Methicillin-resistant Staphylococcus aureus (MRSA), a virulent organism resistant to many drugs, is responsible for most nosocomial and community-acquired infections. It can cause life-threatening diseases, and treatment options are limited. Effective diagnostics is a strategic key element in the campaign against the spread of MRSA, allowing better infection surveillance and control measures as well as more efficient patient treatment and/or isolation options. The MagRSA project aims at the development of a new diagnostics platform that will provide a fast, simple and accurate identification of MRSA from clinical samples.
Methicillin-resistant Staphylococcus aureus (MRSA), an organism resistant to many drugs, is seen with increasing frequency in hospitals and long-term care facilities. It can cause life-threatening disease, and treatment options are limited. MRSA infections are associated with a 40% mortality when found in the blood of patients suffering from severe staphylococcal infection. According to the World Health Organization (WHO), resistance of Staphylococcus aureus to methicillin, its usual antibiotic, increased from 2% in 1975 to 60% today and no new antibiotic is expected on the market before many years. Whereas MRSA is considered as a nosocomial pathogen, recent reports showed an increasing number of outbreaks in the community, despite the absence of known risk factors (prior hospitalization, antibiotic use or household contacts from the healthcare system). A relatively large spread of MRSA strains within the gay community was recently reported several European countries and the United States. Such atypical MRSA is known to produce a potent toxin causing severe skin infections and necrotising pneumonia in both immunocompromised and immunocompetent individuals.
With such critical health issues, an early detection of MRSA carriers is crucial for infection control strategies but also to take appropriate therapeutic decisions, avoiding non-appropriate utilization of last barrier antimicrobial agents. Strategies to fight MRSA transmission are indeed well-described, and can efficiently reduce subsequent colonization and infection. However, for cost issues and better patient management, these strategies need to be focused on patients with confirmed MRSA and defined resistance phenotype.
The MagRSA project aims at the development of a new diagnostics platform that will provide a fast, simple, automated and accurate identification of MRSA from clinical samples.
The diagnostic protocol that we are currently developing relies on a new and clinically validated procedure that consists of a direct one-step enrichment of S. aureus present in either nasal or inguinal swabs, followed by DNA extraction of immunocaptured bacteria and their identification by multiplex sequence amplification using real-time quantitative PCR. This protocol will be implemented with a simple "hands-off" system based on: (1) novel strategies for the integration of full operations required for the entire nucleic acid analysis chain in a microfluidic platform, and (2) advanced microfluidic magnetic nanoparticles manipulation technology allowing efficient capture and extraction of target bacteria and nucleic acids. The separate steps of sample preparation, signal amplification by multiplex PCR, and simultaneous detection of multiple genes, will be performed as one single step using a ready-to-use disposable fluidic chip.
In light of the above, this project aims at providing hospitals and care units with a fast, easy and automated test for the rapid diagnostic of MRSA. Moreover, the simplicity of the proposed technology concept integrating cost effective and widely available components allows providing low cost systems, a prerequisite condition for the large adoption of molecular tests by hospitals. Main development steps occurred during the first half of the current project relies on:
A. Procedure improvement: All steps of the diagnostic protocol were significantly improved in terms of specificity, sensitivity and turn-around time.
Overall, compared to the first protocol, this new procedure is now performed with a turn-around-time of 3 hours instead of 6 hours and relies on the following steps (see Figure 1):
Figure 1: Schematic representation of the procedure (from RICAI 2008 poster by Franois et al. (from left to right). After fast sample conditioning, a single-step immuno-capture is performed before an enzymatic lysis and the DNA purification of captured bacteria. The last step consists in a qPCR and the analysis of results. The whole procedure requires now only 3 hours.
B. Platform testing: The other main domain of activity is the transposition of the assay to other qPCR platforms. Figure 2 shows that 3 different platforms were totally compatible yielding to similar results.
Figure 2: Screenshots of the sensitivity test performed with SDS7700 (left), SDS7500 (middle), or the StepOne (right).
C. Analysis solution: Our informatician team has developed an application allowing the rapid analysis of qPCR results. This development was mandatory based on the fact that the assay will be transposed in routine laboratories. It appears crucial to limit user- or laboratory dependant interpretation of results. All samples are identified either manually or using a barcode reader. The number of samples to be tested is then calculated and the application allows preparing the experiment by calculating all volumes of reagents (primers, probes, enzymatic mixture .) required for the corresponding number of samples to be assessed including the controls (Figure 3A). Following completion of the qPCR run, the results are exported as ".csv file" containing sample identification and fluorescence intensities for the different fluorophores at each cycle. This file is used as an input and the analysis -performed in a few seconds- provides colour-encoded results, as shown on Figure 3B.
Figure 3: Screenshots of the analysis application developed specifically for the qMRSA multiplex analysis. A colour code allows to rapidly detect positive samples containing MRSA (red rectangles), from negative samples (green rectangles) or from samples containing MSSA (orange rectangles) or MRSE (blue rectangles).
The MagRSA project will address the unmet need for new diagnostics tools for management and control of antimicrobial resistance in general and methicillin-resistant Staphylococcus aureus (MRSA) in particular. Moreover, MagRSA project will provide a diagnostics platform with potential applications in molecular diagnostics as the most growing segment within the global in-vitro-diagnostics market.