Abstract
Methicillin-resistant Staphylococcus aureus (MRSA) is a significant cause of hospital-associated pneumonia (HAP). The rapid identification of MRSA would be beneficial for early diagnosis. The study aimed to evaluate a multilocus, fluorescence-based PCR assay based on the detection of mecA and nuc genes for identification of S. aureusin lower respiratory tract (LRT) specimens. Sensitivity and specificity of the PCR assay were analyzed. Clinical evaluation for the assay was performed using LRT specimens from patients with HAP, and the sensitivity, specificity, positive and negative predictive values (PPV and NPV) were evaluated in comparison with semi-quantitative culture methods. The result showed the assay provided positive identification of all MRSA reference strains with a limit of detection for MRSA of 4 × 103 CFU/mL. Compared with semi-quantitative culture, the sensitivity, specificity, PPV and NPV were 100%, 89.6%, 75.0%, and 100%, respectively. A positive correlation between MRSA bacterial colonies and PCR copy number was found. The specificity and PPV reached 96.6% and 89.7% respectively, if the PCR copy number reached a definite positive threshold of 5.96 × 105. It suggested that this novel multilocus, fluorescence-based PCR assay proved to be a fast, sensitive and specific tool for direct detection of MRSA from LRT specimens.
Highlights
Staphylococci bacteria, including Methicillin-resistant Staphylococcus aureus (MRSA), are some of the most common causes of hospital-associated pneumonia (HAP)
China between August 2008 and December 2010 showed that S. aureus was the third most frequent isolate (15.6%) identified in 610 cases of HAP, and MRSA accounted for 87.8% of S. aureus isolates
The presence of the nuc gene identified the presence of S. aureus, and the presence of mecA in S. aureus strains indicated the presence of MRSA
Summary
Staphylococci bacteria, including MRSA, are some of the most common causes of hospital-associated pneumonia (HAP). In a mixed flora clinical sample, MRSA and methicillinresistant CoNS cannot be sufficiently discriminated by detection of the mecA gene [10] When these tests are used for direct detection of MRSA in clinical specimens containing both, the results may be non-specific, resulting in a higher false-positive rate [6]. One approach to address specificity issues with PCR-based assays for detection of MRSA involved the development of a multilocus, fluorescence-based PCR protocol for detection of the mecA and nuc gene combination (Triplex International Bioscience Co. Ltd, Fujian, China), which was found to be suitable for routine use in a variety of clinical specimens, including blood, nasal swabs, sputum and BALF. We report on the development of a multilocus, fluorescence-based PCR assay for direct detection of MRSA and MSSA in clinical sputum specimens from HAP patients. The sensitivity, specificity, PPV and NPV were evaluated in comparison with the standard culture method
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