Selected notes from the peer-review published literature on
culture and other diagnostic methods for
by Marie Kroun, 2013, updated 2016
Comparison of isolation rate of Borrelia burgdorferi sensu lato in two different culture media, MKP and BSK-H.
Ružić-Sabljić E1, Maraspin V, Cimperman J, Strle F, Lotrič-Furlan S, Stupica D, Cerar T. Clin Microbiol Infect. 2014 Jul;20(7):636-41.
PMID: 24237688 DOI: 10.1111/1469-0691.12457 OA-link PDF-link
The aim of the study was to evaluate two culture media for Borrelia
burgdorferi sensu lato isolation from a 5 × 2 × 2 mm skin biopsy that
was dissected into two pieces and inoculated into modified
Kelly-Pettenkofer (MKP) and Barbour-Stoenner-Kelly-H (BSK-H) medium.
Samples were incubated at 33°C for up to 9 weeks. Borrelia species was
determined by MluI-restriction of whole genome or by MseI-restriction
of PCR product. We determined the proportion of isolation rate,
'slow-growers', contaminated specimens and Borrelia species in the two
In each of the two media 235 skin specimens were cultivated. We
found 90/470 (19.1%) contaminated cultures (BSK-H 67/235, 28.5%; MKP
23/235, 9.8%; p <0.0001).
Borrelia growth was ascertained in 59/235
(25.1%) BSK-H and 102/235 (43.4%) MKP cultures (p <0.0001); the
corresponding values for non-contaminated cultures were 59/168 (35.1%)
and 102/212 (48.1%); (p 0.003).
Fourteen specimens were positive only
in BSK-H, 57 solely in MKP, and 43 in both culture media.
was present in 8/59 (13.6%) BSK-H and in 4/98 (4.1%) MKP positive
cultures (p 0.019).
Borrelia afzelii was identified in 44/51 (86.3%)
BSK-H and in 88/98 (89.8%) MKP culture-positive samples; the
corresponding findings for Boreelia garinii and B. burgdorferi sensu
stricto were 6/51 (11.8%) and 9/98 (9.2%), and 1/51 (1.9%) and 1/98
(1.0%), for BSK-H and MKP, respectively. Comparison of MKP and BSK-H
medium for Borrelia culturing from skin specimens of European patients
with erythema migrans revealed the advantage of MKP over BSK-H.
Only BSK-H can currently be bought as a finished ready to use and ab producent quality controlled product from Sigma.
I have asked Sigma (Denmark representative) in 2016, if they could
perhaps make ready to use MKP / MKP-F medium, to increase our chance of
culturing both Borrelia burgdorferi and Borrelia miyamotoi, but with a
very disappointing NO response from Sigma :(
The relapsing fever spirochete Borrelia miyamotoi is cultivable in a modified Kelly-Pettenkofer medium, and is resistant to human complement.
Wagemakers A, Oei A, Fikrig MM, Miellet WR, Hovius JW1. Parasit Vectors. 2014 Sep 4;7:418. doi: 10.1186/1756-3305-7-418.
PMID: 25189195 PMC
Borrelia miyamotoi is a relapsing fever spirochete found in Ixodes
ticks in North America, Europe, and Asia, and has recently been found
to be invasive in humans. Cultivation of this spirochete has not yet
been described, but is important for patient diagnostics and scientific
purposes. Host specificity of Borrelia species is dependent on
resistance to host complement (serum resistance), and since B.
miyamotoi has been identified as a human pathogen we were interested
whether B. miyamotoi is resistant to human complement.
We inoculated B. miyamotoi strains LB-2001 and HT31 in modified-Kelly-Pettenkofer medium with 10% fetal calf serum (MKP-F),
and used standard non-laborious Borrelia culture methods to culture the
spirochetes. Next, we assessed serum sensitivity by a direct killing
assay and a growth inhibition assay.
We were able to passage B. miyamotoi over 10 times using a standard
culture method in MKP-F medium, and found B. miyamotoi to be resistant
to human complement. In contrast to B. miyamotoi, Borrelia anserina--a
relapsing fever spirochete unrelated to human infection--was serum
Using a variation on MKP medium we were able to culture B. miyamotoi,
opening the door to in vitro research into this spirochete. In
addition, we describe that B. miyamotoi is resistant to human
complement, which might play an important role in pathogenesis. We have
also found B. anserina to be sensitive to human complement, which might
explain why it is not related to human infection. Summarizing, we
describe a novel culture method for B. miyamotoi and show it is
resistant to human complement.
conditions for the growth and detection of borrelia from human serum.
Sapi E et al. Int J Med Sci. 2013;10(4):362-76. doi: 10.7150/ijms.5698.
Epub 2013 Feb 18.
PMID: 23470960, Open
* improved sample
of culture media
* use of matrix
The method was first optimized utilizing Borrelia
laboratory strains, and later by demonstrating growth of Borrelia
from sera from fifty seropositive Lyme disease patients
followed by another cohort of 72 Lyme disease patients, all of whom
satisfied the strict CDC surveillance eee definition for Lyme disease.
The procedure resulted in positive cultures in 47% at 6 days and 94% at
week 16. Negative controls included 48 cases.
identification of Borrelia was performed by immunostaining,
PCR, and direct DNA sequencing.
Results from this study showed that by optimizing collection
and transport conditions, media and growth environment, and the
development of a long-term culture method together provided improved
growth conditions for Borrelia. This
is demonstrated by a 94%
detection rate for Borrelia
of Lyme disease patients whose diagnoses were confirmed under the CDC
surveillance testing guidelines.
We have also found significant
sequence variation in these Borrelia samples
which further documents
that the Borrelia grown in this culture
system were not derived from laboratory contamination. The
from sera from healthy controls also argue against laboratory
contamination as being the source of the positive cultures and further
confirm the specificity of the test.
The maximum success rate for Borrelia blood
cultures as outlined in recent publications is in the 40-44% range
after 8-12 weeks of culturing   .
By refining our collection and short-term cultivation methods we were
able to get similar results after just six days in culture.
The highest rate of Borrelia growth previously
reported from clinical samples (up to 88%) involved cultures of skin, a tissue abundant
in collagen  . Such studies revealed a
maximal success rate similar to what we achieved in our long term,
collagen supported in vitro culture system (84% at
8 weeks and 94% at 16 weeks).
Unlike what has been reported by others, 
we found poor results when blood specimens were collected in tubes
containing EDTA. We
found that the addition of BSK-H medium to the whole blood sample at
the time of collection increased spirochete yield. As
previously reported ,
we also noted that allowing
the serum to separate, especially when mixed with BSK-H media, for up
to 24 hours from the time of the blood draw improved the culture
success rate. The BSK-H medium appeared to stabilize and
even draw out the spirochetes from the cellular fraction. Presence of N-acetyl
glucosamine (NAG) in the BSK-H medium, a known chemoattractant for Borrelia , may facilitate this
separation and support the spirochetes during transport.
Presumably, these freely suspended Borrelia were
better able to be cultivated. Interestingly, there was a subset of clinical
samples that grew better in the collection tubes without additional BSK
media. Because of this observation, we utilized both methods in our
experiments to ensure maximal yield.
BSK-H has been the preferred media for isolating and cultivating
spirochetes from human clinical samples  .
We made four changes that
concentration of rabbit serum (12% instead of 6%)
incorporating the observation that cultivation of other Borrelia
strains such as Borrelia hermsii52].
2. Based on the previous observation that DTT added to BSK-H media
helped to isolate Borrelia spirochetes from Ixodes
by testing different concentrations of DTT we determined that a high concentration of
DTT (100 μg/ml) enhanced the growth of Borrelia in our cultures.
3. Since Borrelia species are known to be
microaerophilic, meaning that while some oxygen is needed for
viability, high oxygen concentrations can be inhibitory ,
limit but not eliminate oxygen, such as partially shut lids, nearly
full tubes and use of a CO2 incubator facilitated
concentration of rifampicin (0.4-0.5 µg/ml) prevented secondary
bacterial overgrowth in all but 2 out of 122 cultures used
in this study.
In addition, the use
of two different types and sizes of tubes in the starter culture
The long-term culture system provided the greatest advantage in
achieving a higher success rate. It was found that modified BSK-H media
did not provide an efficient in vitro culture
environment after 8-10 days. We thus investigated whether matrix
protein as a solid support may provide a desirable environment for
growth of Borrelia. -.
Several matrix proteins were tested such as fibronectin, laminin and
hyaluronan, but collagen
gave the best results, consistent with previously published data  . Interestingly,
collagen is present both in skin biopsies and in the novel long-term
culture presented here and may be a common reason for their growth
advantage over other cultures.
This new culture method directly addresses the issue of the low numbers
of Borrelia in clinical samples by amplifying their
quantity through long
term culture in which Borrelia were able to thrive for
as long as eight months (data not shown). The versatility
of this method allows for samples to be harvested from the culture at
any point in time for further study, and it also serves as a source of Borrelia
for a variety of direct detection techniques as well as for additional
research. Finally, this
unique culture method could play an important role in providing useful
diagnostic information for select Lyme disease patients who might have
tested negatively by other methods.
2009 MK personal Borrelia culture pilot study: I
have not done as meticulous a culture study as Sapi el al.
did, but just read the literature sources below and
copycat all the good hints I got from these, but I was also able to
culture spirochetes (and alternative forms)
from samples with visible spirochetes in - I did as follows:
MK: PAR EXCELLENCE INVESTIGATIONs like these, is exactly what every
chronic relapsing borreliosis patient need access to!
- Use a Melag incubat85, set at 35 °C
- why, see my notes on: Borrelia growth optimum
- Use standard BSK-H from Sigma
(complete with 6% rabbit serum
in 100 ml bottle), is send frozen; I thaw the bottle
BSK-H into each of a number of 10 ml red top tubes, which are then
refrozen, as instructed by Sigma. Shortly
before the patient will arrive for sampling, I take tube(s) out of
and put into the incubator, so the tubes get the right
temperature before inoculation. Plasma/serum samples
or culture with spirochetes in may be
frozen (in/with BSK-H medium), and stored for later PCR;
freezing may copycat the
normal lifecycle of spirochetes within in the tick? - after blood
the full tick drops off the host, hide in the moist forest floor or
bracken and begin to lay eggs, if it is an adult female, see
my video of mating tick couple and
egg laying, or the larva/nymph stages molt into the next developmental
stage, which each take at least 6 months, both over summer and during
the winter time, that in our temperate
climate zone include frosty periods, which apparently is no
problem for the spirochetes, that manage to
survive in the ticks gut, and have been able to maintain
their complex infection cycle for thousands of years. Ötzi
the 5300 year old ice mummy had Borrelia DNA and had
repeated illness (3 x Beau's lines on finger mail) within the last six
months of life, and had heart disease and joint
damage (PubMed: 23096483), such changes are well
compatible with chronic borreliosis!
always draw the
blood samples from the patient myself, i.e. my samples have no
travel time, and I know exactly how the samples were handled;
I have used 3-5 of 5 ml EDTA tubes. Barbour
(see below) mention citrate may be better than EDTA and
that adding agarose to the
medium to make it thicker, may
facilitate spirochete growth, but I have not tried yet - or as
Lida say, full blood without additives or serum added
to BSK-H, as Sapi found may be better than EDTA;
my tubes are centrifuged for 20 minutes, and
I extract buffycoat
fraction, as shown in my buffy-coat video
...read more on procedure and stains in why-buffycoat
.. However, I do not make dried blood smears right away,
I do simple wet
drop microscopy first; i.e. after extracting the buffycoat fraction and
mixing the blood cells in the syringe, I have a concentrated
mixture of nearly
ALL the WBCs from that blood sample, and the phagocytes
which "eat" the microbes, is IMO particularly interesting to
observe in the microscope (example videos), some
RBCs (especially those with ringform parasites in should locate
right under the buffycoat layer after centrifugation)
and about 0,5
the platelet rich plasma where the free
microbes including spirochetes accumulate;
then I let the syringe
stand on the plunger with the needle still on, pointing
upwards for a while,
until all the blood cells have sedimented beneath
an the clear plasma fraction is on top, i.e.
I get a nearly free of
cells plasma fraction, that will be
pushed into the
needle first, when pressing the plunger with the needle still
in the upright position; I then drop 2 x 1 drop (circa
50 microliter each) on one object glass and cover each drop separately
with cover glasses of
a suitable size. I can add stain if I wish, a drop of acridine orange or Bb-specific FITC-labelled antibody (KPL) to
the edge of cover glass;
the stain will gradually
diffuse into the plasma under the cover glass and stain what it finds
to bind to; NOTE all fluorescing stains must be well protected
ambient light with aluminium foil while
incubating, or it may soon lose the ability to fluoresce
- For epifluorescence I
use a QBC Paralens (old version,
that can be fitted to any microscope even my old Leitz Wetzlar 1958;
longer observation time (more days), I seal
round the edges of the cover glass with semifluid vaseline, to
out the sample as drying will cause red and white blood cells
fast; this give a barrier, that probably allow some diffusion
and CO2. If watching in 21 °C room temperature, spirochetes
de-granulate and the granules inside cells cease to move in
2-3 days. One hot summer (32-35 °C for a couple of weeks, is
extraordinary hot for Denmark), I found that granules inside a cellular
structure moved continually for at least 8+
since I read 1911-Balfour, 1912-Hindle
and 1914-Nicolle (2001) etc.
aim has been to try to catch on video a full spirochetal life
it was drawn by Hindle 1912, but to do it, I will need to ensure
optimal growth conditions (BSK-H added to serum) and be able
to observe the
same object for 14+ days; I have not been able to do
that yet; the time is not a problem because the
video camera continue to show me the microscope
picture in a window on my computer screen all the time, so it
possible for me to keep an eye on what is going
on and shoot videos
and pictures, whenever I see
something interesting, like movements / development - while if still,
I use my computer for other work, reading, writing;
getting the optimal temperature in the sample has been a
problem; those researchers who studied the spirochetes life cycle 100
ago did their studies in tropical areas. Not being able to
reproduce their findings in temperate climate zone in Europe by similar
microscopy studies (leading to a false conclusion "we dont have it
here, it is only in the tropics") could very well be because of too
temperature during attempts to observe? - I just
found out it is possible to buy a heated thermal stage (fit all
microscopes) that can keep the temperature optimal for observing live
blood, it is a must have for me!
