I wrote a paper for my college course on Lyme serological tests. Here it is, complete with comma splices and some missing information. But I’m a student, so what do you expect? 🙂

The Need for New Borrelia Burgdorferi Serology Tests for Diagnosis

Introduction

In Canada, the number of Lyme Disease cases has been steadily increasing since it became a nationally reportable disease in 2009 (“ Lyme Disease Frequently Asked Questions,” 2013). The bacterial agent for Lyme Disease in North America is known as Borrelia burgdorferi and is a unique and complex bacteria. In Canada, our current serological tests are used to detect the presence of human antibodies to the bacteria. This presents a number of issues, including the inability to detect the bacteria, itself. Being unable to detect if the bacteria is present essentially renders the current serological tests useless in a number of cases, allowing many sufferers to go untreated. This paper will look at how the current serological tests work and how the B. burgdorferi bacteria is able to elude its host, potentially resulting in negative serological tests.

Serology Tests Detect IgG and IgM Antibodies

In order to understand how serological testing works for detecting Lyme Disease, the response of the immune system must first be explored. Initially, when a bacterial agent is introduced to a mammalian host, its immune system responds with specific antigens in order to try and fight off the infection. The first antibody that is released is the IgM antibody, mainly found in blood and lymphatic fluids. After initial infection, it can take weeks or months for the secondary antibody to be released, which is the IgG antibody. Currently, the Public Health Agency of Canada uses a 2-tier serological test for laboratory confirmation, which detects the aforementioned antibodies. Unfortunately, the presence of B. burgdorferi in its host is not always detected by the immune system and thus antibodies might not always be present. This is in large part due to the complexity of the bacteria which allows it to switch off its surface lipoprotein intermittently in order to evade the host’s immune response.

The 2-tier serological tests proposed by the Centers for Disease Control and Prevention (CDC) and adopted by the Public Health Agency of Canada (PHAC) uses an enzyme immunofluorescence assay (ELISA) in order to initially screen potential Lyme Disease patients (“Two-Step Laboratory Testing Process,” 2011). If that serological assay returns positive, it is followed by a western blot. Both of these serological tests must return positive for a laboratory confirmed diagnosis of Lyme Disease. The western immunoblots can be used to detect either IgM or IgG antibodies, depending on the laboratory and which test strip is used. The ELISA can also be used for detecting both the IgM and IgG antibodies, although it has less specificity. Prospective studies have found that the 2-tier system has a 100% sensitivity and a specificity of 99% in patients with later Lyme Disease (Aucott, 2013), which sounds great on paper, but this continues to dismiss that it is detecting an immune response to the bacteria rather than an actual infection. An immune response can be detected for years after an active infection, or can be altogether missed due to the bacteria possibly assuming a cyst formation or using “neutrophil calprotectin [to] induce a dormant state” (Stricker, 2007, p. 150). Some doctors and infectious disease specialists dismiss positive readings based on an assumption that the infection has come and gone already based on faintness of the reactive bands on the test strips, or assume that the disease was successfully treated at an earlier time and thus the infection should no longer be present (Chang, 2012).

The initial serological tests are recommended up to 4 weeks after possible disease onset, however, at least one retrospective study headed by A. Steere suggested that the IgM serology test periods should be extended by several weeks after the study had found patients with Lyme Disease were continuing to receive false negative serological results (Steere, 2008). This simple oversight in the current testing recommendation can easily lead to false negative results for individuals actively suffering from Lyme Disease.

To further evaluate the efficacy of the serological test, an article written by doctors and infectious disease experts John Branda, et al. took blood samples from patients with clinically confirmed Lyme Disease infections and found that the standard 2-tier tests only detected 27% of Borrelia burgdorferi infections in the acute phase of stage 1, 57% in the convalescent phase of stage 1, 40% in stage 2 Lyme Disease, and 100% in the final stage 3 (Branda, 2011, p. 543). This means that 73% of early stage 1 Lyme Disease were missed in this study and 43% of diagnosis were missed in the convalescent phase of stage 1. In stage 2, 60% were missed and it wasn’t until stage 3 that the tests were 100% accurate.

