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Borrelia-ELISA dokumentsamling v/ Alex H. 2015.04.05
Borrelia-ELISA 100%, realitet eller myte ?
Er en ELISA Borrelia-test altid 100% efter 8 ugers infektion – det er spørgsmålet…
Royal Societys motto er ”Nullius in verba” ("tag ingens ord for det"), med det i mente, er
teksten her givet fri
– til personlig fortolkning…
Hop til dokument i denne pdf ved klik på ”dok
i pdf”
se: Serodiagnosis of Erythema Migrans and Acrodermatitis Chronica Atrophicans by the Borrelia burgdorferi
Flagellum Enzyme-Linked Immunosorbent Assay 1989 – Klaus Hansen,
dok i pdf
se: Seronegative Lyme Disease, Dissociation of Specific T- and B-Lymphocyte Responses to Borrelia
burgdorferi 1988 - Raymond J. Dattwyle,
dok i pdf
se: Antibodies against Whole Sonicated Borrelia burgdorferi Spirochetes, 41-Kilodalton Flagellin, P39
Protein in Patients with PCR- or Culture-Proven Late Lyme Borreliosis 1995 - Jarmo Oksi,
dok i pdf
se: Seronegative Lyme Arthritis caused byBorrelia garinii 2002 - H. Dejmkova,
dok i pdf
se: Serologic Tests for Lyme Disease: More Smoke and Mirrors 2008 - Raphael B. Stricker,
dok i pdf
se: More Smoke and Mirrors ref. 2; uddrag, Centers for Disease Control and Prevention (CDC); Case
Definitions for Public Health Surveillance, 1990, Lyme Disease,
dok i pdf
se. More Smoke and Mirrors ref. 3; Let’s tackle the testing - Raphael B. Stricker,
dok i pdf
se: More Smoke and Mirrors ref. 4; The Western Immunoblot for Lyme Disease: Determination of
Sensitivity,Specificity, and Interpretive Criteria with Use of Commercially Available Performance Panels 1997
- Richard C. Tilton,
dok i pdf
se: More Smoke and Mirrors. 5; Evaluation of Two Commercial Systems for Automated Processing,
Reading, and Interpretation of Lyme Borreliosis Western Blots 2008 - M. J. Binnicker,
dok i pdf
se: More Smoke and Mirrors. 6; Detection of Borrelia burgdorferi sensu lato DNA by PCR in serum of
patients with clinical symptoms of Lyme borreliosis 2008 - Iolanda Santino,
dok i pdf
se: Litteraturliste; Seronegativity in Lyme borreliosis and Other Spirochetal Infections 2003 -
Joanne R,
dok i pdf
Her er hvad producenterne af borrelia ELISA-tests selv skriver på deres test kit indlægsbrochure om test-
fortolkning:
se: uddrag, IDEIA Borrelia burgdorferi IgG REF K602911-2 DA, - Thermo Fisher Scientific
v/ Klaus Hansen (den danske ELISA),
dok i pdf
se: uddrag LIAISON® Borrelia IgG ([REF] 310880) – DiaSorin ,
dok i pdf
se: uddrag, Immunetics® C6 B. burgdorferi(Lyme) ELISA™ Kit Cat. No. – DK-E352-096 - Immunetics,
dok i
pdf
Statens Serum Institut om Borrelia burgdorferi
antistof i serum antistoffer og intratekal syntese af
antistoffer:
se: uddrag, Borrelia burgdorferi antistof (serum antistoffer (IgG, IgM) og intratekal syntese af antistoffer) (R-
nr. 302) - Statens Serum Institut,
dok i pdf
 SUU, Alm.del - 2014-15 (1. samling) - Bilag 1413: Henvendelse af 24/5-15 fra Alex Holmstedt vedr. behandling af borrelia
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JOURNAL
OF
CLINICAL
MICROBIOLOGY,
Mar.
1989,
p.
545-551
0095-1137/89/030545-07$02
.00/O
Copyright
©
1989,
American
Society
for Microbiology
Vol. 27, No. 3
Serodiagnosis
of
Erythema
Migrans
and
Acrodermatitis
Chronica
Atrophicans
by
the
Borrelia burgdorferi
Flagellum
Enzyme-Linked
Immunosorbent
Assay
KLAUS
HANSEN'*
AND
EVA
ASBRINK2
Borrelia Laboratory,
Department
of
Treponematoses,
Statens
Seruminstitut,
2300
Copenhagen
S.,
Denmark,'
and
Department of
Dermatology, Sodersjukhuset,
Karolinska Institut,
Stockholm,
Sweden2
Received
15
August
1988/Accepted
28 November
1988
The
diagnostic
performance
of
an
enzyme-linked
immunosorbent
assay
(ELISA)
using
purified
BorrelUa
burgdorferi
flagella
as
test
antigen
was
compared with that
of
a
B.
burgdorferi
sonic
extract
ELISA.
We tested
sera
from
200
healthy
controls,
107
patients
with
erythema migrans
(EM),
50
patients
with
acrodermatitis
chronica
atrophicans
(ACA),
and
98
patients
with
various
dermatological
disorders
without
clinical
evidence
of active
Lyme
borreliosis.
The
flagellum
ELISA
was
significantly
more
sensitive
than
the
sonic
extract
ELISA.
With
sera
from
patients with
EM, the
diagnostic
sensitivity
for
immunoglobulin
G (IgG)
antibody detection
increased from 11.2
to
35.5%
(P
<
0.001)
and
for
IgM
antibody
detection
it
increased
from
16.6
to
44.8%
(P
<
0.001).
In the
flagellum ELISA,
the
number
of
positive
tests
increased
significantly
(P
<
0.005)
when the
duration
of
EM exceeded
month,
but
still
only
about
50%
of patients
with
longstanding
(1
to
12
months)
untreated
EM
were
IgG
seropositive.
Concomitant
general
symptoms
did
not
affect
the antibody
level,
whereas
patients
with multiple erythema
were
more
frequently
seropositive.
Ail
sera
from patients
with
EM
which
were
positive
in the
sonic
extract
ELISA
were
also
positive
in
the
flagellum ELISA.
Not
only
did
the
overall
number
of
positive
tests
increase,
but the
flagellum
ELISA
yielded
a
significantly
better
quantitative
discrimination
between
seropositive
patients
and controls
(P
<
0.002).
IgG
antibodies
to
the B.
burgdorferi
flagellum
were
found
in all
sera
from
patients
with
ACA,
indicating
persistence
of
an
antiflagellum
immune
response
in
late
stages
of
Lyme
borreliosis.
IgM
reactivity
in
sera
from
patients
with
ACA
was
shown
to
be
unspecific
and the
result
of
IgM
rheumatoid factor.
A
rheumatoid
factor
was
detected in
sera
from
32%
of patients
with
ACA,
compared
with
7.5%
of
patients
with
EM.
The improved diagnostic performance,
the
ease
of
standardization
of
the
flagellum
antigen,
and
the
lack
of strain
variation
make
the
B.
burgdorferi
flagellum
a
needed reference
antigen
for
growing
routine serology
in
Lyme
borreliosis.
Serological
tests
for
antibodies
to
Borrelia burgdorferi
have
been
available
since
1982 (9).
The
presently
used
tests,
indirect
immunofluorescence
assay
and
enzyme-linked im-
munosorbent
assay
(ELISA),
use
whole
cells
or
whole-cell
sonic
extracts
as
antigen.
These
tests
are
of limited diagnos-
tic
value
in
early
disease,
since
they
yield
low
diagnostic
sensitivities.
Only
10
to
40%
of
patients
with
erythema
inigrans
(EM)
are
reported
to
be
seropositive
(1,
3,
5,
10,
20-22, 26).
A
low antigen load,
a
late
and
slow
humoral
immune
response,
and
the
quality of
the
test
antigen
used
may
be
responsible.
B.
burgdorferi
shares
immunogenic
antigens
with
many
other
bacteria,
not
only
spirochetes
(7,
14).
The
inclusion
of
such
cross-reactive
antigens
in
the
test
antigen
may
be
responsible
for
the
low
diagnostic specificity
of
the
presently
used
tests.
A
single,
immunodominant,
and
more
specific
Borrelia
antigen
should
be
used for
a
sero-
diagnostic
assay.
Several
Western
(immuno-)
blotting
(WB)
studies
have
shown
that
the human immune
response
in
Lyme
borreliosis
early
and
constantly
recognizes
the
41-
kilodalton band
corresponding
to
the
flagellum (7, 11-13,
17,
25).
In
a
recent
study,
we
showed
that
the
use
of
purified
B.
burgdorferi
flagellum
as
ELISA
antigen
significantly
im-
proved
immunoglobulin
G
(IgG)
and
IgM
serodiagnosis,
especially
in
early
cases
of
lymphocytic meningoradiculitis
(Bannwarth's
syndrome)
(15).
The
aim
of
the
present
study
was
to
investigate
the
diagnostic
efficiency
of
the
B.
burgdorferi
flagellum
ELISA
*
in
patients
with
EM,
the
primary
stage
of
Lyme
borreliosis,
and
in
patients
with the
late
dermatological manifestation
of
B.
burgdorferi infection,
acrodermatitis
chronica atrophi-
cans
(ACA),
which
may
start
0.5
to
8
years
after
EM
(2).
MATERIALS
AND
METHODS
Corresponding
author.
Patients.
A
total
of
107
patients
presenting
with
a
typical,
clinically
uncomplicated
EM
entered
the
study.
There
were
26
males
and
81
females.
They
were
aged
6
to
83
years
(median
age,
54
years).
Ninety-one
patients
had
a
solitary
EM;
sixteen
experienced
multiple erythema.
Thirty-eight
patients reported
accompanying
constitutional
symptoms
(low-grade
fever,
headache,
musculoskeletal
pain),
and
six-
ty-nine
had
only
the
skin
lesion.
The
time
from
onset
of
the
EM
until
blood
sampling
took
place ranged
from
2
days
to
12
months,
with
a
median
duration
of
3.5
weeks.
Another
50
patients
with
ACA,
including
16
males
and
34
females,
were
investigated.
They
were
aged
28
to
89
years
(median
age,
61
years).
The
duration
of
the
ACA
in
these
patients ranged
from
0.5
to
20
years
(median
duration,
2.5
years).
The
diagnosis
of
EM
and
ACA
was
based
on
clinical
evidence and
in
every
case
was
made
by
one
of
us
(E.A.).
The
clinical
diagnosis
of
ACA
was
confirmed
by
histopathol-
ogy
(2).
All
the
patients
were
from
the
Stockholm
area
and
were seen
between
1984
and
1987.
All
serological
measure-
ments
were
done
on
pretreatment
sample
1.
Controls.
Sera
from
200
healthy
Danish
controls
were
used
for determination
of
the
95%
specific
cutoff
level in
both
More likely the case ?
107
19
38
50
Total, all with ME
(38+50=88 likely with borreliosis)
EM only
EM and Low-grade fever, headache, musculoskeletal pain
EM and ACA
According to text
545
107 Total, all with EM
69 EM only,
107-69 = 38 left for other symptoms
38 EM and Low-grade fever, headache, musculoskeletal pain
(the 38 with other symptoms)
50 EM and ACA
(where do they fit? if ME only, 107-69 = 38 left for other symptoms)
 SUU, Alm.del - 2014-15 (1. samling) - Bilag 1413: Henvendelse af 24/5-15 fra Alex Holmstedt vedr. behandling af borrelia
1533935_0003.png
546
HANSEN
AND ÂSBRINK
J.
CLIN.
MICROBIOL.
patients
with various
dermatological
disorders without clinical evi-
dence
of
an
active
B.
burgdorferi
infection.
This
was
to
assure
the
applicability
of
the
cutoff
level
defined
by
the
Danish
controls
to
the
Swedish
patient
population.
Sera
from
patients
and
controls
were
stored
at
-20°C
until
use.
B.
burgdorferi
test
antigens.
A
Swedish
B.
burgdorferi
strain,
ACA-1,
isolated
from
the
skin
of
a
patient
with
ACA
(4)
was
used
for
all
antigen
preparations.
Spirochetes
were
grown
for
5
days
in
BSK
Il medium
(6)
at
32°C
to a
cell
density
of
108/ml.
The
sonic
extract
antigen
for
ELISA
was
prepared
as
described
previously
(15).
The
spirochetes
were
washed three times in
phosphate-buffered
saline
(PBS;
pH
7.4)
and
sonicated
on
ice
by
seven
15-s
blasts with
an
MSE
150
W
ultrasonic
disintegrator
(Manor
Royal,
Crawley,
England).
The
sonic
extract
was
centrifuged
(10,000
x
g,
30
min),
and the
supernatant
primarily
containing
the soluble
antigens
was
used
for
ELISA.
This sonic
extract
contained
flagellar
components
according
to
sodium
dodecyl
sulfate-
Isolation of
B.
burgdorferi
flagellum.
The
purification
pro-
cedure
for
the
B.
burgdorferi
flagellum
was
recently
reported
in
detail
(15).
Briefly,
the
spirochetes
were
subjected
to a
mild-ionic
detergent
for removal
of
the
outer
envelope.
The
detergent-insoluble
material
containing
the
protoplasmic
cyl-
inders with
the
periplasmatic
flagella
attached
was
sheared
in
a
blender
to
achieve
a
mechanical
detachment
of
the
flagella
from
the cell
bodies.
The
sheared
material
was
then
sub-
jected
to
several
differential
centrifugations.
The
final
and
most
important
step
in the
purification
was a
banding
on a
CsCl
density gradient.
Visible
bands
were
isolated
sepa-
rately
and
examined
by
WB
to assess
the
yield
and
purity
of
the
41-kilodalton
flagellum
antigen.
Flagella
containing
bands
were
pooled
and
dialyzed against
PBS.
The
B.
burg-
dorferi
flagellum antigen
prepared
in this
way
proved
to
be
highly
pure
by
electron
microscopy,
WB,
and
crossed
im-
munoelectrophoresis,
as
shown
previously
(15).
ELISA
procedure.
The
B.
burgdorferi
sonic
extract
ELISA
and the
flagellum
ELISA
were
performed
identically
except
for
the
antigen.
Flat-bottomed
polystyrene
microwell
plates
(Immunoplates,
code
2-69620;
Nunc,
Roskilde,
Den-
mark)
were
coated
overnight
at
4°C
with
100
Fil
of
antigen
diluted
in PBS. The
optimal coating
concentration
was
defined
as
the
antigen
dilution
resulting
in the
highest
ratio of
the
optical
densities
(ODs)
between
positive
and
negative
control
sera.
Unspecific
protein
binding
was
blocked
with
1%
(wt/vol)
bovine
serum
albumin in PBS.
The wells
were
washed,
and 100
,ul
of
serum
diluted
1:200 in PBS with
0.5%
(wt/vol)
bovine
serum
albumin and
0.05%
(vol/vol)
Tween 20
was
added
to
the
wells
and
incubated
for
2
h
at
20°C.
After
washing,
100
pl
of
peroxidase
conjugate
was
added,
either
rabbit
anti-human
IgG
or
anti-human
IgM
(codes
P-214
and
P-215;
Dakopats,
Copenhagen,
Denmark)
diluted
1:10,000
and
1:1,000,
respectively,
in
PBS with
0.05%
(vol/vol)
Tween 20.
After
incubation
for
2 h
at
20°C,
the
plates were
washed,
and 200
,ul
of the
substrate
o-phenylenediamine
(0.41
mg/ml;
Sigma
Chemical
Co.,
St.
Louis,
Mo.)
in
citrate
buffer
(pH
5)
with
0.04%
(vol/vol)
H202
was added to each
well.
After
15
min under
protection
from
light, the
enzymatic
reaction
was
stopped by
the
addition
of 50
,ul
of 3 M
H2SO4.
The OD
at
492
nm
was
read
by
a
colorimeter (Immuno
Reader NJ
2000;
Nippon,
InterMed,
Tokyo, Japan). All
washings
were
done
three
times
with
0.56%
(wt/vol)
NaCI
containing
0.05% (vol/vol)
Tween 20.
Positive
and
negative
control
sera
were
included
on
every
plate.
Samples were
tested
in
duplicate,
and
the
mean
value
was calculated. If the
tests.
The
Swedish control
group
consisted of 98
two
values
differed
more
than
10%
from the
mean,
the
sample
was
retested.
To
eliminate
plate-to-plate
and
day-
to-day
variations,
samples
of
three
serum
pools
with known
low,
medium,
and
high
titers
were
included
on
every
plate
for
construction
of
a
standard
curve.
The OD value
of every
sample
was
adjusted
to
this
standard
curve.
Measurement
and
absorption
of
IgM
RF.
All
samples
from
patients
with EM and
ACA
were
investigated
for
IgM
rheumatoid
factor
(RF).
An
ELISA
with
human
IgG
was
used,
and the results
were
evaluated
according
to
the 95%
specific
upper
limit
of the assay, 8
IU/ml
(16).
To
eliminate
unspecific
IgM
reactivity
resulting
from IgM
RF,
all
IgM-
reactive
samples
from
patients
with
ACA
were
subjected
to
IgM
RF
absorption
with
a
commercially
available
kit
(RF
Absorbans;
Behringwerke,
Marburg,
Federal
Republic
of
polyacrylamide
gel electrophoresis
and
WB examination.
Germany).
Statistical
analysis.
The
diagnostic
sensitivities of
the sonic
extract
and
flagellum
ELISAs
were
compared
by
using
McNemar's test,
assuming
a
binominal distribution
of
paired
data.
Nonpaired
data
were
compared
by using
the
chi-square
test.
Furthermore, the
precise
quantitative
discrimination
of
the
two
assays between
controls
and
seropositive
patients
was
estimated.
For each
individual
sample
that
was
positive
in both
ELISAs,
the
distance from
the achieved OD
value
to
the
cutoff
level
was
calculated
for both
assays.
These
differences
were
compared
by using
Wilcoxon's
rank
sum
test
for
paired
data.
RESULTS
Measurement of
IgG
antibodies
to
the
flagellum
of
B.
burgdorferi
in
the
sera
of
200
healthy
controls demonstrated
a
significant
increase in
specificity
compared
with
IgG
mea-
surement
with
a
sonic
extract
ELISA
(Fig. 1).
Using
a
95%
specific upper
limit
in
both
tests,
the
diagnostic
cutoff
level
could
be lowered from
0.400
to
0.160 OD value
in the
flagellum
assay.
For
IgM
antibodies
in
healthy
controls,
there was no
significant increase in
specificity,
since
the
95%
specific
cutoff
levels
were
0.260
and
0.230
OD
values,
respectively
(Fig.
2).
The
investigation
of the Swedish control group
consisting
of
patients
with
various
dermatological disorders
assured
that the
diagnostic
95% specific
cutoff
level
in
both assays
based
on
the Danish
control
population was also repre-
sentative and suitable
when
Swedish patients were
studied
(Fig.
1 and
2).
When IgG and
IgM
antibodies
in sera from
107 EM
patients
were
measured,
the flagellum ELISA showed over-
all
increases
in diagnostic sensitivity from 11.2 to 35.5% (P
<
0.001)
for IgG
and from
16.6
to
44.8% (P
<
0.001) for IgM
detection
compared
with
the sonic extract ELISA (Table
1).
A
separate
evaluation
of the 70
patients with an EM duration
of less
than
1
month and the 37
patients with an EM duration
of
more
than
1
month revealed
similar significant increases in
diagnostic
sensitivity of the IgG and IgM flagellum ELISA
(P
<
0.001) at both
times.
Not only did the
overall
number of
positive
results
increase significantly,
but
so did the quanti-
tative
discrimination
between
controls
and
patients.
This
was the result of a generally
higher
signal
obtained
in
the
flagellum
ELISA in addition to the lower
cutoff
level
(Fig.
3a
and
b).
All
sera that were reactive in
the
sonic
extract ELISA
were also reactive in the
flagellum
ELISA (Fig. 3). The sera
of
69
patients
(64.5%)
were either
IgG
or IgM
reactive
in
the
flagellum
ELISA, compared with
the sera of 28 patients
(26.2%)
in the sonic extract
ELISA.
If the assay
sensitivity
 SUU, Alm.del - 2014-15 (1. samling) - Bilag 1413: Henvendelse af 24/5-15 fra Alex Holmstedt vedr. behandling af borrelia
1533935_0004.png
VOL.
27,
1989
OD
BORRELIA BURGDORFERI FLAGELLUM ELISA
IN EM AND
ACA
OD
547
>1,8
1,7
1,6
1,5
-
1,8
1,7
1,6
1,5
1,4
1,4
1,3
1,3
1
1,2
1,1
1,2
1,1
1,0
1,0
0,9
0,8
0,7
0,9
0,8
0,7
0,6
0,5
1
0
0,6
0,5
0,4
0,3
:1::
'iii'.
...
0,4
0,3
0,2
0,1
S.~ ~ ~
-r
1::1
¶ftulil
-
~
.-U-**ilir
**dii~~~~~~~~~~~~~r.L
ECM
ACA
j"
0,2
0,1
HEALTHY
CONTROLS
ECM
ACA
*.-
.uas.-...
...
HEALTHY
CONTROLS
DERMATOLOGICAL
CONTROLS
DERMATOLOGICAL
CONTROLS
FIG.
1.
IgG
levels
measured
by B.
burgdorferi
sonic
extract
ELISA
(left)
and B.
burgdorferi
flagellum ELISA
(right)
in sera of 107
patients
with
EM
(ECM)
and 50
patients
with ACA.
Serving
as
controls
were 200 healthy
individuals
and 98
patients
with various
dermatological
disorders.
The
horizontal
lines
mark
the diagnostic
95%
specific
cutoff
levels.
is
expressed
as
a
combined
evaluation
of
IgG
and IgM data,
e.g.,
either
IgG
or
IgM
reactivity,
it
must
be remembered
that the
diagnostic
specificity
will
decrease
to
about
90%,
since
10%
of
all controls
were
either
IgG
or
IgM
positive.
The
sera
of
15
patients
(14.0%)
were
both
IgG and
IgM
reactive in
the
flagellum
ELISA,
compared
with the
sera
of
only
2
patients
(1.9%)
in
the
sonic
extract
ELISA.
None
of
the 298
control
individuals
was
IgG
and
IgM
seroreactive.
When the
107
patients
with
EM
were
divided into four
groups
according
to
the
duration of
the
erythema,
the
frequency
of
seropositive
samples increased after
a
disease
duration of
more
than
1
month
(Table
1).
This
increase
was
statistically
significant
only
in
the
IgG
flagellum
ELISA
(P
<
0.005).
There
were
16
patients
with
multiple erythema.
When the
serological
findings
for
these
patients
were
compared
with
those
for
patients
with
a
solitary
EM,
an
increased
IgM
reactivity
was
shown
(68.8
versus
39.5%)
(P
<
0.05)
(Table
2).
Three
patients
with
multiple
erythema
had
a
disease
duration of
more
than
2
months;
they
were
all
IgG
serore-
active.
There
was no
correlation of
the
specific
antibody
level
with
the
occurrence
of
general
symptoms
concomitant
to
the
EM
(Table
2).
Fifty
serum
specimens
from
patients
with
ACA
were
tested.
For
IgG
antibodies,
the
diagnostic
sensitivities of
the
sonic
extract
and
flagellum
ELISA
were
identical,
98
and
100%,
respectively.
As
in
EM,
the
individual
OD
signal
obtained
was
significantly
higher
in
the
IgG
flagellum
ELISA
(Fig.
3c).
The
number
of
IgM-seroreactive
patients
with
ACA
was
reduced
from
22
to
12%
by
the
flagellum
ELISA.
To
rule
out
the
possible
role
of
an
IgM
RF
as
the
cause
of
unspecific
IgM
reactivity,
all
sera
from
patients
with
EM
and
ACA
were
investigated
for
IgM
RF.
A
total
of
16
(32%)
of
50
patients
with
ACA
and 8
(7.5%)
of
107
patients
with
EM
had
IgM
RFs in the
ranges
of
10
to
150 and
10
to
35
IU/ml,
respectively.
Nine
of
eleven
patients
with ACA
whose
sera
were
IgM
reactive
in the
sonic
extract
ELISA
had
detectable
IgM
RF,
including
all
sera
with OD values
of
>0.3.
The
sera
of
all
six
patients
with
ACA
that
were
IgM
reactive
in
the
flagellum
ELISA had
an
IgM
RF.
Absorption
of
the
RF
abolished
IgM
reactivity
in
all
IgM-reactive samples
from
patients
with
ACA
in both
assays.
Since
only
3
of 48
 SUU, Alm.del - 2014-15 (1. samling) - Bilag 1413: Henvendelse af 24/5-15 fra Alex Holmstedt vedr. behandling af borrelia
1533935_0005.png
548
OD
HANSEN
AND
ASBRINK
OD
J.
CLIN.
MICROBIOL.
>1,0
>1,0
-
o,9
0,8
0,9
0,8
0,7
-
-
0,7
0,6
0,6
0,5
0,4
i.
0,5
0,4
0,3
L
-
-
-
*1*
0,3
0,2
0,2
0,1
"SU!~
HEALTHY
~~
..:::::::...SI
ECM
0,1
DERMATOLOGICAL
CONTROL
HEALTHY
CONTROLS
.0
.:IIii!
ACA
CONTROLS
ACA
ECM
DERMATOLOGICAL
CONTROL
FIG.
2.
IgM levels
measured by
B.
burgdorferi
sonic
extract
ELISA
(left)
and
B.
burgdorferi
flagellum
ELISA
(right)
in
sera
of 107
patients
with
EM
(ECM)
and
50 patients
with ACA.
Serving
as
controls
were
200
healthy
individuals
and 98
patients
with
various
dermatological
disorders.
The
horizontal
lines mark
the
diagnostic
95%
specific
cutoff
levels.
IgM-reactive
serum
specimens
from
patients
with
EM
showed
an
IgM RF,
no
absorption
of
the
RF
was
done
with
these
specimens.
DISCUSSION
In
a
previous
study,
the
B.
burgdorferi
flagellum
ELISA
was
shown
to
be clearly
superior
to
a
sonic
extract
ELISA
in
testing
serum
and
cerebrospinal
fluid
samples
from
patients
with
lymphocytic meningoradiculitis
(15).
A
comparable
increase
in the
diagnostic
performance
of
the
flagellum
ELISA
was
found
in
the
present
investigation
of
sera
from
107
patients
with
EM,
the
early
and
first-stage
manifestation
of
Lyme borreliosis.
A
main
advantage
of
the
purified
antigen
was a
greatly
increased specificity
of
the
IgG
ELISA, since
the OD values
in
the
control
groups
were
significantly
lower.
When the
diagnostic
cutoff level
was
adjusted
to
be
95%
specific,
the
gained
specificity
was
converted
into
a
marked
increase
in
diagnostic
sensitivity. The diagnostic
specificity
of the
IgM
flagellum
ELISA
was
almost unaltered,
whereas the
diag-
nostic
sensitivity
increased significantly.
TABLE 1.
Diagnostic
sensitivity of B.
burgdorferi
sonic
extract
and
flagellum
ELISAs
Wk
after
onset
of
EM
(no.
of
patients)
No.
(%)
of
positive
samples
Sonic
extract
ELISA
Flagellum
ELISA
IgG
IgM
IgG
s1 (22)
2-4
(48)
5-12
(32)
>12
(5)
Total
(107)
2
3
6
1
(9.1)
(6.3)
(18.8)
(20.0)
2 (9.1)
11
(22.9)
5
(15.6)
6
12
17
3
(27.2)
(25.0)
(53.1)
(60.0)
IgM
7 (31.8)
22
(45.8)
17
(53.1)
2
(40.0)
12
(11.2)
18
(16.6)
38
(35.5)
48
(44.8)
The
improved diagnostic performance
of the
flagellum
ELISA
compared
with the
sonic
extract
ELISA is
most
likely
the result
of
the
early
and
strong
antibody
response
to
the
41-kilodalton
antigen recognized
in
several
WB
studies
(7, 11-13, 17,
25)
and
the
elimination
of
irrelevant
cross-
reacting
antigens
which
are
contained
in the
whole-cell
sonic
extract.
B.
burgdorferi
shares
antigenic epitopes
with
other
spirochetes
(7,
15,
18)
and
(and
this
may
be
of
more
practical
importance
to
serology)
also
with
many
remotely
related
and
common
bacteria,
including
the
normal
human
flora.
Such
an
antigen
is the
immunogenic
60-kilodalton
protein
of B.
burgdorferi
which
recently
was
shown
to
be
the
widely
cross-reacting
common
antigen (14).
This
protein
is
soluble
and
is
present
in
every
sonic
extract
of
B.
burgdorferi.
A
recent
ELISA
study
(13)
evaluated
a
sonic
extract
antigen
versus a
flagellum-enriched
but
not
purified flagellum
antigen preparation
for
ELISA.
Possibly
because
of
many
residual
antigens
in
the
antigen
preparation,
no
significant
increase
in the
number of
seropositive patients
was
found.
In
agreement
with
two
previous
studies
(11, 15), Grodzicki
and
Steere
(13)
noticed
a
clearly
improved
discrimination
be-
tween
controls
and
patients, since positive
sera
generally
gave
considerably
higher
OD
signals
when
either the
flagel-
lum-enriched
or
purified
flagellum
antigen
was
used. This
observation
is
of
great
importance,
especially
in
growing
routine
serology, since
borderline
values
in sonic
extract
ELISAs
are
frequent and
difficult
or
impossible
to
interpret.
In
patients with
a
disseminated infection,
as
in
lympho-
cytic
meningoradiculitis,
the magnitude
of
the
antibody
response
to
B.
burgdorferi is
correlated
with
the duration
of
the
disease (12, 15,
21, 23). IgM
titers
show
a
maximum
at
5
to
7
weeks
after
onset,
whereas
IgG titers
increase
gradually
and
are
positive
in
almost
every
untreated
case
after
2
months
(15).
In
EM, only the
use
of
the
flagellum
ELISA
showed
a
significant increase
(P
<
0.005)
in
the
number
of
IgG-positive
tests
when the duration
of
an
untreated
EM
Retur til dok liste
 SUU, Alm.del - 2014-15 (1. samling) - Bilag 1413: Henvendelse af 24/5-15 fra Alex Holmstedt vedr. behandling af borrelia
1533935_0006.png
VOL.
27,
1989
OD
1,0
BORRELIA
BURGDORFERI
FLAGELLUM
ELISA IN EM AND ACA
549
a
OD
1,0
b
*
a
0Q9
Q8
07
0,9
0,8
0,7
Q6
C
06
'*.::
*.
:@ *.
0
-
0,5
w
-I
U
0,4
0,3
0,2
-
0,1
1.
.O
.
*
w
.1
0,4
-a
LL.
0
0,3
0,2
102
0,3
O
,
,
,
,
,
,
- 0
.
r*
uj,;i
0,2
0,3
0,4
0,5
0,6
0,7
0,8
se.
0,1
0,9
1,0
*a
'
*,
0,2
0,3
OA 0,5
Bb-SON
ELISA
0,1
Q1
0',6
IgM
0,7
0,8 0,9
1,0
OD
Bb-SON ELISA
IgG
OD
1,8
1,7
-
OD
c
tu
.
u
-
1,6
1,5
-
1,4
1,3
1,2
1,1
1,0
-
-
-
-
oa
0
w
LA.
0,8-
0,7
-
0,6
.
O
0,5
0,4
0,3
0,2
0,1
0,1
0,
2
0,3
0,4
0,5 0,6
0,7
0,8
0,9
1,0
1,1
1,2 1,3
1,4
1,5
1,6
1,7 1,8
Bb-SON
ELISA
IgG
OD
FIG.
3.
Correlation
of
IgG
(a)
and
IgM (b)
measurement
in
sera
of
107
patients
with
EM
and
of
IgG
measurement
in
sera
of 50
patients
with
ACA
(c) by
the
B.
burgdorferi
sonic
extract
and
flagellum
ELISAs.
The vertical
lines
represent the
cutoff
levels
in
the
sonic
extract
ELISA,
and
the
horizontal
lines
mark
the cutofflevels
in
the
flagellum
ELISA.
The
flagellum
ELISA
improved
the
quantitative
discrimination
between
controls
and
seropositive samples
significantly,
as
estimated
by
comparing
the
distances
of the
achieved
OD
values
from
the
cutoff
level
in
each
test
using
Wilcoxon's
rank
sum
test
for
paired
data:
panel
a,
P
=
0.002;
panel
b,
P
=
0.001;
panel
c,
P
=
0.001.
 SUU, Alm.del - 2014-15 (1. samling) - Bilag 1413: Henvendelse af 24/5-15 fra Alex Holmstedt vedr. behandling af borrelia
1533935_0007.png
550
HANSEN
AND
ASBRINK
J.
CLIN.
MICROBIOL.
TABLE
2.
Diagnostic
sensitivity
of
B.
burgdorferi
flagellum
ELISA
for
107
patients
with
EM
%
of
patients
No
con-
Concomitant
comitant
general
Avg
(n=
107)
symptoms
s
general
(n
=t107
(n
=
69)
Positive
reaction
Multiple
erythema
(n
=
16)
68.8
37.5
Solitary
erythema
(n
=
91)
39.5
36.3
sympt38m
44.7
39.5
IgM
IgG
43.5
34.8
44.8
35.5
exceeded
1
month.
Still,
only
slightly
more
than
50%
of the
patients
with
longstanding
EM (1
to
12
months)
were
IgG
seropositive (Table
1).
The
reason
for
this
discrepancy
is
unknown.
It
may
be
because
uncomplicated
EM
is
an
infection
restricted
to
the skin,
whereas
a
disseminated
infection
leads
to
a
more
predictable
and
stronger
immune
response.
This
explanation
is
supported
by
the
observation
of
higher
antibody levels
in
patients
with
multiple
skin
lesions,
which
are
most
likely the
result
of
hematogenous
spread.
The
restriction
of
a
B.
burgdorferi
infection
to
a
solitary
skin
lesion
may
be
controlled
by
host
factors
and
properties
of
the
spirochete
that
determine
its
pathogenicity.
The
spontaneous
course
of
the
infection,
as
well
as
the
production
of
antibodies,
seems
to
be
a
question
not
only
of
disease
duration.
Therefore, the
diagnostic
sensitivity
of
serological
tests
must
be
defined
for
cases
of
EM
and
separately
for
cases
of
systemic
Borrelia
infection
and
not
for
cases
of
unspecified
Lyme
borreliosis.
Many
serological
studies
using
indirect
immunofluores-
cence
assays
or
sonic
extract
ELISAs have been
reported
since
1982.
The
diagnostic
sensitivities
achieved
vary
con-
siderably.
The
results
are
often
not
comparable
for several
reasons:
(i)
unequal selection
or
lack of
classification
of
patients
according
to
the
type
of
manifestation
and
disease
duration;
(ii)
combined
evaluation of
IgM and
IgG
results,
e.g.,
determining
the
number
of
patients
who
are
either IgM
or
IgG
seropositive
without
noting that
this
procedure
re-
duces
the
diagnostic specificity significantly;
expression of
the
diagnostic
sensitivity
of
a
test
as
the
rate
of
either
IgG
or
IgM
reactivity
demands
at
least
a
97.5%
specific
cutoff
level
in
both
the
IgG
and
IgM
assays;
(iii)
inclusion
of
several
samples
from
one
patient
or
consideration
of
only
the
highest
titer
measured
in
a
patient
and
not
the first,
diagnostic,
pretreatment
sample; (iv)
different
criteria
for
size
and
composition
of
the
control
group;
and
(v)
very
different
cutoff
levels.
In
a
diagnostic
or
screening
test,
the
cutoff
level
should
be
at
least 95%
specific
based
on
a
large
and
not
serologically
preselected
control
group.
There
were
no
differences
regarding
the
IgM
or
IgG
antibody
levels
in
200
Danish
and
98
Swedish
controls
representing
two
independent
populations
from
distant
re-
gions.
