Lyme algorithms: stick to standard, move to modified?

Charna Albert

June 2023—For Lyme disease testing, immuno­blots became optional in 2019 when the FDA cleared enzyme immunoassays for use as part of a modified two-tiered testing algorithm. “It was a historic event in the world of Lyme diagnostics,” says Elitza Theel, PhD, D(ABMM), director of the infectious diseases serology laboratory and co-director of the vector-borne pathogens service line at Mayo Clinic.

But the standard two-tiered testing algorithm with its immunoblots hangs on.

Dr. Theel, a member of the CAP Microbiology Committee, which monitors, through the CAP Tick-Transmitted Diseases Survey, how many labs have made the switch to the modified algorithm since the FDA’s approval of multiple ELISAs for use in confirmatory testing, says the number is smaller than expected.

“I suspect it has to do with a number of things: the SARS-CoV-2 pandemic and then mpox, and all the challenges we are experiencing with a limited workforce. In microbiology, like other areas, we continue to experience a shortage of technologists. So you prioritize things that have to get done with the resources you have, and I’m assuming that transitioning to the modified Lyme algorithm is not a priority with everything else going on.”

“So there have been constant pressures and stressors on laboratories over the past few years,” she continues. “And many laboratories rely on reference labs to do their immunoblots rather than run them themselves, and the reference labs continue to offer Lyme blot testing, so if it’s not broken, why fix it?”

A limited understanding of the modified algorithm’s advantages may play a part too.

But immunoblots are immuno­blots. Nobody really wants to do them, she says. “They’re clunky.” Although most labs have transitioned to using automated processors to perform blot testing, “those instruments are typically dedicated to just Lyme blots; they take up precious space and require maintenance,” Dr. Theel says. “And even though we now have optical densitometry readers to help us interpret blot results, some can still be difficult to finalize, despite those readers. So they’re still challenging.”

Under the standard two-tiered testing algorithm, a reactive sensitive enzyme immunoassay is confirmed by supplemental immuno­blot testing for IgM and IgG antibodies to Borrelia burgdorferi.

The modified two-tiered testing algorithm is entirely EIA or chemiluminescent immunoassay-based. “There are still two tiers, but the second-tier assays can be immunoassays rather than blots, with the key requirement that the Borrelia antigens used in the two immunoassay tiers differ,” Dr. Theel says.

With the modified algorithm, a laboratory with an EIA processor can perform first- and second-tier testing on the same instrument and in-house, with a quicker turnaround time to result. When it comes to interpretation, “with the modified algorithm you just get positive or negative, which can be easier to interpret than remembering the number of bands you need to see on immunoblots for them to be considered as either positive or negative.” The Western blot assay is considered IgM-positive if reactivity is observed in at least two of three B. burgdorferi antigens, and IgG blots are positive if reactivity is seen in at least five of 10 antigens. While all laboratories report qualitative result interpretations, some laboratories may also report the bands that are detected for qualitatively negative immuno­blots. The reasoning for this varies, Dr. Theel says, with some clinicians wanting to know what antigens are reactive, regardless of the qualitative interpretation.

“This, however, can lead to both clinician and patient confusion, unfortunately. Patients have a hard time understanding this concept—that even though you have bands there, it doesn’t necessarily mean infection. It could be cross-reactivity or other nonspecific binding. So one of the benefits of the modified algorithm is that it eliminates the challenges of blot banding pattern interpretation and visibility on both the clinician and patient side, which I think is an improvement, particularly for those individuals who are not as familiar with the caveats associated with this sort of testing.”

Though Mayo Clinic has fully transitioned to the modified algorithm (“with a lot of notification and education,” she says), Mayo Clinic Laboratories’ reference lab clients still have the option to order the standard algorithm. “The majority of our Lyme testing is still using the standard algorithm with the immunoblots.” And the lab does report out which specific bands are detected, even if the qualitative result for the blot is negative. “Back in the day, we did not report bands and we got pushback from clinicians who wanted to see how negative was a negative and how positive was a positive, so we decided to report bands out. But now we hear from health care workers who call in and say, ‘Well, you called it negative, but you also list out detected bands, so what does that mean?’” The challenge is ongoing and it is difficult to please everyone, she notes. It’s something that the lab tries to address in its result comments.

“For the Lyme-savvy clinicians, they can look at the banding pattern to see which antibodies are present against which antigens, which in some cases can help provide some information on whether this is a new recent infection versus a more remote infection or possibly cross-reactivity, because you’ll see different antibodies develop to different antigens at different stages of infection. That’s at least partly the reasoning for including bands on negative test results.” Not everyone is aware of those nuances of testing, however. “Ultimately, it causes more confusion than it helps,” in her experience.

Dr. Theel and coauthors evaluated two distinct modified two-tiered testing algorithms using Zeus Scientific Lyme disease EIAs (Sfeir MM, et al. J Clin Microbiol. 2022;60[5]:e02528-21). “Our study,” she says, “was one of the first to look at those assays that had received FDA clearance for use as part of a modified algorithm to see how they performed.” One of the algorithms they evaluated used a VIsE1/pepC10 polyvalent EIA, followed by a whole-cell sonicate polyvalent EIA. The second algorithm used the same first-tier EIA followed by separate IgM and IgG whole-cell sonicate EIAs. In a retrospective phase, the authors compared each modified algorithm to the standard algorithm using archived samples from Lyme disease patients or control subjects.

The first and second modified algorithms were more sensitive (56 percent and 74 percent) than the standard algorithm (41 percent) among 61 patients with acute erythema migrans. In 75 Lyme disease patients with neuroborreliosis, carditis, or arthritis, sensitivity was comparable between algorithms. Compared with the standard algorithm, both modified algorithms provided increased sensitivity in erythema migrans patients, comparable sensitivity in later disease, and non-inferior specificity.