By Ben Butkus
Akonni Biosystems said this week that it has been awarded a $435,000 grant from the National Institutes of Health to begin developing point-of-care diagnostics for drug-resistant tuberculosis strains based on the company's gel-drop PCR microarray biochip technology.
In addition, Akonni is developing tests based on the biochip technology for a number of other infectious agents, and is designing and testing an integrated instrument platform to perform the tests, with the ultimate goal of seeking US regulatory clearance, company officials said this week.
The most advanced of Akonni's diagnostics is a methicillin-resistant Staphylococcus aureus test, for which the company is currently working with several academic collaborators to gather preclinical data and ready a filing with the US Food and Drug Administration for 510(k) clearance, officials added. The company declined to provide a timeline for submission.
"The strategy is that we necessarily have to go to FDA with the assay and an instrument," Darrell Chandler, Akonni's chief scientific officer, told PCR Insider this week. "We cannot decouple them."
As such, each new infectious disease diagnostic project the company tackles for its gel-drop PCR microarray technology, called TruDrop, is following the same course: development and validation of a viable multiplex assay followed by adapting that assay to the testing platform, which the company calls TruDiagnosis.
In December, Akonni won a $3.2 million Phase II Small Business Innovation Research grant from the National Institute of Allergy and Infectious Diseases to develop a test using the TruDrop technology to detect influenzas A and B and their antigenic subtypes, including antiviral-resistant types (PCR Insider, 12/3/2009).
The TruDrop technology, which is based primarily on patents exclusively licensed for diagnostic purposes from Argonne National Laboratory, is a low-density microarray of immobilized three-dimensional 100-by-20-micron drops containing all probes and chemistry necessary for a PCR reaction.
"The TB proposal, and actually the flu proposal as well, is based on doing amplification in an array chamber, followed by a pipette wash step," Chris Cooney, director of engineering, product design, and development at Akonni and principal investigator on the TB grant, told PCR Insider. "So the whole thing is on a valveless biochip [with] a waste chamber and an array chamber … so just the action of adding wash to the chip advances everything into the waste chamber. It allows a somewhat quasi-dry read after the amplification is done.
In parallel with these products, "we're also developing a reader that is based on imaging the arrays," Cooney said. "This imaging is specific for the gel drops because they have higher signal-to-noise ratios and more probes can be packed in per gel drop."
One goal of the flu project, set to run through June 2012, was for Akonni to have prototype instruments and assays ready in the spring of this year to test with collaborators at the Wadsworth Center, Columbia University, Little Company of Mary Hospital, and the US Centers for Disease Control and Prevention.
This week, Chandler said that Akonni is on track with those plans.
"In year one, we spent a lot of time developing a new sample-preparation product called TruTip," Chandler said. "We've got some great data for that and it works well with viruses in clinical samples."
TruTip uses a patented nucleic acid-binding matrix inserted in a pipette tip to extract and purify inhibitor-free DNA or RNA for downstream PCR applications in as few as four minutes, according to the company. For higher-throughput applications, such as preparing samples for analysis on the TruDiagnosis biochips, TruTip can be paired with Eppendorf’s automated epMotion 5070 liquid handling system. Akonni and Eppendorf inked a co-marketing agreement around the technologies in April (PCR Insider, 4/29/10).
Akonni has also developed a prototype TruDrop influenza assay; "and then we've prototyped some of the instruments required to image the array — but the instrument-development effort is really aimed at … integration," Chandler said.
Akonni's goal for the second year of the flu grant is to transfer "all the chemistry, reagents, and tests we developed in year one to our collaborators to do some preclinical testing in real live environments rather than our labs," Chandler said. "And in the meantime Akonni is taking those assays and porting them over to the PCR array platform."
Akonni is working with an undisclosed engineering firm with experience developing products that meet FDA standards to develop its TruDiagnosis system. The platform has "a microfluidic piece that combines the PCR and microarray into one chamber … and another piece that has to do with the thermal cycling around that," Chandler said. "We've got some prototypes that, in principle, provide much more efficient and effective heat transfer and thermal cycling in a microarray type format, and we'll be working on that instrument and prototypes to test that hypothesis specifically."
Assuming the system works as expected, Akonni's flu and TB projects will take on "more of an instrument-development flavor … to bring the PCR array and associated equipment forward for deployment back to our collaborators in year three for another round of preclinical testing and evaluation," he said.
Akonni's test for MRSA, begun before either the TB or influenza projects, "is certainly the most advanced," Chandler said. "That's one of the reasons that the imager is now in true design verification with a qualified IVD engineering firm and manufacturer. Akonni is working on all of the design verification, validation, manufacturing, and infrastructure for the assay to test the microarray portion."
Meantime, "the flu project is going to be drafting behind that," he added. "And we hope to get enough clinical data to support a decision to take it into an FDA trial and clearance."
Under its latest SBIR grant, administered through the National Institute of Biomedical Imaging and Bioengineering, Akonni is working with researchers from Mexico's Laboratorios Medicos Especializados to evaluate the TruTip technology for purifying Mycobaterium tuberculosis samples, and to verify assays run on the PCR biochip.
In parallel, Akonni will expand the multiplexing capacity of the TB arrays, develop a lysis method, and translate the assay to the prototype TruDiagnosis device.
Phase II of the grant aims to further expand the genotyping capacity of the assays and to translate the point-of-care devices to the Laboratorios Medicos Especializados clinic in Juarez, according to the grant's abstract. Akonni has proposed a final test with a consumable cost of $3 operated on a $5,000 instrument.
While the TB project helps secure more funding for Akonni to fully develop its platform for all the diagnostic tests it is developing, it also represents a good fit for the TruDrop technology and is a legacy project from a cadre of Akonni researchers.
"There is a lot of history behind the TB genotyping chip," Cooney said. "Four of our scientists from Russia who [worked at] Argonne and are now with Akonni have been working on TB. In addition, one of the challenges with monitoring TB is these multi-drug-resistant and extreme-resistant strains that are becoming more prevalent and that are hard to detect with real-time assays with low multiplexing."
Lastly, Cooney said, "with this particular project NIH was interested in addressing health disparities. It turns out that TB represents one of the largest disparities between white and minority populations."