Lyme is caused by an infection of the Borrelia spirochete (phylum Spirochetaete) and is the most common vector-borne infectious disease in the United States. With over 300,000 Americans being infected annually, Lyme presents a serious public health concern. Unfortunately, the current testing for Lyme does not account for the wide genetic variability or the multiple variant forms of Borrelia. In addition, the level of spirochetes in the blood can run so low that they may be undetectable at the time of testing.
Currently, there are 112 known Borrelia genomes listed on the Bacterial Bioinformatics Resource Center (PATRIC.) [1] These are divided into twenty-eight Lyme Borrelia [LB] strains and six Relapsing Fever [RF] Borrelia [B] strains. Eight of these LB strains known to infect humans are recognized as B. burgdorferi, B. afzeli, B. garni, B. bavariensis, B. spielmanii, B. valaisiana, B. bissetti, and B. finlandsis. Five of the RF species are recognized as B. recurrentis, B. duttonii, B. crocidurae, B. hermsii, and B. turicatae. Recently, B. miyamotoi, a newly discovered RF species, has been genetically sequenced and has two known genomes.
The Borrelia Data Base
Up until now there has not been a database available to the public dedicated towards cataloging and sharing Borrelia genomes. In June 2014, Stony Brook Dept of Medicine launched a database known as BorreliaBase. [2] BorreliaBase is a phylogenetic database showing the genomic relationships among known and emerging species of Borrelia. This interactive database allows browsing and comparative analysis to scientists throughout the world. [3] Genomic sequencing is the key to unraveling the mystery of Lyme diagnosis and treatment.
As stated in my previous blog, the current 2-tier diagnostic testing for Lyme Disease is about 44% sensitive to Borrelia burgdorferi in the early phase. Accurate testing is critical to ensure the proper diagnosis and early treatment for Lyme. To make matters worse, the current Lyme test is geared towards detecting only one of the 21 known species of Borrelia, B. burgdorferi. It doesn't take a rocket scientist to surmise there are a vast number of patients receiving false-negatives for Borrelia infections.
Stony Brook Direct Sanger DNA
The Department of Pathology at Milford Hospital, CT has developed a new test using direct Sanger DNA sequencing with PCR.[4] This test was able to detect low levels of both B burgdorderi and B miyamotoi with incredible accuracy. [5] At present Milford is the only licensed clinical laboratory in the U.S. to officer a combined DNA sequencing-based diagnostic test. It costs $150 and the results are ready within 5 days of a samples receipt. [6]
During the IDWeek Conference in October of 2014, Dr. Benjamin Luft of Stony Brook University presented the results of an experimental test for Lyme. Using the genomic information from BorreliaBase they were able to isolate 48 antigenic proteins from multiple genospecies of Borrelia. From this they identified 11 individual proteins that were 80% sensitive and 99% specific to B burgdorferi in the early phase of infection. This is almost twice the accuracy of the current FDA approved 2-tier test! Dr. Luft believes that this new test has the potential to become a rapid single-tier test. Using recombinant Borrelia protein, this test has a much broader range of coverage and could possibly detect multiple species of Borrelia. [7] This is exciting news!
Johns Hopkins SYBR Green I/PI assay
Currently researchers at Johns Hopkins Bloomberg School of Public Health are working on a test that will better allow clinicians to check for Borrelia that persist after completion of antibiotic treatment a condition that may lead to the proper diagnosis of "Chronic Lyme" or "Post Treatment Lyme Disease Syndrome." Using the SYBR Green I/PI assay they were able to detect how many bacteria were alive or dead after exposure to various drugs. What they found is that frontline antibiotics such as doxycycline and amoxicillin work well for the growing spirochetal-form of B. burgdorferi, but have limited effectiveness on variant forms--spheroplast (L-form), cyclic (round-form), biofilm-like colonies or persister forms of B. burgdorferi. [8] In fact, researchers found that while Doxycycline reduced the spirochetal form by 90% it increased the number of round body forms about twofold.
IDSA Paper on Metabolic Biosignatures
Just this week, the IDSA has published a study on an emerging test for Lyme. In their paper they acknowledge
July 2015 Update:
-Translational Genomics Research Institute (TGen) Using DNA Sequencing as Diagnostic tool.
