Evaluation of single and double-locus real-time PCR assays for methicillin-resistant Staphylococcus aureus (MRSA) surveillance

Abstract

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) is a human pathogen, representing an infection control challenge. Conventional MRSA screening takes up to three days, therefore development of rapid detection is essential. Real time-PCR (rt-PCR) is the fastest method fulfilling this task. All currently published or commercially available rt-PCR MRSA assays relay on single or double-locus detection. Double-locus assays are based on simultaneous detection of mecA gene and a S. aureus-specific gene. Such assays cannot be applied on clinical samples, which often contain both coagulase-negative staphylococci (CoNS) and S. aureus, either of which can carry mecA. Single-locus assays are based on detection of the staphylococcal cassette chromosome mec (SCCmec) element and the S. aureus-specific orfX gene, assuming that it is equivalent to mecA detection.FindingsParallel evaluation of several published single and double-locus rt-PCR MRSA assays of 150 pure culture strains, followed by analysis of 460 swab-derived clinical samples which included standard identification, susceptibility testing, followed by PCR detection of staphylococcal suspected isolates and in-PCR mixed bacterial populations analysis indicated the following findings.Pure cultures analysis indicated that one of the single-locus assay had very high prevalence of false positives (Positive predictive value = 77.8%) and was excluded from further analysis. Analysis of 460 swab-derived samples indicated that the second single-locus assay misidentified 16 out of 219 MRSA's and 13 out of 90 methicillin-sensitive S. aureus's (MSSA) were misidentified as MRSA's. The double-locus detection assay misidentified 55 out of 90 MSSA's. 46 MSSA containing samples were misidentified as MRSA and 9 as other than S. aureus ending with low positive predicted value (<85%) and very low specificity (<62%).ConclusionThe results indicate that high prevalence of false-positive and false-negative reactions occurs in such assays.

Real‐time multiplex PCR for direct detection of methicillin‐resistant Staphylococcus aureus (MRSA) in clinical samples enriched by broth culture

“…However, Kolman et al. (11) found that 16 of 219 (7.3%) MRSA isolates obtained from clinical samples were false negative using the Huletsky assay. Our protocol, mainly based on the study by Huletsky et al.…”

“…(8), Kolman et al. (11) found that 14 of 50 MSSA were misidentified as MRSA. All false positive samples in our study were found to contain MSSA.…”

“…Additional studies using the same concept have been performed (8–10). However, these reported methods were not able to detect all tested MRSA, and, in addition, the problem with false negative samples remained (11).…”

A novel approach to eliminate detection of contaminating Staphylococcal species introduced during clinical testing

“…However, nucleic acid target amplification presents challenges for dealing with “false-positive” results due to environmental or reagent contaminants introduced during the testing process [ 3 ]. For example, some bacteria, particularly Staphylococcus species, which are a normal microflora of human skin, are ubiquitously present in the environment [ 4 , 5 , 6 , 7 ]. It has been reported that coagulase-negative Staphylococcus (CONS) species are present in the nares of ~ 90% of the human population and are ubiquitously present on skin [ 5 , 6 , 7 ].…”

Microfluidic array chip for highly parallel genomic and transcriptomic applications

“…The mecA gene, which contributes to resistance, is present in both MRSA and MRSE (Figure 7-3). In such a situation, any probe signal that was directed to mecA cannot be linked to presence of MRSA alone.Thus, several multilocus PCR that simultaneously amplify mecA (resistance gene) and a S. aureus species specific gene such as sa442, nuc, femA, femB, clfA and spa were reported[166,168,172,173]. Although multilocus PCR are capable of producing quick and accurate results for blood culture or culture medium enriched samples, they cannot be applied directly to clinical samples as ambiguous results might appear due to presence of mecA-positive CoNS in the mixed clinical samples.…”