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Journal of Clinical Microbiology, April 2000, p. 1648-1650, Vol. 38, No. 4
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Comparison of the Yields of Blood Cultures Using
Serum or Plasma from Patients with Early Lyme Disease
Gary P.
Wormser,1,*
Susan
Bittker,1
Denise
Cooper,1
John
Nowakowski,1
Robert B.
Nadelman,1 and
Charles
Pavia1,2
Division of Infectious Diseases, Department
of Medicine, New York Medical College, Valhalla, New York
10595,1 and New York College of
Osteopathic Medicine Microbiology and Immunodiagnostic Laboratory of
the New York Institute of Technology, Old Westbury, New York
115682
Received 5 November 1999/Returned for modification 20 December
1999/Accepted 2 February 2000
 |
ABSTRACT |
In an initial experiment, culture-grown Borrelia
burgdorferi was added to freshly collected uninfected human
blood. This in vitro study demonstrated that more spirochetes were
distributed into the plasma than into the serum fraction. In a
subsequent clinical study, B. burgdorferi was recovered
from plasma cultures of approximately 50% of 42 patients with early
Lyme disease associated with erythema migrans. The rate of recovery
from plasma cultures was significantly greater than that from serum
cultures (P < 0.001).
| |
TEXT |
The majority of patients with early
Lyme disease present with a single, expanding, circular skin lesion
called erythema migrans. Tests for detection of antibody to
Borrelia burgdorferi are usually negative at this stage of
illness (1). We have recently demonstrated that serum
cultures are positive in approximately 20% of these patients
(17). The volume of serum cultured correlated directly with
yield. The large volume of serum cultured in that study (
9 ml per
patient) is one likely explanation for the higher culture yields
compared to prior studies in which the volume of material cultured was
usually <1 ml (3, 8, 10-12, 14, 15).
The purpose of the present study was to determine if EDTA-preserved
plasma is preferable to serum as a source of culture material for
B. burgdorferi.
Experiment 1.
Experiment 1 was designed to determine if there
is a reduction in the number of B. burgdorferi organisms
detected in serum compared to EDTA-plasma when a large inoculum of
B. burgdorferi is directly introduced into blood samples.
B. burgdorferi strain 297 was grown in 50 ml of BSK medium.
The BSK medium was modified by us (13) from the formula
devised by Barbour (3). This BSK medium formulation did not
contain antibiotics or gelatin and was supplemented with 6% normal
rabbit serum (Sigma Chemical Co., St. Louis, Mo.). When late log phase
was reached, the culture was centrifuged at 2,200 × g
for 30 min to pellet the spirochetes. The pellet was resuspended in 2 ml of BSK medium, and the number of spirochetes present in a 10-µl
aliquot was determined using acridine orange and a microscope equipped
for fluorescence microscopy (see below). The concentration of
spirochetes was adjusted to an inoculum of 108/ml. This
high inoculum concentration was used in order to permit visual
detection of spirochetes when they were introduced into fresh whole
blood (see below).
Whole blood from healthy volunteers seronegative for B. burgdorferi antibodies was obtained by phlebotomy and placed into both 9.5-ml EDTA-blood collection tubes and 9.5-ml blood collection tubes lacking any anticoagulant. (Three volunteers donated blood during
the course of the experiment.) Immediately thereafter, 0.5 ml of the
B. burgdorferi preparation was injected into each blood tube
and the tubes were inverted four or five times to mix the spirochetes
and blood thoroughly. The blood tubes were then placed upright in a
test tube rack at room temperature and allowed to stand for 1.5 to
2 h to simulate clinic procedures. The blood tubes were then
centrifuged at 260 × g for 15 min. Aliquots (10 µl)
were mixed with 10 µl of acridine orange, and the spirochetes were
counted by fluorescence microscopy. The mean number of spirochetes present in five fields at a magnification of ×400 was determined, and
the number of spirochetes per milliliter was calculated based on the
formula devised by Fieldsteel et al. for performing microscopic counts
of spirochetes in liquid medium (7).
Experiment 2.
In EDTA-blood collection tubes, three 3-ml
samples of plasma were collected from untreated adult patients with a
clinical diagnosis of erythema migrans that had been established at the Westchester Medical Center (Valhalla, N.Y.) in 1999. All patients satisfied the surveillance criteria for Lyme disease of the Centers for
Disease Control and Prevention (5). In addition, three 3-ml
samples of serum were collected in sterile blood collection tubes
without anticoagulant. In total, 9 ml of plasma and 9 ml of serum were
collected by a single venipuncture from each patient. Both plasma and
serum were separated by centrifugation at 260 × g for
15 min. Within 3 h of the time of collection, each 3-ml aliquot of
either plasma or serum was inoculated into a 70-ml screw-cap plastic
flask containing 60 ml of BSK medium prepared as described above.
