Evaluación pre-analítica de dos métodos de extracción de ADN para la amplificación del gen de la pneumolisina (PLY) de Streptococcus pneumoniae, en muestras de hemocultivo

Abstract

Background: Streptococcus pneumoniae is a common etiologic agent of invasive respiratory infections among children under 5 years of age and older adults. Isolation rates of S. pneumoniae by traditional culture techniques are low. Aim: To study the sensitivity and specificity of two different DNA extraction methods to amplify the ply gene, applied to three different types of blood culture broths, experimentally inoculated with S. pneumoniae. Material and methods: DNA was ex-tracted from the cultures using an organic method or a technique that consists in dilution, washing with NaOH and concentration of the sample. This was followed by PCR amplification of a 355 pb fragment of the pneumolysin gene (ply). Results: The organic DNA extraction method inhibited the PCR reaction at all concetrations studied (0.6 to 10 6 colony forming units/mL). Using the NaOH extraction, ply gene am-plification was positive in all three blood culture broths, but only at concentrations of 10 3 colony form-ing units/mL or higher. Using the same DNA extraction method, PCR was negative when the broths were inoculated with seven other related bacterial species, which results in a 100% specificity. Conclusions: Detection of S. pneumoniae by amplification of ply gene from blood cultures using the protocol of NaOH for DNA extraction is specific and provides results in a short lapse. However, the diagnostic sensitivity is not optimal, wich limits its clinical use (Rev Méd Chile 2004; 132: 533-8).