Over the past 20 years, climate change has expanded the range of the tick that carries Lyme disease, and reported cases in the United States have nearly doubled. According to the Centers for Disease Control and Prevention (CDC), as many as 30,000 people became infected each year during this 2-decade period. Laboratory tests are a key tool in diagnosing Lyme disease, which if untreated can lead to serious damage to the joints, heart, and nervous system. However, even as this foe of outdoor activities has continued its upward trajectory, diagnostic testing for Lyme disease has not advanced with it.

The current two-tiered algorithm recommended by CDC was developed 2 decades ago. First, an enzyme immunoassay (EIA)—usually an enzyme-linked immunosorbent assay (ELISA)—or more rarely an immunofluorescence assay, is performed to measure a patient’s antibody response to Borrelia burgdorferi, the bacterium that causes Lyme disease. If this yields a reactive or equivocal result, a Western blot is performed (Figure). Whereas the first test is more sensitive, the second is more specific.

“Two-tier testing works really well for disseminated disease,” said Claudia Molins, PhD, a microbiologist and Lyme disease expert at CDC’s Division of Vector-Borne Diseases in Fort Collins, Colorado.

While this algorithm has been adopted widely and has mostly served clinicians and patients well, it also has “a couple of prominent limitations,” according to Elitza Theel, PhD, director of the infectious diseases serology laboratory at the Mayo Clinic in Rochester, Minnesota. One is the approach’s low sensitivity in the first few weeks of infection, before some patients have a chance to develop a robust antibody response. Because only 29% to 40% of patients with early Lyme disease have reactive results using the two-tier testing system, the Infectious Diseases Society of America advises clinicians to treat patients with possible early Lyme disease based instead on clinical findings (Clin Infect Dis 2006;43:1089-1134).

However, some physicians may be unfamiliar with these guidelines or press forward with early stage testing, thereby either not treating some patients who are infected or prescribing antibiotics for others who do not have Lyme disease.

The current testing system is also unable to differentiate between recent and past infections, fur-ther complicating treatment decisions. “Antibodies may persist for many months to years, even fol-lowing successful treatment,” said Theel.

Aware of these limitations, researchers have been developing new tests, and several are now in the pipeline.

Doubling Up on ELISAs

In place of the current two-tier system, some labs are already using two different ELISAs sequentially, according to Eugene Shapiro, MD, a professor of epidemiology at Yale University in New Haven, Connecticut. Using this approach, one ELISA might measure a patient’s antibody response to whole-cell sonicate, whereas the other might target the pathogen’s C6 epitope, providing complementary information.

This approach offers several benefits. First, “Multiple independent studies suggest that the two-ELISA algorithm has increased sensitivity over the standard two-tiered testing algorithm during acute and even during later stages of Lyme disease,” said Theel. Second, because it removes the need for Western blot testing, “it is faster and easier to do, and maybe cheaper,” contended Shapiro.
More research is needed before double ELISAs become de rigueur, Shapiro cautioned. “Maybe this type of testing is marginally more sensitive than standard two-tier testing, but it may be less spe-cific,” he said. “Additional studies performed in real clinical situations are needed to confirm the benefits of this approach.”

Multiplex Makes Its Mark

While the back-to-back ELISA strategy simmers away, a team led by Rafal Tokarz, PhD, an associate research scientist at Columbia University in New York, is exploring a more ambitious approach: the TBD Serochip, which is designed to discriminate between antibody responses to eight different tick-borne pathogens present in the United States. This assay includes roughly 170,000 overlapping, small linear peptides from the protein sequences of each of the pathogens, including Anaplasma phagocytophilum, Babesia microti, Borrelia burgdorferi, Borrelia miyamotoi, Ehrlichia chaffeensis, Rickettsia rickettsii, Heartland virus, and Powassan virus.

The idea is that by measuring antibody responses to such a wide range of epitopes, the test will be able to discriminate between tick-borne infections that might otherwise be hard to distinguish. This approach might also identify characteristic early Lyme disease responses—or reliable differences between pre- and post-treatment samples—that await discovery.

Tokarz and his colleagues reported promising preliminary results earlier this year (Sci Rep 2018;8:3158), and the team is now testing more samples from all stages of Lyme disease, including post-treatment, as well as samples from patients with other tick-borne diseases, such as ehrlichiosis and rickettsiosis, according to Tokarz. In its current form, the TBD Serochip would only be suitable for specialty labs, he indicated.

However, the team is “in the process of simplifying it,” Tokarz added, “and we aim to ultimately provide it as a point-of-care test.” He hopes to make a simplified form avail-able for clinical use within the next 2 years.

Metabolomics Milestone

Molins and her colleagues are working on another type of test that uses an entirely different approach: metabolomics. Inspired by the use of metabolomics to detect biomarkers of cancer, her team’s goal was to develop a test sensitive enough for use in the early stages of Lyme disease. However, by investigating the metabolic biosignature of serum samples from patients with various tick-borne diseases, they also recently found that it is possible to differentiate between early Lyme disease and southern tick-associated rash illness, or STARI (Sci Transl Med 2017;9:eaal2717). These look-alike infections overlap in some sections of the United States and may complicate Lyme disease diagnosis.

Molins said that in addition to detecting early-stage Lyme disease, this approach may help distinguish between current and past infections, as a patient’s metabolic response to the pathogen is not expected to be long lived; however, further testing is needed. At present, the research team is investigating samples from many different stages of Lyme disease, as well as samples from Lyme look a-like diseases. “The test is only as good as the samples you put in to make it,” said Molins. “We want to develop an assay that stands the test of time.” For now, the test closest to completion is the one distinguishing Lyme and STARI, which Molins said may be available for clinical evaluation within the next 2 years.

The biggest drawback of this type of testing is that “the technology used is cumbersome and complex,” said Maria Aguero-Rosenfeld, MD, director of laboratories and microbiology at New York University’s Tisch Hospital. “It uses mass spectrometry, which most likely would not be easily available in many clinical labs.” Molins is interested in simplifying the test to increase its utility but explained that this possibility will depend on how complex it is to detect target metabolites.

Boosting Performance Now

Until these promising alternatives have been validated, experts offered several tips for improving the performance of the two-step algorithm.

First, labs can capitalize on progress made in identifying antigens expressed early in infection. “Antibody tests that use recombinant antigens have better performance than the old antibody as-says,” advised Aguero-Rosenfeld. Second, Shapiro suggested that labs refrain from performing Western immunoblots on samples that do not have a positive or borderline ELISA result. “The specificity of the blot decreases greatly if done without a positive quantitative result,” he added.

Shapiro also emphasized the role of clinical laboratory professionals in educating physicians about when testing is appropriate. “The biggest problem with serologic testing is that people use it for a screening test in patients with a low probability of having Lyme disease—individuals with chronic, nonspecific symptoms, without signs of Lyme disease,” he elaborated. “As a result, the positive predictive value is very poor.”

Kristin Harper is a freelance writer in Seattle, Washington. +Email: [email protected]