Rapid Diagnosis of Prosthetic Joint Infection by Matrix-assisted Laser Desorption

Material and Methods

1. Patients who have a high probability of infection based on the Musculoskeletal Infection Society (MSIS) criteria and are scheduled for debridement only or debridement with implant removal, will be invited to enroll the study after signed informed consent.

   The synovial joint fluid will be sampled before the arthrotomy at the operation room. Aspirates will be collected under an aseptic technique with an 18-Fr sterile syringe with a minimum amount of 14 ccs. The sample will be divided between MALDI-TOF mass spectrometry in standard blood culture bottles (10 ccs), wound culture tube (2 ccs), and synovial fluid analysis (2 ccs). The samples will be delivered to microbiology laboratory within a 2-hour period.

2. Bacterial culture and conventional identification Bacterial identification will be performed by the conventional method using the Vitek 2 system. For the conventional culture, 1 µL of well-mixed synovial joint fluid will be inoculated and spread onto blood agar plates and MacConkey agar plates using a sterile plastic disposable loop. Plates will be incubated in an aerobic atmosphere at 37℃ for 18-24 hr. When bacterial growth is observed, the colonies on blood agar will be counted, and colonies from both types of plates will be identified by using the Vitek 2 system.

3. MALDI-TOF MS identification The suspension obtained following the above sample preparation will be centrifuged at 13,000g for 2 minutes, and the supernatant will be discarded. The pellet will be centrifuged at 13,000g for another 2 minutes prior to the removal of the residual ethanol. Fifty microliters of formic acid (70% v/v) and 50 mL of 100% acetonitrile will be added to the pellet, and mixed thoroughly after each reagent is added. The suspension will be centrifuged again at 13,000g for another 2 minutes, and 1 mL of the supernatant will be spotted onto the steel target plate. Analysis will be performed following air-drying of 1 mL a-cyano-4-hydroxycinnamic acid matrix solution placed onto the dried sample spot in duplicate.

   Mass spectra profiles will be acquired using a microflex LT MALDI-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) following the manufacturer's settings. Spectra will be recorded in the linear positive mode at a laser frequency of 60 Hz within a mass range from 2000 Da to 20,000 Da. All bacteria identifications will be performed by MALDI-TOF Biotyper RTC and the Bruker MALDI Biotyper 3.1 software and library (4613 isolates; Bruker Daltonics). Criteria used for microorganism analysis and identification will be as recommended by the manufacturer.

4. Statistical analysis Time to identification will be determined as the time from colony formation to the time at which the final result is reported to a physician. Statistical analysis will be performed to compare the three methods using Chi-square tests. The level of statistical significance will be set at p \< 0.05.