Fw: culture for Bb

October 14, 1999

culture for Bb

I just emailed the following to someone who is relatively new to Lyme. I reread

this report and just felt a strong need to send it to all of you again. Maybe

you've missed it before. Maybe you're feeling a little crazy because all the

tests are negative. Or maybe you still feel sick after lots of antibiotics and

people are beginning to think it's all in your head. This should help you feel

better. You're not crazy - you just have Lyme.

=)

Robynn

----------------------------

et al: Infection 26 (1998)364-367

A Proposal for the Reliable Culture of Borrelia burgdorferi from Patients with

Chronic Lyme Disease, Even from Those Previously Aggressively Treated.

S. E. , L. H. Mattman, D. Hulinska, H. Moayad

Infection 26 (1998) 364-367

SUMMARY

Since culture of Borrelia burgdorferi from patients with chronic Lyme disease

has been an extraordinarily rare event, clarification of the nature of the

illness and proving its etiology as infectious have been difficult. A method for

reliably and reproducibly culturing B. burgdorferi from the blood of patients

with chronic Lyme disease was therefore sought by making a controlled blood

culture trial studying 47 patients with chronic Lyme disease. All had relapsed

after long-term oral and intravenous antibiotics. 23 patients with other chronic

illness formed the control group. Positive cultures were confirmed by

fluorescent antibody immunoelectron microscopy using monoclonal antibody

directed against Asp A, and Asp A PCR. 43/47 patients (9l%) cultured positive.

23/23 controls (1OO%) cultured negative. Although persistent infection has been,

to date, strongly suggested in chronic Lyme disease by positive PCR and antigen

capture, there are major problems with these tests. This new method for

culturing B. burgdorferi from patients with chronic Lyme disease certainly

defines the nature of the illness and establishes that it is of chronic

infectious etiology. This discovery should help to reestablish the gold standard

in laboratory diagnosis of Lyme disease.

INTRODUCTION

Lyme disease is a multisystem illness caused by infection with Borrelia

burgdorferi. Its manifestations can be myriad. This coupled with problems in

current serologic assays, leads to frequent misdiagnosis at all stages of the

illness. Some investigators believe that Lyme borreliosis is overdiagnosed,

while others maintain that it is underdiagnosed. To further confuse matters, a

significant percentage of patients with Lyme disease relapse despite antibiotic

therapy [1, 2].

Chronic Lyme disease is a controversial topic. Even after extended antibiotic

treatment, persistent infection in chronic Lyme disease has been strongly

suggested by the persistence of borrelial antigen, as demonstrated by polymerase

chain reaction [3, 4]. However, these diagnostic tests are plagued by the

absence of a gold standard. The gold standard for laboratory

diagnosis in the field of infectious diseases has usually involved culturing the

causative organism from the infected host. In the case of Lyme disease, attempts

to do so have been disheartening.

The organism has seldom been cultured from cases of treated, late-stage disease,

and if so, primarily from cerebrospinal or synovial fluid [5-8]. Culture of the

organism from blood has been a rarity, with successful cultures primarily from

cases of untreated, early disease [9, 1O].

We set out to demonstrate a methodology by which we could reliably and

reproducibly culture B. burgdorferi from the blood of patients with chronic Lyme

disease even though they had had extended antibiotic therapy. If this were

successful, it would also provide a unique opportunity to compare the serologic

diagnostic criteria set forth by The Centers for Disease Control (CDC) in

conjunction with The Association of State and Territorial Public Health

Laboratory Directors (ASTPHLD) to what is potentially a gold standard diagnostic

test.

PATIENTS AND METHODS

The study was a multi-center, controlled blood culture trial with an

approximately 2:1 ratio of cases to controls. Patients were selected from

private practices in areas both hyper-endemic and non-endemic for Lyme disease.