Such
differences
could
have
been
the
result
of
re-
gional
differences
in
tick
or
B.
burgdorferi
exposure
of
the
populations
or
differences
in
the
regional
prevalences
of
certain
strains.
Previous
indirect
immunofluorescence
assay
studies
that
tested
sera
with
American
and
European
B.
burgdorferi
strains
did
not
show
any
strain-dependent
differ-
ences
(1, 3,
19,
26).
The
use
of
B.
burgdorferi
flagellum
as
test
antigen
will further
diminish
the
possible
influence
of
strain
variation
on
serological
results,
since
it
is
a
genuswide
antigen
(8).
IgG antibodies
to
the
B.
burgdorferi
flagellum
were
found
in
all
sera
from
patients
with
ACA.
This
proves
the
persis-
tence
of
an
antibody
response
to
the
flagellum
even
into
the
late
stages
of
the disease.
IgM RF
may
cause
false-positive
IgM
reactivity
in
an
indirect
ELISA
(24).
Therefore, samples
from
patients
with
EM
and
ACA
were
investigated
for
an
IgM
RF.
Of
the
samples
from
patients
with
ACA,
32%
had
a
detectable
IgM
RF,
including
9
of
11
serum
samples
that
were
reactive
in the
sonic
extract
ELISA
for IgM
and
6
of
6
samples
that
were
reactive
in
the
flagellum
ELISA for
IgM.
In
accordance
with
a
previous study (26),
pretreatment
of
the
sera
with
IgM
RF
Absorbans
abolished
the
IgM
reactivity
in
all
IgM-reactive
samples
from
patients
with
ACA.
False
IgM
reactivity
caused
by
IgM RF
in
EM
is
probably
rare,
since
only
3
of
48
IgM-reactive
serum
samples
had
a
demonstrable
RF.
IgM
RF
is
frequently
found
in
various
acute
and
chronic
infec-
tious and
immunological
disorders.
For
routine
serology,
we
therefore
recommend
that
the
use
of
the
indirect
IgM ELISA
be
restricted
to
probable
early
cases
of
Lyme
disease
and,
in
particular, to the
investigation of cerebrospinal
fluid
(15).
The B.
burgdorferi
flagellin
(the reduced
protein
subunit of
the
flagellum
antigen)
has
genus-specific
epitopes
(8)
but also
some more
conserved epitopes
cross-reacting with
other
spirochetal
fiagellins,
especially
from
treponemas.
There-
fore, the main
limitation
ofthe
diagnostic specificity
of
the
B.
burgdorferi
flagellum
as
a test
antigen
is
a
partial
cross-
reactivity
with
the Treponema
pallidum flagellum
(15).
The
new test does not
permit serological discrimination
between
patients
with
Lyme
borreliosis and syphilis,
although
sero-
logical cross-reactivity was
reduced
compared
with that
of
the sonic extract ELISA (15). The
magnitude
of
this
problem
in routine serology is limited. During one year, 1987,
when
a
sonic
extract
ELISA
was
still
being
run,
the
Copenhagen
Borrelia Laboratory received 5,264 samples. Only four were
false-positive because
of syphilitic infections. Furthermore,
patients with Lyme
borreliosis
and
syphilis
are
easily
differ-
entiated clinically and serologically by the
nontreponemal
syphilis serological tests (19).
On the basis of the present study
of
sera
of
patients with
EM and ACA and a previous investigation
of
sera
and
cerebrospinal
fluid of patients with lymphocytic
meningora-
diculitis (15), we conclude that the B.
burgdorferi
flagellum
ELISA is for the time being the most sensitive and
specific
quantitative serological test. WB does not seem to be
a
practicable method for routine serology. Neither a single B.
burgdorferi-specific
band nor a combination of such bands
with a high diagnostic sensitivity that would allow the
discrimination of specific and unspecific antibody reactiv-
ities has been identified (13, 17).The flagellum ELISA is of
particular value when the specific antibody level is low, as in
many cases of EM and in the early secondary stage of Lyme
borreliosis (15). A low but specific antibody response is often
missed by a sonic extract ELISA because it is hidden by the
necessarily high
cutoff
level. The antigenic composition of a
whole-cell sonic extract may vary considerably, depending
on the strain and the
preparation
technique. The B. burg-
dorferi flagellum is easy to standardize as a test antigen.
These properties make the B. burgdorferi flagellum a suit-
able and needed
reference
antigen for routine serodiagnosis
in Lyme borreliosis.
ACKNOWLEDGMENTS
Klaus
Hansen
was supported by a grant from the University of
Copenhagen and the Mauritzen la Fontaine foundation. Eva Asbrink
received
a
grant
(no. 7935) from the Swedish Medical Research
Council.
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1533935_0008.png
VOL.
27,
1989
BORRELIA
BURGDORFERI
FLAGELLUM
ELISA
IN EM AND
ACA
551
We
thank
Hanna Hansen
for
perfect technical
assistance;
Karen
Langner for preparing the
manuscript;
Kirsten
Christoffersen,
De-
partment
of Biostatistics, Statens Seruminstitut, for statistical cal-
culations; and Mimi H0ier-Madsen,
Department
of Clinical
Immu-
nology, Statens Seruminstitut,
for
measuring
IgM
RF.
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Vol. 319 No. 22
SERONEGATIVE LYME DISEASE — DATTWYLER ET AL.
1441
SERONEGATIVE LYME DISEASE
Retur til dok liste
Dissociation of Specific T- and B-Lymphocyte Responses to
Borrelia burgdorferi
RAYMOND
J.
DATTWYLER, M.D., DAVID
J.
VOLKMAN, M.D., PH.D., BENJAMIN
J. LUFT,
M.D.,
JOHN
J.
HALPERIN,
M.D.,
JOSEPHINE THOMAS,
B.S.,
AND MARC
G.
GOLIGHTLY, PH.D.
Abstract The diagnosis of Lyme disease often depends
on the measurement of serum antibodies to
Borrelia burg-
dorferi,
the spirochete that causes this disorder. Although
prompt treatment with antibiotics may abrogate the anti-
body response to the infection, symptoms persist in some
patients.
We studied 17 patients who had presented with acute
Lyme disease and received prompt treatment with oral
antibiotics, but in whom chronic Lyme disease subse-
quently developed. Although these patients had clinically
active disease, none had diagnostic levels of antibodies
to
B. burgdorferi
on either a standard enzyme-linked
immunosorbent assay or immunofluorescence assay.
On Western blot analysis, the level of immunoglobulin
reactivity against
B. burgdorferi
in serum from these pa-
MHE
best clinical marker of acute Lyme disease is
tients was no greater than that in serum from normal
controls.
The patients had a vigorous T-cell proliferative re-
sponse to whole
B. burgdorferi,
with a mean (±SEM) stim-
ulation index of 17.8±3.3, similar to that (15.8±3.2) in
18 patients with chronic Lyme disease who had detect-
able antibodies. The T-cell response of both groups was
greater than that of a control group of healthy subjects
(3.1±
-0.5; P<0.001).
We conclude that the presence of chronic Lyme disease
cannot be excluded by the absence of antibodies against
B. burgdorferi
and that a specific T-cell blastogenic re-
sponse to
B. burgdorferi
is evidence of infection in sero-
negative patients with clinical indications of chronic Lyme
disease. (N Engl J Med 1988; 319:1441-6.)
a characteristic skin lesion, erythema chronicum
migrans (ECM).1-4 In many patients, initial infection
progresses to chronic Lyme disease, a spectrum of
clinical signs and symptoms characterized by persist-
ent musculoskeletal, cardiac, and neurologic involve-
ment.4-1° Since
Borrelia burgdorferi
was discovered to be
the etiologic agent of this infectious disease, the dem-
onstration of specific antibodies to this spirochete has
been considered to be the best indicator of exposure to
the organism and has become a prerequisite for the
diagnosis of chronic Lyme disease."5 A vigorous
humoral response against
B. burgdorferi
develops dur-
ing the course of natural infection. In chronic Lyme
disease,
B. burgdorferi
persists, inducing an ongoing
inflammatory response.16'17 The absence of a continu-
ing humoral response in patients treated with anti-
biotics during the ECM phase of Lyme disease has
been believed to indicate that the organism had been
effectively eradicated.18-20 However, many patients
given oral antibiotics at the ECM stage have reported
persistence of symptoms, including severe chronic fa-
tigue, numbness of the extremities, memory loss, and
joint pain.18-2I In the absence of elevated levels of
antibodies to
B. burgdorferi,
these symptoms have been
attributed to a post—Lyme disease syndrome rather
than considered as evidence of persistent infection and
failure of the initial antibiotic regimen to treat the
disease effectively.
In persons exposed to
B. burgdorferi,
an early, vigor-
ous, and sustained specific T-lymphocyte response
develops that often precedes a measurable antibody
response.22'23 In this study we used borrelia-specific
From the Department of Medicine (R.J.D., D.J. V. , B.J.L., J.T.), the Depart-
ment of Pathology (M.G.G.), and the Department of Neurology (J.J.H.), State
University of New York at Stony Brook, School of Medicine, Stony Brook,
N.Y. Address reprint requests to Dr. Dattwyler at the Division of Clinical Immu-
nology, Health Sciences Center, SUNY at Stony Brook, Stony Brook, NY
11794-8161.
Supported in part by grants (P01 AI-16337 and ROI Al-23910) from the U.S.
Public Health Service and by a grant from the Vangee Moore Foundation.
T
-
cell immune responses to document exposure to
B. burgdorferi
in patients with symptoms of chronic
Lyme disease who lacked diagnostic levels of specific
serum antibodies to the organism. It is noteworthy
that all these patients had received oral antibiotics
early in the course of infection. Thus, these findings
exemplify an infectious disease in which removal of
most of the microbial antigens at an early point in
its course appears to abrogate the development of
sustained humoral immunity despite evidence of per-
sistent infection.
METHODS
Subjects
Patients were classified as either seropositive or seronegative on
the basis of their serum antibody reactivity to
B.
burgdorferi in
a
standardized enzyme-linked immunosorbent assay (ELISA). Se-
rum samples were considered positive if the concentrations of anti-
bodies to
B.
burgdorferi,
expressed as optical-density values, were
more than 3 SD above the mean values for a panel of samples
obtained from a group of healthy adults with no history of
B.
burg-
dorferi
infection. Samples with optical-density values less than 3 SD
above the mean were considered negative.
The present study was based on findings in 17 patients with signs,
symptoms, and a history compatible with the diagnosis of chronic
Lyme disease who had specific optical-density values in the negative
range (<3 SD above the mean). All were from Suffolk County, New
York, an area in which Lyme disease is highly endemic. Eighteen
other patients with comparable signs, symptoms, and histories
of chronic Lyme disease who had diagnostic levels of antibodies to
B.
burgdorferi
on ELISA served as positive controls, and 17 healthy
adults with no history of Lyme disease or any other rheumatic or
immune disorders served as negative controls. The clinical manifes-
tations of both the seronegative and seropositive patients are shown
in Table 1. Of the 17 seronegative patients, 15 had well-document-
ed histories of ECM, the best clinical marker of
B.
burgdorferi
infec-
tion. In each of these patients an influenza-like illness had accompa-
nied the ECM. The other two patients had a similar history of an
influenza-like illness that occurred soon after a tick bite. All 17
patients had been treated with oral antibiotics for at least 10 days at
the time of their initial illness. Twelve had received tetracycline
(1 to 2 g per day), four penicillin (1 to 2 g per day), and one eryth-
romycin (1 g per day).
At the time of their initial presentation at the Lyme Disease
Clinic at our institution, all 17 seronegative patients had systemic
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1533935_0010.png
1442
THE NEW ENGLAND JOURNAL OF MEDICINE
Dec. 1, 1988
symptoms, including fatigue, arthralgias, headaches, and cognitive
dysfunction. Fever was not noted. The incidence of arthritis or a
history of arthritis in this group (12 of 17 patients) was similar to
that observed in the seropositive group (15 of 18 patients). In both
the seronegative and the seropositive groups the arthritis was char-
acterized by pain occurring when the affected joint was moved,
periarticular soft-tissue swelling, synovial thickening, or small effu-
sions. In all cases it was mild, oligoarticular, and nonerosive and its
course contained remissions and relapses. The arthritis generally
involved large joints, especially the knee. Nine of the seronegative
patients had peripheral neuropathy. Four had entrapment neuropa-
thies, documented by nerve-conduction studies, and seven had
measurable abnormalities of peripheral nerves other than entrap-
ment neuropathy, also documented by nerve-conduction studies.
The neurophysiologic abnormalities of the peripheral nerves were
indicative of an axonal neuropathic process. Demyelinating neurop-
athy was not observed. None of the patients had a history of recur-
rent infections. At the time of entrance into the study none were
taking glucocorticosteroids or other immunosuppressive agents.
After the 17 seronegative patients received treatment with paren-
teral antibiotics (either penicillin [24 million units per day in divid-
ed doses for 10 days] or ceftriaxone [2 to 4 g per day for 14 days]) , all
had marked improvement. Objective evidence of active disease,
including arthritis, resolved in all patients over a three-month peri-
od, with the exception of peripheral neuropathy, which resolved
more slowly. None of the patients had a recurrent episode of ar-
thritis or a symptomatic relapse during an eight-month follow-up
period.
minetetraacetic acid and 1 nM p-methylsulfonylfluoride and soni-
cated in an Ultra tip Labsonic System (Lab-Line Instruments, Mel-
rose Park, Ill.) for five minutes. The protein concentration was
measured according to the Bradford procedure.' The mixture was
concentrated to 2.5 percent in sodium dodecyl sulfate and 2.5 per-
cent in 2-mercaptoethanol, heated at 100°C for three minutes, and
adjusted to a concentration of 0.4 to 0.6 mg of protein per milliliter.
Sodium dodecyl sulfate—polyacrylamide gel electrophoresis was per-
formed with a Mighty Small II apparatus (Hoeffer, San Francisco)
at 60 V for two hours. Blots were left overnight at 4°C in TRIS
buffer (50 mM TRIS-150 mM sodium chloride) containing 1 per-
cent
bovine serum albumin. They were then incubated with serum
diluted with
TRIS
buffer for two hours at 37°C. Serum was assayed
at dilutions
of both 1:200 and 1:100. The blots
were washed for
10
minutes
with TRIS buffer containing 0.5 percent Tween-20 and
then for 10 minutes in TRIS buffer alone. Bound immunoglobulin
was detected with goat antihuman IgG, IgM, or IgA conjugated to
alkaline phosphatase (Sigma). After a two-hour incubation at
37°C with a 1:5000 dilution of the conjugates, the blots were
again washed in a two-step procedure and then incubated with the
BICIP
-
Substrate System
(5-bromo-4-chloro-3-indole phosphate-
nitroblue tetrazolium; Kirkegaard and
Perry Laboratories,
Gaith-
ersburg, Md.) for 20
minutes. The reaction was stopped by rinsing
the blots in distilled water.
Lymphocyte-Proliferation Assays
The lymphoproliferative response of peripheral-blood mononu-
clear cells to whole
B. burgdorferi
organisms was assessed during the
initial evaluation for chronic Lyme disease. Proliferation assays
were performed as previously described.22 Mononuclear cells were
isolated by Ficoll—Hypaque (Pharmacia, Piscataway, N.J.) density-
gradient centrifugation and washed three times. They were adjusted
to a final concentration of 1 x 106 cells per milliliter in RPMI-1640
supplemented with 10 percent human AB serum, 100 U of penicil-
lin, and 100
p.g
of streptomycin per milliliter, and 2 mM L-gluta-
mine. A total of 1 x 105 cells per well were pipetted into a 96-well
microtiter plate (Costar, Cambridge, Mass.) and stimulated (in
triplicate) with whole
B. burgdorferi
at a concentration of 1 x 106
organisms per well. Control wells received medium only. Cell cul-
tures
were incubated in a humidified atmosphere of 5 percent
car-
bon dioxide at 37°C.
[3H]Thymidine (specific activity, 6.7 Ci per
millimole [New England Nuclear, Boston]) was added to the wells
(1 /Xi
per well) 18 hours before harvesting at five days. Cells were
collected with a cell harvester (Skatron, Sterling, Va.) onto glass-
fiber filters, and the radioactivity associated with the cells was
counted in a liquid scintillation system (LS7500, Beckman In-
struments, Fullerton, Calif.). Counts were expressed as disintegra-
tions
per minute (dpm). The triplicate values were averaged, and a
stimulation index (SI) was calculated for each patient, according
to
the formula, SI = dpm (stimulated)/dpm (unstimulated).
ELISA for Antibodies to
B.
burgdorferi
The ELISA was performed as previously described, I6 with minor
modifications. In brief, 96-well microtiter plates (Flow Laborato-
ries, McLean, Va.) were coated with a sonicate of
B. burgdorferi
(5 ktg per milliliter) for 18 hours in 0.05 M sodium carbonate (pH
9.6) and then washed three times with phosphate-buffered saline
(pH 7.2) containing 0.05 percent Tween 20 (PBS/Tween). Excess
binding sites on the wells were blocked by postcoating the wells with
250 µl of phosphate-buffered saline containing 1 percent bovine
serum albumin. The plates were washed as described above. Serum
from all three study groups was diluted to 1:500 in PBS/Tween.
Aliquots of 0.2 ml were added to duplicate wells and incubated
for one hour at 37°C. The plates were washed, and alkaline phos-
phatase—conjugated goat antihuman immunoglobulin specific for
both light-chain and heavy-chain determinants was added to de-
tect bound immunoglobulin of all isotypes. After incubation for
one hour at 37°C, the wells were again washed three times in
PBS/Tween. p-Nitrophenyl phosphate (Sigma, St. Louis) was add-
ed at a concentration of 0.66 mg per milliliter in 0.15 M sodium
bicarbonate containing 1 mM magnesium chloride, to each well.
Plates were incubated two hours at room temperature, and the
reaction was stopped with 30 la of 3
N
sodium hydroxide. The
optical density of each well at 405 nm was measured on an ELISA
reader (Bio-Tek, Winooski, Vt.).
Statistical Analysis
The Student
one
-
tailed t
-
test
was used
to compare the
T
-
cell
proliferative responses in the three groups tested.
RESULTS
immunofluorescence Assay
Indirect immunofluorescence assays for the detection of both
IgM and IgG antibodies to
B. burgdorferi
were performed according
to standard techniques. In brief, slides coated
with
B. burgdorferi
(Diagnostic Technology, Hauppauge, N.Y.) were incubated with
serum at serial dilutions beginning at 1:64. After 60 minutes at room
temperature, the slides were washed three times in phosphate-buff-
ered saline and then incubated for 30 minutes with fluorescein-
labeled antihuman immunoglobulin, washed, and examined on a
fluorescence microscope (Nikon-Photomat, Garden City, N.Y.).
Electrophoresis and Immunoblotting
Qualitative antibody analyses were performed as previously de-
scribed,' with slight modifications. In brief,
B. burgdorferi
organisms
of the B31 strain were grown in BSK II media25 and washed three
times in phosphate-buffered saline. Spirochetes were resuspended in
2 ml of phosphate-buffered saline containing 10 mM ethylenedia-
At presentation, 12 of the 17 seronegative patients
had evidence of active arthritis on physical examina-
tion, and 9 had a peripheral neuropathy that was con-
firmed by nerve-conduction studies. All 17 reported
that they had had severe chronic fatigue and recurrent
headaches, 16 that they had had arthralgias, and 15
that they had had difficulty with short-term memory
and cognition; all had objective evidence of active
disease (Table 1). Eleven of the patients were evaluat-
ed with an extensive battery of neuropsychological
tests,27 including the California Verbal Learning Test
(a test of memory) and the booklet version of the
Category Test28; all 11 had evidence of intellectual
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Vol. 319 No. 22
SERONEGATIVE LYME DISEASE — DATTWYLER ET AL.
1443
Table 1. Characteristics of Patients Seropositive
and Seronegative for Chronic Lyme Disease.
SEROPOSITIVE
PATIENTS
(N
= 18)
Mean age (yr)
Sex (M/F)
History of ECM
Initial influenza-like illness
Initial treatment
Tetracycline
Penicillin
Cephalexin
Erythromycin
Duration of illness (mo)*
Clinical manifestations
Arthritis
Peripheral neuropathy
Symptoms
36.6 (16-72)
9/9
14
17
1
SERONEGATIVE
PATIENTS
(N = 17)
38.0 (10-65)
11/6
15
17
12
4
1
8.4+2.5
(1-24)
15
7
18
17.615.4
-
(8-72)
12t
9
17
±
'Period (mean - SD) from initial illness to diagnosis of chronic Lyme
disease.
(Thee other patients had a history of arthritis.
tIncludes headaches, fatigue, arthralgia, and cognitive dysfunction.
impairment. The results of serologic tests for antinu-
clear antibodies and rheumatoid factor, VDRL test-
ing, and C 1 q binding assay, and serum protein elec-
trophoresis patterns were all negative or normal.
Table 2 compares the results on ELISA in the sero-
negative and seropositive patients with Lyme disease.
None of the 17 seronegative patients had levels of
antibodies to
B. burgdorferi
that were more than 3 SD
above the mean level in the negative controls. Immu-
nofluorescence assays using whole
B. burgdorferi
organ-
isms were also negative at serum dilutions above 1:64
(minimum positive titers in our laboratory, %1:256).
Western blot analyses of solubilized whole
B. burgdor-
feri
antigens were performed to confirm the absence of
specific anti-borrelia antibodies. Figure IA shows IgG
immunoblots for six seropositive patients. Their se-
rum samples were assayed at a 1:200 dilution; all sam-
ples showed reactivity against distinct borrelia anti-
gens. The samples differed in the number of antigens
detected. The longer the Lyme disease was untreated
in these seropositive patients, the greater the number
of antigens against which their serum samples reacted
(lanes d through f). Figure 1B shows representative
IgG blots for five of the seronegative patients. Lane a
represents serum from a seropositive patient that was
run as a positive control. Lanes b through f represent
samples from seronegative patients that were run at
twice the concentration of the samples from the sero-
positive patients in order to detect low levels of bor-
relia-reactive antibodies. The most consistent indica-
tor reactivity observed in the samples from the
seronegative patients
was
a faint band corresponding
to the 41-kd borrelia flagella antigen. Similar reactiv-
ity was observed
in
blots for 10 other seronegative pa-
tients that
were run in
separate assays (data not
shown). This pattern of reactivity against spirochetal
antigens, especially the 41-kd protein, was also ob-
served in samples from the majority of controls (Fig.
1C). When the blots for the seronegative patients were
stained for IgM or IgA antibodies, little specific
reactivity
was
observed. A faint IgM band of reac-
tivity against the protein associated with the 41-kd
flagella was detectable in 2 of the 14 patients tested.
Faint bands of IgA
reactivity were
also detected
against the 41-kd and 66-kd antigens in all of five
seronegative patients tested. This pattern of low-level
reactivity of IgA and IgM antibodies was also ob-
served in the normal controls and was readily distin-
guishable from the pattern observed in the seroposi-
tive patients, both in terms of the number of antigens
recognized and the intensity of the bands. Thus, im-
munoblot analysis was consistent with the results ob-
tained with the ELISA and the immunofluorescence
assay,
and demonstrated a profoundly blunted re-
sponse to
B. burgdorferi
in the patients with clinically
active disease.
T-cell immune responses specific for
B. burgdorferi
were assessed according to a standard proliferative
assay
using whole
B. burgdorferi
as the stimulatory anti-
gen. These results are shown in Figure 2. Of note is
the finding that the 17 seronegative patients with clini-
cal Lyme disease had proliferative responses that were
virtually identical to those of the seropositive patients.
The stimulation index in the seronegative patients
ranged from 2.7 to 58.0, with a mean (±SEM) of
17.8±3.3. The average number of counts was 19,455
dpm. The group of 18 seropositive patients had sim-
ilar blastogenic responses to
B. burgdorferi,
with a
mean stimulation index of 15.8±3.2. In contrast,
the 17 healthy controls had stimulation indexes that
ranged from 0.5 to 7.8, with a mean of 3.1±0.5, and
an average count of 2932 dpm. Statistical analysis
did not demonstrate any significant difference be-
tween the seronegative and the seropositive patient
groups. However, the proliferative responses in each
patient group were significantly different from those in
the normal control group (P<0.001).
DISCUSSION
Infection with
B.
burgdorferi,
the etiologic agent of
Lyme disease, is associated with the development of a
vigorous T-cell response to this organism.22,23 In this
study we have shown that a specific T-cell response
can occur independently of a diagnostic humoral re-
sponse to
B. burgdorferi.
Although our 17 chronically ill
patients had clinical histories, signs, and symptoms
Table 2. Reactivity for
B.
burgdorferi
Antibodies
on ELISA.*
NORMAL SERONEGATIVE SEROPOSITIVE
PATIENTS
CONTROLS
PATIENTS
(N = 18)
(N = 10)
(N = 17)
Mean serum antibody
concentration
Standard deviation
0.44
0.19
0.55
0.24
3.81
0.62
*Values are expressed as normalized values for optical density (donor
value
in subject ÷
[mean value among normal controls +3 SDI).
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1533935_0012.png
1444
THE NEW ENGLAND JOURNAL OF MEDICINE
Dec. 1, 1988
compatible with the diagnosis of active Lyme disease,
their levels of antibodies to
B.
burgdorferi
as measured
by ELISA or by immunofluorescence assay were in-
distinguishable from those of normal controls. This
lack of reactivity was confirmed by Western blot anal-
ysis. Serum from these 17 patients primarily exhibited
only weak reactivity to the 4 1-kd flagella antigen, an
antigen sharing epitopes with many other spirochetes,
a b cd e f
83
66
41
34,31,29
17,15 AIP
a b cd e f
A
66
41
34,31,29
17,15
B
a b cd e
including both other borrelia species2° and treponema
species. In contrast, 14 of the 17 patients had a
marked T-cell response to this organism. Three pa-
tients had low T-cell responses (stimulation indexes of
2.7, 4.1, and 4.9) that overlapped with the range of the
normal controls. Each of these three patients, how-
ever, had a history of ECM that had been observed by
a
physician, early antibiotic therapy, a clinical course
compatible with Lyme disease, and improvement af-
ter administration of parenteral antimicrobial agents
known to be effective against
B. burgdorferi.
A similar
range of T-cell reactivity was also observed in the
seropositive patients. Once established, this T-cell re-
activity in both seronegative and seropositive individ-
uals was reproducible and persistent. None of the 17
patients had a history suggestive of a primary immune
defect, although the possibility of a specific abnormal-
ity in the ability to respond to discrete spirochetal
antigens or an immunoglobulin-subclass deficiency
was not ruled out.
The disorder in these seronegative patients reflected
a dissociation between T-cell and B-cell immune
responses, in which the cell-mediated arm of the
immune response was intact yet the humoral por-
tion of the response to
B.
burgdorferi
appeared to be
blunted. This diminished antibody response is in con-
trast to the T-cell anergy commonly observed in
several chronic infections (e.g., infection with
Myco-
bacterium leprae
or
M. marinum,
filariasis, and some
chronic fungal infections29-33). It has previously been
found that some patients who received antibiotics
for ECM had low or undetectable levels of anti-
borrelia antibodies.15'2° This condition may be analo-
gous to syphilis, in which early antimicrobial therapy
can abort the development of a measurable humoral
response. Since help from T cells is usually required
to produce a vigorous antibody response to protein
antigens and to augment the response to most car-
bohydrate antigens,34 the development of a measur-
able T-cell response probably occurs before an anti-
body response becomes fully manifest. In previous
studies, we showed that patients with ECM can
mount specific T-cell responses to
B. burgdorferi
before
antibodies to this organism become detectable by
routine ELISA.22'23
Figure
1.
Immunoblots of IgG Antibodies to
B. burgdorferi
Antigens.
B. burgdorferi
proteins were transferred to nitrocellulose and
probed with diluted serum (either 1:200 for ELISA-positive sam-
ples or 1:100 for negative samples). Bound IgG was detected by
goat antihuman gamma-chain antibody conjugated to alkaline
phosphatase. Blots were developed with the
BICIP-substrate system.
Panel A shows blots for six patients with chronic Lyme disease
who were seropositive on ELISA. Panel B shows representative
blots for five of the seronegative patients (lanes b through f), and
Panel C immunoblots of serum from five of the normal controls
(lanes b through f). Lane a in both Panel B and Panel C repre-
sents serum from a seropositive patient, shown for comparison.
Molecular-weight markers are shown to the left.
83
66
41
34,31,29
17,15
C
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1533935_0013.png
a b cde f
83
66
41
34,31,29
it
17,15
A
a bcd e f
66
41
34,31,29
17,15
B
a bcd e f
83
66
41
34,31,29
17,15
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1533935_0014.png
Vol. 319 No. 22
SERONEGATIVE LYME DISEASE — DATTWYLER ET AL.
1445
60
50
STI MULATION
INDEX
a
40
a
30
2
a
-
II-
10
s
1
CONTROL
SEROPOSITIVE
SERONEGATIVE
Figure 2. Lymphoproliferative Responses to
B. burgdorferi.
Isolated peripheral-blood mononuclear cells were stimulated with
whole
B.
burgdorfed
organisms as outlined in the Methods. The
cells were cultured in flat-bottomed microtiter wells, at a concen-
tration of 105 cells per well, pulsed with 1 pCi of [3FIlthymidine for
18 hours, and harvested on day 5. Values for the stimulation
index are shown on the abscissa. The groups studied were pa-
tients with positive values on ELISA for
B.
burgdorferi
(n = 18),
patients without serologic evidence of Lyme disease (n = 17),
and normal controls (n = 17). Bars represent the arithmetic mean
response in each group.
The precise mechanism of the observed humoral
anergy has not yet been delineated. However, each of
the 17 seronegative patients was given antibiotics ear-
ly in the course of infection. Although the standard
antibiotic therapy for Lyme disease appears to eradi-
cate the organism in most instances,'8 we have found
that in some patients the response to treatment is in-
complete and late sequelae develop.2I In the seronega-
tive patients whom we describe, it may well be that
antibiotic therapy resulted in the elimination of most
spirochetes at a critical early stage of the immune
response, before a sustained B-cell response had devel-
oped. A precedent for the abrogation of a mature anti-
body response by early treatment exists not only in
Treponema pallidum
infection35 but also in Rh incom-
patibility, in which the infusion of antibody to the Rh
antigen clears the antigen and selectively inhibits the
maternal antibody response.36 Current concepts of
immune responsiveness hold that T cells initially rec-
ognize foreign antigen presented by cells expressing
Class II major histocompatibility antigens, such as
macrophages and dendritic cells." After recognition,
antigen-specific T cells proliferate and mature to be-
come effector cells. These effector T cells can then
interact with other cells in the immune system that
express the same HLA-D–region gene products. In
antigen-specific systems, an additional requirement
for cell–cell interactions is the presence of antigen to
provide a close physical linkage between the T effector
cell and the other cells. Antigen-specific B cells, hav-
ing both dense Ia and specific antigen receptors on
their surface, are exquisitely suited to present antigen
to T cells.38 In the absence of antigen, cognate recog-
nition is not established and T cells and B cells do not
interact effectively. Thus, according to this model of
immune interaction, after an initial proliferative re-
sponse by B cells that is independent of the response
by T cells, the continued presence of the antigen is
required for the development of a mature humoral
response.
There is an apparent inconsistency in this model of
seronegative Lyme disease and the generally held con-
cept of the pathogenesis of chronic Lyme disease. If
the cognate recognition of B cells by T cells fails to
occur because the spirochetes have been removed,
how can continued disease activity be explained? Al-
though all the patients described were given antibiot-
ics that could effectively eradicate the bulk of
B. burg-
dorferi
organisms from most sites of infection, the
central nervous system and perhaps other privileged
sites may not have received adequate treatment. Cur-
rently recommended regimens of oral tetracycline and
penicillin fail to produce drug concentrations in the
central nervous system that are high enough to reach
the mean inhibitory concentrations for the majority of
strains of
B. burgdorferi. 21,39,4°
Thus, spirochetes reach-
ing this immunologically privileged site may remain
viable despite standard therapy. A similar phenom-
enon has been observed in patients in whom therapy
believed to be curative for
T. pallidum
infection was
administered yet in whom active neurosyphilis later
developed.41-43 Consistent with chronic central nerv-
ous system infection is our recent observation that
some patients with chronic Lyme disease have ap-
preciable abnormalities of the white matter on mag-
netic resonance imaging.27 In addition, three separate
groups of investigators have reported local production
of anti-borrelia antibody in the central nervous system
in patients with neurologic symptoms of Lyme dis-
ease in the absence of diagnostic levels of serum anti-
bodies.44-46
In summary, the diagnosis of chronic
B. burgdorferi
infection should be considered in any patient with
a history of ECM and signs and symptoms compati-
ble with chronic Lyme disease. Serologic assays re-
main the best tests for screening for exposure to
B. burgdorferi.
Although the true incidence of sero-
negative disease is unknown, it is not common. Sero-
negative patients represent less than 5 percent of
the more than 200 patients with Lyme disease who
are referred to our group each year. Assessment of
T-cell blastogenesis should be limited to symptomatic
patients who have been exposed to ticks, who have
objective evidence of active disease, who have re-
ceived antibiotics early in the course of their infection,
and who have nondiagnostic levels of antibody sys-
temically and locally. Although chronic fatigue is
a common finding in chronic Lyme disease, we do not
believe that chronic fatigue as an isolated symptom
should be considered to be indicative of
B. burgdor-
feri
infection. It is important to diagnose chronic
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1446
THE NEW ENGLAND JOURNAL OF MEDICINE
Dec. I, 1988
Lyme disease with its accompanying neurologic and
rheumatologic sequelae correctly, since this systemic
spirochetosis can be effectively treated with appro-
priate intravenous antibiotics.41'47-49 However, it is
also important to remember that immunologic tests,
whether they measure antibodies or cellular immune
responses, are indicators of exposure and do not by
themselves prove that the patient has an active in-
fectious process.
We are indebted to Dr. Peter Gorevic for his helpful comments, to
Ms. Myra Ward and Ms. Maureen Veprek for their help in prepar-
ing the manuscript, and to Ms. Josephine Schultz for her technical
assistance.
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J
OURNAL OF
C
LINICAL
M
ICROBIOLOGY
, Sept. 1995, p. 2260–2264
0095-1137/95/$04.00 0
Copyright 1995, American Society for Microbiology
Retur til dok liste
Vol. 33, No. 9
Antibodies against Whole Sonicated
Borrelia burgdorferi
Spirochetes, 41-Kilodalton Flagellin, and P39 Protein in Patients
with PCR- or Culture-Proven Late Lyme Borreliosis
¨
JARMO OKSI,
1,2
* JAAKKO UKSILA,
2
MERJA MARJAMAKI,
3
JUKKA NIKOSKELAINEN,
1
3
AND
MATTI K. VILJANEN
Department of Medicine, Turku University Central Hospital,
1
Department of Medical Microbiology, Turku University,
2
and National Public Health Institute, Department in Turku,
3
Turku, Finland
Received 26 January 1995/Returned for modification 10 May 1995/Accepted 8 June 1995
The sensitivities and specificities of three enzyme-linked immunosorbent assays (ELISAs) for
Borrelia
burgdorferi
antibodies were compared for 41 patients presenting with symptoms compatible with late Lyme
borreliosis (LB) and 37 healthy controls. All subjects were living in southwestern Finland, where LB is
endemic. Only patients with culture- or PCR-proven disease were enrolled in the study. The antigens of the
ELISAs consisted of sonicated spirochetes, 41-kDa flagellin, and recombinant P39 protein of
B. burgdorferi.
Fifteen patients had strongly or moderately positive results in the serological assay(s), 19 patients had only
weakly positive or borderline antibody levels, and the remaining 7 patients were seronegative by ELISA. The
sensitivities of the ELISAs were 78.0% with sonicate antigen, 41.5% with 41-kDa flagellin, and 14.6% with P39
protein. The specificities of the tests were 89.2, 86.5, and 94.6%, respectively. The sonicate antigen ELISA seems
to be an effective screening method. These results show that antibodies to
B. burgdorferi
may be present in low
levels or even absent in patients with culture- or PCR-proven late LB. Therefore, in addition to serological
testing, the use of PCR and cultivation is recommended in the diagnosis of LB.
