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Serologic Diagnosis of Lyme Disease

Published: May 2008

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Dr. Binnicker will discuss the epidemiology of Lyme disease, review a case of suspected Lyme disease and the Centers for Disease Control and Prevention’s current algorithm for evaluation, and discuss the tests available from Mayo Medical Laboratories and their use.

Presenter: Matt Binnicker, PhD, D (ABMM), from the Division of Clinical Microbiology at Mayo Clinic

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Welcome to Mayo Medical Laboratories' Hot Topics. These presentations provide short discussions of current topics and may be helpful to you in your practice.

Our presenter for this program is Dr. Matthew Binnicker from the Division of Clinical Microbiology at Mayo Clinic. Dr. Binnicker will be discussing Mayo Clinic’s approach to laboratory testing for Lyme disease and the attributes of the currently available tests.

In this presentation we will review the laboratory tests that are available for the diagnosis of Lyme disease and I will place an emphasis on the serologic methods that are most commonly used. In addition we will present the CDC-recommended algorithm for serologic testing and review the situations when laboratory testing is appropriate when evaluating a patient for possible Lyme disease.

Patient Case

What I’d like to do is get started with a patient case that I think will help to illustrate several key points as we move through the presentation. This case is of a 15 year-old boy who was in his normal state of good health when he developed fever, fatigue and myalgia of 3 days duration. Upon review of the patient’s history the primary care provider noted that he had returned with his family from a vacation in northern Minnesota. The boy’s mother indicated that her son had tick exposure during the trip.

The care provider suspects possible Lyme disease in this patient. The question that the care provider was faced with in this case, and the question we will address in this presentation, is what laboratory testing can be used to support a clinical suspicion of Lyme disease?

Lyme Disease

Before we begin to answer this question, I‘d like to briefly review some background information on Lyme disease. Lyme disease is a tick-borne disease that is caused by the spirochete Borrelia burgdorferi. Lyme disease occurs in a specific geographic distribution with the highest incidence occurring in the northeastern and upper Midwestern states. In addition to this specific geographical distribution, most cases occur during the spring, summer or fall months when individuals are outdoors and exposed to the tick factor.

Lyme disease continues to represent a significant public health concern with nearly 20,000 cases being reported to the Centers for Disease Control and Prevention in 2006. However it is probably difficult to accurately estimate the true number of cases of Lyme disease as the clinical presentation is often variable. Patients with early Lyme may present with nonspecific symptoms with or without Erythema migrans which occurs in an estimated 50%-70% of cases. A picture of the classic target lesion is shown on the right hand side of the slide. Patients progressing to later stages of the disease may develop systemic sequelae including arthritic, neurologic or cardiac manifestations.

Lyme Disease - Diagnostic Approach

When a patient presents for evaluation of possible Lyme disease the diagnosis is often clinical and may be based on symptoms and objective clinical findings including the presence of Erythema migrans, facial palsy or arthritis. Laboratory testing is generally not recommended in patients presenting with Erythema migrans as this clinical feature is highly suggestive of Lyme disease. In addition, lab testing should not be performed in patients lacking symptoms, inappropriate exposure history, or in those individuals that reside in a nonendemic region and lack a recent travel history to an endemic region.

For those patients requiring laboratory testing there are several diagnostic approaches available with the most common diagnostic approach being serology. Mayo Medical Laboratories offers a screening immunoassay for Lyme antibody. This is performed by enzyme immunoassay (EIA) and is available on both serum and cerebrospinal fluid. In addition to EIA testing by Western blot for IgM and IgG class antibodies to Borrelia is also available on these specimen types.

In addition to serology I just wanted to mention another diagnostic approach available at Mayo Medical Laboratories that being molecular detection of Borrelia nucleic acid by real-time PCR. This method is available on several specimen types including tissue, synovial fluid, blood and cerebrospinal fluid. Interestingly we have observed the highest detection rates of Borrelia nucleic acid using this method when testing tissue and synovial fluid and we consider these specimens to be the most optimal sources for testing by Lyme real-time PCR.

Patient Case, cont.

Now that we have reviewed the laboratory tests that are available for the diagnosis of Lyme disease, let’s return to our patient case for an update. A serum sample was submitted for Lyme serology and the result of the screening enzyme immunoassay were reported as positive. The physician receives the report of the screening EIA and questions whether additional supplementary tests should be performed to support the diagnosis.

Diagnosis of Lyme Disease

To answer this question I’d like for us to review the algorithm recommended by the CDC for serologic testing.

