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Previous Article | Next Article 
Journal of Clinical Microbiology, August 1998, p. 2346-2348, Vol. 36, No. 8
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Enzyme Immunoassay Detecting Teichoic and
Lipoteichoic Acids versus Cerebrospinal Fluid Culture and Latex
Agglutination for Diagnosis of Streptococcus
pneumoniae Meningitis
Kristin
Stuertz,1
Imke
Merx,1
Helmut
Eiffert,2
Erich
Schmutzhard,3
Michael
Mäder,1 and
Roland
Nau1,*
Departments of
Neurology1 and
Medical
Microbiology,2 University of Göttingen,
Göttingen, Germany, and
Department of Neurology,
University of Innsbruck, Innsbruck, Austria3
Received 1 October 1997/Returned for modification 9 January
1998/Accepted 5 May 1998
 |
ABSTRACT |
A newly developed enzyme immunoassay (EIA) was used to detect the
presence of pneumococcal teichoic and lipoteichoic acids in
cerebrospinal fluid (CSF) from patients with Streptococcus pneumoniae meningitis who were being treated with antibiotics. All initial CSF samples, which on culture grew S. pneumoniae, were positive in the EIA. A total of 14 subsequent
culture-negative samples gave clear signals in the EIA up to day 15 after the onset of antibiotic treatment. For 11 CSF specimens, culture,
microscopy, and latex agglutination were negative while the EIA
detected pneumococcal antigens. The EIA did not react either with CSF
of patients with meningitis caused by bacteria other than S. pneumoniae or by viral pathogens. In conclusion, this EIA can be
a valuable tool for the diagnosis of S. pneumoniae
meningitis from CSF samples in cases in which prior antimicrobial
therapy minimizes the usefulness of culture or other antigen detection
tests.
| |
TEXT |
The management of patients suspected
of having bacterial meningitis depends on their state of consciousness.
Lumbar puncture is performed immediately after admission if the patient
is awake. Antibiotic treatment is started directly thereafter. Patients presenting with coma or focal neurological deficits may have severe edema or mass lesions. In this situation, lumbar puncture may be
harmful because of the potential for cerebral herniation. For this
reason, lumbar puncture is delayed for these patients. Instead, antibiotic treatment is started immediately after samples are taken for
blood cultures (10). Cranial computed tomography is then
performed. Cerebrospinal fluid (CSF) is not obtained by lumbar puncture
if the cranial computed tomograph shows severe brain swelling or mass
lesions. In these cases, CSF is not available for microbiological
cultivation prior to antibiotic treatment, and hence a sensitive test
for the diagnosis of Streptococcus pneumoniae meningitis
after pretreatment of the patient with antibiotics for several days is
highly desirable.
We therefore investigated the specificity and sensitivity of a newly
established enzyme immunoassay (EIA) that recognizes the
phosphorylcholine moieties of pneumococcal teichoic acids (TA) and
lipoteichoic acids (LTA) in patients with culture-proven cases of
S. pneumoniae meningitis. The results were compared with those obtained by microbiological cultivation, CSF Gram stain, and a
latex agglutination assay.
(The data herein were presented in part as a poster (no. D-160) at the
37th Interscience Conference on Antimicrobial Agents and Chemotherapy,
Toronto, Ontario, Canada, 28 September to 1 October 1997.)
Clinical data.
Ten unselected patients treated at our
institutions (Departments of Neurology, Universities of Göttingen
and Innsbruck) for S. pneumoniae meningitis were included in
this study. Prior to admission, three patients received oral antibiotic
treatment (patient B, norfloxacin; patient C, ciprofloxacin; and
patient G, cefixime), and patient J received two 2-g doses of
amoxicillin and two 0.5-g doses of clavulanate prior to lumbar
puncture. In the other patients, intravenous antibiotic therapy was
initiated immediately after lumbar puncture. For empiric therapy of
meningitis, the patients were treated as follows: patients A and B
received intravenous amoxicillin; patients C, D, E, G, and H were
administered amoxicillin plus cefotaxime; patient F was treated with
penicillin G and ceftriaxone; patient I was given amoxicillin and
clavulanate; and patient J received penicillin G alone. The repeat
lumbar punctures were considered necessary by the physicians in charge
of the patients. The authors of this study were not involved in the
timing of repeat lumbar punctures. Patient G had an external
ventriculostomy for treatment of acute occlusive hydrocephalus.
After withdrawal, the CSF was cultured, CSF leukocytes were counted,
and cytocentrifuge preparations were examined microscopically. The CSF
was then centrifuged for 10 min at 3,000 × g, and the supernatant was stored at 4°C for approximately 1 day to allow the
completion of routine analyses. Thereafter, CSF which was not required
for routine analyses was frozen at 
70°C.
EIA.
