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George Mason University Researchers Developing Urine-Based Lyme Disease Test With $1.2M Army Grant


NEW YORK – With the backing of a $1.2 million US Department of the Army grant, researchers at George Mason University are developing a urine-based Lyme disease test that they hope will improve diagnosis by giving earlier answers than immunoassays already on the market.

Alessandra Luchini, systems biology professor and principal investigator on the study, said that the research team intends to develop a mass spectrometry-based test that would be used to identify in urine samples protein biomarkers of the Borrelia organism. That protein signature could eventually be used to create a high-throughput immunoassay, she said.

Luchini noted that very low numbers of Borrelia tend to circulate in a patient's blood in comparison with other types of infectious bacteria, and her team intends to identify proteins from the organism that can be consistently detected in urine.

The project will include evaluating the test using samples collected from acute Lyme patients for the Bay Area Lyme Foundation's Lyme Disease Biobank as well as with banked specimens from Johns Hopkins University, according to an announcement from George Mason University. The researchers also are collaborating in the study with the National Institute of Allergy and Infectious Diseases (NIAID). Luchini said the study will involve the evaluation of samples from a total of 300 Lyme disease patients and controls.

The research team is also developing during that three-year study a collapsible urine sample collection device that uses polymer filaments to capture biomarkers without absorbing water and deliver room-temperature semi-dry samples by mail from patient homes to a diagnostic laboratory.

Lyme disease can be diagnosed through clinical history and evaluation, especially when patients develop the characteristic "bull's-eye" erythema migrans rash and its concentric red rings, as well as with the aid of laboratory testing.

The Centers for Disease Control and Prevention recommends two-step serologic testing for Lyme disease. The standard method involves the use of an enzyme immunoassay or immunofluorescence assay with a confirmatory western blot test. Since 2019, the agency has also endorsed a modified two-tier testing method that uses two immunoassays, a method that laboratories are increasingly using, the CDC said on its website.

However, the agency notes that most Lyme serologic assays are designed to detect antibodies that can develop several weeks after infection, but those tests may deliver false negatives within the first four to six weeks of infection. False positives also can occur in patients with conditions including relapsing fever, syphilis, rheumatoid arthritis, and Epstein-Barr virus infection.

Luchini said that the George Mason researchers hope to develop a standalone biomarker-based test through their analysis of well-characterized samples. However, she said Lyme disease testing is complex, and the study could lead to the development of a two-step testing process.

Luchini coauthored a 2020 article in Scientific Reports that describes the use of pre-analytical concentration, mass spectrometry, and a peptide authentication algorithm to identify and characterize pathogen-specific proteins in urine samples. The authors wrote that they found 160 peptides that were linked with Borrelia infection and they were able to distinguish borreliosis patients from controls by Borrelia peptides in their urine. Among 148 patients with non-acute suspected tick-borne illness, they found at least one Borrelia­-associated peptide in urine samples from 89 patients and two or more in samples from 48 patients.

They also found a correlation between the number of Borrelia peptides in samples from non-acute disease patients and the presence of symptoms of borreliosis such as rash, fatigue, and facial palsy.

Luchini said that the recent Army grant will support additional studies to identify and characterize a far smaller number of targets such as five, 10, or 20 proteins that could be used to identify Borrelia infections. The team has a goal of 95 percent sensitivity and specificity. The urine tests evaluated so far in clinical trials have resulted in 90 percent sensitivity and almost 100 percent specificity, George Mason said in a statement.

Luchini said that she envisions the university eventually developing a laboratory-developed test, with the biomarkers later becoming the basis of a kitted test. Commercialization of the test is beyond the scope of the research project although Luchini said that she thinks George Mason's Office of Technology Transfer would be happy to work with commercial partners who are interested in licensing the technologies and methods developed during the study.

Other Lyme diagnostic and detection tests are already available or being developed, including one from T2 Biosystems, which said this month it had inked a letter of intent with Eco Laboratory for the launch of a laboratory-developed test for the early detection of Lyme disease. DiaSorin said earlier this year that it had submitted for US Food and Drug Administration approval an immunoassay for the early detection of Lyme disease, while Qiagen said that it is planning to soon launch its QuantiFeron Lyme disease test. Kephera Diagnostics said in the fall of 2023 that it had received a $2.8 million grant from the NIAID for the development of its Lyme disease ELISA in a collaboration with three major medical centers.

The existing options also include tests under the two-tier system from the likes of BioMérieux, Revvity's EuroImmun, and Gold Standard Diagnostics. Revvity had also acquired a PCR-based Lyme test with its acquisition of Oxford Immunotec, although the firm said that it no longer offers that test.

Five firms including T2 also won funding awards in February from the US Department of Health and Human Services and the Steven & Alexandra Cohen Foundation through the organizations' LymeX Diagnostics Prize, which is a project that the HHS and the Cohen Foundation launched in 2020 to accelerate innovation in Lyme disease care. The funding is directed at the development of blood- and urine-based tests for the detection of Lyme disease.

The George Mason research team also was one of 10 groups that received in 2022 $100,000 in funding from the LymeX Diagnostics Prize initiative for work by Luchini and Lance Liotta toward the development of a test that could be used to aid the diagnosis, monitoring, and treatment of patients with Lyme disease.

The George Mason team has been developing its urine-based testing methods since about 2010, when internal study results first indicated they could detect Borrelia fragments in urine. Luchini noted that urine samples have already been proven to have diagnostic utility for the detection of tropical diseases such as malaria. While the biomarkers of Lyme disease can fluctuate or diminish in blood, those biomarkers are also filtered in the kidneys, concentrated in the bladder, and shed over time in urine, she said.

While Luchini said that the team is currently detecting those biomarkers using high-complexity mass spectrometry, "we want to translate that to a faster way of detecting the proteins in urine."