Scientists at TGen's Pathogen Genomics Division in Flagstaff, AZ - TGen North- will use the power of targeted DNA sequencing to develop and validate a test to measure the presence and severity of tick-borne Lyme disease at the genomic level.
By analysing a sample of DNA, the new test should be able to pinpoint Lyme disease, identify multiple strains of Lyme, detect other tick-related infections, and show non-Lyme causes of disease. [10]
August 2105 Updates:
-Salivary Diagnostics, developed by Forsyth Institute, is developing a test that will be able to detect Tick-Borne Infections. Founded in 2013 with a $4.2 million dollar grant from the Massachusetts Life Sciences Center, the goal of Forsyth’s Center for Salivary Diagnostics is to revolutionize the medical diagnostics world and replace blood-based tests with salivary tests.
A saliva-based test is being developed for this insidious tick-borne infection. Forsyth investigators have discovered that components of the bacterium that causes Lyme are shed into body fluids including saliva. The test would allow rapid diagnosis and cure with a simple antibiotic regimen, before the disease becomes chronic and debilitating. [11]
September 2015 Update:
-Nanomedical Diagnostics, Inc. based in San Diego, CA has developed a graphene biological field effect transistor (BioFET) technology which will improve the ease, speed and cost of personalized healthcare. The CDC and Nanomedical Diagnostics are entering into a Cooperative Research and Development Agreement to evaluate direct electronic detection of Borrelia burgdorderi antigens for a new Lyme disease diagnostic system. Nanomed has launched AGILE Research, a label-free, quantitative, low-cost biosensor for small molecule and protein analysis. AGILE Research is slated to launch early in 2016 and will be the focal point in the Lyme Disease beta test at Stanford University. [12]
January 2016 Update:
-The IDSA contends that isolation of Borrelia spp by culture is the diagnostic "gold standard" of Lyme testing, however multiple recent studies have proven that Borrelia bacterium is not easily cultured. As highlighted in this article by Dr Emir Hodzic, with the Center for Comparative Medicine, School of Medicine, University of California at Davis, Polymerase Chain Reaction is by far the most accurate method for detecting all species of Borrelia.
July 2016 Update:
IGeneX - Improved Sensitivity of Lyme Disease Western Blots. In an effort to improve the Western Blot sensitivity without decreasing specificity, IGeneX has added two strains of Borrelia Burgdorferi (Bb), B31 and 297, to their immunoblot preparation. They conducted a study with sera from a diverse group of 364 American and European patients and controls. The combined IgG and IgM sensitivity of the Western Blot alone increased from 77.1% to 88.6% by adding 297 (a recombinant OspA antigen) to the standard B31. With the increased sensitivity this test is considered positive with IgG or IgM reactivity with any two of six Bb antigens--23, 31, 34, and 93 (Bb has 3 distinct Outer Surface Protiens (Osp). 23 indicates OspC, 31=OspA, 34= OspB and 93kDa=flagellum antigen specific to Bb) [20] Note: the sensitivity of the CDC recommended combined Two-Tier Lyme test averages just 50%. By removing the 1st tier, the ELISA, the sensitivity of the Lyme test improves dramatically. Using IGeneX in-house WB strips and in-house interpretive criteria the sensitivity increased from 88.6% to 97.1%, however the specificity decreased from 100% to 95%IgG and 93%IgM.
August 2016 Update:
The Chinese CDC, In Collaboration with State Key Laboratory of Infectious Diseases Prevention and Control, have Developed a Novel Test for Borrelia burgdorferi using Recombinase Polymerase Amplification with Lateral Flow Detection. The bacterium that causes Lyme Disease is very difficult to grown in culture making it an unsuitable method for clinical diagnosis. Borrelia has one of the most complex bacterial genotypes around the world. In China, the most common genotypes of Borrelia burgdorferi are B. garinii, B. afzelii, and B. valaisiana. The Chinese recently developed a novel isothermal method of rapidly detecting multiple species of Borrelia using recombinase polymerase amplification with lateral flow detection (LF-RPA). The LF-RPA was found to have similar sensitivity to the real-time qPCR, with the benefit of being able to yield results in less than 30 minutes. For the Study they collected 20 samples from Lyme Borreliosis patients: 18 samples tested positive by LF-RPA, 17 samples were positive by Western Blot, 14 positive by real-time qPCR, and 11 by nested-PCR. This study showed that serum DNA detection was more helpful for diagnosing early infection than the standard methods of antibody detection, although the Western Blot was nearly as sensitive. [21]
As stated in my previous blog, the current 2-tier diagnostic testing for Lyme Disease is about 44% sensitive to Borrelia burgdorferi in the early phase. Accurate testing is critical to ensure the proper diagnosis and early treatment for Lyme. To make matters worse, the current Lyme test is geared towards detecting only one of the 21 known species of Borrelia, B. burgdorferi. It doesn't take a rocket scientist to surmise there are a vast number of patients receiving false-negatives for Borrelia infections.