- My only "special" advantage is that
I act as both being the clinician ordering
and taking samples and as the laboratory worker
who handle the samples and do the microscopic studies, and
I observe patients
participating in my research study via the
Excel(2003) symptom diary! - see
my 2-case presentation from 2007-Leicester - so long distance is no
problem when it comes for me to evaluate if the patient has a relapse
cycle or not; I see and examine blood mainly from
the small group of chronically ill patients, who are displaying a
weekly relapse cycle and have a very high and symptom level ; doing the
symptomdiary cost no money, in danish and english version; LibreOffice
calc is free to download and use, upload of symptomdiary also
do not cost the patient any money!
MY SUGGESTION FOR
FURTHER ENHANCEMENT according to my personal experience is that the
clinician must try to optimize sampling in another way too, by letting
the patient do detailed symptomdiary
and use this to TIME the sampling to START OF A NEW FLARE (patient
do diary for 1-3 months before making appointment for blood
examination; 30 days
is usually enough for me to be able to spot if there is a weekly cycle
about 9 days interval, longer observation is needed when flares comes
at monthly or longer interval, but those patients are doing much better
and may not need blood microscopy, since the long cycle indicate their
immune system do not allow rapid cyst-to spirochete
the patient can be sampled on the first day of a new flare the chance of finding any spirochetes are highest, best about 6-12 hours after the patient begin
to feel very sick again (starting with feeling cold and very
tired, rectal temperature may drop to 36,0 °C or below, but later usually during the
night, temp. increase about 1,5-2 degree C, just before the sweat breaks); I can usually find the first spirochete
within the first
15-60 minutes of microscopy time! - and I could also use Bozsiks double
centrifugation in Dualdur method for this, and BSK-H
medium can be added to that too, he told me, for culture attempt!
I experimented with culturing from 1. full blood EDTA (no growth) 2.
extracted plasma absorbed in a differnt syringe taken out to prepare
the buffy coat fraction (may also use some for serology) 3.
"thick" concentrated platelet rich plasma fraction from the top of the
buffycoat sample - the latter thich with concentrated and visible
spirochetes therein by microscopy, seemed to yield growth faster (in
weeks) than the thin plasma (months); I stopped my experiments at thois
point because I need better microscope with trinocular, darkfield,
thermal stage ..and a better digital camera and program -
because the new camera for Win7 I bought (DinoLite eyepiece 3
Mpixel) is unfortunately not light sensible enough for taking pictures
of acridine orange and FITC stained thin small spirochetes :(.
- When I saw
spirochetes in the microscope they could usually
grow in the BSK-H medium (but I just tried a few samples as preliminary
study), though it could take > 3 months before anything grew, which
showed me I had not got the growth conditions optimal yet! - Sapis work
above explain what else could and should be done to improve the
Because Borrelia is microaerophilic, it is probably quite important to avoid stirring oxygen into the sample, and as I
could not get growth using only 5 ml BSK-H in the 10 ml red top tube,
but could with 7-8 ml, I avoid shaking and turning the BSK-H filled test tube, and
I inoculate deep below the liquid surface to ensure to seed
eventual spirochetes where there is highest chance of optimal oxygen
concentration for Borrelia growth, with the long needle put down to the
bottom of the red top 10 ml tube with BSK-H, and press a bit of the plasma out graually, while pulling
needle of the tube. Growth
always appeared very near the bottom of the tube, as visible
flocculation. I had put the lids firm on. I do not have a CO2
- After having done the wet drop microscopy for moving
spirochetes and inoculated some of the plasma into BSK-H medium, I mix
the rest of the plasma and blood cells in the syringe, and from that I make at least 10-20 blood smears, depending on how much material is left; if there is still a rest of the buffycoat in the syringe I freeze it; can be
used for PCR or inoculation into fresh culture medium after thawing, or
inoculation into animals, perhaps? - read Embers et als. monkey study (PMID: 22253822, PMC).
Well dried blood smear will keep well for many, many years, if
against silverfisk (lepisma) eating the blood, so after chosing some smears for my microscopy, I stack the rest of
bloodsmears and put a clean object glass on top and fix all together
with tape, write
patient ID and date on label, is smart for long term storage of all the
bloodsmears; a tropical medicine colleague took
down from his
20 years old blood smears from a patient with vivax malaria, and
stanied for me to look at and compare with my ringforms,
that was suggested Babesia by US research lab; in these malaria
smears there were lots of
nice well stained ringforms in the
RBCs, many many more than I find in smears from patients with tick
blood smears can be used for PCR according to dr. Shah from
Igenex (Augsburg 2011 conference). That may solve a huge problem for me!?
found that when
wet full blood samples are long time underways to the laboratory
(> 3-4 days) most of the WBC and
some of the RBCs lyse, and on this sort of material it has been
very, very difficult to get a positive PCR, probably due to polymerase inhibitor?
- lysing of the RBCs will spill lots of KCl into the plasma.
Probably therefore I have not yet had any positive PCR results in
any of my PCR test attempts, in any near to Denmark foreign labs
i.e. UK, Sweden (both on full blood EDTA), Finland (urine sample
taken 5 days after antibiotic challenge, why I tried that, see
this, 1992 Lebech PDF);
however a danish patient (sample was not taken by me)
send samples to Brorson in Norway and got a positive PCR on blood
(maybe serum?) with ospA+ospA-reverse primer; so after that, I once
tried sending both plasma and full blood EDTA from same prick as I
found spirochetes in another sample, to Brorson, but again the result
was negative; if I can send dried blood smears for PCR long
will no longer ruin the sample because of inhibitor, because dried
cells attached to object glass do no lyse and leak KCl. Fixation of the
sample may reduce the chance of getting positive PCR, see (1995
Clemmensen PDF). There
is sufficient evidence already, that Borrelia DNA, if present in
the sample, may last very long in dead materials and can be detected by
PCR; PCR was positive for Borrelia DNA on the 5300 year old
frozen Ötzi mummy, as well as in a tick and skin
from embalmed museum animal from late 1800, and in forensic
medicine they can find DNA in blood spatter samples etc. - I need
have to find some money to attempt PCR done on dried bloodsmears, not
only for Borrelia, but also for the most common
coinfections: Babesia, Bartonella, Ehrlichia, Rickettsia ...
- For my microscopy for coinfections I
fix 2-4 dried smears (by methanol in Diff-Quik); stain 2
smears in Diff-Quick and 2 smears in Acridin orange,
i.e. since I got the epifluorescence option, I look at 4 smears from
each sample myself;
I have not yet found any spirochetes free of bloodcells in dried
stained smears, so if there were any in the sample, they have
probably been washed off during the staining process (?), but I
usually finddark-blue staining (DQ) or red/orange (AO, like described by 1998-Brorson) "granules" inside WBCs and I also occasionally find a spirochete like "pearls on a string" configuration within the cytoplasm of DQ stained WBCs, as illustrated in this low quality picture, taken by me in 2003 with
a handheld (in front of the ocular) Minolta Dimage V digital camera,
video max 60 sec in 320 pixel resolution, still picture 1,3
Mpixel) which correspond to later movies of granules inside bloodcells ("GCS"), my first attempt to film what I saw and found intriguing was in 2003 on #18 filmed, but my
microscopy research started in autumn 2000 when I saw similar in my own
blood cells, immediately after I got a microscope,
that prompted me to try to arrange for microscopy - with
specific immune stain for Borrelia at Bowen RTI research
laboratory, was effectuated in March 2001, case#1 presented in York 2004, blood microscopy,
the chronic illness story continue, I had a BIG relapse in
2008; persistence of spirochetal infection were - 8 months
later - verified by Dr. Bozsik with more spirochetes found in Dualdur
by darkfield microscopy (blood sampled two following days, spirochetes
found in both samples! - watch # videos ...
- Another use for cultured spirochetes (or purified isolated
antigens from them) could be smeared onto object glasses or be
placed into wells of ELISA plate and used as antigen for indirect
fluorescence antibody test.
The IFA test technique is very well described by japanese researchers Saito-Ito et al. who were examining a case
with transfusion aquired local japanese Babesia microti
that was PCR positive but serology test negative, when using the
american variant of Babesia microti as test antigen, but the technique
is principally the same for any other microbes that can be
fluorescent-antibody test (IFAT).
An approximately 50% suspension of infected RBCs which had 30 to 50%
parasitemia was prepared in phosphate-buffered saline (PBS; pH 7.2)
containing 50% fetal bovine serum. Roughly 0.3-μl aliquots were spread
into each well of a 24-well HT Coating Slide (MS 342 BL; Bokusui Brown,
Tokyo, Japan) and were then dried. The slides were fixed in acetone for
5 min and were then immediately transferred into PBS to lyse the RBCs.
Following removal of solution by briefly blotting with a filter paper,
the slides were placed in a moisturized chamber, and 15 μl of serial
twofold dilutions of serum specimens, starting from 1:25, was added to
each well. After 1 h of incubation at room temperature, the slides were
washed in PBS, and 15 μl of fluorescein isothiocyanate-labeled protein
A (EY Laboratories, Inc., San Mateo, Calif.) diluted 1:200 in 5% fetal
bovine serum–PBS was added to each well. The slides were incubated at
room temperature for 1 h and washed in PBS. Component A of the Slowfade
antifade kit (Molecular Probes, Eugene, Oreg.) was mounted onto each
well, and cover glasses were placed on the slides. Fluorescent
parasites in RBCs were observed with a fluorescence microscope at a
magnification of 200.
The great advantage is that we can culture the microbes, we can also
use some of them to test if the patient self (or any other) has raised
serum for antibodies against these particular microbes that were
isolated and subcultured, and can be compared genetically to other
isolates in the gene bank.
Using the patients own cultured Borrelia isolate for IFA and immunoblot was exactly what Häupl et al also did, already back in 1993!
- so 20 years ago these researcers showed us the way to go ...
of Borrelia burgdorferi in ligamentous
tissue from a patient with chronic Lyme borreliosis.
T, Hahn G, Rittig M, Krause A, Schoerner C, Schonherr U, Kalden JR,
Burmester GR. Arthritis Rheum 1993
Nov; 36(11): 1621-6 PMID: 8240439
The patient, a 48-year old woman, presented with progressive
disturbance of the
central vision in her right eye. Ophthalmoscopy demonstrated multifocal
choroiditis, with 1 focus involving the macula lutea. The
reported that 2
months previously, a
tick had bitten her left
lower leg, near the ankle, and she had experienced occipital headaches
macular skin lesion. Serologic tests performed at our
titer against B burgdorferi.
diseases, particularly toxoplasmosis, were excluded by laboratory
The patient was treated with 200
of doxycycline (orally) for 6 weeks
(Figure 1). The visual
disturbance was ameliorated, the inflammatory foci of the choroid
and scar tissue formed.
Four weeks after the end of antibiotic therapy, the patient
experience brief episodes of an asymmetric arthritis, primarily
metacarpophalangeal (MCP) and proximal interphalangeal joints. Routine
electrocardiography (EKG) revealed negative P waves, with an ectopic
pacemaker that had not been present on an EKG performed at gall bladder
1 year previously. On ophthalmoscopic examination of the
was no evidence of a recurrence of the choroiditis. .... Therapy was
started with 2 grams of
for 14 days (Figure
1). Within the next 4 weeks, the ectopic atrial rhythm converted to a
normal sinus rhythm, and the arthritis disappeared.
symptoms, the visual disturbances recurred. Ophthalmoscopy showed
of the initial foci of choroiditis. Analysis of CSF
... normal ...
findings on immunofluorescence (IF) analysis for B burgdorferi-specific
antibodies, and a normal
cell count; thus,
there was no
evidence of an inflammatory process involving the central nervous
Antibiotic therapy with a combination of 300 mg of
of sulfamethoxazole, and 320 mg of trimethoprim per day,
described as effective in several cases of advanced Lyme borreliosis
tenosynovitis and mild arthralgia of the patient's
occurred. Despite concurrent antibiotic therapy, "trigger thumb"
developed within 2 weeks, accompanied by pronounced pain of the MCP
surgical splitting of the flexor retinaculum was performed (Figure 1).
After exsanguination of the patient's right arm, surgery was performed
bloodless field. The macroscopic appearance was typical of "trigger
specimen of the altered ligament was obtained, with
attention to avoiding surface contamination of the tissue sample. The
was rinsed several times in saline and medium, and was placed in
modified BSK medium. The patient's postoperative course
complications, and normal functioning of the operated thumb was
weeks after the course of antibiotics, the choroid foci were scarring.
Unfortunately, the patient had an irreversible, 70% reduction of vision
right eye. Approximately 3 weeks later, the arthralgia
Currently, after approximately 2 years of followup, there has been no
evidence of reactivation of the Lyme borreliosis.
assay and findings. The
assay was performed as described earlier, using the German isolate of B
burgdorferi, PKo 2-85 (3,8).
