as previously described. 26 PCR products were genotyped with dot-blot hybridization using type-specific probes for 13 HR and >20 other HPV types, using a numeric measure of signal strength (1-5 from lowest to highest) as a validated semiqualitative measure of viral load. 27 Additionally, samples were also tested with an investigational Luminex assay for HPV16 and HPV18 DNA, which we considered only in supplemental analyses.
Of the 1339 such residual samples, valid PCR results were avail- The first masked comparison generated fair to good agreement (data not shown). There was evidence of competition for reagents in the presence of multiple HPV infections, and slightly diminished sensitivity for some types. We provided the summary results to the Atila Biosystems team and re-randomized the specimens under NCI direct supervision to permit another valid masked testing round. After primer redesign and reagent optimization were completed, the Atila Biosystems team "locked" their new format, which emphasized more sensitive detection particularly of HPV16 and HPV18/45. The masked testing of the specimen set was then completely repeated. This article reports the independent analysis of results from the second round of testing.

| ScreenFire HPV Test
The assay is intended to be performed on "dry swabs"; thus, centrifugation was needed to process the 0.5 mL of cervical cell suspension in Preservcyt. Specifically, the suspension was transferred to a 1.5 mL tube for centrifugation at maximum speed for 20 minutes and the supernatant was discarded, followed by the addition of 50 μL of Â1 lysis buffer and vortexing thoroughly to resuspend the cell pellet. The resuspended cell pellets were then transferred to 96-well PCR plates for incubation at 95 C for 15 minutes, cooled at room temperature and briefly spun. After this initial sample preparation, 5 μL of this prepared specimen was mixed with 20 μL of freshly prepared master mix (including reaction mix and primer mix) into a 96-well PCR plate using hand pipetting. Additionally, positive and negative template controls were included in each 96-well plate as well. Next, the plates were  As a supplementary analysis, the investigational Luminex testing for HPV16 and HPV18 was examined to look for clues as to the meaning of interassay disagreement. Results from this assay are presented because they revealed interesting patterns but are outside the a priori comparison.

| Channel by channel analysis
There was good to excellent agreement between the ScreenFire assay and the PCR assay for all the channels on nonhierarchical analyses (

| Hierarchical analysis
On the hierarchical analysis, which would be useful for risk-based clinical management of the women, there was an overall 96.8% (95% confidence interval [CI]: 95.9%-97.8%) agreement between the ScreenFire and PCR assay (unweighted kappa = 0.89, weighted kappa = 0.90, these statistics are obtained by using sampling weights) ( The assay could help to address an identified public health need.
In resource-limited settings, which account for 90% of cervical cancer deaths, vaccine coverage and particularly organized screening are minimal. 29,30 Cervical cancer prevention efforts are reduced further in the COVID-19 era. 31,32 Even when vaccination reaches reasonable coverage levels among young women, older unvaccinated birth cohorts will remain at risk making secondary prevention efforts essential over the coming decades. 33 WHO recommends either "screen-and-treat" using primary HPV screening and ablation/excision or "screen-triage-treat" As a result of this satisfactory pilot evaluation, the ScreenFire HPV assay is being evaluated in two independent US laboratories, for clinical accuracy against both prior HPV reference testing and histopathologic outcome, based on an extensively-validated set of >2000 provider-collected cervical samples. 35

| CONCLUSIONS
In conclusion, the redesigned ScreenFire HPV assay was shown to provide accurate risk-based genotype grouping. If the ongoing large validation study in two independent laboratories yields similar results, the assay using self-collected dry swabs will be deployed for field evaluation in multiple international cervical cancer screening sites.
The longer-range public health goal is implementation in resourcelimited settings.

AUTHOR CONTRIBUTIONS
The work reported in the article has been performed by the authors,

CONFLICT OF INTEREST
The authors declare no conflict of interest. Atila BioSystems had no role in the design, analysis, interpretation or drafting of this article.

DATA AVAILABILITY STATEMENT
The data that support the findings of our study are available from the corresponding author upon reasonable request.

ETHICS STATEMENT
Both Nigerian and National Cancer Institute (NCI, US) institutional review boards (IRBs) (NCT 00804466) approved the original study protocol that included consent for specimen storage for future research.