All cases had a diagnosis of Lyme disease and had failed or

relapsed after extended oral and intravenous antibiotic therapy. The diagnosis

of Lyme disease was made primarily on clinical grounds. Although almost all

cases had serologic evidence suggestive of infection with B. burgdorferi, few

had positive ELISAs and only a little over half

met CDC serologic criteria for Western blot positivity. 4/47 (9%) were positive

by Lyme ELISA. 3/47 (6%) were equivocal by ELISA. 26/47 (55%) were positive by

CDC criteria for Lyme Western blot. Of these, 2O/26 (77%) were IgM positive,

10/26 (38%) were IgG positive, and 4/26 (15%) were positive for both IgM and

IgG.

To participate in the study, all patients had to have had at least 6 consecutive

weeks of therapy with an intravenous third-generation cephalosporin and a

subsequent relapse. Some patients had had as long as 6 months of intravenous

therapy, with the mean being approximately 3 months.

Controls resided in non-endemic areas and consisted of patients with chronic

illnesses other than Lyme disease.

The following MPM medium was used for this study: To 1 ml of Detroit tap water

was added: proteose peptone 2Og, beef infusion from 1,OOOg, dextrose 10g, sodium

chloride 10g, dipotassium phosphate 4g, sodium thioglycollate lg, purified agar

1g, bacto methylene blue .O04g, sucrose l00g, soluble starch 5g. This was

autoclaved for 15 min at 120 degrees C. For the medium to be used in tube or

slide culture, it had to be refrigerated for 24 h before final preparation.

For the medium to be used in tubes, l0ml of medium were boiled to dissolve the

agar just before use and the following was added to each tube: l ml separately

autoclaved yeast extract from a 1O% solution to give a final concentration of

1%, and 1 ml of sterile 1O% NaHCO3. Since yeast extract may contain

heat-resistant bacilli, it was separately autoclaved for 3O min at 124 degrees C

and batch--tested for sterility. The inoculum was 0.l ml of blood in EDTA to 4

ml of medium in a slender screw-top tube. Incubation was at 3O degrees C under

normal atmospheric conditions for a period of 1-3 weeks.

For the medium to be used in slide culture, it was sterilized in 3O ml amounts

in screw-top tubes. Just before use, the medium was boiled to melt the agar and.

when cool but not solidified, the following was added: 3 ml of separately

autoclaved 1O% yeast ex-tract and l0 ml of sterile 10% NaHCO3. The broth was

then poured aseptically into a sterile plastic Coplin jar. Slides were smeared

with the patient's chosen body fluid. The slides had to be specialized so as not

to require fixative. The smears were dried in an aseptic environment before

being placed in the Coplin jar. Once they were inside, the lid was tightly

closed and incubation was at 3O degrees C under normal atmospheric conditions

for a period of 1-3 weeks.

For the medium to be used for blood agar plates, the broth medium was modified

by adding a total of 16 g of agar. Sixty ml of sheep's blood was added as soon

as the medium was removed from the autoclave, resulting in " chocolate agar. " At

this point, separately autoclaved 1O% yeast extract was added to give a final

concentration of 1%. The medium was then poured into sterile plastic Petri

dishes and stored under refrigeration for 24 h once solidified. The inoculum was

O.5ml of blood in EDTA with incubation at 3O degrees C under normal atmospheric

conditions for a period of 1-3 weeks.

Two blood samples of 5 ml each were collected in EDTA lavender top test tubes

from each patient and control. From these, seven cultures were processed from

each participant. All positive cultures were stained with acridine orange at pH

3.5 4.O and then confirmed by our laboratory with affinity-adsorbed polyclonal

fluorescent antibody to B. burgdorferi (O2-97-91, Kirkegaard &

Laboratories, Gaithersburg, MD, USA).

Further confirmation of positive culture results was accomplished by electron

microscopy. Immunoelectron microscopy utilizing monoclonal antibody directed

against Asp A (monoclonal antibody no. 181, courtesy of Prof. B. Left. Stony

Brook, NY, USA), and plasmid PCR with Asp A primer. The methods employed in

these processes have been previously reported [11, 12].

