NEW YORK – Atila BioSystems, a Mountain View, California-based molecular diagnostics startup, has developed an HPV genotyping test that can detect 15 high-risk human papillomavirus subtypes and allow a single technician to test up to 800 patient samples in one day using a low-cost, portable instrument called AmpFire.
The firm also makes an HPV test for cervical cancer screening that calls out HPV 16/18 and groups other high-risk types. Although there is a paucity of published data on the AmpFire system, it is currently being evaluated by researchers studying HPV epidemiology and cancer prevention, particularly in low- and middle-income countries (LMICs).
Atila was founded in 2009, but only began promoting its testing in the US last year, CEO Youxiang Wang said in an interview. "We're a very small company, and in the past, we were focused on China ... so not that many people have heard about our technology," he said.
Wang, a biochemist with expertise in nucleic acid amplification and lab-on-a-chip technologies, has worked to develop the firm's proprietary nucleic acid amplification technologies and instruments along with a host of oncology assays and non-invasive prenatal tests.
For its HPV testing, Atila uses a multiplexed isothermal assay and real-time fluorescence detection to detect 15 high-risk HPV genotypes using a single tube reaction, Wang said.
The test can also be run as a cervical cancer screening test, and the cost per sample is low enough that it can be used for stat testing without requiring users to wait for a batch to accumulate.
Screening tests typically report out HPV subtypes 16 and 18 — which cause approximately three quarters of all cervical cancers worldwide — and then report the presence of other high-risk subtypes without distinguishing them.
To eliminate cervical cancer by 2030, the World Health Organization is targeting screening 70 percent of women between 35 and 45 years of age globally using "a high-precision test" such as an HPV test. Approximately 90 percent of the nearly 300,000 cervical cancer deaths globally each year occur in LMICs. But testing hundreds of millions of women in low-resource settings could prove costly and time consuming, because using currently available technologies it would require labs with complex equipment and skilled personnel, and clinicians to collect samples.
Globally, as of 2015 there were 193 distinct HPV tests commercially available for cervical cancer screening, mostly using PCR-based detection.
But Wang said Atila is the only company with an isothermal assay for HPV detection, a differentiating factor he attributes to the firm's ability to do multiplex isothermal amplification combined with real-time detection.
The particular isothermal technology Atila is using is proprietary, he said, but he noted that other isothermal methods, like loop-mediated amplification (LAMP), recombinase polymerase amplification (RPA), or helicase-dependent amplification (HDA), are challenging to multiplex beyond two or three targets and can also have issues with primer-dimers because of the low temperature of the amplification.
Competitive HPV screening and genotyping market
The US Food and Drug Administration has cleared the Qiagen Digene Hybrid Capture 2, Roche Cobas HPV, Hologic Aptima HPV, and BD Onclarity HPV. Similar tests, like the Cepheid Xpert HPV and Abbott RealTime High Risk HPV, are also used internationally and for research purposes.
The Qiagen CareHPV test is frequently used in LMICs, Wang said, particularly in rural areas of India and China. The assay is WHO Prequalified and its development was supported by $13 million in funding from the Bill and Melinda Gates Foundation.
But CareHPV requires samples be collected into Qiagen's liquid specimen transport medium (STM), and each run takes nearly three hours with one hour of hands on time, Wang noted. The test is also typically done in 80-sample batches to save costs. Furthermore, it cannot identify the detected high-risk subtypes, and it is not a nucleic acid amplification-based test — rather, it uses full genome RNA probes complementary to the HPV DNA, specific antibodies, and chemiluminescent detection — so it has slightly lower sensitivity, Wang also said.
There are also a handful of HPV assays that can perform full genotyping and detect and distinguish many more of the high-risk HPV subtypes. These are typically used for research purposes and include tests like the Roche Linear Array, which is an industry standard test that detects 37 HPV subtypes and has been widely evaluated in clinical and epidemiological studies.
A test kit from GeneFirst is also a single-tube multiplex assay that detects and distinguishes 14 high-risk subtypes using standard qPCR instruments. A test from Zeesan Biotech detects and distinguishes all 14 high-risk HPV types using a single-tube multiplexed PCR with endpoint multicolor melt-curve analysis and the firm's own real-time PCR instrument called the SLAN-96.
A sequencing-based assay developed by the National Cancer Institute and described this year can detect all 51 high-risk and low-risk subtypes. And a next generation sequencing-based test from BGI Genomics called HPVseq is being incorporated into screening programs in China, and the firm also has a product that has been validated for self-collected patient samples.
