Chronic Lyme Part 2: Testing and Diagnosis

One of the most challenging aspects of chronic Lyme disease is the ability to properly test for and diagnose the disease. There is controversy in the medical community about both testing and how to interpret the results. In this article, we will take a look at different testing options, their validity, and strategies for how best to interpret the results in light of clinical findings.

Chronic Lyme Diagnosis

Chronic Lyme disease is primarily a clinical diagnosis based on a person’s history, symptomatology, and physical examination (1). Laboratory testing is used to confirm clinical findings and presentations. Many, if not most, Lyme tests are not sensitive enough to detect all cases. Diagnosing Lyme – as well as convincing patients and other doctors that Lyme is present – can be difficult, especially when the patient is seronegative. The only definitive way to diagnose Lyme is from the presence of the pathognomonic erythema migrans, or bulls-eye rash. The problem is that up to two-thirds of people infected with Borrelia do not have a rash, or if they do form a rash it is often non-traditional or missed (2).

Timing

The time frame when someone gets tested for Lyme disease impacts the validity of lab results. Only half of people infected produce measurable antibodies to Lyme spirochetes in the first two to four weeks of being infected (2). This means if someone is tested too early after initial infection, they might not show positive results on labs even if they do have Lyme disease. The main antibodies tested, IgM and IgG, are also elevated at different times during the infection. IgM antibodies, which are the first responders and typically present during acute infections, “rise during the third week, peak after four to six weeks, then disappear by week eight,” according to authors Stephen Harrod Buhner and Neil Nathan (2). IgG antibodies, which play a role in long-term immunity, they note, “appear between six weeks and three months of infection and can persist for years or decades, even after successful treatment” (2). 

Other Reasons for Poor Testing Outcomes

False negatives refer to a negative test result even when the patient is clinically positive with Lyme disease. There are several reasons for this. Some individuals have a diminished response to antigens and therefore will not develop enough antibodies for testing to pick up (3). In addition, the bacteria dampens the immune response of the host, further depressing detectable markers. Some testing evaluates the presence of Borrelia DNA, however, spirochete levels may be too low for detection, even by the most sensitive testing (2). This is further complicated when antibiotic therapy is started before testing, lowering the number of spirochetes and further minimizing chances of detection. 

False-positive testing is also a possibility and is due to cross-reactivity of antibody responses to other microorganism proteins, such as the syphilis spirochete and viruses (4). 

There are many species of Borrelia known to cause Lyme disease and other illnesses. Limitations in the testing extend to the lack of specific species identification. Most ELISA and Western blot tests use assays for Borrelia burgdorferi. So if someone is tested for Lyme but has a different species of Borrelia than burgdorferi, a positive diagnosis may be missed. 

Center for Disease Control (CDC) Criteria

A two-tiered testing process is recommended for identifying Borrelia by the CDC (5). First, an ELISA (enzyme immunoassay) or IFA (Indirect immunofluorescence assay) is run. If it is negative, then no other testing is recommended. If found positive or undetermined, then the second-tier Western blot is used to confirm the diagnosis. The problem with this approach is that both types of tests have limitations.

Indirect Testing

Indirect tests, such as the ELISA, IFA, Western blot, and lymphocyte response assays, measure immune response to an infectious agent. When challenged, a healthy immune system reacts to the threat and creates specific antibodies and other immune markers. The problem with using indirect methods is that Lyme disease dampens the immune response. Therefore, results may be negative even in the presence of the disease (1). 

