LAMP Assay Validated for MRSA in Invasive Samples

Staphylococcus aureus is a leading cause of bone and joint infections and one of the most common causative pathogens of bacterial pneumonia in children. An increased risk of mortality from invasive staphylococcal infections has been attributed to methicillin-resistant S. aureus (MRSA).

The detection of pathogens by loop-mediated isothermal amplification (LAMP), which is a rapid, cost-effective, and simple method with high sensitivity, has resolved the major limitations of time-consuming culture-based methods and costly polymerase chain reaction (PCR) molecular techniques.

Molecular microbiologists at the University Hospital of Sant Joan de Déu (Barcelona, Spain) evaluated the analytical and diagnostic performance of the eazyplex MRSA test system for the detection of S. aureus and MRSA in invasive samples such as pleural and synovial fluid, in comparison to the gold standard bacterial culture. A volume of spiked pleural sample (analytical validation) or pleural/synovial fluid (diagnostic validation) was diluted in sterile water and genomic DNA was extracted using the NucliSENS EasyMag system.

The team pipetted 25 mL of a DNA mixture were into the six wells of the eazyplex MRSA strip. This contains a reagent mix and six primers in each cap for the simultaneous but individual amplification of Staphylococcus epidermidis- and S. aureus-specific genes, as well as resistance genes mecA and mecC for the detection of MRSA. Amplification products were generated by isothermal autocycling DNA synthesis and visualized by real-time fluorescence measurement with the Genie II device.

The new system appropriately detected a quality control panel of clinical samples with DNA of methicillin-sensitive S. aureus (MSSA), MRSA, and other pathogens. The limit of detection (LoD) was established at 6.4 × 103 CFU/mL for S. aureus and 1.0 × 104 CFU/mL for MRSA. Diagnostic validation of the eazyplex MRSA assay was performed on 51 prospective clinical invasive samples, resulting in sensitivity and specificity values of 83.3% and 97.8%, respectively, for S. aureus detection. The mean turnaround time was 70 minutes.

The authors concluded that the eazyplex MRSA test system was found to have promising potential as a diagnostic tool for the early detection of S. aureus in samples collected from normally sterile body sites. The study was published on in the June 2017 issue of the International Journal of Infectious Diseases.

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University Hospital of Sant Joan de Déu