[First experience with detecting bacterial DNA in the cerebrospinal fluid of patients with purulent meningitis using broad spectrum multiplex nested PCR].
OBJECTIVE
To propose and verify a PCR assay for detecting Escherichia coli, Haemophilus influenzae, Listeria monocytogenes, Staphylococcus species, Streptococcus pneumoniae and Neisseria meningitidis serogroups B and C in a single sample of the cerebrospinal fluid of patients with purulent meningitis.


METHODS
DNA from the cerebrospinal fluid was isolated using the QIAamp DNA Mini Kit. PCR was performed as two-step amplification (nested PCR). For E. coli, H. influenzae, L. monocytogenes, S. species and S. pneumoniae, universal and species-specific primers encoding bacterial 16S rDNA were used in the first and second reaction, respectively. For N. meningitidis serogroups B and C, an amplification system with primers for the SiaD gene was utilized.


RESULTS
Of 25 patients examined at the beginning of their treatment, bacterial DNA was detected in the cerebrospinal fluid of 17 (68 %) of them. Those were six cases of N. meningitidis serogroup B, four of N. meningitidis serogroup C, five of S. pneumoniae, one of H. influenzae and one of L. monocytogenes. Of 7 patients in whom antibiotic therapy was initiated prior to diagnostic lumbar puncture, PCR was positive in four cases.


CONCLUSIONS
The proposed nested PCR approach is faster than traditional culture methods and suitable for early laboratory diagnosis of infectious agents. When compared to culture methods, the technique offers slightly higher positivity (by 16 %). This is similar in samples analyzed after the initiation of antibiotic therapy. The PCR method never detected other bacteria than the cultured ones.
Moravcová
Lenka
L
Univerzita Karlova, 2. lékarská fakulta, 1. infekcní linika, FN Bulovka, Praha. moravcol@fnb.cz
Pícha
Dusan
D
Zdárský
Emanuel
E
Holecková
Daniela
D
Dzupová
Olga
O
Lásiková
Sárka
S
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002648:Child; D002675:Child, Preschool; D004269:DNA, Bacterial / Q000134:cerebrospinal fluid*; D005260:Female; D006801:Humans; D007223:Infant; D008297:Male; D016920:Meningitis, Bacterial / Q000175:diagnosis* / Q000382:microbiology; D008875:Middle Aged; D016133:Polymerase Chain Reaction
D004269:DNA, Bacterial