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Journal of Clinical Microbiology, February 2000, p. 855-857, Vol. 38, No. 2
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Simultaneous Approach for Nonculture PCR-Based Identification
and Serogroup Prediction of Neisseria
meningitidis
Muhamed-Kheir
Taha*
Unité des Neisseria and Centre National
de Référence des Meningocoques, Institut Pasteur, 75724 Paris cedex 15, France
Received 27 May 1999/Returned for modification 12 August
1999/Accepted 4 November 1999
 |
ABSTRACT |
A nonculture PCR-based method to characterize Neisseria
meningitidis was used to test 225 clinical specimens. PCR
correctly identified and predicted the serogroups of N. meningitidis of culture-proven meningococcal diseases and
confirmed this diagnosis in 35% of suspected samples. This approach
could be useful when culture fails to isolate N. meningitidis.
| |
TEXT |
Meningococcal infections (septicemia
and meningitis) are usually suspected on the basis of clinical symptoms
such as fever, vomiting, neck stiffness, and skin rash (purpura).
An etiologic diagnosis is confirmed by the isolation of
Neisseria meningitidis from cerebrospinal fluid (CSF),
blood, or other body fluids. However, this diagnosis is hindered by the
failure to isolate bacteria following early treatment
(3). Prophylactic measures (vaccination and/or
chemoprophylaxis) should be undertaken among contacts of the patient to
prevent secondary cases. Vaccines are available against strains of
N. meningitidis belonging to serogroups A, C, Y, and
W135 but not against strains of serogroup B. Hence, serogrouping is
necessary to better apply preventive measures. The aim of this
prospective study was to establish and to assess a nonculture
approach for identification and characterization of N. meningitidis.
Biological samples (n = 225) were obtained from 150 different patients who were admitted to several hospitals (n = 25) in France due to suspected meningococcal infection (Table
1). The hospitals enrolled in this study
all have pediatric departments. Patient clinical histories included two
or more of the following symptoms and signs: fever, meningism, purpura,
and arthralgia. Patients were classified into five categories on the
basis of laboratory findings. In patient category 1 (n = 33), culture was positive for N. meningitidis in CSF,
blood, or other body fluids (culture-proven meningococcal infection).
N. meningitidis was identified and systematically verified
using the Api-NH system (Biomérieux). Serogrouping for all
strains isolated was determined using group-specific immune sera
(Sanofi Diagnostics Pasteur). In patient category 2 (n = 20), culture was negative but direct smear and/or antigen
detection for N. meningitidis was positive (suspected
meningococcal disease). Antigen detection was performed using the
Pastorex latex agglutination kit (Sanofi Diagnostics Pasteur). In
patient category 3 (n = 147), culture was negative. Direct smear and antigen detection were negative or not done. However,
the CSF was abnormal, with a high cell count (>100 cells/µl of CSF),
decreased glucose content, or increased protein (possible meningococcal
disease). In patient category 4 (n = 10), culture was
positive for other bacterial species (other known bacterial meningitis). The diagnoses included pneumococcal meningitis
(n = 1), staphylococcal meningitis (n = 4), Listeria monocytogenes meningitis (n = 2), N. sicca meningitis (n = 1),
Bacteroides meningitis (n = 1), and
Mycobacterium tuberculosis meningitis (n = 1). In patient category 5 (n = 15), culture,
direct smear, and antigen detection were negative, with a low leukocyte
count (<100 cells/µl) and normal glucose and protein in the CSF
(clinically diagnosed viral meningitis).
To identify N. meningitidis, regardless of its serogroup, a
PCR screen was designed that amplified a conserved regulatory gene in
N. meningitidis, crgA, which was recently
identified in this laboratory. This gene is involved in the
regulation of adhesion of N. meningitidis to target cells
(A. Deghmane, S. Petit, A. Topilko, Y. Pereira, D. Giorgini, M. Larribe, and M.-K. Taha, unpublished data). For serogroup
prediction (A, B, C, Y, and W135), a multiplex PCR was performed
simultaneously with oligonucleotides in the siaD gene
(serogroups B, C, Y, and W135) (4) and in orf-2
of a gene cassette required for the biosynthesis of the capsule of
serogroup A (13) (Table 2).
The sizes of the expected amplicons from this multiplex PCR are 450 bp
(serogroup B), 400 bp (serogroup A), 250 bp (serogroup C), and 120 bp
(serogroups Y and W135) (Fig. 1). A PCR
for each serogroup was further realized to confirm the result and to
discriminate serogroups W135 and Y, for which the amplicon size was 120 bp (Table 2). Oligonucleotides used for the prediction of serogroups
were first tested on a collection of meningococcal strains of known
serogroups (15 strains of each serogroup were tested). These
oligonucleotides were specific for the corresponding serogroup, and no
cross-reactivity was observed between meningococcal strains of
different serogroups or with strains belonging to other bacterial
species (7 strains of N. gonorrhoeae, 2 strains of L. monocytogenes, 2 strains of Streptococcus pneumoniae, and 1 strain of N. lactamica) (data not
shown). Samples were subjected to one freeze-thaw cycle, heated at
100°C for 3 min, and then centrifuged for 5 min at 10,000 × g. In each assay, the final 50-µl reaction mixture contained
15 µl of each sample, 60 mM Tris-HCl (pH 8.8), 17 mM
(NH4)2SO4, 5 mM MgCl2,
0.5 mM each deoxynucleoside triphosphate, the corresponding
oligonucleotides (Table 2) at 0.3 µM, and 1 U of Taq
polymerase (Promega). The PCR assays were performed in a DNA thermal
cycler (Techne) with the following parameters: a first cycle of
denaturation at 94°C for 3 min, annealing at 55°C for 30 s,
and polymerization at 72°C for 20 s. The subsequent 35 cycles
were as follows: 92°C for 40 s, annealing at 55°C for 30 s, and polymerization at 72°C for 20 s. A final cycle of
polymerization at 72°C for 10 min was then performed. Amplicons were
analyzed by electrophoresis on a standard 2% agarose gel. In order to
test whether PCR was feasible for a given sample, a separate PCR was
performed after the addition of 3 µl of a suspension of N. meningitidis with a known concentration.
