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Volume: 2
Issue: 20
Date: 02-Dec-94

Table of Contents:

I.    NIAID: Summary Of Second Consultation On Chronic LD
II.   NIAID: Recent Projects at Rocky Mountain Labs
      vancomycin against the spirochete Borrelia burgdorferi
IV.   About The LymeNet Newsletter


*                  The National Lyme Disease Network                  *
*                         LymeNet Newsletter                          *

IDX#                Volume 2 - Number 20 - 12/02/94
IDX#                            INDEX
IDX#  I.    NIAID: Summary Of Second Consultation On Chronic LD
IDX#  II.   NIAID: Recent Projects at Rocky Mountain Labs
IDX#        vancomycin against the spirochete Borrelia burgdorferi
IDX#  IV.   About The LymeNet Newsletter

I.    NIAID: Summary Of Second Consultation On Chronic LD
Sender: Edward McSweegan <[email protected]>

On October 18, NIAID convened a second informal meeting on chronic
Lyme disease (LD).  The discussion focused on possible clinical
trial designs for chronic LD, infectious and non-infectious aspects
of chronic LD, and potentially important co-infecting microorganisms
in some cases of chronic LD.  The meeting also served to provide the
NIAID with a better understanding of the biomedical, logistical and
analytical issues involved in carrying out a treatment trial for
chronic LD.  As noted at the first meeting on January 31, a
fundamental question about chronic LD is whether the lingering signs
and symptoms of post-treatment LD are due to persistent infection by
Borrelia burgdorferi or to non-infectious sequelae.  Furthermore, if
persistent infection is common, is it antibiotic-responsive?  
The answers to these questions are critical to guiding the care and
treatment of patients with evidence of chronic LD.    

For purposes of the meeting, the term Post-Lyme Disease Syndrome
(PLDS) was used to describe a condition of chronic or intermittent
signs and symptoms coincident with the time of infection and
persisting for six months to years despite documentation of
appropriate antibiotic therapy.  PLDS was further characterized by
symptoms of cognitive disturbances (encephalopathy), chronic fatigue
and malaise, headache, and joint and muscle pain.  The PLDS concept
was useful for discussing chronic LD and for outlining various
etiologic considerations, including; persistent infection, incorrect
diagnosis, slow resolution of signs and symptoms, residual damage
from infection, re-infection, unmasking of a prior pathology, and a
post-infectious immune or inflammatory process.    

Given the varied clinical presentations and possible etiologies
associated with PLDS, most of the meeting participants concluded
that an initial trial should focus on a well-defined patient
population with probable infection that might respond to antibiotics.  
The use of an antibiotic-responsive or microbiologic case definition
in an antibiotic treatment trial would also simplify entry criteria,
endpoint and outcome measures, and reduce the necessary study size.  
Entry criteria for such a treatment trial might include objective
measures such as positive serologies, a history of tick bite or EM
rash, compatible clinical signs and symptoms, repetitive positive
PCRs, positive culture, and severe fatigue (> 6 months) after
appropriate antibiotic therapy.    

The use of a placebo was also thought by many participants to be
essential in measuring the efficacy of an antibiotic treatment.  
The use of a double-blind, randomized, placebo-controlled trial
would allow investigators to determine if the antibiotic-treated
group improved significantly over the placebo group.  Subsequent to
such a determination it would be possible to ask other questions
about optimal treatment duration, optimal drug route and optimal
drug choice.

The meeting participants noted, however, that it would be
unacceptable to enroll a placebo group into a treatment trial,
based on their likelihood of being infected, without eventually
treating them as aggressively as the antibiotic-treated group.  
Various approaches, such as a cross-over and parallel track trial
designs, were discussed as ways of ensuring proper patient care
throughout the duration of a treatment study.    

An initial well-designed treatment trial involving a select
population of PLDS patients would provide new information about
treating chronic LD and suggest additional avenues of patient care
and research.  Most investigators recognized that in the absence of
a sufficient number of infected patients who had never been treated,
no particular group of LD patients would be uniform and ideal for a
given treatment trial.  It was also noted that a patient group with
probable infection represented only the apex of a pyramid of PLDS
patients with varying clinical presentations and possible etiologies.
However, the intention of a first treatment trial would be to ask
treatment and diagnostic questions in one well-defined patient group
in the hope that the resulting answers could eventually be applied
to others.    

