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Lucigen Enters Phase II of Grant for POC Hemorrhagic Fever Test; Targets C. diff, Pig Pathogen MDx

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NEW YORK (GenomeWeb) — Lucigen has entered phase two of a five-year, $1.7 million, Small Business Innovation Research grant from the National Institute of Allergy and Infectious Diseases to develop a nucleic acid test for three types of viral hemorrhagic fever: Ebola, Lassa, and Marburg.

Based on the firm's OmniAmp polymerase technology, the test will be a single tube reaction with multiplexed lateral flow readout. In an interview with PCR Insider late last week, Hemanth Shenoi, director of business development at Middleton, Wis.-based Lucigen, said that OmniAmp, the firm's patented thermostable polymerase with innate reverse transcriptase activity, is currently available for sale as a research-use-only product.

But Lucigen is taking the core technology — which was discovered in a screen of viral metagenomes obtained from a Yellowstone National Park hot spring known for its diverse populations of thermophillic microbes and thick microbial mats — and developing it specifically for point-of-care tests for animal and human infectious diseases. In addition to the viral hemorrhagic fever test, the company is developing diagnostics for Clostridium difficile and porcine epidemic diarrheal virus, or PEDv.

The company achieved ISO 13485 status about six months ago, which will likely be important to the development of these diagnostics.

The Ebola/Lassa/Marburg diagnostic might be useful for biodefense applications, should hemorrhagic fever viruses ever be weaponized, according to the NIAID grant application. However, Lucigen is also developing the test to be used in low-resource settings in endemic areas of Africa.

"In those countries, the health sector is not very well developed, and diagnostic facilities are just next to none," said Yogesh Chander, a senior scientist at Lucigen and one of the researchers actively developing the VHF diagnostic.

"Testing of these viral agents is not trivial, because you have to collect the sample and ship it to central facilities where they are equipped to do real-time PCR and have all the sophisticated instruments. The time lag and the back up are very long," he said.

Chander told PCR Insider that Lucigen has already developed the rapid sample prep method and showed its feasibility with spiked blood samples. It also determined the optimal method to dry the reagents, and discovered that lyophilized OmniAmp is "stable for more than 120 days, even at 37 degrees Celsius," he said.

The next step is visualization of the assay. Because the test uses loop-mediated isothermal PCR, Chander explained that it is not multiplexed. Each of the three targets will be detected in a separate reaction. However, the results, which will be detected using a lateral flow method, will run together, "and that will tell you which agent is present," he said.

Chander stressed that the company does not use hemorrhagic fever viruses at its labs in central Wisconsin. Instead, they have an ongoing collaboration with Thomas Geisbert and the Biosafety Level 4 facilities of Galveston National Labs, which is part of the University of Texas Medical Branch. "We don't even go near the VHF agents," Chander said. "We do our work here and then send it to them, and when the time comes, they [run the assay] with the real viruses."

A point-of-care test that can distinguish and detect these particular hemorrhagic fever viruses might help control outbreaks and prevent losing patients who can't or don't follow up, Chander suggested.

"In Africa and Asia, people have to travel many miles to reach the healthcare facilities. You cannot expect them to come back for the treatment," he said. "That's where these tests come into the picture. The total time taken will be about 30 to 40 minutes for this test, right from the sample prep to the answer. So, while the person is waiting in the clinic, you can have the result and start the treatment."

In the case of Ebola and Marburg viruses, many treatments are currently being studied, including early post-exposure interferon beta treatment, and a recently patented gene silencing method developed by Geisbert. UTMB has also partnered with Tekmira to develop this technology, and the collaboration was awarded a one-year, $1.4 million grant from the National Institutes of Health in March of this year.

However, at the moment there is no cure for VHF infections, so isolating infected patients is critical. "If you let the person go back without treating, he becomes a carrier, and in many cases he might start spreading the disease," Chander said.

The Ebola outbreak currently raging in Africa is the largest to date, according to data from the US Centers for Disease Control and Prevention. It began in March and has claimed 603 lives in Guinea, Sierra Leone, and Liberia as of July 12.

Chander estimated Lucigen's VHF diagnostic — with a lateral flow detection cartridge and fully integrated detection system — may be realized as early as the end of 2016.

Beyond VHF

Beyond the VHF test, Lucigen is developing an instrument for the detection of C. difficile bacteria, as described previously in PCR Insider.

That diagnostic device is based on the capabilities of the OmniAmp polymerase, "but it is a full instrument, complete with a sample collection module, cartridge to run the assay, and the detection platform," Chander said.

"We expect to talk more about that publicly towards the end of the year, with the intention of starting the clinical trial early next year," Shenoi explained.

In parallel to developing diagnostics for human pathogens, Lucigen is also working on an assay for the animal market. It is targeting PEDv, a coronavirus with an RNA-based genome. This pathogen sickens adult pigs and is extremely deadly to piglets. Some estimate as many as 8 million pigs have died since the virus first showed up in US herds 14 months ago.

Chander said Lucigen now aims to take a PEDv molecular diagnostic "to penside" by combining sample preparation, amplification, and detection in a 30-minute, sample-in, answer-out format.

Running point-of-care molecular diagnostics on the farm presents some atypical challenges, however. The test format must be simple and easy to use for non-laboratory staff. The assay must be robust, able to be stored at ambient temperature and humidity, and be a "no mix and no measure" format so the operator has minimal chance of error, Chander said.

However, he added, "the OmniAmp polymerase used in the assay is resistant to many inhibitors of PCR and can tolerate a relatively crude sample prep method. Unlike other LAMP polymerase enzymes, OmniAmp can be lyophilized, creating extremely stable reaction components that retain 100 percent activity once rehydrated."

Chander said he is aware of a few other companies who have developed PCR-based molecular diagnostic tests and platforms for animal health, in general, but, "none offer the ease of use, and potential for penside use by non-laboratory staff, provided by our test methods."

For this test, Chander said Lucigen has already completed initial development of the PEDv LAMP assay, as well as the rapid sample preparation methods. It is currently focused on validating the assay with real clinical samples.

Lucigen plans to present initial results at the annual meeting of the American Association of Veterinary Laboratory Diagnosticians in October 2014, Chander said. "We are looking for partners to help with completing the USDA licensing process and commercialization of this test," he added.

In terms of other commercial partnerships, Shenoi said that the company has "a lot of active discussions going on, but currently no licensing relationships that we can divulge at this time."

"I would say that what's very interesting is that those discussions are going on both in the animal health as well as the human health side, so they are very well aligned with the research program that Yogesh and our other scientists are focusing on," he said.

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