ELISA Testing

The ELISA test detects the presence of antibodies to Borrelia organisms. It is poorly specific as the assays are prepared from whole-cell cultured Borrelia, which lacks the reactive antigens that smaller, more specific antigens provide (6). Additionally, the assays express other antigens that can bind to non-Borrelia antibodies leading to cross-reactivity (7). Although claims are made that ELISA testing is adequate, it is not as sensitive as it should be to be considered a screening test (8). Studies have shown that only 65-70% of patients who tested positive for Lyme with culture showed an antibody response. This means 30-35% of these confirmed cases would have been missed with ELISA testing alone. There have been some advances in ELISA testing for Lyme. The C6 peptide ELISA test detects more specific proteins to B. burgdorferi. This test has been found to have high specificity with low cross-reactivity to other pathogens and has proven to be virtually equivalent to the two-tier protocol (15). It can detect early exposure to Borrelia and some studies suggest it can be used to track progression of the disease and treatment, as it has been seen to decrease with successful treatment (16).

Western Blot

The Western blot, although known to be more specific than the ELISA test, is only half as sensitive, especially in early onset of the disease (9). Western blot assays use outer protein fragments of the Borrelia bacteria and assess reactivity to these antigens by the immune system. Since proteins of the same molecular size cluster together in what are called bands, a blood specimen is tested to see how many bands the blood reacts with (2). The more bands, the more specific the diagnosis. 

According to the CDC standardized guidelines, two of three particular bands are required for a positive IgM blot result, and five out of 10 bands are required for a positive IgG blot result (6). There 

is disagreement, however, as to which bands are clinically significant as well as the number of bands needed to identify positive results. This dispute lies in the fact that the standardized guidelines were originally put in place for surveillance, not for clinical diagnosis (1). Other private and specialized lab companies consider not only IgM bands of 23-25 kDa, 39 kDa, and 41 kDa to be clinically significant, but also 31 kDA, 34 kDA, and 83-93 kDa. As for clinically relevant IgG bands, there are 10 bands tested, and of these, the CDC does not include bands 31 kDa or 34 kDa, which have both been seen to appear later in immune response and are highly Lyme specific (2). 

Cross-reactivity can also be a problem leading to false positives with the Western blot test. The 41 kDa band is often the first to react with blood but can also cross-react with other flagella-like organisms. So even though this band is considered significant, it should be considered in combination with other bands for greater reliability (1). 

There is also disparity in how the results are reported. Some labs will simply report positive or negative, while other, more specialized laboratory companies will report which bands were positive, negative, or indeterminate. Additionally, some labs quantify how strong the reaction is by indicating a single + up to multiple ++++. Having information about each band and the degree to which it was reactive is extremely helpful as it can aid Lyme-literate doctors in reading between the lines. Even in an overall “negative” Lyme result, there may be enough bands present and reactive to indicate there is some immune response to Borrelia. This type of result can be used in conjunction with the patient’s history and clinical presentation to determine if Lyme is present. Again, since the Borrelia bacteria hinders immune response, often the sickest patients with Lyme disease have negative or equivocal results (1). 

The Western blot test also has limitations in its ability to identify active versus past infection (6). Although it can provide information about the duration of infections over time, there are cases where seroconversion from IgM to IgG does not happen, even in chronic conditions. This is due to the ability of the bacteria to change its outer protein surface markers making it difficult to detect by testing and confusing the immune system into thinking it is confronting a new infection. The CDC specifies that IgM positive results alone on Western blot should only be used within the first month of infection (6). 

Some advances such as the Lyme ImmunoBlot offered by IGeneX, have increased specificity and sensitivity to 98.7% and 90.9% respectively (13). The ImmunoBlot is similar to the Western blot but rather than proteins separated by size, recombinant B. burgdorferi species antigens are sprayed at specific positions on the blot. It is designed to detect antibodies to B. burgdorferi variants, including the eight most common Borrelia strains found in North America and Europe (10).

According to a meta-analysis on test accuracy, the mean sensitivity for all tests and all samples was 59.5% and 53.7% when the two-tier methodology was used (14). All things considered – different degrees of sensitivity, specificity, criteria, and testing methods – these indirect tests have a wide range for interpretation making them unreliable for a clear diagnosis of Lyme disease. 

