Screening for methicillin-resistant Staphylococcus aureus in clinical swabs using a high-throughput real-time PCR-based method

Abstract

The presence of methicillin-resistant Staphylococcus aureus (MRSA) in hospitals and the community is a serious problem. Accordingly, a comprehensive plan has been implemented in the County of Vejle, Denmark, to identify colonised and/or infected individuals and to control the spread of MRSA. Since 2005, all patients and healthcare personnel have been screened for MRSA colonisation, involving analysis of 300-400 samples daily. To deal with this number of samples, a PCR-based method customised for high-throughput analysis and a system for fast reporting of MRSA carrier status were developed. Swab samples were incubated overnight in a selective tryptone soya broth and were analysed by PCR the following day. Using this strategy, non-colonised individuals were identified within 24 h, while MRSA-positive samples were analysed further by traditional microbiological methods to determine the resistance pattern. This is a cost-effective approach, as the greatest expense in hospitals involves the isolation of patients of unknown MRSA status. The method was evaluated by testing 2194 clinical samples, with a sensitivity and specificity of 100% and 94%, respectively. The analytical sensitivity was 97%, with 161 of 166 different MRSA strains and isolates generating positive results according to PCR analysis. Using four control strains, the inter-assay variation was revealed to be a maximum of 2.6%, indicating good reproducibility.

Real‐time multiplex PCR for direct detection of methicillin‐resistant Staphylococcus aureus (MRSA) in clinical samples enriched by broth culture

“…Our protocol, mainly based on the study by Huletsky et al. (7), but with the addition of primer 85/1940 (9) and an extra probe, did not demonstrate any false negative isolates when analyzing the clinical samples and collection of MRSA investigated here.…”

“…A PCR assay for direct detection of MRSA using primers and probes for SCC mec ‐specific sequences in combination with orfX (SCC :orfX ) was described for the first time in 2004 (7). Additional studies using the same concept have been performed (8–10). However, these reported methods were not able to detect all tested MRSA, and, in addition, the problem with false negative samples remained (11).…”

Detection of novel chromosome-SCCmec variants in Methicillin Resistant Staphylococcus aureus and their inclusion in PCR based screening

“…Indeed, an isogenic pair of MSSA and MRSA was recently described that supports the occurrence of horizontal transfer of SCC mec between Staphylococcal species [15]. The 86 strains not detected as being MRSA in other laboratories by commercially available tests raises concern about their performance [16,17]. Exploiting the SCC mec junction as target in PCR used for detection of MRSA requires continuous awareness of possible variants.…”

The search and destroy strategy prevents spread and long-term carriage of methicillin-resistant Staphylococcus aureus: results from the follow-up screening of a large ST22 (E-MRSA 15) outbreak in Denmark

“…TSB SSI broths were incubated overnight at 35°C and PCR for MRSA was performed with the broth cultures obtained from all human swabs [9]. PCR-positive broths were sub-cultured on blood agar for final identification (ID) and antimicrobial susceptibility testing.…”