New PCR primers for the sensitive detection and specific identification of group B beta-hemolytic streptococci in cerebrospinal fluid.

Group B beta-hemolytic streptococci (GBS) are a major cause of sepsis and meningitis in newborn babies. Although penicillin remains an effective treatment, there has been no decline in mortality. The rapid identification of GBS in cerebrospinal fluid (CSF) would improve the diagnosis of meningitis, but data from several previous studies indicate that the sensitivity of polymerase chain reaction (PCR) is not better than culture. Following extraction and precipitation of DNA, we have used semi-nested PCR to amplify a 450 base-pair and a 265 base-pair product of the 16S rRNA gene. This method has a lower detection limit of 50 fg of DNA and six CFU of GBS per millilitre. Specificity was confirmed by analysing a range of Gram-positive and Gram-negative organisms and pediatric isolates. Polymerase chain reaction analysis of 56 cerebrospinal fluid specimens from infants under 1 year of age was performed and compared with culture. False-negative results were only encountered when processed CSF supernatant was analysed. False-positive results were obtained when DNA from Streptococcus porcinus was amplified, however this is a rare organism which has yet to be isolated as a cause of neonatal meningitis. The data indicate that semi-nested PCR is a rapid tool which might be used to confirm a diagnosis of GBS meningitis in infants and newborn babies.