NEW YORK – A University of California, Los Angeles-led team of researchers recently reported that they have developed a 20-minute Lyme disease immunoassay that could be used at the point of care to help deliver a diagnosis more quickly than today's two-test standard.
Reporting last month in Nature Communications, the team of researchers from UCLA, New York Medical College, Connecticut-based firm Biopeptides, and the Lyme Disease Biobank in Portland, Oregon, said that their serologic test was used to detect antibodies to Borrelia burgdorferi in a serum sample, with 95.5 percent sensitivity and 100 percent specificity in a 30-sample validation set with equal numbers of Lyme-positive patients and controls. They added that the low-cost test could be used during a patient visit to aid the rapid diagnosis and treatment of Lyme disease.
The researchers designed the test as a vertical flow assay that employed three synthetic peptides that were distributed in an array of immunoreaction spots and was used for the simultaneous detection of IgM and IgG antibodies. The assay was inserted into a portable reader and illuminated with green LEDs, and the results are captured using a smartphone and analyzed remotely through a machine learning-developed algorithm.
Dino Di Carlo, coauthor of the article and chair of the bioengineering department at the UCLA School of Engineering, said that the immune response to Lyme disease is complicated and variable person to person, and he noted that IgM and IgG levels rise and fall during different stages of immune response. The algorithm that was developed as part of the test is used to detect the intensity of reactions to the peptides and controls that are incorporated into the array.
"Each patient will have their own repertoire of antibodies that they'll develop," he said.
The vertical flow format of the UCLA test allows for the development of a multiplex assay with simultaneous reactions that can be used to analyze a patient's immune response, Di Carlo said, adding that the standard two-tier testing format, in comparison, typically involves a first test that has low specificity followed by a more complex second test with higher specificity, a process that increases costs and introduces delays.
The UCLA team's study results, he said, suggest that those steps could be combined into a single point-of-care test with equivalent performance.
"It is a single test, but it performs at the same level as the two tiers of the traditional test," he said. "What we do that allows us to have that performance is we have these arrays of peptides that give us specificity to the Lyme pathogen instead of other related pathogens, and that was usually done in the second-tier western blot, where you're looking at IgM and IgG immune responses to many of these different antigens."
The researchers used machine learning to train the diagnostic model using 20 serum samples from patients with early-stage Lyme disease and 20 controls. All of those samples came from patients who had reported tick bites between one and four weeks prior to the blood draw.
The team validated the model using three peptides, modVlsE-FlaB, Var2FlaB, and OppA4, and tested it in triplicate on 30 samples from the Bay Area Lyme Disease Biobank, resulting in two false negatives and no false positives.
They reported that the test was further evaluated using 32 samples from the US Centers for Disease Control and Prevention's Lyme serum repository research panel, with the vertical flow assay results matching lab-based two-tier test results. The samples consisted of samples from 12 patients with Lyme disease, 12 with look-alike bacterial infections that are known to be cross-reactive with some Lyme disease tests, and eight controls.
Di Carlo said that the test could help healthcare providers to identify infections more quickly and allow earlier treatment selection.
The researchers noted that Lyme treatments are most effective during early stages of the disease. However, the CDC notes that serology assays are prone to delivering false negative results for up to six weeks after the initial infection, and the small, transient numbers of Borrelia bacteria circulating in blood have been difficult to detect through PCR and culture-based testing.
Liz Horn, a Bay Area Lyme Foundation researcher who helped found the organization's Lyme Disease Biobank, said that Lyme disease also can be difficult to detect in patients who don't develop the characteristic erythema migrans rash with its "bull's-eye" appearance of concentric red circles. According to the CDC, the rash forms an average of seven days after a tick bite but may take up to 30 days after a bite to form.
"We need better diagnostics, and we really need better diagnostics in early disease," she said.
Di Carlo said that the research team has not yet conducted the studies to determine how soon following a tick bite the vertical flow assay could be used to detect B. burgdorferi antibodies, but he expects that those antibody responses would be detectable in less than one week.
Horn said that the Bay Area Lyme Foundation has a long-term collaboration with the UCLA researchers and helped to connect them with the New York Medical College researchers who developed the peptides that were used in the study. She said that the foundation also provided well-characterized samples from its biobank for the recent study along with clinical information and test results from the patients.
Meanwhile, other firms have taken different approaches to creating new tests that are meant to hasten a Lyme disease diagnosis. Startup En Carta Diagnostics has been developing a microneedle patch-based molecular test for the detection of Borrelia bacteria in an interstitial fluid sample, while T2 Biosystems has announced plans to launch a Lyme disease panel for the detection of tick-transmitted pathogens within the first 30 days of infection. DiaSorin said early this year that it had submitted for US Food and Drug Administration approval an immunoassay for the early detection of Lyme disease, while Kephera Diagnostics has secured millions of dollars in federal support for an ELISA-based standalone test.
The US Department of Human Services and the Steven & Alexandra Cohen Foundation also announced early this year $2 million in awards for the development of Lyme disease tests.
Di Carlo said that the results that were reported in Nature Communications were the product of five years of studies into the use of peptide-based tests to aid Lyme diagnosis as well as the team's previous work on vertical flow assay development and whole protein-based Lyme tests. In the recent study, the researchers selected for peptides that could provide high specificity and perform well in a paper-based vertical flow assay format so that the resulting test would deliver fast, accurate results with a low cost of materials.
The article's authors estimated that the material costs for a single test was less than $3.
Di Carlo said that the research team is now studying the performance of the test using whole-blood samples. The recent study used serum samples that were easily accessible through biobanks, whereas using fingerprick blood samples or other blood samples would allow the use of the test at the point of care or in homes.
Using whole blood, however, will require overcoming the challenge of incorporating filters into the test to remove blood cells and allowing the flow of antibodies and serum to the reaction sites, he said.
Meanwhile, Di Carlo said that the researchers are looking for partners who can help to evaluate the Lyme disease test in clinical settings as well as to help bring the test to market. That could include licensing the technologies for further development, he said.
"We're trying to find the right partners to commercialize the technology, and that would lead to regulatory-type clinical trials," he said.