Evaluation of a nested-PCR assay for Streptococcus pneumoniae detection in pediatric patients with community-acquired pneumonia.

ABSTRACT

The aim of the present work was to evaluate the usefulness of a simplified method for DNA extraction coupled to a nested-PCR protocol, based on the amplification of pneumolysin gene fragments for the diagnosis of pneumococcal pneumonia in pediatric patients with clinical and radiological evidence of bacterial infection. Bacterial DNA was extracted from sera by boiling and used without further purification in the PCR for the pneumolysin gene. None toxic reagents were used and the necessary steps to obtain the DNA were left at a minimum; furthermore, it overcomes the use of expensive commercial kits for DNA purification. The total procedure can be completed the same day of sampling and, most important, it avoids the use of sophisticated technology. Both in vitro analytical specificity and sensitivity (10 CFU/ml) of the assay were similar to those previously reported. When clinical samples were tested, the rate of positivity was shown to be 83.3