This story has been updated to include addtional comments on limits of detection.
NEW YORK – An assay to detect COVID-19 from Atila Biosystems that was granted US Emergency Use Authorization this week is getting positive reviews from early evaluators.
The moderate-throughput test uses a proprietary isothermal multiplex real-time reverse-transcriptase PCR technology to detect SARS-CoV-2 virus directly from dried swabs in less than 90 minutes. It is supplied with the necessary swabs, and does not require a separate extraction kit, so it could be particularly useful for labs experiencing supply shortages. It is also plate-based and can process up to 94 samples per run.
Evaluations of Atila's EUA iAMP COVID-19 Detection Kit are underway in labs at the Medical College of Wisconsin and Dartmouth-Hitchcock Medical Center, and preliminary results suggest the test could help to quickly expand testing capacity.
In limit of detection studies performed for the US Food and Drug Administration EUA, the iAMP COVID-19 detection kit was fairly sensitive — with a reported LOD of 4 viral copies per microliter, or 4,000 per milliliter.
Founder and CEO of Atila, Youxiang Wang, noted however that the Atila test is likely to be much more sensitive in practice than the LOD reported in the EUA documents might indicate. In part, this is because the sample the test uses is a dried swab that is placed directly into a small volume of lysis buffer, as opposed to a fresh sample placed in transport media, which most tests use. Essentially, the Atila workflow means more copies of the virus will be available to start the amplification reaction, which increases sensitivity.
By comparison, the Abbott RealTime SARS-CoV-2 assay on the lab-based m2000 system has a reported LOD of about 100 viral copies per milliliter, while the point-of-care Abbott ID Now test is perhaps slightly less sensitive with an LOD of 125 genome equivalents per milliliter.
And, the US Centers for Disease Control and Prevention assay N1 and N2 targets have LODs of 1,000 and 300 RNA copies per milliliter, respectively, while the BioFire COVID-19 Test LOD is 330 viral copies per milliliter.
Wang said in an interview that the iAMP test has also been evaluated with clinical samples, unlike many other EUA tests. Through a connection in China the firm was able to evaluate the iAMP on 890 negative and 125 positive clinical samples there. It found 100 percent agreement with COVID-19 diagnosis compared to a China FDA-approved PCR kit from DaAn Gene coupled with clinical diagnostic criteria.
Wang said that Mountain View, California-based Atila has the manufacturing capacity to make approximately 100,000 tests per week. This is due in part to the fact that the firm's proprietary isothermal method does not compete for the same reagents and sample collection supplies that other qPCR tests need, he said.
It is also a kit, rather than a cartridge-based test, so it may be somewhat easier to make in large volumes. Integrated DNA Technologies is the supplier of the primers and probes in the kit to detect the nucleocapsid (N) gene and the ORF-1ab gene, but Wang declined to name the supplier of the enzymes used for the isothermal multiplex RT-qPCR, except to say it is a US-based company. Manufacturing could be scaled up by placing larger orders of reagents if demand should warrant it, Wang suggested.
Most rapid EUA COVID-19 tests are intended for the point of care and process one or a few samples at a time in each testing instrument. The Atila iAMP uses 96-well plates, with two wells taken up by controls, so it has significantly higher throughput.
The iAMP assay takes between one hour and 90 minutes to run, and it does not require other equipment for extraction, Wang emphasized. The test set up involves placing a dried sample swab in lysis buffer, vortexing the tube, and waiting 15 minutes. Then, it is ready to be aliquoted into the plate and go on the qPCR instrument for approximately 50 minutes.
Like the firm's human papillomavirus testing, the COVID-19 test was designed to be easy to use. "Any person that knows how to do pipetting will know how to run our assay," Wang said.
Although the firm also makes assays that run on its AmpFire system, the iAMP COVID-19 kit was granted EUA to run on the Bio-Rad CFX96 Real-Time PCR System. The firm is planning to file an amendment with the FDA to add other validated qPCR equipment in the near future, Wang said, as the test can be run on any qPCR instrument with two-color fluorescence detection capabilities.
Atila has also written software to report the results from the Bio-Rad instrument, so users just "put the plate in the equipment, and the results come out," Wang said. The firm is working on writing report-generating software for the other instruments it is validating as well.
The iAMP kit is also reportedly low cost, but Wang declined to disclose the exact cost of the firm's test.
In-process iAMP validations
The Atila technology was recently evaluated for HPV testing in a study published in the Journal of Molecular Diagnostics, as previously reported, as well as in a more recent Journal of Virological Methods study.
For the iAMP COVID-19 test, preliminary results from end users appear to support the claims Atila has made.
Juan Felix, director of surgical pathology at the Medical College of Wisconsin, has been validating Atila's low-cost HPV assay a little more than a year.
"When the pandemic became evident, [Atila] contacted me informing me that they had a SARS-CoV-2 assay. I immediately asked if we could validate it," Felix said in an email, noting that the Atila technology in general has advantages in terms of simplicity, speed, and low cost.
The lab is currently validating the Atila iAMP COVID-19 assay and plans to incorporate it into the clinical workflow. The lab has contracts with several other vendors for minimum test volumes that it is not currently exceeding, Felix said, but he added that with anticipated increased testing requirements he expects the lab will ultimately be using the Atila test as well.