Cultures were incubated at 32 to 33°C for up to 12 weeks. The
cultures were examined by fluorescence microscopy at 2 weeks
and
thereafter at 2- to 4-week intervals. Sampling for each culture
was
done as follows. A 10-µl aliquot of culture material was mixed
with
10 µl of an acridine orange staining solution (100 µg/ml
in
phosphate-buffered saline [pH 7.41]). Ten microliters of this
mixture
was placed on a slide overlaid with a coverslip and examined
microscopically (magnification, ×400). A minimum of 20 high-power
fields were viewed for the presence of spirochetes. Confirmation
that
the visualized spirochetes were
B. burgdorferi was done by
performing PCR on a sample of the culture medium as previously
reported
(
16).
To remove nonborrelial microorganisms selectively, contaminated
cultures were filtered twice, first through a 0.45-µm-pore-size
filter (Nalgene; Nalge Co., Rochester, N.Y.) and then through
a
0.2-µm-pore-size filter (Nalgene) (
9).
Statistics.
Fisher's exact test, two tailed, was used for
comparisons of proportions. The Wilcoxon signed-ranks test was used for
paired data that were not normally distributed.
In experiment 1, in which blood samples were spiked with high
concentrations of
B. burgdorferi, the number of
microorganisms
observed in plasma was significantly higher than that
observed
in serum (Table
1) (
P = 0.002). The number of
B. burgdorferi bacteria in
plasma was 1.9 to 125 times larger than the number
in serum. The median
plasma-to-serum ratio of the number of
B. burgdorferi
bacteria per milliliter was 42.
In experiment 2, 20 (48%) of 42 patients had a positive serum or
plasma blood culture, including 8 (67%) of the 12 patients
with
multiple erythema migrans lesions and 12 (40%) of the remaining
30 patients with a solitary skin lesion (
P = 0.17). Serum
was
culture positive for 7 (17%) of the 42 patients, whereas
EDTA-plasma
cultures yielded
B. burgdorferi for 20 (48%)
patients (
P = 0.005)
(Table
2). Forty-two (33%) of the 126 3-ml
plasma samples were
culture positive for
B. burgdorferi,
compared to 16 (13%) of 126
3-ml serum samples (
P < 0.001). All culture-positive patients
had a positive plasma
culture, and plasma was the only culture-positive
sample for 13 (31%)
of the 42 patients. None of the plasma or
serum cultures was
contaminated.
View this table:
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|
TABLE 2.
Comparison of plasma (EDTA) and serum cultures for
recovery of B. burgdorferi in 42 adult patients with
erythema migransa
|
|
Of the 20 patients with positive plasma cultures, 7 (35%) were
positive on only a single 3-ml plasma sample. This finding
suggests
that culturing of more than 9 ml of plasma per patient
may be
associated with even higher rates of recovery of
B. burgdorferi.
These experiments have shown that 3-ml aliquots of EDTA-plasma from
untreated adult patients with erythema migrans are significantly
more
likely to yield
B. burgdorferi on culture than 3-ml samples
of serum (
P < 0.001). The most likely explanation for
the lower
rate of recovery from serum is that more spirochetes are
trapped
in a blood clot than in centrifuged erythrocytes. Consistent
with
this hypothesis, a greater-than-1-log reduction in the number
of
B. burgdorferi bacteria was demonstrated in serum compared
to plasma in an in vitro study using spiked samples of fresh whole
blood. Whether spirochete trapping during clot formation is merely
a
mechanical event or is related to binding of
B. burgdorferi to activated platelets (
6) or to another specific clot
component
is unknown. Alternatively, substances released during the
clotting
process might adversely affect the viability of
B. burgdorferi.
The higher yield of plasma cultures compared with serum cultures is
consistent with prior observations on detection of DNA
of
B. burgdorferi in blood components by PCR. Goodman et al.
(
8)
studied 76 patients with erythema migrans and were able
to amplify
B. burgdorferi DNA in 0.5 ml of EDTA-plasma of 14 (18.4%) of the
patients. PCR testing of serum, however, was negative
in at least
9 of these 14
patients.
In summary, our results demonstrate that the yield of blood plasma
cultures in early Lyme disease is approximately 50%, a
rate comparable
to or higher than those of many other common infectious
diseases, such
as pneumococcal pneumonia (
2). Plasma is preferable
to serum
as a source of culture
material.
| |
ACKNOWLEDGMENTS |
We gratefully acknowledge the assistance of Daniel W. Byrne,
Diane Holmgren, and Eleanor Bramesco.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Infectious Diseases, Room 209SE, Macy Pavilion, Westchester Medical
Center, Valhalla, NY 10595. Phone: (914) 493-8865. Fax: (914) 493-7289. E-mail: Gary_Wormser{at}NYMC.EDU.
| |
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Journal of Clinical Microbiology, April 2000, p. 1648-1650, Vol. 38, No. 4
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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