RESULTS

Of the 47 patients with chronic Lyme disease, 43 (91%) cultured positive for B.

burgdorferi, while 23/23 (IOO%) of the controls cultured negative. Many of the

cultures were clearly spirochetes when examined under light microscopy (Figures

1-3). Immunoelectron microscopy and Osp A PCR confirmation provided additional

confirmatory evidence as to the identity of the spirochetes (Figures 4-7). The

slide cultures consistently demonstrated the fastest and most abundant yields.

With this technique, placement in the Coplin jar allows for varying gradations

of oxygen tension. Sometimes spirochetal growth can be seen after as little as

2O h. appearing as a band near the upper end of the smear.

DISCUSSION

An attempt to culture B. burgdorferi from the blood of previously aggressively

treated chronic Lyme disease patients seemed at first a monumental task. Before

undertaking this effort, we therefore had to be as sure as possible that the

organisms were indeed present in the blood of these patients. As a first step,

we scrutinized a report where B. burgdorferi had been cultured from the blood of

patients with early, untreated disease. From this group of patients it had been

noted in follow-up that subsequent blood cultures became routinely negative

after antibiotic therapy, despite 71% of the patients remaining symptomatic [9].

Three possibilities readily come to mind for the explanation of this paradox:

either 1) the infection is cleared, but a post-infectious process continues, or

2) the organism is cleared from the blood rapidly, but finds a pathogenic harbor

elsewhere, or 3) the organism is maintained in the blood in an altered state

which cannot be cultured on routine media.

In response to the first possibility, the notion of a post-Lvme syndrome has

countless flaws. A post-infectious syndrome could not explain the observation

that patients with " post-Lyme " or " post-Lyme fibromyalgia " responded to

retreatment with antibiotics, only to relapse with its discontinuation [13-15].

With the advent of PCR, antigen capture, and the benefit of those rare

successful culture experiments even in the face of prior " curative treatment "

[3-8] the notion of " post-Lyme " should have been dismissed long ago. In response

to the second possibility, given the common finding of circulating immune

complexes with Lyme disease, we thought this unlikely [16]. Thus we were left

with the third and most logical possibility. Specifically, we chose to pursue

the organism in its cell wall-deficient state, i.e. L-forms, as previously

reported [17].

Although L-forms will complex with fluorescent antibody to B. burgdorferi, only

as they revert to classic parent forms can the typical spirochetal morphology be

seen. There has been a considerable spectrum of cell wall-deficiency

demonstrated in our laboratory. B. burgdorferi may exist in various forms

depending on its environment. In addition to the spirochetal form, we have

demonstrated its growth both as amorphous L-forms and rounded giant L-bodies

which have been previously described as cystic forms [11, 18]. As B. burgdorferi

reverts from cell wall deficiency with the rebuilding of its cell wall, classic

spirochetal forms can be seen. Most often, in our cultures, B. burgdorferi can

be seen in varying stages of reversion, i.e. some L-dependent spirochetal forms

within an L-form colony.

The L-form variants, cosmotically fragile by nature, require precise conditions

to grow in culture. Our medium and methodology are specifically designed for the

fostering of cell wall-deficient organisms and their reversion to classic parent

forms. In. most instances, the methods must be followed precisely. Even small

variations produce no growth. For example, 2% yeast extract instead of 1% is

inhibitory. Or if the yeast extract is autoclaved with the rest of the medium

instead of separately, that too will be inhibitory. However, there is one aspect

of B. burgdorferi's growth characteristics which we found to be remarkably

nonfastidious. The organism can be easily grown throughout a wide range of pH,

from 6.8-7.8. This explains the different ratios of NaHCO3 used in the various

types of culture mediums. We are still not sure about the optimal pH for

culture. Future research will address that question more specifically.

It should be noted from this study that currently accepted standards for

serologic diagnosis seem to be inadequate. Only a small minority of participants

in the study had positive Lyme ELISAs. Under the current recommendations for

two-tier testing by the CDC/ASTPHLD, 91% of the patients in the study would have

been misdiagnosed as not having Lyme borreliosis.