A report of the October 18 meeting will be presented to the NIAID
Council at its next meeting in February 1995.    

       Chronic Lyme Disease: Clinical Trial Considerations
Chairman - Steve Schutzer, M.D., Dept. of Medicine, UMDNJ. Med. Sch.
Janice I. Beers, J.D., L.D. Assoc. of the U.S.
William Blackwelder, Ph.D., Chief, Biometry, DMID/NIAID, NIH
Carl Brenner, L.D. Coalition of NY
Patricia Coyle, M.D., Dept. of Neurology, SUNY
George Curlin, M.D., Deputy Director, DMID/NIAID, NIH
Raymond Dattwyler, M.D., Div. Allergy, Rheum. & Clinical Immuno.
 SUNY Sch. of Med.
Charlene DeMarco, M.D., Private Practice
David Dennis, M.D., Div. Vector-Borne Inf. Dis., CDC/NCID
Jesse Goodman, M.D., Div. of Medicine, U. of Minnesota
Stephen Heyse, M.D., Director, OPECA/NIAMS, NIH
Benjamin Luft, M.D., Div. of Inf. Dis., SUNY Sch. of Med.
Robin McKenzie, M.D., Lab. Clin. Invest., NIAID, NIH
David H. Persing, M.D., Lab. Med. Patho., Mayo Foundation
Robert Quackenbush, Ph.D., Chief, BMB/DMID/NIAID, NIH
Alfred J. Saah, M.D., School of Hygiene & Pub. Hlth, Johns Hopkins U.
Susana Serrate-Sztein, Ph.D., NIAMS, NIH

Janice M. Soreth, M.D., Div. Anti-Infective Drug Prod., NIH
Allen Steere, M.D., N.E. Med. Ctr., Rheumatology/Immunology
David L. Weld, Exec. Dir., American Lyme Disease Foundation Inc.
Edward McSweegan, Ph.D., NIAID

CONTACT: Edward McSweegan, Ph.D.
Lyme Disease Program Officer
National Institute of Allergy and Infectious Diseases
Solar Bldg., Rm. 3A34
Bethesda, MD 20892-7630
301-402-2508 (fax)
[email protected]  


II.   NIAID: Recent Projects at Rocky Mountain Labs
Sender: Edward McSweegan <[email protected]>


The objectives of this project are to (1) use recombinant DNA
techniques to express specific antigens of Borrelia burgdorferi
to improve the serodiagnosis of Lyme disease, (2) characterize at
the molecular level, isolates of the Lyme disease spirochete from a
wide range of biological and geographical sources, and (3) to use
animal models to help understand the pathogenesis of Lyme disease
and to examine serological responses to infection.  
During the last year we entered into a collaboration with Korean
scientists to examine the potential for human Lyme disease in Korea.
Lyme disease spirochetes, Borrelia burgdorferi sensu lato, were
identified and characterized for the first time from Korea.  Four
isolates, designated Konkuk-1, Konkuk-2, Kangwon-3, and KM-4 were
made from midgut suspensions of three Ixodes ticks and heart tissue
from one mouse, Apodemus agrarius, collected from Chungbuk and
Kangwon Provinces.  The four Korean isolates and B. burgdorferi
sensu lato from other geographic areas and biological sources were

compared using sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) for protein profiles, Western immunoblot
analysis for reactivities with monoclonal and polyclonal antibodies,
and agarose gel electrophoresis for plasmid profiles.  Two typing
schemes using the polymerase chain reaction (PCR) identified three
of the isolates as members of group VS461 and one, Kangwon-3, as
B. garinii.  These results demonstrate the potential for human Lyme
disease to occur in some provinces of Korea.  
Previous studies have demonstrated that the urinary bladder is a
consistent source for isolating the Lyme disease spirochete,
Borrelia burgdorferi, from both experimentally infected and
naturally exposed rodents.  We examined histopathological changes in
the urinary bladder of different types of rodents experimentally
infected with Lyme spirochetes, including BALB/c mice (Mus
musculus), nude mice (M. musculus), white-footed mice (Peromyscus
leucopus), and grasshopper mice (Onychomys leucogaster).  Animals