Enabling more self-collected sample testing
Sample preparation is a key bottleneck in HPV testing. Because extraction efficiency can vary, most systems need to use automated methods to maintain sensitivity, and this increases the cost of the capital equipment, Wang said.
The sample prep for the Atila test, on the other hand, involves placing a dry sample swab in a lysis buffer and shaking it, followed by adding primer mix and master mix, which any technician can typically do without training.
Efficiently detecting HPV in samples that patients collected themselves at home is somewhat dependent on using a test that performs amplification. This is because there may be a lower concentration of virus in vaginal samples, which is what a patient typically collects, compared to endocervical samples that can be collected by clinicians.
Self-collected samples can be a benefit for screening programs, as they enable testing of women who would otherwise avoid being examined. And, in one study of patients in Papua New Guinea, for example, using this type of screening and the Cepheid GeneXpert, self-collection produced results similar to physician-collected samples.
Typically, vaginal and endocervical samples must be collected into a liquid transport medium containing methanol, a flammable and hazardous substance. This is in part a vestige of pathology co-testing in which a sample is collected into liquid-based cytology medium, typically Becton Dickinson's SurePath or Hologic's ThinPrep PreservCyt solutions.
But because the Atila test can be run from dry swab samples, the sample can safely be collected by the patient at home, and the dried collection brush can be simply placed in the mail, Wang said.
Jerome Belinson, a specialist in gynecological cancer who began evaluating the Atila AmpFire HPV test about a year ago, confirmed in an interview that Atila's extraction-free workflow enables testing directly from raw samples, a feat he had been trying for years to perfect.
In addition to serving as a professor of surgery at the Cleveland Clinic and president of Preventive Oncology Interational — a non-profit organization working to provide humanitarian care and to help develop new cancer screening technologies — Belinson has devoted efforts to bringing HPV testing to LMICs. He had previously developed a method to ship dried specimens by applying them to a card, and his group had also evaluated at-home self-collection of samples into mouthwash, "an acceptable nontoxic preservation medium for HPV DNA testing," according to one published study.
But, he said, other technologies in development had issues when the flora in a raw sample would interfere with test results. With the Atila technology, a case of bacterial overgrowth "doesn't affect this at all," he said.
Belinson said he had been "focusing on screening for 22 years, all around the world, and there is nothing that compares to this assay,” he said.
Currently, his team is collaborating with researchers in China using the BGI next-generation sequencing-based HPV assay for genotyping. They recently used the BGI test to evaluate 188,000 patients in 29 days using self-collected samples, he said, and the group recently presented data at the American Society for Colposcopy and Cervical Pathology annual meeting describing 10,885 patients in which the BGI SeqHPV test was compared to the Cobas 4800 HPV test for both self- and direct-collected liquid specimens, showing the two methods have similar sensitivities and specificities for diagnosing a pre-cancerous level of cervical dysplasia known as CIN 2+.
The BGI testing is great, Belinson said, but to his mind it may be limited because it can essentially only be run at BGI. "It is great for nation-wide screening in China, but I can't see it applied virtually anywhere else right now," he said.
On the other hand, Belinson said he is aware that Atila has recently demonstrated its technology at places like the US Centers for Disease Control and Prevention, and that it has also been evaluated by other HPV experts.
"Everybody is crazy about it," he said, in part because it is "very different than anything else" and "extraordinarily simple." The system solves many of the problems currently plaguing researchers who want to quickly do HPV genotyping in a cost-effective way on large groups of people in places without sophisticated labs, Belinson said.
Other early users
He has used the AmpFire HPV genotyping system, specifically to genotype anal specimens for research purposes at sites such as the Rwanda Military Hospital in Kigali, Rwanda, and the University of Zimbabwe College of Health Sciences.
In an interview, Palefsky said he and his team chose the AmpFire system over others because it provides full genotyping and is more cost-effective compared to other HPV genotyping methods.
It is "relatively inexpensive, easy to do, pretty reliable, and it's quick," he added.
The group has also set up the system in labs close to the patient populations being studied. "We are going to be starting a new project in Latin America where our hope is to actually place the machine in clinics to provide real-time information on patients," he said, with the potential of providing same-day results.
Palefsky and his team collects sample into liquid medium so they can also do cytology. "If that isn't needed, then I think [Atila's] dried swab approach is a good one because it is simpler and less costly for shipping," Palefsky said. In contrast, the Roche Linear Array genotyping test is "too expensive for routine use," he said.
Palefsky became aware of the Atila system at a conference and approached Wang. "It's a small company with a very big agenda," he noted.
