CULTURING BORRELIA

Report
CULTURING BORRELIA
A NEW GOLD STANDARD IN
LYME BORRELIOSIS TESTING
Joseph J. Burrascano Jr. M.D.
DISCLAIMER STATEMENT
• 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
• My compensation is not affected by sales at
any of our clients, including Advanced Labs
WHY IS LYME SUCH A DIFFICULT
ILLNESS TO TREAT?
• Diagnostic tests are a disaster- insensitive and
indirect
• 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 know whether symptoms that persist
or recur after treatment represent treatment
failure or another process
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).
• 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).
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)
• 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)
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!
SEROLOGIC TESTING IS
NOT INFORMATIVE
SEROLOGIES ARE INDIRECT TESTS• 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
NUCLEIC ACID TESTING (PCR)
• Sensitivities are low (no better than 30% for peripheral
blood; slightly higher for synovial biopsy)
• 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”
• Can a positive PCR reflect “old” DNA, and not currently
living Borrelia?
• Not accepted as proof of infection by CDC or insurance
carriers
LITERATURE ON PROBLEMS WITH PCR
• 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).
OTHER TECHNOLOGIES
URINE ANTIGEN CAPTURE
• Is a direct test of antigen spillage
• Not known whether all pathogenic strains of Bb will be detected
• Antigen spillage is not constant- may vary day to day
• Sensitivity is low (30%), similar to PCR
• Not accepted as proof of infection by CDC or insurance carriers
T-CELL STIMULATION ASSAYS
• Are several methods in use
• Not known whether all pathogenic strains of Bb will be detected
• No large scale clinical studies have been published on sensitivity
and specificity
• Apparently are significant numbers of false positives and false
negatives
• Also not accepted as proof of infection by CDC or insurance
carriers
WHY THESE PROBLEMS- 1
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Borrelia have many variants
Borrelia change over time
Borrelia adapt to changing environments
Borrelia contain a large amount of highly
complex genetic material
• Clinical, wild-type Borrelia specimens are
quite different from laboratory strains
• Borrelia have been very difficult to culture
WHY THESE PROBLEMS- 2
LYME- MORE THAN ONE SPIROCHETE
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B. burgdorferi
B. afzelii
B. garinii
B. spielmanii
B. lonestari
B. bissetti
B. carolinensis
B. americana
B. andersonii
B. kurtenbachii
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B. lusitaniae
B. valaisiana
B. sinica
B. bavariensis
B. japonica
B. miyamoti
B. yangtze
B. tanukii
B. turdi
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.
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.
• “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).
CULTURING BbADVANTAGES
• 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 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
• If combined with DNA sequencing, the exact
identity of the Borrelia can be ascertained
CULTURING BbDISADVANTAGES
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Expensive
Peripheral blood only at this time
Large volume required
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
• As with all blood cultures, may need several sets to
be sure
CULTURE SUCCESS!
Now available for clinical use
CONCEPT OF Bb AS A SYMBIONT
EXPLAINS PREVIOUS CULTURE FAILURES
TWO CLASSES OF SPIROCHETES:
• Free-living (soil, ponds)
• 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)
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 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
OPTIMIZING CULTURE CONDITIONS
Development of mBSK
• Began with BSK- proven track record and accepted
• 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, CO2
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
THE RESULT:
FAILURE!
• 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
• In addition, several solid BSK-based media and PMRagar 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 longterm Borrelia growth
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)
• Agar (old fashioned but useful in many bacterial cultures)
• Alginate (major component of Borrelia biofilm)
• Connective tissue components (Bb lives in joints)
• Tick shell components (Bb lives in ticks for years)
• Different composition of the tubes, dishes and slides
• Different surface treatment of the tubes, dishes and slides
• Others….
PHYSICAL PREFERENCES
OF Bb in vitro
• Prefer to grow on a solid surface
• Prefer frosted slides over smooth ones
• Prefer glass over plastic
• Collagen supports growth
FINAL METHOD:
• Begin with a starter culture in mBSK then after 6
days transfer into tubes that contain collagencoated slides
SUCCESS !!!!
94% sensitivity
100% specificity
• Note that in mammals, Bb are most often found
associated with collagen
• 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%
– Similar to overall success rate of this system
• the key may be that skin contains a large quantity
of collagen
HIGHLY SENSITIVE
• Spiking experiments using lab strain
B31:
– Able to obtain positive cultures when as
little as ONE Bb organism was added
• 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
VALIDATION STUDY
72 CDC-positive Lyme disease patients
• 34 of the 72 cultures were positive after 6 days in
culture (47%).
• An additional 26 samples were positive at 8 weeks
(83%)
• At 16 weeks an additional 8 samples became positive
(total 94%).
• 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).
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 variation was identified at the CTP synthase
locus, suggesting that each sample was derived from
an independent source and not from laboratory
contamination.
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).
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
• 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
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 may also allow an earlier reported positive result, as
PCRs are performed early on all specimens
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
• A culture that is positive post treatment indicates ongoing
infection
• 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
• 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
– Any positive test is diagnostic, even if it is the only positive out of a
set of three
ACTUAL CULTURE RESULTS
CONCLUSIONS
REMARKABLE, LANDMARK ACHIEVEMENT!
• 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
• 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.
• Culturing should be utilized in thoughtfully designed
clinical studies to generate meaningful data and to end
the current reliance on anecdotal and uncontrolled
reports
Thank you!
J. J. Burrascano Jr. M.D.
New York

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