Other Indirect Tests

Immunofluorescence Assay (IFA) detects the combination of antibodies (IgM, IgG, and IgA) against B. burgdorferi. It is still looking at immune response, but the benefit is that it is not as dependent on the timing of testing. If the antibodies are present, the slides will fluoresce bright green when viewed with a fluorescent microscope (12). It may be used in conjunction with the Western blot but should not be used as a stand-alone test.

The ELISPOT (Enzyme-Linked ImmunoSpot) assay detects T cell activation to Borrelia-specific antigens. The benefits of this test are that it can detect this T cell reaction early on, before antibodies may be detected, or even later in the disease stage when antibodies are low (10). This makes this test especially helpful for those who are seronegative yet still suspicious of Lyme disease. Additionally, it can demonstrate quantitative aspects of the disease as it measures the amount of T cell reactivity, allowing us to assess the level of immune response, and predict disease severity (11).

Direct Tests

Direct tests are those such as microscopy, culture, and molecular methods of measurement such as Polymerase Chain Reaction (PCR), which detect the presence of the actual microbe (3). These tests can be much more definitive in identifying Lyme and other infections than indirect methods. Microbial culture is considered the “gold standard” of direct-testing methods, although not all organisms may effectively be cultured.

PCR

PCR evaluates the presence of bacterial DNA in serum or other body fluids (1). This test is very specific, although false positives are possible as DNA and mRNA can be detected in samples after spirochetes are no longer present (2). Additionally, Borrelia hide deep in the tissues and cells and are often not found in the bloodstream. PCR may be more helpful in early stages of the disease when spirochetes are prevalent in the blood (1).

Culture

Culturing refers to growing bacteria or organisms from a blood sample. The blood sample is placed on a special medium that promotes growth. If the microbe is present, it will reproduce. It can then be identified by cell formation and growth characteristics and further confirmed by immune-staining or PCR (1). This test is more reliable than the Western blot and more sensitive than PCR, however, it can take weeks or even months to produce bacterial growth. Some organisms do not grow well on the medium and spirochetes with a low concentration in the blood may be hard to find. If the sample being used does not have spirochetes present, then there will be no growth (2).

Direct Antigen Test

The Direct Antigen test looks for the Borrelia bacteria or antigens of the bacteria in urine (12). The antigens are combined with anti-B. burgdorferi-specific antibodies and treated with a stain that makes them change color for identification. This test is often done with antibiotic or antimicrobial provocation to enhance the presence of antigens in the urine (1). It can be helpful when a patient is seronegative and PCR negative, but Lyme is still suspected. 

Testing for Coinfections

Because coinfections are common in Lyme disease, it is important to test for them to understand the disease complex and guide treatment approaches. If coinfections are present, then appropriate antimicrobials must be used to address them. The clinical picture which includes history, symptoms, and physical exam should be taken into consideration when trying to identify coinfections. Like Lyme, testing can come back negative, even if a patient has the suspected infection. Some of the most common tests for coinfections include antibody testing, Fluorescent In-Situ Hybridization (FISH) tests, and PCR tests (1). The FISH test is a direct test that uses a blood smear with fluorescent dye to identify the microbe (12). If the organism is present it will fluoresce. The FISH test for Babesia and Bartonella can identify different strains of each infection and has a high level of specificity with very few false positives. It can be falsely negative, however, if there were no pathogens in that particular sample. 

Conclusion

Testing for Lyme disease is as complicated as the disease itself. Except for the presence of an erythema migrans rash, there is no guaranteed way to diagnose Lyme disease. Laboratory testing is used as a way to help clarify the possible diagnosis of infection and coinfections. Lab results should be viewed in light of their limitations and interpreted in combination with clinical presentations, physical exams, and response to treatment. 