Felix highlighted that dry swab collection and the removal of the requirement for a separate extraction kit saves time and reagents. However, one potential disadvantage might be that the current workflows in hospitals tend to involve collecting samples into viral transport media, so there could be an extra step required in the lab to separate the sample, which could add about one hour to the turnaround time.
The MCW lab is now performing a non-inferiority clinical trial to assess iAMP more carefully, likely using hundreds of patient samples with the CDC test as comparator, Felix said.
So far, "We find the test to be exquisitely sensitive, rivaling the CDC approved test," he said. "This test would be our preferred test, because of its speed, accuracy, and low cost, however, prior contractual agreements will make it an overflow test in our lab."
Feedback from an ongoing clinical lab evaluation at Dartmouth-Hitchcock Medical Center has been similarly positive.
Joel Lefferts, the assistant director of molecular pathology at Dartmouth-Hitchcock, is validating the Atila iAMP now.
"We have gotten some very promising results using residual clinical specimens collected in transport media, but this assay is designed to work with dry swabs," Lefferts said in an email.
The lab is in the process of collecting dry swabs to confirm the assay's performance. The positivity rate for COVID-19 infection is still relatively low in New Hampshire, he said, "so it may take some time for us to collect enough of these specimens." The lab plans to run tens of actual or contrived specimens to validate the test, but its initial work on four positive and four negative specimens in transport media showed 100 percent concordance.
The high-throughput assay that the Dartmouth lab currently uses requires batch testing, with a total test time of between five and seven hours. So, the typical turnaround time for COVID-19 testing is 12 to 24 hours, Lefferts said.
"We are eager to have another option for testing that would complement our higher-throughput test. Atila would fit this need nicely," he said, as it is relatively fast and scalable for low- to medium-throughput. One drawback, however, is that it is not automated.
"Without automation, it would be challenging to use this assay exclusively for the Dartmouth-Hitchcock Health System, but I anticipate that it will be a very important component of our overall testing strategy," Lefferts said.
He also anticipates simplicity might mean Atila will not face the manufacturing challenges that have stymied other molecular test makers. "Unfortunately, but understandably, many of the manufacturers of SARS-CoV-2 kits that have obtained EUAs have had difficulties meeting the demand for testing across the country," Lefferts said.
Jerome Belinson, president of Preventive Oncology International and an early adopter of the Atila technology for HPV testing, emphasized in an email that with a single standard qPCR reader a lab could process more than 2,000 suspected COVID-19 samples in 24 hours using the iAMP test. And, the actual lab work only requires basic skills, he said.
The workflow could also provide unique benefits in the current crisis, Belinson said. "Whether manual or automated, most tests require extraction kits, which are back ordered. The iAMP, similar to the [Atila] AmpFire HPV assay, needs only raw sample — add reagent to a dry brush, shake, and put the sample in the qPCR reader," he said.
The test is authorized for nasal, nasopharyngeal, or oropharyngeal swabs samples. There are 100 synthetic fiber swabs supplied in the Atila kit, which are standard swabs used for respiratory specimen collection. But the firm also validated the test with the 9-micron soft nylon brushes that are used for cervical cancer screening, which Belinson noted are widely available. These had improved comfort for patients and yielded higher sensitivity compared to typical respiratory swabs, he said.
In addition to the US, Atila is also supplying its iAMP test to low- and middle-income countries (LMICs), particularly ones that have already adopted its HPV tests and AmpFire system, Wang said.
Labs in Haiti, Zimbabwe, Nigeria, Kenya, and South Africa have already placed orders, he said, and they will be able to run the COVID-19 test on the Atila AmpFire instrument using a very similar protocol.
A non-profit in Pittsburgh, Basic Health International, has helped validate the Atila HPV test in El Salvador and is also reportedly working to expand COVID-19 test capacity there using the iAMP assay. Atila is now pursuing funding from the Biomedical Advanced Research and Development Authority (BARDA) in collaboration with BHI, Wang said.
Furthermore, the World Health Organization is looking at the Atila assay for use in LMICs, he said, and the Foundation for Innovative New Diagnostics has ordered 300 tests for an evaluation study.
For a startup, having a test evaluated head-to-head with more established technologies could be risky, particular with an unprecedented amount of public attention currently on testing. "We're a small company, so we always have people question and challenge us," Wang said.
But, he said is confident in the iAMP technology, and the fact that the test is being used by Dartmouth and MCW, and was also recently evaluated by a lab at Stanford that will be publishing its study soon, reinforces that confidence.
In the past, pursuing an EUA for a diagnostic test for the Ebola or Zika emergency carried a risk of investing a lot of development effort and funds for a low volume of sales. An offsetting factor, besides the humanitarian benefit, was perhaps a leg up in the regulatory process for an instrument or assay in the future.
This outbreak is different, affecting the world on a much larger scale. Wang noted that large test manufacturers are being stretched thin in just supplying tests to big labs, and small labs and LMICs are being left out. That said, early testing and contact tracing may still have the potential of heading off an outbreak in rural places in the US and in countries without extensive healthcare infrastructure.