LTA was prepared from S. pneumoniae R6
(1). Polyclonal antibodies were raised in two New Zealand
White rabbits immunized subcutaneously with 500 µg of LTA mixed with
an equal volume of incomplete Freund's adjuvant. Immunization was
repeated every 4 weeks until high titers (
1:32,000) were obtained.
The sera were preserved at 20°C. The EIA used the commercially
available monoclonal immunoglobulin A (IgA) antiphosphorylcholine
antibody TEPC-15 (Sigma, Deisenhofen, Germany) as the
capture antibody and the polyclonal antiserum raised against LTA as the
detector antibody. Ninety-six-well microtiter plates were coated with
TEPC-15 monoclonal antibody at a concentration of 5 µg/ml. Blocking,
as well as the subsequent steps, was carried out with 5% fetal calf serum in phosphate-buffered saline at room temperature. CSF samples diluted 1:2 or higher in blocking buffer were incubated for 2 h;
this was followed by a 2-h incubation with the polyclonal antiserum (1:4,000). Bound rabbit antibodies were detected with
peroxidase-conjugated goat anti-rabbit IgG antibodies (1:8,000;
Dianova, Hamburg, Germany). Enzyme activity was determined with a
1-mg/ml ABTS solution [2,2'-azino-di(3-ethylbenzthiazoline sulfonate)
in 50 mM phosphate-citrate buffer (pH 4.4) containing 3 mM sodium
perborate]. The absorbance at 405 nm was determined after 30 min of
incubation. The assay was calibrated with a standard LTA preparation
(1) at concentrations ranging from 0.8 to 50 ng/ml and did
not differentiate between TA and LTA. The quantification limit of the
assay for LTA-spiked CSF from patients without evidence of bacterial
infection was 3.1 ng/ml. At room temperature and at 4°C, levels of
LTA in the spiked CSF were stable for 14 days. After 70 days at 4°C,
the LTA concentrations in these CSF samples still exceeded 50% of the
initial levels. No reduction of the LTA concentrations in CSF was
observed after several months of storage at 20°C.
Routine CSF analysis.
Standard procedures were used for Gram
staining, cultivation of microbes from CSF and blood, and typing of
bacteria. The commercially available S. pneumoniae-specific
Wellcogen latex agglutination test (Murex Diagnostika GmbH, Burgwedel,
Germany) was performed as recommended by the manufacturer.
In Table 1, the results of microbial
cultivation, CSF Gram stain, and detection of pneumococcal antigens, by
EIA and latex agglutination, in the CSFs of 10 patients with
culture-proven S. pneumoniae meningitis are shown. For the
majority of the patients, the first lumbar puncture was performed
before the initiation of antibiotic treatment, and S. pneumoniae could be cultured from CSF in all of these patients and
from blood in five cases. In 9 of 10 patients, the second CSF specimen
obtained was sterile, although gram-positive cocci were still visible
in the CSFs of 3 patients up to the 4th day of antibiotic treatment.
The CSF culture for patient J still was positive for S. pneumoniae 1 day after initiation of treatment. The samples
obtained on all subsequent days were culture negative.
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TABLE 1.
Detection of S. pneumoniae by culture, EIA,
and latex agglutination in consecutive CSF specimens from 10 patients
with culture-proven S. pneumoniae meningitis
|
|
On the first day, the latex agglutination assay detected pneumococcal
antigen in 8 of the 10 culture-positive CSF specimens. Agglutination of
the latex particles was observed in the sterile CSFs of two patients (E
and F) from the 3rd day of antibiotic therapy. The latex agglutination
assay was negative for three culture-positive CSF samples from two
patients (C and J).
All initial CSF samples, which on culture grew S. pneumoniae, were positive in the EIA. A total of 14 subsequent
culture-negative CSF samples from seven patients gave clear signals in
the EIA up to day 15 after the onset of antibiotic treatment. In 11 CSF specimens drawn during antibiotic therapy, all of which were negative by culture, microscopy, and latex agglutination, the EIA detected pneumococcal antigens. The time course of LTA and TA concentrations in
the CSFs of two patients who underwent repeated lumbar puncture is
shown in Fig. 1.
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|
FIG. 1.
Concentrations of LTA and TA detected by EIA in repeat
CSF samples from two patients with pneumococcal meningitis.  , EIA,
latex agglutination, and culture positive; +, EIA and latex
agglutination positive.
|
|
Control CSF samples from patients with viral meningitis
(n = 6) or bacterial meningitis caused by
Borrelia burgdorferi (n = 3),
Corynebacterium spp. (n = 1),
Enterococcus faecalis (n = 1),
Listeria monocytogenes (n = 3),
Mycobacterium tuberculosis (n = 2),
Neisseria meningitidis (n = 1),
Staphylococcus aureus (n = 2), or
Streptococcus agalactiae (n = 1) did not
cross-react in the EIA. Similarly, no cross-reactions were observed
with supernatants from cultures of Corynebacterium xerosis,
Enterobacter cloacae, Enterobacter sakazakii,
Enterococcus faecalis, Escherichia coli, Haemophilus influenzae, Listeria monocytogenes,
Proteus sp., Pseudomonas aeruginosa,
Salmonella enteritidis, Staphylococcus
epidermidis, and viridans streptococci killed by exposure to
bactericidal antibiotics. In broth, the EIA reacted with protein A
released by Staphylococcus aureus; this reaction could
easily be eliminated by adding solutions containing human IgG (e.g.,
CSF).