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|
FIG. 1.
PCR amplification of the siaD (serogroups B,
C, and Y and W135) and orf-2 (serogroup A) genes from
strains belonging to serogroups A (strain LNP10824), B (strain
LNP10846), C (strain LNP13331), and Y and W135 (strains LNP13145 and
LNP13230, respectively) and from three clinical specimens (lanes 1, 2, and 3). The strains used were previously described (6).
Lane  is the negative control (no bacteria). Electrophoresis was
done on a 2% agarose gel. Size markers (bacteriophage  X174 digested
by HaeIII) are indicated in base pairs at the right.
|
|
PCR correctly identified and predicted the serogroups of N. meningitidis in 32 of 33 samples that were also positive for this bacterium by culture (Table 1). The failure of PCR in one
culture-proven case (CSF but no serum was available) could be due to
the presence of a substance inhibitory for PCR and/or to the small
amount of meningococci in that specific CSF sample. Indeed, PCR was
less efficient with this specimen when external meningococci were added (data not shown). Moreover, this specimen was from a 2-year-old child
who died of meningococcal septicemia. Only 1 CFU was isolated from this
sample (200 µl). To circumvent such a problem, both CSF and serum
samples should be tested by PCR.
Specimens of category 2 that were positive by both direct observation
and antigen detection were also positive by PCR (Table 1).
Serogroup prediction by PCR matched that obtained by antigen assays. Three specimens were only positive by antigen detection for
serogroup B and were negative by PCR (Table 1). It is noteworthy that
cross-reactions between N. meningitidis and several other bacterial species were reported in antigen detection assays, in particular with serogroup B (7).
All samples obtained from other cases of nonmeningococcal bacterial
meningitis (n = 10) were negative by PCR for the
presence of meningococcal DNA. Samples from patients in category 5 (clinically diagnosed viral meningitis) were all negative by PCR,
except for one case (Table 1). The false-positive sample was CSF from a 5-year-old child who was hospitalized and treated for meningococcal meningitis. However, viral meningitis (category 5) was later kept as a
final diagnosis by clinicians, as CSF protein and glucose were normal.
The leukocyte count in the CSF was 28 cells/µl (63% polymorphonuclear cells). Several cases of culture-proven meningococcal disease reported in this study had similar CSF findings.
Sensitivity (93%), specificity (96%), positive predictive value
(98%), and negative predictive value (86%) were calculated using a
two-way table (Table 3) (14).
Positive cases (culture or direct smear and/or antigen detection)
corresponded to categories 1 (n = 33) and 2 (n = 20). Negative cases, which represent a rather small number, corresponded to categories 4 (n = 10) and
5 (n = 15) (Tables 1 and 3).
For possible meningococcal diseases (category 3), 51 (35%) specimens
were confirmed by PCR. These cases were undiagnosed by currently used
methods. This percentage is comparable to that (31%) obtained by
Newcombe et al. using PCR in suspected cases of meningococcal disease
(12).
Other methods for nonculture diagnosis of N. meningitidis
have been recently reported. They are based on the amplification of the
16S rRNA gene, IS1106, or the porB gene (8,
10, 12, 15). However, these methods usually identify the presence
of N. meningitidis without prediction of the serogroup or
only predict two serogroups (1). To the best of my
knowledge, no method is available for the prediction of serogroup A,
which is the serogroup encountered in the major epidemics in Africa.
Moreover, false-positive diagnosis of meningococcal infection by the
IS1106 PCR has been reported (2). The
amplification of two different meningococcal genes, as reported here,
should limit such a false-positive response. The identification and the
prediction of serogroups by this method can be performed rapidly.
Subsequently, it could be combined with our previously described
nonculture N. meningitidis typing method, which is based on
analysis of the polymorphism of a meningococcal gene (pilA)
(5). The distribution of pilA alleles among
different strains was previously shown to correlate well with the
typing obtained with the reference typing method, multilocus enzyme
electrophoresis (6, 9, 11). When this technique (the
polymorphism of pilA) was applied to samples which were
positive by PCR, different alleles of pilA were observed,
indicating different genetic lineages among the strains incriminated.
This result is in agreement with an endemic situation of meningococcal
disease in France. PCR assays should not be developed as an alternative
to culture. The development of specific and sensitive PCR methods
represents an efficient approach in the surveillance of meningococcal
disease when culture fails to isolate N. meningitidis.
| |
ACKNOWLEDGMENTS |
I am grateful to Jean-Michel Alonso for his great support, to
Edouard Bingen for encouragement, and to all of the biologists and
physicians who sent samples, strains, and clinical information to the
Centre National de Référence des Méningocoques. I
thank Dario Giorgini for technical help and Stewart Cole and Alexander Pym for critical reading of the manuscript.
| |
FOOTNOTES |
*
Mailing address: Unité des Neisseria and Centre
National de Référence des Méningocoques, Institut
Pasteur, 28 rue du Dr Roux, 75724 Paris cedex 15, France. Phone: 33 1 45 68 84 38. Fax: 33 1 45 68 83 38. E-mail:
mktaha{at}pasteur.fr.
| |
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Journal of Clinical Microbiology, February 2000, p. 855-857, Vol. 38, No. 2
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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