Comparative Evaluation of Traditional Susceptibility Testing for MRSA with the PCR Approach

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) is a multi-drug resistant pathogen, which is responsible for increasing cases of serious diseases, including life-threatening diseases and nosocomial and community-acquired infections. Laboratory identification of MRSA is crucial and essential both for initiation of appropriate antimicrobial therapies and for effective infection control strategies that are designed to limit the spread of MRSA. In spite of the availability of commercial kits for MRSA detection in the market, the Clinical and Laboratory Standards Institute (CLSI) recommends the use of phenotypic methods, such as the disk diffusion method with oxacillin or with cefoxitin, as well as a serial dilution method with oxacillin. Nevertheless, some studies have shown that results obtained with such phenotypic methods are controversial. The aim of the study described in this paper was to comparatively evaluate the traditional susceptibility testing for MRSA with PCR as the gold standard assay. Analysis of collection (n = 68) isolates of Staphylococcus aureus revealed that the serial dilution method with oxacillin possessed the highest sensitivity (at 100%). In contrast, the disk diffusion methods with oxacillin and cefoxitin showed lower sensitivity (95.83%, 95% CI (78.81% -99.30%)). Furthermore, the borderline value of zone inhibition diameters for cefoxitin might be considered as a risk, and they may give false-susceptible result.

An integrated closed-tube 2-plex PCR amplification and hybridization assay with switchable lanthanide luminescence based spatial detection

“…2 These trends have directed the focus towards multiplexed lab-on-a-chip devices, where several nucleic acid targets can be amplified and characterized in one sealed reaction unit in a separation-free manner. 3 Integrated multiplexed processing and handling of small samples and reagent volumes in a disposable reaction unit would make the biological analysis more cost-efficient and user friendly and less prone to human error and contamination. Thus far multiplexed nucleic acid assays have mostly been used in gene expression profiling and genotyping applications, but their importance in microbial diagnostics is growing fast 4 especially in pathogen identification and multidrug resistance screening.…”

“…Thus far multiplexed nucleic acid assays have mostly been used in gene expression profiling and genotyping applications, but their importance in microbial diagnostics is growing fast 4 especially in pathogen identification and multidrug resistance screening. 3 Numerous efforts have been made to develop a relatively fast multiplexed nucleic acid amplification test in a closedtube format for microbial point-of-care testing. The polymerase chain reaction (PCR) is still the most widespread nucleic acid amplification method due to its sensitivity, effectiveness and capacity despite the requirement of special equipment.…”

“…5 Since the introduction of PCR several multiplexed PCR assays have been developed and reached the market. So far most methods have needed separate post-PCR steps, such as agarose gel electrophoresis, 3,6,7 restriction enzyme analysis or hybridization assay for analyzing the amplification products [8][9][10][11][12] which remarkably increases the risk of contamination of the testing area with the amplification products. The importance of a multiplexed test lies in its ability to detect and identify a broad spectrum of pathogens at a time, which is important when a bacterial group is being screened instead of a single specific pathogen.…”

“…The importance of a multiplexed test lies in its ability to detect and identify a broad spectrum of pathogens at a time, which is important when a bacterial group is being screened instead of a single specific pathogen. 3 Several hybridization assays with a relatively high multiplexing capacity have been developed, such as the commercially available QuantiGene DNA Multiplex Assay by Affymetrix® (http://www.affymetrix. com) which can multiplex up to 33 different DNA targets, the Prove-it Sepsis assay from Mobidiag (http://www.mobidiag.…”

Advances in Rapid Pathogen Detection

“…qPCR looks at the amplification of DNA, through the generation of thousands to millions of copies of a particular DNA sequence, in real time and can detect down to one microbial cell. 10 This allowed a limit of detection of down to one cell to be achieved. In trials, the research group successfully identified a specific strain of Salmonella (S. enterica), selected from soil samples, and Pseudomonas aeruginosa from samples of water.…”