It is hoped that our work will help to end a medical controversy which has been

going on for far too long. This study proves that chronic Lyme disease is of

chronic infectious etiology, and that even antibiotic treatment well in excess

of current recommendations is not necessarily curative. Given the flaws in

currently accepted serologic diagnostic criteria, it is likely that Lyme

borreliosis is vastly underdiagnosed. May this research help to focus the

scientific community on effective curative therapies for patients with chronic

Lyme disease. It should also be noted that, in addition to its utility in

growing B. burgdorferi, the MPM medium may be useful for culturing a variety of

other spirochetes from patients.

REFERENCES

1. Krupp, L. B., Maser, V., Schwartz, J., Doyle, P. K., Langenback, L. J.,

Fernquist, S. R.: Cognitive functioning in late Lyme borreliosis. Arch. Neurol.

48 (1991) 1125-1129.

1.. Logigian. E. L., Kaplan. R. F., Steere, A. C.: Chronic necrologic

manifestations of Lyme disease. N. Engl J. Med. 323 (1990) 143-1444.

2.. Bayer. M. E., Zhang, L., Bayer, M. H.: Borrelia burgdorferi DNA in the

urine of treated patients with chronic Lyme disease symptoms: A PCR study of 97

cases. Infection 24 (1996) 347-353.

3.. Nocton, J. J., Dressier. F., Rutledge, B. J., Rys, P. N., Persing, D. H.,

Steere, A. C.: Detection of Borrelia burgdorferi DNA by polymerase chain

reaction in svnovial fluid from patients with Lyme arthritis. N. Engl. J. Med.

330 (1994) 229-234.

4.. Preac Mursic, V., Weber, K., Pfister, H. W., Wilske, B., Gross, B.,

Baumann, A., Prokop, J.: Survival of Borrelia burgdorferi in antibiotically

treated patients with Lyme borreliosis. Infection 17 (1989) 355-3S9.

5.. Schmidli, J., Hunzicker, T., Moesli, P., Schasd, U. B.: Cultivation of

Borrelia burgdorferi from joint fluid three months after treatment of facial

palsy due to Lyme borreliosis. I. Infect. Dis. 158 (1988) 905-906.

6.. Pfister, H. W., Preac Mursic. V., Wilske, B., Schielke, E., Sorgel, F.,

Einhaupl, K. M.: Randomized comparison of ceftriaxone and cefotaxime in Lyme

neuroborreliosis. J. Infect. Dis. 163 (1991) 311-318.

7.. Hassler, D., Riedel. K., Zorn, J., Preac Mursic, V.: Pulsed high-dose

cefotaxime therapy in refractory Lyme borreliosis (letter). Lancet 338(1991)

193.

8.. Nadelman, R. B., Pavia, C. S., Magnarelle, L. A., Wormser, G. P.:

Isolation of Borrellia burgdorferi from the blood of seven patients with Lyme

disease. Am. J. Med. 88 (199O) 21-26.

9.. Berger, B. W., . R. C., Kodner, C., , L.: Cultivation of

Borrelia burgdorferi from the blood of two patients with erythema migrans

lesions lacking extracutaneous signs and symptoms of Lyme disease. J. Am. Acad.

Dermatol. 3O (1994) 48-51.

10.. Hulinska, D., Bartak, P., Hercogova, J., Hancil, J., Basta, l.,

Schramlova, J.: Electron microscopy of Langerhans cells and Borrelia burgdorferi

in Lyme disease patients. Zbl. Bakt 28O (1994) 348-359.

11.. Hulinska, D., Krausova, M., Janovska, D., Rohacova, H., Hancil, J.,

Mailer, H.: Electron microscopy and the polymerase chain reaction of spirochetes

from the blood of patients with Lyme disease. Cent. Eur. J. Public Health J.

(1993) 81-85.

12.. Sigal, L. H., Patella. S. J.: Lyme arthritis as the incorrect diagnosis

in pediatric and adolescent fibromyalgia. Pediatrics 9O (1992) 523-528.