Serum samples were
preabsorbed with Treponema phagedenis lyophilysate (Behringwerke,
For the detection of specific IgM antibodies, a second absorption step
absorbent (Behringwerke) was performed. Using this technique, titers in
sera were <1:16. In parallel, the serum samples were analyzed
commercial B burgdorferi enzyme-linked immunosorbent assay (ELISA) kit
(Viramed, Munich, Germany,
with a protein
preparation of B31 Borrelia,
as antigen. A
a negative, and a borderline control specimen served as internal
ratio between the optical density
versus that of the borderline specimen was used to quantitate the
Ratios >1.0 were considered positive; those <1.0 were
analysis revealed positive titers of IgG, but not IgM, antibody
against B burgdorferi only during the early stage of infection when the
presented with the first episode of choroiditis. Analysis
comparable results, with specific IgG ratios of 1.15 at the onset of
disease and 0.85 in
the later stage. The IgG titer
rapidly decreased within a few weeks
first antibiotic therapy, and remained negative in both the IF and
evaluations, despite progression of the disease.
analysis and findings.
analysis, we used a method previously described (9), in which lysed
of whole B burgdorferi strain PKo 2-85 and LW2, the isolate from the
(see below) (protein concentration 100 µg/ml), were separated by sodium
sulfate-polyacrylamide gel electrophoresis (5 µg of protein per lane)
in a 10%
gel. Proteins were transferred to nitrocellulose and incubated with the
a dilution of 1:100, which we had previously found yielded optimum
Bound immunoglobulins were visualized by application of
reagents. Polyclonal antisera from patients with Lyme disease and
antibodies against outer surface protein A (OspA) and flagellin (the
kindly provided by M. D. Kramer, Institute of Immunology and Serology,
University of Heidelberg, Germany) were used to compare the isolated
LW2 and the B burgdorferi strain PKo 2-85 by immunoblot.
serum samples from the patient were tested against protein
both the LW2 and the PKo 2-85 strain, on immunoblots. There were no
differences in reactivity to the isolates. Upon repeated analysis of
consecutive serum samples, only nonspecific faint bands (75 kd,
41 kd, and 15 kd) were revealed, demonstrating a pattern different from
found in typical patients with stage III disease (10).
of mononuclear cells and
proliferation assay. Peripheral blood
and antigen stimulation were performed as described previously (8.9)
isolated cells (105/well) were stimulated in
triplicate with either 105
PKo 2-85 B burgdorferi per well, 10 µg/ml of recombinant OspA, 10 µg/ml
recombinant flagellin (41-kd protein) from B burgdorferi (both
purified as described elsewhere ), or 7 µg/well of T phagedenis
lyophilysate. Previous studies had shown these to be optimal
Control wells received
either medium alone or tetanus
toxoid (10 µg/ml; Behringwerke). Samples from normal blood donors were
each assay to exclude any nonspecific response. It
has been clearly
by appropriate separation experiments (9) that reactivity to Borrelia
under these conditions is T cell derived.
Stimulation of the patient's PBMC with the 2 strains of B burgdorferi
as with OspA
resulted in significantly elevated 3H-thymidine uptake at
times tested (compared with normal PBMC) (Figure 1). Tetanus toxoid
high levels of 3H-thymidine uptake, between 143,000 and 181,000 Dcounts
minute (stimulation values in the presence of antigen minus those in
absence of antigen). In the
earlier disease stages and prior to
to Borrelia antigens corresponded well to the
course, despite negative findings on IF and ELISA testing, reaching
at peak disease activity, as manifested by intermittent arthritis and
involvement (Figure 1). After the ceftriaxone therapy and
symptom-free period, PBMC proliferation decreased, but during a period
inflammatory reactivation of the disease, was still elevated.
symptoms at that time were choroiditis, mild arthralgia, and the
development of "trigger finger." B.
burgdorferi was isolated from
and characterization of B burgdorferi strain LW2. Tissue
the flexor retinaculum of the patient's right thumb were taken during
for the "trigger finger." Samples
were cultured at 37 °C
conditions, in modified BSK medium. Cultures were
evaluated weekly for
spirochetes in the supernatant, as detected by darkfield microscopy.
weeks, viable spirochetes were seen. This
LW2, was used as antigen for immunoblot. B
monoclonal antibodies against OspA and flagellin stained protein bands
pattern comparable to that of the PKo 2-85 bacterial antigen (data not
An aliquot of
this supernatant was examined for B burgdorferi-specific
sequences by polymerase chain reaction (PCR) amplification and Southern
and Southern blot techniques.
from the patient's tissue specimen were investigated by standard PCR
(11). We used primers which amplified a 276-basepair segment
BRL Life Technologies. Munich,
41-kd flagellin protein of B burgdorferi (primer
5'-TTCAGGGTCTCAAGCGTCTTGGACT-3'; reverse primer
amplification protocol consisted of 40 cycles: 1-minute denaturation at
940C, 30-second annealing at 500C, and -minute extension at 670C.
An amplification product, which could be hybridized by Southern blot
with the 32P-g-ATP-labeled probe
(13) using Hybond-N nylon-blotting membranes (Amersham, Amersham, UK),
obtained. The amplimer, which was characterized by Picken (13 [OA-PDF]),
the central, nonhomologous region of the flagellin gene. It has been
be both specific for B burgdorferi and discriminatory for 3
spirochete, based on the nucleic acid sequence.
Water, synovial tissue
patient with rheumatoid arthritis, and B burgdorferi strain PKo 2-85
as negative and positive controls for the studies.
microscopy techniques and
Transmission electron microscopy was performed on the ligament tissues.
specimen was removed from the culture medium and prepared and analyzed
described previously (14). Semithin sections were cut from the plastic
for the light microscopic evaluation. Thin sections were cut from
areas and placed onto copper grids (Polysciences, St. Goar,
After counterstaining with 10% uranyl acetate, followed by 2.8% lead
(both from Merck, Darmstadt, Germany), sections were studied using
101 (Munich, Germany) and Zeiss EM 902 (Wetzlar, Germany) electron
found to be heavily infiltrated by spirochetes.
the organisms lay between unaltered collagen fibers (Figure 2A); others
closely attached to the cell surface of the fibroblasts (Figure 2B).
numerous fibroblasts deeply invaginated by the spirochetes, thereby
membrane-bound cavities. These cavities appeared as vacuoles in
The patient whose case is presented herein had relapsing Lyme
choroiditis, arthritis, carditis, and tendinitis. The humoral [antibody] immune
correlated with neither the cellular reactivity in vitro nor the
activity of the disease manifestations. Repeated
necessary to stop the progression of disease, but obviously did not
eliminate B burgdorferi from all sites of infection. This was confirmed
culture of viable B burgdorferi from a ligament sample obtained
This organism characterized by molecular biology studies by our group,
subsequently evaluated in a genomic comparison study of other isolates.
study, Wallich et al identified the genogroup AAA (flagellin type at,
shock protein [HSP] 60 type A, and HSP 70 type A), and OspA genotype I
Electron microscopy of the ligament revealed spirochetes situated
collagen fibers or associated with fibroblasts, deeply invaginating
these cells. This is the first time that B burgdorferi
was isolated from human ligamentous material.
data indicate that vital
B burgdorferi persisted (a) despite
courses of antibiotic therapy, (b) even when clinical symptoms
(c) even when
no humoral immune response was detectable by ELISA or by
IF. Therefore. the
hypothesis may be
raised that an inadequate immune response as well as an evasion into
immunologically privileged sites may be the mechanisms of microbial
in patients with chronic Lyme borreliosis.
The specific humoral and cellular immune responses to B burgdorferi,
elevated during early disease manifestations, apparently were not
eliminate the pathogen. In the later stage, these
became discordant, with negative humoral and positive cellular
immunity, as has
been described in another cohort with chronic disease (16) [Dattwyler
RJ et al.
Seronegative Lyme disease: dissociation of specific T- and B-lymphocyte
response to Borrelia burgdorferi, NEJM 1988; 319:1441-46. PMID: 3054554
immune responses were also directed against
surface protein OspA during each recurrence of clinical symptoms, even
antibodies were not detectable by immunoblot.
Interpretation of this
dissociation of the humoral and cellular immune responses is difficult
requires further investigation. Initial experiments with T cell clones
patients with chronic Lyme disease (17) [Yssel H et al. Borrelia
activates a T helper type 1-like T cell subset in Lyme arthritis. J Exp
1991 Sep 1; 174(3): 593-601] suggest that selective activation of a T
subset may occur, producing a restricted pattern of cytokines which are
incompetent to activate B cells.
Even in the presence of an ineffective immune response, antibiotic
should have eradicated the spirochetes and stopped the disease
our patient. However, several of the treatment regimens recommended in
then-current literature, including combination therapies which have
described as effective in several refractory cases of advanced-stage
(6,7), did not eliminate the pathogen. Of interest, our patient showed
(split of DR2) HLA type (possibly even homozygous), which has been
shown to be
associated with a poor response to antibiotic therapy in chronic B
infection (4) [Steere Ac et al, Association of chronic Lyme arthritis
HLA-DR4 and HLA-DR2 alleles, NEJM 1990; 323:219-23]. Possible
the persistence of Borrelia are that spirochetes either develop
the antibiotics (though not experimentally documented so far) or escape
sites at which drug levels are ineffective. The detection of
between collagen fibers of bradytrophic dense connective tissue
second hypothesis. Moreover, the motility of spirochetes has been shown
enhanced in fluids as viscous as the extracellular matrix (18) [Kimsey
RB et al
Motility of Lyme disease spirochetes in fluids as viscous as the
matrix. JID 1990; 162: 1205-8].
evasion supports the use of more aggressive therapy as described in
reports (19), in which 3-4 weeks of intravenous antibiotics was
first-line treatment when systemic manifestations develop, such as the
choroiditis in our patient.
Although our electron microscopic studies were of a subcultured
specimen, and therefore in vitro effects cannot be excluded, it is of
interest that spirochetes penetrated into the extracellular matrix
causing apparent destruction. Spirochetes had also deeply invaginated
fibroblasts, thereby suggesting transcellular passage. Penetration of
monolayers by B burgdorferi has been demonstrated (20) [Comstock LE et
Penetration of endothelial cell monolayers by Borrelia burgdorferi.
Immun 1989; 57:1626-28]. In conclusion, an inappropriate immune
well as the evasion of B burgdorferi into specific sites that are only
accessible to antibiotics and immunologic attack, may be mechanisms
to chronic infection with B burgdorferi.
Intracellular location protect Bb from antibiotics that do not cross
cell-membranes ex. penicillin, cephalosporin [Georgilis K et al.
protect the Lyme disease spirochete, Borrelia burgdorferi, from
vitro. J Infect Dis 1992 Aug; 166(2): 440-4]
False negative serology can
and must never be used to outrule the possibility of persistent intermittendly active
/ relapsing and chronic borreliosis int he patient with relapsing - usually multiorgan system symptomatology!
diagnostic techniques should be developed and available for all other
possible tickborne co-infections, so the chronically ill patients can
be examined properly for these infections also, as the majority (3/4 in
my little study on 50 patient from 2001-6) of patients, who relapsed
after penicillin / ceftriaxon, had sign of co-infection(s) by SIMPLE
microscopy of stained blood smears, investigations that any
microbiology department should be able to do, really!
DETECTION of SPIROCHETES CAN BE DONE and it is not that difficult, really!
- so why do our local microbiologists refuse to attempt culture, microscopy etc.?
Why in earth are they NOT INTERESTED - this was stated in written answer from
the local university hospitals leading microbiologist, when I in 2008
asked for PCR/culture of my blood for Borrelia, after I had found spirochetes in my
blood by simple microscopy?
DID NOT GET ANY DIAGNOSTIC HELP WHATSOEVER FROM ANY DANISH
MICROBIOLOGISTS IN MY OWN CASE, apart from some positive Borrelia
IgMs in 1996-8, nor from the ID doctors in the local
university hospital (OUH)
- likewise many other of my project
patients were not helped by them either, nor by other ID departments in
DK university hospitals!
The local professor in ID (at the
time) would not offer me any antibiotic (re-) treatment for
finding Borrelia antigen by direct immunostain in 2001, nor by finding
moving spirochetes in the blood, alone by simple microscopy in 2008 (I
asked generally, not telling the ID who the patient(s) were!)
They demand ALL findings MUST FIRST be confirmed by a DANISH MICROBIOLOGIST,
but the microbiologist have consistently refused to do any of the
direct test - microscopy with specific immune stain for Borrelia
(which I can now do myself), culture (which I can now do myself),
nor PCR (which I can not do myself)
- methods I have asked
for more times, since I relapsed after IV ceftriaxone in 1998 -
nor would they test me for tickborne co-infections!
I HAD DO DO IT ALL INVESTIGATIONs MYSELF, for money paid of own pocket, instead of by the public health insurance!
were they interested nor the least bit helpfull in my familys cases,
#18 - is my husband - was refused treatment for his Babesia by ID, OUH
- read his history and blood#18 was presented by me in York 2004; I could unfortunately not offer my husband treatment for Babesia, because I had, at
the time applied for, but was just been refused permission to import atovaquone suspension for treatment of project
participants with ringforms in early 2002 by the danish drug adminstration
, who ordered
me to send patients with ringforms for evaluation by the ID
department in the various university hospitals, they have
never been able to confirm anything, I guess it is because they
are not optimizing their diagnostic methods and have
not much experience and little knowledge on the tickborne infections!?
- in my husbands case they took a set of thin and thick bloodsmear
"malaria smear" from earprick, that disappeared mysteriously, another
set was taken result negative; the ID doctor refused to tell
me who had done the microscopy, so I could not ask about
technique (how long time, at which magnification, stain etc.?) - I
could not find anything either that day, but could refind ringforms
again later! - just as it is wellknown from malaria cases, blood
smears are not always positive for intracellular babesia parasites all
it was a total waste of us parents time to go to the university
hospital ID department, we have NOT wanted our daughter AP91
to be seen by them - it would just cause stress due to frustration and
anger, that does not help!
