Lyme Disease ddPCR Test Detects Active Infection with Over 90% Sensitivity

Researchers at Dartmouth Hitchcock Medical Center have developed a new test that could diagnose Lyme disease earlier and more accurately than currently available testing methods.

Results presented today at the Association for Molecular Pathology (AMP) 2025 Annual Meeting & Expo show this new test achieved 90.9% sensitivity in a small set of samples. This represents a major improvement when compared to two-tiered serology testing, the current diagnostic standard for Lyme disease, which typically has a sensitivity lower than 50% during early stages, leading to many false negative results in the first six weeks of infection. 

Early diagnosis of Lyme disease can significantly reduce the risk of long-term complications affecting the skin, joints, heart, and nervous system. However, many patients receive a diagnosis months or even years later. This is partly because only about 25% of infections with Borrelia burgdorferi bacteria result in the characteristic bull’s eye rash that can help doctors diagnose Lyme disease early. Many other patients develop a range of skin lesions that can be easily mistaken for other conditions, and more than half of them will receive a false negative result due to the limitations of current testing methods. 

This was precisely the case of a 73-year old woman seeking care at the Dartmouth Hitchcock Medical Center for progressively worsening symptoms including skin hardening and joint immobility. While her doctors initially suspected she had an unrelated skin condition, she was eventually found to respond well to medication for Lyme disease. Because antibody serology testing had not shown evidence of an active Borrelia infection, it took more than four years for the patient to receive a Lyme disease diagnosis. 

To improve the care of future patients, researchers at Dartmouth Hitchcock Medical Center were tasked with the development of a new molecular test to confirm the presence of B. burgdorferi in a patient sample. To achieve high sensitivity, the team employed droplet digital PCR (ddPCR) technology, which can achieve high accuracy in smaller sample sizes.

The resulting test involves three ddPCR assays: one to detect all species of Borrelia, another that detects species that cause Lyme disease, and another that specifically targets B. burgdorferi, which is responsible for most cases of Lyme disease in the U.S.

“Using the ddPCR assay, we successfully detected B. burgdorferi DNA in this patient’s skin biopsy,” said Guohong “Grace” Huang, PhD, genomic scientist at the department of pathology and laboratory medicine in Dartmouth Hitchcock Medical Center. “This finding was further confirmed by DNA sequencing, supporting the diagnosis of chronic Lyme disease.”

The assay was then tested on a small group of patients with either confirmed or suspected Lyme disease, showing high accuracy even when detecting minimal amounts of bacteria in the sample, as low as five to ten single bacterial cells. The test showed 90.9% sensitivity in fixed tissue samples, a figure the researchers expect to see increase when using fresh or frozen tissue samples, which preserve DNA better. 

Unlike antibody tests used today to diagnose Lyme disease, the new test developed by Huang and colleagues could distinguish between patients with an active Borrelia infection and those who have been exposed in the past. “This is another clinical scenario where the ddPCR assay offers a clear advantage by detecting active bacterial presence rather than relying on indirect immune markers,” said Huang.

Following further development and validation work, she hopes this test could one day save patients years of waiting for a diagnosis, reducing long-term complications by enabling early treatment for a higher number of patients: “To advance this work, the next step is to expand testing to a large number of cases and explore strategies to further enhance the assay sensitivity.”