Summary Points
- Lyme disease (LD) is a growing public health burden and in a select group of patients may lead to post-treatment Lyme disease syndrome, which can persist for years.
- There are many LD tests, but the two-tier serologic test is still considered the most accurate and useful.
- Patients can test positive on two-tier testing for both IgG and IgM antibodies even after they have been successfully treated for LD.
- The use of alternative LD testing or "home brew" testing is not reliable and should be avoided due to the increased number of false positives.
By Traci Pantuso ND, MS
Adjunct Clinical Faculty Bastyr University, and Owner/Physician, Harbor Integrative Medicine, Bellingham, WA
Dr. Pantuso reports no financial relationships relevant to this field of study.
In August 2013, the Centers for Disease Control and Prevention (CDC) reported that 300,000 cases of Lyme disease (LD) are diagnosed each year and that the 30,000 reported cases is 2008 are a gross underestimation. The CDC investigated laboratory reports and clinical records and surveyed the public to collect these data.1,2 Unfortunately, the entirety of the research results have yet to be published to shed further light on the discrepancy of the LD statistics.1,2
This August 2013 announcement came in the wake of an April 2013 CDC publication recommending that clinicians order the standard of care test (two-tier serologic testing) for LD when indicated and to not use alternative laboratory testing also known as "home brew" testing for LD that has not been approved by the FDA.3,4 The article discussed a recent investigation into a novel laboratory-developed test that claimed to be able to identify Borrelia burgdorferi (Bb) through a two-step pre-enrichment process followed by immunostaining with or without polymerase chain reaction (PCR) to identify the bacterium and be considered "culture positive." The CDC reports that there was a concern regarding increased false positives due to laboratory contamination leading to the misdiagnosis of LD in patients.3,4
Unfortunately, it is perceived that there are limitations in the CDC recommended two-tier serologic LD testing, leading to the use of unapproved LD testing by patients and clinicians.1-3,5,6 However, the use of non-validated LD tests also has issues; there may be false-positive results and unnecessary antibiotic use.3,7 This article will review some of the details about LD testing as a guide for clinicians whose patients are trying to negotiate this often confusing topic.
Background
LD is a tick-transmitted infection caused by the spirochete bacteria Bb.1 Bb was first described in the United States in 1975 by Steere et al.1,8 Bb was reported to be the causative agent of LD disease in 1983.8 The symptoms of LD were reported for many years in Europe prior to the discovery in the United States.8 LD is the most common vector-borne illness in the United States.1 In 2013, 27,203 confirmed cases of LD were reported in the United States and more than 80,000 cases in Europe.1,9
Symptoms of LD may include fatigue, headache, arthralgia, myalgia, and fever. The most common sign of Bb infection, occurring in 80% of infected individuals, is the erythema migrans (EM) rash that appears 1-2 weeks after a tick bite, at the site of the tick bite.1,5-7 EM is also referred to as the "bulls-eye rash" even though two-thirds of EM are uniformly erythematous or will have an enhanced central erythema without clearing around it.1,5-7 Other symptoms that may occur after Bb disseminates into other tissue are cranial nerve palsy, meningitis, and cardiac manifestations such as atrio-ventricular block.1,5-7
Arthritis is a symptom of a later stage of disseminated LD and occurs in < 10% of all cases, partly because most patients are treated earlier.1,5-7 The majority of patients respond favorably to antibiotic treatment.1,5-7 Despite being treated for LD, approximately 10-20% of patients report having post-Lyme disease symptoms such as musculoskeletal pain, short-term memory and concentration difficulties, and fatigue; if these symptoms last for 6 months or longer, the patient is then considered to have post-treatment-Lyme disease syndrome (PTLDS).1,7 The etiology of PTLDS is not currently known and may be due to systemic inflammation or autoimmune pathologies occurring after Bb exposure.1,5-7
Bb Genospeices
A number of Bb species are known to cause disease (see Table 1).6 Also, although EM usually occurs in the early phase of infection while arthritis occurs later, some of these symptoms have also been linked to specific Bb species (see Table 2).6 Only Bb is known to cause human LD in North America.