Could lack of interest in
improving Borrelia diagnosis via DIRECT TEST METHODS be
because somebody still has an economic conflict of interest in the
IDEIA ELISA flagella antibody serology test, that was developed in-house in the danish state microbiology reference laboratory i.e. Statens Serum Institut (ssi.dk)?
are the microbiologist afraid, that if they do offer us culture
for Borrelia, and find some positive, they might find some culture
positive, but seronegative late/chronically infected Borrelia cases,
which will show for 100% certain that their serology tests fails,
as was shown by Oksi in JCM 1995 (PDF) ..
needed to study the direct diagnostic methods for detection of
Borrelia, because when the danish microbiologist would
not not help me/us, I have to do what I can.
My plan is, I
will try to examine concurrent serum samples from some microscopy
and culture spirochete positive chronically ill patients, for
comparison of their serology results, measured by different test
methods, of course the danish flagel-ELISA, compared with Line
immunoblot (and if possible with any other commercial Borrelia serology
test, done in labs within the EU, with any commercial
tests), plus LTT ..
- that is if we can find the money for doing the study; research donations are wellcome and can be paid to http://Daninfekt.dk
Laboratory aspects of
Barbour AG. Clin Microbiol Rev 1988 Oct; 1(4):
PMID: 3069200 PDF - excerpts:
With the microhematocrit
, in which the
cellular elements at the interface between plasma and packed
erythrocytes are examined, the limit of detection might be as low as 103
... By either phase-contrast or
microscopy of live organisms or standard light microscopy
, B. burgdorferi can usually be
distinguished from other
borreliae by its looser and more irregular coiling.
borreliae are gram
. However, the Gram stain is not nearly as
sensitive as Giemsa
(39, 40) and silver (31, 58, 61) stains for
demonstrating the organisms. Acridine
was used to detect
spirochetes in phagocytic cells (29) and in the CSF of a patient with
Bannwarth's syndrome (36). This dye was also used to stain what
appeared to be spirochetes in the urine of field mice (35).
and modified Dieterle
have been used to
reveal the spirochetes in a variety of biopsy and autopsy materials
that have been Formalin fixed and embedded in paraffin (31, 61, 62, 67,
99, 123, 130, 146). According to Duray and Johnson, a modified Dieterle
stain is easier to perform than the Warthin-Starry stain
various reports, silver
have been used successfully to detect
spirochetes in <1 to 100% of ECM lesion biopsies (31, 32, 63,
130); for most investigators the success has been about 40 to 50%. The
spirochetes are most easily found if the biopsy is taken from the
advancing edge of the ECM lesion and the papillary dermis is examined.
Usually fewer spirochetes are located in the center of the lesion and
in the epidermis. A "positive control" slide prepared from B.
burgdorferi cells suspended in an agar block should be included when
biopsy material is examined by silver stains (50).
cases, spirochetes have also been detected in synovial tissue biopsies
with either the standard or modified Dieterle silver stain (63, 98).
When seen, the numbers of spirochetes present were very low. The
borreliae were seen within and close to small vessels displaying
microangiopathic changes (98).
A modification of the Steiner
is reported to further improve the sensitivity of
detection (58). In this method the tissues are treated with amylase
after they have been fixed in Formalin and before the immersion in
silver nitrate. When compared with the Warthin-Starry stain, this
method has been reported to provide greater contrast between the
spirochetes and the background tissues. Using this modified Steiner
stain (Bosma-Steiner), de Koning et al. reported 100% sensitivity in
demonstrating spirochetes in skin biopsies of ECM patients and in
synovial biopsies of Lyme arthritis patients (58).
antibodies have been used successfully in immunohistologic studies to
demonstrate spirochetes in tissues (35, 102). However, with monoclonal
antibodies not only are spirochete structures demonstrated, but also
the particular type of spirochete can be determined (18, 20, 21).
a monoclonal antibody to a borrelial flagellar antigen, Park et al.
demonstrated spirochetes in a frozen section of skin biopsy from a
patient with ECM (133). Monoclonal
antibodies were also used by
MacDonald and Miranda to reveal spirochetes in touch preparations of
unfixed human brain tissues from an autopsy specimen (115)
Magnarelli et al. to identify B. burgdorferi in the kidney tissues of a
dog with renal disease (118), and by Burgess et al. to show borreliae
in organs of an infected cow (47). Direct and indirect
immunofluorescence assays with antiborrelial antibodies have been used
to determine the prevalence of infected ticks in different geographic
areas (7, 43). Although this approach has proved useful in field
studies, laboratory experiments with ticks have shown that some
borreliae in the ticks may either not react at all with certain
monoclonal antibodies or react more weakly than they usually do with
polyclonal antisera (43, 110). This phenomenon suggests that antigenic variation
Koch's postulates are partially fulfilled by isolating the offending
organism from the affected patient; this has been done in several cases
of Lyme borreliosis as described below. The culture medium is complex
and expensive and has a short shelf life. Only a minority of cultures
from definite cases of Lyme borreliosis yield spirochetes. Under these
circumstances, B. burgdorferi cultivation can hardly be considered the
diagnostic method of choice, but
remains the only way to confirm a diagnosis.
B. burgdorferi from a patient indicates an active or latent
state and not simply an inconsequential
We first recovered a spirochete from I. dammini ticks by using
Stoenner's version of Kelly's medium (39). This, or a closely related
formulation, was then used to recover identical spirochetes from the
blood, skin, and CSF of patients with
Lyme disease (26, 161). By
additional modifications of Stoenner-Kelly medium to improve the
buffering capacity and make preparation easier (BSK medium), we were
able to isolate a borrelia from I. ricinus ticks of Europe and to grow
B. burgdorferi from a single organism (15). This culture capability
allowed us to clone the newly isolated spirochetes by limiting
dilution. For details of the current medium formulation used in our
laboratory, BSK II, see reference 11. Kanamycin and 5-fluorouracil have
been added to BSK
medium for the selective isolation of the spirochetes from ticks (96).
Others have used neomycin, gentamicin, rifampin, or kanamycin alone to
reduce contamination (9, 42, 43, 130, 161). We currently use rifampin
(50 pug/ml) and phosphomycin (100 pg/ml) to prevent the growth of other
bacteria (A. Barbour and A. MacDonald, unpublished results). ...
burgdorferi is grown at temperatures between 30 and 37 °C in the
laboratory. [see also http://lymerick.net/Borrelia-growth-optimum.html
At temperatures above 38°
C, borrelial growth slows
substantially (11). Most investigators use temperatures of 32 to 34 °
The cap or lid of the
culture vessel is usually tight or sealed to
prevent loss of carbon dioxide from the medium.
generation time is
8 to 24 h, and culture-adapted strains achieve cell densities of about
108 spirochetes per ml (11). The
character of the
borrelia is indicated by its preference for the bottom portion of the
culture medium during initial growth (11, 92).
concentrations (0.1 to 0.2%) of agarose to further thicken the medium
improves the recovery of B. burgdorferi from animal fluids and tissues
(4, 6, 94, 95).
... Our early studies showed that B. burgdorferi
would grow as a lawn on BSK medium containing 0.8% agarose (11). Kurtti
and colleagues subsequently grew these organisms as isolated colonies
by increasing the agarose concentration to 1.3% and doubling the amount
of gelatin (109). The plates were incubated for 2 to 3 weeks in a
candle jar. Using this solid medium, these investigators demonstrated
growth of at least two different colony types of B. burgdorferi.
burgdorferi can survive in citrated blood stored at 4 °C for 25 days
(G. Baronton and I. Saint-Girons, Abstr. Int. Conf. Lyme Dis., abstr.
no. 55, 16 Sept. 1987), but most cultures have been inoculated soon
after blood collection. Citrated
or heparinized blood is lightly
centrifuged to separate the plasma from the cellular blood elements.
The plasma is then centrifuged at a higher force, and the plasma pellet
is suspended in growth medium.
platelets as borrelia
are usually found in the platelet-rich fraction
Cells in the CSF have also been concentrated by
centrifugation to improve the odds of recovery (136, 138, 139).
frequency of recovery of borrelia from skin biopsies
ranged from about 6 to 45% (8, 32, 139, 140, 160). Most biopsies
yielding positive cultures have been taken from the expanding edge of
the ECM lesion where histologic stains have shown that the
spirochetes are in highest numbers. B. burgdorferi has also been
isolated from skin biopsies of patients with acrodermatitis chronica
atrophicans of several years duration (9, 130) and patients with
(86). A skin biopsy was
stored frozen at -80'C for 2 years before
successful recovery of borreliae in culture medium (130).
agent has been isolated only infrequently from affected joints (140,
153); synovial fluid cultures of humans and dogs with Lyme arthritis
have usually been negative (83, 104, 160). Nevertheless, the
ameliorating effect of antibiotics on Lyme arthritis strongly suggests
that viable organisms are required for disease progression (64, 83,
B. burgdorferi has been isolated from Ixodes spp. ticks
7, 15, 39, 43, 96, 161). The culture success rate is close to
prevalence of infection in the tick population as established by
. Although organisms have been isolated
from whole ticks ground up and inoculated into culture medium, the
midgut is the site most likely to contain cultivable spirochetes (39,
40, 43). Dissection of the midgut out of the tick reduces the chance of
contamination of the cultures (15, 39). Preparing serial dilutions
of the inoculated medium can also reduce the chance of contamination;
enough spirochetes are usually present in the tick to permit up to
1,000-fold dilutions of the original inoculum (15). In some studies,
antibiotics have been added to the medium to prevent overgrowth of
bacteria colonizing the exterior and interior of the tick (43, 96).
in culture the borreliae may undergo change in one or more traits.
Alteration in both the size of outer membrane proteins and the
reactivity of these proteins with monoclonal antibodies has been seen
after an isolate has been passaged as few as 10 to 20 times (70 to 140
generations) in the laboratory (20, 151, 176).
studies have been carried out on the role of cell-mediated immunity in
Lyme disease and, specifically, the interaction between B. burgdorferi
cells and the cellular elements of the immune system. Although much of
does not have immediate relevance for clinical
microbiology, a brief consideration of pertinent immunologic findings
might provide useful background for understanding the disease and
burgdorferi cells associate with macrophages and polymorphonuclear
leukocytes even in the absence of immune serum (29, 135).
Phagocytosis has been demonstrated under these conditions, but it is
still unclear whether the association between the borreliae and the
macrophages in this situation is entirely due to uptake of the
spirochetes into the phagocyte. The spirochetes that are taken up may
be those already damaged during the centrifugation and washing steps of
cell preparation. Moreover,
finding that killed B. burgdorferi cells adhere to T- and B-cell
lymphocytes suggests that the spirochete has binding sites on its
surface for eucaryotic
cell ligands (68)
The addition of specific antiserum significantly increases the
association of the spirochetes with macrophages and other phagocytic
cells; this appears to be due to phagocytosis of the borreliae
borreliae appear to be killed once inside a phagocytic cell (29).
[killing rate not 100% PMID: 8423346
] Uptake appears not to be
heat-labile plasma components such as complement (29, 135).
of cell-mediated immunity showed a specific response by T cells of Lyme
borreliosis patients to B. burgdorferi antigens (57, 124, 132, 152).
This response has been demonstrated for T cells obtained from the
peripheral blood, the diseased synovium, and the CSF. The specific
proliferative response of T cells from either CSF or synovial fluid is
greater than that of peripheral blood T cells obtained at the same time
from a Lyme disease patient (132, 152). The greatest reactivity is at
the site of the localized infection, be it the nervous system or a
joint. Antigen-specific T-cell responses have also been demonstrated in
the synovial fluids of patients with postinfectious Reiter's syndrome
(66). T-cell clones that
specifically to B. burgdorferi antigens have been recovered from the
CSF of a patient with Lyme borreliosis (124).
Patient's T cells were
significantly stimulated by whole cells as well as soluble components
of the spirochete (57, 132, 152).
T-cell blastogenesis was more vigorous in the presence of whole
borreliae than when a sonicated supernatant was used (R. J. Dattwyler,
D. J. Volkman, J. Thomas, P. A. Falldorf, and M. G. Golightly, Ann.
N.Y. Acad. Sci., in press). The
active response to whole cells of B. burgdorferi indicates that
patients are responding to cell surface components.
The stimulation index of T-cell responsiveness to whole borreliae has
been used at one institution to confirm the diagnosis of Lyme
borreliosis (57). Some
patients demonstrate a significant cell-mediated immune response to the
borreliae when they have only borderline or slightly elevated antibody
titers to the organisms (57).
[probably due to formation
immune complexes that traps many/most/all formed antibodies which are
becoming bound to antigen only whenever there are many Borreliae
currently active]. Family members of Lyme disease patients
higher stimulation indices than unrelated controls, indicating either a
hereditary predisposition or shared exposure to the infectious agent
responses of patients show less apparent cross-reactivities than the
corresponding antibody responses to relapsing fever borreliae antigens
In affected tissues and
organs, there is usually a prominent lymphocytic infiltration. Among T cells, there are more
helper/inducer cells than cytotoxic/suppressor cells (57, 127,
this is also the case in joint fluids in rheumatoid arthritis patients
(72). In the skin, large numbers of dendritic Langerhans cell have also
been seen (37). In biopsies of ECM lesions, HLA-DR markers have been
found on keratinocytes; patients acrodermatitis chronica atrophicans
have both HLA-DR and HLA-DQ markers displayed on keratinocytes in their
chronic skin lesions (172).
