Quantitative PCR for Etiologic Diagnosis of Methicillin-Resistant Staphylococcus aureus Pneumonia in Intensive Care...

Ventilator-associated pneumonia (VAP) requires prompt and appropriate treatment. Since methicillin-resistant Staphylococcus aureus (MRSA) is a frequent pathogen in VAP, rapid identification of it, is pivotal. Our aim was to evaluate the utility of quantitative polymerase chain reaction (qPCR) as a useful method for etiologic diagnoses of MRSA pneumonia.

We performed qPCR for mecA, S. aureus-specific femA-SA, and S. epidermidis-specific femA-SE genes from bronchoalveolar lavage or bronchial washing samples obtained from clinically-suspected VAP. Molecular identification of MRSA was based on the presence of the mecA and femA-SA gene, with the absence of the femA-SE gene. To compensate for the experimental and clinical conditions, we spiked an internal control in the course of DNA extraction. We estimated number of colony-forming units per mL (CFU/mL) of MRSA samples through a standard curve of a serially-diluted reference MRSA strain. We compared the threshold cycle (Ct) value with the microbiologic results of MRSA.

We obtained the mecA gene standard curve, which showed the detection limit of the mecA gene to be 100 fg, which corresponds to a copy number of 30. We chose cut-off Ct values of 27.94 (equivalent to 1×10(4) CFU/mL) and 21.78 (equivalent to 1×10(5) CFU/mL). The sensitivity and specificity of our assay were 88.9% and 88.9% respectively, when compared with quantitative cultures.

Our results were valuable for diagnosing and identifying pathogens involved in VAP. We believe our modified qPCR is an appropriate tool for the rapid diagnosis of clinical pathogens regarding patients in the intensive care unit.