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No-blot testing charts new course for Lyme Dx

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Anne Paxton

January 2020—“You can eliminate blots altogether!” may sound like a pitch for a cleaning product. But it’s a line from a webpage of Zeus Scientific touting the new algorithm for Lyme disease testing that ditches Western immunoblot in favor of a two-enzyme immunoassay test sequence.

The new algorithm, which the CDC recommended last year, is the first approved alternative to the standard algorithm in 25 years.

“The big picture is, it’s a change,” says Carol A. Rauch, MD, PhD, adjunct associate professor of pathology, microbiology, and immunology at Vanderbilt University School of Medicine. “And by doing it differently, the components of possibly unintended Western blot testing and unintended interpretation of bands as positive results when they are actually negative, and people reacting to bands at all, may be minimized. I’d like it to be headed in a healthier direction so that we can get everybody the right diagnosis.”

The update in the CDC’s guidelines is significant for a variety of reasons, says John A. Branda, MD, associate director of the microbiology laboratory at Massachusetts General Hospital. “The modified algorithm may bring only an incremental improvement in Lyme disease diagnosis, but it’s an important change.”

Since 1994, the strategy of testing for Lyme disease has been to use EIA and a confirmatory Western immunoblot to achieve high sensitivity and specificity. But this standard algorithm has weaknesses that include insensitivity during the first weeks of Lyme disease and a confirmatory test with its own limitations. Lyme is also one of the rare diseases over which there is a running public controversy about how the diagnostic test should be interpreted.

The CDC’s recommendation followed the FDA’s clearance of Zeus’ existing Lyme disease ELISAs for use in the modified two-test method. With improved EIA design and development, studies have shown, a single multiplex or a two-tiered strategy involving two different EIAs performs as well as or better than the traditional algorithm.

By eliminating the need for the confirmatory Western immunoblot test, experts in Lyme disease diagnostics suggest, the modified algorithm is likely to speed test results and lead to fewer reference lab tests. It may also lower costs, reduce false-negative results in recently infected patients, and improve time to treatment. And there are hopes that the modified algorithm could reduce misinformation among the public about Lyme disease diagnosis.

Four Zeus ELISAs already approved for Lyme disease testing but typically used only for the first tier of the standard algorithm are now CDC endorsed for use in both tiers of the modified algorithm: Zeus ELISA Borrelia VlsE1/pepC10 IgG/IgM Test System, Zeus ELISA Borrelia burgdorferi IgG/IgM Test System, Zeus ELISA Borrelia burgdorferi IgM Test System, and Zeus ELISA Borrelia burgdorferi IgG Test System.

Under the standard algorithm, a reactive result from one of those tests was reflexed to a Western blot test for confirmation. The CDC has now okayed conducting a second ELISA—or in some cases two more ELISA tests—instead, explains Elitza S. Theel, PhD, director of the infectious diseases serology laboratory at Mayo Clinic in Rochester, Minn.

“Zeus has put forth two different versions of the modified algorithm using these ELISAs, which may appear to be more complicated,” Dr. Theel says. “Regardless of which modified algorithm version you choose to use, however, Zeus indicates to start with the VlsE1/pepC10 IgG/IgM ELISA as the first-tier test and then, if that’s reactive—positive or equivocal—you can do one of two things.”

‘In my opinion, it’s more helpful to have that differentiation
for second-tier test results.’ — Elitza Theel, PhD

“In the first version, the second-tier assay is the Zeus whole-cell antigen IgM/IgG ELISA, which doesn’t differentiate between which class of antibody is present. Alternatively, the other version of the modified algorithm involves performing two separate ELISAs for second-tier testing. These two ELISAs are also based on whole-cell antigen material, but they test for IgM and IgG separately, so you would be able to tell which class of antibody you’re detecting.”

For diagnosis, it’s important to know whether the antibodies are IgG or IgM, she says. “The current CDC guidelines state that if the patient has had more than 30 days of symptoms at the time of testing, only the IgG component should be considered. On the other hand, if the patient has had less than 30 days of symptoms, you’d want to look at that IgM antibody result as well. In my opinion, it’s more helpful to have that differentiation for second-tier test results. Are we just detecting IgG or just IgM, or is it both? With the total IgM/IgG ELISA, you can’t answer that question.”

Knowing the antibody class would not necessarily have treatment implications. “It would help guide clinicians on whether the patient has an acute or recent infection, or if just IgG-class antibodies are present, it might be more suggestive of a past infection given that IgG-class antibodies to Lyme disease can persist for months or years.” In such a case, “the clinician has to interpret the results in context with the patient’s presentation and duration of symptoms at the time of testing.”

Testing for antibodies to Borrelia burgdorferi, the causative agent of Lyme disease, is typically performed on routinely collected serum samples, or testing can be added on to already collected samples. But the standard algorithm has multiple weaknesses, Dr. Theel says. “First, its sensitivity in the early stage of Lyme disease is pretty poor, reported as about 40 to 60 percent, depending on the study. The immunoblot test can also be challenging for some laboratories to implement. And interpretation of the banding patterns, by the lab, clinicians, and increasingly also by patients, can be confusing.”

