NEW YORK – Aided by more than a dozen federal grants, startup Kephera Diagnostics has been assembling a menu of immunoassays for infectious diseases including a point-of-care Chagas disease tests that could provide more accurate identification of active infections than current testing methods.
Since its founding in 2016, Kephera has secured federal grants and contracts to develop tests also for Zika virus infection, Taenia solium tapeworm-related neurocysticercosis and taeniasis, Lyme disease, liver fluke infection, and hookworm infection. According to CEO Andrew Levin, the company has accumulated about $15 million in federal funding with most of that coming from the US National Institutes of Health and Centers for Disease Control and Prevention.
Earlier this year, the firm won a $3 million grant from the NIH to finish work on its point-of-care rapid antigen test for Chagas disease, or American trypanosomiasis, a disease caused by infection with the parasite Trypanosoma cruzi. It is also a founding member of the Harvard University-based Wyss Institute's Diagnostics Accelerator Industrial Participant Program, which Levin said has already helped the firm secure a research agreement with the US Armed Forces Research Institute of Medical Sciences toward the development of ELISA and lateral flow tests for infections with Clonorchis sinensis and Opisthorchis viverrini liver flukes that are associated with an increased risk of a form of bile duct cancer called cholangiocarcinoma.
Levin, a molecular biologist, founded Kephera in 2016 with three people he brought with him from Immunetics, a testing startup he sold to Oxford Immunotec. By the summer of 2019, the firm had built out its current laboratory in Framingham, Massachusetts, where the firm can develop and manufacture tests.
In that lab, Levin said his team is developing its Chagas test that shows promise of high accuracy through the detection of antibodies to trypanosoma excreted/secreted antigen (TESA) produced by cultured live T. cruzi parasite. He said early results so far indicate that test can provide more consistent results across T. cruzi strains than competing serology-based tests currently on the point-of-care market. The firm plans to soon begin a multicenter clinical trial for the test.
The World Health Organization estimates upward of 7 million people worldwide are infected with T cruzi, a vector-borne parasite that is endemic in 21 Latin American countries. The CDC estimates more than 300,000 people in the US are infected with T. cruzi, although almost all were infected in Latin America.
Infections can occur when blood-sucking triatomine bugs carrying the parasite bite a person's face and defecate, shedding protozoa that can be rubbed or scratched into the bite wound or the person's eyes or mouth. Chronic infections can lead to cardiac complications such as an enlarged heart and heart failure, as well as gastrointestinal problems such as an enlarged esophagus or colon.
Acute Chagas disease can be diagnosed through methods including identification of T. cruzi in blood by microscopy, but chronic disease is often diagnosed with the help of serologic tests for antibodies to the parasite. According to the CDC, no one serologic test has sufficient sensitivity to confirm chronic T. cruzi infection, and the standard approach is to perform at least two different types of antigen tests and consider the results along with patient history. For its own testing, the agency uses an enzyme immunoassay and an immunoblot assay as the two first-line tests, and an immunofluorescence assay as a tiebreaker if tests on multiple specimens return discordant results.
Study results published in late 2019 found that two existing rapid tests used for patient screening, InBios International's Chagas Detect Plus and Chembio's Chagas Stat Pak Assay, produced highly sensitive and specific results. Levin, however, said the advantage of Kephera's TESA-based lateral flow assay is its consistent results across T. cruzi strains from patients in South America, Central America, and Mexico, though the results "will need to be verified in the future clinical trial on the final version of the assay and on larger numbers of patients."
Though he expects Kephera's assay will be used in the current two-test protocol along with serological tests, he said the TESA-based assay's performance in clinical trials could determine whether it has the potential to be used alone. The firm plans to eventually seek US Food and Drug Administration 510(k) clearance for that test.
While other firms have developed highly sensitive and specific TESA-based immunoblot assays, Levin said Kephera's innovation is to apply that technology to a rapid, point-of-care assay. He was not ready to release sensitivity and specificity data for the Chagas test but said its performance will be reported following the multicenter clinical trial.
"We are aiming for high sensitivity and specificity, but Chagas is a tricky field and continues to be very challenging to assay developers, including us," he said.
Sue Montgomery, veterinary medical officer in the CDC's parasitic diseases branch, said in an email that most people with Chagas disease have chronic infections and diagnosis of those chronic infections depends on detecting antibodies to the T. cruzi parasite.
