NEW YORK – A new sample preparation approach developed by Atila BioSystems could potentially enable affordable, easy-to-use, and high-throughput global infectious disease testing.
Atila's Biodome technology is a novel hydrophobic gel matrix that sequesters liquid PCR master mix, primers, and enzymes within a single reaction tube so that they don't mix during shipping yet melt during thermocycling and float to the surface of the reaction.
The tech has been adopted by a team based in the Feinberg School of Medicine at Northwestern University and is now helping global health researchers broaden their screening and cancer prevention programs in low- and middle-income countries while also reducing expert staffing and downtime caused by contamination.
"It really is a game changer," said Mamoudou Maiga, director of translational research for Northwestern's Center for Innovation in Global Health Technologies (CIGHT), who has been using Biodome for human papillomavirus screening in Mali and Nigeria. "It's a very simple thing that solves big issues," he said.
Contamination is a problem for any clinical microbiology lab since each PCR reaction generates millions of amplicons that can potentially interfere with subsequent runs of extremely sensitive molecular tests.
"It makes the space very dirty quickly, and there is no way to avoid that," Maiga explained. Labs typically have separate clean lab and dirty lab spaces to try to prevent contamination, but contamination looms at each step in the pre-amplification workflow. Lyophilizing nucleic acid amplification reagents, meanwhile, can help increase their shelf life and allow for room-temperature shipping but requires clean, nuclease-free water for rehydration.
Biodome hermetically seals reagents, however, so they can be shipped at room temperature while technicians need only pipette their sample on top prior to testing.
The semisolid gel matrix melts above 45 degrees Celsius, and because it is hydrophobic, it then floats to the surface to mix the reagents as thermal cycling begins. It also solidifies on the surface at the end of PCR, re-sealing the reaction and preventing post-amplification contamination.
Lifang Hou, the director of the Center for Global Oncology at Northwestern's Institute for Global Health, said troubleshooting lab contamination is a costly, time-consuming drain on reagents and resources. It also pauses study recruitment, which is particularly painful when one is trying to catch infections that can cause fatal cancers.
Maiga, Hou, and their global health colleagues used assays from Cepheid in research published last year, showing that for a sample of 96 women living with HIV, 42 percent were also infected with high-risk HPV, a prevalence generally consistent across sub-Saharan Africa. It is now known that almost 99 percent of cervical cancer cases are linked to HPV infection, Maiga said, and "without HPV, there is no cervical cancer."
Unlike lower-throughput methods that prevent contamination through the use of cartridges, the Atila HPV testing approach enables high-throughput testing at a lower cost.
The Biodome tech is available in tube strips or a plate-based format and can be used with Atila's AmpFire and ScreenFire diagnostics systems though it is essentially instrument-agnostic, Maiga and Hou said, and they have tested it with four other thermal cyclers.
HPV screening and beyond
Awarded nearly $2 million to date by the National Cancer Institute for studies of HPV screening and self-sampling in western Africa, Maiga and Hou have found that allowing people to collect their own vaginal swabs overcomes many cultural and religious barriers to gynecological exams.
A single technician can collect swabs from 100 women in five minutes, Maiga noted, and the 96-well Atila test format facilitates more than 1,000 tests per day. This allows technicians to triage the 20 percent or so of women who have high-risk HPV for additional care while sending the rest home.
High-risk HPV-positive women require visual inspection of the cervix (VIA), Hou said, which demands clinician time and a private exam room. "You can imagine if you use Atila's testing to understand who is in need of VIA, how much public and clinical resources will be saved," she said.
Lives will also be saved, she suggested, and the fact that HPV is now treatable makes ongoing high cervical cancer deaths rates globally an issue of health equity and justice. "That is why we are so devoted to this work," Hou said.
The team plans to port its HPV screening efforts to mobile clinics using two Atila systems and the Biodome technology. The method isn't just for HPV, Hou said, adding her team is using it for malaria testing in Mali and will soon use it for tuberculosis testing.
