Rapid detection of methicillin-susceptible and methicillin-resistant Staphylococcus aureus directly from positive BacT/Alert blood culture bottles using real-time polymerase chain reaction: evaluation and comparison of 4 DNA extraction methods.
Although the introduction of automated blood culture systems has dramatically reduced laboratory personnel bench time, 48 to 72 h is still required for the identification of pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) and the accurate determination of antimicrobial sensitivities for prompt optimal patient therapy and infection control initiatives. The following 4 DNA blood culture extraction methods were compared: (a) organic, (b) differential centrifugation and lysis, (c) alkali wash/lysis, and (d) Qiagen lysis/filtration (QIAGEN, West Sussex, UK). The benzyl alcohol extraction method (a) was found to be the most optimal method having a reasonable extraction time of 1.8 h and 100% correlation with the "gold standard" laboratory culture. A "dual locus" real-time polymerase chain reaction (PCR) targeting the S. aureus-specific thermonuclease nuc gene and the staphylococcal methicillin resistance determinant mecA gene were used as a reliable indicator of the presence of MRSA. In conjunction with the DNA extraction method (a), detection time for MRSA/MSSA isolates from positive blood cultures was dramatically reduced from at least 24 to 48 h to approximately 3 h.
Hogg
Graham Michael
GM
Department of Microbiology, Belfast Health and Social Care Trust, Kelvin Building, Belfast BT12 6BA, Northern Ireland, United Kingdom. graham.hogg@belfasttrust.hscni.net
McKenna
James Patrick
JP
Ong
Grace
G
D001769:Blood / Q000382:microbiology*; D004269:DNA, Bacterial / Q000235:genetics*; D006801:Humans; D016106:Methicillin Resistance; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013203:Staphylococcal Infections / Q000175:diagnosis*; D013211:Staphylococcus aureus / Q000235:genetics / Q000302:isolation & purification*; D013997:Time Factors
D004269:DNA, Bacterial