title: Enrichment of bacteria samples by centrifugation improves the diagnosis of orthopaedics-related infections via real-time PCR amplification of the bacterial methicillin-resistance gene.
abstract: BACKGROUND
  To effectively treat orthopaedic infections by methicillin-resistant strains, an early diagnosis is necessary. Bacterial cultures and real-time polymerase chain reaction (PCR) have been used to define methicillin-resistant staphylococci. However, even when patients display clinical signs of infections, bacterial culture and real-time PCR often cannot confirm infection. The aim of this study was to prospectively compare the utility of real-time PCR for the mecA gene detection following centrifugation of human samples with suspected orthopaedic infections.
  
  
  RESULTS
  In addition to the conventional real-time PCR method, we performed real-time PCR following centrifugation of the sample at 4,830×g for 10 min in a modified real-time PCR (M-PCR) method. We suspended cultured methicillin-resistant Staphylococcus aureus and generated standard dilution series for in vitro experiments. The in vitro detection sensitivity of the M-PCR method was approximately 5.06 times higher than that of the conventional real-time PCR method. We performed bacterial culture, pathological examination, real-time PCR, and M-PCR to examine the infectious fluids and tissues obtained from 36 surgical patients at our hospital. Of these, 20 patients who had undergone primary total hip arthroplasty were enrolled as negative controls. In addition, 15 patients were examined who were clinically confirmed to have an infection, including periprosthetic joint infection (eight patients), pyogenic spondylitis (two patients), infectious pseudoarthrosis (two patients), and after spine surgery (three patients). In one sample from a patient who developed infectious pseudoarthrosis and two samples from surgical site infections after spine surgery, the mecA gene was detected only by the M-PCR method. In one patient with infectious pseudoarthrosis, one patient with infection after arthroplasty, and two patients with purulent spondylitis, the detection sensitivity of the M-PCR method was increased compared with PCR (clinical sample average: 411.6 times).
  
  
  CONCLUSIONS
  These findings suggest that the M-PCR method is useful to detect methicillin-resistant strains infections. In addition, the centrifugation process only takes 10 min longer than conventional real-time PCR methods. We believe that the M-PCR method could be clinically useful to detect orthopaedic infections caused by methicillin-resistant strains.
authors.affiliation: Department of Orthopaedic Surgery, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan. a-tsuru@m2.kufm.kagoshima-u.ac.jp.
authors.forename: Arisa
authors.initials: A
authors.lastname: Tsuru
authors.affiliation: The Near-Future Locomotor Organ Medicine Creation Course (Kusunoki Kai), Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima, 890-8520, Japan. setoro@m2.kufm.kagoshima-u.ac.jp.
authors.forename: Takao
authors.initials: T
authors.lastname: Setoguchi
authors.affiliation: Department of Orthopaedic Surgery, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan. kawa-bacho@hotmail.co.jp.
authors.forename: Naoya
authors.initials: N
authors.lastname: Kawabata
authors.affiliation: Department of Orthopaedic Surgery, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan. masa-h@m2.kufm.kagoshima-u.ac.jp.
authors.forename: Masataka
authors.initials: M
authors.lastname: Hirotsu
authors.affiliation: Department of Orthopaedic Surgery, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan. tyamamo@m2.kufm.kagoshima-u.ac.jp.
authors.forename: Takuya
authors.initials: T
authors.lastname: Yamamoto
authors.affiliation: Department of Orthopaedic Surgery, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan. naga@m2.kufm.kagoshima-u.ac.jp.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Nagano
authors.affiliation: Department of Orthopaedic Surgery, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan. masahiro@m3.kufm.kagoshima-u.ac.jp.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Yokouchi
authors.affiliation: Department of Orthopaedic Surgery, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan. kakoi@m2.kufm.kagoshima-u.ac.jp.
authors.forename: Hironori
authors.initials: H
authors.lastname: Kakoi
authors.affiliation: Infection Control Team, Kagoshima University Hospital, Kagoshima, Japan. hidekik@m2.kufm.kagoshima-u.ac.jp.
authors.forename: Hideki
authors.initials: H
authors.lastname: Kawamura
authors.affiliation: Department of Medical Joint Materials, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan. ishidou@m2.kufm.kagoshima-u.ac.jp.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Ishidou
authors.affiliation: Molecular and Cellular Pathology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan. akit09@m3.kufm.kagoshima-u.ac.jp.
authors.forename: Akihide
authors.initials: A
authors.lastname: Tanimoto
authors.affiliation: Department of Orthopaedic Surgery, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan. skomiya@m3.kufm.kagoshima-u.ac.jp.
authors.forename: Setsuro
authors.initials: S
authors.lastname: Komiya
mesh_terms: D019644:Arthroplasty, Replacement, Hip; D001426:Bacterial Proteins / Q000235:genetics*; D002498:Centrifugation; D006622:Hip Prosthesis / Q000382:microbiology*; D006801:Humans; D016106:Methicillin Resistance / Q000235:genetics; D055624:Methicillin-Resistant Staphylococcus aureus / Q000235:genetics / Q000302:isolation & purification*; D046915:Penicillin-Binding Proteins / Q000235:genetics*; D011542:Pseudarthrosis / Q000382:microbiology* / Q000473:pathology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D013203:Staphylococcal Infections / Q000175:diagnosis* / Q000382:microbiology / Q000473:pathology
chemical_list: D001426:Bacterial Proteins; D046915:Penicillin-Binding Proteins; C487789:mecA protein, Staphylococcus aureus