Evaluation of a real-time PCR assay and an multiplex-reverse hybridisation system for the detection of methicillin-resistant Staphylococcus aureus

Abstract number: 1733_393

Ieven M., Michiels M., Jansens H., Goossens H.

Objective: To evaluate the performance of a commercial multiplex PCR – reverse hybridisation system, Hyplex StaphyloResist® (BAG Germany distributed in the Benelux by AlphaOmega instruments), and a real-time PCR assay for the detection of MRSA from 24 hours enrichment broths compared to the routine culture method.

Methods: A total of 500 enrichment broths from MRSA screenings were included in the study. On arrival in the lab, swabs were cultured by direct plating and subsequently put in MRSA enrichment broth. After 24 hours incubation the broth was subcultured on routine media and MRSA was identified by conventional methods. A broth sample was taken for DNA extraction and two PCR assays. The Hyplex StaphyloResist® consists of a multiplex PCR amplifying simultaneously mecA gene, coagulase gene and a ``housekeeping'' gene. Specimens were processed according to the manufacturer's instructions. For the real-time PCR assay specimens were extracted using the Qiagen blood mini kit with consecutive detection of mecA, nuc and SCCmec genes, according to a modified protocol by Huletsky et al (JCM 2004; 42: 1875–84). PCR results were considered positive if confirmed by culture or the second PCR.

Results: In total 78 (16%) of the 500 MRSA enrichment broths tested were culture and both PCRs positive, 6 were culture negative and both PCRs positive, 1 was culture positive and both PCRs negative. 6 broths were culture positive and one PCR negative (5 real-time PCR assay, 1 Hyplex StaphyloResist® assay) and 28 broths were culture negative and one PCR negative (14 real-time PCR assay, 14 Hyplex StaphyloResist® assay). The sensitivity, specificity and negative and positive preditive values of the Hyplex StaphyloResist® assay for the detection of MRSA from broth were 98%, 96%, 99% and 83% and 93%, 96%, 98% and 83% for the real-time PCR assay, respectively. Both PCR assays provided same day results on 24 hours enrichment broths compared to 48 to 96 hours for routine culture method. The time needed to complete the Hyplex StaphyloResist® and real-time PCR assays were 3 hrs 25 min and 2 hrs 30 min with a hands-on time of 1 hrs 30 min and 1 hrs 15 min, respectively.

Conclusion: There is no significant difference in sensitivity and specificity between the Hyplex StaphyloResist® assay and the real-time assay for the detection of MRSA from enrichment broth. More than 90% of MRSA positives can be identified after one day by either PCR if performed on MRSA enrichment broth.

Session Details

Date: 31/03/2007
Time: 00:00-00:00
Session name: European Society of Clinical Microbiology and Infectious Diseases
Location: ICC, Munich, Germany
Presentation type:
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