NEW YORK – Validations of molecular tests from Atila Biosystems have been steadily accumulating as the firm continues to support the expansion of molecular diagnostics into low-resource settings. The Sunnyvale, California-based maker of nucleic acid amplification technologies and diagnostics instruments intends to pursue US regulatory approval for its rapid cervical cancer screening assay and has also recently been awarded new funding to propel it on the pathway toward business expansion.
Atila Founder and CEO Youxiang Wang is the co-creator of the firm's two core technologies, called Omega amplification and 3N chemistry. Omega amplification is a proprietary rapid isothermal amplification method, while the 3N technology creates primers using only three of the four conventional nucleotides, which prevents them from stably hybridizing with each other, thus eliminating primer-dimer formation and enabling higher multiplexing.
Atila uses these core technologies to develop isothermal, qPCR, and digital PCR molecular assay kits that can be used on standard lab equipment or on the firm's own instruments.
It offers two different molecular diagnostic instruments for low-cost and portable molecular testing. The Power-Gene 9600 is a standard Peltier thermal cycler that can perform qPCR on 96 samples with five-channel detection, while the iAMP is a portable system that shares the same throughput and optical specs. Atila is also developing a fully automated point-of-care instrument, Wang said.
Isothermal amplification chemistries typically have the advantage of being faster than standard PCR thermal cycling, but, at least for the ever-popular loop mediated isothermal amplification (LAMP) approach, primer design can be tricky since each target requires six. This, in turn, can make single-tube multiplexing even trickier.
Using the 3N and Omega methods, however, has allowed Atila to develop single-tube isothermal assays with as many as six targets. They also allow the isothermal reactions to produce real-time results with high specificity, so that the curves generated look nearly identical to qPCR, Wang said, but only take 20 to 30 minutes.
"Our goal is to be able to convert any qPCR assay to an isothermal assay and have even better performance than the gold standard," he said.
By way of example, he cited a 2021 study showing that the firm's AmpFire isothermal HPV genotyping assay had good inter-assay agreement with the Roche Linear Array test. In that study, Atila's test also detected a broader range of subtypes for one of the rarer high-risk HPV strains and detected another strain without the need for confirmatory testing.
At the start of the pandemic, Atila also quickly developed a SARS-CoV-2 assay and obtained Emergency Use Authorization from the US Food and Drug Administration, and the test was subject to an initial validation study in April 2020.
But Atila's isothermal technology has seen its greatest uptake to date in HPV testing, and the firm is on a mission to increase global access to cervical cancer screening in low-resource settings, Wang said.
Almost entirely caused by prior HPV infection, cervical cancer deaths in high-income countries have been dramatically reduced in the past few decades by screening and vaccination campaigns. Now, nearly 90 percent of cases occur in low- and middle-income countries, according to the World Health Organization, resulting in the death of more than 300,000 women in LMICs in 2020.
Atila's AmpFire HPV multiplex isothermal test detects 15 high-risk strains. Since an initial validation of the technology in early 2020, at least 16 additional peer-reviewed studies have been published. Many focus on validating Atila's HPV testing in local contexts, but taken together, they demonstrate that it is capable of performing accurately for a variety of settings and sample types.
The AmpFire HPV test was also redesigned in partnership with researchers at the US National Cancer Institute along with collaborators in Nigeria, Spain, and New York. As described in the International Journal of Cancer in 2022, this version of the assay, called ScreenFire HPV, eliminates detection of two strains that can cause lesions which appear to be high-grade but that rarely lead to cancer.
ScreenFire HPV is multiplexed with 13 high-risk HPV strains binned into four groups with differential cervical cancer risk so that it can be run alongside a control on Atila's five-channel iAMP to support an approach called risk-based screening and management. The test detects HPV 16 in a single channel, designates a channel for combined HPV 18 and HPV 45 targets, then one channel for HPV31/33/35/52/58 and one for HPV39/51/56/59/68.
Part of the approach is also to adapt the target sensitivity to the epidemiology in a hierarchical fashion, Wang noted.