Stony Brook Direct Sanger DNA
The Department of Pathology at Milford Hospital, CT has developed a new test using direct Sanger DNA sequencing with PCR.[4] This test was able to detect low levels of both B burgdorderi and B miyamotoi with incredible accuracy. [5] At present Milford is the only licensed clinical laboratory in the U.S. to officer a combined DNA sequencing-based diagnostic test. It costs $150 and the results are ready within 5 days of a samples receipt. [6]
During the IDWeek Conference in October of 2014, Dr. Benjamin Luft of Stony Brook University presented the results of an experimental test for Lyme. Using the genomic information from BorreliaBase they were able to isolate 48 antigenic proteins from multiple genospecies of Borrelia. From this they identified 11 individual proteins that were 80% sensitive and 99% specific to B burgdorferi in the early phase of infection. This is almost twice the accuracy of the current FDA approved 2-tier test! Dr. Luft believes that this new test has the potential to become a rapid single-tier test. Using recombinant Borrelia protein, this test has a much broader range of coverage and could possibly detect multiple species of Borrelia. [7] This is exciting news!
Johns Hopkins SYBR Green I/PI assay
Currently researchers at Johns Hopkins Bloomberg School of Public Health are working on a test that will better allow clinicians to check for Borrelia that persist after completion of antibiotic treatment a condition that may lead to the proper diagnosis of "Chronic Lyme" or "Post Treatment Lyme Disease Syndrome." Using the SYBR Green I/PI assay they were able to detect how many bacteria were alive or dead after exposure to various drugs. What they found is that frontline antibiotics such as doxycycline and amoxicillin work well for the growing spirochetal-form of B. burgdorferi, but have limited effectiveness on variant forms--spheroplast (L-form), cyclic (round-form), biofilm-like colonies or persister forms of B. burgdorferi. [8] In fact, researchers found that while Doxycycline reduced the spirochetal form by 90% it increased the number of round body forms about twofold.
IDSA Paper on Metabolic Biosignatures
Just this week, the IDSA has published a study on an emerging test for Lyme. In their paper they acknowledge
"two-tier serology-based assays yield low sensitivities (29-40%) for early infection. The lack of an accurate laboratory test for early Lyme disease contributes to misconceptions about diagnosis and treatment, and underscores the need for new diagnostic approaches."Using Liquid chromatography-mass spectrometry (LC-MS) they were able to identify 44 metabolic biosignatures that allow for detection of "early" Lyme with a sensitivity of 88%, and a specificity of 95%. Also, important, the metabolic biosignature was able to correctly identify 77-95% of the Sero-negative Lyme patients. [9]
July 2015 Update:
-Translational Genomics Research Institute (TGen) Using DNA Sequencing as Diagnostic tool.
Scientists at TGen's Pathogen Genomics Division in Flagstaff, AZ - TGen North- will use the power of targeted DNA sequencing to develop and validate a test to measure the presence and severity of tick-borne Lyme disease at the genomic level.
By analysing a sample of DNA, the new test should be able to pinpoint Lyme disease, identify multiple strains of Lyme, detect other tick-related infections, and show non-Lyme causes of disease. [10]
August 2105 Updates:
-Salivary Diagnostics, developed by Forsyth Institute, is developing a test that will be able to detect Tick-Borne Infections. Founded in 2013 with a $4.2 million dollar grant from the Massachusetts Life Sciences Center, the goal of Forsyth’s Center for Salivary Diagnostics is to revolutionize the medical diagnostics world and replace blood-based tests with salivary tests.