Infected humans produce
(14, 54, 82, 116, 161, 168, 178), and IgE antibodies
(30) that recognize B. burgdorferi
. There does not seem to be a significant specific
IgA response (82). The bulk of the IgG-reactive antibodies are of the IgG1 and IgG3 subclasses
(B. Vandvik, Abstr. Int. Conf. Lyme Dis., abstr. no. 43, 16 Sept. 1987;
K. E. Hechemy, H. L. Harris, and M. J. Duerr, Abstr. Int. Conf. Lyme
Dis., abstr. no. 18, 14 Sept. 1987).
In the IFA [indirect
immunofluorescent antibody], whole borrelial cells are invariably used.
The cells are dried on the slide with or without yolk sac material. Our
laboratory uses washed sheep erythrocytes mixed with the borreliae to
evenly distribute the spirochetes on the smear and to provide a
convenient reference point for microscope focusing (14, 21). Once dried
on the slide, the spirochetes are fixed with methanol or acetone; some
investigators freeze the slides without the organic solvent fixation
While many laboratories use the original Shelter Island,
N.Y., isolate of B. burgdorferi, strain B31 (ATCC 35210), several other
isolates are also used. This practice of using different strains may
have little consequence for testing
within North America, where
strains are very similar to one another in their antigenic makeup (12,
20, 43). On the other hand, European strains are more heterogeneous in
the types of major outer membrane proteins they possess (Fig. 3) (19,
155, 176; B. Wilske, V. Preac-Mursic, G. Schierz, R. Kuhbeck, A. G.
Barbour, and M. Kramer, Ann. N.Y. Acad. Sci., in press). The
differences between strains are not great enough to completely
invalidate use of one strain, even a North American one, like B31, in
all geographic areas. This strain has been used successfully in Europe
for serologic testing (2). Nevertheless,
a one- or two-tube difference between the reactivity of a particular
immune serum against one strain versus another could, in some
instances, mean that a sample could be called falsely negative.
There is also the question whether continuous passage of the test
strain could result in loss of certain critical antigens for IFA and
ELISA. Changes in the major outer membrane proteins OspA and OspB (Fig.
3) have been noted during serial in vitro cultivation (20, 33, 151,
l51a, 176; Wilske et al., in press).
False-positive IFA and
can occur in patients with syphilis or
relapsing fever, two other spirochetal diseases (89, 116, 117). Lyme
borreliosis patients occasionally have reactive fluorescent treponemal
antibody absorption and treponemal agglutination tests for syphilis
(89, 117, 128)
; in these as well as treponemal antibody
cases, reagin antibody-assays, such as the Venereal Disease Research
Laboratory and rapid plasma reagin tests, are negative. As
demonstrated by IFA, syphilis, yaws, and pinta patients have had high
titers of antibodies that react with B. burgdorferi (14, 117, 126)
of sera from syphilis patients are qualitatively different from those
seen with sera from Lyme disease patients. The cross-reactive
antibodies of syphilis patients do not bind to the outer membrane blebs
of the spirochete,
and, consequently, the stained spirochetes appear
thinner and less irregular than fixed organisms bound by antibodies
recognizing outer membrane antigens (14). In serologic tests, some Lyme
disease patients have equivalent titers to B.
burgdorferi and B.
hermsii, a relapsing fever agent in North America (117). Patients with
tick-borne and louse-borne relapsing fever have cross-reactive
antibodies to B. burgdorferi (117). Although this is a potential
problem, the clinical
presentation and the epidemiologic features of
the case would usually allow discrimination between Lyme borreliosis
and relapsing fever. There is much less cross-reactivity between B.
burgdorferi and the leptospires (14, 117). Only a
few serovars of Leptospira interrogans seem to be weakly cross-reactive
with B. burgdorferi (118).
patients with rheumatic diseases, such as rheumatoid arthritis and
systemic lupus erythematosus, which the physician may be trying to
distinguish from Lyme borreliosis, have false-positive reactions in B.
tests (14, 53, 116, 144). These reactions
appear due to the nonspecific sticking of rheumatoid factor aggregates
or immune complexes to the borrelial antigens. One indicator of a
false-positive IFA reaction among rheumatic disease patients is the
beaded appearance of the spirochetes (128). This type of staining
reaction is also seen in false-positive fluorescent treponemal
antibody-absorption test results of patients with systemic lupus
erythematosus and other autoimmune diseases (125).
determinations tend to be less specific than those for IgG antibodies,
but may be useful in early disease or when reactivation or reinfection
is suspected (54, 126). Patients with infectious mononucleosis often
have falsepositive IgM tests (126, 161, 168). Magnarelli and Johnson
found that 5 of 16 patients with Rocky Mountain spotted fever and three
of 7 patients with rheumatoid arthritis had a positive IgM-specific
ELISA for B. burgdorferi (116).
While patients with second- or
third-stage Lyme borreliosis almost always have elevated IgG titers,
those with early disease often have serum antibody titers below the
diagnostic threshold for 6 weeks or more after onset (2, 54). Only
about 50 to 60% of patients with early disease, i.e., ECM, have
diagnostic titers as measured by either IFA or ELISA (2, 14, 54, 126).
Antibiotic therapy of first-stage disease may blunt the immunoglobulin
response to the point that diagnostic thresholds are never
(161). In cases of reinfection, the antibody titers to B. burgdorferi
may show a fourfold rise from the previous convalescent value (137).
patients with a neurologic disorder attributable to Lyme borreliosis,
the antiborrelia antibody concentrations in the CSF are usually higher
than could be accounted for by leakage of circulating antibodies into
the CSF. Any CSF titer above 5 is probably significant (168). However,
antibodies may be present in the CSF as a consequence of disturbance of
the blood-brain barrier. One indication of nervous system involvement
is the presence of oligoclonal immunoglobulin peaks in the
but not in the serum (82, 84, 129, 143). To further establish that the
antiborrelia antibodies were produced intrathecally, a comparison of
serum antibodies and CSF antibodies can be carried out.
CSF/serum-specific antibody ratios can be adjusted by using factors
into account the total IgG, IgM, or albumin concentrations
in the CSF and serum. The resultant indices serve to identify those
patients with antibody produced locally in the central nervous system
(82, 84, 85, 105, 168, 170, 178). Calculations of such an index may be
needed, for example, to accurately diagnose the disease in a patient
who has a neurologic disorder resembling multiple sclerosis and an
elevated titer to B. burgdorferi in the serum.
Western blot (immunoblot)
assays have been performed on a research basis to determine to which
protein antigens patients are responding with antibody (12, 14, 51, 53,
74, 176, 178; Wilske et al., in press). These studies have confirmed
the finding of IFA and ELISA studies that there is a delay in
production of detectable amounts of antibody to the borreliae. Once
antibody production begins, it is usually in the form of IgM antibody
to flagellin (18), a 41,000-dalton (41-kilodalton [kDa]) protein that
is the predominant component of the flagella (53, 74). With
both IgM and IgG antibodies to a variety of other antigens appear;
these include proteins with apparent molecular weights of 15,000,
27,000, 55,000, 60,000, 66,000, and 83,000 (12, 14, 53, 177,
The 66-kDa protein appears to be another protein associated with the
outer membrane (20, 51). The more chronic and complicated the disease,
the greater the number of antigens to which the patients respond.
Almost all patients with Lyme disease of more than a few weeks duration
have IgG antibody to the 41-kDa flagellin protein (14, 51, 53, 74).
Some patients have IgE antibodies to the 41-kDa protein (30).
abundant proteins of the B. burgdorferi cell are the major
surface-exposed proteins OspA and OspB (87, 88). In most North American
strains, the apparent molecular weights of these proteins are 31,000
and 34,000, respectively (Fig. 3) (20, 21). These proteins
to be highly immunogenic in experimental animals that have been
injected with whole organisms (20, 21; unpublished observations).
Paradoxically, humans with Lyme borreliosis develop antibody against
OspA and OspB, if they develop them at all, only late in the course of
the disease (14, 51, 53). Sera from patients with other spirochetal
disease have shown cross-reactions in Western blots to the 41- and
60-kDa proteins of B. burgdorferi (14, 74, 176). Considering the known
antigenic relatedness between the flagella of the different Borrelia
spp. (18), one might expect some degree of cross-reactivity to the
flagellin protein. Epitopes of the 60-kDa protein antigen appear to be
conserved among various spirochetes (75a, 176).
as well as quantitative differences may be seen in Western blots and
other immunoblotlike assays that use serum and CSF obtained from the
same patient with neurologic involvement (129, 178).
these differences have not been noted when paired serum and synovial
fluid specimens from patients with arthritis have been examined (53;
unpublished observations). The B-cell response to B. burgdorferi has
been demonstrated by studies of immunoglobulin synthesis and
specificity on an individual B-cell level (111).
The finding of
almost universal responsiveness to the 41-kDa flagellar protein has
been used by investigators as a point of departure for studies of
subunit components of B. burgdorferi. Coleman and Benach used purified
flagellin protein eluted from sodium dodecyl sulfate-polyacrylamide
electrophoresis gels (51); the protein presumably was denatured during
purification. These investigators found that an ELISA based on a cruder
but undenatured "flagellin-enriched" fraction was more sensitive than
an ELISA that used purified flagellin eluted from a gel. Hansen
isolated whole flagella through mild detergent disruption of the cells
and subsequent density gradient ultracentrifugation (76); flagella
remain intact by this method (18). This
group used the isolated flagella as the antigen in ELISA testing and
found improved sensitivity in serologic tests of patients with early
disease when compared with a standard ELISA (76). The
heightened sensitivity was due in part to lowering of the cutoff point
between positive and negative reactions.
The assay of Hansen and co-workers appeared to provide greater
discrimination between patients with Lyme borreliosis and those either
without disease or with nonspirochetal disorders.
The outer membrane
OspA and OspB proteins are other isolated components of the borrelial
cell that have been examined by Coleman and Benach for use in
immunoassays (51). This study, which used eluted proteins in an ELISA,
confirmed the Western blot analyses that showed antibodies against
these antigens appearing later in the course of the disease. Although
these outer membrane proteins may not be useful for immunodiagnosis of
early disease, they could have a role as components of a very specific
assay in secondary or tertiary disease in humans. A patient with Lyme
arthritis had antibodies that bound to recombinant OspA and OspB
proteins (88), and, thus, it is likely that patients are responding to
the proteins themselves and not carbohydrate or glycolipid moieties
that might be associated with them.
Western blot analysis has been proposed as a practical clinical
laboratory test for Lyme borreliosis (74). The advantage to this
procedure is that the response to individual components can be examined.
Grodzicki and Steere found
Western blot to be the most sensitive test in early Lyme borreliosis
(74). Kirsch et al. used the Western blot to diagnose Lyme disease in a
patient with a fatal illness (102). Almost all immunodiagnostic assays
reported on have used culture-grown borreliae that were washed and
centrifuged at least twice before use in the assay. Whether or not
loosely associated spirochetal antigens, such as a slime layer, could
be dislodged from the cell surface during antigen preparation is not
known. Neubert and colleagues used borreliae obtained directly
from the blood of an infected mouse for their IFA; however, the
Borrelia species used in the test was not B. burgdorferi (130). Another
area that has been little investigated is whether there are
important nonproteinaceous antigens of B. burgdorferi.
the number of isolates of B. burgdorferi from different human and
animal sources and from different parts of the world increases, greater
attention is being paid to strain distinctions. Several options are
available, including poly-acrylamide gel electrophoresis profiles of
cellular proteins, reactivities of monoclonal antibodies, and plasmid
analysis. The initial isolates of B. burgdorferi from the United States
were almost identical in their polyacrylamide gel electrophoresis
profiles (12, 20, 21, 43). They all had major proteins of 31 (OpsA) and
41 (flagellin) kDa. A large majority had an abundant 34-kDa surface
protein, OspB, but some isolates either lacked this protein or had an
OspB with a slightly different electrophoretic migration (12, 20). As
more isolates from Europe were examined, differences in the
and OspB proteins were noted (19, 155, 176; Wilske et al., in press).
The OspA-like proteins varied from approximately 30 to 33 kDa in
apparent size. OspB-like proteins also varied; some European
strains had no major protein that could be considered the equivalent of
OspB. Some strains, especially those from regions of Germany, Austria,
and Scandinavia, lacked even an OspA-like protein. Instead, they had a
major protein of about 22 kDa. This protein has been designated "pC" by
Wilske et al. until its surface localization can be confirmed (176; in
press). A single United States strain with a major surface protein of
about the same size as pC has been isolated from a tick in California
When antisera prepared
against whole cells of different strains have been compared by IFA, too
few differences in the reactivities of the various isolates have been
seen to justify a serologic typing scheme based on use of antisera
against whole cells (7).
Polyclonal antibodies to isolated
components, such as OspA and pC protein, offer better discrimination
between strains (Wilske et al., in press), as do monoclonal antibodies.
The monoclonal antibodies are directed against single epitopes in one
protein, usually either OspA- or OspB-like proteins (20, 21; Wilske et
al., in press). Using criteria of polyacrylamide gel electrophoresis
profiles, polyclonal antisera reactivities, and monoclonal antibody
binding, Wilske et al. (in press) identified seven distinct types of B.
burgdorferi among a panel of European strains.
Another way to
characterize B. burgdorferi isolates is to analyze their plasmid
content; both circular and linear plasmids have been identified (13,
16, 90, 151a). A relatively simple extraction procedure can be used to
enrich for plasmids in the DNA preparation (13, 16). The plasmid
species are then separated on low-percent agarose gels. Analyses have
shown considerable heterogeneity in plasmid profiles among strains,
even those from North America (13). Plasmids
either undergo rearrangement or are lost from the cell during serial in vitro
cultivation (13, 90, 151a).
