First experience with detecting bacterial DNA in the cerebrospinal fluid of patients with purulent meningitis using broad spectrum multiplex nested PCR
Abstract
Objectives: To propose and verify a PCR assay for detecting Escherichia coii, 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. Material and methods: DNA from the cerebrospinal fluid was isolated using the QIAamp DNA Mini Kit.
PCR was performed as twostep amplification (nested PCR). For E. coli, H. influenzae, L. monocytogenes, S. species and S. pneumoniae, universal and speciesspecific 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. Resiilts: 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.