New Real-Time PCR Assay for Rapid Detection of Methicillin- ResistantStaphylococcus aureusDirectly from Specimens Containing a Mixture of Staphylococci

Abstract

Molecular methods for the rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) are generally based on the detection of an S. aureus-specific gene target and the mecA gene. However, such methods cannot be applied for the direct detection of MRSA from nonsterile specimens such as nasal samples without the previous isolation, capture, or enrichment of MRSA because these samples often contain both coagulasenegative staphylococci (CoNS) and S. aureus, either of which can carry mecA. In this study, we describe a real-time multiplex PCR assay which allows the detection of MRSA directly from clinical specimens containing a mixture of staphylococci in <1 h. Five primers specific to the different staphylococcal cassette chromosome mec (SCCmec) right extremity sequences, including three new sequences, were used in combination with a primer and three molecular beacon probes specific to the S. aureus chromosomal orfX gene sequences located to the right of the SCCmec integration site. Of the 1,657 MRSA isolates tested, 1,636 (98.7%) were detected with the PCR assay, whereas 26 of 569 (4.6%) methicillin-susceptible S. aureus (MSSA) strains were misidentified as MRSA. None of the 62 nonstaphylococcal bacterial species or the 212 methicillin-resistant or 74 methicillinsusceptible CoNS strains (MRCoNS and MSCoNS, respectively) were detected by the assay. The amplification of MRSA was not inhibited in the presence of high copy numbers of MSSA, MRCoNS, or MSCoNS. The analytical sensitivity of the PCR assay, as evaluated with MRSA-negative nasal specimens containing a mixture of MSSA, MRCoNS, and MSCoNS spiked with MRSA, was ϳ25 CFU per nasal sample. This real-time PCR assay represents a rapid and powerful method which can be used for the detection of MRSA directly from specimens containing a mixture of staphylococci.

Detection of Methicillin-Resistant Staphylococcus aureus by a Duplex Droplet Digital PCR Assay

“…However, it was possible to evaluate the remaining DNA samples for the presence of the mecA gene using an independent set of primers. Of 17 discordant samples tested, seven were ultimately shown to contain mecA by sequence analysis and were not empty-cassette variants, as has sometimes been noted (23,34). If these additional seven samples do indeed represent true-positive samples, then the sensitivity and specificity of our assay should improve compared to the GeneXpert MRSA assay.…”

Molecular diagnostics and genotyping of MRSA: an update

“…The major limitations of this PCR strategy for direct testing in clinical samples were the necessity of enrichment and the inability to directly link identification of S. aureus with mecA gene detection because of the confounding effect caused by MRCoNS. In 2004, Huletsky et al described a novel real-time PCR assay [33]. This assay is able to distinguish between MRSA and mixtures of MSSA and MRCoNS and, therefore is suitable for direct identification of MRSA in clinical specimen.…”

Diagnostic dilemmas in retinitis and endophthalmitis

“…2,[29][30][31][32][33][34] Concordance with culture is close to 100%, with greater sensitivity with PCR testing. 29,34 The presumption is that PCR will eventually replace culture and sensitivity testing (by amplifying loci known to determine resistance to antibiotics 35 ) and enable the detection of unsuspected, fastidious, or previously unknown pathogens. 9,36,37 Intraocular Whipple disease is diagnosable by PCR of aqueous humor or vitreous.…”