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CULTURING BORRELIA A NEW GOLD STANDARD IN LYME BORRELIOSIS TESTING Joseph J. Burrascano Jr. M.D

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Text of CULTURING BORRELIA A NEW GOLD STANDARD IN LYME BORRELIOSIS TESTING Joseph J. Burrascano Jr. M.D

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CULTURING BORRELIA A NEW GOLD STANDARD IN LYME BORRELIOSIS TESTING Joseph J. Burrascano Jr. M.D. Slide 2 DISCLAIMER STATEMENT I am not an employee or direct consultant to any commercial or research laboratory I am not an employee or direct consultant to any commercial or research laboratory I am a full time employee of Apogenics Inc. which has consulted with Advanced Labs I am a full time employee of Apogenics Inc. which has consulted with Advanced Labs My compensation is not affected by sales at any of our clients, including Advanced Labs My compensation is not affected by sales at any of our clients, including Advanced Labs Slide 3 WHY IS LYME SUCH A DIFFICULT ILLNESS TO TREAT? Diagnostic tests are a disaster- insensitive and indirect Diagnostic tests are a disaster- insensitive and indirect Many patients with chronic multisystem illnesses that could be Lyme have negative or inconclusive serologies Many patients with chronic multisystem illnesses that could be Lyme have negative or inconclusive serologies No way to accurately assess whether treatment has cleared the infection No way to accurately assess whether treatment has cleared the infection No way to know whether symptoms that persist or recur after treatment represent treatment failure or another process No way to know whether symptoms that persist or recur after treatment represent treatment failure or another process Slide 4 LITERATURE CITATIONS ON THE NEED FOR A BETTER TEST Many cases of Lyme disease go undiagnosed and untreated, putting an infected patient at risk for developing a debilitating long-term illness (Liegner 1992, Klempner 2001, Dumber 2001, Nelson 2005, Augero 2005, Wormser 2006). Many cases of Lyme disease go undiagnosed and untreated, putting an infected patient at risk for developing a debilitating long-term illness (Liegner 1992, Klempner 2001, Dumber 2001, Nelson 2005, Augero 2005, Wormser 2006). The signs and symptoms of disseminated Lyme disease are shared with many other diseases (Wormser 2006). The signs and symptoms of disseminated Lyme disease are shared with many other diseases (Wormser 2006). A highly sensitive and specific assay for Borrelia spp is needed to assist in the accurate detection of these undiagnosed infections. (Augero 2005, Wormser 2006). A highly sensitive and specific assay for Borrelia spp is needed to assist in the accurate detection of these undiagnosed infections. (Augero 2005, Wormser 2006). Slide 5 SEROLOGIC TESTING IS INSENSITIVE All are based on only one or two lab strains! Low Sensitivity- ELISA- Sensitivities range from 29% to 68% (Stricker, BMJ 2007; 335 (7628): 1008) ELISA- Sensitivities range from 29% to 68% (Stricker, BMJ 2007; 335 (7628): 1008) C6- ELISA assay- as bad as the standard ELISA C6- ELISA assay- as bad as the standard ELISA Western blot- sensitivities range from 46% to 50% for commercial kits (Goossens, Eur J Clin Microbiol Infect Dis. 1999; 18: 551-560) and no more than 80% for reference tests (Donta, Clin Infect Dis. 2007; 44(8): 1134-1135) Western blot- sensitivities range from 46% to 50% for commercial kits (Goossens, Eur J Clin Microbiol Infect Dis. 1999; 18: 551-560) and no more than 80% for reference tests (Donta, Clin Infect Dis. 2007; 44(8): 1134-1135) False positive IgMs in presence of EBV and Parvovirus (percentage unknown) Spinal tap- Only 9% have + CSF antibodies (Coyle, SUNY at Stony Brook) PARADOX- the more ill the patient, the weaker the serologic response and the LESS likely you are to get a positive test ! Slide 6 SEROLOGIC TESTING IS NOT INFORMATIVE SEROLOGIES ARE INDIRECT TESTS- Serologies do not detect an active infection, only prior exposure Serologies do not detect an active infection, only prior exposure Serologies may be non-reactive despite an active infection Positive serologies may remain positive post treatment even if the infection has cleared Are therefore are useless to assess treatment efficacy Are therefore are useless to assess treatment efficacy Slide 7 NUCLEIC ACID TESTING (PCR) Sensitivities are low (no better than 30% for peripheral blood; slightly higher for synovial biopsy) Sensitivities are low (no better than 30% for peripheral blood; slightly higher for synovial biopsy) PCR Primers- Trade-off between sensitivity and specificity PCR Primers- Trade-off between sensitivity and specificity Broad primer sets may include spirochetes other than pathogenic Borrelia More focused primer sets may miss some important Bb strains Solution requires doing a series of PCRs Positive PCRs are often dismissed as contaminated Positive PCRs are often dismissed as contaminated Can a positive PCR reflect old DNA, and not currently living Borrelia? Can a positive PCR reflect old DNA, and not currently living Borrelia? Not accepted as proof of infection by