13.. Dinerman, H., Steere, A. C.: Lyme disease associated with fibromyalgia.

Ann Intern Med. 117 (1992) 281-285.

14.. Steere, A. C., , E., McHugh, G. L., Logigian, E. L.: The

over-diagnosis of Lyme disease JAMA 269 (1993) 1812-1816.

15.. Schutzer, S. E., Coyle, P. K., Belman, A. L., Golightly, M. G., Drulle,

J: Sequestration of antibody to Borrelia burgdorferi in immune complexes in

seronegative Lyme disease. Lancet 33S (1990) 312-315.

16.. Preac Mursis V., Wanner, G., Reinhardt, S., Wilske. B., Busch, U.,

Marget, W.: Formation and cultivation of Borrelia burgdorferi spheroplast L-form

variants. Infection 24 (1996) 218-226.

17.. Brorson, 0, Brorson, S. H.: Translormation of cystic forms of Borrelia

burgdorferi to normal. mobile spirochetes. Infection 2S (1997) 240-246.

Received: 27 September 1997/Revision accepted: 3 September 1998.

S. E . M. D., Greenwich Hospital, S Ridge Rd., Greenwich, CT

O683O: L. H. Mattman, Ph. D., Spirotech Institute, Empire State Bldg., 350 Fifth

Ave., Suite 61OI, New York, NY 10118: H. Monyad. D. O., Columbia North Hills

Medical Center, 4401 Booth Calloway Rd., North Richland Hills, TX 76180, USA.

Dagmar Hulinska, Ph. D., National Institute of Public Health. GEM-ELM. Srobarova

48. l0042 Praha IO. Czech Republic.

Correspondence to: Dr. S. E. . 10 Lane. Suite 2. Ridgefield. CT

06877. USA.

October 15, 1999

when you read this article, don't forget to also read the article entitled : The

Bacteria Revolution

It mentions the Culture article in it and much much more. Sorry for those I've

already said this too. But this is a fascinating article and well worth reading.

You can skip all the other links in it, except the culture one. I

printed it because it is long and hard to read on the screen. The site is: (oh

you might have to copy this address and paste it on the internet " address " bar:

http://www.radio.cbc.ca/programs/ideas/shows/bacteria/bacttext.html Somebody

else posted this earlier, I can't remember who, but whoever you are Thank you.

It gave me hope that the Scientists are at least looking for updated methods of

searching for better methods to find hidden spirochetes. Please read this.

Vicki

[Lyme-aid] Fw: culture for Bb

culture for Bb

I just emailed the following to someone who is relatively new to Lyme. I

reread this report and just felt a strong need to send it to all of you again.

Maybe you've missed it before. Maybe you're feeling a little crazy because all

the tests are negative. Or maybe you still feel sick after lots of antibiotics

and people are beginning to think it's all in your head. This should help you

feel better. You're not crazy - you just have Lyme.

=)

Robynn

----------------------------

et al: Infection 26 (1998)364-367

A Proposal for the Reliable Culture of Borrelia burgdorferi from Patients

with Chronic Lyme Disease, Even from Those Previously Aggressively Treated.

S. E. , L. H. Mattman, D. Hulinska, H. Moayad

Infection 26 (1998) 364-367

SUMMARY

Since culture of Borrelia burgdorferi from patients with chronic Lyme

disease has been an extraordinarily rare event, clarification of the nature of

the illness and proving its etiology as infectious have been difficult. A method

for reliably and reproducibly culturing B. burgdorferi from the blood of

patients with chronic Lyme disease was therefore sought by making a controlled

blood culture trial studying 47 patients with chronic Lyme disease. All had

relapsed after long-term oral and intravenous antibiotics. 23 patients with

other chronic illness formed the control group. Positive cultures were confirmed

by fluorescent antibody immunoelectron microscopy using monoclonal antibody

directed against Asp A, and Asp A PCR. 43/47 patients (9l%) cultured positive.

