One-step heminested PCR for amplification of Neisseria meningitidis DNA in cerebrospinal fluid.
A one-step polymerase chain reaction (Heminested-PCR) was designed to target the 16S rRNA fragment simultaneously using a set of primers for the universal bacterial group and a Neisseria meningitidis species-specific sequence for diagnostic purposes. The diagnostic features of the Heminested-PCR were evaluated in the study of 168 cerebrospinal fluid (CSF) specimens from 84 patients with a N. meningitidis infection, meningitis caused by unrelated bacteria and other etiologies (57 patients), or suspicious cases (27 patients) with clinical symptoms of bacterial meningitis but with negative results from bacteriological procedures. About 90% of patients with bacterial meningitis, including those suspicious cases, had prior antibiotic therapy. The sensitivity, specificity, positive, and negative predictive values found in relation to culture and/or microscopy were 91.7, 100, 100, 100, and 90.5%, respectively. In patients suspected of having bacterial meningitis, the Heminested-PCR revealed 51.9% (14 patients) positive for N. meningitidis infection and 40.7% (11 patients) positive for unrelated bacterial infections. The agreement of the Heminested-PCR with culture and/or microscopy was high and ranked as almost perfect (kappa indices > 0.856), in contrast to its agreement with other techniques. These findings speak in favor of the molecular diagnosis of meningococcal meningitis in patients who are culture- and/or microscopy-negative, due to their prior antibiotic treatment.
Atobe
J H
JH
Pharmaceutical Science Faculty, Department of Clinical and Toxicological Analysis, University of São Paulo, Brazil. atobe@usp.br
Hirata
M H
MH
Hoshino-Shimizu
S
S
Schmal
M R
MR
Mamizuka
E M
EM
D017931:DNA Primers; D004269:DNA, Bacterial / Q000134:cerebrospinal fluid*; D006801:Humans; D008585:Meningitis, Meningococcal / Q000134:cerebrospinal fluid / Q000175:diagnosis*; D008828:Microbiological Techniques; D009345:Neisseria meningitidis / Q000235:genetics* / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011237:Predictive Value of Tests; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S