In this way, the component assay with the highest sensitivity detects the highest-risk target, HPV 16, and the next, most-sensitive detects the HPV 18/45 target. Wang said people can be infected by multiple high-risk subtypes, which can then compete in the test reaction, but that detecting the highest-risk subtypes is most critical. The approach might be particularly useful because, "in low- and middle-income countries, most women have only one time in their lifetime to be screened," he said.
Using ScreenFire HPV, the NCI scientists and their international collaborators assessed 2,076 stored specimens. As described online in September in the International Journal of Cancer Research, they found the isothermal test compared favorably with reference results using Roche's LinearArray and NCI's TypeSeq – both of which are lab-based PCR tests.
Specifically, ScreenFire's overall clinical sensitivity for cervical intraepithelial neoplasia 3+ (CIN3+) lesions was 94.7 percent, similar to the Linear Array and TypeSeq, the authors wrote, with a high sensitivity for all types and channels.
A forthcoming study being led by researchers at the NCI, called PAVE, will also assess a novel cervical cancer screening-triage-treatment strategy that includes self-sampled HPV testing and the Atila ScreenFire test.
The NCI has previously declined to provide comments in order to avoid the appearance of endorsing a specific system or product, but a number of the research studies of Atila's technology published in the past few years have been co-authored by scientists at the institute.
An international team of researchers, including members of the NCI team that redesigned Atila's test, also used AmpFire HPV paired with a self-sampling strategy to screen and treat 3,345 women in the Inner Mongolia region of China over a five-day campaign.
In a study published in late 2020 in Infectious Agents and Cancer, the researchers said that China has no national screening program, cervical cancer causes 30,000 deaths there each year, and mortality rates are nearly 50 percent higher in rural areas.
They found the Atila AmpFire HPV system to be "highly portable, simple, rapid … and economical," with an ability to test 92 specimens per instrument every 65 minutes. They also said the system was minimally labor intensive, did not require a high degree of technical expertise to run, and had a total cost of $7 per specimen.
Cost of testing, particularly of WHO prequalified HPV assays, is the greatest barrier to molecular-based cervical cancer screening in LMICs, according to a research team from Harvard and Botswana.
In May 2023, this team published a study demonstrating that AmpFire HPV had a high positive percent agreement with the prequalified Cepheid Xpert HPV test, stating that the $9 per-test cost for the Atila 15-target genotyping assay is more affordable than most available HPV tests.
"These results show that an affordable and accurate HPV assay with individual HPV genotyping is within reach of the global market," the authors concluded. "Because of its simplicity, low cost, and ease of use, AmpFire can be easily implemented in facilities that currently have no HPV testing," they also said.
Adding the Atila test to the list of WHO prequalified HPV screening assays would likely help to increase uptake, these authors also noted, adding that "Expedited approval is essential to rapidly scale up affordable HPV primary screening for cervical cancer."
This is because, "Currently WHO recommends primary cervical screening with HPV assays with particular regulatory approvals," they wrote, citing an International Papillomavirus Society position statement which guides that only clinically validated HPV assays which have fulfilled "internationally accepted performance criteria" be used for primary cervical cancer screening.
The WHO recommends using HPV DNA detection as the primary screening test for cervical cancer, and its Prequalification of In Vitro Diagnostics program is intended to enhance the quality of testing for certain high-risk conditions in LMICs. Currently only the Cobas HPV Assay from Roche Molecular Systems and the BD Onclarity HPV Assay from Becton Dickinson are cleared for primary HPV screening, while the Digene HC2 High-Risk HPV DNA Test from Qiagen, the Cervista HPV HR Assay, and the Aptima HPV Assay from Hologic are cleared for reflex testing in conjunction with cytology.
Although WHO prequalification does not absolutely require FDA approval as a prerequisite, Wang said that Atila is nevertheless pushing forward on US Food and Drug Administration Premarket Approval for its HPV assay.
"The main reason is that the US public health also needs a test that is simple, fast, easy and affordable," he said, adding that there is no such test available in the US at the moment.