Currently the CDC recommends a two-step approach for the serologic diagnosis of Lyme disease. In the first tier of testing the specimen is tested by a highly sensitive screening assay such as enzyme immunoassay. If the result of the screening EIA is negative, the report should go out as negative and no further testing is generally required with the present specimen. However, in the case of short disease duration it may be beneficial to submit a follow-up specimen in 7 to 14 days if clinically indicated.

If, on the other hand, the result of the screening assay is positive or equivocal, the specimen should then be tested by Western blot for IgM and IgG class antibodies. Currently the criteria for determining whether a Lyme Western blot is positive or negative depends on the total number of diagnostic bands determined to be present on the test strip.

For instance, an IgM Western blot is positive if at least 2 of the 3 diagnostic bands are present and for IgG the laboratory must observe at least 5 of the 10 diagnostic bands for a positive result. Specimens meeting these criteria are reported as positive and these results would support a clinical suspicion of Lyme disease.

If these criteria are not met, the result is reported as negative and no further testing is required except again in the setting of short disease duration. If this is the case a follow-up specimen could be tested in 1 – 2 weeks if clinically indicated.

I just want to empathize one additional point regarding Lyme Western blot before we move on. The criteria for Lyme Western blot were established to guide the interpretation of serum samples and should not be applied when interpreting CSF results. To assist in the interpretation of CSF Western blot results, we recommend that a serum sample collected at or near the time of the spinal tap be submitted along with the CSF specimen so that both may be tested in parallel.

Lyme Disease - Serologic Assays

Although Lyme serology is a reliable method to assist in the diagnosis, there are several important limitations that we should discuss. Regarding the screen enzyme immunoassay the sensitivity of the assay may vary with the disease stage. As we can see in this table, the sensitivity of the screening EIA in stage 1 of the disease may be as low as 70%-75%. In contrast when testing is performed during stages 2 and 3 of the disease, the sensitivity increases to nearly 100%.

An additional point to consider is that early administration of antibiotics may weaken or delay the antibody response and lower the sensitivity of the EIA to 60% or 70%.

As we’ve already reviewed, specimens testing positive or equivocal by the screening method should be tested by Western blot. Lyme Western blot assays test for IgM and IgG class antibodies to Borrelia burgdorferi. Western blot is considered a supplementary serologic approach and not confirmatory due to the potential for false positives and inter and intra laboratorial result variability with this method.

As we’ve discussed, the result of Western blot should be interpreted according to the current CDC criteria and testing by Western blot is not recommended in those patients testing negative by the screen EIA.

As with the screening test, the sensitivity of Lyme Western blot is also dependent on the stage of the disease when testing is performed. During Stage 1, Western blot may demonstrate a sensitivity of 20%-50% with the majority of antibody detected being of the IgM class. In stages 2 and 3, of the disease the sensitivity of Western blot may increase to 70%-100% and we typically observe a switch in the prevalence of antibody to that of the IgG class.

It is important to point out that although a positive IgM Western blot generally indicates recent infection IgM class antibodies may persist for months to years following infection and because of this a positive IgM does not always serve as an indicator of recent acute infection.

Patient Case, cont.

If we now return to our patient case you’ll remember that the screen EIA was positive so the testing laboratory appropriately reflects the specimen to Western blot for supplementary testing. The results of the Western blot were as follows: The IgM was interpreted as positive and the IgG was reported as negative.

A common question following a result profile such as this is whether any further follow-up testing should be performed. For those patients testing positive by IgM Western blot alone, the general recommendation is to repeat serologic testing on a new specimen after 14-21 days. After this time, we should be able to demonstrate seroconversion of IgG which would then provide strong support of evidence of infection.

A few more points regarding Lyme Western blot. If testing is performed within 4 weeks of symptom onset, it is recommended that both IgM and IgG Western blot be performed. However, in patients with disease duration of greater than 1 month, IgM Western blot should not be performed. A positive IgM alone in this setting should not be used to diagnose infection due to the increased possibility of false positive results during the later stages of disease.

Returning to our patient case to wrap up, a second serum specimen was submitted 10 days later for follow-up Lyme serology. The result of the screen EIA was again positive and Lyme Western blot was determined to be positive for both IgM and IgG. These laboratory results provided strong supportive evidence that this patient had been recently infected with Borrelia burgdorferi.

Lyme Disease - Conclusions

In conclusion, the laboratory diagnosis of Lyme disease is most commonly achieved by serologic testing which should be limited to those persons with an appropriate exposure history and objective clinical findings. When serology is ordered, testing should be performed using the two step approach as recommended by the CDC, in which specimens are first tested by a highly sensitive screening method such as EIA with all screen positive or equivocal specimens being tested by Western blot. Finally, the result of Lyme serology should be used to support a clinical diagnosis in those patients being evaluated for Lyme disease.