Borrelia burgdorferi
is difficult to isolate from body fluids,
because the numbers of spirochetes are low and bactericidal
factors may suppress their growth (1, 20, 34). PCR is extremely
sensitive, detecting even dead spirochetes (25). However, suit-
able body fluids or tissue biopsy specimens are not always
available for PCR or culture. Therefore, serological tests re-
main important screening methods in the diagnosis of Lyme
borreliosis (LB) (5, 17).
B. burgdorferi
is an antigenically complex microorganism
with epitopes common to other microbes (18) and host tissue
components (31). On the other hand, antibody responses to
B.
burgdorferi
can be weak, delayed, or even absent during differ-
ent stages of the disease (3, 4, 7, 20, 34, 38). The 41-kDa
flagellin has shown promise in the diagnosis of early and also
late stages of LB (9, 11–13, 34). Furthermore, recent reports
have suggested that antibodies to the 39-kDa antigen of
B.
burgdorferi
may be important markers for LB (8, 32, 33).
In a recent study, Mitchell et al. compared results of immu-
noserologic assays for patients with culture-positive erythema
migrans (EM) and found that an immunoglobulin M (IgM)
indirect fluorescent-antibody assay detected antibodies to
B.
burgdorferi
in 78% of cases, while other assays tested were
substantially less sensitive (21). The diagnostic sensitivity of
serological tests is considered to be better in later stages of LB
than in early disease. However, several reports about seroneg-
ative patients with definite late LB have been published (6, 7,
10, 16, 24, 26, 29). In the present study, we concentrated on
testing the sensitivities of three enzyme-linked immunosorbent
assays (ELISAs) in late LB. Only PCR- or culture-positive
patients from a larger group of Lyme disease patients were
included. This enabled us to measure antibody levels in pa-
tients who have live spirochetes or borrelia DNA in their
* Corresponding author. Mailing address: Department of Medical
Microbiology, Turku University, Kiinamyllynkatu 13, FIN-20520
Turku, Finland. Fax: 358-21-2330008.
2260
bodies and to avoid patients with post-Lyme syndrome and
patients with old serological scars months or years after infec-
tion.
(Condensed parts of this paper were presented at the 33rd
Interscience Conference on Antimicrobial Agents and Chemo-
therapy, New Orleans, La., 17 to 20 October 1993.)
MATERIALS AND METHODS
Patients.
The study included 41 patients with late LB from southwestern
Finland and 37 healthy controls from the same region. Table 1 shows the
demographic data for the patients and controls. All subjects were living in an
area where LB is endemic. Thirty-four of the patients were seen by one of the
authors (J. Oksi) and seven were seen by other clinicians at Turku University
Hospital in 1991 to 1992. The diagnosis of LB was based on clinical symptoms
and positive culture and/or PCR. Past histories and clinical manifestations (Table
1) indicated that 26.8% of the patients recalled a tick bite(s), and 31.7% had a
history of EM. Two patients had developed EM around the area of a fly bite. All
patients had been suffering from LB symptoms for more than 3 months, 78.0%
had had LB symptoms for more than 6 months, 53.7% had had LB symptoms for
more than 1 year, and many patients had had symptoms for several years. The
clinical criteria for LB in our patients follow the case definition for Lyme disease
developed by the Centers for Disease Control and Prevention (2, 27). Table 1
shows the manifestations and their frequencies. Detailed case reports for two of
these patients have been previously published (23, 35).
All patients were positive by culture and/or PCR (12 positive by culture, 39
positive by PCR, and 10 positive by both tests). The numbers of specimens
positive by culture and/or PCR were as follows: peripheral blood, 14; serum, 15;
cerebrospinal fluid, 11; aspirates, 5 (including 4 synovial fluid samples and 1 bone
marrow aspirate); and biopsy material, 6. For six patients, PCR or culture was
positive for two or more body fluid or biopsy specimens. Plasma or serum
samples were positive by culture or PCR for 28 patients (68.3%).
The control material consisted of 37 serum specimens. Of these, 30 were
obtained from healthy blood donors and 7 were obtained from patients with
suspected allergy but with negative radio-allergo-sorbent tests.
ELISAs.
IgM and IgG antibodies against whole-cell sonicated
B. burgdorferi
were measured by an in-house ELISA (SA-ELISA) (36).
B. burgdorferi
B31
(ATCC 35210; high passage) was grown in BSK-II medium, harvested by cen-
trifugation (10,000
g
for 30 min), washed with 5 mM MgCl
2
in phosphate-
buffered saline (PBS), and sonicated in an ice bath four times for 30 s at 30 W
(Sonifier cell disrupter model B15; Branson Sonic Co. Danbury, Conn.). The
protein concentration of the sonicate was adjusted to 20 g/ml in 0.05 M PBS for
attachment to microwells. All steps of the SA-ELISA, including coating of the
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V
OL
. 33, 1995
TABLE 1. Demographic data and clinical symptoms of patients
with culture- or PCR-proven late LB and control subjects
Value for group
Characteristic
Patients
(n 41)
Controls
(n 37)
ANTIBODIES IN LATE LYME DISEASE
2261
Men/women
Mean age, yr (range)
Tick bite (%)
History of EM (%)
Tick bite and/or EM (%)
Other manifestations (%)
Skin, other than EM
a
Musculoskeletal
b
Neurological
c
Cardiac
d
Ocular
e
Recurrent fever episodes
Hepatitis
Other symptoms or findings
f
a
19/22
37.6 (4–76)
11 (26.8)
13 (31.7)
17 (41.5)
10 (24.3)
31 (75.6)
24 (58.5)
6 (14.6)
5 (12.2)
19 (46.3)
2 (4.9)
5 (12.2)
12/25
32.2 (1–59)
Cultivation.
The specimens (e.g., skin biopsy specimens, cerebrospinal fluids,
or blood or serum samples) were inoculated into tubes containing BSK-II me-
dium and incubated at 30 C. The tubes were examined macroscopically twice a
week and passaged once a week for at least 2 months. Dark-field microscopy was
carried out if the color of the culture medium indicated growth. The final
identification of cultured spirochetes was based on PCR.
Extraction of DNA for PCR.
One milliliter of sample (plasma, serum, cere-
brospinal fluid, or synovial fluid) was centrifuged (Eppendorf Microfuge, 13,000
rpm, 10 min), 800 l of supernatant was removed, and the remaining 200 l was
mixed with 300 l of sodium dodecyl sulfate (SDS) solution (0.1 M NaOH, 2 M
NaCl, and 0.5% SDS). After incubation at 95 C for 15 min, 200 l of 0.1 M
Tris-HCl (pH 8) was added. After SDS treatments, DNA was extracted with
phenol-chloroform, precipitated with ethanol, and finally dissolved in water.
PCR.
A 5- l volume of extracted DNA was added to the reaction tube. Our
target sequence for the PCR was the
fla
gene. The PCR was run in two steps, first
with external primers prB31/41-4 and prB31/41-5 (37), resulting in a 730-bp PCR
product, and then with nested primers WK1 and WK2 (14), resulting in a 290-bp
fragment. Each PCR run included a positive control containing DNA extracted
from reference strain B31 of
B. burgdorferi
(ATCC 35210). Furthermore, every
fifth tube of each run was used as a negative control subjected to all sample
treatment procedures. The PCR products were detected by gel electrophoresis
on a 1.5% agarose gel with ethidium bromide staining.
Numbers of patients (n) with the indicated symptoms: panniculitis, 3; unspe-
cific dermatitis, 1; vasculitis, 2; secondary EM, 2; and chronic urticaria, 2.
b
n
for the following symptoms: arthritis, 17; arthralgia, 9; myositis, 4; myalgia,
4; multiple-site osteomyelitis, 1; tendinitis, 3; and fibromyalgia, 1.
c
n
for the following symptoms: meningitis, 2; myelitis, 1; radiculitis, 1; pares-
thesia, 3; encephalitis, 2; dizziness, 7; memory impairment or encephalopathy, 7;
epilepsy, 1; multiple cerebral infarcts, 1; transient hemiparesis, 1; hemisyndrome,
1; ataxia, 2; cephalalgia, 6; involvement of III, VI, or VII cranial nerve, 6,
including 1 case of bilateral facial palsy; dementia, 2; and brain abscess, 1.
d
n
for the following symptoms: transient third-degree atrioventricular block,
1; myocarditis, 2; pancarditis, 1; endocarditis, 1; and cardiomyopathy, 1.
e
n
for the following symptoms: iritis, 3; chorioretinitis, 1; (kerato)conjuncti-
vitis, 2; and chronic photophobia, 1.
f
n
for the following symptoms: lymphadenopathy, 1; and prolonged fatigue, 4.
RESULTS
In determining the sensitivities and specificities of the three
ELISAs, three different levels of positivity (weakly positive,
positive, and strongly positive) were considered positive re-
sults. Table 2 shows ELISA results with three different anti-
gens for culture-positive patients, only-PCR-positive patients,
and control subjects. For all 41 patients presenting with symp-
toms compatible with late LB, the sensitivity of SA-ELISA was
78.0%, that of FL-ELISA was 41.5%, and that of P39-ELISA
was 14.6% (Table 2). Both FL-ELISA and P39-ELISA de-
tected only one positive specimen which had gone undetected
by the other two tests. In the analysis of 37 healthy controls, the
test specificities were 89.2% for SA-ELISA, 86.5% for FL-
ELISA, and 94.6% for P39-ELISA (Table 2). The respective
positive predictive values were 88.9, 77.3, and 75.0%. The
respective negative predictive values were 78.6, 57.1, and
50.0%.
The sensitivities achieved with different combinations of the
three tests were as follows: SA-ELISA plus FL-ELISA, 80.5%;
SA-ELISA plus P39-ELISA, 80.5%; FL-ELISA plus P39-
ELISA, 51.2%; and SA-ELISA plus FL-ELISA plus P39-
ELISA, 82.9%. Thus, for 34 of the 41 patients, diagnosis could
be confirmed by serological tests.
Although all patients suffered from late LB, 10 (24.4%) and
8 (19.5%) of the patients had only IgM antibodies as measured
by SA-ELISA and FL-ELISA, respectively. The corresponding
figures for IgG antibodies were 17 (41.5%) and 4 (9.8%). Both
IgM and IgG antibodies were detected in 5 patients (12.2%) by
both SA-ELISA and FL-ELISA. The levels of antibodies
against
B. burgdorferi
in patients and controls are shown in
Fig. 1.
solid phase with antigen, were carried out automatically by an Auto-EIA II
instrument (Labsystems, Helsinki, Finland). Serum samples were tested at a
dilution of 1:100. Alkaline phosphatase-conjugated swine anti-human IgG or
IgM antibodies (Orion Diagnostica, Espoo, Finland) and
p-nitrophenylphos-
phate substrate were used for detection of bound antibodies. A standard curve
was drawn by using a strongly positive patient serum specimen as a standard. The
results were expressed as relative ELISA units. Seropositivity was determined by
comparing antibody results for test serum samples with those for 110 healthy
controls. The cutoff values were the mean 2 standard deviations (SD) of the
controls for weakly positive results, the mean 4 SD for positive results, and the
mean 6 SD for strongly positive results.
Commercial kits were used for measurement of antibodies against 41-kDa
flagellin of
B. burgdorferi
(FL-ELISA) (Lyme borreliosis ELISA kit, second
generation; DAKO A/S, Glostrup, Denmark) (11) and against recombinant P39
protein (P39-ELISA) (ImmunoWeLL Recombinant P39 [Lyme] test; General
Biometrics, Inc., San Diego, Calif.) (33). FL-ELISA measures both IgM and IgG
antibodies, whereas P39-ELISA does not differentiate between immunoglobulin
isotypes. Interpretation of the results obtained by the kits was done as instructed
by the manufacturers. Borderline or positive results with FL-ELISA were re-
corded as weakly positive, strongly positive ones were considered positive, and
very strongly positive ones were classified as strongly positive. Results with
P39-ELISA were classified as negative, weakly positive, or positive.
TABLE 2. Results of SA-ELISA, FL-ELISA, and P39-ELISA for patients with culture- or PCR-proven late LB
No. of results
Group
SA-ELISA
(IgM, IgG, or both)
Positive
Negative
FL-ELISA
(IgM, IgG, or both)
Positive
Negative
Positive
P39-ELISA
Negative
Patients
Culture positive (n 12)
Only PCR positive (n 29)
Total
Controls (n
37)
11
21
32
4
1
8
9
33
6
11
17
5
6
18
24
32
5
1
6
2
7
28
35
35
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1533935_0018.png
2262
OKSI ET AL.
J. C
LIN
. M
ICROBIOL
.
FIG. 1. Levels of IgM and IgG antibodies in patients with PCR- or culture-proven late LB (squares) and control subjects (circles) as measured by SA-ELISA and
FL-ELISA. Closed squares and closed circles represent sera which had borderline or positive results by P39-ELISA.
DISCUSSION
This study shows that patients with late LB who have live
spirochetes or borrelial DNA in their body fluids may have low
or negative levels of borrelial antibodies in their sera. This
emphasizes that an efficient diagnosis of LB has to be based on
culture, PCR, and serology, because even the combined sensi-
tivity of the three ELISA modifications tested was only 83%. If
serological means alone are used, a considerable proportion of
LB patients may not be diagnosed and treated. It might be
possible that LB patients with weak or no humoral immune
responses against the spirochete develop even more serious
disease than the patients with strong antibody responses (15).
Our results also show that plasma and serum samples are
suitable specimen types for the detection of circulating spiro-
chetes or their structures also in late disease.
In this study, multiple organs were frequently involved. Re-
current fever episodes were seen in nearly half of the patients,
neurological symptoms were seen in more than half of the
patients, and musculoskeletal manifestations were seen in
three-fourths of the patients. Moreover, most of these mani-
festations were long-lived. In spite of this, several patients were
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ANTIBODIES IN LATE LYME DISEASE
2263
seronegative and most seropositive patients had only weakly
positive antibody levels.
Several antigenic components of
B. burgdorferi
have been
tested in an attempt to improve the diagnostic efficacy of se-
rological tests. We tested one purified borrelia antigen, 41-kDa
flagellin, and one recombinant borrelia antigen, P39 protein, in
serological diagnosis of late LB. These antigen types have
recently shown promise in the diagnosis of early and late stages
of LB (8, 9, 11, 12, 22, 32–34). Compared with the results of
these earlier studies, our results are disappointing. In our
study, SA-ELISA was far more sensitive than FL-ELISA. The
sensitivity of P39-ELISA was 14.6%, which is substantially
lower than those in one earlier study with this commercial kit
giving sensitivities of 8% for early and 39% for late disease
(28). However, in the culture-positive subgroup of our pa-
tients, 5 of 12 (41.7%) had borderline or positive results as
determined by P39-ELISA. Our results with P39-ELISA were
not due to any technical errors, because the positive controls of
the kits repeatedly gave absorbance values within the limits
indicated by the manufacturer. Furthermore, the specificity
advantage obtained by the components was limited.
One reason for the disappointingly low sensitivity obtained
by the two separate component antigen tests may be the dif-
ference in expression of antigens by various strains and, pos-
sibly even more importantly, the differences in the host ability
to develop an immune response to a given antigen (i.e., the
immunogenicity of the antigen). It is also evident that tests
relying upon single antigenic components are far more sensi-
tive to the differences in host immune responses to those an-
tigens than tests using crude antigen extracts. Crude extracts
always contain such a broad spectrum of antigens that differ-
ences in host immune reactions to some antigens do not affect
test sensitivity. In fact, this hypothesis is supported by the study
of Magnarelli et al. in which roughly similar antibody titers
were obtained by a whole-cell ELISA using different
B. burg-
dorferi
strains (19). However, the present study design may give
a too pessimistic estimation of the sensitivity of the serological
techniques tested, because we excluded patients with diagnoses
based only on serological laboratory evidence.
Low or even undetectable antibody levels in late LB may be
caused by formation of circulating immune complexes (29).
Immune complexes are formed especially in the presence of
excess antigen. Furthermore, circulating immune complexes
may be a sign of active disease. Schutzer and coworkers dem-
onstrated complexed antibody against
B. burgdorferi
in almost
all of their patients with active symptoms of LB and its absence
in a group of recovering patients (30). We did not assess
circulating immune complexes or antibodies after dissociation
of immune complexes. However, 68% of our patients had
borrelia DNA or cultivatable spirochetes in the serum or
plasma. This shows that borreliae or their structures are fre-
quently present in the circulation of patients with late LB,
permitting complex formation. It is possible that for patients
without borrelia DNA in their circulation, the sensitivity of
serology may be greater than observed in our study.
Elevated IgM antibody levels without a concomitant rise in
IgG levels are generally considered a sign of a primary immune
response. Our results provide further support for earlier stud-
ies showing that antibody responses against
B. burgdorferi
can
be restricted to IgM even in late LB (23, 35). The persistence
of the IgM response can be explained either by a disability to
switch antibody production from IgM to IgG or by a continu-
ous appearance of new antigenic epitopes on the spirochetes
during the infection (4). The isolated occurrence of IgM with-
out the presence of IgG was also detected by FL-ELISA. This
indicates that host factors are more important than microbial
factors for IgM persistence, because there are no data showing
antigenic variation in flagellin, whereas the variation of the
whole antigenic mosaic of
B. burgdorferi
can be abundant.
We conclude that antibodies to
B. burgdorferi
often are
present in only low levels or are even absent in culture- or
PCR-positive patients who have been suffering for years from
symptoms compatible with LB. Therefore, in addition to sero-
logical testing, the use of PCR and cultivation in the diagnosis
of LB is recommended. Furthermore, the use of the two kits
using component antigens tested in this study does not solve
the problems of serological diagnosis of LB, at least in north-
ern Europe. SA-ELISA seems to be an effective method for
screening purposes, especially in late disease.
ACKNOWLEDGMENTS
This study was supported by the Emil Aaltonen Foundation, the
Maud Kuistila Foundation, the Orion Corporation Research Founda-
tion, the Turku University Society, and the Turku University Founda-
tion.
The language of the manuscript was revised by Simo Merne.
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Clin Rheumatol (2002) 21:330–334
ß
2002 Clinical Rheumatology
Clinical
Rheumatology
Case Report
Seronegative Lyme Arthritis caused by
Borrelia garinii
H. Dejmkova
1
, D. Hulınska
2
D. Tegzova
1
, K. Pavelka
1
, J. Gatterova
1
and P. Vavr´k
3
´
´
´
´
ˇı
Institute of Rheumatology, Prague;
2
National Institute of Public Health, Reference Laboratory on Borreliosis and Electron
Microscopy, Prague;
3
I Orthopaedic clinic, First Medical Faculty, Charles University, Prague, Czech Republic
1
Abstract:
A case of a female patient suffering from
Lyme arthritis (LA) without elevated antibody levels to
Borrelia burgdorferi
sensu lato is reported. Seronegative
Lyme arthritis was diagnosed based on the classic
clinical manifestations and DNA-detected
Borrelia
garinii
in blood and synovial fluid of the patient, after
all other possible causes of the disease had been ruled
out. The disease was resistant to the first treatment with
antibacterial agents. Six months after the therapy,
arthritis still persisted and DNA of
Borrelia garinii
was repeatedly detected in the synovial fluid and the
tissue of the patient. At the same time, antigens or parts
of spirochaetes were detected by electron microscopy in
the synovial fluid, the tissue and the blood of the patient.
The patient was then repeatedly treated by antibiotics
and synovectomy has been performed.
Keywords:
Borrelia garinii;
Lyme arthritis; Therapeutic
failure
Introduction
Lyme borreliosis (LB) is a multisystemic disease caused
by a Gram-negative spirochaete,
Borrelia burgdorferi
sensu lato (B.
burgdorferi).
The infection is usually
transmitted through an infected tick bite [1]. The first
common sign of the disease, so-called erythema migrans,
is localised on the skin. After several days or even
weeks, dissemination of the infection to various organs
is observed. Several months or years later, the organs in
which the infection had been previously localised, show
Correspondence and offprint requests to:
Professor K. Pavelka,
Institute of Rheumatology, Na Slupi 4, 12850 Prague 2, Czech
Republic.
a chronic involvement. The nervous system and joints
are affected most often. Rarely, the involvement of other
organs has also been reported [2].
The development of species-specific DNA amplifica-
tion methods has made it possible to differentiate eight
genotypes of
B. burgdorferi
sensu lato. It has been
suggested so far that LB can be caused by
B. burgdorferi
sensu stricto,
Borrelia afzelii, Borrelia garinii
and
Borrelia valaisiana.
In other species, no association,
with LB has been proven [3–5]. It is not clear whether
individual species have a special affinity for different
organs [6,7]. It is commonly stated that
Borrelia afzelii
is responsible for cutaneous manifestations,
Borrelia
garinii
is often associated with neurological affections,
and
B. burgdorferi
sensu stricto affects joints [8]. The
results of the majority of European works studying the
representation of individual species in LB patients with
joint involvement suggest that
B. burgdorferi
sensu
stricto prevails in these patients [9–11].
In the initial phase LA is associated with migrating
arthralgia and/or arthritis. In the course of the disease, an
intermittent monoarthritis or oligoarthritis may develop
and progress into a chronic arthritis. The classic clinical
picture of LA is represented by monoarthritis of the
knee, which occurs in more than 85% of patients. The
diagnosis of LA is made based on classic-clinical
manifestations, endemic area exposure and on laboratory
findings of elevated anti-Borrelia IgG antibodies in the
patient’s serum [12]. The prognosis of patients with LA
is usually good, although in 10%–20% arthritis is
resistant to treatment with antibiotics [13–15].
We present a case of a female patient diagnosed with a
seronegative LA. The diagnosis was based on a positive
epidemiological history, typical clinical manifestations
and repeated evidence of
Borrelia garinii
DNA in the
blood and the synovial fluid of the patient. The first
treatment with antibiotics proved unsuccessful. The
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Seronegative Lyme Arthritis caused by
Borrelia garinii
331
failure of the treatment could be established with regard
to the clinical state of the patient, as well as the presence
of
Borrelia garinii
in the synovial fluid and synovial
tissue of the patient 6 months after therapy. At the same
time spirochaetes were found in the patient’s synovial
fluid, synovial tissue and blood.
Case Report
A 27-year-old agricultural engineer presented with
recurrent attacks of oligoarthritis limited in time, with
a dominant affection of the knee joints, and was admitted
to the Institute of Rheumatology, Prague, to establish the
diagnosis.
Her past history indicated that she worked in the
country. Contact with ticks cannot be ruled out, as the
patient had been repeatedly exposed but was not aware
either of a tick bite or of erythema migrans. She reported
that she repeatedly suffered from gynecological and
urological infections which were treated with antimicro-
bial agents. No medical documentation from this period
is available and information about causative agents from
that time is also missing. No other serious conditions
were reported.
The present disease began in 1992 and presented with
painful swellings of the right wrist and both knee joints.
The patient was treated with amoxycillin and non-
steroidal anti-inflammatory drugs on a short-term basis
and her condition gradually improved. In 1993, arthritis
of the knee joints recurred and lasted 2 months. Since
then, until the year 2000, bilateral gonitis recurred
several times; in between the attacks the patients had no
complaints. The patient had been treated by her
rheumatologist and orthopaedist with non-steroidal
anti-inflammatory drugs, and repeatedly with intra-
articular corticosteroids. Sulfasalazine was also adminis-
tered for 9 months. However, the condition did not react
to the treatment. The medical documentation from that
period is not available and we presume that the therapy
with sulfasalazine was introduced as a therapeutic trial
which was ineffective.
In July 2000 the patient was admitted to our clinic. At
that time she was experiencing a disturbing pressure in
her knee joints, without any other complaints. physical
examination showed an effusion in the knee joints, as
well as mild warmth and painful sensations on move-
ment to border positions. Both popliteal areas showed a
swelling indicative of Baker’s cysts. Other findings were
normal. Laboratory data showed normal values of acute-
phase reactants, and the blood picture and clinical
chemistry were normal. Serum examination detecting
antibody response to arthritogenic agents, anti
Yersinia
anti-Salmonella and anti-Chlamydia
trachomatis
anti-
bodies was negative and no
Chlamydia trachomatis
on
other pathogens were found in urine or cervical smears.
Immunological tests were normal, apart from one finding
of a slightly increased titre of antinuclear antibodies,
which was not confirmed when repeated. HLA-B27
antigen was not detected. HLA-DR4 and HLA-DR2,
which are assumed to be associated with resistance to
treatment in LA, were not detected [13]. The patient has
HLA-DR 01 and HLA-DR 08 alleles.
ELISA did not show an elevation in serum levels of
antibodies to
B. burgdorferi.
Immunoblot analysis only
confirmed a borderline reactivity against
B. garinii
(Western blot IgM and IgG anti-B.
garinii,
Biowestern
dg.and Immunoblot
B. garinii
IgG, Euroimmun, Ltd) in
the IgG class (P83+, P60+, P56+, Osp C+, P14+).
Cytology of the synovial fluid revealed signs of mild
inflammation (leukocytes 1.3
6
10
9
/l, erythrocytes 0.1
6
10
9
/l, leukocyte count showed 2% neutrophil
segments, 60% lymphocytes and 38% monocytes).
Cultivation of the synovial fluid for the detection of a
specific and non-specific infection proved negative. The
examination of anti-Borrelia antibodies using an
immunoblot analysis (Biowestern) was negative in the
right knee joint and borderline IgG reactivity was
detected in the left knee joint (83+, Osp A+). With
regard to the clinical manifestations indicative of LA, we
proposed a polymerase chain reaction (PCR) examina-
tion of the DNA of
B. burgdorferi
sensu lato with
species-specific primers to amplify the Osp As of strains
[16,17] in the patient’s synovial fluid and blood. This
examination proved positive both in the synovial fluid
and the blood, and indicated the presence of DNA of
Borrelia garinii
Osp A-type 5. PCR fragments were
purified and directly sequenced by the dideoxychain
termination procedure using CEQ 2000 Dye terminator
Cycle (Hulınska, unpublished).
´ ´
X-rays of the knees, hands, feet and sacroiliac joints
were normal. Ultrasound examination of the knee joints
showed hypertrophy of synovial tissue, synovial fluid
excess and bilateral Baker’s cyst. The findings were
more pronounced in the left knee. Findings from MRI
examination of the knee joints correlated with the
ultrasound examination. Electrocardiography and echo-
cardiography findings were normal.
With regard to the above-mentioned findings, the
diagnosis of LA was made and intravenous ceftriaxon
administered for 14 days in a daily dose of 2 g. After the
treatment, a gradual improvement of the condition in the
right knee joint was observed, but the left-sided gonitis
persisted.
In January 2001 the patient was again hospitalised.
Arthritis of the left knee persisted and the condition was
confirmed by ultrasound examination.
Borrelia garinii
Osp A-type 5 was again detected in the synovial fluid.
Also, in the blood and synovial fluid antigens and
altered parts of spirochaetes were detected using mono-
clonal antibody in immunosorbent method (ISEM) and
negative staining on electron microscopy (Figs 1 and 2).
Treatment with intravenous ceftriaxon was initiated,
followed by oral administration of doxycyclin in standard
dosage. Synovectomy was indicated for the left knee
joint. Histology of the synovial tissue showed a non-
specific synovitis. In the synovial tissue sections,
spirochaetes as partly helical or convoluted forms were
found, using electron microscopy (Figs 3 and 4). PCR
examination detected DNA of
Borrelia garinii
in the
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332
H. Dejmkova et al.
´
Fig. 1.
Immunocytochemistry of human blood sample with
monoclonal antibody anti-Osp A
Borrelia garinii
– anti-mouse,
labelled with gold (Janssen GoAM/IgGAu). Expression of antigens on
surface of alterated spirochaetes. Particle size 10 nm. (Negative
staining with 1% phosphotungstic acid, magnification 82.0006.)
Fig. 4.
Synovial tissue. Tangential section of apical site of synovial
membrane shows central and apical parts of spirochaetes. Alteration
of synovial cell and partial lysis of reticular fibrils but no borrelial
cells are seen. (UaLc, magnification 72.0006.)
synovial tissue with different set of primers instead of the
target Osp A gene (Hulınska, unpublished).
´ ´
Six months after the second treatment, the patient’s
condition showed no signs of clinical or laboratory
recurrence of the disease.
Discussion
We present the case of a female patient with LA that
showed certain interesting features. The first surprising
finding was a long-term absence of antibody reactivity
against
B. burgdorferi
sensu lato antigens in the patient’s
serum and synovial fluid that for a long time made it
difficult to diagnose the condition. It is anticipated that
antibody response to the
Borrelia
antigens plays an
important role in the pathogenesis of LA [12]. However,
a number of studies suggest that in patients insufficiently
treated with antibacterial agents a seronegative LA
develops. This therapy may suppress the immune
response of the affected body, and the infection itself
may persist [18]. This may explain the seronegativity of
our patient, who had been repeatedly treated with
antibiotics for short periods of time owing to the
urogenital infections. Because of the patient’s exposure
and the clinical finding of arthritis of the knee joints with
effusions causing popliteal cysts, the diagnosis of LA
could not have been omitted in differentials, leading to
the use of a direct method capable of detecing the
Borrelia
infection. The results of the examination were
positive in both the synovial fluid and the blood of the
patient.
It is also interesting to note that arthritis in this patient
has been associated with
Borrelia garinii,
as there has
been only rare evidence of this causality in connection
with arthritis in Europe [7,16].
The first treatment with antibiotics proved unsuccess-
ful. Insufficient response to the therapy may have a
Fig. 2.
Altered spirochaete coiled inside granular material, which
could be debris of a human cell or rest of cyst in synovial fluid sample.
(1% phosphotungstic acid, magnification 25.2006.)
Fig. 3.
Electron microscopy of synovial tissue samples stained with
uranyl acetate and lead citrate (UaLc). Tissues were fixed in 6%
glutaraldehyde; after fixation the tissues were treated with 1% OsO4.
Cross-section of convoluted spirochaete (Magnification 58.0006.)
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Seronegative Lyme Arthritis caused by
Borrelia garinii
333
4. Canina MM, Nato F, du Merle L, Mazie JC, Baranton G, Postic
D. Monoclonal antibodies for identification of
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sp.
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Borreliosis. Scand J Infect Dis 1993;25:441–48.
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phenotypic analysis of
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sp.nov. (Borrelia
genomic groups VS 116 and M19). Int J Syst Bacteriol
1997;47:926–32.
6. Van Dam AP, Kuiper H, Vos K et al. Different genospecies of
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are associated with distinct clinical
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Ann Reum Dis
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8. Mateicka F, Kmety E. Lyme borreliosis: open questions and
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problems at the turn of the millenium. Bratisl.lek. Listy
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Borrelia burgdorferi
sensu stricto by reverse line blot in the joints
of Dutch patients with Lyme arthritis. Arthritis Rheum
1999;42:1473–80.
10. Priem S, Lunemann JD, Zarmas S et al. Rapid subtyping of
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species in bacterial cultures and clinical
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(abstract) Arthritis Rheum 1999;42(Suppl 9):338.
11. Jaulhac B, Heller R, Limbach FX et al. Direct molecular typing of
Borrelia burgdorferi
sensu lato species in synovial samples from
patients with Lyme arthritis. J Clin Microbiol 2000;38:1895–900.
12. Steere AC. Musculoskeletal manifestations of Lyme disease. Am
J Med 1995;98(Supl 4A):4A–44S.
13. Steere AC, Dwyer E, Winchester R. Association of chronic Lyme
arthritis with HLA-DR4 and HLA-DR2 alleles. N Engl J Med
1990;323:219–223.
14. Kalish RA, Leong JM, Steere AC. association or treatment-
resistent chronic lyme arthritis with HLA-DR4 and antibody
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Immun 1993;61:2774–9.
15. Huppertz HI, Karch H, Suschke HJ et al. Lyme arthritis in
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1995;38:361–8.
16. Hulınska D, Votypka J, Valesova M, Pesistence of
Borrelia
´ ´
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garrinii
and
Borrelia afzelii
in patients with Lyme arthritis.
Zentralbl Bakteriol 1999;289,3:301–18.
17. Vasiliu V, Herzer P, Rossler D, Lehnert G, Wilske B.
Heterogenity of
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sensu lato demonstrated
by an OspA-type-specific PCR in synovial fluid from patients
with Lyme arthritis. Med Microbiol Immunol 1998;197:97–102.
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Golingt MG. Dissociation of specific T- and B lymphocyte
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N Engl J Med 1989;819:1441–
6.
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Status of
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and effects of corticosteroids: An experimental study. J Infect Dis
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Intracellular persistence of
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in human
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to normal, mobile spirochetes. Infection
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Persistence of
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in experimentally infected
dogs after antibiotic treatment. J Clin Microbiol 1997;35:111—16.
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Borrelia
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treatment. J Infect Dis 1994;170:1312–16.
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AC. Detection of
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number of reasons, one of which may be a repeated
intra-articular application of corticosteroids into the
knees in the course of the treatment. Another possibility
may be the choice of antibiotics. The treatment with
ceftriaxon, which possesses mostly extracellular activity,
may have led to a temporary eradication of the infection
while intracellular infection persisted. Experimental
studies suggest that
B. burgdorferi
sensu lato may
penetrate into cells, including synovial cells. This
mechanism protects
Borrelia
against the effect of
prevalently extracellular antibiotics [19,20]. Also, a
number of other factors may be responsible for the
failure of the antibiotic treatment. Experimental studies
in vitro document the potential of
B. burgdorferi
sensu
lato to transform itself into a metabolically inactive
cystic form under unfavourable conditions. Antibiotic
therapy can represent such an unfavorable condition, and
it is anticipated that under the influence of antibiotics
B.
burgdorferi
sensu lato is able to transform into a cystic
forms. However, active bacterial metabolism is a
prerequisite for an effective antimicrobial treatment.
The above-mentioned mechanism is probably used by
some spirochaetes to avoid the effect of antibiotics.
These metabolically inactive forms are capable of
transforming into a metabolically active form under
certain favourable conditions, and this may be respon-
sible for treatment-resistant conditions in some patients
with LB [21]. Animal studies also document the
possibility of survival of
Borrelia
infection after
antimicrobial treatment. In experimentally infected
animals the antibiotic therapy suppresses clinical
manifestations; nevertheless, the DNA of
B. burgdorferi
sensu lato can be isolated from the tissues of some
animals several months after treatment [19,22,23]. Also,
in human there is evidence that in a number of patients
with chronic arthritis the DNA of
B. burgdorferi
sensu
lato can be detected in joints despite repeated antibiotic
therapy [24,16]. However, this does not necessarily
mean that
Borrelia
is still alive. The vitality of the strain
can only be detected by cultivation, but this method has
an extremely low sensitivity. It is also very difficult to
detect an intra-articular infection caused by
B. burgdor-
feri
by means of electron microscopy [25,26,16]. There
exist only few reports documenting success in detecting
the presence of spirochaetes in the synovial fluid of a
patient using electron microscopy [26,27].
The presented case documents the possible persistence
of
B. burgdorferi
sensu lato (Borrelia
garinii
in this
case) in joints after ceftriaxon treatment.
