Direct Detection of Methicillin-Resistant Staphylococcus Aureus in Sputum Specimens from Patients with Hospital-Associated Pneumonia Using a Novel Multilocus Pcr Assay

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.

Characterization and Detection of mecA gene in Different Species of Staphylococcus, Streptococcus and Kocuria Which Isolated from Thalassemia Patients in Erbil City

“…All isolated bacteria which they are methicillin resistant and methicillin sensitive were tested for mecA gene by using PCR. The results of mecA of present study indicated that all isolated Staphylococci speceis were harboring mecA gene with the product size 533 bp except S. vitulinus as shown in figure (1) and these results were in agreement with [31]. In addition to above results, the results showed that mecA gene was not found in other isolated bacteria except one isolates of K. rosea with same mecA (533bp) as appeared in figure (2).…”

Detection of Methicillin Resistance Gene in S. Aureus by PCR

“…According to CLSI guidelines MRSA can be confirmed by detection of mecA gene. PCR based amplification of mecA is commonly used for detection of MRSA 14 . FDA approved PCR methods 6 have sensitivity in the range of 82 to 100 %.…”

“…A study 14 identified a novel multi locus fluorescence based PCR where mecA and nuc gene were detected simultaneously. The specificity of this PCR was shown to be over 96 % if PCR copy number reaches certain threshold.…”

New Strategies Targeting Virulence Factors of Staphylococcus aureus and Pseudomonas aeruginosa

“…93 Advances in molecular diagnostics, such as multivalent polymerase chain reaction analysis or automated microscopy, offer the ability to perform rapid real-time identification of pathogens, which can be confirmed with companion microbiological methods to measure antibiotic susceptibility profiles and the spectrum of pathogens. [94][95][96] In conjunction with rapid diagnosis, targeting specific virulence factors with AVAs may provide increased potential for individualized therapy in a bench-to-bedside approach to therapy. Early diagnosis may also open the door for prophylaxis and the potential to avoid inappropriate use of broadspectrum antibiotics before or at the time of clinical diagnosis of pneumonia.…”