And, Atila already has regulatory approval in many LMICs, including Kenya, Argentina, the Philippines, and Indonesia, Wang said. In addition, roughly 20 countries in Africa are currently using Atila's technology, and Wang frequently travels to these locations to help with the initial set-up of the system.
"For all of them, we hardly do any advertisement," he said. "They find us through word of mouth."
Global nonprofit HPV testing
Cervical cancer is essentially "100 percent preventable," so it is a travesty that nearly 350,000 women worldwide die each year from this disease, said Rebecca Lepsik, executive director at the Los Angeles-based nonprofit CureCervicalCancer that uses Atila's HPV testing technology.
Since its founding in 2013, CureCervicalCancer has trained 766 healthcare professionals and 1,761 community health workers, establishing more than 100 sustainable cervical cancer prevention clinics, Lepsik said. The group has worked with communities in China, Ethiopia, Guatemala, Haiti, Kenya, Lebanon, Nigeria, Tanzania, Uganda, and Vietnam. In total, this team has screened 188,603 women for cervical cancer and treated 12,828 women for precancerous lesions.
Initially, CureCervicalCancer relied on a "see and treat" protocol involving detection of lesions by application of a vinegar solution during a gynecological exam. But testing for high-risk HPV strains is now considered the gold standard for cervical cancer screening by the WHO, Lepsik said. Combined with self-sampling, this molecular approach is potentially faster and less invasive for women and can therefore increase access and uptake of screening in low- and middle-income countries.
Two years ago, CureCervicalCancer launched Mobile Health for Mamas, a community-based screening program in Kenya, and hoped to deploy molecular HPV testing.
"We were looking for something portable, highly durable, simple to learn and operate, with a quick processing time," Lepsik said. The team was also considering the cost of the HPV assays, as they wanted to reach as many women as possible and show that HPV testing can be cost effective compared to the visual approach.
"Atila Biosystems was the obvious choice for us," she said, adding, "We have never been disappointed." Atila's commitment to troubleshooting and continual test system and assay improvement, along with Wang's personal commitment to the cause, have been helpful, as well, she said.
The CureCervicalCancer team has incorporated both the Atila PowerGene 9600 and iAMP-PS96 isothermal qPCR readers into its mobile lab, using solar power to overcome unreliable electricity supply in some areas of Kenya, Lepsik said.
So far, the mobile program has provided free screening for more than 20,000 women using Atila HPV testing and self-collected samples, and the team plans to expand into another region of Kenya to reach an additional 25,000 women.
Ultimately, "we want to demonstrate an effective and scalable model for implementing HPV testing in low-resource settings," Lepsik said.
Self-sampling is one of the keys to these cost savings, and Atila has been attempting to address this: In July the firm was awarded a 12-month, $522,460 grant from the Gates Foundation to support evaluation and development of the ScreenFire HPV test paired with self-sampling. The self-sampling approach is also an aim in Atila's two-year Phase II small business innovation grant totaling $1,011,310 that was awarded by the National Institutes of Health in 2022.
For the Atila test, a self-sample is collected in a dry tube with no medium required, which reduces cost and also allows the team to bring the mobile service to more remote regions. Similar work in Papua New Guinea in 2016 demonstrated that self-sampling paired with molecular testing using the Cepheid system could potentially improve uptake of screening.
Overall, "because the testing system is fairly 'low-maintenance,' it's perfect for a mobile laboratory," Lepsik said, while the processing time of approximately 90 minutes allows the option for same-day testing and treatment.
Maggie Carpenter, executive director and founder of the cervical cancer nonprofit Go Doc Go, has also deployed the Atila HPV testing system in her work in Senegal and Gambia because of the relatively low cost, fast processing speed, and ability to test fewer samples per run if needed.
In 2019, Wang also accompanied her team to help set up testing at three community clinics. "He's the real deal," Carpenter said.
During the first trip to Saint-Louis, Senegal, to set up testing in a clinic, Carpenter said the head midwife there volunteered to be the first to be screened. Carpenter explained that the testing uses self-sampling. When the midwife returned with her sample, she went to get on the exam table, and Carpenter said that wasn't necessary. "She gave me a huge hug," Carpenter said.