Borrelia Burgdorferi as a Bacteria

Borrelia burgdorferi is a spirochete bacteria most commonly transmitted to humans in North America through the bite of an ixodes scapularis, also known as a deer or blacklegged tick. Borrelia burgdorferi can cause chronic infection, persisting in an infected human despite humoral and cellular immune response. The bacteria is understood to have the ability to evade the immune response for long periods of time, however, it is not known exactly how the bacteria is capable of doing so. Numerous outer surface proteins of B. burgdorferi have been identified over the past two decades, which are likely contributing factors to its ability to evade its hosts’ immune response (Seemanapalli, 2010).

Borrelia burgdorferi is unique in its extensive amount of genetic sequences and numerous outer surface proteins. The pure abundance of its gene sequences—over 1,500—allows it to continue a fluctuation of outer surface proteins, continuing its ability to evade the hosts’ humoral response, sometimes even evading detection for extended periods of time. The outer surface protein C (OspC) is expressed upon infection of a mammalian host, which is theorized to aide the bacteria in evading humoral response. After establishing itself in the host, Borrelia burgdorferi then switches its surface lipoproteins, including the VIsE and BBF01 while also down-regulating its OspC, allowing it to further evade the mammalian’s humoral response (Seemanapalli, 2010, p. 6).

Due to its ability to evade the humoral and cellular immune response of its host, B. burgdorferi is not easily detected, especially in serological testing where detection of the immune response is required for positive readings. Despite this newer research, the blood tests which were recommended in 1995—the 2-tier serological tests—are still used for diagnosis of Borrelia burgdorferi infection before treatment can begin.

Lyme Disease Stages

Lyme Disease is divided into 3 main stages: Stage 1 includes an acute stage and a convalescent stage which occurs a few days to a few weeks after being infected. Stage 2 starts once the disease begins to spread throughout the body, infecting spinal fluid, blood, parasympathetic nervous system, the central nervous system, muscle tissue, and/or joints. The last stage, Stage 3, is when the bacteria has successfully spread throughout the body.

In stage 1, sufferers may not know they have been infected. This stage is where the typical bulls-eye rash shows in patients, however, 16% of early Lyme disease patients will not present with this rash (Aucott, 2012). Along with the potential erythema migrans rash, sufferers will likely experience flu-like symptoms during this stage of infection. The current 2-tier serological testing will only diagnose 27-56% of these patients (Branda, 2011). If the infection is caught during the first stage, antibiotic treatment will more likely be successful as the disease is still localized and has not yet begun to spread from the site of infection to the rest of the body. In stage 2, the disease begins to invade other areas of the body. As it spreads, the bacteria can cause its sufferers meningitis, paralysis of facial nerves, headaches, radicular neuropathy, cardiovascular issues of varying degrees, muscle pain, joint stiffness, joint swelling, and the flu-like symptoms of stage 1 may continue. Now that the bacteria has begun to spread, antibiotic treatments might not be as effective. Stage 3 is the last specified stage of Lyme disease. In this stage, the B. burgdorferi bacteria has successfully infiltrated the host’s body. Once the infection gets to this point, it is referred to as Late Lyme Disease and can lead to symptoms that may not be reversible.

Lyme Disease patients in Aucott’s Post-Treatment Lyme disease syndrome study found patients to have increasing symptoms after treatment once they got to stage 2 and 3 of Lyme disease. Although Aucott and his team of researchers discovered a drop in symptoms in their stage 2 & 3 Lyme disease patients during treatment, within 4 weeks the patient-reported symptoms began rising. By 6 months, the patients were reporting symptoms higher than before treatment began (Aucott, 2013, p. 80).