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Received for publication 28 August 2001
Accepted in revised form 1 January 2002
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hemoglobin levels, in our view, is the clin-
ical significance of any toxicity. The ab-
normalities were reversible. In the patients
for whom 4-week values were available,
median creatinine and hemoglobin values
were the same for patients in both trial
arms. Of the 5 patients in the high-dose
arm who discontinued study drugs early,
4 were alive and well and receiving anti-
retroviral therapy at 6 months, and 1 died
of culture-proven tuberculosis.
For settings in developed countries, the
current Infectious Diseases Society of
America guidelines [3] for treatment of
HIV-associated cryptococcal meningitis
suggest a dose range for AmB of 0.7–1 mg/
kg per day combined with flucytosine. The
updated guidelines will retain this dose
range (J. R. Perfect, personal communi-
cation). Our study [2] provides the first
comparative data for making a choice of
dose within this range. At the higher dose,
clinicians will know that they can achieve
more-rapid clearance of infection. In ad-
dition, complementary data on the tox-
icity of AmB at 1 mg/kg per day for a
larger number of patients will be available
from a trial in Vietnam in which all pa-
tients received the higher dose [4].
In the many settings in which flucyto-
sine is not yet generally available—and re-
source limitations may make a full 2 weeks
of induction treatment difficult—toxicity
issues may be reduced, and the impor-
tance of more-rapid initial clearance, in
the absence of flucytosine, is increased. In
such settings, our study [2] and an earlier
study [5] provide evidence to support the
use of the 1 mg/kg dose of AmB. Thus,
South African guidelines advocate AmB at
1 mg/kg per day for 7–14 days [6].
Routine, frequent monitoring and sa-
line fluid loading during administration of
AmB, at any dose, are essential. In Kam-
pala, Uganda, with use of AmB at 0.7 mg/
kg per day in 2 observational study cohorts
in 2001 and 2006, 2-week mortality was
reduced from 42% in 2001 to 20% in
2006, a reduction that may have been as-
sociated, at least in part, with more-fre-
quent monitoring (3 times weekly vs. once
weekly) and routine fluid loading in the
later cohort [7]. Indeed, in settings in
which frequent routine monitoring and
transfusion, if occasionally needed, are not
available, it is possible that an optimized
oral treatment regimen could give results
comparable to the results of treatment
with AmB. In response to specific ques-
tions, none of our patients were excluded
on the basis of previous adverse reactions
to AmB, saline preloading was given for
all doses, and AmB infusions were over
4 h.
Acknowledgments
Potential conflicts of interest.
All authors: no
conflicts.
Tihana Bicanic,
1,4
Robin Wood,
1
Graeme Meintjes,
2,3
Kevin Rebe,
2,3
Annemarie Brouwer,
4,6
Angela Loyse,
1,4
Linda-Gail Bekker,
1
Shabbar Jaffar,
5
and Thomas Harrison
1,4
Desmond Tutu HIV Centre, Institute of Infectious
Disease and Molecular Medicine, and
2
Department
of Medicine, University of Cape Town, and
3
Infectious Diseases Unit, GF Jooste Hospital, Cape
Town, South Africa;
4
Centre for Infection,
Department of Cellular and Molecular Medicine, St.
George’s University of London, and
5
Department of
Epidemiology and Population, London School of
Hygiene and Tropical Medicine, London, United
Kingdom; and
6
Department of Internal Medicine
and Infectious Diseases, University Medical Centre
Nijmegen, The Netherlands
References
1. Pasqualotto AC. Amphotericin B: the higher
the dose, the higher the toxicity. Clin Infect Dis
2008;
47:1110 (in this issue).
2. Bicanic T, Wood R, Meintjes G, et al. High-
dose amphotericin B with flucytosine for the
treatment of cryptococcal meningitis in HIV-
infected patients: a randomized trial. Clin Infect
Dis
2008;
47:123–30.
3. Saag MS, Graybill RJ, Larsen RA, et al., for the
Mycoses Study Group Cryptococcal Subpro-
ject. Practice guidelines for the management of
cryptococcal disease. Clin Infect Dis
2000;
30:
710–8.
4. Current Controlled Trials Web site. Trial
ISRCTN95123928. Available at: http://www
.controlled-trials.com/. Accessed 22 August
2008.
5. Bicanic T, Meintjes G, Wood R, et al. Fungal
burden, early fungicidal activity, and outcome
in cryptococcal meningitis in antiretroviral-
naive or antiretroviral-experienced patients
treated with amphotericin B or fluconazole.
Clin Infect Dis
2007;
45:76–80.
6. McCarthy K, Meintjes G. Arthington-Skaggs B,
1
et al. Guidelines for the prevention, diagnosis
and management of cryptococcal meningitis
and disseminated cryptococcosis in HIV-in-
fected patients. Southern African J HIV Med
2007;
8:25–35.
7. Kambugu AD, Meya DB, Rhein J, et al. Out-
comes of cryptocccal meningitis in Uganda be-
fore and after the availability of highly active
antiretroviral therapy. Clin Infect Dis
2008;
46:
1694–701.
Reprints or correspondence: Dr. Tihana Bicanic, Div. of Infec-
tious Diseases, Dept. of Cellular and Molecular Medicine, St.
George’s Hospital Medical School, Cranmer Terrace, London
SW17 ORE, United Kingdom ([email protected]).
Clinical Infectious Diseases 2008; 47:1110–1
2008 by the Infectious Diseases Society of America. All
rights reserved. 1058-4838/2008/4708-0022$15.00
DOI: 10.1086/592118
Serologic Tests for Lyme
Disease: More Smoke and
Mirrors
To the Editor—The
article by Steere et
al. describing serologic testing for Lyme
disease contains the following conclusion:
“the sensitivity of 2-tier testing in patients
with later manifestations of Lyme disease
was 100%, and the specificity was 99%”
[1, p. 192]. This conclusion is both dis-
ingenuous and misleading.
Steere et al. [1] classified 44 patients as
having disseminated (stage 2) or persistent
(stage 3) infection due to
Borrelia burg-
dorferi,
the spirochetal agent of Lyme dis-
ease. The mandatory inclusion criteria for
these categories were neurologic, cardiac,
or joint involvement and a serologic result
positive for
B. burgdorferi
by ELISA and
Western blot [2]. Thus, by definition, all
patients with disseminated or persistent
Lyme disease were required to have a pos-
itive serologic test result. It is disingenuous
to define a condition by a positive test
result and then state that the test has 100%
sensitivity. The true sensitivity of the 2-
tier test system has been estimated to be
44%–56% when standard commercial
Lyme testing was evaluated in clinical
practice [3–5]. In fact, on the basis of a
recent molecular diagnostic study, the sen-
sitivity of this testing approach may be as
low as 7.5% [6]. Thus, the sensitivity data
presented by Steere et al. [1] is not realistic.
In the study by Steere et al. [1], 14 pa-
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tients were classified as having “post–Lyme
disease symptoms,” with persistent symp-
tomatic manifestations after receiving
“recommended antibiotic therapy” for
Lyme disease. Among these patients, 36%
had serologic evidence of persistent infec-
tion due to
B. burgdorferi,
as defined by
the Centers for Disease Control and Pre-
vention criteria of positive results of ELISA
and IgG Western blot [2]. Recent studies
have revealed that “post–Lyme disease
symptoms” may represent failure of short-
course antibiotic therapy and persistent
infection due to the Lyme spirochete, and
this chronic illness may respond to a
longer duration of antibiotic treatment
[7–11]. Thus, the test results in patients
with “post–Lyme disease symptoms” may
reflect the true sensitivity of 2-tier testing
for persistent Lyme disease, and the 36%
sensitivity reported by Steere et al. [1] is
consistent with the poor results of previ-
ous studies [3–5]. The VlsE C6 peptide
ELISA was not significantly better, with a
test sensitivity of only 43% for patients
with persistent Lyme disease symptoms.
In summary, the sensitivity data pre-
sented by Steere et al. [1] reflect both cir-
cular reasoning in the context of dissem-
inated infection and poor results in the
context of persistent Lyme disease. Better
tests are needed for diagnosis of this elu-
sive tick-borne illness.
Acknowledgments
Potential conflicts of interest.
R.B.S. has
served without compensation on the medical ad-
visory panel for QMedRx. L.J.: no conflicts.
Raphael B. Stricker and Lorraine Johnson
International Lyme and Associated Diseases
Society, Bethesda, Maryland
References
1. Steere AC, McHugh G, Damle N, Sikand VK.
Prospective study of serologic tests for Lyme
disease. Clin Infect Dis
2008;
47:188–95.
2. Centers for Disease Control and Prevention.
Case definitions for public health surveillance.
MMWR Morb Mortal Wkly Rep
1990;
39:
1–43.
3. Stricker RB, Johnson L. Lyme wars: let’s tackle
the testing. BMJ
2007;
335:1008.
4. Tilton RC, Sand MN, Manak M. The Western
immunoblot for Lyme disease: determination
5.
6.
7.
8.
9.
10.
11.
of sensitivity, specificity, and interpretive cri-
teria with use of commercially available per-
formance panels. Clin Infect Dis
1997;
25(Suppl 1):S31–4.
Binnicker MJ, Jespersen DJ, Harring JA, Rol-
lins LO, Bryant SC, Beito EM. Evaluation of
two commercial systems for the automated
processing, reading and interpretation of Lyme
Western blots. J Clin Microbiol
2008;
46:
2216–21.
Santino I, Berlutti F, Pantanella F, Sessa R, del
Piano M. Detection of
Borrelia burgdorferi
sensu lato DNA by PCR in serum of patients
with clinical symptoms of Lyme borreliosis.
FEMS Microbiol Lett
2008;
283:30–5.
Phillips SE, Burrascano JJ, Harris NS, Johnson
L, Smith PV, Stricker RB. Chronic infection
in “post-Lyme borreliosis syndrome.” Int J Ep-
idemiol
2005;
34:1439–40.
Hodzic E, Feng S, Holden K, Freet KJ, Bart-
hold SW. Persistence of
Borrelia burgdorferi
following antibiotic treatment in mice. Anti-
microb Agents Chemother
2008;
52:1728–36.
Stricker RB, Johnson L. Searching for auto-
immunity in “antibiotic refractory” Lyme ar-
thritis. Mol Immunol
2008;
45:3023–4.
Stricker RB. Counterpoint: long-term antibi-
otic therapy improves persistent symptoms as-
sociated with Lyme disease. Clin Infect Dis
2007;
45:149–57.
Fallon BA, Keilp JG, Corbera KM, et al. A
randomized, placebo-controlled trial of re-
peated IV antibiotic therapy for Lyme en-
cephalopathy. Neurology
2008;
70:992–1003.
Reprints or correspondence: Dr. Raphael B. Stricker, 450
Sutter St., Ste. 1504, San Francisco, CA 94108 (rstricker
@usmamed.com).
Clinical Infectious Diseases 2008; 47:1111–2
2008 by the Infectious Diseases Society of America. All
rights reserved. 1058-4838/2008/4708-0023$15.00
DOI: 10.1086/592121
Reply to Stricker and
Johnson
To the Editor—Stricker
and Johnson
[1] maintain that the frequency of sero-
positivity among patients with dissemi-
nated or persistent Lyme disease is lower
than the frequency reported in our pro-
spective study of serologic testing for this
infection. As stated in our article [2], it is
problematic to determine the frequency of
seroreactivity among patients with neu-
rologic, cardiac, or joint manifestations of
Lyme disease, because serologic confir-
mation is a part of the case definition [3].
However, it has not been possible to con-
firm
Borrelia burgdorferi
infection by other
methods, such as culture or PCR, in all
patients with the aforementioned mani-
festations of Lyme disease. Nevertheless,
B. burgdorferi
DNA can be detected by
PCR of joint fluid specimens obtained be-
fore antibiotic therapy for the majority of
patients with Lyme arthritis [4, 5], and it
has been detected by culture or PCR in
some patients with neuroborreliosis [6, 7].
In our experience, all such patients have
had samples that were seropositive for
B.
burgdorferi.
Moreover, in animal models
of Lyme disease, spirochetes have been
seen in and cultured from CNS, heart, or
joint lesion specimens, and animals with
spirochetes were seropositive for
B. burg-
dorferi
[8, 9]. Therefore, on the basis of
current knowledge, all patients with ob-
jective neurologic, cardiac, or joint ab-
normalities of Lyme disease have serologic
responses to
B. burgdorferi.
Serologic testing for Lyme disease is in-
sensitive during the first several weeks of
infection in patients with the initial skin
lesion erythema migrans, but the fre-
quency of seropositivity is low during this
period only [10, 11]. In our study, 29%
of patients with erythema migrans had
acute-phase samples with positive IgM or
IgG antibody responses to
B. burgdorferi,
and 64% had convalescent-phase samples
with positive responses 3–4 weeks later
[2]. After that time, the sensitivity of 2-
tier testing (ELISA and Western blot) for
patients with disseminated or persistent
Lyme disease was 100%, and the specificity
was 99% [2]. Others have reported similar
results [11], and similar results were found
with a newer serologic test, the VlsE C6
peptide ELISA [2, 11, 12].
In our study, 36 (47%) of the 76 pa-
tients with erythema migrans had blood
samples obtained during the acute phase
of the illness that were positive for
B. burg-
dorferi
by PCR [2]. Other researchers have
found similar results of blood cultures for
patients with erythema migrans [13]. After
this early period, results of culture and
PCR testing of blood samples for
B. burg-
dorferi
DNA are almost always negative.
Finally, 10 (71%, not 36%) of 14 pa-
tients with post–Lyme disease symptoms
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Uddrag fra Centers for Disease Control and Prevention (CDC):
Case Definitions for Public Health Surveillance, 1990, Lyme Disease
Retur til dok liste
October 19, 1990
20
MMWR
Listeriosis
Clinical description
Infection caused by
Listeria monocytogenes,
which may produce any of several
clinical syndromes, including stillbirths, listeriosis of the newborn, meningitis, bac-
teremia, or localized infections
Laboratory criteria for diagnosis
•
Isolation of
L. monocytogenes
Case classification
from a normally sterile site
Confirmed:
a clinically compatible case that is laboratory confirmed
Lyme Disease
Clinical description
A systemic, tick-borne disease with protean manifestations, including derma-
tologic, rheumatologic, neurologic, and cardiac abnormalities. The best clinical
marker for the disease is the initial skin lesion, erythema migrans, that occurs
among 60%-80% of patients.
Clinical case definition
•
Erythema migrans, or
•
At least one late manifestation, as defined below, and laboratory confirmation of
infection
Laboratory criteria for diagnosis
•
Isolation of
Borrelia burgdorferi
serum or CSF, or
from clinical specimen, or
Unlikely
•
Demonstration of diagnostic levels of IgM and IgG antibodies to the spirochete in
•
Significant change in IgM or IgG antibody response to
B. burgdorferi
acute- and convalescent-phase serum samples
Case classification
Confirmed:
a case that meets one of the clinical case definitions above
Comment
This surveillance case definition was developed for national reporting of Lyme dis-
ease; it is NOT appropriate for clinical diagnosis.
Definition of terms used in the clinical description and case definition:
in paired
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LETTERS
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2
3
4
5
LYME WARS
Let’s tackle the testing
The two tier testing system endorsed
by the Centers for Disease Control and
Prevention (CDC) has a high specificity
(99%) and yields few false positives. But
the tests have a uniformly miserable
sensitivity (56%)—they miss 88 of every
200 patients with Lyme disease (table).
By comparison, AIDS tests have a
sensitivity of 99.5%—they miss only
one of every 200 AIDS cases. In simple
terms, the chance of a patient with
Lyme disease being diagnosed using
the commercial tests approved by the
Food and Drug Administration and
sanctioned by the CDC is about getting
heads or tails when tossing a coin, and
the poor test performance assures that
many patients with Lyme disease will go
undiagnosed.
Sensitivity and specificity of commercial two tier testing for
Lyme disease
Study
Schmitz et al.
Eur J Clin
Microbiol Infect Dis
1993;12:419-24
Engstrom et al.
J Clin Microbiol
1995;33:419-27
Ledue et al.
J Clin Microbiol
1996;34:2343-50
Trevejo et al.
J Infect Dis
1999;179:931-8
Nowakowski et al.
Clin Infect
Dis
2001;33:2023-7
Bacon et al.
J Infect Dis
2003;187:1187-99
Mean of all studies
Sensitivity
66%
Specificity
100%
ALCOHOL CONFUSION
What is a unit?
A recent
BMJ
editorial
1
discussed the World
Cancer Research Fund (WCRF) report on
cancer
2
and commented on the report’s
recommendation that men should drink
no more than two units of alcohol a day
and women no more than one unit a day.
These recommendations are much lower
than current government advice in Britain.
2
This highlights a widespread confusion
regarding units of alcohol and “standard”
drinks—WCRF “drinks” contain 10-15 g of
ethanol and British units contain 8 g.
Although a unit is often taken as one drink
(half a pint of beer or one glass of wine),
this is not the case. One pint of beer (4.2%)
contains 2.4 units and a 175 ml glass of wine
(12%) contains 2.1 units. The Department
of Health leaflet,
How Much is Too Much?,
promises information on the number of units
in alcoholic drinks. It advises using smaller
glasses, stating that a 125 ml glass of wine
contains one unit.
3
This would be true if the
alcohol content was 8%, but at a more typical
12% it contains 1.5 units.
Furthermore, the standard drink varies
across the world. The WCRF report is
an international publication, which may
explain the wide range of ethanol contents
per drink (10-15 g) in the recommendation.
In 1991, Miller et al issued “a plea for
consistency” regarding alcohol content.
4
Given the ambiguity present in the
WCRF report, and the confusion evident
even in a
BMJ
editorial, it is surely time to
heed that plea.
Rachel Seabrook
research manager, Institute of Alcohol
Studies, St Ives, Cambridgeshire PE27 5AR
[email protected]
Competing interests:
None declared.
1
2
Key T. Diet and the risk of cancer.
BMJ
2007;335:897.
World Cancer Research Fund/American Institute for
Cancer Research.
Food, nutrition, physical activity,
and the prevention of cancer: a global perspective.
Washington DC: AICR, 2007.
Department of Health.
How much is too
much?
2006. London: DoH. www.dh.gov.
uk/en/Publicationsandstatistics/Publications/
PublicationsPolicyAndGuidance/DH_4139672.
Miller WR, Heather N, Hall W. Calculating standard
drink units: international comparisons.
Br J Addiction
1991;86:43-7.
BMJ
|
17 NOVEMBER 2007
|
VOLUME 335
James E Griffin
specialist registrar in haematology, Bristol
Haematology and Oncology Centre, Bristol BS2 8ED
[email protected]
Competing interests:
None declared.
1
White C. Cardiopulmonary resuscitation decisions should
be extended to nurses.
BMJ
2007;335:901. (3 November.)
RESISTANCE TO HIV DRUGS
Detainees are affected
The problem of discontinuous antiretroviral
therapy in the prison healthcare system
leading to increased viral resistance applies
also to detainees held by the immigration
authorities. Many such detainees are held
for a long time after the initial decision
to deport, pending the resolution of legal
challenges.
1
Detainees are often moved
between detention centres and immigration
removal centres as their cases are considered,
and their antiretroviral drugs often do not
follow them. It may be weeks before the
local genitourinary clinic is made aware that
a transfer has taken place, which causes a
large gap in treatment.
Consequently, when these patients are
eventually deported, they have developed
a resistance pattern that requires second or
third line antiretrovirals, which may not be
available in the countries to which they are
deported, even if those countries have some
provision of antiretrovirals. It is a serious
failure of the healthcare systems provided
by the Border and Immigration Agency that
the treatment of HIV positive detainees is
undermined.
Edward Costar
foundation year 1 doctor, Kent and
Canterbury Hospital, Canterbury CT1 3NG
[email protected]
Jillian Pritchard
consultant in genitourinary medicine, St
Peter’s Hospital, Chertsey
Competing interests:
None declared.
1
Laurent C. Prisoners are developing resistance
to HIV drugs because their care is fractured.
BMJ
2007;335:583. (22 September.)
55%
50%
29%
66%
68%
56%
96%
100%
100%
99%
99%
99%
Until we scrap the worthless
commercial tests for Lyme disease and
find a better way to make the diagnosis of
this protean illness, the “Lyme wars” will
continue unabated.
1
Raphael B Stricker
past president, International Lyme and
Associated Diseases Society, San Francisco, CA 94108, USA
[email protected]
Lorraine Johnson
executive director, California Lyme Disease
Association, Los Angeles, CA 90068, USA
Competing interests:
RBS serves on the advisory panel
for QMedRx.
1 Tonks A. Lyme wars.
BMJ
2007;335:910-2. (3 November.)
1008
3
4
COMSTOCKCOMPLETE.COM
Wald DS, Bestwick JP, Wald NJ. Child-parent screening
for familial hypercholesterolaemia: screening strategy
based on a meta-analysis.
BMJ
2007;335:599. (22
September.)
Is cascade testing a sensible method of population
screening?
J Med Screen
2004;11:57-8.
Morris JK, Law MR, Wald NJ. Is cascade testing a
sensible method of screening a population for
autosomal recessive disorders?
Am J Med Genet
2004;128A:271-5.
Hopcroft KA. Child-parent screening may have adverse
psychological effects.
BMJ
2007:335:683. (6 October.)
RESUSCITATION DECISIONS
Yes, but who is in charge?
I definitely support nurses’ involvement
in resuscitation decisions.
1
I have worked
on teams where nursing staff were always
involved before a “not for resuscitation”
decision was made, and if they did not agree
the patient would remain “for resuscitation.”
I have also worked for consultants who will
not make patients not for resuscitation.
What will happen if the consultant
responsible for a patient wishes them
to remain for resuscitation but a senior
nurse feels that they should not be for
resuscitation? Other doctors may agree with
the nurse’s decision but in the end I would
want clarification as to who has the final say.
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1533935_0030.png
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S31
The Western Immunoblot for Lyme Disease: Determination of Sensitivity,
Specificity, and Interpretive Criteria with Use of Commercially Available
Performance Panels
Richard C. Tilton, Mary N. Sand, and Mark Manak
From BBI Clinical Laboratories, Inc., New Britain, Connecticut; and
Biotech Research Laboratory, Rockville, Maryland
Recent recommendations for the serological diagnosis of Lyme disease include statements on
quality assurance and the use of performance panels to assess laboratory competency. We used two
performance panels — one from the Centers for Disease Control and Prevention (CDC) and one
from Boston Biomedica Inc. (West Bridgewater, MA) — to evaluate the sensitivity and specificity of
four western blot kits. We used the same panels to compare the interpretive criteria for western
blots as proposed by participants in the Centers for Disease Control and Prevention, Association of
State and Territorial Public Health Laboratory Directors Conference and those proposed by BBI
Clinical Laboratories (BBICL; New Britain, CT). Our results indicated that the BBICL western
blots were more sensitive than those of the CDC, MarDx (Carlsbad, CA), or Cambridge Biotech
(Rockville, MD). However, use of the CDC criteria with the BBICL western blots increased specificity
to 100% but reduced sensitivity to 74.3%. A sample table is provided as an example of the test
results obtained with the BBI performance panel. Obviously, this work should be confirmed by other
investigators.
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In response to numerous reports on problems associated with
Lyme disease testing [1 – 3] , participants in the recent Centers
for Disease Control and Prevention (CDC), Association of State
and Territorial Public Health Laboratory Directors
(ASTPHLD) Conference on the Serological Diagnosis of Lyme
Disease [4] made several recommendations including:
(1)
Lyme disease testing should be performed only in
laboratories that have comprehensive quality assur-
ance programs.
Serum samples used to evaluate screening tests or
western blots in proficiency testing should cover all
stages of Lyme disease, and samples should be repre-
sentative of the target population. Each sample
should be from a single donor.
A repository of serum specimens from patients with
well-characterized
Borrelia burgdorferi
infections
(early and late), other spirochetal infections, other
infections and inflammatory disorders that have
shown cross-reactivity in Lyme disease testing, and
normal serum samples from areas of nonendemicity
should be maintained by the CDC. Industry should
provide resources to develop appropriate serum pan-
els. These panels should be made available to re-
search and development laboratories and to testing
laboratories for validation studies. At least two such
panels are currently available: one, which comprises
a 45 – 47-member panel, is available from the CDC,
and the other, which comprises a 15-member mixed
titer panel, is available from Boston Biomedica
(West Bridgewater, MA).
Materials and Methods
The CDC performance panel was used to evaluate the sensi-
tivity and specificity of three western blot products (BBI Clini-
cal Laboratories [BBICL; New Britain, CT], MarDx [Carlsbad,
CA], and Cambridge Biotech [Rockville, MD]). In a separate
evaluation, the CDC panel was also used to compare the BBICL
western blot and the CDC western blot. The Boston Biomedica
Lyme Disease Mixed Titer Performance Panel can also be used
to validate new Lyme disease antibody tests and to compare
the sensitivity and specificity of a newly adopted antibody test.
Each of the serum samples in the CDC panel has limited
clinical classification, including presence/absence of erythema
migrans (EM), culture results, and whether the patient was
IgG/IgM reactive or seronegative. The western blot and ELISA
results on this panel are not available to the purchaser until the
testing has been performed and sent to the CDC for analysis;
only then are the reference results released. Hence, use of the
panel is blinded. While clinical characterization is provided,
there are no data available on when the specimens were col-
lected in reference to the appearance of EM or a culture positive
for
B. burgdorferi.
We compared three western blot kits for the detection of
IgM and IgG antibodies to
B. burgdorferi.
They included the
BBICL western blot kit, made by Biotech Research Labora-
tories (BBI), the MarDx kit, and the Cambridge Biotech kit.
(2)
(3)
Reprints or correspondence: Dr. R. Tilton, BBI Clinical Laboratories, Inc.,
75 North Mountain Road, New Britain, Connecticut 06053.
Clinical Infectious Diseases 1997;25(Suppl 1):S31–4
1997 by The University of Chicago. All rights reserved.
1058–4838/97/2501–0005$03.00
/ 9c34$$jy11
06-06-97 19:58:41
cida
UC: CID
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S32
Tilton, Sand, and Manak
CID 1997;25 (Suppl 1)
Table 1.
Comparison of three western blot kits for detection of IgM
antibody to
Borrelia burgdorferi
with use of the performance panel
of the Centers for Disease Control and Prevention (CDC).
Western blot product
Result
Sensitivity (%)
Specificity (%)
BBICL
90.0
86.4
MarDx
78.9
100
Cambridge Biotech
64.3
68.2
NOTE. BBICL
Å
BBI Clinical Laboratories (New Britain, CT).
Each assay was performed by using the procedure described
in the manufacturer’s product literature.
The BBICL criteria are:
IgM-significant bands — 23, 39, 41, 83 kD*
Reactive — two of the following four bands (23, 39, 41,
83 kD) must be present.
Equivocal — one of the following bands (23, 31, 34, 37,
39, 41, 83 kD) must be present.
Nonreactive — no Lyme-specific bands are present.
IgG-significant bands — 20, 23, 31, 34, 35, 39, 83 kD
Reactive — three of the following bands (20, 23, 31, 34,
35, 39, 83 kD) must be present.
Equivocal — one or two of the following bands (20, 23,
31, 34, 35, 39, 83 kD) must be present.
Nonreactive — no Lyme-specific bands are present.
The CDC/ASTPHLD criteria are:
IgM-significant bands
Reactive — two of the following three bands (23, 39, 41
kD) must be present.
Nonreactive — fewer than two bands are present.
IgG-significant bands
Reactive — five of the following bands (18, 21, 28, 30,
41, 45, 58, 66, 93 kD) must be present.
Nonreactive — fewer than five bands are present.
* The 83 and 93 kD bands are equivalent.
Interpretive criteria for the BBICL western blot included
both the BBICL criteria and the CDC/ASTPHLD criteria [4].
The results obtained with use of the other two kits were inter-
preted on the basis of the CDC/ASTPHLD criteria. Each blot
was read independently by two technologists and then validated
by a director, all of whom were employees of BBICL. Both
the technologists and the director were blinded as to the blot
manufacturer, and the specimens were coded.
Results and Discussion
Table 1 shows the results of our comparison of the three
western blot products for detecting IgM, based on the CDC/
ASTPHLD performance panel criteria. The BBICL IgM west-
ern blot is more sensitive than either the MarDx or Cambridge
Biotech IgM western blot and slightly less specific than the
MarDx IgM western blot. The BBICL criteria for IgM western
blot are virtually identical to the proposed CDC/ASTPHLD
criteria, except for the inclusion of the 83/93-kD band and an
indeterminate category. Thus, differences in performance of
the kits are probably product related and not due to differences
in interpretive criteria.
Table 2 shows the results of our comparison of the three
western blot products for detecting IgG with use of the CDC
performance panel. On the basis of the CDC/ASTPHLD crite-
ria, the BBICL IgG western blot is more sensitive than and as
specific as the other two IgG western blot products. However,
if the BBICL criteria are applied, the sensitivity of the BBICL
IgG western blot increases to 87%, but its specificity is reduced.
The reduced sensitivities of all IgM western blot kits, particu-
larly those of MarDx and Cambridge Biotech, could well reflect
the time at which the specimens were drawn for the panel. If
the patients donated
§1
unit of plasma months after the initial
acute episode of Lyme disease, then the IgM titer would be
expected to be diminished.
The second study was also done in blinded fashion at BBICL.
IgG and IgM western blots were performed on the 46-member
CDC panel, and the results were sent to the CDC for analysis.
A comparison of results obtained with BBICL or CDC western
blots and clinical information are shown in figures 1 and 2.
BBICL IgM western blots classified 28 of 30 patients with Lyme
disease as IgM positive (figure 1). The diagnosis was missed in
two patients (6%). The CDC reported apparently false-negative
IgM western blots for 10 patients, all of whom were symptom-
atic. Of these 10 patients who were negative by the CDC IgM
western blot and positive or equivocal by BBICL IgM western
blot, the one patient who was positive by the BBICL IgM west-
ern blot had confirmed EM and was culture positive for
B. burg-
dorferi.
Of the nine patients who were negative by the CDC
western blot and equivocal by the BBICL western blot, seven
were positive for IgG antibodies to
B. burgdorferi
by western
blot in both laboratories, and two were negative for IgG antibod-
ies in both laboratories. All nine patients had confirmed EM and
cultures positive for
B. burgdorferi.
BBICL reported that 35 of 46 patients had either positive (19
patients) or equivocal (16 patients) IgG western blots (figure 2).
Of these 35 patients with clinically defined Lyme disease, 23
were found to be seropositive with use of the CDC western
blot. There were 12 false-negative results. One of the specimens
negative for IgG by the CDC western blot but positive by the
BBICL western blot was from a
B. burgdorferi–infected
patient
whose IgM western blot was found to be positive in both labora-
tories. The 12 patients who were negative by the CDC/
ASTPHLD criteria but had confirmed Lyme disease were equivo-
cal by the BBICL criteria. Seven of these
B. burgdorferi–infected
patients had a positive IgM western blot in both laboratories.
Four patients had a history of tick bite, EM, and positive
cultures. There were no clinical data for one patient. There
were five patients who were positive by the CDC/ASTPHLD
criteria and equivocal by the BBICL criteria. There were multi-
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CID 1997;25 (Suppl 1)
Western Immunoblot for Lyme Disease
S33
Table 2.
Comparison of three western blot kits for the detection of IgG antibody to
Borrelia burgdorferi
with use of the performance panel of the Centers for Disease Control and Prevention (CDC), according
to the criteria of the BBI Clinical Laboratories or the criteria of the CDC.
Cambridge
Biotech
(CDC criteria)
43.6
100
Result
Sensitivity (%)
Specificity (%)
BBICL
(BBICL criteria)
87.2
60.0
BBICL
(CDC criteria)
74.3
100
MarDx
(CDC criteria)
47.0
100
ple bands on both western blots for all five of these patients,
but there were not enough bands to fulfill the BBICL criteria
for positivity. Thus, BBICL classified all (35) patients who
met the CDC criteria for Lyme disease as either positive or
equivocal, while the CDC western blot results indicated that
12 patients who met the CDC criteria for Lyme disease were
negative. The case of one patient who was western blot – posi-
tive for IgM at the CDC and negative for IgM with use of
the BBICL western blot is still unresolved. In addition, five
specimens were equivocal with use of the BBICL IgG western
blot but positive with use of the CDC IgG western blot.
The BBICL western blot appears to be more sensitive than
the CDC western blot, the MarDx western blot, or the Cam-
bridge Biotech western blot. Of course, the issue is whether a
western blot should be more sensitive than specific, or vice
versa. If the western blot is to be used solely for confirmation
of the results of ELISA, then specificity may be more desirable
than sensitivity. However, a specific western blot with low
sensitivity may invalidate a sensitive and specific ELISA. A
highly sensitive and specific western blot is desirable for a
two-tiered test system. Of major significance is the fact that
despite the CDC recommendation for two-tiered testing, many
physicians who treat patients with Lyme disease do not believe
that an ELISA is an appropriate screening test and consequently
use the western blot as a primary test for Lyme disease or
request that both ELISA and western blot be done.
If the western blot is to be used in this manner, then sensitiv-
ity may be preferred over specificity, particularly for patients
with acute Lyme disease or for those with suspected persistent
disease and symptoms that are not commonly observed. We
recognize, however, that reduced specificity may further com-
plicate the serodiagnosis of Lyme disease because of the poten-
tial for increased numbers of false-positive tests.
Workers at Boston Biomedica have assembled a set of 15
aliquots of frozen serum and plasma units with reactivity to
B. burgdorferi
ranging from negative to strongly positive when
used with a variety of currently available test methodologies.
Samples have been selected to demonstrate IgG and/or IgM
reactivity. In addition, one negative plasma unit has been in-
cluded as a nonreactive control. These specimens are undiluted
aliquots from plasma and serum units collected from 1994
to 1995. The units were processed by sterile filtration. No
preservatives were added. Clinical information on panel mem-
bers was included when available. The purpose of this perfor-
mance panel of naturally occurring serum and plasma samples
is to enable manufacturers and diagnostic laboratories to evalu-
ate their tests for detection of antibodies to
B. burgdorferi
with
characterized samples and to provide comprehensive data for
comparative analysis.
The tables provided in the Panel give results from both
commercially available test kits and in-house procedures per-
formed at BBICL, Boston Biomedica, and internationally rec-
ognized reference laboratories. Product numbers are indicated
for identification of each method. Numeric results are the means
of duplicate tests. Some results are expressed as signal-to-cutoff
ratios to facilitate comparisons among kits; ratios of
§1.0
are
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Figure 1.
Comparison of the sensitivity and specificity of BBI Clin-
ical Laboratory (New Britain, CT) and Centers for Disease Control
and Prevention western blots for IgM antibody to
Borrelia burgdorferi
with that of clinical information (culture results, the presence of ery-
thema migrans [EM], and serology) in the detection of
B. burgdorferi
infection.
Figure 2.
Comparison of the sensitivity and specificity of BBI Clin-
ical Laboratory (New Britain, CT) and Centers for Disease Control
and Prevention western blots for IgG antibody to
Borrelia burgdorferi
with that of clinical information (culture results, the presence of ery-
thema migrans [EM], and serology) in the detection of
B. burgdorferi
infection.
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1533935_0033.png
S34
Tilton, Sand, and Manak
CID 1997;25 (Suppl 1)
Figure 3.
A representative sample
of results for panel members of a
BBI Performance Panel for a MarDx
(Carlsbad, CA) western blot kit.
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considered reactive. Results with use of indirect fluorescent
antibody are endpoint dilutions.
There are no universally accepted criteria for western blot
interpretation; therefore, the interpretation of the band pattern
was based on the manufacturers’ criteria for their kits and the
in-house criteria (BBICL) for the in-house methods. Figure 3
shows a representative sample of the results provided with the
panel, in this case western blot results for panel members of a
MarDx western blot kit. This performance panel will be invalu-
able to both kit manufacturers and hospital laboratory personnel
who wish to validate their diagnostic procedures for Lyme
disease. While this Lyme disease panel is antibody based, PCR
is becoming more widely used for the laboratory diagnosis of
Lyme disease [5]. Molecular panels are now needed for the
diagnosis of Lyme disease.
