qMRSA: Rapid Molecular Detection of Methicillin-Resistant Staphylococcus aureus (MRSA) Directly from Clinical Swab Samples

Research Program Rewarded by the 2004 Pfizer Award

A rapid procedure was developed for the detection and identification of methicillin-resistant Staphylococcus aureus (MRSA) directly from sterile sites or mixed flora samples (e.g. nares or inguinal swabs).

After a rapid conditioning of samples, the method consists of three main steps:

1. immunomagnetic enrichment in S. aureus;
2. rapid DNA extraction;
3. amplification/detection profile on DNA extracts using multiplex quantitative PCR.

This assay was optimized to run either on the SDS 7700 TaqMan (Applied Biosystems®) or a random access machine (SmartCycler®, Cepheid®).

The triplex qPCR assay measures simultaneously the following targets :

1.  mecA gene, conferring methicillin resistance, common to both S. aureus and S. epidermidis;
2.  femA gene from S. aureus;
3.  femA gene from S. epidermidis.

This quantitative approach allows discriminating the origin of the measured mecA signal. qPCR data are calibrated using two reference strains (methicillin-resistant S. aureus and S. epidermidis) processed in parallel to clinical samples.

We prospectively tested Intensive Care Unit (ICU) patients whose 483 samples originated from four different sources (nares, inguinal, wound or pooled swabs). MRSA prevalence was 16.3% based on culture results. Compared to an optimized culture procedure including broth enrichment, molecular testing yielded a sensitivity of 81% and a specificity of 92%. Sensitivity reached 100% when a larger fraction of the sample extract was subjected to qPCR (n=49).

The complete protocol may provide results in less than 4h (while standard procedure needs 2-3 days), thus allowing prompt and cost-effective implementation of contact precautions.

Immunocapture from mixed cultures

Immunocapture of biotinylated monoclonal anti-protein A antibodies (anti-spa) using streptavidin-coated magnetic beads Optimized anti-spa titres to immunocapture <5 CFU of MRSA MRSA recovery >85% even with a 1’000 fold excess amount of Methicillin-Resistant Staphylococcus epidermidis (MRSE) Excellent recovery rates observed for 5-1’000 CFU of MRSA Similar recovery rates using two Pastorex-negative MRSA strains

Linearity & detection limits of the triplex qPCR assay

Detection was linear across >6 orders of magnitude Lower limits of detection: 5 fg genomic DNA, this is equivalent to 1-2 S.aureus cells Similar results were obtained with the two femA targets * Value excluded from linear regression ** Amounts of input DNA leading to irregular signal detection

Effect of the immunocapture on the qPCR assay

	MRSA (103 CFU) was mixed with MRSE as indicated. Without immunocapture: MRSA signals were reduced by a factor >30, when mixed with a 10 fold excess amount of MRSE In the presence of a >100 fold excess amount of MRSE, MRSA signals were no longer detected (NA) Following immunocapture: MRSA detection was not affected by the presence of a 100 fold excess amount of MRSE

qMRSA versus culture in a prospective clinical study

483 samples were prospectively collected in two ICU wards from the four following body sites: nares (193), inguinal (193), pooled swabs (69), or wounds (28). MRSA prevalence was 16.3% . Cultures were performed after a 24h enrichment in CS broth: BHI supplemented with colistin (10 µg/ml) and 2.5% NaCl. Sensitivity: In this study, only 10% of the sample extract were subjected to qPCR. When using the complete sample extract, the sensitivity increased from 81% to 100% (similar population and MRSA prevalence, n=49). Specificity: A retrospective analysis revealed that the majority of the 32/483 (8%) false positive cases were either previously known as MRSA carriers and/or were screened as MRSA culture-positive at any other sampling site during the same hospital stay.

Recently, an absolute quantification assay of qMRSA was developed. The robustness of the assay was evaluated on bronchoalveolar lavage (BAL) to identify and quantify methicillin-resistant Staphylococcus aureus (MRSA) in ventilator associated pneumonia. Absolute qPCR for MRSA was able to distinguish MRSA from MRSE, MSSA, MSSE based on three genes amplification profiles and detect mecA gene down to a copy number of 3 with a high degree of linear correlation (R2=0.999). Among nucleic acid extracts from BAL specimens, 19 specimens showed the presence of MRSA, 5 specimens showed MRSA and MRSE, 8 showed only MRSE, 1 showed MSSA, 3 showed MSSE and 62 samples were negative.

Using quantitative culture results as a  gold standard for MRSA diagnosis, the sensitivity and specificity of absolute MRSA qPCR were 90.45% and 94.47% respectively. When using samples >100 copies/ml mecA gene as cutoff, the positive predictive value increases from 82.61% to 94.12% without significantly sacrifice the negative predictive value (97.26% to 96.20%).

MRSA absolute qPCR is fast, highly sensitive, specific and quantitative assay for detection of MRSA in VAP by using mini-BAL specimens. See the poster presented by Dr. Xue-Ping Wang at the Association of Biomolecular Resource Facilities 2007 meeting or visit the website.

Conclusions

Early detection of MRSA-carriers is crucial for infection control strategies but also for therapeutic decisions. The specificity of qMRSA molecular identification is based on:

 
1. The presence of the mecA gene;
 
2. and the presence of a S. aureus-specific femA signal that does not cross-react with other bacterial species including S. epidermidis.

This novel qPCR assay allows detection and identification of MRSA in less than 4 hours with an exquisite sensitivity. Very high negative predictive values should allow the implementation of prompt and cost-effective infection control measures. A large-scale prospective comparative study was initiated in our institution to evaluate the performance of qMRSA and the impact of single-day MRSA identification (Francois et al., J. Clin. Microbiol. Jan 2003). This assay has been recently evaluated in the Geneva hospitals medical ICU and proved useful to reduce hospital-acquired infection rates (Harbarth et al., Crit Care. 2006 Feb ).

References

Evaluation of rapid screening and pre-emptive contact isolation for detecting and controlling methicillin-resistant Staphylococcus aureus in critical care: an interventional cohort study.
Harbarth S, Masuet-Aumatell C, Schrenzel J, Francois P, Akakpo C, Renzi G, Pugin J, Ricou B, Pittet D.
Crit Care. 2006 Feb 6;10(1):R25
[Abstract] - [PubMed]

Rapid detection of methicillin-resistant Staphylococcus aureus directly from sterile or nonsterile clinical samples by a new molecular assay.
Francois P, Pittet D, Bento M, Pepey B, Vaudaux P, Lew D, Schrenzel J.
J. Clin. Microbiol. Jan 2003. 41:254-60
[Abstract] - [PubMed]

Download the Poster presented for the RICAI 2007 (Paris - France).