Ultra fast protocol for direct detection of MRSA from swabs

Abstract number: P1963

Eigner U., Fahr A., Weizenegger M.

Objectives: We modified and validated the protocol of the GenoQuick^®MRSA direct version 2 beta (Hain Lifescience, Nehren, Germany) by introduction of a high speed Taq-Polymerase (SpeedSTAR™ HS-DNA-Polymerase (TaKaRa, Otsu, Japan). The GenoQuick^® MRSA assay is a PCR based assay targeting the staphylococcal cassette (SCCmec). For the detection of the MRSA specific amplicon and the "amplification control" (AC) a lateral flow dipstick is used.

Methods: For nucleic acid based identification of MRSA from swabs the prelaunched GenoQuick MRSA version 2 beta was used. The assay includes a lysis mix for isolation of the staphylococcal DNA, a PCR based amplification mix and a dipstick for detection. 329 DNA isolates from swabs sent for routine diagnostic were investigated. DNA-isolates (frozen at -70°C for several weeks) were taken from a previous study. The results were compared to routine testing including a chromogenic agar (CHROMagar MRSA, Becton Dickinson, Heidelberg, Germany) and a trypticase/soy broth. Cultured MRSA strains were confirmed with the GenoType MRSA (Hain Lifescience) test.

The PCR protocol recommended by the manufacturer was shortened to 15 min, 95°C, 40 cycles of 5 sec 95°C, 10 sec 55°C, 10 sec 72°C and for final denaturation 1 cycle of 2 min 95°C and 5 min 20°C. Samples run on a PE 2720 thermo cycler (Applied Biosystems, Darmstadt, Germany).

Results: 48 isolates had a congruent positive result and 277 a congruent negative result (sensitivity 96.1%, specificity 99.3%, positive predictive value 96.1% and negative predictive value 99.3%. Two samples of culture positive MRSA were missed. Two specimens had a false positive result with the GenoQuick assay. These two false positives were identified as meticillin (oxacillin) susceptible Staphylococcus aureus (MSSA) bearing a SCCmec missing the mecA gene. Six culture negative samples resulted weak positive with the PCR-test. All converted negative when repeated.

Conclusions: The modified GenoQuick^®MRSA assay adapted to the SpeedSTARTMHS-DNA-Polymerase shortens the time to result for the detection of MRSA from swabs from 2.5 to 1.5 hours. Together with the quick DNA-isolation procedure and an efficient manual hands on time this assay represents a very time optimised tool for direct detection of MRSA. Compared to the manufacturer's protocol no decline of the sensitivity and specificity values were observed.