ASTPHLD interpretive criteria to the BBICL results increased
specificity but reduced sensitivity. Sample data are also pro-
vided from a commercially available Lyme disease antibody
performance panel. Use of such a panel should enable labora-
tory personnel to compare results with their currently used test
kits to those obtained with a wide variety of kits and methods.
References
1. Bacon LL, Case KL, Callister SM, et al. Performance of 45 laboratories
participating in a proficiency testing program for Lyme disease serology.
JAMA
1992;
268:891 – 5.
2. Magnarelli LA, Meegan JM, Anderson JF, et al. Comparison of an indirect
fluorescent antibody test with an enzyme-linked immunosorbent assay
for serological studies of Lyme disease. J Clin Microbiol
1984;
20:
181 – 4.
3. Fister RD, Weymouth LA, Mardi JC, et al. Comparative evaluation of three
products for the detection of
Borrelia burgdorferi
antibody in human
serum. J Clin Microbiol
1989;
27:2834 – 7.
4. Centers for Disease Control and Prevention, Association of State and Terri-
torial Public Health Laboratory Directors. Proceedings of the Second
National Conference on Serologic Diagnosis of Lyme Disease (Dearborn,
MI). Washington, DC: Association of State and Territorial Public Health
Laboratory Directors.
1994.
5. Schmidt BL. Laboratory diagnosis of human
Borrelia burgdorferi
infec-
tions. Clin Microbiol Rev
1997;
10:185 – 201.
Conclusion
The results of our comparative testing of available western
blot kits with use of a CDC performance panel indicated that
BBICL western blots were more sensitive than those of compet-
ing manufacturers. However, application of the CDC/
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Retur til dok liste
J
OURNAL OF
C
LINICAL
M
ICROBIOLOGY
, July 2008, p. 2216–2221
0095-1137/08/$08.00 0 doi:10.1128/JCM.00200-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 46, No. 7
Evaluation of Two Commercial Systems for Automated Processing,
Reading, and Interpretation of Lyme Borreliosis Western Blots
M. J. Binnicker,
1
* D. J. Jespersen,
1
J. A. Harring,
1
L. O. Rollins,
1
S. C. Bryant,
2
and E. M. Beito
1
Division of Clinical Microbiology and Department of Laboratory Medicine and Pathology
1
and Division of Biostatistics,
2
Mayo Clinic and Mayo Clinic College of Medicine, Rochester, Minnesota 55905
Received 1 February 2008/Returned for modification 27 March 2008/Accepted 29 April 2008
The diagnosis of Lyme borreliosis (LB) is commonly made by serologic testing with Western blot (WB)
analysis serving as an important supplemental assay. Although specific, the interpretation of WBs for diag-
nosis of LB (i.e., Lyme WBs) is subjective, with considerable variability in results. In addition, the processing,
reading, and interpretation of Lyme WBs are laborious and time-consuming procedures. With the need for
rapid processing and more objective interpretation of Lyme WBs, we evaluated the performances of two
automated interpretive systems, TrinBlot/BLOTrix (Trinity Biotech, Carlsbad, CA) and BeeBlot/ViraScan
(Viramed Biotech AG, Munich, Germany), using 518 serum specimens submitted to our laboratory for Lyme
WB analysis. The results of routine testing with visual interpretation were compared to those obtained by
BLOTrix analysis of MarBlot immunoglobulin M (IgM) and IgG and by ViraScan analysis of ViraBlot and
ViraStripe IgM and IgG assays. BLOTrix analysis demonstrated an agreement of 84.7% for IgM and 87.3% for
IgG compared to visual reading and interpretation. ViraScan analysis of the ViraBlot assays demonstrated
agreements of 85.7% for IgM and 94.2% for IgG, while ViraScan analysis of the ViraStripe IgM and IgG assays
showed agreements of 87.1 and 93.1%, respectively. Testing by the automated systems yielded an average time
savings of 64 min/run compared to processing, reading, and interpretation by our current procedure. Our
findings demonstrated that automated processing and interpretive systems yield results comparable to those
of visual interpretation, while reducing the subjectivity and time required for Lyme WB analysis.
Lyme disease is a multisystem, tick-borne disease caused by
the spirochete
Borrelia burgdorferi.
In 2006, the Centers for
Disease Control and Prevention (CDC) reported 19,931 cases
of Lyme disease in the United States (7), confirming that the
disease continues to represent a significant public health
threat. The clinical manifestations of early localized disease
range from nonspecific sequelae, including malaise, myalgia,
and lymphadenopathy, to more characteristic findings, such as
erythema migrans (EM). In the absence of appropriate ther-
apy, disease progression may lead to significant complications,
including rheumatologic, neurologic, or cardiac manifestations
(15, 16).
The diagnosis of Lyme borreliosis (LB) can be made clini-
cally when patients from regions where the disease is endemic
present with EM (5, 8, 18). However, in patients without EM
but with objective clinical findings suggestive of disseminated
LB, serologic testing is an important diagnostic approach. Ap-
propriate serologic testing should follow the two-tier algorithm
recommended by the CDC (6), consisting of initial testing with
a sensitive screening assay (e.g., enzyme immunoassay) with
positive or equivocal specimens to be tested by Western blot
(WB) analysis. Current CDC criteria for WB interpretation
recommend that 2 bands on the immunoglobulin M (IgM)
WB or 5 bands on the IgG WB be present for the immuno-
blot to be considered positive (6, 9). Although WB is consid-
ered to be highly specific, current testing protocols in most
clinical laboratories rely on visual reading and interpretation of
* Corresponding author. Mailing address: Mayo Clinic, 200 First
Street SW Hilton 860A, Rochester, MN 55905. Phone: (507) 538-1640.
Fax: (507) 284-4272. E-mail: [email protected].
Published ahead of print on 7 May 2008.
2216
WB strips. These procedures require the laboratory technolo-
gist to visually compare band intensities on the patient strip to
those of a weakly reactive control. This approach is labor-
intensive, time-consuming, and subjective, allowing for poten-
tial intra- and interlaboratory variation in WB reading and
interpretation. Previous studies analyzing the performance of
LB serologic tests among testing laboratories have demon-
strated significant variation in results, even for more objective
methods, such as enzyme immunoassay (2, 3, 10). Therefore,
given the inherent subjectivity in reading and interpreting WBs
for diagnosis of LB (i.e., Lyme WBs), one would expect to
observe significant variation in WB results, with potentially
adverse effects on the laboratory diagnosis of Lyme disease and
subsequent patient management decisions. Due to the need for
more objective and consistent interpretation of Lyme WBs, we
undertook a study to evaluate and compare two systems (Trin-
Blot/BLOTrix [Trinity BioTech, Carlsbad, CA] and BeeBlot/
ViraScan [Viramed Biotech AG, Munich, Germany]) designed
for automated processing, reading and interpretation of Lyme
WBs. The goal of the present study was to determine whether
automated systems yield comparable results to visual reading
and interpretation while reducing the subjectivity and time
required for Lyme WB analysis.
MATERIALS AND METHODS
Serum specimens.
A total of 518 consecutive, unique serum specimens sub-
mitted to our reference laboratory for routine LB serologic testing between June
and September 2007 were included in the study. The specimens were submitted
without accompanying clinical information. The study protocol was reviewed and
approved by the institutional review board of the Mayo Clinic.
In addition, two Lyme WB performance panels consisting of 55 clinically
characterized and laboratory-characterized serum specimens were purchased
from the CDC and Boston Biomedica, Inc. (BBI; West Bridgewater, MA).
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V
OL
. 46, 2008
LYME WESTERN BLOT INTERPRETIVE SYSTEMS
2217
FIG. 1. Study design.
Study design.
Each specimen was processed by our current procedure, per-
formed according to the manufacturer’s instructions for processing MarBlot IgG
and IgM strips (MarDx Diagnostics, Carlsbad, CA) using the Autoblot 6000
instrument (MedTec, Inc., Hillsborough, NC). The strips were visually inter-
preted, and the results were recorded manually. Each specimen was also tested
by MarBlot IgG and IgM strips using the automated TrinBlot processor (Bee
Robotics, Caernarfon Gwynedd, United Kingdom) with subsequent scanning
and analysis by the BLOTrix interpretive software. In addition, each specimen
was tested by the ViraBlot and ViraStripe IgG and IgM strips (Viramed Biotech
AG) on the automated BeeBlot processor (Bee Robotics) with subsequent anal-
ysis by the ViraScan interpretive software. A laboratory technologist blinded to
the results of visual interpretation in the laboratory then reviewed the BLOTrix
and ViraScan software interpretive results for each specimen (Fig. 1). The
laboratory technologist, when reviewing the software interpretive results for each
strip, (i) ensured that the software had analyzed only bands demonstrating
uniform intensity across the entire width of the strip, (ii) checked that any
background intensity (non-band intensity) had been accounted for, (iii) verified
that the software had properly aligned test bands on the patient strip with bands
on the serum band locator control strip, and (iv) ensured that the densitometric
read was focused on the center of each test band.
WB assays.
MarDx MarBlot IgM and IgG assays (Fig. 2) utilize antigens of
B.
burgdorferi
strain B31 for Western blot analysis. The antigens are separated by
electrophoresis through a sodium dodecyl sulfate-polyacrylamide gel. The re-
solved antigens are then transferred to a nitrocellulose membrane. Similarly, the
ViraBlot and ViraStripe IgM and IgG assays use strain
B. burgdorferi
B31 as the
source of antigen for Western blot analysis. ViraBlot IgM and IgG assays (Fig.
2) are manufactured by separation of antigens by gel electrophoresis, with sub-
sequent transfer to nitrocellulose. In contrast, the ViraStripe IgM and IgG assays
(Fig. 2) are generated by “printing” highly purified antigens at a defined location
with standardized concentrations on a nitrocellulose membrane. The ViraStripe
IgM and IgG assays are currently prototype Lyme WB strips, while the MarBlot
and ViraBlot assays are both Food and Drug Administration cleared.
WB strip processing, reading, and interpretation.
For each specimen tested by
our current Lyme WB procedure, 80 l of the serum band locator, 20 l of the
weakly reactive and negative controls, and 20 l of patient serum were added to
the appropriate channels of the Autoblot 6000 instrument. After being incubated
and washed, strips were air dried and mounted for visual reading and interpre-
tation. Each test band was visually compared to the 41-kDa band on the weakly
reactive control strip and was considered present if the intensity was equal to or
greater than that of the weakly reactive control. The IgM assay was considered
positive if two of the following three bands were present: 23, 39, and 41 kDa. The
IgG assay was considered positive if five of the following ten bands were present:
18, 23, 28, 30, 39, 41, 45, 58, 66, and 93 kDa.
Each specimen was also tested by the ViraBlot and ViraStripe IgM and IgG
assays, performed according to the manufacturer’s instructions on the BeeBlot
automated processor. In brief, strips were added to the incubation tray and
allowed to presoak in 1.5 ml of wash buffer for 5 min. Next, 20 l of patient
serum or 100 l of control was added to the appropriate channel of the incuba-
tion tray. After being incubated and washed, strips were air dried in the incu-
bation tray, scanned by using the ViraCam scanner (Viramed Biotech, AG), and
analyzed by the ViraScan analysis software version 2.01. Based on densitometric
analysis as described by Nishizuka et al. (12), 8-bit, grayscale images at a reso-
lution of 220 dots per inch were stored in bitmap format for subsequent analysis
by ViraScan. With the aid of predefined band-locator images, ViraScan locates
and measures the immunospecific banding patterns on each patient strip. Each
band is then analyzed for band location and maximum intensity. After a sub-
traction of background intensity (the average non-band intensity), the software
assigns a numerical value to each band. The software then divides the numeric
value of each test band by the numeric value assigned to a separate calibrator
control band to determine a relative intensity. For the present study, a test band
was considered present if the relative intensity met or exceeded the following
manufacturer’s recommended cutoff settings: ViraBlot IgG, 75%; ViraBlot IgM,
70%; ViraStripe IgG, 85%; and ViraStripe IgM, 60%.
Each specimen was also tested by the MarDx MarBlot IgM and IgG assays,
performed according to the manufacturer’s instructions using a TrinBlot auto-
mated processor. After automated processing, the strips were air dried in the
incubation tray, scanned, and analyzed by the BLOTrix analysis software version
2.6. Similar to ViraScan, BLOTrix software utilizes densitometric analysis to
compare the intensity of each test band to that of a separate calibrator control
band and calculate a relative percent intensity. For the present study, a test band
was considered present if the calculated relative intensity met or exceeded the
following manufacturer’s recommended cutoff settings: TrinBlot IgG, 90%; and
TrinBlot IgM, 90%.
Statistics.
Statistical analyses were performed by using statistical analysis soft-
ware (SAS Institute Inc, Cary, NC). In addition to the percent agreement, kappa
coefficients were determined as a secondary measure of agreement. Result
agreements by kappa values are categorized as near perfect (0.81 to 1.0), sub-
stantial (0.61 to 0.80), moderate (0.41 to 0.60), fair (0.21 to 0.40), slight (0 to
0.20), or poor ( 0) (11).
Workflow analysis.
The average assay time for testing by our current proce-
dure was calculated by timing three separate runs (40 specimens/run) from the
addition of strips to the incubation tray through the manual recording of results
by the technologist. The average assay time for testing by the automated systems
was calculated by timing three separate runs (40 specimens/run) from the addi-
tion of strips to the incubation tray through the review of software results by the
laboratory technologist.
RESULTS
Agreement between automated and visual interpretation.
To
assess agreement, the qualitative results (positive or negative
based on CDC criteria) were compared after the testing of 518
consecutive serum specimens. Among the 518 specimens, 74
(14.3%) were positive for IgG, and 191 (36.9%) were positive
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1533935_0036.png
2218
BINNICKER ET AL.
J. C
LIN
. M
ICROBIOL
.
FIG. 2. Comparison of WB strips for a single patient specimen. The same patient specimen was tested by the MarBlot, ViraBlot, and ViraStripe
assays. Strips were scanned by their respective systems, and the images were captured in tag image file format (TIFF). The migration positions of
bands used in the CDC interpretation criteria are indicated by molecular mass (in kilodaltons). PC, positive control; T, test (patient) sample.
for IgM by our current procedure. BLOTrix analysis of the
MarBlot assays demonstrated an agreement of 84.7% (439/
518) for IgM and 87.3% (452/518) for IgG compared to results
obtained by routine testing (Table 1). ViraScan analysis of the
ViraBlot IgM and IgG assays showed 85.7% (444/518) and
94.2% (488/518) agreement, respectively, while analysis of the
ViraStripe assays demonstrated 87.1% agreement (451/518)
for IgM and 93.1% agreement (482/518) for IgG (Table 1). A
technologist blinded to the results of routine testing then re-
viewed the BLOTrix and ViraScan interpretive results for each
TABLE 1. Agreement of Lyme WB strip results obtained by visual interpretation, automated software, and technologist-adjusted software
interpretation among prospective serum specimens (n 518)
a
Assay
b
Software alone
% Agreement (95% CI)
K
c
Technologist adjusted
% Agreement (95% CI)
K
c
K
d
MarBlot
IgM
IgG
ViraBlot
IgM
IgG
ViraStripe
IgM
IgG
a
b
84.7 (80.3–88.6)
87.3 (84.0–90.0)
85.7 (81.3–89.6)
94.2 (90.9–96.9)
87.1 (82.6–90.9)
93.1 (89.7–95.7)
0.69
0.57
0.70
0.75
0.72
0.72
87.1 (82.6–90.9)
92.3 (89.0–95.0)
85.9 (81.5–89.8)
94.8 (90.5–96.5)
86.7 (82.3–90.6)
91.7 (88.4–94.4)
0.73
0.72
0.71
0.75
0.71
0.69
0.86
0.77
0.97
0.90
0.97
0.89
Percent agreement refers to percent agreement with visual interpretation of MarDx MarBlot strips.
MarBlot results were analyzed by using BLOTrix software application; ViraBlot and ViraStripe results were analyzed by using the ViraScan software application.
c
Kappa coefficient for comparison with visual interpretation.
d
Kappa coefficient, software-alone interpretation versus technologist-adjusted software interpretation.
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1533935_0037.png
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LYME WESTERN BLOT INTERPRETIVE SYSTEMS
2219
TABLE 2. Agreement of Lyme WB strip results obtained by visual interpretation and technologist-adjusted software interpretation among
the BBI and CDC serum performance panels
% Agreement (95% CI)
a
Panel
MarBlot*
MarBlot†
ViraBlot
ViraStripe
BBI
IgM
IgG
CDC
IgM
IgG
a
73.3 (46.0–91.1)
86.7 (57.0–100.0)
NA
NA
73.3 (46.0–91.1)
86.7 (57.0–100.0)
80.0 (62.6–92.1)
90.0 (71.8–100.0)
93.3 (62.0–100.0)
86.7 (57.0–100.0)
67.5 (51.2–80.7)
92.5 (74.1–100.0)
80.0 (51.3–96.4)
80.0 (51.7–96.1)
82.5 (64.9–94.4)
90.0 (71.8–100.0)
The percent agreement with reference WB results provided by BBI or the CDC. *, tested by the current procedure with visual reading and interpretation; †, analyzed
by using the BLOTrix software application. ViraBlot and ViraStripe results were analyzed by using the ViraScan software application. NA, not applicable (inadequate
specimen volume to be tested by current lab procedure with visual interpretation).
specimen. The technologist-adjusted BLOTrix and ViraScan
results showed substantial agreement (0.61
0.80) with
results obtained by routine testing with visual interpretation
(Table 1).
In addition to the analysis of 518 consecutive serum speci-
mens, two performance panels (BBI and CDC) consisting of 55
serum specimens were tested by the automated systems. BBI
serum specimens were also tested by our current Lyme WB
procedure. Agreement was assessed by comparing the results
of testing to the reference WB results provided with the per-
formance panels (Table 2). Interestingly, the technologist-ad-
justed ViraScan results of the ViraBlot and ViraStripe IgM
assays showed closer agreement (93.3 and 80%, respectively)
with the reference BBI WB results than did the results ob-
tained by routine testing (73.3%) (Table 2).
Sensitivity and specificity of automated systems.
After the
testing of 518 prospective specimens, the results were analyzed
by using the visual interpretation of the MarDx assays as the
“gold standard.” Although the automated systems demon-
strated comparable performances overall, BLOTrix analysis
showed higher sensitivities for IgM and IgG (93.3 and 81.1%,
respectively) than did ViraScan analysis of the ViraBlot (86.1
and 74.3%, respectively) or ViraStripe (78.9 and 77.0%, re-
spectively) strips (Table 3). In contrast, ViraScan analysis of
the ViraStripe IgM and IgG strips showed the highest speci-
ficity (92.0 and 95.7%, respectively) compared to visual inter-
pretation of the MarDx MarBlot assays (Table 3).
Adjusted sensitivities and specificities were then calculated
after visual review of the automated software results by a
laboratory technologist (Table 3). A total of 37/518 (7.1%)
MarBlot IgM and 40/518 (7.7%) MarBlot IgG results were
adjusted after visual review of the BLOTrix analyses. The
majority of the changes (56/77 72.7%) made to the BLOTrix
interpretations were from positive to negative. These adjust-
ments yielded a marginal increase in specificity for the MarBlot
assays (Table 3). In contrast, a total of 8/518 (1.5%) ViraBlot
IgM, 12/518 (2.3%) ViraBlot IgG, 8/518 (1.5%) ViraStripe
IgM, and 15/518 (2.9%) ViraStripe IgG results were adjusted
after review of the ViraScan analyses. The majority of changes
(35/43 [81.4%]) made to the ViraScan interpretations were
from negative to positive, resulting in the adjusted sensitivities
and specificities outlined in Table 3.
The clinical sensitivity and specificity of the automated sys-
tems were further evaluated by using the 40-member CDC
serum performance panel. Specimens were categorized by clin-
ical diagnosis according to detailed histories included with the
panel, and WB results were analyzed by comparison to the
clinical findings. Reference CDC WB results showed a sensi-
tivity of 51.4% (18/35) for IgM and 48.6% (17/35) for IgG in
patients with confirmed or probable Lyme disease (Table 4).
TABLE 3. Sensitivity and specificity of automated software or technologist-adjusted software interpretation in prospective
serum specimens (n 518)
a
Sensitivity (95% CI)
Assay
b
Software alone
Technologist adjusted
Software alone
Technologist adjusted
Specificity (95% CI)
No. of technologist
adjustments (% of
total tests)
MarBlot
IgM
IgG
ViraBlot
IgM
IgG
ViraStripe
IgM
IgG
a
b
93.3 (88.9–96.0)
81.1 (70.7–88.4)
86.1 (82.3–88.5)
74.3 (66.3–82.9)
78.9 (72.6–84.0)
77.0 (66.3–85.1)
92.8 (88.3–95.7)
86.5 (76.9–92.5)
86.1 (82.3–88.5)
81.1 (70.7–88.4)
77.8 (73.7–80.9)
81.1 (70.7–88.4)
79.6 (75.8–82.8)
88.3 (85.0–91.0)
85.5 (81.2–88.9)
97.5 (95.6–98.6)
92.0 (88.5–94.5)
95.7 (93.4–97.2)
83.6 (79.2–87.3)
93.2 (90.5–95.2)
85.8 (82.4–88.4)
96.0 (93.7–97.4)
92.0 (89.1–93.9)
93.5 (90.8–95.4)
37 (7.1)
40 (7.7)
8 (1.5)
12 (2.3)
8 (1.5)
15 (2.9)
Visual interpretation of MarDx MarBlot strips served as the gold standard for sensitivity and specificity results.
MarBlot results were analyzed by using the BLOTrix software application; ViraBlot and ViraStripe results were analyzed by using the ViraScan software application.
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2220
BINNICKER ET AL.
J. C
LIN
. M
ICROBIOL
.
TABLE 4. Clinical sensitivity and specificity of technologist-adjusted software interpretation among the CDC serum
performance panel (n 40)
No. (%) positive
a
by:
Clinical diagnosis
No. of
patients
CDC reference WB*
IgM
IgG
MarBlot (BLOTrix)†
IgM
IgG
ViraBlot (ViraScan)†
IgM
IgG
ViraStripe (ViraScan)†
IgM
IgG
Confirmed Lyme disease
b
Probable Lyme disease
c
Subtotal
Healthy donor
Clinical sensitivity
d
(%)
Clinical specificity
e
(%)
a
b
28
7
35
5
16 (57.1)
2 (28.6)
18
0 (0)
51.4
100.0
10 (35.7)
7 (100.0)
17
0 (0)
48.6
100.0
12 (42.9)
2 (28.6)
14
0 (0)
40.0
100.0
6 (21.4)
7 (100.0)
13
0 (0)
37.1
100.0
27 (96.4)
3 (42.9)
30
1 (20.0)
85.7
80.0
8 (28.6)
6 (85.7)
14
0 (0)
40.0
100.0
10 (35.7)
1 (14.3)
11
0 (0)
31.4
100.0
11 (39.3)
6 (85.7)
17
0 (0)
48.6
100.0
*, MarDx MarBlot (visual interpretation at the CDC); †, results obtained after technologist-adjusted software interpretation.
Physician-documented EM and/or positive culture for
Borrelia burgdorferi.
c
Positive LB serology and meeting CDC case definition.
d
The subtotal number compared to the total number of confirmed and probable Lyme disease cases (n 35).
e
The number of negative healthy donor results compared to the total number of healthy donors (n 5).
The technologist-adjusted BLOTrix results showed a sensitiv-
ity of 40.0% (14/35) for IgM and a sensitivity of 37.1% (13/35)
for IgG. In contrast, ViraScan analysis of the ViraBlot IgM and
IgG strips demonstrated sensitivities of 85.7% (30/35) and
40.0% (14/35), respectively, while the ViraStripe assays showed
sensitivities of 31.4% (11/35) for IgM and 48.6% (17/35) for
IgG. Each of the systems demonstrated 100% specificity for
IgM and IgG with the exception of the ViraBlot IgM assay,
which showed a specificity of 80% (4/5) (Table 4).
DISCUSSION
It is estimated that over 2.5 million LB serology tests are
performed annually in the United States (1, 18). In 2006, our
laboratory at the Mayo Clinic performed 75,478 LB serology
tests, with 37,338 (49.5%) of these being done by Lyme WBs.
These numbers indicate that LB serology continues to play an
important role in the diagnosis of the disease. Furthermore,
these data emphasize the need to improve the efficiency of
Lyme WB processing, reading, and interpretation due to the
significant time and effort required by laboratory personnel.
A significant limitation of current Lyme WB testing is the
subjectivity involved in the visual reading and interpretation of
test strips. Preliminary studies in our laboratory have demon-
strated considerable variation in Lyme WB results when strips
are visually read by different laboratory technologists (M. Bin-
nicker, unpublished data). This variation in WB results may
contribute to inaccurate diagnoses, resulting in various conse-
quences to patients, as described in past studies (4, 13, 14, 17).
Therefore, an important need of clinical laboratories is to
enhance the objectivity and consistency of Lyme WB interpre-
tation.
Our findings show substantial agreement between the results
of automated and visual interpretation of Lyme WBs. Both
systems we evaluated demonstrated comparable results, excel-
lent reproducibility (data not shown), and similar features and
total average assay times (Table 5). We should emphasize that
both systems are designed to aid in band identification and
result interpretation and yet require a laboratory technologist
to review and verify results prior to reporting. In our experi-
ence, the ViraScan software application was more intuitive to
operate and required fewer result modifications by the review-
ing laboratory technologist (Table 3). This difference may be
due, in part, to the specific manufacturer’s recommended cut-
off settings used in our evaluation. Clinical laboratories should
perform their own thorough evaluation prior to implementing
an automated system, since the appropriate cutoff settings may
differ between regions where Lyme disease is and is not
endemic.
The present study has several additional limitations. First,
the number of clinically characterized specimens tested was
limited and, therefore, no firm conclusions can be made re-
garding the accuracy of the automated interpretive systems.
Future studies should test a large panel of clinically defined
and laboratory-defined specimens in order to more accurately
determine the clinical sensitivity and specificity. Second, the
data presented here compare the qualitative interpretations
(positive versus negative) using the current CDC criteria and
do not focus on a direct comparison of individual bands. How-
ever, we observed very good correlation, overall, for the de-
tection of specific bands between automated and visual inter-
pretation. Interestingly, correlation seemed to be lowest for
the 41- and 58-kDa bands (data not shown), and this may be
due, in part, to their migration proximity to the 39- and 60-kDa
TABLE 5. Comparison of features between automated systems
System feature
TrinBlot/BLOTrix
BeeBlot/ViraScan
Software application
Total assay time/run (h)
a
Maximum run size
c
Scanning resolution (dpi)
d
IgG and IgM in single run
Electronic interface with LIS
e
Serum and conjugate control
bands on assay strip
a
BLOTrix
2.62
50
104
No
Yes
Yes
ViraScan
2.65
b
50
220
Yes
Yes
Yes
This result represents the average time of three separate runs (adding strips
to the incubation tray through technologist review and verification of software
results; 40 specimens/run). The total assay time for the current protocol using
MarBlot strips (adding strips to the incubation tray through visual reading and
interpretation; 40 specimens/run) was 3.7 h.
b
The total assay time for either ViraBlot or ViraStripe.
c
The maximum run size for the current protocol (MarBlot strips on AutoBlot
6000) 60 strips.
d
dpi, dots per inch.
e
LIS, Laboratory Information System.
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V
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. 46, 2008
LYME WESTERN BLOT INTERPRETIVE SYSTEMS
REFERENCES
2221
antigens, respectively. These antigens are often difficult to dis-
tinguish by visual interpretation and may also require a more
detailed verification following the automated software inter-
pretation. A third limitation of the present study is that the
same laboratory technologist reviewed and verified the soft-
ware results for each specimen. Although this approach was
used for consistency, the decision to manually adjust software
results may vary between technologists. In order to minimize
subjectivity and result variability, it will be essential for testing
laboratories to establish specific criteria to guide and regulate
the modification of software results. A point of interest for
future studies will be to determine whether automated systems
decrease inter- and intralaboratory result variability in com-
parison to visual reading and interpretation.
In summary, automated systems showed results comparable
to those obtained by routine testing, while demonstrating sev-
eral advantages. First, the average turnaround time was re-
duced by 64 min/run, translating into a time savings of approx-
imately 1,000 h/year for clinical laboratories testing 37,000 to
40,000 specimens by WBs. Second, automated systems yield an
approximate savings of 0.3 full-time equivalent in comparison
to routine testing with visual interpretation. Additional bene-
fits include the ability to electronically store data, share results
with clinicians, and interface results with the Laboratory In-
formation System. Finally, automated systems allow for a more
objective interpretation of test strips, which may prove to sig-
nificantly enhance the consistency of Lyme WB results.
ACKNOWLEDGMENTS
We thank the laboratory technologists and assistants in the Infec-
tious Diseases Serology laboratory at the Mayo Clinic, who provided
excellent laboratory and technical support during this study. We also
thank Joseph Yao for critical review of the manuscript. The reagents
and kits used in this study were provided by Trinity Biotech and
Viramed Biotech AG.
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1533935_0040.png
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RESEARCH LETTER
Detection of
Borrelia burgdorferi sensu lato
DNA by PCR in serum
of patients with clinical symptoms of Lyme borreliosis
Iolanda Santino, Francesca Berlutti, Fabrizio Pantanella, Rosa Sessa & Massimo del Piano
Department of Public Health Sciences, Sapienza University of Rome, Rome, Italy
Correspondence:
Iolanda Santino,
Department of Public Health Sciences,
Sapienza University of Rome, P.le Aldo Moro
5, 00185 Rome, Italy. Tel.:
139
6 49914622;
fax:
139
6 49914634; e-mail:
[email protected]
Received 30 October 2007; accepted 15
February 2008.
First published online 1 April 2008.
DOI:10.1111/j.1574-6968.2008.01134.x
Editor: Reggie Lo
Keywords
Borrelia burgdorferi
; PCR; serum samples;
diagnosis.
Abstract
Lyme borreliosis is a disease caused by spirochaetes belonging to the genospecies
complex
Borrelia burgdorferi sensu lato
(s.l.) transmitted by
Ixodes
ticks. At present,
serology remains the main diagnostic tool for laboratory diagnosis of Lyme
borreliosis. Recently, the PCR technique has been applied for diagnosis of
B. burgdorferi s.l.,
but, until now, a reliable, easy-to-perform and sensitive method
has not been described. Here we present a new PCR-based method for the detection
of both
B. burgdorferi s.l.
and
Borrelia
genospecies DNAs in serum samples collected
from patients showing Lyme disease symptoms. Of 265 serum samples of patients
included in this study, 7.5% were positive, 1.9% was borderline and 90.6% were
negative for antibodies against
B. burgdorferi
by enzyme-linked immunosorbent
assay and Western blotting. The
B. burgdorferi s.l.
16S rRNA gene was detected by
PCR in all serum-positive and in two borderline samples. None of the serum-
negative samples nor serum samples collected from healthy subjects gave positive
PCR reactions. Of PCR-positive serum samples, 50% gave a positive reaction for
Borrelia afzelii,
18% for
Borrelia garinii
and 23% for two
Borrelia
species. Two
samples (9%) were not identified to species level. The new protocol could be
considered to be reliable as neither false-positive nor false-negative reactions were
recorded, and to be sensitive as it detects DNA from one bacterial cell.
Introduction
Lyme borreliosis is an infectious disease caused by
spirochaetes belonging to the genospecies complex
Borrelia burgdorferi sensu lato
(s.l.) transmitted by
Ixodes
ticks.
Three genospecies,
Borrelia afzelii, Borrelia garinii
and
B. burgdorferi sensu stricto
(s.s.), are widely distributed in
Europe causing human borreliosis (van Dam
et al.,
1993).
Each species is correlated with distinct clinical manifesta-
tions:
B. garinii
is predominantly associated with neurologi-
cal symptoms,
B. afzelii
with late skin manifestations and
B. burgdorferi s.s.
with arthritis. Other
Borrelia
genospecies,
such as
Borrelia valaisiana,
have been isolated from ticks in
Europe and are involved in human Lyme borreliosis (Escuredo
et al.,
2000). In addition,
Borrelia spielmanii
has been
isolated from a patient affected by erythema migrans (Wang
et al.,
1999) and
Borrelia lusitaniae
has been isolated from a
patient for the first time in 2004 in Europe (Collares-Pereira
et al.,
2004).
c
Journal compilation

2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. No claim to original Italian government works
At present, borreliosis is diagnosed mainly on the basis of
clinical symptoms and serological tests. These tests, based on
demonstration in human serum of anti-B.
burgdorferi s.l.
IgG and IgM antibodies, are usually carried out by enzyme-
linked immunosorbent assay (ELISA) and Western blot tests
(Aguero-Rosenfeld
et al.,
2005). Unfortunately, serological
tests have a poor reliability for the identification of different
Borrelia
genospecies, such as
B. afzelii, B. garinii
and
B. burgdorferi s.s.
Misdiagnosis (both false-positive and
false-negative) is frequent due to technical reasons
(Niscigorska
et al.,
2003).
The PCR technique has been applied for the diagnosis of
B. burgdorferi s.l.
in human serum samples (Schmidt, 1997).
Although the recovery of
B. burgdorferi s.l.
DNA in clinical
samples represents an appealing alternative tool, the low
number of spirochetes in blood, plasma and serum could
represent a serious problem for PCR reliability (Aguero-
Rosenfeld
et al.,
2005).
In order to develop a reliable PCR-based method,
widely applicable to the identification of various
Borrelia
FEMS Microbiol Lett
283
(2008) 30–35
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Detection of
Borrelia burgdorferi
by PCR
31
genospecies, several protocols have been attempted and
DNA target sequences have been used (Portnoi
et al.,
2006).
However, until now, an easy-to-perform and sensitive
method has not been described.
Here we present a new PCR protocol able to detect both
B. burgdorferi s.l.
and
Borrelia
genospecies DNAs in serum
samples collected from patients showing symptoms of Lyme
disease. In particular, the new protocol demonstrated both
high sensitivity, giving a PCR-positive reaction with
Borrelia
DNA extracted from human serum samples containing
1 bacterium mL
À1
, and 100% specificity, as neither false-
negative nor false-positive reactions were detected.
Serological tests
The presence of specific IgM and/or IgG antibodies against
B. burgdorferi s.l.
was determined by ELISA (recomWell
IgM/IgG, Mikrogen, Neuried, Germany) and Western blot
(ViraBlot IgM/IgG, Viramed Biotech AG) tests. Recombi-
nant proteins specific for
B. burgdorferi s.l.
were used as
positive control.
ELISA tests were considered negative, positive or border-
line when values were lower than 20, higher than 24 or
between 20 and 24 U mL
À1
, respectively, as suggested by the
manufacturer.
Detection of
B. burgdorferi s.l
. DNA by PCR
Materials and methods
Bacterial isolates and culture conditions
Borrelia burgdorferi
B31,
B. afzelii
and
B. garinii,
kindly
provided by the Department of Infectious, Parasitic and
`
Immune-Mediated Diseases, Istituto Superiore di Sanita,
Rome, Italy, were used in this study. All strains were cultured
in BSKII medium (Sigma Chemical Co., St. Louis, MO) at
34
1C.
Growth was checked by dark-field microscopy.
