Antimicrobial Susceptibility Studies
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

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Abstract

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.

Introduction

Blood culture is the mainstay laboratory procedure for the prompt diagnosis and effective management of bacteremic/septicemic patients. Automated blood culture systems still require approximately 48 h to identify the pathogen and determine antimicrobial sensitivities to allow prompt and optimal chemotherapy of such significant pathogens as methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) (Brown et al., 2005, Gould, 2005). The management of continuously monitoring blood culture systems can be dramatically improved by considering the rapid identification of such blood culture isolates.

Molecular-based diagnostic methods, such as real-time polymerase chain reaction (PCR), is now becoming established in clinical microbiology laboratories because this technology has the potential to deliver rapid, specific, and sensitive detection of microbial pathogens from blood cultures within 1 working day (Espy et al., 2006, Paule et al., 2005, Tan et al., 2001). It is essential, however, that the DNA extraction procedure produces DNA of sufficient quality for PCR where the presence of PCR inhibitory substances, for example, sodium polyanetholesulfonate (SDS), found in blood culture media can be successfully removed (Al-Soud and Rådström, 2001, Fredricks and Relman, 1998, Wilson, 1997). To fit into a busy diagnostic laboratory, this extraction procedure must be relatively simple, fast, and sensitive yet robust, reproducible, and cost-effective.

In this study, 4 blood culture DNA extraction methods were compared when used in a rapid specific real-time PCR assay targeting the Staphylococcus species-specific nuclease gene (nuc) and the methicillin resistance gene (mecA) (Costa et al., 2005).

Section snippets

Clinical specimens

Twenty-nine blood culture-positive (for MRSA/MSSA) (BacT/Alert® FA [n = 15], BacT/Alert® SN [n = 13], BacT/Alert® PF [n = 1]; Organon Teknina Corp., Durham, NC) bottles were collected and nucleic acid extracted using the 4 methods described below. Uninoculated blood culture bottles were included as extraction controls. All DNA extracts were subsequently tested in duplicate by real-time PCR using established in-house mastermixes with the inclusion of relevant positive and negative controls.

a) Organic extraction

This

Results

The study results are presented in Table 2, Table 3. Table 2 lists the breakdown of the real-time PCR results for each DNA extraction method. Table 3 shows the comparison and the total score outcomes for each DNA extraction method.

Discussion

Blood cultures are among the most important laboratory tests used for the prompt diagnosis and effective management of septicemia. The introduction of automated blood culture systems dramatically reduced laboratory personnel bench time; however, it still requires 48 to 72 h for the identification of pathogens such as MRSA/MSSA and the accurate determination of antimicrobial sensitivities for prompt optimal patient therapy and infection control initiatives (Brown et al., 2005). Because

Conclusion

Using the DNA organic extraction method with our real-time PCR assay, which had a specificity and sensitivity for MRSA of 99.2% and 100%, respectively (unpublished data), detection time for MRSA/MSSA isolates from positive BacT/Alert® blood cultures was dramatically reduced from at least 24 to 48 h to approximately 3 h.

The Qiagen® kit (method d), the commercial DNA extraction system routinely used in our laboratory, was found to be less successful in DNA extraction from BacT/Alert blood

Acknowledgments

The authors would like to acknowledge the financial support from Antimicrobial Action Plan for Northern Ireland (AMRAP).

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