Conclusion

It was discussed how the early stages of Borrelia burgdorferi is difficult to detect with the currently recommended 2-tier blood tests. Statistically, the tests are 27-56% effective in stage 1 of Lyme Disease. As outlined, Borrelia burgdorferi behaves in a unique way that allows it to evade immune response, likely causing the low efficacy of the serological tests. In continuing to use the recommended 2-tier test, doctors are unwittingly allowing stage 1 Lyme Disease to progress into stage 2 and 3. Once in stages 2 or 3, the bacteria is already infiltrated into multiple body systems and treatment is less likely to keep symptoms at bay, leaving patients to suffer from prolonged symptoms and sometimes even developing “substantial disability” (Bratton, 2008).

With a potential for 100,000 new cases per year in North America (Aucott, 2013), having better serological testing is important for early treatment and prevention of numerous patients suffering needlessly for prolonged periods of time.

References

Aucott, J., Crowder, L., Kortte, K., & Rebman, A. (2013). Post-treatment Lyme disease syndrome symptomatology and the impact on life functioning: is there something here?. Quality Of Life Research, 22(1), 75-84. doi:10.1007/s11136-012-0126-6

Aucott, J., Seifter, A., & Rebman, A. (2012). Probable late lyme disease: a variant manifestation of untreated Borrelia burgdorferi infection.BMC Infectious Diseases, 12173.

Branda, J., Linskey, K., Kim, Y., Steere, A., & Ferraro, M. (2011). Two-tiered antibody testing for Lyme disease with use of 2 enzyme immunoassays, a whole-cell sonicate enzyme immunoassay followed by a VlsE C6 peptide enzyme immunoassay. Clinical Infectious Diseases: An Official Publication Of The Infectious Diseases Society Of America, 53(6), 541-547. doi:10.1093/cid/cir464

Bratton, R., Whiteside, J., Hovan, M., Engle, R., & Edwards, F. (2008). Diagnosis and treatment of Lyme disease. Mayo Clinic Proceedings, 83(5), 566-571.

Chang, Z., Cox, M., Ndukwe, N., Seriburi, V., & Wormser, G. (2012). High frequency of false positive IgM immunoblots for Borrelia burgdorferi in clinical practice.

Kenedy, M., Lenhart, T., & Akins, D. (2012). The role of Borrelia burgdorferi outer surface proteins. FEMS Immunology And Medical Microbiology, 66(1), 1-19. doi:10.1111/j.1574- 695X.2012.00980.x

Lyme Disease Frequently Asked Questions (2013, August 15).
Retrieved from http://www.phac-aspc.gc.ca/id-mi/lyme-fs-eng.php

Ogden, N., Lindsay, L., Morshed, M., Sockett, P., & Artsob, H. (2009). The emergence of Lyme disease in Canada. CMAJ: Canadian Medical Association Journal = Journal De L’association Medicale Canadienne, 180(12), 1221-1224. doi:10.1503/cmaj.080148

Seemanapalli, S. V., Qilong, X., McShan, K., & Fang Ting, L. (2010). Outer Surface Protein C Is a Dissemination-Facilitating Factor of Borrelia burgdorferi during Mammalian Infection. Plos ONE, 5(12), 1-8. doi:10.1371/journal.pone.0015830

Steere, A., McHugh, G., Damle, N., & Sikand, V. (2008). Prospective study of serologic tests for lyme disease. Clinical Infectious Diseases: An Official Publication Of The Infectious Diseases Society Of America, 47(2), 188-195. doi:10.1086/589242

Stricker, R. B. (2007). Counterpoint: Long-Term Antibiotic Therapy Improves Persistent Symptoms Associated with Lyme Disease. Clinical Infectious Diseases, 45(2), 149-157. doi:10.1086/518853

Two-Step Laboratory Testing Process (2011, November 15). Retrieved from http://www.cdc.gov/lyme/diagnosistesting/LabTest/TwoStep