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Mayo Clinic Lab Discovers New Lyme Disease-Causing Bacterium Using Routine Melt Curve Analysis

Spirochetes of B. mayonii. (Used with permission: Mayo Foundation for Medical Education and Research. All rights reserved.)

NEW YORK (GenomeWeb) – In the process of routinely evaluating samples for Lyme disease, researchers at the Mayo Clinic have discovered a new strain of the spirochete that causes the disease.

While there are four strains of the tick-borne bacteria globally that cause Lyme, only one — Borellia burgdorferi — was previously known to be endemic in the US.

There are no US Food and Drug Administration-approved, PCR-based commercial assays for Lyme disease bacteria. But, using a PCR-based assay specific for the plasminogen-binding protein gene, oppA1, previously developed at Mayo's international reference lab, the researchers began seeing an atypical result in their standard endpoint melting temperature analysis.

It started in the summer of 2013 with a single sample, Bobbi Pritt, director of Mayo's Clinical Parasitology Laboratory and first author on a report describing the new species published in The Lancet last week, told GenomeWeb.

Over the next year and a half, the lab got four additional positives. That prompted a retrospective analysis of 100,545 specimen results dating back to 2003, from which the group discovered one additional result from 2012.

"Our assay uses primers and probes, so it's specific, but in addition to an amplification curve we also use the melting temperature to confirm what we've detected is what we were expecting to detect," Pritt explained. Hybridization probes are very good for organisms that tend to mutate, like RNA viruses, she said, and in the atypical results the melt temp was about 10 degrees lower than expected.

From this, Pritt and her team were able to infer that it was a positive result. It amplified with the primers and was detected by the probes, but it was not a perfect match for the probes, suggesting it was closely related to B. burgdorferi, but not identical. They then sequenced the bacterium to discover that it was a previously-unidentified species.

The group has tentatively named the species Borrelia mayonii, and also demonstrated its presence in Ixodes scapularis ticks collected in regions of Minnesota and Wisconsin where suspected patient exposures occurred, possibly associated with summertime camping and outdoor activities.

The Mayo lab extracted DNA from specimens using the Roche MagNaPure system. It then used the Roche Light Cyler 2.0, which has the capacity to do HRM. "It is one of the primary instruments used by our lab, because not all real-time PCR devices have that ability," Pritt said.

"If you were using shorter primers and a probe that wouldn't anneal if there was a mutation, then you would never detect it, it would just show up as a negative result," she said. "If you're using an assay that is really unforgiving when comes to any mutations, then you're not going to detect new things," she added.

"I think it shows how melting temperature is a really powerful technology for picking up variants," she said.

The group also described a possibly more virulent disease induced by the new bacteria. For example, there has never been reported visualization of B. burgdorferi spirochetes in microscopic analysis of blood samples. But spirochetes were visible in blood of one B. mayonii-infected patient, and among five blood specimens the median oppA1 copy number was 180 times higher compared to 13 specimens with B. burgdorferi.

"I think the fact that you get these higher levels of spirochetes in the blood is interesting, and our patients seemed like they were really sick — we only had six of them, but two were hospitalized, which you just don't see that often with Lyme disease," Pritt said.

Also, this potentially more severe illness might be harder to spot, since most of the patients did not have the classic bulls-eye rash. Because of this, even with non-classic symptoms, physicians should consider, "Could this be infection with this novel Borellia species?"

Authors of an accompanying comment in the journal also noted, "The discovery of a novel tick-borne pathogen poses new challenges to diagnostics, therapy, and public health surveillance." The Mayo PCR assay, meanwhile, was published a few years ago, and "anyone can bring this assay in if they wanted to use it," Pritt said.

Remarkably, this isn't the first time the Mayo lab has found previously unknown organisms. "This is actually the second new species that I've detected in my laboratory, and my colleagues in my lab have detected a couple other species this way too," Pritt said.

In 2011, they described a new species of Ehrlichia in a New England Journal of Medicine article. It was almost the same story, Pritt said, in that the lab was performing routine clinical testing and a blood specimen from a patient was positive but had a melting temperature about 10 degrees cooler.

Pritt was also an author on a study describing a novel probe binding-site polymorphism in HSV 1/2 detectable by melt curve analysis, as well as a report describing mutability of the H1N1 virus during the 2009 epidemic, as reported by GenomeWeb.

In the latter case, "half the assays that were on the market didn't detect [H1N1] because they were TaqMan-based probe assays and they were very specific," Pritt noted. The group's FRET probe-based assay, however, was able to still detect H1N1 as mutations crept in over a three-month time period. 

"We really like the extra step of having the melting temperature," Pritt said. "It just gives us that extra layer of specificity — we consider what is generated by the melting temperature to be our final result."

She also said a more flexible assay does not lower the specificity per se. "If you validated your assay and knew that positives always melted within, say, a five-degree temperature range, then you would know that anything within that range is what you've validated your assay for — if it starts shifting, then you would want to investigate those." This gives the flexibility to still detect things outside the range, but also know they were something other than what the assay was designed for.

Overall, Pritt said it might be somewhat rare for clinical labs to discover new species. "Being in the Upper Midwest, we have a lot of tick-borne diseases, so, the fact that we've detected two is pretty amazing," she said, adding, "We were just very lucky, we were in the right place at the right time and we were using assays that allowed us to detect it."