Tuberc Respir Dis.  2012 Mar;72(3):293-301.

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

• Kwon SJ
• Jeon T
• Seo D
• Na M
• Choi EG
• Son JW
• Yoo EH
• Park CG
• Lee HY
• Kim JO
• Kim SY
• Kang J

Abstract

BACKGROUND
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.
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 1x10(4) CFU/mL) and 21.78 (equivalent to 1x10(5) 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.

Keyword

Methicillin-Resistant Staphylococcus aureus; Real-Time Polymerase Chain Reaction; Pneumonia, Ventilator-Associated

MeSH Terms

Adenosine
Bronchoalveolar Lavage
Coat Protein Complex I
DNA
Humans
Critical Care
Intensive Care Units
Limit of Detection
Methicillin Resistance
Methicillin-Resistant Staphylococcus aureus
Pneumonia
Pneumonia, Ventilator-Associated
Polymerase Chain Reaction
Real-Time Polymerase Chain Reaction
Sprains and Strains
Stem Cells
Adenosine
Coat Protein Complex I
DNA

Figure

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  Figure 1 Correlation between input DNA amount and Ct values. MRSA: methicillin-resistant Staphylocccus aureus.

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  Figure 2 Box-plot of Ct values from methicillin-resistant Staphylocccus aureus (MRSA) cultured specimens. Of the total samples, we created 4 groups based on the colony-forming units (CFU) results from the quantitative culture. Group 1 (n=56) contained samples with negative cultures, group 2 (n=0) contained samples with cultures of ≥103 to <104 CFU/mL, group 3 (n=8) contained samples with cultures of ≥104 to <105 CFU/mL, and group 4 (n=8) had samples with a culture of ≥105 CFU/mL. Boxes represent the 25th to 75th percentile interquartile range, with the line in the middle representing the median. The upper horizontal line represents the cut-off value of 1×104 CFU/mL, and the lower horizontal line represents the cut-off value of 1×105 CFU/mL.

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