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

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

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

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).

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 !

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

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

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).

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

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

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

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.

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).

14. CULTURING Bb- ADVANTAGES Directly detects living Bb- if the patient’s 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 patient’s 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 designed to detect all relevant clinical strains of Borrelia, and not just lab strains If combined with DNA sequencing, the exact identity of the Borrelia can be ascertained If combined with DNA sequencing, the exact identity of the Borrelia can be ascertained

15. CULTURING Bb- DISADVANTAGES Expensive Expensive Peripheral blood only at this time Peripheral blood only at this time Large volume required Large volume required May take several weeks for Bb to grow May take several weeks for Bb to grow – Overall, of all positives, 48% can be reported within first week; rest take 8-16 weeks Must be off all antibiotics for at least four weeks prior to specimen collection Must be off all antibiotics for at least four weeks prior to specimen collection As with all blood cultures, may need several sets to be sure As with all blood cultures, may need several sets to be sure

16. CULTURE SUCCESS! Now available for clinical use

17. CONCEPT OF Bb AS A SYMBIONT EXPLAINS PREVIOUS CULTURE FAILURES TWO CLASSES OF SPIROCHETES: Free-living (soil, ponds) Free-living (soil, ponds) Symbionts (only exist within other living hosts) Symbionts (only exist within other living hosts) B. burgdorferi is a symbiont Compared to free-living spirochetes, symbionts (T. pallidum and B. burgdorferi) have lost 80% of the genes necessary for free living (Margulis, Symbiosis, 2009; (47): 51-58) Compared to free-living spirochetes, symbionts (T. pallidum and B. burgdorferi) have lost 80% of the genes necessary for free living (Margulis, Symbiosis, 2009; (47): 51-58)

18. PREVIOUS Bb CULTURE FAILURES Many culture efforts hit a dead end- Bb may remain viable for a period of time, but then stop dividing or divide slowly Many culture efforts hit a dead end- Bb may remain viable for a period of time, but then stop dividing or divide slowly Many of these later cultures contain only dead Borrelia, or altered forms of Bb, usually L-forms and cysts, possibly reflecting an insufficient growth environment Many of these later cultures contain only dead Borrelia, or altered forms of Bb, usually L-forms and cysts, possibly reflecting an insufficient growth environment The key is to develop a culture system that has the nutrients and microenvironmental conditions that mimic what exists in animal and arachnid hosts The key is to develop a culture system that has the nutrients and microenvironmental conditions that mimic what exists in animal and arachnid hosts

19. OPTIMIZING CULTURE CONDITIONS Development of mBSK Began with BSK- proven track record and accepted Began with BSK- proven track record and accepted Many parameters were explored and optimized- Many parameters were explored and optimized- – Collecting and transporting blood with and without culture media- must do both to maximize yield – Adding serum from antibiotic-free animals, and increasing the concentration – Optimizing culture conditions (full tubes, loose caps, CO 2 in the incubator because Bb is microaerophilic) – Use of two different culture tube sizes- must use both – Adding small concentrations of rifampin to prevent bacterial overgrowth – Adding DTT- a strong antioxidant

20. THE RESULT: FAILURE! Although spirochetes grew much more quickly and in increased numbers, after 10-14 days they converted into different morphological forms Although spirochetes grew much more quickly and in increased numbers, after 10-14 days they converted into different morphological forms All attempts to revert them back to spirochetal form failed regardless of the use of several different liquid media All attempts to revert them back to spirochetal form failed regardless of the use of several different liquid media In addition, several solid BSK-based media and PMR- agar media also failed In addition, several solid BSK-based media and PMR- agar media also failed In summary, while modifications to BSK had a positive effect on the initial Borrelia cultures, additional parameters needed to be optimized to permit long- term Borrelia growth In summary, while modifications to BSK had a positive effect on the initial Borrelia cultures, additional parameters needed to be optimized to permit long- term Borrelia growth

21. WHAT EXISTS IN LIVING HOSTS THAT IS ABSENT FROM PREVIOUSLY USED CULTURE SYSTEMS? Many theories were considered and some were even attempted: Sonicated Borrelia (should provide everything a culture medium might need) Sonicated Borrelia (should provide everything a culture medium might need) Agar (old fashioned but useful in many bacterial cultures) Agar (old fashioned but useful in many bacterial cultures) Alginate (major component of Borrelia biofilm) Alginate (major component of Borrelia biofilm) Connective tissue components (Bb lives in joints) Connective tissue components (Bb lives in joints) Tick shell components (Bb lives in ticks for years) Tick shell components (Bb lives in ticks for years) Different composition of the tubes, dishes and slides Different composition of the tubes, dishes and slides Different surface treatment of the tubes, dishes and slides Different surface treatment of the tubes, dishes and slides Others…. Others….

22. PHYSICAL PREFERENCES OF Bb in vitro Prefer to grow on a solid surface Prefer to grow on a solid surface Prefer frosted slides over smooth ones Prefer frosted slides over smooth ones Prefer glass over plastic Prefer glass over plastic Collagen supports growth Collagen supports growth FINAL METHOD: Begin with a starter culture in mBSK then after 6 days transfer into tubes that contain collagen- coated slides Begin with a starter culture in mBSK then after 6 days transfer into tubes that contain collagen- coated slides

23. SUCCESS !!!! 94% sensitivity 100% specificity Note that in mammals, Bb are most often found associated with collagen Note that in mammals, Bb are most often found associated with collagen Previous published reports showed maximal success rate in liquid media was 44% Previous published reports showed maximal success rate in liquid media was 44% – Similar to results after day 6 However, skin cultures had a maximal success rate of 88% However, skin cultures had a maximal success rate of 88% – Similar to overall success rate of this system the key may be that skin contains a large quantity of collagen the key may be that skin contains a large quantity of collagen