Bb Antigenicity
Numerous immunogenic Bb surface, cytoplasmic, and membrane antigens have been described. Bb does have a number of outer surface proteins (OSP).5-7,10-12 These OSPs are differentially expressed among the different genospecies and environmental triggers.10 OSPs are upregulated and important to Bb establishing an infection in the host.13 The cell envelope of Bb differs from other bacteria such as Escherichia coli, as it has an outer layer that is fluid and is bilayer with phosphatidylcholine, phosphatidylglycerol, and cholesterol glycolipids.10 There are also numerous lipoproteins on the surface of the bacterium. Bb also has flagella that are immunogenic. Antibody responses are elicited in most patients with early production of immunoglobulin M (IgM) reducing the burden of the spirochete. Immunoglobulin G (IgG) production by B cells is usually measurable by the second week of Bb infection.10 Bb evades antibodies by changing expression of antigenic molecules and may suppress host inflammation to evade detection.10,14
Laboratory Testing
Because of the different species of Borrelia and the mechanisms that the bacterium uses to evade immune detection, clinical laboratory diagnosis is challenging. Antibody production to the Borrelia bacterium vary by individual and isolating the Borrelia bacterium is also challenging.11,15 The two broad categories of laboratory testing used to identify infection with Bb are direct and indirect detection.
|
Table 1: Pathogenic Borrelia Species and Location |
|
|
Borelia |
Continent |
|
Borrelia burgdorferi |
North America |
|
Borrelia afzelii, Borrelia garinii, Less common but have demonstrated pathogenicity in some patients: Borrelia bissettii, Borrelia lusitaniae, and Borrelia valaisiana |
Europe |
|
Borrelia garni |
Asia |
|
Table 2: Pathogenic Borrelia and Association with Symptoms10 |
|
|
Borelia |
Symptoms |
|
B. afzelii |
Skin manifestations |
|
B. garinii |
Neurotrophic |
|
B. burgdorferi |
Arthritogenic |
The direct types of laboratory testing include culture, microscopic assays, and Lyme-specific proteins and/or nucleic acids using tests such as PCR. The most specific and conclusive test is a positive culture of the Bb clinical isolate, but it has drawbacks (culture difficulty and low concentration of Bb in specimens), limiting its clinical usefulness.11 However, it is possible for Bb to be cultured from skin biopsies of the EM lesion. In a study investigating 21 patients in the United States with EM that were biopsied, recovery of Bb was 86%.16 In patients who are being treated with antibiotics or the EM has resolved, Bb is rarely recovered from the site.17 Bb can also be isolated and cultured from plasma or serum; however, a large amount of plasma is required to isolate a significant number of organisms to culture.11 Clinical isolates of possible Bb infection require up to 12 weeks of culture to determine that the sample is in fact negative.11 A positive culture can be identified at 4 weeks, according to some researchers’ unpublished data.11
PCR
PCR techniques can be useful in the diagnosis of LD because they can be used on clinical samples with few Bb organisms. The quality of the PCR results are dependent on the different primers that are used and host tissue can also interfere with the results.18 In blood and CSF, PCR testing for Bb DNA is not recommended due to low sensitivity.19 PCR has an 80% positive result rate when used to test synovial fluid samples for Bb DNA in untreated LD arthritis patients.
Indirect Methods for LD Testing
Immunofluorescent-antibody assays (IFA), enzyme-linked immunosorbent assay (ELISA), and western blot (WB) are all indirect methods that are dependent on the host antibody response to Bb. The two-tier serologic testing for Bb antibodies uses either IFA or ELISA followed by a WB. The currently recommended lab test for LD is the two-tier serologic testing for antibodies to Bb.
If the IFA or ELISA (first-tier) test results are equivocal or positive, then an IgG and IgM WB is performed on the same serum sample.1 Most patients who have had a demonstrated tick bite with EM and other non-specific symptoms of infection for at least 4 weeks will have a positive IgG. Due to a natural delay in antibody response, in the first 2 weeks after infection, the two-tier testing for LD is considered insensitive and unreliable.
