NEW YORK ─ Researchers at the Tufts University School of Medicine and the National Institute of Allergy and Infectious Diseases at the National Institutes of Health (NIH) are developing a test that uses phospholipid biomarkers to enable earlier diagnosis and treatment of Lyme disease.
The test detects antibodies against phospholipids that the Lyme bacteria Borrelia burgdorferi acquires from the host to grow, said Peter Gwynne, a molecular biology and microbiology researcher at Tufts University School of Medicine and one of the test's developers.
"With Lyme disease, we're limited to using antibody tests to measure the immune system's response to past infection because there's not enough bacteria to do direct detection even if you're in the middle of an acute infection," he said.
However, unlike the immunoglobulin M and immunoglobulin G antibodies used in current tests, antiphospholipid antibodies may enable early detection, Gwynne added.
The researchers recently described a proof-of-concept test and its preliminary validation in the Journal of Clinical Investigation, saying that the elevation of antiphospholipid antibodies in an ELISA assay predicted early Lyme disease infection with better sensitivity than the standardized two-tier testing approach currently used to diagnose the disease.
In the two-tier approach, which is recommended by the US Centers for Disease Control and Prevention, specimens are first tested by an enzyme immunoassay and then by an immunoblot assay to confirm positive results or evaluate unequivocal results.
In the new study, antiphospholipid antibodies were "elevated faster and declined faster than the antibodies used by the current Lyme disease tests," which has implications for diagnosis, Gwynne said.
Extensive validation is needed to move the test toward commercialization, but the antiphospholipid ELISA method has the potential to become part of future Lyme disease testing workflows, providing clinical utility in early diagnosis, the tracking of response to therapy, and the diagnosis of reinfection, Gwynne said.
In the study, he and his colleagues used a culture medium to first demonstrate that exogenous lipids are essential to support the growth of B. burgdorferi. They showed that the bacterium acquired three phospholipids — phosphatidylserine acid, phosphatidic acid, and borrelial phosphatidylcholine — and then studied antibody responses to them.
Sera were collected from 12 untreated individuals at the time of diagnosis, between one and 75 days after the onset of symptoms, representing the early stages of infection when current antibody-based diagnostic tests have low sensitivity, the researchers said. Sera were also collected from 12 treated individuals at least one year from the initial diagnosis and treatment.
The cutoff value to define a positive sample was established using a panel of 12 negative controls from healthy donors, and the three phospholipids that were elevated during infection were assessed for diagnostic value.
In the untreated group, when the three biomarkers were combined in a panel, it accurately detected Lyme disease in 11 of the 12 samples, Gwynne said.
Overall, they found that the phospholipid antibodies were elevated early during the infection and declined in post-treatment samples, Gwynne said, adding, "This means a commercial test using this approach could be more sensitive earlier and could be used to monitor individuals following treatment."
Wendy Adams, research grant director at Bay Area Lyme Foundation, which collaborates with scientists and institutions seeking medical breakthroughs for Lyme disease, said that "antiphospholipid antibodies are found in several different infections and autoimmune diseases" and added that she finds the Tufts group's preliminary data promising.
"This group’s novel translation of bacterial metabolism mechanisms into potential infection biomarkers is encouraging, and time will tell if it bears out when data from studying a larger, more heterogeneous group of patients is available," said Adams, who is not involved in developing the test.
Overall, diagnosing Lyme disease is so problematic that ultimately several different diagnostic strategies may be needed "to diagnose an infection or [confirm] its resolution depending on duration of symptoms, geographic area, and clinical presentation," said Adams.
The disease, caused by a bite from an infected tick, can lead to long-term complications including arthritis, fatigue, and mental impairment, and though the illness can be eliminated with antibiotics, it can also persist for many years.
"Studies show that a delay in diagnosis, using ineffective antibiotics, early steroid treatment, and a lackluster immune response put a patient at a much higher risk of persistent symptoms," Adams said.
While antibody tests remain the only US Food and Drug Administration-cleared option to help with a diagnosis, they have trouble differentiating current from past infection and can't prove that an infection has been eliminated, she added.
Given the challenges with early detection of Lyme disease, numerous companies are exploring different testing methods.
Adaptive Biotechnologies' T-Detect Lyme test, poised to debut this year, uses T cells to detect the early onset of Lyme disease and, according to the firm, may enable accurate detection after a tick bite or with the first sign of symptoms.
Last April, DiaSorin and Qiagen announced they had received CE marking for the Liaison LymeDetect assay based on QuantiFeron interferon gamma release assay technology for the early diagnosis of Lyme disease.
And last March, researchers from the University of Leicester in the UK published a study describing a test that uses quantitative PCR to target genes encoded by prophages — viruses present in Lyme-causing bacteria that can be easier to detect than the bacteria itself.
While the current antibody tests "are very specific, their sensitivity rates are poor among subjects who have recent exposure to a tick bite and when the disease is in earlier stages and Borrelia-specific antibodies have not yet developed," said Gunjan Arora, an associate research scientist at Yale University School of Medicine.
With colleagues, Arora and fellow Yale researchers wrote an accompanying commentary about the Tufts-NIH Lyme disease diagnostic test in the same issue of the Journal of Clinical Investigation.
Arora and his colleagues, who are not affiliated with the test's development, said the study findings "suggest that B. burgdorferi–induced anti-lipid antibodies in conjunction with a careful clinical assessment may aid in the diagnosis of Lyme disease."
However, in an interview, Arora also cautioned that the results were obtained from in vivo studies using a mouse model and a small number of human clinical specimens from patients with early Lyme disease.
As a result, the diagnostic potential of phospholipid antibodies against Lyme disease "needs further investigation in single-blind and double-blind studies using a large number of clinical specimens," Arora said.
Gwynne noted that it could take a few years to commercialize its test but said work is underway to test larger numbers of samples and to establish the test's sensitivity and specificity. The group is planning to validate the test in hundreds of samples and include a wide number of possible conditions including Lyme disease and similar diseases, he said.
The researchers are automating the test to make it more suitable for clinical use, but to enable its commercialization they would need to attract the interest of a diagnostic testing partner, Gwynne said.
Nonetheless, they see the potential to commercialize an assay that runs on widely available laboratory-based ELISA platforms, as well as a point-of-care version that uses lateral flow or microfluidic technology for use in physician offices, Gwynne said.