NEW YORK (GenomeWeb) – Rockville, Md.-based diagnostics firm Tetracore has been awarded a one-year grant from the National Institute of Allergy and Infectious Diseases to develop a point-of-care, real-time reverse-transcriptase PCR assay that detects and distinguishes dengue and chikungunya viruses.
The new assay will run using a direct-to-test sample collection system and a miniaturized PCR platform the firm has also developed. The company intends to seek US Food and Drug Administration approval and CLIA waiver for the assay and platform combination, and is concurrently developing a broader menu of molecular diagnostic tests for point-of-care use.
A small, privately-held company, Tetracore currently markets lateral flow immunoassays for biothreats, including an anthrax test that was cleared by the FDA in 2003. Although it manufactures RT-qPCR assays for veterinary and biodefense applications, a medical test on the new platform would mark its first entry in the commercial human molecular diagnostics market.
Dengue and chikungunya are mosquito-borne arboviruses with RNA-based genomes, and infections present with very similar symptoms. They are also both endemic to tropical regions, and feared to be moving into more temperate areas, like the continental US, due to climate change. The first cases of local chikungunya transmission in the US were reported last summer, and health authorities are bracing for a wider spread, according to a report in the Journal of the American Medical Association.
Tetracore's newly funded assay is based on a dry-format, field-deployable dengue test the company developed in collaboration with the Naval Medical Research Center, Bill Nelson, President and CEO of Tetracore and a retired US Naval Commander told GenomeWeb in an interview this week.
The former assay was about 99 percent sensitive and 100 percent specific in a small study of patient samples, and was designed to run on the Cepheid SmartCycler platform.
The new assay "is mulitplexed in multiple ways," Nelson said. Dengue detection is accomplished with multiplexed RT-qPCR to detect all serotypes of the virus. Chikungunya, however, is an emerging pathogen, so viral detection is multiplexed to pick up many genomic regions in case the virus mutates. The one-year, $169,000 grant is phase one, and the firm will then use the data to seek phase two funding.
The test is now up and running in the lab, and takes about one hour, Nelson said. It will be amenable to point-of-care use because it runs on a PCR platform the firm has developed in house, he said.
Called the T-Cor 8, the platform is a miniaturized thermocycler that meets many of the requirements for portable thermocyclers described by Nelson and his Tetracore colleagues in a review published in 2013 in Chemical Society Reviews.
Namely, the T-Cor 8 is battery- and touchscreen operated, weighs less than 4.5 kilograms, and is "cloud ready." It can be used in a very simple mode by technical staff, but granular data on cycle thresholds and curves can also be accessed remotely, in real time. The latter might be particularly useful for POC molecular testing in rural or low-resource settings.
Nelson said he personally monitors instruments in his lab while he is out of the country, so, for example, "an administrator for a set of hospitals or labs could be in a central location and have access to every machine." There is no software required to connect with the instrument; "You just have to pull it up on your browser," he said. Users can also download data reports via a USB port on the instrument.
Importantly, Nelson said the T-Cor 8 runs eight samples at a time, but can run them as eight separate PCR reactions, where each unit in the block is set to different thermocyling programs. The platform currently has four-color capabilities, is equipped to run any standard PCR test, and is both barcode-driven and programmable.
The platform uses fluorescent and real-time TaqMan probe chemistries. "It does many of the fluorescent isothermal assays as well," Nelson noted, adding that the firm has run TwistDx recombinase polymerase amplification assays on it, for example. It can also run both RNA and DNA assays.
The four channels on the T-Cor 8 are common to other laboratory-based thermocyclers, so users can port assays "without a significant effort," Nelson said. "If you ran an assay on the ABI 7500 – which is one of the main instruments used both in clinical labs and in veterinary labs – you could put that assay on [the T-Cor 8] and it would run exactly the same," he said.
"We picture this instrument both as a point-of-care and a laboratory-based instrument" Nelson said. Individually programmable wells could be useful to clinical labs that usually do batch processing but may need to run one-off PCRs, as well. "It is a stand-alone instrument that can be utilized by the laboratory as needed, well by well," he said, explaining that "each one of the fluorimeters is in essence a separate instrument."
The firm's initial foray into instrument development came from its biodefense work and interest on the government's part, Nelson said. A previous incarnation of the platform, the T-Cor 4, was a "much more hardened, biodefense-related instrument" that ran two colors and had four wells, he said.
For the T-Cor 8, the firm funded development internally, and finished a small production run for validation testing about three months ago. "We're now in low-level production," said Nelson.
Tetracore is currently negotiating distribution agreements with an undisclosed party "on the human side," he said.
Its goal is to pursue FDA approval and CLIA waiver for this platform to enable its use in physicians' offices and pharmacy-based "minute clinics," Nelson said.
This development pathway is much like Alere's new molecular platform, he noted, which was recently cleared by the FDA. Earlier this month, the Alere i isothermal platform became the first point-of-care nucleic acid amplification assay ever to receive CLIA waiver, which it did for a 15-minute molecular influenza test that may soon appear in CVS clinics and other low-complexity settings, as reported by GenomeWeb.
In order to enable a turnaround time that fits within a typical patient encounter, Nelson said Tetracore's new tests will also use the firms sample preparation system, called Collect to Test, or C2T.
The C2T system includes a simple blood collection and processing method, requires no pipetting or sample manipulation, and is combined with a cartridge containing the dried-down reagents for simultaneous sample preparation and template amplification, according to the new grant's abstract.
The firm has spent the last year or so demonstrating to other companies that the T-Cor 8 platform can run their tests, Nelson said. In house, it is also developing an expanded menu of molecular diagnostics that will rely on the C2T sample prep and T-Cor 8 thermocycling.
Specifically, the company has developed a Group A Strep assay that runs in less than half an hour, Nelson said, and will likely be the first the firm takes through the FDA process.
Tetracore is also currently validating an Ebola assay in collaboration with the Department of Health and Human Services, and it is developing a combined influenza A/B and respiratory syncytial virus assay. Although the latter might bring Tetracore into a crowded flu market, Nelson said a partnership could help in taking on that challenge.
The firm developed the T-Cor 8 using in-house engineers and software experts, and its own funds. Aspects like injection molding are contracted out, and "we have hired consultants for some of the trickier things," said Nelson, but otherwise this process has been internal.
Units from the current low-level production of the T-Cor 8 currently cost around $28,000, Nelson said, which is on par with lab-based instruments, and that price could decrease in the future.
Other miniaturized PCR platforms recently described by GenomeWeb include Amplyus' mini-PCR, Chai Biotechnologies' Open qPCR, and Ubiquitome's Freedom4. The first two are being marketed for educational and home use, although the mini-PCR is also used in research labs. The Freedom4 is field-operable and has basic biology applications, but can also run molecular diagnostics, as evidenced by that company's pursuing Emergency Use Authorization for an Ebola test.
These thermocyclers heat and cool samples using a single block, Nelson explained. While Amplyus' device is made miniature via special thin-film heating and convection cooling, Nelson said that the T-Cor 8 is Peltier driven.
There are also other firms with sample collection and preparation systems similar to the C2T. DxTerity, for example, recently launched DxCollect, a finger-stick device containing RNA stabilization buffer.
Meanwhile, in the US, Focus Diagnostics, a Quest Diagnostics subsidiary, is currently the only firm that has a molecular test for chikungunya, and that company's Simplexa Dengue test was recently validated in an independent study, as reported by GenomeWeb. It is currently developing a one-hour chikungunya assay on the Simplexa platform.