DNA hybridization of whole chromosomal DNA has shown that B.
burgdorferi is a distinct species in the genus Borrelia (90, 92, 93,
148) and that strains within the species differ in the amount of DNA
relatedness. These differences may not be great enough, however, to use
genomic DNA hybridization as a routine typing procedure for B.
burgdorferi. Its most appropriate use is still as a tool for
determining whether an unknown arthropod-associated spirochete is a
member of the genus Borrelia and to what species it is most closely
related. Use of DNA probes for specific genes, such as the ospA gene,
may offer more advantages for distinguishing between strains within the
B. burgdorferi is a
blood-borne pathogen of humans and domestic animals that can cause
significant and prolonged disease. It may be confused with a variety of
other chronic, noninfectious disorders. B. burgdorferi, like the
relapsing fever borreliae, has been considered a biocontainment level 2
organism, and it is appropriate to continue to treat it as such.
Although there have been no documented examples of laboratory-acquired
Lyme borreliosis in humans*, there clearly has not been enough
experience with the organism to be complacent about its risk to
laboratory workers. The
most likely routes of infection would be through a break in the skin,
the conjunctiva*, and the oral mucosa
; experience with the
closely related relapsing fever borreliae indicates
that infection can occur through these routes (17). Infected
blood and cultures pose the greatest potential risk, but animal and
laboratory workers may also be infected through contact with urine of
infected animals (35, 46) and through handling live ticks.
There is probably little chance of infection through aerosolization or
contact with spirochetes that have dried on animate or inanimate
*) 1996 PMID: 8817171 describe
2 cases of direct transmission of Borrelia into the eye
via blood spatter accidents i.e. in the laboraty setting - so
wear protection like glasses, mask and gloves, when handling
possibly infected blood samples and other material, both for
own safety reason and to avoid contamination of the sample!
Isolation of Borrelia burgdorferi from biopsy specimens taken from
healthy-looking skin of patients with Lyme borreliosis. Kuiper H, van
Dam AP, Spanjaard L, de Jongh BM, Widjojokusumo A, Ramselaar TC, Cairo
I, Vos K, Dankert J. J Clin Microbiol 1994 Mar; 32(3): 715-20.
PMID: 8195384 PDF
six patients, a skin biopsy specimen was taken at the site of a
previous erythematous skin lesion 1 to 6 months after disappearance of
the lesion. Four of them presented with early disseminated Lyme
borreliosis. In one additional patient with early disseminated Lyme
borreliosis, the site of a previous tick bite was biopsied. None of these patients
had been treated with antibiotics before presentation.
cultures of the skin biopsy specimens of the seven patients showed
growth of Borrelia species. By rRNA gene restriction analysis and
genospecies-specific PCR, six isolates were classified as Borrelia garinii
and one as Borrelia group VS461 [Borrelia
Excerpt on procedure:
A 4-mm-diameter biopsy was taken with a biopsy punch (Disposable Biopsy
Punch; Stiefel, Wachtersbach, Germany) after the skin was treated with
either 1% iodine (wt/vol) or 0.5% chlorhexidine in 70% (vol/vol)
... Culture method for B. burgdorferi.
specimens were transferred to 5.5
ml of modified Kelly's medium
(28) in 7-ml Duran borosilicate glass disposable culture tubes (Schott,
Mainz, Germany). Prior to use, the tubes were rinsed 10 times with
distilled water and subsequently sterilized with heated air at 180 °C
for 2 h. Rifampin (final concentration of 50 mg/liter) and fosfomycin
(final concentration of 100 mg/liter) were added to the medium to
inhibit the growth of contaminating bacteria. Preliminary experiments
had shown that these concentrations of antibiotics did not inhibit the
growth of B. burgdorferi
culturing of CSF specimens, approximately 2 to 4 ml of CSF was
centrifuged at 5,000 x g for 20 min. The pellet was transferred to 5.5
ml of modified Kelly's medium without antimicrobial agents. Incubation
at 33 °C
for 8 weeks. The presence of spirochetes was determined by dark-field
microscopy weekly. A culture was considered positive when motile
spirochetes were seen. All isolates were preserved at - 70 °C with 50%
glycerolpeptone for cryoprotection.
... 57 consecutive patients
referred to our hospital. All these patients were examined by a
dermatologist (Toni Ramselaar or Irina Cairo) and met the Centers for
Disease Control case definition criteria for EM. The recovery rate of B. burgdorferi from skin biopsy specimens from
patients with EM was 86%
Four of them (patients 1, 3, 4, and 5) had neuroborreliosis, as shown
by an increase of the leucocyte count and protein level in the CSF.
Patient 2 had clinical symptoms of arthritis and cardiac involvement.
... Three of the four patients with neuroborreliosis had also
against B. burgdorferi in the CSF; one patient (patient 4), who only
had immunoglobulin M (IgM) antibodies against B. burgdorferi in her
serum, had no CSF antibodies against B. burgdorferi.
Two other patients were included in the study, since they reported
previous erythematous lesions and had antibodies against B. burgdorferi
in their sera. One
them (patient 6) [sampled 60 days after disapperance of erythema
IgM+IgG serum antibody positive] had mild myalgia at the time of skin
biopsy, whereas the other patient (patient 7) [sampled 180 days after
disappearance of erythema, IgM negastive, IgG positive] was
...None of the cultures became contaminated
with other microorganisms. From patient 7, a culture from a second skin
biopsy specimen taken at a site without a previous skin lesion remained
... It was concluded that the strains from patients 4 and 6 were B. garinii
and the strain from patient 7 was group VS461and that an additional
typing method was necessary for the other four strains. Therefore,
isolates were also genotyped by genospecies-specific PCR (18).
Using the primer pair specific for B. garinii
a 527-bp fragment was amplified from the strains recovered from
patients 1 through 6 (Fig. 4). A 591-bp fragment was amplified from the
isolate from patient 7 by the primer pair specific for group
we conclude that the strains from patients 1 through 6 were B. garinii and the
strain from patient 7 belonged to group VS461 [~Borrelia afzelii].
of antibodies to B. burgdorferi. Antibodies to B. burgdorferi in serum
and CSF samples were detected by flagellum-enzyme-linked immunosorbent
assay (ELISA) (Dakopatts
A/S, Glostrup, Denmark) (13).
1: One patient (#4) with radiculopathy and CSF pleocytosis of
and Sp-protein 0.69 g7l was IgM positive, but IgG negative for
Borrelia antibodies in serum at time of biopsy 32 days after
disappearance of the skin lesion; all the others
serum antibody positive.
... Interestingly, at
42 days after the onset of disease one of the seven patients (patient
4) had developed only IgM but no IgG antibodies against B. burgdorferi.
is unusual, although such patients were also described by Hansen and
Lebech (14), who reported that 44 of 187 patients with neuroborreliosis
had only IgM antibodies against B. burgdorferi; the range of their
disease duration was 6 to 54 days, with a median of 19 days.
Hansen K, Lebech AM. The clinical and epidemiological profile
Lyme neuroborreliosis in Denmark 1985-1990. A prospective study of 187
patients with Borrelia burgdorferi specific intrathecal antibody
production. Brain 1992;115:399-423.
We cultured B. burgdorferi from CSF samples from two of
four patients included in this study
(50%), but CSF cultures from 13 other patients with neuroborreliosis
seen in our hospital remained negative. Our total recovery rate from
CSF specimens from patients with neuroborreliosis was therefore 3 of 17
(18%), which is similar to the recovery rate reported in the
above-cited studies. The rather high recovery rate from CSF specimens
from the four patients included in this study could possibly be
explained by the recent onset of neuroborreliosis in them; also,
Karlsson et al. reported that CSF cultures were most successful for
patients with a recent onset of neurological symptoms (15).
patients with EM are treated with an antibiotic, since such treatment
is generally effective in preventing the onset of early disseminated or
chronic LB (4). Our
patients had no antibiotic treatment because their skin lesions had not
been recognized as EM by themselves or by their general physicians.
Whether antibiotic treatment always leads to elimination of B. burgdorferi
from the skin has been studied by culturing posttreatment skin
biopsy specimens taken from healthy-looking skin from the site
from which B.
been cultured before treatment (8, 23). In total, 26 patients were
involved in these studies, and cultures were negative in all cases. However, B.
has been cultured from skin biopsy specimens obtained at the sites of
previous erythematous lesions from three patients after antibiotic
Preac-Mursic V, Weber K, Pfister HW, Wilske B, Gross
B, Baumann A, Prokop J. 1989. Survival of Borrelia burgdorferi
in antibiotically treated patients with Lyme borreliosis. Infection
17:355-359. PMID: 2613324
vitro studies showed that human skin fibroblasts can protect B.
burgdorferi from antibiotic treatment (11). Invasion of spirochetes
into fibroblasts has been documented and could serve as a possible
mechanism by which spirochetes
can escape from antimicrobial actions of antibiotics (16). Intracellular
persistence of spirochetes
could also lead to a decrease of the inflammatory skin reaction and to
fading of the erythema. Alternatively, a down-regulation of the cellular
response, which has been documented for the related microorganism
Treponema pallidum (10), could explain the disappearance of the
erythema during persistence of microorganisms in the skin.
Tilton 2001. Culture of Borrelia burgdorferi. (PDF)
- a rather bad attempt to compare MPM and BSK medium for culture of
Borrelia from patients blood.
Reed 2002. Laboratory Testing for Lyme Disease: Possibilities and
Both serum and whole blood
were collected from 25 patients who presented with signs and symptoms of persistent Lyme
and whose disease was confirmed clinically and by
of whole blood
were added to 10-ml tubes containing 5.0 ml of MPM
medium and to 10-ml tubes containing 5.0 ml of BSK-H medium (complete
medium; Sigma, St. Louis, Mo.).
The MPM tubes were incubated for 4
weeks at 30 °C
BSK-H tubes were incubated for 4
weeks at 35 °C.
Aliquots of a control culture of B. burgdorferi 2591 were added to
replicates containing either MPM or BSK-H medium and incubated
similarly. All tubes were periodically sampled, and the slides were
stained with acridine orange (AO) stain. A terminal PCR targeting the
OspA gene (1) was performed on all patient cultures and controls. None
of the patient samples showed growth of B. burgdorferi.
culture in BSK-H grew luxuriantly, as evidenced by the appearance of
spirochetes in the AO stain and a positive PCR result. The control
organism failed to grow in MPM medium.
These authors do not state exactly how they
diagnosed their patients to have persistent Lyme
symptomatology, if the blood specimens were collected during current
flare activity and if the blood samples were cultured immediately after
blood draw or there had been an interval between sampling and culture
(sending samples by mail for instance, time without media, freezing,
cooling of samples ... conditions that may affect the time to succefull
spirochetes can only be found in the blood for at very short time
window of only about 24 hours in the beginning of a new flare, for
instance by simple microscopy using the microhematocrit technique as
described by Barbour above already in 1988 (and by others before
improving diagnosis of RF Borrelia); phase contract microscopy of the
buffy-coat fraction (WMV
Febr. 6, 2008 by Marie Kroun)
and darkfield microscopy after double centrifugation in
Dualdur reagent (WMV
Oct. 16, 2008 by Bela Bozsik), shows two illustrative examples of
spirochetes detected in blood by simple microscopy from the same
patient with persistent borreliosis, the samples were taken 8 months
apart and no antibiotic treatment was given to the patient in between
the samples, because first the patient was trying to persuade her GP to
ask the microbiology department for attempting culture for Borrelia and
that took time and then she was waiting for several months after the
first microscopy for the answer from the microbiologist, if they would
try to culture her blood and had to abstain from
treatment because that would have ruined the option of getting
confirmation of persistent borrelia infection! - culture was
unfortunately denied, but in the meantime the patient
had - as she
usually does during summer time - experienced a sponataneusly
improved period; symptoms of persistent Borrelia
flares more and for longer time (a few weeks to 3-4 months) during
spring and autumn, corresponding to the blood-meal seeking
tick seasons out in nature! - thus she waited until the next big autumn
flare hit in order to get re-microscopy confirmation by dr. Bozsik!
is thus very important to time the bloodsampling for
culture according to the individual patients CURRENT FLARE
, it is far the best to sample blood for culture
within the first day of a new flare, since the
chance of finding visible spirochetes in the blood outside this narrow
time window can be calculated to be only around 3-10%, when not taking
the cyclical relapse pattern into consideration for optimal sampling
when the patient experiences a monthly (i.e. flares comes at about 4
weeks intervals, 1/30 days ~ 3% chance) versus a weekly (i.e. flares
comes at 8-10 days intervals, 1/10 ~ 10% chance) - this easily explains
why many researchers who have NOT taken the cyclicity pattern into
consideration when sampling, has so low luck in finding spirochetes in
blood from patients with long term persistent Lyme borrelia infection!
the patient has mounted a detectable amount of ANTIBODY no matter if it
is being measured by conventional ELISA or by Blot methods - even the
new variants (Line blot) that are based on recombinant selected diagnostic
relevant Borrelia antigens from more strains - the finding of
CURRENTLY SEROPOSITIVE STATUS indicates at the same time that there
must be low or no level of Borrelia ANTIGEN
circulating in the blood!
hence it is quite unlikely to get a positive culture (or microscopy or
PCR) of Borrelia from blood from currently SEROPOSITIVE individuals.
from whose blood one will attempt to culture
Borrelia must optimally be
selected from the group of patients displaying A CURRENT (WEEKLY)
RELAPSING PATTERN and sampling must preferentially be done on
of a new flare, and selectively attempted in those patients, who are
currently seronegative - or who recently turned from higher positive
level to current lower positive
or seronegative indicating that more of
the formed antibodies are now again being used up quickly by binding to
proliferating and in blood circulating ANTIGEN - THIS IS THE TIME TO
HAVE A LOOK; therefore the patient must be urged to keep a very
detailed daily symptom log like the Excel symptom-diary (Marie Kroun
Leicester 2007, UK powerpoint
) - so cyclicity can be
overviewed and detected fast and sampling can be planned to start of
the next expected flare start!
weeks is a rather short time considering the slow growth rate of
Borrelia and knowledge was present aldready in 2001, that under
adverse conditions for growth of the spirochete form the
quickly turn into their alternate forms (cysts, spheroblasts,
granules, blebs - many names associated with these structures,
see 100 years pictorial
that it may take between 9 days and 4 weeks or longer for the "cyst"
form to return to (produce several new baby-)
according to 1998-Brorson
... already during skin transmission experiments done back in
1950'ies showed that a
certain maturation time of 8-10 weeks of the clinically
material was needed before successful transfer of the infection could
occur [Pashoud JM. Hautarzt 1957;8:197-211, 1958;
9:263-269 - 1958;9: 311-315.]