23/23 controls (1OO%) cultured negative. Although persistent infection has been,

to date, strongly suggested in chronic Lyme disease by positive PCR and antigen

capture, there are major problems with these tests. This new method for

culturing B. burgdorferi from patients with chronic Lyme disease certainly

defines the nature of the illness and establishes that it is of chronic

infectious etiology. This discovery should help to reestablish the gold standard

in laboratory diagnosis of Lyme disease.

INTRODUCTION

Lyme disease is a multisystem illness caused by infection with Borrelia

burgdorferi. Its manifestations can be myriad. This coupled with problems in

current serologic assays, leads to frequent misdiagnosis at all stages of the

illness. Some investigators believe that Lyme borreliosis is overdiagnosed,

while others maintain that it is underdiagnosed. To further confuse matters, a

significant percentage of patients with Lyme disease relapse despite antibiotic

therapy [1, 2].

Chronic Lyme disease is a controversial topic. Even after extended

antibiotic treatment, persistent infection in chronic Lyme disease has been

strongly suggested by the persistence of borrelial antigen, as demonstrated by

polymerase chain reaction [3, 4]. However, these diagnostic tests are plagued by

the absence of a gold standard. The gold standard for laboratory

diagnosis in the field of infectious diseases has usually involved culturing

the causative organism from the infected host. In the case of Lyme disease,

attempts to do so have been disheartening.

The organism has seldom been cultured from cases of treated, late-stage

disease, and if so, primarily from cerebrospinal or synovial fluid [5-8].

Culture of the organism from blood has been a rarity, with successful cultures

primarily from cases of untreated, early disease [9, 1O].

We set out to demonstrate a methodology by which we could reliably and

reproducibly culture B. burgdorferi from the blood of patients with chronic Lyme

disease even though they had had extended antibiotic therapy. If this were

successful, it would also provide a unique opportunity to compare the serologic

diagnostic criteria set forth by The Centers for Disease Control (CDC) in

conjunction with The Association of State and Territorial Public Health

Laboratory Directors (ASTPHLD) to what is potentially a gold standard diagnostic

test.

PATIENTS AND METHODS

The study was a multi-center, controlled blood culture trial with an

approximately 2:1 ratio of cases to controls. Patients were selected from

private practices in areas both hyper-endemic and non-endemic for Lyme disease.

All cases had a diagnosis of Lyme disease and had failed or

relapsed after extended oral and intravenous antibiotic therapy. The

diagnosis of Lyme disease was made primarily on clinical grounds. Although

almost all cases had serologic evidence suggestive of infection with B.

burgdorferi, few had positive ELISAs and only a little over half

met CDC serologic criteria for Western blot positivity. 4/47 (9%) were

positive by Lyme ELISA. 3/47 (6%) were equivocal by ELISA. 26/47 (55%) were

positive by CDC criteria for Lyme Western blot. Of these, 2O/26 (77%) were IgM

positive, 10/26 (38%) were IgG positive, and 4/26 (15%) were positive for both

IgM and IgG.

To participate in the study, all patients had to have had at least 6

consecutive weeks of therapy with an intravenous third-generation cephalosporin

and a subsequent relapse. Some patients had had as long as 6 months of

intravenous therapy, with the mean being approximately 3 months.

Controls resided in non-endemic areas and consisted of patients with chronic

illnesses other than Lyme disease.

The following MPM medium was used for this study: To 1 ml of Detroit tap

water was added: proteose peptone 2Og, beef infusion from 1,OOOg, dextrose 10g,

sodium chloride 10g, dipotassium phosphate 4g, sodium thioglycollate lg,

purified agar 1g, bacto methylene blue .O04g, sucrose l00g, soluble starch 5g.

This was autoclaved for 15 min at 120 degrees C. For the medium to be used in

tube or slide culture, it had to be refrigerated for 24 h before final

preparation.

