Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Sun, 02 Dec 2018 01:35:45 J. Med. Microbiol. - Vol. 47 (1998), 607-613 1998 The Pathological Society of Great Britain and Ireland MOLECULAR DIAGNOSIS Development and evaluation of a PCR-based immunoassay for the rapid detection of methicillin-resistant Staphylococcus aureus K. J. TOWNER, D. C. S. TALBOT”, R. CURRAN, C. A. WEBSTER and H. HUMPHREYS Department of Microbiology and PHLS Laboratory, University Hospital, Queen’s Medical Centre, Nottingham NG7 2UH and * Biorecognition, Unilever Research, Colworth Laboratory, Sharnbrook, Bedfordshire MK44 1 LO A multiplex polymerase chain reaction (PCR), involving detection of the mecA and femB genes, was combined with a novel immunoassay system capable of detecting specific PCR products. The resulting PCR-immunoassay was evaluated in comparison with conventional microbiological techniques used in the routine diagnostic laboratory for the rapid identification of methicillin-resistant Staphylococcus aureus (MRSA), either in pure culture or in overnight broth cultures obtained following enrichment of patient screening swabs. Among the 480 purified isolates of staphylococci and 246 enrichment broths examined, only one ‘false-negative’ result was obtained by PCR, compared with 18 ‘false-negative’ results obtained by conventional methodology and demonstrated by further conventional examination. Five demonstrable ‘false-positive’ results were obtained by conventional methodology, compared with a possible 10 by the PCR- immunoassay, although it was not certain that these 10 PCR results were true ‘false positives’ as, by definition, MRSA could not be isolated by conventional methodology. The results indicated that the routine diagnostic laboratory was encountering difficulties in identifying MRSA correctly, and that the conventional microbiological techniques lacked sensitivity. Overall, the PCR technique was more accurate and sensitive than conventional methodology in detecting MRSA, and results were available within 24 h of screening swabs arriving in the laboratory, compared with a minimum of 48-72 h by conventional techniques. The immunoassay system added to the usefulness of the method by allowing the detection of specific PCR products within 5 min of completing the PCR, without the normal additional step of agarose gel electrophoresis. Introduction Staphylococcus aureus is found in the nose and on the skin of a variable number of healthy individuals, and is an opportunist pathogen in patients with lowered host resistance. The isoxazolylpenicillins ( e g , methicillin, cloxacillin, flucloxacillin, etc.) have been the mainstay of treatment for well over 25 years, but the emergence of strains (referred to as methicillin-resistant S. aureus; MRSA) resistant to this group of compounds and all other P-lactam agents poses increasing problems, especially in hospitals, because of the propensity of certain epidemic MRSA strains to spread and colonise Received 4 Sept. 1997; revised version accepted 8 Nov. 1997. Corresponding author: Dr K. J. Towner. E-mail : Kevin. Towner@nott . ac .uk debilitated patients. Such strains may or may not be resistant to multiple antibiotics, leading to potential difficulties in treating systemic infections [ 1, 21. In the UK, epidemic strains have been numbered sequentially on the basis of phage typing (i.e., EMRSA-1, EMRSA-2, etc.), and three well-defined strains (EMRSA-3, EMRSA- 1 5 and EMRSA- 16) continue to affect hospitals in England and Wales, with the incidence of EMRSA-15 and EMRSA-16 increasing in particular [3]. The identification of MRSA in a hospital ward may warrant immediate patient isolation (and occasional ward closure), screen- ing of patient contacts and staff, and appropriate disinfection measures. Screening for carriers, rather than simply identifying infected patients, has a major role in control of an outbreak and reduces the number of infections [3]. However, the results of screening may not be available for at least 48 h, resulting in