Patient samples
A total of 265 human serum samples were collected during
January 2005 and April 2007 at the Policlinico Umberto I
Hospital, Sapienza University of Rome, from patients pre-
senting clinical manifestations of Lyme borreliosis, includ-
ing early manifestations (erythema migrans, fever, malaise,
fatigue, skin rash, arthralgia, myalgia) or later manifesta-
tions of Lyme disease (severe arthritic, neurologic and
cardiac manifestations). Twenty serum samples collected
from healthy subjects were used as negative controls. All
serum samples were stored at
À
20
1C
until use.
Borrelia
DNA was extracted from human serum samples
according to the following experimental protocol (Protocol
A): 100
mL
of human serum samples was incubated in the
presence of 200
mL
ammonium hydroxide (0.7 M) at 100
1C
for 5 min in a 1.5-mL tube, followed by 10 min at 100
1C
with the tube open.
Borrelia
DNA precipitation was ob-
tained by adding 2 volumes of ethanol and 0.1 volume of
3 M sodium acetate. The samples were centrifuged at
12 000
g
for 15 min and DNA rinsed with 70% ethanol. After
centrifugation, DNA samples were air dried, suspended in
100
mL
Tris-EDTA (TE) buffer and stored at
À
70
1C
until
use. In comparative experiments
Borrelia
DNA was ex-
tracted from human serum samples following the protocol
of Guy & Stanek (1991) (Protocol B). PCRs were performed
using 16S rRNA gene PCR primers described by Marconi &
Garon (1992) and Liebisch
et al.
(1998). In particular, the
primer sets LD, BB, BG, BA and BV were specific for
B. burgdorferi s.l., B. burgdorferi s.s., B. garinii, B. afzelii
and
B. valaisiana,
respectively (Table 1). PCR reactions were
performed in a reaction volume of 25
mL
containing
12.5 pmol of appropriate primer set and 1
mL
of bacterial
DNA extracted from human serum samples as described
above. Companion PCR experiments were carried out using
Table 1.
PCR primer sets used in this study
Primer set
LD
BB
BG
BA
BV
Sequence (5 –3 )
ATGCACACTTGGTGTTAACTA
GACTTATCACCGGCAGTCTTA
GGGATGTAGCAATACATTC
ATATAGTTTCCAACATAG
GGGATGTAGCAATACATCT
ATATAGTTTCCAACATAGT
GCATGCAAGTCAAACGGA
ATATAGTTTCCAACATAGC
GCAAGTCAAACGGGATGTAGT
GTATTTTATGCATAGACTTATATG
0
0
Amplimer (bp)
357
574
574
591
549
Annealing
temperature (1C)
50
50
44
46
49
Reference
Marconi & Garon (1992)
Marconi & Garon (1992), erratum (1993)
Marconi & Garon (1992)
Marconi & Garon (1992), erratum (1993)
Liebisch
et al.
(1998)
LD,
Borrelia burgdorferi sensu lato;
BB,
Borrelia burgdorferi sensu stricto;
BG,
Borrelia garinii;
BA,
Borrelia afzelii;
BV,
Borrelia valaisiana.
FEMS Microbiol Lett
283
(2008) 30–35
c
Journal compilation

2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. No claim to original Italian government works
 SUU, Alm.del - 2014-15 (1. samling) - Bilag 1413: Henvendelse af 24/5-15 fra Alex Holmstedt vedr. behandling af borrelia
1533935_0042.png
32
I. Santino
et al.
as template DNA extracted from
Borrelia
strains, i.e.
B.
burgdorferi
B31,
B. afzelii, B. garinii. Borrelia
strains were
cultured in BSK-II medium for 3 days at 34
1C.
After incuba-
tion, bacterial cells were treated for DNA extraction according
to the protocol outlined above (Rosa & Schwan, 1989).
PCR amplifications were performed using a Perkin-Elmer
Cetus thermocycler by denaturing the template DNA for
1 min at 94
1C,
with extension for 1.5 min at 72
1C
for 35
cycles. Annealing temperatures ranged from 44 to 50
1C
for
the different primer sets as detailed in Table 1. Amplification
products were visualized after electrophoresis with 10
mL
of
the PCR reaction volume in 1.0% agarose gels in TAE buffer
[40 mM Tris-acetate, 2 mM EDTA (pH 8.5)] containing
ethidium bromide at 0.5 pg mL
À1
.
In order to confirm the PCR-based species attribution,
amplimers obtained with species-specific primer sets were
digested with HindIII and SacII restriction enzymes.
To determine PCR sensitivity, serum samples from two
healthy subjects were pooled and divided into 1-mL ali-
quots. Each aliquot was infected by 10-fold serial dilutions
of
B. burgdorferi
B31 live cells. DNA was extracted from
100
mL
infected serum samples following the methods
described above (protocols A and B) and PCRs were
performed using the LD (B.
burgdorferi s.l.)
primer set.
Table 2.
PCR sensitivity of
Borrelia burgdorferi
B31 DNA detection in
human serum samples
B. burgdorferi
Serum sample
DNA used
BB primer set PCR
B31 (CFU mL
À1
treated for DNA as PCR
of serum)
extraction (mL)
template (mL) Protocol A Protocol B
100 000
10 000
1000
100
10
1
None
100
100
100
100
100
100
100
1
1
1
1
1
1
1
1
1
1
À
À
À
À
1
À
À
À
À
À
À
The 16S rRNA gene BB primer set specific for
B. burgdorferi s.s.
was
employed (see Table 1).
Table 3.
PCR detection of
Borrelia burgdorferi s.l.
DNA in human serum
samples
PCR
Serological
tests
No. tested Positive Negative
20
10
5
20
1
20
0
2
0
1
0
10
3
20
0
Subjects
With clinical manifestations Positive
Ã
Negative
Borderline
Healthy subjects
Negative
Positive control
Ã
Positive,
4
24 U mL
À1
; negative,
o
20 U mL
À1
; borderline, between 20
Results
To confirm PCR reliability, preliminary experiments were
carried out using DNA samples extracted from reference
Borrelia
strains. As expected, PCRs performed using ex-
tracted DNA from cultured strains and LD and genospecies-
specific primer sets were positive (data not shown).
To test PCR sensitivity,
Borrelia
DNA was extracted from
serial dilutions of
B. burgdorferi
B31 culture in serum
samples following both protocols A and B. PCRs were
performed using the BB primer set (Table 2). Using DNA
extracted following Protocol A, PCR reactions were positive
on serum samples infected with 10
5
, 10
4
and 10
3
CFU mL
À1
;
following Protocol B, PCR was positive on serum sample
infected with 10
5
UFC mL
À1
. As PCRs were performed using
1
ml
of extracted DNA, the sensitivity was equal to 1 and 100
bacterial cells for protocols A and B, respectively.
In order to investigate the possibility of increasing the
sensitivity of Protocol A, a further set of experiments were
carried out. The amount of serum to be treated was raised to
200
mL
and the volume of TE to suspend
Borrelia
DNA was
reduced to 25
mL.
The results did not indicate a significant
increase in PCR sensitivity (data not shown).
Of the 265 patient samples included, 20 (7.5%) were positive,
five (1.9%) were borderline and 240 (90.6%) were negative for
antibodies against
B. burgdorferi
by ELISA and Western blotting.
Twenty seropositive, five borderline and nine seronegative
samples, as well as 20 serum samples collected from healthy
c
Journal compilation

2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. No claim to original Italian government works
and
Z24
U mL
À1
.
subjects were analyzed for the presence of
B. burgdorferi s.l.
DNA by PCR (Table 3).
Borrelia burgdorferi s.l.
DNA was
found in all 20 seropositive and in two of the five borderline
serum samples. All nine seronegative as well all 20 serum
samples from healthy subjects gave negative PCRs.
PCR reactions with species-specific primers for
B. burg-
dorferi s.s., B. afzelii, B. garinii
and
B. valaisiana
were carried
out on the 22
B. burgdorferi s.l.
PCR-positive serum samples.
Fifteen samples (68%) contained DNA from a single
Borrelia
genospecies, and five samples (23%) showed the presence of
DNA from two different genospecies. The genospecies
attribution could not be made for two serum samples (9%)
positive for
B. burgdorferi s.l.
as PCRs performed with
genospecies-specific primers were negative (Fig. 1). None
of the serum sample was positive for
B. burgdorferi s.s.
In order to confirm the PCR-based species attribution,
amplimers obtained with species-specific primer sets were
digested with HindIII and SacII restriction enzymes. Results
showed restriction fragments consistent with species-
specific 16S rRNA gene sequences (data not shown).
The distribution of
Borrelia
genospecies in relation to
clinical manifestations is shown in Table 4. Of particular
note, 16 patients had early clinical manifestations and six
late clinical manifestations.
FEMS Microbiol Lett
283
(2008) 30–35
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1533935_0043.png
Detection of
Borrelia burgdorferi
by PCR
33
B. afzelii
50%
B. garinii
18%
Unknown
9%
Borrelia
spp.
coinfection
23%
Fig. 1.
Borrelia burgdoferi
DNA genospecies recovered in human serum
samples.
Table 4.
Distribution of
Borrelia
genospecies in human serum samples
in relation to clinical manifestations
Genospecies
Clinical manifestation
Early
B. afzelii
B. garinii
B. afzelii
and
B. garinii
B. afzelii
and
B. valaisiana
B. garinii
and
B. valaisiana
Unknown
Total
7
4
3
À
1
1
16
Late
4
À
À
1
À
1
6
Total
11
4
3
1
1
2
22
Discussion
The aim of the present study was to improve the PCR-based
protocol for identification of
B. burgdorferi s.l.
from human
serum samples and to compare the diagnostic value of PCR
with that of serology. At present, serology remains the main
diagnostic tool for laboratory diagnosis of Lyme borreliosis.
However, in the early phase of infection, the level of
antibodies against
B. burgdorferi
is low. False-negative
results occur primarily during the first weeks of infection
(Hofmann, 1996). Culturing
B. burgdorferi
from body fluids
is difficult because of the long incubation period, the high
cost of culture medium and small proportion of positive
results (Aguero-Rosenfeld
et al.,
2005). Therefore, the need
for a reliable diagnostic method is evident (Hofmann,
1996). As PCR has been considered as a sensitive tool for
identification of fastidious microorganisms that are difficult
to culture, attempts have been made to apply this technique
to detect
Borrelia
DNA in human serum (Niscigorska
et al.,
2003).
Here we present a simple, easy-to-perform, and reliable
PCR protocol for the identification of
Borrelia
from human
serum samples. The protocol involves a simple method to
extract
Borrelia
DNA from human serum samples. Several
PCR methods do indeeds yield acceptable results when
Borrelia
DNA is extracted from skin biopsy of patients with
FEMS Microbiol Lett
283
(2008) 30–35
Erythema chronicum migrans (ECM) or synovial fluid in
patients with Lyme arthritis (Aguero-Rosenfeld
et al.,
2005).
In the present study, the extracted DNAs were used to
perform PCRs using different primer sets to identify
Borrelia
to genospecies level. We have chosen to employ the pre-
viously described primer sets that amplify the 16S rRNA
gene. It is well known that these primer sets are specific
for
B. burgdorferi s.l.
and
B. burgdorferi
genospecies
(Liebisch
et al.,
1998; Marconi & Garon, 1992). Our proto-
col has been shown to be more sensitive than a previously
published method, giving a positive PCR reaction using
DNA from 1 as compared with 10
3
bacterial cells (Portnoi
et al.,
2006). Sensitivity similar to that for our protocol has
been reported by Joss
et al.
(2008), who described a real-
time PCR method to detect
Borrelia
from serum samples.
However, it is well known that real-time PCR is costly,
laborious to perform and, moreover, requires the use of
sophisticated apparatus.
The protocol proposed shows 100% reliability: no false-
positive or false-negative reactions were recorded. In fact,
our protocol recognized as positive only those serum
samples positive by ELISA and Western blotting and two
serum samples from subjects with early symptoms that give
borderline reactions in serological tests. This result confirms
the poor reliability of serological tests in early
Borrelia
infections (Riesbeck & Hammas, 2007). Moreover, the
protocol we have described allowed us to identify at species
level
Borrelia
DNA in human serum samples. To confirm the
reliability of the proposed method, we have also analyzed the
PCR amplimers obtained by digesting them with restriction
enzymes. As expected, DNA fragments obtained by restric-
tion enzymes were consistent with PCR amplification and
confirmed the PCR-based
Borrelia
identification.
In the present study, 7.5% of the patients with suspected
Lyme borreliosis had antibodies against
B. burgdorferi
in
blood serum.
Borrelia burgdorferi
DNA was detected by PCR
in all serum samples from seropositive patients (100%) and
from two (22%) seronegative subjects. Of note, 72.7% of
patients with positive PCR showed symptoms referable to
early borreliosis, while only 27.3% of PCR-positive patients
showed late borreliosis. Moreover, two borderline patients
manifesting early symptoms as erythema migrans were PCR
positive. These results are in agreement with those of Guy &
Stanek (1991), underlining that the PCR method can con-
tribute to the reliability of diagnosis during early stages of
infection.
In the present study, the frequency of recovery of
Borrelia
DNA was equal to 8.3% (22 positive of 265 serum samples).
Variable frequency of recovery values have been reported in
the past, ranging from 76 to 3.8% (Oksi
et al.,
1999, 2001;
Kondrusik
et al.,
2004; Chmielewska-Badora
et al.,
2006).
These discrepancies are probably due to the frequency of
infection and to the different protocols used in performing
c
Journal compilation

2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. No claim to original Italian government works
 SUU, Alm.del - 2014-15 (1. samling) - Bilag 1413: Henvendelse af 24/5-15 fra Alex Holmstedt vedr. behandling af borrelia
34
I. Santino
et al.
PCR reactions, underlining the need to develop a reliable
PCR protocol.
Regarding genospecies identification,
B. afzelii
was the
most frequent species (50%, 11 of 22 serum samples) while
B. burgdorferi s.s.
DNA was not recovered. Interestingly, 23%
of the samples were positive for two different
Borrelia
species. Several studies indicate that different
Borrelia
spe-
cies may be associated with specific reservoir hosts and with
distinct clinical manifestations of Lyme disease (van Dam
et al.,
1993; Balmelli & Piffaretti, 1995). Therefore,
Borrelia
identification at genospecies level in patients with Lyme
borreliosis appears to be of epidemiological, pathogenetic
and diagnostic importance.
Mixed infections with two or more
Borrelia
species are
frequent in ticks (Santino
et al.,
1998; Santino
et al.,
2003)
and have been reported also in human patients
(Ruzic-Sablijic
et al.,
2005). The present study indicates that
human patients with Lyme borreliosis can simultaneously
harbor different
B. burgdorferi s.l.
strains and that these
infections are mainly double infections with the presence of
B. garinii
and
B. afzelii
or
B. valaisiana.
In conclusion, the protocol described here could be
usefully employed in PCR-based
Borrelia
identification from
human serum samples.
Acknowledgements
This work was supported by Faculty 60% funds granted to
I.S. We thank Lorenzo Ciceroni of the Department of
Infectious, Parasitic and Immune-Mediated Diseases, Istitu-
`
to Superiore di Sanita, for kindly providing some of the test
isolates. We also thank Cristina Iori for excellent technical
assistance.
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c
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Retur til dok liste
Seronegativity in Lyme borreliosis and Other Spirochetal Infections
16 September 2003
“If false results are to be feared, it is the false negative result
which holds the greatest peril for the patient.”
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Author
Year
Title
Journal
Borrelia burgdorferi
1.
Dejmkova H;
Hulinska D;
Tegzova D;
Pavelka K;
Gatterova J;
Vavrik P.
2002
Seronegative Lyme arthritis caused by Borrelia garinii.
Clinical Rheumatology, 21(4):330-4
[From the abstract:] “A case of a female patient suffering from Lyme arthritis (LA) without elevated antibody levels to Borrelia burgdorferi sensu lato is reported.
Seronegative Lyme arthritis was diagnosed based on the classic clinical manifestations and DNA-detected Borrelia garinii in blood and synovial fluid of the patient,
after all other possible causes of the disease had been ruled out. The disease was resistant to the first treatment with antibacterial agents. Six months after the therapy,
arthritis still persisted and DNA of Borrelia garinii was repeatedly detected in the synovial fluid and the tissue of the patient. At the same time, antigens or parts of
spirochaetes were detected by electron microscopy in the synovial fluid, the tissue and the blood of the patient. The patient was then repeatedly treated by antibiotics
and synovectomy has been performed.”
2002
Wien Klin Wochenschr, 114(13-14):601-5
2.
Tylewska-
Wierzbanowska S;
Chmielewski T;
Limiation of serologic testing for Lyme borreliosis: evaluation of ELISA and western blot in comparison
with PCR and culture methods.
[From the abstract:] “No correlation was found between levels of specific B. burgdorferi antibodies detected with a recombinant antigen ELISA and the number of protein
fractions developed with these antibodies by immunoblot. Moreover, Lyme borreliosis patients who have live spirochetes in body fluids have low or negative levels of borrelial
antibodies in their sera. This indicates that an efficient diagnosis of Lyme borreliosis has to be based on a combination of various techniques such as serology, PCR and
culture, not solely on serology.” [Testing was performed on samples from 90 patients.]
3.
Breier F;
Isolation and polymerase chain reaction typing of Borrelia afzelii from a skin lesion in a
2001
Br J Dermatol, 144(2):387-392
Khanakah G;
seronegative patient with generalized ulcerating bullous lichen sclerosus et atrophicus.
Stanek G; Kunz G;
Aberer E;
[From the abstract:] “Spirochaetes were isolated from skin cultures obtained from enlarging LSA lesions. These spirochaetes were identified as Borrelia afzelii
by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and polymerase chain reaction (PCR) analyses. However, serology for B. burgdorferi
Schmidt B;
sensu lato was repeatedly negative."
Tappeiner G.
2001
J Immunol Methods, 249(1-2):185-190
4.
Brunner M.
New method for detection of Borrelia burgdorferi antigen complexed to antibody in
seronegative Lyme disease.
[From the abstract:] "...serologic tests for early Lyme disease can be falsely negative due to lack of sensitivity of ELISAs and Western blots. Most routine antibody tests are
designed to detect free antibodies, and in early, active disease, circulating antibodies may not be free in serum but sequestered in complexes with the antigens which
originally triggered their production. This difficulty may be overcome by first isolating immune complexes (IC) from the serum and using this fraction for testing. Free Borrelia-
specific antibodies can then be liberated from the immune complexes which may enhance test sensitivity in patients with active disease. We developed a technique that
captures the antibody component of IC on immunobeads, and subsequently releases the antigen component of IC. Immunoblotting with monoclonal antibody detected at least
one antigen to be OspA, thus definitively demonstrating a Borrelia-specific antigen in circulating IC in early Lyme disease. This test is also useful in demonstrating Bb antigen
in otherwise seronegative Lyme disease patients."
Page 1 of 17
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1533935_0047.png
Author
5.
Wang P;
Hilton E.
Year
2001
Title
Contribution of HLA alleles in the regulation of antibody production in Lyme disease.
Journal
Front Biosci, 6:B10-B16
"Of eighteen seronegative LD patients, 14 were OspA PCR positive on mononuclear cells and 5 were positive on CSF. ...The presence of certain HLA alleles with
seronegativity to disease has been reported in malaria (10), HIV (16,17), rheumatoid arthritis (RA) (18) and spondylarthropathies (SpA) (19). ...Our results provide
evidence of a correlation between certain HLA genotypes and the ability to mount an antibody response to Bb. In this study, 9 of 22 (40.9%) seropositive LD patients
and only 1 out of 18 (5.6%) seronegative LD patients had HLA-DR7 alleles. ...
Our study provides evidence that HLA alleles are involved in antibody responsiveness or non-responsiveness to Bb infection. A low frequency of HLA-DR7 alleles
and HLA-DR6 alleles and a high frequency of HLA-DR1 alleles may contribute to non-responsiveness of antibody production in LD patients. Thus, genetic
predisposition may be a critical factor in the regulation of the host immune response and the diagnosis and prognosis of Lyme disease.”
6.
Grignolo MC;
Buffrini L;
Monteforte P;
Rovetta G.
2001
Reliability of a polymerase chain reaction (PCR) technique in the diagnosis of Lyme
borreliosis.
Minerva Med, 92(1):29-33
[From the abstract:] "50% of the PCR positive results, obtained with serum and cerebrospinal fluid samples corresponded to patients who were true
positives at clinical examination but negatives at serologic tests. 62.5% of urine samples positive results belonged to tp patients who had negative serologic
and serum PCR RESULTS. CONCLUSIONS: The obtained results suggested a good reliability of positive results obtained with the PCR technique used in
this study and allowed the false negatives of serologic tests to be detected, more specifically when urine samples were used."
2001
Intralaboratory reliability of serologic and urine testing for Lyme disease.
American Journal of Medicine, 110(3):217-19
7.
Klempner MS;
Schmid CH; Hu L;
Steere AC;
Johnson G;
McCloud B;
Weinstein A.
Honegr K;
Hulinska D;
Dostal V;
Gebousky P;
Hankova E;
et al.
“
In the 21 patients with Lyme disease, the results of the initial western blot analysis were positive in 14 cases and negative in 7. ...
Repeat testing of the 7 seronegative samples showed fewer than 5 reactive bands in all samples.”
2001
[Persistence of Borrelia burgdorferi sensu lato in patients with Lyme borreliosis].
Epidemiol Mikrobiol Imunol, 50(1):10-6
8.
[From the abstract:] “In 18 patients with Lyme borreliosis the authors proved the persistence of Borrelia burgdorferi sensu lato by detection of the causal agent by
immune electron microscopy or of its DNA by PCR in plasma or cerebrospinal fluid after an interval of 4-68 months. ...Examination of antibodies by the ELISA method
was negative in 7 of 18 patients during the first examination and in 12 of 18 during the second examination. In all negative examinations the specific antibodies were
assessed by the Western blot or ELISA method after liberation from the immunocomplexes."
2001
MMW Fortschr Med, 143(6):17
9.
Paul A.
[Arthritis, headache, facial paralysis. Despite negative laboratory tests Borrelia can still be
the cause.]
10.
Pleyer U; Priem S;
Bergmann L;
Burmester G;
Hartmann C;
Krause A.
2001
Detection of Borrelia burgdorferi DNA in urine of patients with ocular Lyme borreliosis.
Br J Ophthalmol, 85(5):552-5
[From the abstract:] "RESULTS: Only four of six uveitis patients suspected for Lyme borreliosis were ELISA positive, while all six subjects showed a positive western blot.
B burgdorferi PCR was positive in all of these six patients. Whereas two of the 30 controls had a positive Lyme serology, B burgdorferi DNA was not detectable by PCR in any
sample from these patients. CONCLUSIONS: PCR for the detection of B burgdorferi DNA in urine of uveitis patients is a valuable tool to support the diagnosis of ocular Lyme
borreliosis. Moreover, these patients often show a weak humoral immune response which may more sensitively be detected by immunoblotting.”
2001
[Lyme neuroborreliosis in More and Romsdal].
Tidsskrift for Den Norske Laegeforening,
121(17):2008-11.
11.
Eldoen G;
Vik IS; Vik E;
Midgard R.
[From the abstract:] "Fourteen of 25 (56%) patients had positive Borrelia burgdorferi-IgM and IgG titres in cerebrospinal fluid despite negative tests in serum."
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Author
12.
Brunner M;
Sigal LH.
Year
2000
Title
Immune complexes from serum of patients with Lyme disease contain Borrelia burgdorferi
antigen and antigen-specific antibodies: potential use for improved testing.
Journal
Journal of Infectious Diseases, 182(2):534-9
[From the abstract:] "We report sequestration of specific IgM anti-Borrelia burgdorferi (Bb) and Bb antigens within immune complexes (ICs) isolated from serum of patients
with Lyme disease (LD). ...Immunoblot demonstrated that ICs contained antibodies against specific Bb proteins, whereas reactivity was absent or significantly lessened in
unprocessed serum."
13.
Kaiser R.
2000
False-negative serology in patients with neuroborreliosis and the value of employing of different
borrelial strains in serological assays.
J Med Microbiol, 49(10):911-5.
[Abstract:] “The risk of obtaining false-negative results in serological assays in serum and CSF specimens with only one strain of Borrelia burgdorferi sensu lato as
antigen was investigated in 79 patients with neuroborreliosis with specimens obtained at initial presentation. Serum antibodies were assessed by immunoblotting; the
criteria of Hauser et al. were used to evaluate the test. The intrathecal synthesis of borrelial-specific IgM and IgG antibodies was examined by enzyme immunoassay
(EIA). Strains of B. burgdorferi sensu stricto (BbZ160), B. garinii (Bbii50) and B. afzelii (PKO) served as sources of antigen in both assays. All patients produced
either a positive IgM or IgG test in serum with at least one strain of B. burgdorferi sensu lato. Reactivity of IgM or IgG antibodies, or both, with antigens of all three
strains was demonstrated in 67 (85%) of 79 sera. The correlation of results of immunoblotting with different strains was significantly better for IgG (85%) than for IgM
antibodies (54%). The variability of positive IgM reactions in 18 specimens was mainly due to the fact that the antibodies were directed to the relevantvariable outer-
surface protein C (p23). Intrathecal synthesis of IgG antibodies was demonstrated in 58 patients (81%) of 72 and of IgM antibodies in 25 of 58 patients. No patient
had isolated intrathecal synthesis of IgM antibodies. The majority of CSF samples (56 of 58) were assessed as IgG antibody-positive, independent of the borrelial
strain used as antigen in EIA, whereas only 10 of 25 IgM antibody-positive CSF specimens reacted with all three strains. All patients in the study had intrathecal
antibody synthesis demonstrable at 6-week follow-up. From this study it is concluded that there is a small, but real, risk of false-negative serological findings at the
time of initial clinical presentation in patients with typical symptoms of neuroborreliosis. In these patients a negative serological result with one strain should prompt
the repetition of the test with other strains of B. burgdorferi sensu lato.”
14.
Kmety E.
2000
[Dynamics of antibodies in Borrelia burgdorferi sensu lato infections.]
Bratisl Lek Listy, 101(1):5-7
[From the abstract:] "...During 1994-1998 at least two serum samples were submitted for serological testing from more than 1200 patients. An
immunofluorescence test was performed paralelly [sic] with two pools of antigen (B. bg.s.s. + B. afzelii, and two serological different strains of B. garinii, all of local origin).
In 92-96% of patients no change of antibody level was found in repeated tests, about 20% of them being negative (< 1:512).
...Only in 9 cases a rise of the titer appeared during 3 weeks after the first negative sample, at contrary in 7 cases no rise of the titer was seen in that time. 2 patients
were still after 1 month, 3 after 3 months and 1 even after 7 months (patient with a positive CSF culture) serologically negative."
15.
Wilke M;
Eiffert H;
Christen HJ;
Hanefeld F.
2000
Arch Dis Child, 83(1):67-71.
Primarily chronic and cerebrovascular course of Lyme neuroborreliosis: case reports and literature review.
"In this context, even the complete absence of specific antibodies has been observed; in a girl diagnosed as having focal vasculitis through CNS biopsy, the presence
of B burgdorferi in CSF was confirmed by polymerase chain reaction. No specific antibodies were detectable. In three other children, B. burgdorferi could be cultured
from CSF in the absence of specific antibodies in CSF or blood."
2000
Evaluation of a commercial enzyme-linked immunosorbent assay for detection of Borrelia
burgdorferi exposure in dogs.
J Am Vet Med Assoc, 216(9):1418-22
16.
Sheets JT; Rossi
CA; Kearney BJ;
Moore GE.
"The commercial ELISA kit evaluated in this study appeared to lack adequate sensitivity for detecting all potential cases of borreliosis in dogs.”
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Author
Year
Title
Journal
Detection of Borrelia DNA in circulating monocytes as evidence of persistent Lyme disease.
17.
Wang P;
2000
J Spirochetal and Tick-borne Diseases,
Gartenhaus R;
7(1):16-19
Sood SK; DeVoti J;
[Abstract:] "We report the detection of Borrelia burgdorferi DNA in circulating monocytes in a 31-year-old female who presented with a flu-like syndrome followed by
Singer C; et al.
neurological abnormalities after a trip to Southampton, Long Island, New York. ELISA and Western blot were negative. Lymphocyte proliferation assay to Borrelia
burgdorferi was positive. Borrelia burgdorferi DNA was detected in circulating monocytes using a nested polymerase chain reaction (PCR). Treatment with parenteral
ceftriaxone resulted in clinical improvement and repeat PCR on monocytes was negative. The use of detecting DNA by PCR from circulating monocytes may be
useful in evaluating seronegative patients with a high suspicion of Lyme disease."
18.
Brown SL;
Hansen SL;
Langone JJ.
1999
Role of serology in the diagnosis of Lyme disease.
(FDA Medical Bulletin)
JAMA, 282(1): 62-65
"The
Food and Drug Administration (FDA) is concerned about the potential for misdiagnosis of Lyme disease based on the results of commonly marketed tests for
detecting antibodies to Borrelia burgdorferi, the organism that causes Lyme disease. It is important that clinicians understand that a positive test result does not
necessarily indicate current infection with B. burgdorferi, and a patient with active Lyme disease may have a negative test result.
The tests should be used only to support a clinical diagnosis of Lyme disease and should never be the primary basis for making diagnostic or treatment decisions.”
19.
Bertrand E; Szpak
GM; Pilkowska E;
Habib N; et al.
1999
Central nervous system infection caused by Borrelia burgdorferi.
Clinico-pathological correlation of three post-mortem cases.
Folia Neuropathol, 37(1):43-51
"Case 1: ...Specific borrelia IgM and IgG value in serum and CSF were normal (<250). However, on microscopical examination the spirochete B.
burgdorferi was demonstrated in serum and CSF. The bacteria were cultured both from blood and from CSF, in CSF they were also identified by PCR."
20.
Mikkila H, Karma
A, Viljanen M,
Seppala I.
1999
[The laboratory diagnosis of ocular Lyme borreliosis.]
Graefes Arch Clin Exp Ophthalmol,
237(3):225-30
"Seven patients, including two with negative ELISA, had a positive immunoblot. Seven of the 13 patients in whom PCR was examined during clinically active disease
had a positive PCR result. Immunoblot analysis gave a negative result from the sera of five PCR-positive patients. CONCLUSIONS: For efficient diagnosis of ocular
Lyme borreliosis, immunoblot analysis and PCR should be used in addition to ELISA."
21.
Oksi J;
Marjamaki M;
Nikoskelainen J;
Viljanen MK.
1999
Borrelia burgdorferi detected by culture and PCR in clinical relapse of disseminated Lyme
borreliosis.
Annals of Medicine, 31(3):225-32
"Three of the 13 patients had only IgM antibodies against B. burgdorferi, and one culture-positive patient was seronegative despite the disseminated stage of the disease.
The reason for the lack of IgG antibodies, or of both IgM and IgG antibodies, was not restriction of the infection to privileged sites, as all these patients had a multiorgan
disease. We have previously shown that patients with late LB with live spirochetes or borrelial DNA in their body fluids may have low or negative serum borrelia antibody
levels."
1998
Culture-positive Lyme borreliosis.
Med J Aust, 168(10):500-2
22.
Hudson BJ;
Stewart M;
Lennox VA;
et al.
[From the abstract:] "We report a case of Lyme borreliosis. Culture of skin biopsy was positive for Borrelia garinii, despite repeated prior treatment with
antibiotics.'
"The results of conventional serological and histopathological tests were negative, despite an illness duration of at least two years."
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Author
23.
McCaulley,
Mark E., M.D.
Year
1998
Title
Guidelines for the clinical diagnosis of Lyme disease.
Journal
Annals of Internal Medicine, 129(5): 422-423
[Letter to the Editor:] "The position paper on laboratory diagnosis of Lyme disease is based on a widely accepted paradigm that is inconsistent with a growing body
of medical literature. According to this paradigm, cases of Lyme disease are overwhelmingly seropositive and are unlikely to be associated with persistent symptoms
after presumed adequate therapy. In addition, any patients remaining persistently symptomatic are presumed to no longer have Lyme disease at all but rather to have
such conditions as fibromyalgia, depression, or the chronic fatigue syndrome and, as a result, to be unlikely to respond to additional antibiotic therapy. Such
presumptions are inconsistent with an increasing number of reports.
A 1994 article reports the increased frequency of multiple symptoms in previously treated patients with Lyme disease compared with controls. Antibodies on ELISA
were found in less than half of the patients with Lyme disease. Re-treatment was associated with improvement in half of re-treated patients. Had the guidelines been
followed in a clinical evaluation of these or similar patients, Lyme disease would have been diagnosed in few of them.
In a 1996 report, Borrelia burgdorferi plasmid DNA was detectable by polymerase chain reaction assay only in a subset of patients with Lyme disease who were
seronegative. Many case reports have described patients with Lyme disease who remain antigen positive and symptomatic despite intensive antibiotic treatment.
I suggest the acceptance of a new paradigm that incorporates the above information. Physicians involved in the treatment of Lyme disease should consider that 1)
Patients with Lyme disease, especially those in late stages of the disease, are frequently seronegative; 2) the persistence of symptoms, which may be vague, is
common and may respond to additional antibiotic therapy; and 3) there is much to be learned about the optimal treatment of Lyme disease at any stage."
24.
Petrovic M;
Vogelaers D; Van
Renterghem L;
Carton D; De
Reuck J;
Afschrift M.
1998
Lyme borreliosis - a review of the late stages and treatment of four cases.
Acta Clinica Belgica, 53(3):178-83
[From the abstract:] "Difficulties in diagnosis of late stages of Lyme disease include low sensitivity of serological testing and late inclusion of Lyme disease in the
differential diagnosis. Longer treatment modalities may have to be considered in order to improve clinical outcome of late disease stages...The different clinical cases
illustrate several aspects of late borreliosis: false negative serology due to narrow antigen composition of the used ELISA format, the need for prolonged antibiotic
treatment in chronic or recurrent forms and typical presentations of late Lyme disease, such as lymphocytic meningo-encephalitis and polyradiculoneuritis."
1997
http://www.medscape.com/CPG/ClinReviews/1997
/v07.n06/c0706.cnu/c0706.cnu.html#Lyme
25.
American
Academy of
Neurology 49th
Annual Meeting
April 12-19.
Lyme encephalopathy may surface despite antibiotic treatment.
"Of the 8 patients with CNS infection, only 2 were seropositive on both the ELISA and Western blot tests. Four had indeterminate ELISA results and a
negative Western blot, and 2 had negative results on both the ELISA and the Western blot. Neither of the 2 seropositive patients had received antibiotics during the
first month of infection for early localized or disseminated disease," said the Boston researchers. Of the 6 seronegative patients with CNS infection, however, 5 (84%)
had received a recommended course of oral or intravenous antibiotics during the first month of infection.'"
PCR evidence for Borrelia burgdorferi DNA in synovium in absence of positive serology.
26.
Branigan P; Rao J;
1997
American College of Rheumatology,
Rao J; Gerard H;
Vol 40(9), Suppl:S270
Hudson A;
Williams W;
"PCR evidence for Borrelia has been identified in synovial biopsies of patients with clinical pictures that had not initially suggested Lyme disease.
Arayssi T; Pando
All 6 PCR-positive] patients were negative for antibodies to Borrelia and some were PCR positive in synovium despite previous treatment with antibiotics."
J; Bayer M;
Rothfuss S;
Clayburne G;
Sieck M;
Schumacher HR.
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Author
27.
Donta ST.
Year
1997
Title
Tetracycline therapy for chronic Lyme disease.
Journal
Clin Infect Dis, Jul;25 Suppl 1:S52-6
"Treatment outcomes for seronegative patients (20% of all patients) were similar to those for seropositive patients. Western immunoblotting showed reactions to one
or more Borrelia burgdorferi-specific proteins for 65% of the patients for whom enzyme-linked immunosorbent assays were negative."
28.
Hauser U;
Wilske B.