Cultivation of S. pneumoniae from a CSF specimen is
sometimes compromised by spontaneous autolysis or antibiotic treatment which decreases the concentration of living bacteria below the critical
threshold for culturing (2, 3). Moreover, bacterial identification by culture takes at least 24 h. Thus, a rapid and reliable alternative means of diagnosis, like capsular antigen detection, is of clinical importance. Several approaches to diagnosing S. pneumoniae meningitis based on antigen detection have
been pursued. Latex agglutination proved to be more sensitive than counterimmunoelectrophoresis or coagglutination (6, 7, 9). Latex agglutination is very simple to carry out and rapid, and special
equipment is not required. Several latex agglutination kits are
commercially available.
In this study, a newly developed EIA specific for the common
pneumococcal antigens TA and LTA was compared with microbiological cultivation of bacteria and with the Wellcogen latex agglutination test. In contrast to the other methods, the EIA allowed the detection of antigens from S. pneumoniae up to 15 days after the onset
of antibiotic therapy. For 11 CSF specimens drawn during antibiotic therapy, only the EIA was able to establish the diagnosis of S. pneumoniae meningitis. Holloway et al. (5) reported
that bacterial concentrations of 106 to 107
CFU/ml were necessary to consistently detect pneumococcal antigen by
latex agglutination. Yolken et al. (11) found a limiting concentration of 103 CFU/ml to be necessary for a positive
response in their EIA. These authors (11) used a polyclonal
antibody raised against the C polysaccharide (i.e., TA covalently
linked to peptidoglycan) as the capture reagent and a polyclonal
antibody against capsular polysaccharides from all 83 known serotypes
of S. pneumoniae (Omniserum; Statens Serum Institut,
Copenhagen, Denmark) as the detector antibody. Therefore, this assay
detects large cell wall fragments containing TAs together with capsular
polysaccharide linked by peptidoglycan (11). In addition to
recognizing larger pneumococcal fragments, the EIA developed by us also
recognizes free TA and LTA released by bacteria treated with
antibiotics (4). This enlarges the spectrum of detectable
antigens. The fact that small soluble antigens probably are not
phagocytosed qualifies our assay to recognize S. pneumoniae
antigens for several days after initiation of antibiotic treatment. The
EIA reacted with all samples containing culturable S. pneumoniae, whereas three false-negative results were obtained with the latex agglutination assay. Furthermore, as a quantitative assay, this EIA can be used for scientific purposes, e.g., to correlate
the CSF LTA and TA concentrations with the clinical outcome.
The EIA is, however, more time-consuming and expensive than latex
agglutination. Due to its greater demand for equipment compared with
culture and latex agglutination, the EIA will not replace these methods
in routine clinical circumstances. Nevertheless, its use is indicated
when the routine methods fail to identify the causative agent of
meningitis. When quantification of the concentration of LTA and TA is
not necessary, the EIA can be performed with a reduced standard curve:
blank wells lacking LTA, wells containing LTA at 3.1 ng/ml (i.e., the
quantification limit), and wells containing LTA at 50 ng/ml. This
strategy reduces the demands for time, reagents, and equipment.
Sørensen et al. (8) found that components of several
Streptococcus mitis strains cross-reacted in the
coagglutination assay. A cross-reaction with S. mitis was
also found by us when culture supernatants of S. mitis
killed by heat or by antibiotics were tested in the EIA. However,
bacteria that typically cause meningitis did not produce false-positive
results in this EIA.
In conclusion, due to its high level of sensitivity and rapidity of
performance, this EIA can be a valuable tool for the diagnosis of
S. pneumoniae meningitis (i) from CSF specimens of patients with severe brain edema, on whom lumbar puncture cannot be performed during the first few days of treatment, or (ii) from CSF samples in
cases for which prior antimicrobial therapy minimizes or negates the
usefulness of culture or other antigen detection tests.
| |
ACKNOWLEDGMENTS |
This work was supported by the Deutsche Forschungsgemeinschaft (Na
165/2-2).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Dept. of
Neurology, University of Göttingen, Robert-Koch-Str. 40, D-37075
Göttingen, Germany. Phone: 49-551-398455 or 396684. Fax:
49-551-398405. E-mail: mau{at}gwdg.de.
| |
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Journal of Clinical Microbiology, August 1998, p. 2346-2348, Vol. 36, No. 8
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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