Finally why did the authors culture at different
temperatures in MPM and BSK? ---when comparing two media for culture,
all other conditions should have been kept equal; a difference in time
to culture Borrelia, with lower chance of positive culture in MPM at
suboptimal temperature compared to in BSK at a more optimal temperature
authors chose to attempt culture in WHOLE BLOOD despite Barbour already
in 1988 had recommended incubation of the pellet residue (after of
double-centrifugation of blood then serum, like dr. Bozsik
does!) re-suspended in culture medium; my own culture
support Barbours suggestion, since there in my first attempt was
quicker growth in sample from buffy-coat fraction with large
serum, but not quick growth in full blood sample not in serum fraction
taken from way above the buffy-coat layer all from the same
Culture of B. burgdorferi
involves incubating a specimen in Barbour-Stoenner-Kelly medium (BSK)
(or modifications of BSK) and detecting the presence of characteristic
spirochetes by dark-field microscopy or by fluorescent
acridine orange or a specific
fluorescent antibody (FA).
Some microbiologists consider Borrelia culture to be too expensive and
tedious to be practical for many clinical laboratories. In reality,
isolation of B. burgdorferi sensu lato is no more difficult and is
probably easier than recovering and identifying other fastidious
microorganisms such as Legionella or Mycobacteria spp. from clinical
specimens. BSK is
commercially available*, and the only specialized equipment required is
a fluorescent microscope or a light microscope equipped with a
Time to detection of a positive specimen can be within a
relevant time frame, provided cultures are examined for the presence of
spirochetes at frequent intervals, especially during the first 2 weeks
of incubation. Of 90 specimens that were submitted to Marshfield
Laboratories from 1991 to 1997 and that turned out to be culture
positive, 74 (82%) were identified as positive for Borrelia within the
first week of incubation and 32 (35%) were identified within the first
3 days, with the longest time to detection of a positive culture being
16 days (Fig. 1).
sells ready to use BSK-H
(after thawing) with a
guaranteed durability of 9 months in frozen condition, and in
small amounts of 100 ml, thus the laboratory will not risk loosing
money on buying a large amount of BSK-H medium that are not used up
before the expiration date.
**) also needed is an incubator in order to keep a constant optimal temperature for growth
of Borrelia around 35 °C
- otherwise it may
take very long time to get growth of Borrelia!
2005 Youtube video http://www.youtube.com/watch?v=WozrCFW0mRM
(put up byTrevor Marshall) Lida
Mattman's presentation at the conference held in Chicago in 2005 by the
Autoimmunity Research Foundation. This is Lida's second to
last presentation. Excerpted transcripts:
The term cell
wall diverse / divergent forms, should be used
instead of calling them cell wall deficient .. 03:00 pictures of
pleomorphic forms of Borrelia burgdorferi ...
12:20 Xylol float
(described in her
book) .. take 1-2 ml of blood culture and you put
1/10 ml of Xylol,
shake for about 5 minutes, the leave it for an hour .. the cells wall
deficient forms .. will move into the Xylol phase
28:02 show "fluorescent spirochetes in a pleiomorphic
colony in semen
so if a male is infected, his semen is very apt to be
and this is now the most important cause of congenital
.. not syphilis which used to be true, but the organism of
28:36 she talks about congenital borreliosis
"this is data from just one hospital saying that 1/6 of their
patients who were newborn
fatalities had died because of Lyme .. these are autopsy findings"
Tabulation of Southampton Hospital
Autopsier showing evidence of Lyme borreliosis
- ventricular septum defect VSD (AV canal)
- neural tube defect (hydroceph. meningocele)
abscence of left hemidiaphragm
29:05 Then we get to .....
Report of spirochetes
stained in CNS tissue or spinal fluid
- Guirand P 1931
- Austregesilo A 1933
- Blackman N 1936
- Rogers N 1932
- Hassin G 1939
- Marburg D 1942
- Mattman L 1993
-Steiner G 1927
- Schrinken I 1937
For complete references se Marshall
V, Medical hypothesis 88, 1988
".. a different topic .. and this I think is too bad and perplexing .. it
takes an awful long time for the world to accept anything new; a
derogatory article will be published by any journal right away, a
negating article, but one with any new, they are scared to death to
publish and .. but Vincent Marshall, who somebody of you
of, was a veterinarian, who studied a lot of things, including the
spirochetes in his vet lab and this is from an article he wrote, and
the same thing is in my book, but look at all the countries, where they
that there was a spirochete in multiple sclerosis, so this
is MS, from France and from the United States ..? .. and in Germany,
only did they the spirochetes in MS, but they were able to do
Kocks postulates ..
think you all know what Kocks postulates are .. that you have a
culture, that you got from a patients specimen, you put it into an
experimental animal .. you get a disease, you get illness, and you take
it out out and you find what you injected, you know that it was the
pathogen, and so often you get the same kind of disease ..
confirmed it in monkey and Vincent Marshall did it in all his small
animals, mice, and rats and hamsters, and Steiner did it using a silver
stain, he looked at autopsy
tissue from the brain of MS patients
and that has been confirmed in Austria and many parts of Europe - so
why on Earth don't people know that MS is not due to a virus, it is due
to a spirochete!
- and I am going to show you what the spirochetes
looks like, and how easy it is to show that .. this is uncentrifuged
spinalfluid, it is so loaded that you see the spirochete in MS
31:35 .. "you know that is one good thing about this, I
said we have a happy note here, anyway .. so very often there are so
many organisms present in these spirochetal diseases, that all you need
to do is incubate the sample, it is the best culture medium, what can
be more simple and it even go into the refrigerator sometimes, but it
is best to put it at 35 degrees, so you don't have to say .. oh I could
not make up that fuzzy medium .. all you need to do is to do is take
one ml of it and incubate it and see what grows .. you know the
textbook say, don't even try to grow spirochetes, you will just mistake
fibrin for spirochetes .. and you
can tell fibrin from spirochetes by using acridine orange,
I will show you that in a minute .. this is fluorescent antibody ..
this is blood right, you can see it better [audience: it says
refrigerated blood] .. yes it was just blood that was stored
and they didnt even need 35 degrees, it grew .. and this is
antibody to Borrelia burgdorferi- so why on Earth won't people accept
33:30 about Brorsons work .. "you can learn a great deal by looking for
the cystic form, not the spirochete .. the spirochetes all look pretty
much alike" .. "he found these cysts in multiple sclerosis in the spinal
fluid directly, and in spinal fluid cultures in BSK medium"
"and this brings me to a sad point, something that made me cry, it has
been quite a while ago, but when we published our paper showing we
could grow spirochetes, something came out from a prestigious source,
that said that said that they could not grow any spirochetes
.. you know
there is nothing easier in the world thant not growing them, if you
don't do it right" .. laughter from audience .. " so why did they not call us and ask?"
published .. Steve Phillips was the senior author .. I don't mean to
sound like paranoid, but to get an article published in a medical
journal in the United States, the senior author should be an
MD male .. // .. so we published this paper on our wonderful MPM medium
to grow spirochetes ... and article came out .. we
not grow anything, we tried and tried .. we held it for weeks
weeks and we looked for spirochetes and did not grow any ..
Why did they not call us? ..
Another lab had a negative finding and they called dr.
Phillips and he said it is probably the final pH, you don't have it quite
right, it has got to be alkaline, and so
they called him a few weeks later and said, they are growing like crazy
So why did they not call me and get it right straightened out?
for all those who have asked, I have made copies of our medium for
anyone who wants to grow spirochetes, also the precautions, there are
about 8 things you can do wrong .. and one of them is to use
medium when it first comes out of the autoclave
..// .. we think that it is hydrogen peroxide that forms and it wont
let anything grow .. one day later you can use it .. but you will never
grow spirochetes on a medium freshly autoclaved"
"... so we
confirmed the work of the Brorsons, but we carried it a bit
further because our MPM medium is better than BSK, because it will grow
them from the blood of an MS case .. who likes to
spinal cord punctured .. people object to a spinal punctures,
everyone is used to an iv .. so we can grow them from the
using our medium and don't have to use spinal fluid ..
looked at 40 MS cases, and the blood cultures on 40, and they all have
a spirochete that reacts with the antibody to Borrelia burgdorferi, but
the fact that they make these cysts [which Lida call backpacks because
they have material on the outside of the cyst wall] show that
are not [Lyme] Borrelia because the cysts of Borrelia look different
and I will show you some"
In Lyme disease there are two
kinds of cysts .. rectangular things attached to
with fibriae.. [another picture show some] round forms
"do not in any way resemple the cyst of MS, does it?"
(electron microscopy were done by Carol Ayala - for her
info (2012/10) see http://lifespan.org ) ..
39:45 "This is another interesting disease .. this is what you will
find in the blood of any Lou
Gehrig disease patient (ALS)
and this will grow in 48 hours as sure as the sun comes up in the
morning and sets in the evening .. but you have to
have precautions and inoculate it right and do everything
correctly .. but once you learn how there is nothing easier to do ..
Hyphenated (segmented) Fimbriae grew in blood culture of fours ALS
I have a cigarette cought and I never smoked, it is not fair ..
Blood culture of ALS patient has pleomorphic spirochete colonies
staining with fluorescent Lyme antibody ..
(only 5 cases, luckily Parkinson is rare, two types of cysts, looking
different from Lyme borrelia)
goldlabelled: first treated with unlabelled Borrelia antibody (made in
rabbits), after wash edsurplus antibody away then gold
anti-IgG antibody to rabbit serum is added, which binds to the
rabbit borrelia antibody bound to the structures .. i.e. indirect
"I like fluorescent better"
... the pleomorphic
forms [of ALS,
MS and Parkinsons] react with Borrelia burgdorferi antibody,
but their cysts show they
are different .. so they are in the same genus, but different species
- they are all in the Borrelia genus!
Other stains: carbol
fuchsin (negative staining); acridin orange
(stains RNA red-orange (live active, while resting stain yellow-green, AO do NOT stain fibrin
show pictures of colonie in blood and in urine (x 1500) from af Lyme
patient stained with AO (fluffy background looks like biofilm?) ..
Sudan black B
(wiki) for mycobacteria
47:30 About transmission .. most of the people who get Lyme
disease have never seen a tick ... We know now it is in TEARS,
and people wipe their eyes and then you shake hands with them
and it is spread by mosquitoes and who haven't had a mosquito bite ..
we tested the mosquitoes in MIchigan and sure enough they can carrythe
13:50 Lida use a Coplin jar to grow bacteria on slides
... so you flame the slide to sterilise it, and you spread a loop very
nicely .//.. or a whole drop, not too thick, and then put in culture
... the first time we tried this for TB we tried
to stain with auramine-rhodamin (AR) .. but
the L-forms of TB
don't stain with AR, but the stupid things grow on the slide and get
stained .. so you can use a medium with AR stain ...
Coplin jar can grow all kinds of L-forms overnight ..
love to grow borrelia in it .. it is not an anaerobe, but is not an
aerobe either it is an microaerophile, so you will se an area
L-forms growing way down [where the xygen amount is
Lidas method growing L-forms give RAPID DIAGNOSIS only 48 hours!
Whenever you have a
serious infection, there will be L-forms in the blood.
If anyone in the lab want to see what cell wall deficient forms look
like, just look at early growth, don't wait 18 hours, look at the
growth as they develop .. or grow on plate with antibiotic ..
zone look like complete antibiotic inhibition .. but if you
closely very frequently you will see some intermediate concordial
growth, and that will be the variants .. so it is not too hard to find
MPM medium: http://lymerick.net/MPM-2001-medium-Bb.html
LOTS of tips to try out!
Validation of cultivation and PCR methods for diagnosis of Lyme
neuroborreliosis. Cerar T, Ogrinc K, Cimperman J, Lotric-Furlan S,
Strle F, Ruzić-Sabljić E. J Clin Microbiol. 2008 Oct;46(10):3375-9.
Epub 2008 Aug 20. PMID: 18716226 PDF
Borrelial infection may manifest with a wide range of clinical
signs, and in many cases, microbiological findings are essential for a
proper diagnosis. This study included 48 patients with a working
clinical diagnosis of Lyme neuroborreliosis, 45 patients with a working
clinical diagnosis of suspected Lyme neuroborreliosis, and a control
group comprising 42 patients with tick-borne encephalitis and 21
neurosurgical patients. The aim of the study was to analyze and compare
findings of two PCR methods and Borrelia burgdorferi sensu lato culture
results by examination of prospectively collected cerebrospinal fluid
(CSF) and blood specimens from patients with clinical features of Lyme
Borrelial DNA was detected with at least one of the
PCR approaches in 16/135 (11.9%) blood samples and 24/156 (15.4%) CSF
samples. Using MseI restriction of PCR products of the
rrf-rrl region, we
identified the majority of strains as Borrelia
were isolated from 1/135 (0.7%) blood samples and
from 5/156 (3.2%) CSF specimens. Using MluI restriction
characterization of isolated strains, Borrelia
in all CSF isolates. Our study revealed that different
direct demonstration of borrelial infection give distinct results, that
there is an urgent need for standardization of the methods for direct
detection of borrelial infection, and that the design of studies
evaluating the validation of such methods should include appropriate
control group(s) to enable assessment of both sensitivity and
Material and Methods:
Blood (5 ml)
and CSF (1 ml)
samples were obtained at initial examination from all patients with a
working clinical diagnosis of Lyme neuroborreliosis, suspected Lyme
neuroborreliosis, and TBE.
