Cost-effective modification of a commercial PCR assay for detection of methicillin-resistant or -susceptible Staphylococcus aureus in positive blood cultures

Abstract

Real-time detection of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) in cases of clinical bacteremia may promote appropriate antimicrobial therapy and infection control. Expense inherent to molecular diagnostics may prevent laboratories from utilizing real-time PCR for this purpose. BD GeneOhm StaphSR assay master mix was reconstituted and aliquoted into SmartCycler tubes in 25-mul volumes (freshly reconstituted master mix), with a portion being frozen at -70 degrees C (frozen master mix). Incubation of 40 previously analyzed lysates from positive BacT/Alert SA and SN blood culture bottles (identified as 10 MRSA strains, 10 MSSA strains, 12 coagulase-negative Staphylococcus strains, and 8 Micrococcus strains) in freshly reconstituted master mix and master mix frozen between 1 week and 6 months generated the expected results in all PCRs. Similarly, positive- and negative-control reagents stored frozen at -70 degrees C for up to 18 weeks yielded the expected reactions. Prospective analysis of 244 positive blood culture samples utilizing 1-week-frozen master mix and freshly reconstituted master mix yielded a 1.2% discordant rate upon initial testing due to three unresolved results (two unresolved results for freshly reconstituted master mix and one unresolved result for frozen master mix). Repeat testing produced a final 100% concordance rate between the two master mix preparations. Use of master mix that was frozen up to 6 months did not compromise performance of the BD GeneOhm StaphSR assay. This modification, resulting in less reagent waste, may allow a greater number of laboratories to consider real-time PCR methodology for detection of bacteremia caused by MRSA and MSSA.

FIG. 1.

(A and B) Cycle threshold (C_T) values for BD GeneOhm StaphSR assay master mix frozen at variable intervals prior to incubation with lysates derived from clinical blood cultures yielding methicillin-resistant Staphylococcus aureus (A) and methicillin-susceptible Staphylococcus aureus (B). Data from two representative lysates are exhibited per panel and are shaded accordingly. (C) Internal-control C_T for frozen master mix incubated with representative coagulase-negative Staphylococcus lysates (solid black bars) and Micrococcus lysates (gray dotted bars).