were inoculated intraperitoneally, subcutaneously, or intranasally
with low-passaged spirochetes, high-passaged spirochetes, or
phosphate-buffered saline.  At various times following inoculation,
animals were sacrificed and approximately one-half of each urinary
bladder and kidney were cultured separately in BSK-II medium while
the other half of each organ was prepared for histological
examination.  Spirochetes were cultured from the urinary bladder of
all 35 mice inoculated with low-passaged spirochetes while we were
unable to isolate spirochetes from any kidneys of the same mice.  
The pathological changes observed most frequently in the urinary
bladder of the infected mice were the presence of lymphoid
aggregates, vascular changes including an increase in the number of
vessels and thickening of the vessel walls, and perivascular
infiltrates.  Our results demonstrate that nearly all individuals
(93%) of the four types of mice examined had a cystitis associated
with spirochetal infection.  

Our broad objective is to understand the means by which Borrelia
burgdorferi establishes an infection: transmission cycle between the
tick vector and mammalian reservoir host, both of which are needed
to maintain the spirochete in nature.  The following research efforts
relate to this goal.  
l. Outer surface protein variation.  The outer membrane of B.
burgdorferi contains several abundant proteins (Osps) that are
variable in size and expression and of unknown function.  It is
likely that the different Osps confer distinct properties on the
spirochete that are pertinent to the disparate environments in which
it must survive.  Rosa and Hogan have identified homologous
recombination between DSP genes in natural populations of B.
burgdorferi in ticks.  Dr. Margolis has also determined that
transcriptional regulation or mRNA stability is responsible for
different patterns of Osp synthesis among clonal variants.  He has

cloned an osp gene that is variably expressed in B. burgdorferi in
culture and is investigating the sequences and factors involved in
the regulation of its expression.  

2. Linear plasmid replication.  An understanding of the unusual
linear structure of the Borrelia chromosome and plasmids is of
intrinsic interest and practical merit.  Dr. Tilly has isolated
homologs of heat shock genes that are involved in plasmid
replication in other systems.  These genes are induced by a
temperature upshift as would be experienced upon transfer from a
tick to a mammal.  She has isolated and sequenced the gene of the
Borrelia IHF homolog, another candidate gene that may be involved in
linear plasmid replication, and is characterizing its biological
activities in E.  coli.  She has identified unique concatemeric or  
circular forms of the linear plasmids that could represent
replication  intermediates.  


III.  ANTIMICROB AGENTS CHEMOTHER:  In vitro activity of vancomycin
     against the spirochete Borrelia burgdorferi
AUTHORS: Dever LL, Jorgensen JH, Barbour AG
REFERENCE: Antimicrob Agents Chemother; 1993: 37, 1115-1121

Borrelia burgdorferi, a spirochete and the causative agent of Lyme
disease, has been reported to be susceptible to a variety of
antimicrobial agents.  In this investigation, the action of
vancomycin, a glycopeptide antibiotic not previously known to have
activity against spirochetes, against borrelias was examined.  The
in vitro activity of vancomycin against a variety of strains of B.
burgdorferi and one strain of Borrelia hermsii was determined by use
of a microdilution MIC method (L.L. Dever, J.H. Jorgensen, and A.G.
Barbour, J. Clin. Microbiol. 30:2692-2697, 1992).  MICs ranged from
0.5 to 2 micrograms/ml.  MICs of the glycopeptides ristocetin and
teicoplanin and the lipopeptide daptomycin against strain B31 of B.
burgdorferi were all > or = 8 micrograms/ml.  Subsurface plating,
time- kill studies, synergy studies, and electron microscopy were
used to investigate further the activity of vancomycin against B31.
The MBC of vancomycin was 2 micrograms/ml.  Time-kill curves
demonstrated > or = 3-log10-unit (99.9%) killing of the final

inoculum after 72 h by vancomycin concentrations twice the MIC.  
Synergy between vancomycin and penicillin was demonstrated at
concentrations one-fourth the MIC of each drug.  In electron
microscopy, B31 cells exposed to vancomycin showed a disruption of
cellular integrity and were indistinguishable from those exposed to
penicillin.  These studies demonstrate another class of
microorganisms susceptible in vitro to vancomycin.


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