"All currently available serological tests have performance limitations," she said. "Reasons for those limitations include the relatively low prevalence of Chagas disease in the patient population, individual patient variation in antibody responses, and differences in the discrete typing units (strains) of T. cruzi."
Any new test is only better if it's accessible, and access depends in part on healthcare providers' knowledge of Chagas disease, she noted, adding there is a need for tests that would indicate when an infection has been eliminated through treatment.
Toward that goal, Kephera is trying to develop another NIH-funded test to monitor patient response to Chagas treatment using antibody titer concentrations in blood. Levin said the firm is preparing to publish some early, but promising, results from its efforts to discover antigens that could serve as biomarkers for treatment response and give physicians actionable information during the first year of treatment.
"With current assays that are used to diagnose the disease, you might spend five or 10 years waiting to see a decrease in antibody titer," he said.
Levin said Kephera is close to commercial release of its ELISA test for Zika, the mosquito-borne disease that was the target of the first grant received by the firm, a $2 million Phase II Small Business Innovation Research grant awarded in 2020 by the National Institute of Allergy and Infectious Diseases to continue development and launch of a test that could detect Zika antibodies without cross-reactivity with dengue virus. Zika and dengue are both mosquito-transmitted flaviviruses. Between 2015 and 2016, a Zika epidemic swept through Brazil and other parts of South and North America, and though Zika was no longer a public health threat by the time of the NIAID grant, the firm said at the time that a test could be an important tool for public health if Zika were to reemerge in a more virulent form.
The firm continues work on hookworm infection and Lyme disease ELISAs, the latter of which Kephera intends to be accurate enough to deliver reliable results in a single test compared to the current recommended two-step testing protocol of an enzyme immunoassay or immunofluorescence assay followed by confirmatory immunoblot. The firm is also working on point-of-care immunoassays for neurocysticercosis, taeniasis, and liver fluke infection.
Sharon Roy, medical epidemiologist with the CDC's parasitic diseases branch, said in an email that early-stage infections with Clonorchis and Opisthorchis liver flukes are often mild or asymptomatic, and diagnosis typically involves microscopy to identify parasite eggs. But, as with tapeworm eggs, liver fluke eggs can be missed in a single test and repeated testing improves sensitivity. Abdominal ultrasonography is used to evaluate the biliary tree, she said.
In one federal grant document, Kephera said its upcoming test for neurocysticercosis, an infection with epilepsy-causing T. solium pork tapeworms, would use recombinant and synthetic T. solium antigens to provide a rapid, commercially available point-of-care alternative to serologic tests such as the CDC's laboratory-based enzyme immunotransfer blot test. The firm separately said it plans to develop the first highly accurate and commercially available assays for taeniasis, which is also caused by T. solium tapeworms.
Jill Weatherhead, assistant professor of tropical medicine and infectious diseases at Baylor University's College of Medicine, said there is a need for better diagnostics for most parasitic infections. Diagnosing those infections, especially in low-resource areas, typically relies on microscopy or serology, each of which has shortcomings.
Identifying eggs or ova of a particular parasite can be difficult, especially in a stool sample, unless a patient has a high parasite burden or the microscopist is highly skilled and experienced at looking for that target, she said. Many IgG-based serology tests only tell whether a patient has been exposed to a particular parasite during their lifetime and do not distinguish between acute and chronic infections.
"You don't know if that infection is something that's happening right now or if they had it when they were a child or decades ago," she said.
Levin said it will take several years to develop his firm's tests into commercial kits and then gain regulatory clearances in the US, Europe, and countries in Southeast Asia, where Levin hopes to sell the liver fluke tests. In the meantime, the firm plans to start bringing in revenue through its CLIA lab with a pair of Chagas tests: France-based Infynity Biomarkers' Multi-Cruzi multiplex immunoassay and Argentina-based Wiener Laboratories' Chagatest ELISA Recombinante v.3.0. The firm also plans to offer its validated ELISAs for Lyme disease, neurocysticercosis, and C. sinensis liver fluke parasites.
Levin estimated the point-of-care Zika test will be available later this year, following a third-party study on the test's performance. He said the price of that test will be competitive with Zika assays already on the market. He added that it's too early to announce pricing for the point-of-care Chagas tests but they, too, will be competitive with other commercial tests.
Levin said the company plans to eventually form partnerships for commercial distribution of the tests in the US and globally as well as connect with public health agencies that procure tests for distribution where they are needed.