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Background: The BD GeneOhm StaphSR real-time polymerase chain reaction (RT-PCR) assay (SR) was designed to rapidly identify and differentiate methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA), and S. aureus (SA)-negative blood cultures. SA is a common pathogen in skin and soft tissue (SST) and surgical site infections. Rapid detection of MRSA/MSSA in wound/SST specimens by RT-PCR would guide antimicrobial therapy decisions faster than culture.
Objective: To perform a laboratory validation of the BD GeneOhm StaphSR assay on wound/SST specimens.
Methods: The limit of detection (LOD) of SR for detecting MRSA and SA was determined by testing serial dilutions of MRSA and MSSA strains in trypticase soy broth (TSB) or pooled culture- and SR-negative wound broths and plotting cycle threshold (CT) values against colony forming units (CFU) per ml. Consecutive wound/SST swabs were eluted in TSB; 50ul of wound broth was lysed and SR performed per protocol. Sensitivity and specificity of SR for detecting MRSA and MSSA were calculated, using the cut-off CT value determined in the LOD studies. For SR results incongruent with culture, broth culture amplification for SA by incubation in TSB + 10% NaCl and PCR for femA and mecA genes was performed. femA (+) specimens were considered SA (+) and mecA (+) SA isolates were considered MRSA.
Results: Average LOD for MRSA and SA of 260 and 390 CFU/ml respectively consistently generated SR CT values ≤39. Two hundred sixty-four wound/superficial SST specimens were tested (table). The sensitivity and specificity of SR for MRSA alone was 93.3% (CI, 85.5-97.3%) and 97.7 (CI, 93.8-99.3%) respectively, and 87.3% (CI, 77.5-93.4%) and 96.8% (CI, 92.7-98.7%) respectively for MSSA alone. Twenty seven (10.3%) SR results that were incongruent with routine culture were further investigated. Three specimens, culture (+) for MSSA, were misidentified by SR as MRSA and were mecA (-). Two specimens, culture (+) for MRSA, were misidentified as MSSA by SR and were mecA (+). Of 27 incongruent results, 5 (1.9%) were misidentified by SR due to altered genetic targets, 13 (4.9%) were false SR results not attributable to altered genetic targets, and 8 (3.0%) were MRSA/MSSA (+) by SR and confirmatory tests but MRSA/MSSA (-) by routine culture. Six of 12 (50%) false negative specimens grew only rare SA on culture or grew other additional non-SA organisms.
Conclusions: A high rate of incongruence between SR and culture was observed when validating SR on wound/SST swab specimens. SR identified several MRSA/MSSA (+) specimens that were not detected by routine culture. However, SR was not reliable for use on wound/SST specimens due to the high rate of false positive and false negative results and the existence of certain SA clones whose methicillin susceptibility cannot be accurately assessed.