CDC or insurance carriers Not accepted as proof of infection by CDC or insurance carriers Slide 8 LITERATURE ON PROBLEMS WITH PCR Attempts at using PCR have been disappointing in general (Wallach 1993, Nocton 1996, Rauter 2005, Augero 2005, Marques 2010). Attempts at using PCR have been disappointing in general (Wallach 1993, Nocton 1996, Rauter 2005, Augero 2005, Marques 2010). Concerns over false positives have been raised (Klempner 2001, Klempner 2001, Molloy 2001). Concerns over false positives have been raised (Klempner 2001, Klempner 2001, Molloy 2001). Slide 9 OTHER TECHNOLOGIES URINE ANTIGEN CAPTURE Is a direct test of antigen spillage Is a direct test of antigen spillage Not known whether all pathogenic strains of Bb will be detected Not known whether all pathogenic strains of Bb will be detected Antigen spillage is not constant- may vary day to day Antigen spillage is not constant- may vary day to day Sensitivity is low (30%), similar to PCR Sensitivity is low (30%), similar to PCR Not accepted as proof of infection by CDC or insurance carriers Not accepted as proof of infection by CDC or insurance carriers T-CELL STIMULATION ASSAYS Are several methods in use Are several methods in use Not known whether all pathogenic strains of Bb will be detected Not known whether all pathogenic strains of Bb will be detected No large scale clinical studies have been published on sensitivity and specificity No large scale clinical studies have been published on sensitivity and specificity Apparently are significant numbers of false positives and false negatives Apparently are significant numbers of false positives and false negatives Also not accepted as proof of infection by CDC or insurance carriers Also not accepted as proof of infection by CDC or insurance carriers Slide 10 WHY THESE PROBLEMS- 1 Borrelia have many variants Borrelia have many variants Borrelia change over time Borrelia change over time Borrelia adapt to changing environments Borrelia adapt to changing environments Borrelia contain a large amount of highly complex genetic material Borrelia contain a large amount of highly complex genetic material Clinical, wild-type Borrelia specimens are quite different from laboratory strains Clinical, wild-type Borrelia specimens are quite different from laboratory strains Borrelia have been very difficult to culture Borrelia have been very difficult to culture Slide 11 WHY THESE PROBLEMS- 2 LYME- MORE THAN ONE SPIROCHETE B. burgdorferi B. burgdorferi B. afzelii B. afzelii B. garinii B. garinii B. spielmanii B. spielmanii B. lonestari B. lonestari B. bissetti B. bissetti B. carolinensis B. carolinensis B. americana B. americana B. andersonii B. andersonii B. kurtenbachii B. kurtenbachii B. lusitaniae B. lusitaniae B. valaisiana B. valaisiana B. sinica B. sinica B. bavariensis B. bavariensis B. japonica B. japonica B. miyamoti B. miyamoti B. yangtze B. yangtze B. tanukii B. tanukii B. turdi B. turdi Slide 12 WHY THESE PROBLEMS- 3 BORRELIA: A CHANGELING Falsenfeld, 1971: Borrelia adapt to their local environment and vectors, and many new varieties and even genospecies arise: Variations of Borrelia due to regional vectors could lead to new, as yet unseen forms of the disease. Pachner, 1989: Experimentally infected a mouse and then isolated the Bb- the brain isolate was different from those in the blood. Presumably the different environment affected Bb gene expression, inducing a change in surface antigens. A similar change occurs as Bb passes from the tick into the human host. Slide 13 CULTURE: THE IDEAL LYME DISEASE TEST A reliable, sensitive, and specific direct test that can inform whether the patient is currently infected with live bacteria. In other types of bacterial infections, typically a culture is taken, and growth indicates infection and possibly what specific bacterium is causing it. Examples include urine and throat cultures. In other types of bacterial infections, typically a culture is taken, and growth indicates infection and possibly what specific bacterium is causing it. Examples include urine and throat cultures. For Lyme disease, a positive culture result would provide direct evidence of an active infection and would be beneficial to assess the accuracy of other diagnostic tests (Auero-Rosenfeld 1996). For Lyme disease, a positive culture result would provide direct evidence of an active infection and would be beneficial to assess the accuracy of other diagnostic tests (Auero-Rosenfeld 1996). Slide 14 CULTURING Bb- ADVANTAGES Directly detects living Bb- if the patients culture is positive, then the patient had an active infection with Bb at the time the specimen was taken Directly detects living Bb- if the patients culture is positive, then the patient had an active infection with Bb at the time the specimen was taken Can be used for diagnosis Can be used for diagnosis Can be used post-treatment to see whether the treatments have eliminated the infection Can be used post-treatment to see whether the treatments have eliminated the infection Can be designed to detect all relevant clinical strains of Borrelia, and not just lab strains Can be desig