Evaluation of 2 real-time PCR assays for the investigation of mecA gene in clinical isolates of MRSA in Western Saudi Arabia.
OBJECTIVE
In diagnostic microbiology laboratories, Methicillin resistant Staphylococcus aureus (MRSA) is identified by positive coagulase test and positive deoxyribonuclease (DNase) activity followed by demonstration of oxacillin resistance on susceptibility testing on agar plate. This usually takes an approximately 48-72 hours. The purpose of this study is to evaluate 2 real-time polymerase chain reaction (PCR) assays for the presence of mecA gene in a population of MRSA strains circulating in Jeddah, Western Saudi Arabia, in order to determine their usefulness in the speedy diagnosis of MRSA in our clinical setting and their contribution to optimal patient management.


METHODS
Ninety MRSA isolates obtained from clinical samples were identified by using conventional methods. They were collected between February 2004 and August 2004, from 2 major hospitals in Jeddah; King Abdul-Aziz University Hospital, Jeddah (50 strains) and King Khalid National Guard Hospital, Jeddah (40 strains). All isolates were confirmed as MRSA using Gram stain, catalase and coagulase activity, confirmatory DNAse activity and Kirby Bauer disc diffusion method with resistance to oxacillin by the agar disc method. The DNA extract was tested by 2 assays. The first was the commercial IVD Roche kit, which detects the mecA gene using the Light Cycler system. The other method employs multiplex PCR which detects As442 fragment and mecA optimized for the Smart Cycler system (Cephied). The length of time taken to perform the assays was recorded.


RESULTS
All isolates were positive for Sa442 fragment and the coa gene specific for Staphylococcus aureus (S. aureus). However, 88/90 isolates (97.7%) tested were positive for mecA gene with both systems. The amplification, detection and melting curve analysis took 59.2 minutes for 32 samples on the Light Cycler and 46.7 minutes for 16 samples on the Smart Cycler.


CONCLUSIONS
The 2 methods studied were equally specific and sensitive for the detection of mecA gene in confirmed S. aureus isolates and capable of identifying MRSA much earlier than conventional methods. The detection of 2 targets in the multiplex PCR assay reduces the 2-hour time required for DNase testing and may be used as a primary screening test for the detection of MRSA in clinical samples, such as blood cultures and sterile body fluids.
Al-Haj-Hussein
Baraa T
BT
Molecular Biology Unit, Division of Microbiology, King Khalid National Guard Hospital, Jeddah, Kingdom of Saudi Arabia.
Al-Shehri
Mohammed A
MA
Azhar
Esam A
EA
Ashankyty
Ibraheem M
IM
Osoba
Abimbola O
AO
D017931:DNA Primers; D024881:Drug Resistance, Bacterial; D005798:Genes, Bacterial; D006801:Humans; D016106:Methicillin Resistance / Q000235:genetics*; D046915:Penicillin-Binding Proteins; D016133:Polymerase Chain Reaction; D012529:Saudi Arabia; D013203:Staphylococcal Infections / Q000382:microbiology; D013211:Staphylococcus aureus / Q000145:classification / Q000187:drug effects* / Q000235:genetics; D013997:Time Factors
D017931:DNA Primers; D046915:Penicillin-Binding Proteins