title: Identification of orthopaedic infections using broad-range polymerase chain reaction and reverse line blot hybridization.
abstract: BACKGROUND
  Culture remains the gold standard in the diagnosis of bacterial infection, but molecular biological techniques have yielded promising results. In this study, we validated a combined polymerase chain reaction and reverse line blot hybridization protocol for identifying musculoskeletal infections.
  
  
  METHODS
  Samples were obtained from seventy-six patients undergoing orthopaedic surgery for various aseptic and septic indications. The diagnosis of infection was based on a review of all available clinical and culture data. In addition to routine culture for aerobic and anaerobic growth, samples were analyzed with a broad-range 16S rRNA polymerase chain reaction and subsequent reverse line blot hybridization with use of twenty-eight group, genus, and species-specific oligonucleotide probes.
  
  
  RESULTS
  An infection was diagnosed on the basis of patient data in thirty-one patients. All but one of the patients with a clinical diagnosis of infection had a positive result of the polymerase chain reaction-reverse line blot hybridization. Five of the forty-five patients in whom an infection was not suspected on the basis of patient data had at least one positive result of the polymerase chain reaction-reverse line blot hybridization. Cultures demonstrated microorganisms in twenty-five patients with an infection and in two patients in whom an infection was not suspected on the basis of the patient data. Staphylococcus aureus was the most common organism grown on culture. The species identified by the polymerase chain reaction-reverse line blot hybridization was in full accordance with that grown on culture in all but one patient.
  
  
  CONCLUSIONS
  Polymerase chain reaction-reverse line blot hybridization performed well in detecting and identifying the various bacterial species and was more sensitive than routine culture. It identified Staphylococcus aureus as the most frequently found microorganism. Five patients in whom an infection was not suspected on the basis of the patient data had a positive result of the polymerase chain reaction, which may have been caused by contamination of the samples. However, three of these patients had aseptic loosening of a total hip prosthesis, suggesting the presence of a low-grade bacterial infection that remained undetected by the culture but was detected by the polymerase chain reaction-reverse line blot hybridization.
  
  
  METHODS
  Diagnostic Level III.
authors.affiliation: Department of Orthopaedics, University Medical Center Utrecht, P.O. Box 85500, 3508GA Utrecht, The Netherlands. D.J.F.Moojen@umcutrecht.nl
authors.forename: Dirk Jan F
authors.initials: DJ
authors.lastname: Moojen
authors.forename: Sanne N M
authors.initials: SN
authors.lastname: Spijkers
authors.forename: Corrie S
authors.initials: CS
authors.lastname: Schot
authors.forename: Marc W
authors.initials: MW
authors.lastname: Nijhof
authors.forename: H Charles
authors.initials: HC
authors.lastname: Vogely
authors.forename: André
authors.initials: A
authors.lastname: Fleer
authors.forename: Abraham J
authors.initials: AJ
authors.lastname: Verbout
authors.forename: René M
authors.initials: RM
authors.lastname: Castelein
authors.forename: Wouter J A
authors.initials: WJ
authors.lastname: Dhert
authors.forename: Leo M
authors.initials: LM
authors.lastname: Schouls
mesh_terms: D001178:Arthroplasty; D001424:Bacterial Infections / Q000175:diagnosis*; D020878:Device Removal; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods*; D009985:Orthopedics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011183:Postoperative Complications / Q000175:diagnosis; D016459:Prosthesis-Related Infections / Q000175:diagnosis*; D012329:RNA, Bacterial / Q000302:isolation & purification; D012336:RNA, Ribosomal, 16S / Q000302:isolation & purification; D012086:Reoperation; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013211:Staphylococcus aureus / Q000235:genetics
chemical_list: D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S