A saliva-based test is being developed for this insidious tick-borne infection. Forsyth investigators have discovered that components of the bacterium that causes Lyme are shed into body fluids including saliva. The test would allow rapid diagnosis and cure with a simple antibiotic regimen, before the disease becomes chronic and debilitating. [11]
September 2015 Update:
-Nanomedical Diagnostics, Inc. based in San Diego, CA has developed a graphene biological field effect transistor (BioFET) technology which will improve the ease, speed and cost of personalized healthcare. The CDC and Nanomedical Diagnostics are entering into a Cooperative Research and Development Agreement to evaluate direct electronic detection of Borrelia burgdorderi antigens for a new Lyme disease diagnostic system. Nanomed has launched AGILE Research, a label-free, quantitative, low-cost biosensor for small molecule and protein analysis. AGILE Research is slated to launch early in 2016 and will be the focal point in the Lyme Disease beta test at Stanford University. [12]
January 2016 Update:
-The IDSA contends that isolation of Borrelia spp by culture is the diagnostic "gold standard" of Lyme testing, however multiple recent studies have proven that Borrelia bacterium is not easily cultured. As highlighted in this article by Dr Emir Hodzic, with the Center for Comparative Medicine, School of Medicine, University of California at Davis, Polymerase Chain Reaction is by far the most accurate method for detecting all species of Borrelia.
"Having worked with for over 20 years, it is apparent that not all isolates or strains can be easily cultured, and this is especially apparent during long-term infection. Thus, culture cannot be relied upon as a gold standard of viability. As noted above, our studies and those of others in mice, dogs and non-human primates have all reached similar conclusions: spirochetes are persisting, but are paradoxically non-cultivable." [13]-Real-Time PCR: UC Davis Vet. Med's Website has a great explanation of the difference between Conventional PRC, RT-PCR and how that differs from serology here. [14] Hodzic and Barthold's studies differentiating between the presence of bacterial DNA versus live bacteria use this method.
"Real-time PCR has a lower limit of detection of 5 molecules and has better analytical sensitivity than conventional PCR with eliminated risk of contamination."-Broad-Range PCR with Electrospray Ionization Mass Spectrometry: This unique test is able to simultaneously detect a wide range of vectorborne microorganisms in a single test. It is available at Ibis Biosciences, Inc. and Abbott Company in Carlsbad CA. [15] Not only is this Broad-Range PCR able to detect multiple infections with a single test, it is able to detect early Lyme disease with much greater accuracy than the standard 2-tier test.
"The combined results of our direct detection assay with initial physician visit serology resulted in the detection of early Lyme disease in 19 of 21 (90%) of patients at the initial visit. In 5 of 21 cases we demonstrate the ability to detect B. burgdorferi in early Lyme disease directly from whole blood specimens prior to seroconversion."[16]-Plasminogen-binding Protein PCR: This test was used by the Mayo Clinic in 2015 to detect a new strain of Lyme Borrelia Mayonii that was reported by Dr. Alan Barbour at the 14th International Conference of Lyme Borreliosis. The Mayo Clinic recommends PCR because
"serology may not be positive until 2 to 4 weeks after onset of ECM, direct detection of Borrelia burgdorferi-specific target DNA sequences using PCR is a promising adjunct to existing diagnostic tests." [17]
-Next Generation DNA Sequencing for Rapid Diagnosis. This is a powerful method that was developed by Dr. Charles Chiu at UCSF. It is a rapid test that uses genetic sequencing and cloud-compatible bioinformatics and is capable of detecting all pathogens - viruses, bacteria, fungi and parasites - with a single test. [18] In 2013 Dr. Chiu and his team used advanced DNA sequencing technology to save a 14-year-old boy with bacterial encephalitis. [19] Knowing that ticks carry a multitude of pathogens, and at least 20% of patients remain ill following standard antibiotic treatment, I believe this test will prove to be very useful in diagnosing those who remain severely ill with Post-Treatment Lyme Disease Syndrome.
July 2016 Update:
IGeneX - Improved Sensitivity of Lyme Disease Western Blots. In an effort to improve the Western Blot sensitivity without decreasing specificity, IGeneX has added two strains of Borrelia Burgdorferi (Bb), B31 and 297, to their immunoblot preparation. They conducted a study with sera from a diverse group of 364 American and European patients and controls. The combined IgG and IgM sensitivity of the Western Blot alone increased from 77.1% to 88.6% by adding 297 (a recombinant OspA antigen) to the standard B31. With the increased sensitivity this test is considered positive with IgG or IgM reactivity with any two of six Bb antigens--23, 31, 34, and 93 (Bb has 3 distinct Outer Surface Protiens (Osp). 23 indicates OspC, 31=OspA, 34= OspB and 93kDa=flagellum antigen specific to Bb) [20] Note: the sensitivity of the CDC recommended combined Two-Tier Lyme test averages just 50%. By removing the 1st tier, the ELISA, the sensitivity of the Lyme test improves dramatically. Using IGeneX in-house WB strips and in-house interpretive criteria the sensitivity increased from 88.6% to 97.1%, however the specificity decreased from 100% to 95%IgG and 93%IgM.
