title: Development of a molecular methodology to quantify Staphylococcus epidermidis in surgical wash-out samples from prosthetic joint replacement surgery.
abstract: Prosthetic joint infection is a serious complication of total joint arthroplasty that causes great morbidity in affected individuals. The most common cause of prosthesis associated infections are members of Staphylococcus spp., including Staphylococcus epidermidis. Culture has served as the gold standard for diagnosis, despite obvious shortcomings in terms of sensitivity and time. Bacterial genomic DNA extraction methodologies were evaluated for optimal recovery of genomic DNA from sterilised wash-out samples, spiked with S. epidermidis. Real time Polymerase chain reaction (PCR) assays targeting the S. epidermidis specific gseA gene were designed to reliably detect and quantify S. epidermidis. Sixty post-operative wash-out samples from primary hip and knee arthroplasties were taken aseptically. All were shown to be culture negative using the culture-dependent approach. These were samples were subjected to S. epidermidis-specific real time PCR. Standard curve showed good linearity. Sensitivity limit of the assay was <10 CFU S. epidermidis per sample. Reproducibility of the assay was confirmed. S. epidermidis was not identified in any of these samples using the novel species specific SYBR Green real time PCR technique. Results indicated that wash-out samples were true negatives and did not harbour S. epidermidis. To support this, patients displayed no symptoms of infection. To illustrate the full effectiveness of the novel real time PCR assay, a larger number of samples need to be tested (>1,000 patients).
authors: Orthopaedics Department, Merlin Park Regional Hospital, Galway, Ireland. fergbyrne@gmail.com.
authors: Fergus J
authors: FJ
authors: Byrne
authors: Teagasc, Animal Reproduction Research Centre, Mellows Campus, Athenry, Co., Galway, Ireland.
authors: Sinéad M
authors: SM
authors: Waters
authors: National University of Ireland, Galway, Ireland.
authors: Peadar S
authors: PS
authors: Waters
authors: Orthopaedics Department, Merlin Park Regional Hospital, Galway, Ireland.
authors: William
authors: W
authors: Curtin
authors: Department of Surgery, Clinical Science Institute, University College Hospital, Galway, Ireland.
authors: Michael
authors: M
authors: Kerin
mesh_terms: D013212:Staphylococcus epidermidis / Q000302:isolation & purification / Q000235:genetics; D006801:Humans; D016459:Prosthesis-Related Infections / Q000382:microbiology / Q000175:diagnosis; D019645:Arthroplasty, Replacement, Knee / Q000009:adverse effects; D013203:Staphylococcal Infections / Q000175:diagnosis / Q000382:microbiology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D019644:Arthroplasty, Replacement, Hip / Q000009:adverse effects; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D004269:DNA, Bacterial
keywords: 
                Staphylococcus 
                epidermidis
              ; Bacterial infection; Joint replacement; Prosthesis; Real time PCR