For the medium to be used in tubes, l0ml of medium were boiled to dissolve

the agar just before use and the following was added to each tube: l ml

separately autoclaved yeast extract from a 1O% solution to give a final

concentration of 1%, and 1 ml of sterile 1O% NaHCO3. Since yeast extract may

contain heat-resistant bacilli, it was separately autoclaved for 3O min at 124

degrees C and batch--tested for sterility. The inoculum was 0.l ml of blood in

EDTA to 4 ml of medium in a slender screw-top tube. Incubation was at 3O degrees

C under normal atmospheric conditions for a period of 1-3 weeks.

For the medium to be used in slide culture, it was sterilized in 3O ml

amounts in screw-top tubes. Just before use, the medium was boiled to melt the

agar and. when cool but not solidified, the following was added: 3 ml of

separately autoclaved 1O% yeast ex-tract and l0 ml of sterile 10% NaHCO3. The

broth was then poured aseptically into a sterile plastic Coplin jar. Slides were

smeared with the patient's chosen body fluid. The slides had to be specialized

so as not to require fixative. The smears were dried in an aseptic environment

before being placed in the Coplin jar. Once they were inside, the lid was

tightly closed and incubation was at 3O degrees C under normal atmospheric

conditions for a period of 1-3 weeks.

For the medium to be used for blood agar plates, the broth medium was

modified by adding a total of 16 g of agar. Sixty ml of sheep's blood was added

as soon as the medium was removed from the autoclave, resulting in " chocolate

agar. " At this point, separately autoclaved 1O% yeast extract was added to give

a final concentration of 1%. The medium was then poured into sterile plastic

Petri dishes and stored under refrigeration for 24 h once solidified. The

inoculum was O.5ml of blood in EDTA with incubation at 3O degrees C under normal

atmospheric conditions for a period of 1-3 weeks.

Two blood samples of 5 ml each were collected in EDTA lavender top test

tubes from each patient and control. From these, seven cultures were processed

from each participant. All positive cultures were stained with acridine orange

at pH 3.5 4.O and then confirmed by our laboratory with affinity-adsorbed

polyclonal fluorescent antibody to B. burgdorferi (O2-97-91, Kirkegaard &

Laboratories, Gaithersburg, MD, USA).

Further confirmation of positive culture results was accomplished by

electron microscopy. Immunoelectron microscopy utilizing monoclonal antibody

directed against Asp A (monoclonal antibody no. 181, courtesy of Prof. B. Left.

Stony Brook, NY, USA), and plasmid PCR with Asp A primer. The methods employed

in these processes have been previously reported [11, 12].

RESULTS

Of the 47 patients with chronic Lyme disease, 43 (91%) cultured positive for

B. burgdorferi, while 23/23 (IOO%) of the controls cultured negative. Many of

the cultures were clearly spirochetes when examined under light microscopy

(Figures 1-3). Immunoelectron microscopy and Osp A PCR confirmation provided

additional confirmatory evidence as to the identity of the spirochetes (Figures

4-7). The slide cultures consistently demonstrated the fastest and most abundant

yields. With this technique, placement in the Coplin jar allows for varying

gradations of oxygen tension. Sometimes spirochetal growth can be seen after as

little as 2O h. appearing as a band near the upper end of the smear.

DISCUSSION

An attempt to culture B. burgdorferi from the blood of previously

aggressively treated chronic Lyme disease patients seemed at first a monumental

task. Before undertaking this effort, we therefore had to be as sure as possible

that the organisms were indeed present in the blood of these patients. As a

first step, we scrutinized a report where B. burgdorferi had been cultured from

the blood of patients with early, untreated disease. From this group of patients

it had been noted in follow-up that subsequent blood cultures became routinely

negative after antibiotic therapy, despite 71% of the patients remaining

symptomatic [9]. Three possibilities readily come to mind for the explanation of

this paradox: either 1) the infection is cleared, but a post-infectious process

continues, or 2) the organism is cleared from the blood rapidly, but finds a

pathogenic harbor elsewhere, or 3) the organism is maintained in the blood in an

altered state which cannot be cultured on routine media.