Isolation of Borrelia
strains from blood and CSF.
One milliliter of CSF,
obtained by lumbar puncture, was immediately inoculated into a tube
with 5 ml of MKP
medium and promptly transported to the laboratory (16). Five milliliters of blood
obtained by venipuncture was placed in a tube containing sodium citrate
and centrifuged at 100 X g for 5 min, and the supernatant was
inoculated into two or more tubes of MKP medium (16, 21,
23). Samples were cultivated at the Institute of Microbiology and
Immunology of the Faculty of Medicine Ljubljana at 33 °C as
described previously (16) and were examined
once a week
for the presence of spirochetes by using dark-field microscopy. Samples
were considered negative if no growth was detected after 9 weeks of
incubation for CSF and 12 weeks for blood (16).
short culture observation time! .. and 2 degree lower temp. than what
most other report to be the optimal temp. for culture]
targeting ospA detected the presence of borrelial DNA in
14/135 (10.4%) blood
samples and in 19/156
(12.2%) CSF specimens.
Blood samples were positive in 7/48 (14.6%) patients with a working
clinical diagnosis of Lyme neuroborreliosis, in 5/45 (11.1%) patients
with suspected Lyme neuroborreliosis, and in 2/42 (4.8%) of patients
with TBE (nonsignificant differences). The corresponding results for
CSF samples were 10/48 (20.8%), 7/45 (15.6%), and 2/42 (4.8%),
respectively. PCR targeting ospA found no borrelial DNA in the CSF
samples of the 21 patients in the neurosurgical control group (Table 1
and Fig. 1). ...
targeting the rrf-rrl region, 14/135 (10.4%) blood samples and
19/156 (12.2%) CSF samples tested positive.
Blood samples were positive in 5/48 (10.4%) patients with a working
clinical diagnosis of Lyme neuroborreliosis, in 7/45 (15.6%) patients
with suspected Lyme neuroborreliosis and in 2/42 (4.8%) patients with
TBE (nonsignificant differences). The corresponding findings for CSF
were 10/48 (20.8%), 8/45 (17.8%) and 1/42 (2.4%), respectively (Table
1). Absence of borrelial DNA in the CSF of the neurosurgical patients
was established with PCR targeting the rrf-rrl region.
simultaneous presence of borrelial DNA in both blood and CSF was
established in only three patients—in two with working
clinical diagnosis of Lyme neuroborreliosis and in one with suspected
MseI restriction of the PCR product of the amplified intergenic rrf-rrl
region, we were able to characterize 13/14 (92.9%) DNA-positive blood
samples and 18/19 (94.7%) DNA-positive CSF samples; the majority of
strains were found to be B.
afzelii (Table 2).
were isolated from 1/135 (0.7%) blood samples and from 5/156 (3.2%) CSF
specimens. Four out of the five CSF isolates were from patients with a
clinical diagnosis of Lyme neuroborreliosis; the fifth was from a
patient with suspected Lyme neuroborreliosis. Borreliae were not
isolated from CSF samples of patients without signs of borrelial
infection (control group of TBE and neurosurgical patients).
were able to identify to the species level all five strains isolated
from CSF and the single strain isolated from blood (Table 3). Using
pulsed-field gel electrophoresis after MluI restriction, four of the CSF isolates (all
from patients with a working clinical diagnosis of Lyme
neuroborreliosis) were found to be B.
garinii (three typed as Mlg2, one as Mlg3); the fifth CSF isolate
(from a patient with suspected Lyme neuroborreliosis) was B. afzelii
(typed as Mla1). The
strain isolated from the blood of a patient with suspected Lyme
neuroborreliosis was also found to be B.
(typed Mla1). In only half of the culture-positive CSF and blood
specimens were we able to demonstrate the presence of borrelial DNA
directly in the corresponding samples with at least one of the two PCR
approaches (Table 3).
main advantage of targeting the rrf-rrl region rather than ospA is the
possibility of identifying the borrelial species. In
this way, we were able to characterize all but two (31/33 [94%])
PCR-positive blood and CSF samples (Table 2). The two specimens that
were not characterized had an unusual restriction pattern that will be
investigated further using sequence analysis.
was the species most frequently identified, although it is not the
principal causative agent of Lyme neuroborreliosis in Europe (23).
sensitivity of borrelia detection in the blood of patients with
(suspected) Lyme neuroborreliosis (Table 1) could have been the
consequence of a transitional spirochetemia, which in the majority of
patients most probably ended at the time of central nervous system
involvement, and/or the result of low numbers of
spirochetes in the blood. With regard to the method
itself, possible inhibitors
in host blood
can diminish positive findings (1). Although we predicted a low
proportion of PCR-positive blood samples, we were surprised to find an
almost equal CSF-positive rate (Table 1). According to literature
reports, the sensitivity of PCR in CSF ranges from 12% to 100% (median,
23%) and depends upon numerous factors, including method, origin of
samples, and the differing approaches used in individual studies (1)
significant differences were established when comparing PCR results for
blood and CSF samples from patients in the Lyme neuroborreliosis group
(and to a lesser extent in those constituting the suspected Lyme
neuroborreliosis group) with findings for the control group(s), and it
was somewhat surprising that borrelial DNA was detected in some of the
patients with TBE. We emphasize that all contamination precautions were
strictly followed, that all routine negative controls gave negative
results, and that the quality of the samples was monitored. To
determine the specificity of the methods, we also used CSF samples of
neurosurgical patients who had no signs of borrelial infection and no
known (recent) exposure to ticks; the results of all tests were
negative, indicating high specificity of the utilized methods.
Slovenia, approximately 30% of Ixodes ricinus ticks are infected with
Borrelia burgdorferi sensu lato (data based on tick culture) and 0.3%
to 1.2% with TBE virus (15; N. Knap, unpublished results) ... It is
also quite possible that not all borrelia infections, even those that
can be demonstrated in CSF and/or blood by PCR, result in an illness.
findings emphasize the importance of appropriate design of studies
evaluating the validity of PCR approaches for the detection of borrelia
infection; the design should include not only the assessment of
sensitivity but also an appropriate control group for determination of
specificity. Interpretation of the results of published studies that
have used differing PCR approaches for the demonstration of borrelial
DNA would be much safer with the inclusion of an appropriate control
group that would enable assessment of both specificity and sensitivity.
of blood samples gave an extremely low yield; we were able to isolate
borreliae from only 1 out of 135 blood samples. We had
to isolate more borrelia strains, although, in Europe, the recovery
rate has generally been low (<10% in patients with early Lyme
borreliosis manifested by erythema migrans) using volumes of blood
similar to those in the present study (1, 19). The explanations for the
low recovery rate could be the same as those for the low proportion of
positive PCR findings in blood (4, 12, 13).
isolation of the etiological agent from CSF samples is the most
reliable method for diagnosis of borrelial central nervous system
infection in patients with suspected Lyme neuroborreliosis and also
provides live organisms that can be further characterized (23).
However, this is a low-yield procedure that takes several weeks. In
patients with proven Lyme neuroborreliosis, the reported recovery rate
from CSF is <10% (23). In the present study, isolation from CSF
successful in 3.7% (5/135) of samples, but the isolation rate in the
group of patients with a working clinical diagnosis of Lyme
neuroborreliosis was 8.3% (4/48).
We were able to characterize all the isolated borrelia strains (Table
3). Using MluI restriction for borrelia characterization, B. garinii was
identified in all CSF isolates of patients with Lyme neuroborreliosis,
whereas in a patient with suspected Lyme
neuroborreliosis who did not have pleocytosis, the isolated species was
These findings corroborate the results of previous studies
on the predominance of B.
garinii strains isolated from CSF samples of patients with
Lyme neuroborreliosis (3, 16, 23) and on the uncertain value of B. afzelii as the causative agent of Lyme
neuroborreliosis (23). However, PCR performed
directly on CSF samples revealed a predominance of B.
Comparison of the results of cultivation and the two PCR methods
revealed that several
culture-positive samples were PCR negative and vice versa.
An explanation for the finding that an individual CSF sample may be
positive only with cultivation but not with PCR could be the presence
of a low number of borrelia cells in CSF; this may not allow the
detection of borrelial DNA by PCR but would still allow proliferation
of borrelia during cultivation. In patients with CSF samples positive
with PCR only, negative culture results could be explained by the
influence of the immune response and also by the fact that the borreliae must adapt to an
artificial culture medium,
which probably limits the isolation rate, whereas their DNA, not only
from living but also from dead cells, can be detected with PCR.
[MK: why did
these authors use MKP meidum instead of BSK-H? - and why culture at 33 degree C
instead of at 35 degree C, which others have reported the
optimal temperature for growth of Borreliae? - how about CO2
and O2 concentration, how about pH?
...which is not mentioned in the text thus apparently not
taken into consideration?
could sub-optimal conditions for growth perhaps explain the relatively
low positive culture rate compared to they got an expectable rate of
positive PCRs in blood and CSF, considering the authors did apparently
not take cyclical relapse pattern into account when sampling for
... if - at it seems from my studies - Borrelia
spirochetes only wanders from the tissues to the blood stream at
intervals of 8-10 days, and Borrelia within 1-24 hours under
unfavourable growth conditions for the spirochete form
"cyst" form, that take at least 9 days to revert back to spirochete
form, and that reversal may perhaps not happen ever,
the microbes are given the very best conditions for survival of the
spirochete form, i.e. optimal growth medium, optimal temperature at 35
degree C, optimal pH, optimal CO2 and O2
concentration ... not taking care of all this could result in
low recovery rate of Borrelia by culture!
When findings show that Borrelia
is often detectable in the CSF and may cause symptoms of
neuroborreliosis with same exact degree of disease / severity of
debilitating symptoms as patients infected which Borrelia garinii
since in ref. 23 the only significant difference in the patients
symptoms was the presence of "typical radicular pain"
only 65% of Borrelia
garinii patients and abscence of this symptom in the Borrelia afzelii
i.e. that particular sypmtom, which is usually put very much weight on
in the clinical diagnosis of "typical neuroborreliosis", but which can
not be a requirement for the diagnosis of neuroborreliosis, because
certainly even not all Borrelia
garinii patients had it!
and in the Borrelia afzelii group often was abscence of the
laboratory signs considered "typical" and normally
required for the diagnosis of
Lyme neuroborreliosis, i.e. pleocytosis, increased protein and
seropositive Borrelia index status ...
- is NOT fulfilled for all patients in which Borrelia burgdorferi sensu
lato can be detected
- has the time then not come NOW, to change the "common view" on the
definition of Lyme neuroborreliosis definition?
- and accept that this particular picture only seems partially
true for Borrelia
garinii, but certainly is not true for most cases of Borrelia afzelii
Since patients with Borrelia
afzelii infection may be just as sick and may have Borrelia afzelii
in their CSF and or blood - whether they express the usual
laboratory signs of "certain neuroborreliosis" or not, it should be
clear to all that the patients infected with Borrelia afzelii
also deserve to get the correct diagnosis and treatment for their
It seems that Borrelia
afzelii is apparently less immunogenic / more stealth in
its nature than Borrelia
so correct diagnosis is not made by the conventional test methods, are
not made early at the time when there is great chance of cure by short
term antibiotic treatment - because the patients are not diagnosed
and treated as early they have a worse long term prognosis and
not cured by conventional treatment for Borrelia, resulting in long
term debilitating sickness, and inability of patients to work etc. etc
... the impact on the society of undiagnosed Borrelia afzelii
infection with or without sign of neuroborreliosis is probably much
more common than hitherto beleived, because usual tests failed to give
the correct diagnosis, a much larger proportion of chronic illness
could be due to undetected and untreated Borrelia afzelii
infection, compared to typical Borrelia
neuroborreliosis infection, that usually raise a quick and high immune
response in its victims ... leading to fast diagnosis and treatment and
good chance of cure by early treatment!? .... WE NEED ACCESS TO DIRECT
DETECTION METHODS, must find what is the OPTIMAL CULTURE CONDITIONS! -
by systematic comparison of all known methods and media!]
pathogens of humans and domestic animals
depend on hematophagous arthropods to transmit them from one vertebrate
reservoir host to another
and maintain them in an environment. These pathogens use antigenic
variation to prolong their circulation in the blood and thus increase
of transmission. By convergent evolution, bacterial and protozoal
vector-borne pathogens have acquired similar genetic mechanisms for
antigenic variation. Borrelia spp. and Anaplasma
marginale (among bacteria) and African trypanosomes, Plasmodium
falciparum, and Babesia bovis
(among parasites) are examples of pathogens using these mechanisms.
Antigenic variation poses a challenge in the development of vaccines
Compared with Slovenian patients, U.S. patients had erythema migrans
for a briefer duration (median duration, 4 days compared with 14 days;
P , 0.001) but were more likely to have systemic symptoms (P 5 0.01),
abnormal findings on physical examination (P , 0.001), and
seroreactivity (P , 0.001). Central clearing of erythema migrans
lesions was more likely in Slovenian patients (P ,0.001).
Erythema migrans caused by B. afzelii in Slovenia and erythema migrans
caused by B. burgdorferi in New York have distinct clinical
presentations. Caution should be used when clinical and laboratory
experience from one side of the Atlantic is applied to patients on the
WORK IN PROGRESS ....