The team runs a training program for clinicians in Mali and Nigeria, as well as a network of HPV cervical cancer programs in seven countries in Africa, including Burkina Faso, Senegal, Guinea, Nigeria, Tanzania, Rwanda, and Kenya, Maiga said. In these programs, too, Maiga and his colleagues are expanding their portfolio to diseases beyond HPV using the Atila system.
Populations in the region, "want to do affordable, scalable molecular testing in their own country, not sending tests to Europe," Hou said.
Maiga and Hou recently validated the Biodome tech on samples from Mali and Nigeria processed at their labs at Northwestern, with results reported in an Infectious Agents and Cancer study published late last year. Specifically, the team compared the Biodome format in Atila's isothermal ScreenFire HPV RS assay to the results of using the ScreenFire HPV RS assay standard format on Bio-Rad CFX96 real-time PCR instrument.
ScreenFire HPV RS employs Atila's proprietary isothermal amplification and 96-well format to detect high-risk HPV with a sensitivity of approximately 95 percent, as previously reported.
Specifically, the test uses four fluorescent detection channels with sensitivity hierarchically tailored to cancer risk, such that the most sensitive component assays detect HPV16 and HPV18/45, while less sensitive assays detect HPV strains 31/33/35/52/58 and HPV strains 39/51/56/59/68.
However, without the Biodome sample prep tech, ScreenFire HPV RS still requires manual preparation of master mix reagents, the team wrote in the study.
For the HPV RS test, Atila developed what it calls Zebra Biodome — a preloaded format that stacks and separates the reagents needed for its proprietary tests.
In the study, the Northwestern team set up a Bio-Rad Laboratories CFX96 real-time PCR machine with a test program of one minute per cycle at 60 C for 60 cycles. They found the HPV screening workflow using Zebra Biodome had approximately 98 percent agreement with the standard workflow but reduced the number of pipetting steps from three to one.
In an assessment of 173 banked samples of extracted nucleic acids, only seven samples showed inconsistent HPV genotyping results between the two tests. These were later found to have viral loads near the limit of detection, and increasing the sample volume from 5 microliters to 10 microliters led to complete agreement between the two tests.
In addition, the Northwestern team validated the Biodome tech in 46 crude samples that had been collected previously. They added vaginal swabs to lysis buffer before pipetting into the assays, finding 100 percent agreement between Biodome and the standard format.
Other work published in the Journal of Clinical Microbiology in December by researchers based at the National Cancer Institute and in Nigeria showed similar agreement between Biodome and the standard Atila formats. This validation study built upon prior work comparing Atila's HPV testing approach to the Cepheid GeneXpert, as well, showing a $6 per test cost compared to $15 per test, and noting that the technology can have broad applications for other DNA amplification assays beyond HPV.
Atila's plans
Youxiang Wang, CEO and founder of Atila, said in an interview that the company spent about two years developing Biodome and now hopes to sell preloaded Zebra Biodome assays going forward as well as offer a service for assay developers to port their tests to the universal Biodome format.
"My vision is that this format will replace lyophilization," Wang said, a process that he noted is tedious and impacts production capacities and timelines. The addition of Biodome matrix to tubes or plates is automated, he said, and only adds a few cents to the cost of an assay.
Atila also hopes to partner with larger companies to make the Biodome technology more widely available in their consumables, he said, so "then they never need to worry about lab contamination."
Atila was recently granted breakthrough device designation by the US Food and Drug Administration to advance its use of the ScreenFire HPV RS assay in high-grade squamous intraepithelial lesion testing of anal specimens and anal cancer screening for HIV-positive individuals over 35 years of age.
The tech may also help meet global goals to reduce cancer rates in LMICs. With a low throughput, "point-of-care testing is never going to finish screening 70 percent of the women in the world," Wang said, adding that cartridge-based tests prevent contamination but are typically more costly than lab-based testing. Yet, throughput using Atila's approach is also modifiable, he said, as users can trim down tube strips to run just two samples if needed.
"This innovation is going to change the field," Wang said.