Patients who are testing seronegative for 6-8 weeks and have not been treated for LD are unlikely to have LD.1,6 Specificity of the two-tiered testing after 30 days has been reported to be 98-100%; however, this is dependent on the control serum that is used during testing.9,15 In regions where LD is endemic to the population, there is a high seropositive background rate, which can confound testing. A positive LD serological test does not mean that the patient has an active infection. IgG and IgM antibodies to Borrelia persist in patients for years after resolution of LD The CDC recommends only performing IgG and not IgM WB if the patient has been symptomatic and there is a clinical suspicion of LD for 30 days or more to prevent false positive results.1 Related to this is the caveat that the IgM WB test has an increased false positive rate after 30 days of infection.
Some nuances to the use of indirect LD testing
ELISA, IFA, and WB are all dependent on the host antibody response to Bb, which affects the testing sensitivity and specificity. The timing of these indirect tests is important, as is a competent immune system. Independent variability in antibody responses to the Bb infection can further complicate the indirect method of testing.
Patients who are treated in the acute phase may not test positive on the two-tier serologic assay. Such patients may continue to have false-negative results because they were treated appropriately and did not develop a strong antibody response.1
Alternative Testing Regimens
There are a number of alternative testing regimens offered by various laboratories that are not FDA approved. These non-FDA approved Bb tests are not recommended by the CDC and are considered "home brew," which means that they use in-house validation and different interpretation criteria for their laboratory tests. These tests rarely have data published comparing them to the two-tier serologic testing standard. The CDC recommends avoiding the following tests:
- Urine antigen capture tests
- Lymphocyte transformation tests
- Quantitative CD57 lymphocyte tests
- "Reverse Western Blots"
- WB using in-house interpretation criteria
- Measurements of antibodies from joint synovial fluid
- WB IgG/IgM without first performing an ELISA or IFA assay
- Cell-wall deficient or cystic forms of B. burgdorferi cell sorting
- Immunofluorescent staining
There is disagreement between the CDC and the IDSA with the International Lyme and Associated Diseases Society (ILADS) with respect to some of these "alternative" LD tests.1,21,22 The ILADS is a group of medical doctors and researchers who have published evidence-based guidelines regarding antibiotic treatment for persistent, recurrent, and refractory symptoms, postulated to be due to chronic LD.22 Because the ILADS protocols veer away from standard of care, many health care providers are wary about following the type and long duration of antibiotic treatment recommended.
CO-INFECTION TESTING
Tick co-infections can include Babesia microti, which is the primary cause of babesiosis, and Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis (HGA). HGA is formerly known as human granulocytic ehrlichiosis. Both of these co-infections can be symptomatic and should be tested for in patients who are infected with LD and present with the following per IDSA guidelines:
- High-grade fever for > 48 hours despite antibiotic therapy for LD
- Acute onset of fever, chills, and headache
- Unexplained leukopenia
- Thrombocytopenia
- Increased liver enzymes
- Anemia
- Resolution of EM and yet worsening or no improvement in viral like symptoms
- In a geographic areas known to have B. microti and A. phagocytophilum
HGA Diagnostic Testing
HGA can be detected on blood smears; however, the best test is the indirect fluorescent testing in both the acute phase and in the convalescent phase. Treatment can be started per IDSA guidelines without testing results in symptomatic patients. Treatment in seropositive but asymptomatic individuals is not recommended due to the fact that such mild co-infections are usually self-limiting.21
Babesiosis Diagnostic Testing
Active Babesia species causing babesiosis require treatment. The IDSA recommends diagnostic testing through the identification of the Babesial parasites in a blood smear or through PCR. If the patient is asymptomatic and no Babesial parasites are noted on the blood smear but the patient is seropositive for Babesial antibodies, he/she should not be treated.
Treatment is not recommended in asymptomatic individuals unless they have positive Babesial smears and or PCR that are positive for > 3 months. One caveat with Babesia testing is that there are a number of species in the genus and there are specific antibodies for certain species, which can confound testing. Additional information is available through IDSA guidelines.21
To test or not to test
Beyond the scope of this article is a complete review of the signs and symptoms of LD infection and the clinical decision-making relevant to whether testing is even indicated. For example, there are a subset of patients for whom treatment is indicated and testing is not, including those who have a typical EM rash. The references provided in this article provide information relevant to testing in the context of a given clinical scenario.