REFERENCES

1. “Testing For Lyme .” The Beginner’s Guide to Lyme Disease: Diagnosis and Treatment Made Simple, by Nicola McFadzean, BioMed Publishing Group, 2013, pp. 79-99. 
2. “Lyme Disease and Other Borrelial Infections .” Healing Lyme: Natural Healing of Lyme Borreliosis and the Coinfections Chlamydia and Spotted Fever Rickettsioses, by Stephen Harrod Buhner and Neil Nathan, 2nd ed., Raven Press, 2015, p. 46-56. 
3. Info. “Lyme Disease and Detection.” Fry Laboratories, L.L.C, Fry Laboratories, L.L.C, 25 July 2019, frylabs.com/resources/lyme-disease-and-detection/. 
4. Naesens R, Vermeiren S, Van Schaeren J, Jeurissen A. False positive Lyme serology due to syphilis: report of 6 cases and review of the literature. Acta clinica Belgica. Jan-Feb 2011;66(1):58-59.
5. “Diagnosis and Testing.” Centers for Disease Control and Prevention, Centers for Disease Control and Prevention, 20 Nov. 2019, www.cdc.gov/lyme/diagnosistesting/index.html. 
6. Branda, John A, et al. “Advances in Serodiagnostic Testing for Lyme Disease Are at Hand.” OUP Academic, Oxford University Press, 7 Dec. 2017, academic.oup.com/cid/article/66/7/1133/4706288. 
7. “Updated CDC Recommendation for Serologic Diagnosis of Lyme Disease.” Centers for Disease Control and Prevention, Centers for Disease Control and Prevention, 15 Aug. 2019, www.cdc.gov/mmwr/volumes/68/wr/mm6832a4.htm?s_cid=mm6832a4_w. 
8. Bakken, L L, et al. “Interlaboratory Comparison of Test Results for Detection of Lyme Disease by 516 Participants in the Wisconsin State Laboratory of Hygiene/College of American Pathologists Proficiency Testing Program.” Journal of Clinical Microbiology, vol. 35, no. 3, 1997, pp. 537–543., doi:10.1128/jcm.35.3.537-543.1997. 
9. VK;, Steere AC;McHugh G;Damle N;Sikand. “Prospective Study of Serologic Tests for Lyme Disease.” Clinical Infectious Diseases : an Official Publication of the Infectious Diseases Society of America, U.S. National Library of Medicine, pubmed.ncbi.nlm.nih.gov/18532885/. 
10. “IGeneX Inc. Introduces New Diagnostic Tests for Lyme Disease and Tick-Borne Relapsing Fever.” IGeneX, 29 July 2019, igenex.com/press-release/igenex-inc introduces-new-diagnostic-tests-for-lyme-disease-and-tick-borne-relapsing-fever/. 
11. Slota, Meredith, et al. “ELISpot for Measuring Human Immune Responses to Vaccines.” Expert Review of Vaccines, vol. 10, no. 3, 2011, pp. 299–306., doi:10.1586/erv.10.169. 
12. “Test Methodologies.” IGeneX, 23 Apr. 2020, igenex.com/test-methodologies/. 
13. Lyme Immunoblot IgM and IgG, IGeneX, igenex.com/wp-content/uploads/LymeImmunoBlot-DataSheet.pdf. 
14. Cook, Michael, and Basant Puri. “Commercial Test Kits for Detection of Lyme Borreliosis: a Meta-Analysis of Test Accuracy.” International Journal of General Medicine, Volume 9, 2016, pp. 427–440., doi:10.2147/ijgm.s122313. 
15. C6 Lyme ELISA, Oxford Immunotec, 2017, www.itk.nl/files/images/ITK%20-%20C6%20Lyme%20ELISA%20Brochure_v6.pdf. 
16. Philipp, M. T., et al. “C6 Test as an Indicator of Therapy Outcome for Patients with Localized or Disseminated Lyme Borreliosis.” Journal of Clinical Microbiology, vol. 41, no. 11, 2003, pp. 4955–4960., doi:10.1128/jcm.41.11.4955-4960.2003.