24. HIGHLY SENSITIVE Spiking experiments using lab strain B31: Spiking experiments using lab strain B31: – Able to obtain positive cultures when as little as ONE Bb organism was added Clinical specimens: Clinical specimens: – Some cultures demonstrated detectable growth in as little as SIX DAYS of culturing – Of all cultures that eventually became positive, 48% were positive at day 6

25. VALIDATION STUDY 72 CDC-positive Lyme disease patients 34 of the 72 cultures were positive after 6 days in culture (47%). 34 of the 72 cultures were positive after 6 days in culture (47%). An additional 26 samples were positive at 8 weeks (83%) An additional 26 samples were positive at 8 weeks (83%) At 16 weeks an additional 8 samples became positive (total 94%). At 16 weeks an additional 8 samples became positive (total 94%). All samples stained positively with both polyclonal and monoclonal antibodies. All samples stained positively with both polyclonal and monoclonal antibodies. PCRs were positive for all the positive cultures (tested at either the 16S rDNA or CTP synthase loci- most were tested at both). PCRs were positive for all the positive cultures (tested at either the 16S rDNA or CTP synthase loci- most were tested at both).

26. SPECIFICITY 100% 48 negative controls: – All 48 negative control samples were negative by dark field microscopy and by anti-Borrelia monoclonal and polyclonal antibody staining at all time points examined (6 days, 8 weeks and 16 weeks; 100%). Sequence analyses of each PCR product from the positive cultures confirmed that the DNA was derived from Borrelia, and sequencing showed a 93 to 100% similarity to B31/N40 strains of Bb. Sequence analyses of each PCR product from the positive cultures confirmed that the DNA was derived from Borrelia, and sequencing showed a 93 to 100% similarity to B31/N40 strains of Bb. Sequence variation was identified at the CTP synthase locus, suggesting that each sample was derived from an independent source and not from laboratory contamination. Sequence variation was identified at the CTP synthase locus, suggesting that each sample was derived from an independent source and not from laboratory contamination.

27. SEQUENCE TREE Figure 9. shows a neighbor-joining phylogenetic analysis of 600 bp alignment of the CTP synthase gene (pyrG) of the 51 Borrelia clinical isolates obtained from our validation study using MEGA 5 Evolutionary Genetics Analysis program [36]. Alphanumeric codes represent GenBank (NCBI) ID numbers. Nucleotide sequences for CTP synthase locus for 51 clinical isolates have the following sequential accession numbers: JX867374 through JX867424. Numbers at nodes indicate bootstrap support values (500 replicates).

28. OPTIONS FOR THE PRACTITIONER Standard procedure: stain with polyclonal immunostain Has been shown to detect B. burgdorferi s.s., B. garinii, B. afzelii and B. hermsii Has been shown to detect B. burgdorferi s.s., B. garinii, B. afzelii and B. hermsii Will not detect Treponema denticola Will not detect Treponema denticola May instead stain with a monoclonal Will only detect Bb s.s., and not afzelii, garinii, or hermsii, nor will it detect T. denticola Will only detect Bb s.s., and not afzelii, garinii, or hermsii, nor will it detect T. denticola May add-on, to either of the above, DNA PCR combined with DNA sequencing This allows a second layer of confirmation of the culture This allows a second layer of confirmation of the culture This may also allow an earlier reported positive result, as PCRs are performed early on all specimens This may also allow an earlier reported positive result, as PCRs are performed early on all specimens

29. Bb CULTURE- NUTS AND BOLTS A POSITIVE CULTURE INDICATES THAT AN ACTIVE INFECTION WAS PRESENT AT THE TIME THE SPECIMEN WAS TAKEN Cultures may be positive in an infected patient who is seronegative Cultures may be positive in an infected patient who is seronegative A culture that is positive post treatment indicates ongoing infection A culture that is positive post treatment indicates ongoing infection Culture positivity fulfills even the strict CDC surveillance case definition Culture positivity fulfills even the strict CDC surveillance case definition MY CLINICAL EXPERIENCE: Highest sensitivity if the patient is symptomatic at the time of blood draw, and best to draw blood in early afternoon Highest sensitivity if the patient is symptomatic at the time of blood draw, and best to draw blood in early afternoon Must not have been exposed to antibiotics for at least four weeks- six is better- prior to drawing the blood specimen Must not have been exposed to antibiotics for at least four weeks- six is better- prior to drawing the blood specimen As with all blood cultures, may need several sets to be sure As with all blood cultures, may need several sets to be sure – Any positive test is diagnostic, even if it is the only positive out of a set of three

30. ACTUAL CULTURE RESULTS

31. CONCLUSIONS REMARKABLE, LANDMARK ACHIEVEMENT! Able to identify Borrelia species; further species information is possible by adding a monoclonal immunostain Able to identify Borrelia species; further species information is possible by adding a monoclonal immunostain Is the most sensitive and specific test for Borrelia in human clinical samples currently available Is the most sensitive and specific test for Borrelia in human clinical samples currently available Can be used for diagnosis, to indicate treatment efficacy, and can form the basis for a variety of future studies Can be used for diagnosis, to indicate treatment efficacy, and can form the basis for a variety of future studies HOWEVER, is only a laboratory tool. Clinical assessment will remain the primary informational tool for LLMDs. HOWEVER, is only a laboratory tool. Clinical assessment will remain the primary informational tool for LLMDs. Culturing should be utilized in thoughtfully designed clinical studies to generate meaningful data and to end the current reliance on anecdotal and uncontrolled reports Culturing should be utilized in thoughtfully designed clinical studies to generate meaningful data and to end the current reliance on anecdotal and uncontrolled reports

32. Thank you! J. J. Burrascano Jr. M.D. New York