CONCLUSION
When LD testing is indicated, the current standard of care is the FDA-approved and CDC-recommended two-tier serologic testing. There is a lack of evidence for using alternative or "home brew" laboratory tests, as illustrated by the recent April 2014, CDC alert regarding the importance of not ordering unapproved or unvalidated LD tests. Furthermore, the FDA announced on July 31, 2014, that it will begin to start regulating the field of "home brew" laboratory testing.12 Hopefully, these new regulations will assist in the development of novel, fast, and accurate LD tests for patients with an unclear diagnosis or cause of lingering symptoms.
REFERENCES:
- Centers for Disease Control and Prevention. Lyme Disease. Reported cases of Lyme disease by state or locality, 2004-2013. Available at: http://www.cdc.gov/lyme/stats/chartstables/reportedcases_statelocality.html. Accessed Oct. 14, 2014.
- Kuehn BM. CDC estimates 300,000 US cases of Lyme disease annually. JAMA 2013;310:1110.
- CDC. Notice to readers: Caution regarding testing for Lyme disease. MMWR 2005;54:125. Available at: http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5405a6.htm. Accessed Oct. 14, 2014.
- Johnson BJ, et al. Assessment of new culture method for detection of Borrelia species from serum of lyme disease patients. J Clin Microbiol 2014;52:721-724.
- Shapiro ED. Clinical practice. Lyme disease. N Engl J Med 2014;370:1724-1731.
- Stanek G, et al. Lyme borreliosis. Lancet 2012;379:461-473.
- Feder HM, et al. A critical appraisal of "chronic Lyme disease." N Engl J Med 2007;357:1422-1430.
- Sigal LH. Lyme disease: A review of aspects of its immunology and immunopathogenesis. Annu Rev Immunol 1997;15:63-92.
- Wormser GP, et al. Utility of serodiagnostics designed for use in the United States for detection of Lyme borreliosis acquired in Europe and vice versa. Med Microbiol Immunol 2014;203:65-71.
- Radolf JD, et al. Of ticks, mice and men: Understanding the dual-host lifestyle of Lyme disease spirochaetes. Nat Rev Microbiol 2012;10:87-99.
- Aguero-Rosenfeld ME, et al. Diagnosis of lyme borreliosis. Clin Microbiol Rev 2005;18:484-509.
- No authors listed. Home-brew tests need regulation. Nature 2014;512:5.
- Seemanapalli SV, et al. Outer surface protein C is a dissemination-facilitating factor of Borrelia burgdorferi during mammalian infection. PLoS One 2010;5:e15830
- Norris SJ. Antigenic variation with a twist—the Borrelia story. Mol Microbiol 2006;60:1319-1322.
- Molins CR, et al. Collection and characterization of samples for establishment of a serum repository for lyme disease diagnostic test development and evaluation. J Clin Microbiol 2014;52:3755-3762.
- Berger BW, et al. Cultivation of Borrelia burgdorferi from erythema migrans lesions and perilesional skin. J Clin Microbiol 1992;30:359-361.
- Berger BW, et al. Failure of Borrelia burgdorferi to survive in the skin of patients with antibiotic-treated Lyme disease. J Am Acad Dermatol 1992;27:34-37.
- Molloy PJ, et al. False positive results of PCR testing for Lyme disease. Clin Infect Dis 2001;33:412-413.
- Dumler JS. Molecular diagnosis of Lyme disease: Review and meta analysis. Mol Diagn 2001;6:1-11.
- Steere AC. Reinfection versus relapse in Lyme disease. N Engl J Med 2012;367:1950-1951.
- Wormser GP, et al. The clinical assessment, treatment, and prevention of Lyme disease, human granulocytic anaplasmosis and babesiosis: Clinical practice guidelines by the Infectious Diseases Society of America. Clin Infect Dis 2006;43:1089-1134.
- Cameron D, et al; ILADS Working Group. Evidence-based guidelines for the management of Lyme disease. Expert Rev Anti-infect Ther 2001:2(1 Suppl):S1-13.
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