Xavier Bosch and his team at the Cancer Epidemiology Research Program of the Catalan Institute of Oncology, initially established a collaboration with Atilla about two years ago.
The lab chief in Bosch's lab, Miquel Pavón Ribas, said in an email that cost and complexity of other tests make them difficult to implement in LMICs, which is a challenge in the face of the WHO call for the elimination of cervical cancer.
"It is essential that we have more options for the detection of HPV," he said, and noted that the AmpFire test uses a simple protocol and has a lower price.
At the Catalan Institute of Oncology's Infection and Cancer Laboratory, Ribas uses HPV tests for cervical cancer screening, diagnosis of HPV-associated tumors, clinical care, and epidemiological research
"We use different technologies accordingly on their characteristics, cost, or genotypes that we need to detect," he said, and added, "Our experience using the Atila system shows that this technology could be a good choice for laboratories or clinical departments with basic or limited infrastructures." The system could be "easily implemented" in LMICs, but also could be used in decentralized settings for testing clost to the target population, Ribas also said.
The lab has continued working to validate the use of the technology in other contexts beyond cervical cancer screening and cytology sample analysis, he said, including using it on formalin-fixed paraffin-embedded samples from oropharyngeal tumors.
"In this context it is particularly valuable to have a technology to analyze a low number of tumors per run but using a quick method, as the result can be used to help clinical decision making," he said.
Atila has also been working with other groups to validate and disseminate its HPV testing technology. The AmpFire system is under evaluation at places like the Stem Cell and Cancer Institute in Jakarta Timur, Indonesia and the Addis Ababa University College of Health Sciences in Ethiopia.
And, at Battor Catholic Hospital in the North Tongu district of Ghana’s Volta region, the AmpFire instrument was donated in June to the Cervical Cancer Prevention and Training Centre by Okudzeto Ablakwa, a local member of parliament.
This has attracted a lot of press attention in the region, Wang said, with news reports also incidentally promoting the same-day screening and self-sampling for cervical cancer screening that are enabled by the test system. Ablakwa covered the $16,000 cost of the AmpFire instrument, which meant the hospital did not have to transfer the capital costs to women being screened. This brought the cost women must pay for screening down from the equivalent of approximately $28 to about $19.
And, since the assay is isothermal and runs on what is essentially a basic qPCR thermal cycler instrument with five fluorescence channels, it can also run other testing.
Belinson noted that the ability to run other types of sexually transmitted infection testing, in addition to HPV, is particularly useful in LMICs. For example, Atilla sells a single-tube isothermal multiplex test for four STI pathogens — Chlamydia trachomatis, Neisseria gonorrhea, Trichomonas vaginalis, and Mycoplasma genitalium.
The AmpFire instrument is also portable, insofar as it can be moved on a cart and does not require recalibration once it is moved, Wang said. The firm also has developed a four-channel isothermal instrument with 96 wells, but has not begun manufacturing it yet, he said.
The HPV test is the most mature product, Wang said. It has been CE marked and approved by the China Food and Drug Administration. "We've sold more than 1 million tests in China already," he said.
Additionally, the firm has developed a number of other assays and kits, including a multiplexed non-invasive prenatal test which Wang said is the first of its kind.
There are no published peer-reviewed descriptions of Atila's technology or clinical evaluations of the AmpFire HPV assays to date, only a few poster abstracts, and ulitmately prospective clinical trials will be needed to know whether it is actually useful in real-world clinical settings.
Belinson has run a retrospective study using 2,000 patient samples from a village in China. The samples were originally collected as part of trial in which patients had Roche Cobas HPV testing along with cytology, BGI's SeqHPV test, and provided self-collected as well as clinician-collected samples, he said.
"Those 2,000 patients essentially gave us 4,000 samples — we tested them all with the AmpFire and it performed extremely well," Belinson said. However, to increase the number of positive samples, he and his colleagues in China are now testing an additional 8,000 samples collected from patients in two other villages. If the results come back and show the test is as accurate as he hypothesizes it is, Belinson said he will definitely adopt the test. "I will use it as much as I can," he said, noting that he plans to publish the results of the study soon.
Going forward, Wang is considering ways to get non-governmental organization funding to further the development of the system. This month he will be meeting with PATH — a global health organization that helped develop CareHPV and has an ongoing scale-up project to expand access to HPV DNA testing, particularly in Central America. He is also having conversations with researchers at NCI, and the company has applied for funding through the Johns Hopkins Point of Care Technology Research Networkto further develop its STI testing.