M. Duplessis et al., RAPID DETECTION OF PENICILLIN-RESISTANT STREPTOCOCCUS-PNEUMONIAE IN CEREBROSPINAL-FLUID BY A SEMINESTED-PCR STRATEGY, Journal of clinical microbiology, 36(2), 1998, pp. 453-457
A seminested-PCR assay, based on the amplification of the pneumococcal
penicillin-binding protein 2B gene (pbp2B), was developed for the det
ection of penicillin-resistant and -susceptible pneumococci in cerebro
spinal fluid (CSF) specimens. Species-specific primers (P5 and P6) whi
ch amplified a 682-bp conserved region of the transpeptidase-encoding
region of the pbp2B gene were used. Four ''resistance'' primers were d
esigned to bind to altered areas of the pbp2B gene identified in penic
illin-resistant South African wild-type strains. Together with the dow
nstream primer P6, the upstream resistance primers amplified fragments
which were used to detect the presence of penicillin resistance. This
system identified all 35 of the S. pneumoniae isolates evaluated, inc
luding strains of 11 different serotypes and a range of penicillin-res
istant and -susceptible strains. The specificity of the assay was demo
nstrated by its inability to amplify DNA from other bacterial species
which commonly cause meningitis. It was possible to detect pneumococca
l DNA from culture-negative CSP inoculated with 2.5 pg of purified DNA
or 18 CFU. Analysis of 285 CSF specimens showed that PCR detected the
pneumococcus in 18 samples positive by culture, including the identif
ication of four penicillin-resistant isolates. The positive predictive
value and the negative predictive value of the assay were each 100%.