Eur J Orthop Surg Traumatol (2007) 17:449–456 DOI 10.1007/s00590-007-0206-4 123 ORIGINAL ARTICLE Development of a molecular methodology to quantify Staphylococcus epidermidis in surgical washout samples from prosthetic joint replacement surgery Fergus J. Byrne · Sinéad M. Waters · Peadar S. Waters · William Curtin · Michael Kerin Received: 4 December 2006 / Accepted: 11 January 2007 / Published online: 8 February 2007 © Springer-Verlag 2007 Abstract Prosthetic joint infection is a serious com- plication of total joint arthroplasty that causes great morbidity in aVected individuals. The most common cause of prosthesis-associated infections is members of Staphylococcus spp., including Staphylococcus epide- rmidis. Culture has served as the gold standard for diagnosis, despite obvious shortcomings in terms of sensitivity and time. Bacterial genomic DNA extrac- tion methodologies were evaluated for optimal recov- ery of genomic DNA from sterilised washout samples, spiked with Staphylococcus epidermidis. Real time PCR assays targeting the Staphylococcus epidermidis speciWc gseA gene were designed to reliably detect and quantify S. epidermidis. Sixty postoperative washout samples from primary hip and knee arthroplasties were taken aseptically. All were shown to be culture negative using the culture-dependent approach. These samples were subjected to S. epidermidis-speciWc real time PCR. Standard curve showed good linearity. Sensitivity limit of the assay was less than 10 CFU S. epidermidis/sample. Reproducibility of the assay was conWrmed. Staphylo- coccus epidermidis was not identiWed in any of the samples taken using the novel species-speciWc SYBR green real time PCR technique. Results indicated that washout samples were true negatives and did not harbour S. epidermidis. To support this, patients dis- played no symptoms of infection. To illustrate the full eVectiveness of the novel real time PCR assay, a larger number of samples need to be tested (>1,000 patients). Keywords Staphylococcus epidermidis · Real time PCR · Bacterial infection · Prosthesis · Joint replacement Mise au point d’une méthode moléculaire pour quantiWer le Staphylocoque epidermidis dans les produits de lavage en chirurgie prothétique Résumé L’infection prothétique est une complication sérieuse des arthroplasties totales total et entraîne une grande morbidité chez les patients qui en sont att- eints La cause la plus commune des infections liées aux prothèses sont dues à diverses espèces de staphy- locoque, y compris le staphylocoque epidermidis. La culture a servi de gold standard au diagnostic, en dépit des imperfections évidentes en termes de sen- sibilité et de temps. Des méthodologies géniquo-bactéri- ennesd’extraction d’ADN ont été évaluées pour le rétablissement optimal de l’ADN génique des échantil- lons stérilisés de lavage, inoculés avec le staphylocoque epidermidis. Des analyses en temps réel de PCR visant le gène spéciWque du gseA du staphylocoque epidermi- dis ont été conçues pour détecter de façon crertaine et pour quantiWer le staphylocoque epidermidis. Soixante F. J. Byrne (&) · W. Curtin Orthopaedics Department, Merlin Park Regional Hospital, Galway, Ireland e-mail: fergbyrne@gmail.com S. M. Waters Animal Reproduction Research Centre, Teagasc, Mellows Campus, Athenry, Co., Galway, Ireland P. S. Waters National University of Ireland, Galway, Ireland M. Kerin Department of Surgery, Clinical Science Institute, University College Hospital, Galway, Ireland