A Negative Lyme Test Does't Exclude the Diagnosis of Borreliosis

Lyme disease, caused by the Borrelia Burgdorferi spirochete, is the most common vector borne disease in the United States. There is considerable debate regarding the accuracy of current testing for Lyme Diseaes.

Obtaining a positive confirmation for Lyme is hampered by several factors:

1. Antibodies against Borrelia burgdorferi [Bb] are slow to develop (1-4 weeks for early antibodies (IgM) & 4-6 weeks or more for mature antibodies (IgG)); 
2. Co-infection with other spirochetes can yield false positives; 
3. Co-infection with certain viruses can yield false negatives;
4. Co-infection with other tick-borne disease can yield false negatives;
5. Immunosupression (lack of antibodies) can yield false positives; 
6. Immunoblots (a test that looks for antibodies) are subject to human interpretation and error;
7. No single Enzyme Immunoassay Test (EIA) currently exists to detect all 21 strains of Borrelia; 
8. Early antibiotic use can yield false negatives;
9. Many doctors don't know the reason's for false negative tests, and fail to repeat them.

Unless a patient has a typical bulls-eye rash [EM-erythema migrans], most diagnoses of Lyme borreliois requires a positive laboratory test. The gold-standard for isolation of Borrelia has been by culture with confirmation by PCR. Unfortunately, PCR takes 2-6 weeks and is only 40-70% sensitive for EM, 3-17% for cerebral spinal fluid, and lower than that for synovial fluid or tissue samples. Therefore, a negative test for Lyme does not exclude the diagnosis of Lyme borreliosis. [1 ]

In U.S. patients with Erythema Migrans, serologic blood tests are only 45% accurate in the Early Phase of Lyme and only .07-7.7%  accurate in European patients. [2] 

Current Types of Lyme Testing and Their Corresponding Sensitivity: 

• Culture Tests
• Large-volume blood cultures - 45% Sensitivity
• Skin culture - 51% 
• Antibody Tests 
• 2-stage antibody testing (with interpretation of immunoblots by CDC criteria) 
• 1st step- ELISA IgM and IgG, 
• 2nd Second step-Western Blot - 29-68% (see chart below [3])
• C6 peptide ELISA using V1sE protein specific to Bb - 74-100%
• Polymerase Chain Reaction (PCR) 
• Nested PCR with amplification - 64% 
• Quantitative PCR with amplification - 81% 

U.S. Lyme Tests Kits Detect Only One Species of Borrelia--There Are Dozens of Species. 
U.S. Lyme tests kits were originally designed to detect one species of Borellia--B.burgdorferi Sensu stricto. Recently, B. bissettii-like DNA was detected in the blood serum of Northern California residents. B.bissettii was previously only associated with Lyme in Europe. [4]  In addition, a recent study of the San Francisco Bay area found B. Miyamotoi, which was just discovered in 2013 to infect humans. [5] Currently there is no commercially available test for B. Miyamotoi in the U.S. This raises the obvious question of the number of patients with Lyme-like symptoms who have gone undiagnosed, especially in California. 

To complicate matters further Dr. George Chaconas has a dramatic new paper on the inner workings of antigen variation of Borrelia burgdorferi. The study demonstrates that during the infection of a vertebrate, B.burgdorferi undergoes antigenic variation by DNA recombination allowing it to persist in the host. [6]  In another study, scientist were able to identify Borrelia spirochetes in the tissue of mice 12 months after completing a 30 day course of Ceftriaxone. Unfortunately, Borrelia was non-cultivable in these same mice. Which means a standard Lyme tests would be negative for these visible spirochetes. (see photo below) [7]

Sensitive and reliable tests for all Borrelia species is needed for timely diagnosis. The sooner a patient gets a proper diagnosis and begins antibiotic treatment the fewer complications and better chance for recovery there will be. The Department of Pathology at Milford Hospital, CT has developed a new test using direct Sanger DNA sequencing with PCR. Incredibly, this test was able to detect low levels of both B.burgdorferi and B.miyamotoi with a high level of accuracy. [8] (I will address emerging tests in the next post)

New CDC Guidelines are Too Narrow

In 2014 the CDC announced new guidelines for Lyme testing. Two-tier testing consists of an FDA-cleared enzyme immunoassay (EIA) that, if positive or equivocal, is followed by an FDA-cleared immunoblot test, commonly known as a "Western blot" test. Results are considered positive only when both the EIA and Western blot are positive. The ELISA test is very sensitive to one genotype of Bb- specifically the B31 which is found in the Northeast U.S. I do not know the sensitivity of standard tests for other species of Lyme (B.b Sensu lato)?  The Western Blot is equally narrowed. The CDC recommends culture and PCR under vary rare circumstances.[9]

Immediate Change is Needed

One thing that could be done immediately to improve the accuracy of Lyme testing in the U.S. is to stop relying on the ELISA and begin using an improved Western Blot that allows detection with more accuracy.  My second hope is that science will develop a rapid PCR or Urine test, similar to that used for AIDS, that is capable of detecting all Borrelia species. Now. 

References:

1. (a)European Lyme Borreliosis: 231 Culture Confirmed Cases
    (b)Lyme Disease: A rigorous review of diagnostic criteria and treatment
2. Clinical Infectious Disease 26 March 2001. Laboratory Diagnostic Techniques for Patients with Early Lyme Disease Associated with Erythema Migrans: A Comparison of Different Techniques. 

3.  LymeDisease.org The CDC, the FDA and Lyme Disease

4.  Journal of Clinical Microbiology March 2011. Genetic Diversity of Borrelia burgdorderi and Detection of B.bissettii-Like DNA in Serum of North-Coastal California Residents. 

5. Stanford News 18 February 2014. Stanford study says ticks may cause double trouble. 

6. University of Calgary April 2013. Genetic analysis and the effect upon mouse infection of nucleic acid metabolizing genes in Lyme disease spirochete. 

7. PlosOne: 23 January 2014. Resurgence of Persisting Non-Cultivable Borrelia burdorferi Following Antibiotic Treatments in Mice. 
8. International Journal of Molecular Science 12 May 2014. DNA Sequencing Diagnosis of Off-Season Spirochetemia with Low Bacterial Density in Borrelia burgdorferi and Borrelia miyamotoi Infections.

9. CDC.gov Lyme Disease: Two-Step Laboratory Testing Process 

Additional Reading

• A European Multicenter Study of Immunoblotting in Serodiagnosis of Lyme Borreliosis.
• Sensitive and Specific Serodiagnosis of Lyme Disease by Enzyme-Linked Immunosorbent Assay with a Peptide Based on an Immunodominant Conserved Region of Borrelia burgdorferi    VIsE
• Performance of Uninted States Serologic Assays in the Diagnosis of Lyme Borreliosis Acquired in Europe.    
• Improvement of Lyme Borreliosis Serodiagnosis by a Newly Developed Recombinant Immunoglobulin G (IgG) and IgM Line Immunoblot Assay Addition of VIsE and DbpA Homologues
• Laboratory diagnosis of Lyme Neuroborreliosis: a comparison of three CSF anti-Borrelia antibody assays. 
• Molecular Analysis of decorin-binding protein A (DbpA) Reveals Five Major Groups among European Borrelia burgdorferi sense alto strains, with impact for development of serological assays and indicates lateral gene transfer of the dbpA gene. 

Note: I am a licensed Health Professional, but I AM NOT A DOCTOR. I cannot diagnose nor can I prescribe treatment. While I will attempt to provide citations the basic information contained in this blog is simply my opinion.