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New PCR primers for the sensitive detection and specific identification of group B beta-hemolytic streptococci in
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Rapidly identify Group B Streptococcus (GBS) in newborns using semi-nested PCR. This molecular test offers a faster, sensitive method for diagnosing GBS meningitis in infants, potentially improving outcomes.
Area of Science:
- Microbiology
- Molecular Biology
- Neonatal Medicine
Background:
- Group B Streptococcus (GBS) is a leading cause of neonatal sepsis and meningitis.
- Penicillin is effective, but mortality rates have not decreased, highlighting diagnostic challenges.
- Current diagnostic methods lack rapid turnaround for timely intervention.
Purpose of the Study:
- To evaluate the sensitivity and specificity of a semi-nested PCR assay for GBS detection in cerebrospinal fluid (CSF).
- To assess the utility of PCR as a rapid diagnostic tool for GBS meningitis in infants.
Main Methods:
- DNA extraction and precipitation followed by semi-nested PCR amplification of the 16S rRNA gene.
- Detection of 450 bp and 265 bp products with a lower limit of detection of 50 fg DNA and 6 CFU/mL.
- Comparison of PCR results with conventional culture methods in 56 infant CSF samples.
Main Results:
- The semi-nested PCR demonstrated a high specificity, with no cross-reactivity with common Gram-positive and Gram-negative organisms.
- A low detection limit of 6 CFU/mL was achieved, surpassing culture sensitivity in some cases.
- False-negative results occurred with processed CSF supernatant, while rare Streptococcus porcinus DNA caused false positives.
Conclusions:
- Semi-nested PCR is a rapid and sensitive method for confirming GBS meningitis in newborns and infants.
- This molecular technique can significantly aid in the timely diagnosis and management of neonatal meningitis.
Abstract:
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.