The modified algorithm has been winning support in part because the EIA method provides increased sensitivity during the early stage of disease. “Depending on the study and the combination of ELISAs used, sensitivity increases from 10 to maybe 20 percent for modified algorithms versus the standard algorithm.” In later stages of infection, when the patient might have Lyme-associated arthritis or arthralgia, the test sensitivity is about the same whether the standard or modified algorithm is used, Dr. Theel says.

The Western immunoblot has limitations as well. Originally—and some labs may still do it this way—the test is performed manually and the presence or absence of bands is interpreted visually. “That comes with quite a bit of subjectivity and variability in interpretation. Other labs, including many reference labs, have moved to processing these blots using an automated system, and band presence is determined by taking advantage of scanning software to provide more objective and standardized results.”

“While this has somewhat improved the subjectivity associated with interpretation of blot results, there are still challenges associated with blot testing, including false-positive results, particularly with IgM blots. Also, from a lab perspective, we have to maintain competency in blot testing, and for smaller hospital labs that send their blot testing to commercial labs, the turnaround time to results is significantly longer.”

The three most common causes of false-positive blot test results are syphilis, infectious mononucleosis, and multiple sclerosis, Dr. Theel notes. Fortunately, the typical treatment for Lyme, a course of doxycycline, doesn’t subject patients who are being treated unnecessarily to too many side effects. “But the bigger problem is that you are missing the true diagnosis and unnecessarily administering antibiotics.”

Large numbers of providers order the test for multiple reasons, she says. “Lyme symptoms are fairly nonspecific: fever, fatigue, headache, myalgia, arthralgia. So if the patient presents with those symptoms, in a Lyme disease-endemic area in the right season, chances are they’re going to get Lyme disease testing.”

Lyme disease’s prominence in the media enters into the test-ordering decision too, Dr. Theel says. “Frequently, we see patients who come in and specifically request testing for Lyme disease, so there’s some pressure on providers to perform the testing even though the patient may not meet the criteria or epidemiologic factors for Lyme disease. This can lead to overtesting for Lyme disease, which in low-risk individuals equates to a higher risk for false-positive results.”

People sometimes misunderstand Lyme disease and how it can be diagnosed and treated. “There have been a lot of good public health campaigns to make people more aware,” says Laura Gillim-Ross, PhD, discipline director for infectious disease immunology at LabCorp and a director in the Department of Science and Technology. “What I try to stress when I talk to providers is that the laboratory test is just a tool to aid in the diagnosis and should be considered in conjunction with clinical presentation and epidemiological factors.”

LabCorp and other labs cannot police whether a patient has symptoms, she notes. “We test what we receive. And we—and I’m sure all labs—receive a fair number of samples from patients who do not have travel history or known tick bite or anything else that would connect them with Lyme. Clinicians at times may test for numerous infectious diseases, especially if they aren’t savvy enough about the testing’s weaknesses.”

In Dr. Gillim-Ross’ view, the modified algorithm should make interpretation easier, “as long as the sample is being submitted on a patient who meets the recommended testing criteria.”

Two-tier serologic testing has an edge over the standard algorithm for Lyme testing because of performance and practical advantages, Dr. Branda says. “What made it feasible, and what allowed CDC to endorse this test, was that if you use two different EIAs sequentially or concurrently, you can achieve specificity equal to that of conventional two-tiered testing with ELISAs followed by Western immunoblots.”

“In very early infections, the sensitivity of the modified approach is significantly better than with the conventional testing because ELISAs tend to be more sensitive.”

The main practical advantage, Dr. Branda says, is that ELISA tests are much more approachable and feasible for typical clinical laboratories than Western blots. “You can do two ELISAs, which are objectively interpreted, tend to have very high throughput, can easily be automated, and, for all those reasons, tend to have short turnaround time.” It’s also generally less costly to perform two ELISAs than it is to perform an ELISA followed by an IgM/IgG Western blot.

For straightforward cases of suspected Lyme handled by non-Lyme disease experts, he says, “a lot of the information that is lost by doing the modified approach may not have been well used in the first place and may even have sometimes been misinterpreted.”

That is, “one does lose some information by replacing Western blotting with another ELISA because Western blotting not only gives us detailed information about the presence of an antibody response, and whether it is a predominantly IgM or IgG antibody response, but also tells us something about the maturity and breadth of the antibody response.”

But the modified two-tier testing has the potential to simplify interpretation in the vast majority of cases, Dr. Branda says. “On the flip side, if the case is difficult and challenging and more information is needed than can be acquired through ELISA, then having only the modified two-tier testing will make interpretation more difficult in a way. In those cases one would still want to pursue Western blotting to get as much information as possible.”

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