1997
Enzyme-linked immunosorbent assays with recombinant internal flagellin fragments
derived from different species of Borrelia burgdorferi sensu lato for the serodiagnosis of
of Lyme.
Medical Microbiology & Immunology.
186(2-3):145-51
[From the abstract:] "The serodiagnosis of early Lyme neuroborreliosis is hampered by false negative results and one of the reasons could be the heterogeneity of
strains of Borrelia burgdorferi sensu lato."
29.
Pradella SP;
Krause A;
Muller A.
1997
Acute Borrelia infection. Unilateral papillitis as isolated clinical manifestation.
Ophthalmologe, Aug;94(8):591-4
[From the abstract:] "Seronegative values in subjects strongly suspected of having Lyme disease do not necessarily exclude the diagnosis of Lyme disease."
30.
Schumacher HR.
1997
PCR evidence for Borrelia burgdorferi DNA in synovium in absence of positive serology.
Abstract ACR 61st National Scientific Meeting
November 8-12
31.
Aberer E;
Kersten A; Klade
H; Poitschek C;
Jurecka W.
1996
Heterogeneity of Borrelia burgdorferi in the skin.
American Journal of Dermatopathology,
18(6):571-9
"Neuralgias arising 6 months after ECM in spite of antibiotic therapy were evident in a seronegative patient who showed perineural rod-like borrelia structures."
"The morphological forms of borreliae seen in biopsies were correlated with clinical findings. Seropositive patients showed clumped and agglutinated borreliae
in tissue, whereas seronegative patients exhibited borreliae colony formation (n=2). ...the behavior of borreliae within collagen fibers is strongly influenced by
immune recognition by the patient. Borrelia may escape immune surveillance by colony formation and masking within collagen, resulting in seronegativity."
32.
Breier P; Klade H;
Stanek G;
Poitschek C;
Kirnbauer R;
Dorda W;
Aberer E.
1996
Lymphoproliferative responses to Borrelia burgdorferi in circumscribed scleroderma.
Br J Dermatol, 134(2):285-91
"These findings show that the pattern of Bb-specific immune responses is more complex than previously thought, and underscore the importance of lymphocyte
function assays in evaluating the diagnosis of potential Bb infection in seronegative patients."
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Author
33.
Huppertz HI;
Mosbauer S;
Busch DH;
Karch H.
34.
Luft BJ.
Year
1996
Title
Lymphoproliferative responses to Borrelia burgdorferi in the diagnosis of Lyme arthritis in
children and adolescents.
Journal
Eur J Pediatr, 155(4):297-302
"In one patient with seronegative LA [Lyme arthritis] specific lymphocyte proliferation and polymerase chain reaction for borrelial fla sequences in
urine were positive."
1996
Chronic Lyme disease: an evolving syndrome.
9th Annual International Scientific Conference on
Lyme Disease & Other Tick-Borne Disorders,
Boston, MA, April 19-20
[From the abstract:] "In the case of the ticks, environmental factors such as temperature, humidity and source of blood meal may alter the major outer surface proteins
(Osp) of the spirochete within the tick vector. ...Humans with chronic arthritis are more likely to show an immune response to Osp A."
[Seronegativity:] "Chronic Lyme disease patients may be seropositive or seronegative with or without a documented history of Lyme disease."
[Diagnosis:] "Since Lyme disease is a clinical diagnosis, research must continue to improve diagnostic assays using recombinant proteins which are more sensitive
and specific than the whole organism sonicate used for both ELISA and Western blots."
35.
Luft BJ; Dattwyler
RJ; Johnson RC;
Luger SW; Bosler
EM; Rahn DW;
et al.
36.
Mouritsen CL;
Wittwer CT;
Litwin CM; Yang
L; Weis JJ;
Martins TB;
Jaskowski TD;
Hill HR.
1996
Azithromycin compared with amoxicillin in the treatment of erythema migrans.
A double-blind, randomized, controlled trial.
Annals of Internal Medicine, 124(9):785-91
"Fifty-seven percent of patients who had relapse were seronegative at the time of relapse."
1996
Polymerase chain reaction detection of Lyme disease: correlation with clinical
manifestations and serologic responses.
American Journal of Clinical Pathology.
105(5):647-54
[From the abstract:] "...nine serum samples and one synovial fluid from patients with definite clinical features of Lyme disease were found to be negative by EIA and
Western blot analysis for IgG and IgM antibody, but contained B burgdorferi DNA, as detected by PCR. Polymerase chain reaction analysis of serum and synovial
fluid may be of significant diagnostic value in Lyme disease, especially in the absence of a serologic response in early, partially treated and seronegative chronic
disease....This is the first study to report an association between PCR positivity and the absence of a serologic response to Lyme borreliosis."
1996
Formation and cultivation of Borrelia burgdorferi spheroplast L-form variants.
Infection, 24(3):218-26
37.
Mursic VP;
Wanner G;
Reinhardt S;
Wilske B; et al.
This study investigated In vitro morphological variants of B. burgdorferi, in an effort to explain the clinical persistence of active Lyme borreliosis despite antibiotic
therapy. The authors suggest that these atypical forms may allow Borrelia to survive antibiotic treatment.
"Penicillin G was the most effective inducer of SL-forms [spheroplast-L-forms). The reversion of this form to the helical parental forms was mostly achieved by
cultivation of isolated SL-colonies in penicillin G-free medium. The atypical forms isolated from patients treated with antibiotics show similar features. The same
effect is probably obtained with all other ß-lactam antibiotics."
"With regard to the polyphasic course of Lyme borreliosis, these forms without cell walls can be a possible reason why Borrelia survive in the organism
for a long time (probably with all beta-lactam antibiotics) [corrected] and the cell-wall-dependent antibody titers disappear and emerge after reversion."
38.
Preac Mursic V;
Marget W; Busch
U; Pleterski
Rigler D; Hagl S.
1996
Kill kinetics of Borrelia burgdorferi and bacterial findings in relation to the treatment of Lyme
borreliosis.
Infection, 24(1):9-16
“The patients had clinical disease with or without diagnostic antibody titers to B. burgdorferi."
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Author
39.
Pachner A.
Year
1995
Title
Early disseminated Lyme disease.
Journal
Am J Med, 98 (suppl 4A):4A-30S-51S – Discussion.
“The correlation between a positive Western blot and Lyme arthritis is probably the best of almost any Western blot and any Lyme disease manifestation. With neurologic
disease, I have had a lot of patients who don't have a positive Western blot; they just have not developed a peripheral antibody response, for whatever reason.”
40.
Coyle PK;
Schutzer SE;
Deng Z; Krupp
LB; Belman MD;
Benach JL;
Luft BJ.
1995
Detection of Borrelia burgdorferi-specific antigen in antibody negative cerebrospinal fluid in
neurologic Lyme disease.
Neurology, 45:2010-2014
[From the abstract:] " RESULTS: Of the 35 of 83 (42%) patients who were positive for OspA antigen in their CSF, 15 (43%) were antigen positive despite being
antibody-negative in CSF. Seven of these 15 (47%) had otherwise normal routine CSF analyses. Six of these 15 (40%) patients met strict CDC surveillance criteria
for Lyme disease: four (27%) patients had seroconversion coincident with new neurologic problems; and three (20%) with characteristic syndromes for Lyme disease
were seronegative, but had complexed antibody to B. burgdorferi. The final two patients (13%) were seropositive and had unexplained neurologic problems not
characteristic of Lyme disease. CONCLUSIONS: B. burgdorferi antigen can be detected in CSF that is otherwise normal by conventional methodology, and can be
present without positive CSF antibody. Since CSF antigen implies intrathecal seeding of the infection, the diagnosis of neurologic infection by B. burgdorferi should
not be excluded solely on the basis of normal routine CSF or negative CSF antibody analyses."
[From the article:] "Prompt and precise diagnosis is difficult because basic microbiologic tests such as culture and staining have not been useful, on a broad scale, to
document the presence of the spirochete in a body fluid. Instead, detection of specific antibodies to B burgdorferi in blood and CSF is commonly used to support
or refute a clinical suspicion of infection. Many of the commercially available assays have been plagued by lack of sensitivity, specificity, and reproducibility.
Furthermore, the absence of free antibodies to B burgdorferi components has been documented in well-characterized erythema-migrans-positive cases of Lyme
disease, including those with prominent neurologic involvement."
41.
Karma A; Seppala
I; Mikkila H;
Kaakkola S;
Viljanen M;
Tarkkanen A.
1995
Diagnosis and clinical characteristics of ocular Lyme borreliosis.
American Journal of Ophthalmology,
119(2):127-35
[From the abstract:] “Results of ELISA disclosed that five patients [out of ten] were seropositive, two patients showed borderline reactivity, and three
patients were seronegative. Four of the five patients with borderline or negative results by ELISA had a positive result by western blot analysis. ...
CONCLUSIONS: Late-phase ocular Lyme borreliosis is probably underdiagnosed because of weak seropositivity or seronegativity in ELISA assays."
1995
Seronegative chronic relapsing neuroborreliosis.
European Neurology, 35(2):113-7
42.
Lawrence C;
Lipton RB; Lowy
FD; Coyle PK.
[From the abstract:] This article reports a Lyme disease patient "who experienced repeated neurologic relapses despite aggressive antibiotic therapy." The patient
was seronegative. "Although the patient never had detectable free antibodies to B. burgdorferi in serum or spinal fluid, the CSF was positive on multiple occasions for
complexed anti-B. burgdorferi antibodies, B. burgdorferi nucleic acids and free antigen."
43.
Millner M.
1995
Neurologic manifestations of Lyme borreliosis in children.
Wiener Medizinische
Wochenschrift,145(7-8):178-82
"Our own observations in children which suffered from an acute neuroborreliosis (NB) showed the following:... Indeed, there is a seronegative NB also in children."
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Author
44.
Oksi J; Uksila J;
Marjamaki M;
Nikoskelainen J;
Viljanen MK.
Year
1995
Title
Journal
Journal of Clinical Microbiology, 33(9):2260-4
Antibodies against whole sonicated Borrelia burgdorferi spirochetes, 41-Kilodalton flagellin,
and P39 protein in patients with PCR- or culture-proven late Lyme borreliosis.
[From the abstract:] "These results show that antibodies to B. burgdorferi may be present in low levels or even absent in patients with culture- or PCR-proven late LB
[Lyme borreliosis]. Therefore, in addition to serological testing, the use of PCR and cultivation is recommended in the diagnosis of LB."
45.
Skripnikova IA;
Anan'eva LP;
Barskova VG;
Ushakova MA.
46.
Schubert HD;
Greenebaum E;
Neu HC.
1995
The humoral immunological response of patients with Lyme disease.
Ter Arkh, 67(11):53-6
"Both acute and chronic borreliosis can be seropositive or seronegative."
1994
Cytologically proven seronegative Lyme choroiditis and vitritis.
Retina, 14(1):39-42
[From the abstract:] "RESULTS: Intravitreal spirochetes consistent with Borrelia burgdorferi were found in this seronegative patient. CONCLUSION: Vitreous specimens of
patients with choroiditis and vitritis of unknown cause should be examined cytologically, particularly when serologic results do not corroborate the clinical findings of Lyme
disease."
47.
Sigal LH.
1994
The polymerase chain reaction assay for Borrelia burgdorferi in the diagnosis of Lyme
disease.
Annals of Internal Medicine, 120(6):520-521
"Polymerase chain reaction may be more sensitive than antibody detection techniques in human Lyme neuroborreliosis [17,19] and the murine experimental model
[22] and clearly is more sensitive than current culture techniques. Our experience suggests that a few patients may be positive by PCR despite negative
immunologic assay results in inflammatory fluid and blood (Sigal LH and Liebling M. Unpublished observation)."
48.
Bojic I;
Mijuskovic P;
Dokic M; Nozic D;
Lako B; et al.
49.
Coyle PK.
1993
Clinical characteristics of Lyme disease.
Vojnosanit Pregl, 50(4):359-64
[From the abstract:] "Clinical characteristics of Lyme disease were analysed in 22 patients. Erythema migrans was found in 20 (91%), arthralgia in 18 (81%),
neuralgia in 8 (36%), encephalitis in 3 (13%), carditis in 2 (9%) and arthritis in 2 (9%) patients. The positive antibody titer was found in 14 (63%) patients.
1993
Antigen detection and cerebrospinal fluid studies.
In "Lyme Disease," ed. P. Coyle, p.143
"...spirochetes show a peculiar feature compared to other bacterial neurologic infections: the organisms can be present in CSF without inducing inflammatory
changes. This is well-documented for neurosyphilis, leptospirosis, and relapsing fever, and appears to be occasionally true for Lyme disease as well. In
Europe, B. burgdorferi has been cultured from otherwise normal CSF."
Persistence of Borrelia burgdorferi in ligamentous tissue from a patient with chronic Lyme
50.
Häupl T; Hahn G;
1993
Arthritis & Rheumatism, 36(11):1621-6
Rittig M; Krause
borreliosis.
A; Schoerner C;
Schonherr U; et al.
[From the abstract:] "The initially significant immune system activation was followed by a loss of the specific humoral immune response and a
decrease in the cellular immune response to B burgdorferi over the course of the disease." [From the article:] "Interestingly, the cellular immune responses were also
directed against the surface protein OspA during each recurrence of clinical symptoms, even though anti-OspA antibodies were not detectable by immunoblot."
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Author
51.
Hulinska D;
Krausova M;
Janovska D;
et al.
52.
Kazakoff MA;
Sinusas K;
Macchia C.
53.
Liegner KB;
Shapiro JR;
Ramsay D;
Halperin AJ;
Hogrefe W;
Kong L.
Year
1993
Title
Electron microscopy and the polymerase chain reaction of spirochetes from the blood of
patients with Lyme disease.
Journal
Central European Journal of Public Health.
1(2):81-5
[From the abstract:] "Results of studies using direct antigen detection suggest that seronegative Lyme borreliosis is not rare and support the hypothesis that Borrelia
antigens can persist in humans."
1993
Liver function test abnormalities in early Lyme disease.
Arch Fam Med, 2(4):409-13
[From the abstract:] "PATIENTS: Thirty-seven female and 36 male patients with erythema migrans who had not yet been treated with antimicrobial agents. ...
Only seven patients (9%) had a positive titer in response to the enzyme-linked immunosorbent assay for Lyme disease.”
1993
Recurrent erythema migrans despite extended antibiotic treatment with minocycline in a
patient with persisting Borrelia burgdorferi infection.
Journal of the American Academy of Dermatology,
28(2 Pt 2):312-4
[Abstract:] "Erythema migrans recurred in a patient 6 months after a course of treatment with minocycline for Lyme disease. Polymerase chain reaction on
heparinized peripheral blood at that time demonstrated the presence of Borrelia burgdorferi-specific DNA. The patient was seronegative by Lyme enzyme-linked
immunosorbent assay but showed suspicious bands on Western blot. Findings of a Warthin-Starry stain of a skin biopsy specimen of the eruption revealed a
Borrelia-compatible structure. Reinfection was not believed to have occurred. Further treatment with minocycline led to resolution of the erythema migrans."
1993
Seronegative Lyme disease.
In "Lyme Disease," ed. P. Coyle, p.192
54.
Schutzer SE.
"The number and percentage of seronegative Lyme disease cases remain controversial. At some academic centers the estimate is 5%, and in certain private settings
the number may be higher. There is little question that seronegative Lyme disease can exist."
55.
Sigal LH.
1993
Lyme disease: testing and treatment. Who should be tested and treated for Lyme disease
Rheum Dis Clin North Am, 19(1):79-93
[From the abstract:] "LD is not a diagnosis that can be made on the basis of serologic testing. By this is meant that vague symptoms plus a positive serologic test do
not assure that the patient has LD. On the other hand, a patient with ECM or other manifestations of LD may still be seronegative."
56.
Steere AC.
1993
Seronegative Lyme disease.
JAMA, (270):1369.
"The number and percentage of seronegative Lyme disease cases remain controversial. At some academic centers the estimate is 5%, and in certain private settings
the number may be higher. There is little question that seronegative Lyme disease can exist."
First isolation of Borrelia burgdorferi from an iris biopsy.
57.
Preac-Mursic V;
1993
J Clin Neuroophthalmology, 13(3):155-61; discussion 162.
Pfister HW;
Spiegel H; Burk R;
[From the abstract:]
“
Antibiotic therapy may abrogate the antibody response to the infection as shown by our results. Patients may have subclinical or clinical
Wilske B; el al.
disease without diagnostic antibody titers. Persistence of B. burgdorferi cannot be excluded when the serum is negative for antibodies against it."
58.
Oksi J; Viljanen
MK; Kalimo H;
Peltonen R;
Marttia R;
Salomaa P;
et al.
1993
Fatal encephalitis caused by concomitant infection with tick-borne encephalitis virus and
Borrelia burgdorferi.
Clinical Infectious Diseases, 16(3):392-6
"Serology for Borrelia was negative. Autopsy revealed necrotizing encephalitis and myelitis with involvement of the dorsal root ganglion. With use of
polymerase chain reaction tests, segments of two separate genes of B. burgdorferi were amplified from the patient's CSF."
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Author
59.
Keller TL;
Halperin JJ;
Whitman M.
Year
1992
Title
PCR detection of Borrelia burgdorferi DNA in cerebrospinal fluid of Lyme neuroborreliosis patients.
Journal
Neurology, 42(1):32-42
"PCR detected B burgdorferi DNA in the CSF of seven patients whose blood serology ...failed to demonstrate prior exposure to the organism. The failure of existing
diagnostic methods to detect patients exhibiting well-recognized manifestations of Lyme disease has been described. This has generally been attributed to abrogation of the
host immune response by noncurative antimicrobial treatment. This explanation seems unlikely in at least four of the seven patients who had no recollection of having
received antibiotics. "
1992
Difficulties with Lyme serology.
J Am Optom Assoc, 63(2):135-9
60.
Banyas GT.
[From the abstract:] "A major problem has been seronegativity in persons possessing the disease (false negatives). At present, seronegativity in persons strongly
suspected of having Lyme disease does not necessarily exclude the diagnosis of Lyme disease. The clinician must recognize this in patients who may have Lyme
disease or a recurrence of the disease."
61.
Dinerman H;
Steere AC.
1992
Lyme disease associated with fibromyalgia.
Annals of Internal Medicine, 117:281-5
"The small percentage of patients who are seronegative by enzyme-linked immunosorbent assay (ELISA) later in the illness usually have positive
Western blots or cellular immune responses to borrelial antigens (9,10)."
62.
Fraser DD; Kong
LI; Miller FW.
Clinical & Experimental Rheumatology,
10(4):387-90.
[From the abstract:] "Antibiotic therapy was reinstituted after Borrelia burgdorferi was detected in the patient's peripheral blood leukocytes by the polymerase chain
reaction (PCR). All serologic, T-cell stimulation, and western blot analyses, however, were negative... In addition, this case emphasizes the potential clinical utility of
PCR technology in evaluating the persistent sero-negative Lyme disease which may occur in immunocompromised individuals."
Molecular detection of persistent Borrelia burgdorferi in a man with dermatomyositis.
1992
PCR detection of Borrelia burgdorferi DNA in cerebrospinal fluid of Lyme neuroborreliosis
patients.
Neurology, 42(1):32-42
1992
63.
Keller TL;
Halperin JJ;
Whitman M.
[Abstract:] "We used the polymerase chain reaction (PCR), a method useful in the detection of Borrelia burgdorferi in vitro, to evaluate CSF in patients thought to have
neuroborreliosis. Nested pairs of oligonucleotide primers were designed to recognize the C-terminal region of B burgdorferi OspA. CSF samples were obtained from
(1) patients with immunologic evidence of systemic B burgdorferi infection and clinical manifestations suggestive of CNS dysfunction, (2) seronegative patients with
clinical disorders consistent with Lyme borreliosis, and (3) patient and contamination controls; all were analyzed in a blinded fashion. PCR detected B burgdorferi
OspA DNA in CSF of (1) 10 of 11 patients with Lyme encephalopathy, (2) 28 of 37 patients with inflammatory CNS disease, (3) seven of seven seronegative patients
with Lyme-compatible disorders, and (4) zero of 23 patient controls. Zero of 83 additional contamination controls were PCR-positive”
64.
Faller J;
Thompson F;
Hamilton W.
1991
PCR detection of Borrelia burgdorferi DNA in cerebrospinal fluid of Lyme neuroborreliosis
patients.
Foot & Ankle, 11(4):236-238.
"Patient 8 had several negative ELISA assays. She then had a lymphocyte reactivity test for cell mediated immune (CMI) response to Borrelia burgdorferi antigen. Her
peripheral blood lymphocytes were markedly responsive to the spirochete, with an index of 46 (18 is three standard deviations above the controls.)"
65.
Reik L, Jr.
1991
Lyme Disease and the Nervous System.
New York: Thieme Medical Publishers, Inc.
“In some cases, specific serum antibody is present but sequestered in immune complexes, and therefore not measurable by routine ELISA.”
“...test results for serum antibodies are not always positive when neurologic abnormalities develop, especially in stage 2.”
66.
Havlik J;
Rohacova H;
Hulinska D; et al.
67.
Nields JA;
Kueton JF.
68.
Steere AC.
1991
Seronegative Lyme borreliosis.
The 5th European Congress of Clinical Microbiol.
and Infect. Diseases, Sept. 8-11, Oslo, Norway:1385.
Lancet, 338(8759):128-9
Rheumatology News
1991
1991
Tullio phenomenon and seronegative Lyme borreliosis.
Rheumatology research in the 90s.
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Author
69.
Nadelman RB;
Pavia CS;
Magnarelle LA;
Wormser GP.
Year
1990
Title
Isolation of Borrelia burgdorferi from the blood of seven patients with Lyme disease.
Journal
American Journal of Medicine, 88:21-6
"The absence of significant antibody titers to B. burgdorferi is not uncommon in Lyme disease, especially in early disease. ...Although it was initially believed
that patients with neurologic Lyme disease generally have antibodies to B. burgdorferi, this may not always be the case. ...We would advise that in an endemic
area, the differential diagnosis of nonspecific muscle and joint aches without rash should include Lyme disease--even in the absence of antibodies to B. burgdorferi.”
70.
Schutzer SE;
Coyle PK; Belman
AL; Golightly MG;
Drulle J.
71.
Bianchi G;
Rovetta G;
Monteforte P;
Fumarola D;
et al.
72.
Dieterle L; Kubina
FG; Staudacher T;
Budingen HJ.
1990
Sequestration of antibody to Borrelia burgdorferi in immune complexes in seronegative
Lyme disease.
Lancet, 335(8685):312-5
[From the abstract:] "Apparent B burgdorferi seronegativity in serum immune complexes may thus be due to sequestration of antibody in immune complexes."
1990
Articular involvement in European patients with Lyme disease. A report of 32 Italian patients.
British Journal of Rheumatology, 29(3):178-80
[From the abstract:] “In addition, interpreting serological tests for antibodies against B. burgdorferi and the real prevalence of arthritis in LD [Lyme disease] is
complicated by the possible existence of seronegative LD and by the effect of early antibiotic treatment."
1989
Neuro-borreliosis or intervertebral disk prolapse?
Dtsch Med Wochenschr, 114(42):1602-6
[From the abstract "Between September 1986 and November 1988, 17 patients were hospitalized and treated for neuro-borreliosis. ...Three of 14 patients had no IgG
antibodies against Borrelia, either in serum or cerebrospinal fluid at the initial examination, two had positive titres in serum only.
73.
Guy EC;
Turner AM.
1989
Seronegative neuroborreliosis.
Lancet, 1:441
“We wish to report a case with a clinical diagnosis of acute Lyme neuroborreliosis for whom negative serology was reported by a Lyme disease referral centre using
ELISA. ...
Western blot analysis of sera revealed both IgM and IgG binding to several B burgdorferi proteins, compatible with acute Lyme disease. Antibody binding to a larger
number of B burgdorferi proteins was found when the CSF was tested similarly. Our observation of a more diverse antibody response in CSF compared with serum is
consistent with the findings of a previous study which showed 44% of patients with neurological manifestations of Lyme disease to have positive CSF antibody titres
but negative serum titres when measured by ELISA. Analysis of CSF for the detection of IgM and IgG binding to B burgdorferi is essential if seronegative cases of
acute Lyme neuroborreliosis are to be identified.”
74.
MacDonald AB.
1989
Gestational Lyme borreliosis. Implications for the fetus.
Rheum Dis Clin North Am, 15(4):657-77
"From a biologic perspective, most of the fatal cases of LB [Lyme borreliosis] in pregnancy were reactive either in titers in the borderline region or were completely
nonreactive in serologic tests. The tendency toward seronegativity in pregnancy makes maternal serology a less satisfactory discriminator of maternal infection and
useless as a practical tool to predict the actual state of the fetus..."
75.
Trock DH; Craft
JE; Rahn DW.
1989
Clinical manifestations of Lyme disease in the United States.
Connecticut Medicine, Vol 53, No. 6
"...cases of seronegative Lyme disease have been reported..."
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Author
76.
Dattwyler RJ
;
Volkman DJ;
Luft BJ.
77.
Preac-Mursic V;
Weber K; Pfister
HW; Wilske B;
Gross B;
Baumann A;
Prokop J.
Year
1989
Title
Journal
Rev Infect Dis, II(Suppl 6):S1494-98.
Immunologic aspects of Lyme borreliosis.
"Three separate groups of investigators have reported individuals who lacked diagnostic levels of specific antibody in their serum, yet had neurologic involvement and
diagnostic levels of antibody in their CSF. We have confirmed this finding in our laboratory.”
1989
Survival of Borrelia burgdorferi in antibiotically treated patients with Lyme borreliosis.
Infection, 17(6):355-9
[From the abstract:] "We conclude that early stage of the disease as well as chronic Lyme disease with persistence of B. burgdorferi after antibiotic therapy cannot
be excluded when the serum is negative for antibodies against B. burgdorferi."
[Seronegativity:] "As shown, negative antibody-titers do not provide evidence for successful therapy; antibody-titers may become negative despite persistence of
B. burgdorferi." (p.358)
78.
Berger BW;
MacDonald AB;
Benach JL.
1988
Use of an autologous antigen in the serologic testing of patients with
erythema migrans of Lyme disease.
Journal of the American Academy of Dermatology,
18(6):1243-6
[Abstract:] "We attempted to detect an early rise in antibody titers to Borrelia burgdorferi in the serum of patients with erythema migrans of Lyme disease by utilizing
B. burgdorferi isolates obtained from patients' own skin lesions instead of the B31 reference strain. B. burgdorferi was isolated from nine of 23 skin biopsy
specimens submitted for culture. Elevated antibody titers were not detected in any of the 23 acute serum samples by immunofluorescence assay. The antigens
derived from patient isolates were no more effective than the reference strain in detecting antibodies in patients with early Lyme disease."
79.
Dattwyler RJ;
Volkman DJ; Luft
BJ; Halperin JJ;
Thomas J;
Golightly MG.
1988
Seronegative Lyme disease. Dissociation of specific T- and B-lymphocyte responses to
Borrelia burgdorferi.
New England Journal of Medicine,
1;319(22):1441-6
[From the abstract:] "Although these patients had clinically active disease, none had diagnostic levels of antibodies to B. burgdorferi on either a standard enzyme-
linked immunosorbent assay or immunoflourescence assy. ...We conclude that the presence of chronic Lyme disease cannot be excluded by the absence of
antibodies against B. burgdorferi and that a specific T-cell blastogenic response to B. burgdorferi is evidence of infection in seronegative patients with clinical
indications of chronic Lyme disease."
1988
Borrelia burgdorferi in the nervous system: the new "great imitator".
Annals of the New York Academy of Sciences,
539:56-64
80.
Pachner AR.
"The antibody response in serum in CNS Lyme disease seems to be related to the presence of other manifestations; patients who have had both arthritis and CNS
disease have quite high titers, while those with only CNS disease sometimes do not."
81.
Lavoie PE;
Lattner BP;
Duray PH;
Barbour AG;
Johnson HC.
1987
Culture positive seronegative transplacental Lyme borreliosis infant mortality.
Arthritis Rheum, Vol 30 No 4, 3(Suppl):S50
“We report a culture positive neonatal death occurring in California, a low endemic region. ...Bb was grown from a frontal cerebral cortex inoculation. The spirochete
appeared similar to the original Long Island tick isolate. Silver stain of brain & heart was confirmatory of tissue infection. The mother had been having migratory
arthralgias and malaise since experiencing horse fly & mosquito bites while camping on the Maine coast in 1971. The family was seronegative for LB by ELISA at
Yale. Cardiolipin antibodies were also not found."
Journal of Clinical Neuro-Ophthalmology,
7(4):185-90
[From the abstract:] "Potential pitfalls in the diagnosis of Lyme disease with an emphasis on false negative serology and currently available
diagnostic modalities are presented."
Lyme disease. A neuro-ophthalmologic view.
1987
82.
MacDonald AB.
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Author
83.
Hederstedt B;
Hovmark A;
Stiernstedt G;
Asbring E.
Year
1986
Title
[Borrelia-diagnos aktuell aret om visar serologisk undersokning av 1985 ars fall.]
Journal
Lakartidningen, 83:3987-89
[According to Guy & Turner, 1989, this study found that 44% of patients with neurological Lyme disease had positive CSF antibody titres but negative serum titres
when measured using ELISA testing.]
84.
Schmidt R;
Kabatzki J;
Hartung S;
Ackermann R.
1985
[Erythema migrans borreliosis in the Federal Republic of Germany.
Epidemiology and clinical aspects.]
Deutsche Medizinische Wochenschrift,
110(47):1803-7
[Abstract:] "A positive antibody titre against Ixodes-ricinus-Borrelia (burgdorferi), using indirect immunofluorescence or ELISA, could be detected in serum and (or)
liquor of 935 (32%) out of a total of 2955 patients between January 1984 and July 1985. In 289 of these cases the typical clinical manifestations were lacking whereas
a characteristic disease picture enabled a diagnosis to be made in 171 patients with negative or borderline antibody titres. The 1106 cases of infection observed
covered all regions of the country. A typical clinical syndrome was seen in 817 (74%) of these. Most common were erythema chronicum migrans (n = 458) and
meningopolyneuritis Garin-Bujadoux-Bannwarth (n = 404); in 42% of the cases meningopolyneuritis was preceded by an erythema. Arthritis (n = 63), acrodermatitis
chronica atrophicans (n = 72), carditis (n = 13) and lymphadenosis benigna cutis (n = 5) were much less common. Chronic Borrelian encephalomyelitis (n = 45)
appeared surprisingly often (n = 45). The fact that in 73% of cases the various syndromes appeared alone, were double in 24% and combined only in 3%, illustrates
the polymorphic nature of this disease."
Other Spirochetes
85.
Stephan C;
Hunfeld KP;
Schonberg A;
et al.
86.
Jacobs R.
2000
[Leptospirosis after a staff outing].
Dtsch Med Wochenschr, 125(20):623-627.
“[From the abstract:] "The symptoms are non-specific and, moreover, in some cases the laboratory tests are negative, so that clinical diagnosis remains crucial.”
1999
Current Medical Diagnosis & Treatment 1999.
38th Edition. Appleton & Lange. Stamford, CT.
Ed. Tierney LM Jr; McPhee SJ, Papadakis MA.
Infectious Diseases: Spirochetal. Syphilis.
“The VDRL titer is usually high (>1:32) in secondary syphilis and tends to be lower (< 1:4) or even negative in late forms of syphilis.”
87.
Vecsei AK,
Vecsei PV,
Dangl-Erlach E,
1999
Wien Klin Wochenschr, 111(10):410-3
[Congenital syphilis: late diagnosis in spite of screening].
[Abstract:] "We report the case of an infant in whom congenital syphilis was diagnosed at the age of 5 weeks. The case is remarkable because of (a) the negative
venereal disease laboratory test from the cord blood, (b) the incidental diagnosis of the disease in the fifth week of life, (c) pneumonia alba being one of the
symptoms, (d) the occurrence of a mild Jarisch-Herxheimer reaction after initiation of penicillin therapy and (e) the successful treatment of infection related anaemia
with recombinant human erythropoietin."
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Author
88.
Uribe CS;
Garcia FA.
Year
1998
Title
Journal
Rev Neurol, 27(160):970-2
[Neurosyphilis and the prozone effect].
[Abstract:] "INTRODUCTION: Neurosyphilis (NS) is an entity which still frequently presents to our Neurology Department. The prozone phenomenon occurs in
approximately 2% of all cases of late primary syphilis or secondary syphilis; we have found no cases described of prozone and neurosyphilis occurring together.
CLINICAL CASE: We present the unusual case of a 44 year old patient with NS and dementia PGP (progressive general paralysis). Initially serum VDRL was
negative, but in CSF reacted at dilutions of 1:32. When serum VDRL was repeated using dilutions, it was reactive 1:128 and serum FTA was also reactive. The patient
was treated with i.v. crystalline penicillin, after which his condition improved. CONCLUSIONS: We wish to draw attention to the possibility that patients with a
dementia syndrome and negative serum VDRL may have the prozone phenomenon, and the laboratory should therefore be asked to do serial dilutions."
89.
Tikjob G; Russel
M; Petersen CS;
Gerstoft J;
Kobayasi T.
90.
Berkowitz K;
Baxi L; Fox HE.
1991
Seronegative secondary syphilis in a patient with AIDS: identification of Treponema pallidum
in biopsy specimen.
Journal of the American Academy of Dermatology,
24(3):506-8
1990
False-negative syphilis screening: the prozone phenomenon, nonimmune hydrops, and
diagnosis of syphilis during pregnancy.
Am J Obstet Gynecol, 163(3):975-7
[From the abstract:] "Recently we encountered four cases of false-negative syphilis serologic results in women who gave birth to infants with congenital syphilis. The
false-negative results were caused by the prozone phenomenon. The prozone phenomenon, seen during primary and secondary syphilis, occurs because a higher
than optimal amount of antibody in the tested sera prevents the flocculation reaction typifying a positive result in reagin tests. Serum dilution is necessary to make the
correct diagnosis. We recommend that for any pregnant woman with apparently negative syphilis serologic results in whom fetal compromise of unknown etiology
exists, particularly nonimmune hydrops, nontreponemal testing should be repeated using serum dilutions to prevent a missed diagnosis of syphilis. We further
recommend serum dilution as a routine procedure for all pregnant women in areas of high syphilis prevalence."
91.
Borisenko KK;
Vinokurov IN;
Toporovskii LM.
1989
[Characteristics of the course of latent forms of syphilis].
Vestn Dermatol Venerol, (11):25-9
[Abstract:] "Seven patients with latent syphilis are described, in whom the routine serologic tests (RST) were negative during the first examination and over the course
of therapy, and the specific tests (T. pallidum immobilization and immunofluorescence) were repeatedly positive before therapy. Early latent seropositive recurrent
forms of syphilis were detected in the majority of these patients' sexual partners. The patients were not administered antisyphilis therapy before. The diagnosis of
latent seronegative early syphilis negative in the RST is epidemiologically significant, for it helps timely carry out the necessary treatment and prophylaxis measures
to prevent the disease dissemination."
92.
Ovcinnikov NM.
1981
Vestn Dermatol Venerol, 8:22-26
[Important problems in the serodiagnosis of syphilis.].
[According to Mattman L., 1993: "It is thought [by Ovcinnikov] that false negative serological tests for syphilis may be explained because cystic and granule stages of the
treponeme have not stimulated antibody reactive with the spirochetal stage."]
93.
Il'in II.
Pakhomova LV.
1981
[Seronegative forms of latent syphilis].
Vestnik Dermatologii i Venerologii. (1):66-9
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Author
94.
Sparling PF.
Year
1971
Title
Journal
New England Journal of Medicine, 284: 642-653
Diagnosis and treatment of syphilis.
Some infected patients had negative or equivocal serologic tests for syphilis. “These studies [reviewed] emphasize the fact that late syphilis can occur
even if all serologic tests are negative.”
