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KMID : 0383820120720030293
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Tuberculosis and Respiratory Diseases
2012 Volume.72 No. 3 p.293 ~ p.301
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Quantitative PCR for Etiologic Diagnosis of Methicillin-Resistant Staphylococcus aureus Pneumonia in Intensive Care Unit
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Kwon Sun-Jung
Jeon Tae-Hyeon
Seo Dong-Wook
Na Moon-Joon
Choi Eu-Gene
Son Ji-Woong
Yoo Eun-Hyung
Park Chang-Gyo
Lee Hoi-Young
Kim Ju-Ock
Kim Sun-Young
Kang Jae-Ku
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Abstract
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Background : Ventilator-associated pneumonia (VAP) requires prompt and appropriate treatment. Since methicillinresistant 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.
Materials and Methods : 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.
Results : 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 1×104 CFU/mL) and 21.78 (equivalent to 1×105 CFU/mL). The sensitivity and specificity of our assay were 88.9% and 88.9% respectively, when compared with quantitative cultures.
Conclusion : 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.
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KEYWORD
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Methicillin-Resistant Staphylococcus aureus, Real-Time Polymerase Chain Reaction, Pneumonia, Ventilator-Associated
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