August 2016 Update:
The Chinese CDC, In Collaboration with State Key Laboratory of Infectious Diseases Prevention and Control, have Developed a Novel Test for Borrelia burgdorferi using Recombinase Polymerase Amplification with Lateral Flow Detection. The bacterium that causes Lyme Disease is very difficult to grown in culture making it an unsuitable method for clinical diagnosis. Borrelia has one of the most complex bacterial genotypes around the world. In China, the most common genotypes of Borrelia burgdorferi are B. garinii, B. afzelii, and B. valaisiana. The Chinese recently developed a novel isothermal method of rapidly detecting multiple species of Borrelia using recombinase polymerase amplification with lateral flow detection (LF-RPA). The LF-RPA was found to have similar sensitivity to the real-time qPCR, with the benefit of being able to yield results in less than 30 minutes. For the Study they collected 20 samples from Lyme Borreliosis patients: 18 samples tested positive by LF-RPA, 17 samples were positive by Western Blot, 14 positive by real-time qPCR, and 11 by nested-PCR. This study showed that serum DNA detection was more helpful for diagnosing early infection than the standard methods of antibody detection, although the Western Blot was nearly as sensitive. [21]
1. PATRIC: Bacterial Bioinformatics Resource Center, an NIH/NIAID-funded project of the University of Chicago.
2. BCM Bioinformatics, 3 July 2013. BorreliaBase: A phylogeny-centered browser of Borrelia genomes.
3. BorreliaBase: A Phylocentric Browser for Borrelia Genomes
4. Int. J. Mol. Sci 2014, DNA Sequencing Diagnosis of Off-Season Spirochetemia with Low Bacterial Density in Borrelia burgdorferi and Borrelia miyamotoi Infection.
5. Milford Medical Laboratory Molceular Diagnostics Department Bridgeport Ave Milford, CT.
6. Milford Medical Laboratory: Request a Lyme Disease DNA Test.
7. IDWeek October 2014: Next Generation Diagnostic Test for Lyme Disease. Poster Abstract Session: Diagnostic Microbiology: Bacterial Infections.
8. PLOS|one: 3 November 2014. An Optimized SYBR Green I/PI Assay for Rapid Viability Assessment and Antibiotic Susceptibility Testing for Borrelia burgdorferi
9. Clin Infect Dis. 2015 Mar 11. Development of a Metabolic Biosignature for Detection of Early Lyme Disease.
10. TGen developing quick, affordable and accurate test to diagnose debilitating Lyme disease.
11. Salivary Diagnostic: Forsyth Institute, Office of Technology Development, Cambridge, MA
12. Announcing Launch of Nanomedical Diagnostics, Inc., a Company Releasing the World's First Commercialized Graphene Biosensor.
13. Lyme Borreliosis: is there a preexisting (natural) variation in antimicrobial susceptibility among Borrelia burgdorferi strains.
14. Real-Time PCR Research Diagnostics Core Facility at UC Davis Veterinary Medicine.
15. Identification of endosymbionts in ticks by broad-range polymerase chain reaction and electrospray ionization mass spectrometry.
16. Direct molecular detection and genotyping of Borrelia burgdorferi from whole blood of patients with early Lyme disease.
17. Mayo Clinic Medical Laboratories: Lyme Disease (Borrelia burgdorferi), Molecular Detection, PCR, Blood.
18. A cloud-compatible bioinformatics pipeline for ultra rapid pathogen identification from next-generation sequencing of clinical samples.
19. A Diagnosis Just in Time.
20. Improved Sensitivity of Lyme Disease Western Blots Prepared with a Mixture of Borrelia Burgdorferi Strains 297 and B31.
21. A Novel Isothermal Assay of Borrelia burgdorferi by Recombinase Polymerase Amplification with Lateral Flow Detection.
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.
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