Includes a review of recent [as of 1971] evidence indicating that penicillin treatment is not always curative in patients with late syphilis. "Penicillin therapy of
neurosyphilis has not been as effective [as in early syphilis]. Several studies have reported relapses... Clinical progression of symptomatic neurosyphilis is relatively
common despite antibiotics." (p.650)
95.
Ch'ien L.
Hathaway BM.
Israel CW.
1970
Seronegative dementia paralytica: report of a case.
Journal of Neurology, Neurosurgery & Psychiatry.
33(3):376-80
96.
Hallock J;
Tunnessen WW.
97.
Roitburd MF.
1968
Congenital syphilis in an infant of a seronegative mother.
Obstet Gynecol, 32(3):336-8
1968
[2 cases of seronegativity in secondary syphilis].
Vestn Dermatol Venerol, 42(8):82-3
98.
Smith JL.
1968
Spirochetes in late seronegative syphilis, despite penicillin therapy.
Med Times, 96(6):611-23
99.
Smith JL;
Israel CW.
1967
Spirochetes in the aqueous humor in seronegative ocular syphilis. Persistence after
penicillin therapy.
Archives of Ophthalmology, 44:474-477
"The purpose of this communication is to report the presence of spirochetes in the aqueous humor both before and after the intramuscular administration of
9,200,000 units of long-acting penicillin for seronegative ocular syphilis. The treponemes were found by the flourescein antibody technique, in which the spirochetes
stained with flourescein tagged anti-Treponema pallidum globulin when viewed with ultraviolet microscopy. ...
Spirochetes have now been found in aqueous humor, cerebrospinal fluid, liver, and lymph nodes in several patients with late seronegative syphilis."
100.
Smith JL;
Israel CW.
1967
The presence of spirochetes in late seronegative syphilis.
JAMA, 199(13):980-984
[Abstract:] "Late seronegative syphilis refers to clinical signs of ocular or neurosyphilis in a patient whose routine blood (reagin) test is nonreactive, but in whom a
specific treponemal test is reactive. This report documents the presence of spirochetes in aqueous humor, cerebrospinal fluid, and at liver biopsy in such patients.
Spirochetes have been found in the aqueous humor with no biomicroscopic abnormality and in cerebrospinal fluids which had normal cell counts, protein levels, and
reagin and colloidal gold test results. Identification of the organisms depends on use of the flourescein antibody technique, in that spirochetes stain with
flourescein-tagged anti-Treponema pallidum globulin on ultraviolet microscopy."
[From the article:] "The finding of motile spirochetes in the aqueous humor of animal eyes which showed no clinical signs of inflammation at the time of paracentesis
led to the initial clinical studies reported here. It must be emphasized that several patients were found to have negative findings in darkfield examinations of aqueous
humor and cerebrospinal fluid, in which later study with the fluorescent antibody technique revealed morphologically typical spirochetes which stained with anti-T
pallidum globulin. Darkfield examination of CSF requires that the fluid be centrifuged and examined within ten minutes after lumbar puncture in order to see motile
treponemes."
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1533935_0062.png
Author
101.Smith
JL; Singer
JA; Moore MB, Jr;
Yobs AR.
Year
1965
Title
Sero-negative ocular and neuro-syphilis.
Journal
American Journal of Ophthalmology, 59:753-762
102.
Taylor WH; Smith
JL; Singer JA.
1965
Experimental seronegative syphilis.
American Journal of Ophthalmology,
60:1093-1098
103.
McCord.
1929
Study of two hundred autopsies made on syphilitic foetuses.
Am J Obst & Gynec, 18:597
Original file available at www.lymeinfo.net
Page 17 of 17
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1533935_0063.png
10.4. RESUMÉ AF IDEIA
BORRELIA BURGDORFERI IgG
Uddrag fra test kit indlægsbrochure:
-ANALYSEPROCEDUREN
Sørg for, at reagenserne har nået stuetemperatur (15–30°C) inden
brug
11.4.2
Semikvantitativ fortolkning (arbitrære enheder)
IDEIA Borrelia burgdorferi IgG REF K602911-2 DA
Inden for området af OD-værdier mellem OD
IgG Cut-Off
and OD
IgG Positiv
svarer OD-værdierne direkte til logaritmen af arbitrære enheder
af specifikt antistof i prøven. Dette er illustreret i figur 1, der viser
resultater fra et IgG anti-B. burgdorferi-positivt serum fortyndet
serielt i negativt serum.
2.500
2.000
OD
1.500
1.000
0.500
IgG-Cut-off-kontrol
Positiv IgG-kontrol
14. SÆRLIGE EFFEKTIVITETSKARAKTERISTIKA
14.1. SPECIFICITET
Specificiteten af IDEIA Borrelia burgdorferi, IgG blev evalueret
på et uafhængigt rutinediagnostiklaboratorium i Sverige. Studiet
blev udført på et panel af serumprøver udtaget fra formodentligt
raske bloddonorer, der boede i et område med endemisk Lyme
borreliosis.
Specificitet
Forventet
Evaluering*
98%
98.5%
*Tvivlsommer resultater er fortolket som negative.
14.2. DIAGNOSTISK SENSITIVITET
Den diagnostiske sensitivitet af IDEIA Borrelia burgdorferi, IgG blev
evalueret på et uafhængigt rutinediagnostiklaboratorium i Sverige.
Studiet blev udført på tre paneler af serumprøver fra patienter
med nogle af de mest almindelige kliniske manifestationer af
infektion med B. burgdorferi: 45 serumprøver fra patienter
med erythema migrans, 38 serumprøver fra patienter med
lymfocytisk meningoradiculitis og 20 serumprøver fra patienter
med acrodermatitis chronica atrophicans. Resultaterne er
sammenlignet med tidligere rapporterede forventede værdier
6,8
.
Diagnostisk sensitivitet
Klinisk manifestation
Forventet Evaluering*
Erythema migrans
36%
38%
Lymfocytisk meningoradiculitis
77%
79%
Acrodermatitis chronica
100%
100%
atrophicans
*Tvivlsommer resultater er fortolket som negative.
14.3. KRYDSREAKTIVITET
Fortynd serumprøverne 1/200 ved tilsætning af 10µL serum til
2mL prøvediluent.
100µL kontrol eller testprøve
Tildæk og inkuber
ved 20–25°C under
omrystning i 60 minutter
Vask (x4)
Tilsæt 100µL anti-IgG-konjugat
Tildæk og inkuber
ved 20–25°C under
omrystning i 60 minutter
0.000
1
10
100
Log af fortynding
1000
Vask (x4)
Tilsæt 100µL substrat
Tildæk og inkuber
ved 20-25°C uden
omrystning i 10 minutter
Tilsæt 100µL stopopløsning
Aflæs absorbansen fotometrisk ved
450nm (reference, 620-650nm)
11. KVALITETSKONTROL
TESTRESULTATERNE
11.1. PRØVEDILUENT
OG
FORTOLKNING
AF
Figur 1
Resultater af en serie 2-gangefortyndinger af et
serum positivt for IgG-antistoffer mod B. burgdorferi i negativt
serum. Endvidere er OD-værdierne for IgG-Cut-Off-kontrollen og
den positive IgG-kontrol vist.
Niveauet af specifikke antistoffer i IgG-Cut-Off-kontrollen er
defineret som 1 (U
IgG Cut-Off
= 1 enhed). Niveauet af specifikke
antistoffer i den positive IgG–kontrol er justeret til 8 x U
IgG Cut-Off
(U
IgG Positiv
= 8 enheder).
For en prøve kan de arbitrære enheder af specifikke antistoffer
(Uprøve) beregnes ved at benytte formlen:
DO
prøve
- DO
IgG Cut-off
DO
IgG Positiv
- DO
IgG Cut-off
U
prøve
=10
a
, a =
x 0,9*
OD-værdien af brønden med prøvediluent skal være mindre
end 0,100, men større end 0,000 (dobbelt bølgelængde). Hvis
værdien er over 0,100, kan utilstrækkelig vask eller kontaminering
af substratet være årsagen. Hvis værdien er under 0,000, bør
pladeaflæseren nulstilles mod luft på ny, og mikrobrøndene
aflæses igen.
Hvis kvalitetskontrolkravene ikke er opfyldt, er testresultaterne
ugyldige, og analysen skal gentages.
11.2. IgG-CUT-OFF-KONTROL OG POSITIV IgG-KONTROL
Beregn
middel-OD-værdierne
for
de
3-IgG-Cut-Off-
kontrolmikrobrønde (OD
IgG Cut-Off
) og for de 2 positive IgG-
kontrolmikrobrønde (OD
IgGPositiv
). De enkelte OD-værdier bør
ikke afvige mere end 25% fra middel-OD-værdien. Hvis en af
OD-værdierne for IgG-Cut-Off-kontrollen afviger mere end 25%
fra middel-OD-værdien, bør den udelukkes fra beregningen, og
middelværdien skal beregnes igen.
Forskellen mellem OD-værdien for Cut-Off-kontrol og positiv
IgG-kontrol skal være mindst 0,500. Hvis forskellen er mindre
end 0,500, kan det skyldes utilstrækkelig vask, utilstrækkelig
omrystning under inkuberingerne eller for lav omgivende
temperatur navnlig under inkubation med substratet.
Hvis kvalitetskontrolkravene ikke er opfyldt, er testresultaterne
ugyldige, og analysen skal gentages.
*logU
IgG Positiv
-logU
IgG Cut-off
= log8 - log1 = 0,9
Tvivlsommer resultater
Grænsen for påvisning af IgG-antistof svarer til 0,9 enheder. Det
niveau over hvilket, at antistoffet ventes at være tilstede på grund
af aktiv infektion, svarer til 1,1 enhed. Prøver med mindre end 0,9
enhed specifikt antistof fortolkes som negative for IgG-antistoffer
mod B. burgdorferi. Prøver med mellem 0,9 og 1.1 enheder
angiver tilstedeværelsen af lavt antistofniveau, som skal fortolkes
med forsigtighed, og der anbefales opfølgende prøvetagning
efter to uger for at bekræfte patientstatus. Prøver med 1,1 eller
flere enheder specifikt antistof fortolkes som positive for IgG-
antistoffer mod B. burgdorferi.
For prøver med OD-værdier over OD
IgG Positiv
bør de rapporterede
enheder af specifikke antistoffer mod B. burgdorferi være højere
end 8.
En ændring af en patients specifikke antistofniveau kan betragtes
som signifikant, når de arbitrære enheder i en efterfølgende
prøve er enten fordoblet eller halveret. Dette er udelukkende
vejledende.
Sera fra patienter med syfilis og inflammatoriske sygdomme
(positive for rheumatoid faktor (RF)) blev testet med IDEIA
Borrelia burgdorferi IgG med følgende resultater:
Patientsera
Antal sera
Antal positive*
Syfilis
25
0
FR
18
1
*Tvivlsommer resultater er fortolket som negative.
15. REFERENCER
1. Burgdorferi W, Barbour AG, Hayes SF, Benach JL, Grunwaldt E, Davis JP.
(1982) Lyme disease - a tick-borne spirochetosis? Science 216: 1317-9.
2. Steere AC, Grodzicki RL, Kornblatt AN, Craft JE, Barbour AG, Burgdorferi
W, et al. (1983) The spirochetal etiology of Lyme disease. N Engl J Med 308:
733-40.
3. Steere AC. (1989)Lyme disease. N Engl J Med 321: 586-96.
4. Craft JE, Duncan KF, Shimamoto GT, Steere AC. (1986) Antigens of
Borrelia burgdorferi recognized during Lyme disease. Appearance of a
new immunoglobulin M response and expansion of the immunoglobulin G
response late in the illness. J Clin Invest 78: 934-9.
5. 5.Zöller L, Burkard S, Schäfer H. (1991) Validity of Western immunoblot
band patterns in the serodiagnosis of Lyme borreliosis. J Clin Microbiol 29:
174-82.
6. Hansen K, Pii K, Lebech A-M. (1991) Improved immunoglobulin M
serodiagnosis in Lyme borreliosis by using a µ-capture enzyme-linked
immunosorbent assay with biotinylated Borrelia burgdorferi flagella. J Clin
Microbiol 29: 166-73.
11.3. PATIENTPRØVER
Beregn middel-OD-værdien for hver patientprøve (OD
prøve
).
De enkelte OD-værdier bør ikke afvige mere end 25% fra
middelværdien. Alle sådanne prøver bør analyseres igen. Hvis
imidlertid begge testmikrobrønde viser et negativt resultat, kan
en forskel på mere end 25% være acceptabelt uden ny analyse,
da lave OD-værdier måles med mindre præcision.
11.4. FORTOLKNING AF RESULTATER
Testen IDEIA Borrelia burgdorferi IgG omfatter en cut-off-kontrol
indeholdende en bestemt mængde af IgG-antistoffet Borrelia
burgdorferi. Det niveau af antistoffet, som påvises over cut-off,
er i høj grad indikativ for aktiv Borrelia burgdorferi-infektion.
IDEIA Borrelia burgdorferi IgG-kittet kan påvise antistoffet ved
et niveau under cut-off, og antistoffet kan være tilstede i form af
latent antistof fra tidligere infektion eller et meget lavt niveau af
antistof straks efter en nylig infektion. Det lave antistofniveau skal
fortolkes forsigtigt og det anbefales at der for at være sikker på
betydningen af det lave antistofniveau følges op med prøvetagning
hos patienterne efter mindst 2 uger for at identificere forandring i
antistofniveauet, der bedre kan angive betydningen af antistoffet.
11.4.1
Kvalitativ fortolkning
Grænsen for påvisning (OD
IgG DETECTION
) for IgG-antistof vedr.
Borrelia burgdorferi beregnes som OD
Cut-Off
x 0,5.
Det niveau, over hvilket antistoffet ventes at være tilstede på
grund af aktiv infektion, svarer til OD
IgG Cut-Off
.
Prøve-OD mellem de to niveauer angiver tilstedeværelsen af et
lavt antistofniveau, som skal fortolkes forsigtigt. Fortolkes som
følger:
11.4.3
Kommentarer til resultatfortolkninger
Negative resultater
Et negativt resultat udelukker ikke eksponering mod B.
burgdorferi. Hvis der stadig er mistanke om Lyme borreliosis, bør
der udtages en yderligere prøve på et senere tidspunkt.
Positive resultater
Et positivt resultat indikerer nylig eksponering mod B. burgdorferi.
Tvivlsomme resultater
Alle resultater inden for ±20% af OD-IgG-Cut-off bør betragtes
som tvivlsomme og fortolkes med forsigtighed. Det anbefales at
gentage analysen af sådanne prøver.
Et tvivlsomt resultat bør føre til, at der inden for 2 uger udtages en
yderligere prøve til analyse. Hvis begge (eller yderligere senere)
prøver giver tvivlsomme resultater, kan patienten betragtes at
være IgG-negativ.
12. TESTENS BEGRÆNSNINGER
12.1. Et negativt resultat udelukker ikke muligheden for
B. burgdorferi-infektion hos patienten. Manglende
påvisning af B. burgdorferi kan være resultat af sådanne
faktorer som udtagning af prøven på et forkert tidspunkt
inden fremkomsten af detekterbare antistoffer, forkert
prøveudtagning eller forkert håndtering af prøven. Tidlig
antibiotikabehandling kan undertrykke antistofreaktionen,
og nogle patienter producerer muligvis ikke antistoffer i et
detekterbart niveau.
12.2. Et positivt resultat indikerer en tidligere immunologisk
eksponering og er ikke et bevis på aktiv infektion.
12.3. Alle positive resultater skal fortolkes i forbindelse med
patientrelateret klinisk information og epidemiologiske data
og retfærdiggør ikke behandling af en patient. Muligheden
for eksponering mod flåtbid bør altid tages i betragtning.
12.4. Testens resultat bliver påvirket, hvis reagenserne
modificeres eller opbevares under forhold, som afviger fra
de i afsnit 5.2. anførte.
13. FORVENTEDE VÆRDIER
Niveauet af IgG-Cut-Off-kontrollen bør justeres til en specificitet
på 98% for normale sera. Antistofreaktionen mod B. burgdorferi-
flageller afhænger af infektionens kliniske manifestation og
sygdommens varighed. Ved nogle af de mest almindelige
kliniske manifestationer af infektion med B. burgdorferi blev den
diagnostiske sensitivitet af en indirekte IgG-analyse anvendende
oprensede, native B. burgdorferi-flageller som testantigen
rapporteret som følger:
6,8
Klinisk manifestation
Erythema migrans
Lymfocytisk meningoradiculitis
Acrodermatitis chronica atrophicans
Diagnostisk sensitivitet
36%
77%
100%
7. Hansen K, Hindersson P, Pedersen NS. (1988) Measurement of
antibodies to the Borrelia burgdorferi flagellum improves serodiagnosis in
Lyme disease.J Clin Microbiol 26: 338-46.
8. 8.Karlsson M. (1990) Western immunoblot and flagellum enzyme-linked
immunosorbent assay for serodiagnosis of Lyme borreliosis. J Clin Microbiol
28: 2148-50.
Retur til dok liste
OD
prøve
<OD
IgG-påvisning
Negativ for IgG-antistoffer
mod B. burgdorferi:
Antistof tilstede.
OD
prøve
>OD
IgG-påvisning
and <OD
IgG-cut-off
Der anbefales opfølgende prøvetagning efter to uger for at
bekræfte patientstatus
OD
prøve
>OD
IgG-cut-off
Positiv for aktiv produktion af
IgG-antistof til B. burgdorferi
X7841 revideret oktober 2011
OXOID Limited,
Wade Road, Basingstoke,
Hampshire, RG24 8PW,
Verenigd Koninkrijk
Ved alle henvendelser, kontakt venligst Deres
lokale Oxoid filial eller forhandler
 SUU, Alm.del - 2014-15 (1. samling) - Bilag 1413: Henvendelse af 24/5-15 fra Alex Holmstedt vedr. behandling af borrelia
1533935_0064.png
Uddrag fra test kit indlægsbrochure:
LIAISON® Borrelia IgG ([REF] 310880)
Retur til dok liste
12. QUALITY CONTROL
LIAISON
®
controls should be run in singlicate to monitor the assay performance. Quality control must be performed by
running LIAISON
®
Borrelia IgG and Borrelia IgG Liquor controls
(a) at least once per day of use,
(b) whenever a new reagent integral is used,
(c) whenever the kit is calibrated,
(d) whenever a new lot of Starter Reagents is used,
(e) to assess adequacy of performance of the open integral beyond four weeks, or in agreement with guidelines or
requirements of local regulations or accredited organizations.
Control values must lie within the expected ranges: whenever one or both controls lie outside the expected ranges,
calibration should be repeated and controls retested. If control values obtained after successful calibration lie repeatedly
outside the predefined ranges, the test should be repeated using an unopened control vial. If control values lie outside the
expected ranges, patient results must not be reported.
The performance of other controls should be evaluated for compatibility with this assay before they are used. Appropriate
value ranges should then be established for quality control materials used.
13. INTERPRETATION OF RESULTS IN SERUM OR PLASMA
13.1. Borrelia IgG test
(serum or plasma samples)
The analyzer automatically calculates
Borrelia burgdorferi
IgG antibody concentrations expressed as arbitrary units (AU/mL)
and grades the results. For details, refer to the analyzer operator’s manual.
Calibrators and controls may give different RLU or dose results on LIAISON
®
and LIAISON
®
XL, but patient results are
equivalent.
Assay range.
5 to 240 AU/mL
Borrelia burgdorferi
IgG.
Samples containing antibody levels above the assay range may be prediluted by the Dilute function of the instrument and
retested (the recommended dilution factor is 1:10). The results will then be automatically multiplied by the dilution factor to
obtain the antibody levels of the neat specimens. The specimen diluent excess available in the reagent integral allows up to
10 sample predilutions to be performed.
Sample results should be interpreted as follows:
Samples with
Borrelia burgdorferi
IgG concentrations below 10 AU/mL should be graded
negative.
Samples with
Borrelia burgdorferi
IgG concentrations ranging between 10 and 15 AU/mL should be graded
equivocal.
Equivocal samples must be retested in order to confirm the initial result. Samples which are positive at the second test
should be considered positive. Samples which are negative at the second test should be considered negative. A second
sample should be collected and tested no less than one week later when the result is repeatedly equivocal.
Samples with
Borrelia burgdorferi
IgG concentrations equal to or above 15 AU/mL should be graded
positive.
13.2. Interpretation of results for serum or plasma samples
A negative result for IgM and/or IgG antibodies to
Borrelia burgdorferi
generally indicates that the patient has not been
infected, but does not always rule out acute borreliosis, because the infection may be in its very early stage and the patient
may be still unable to synthesize
Borrelia burgdorferi
specific antibodies, or the antibodies may be present in undetectable
levels. Specific IgM antibodies are more easily detected in the early stages of infection; in later stages they progressively
decline. It should be underlined that the test scores negative during the first weeks after infection. If clinical exposure to
Borrelia burgdorferi
is suspected despite a negative or equivocal finding, a second sample should be collected and tested
for IgM and IgG later during the course of infection.
A positive result for IgM and/or IgG antibodies to
Borrelia burgdorferi
generally indicates exposure to the pathogen (acute or
past infection). A single specimen, however, can only help estimate the serological status of the individual. An isolated
positive IgM result is observed relatively often in the early stages of the disease, but rarely in the later stages. An isolated
positive IgG result may indicate either active Lyme disease or past infection with persisting antibodies. The following table
summarizes the different immunological pictures. Results were obtained using LIAISON
®
Borrelia assays.
Borrelia burgdorferi
IgM result
negative
positive
negative
positive
Borrelia burgdorferi
IgG result
negative
negative
positive
positive
Interpretation
No evidence of infection. In case of clinical uncertainty (presence of tick bite or neu-
rological symptoms), the patients should be followed up during time.
Probable infection at an early stage.
Probable infection at any stage.
Probable acute infection.
Borrelia-G-en.fm
November 19, 2014 10:21 am
5 / 11
LIAISON
®
Borrelia IgG ([REF] 310880)
EN - 200/007-881, 03 - 2014-11
 SUU, Alm.del - 2014-15 (1. samling) - Bilag 1413: Henvendelse af 24/5-15 fra Alex Holmstedt vedr. behandling af borrelia
1533935_0065.png
14. INTERPRETATION OF RESULTS IN CEREBROSPINAL FLUID
14.1. Borrelia IgG test
(cerebrospinal fluid samples)
The analyzer automatically calculates
Borrelia burgdorferi
IgG antibody concentrations expressed as arbitrary units (AU/mL)
and grades the results. For details, refer to the analyzer operator’s manual.
Calibrators and controls may give different RLU or dose results on LIAISON
®
and LIAISON
®
XL, but patient results are
equivalent.
Assay range.
0.2 to 240 AU/mL
Borrelia burgdorferi
IgG.
Samples containing antibody levels above the assay range may be prediluted by the Dilute function of the instrument and
retested. The recommended dilution factor is 1:10; when the diluted samples still score above the assay range, the test
should be repeated after prediluting the samples 1:100. The results will then be automatically multiplied by the dilution factor
to obtain the antibody levels of the neat specimens. The specimen diluent excess available in the reagent integral allows up
to 10 sample predilutions to be performed.
Sample results should be interpreted as follows:
Samples with
Borrelia burgdorferi
IgG concentrations below 4.5 AU/mL should be graded
negative.
Samples with
Borrelia burgdorferi
IgG concentrations ranging between 4.5 and 5.5 AU/mL should be graded
equivocal.
Equivocal samples must be retested in order to confirm the initial result. Samples which are positive at the second test
should be considered positive. Samples which are negative at the second test should be considered negative. A second
sample should be collected and tested no less than one week later when the result is repeatedly equivocal.
Samples with
Borrelia burgdorferi
IgG concentrations equal to or above 5.5 AU/mL should be graded
positive.
14.2. Interpretation of results for cerebrospinal fluid samples
A negative result for IgG antibodies to
Borrelia burgdorferi
indicates unlikely intrathecal synthesis of
Borrelia burgdorferi
antibodies. If neuroborreliosis is strongly suspected despite a negative finding, further diagnostic investigation is suggested.
A positive result for IgG antibodies to
Borrelia burgdorferi
suggests possible intrathecal synthesis of
Borrelia burgdorferi
antibodies: neuroborreliosis is therefore suspected.
Positive results may be observed in patients with extremely high levels of circulating
Borrelia burgdorferi
antibodies as well
as in patients positive for
Borrelia burgdorferi
serum antibodies associated with high albumin concentrations in cerebrospinal
fluid. The latter finding suggests a possible damage to the blood/cerebrospinal fluid barrier.
Intrathecal presence of specific
Borrelia burgdorferi
antibodies should be evaluated taking into due consideration basic
cerebrospinal fluid variables, such as increased cell count, total IgG concentration, total protein concentration. For more
reliable quantification of intrathecal immunoglobulin synthesis, a hyperbolic function should be referred to for discriminating
between brain- and blood-derived protein fractions present in cerebrospinal fluid (according to H. Tumani, G. Nölker,
H. Reiber, 1995). The cerebrospinal fluid to serum volume ratio used in the LIAISON
®
Borrelia IgG test is 10.
15. LIMITATIONS OF THE PROCEDURE
Assay performance characteristics have not been established when any LIAISON
®
Borrelia test is used in conjunction with
other manufacturers’ assays for detection of specific
Borrelia burgdorferi
serological markers. Under these conditions, users
are responsible for establishing their own performance characteristics.
A skillful technique and strict adherence to the instructions are necessary to obtain reliable results.
Bacterial contamination or heat inactivation of the specimens may affect the test results.
Test results are reported quantitatively as positive or negative for the presence of
Borrelia burgdorferi
IgG. However,
diagnosis of infectious diseases should not be established on the basis of a single test result, but should be determined in
conjunction with clinical findings and other diagnostic procedures as well as in association with medical judgement.
Antibiotic therapy during the early stages of the disease often prevents development of antibody response.
Integrals may not be exchanged between analyzer types (LIAISON
®
and LIAISON
®
XL). Once an integral has been
introduced to a particular analyzer type, it must always be used on that analyzer until it has been exhausted. Due to
traceability issues resulting from the above statement, patient follow-ups may not be concluded between analyzer types.
These must be accomplished on one particular analyzer type (either LIAISON
®
or LIAISON
®
XL).
Borrelia-G-en.fm
November 19, 2014 10:21 am
6 / 11
LIAISON
®
Borrelia IgG ([REF] 310880)
EN - 200/007-881, 03 - 2014-11
 SUU, Alm.del - 2014-15 (1. samling) - Bilag 1413: Henvendelse af 24/5-15 fra Alex Holmstedt vedr. behandling af borrelia
1533935_0066.png
Assay Procedure
Uddrag fra test kit indlægsbrochure:
Immunetics® C6 B. burgdorferi(Lyme) ELISA™ Kit Cat. No. – DK-E352-096
Retur til dok liste
Bring all assay reagents to room temperature
(20 – 25°C) before beginning the assay. All steps are performed at room
temperature (20 – 25°C).
1. Record the sample identity for each well on the provided record sheet to determine the number of strips necessary to perform the assay.
Five wells will be needed for controls and calibrators. One well will be needed for each sample.
2. Remove the microplate frame containing the microplate strips from the foil pouch. Remove unneeded microplate strips from the frame and
reseal unused strips in the foil pouch with desiccant. Microplate frame should be retained at the end of the assay to be used with the
remaining microplate strips.
3. Add 100_L of diluted Positive Control to one microwell and 100_L of diluted Negative Control to another microwell.
4. Add 100_L of diluted Calibrator to each of three microwells.
5. Add 100_L of each diluted patient sample to microwells.
6. Incubate for 30 minutes.
7. Aspirate wells. If using manual or semi-automated washing manifold, wash three times as follows. Dispense approximately 150_L (half of
well volume) of 1X Wash Buffer into each well, then aspirate. Refill wells with approximately 300_L of 1X Wash Buffer (full volume of well)
and aspirate a second time. Refill wells with 300_L of 1X Wash Buffer and aspirate a third time. Make sure that all wells have been
aspirated after the third (final) wash step. (If an automated plate washer is used, wash four times with each wash consisting of 300-350_L
of 1X Wash Buffer. After the final wash for both manual and automated washing, tap the plate on absorbent towels to remove all residual
liquid.)
8. Dispense 100_L of Conjugate into each well.
9. Incubate for 20 minutes.
10. Aspirate wells. Perform four wash steps with 1X Wash Buffer as in step 8 above.
11. After the final aspiration, invert, shake out and blot the plate against absorbent towels to remove all residual liquid.
12. Dispense 100_L of TMB ELISA Substrate into each well.
13. Incubate for 4 minutes. Please note: Optimal assay performance requires precise timing of the TMB ELISA Substrate incubation step.
14. Dispense 100_L Stop Solution into each well in the same order as the TMB ELISA Substrate was dispensed in the previous step. Tap the
plate gently to mix contents of wells. Read absorbance values within 5 minutes.
15. Read Absorbance at 450nm with a reference filter of 650nm using an ELISA plate reader. If the reader is not equipped with a 650nm filter,
the use of an alternate filter between 590 – 650nm will provide equivalent results.
Quality Control
1.
2.
3.
4.
5.
Control values must be within the following ranges in order for the assay to be considered valid:
Negative Control A
450
must be <0.18
Each Calibrator A
450
must be between 0.400 and 2.00
Positive Control A
450
must be >1.2
If any control A
450
value is not within the above ranges, the assay should be repeated.
Calculations
1. Calculate the mean value for the three Calibrator Controls. If any Calibrator absorbance value differs by more than 0.1 absorbance units
from the mean, discard the data point that is farthest from the mean. Recalculate the mean from the two remaining data points. If the
Calibrator absorbance values still differ by more than 0.1 absorbance units from the mean, the assay is invalid and must be re-run. The
mean Calibrator absorbance value must be between 0.4 and 2.0 absorbance units.
2. Calculate the assay cutoff value by dividing the mean Calibrator value by 2.150 (Correction Coefficient).
3. Calculate the Lyme Index value (LI) for each patient sample by dividing the A
450
of the sample by the cutoff value.
Interpretation of Results
Lyme Index
≤
0.90
Interpretation
Negative result. No antibody to
B. burgdorferi
detected in the present assay. This result does not exclude the
possibility of
B. burgdorferi
infection, and where early Lyme disease is suspected, a second sample should be
drawn 2 – 4 weeks later and re-tested.
Equivocal result. The imprecision inherent in any method implies a lower degree of confidence in the interpretation
of samples with A
450
values very close to the calculated cutoff value. For this reason an equivocal category has
been designated. Equivocal samples should be tested with a supplemental assay such as a standardized Western
Blot test in accordance with CDC/ASTPHLD recommendations.
Positive result. Antibody to
B. burgdorferi
detected in the present assay. All positive results should be
supplemented by re-testing the corresponding serum samples on a standardized Western Blot test in accordance
with CDC/ASTPHLD recommendations
12
.
0.91 – 1.09
≥
1.10
The cutoff is determined for each assay run by dividing the mean calibrator value by the correction coefficient. In this way, the cutoff is
intended to compensate for run-to-run assay variations, which might otherwise affect sensitivity and specificity. The calibrator has been
designed to yield an absorbance value in the linear portion of the C6 ELISA dose-response curve. The correction coefficient was determined
by analysis of C6 ELISA results for 131 normal donors and 108 well-characterized Lyme disease patients. In assay runs using two separate kit
lots, the correction coefficient (2.15) was determined as the value which yielded a cutoff which minimized the number of false positive and of
false negative results.
Limitations
1. A negative result does not exclude the possibility of infection with
B. burgdorferi.
Patients in early stages of Lyme disease and those who have been
treated with antibiotics may not exhibit detectable antibody titers. Patients with clinical history, signs or symptoms suggestive of Lyme disease
should be re-tested in 2-4 weeks in the event that the initial test result is negative.
2. A positive result is not definitive evidence of infection with
B. burgdorferi.
It is possible that other disease conditions may produce artifactual
reactivity in the assay. All equivocal or positive results should be supplemented by re-testing the corresponding serum samples on a standardized
12
Western Blot test in accordance with CDC/ASTPHLD recommendations .
CF-E352-807
Effective Date: 29 December 2005
Page 3 of 8
 SUU, Alm.del - 2014-15 (1. samling) - Bilag 1413: Henvendelse af 24/5-15 fra Alex Holmstedt vedr. behandling af borrelia
1533935_0067.png
Uddrag: Borrelia burgdorferi antistof (Rnr. 302) - Statens Serum Institut
URL: http://www.ssi.dk/Diagnostik/DiagnostiskHaandbog/300-399/302.aspx
1 – 9 = positivt
≥
10 = stærkt positivt
Intratekal syntese angives som et antistof indeks.
Retur til dok liste
Svartid
Tolkning og
reference-
værdier/interval
Analysen udføres mindst 1 gang ugentligt, og svar afgives umiddelbart
herefter.
Diagnostik af de forskellige former for Borrelia infektion kan
erfaringsmæssigt volde problemer, fordi der ofte er en del
differentialdiagnostiske muligheder.
Antistoffer i serum: Kun c a. halvdelen af patienter med ubehandlet
erythema migrans (1. stadium) har antistoffer når de opsøger en læge. En
del af disse patienter forbliver seronegative.
Ca. 70 % af patienterne med ubehandlet neuroborreliose (2. stadium) er
seropositive efter 2 ugers sygdomsvarighed. 100 % er seropositive efter 8
uger.
IgG resultatet er altid positivt ved 2. og 3. stadie manifestationer med
sygdomsvarighed > 3 måneder. Ved acrodermatitis chronica atrophic ans
findes sædvanligvis meget høje IgG antistoffer og ingen IgM antistoffer.
Testen for serum antistoffer er for IgG og IgM indstillet således, at
henholdsvis 95 % og 98 % af raske kontrolpersoner giver et negativt
resultat.
Efter rekommanderet behandling og klinisk remission kan IgG syntese
fortsætte i lang tid (år). Syntese af IgM ophører oftest i løbet af 6
måneder, men kan vare noget længere uden kliniske tegn på terapisvigt.
Ved gentagne reinfektioner produceres der efterhånden ikke IgM
antistoffer. Mangel på IgM udelukker ikke aktiv infektion.
Intratekal antistofsyntese: Syntesen af antistoffer mod Borrelia begynder i
løbet af 2 uger hos ca. 80 % af patienterne med ubehandlet
neuroborreliose og kan altid påvises ved sygdomsvarighed over 6 - 8 uger.
Efter rekommanderet behandling og klinisk remission kan IgG syntese
fortsætte i lang tid (år). Syntese af IgM ophører oftest i løbet af 6
måneder, men kan vare noget længere uden kliniske tegn på terapisvigt.
?
?
Bemærkning
Falsk positiv IgM i serum kan ses ved bl.a. mononukleose og autoimmune
sygdomme.
De anvendte testmetoder måler antistoffer mod de tre kendte europæiske
genospecies af
Borrelia burgdorferi
sensu lato, der er årsag til human
sygdom (B.
afzelii, B. garinii, B. burgdorferi
senso stricto).
Syfilis antistoffer kan give falsk positive fund.
?
Relaterede
undersøgelser
Undersøgelsens
princip
Borrelia burgdorferi group (DNA) (R-nr. 1083)
IgG: indirekte ELISA, IgM: µ-capture ELISA. Som antigen anvendes
oprenset nativt flagel protein fra
B. afzelii
strain DK1.
Intratekal syntese af antistoffer: Samtidigt udtagen spinalvæske- og
serumprøve undersøges ved en særlig c apture ELISA for indhold af IgG og
IgM Borrelia antistof med flagel fra
B. afzelii
strain DK1 som antigen. Ud fra
mængderne i spinalvæske og serum beregnes separat for IgG og IgM et