Laboratory Testing for Lyme Disease


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Section 9: Lab Testing for Lyme Disease

The lab testing for Lyme Disease has been a source of great debate for as long as the tests have been available. Ideally, one would prefer to culture the organism from tissue or blood (the culture medium of choice is Barbour-Stoenner-Kelley medium), but this has proved highly impractical due to the low recovery rates typically observed, in part due to the paucity of organisms involved. A surrogate method, commonly used for other pathogens, is the polymerase chain reaction (PCR), which utilizes techniques to magnify limited amounts of antigen. While quite useful and specific when positive, this technique is also limited by the paucity of organisms in blood, urine and infected tissues. Other techniques for antigen capture, such as the PCR urine panel and Lyme Dot-Blot assay (LDA) from Irene Lab, are now under evaluation at the Jemsek Specialty Clinic, possibly later will be used both for screening and confirmation of state of disease.

When dealing with antibody responses to Bb or any other infection, one is always at the mercy of the vagaries of the immune system response involved. Serologic or antibody testing has always been regarded as problematic at best, with the caveat that one may “live or die” by the report. The basic variables in testing include the timing and degree of antibody response. To be useful to the clinician, these disease specific results must be easily quantifiable and reported on a scale or measurement that embodies a meaningful correlation with a clinical entity or fact. In some systems like the Immunoblot assays, however, the test is either positive or negative depending on whether a certain marker is present. The important concept about serologic testing is that no test has intrinsic value in and of itself. It is the interpretation of the test report that makes things interesting, and often there is more art than science when guidelines are being drawn. After this deed is done, both clinicians and patients have to live with what has been decided, until the next revision happens.

In virtually all infections, the IgM class of antibody (immunoglobulin) appears first and therefore represents a marker for an early infection. In most immune models, the IgM antibody gives way to the well-known IgG antibody class, usually regarded as the major enduring antibody response in chronic infectious diseases or other immune models. In almost all infection models, after just a few weeks, the IgM antibody level wanes to the point of being non-detectable and does not recur. However, a confounding fact in Bb infections is that the IgM antibody may persist for years, a very unusual situation in most disease states. Logically, one would have to conclude that this reflects an ongoing reactivation or persisting and continually renewing infectious state of Bb infection. Most active diagnostic laboratories involved with LD tend to agree with this notion. Therefore, the Jemsek Specialty Clinic acknowledges that a persistently positive IgM antibody response may qualify as laboratory confirmation for an active LD case. As discussed below, this most likely will be a Western Blot test. The presence of either a positive IgG ELISA or a positive IgG Western Blot test does not confirm active disease, and may only signify dormant or inactive (suppressed) infection.

There are various techniques accepted for screening for both types of antibodies, most notably the ELISA or IFA (Enzyme Linked Immunosorbant assay and Indirect fluorescent antibody), known for their widespread applicability and high level of sensitivity. In contrast, the Immunoblot assays are generally regarded as insensitive but highly specific since a specific set of proteins are isolated from Bb (such as outer coat antigens Osp A and Osp C) and set up in an agar electrophoresis field to be mixed with serum from the host. A positive reaction to one of the proteins is called a “positive band” and requires visual interpretation of a line or band in the agar by the laboratory personnel. In LD, by protocol per the CDC and another group called the Association of State and Territorial Public Health Laboratory Directors (ASPHLD), there are 10 proteins set up for IgG testing and 3 proteins set up for IgM testing.

Based on this information, one would naturally conclude that the ELISA would make an excellent screening test for LD and that the Immunoblot Test, in this case, the Lyme Western Blot (WB) Test, would be an insensitive, but highly specific and confirmatory assay. This is precisely the case for other models of infection, a notable example being testing for the Human Immunodeficiency Virus (HIV), a retrovirus, where this two-tier system of screening with ELISA and confirming with Western Blot technology works beautifully. In 1995, a CDC/ASPHLD joint committee apparently felt the same approach would apply for LD as they introduced a two-tier system for serologic confirmation, i.e. a positive ELISA or IFA followed by a confirmatory positive WB. Unfortunately, almost one/third patients with LD are IgG seronegative during the first year and later the percent negative by ELISA only increases, up to 50% at two years. Clearly, the next generation ELISA test will need to increase sensitivity for Bb by incorporating more unique and specific antigens in their protocol, similar to what one finds in the WB analysis.

To compound matters further, this committee also deemed that, for a WB to be positive, 5 of the10 listed bands must be reactive (qualifying bands include 23-25, 39, 31, 41,45, among others) in order to be IgG positive, a situation that merely indicates that the subject was infected sometime in the past. In order to be positive for IgM WB testing, 2 of 3 listed bands positive for IgM (qualifying bands include 23, 39, and 41) must be present. Of interest is the fact that bands 31(OspA), 34(OspB), and 93 are considered highly specific for Bb but are excluded as qualifying bands, according to current criteria. It is equally curious that the antigen for band 31(OspA) was included in the ill-fated LymeRIX vaccine, but apparently does not merit any respect from the creators of the diagnostic criteria (106).


It is curious to note that some of our patients only convert to a laboratory positive after they have received antimicrobial therapy, whether it is oral, intravenous, or a combination of the two. We suspect this phenomenon stems from Bb die-off on therapy, with an ensuing boosted immune response. As we have stated, fully one-third or more of all patients with active Bb infection will test negative with current methods (far more will be undiagnosed employing Lyme disease illiterate MDs), and so, as much as those of us in Infectious Disease would like a positive or confirmatory laboratory diagnostic report to comfort us, and we will continue to strive for this piece of paper to “soothe our souls”, we agree with the CDC that neuroborreliosis remains a clinical diagnosis.

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