Prospective study on the diagnostics and susceptibility testing of infections associated with osseointegrated percutaneous implants

摘要

Introduction: Biomaterial-associated infections (BAI) are among the main reasons for implant failure, often leading to long-term antimicrobial treatment and implant removal. BAI are mainly caused by biofilm producing bacteria with increased antimicrobial resistance. Proper and rapid diagnosis is important for successful treatment. Sonication is an alternative method for the diagnosis of prosthetic infections with higher sensitivity than standard tissue cultures. The aims of the study were to: ⅰ) identify and quantify bacteria causing osteomyelitis associated with osseointegrated percutaneous amputation prostheses by sonication of explanted implants; ⅱ) characterise the isolated strains by their biofilm production abilities and antimicrobial susceptibility. In parallel, the diagnostic laboratory at the hospital performed standard culturing techniques of bone tissue and blood marrow samples. Materials and Methods: A bone-anchored part (fixture) of a prosthesis was extracted due to suspicion of infection according to clinical evaluation. The retrieved fixture was placed in sterile saline, sonicated at 47 KHz for 5 min and vortexed for 1 min. The implant was removed, the remaining solution was centrifuged, pellet was re-suspended in saline and quantitative cultures were made on general purpose agar (blood agar for aerobic and brucella agar for anaerobic cultures) and selective agar plates (staphylococci agar, enterococci agar, streptococci agar, chromogenic orientation agar and pseudomonas agar). Crystal violet (CV) staining and Congo Red (CR) agar plate test was performed to evaluate the biofilm production. Identification of the staphylococcal strains was performed using API Staph. The Calgary Biofilm Device and a custom-made antimicrobial susceptibility plate were used to determine minimum biofilm eradication concentrations (MBEC) and minimum inhibitory concentrations (MIC) to 10 antimicrobial agents. Results and Discussion: We report the first results of an ongoing prospective study where the method has been tested real-time. In the first patient, Enterococcus faecalis (5×10~4 CFU/implant) and Escherichia coli (10~2 CFU/implant) were detected after sonication. These strains were both biofilm-producing strains according to CV and CR tests. Biofilms showed increased tolerance to the antimicrobials compared with the standard planktonic MIC values (Table 1). Escherichia coli was the only species detected by standard tissue cultures; in contrast, by the use of sonication, Enterococcus faecalis was further identified indicating the possibility of a polymicrobial infection with potential implications for the choice of the optimal treatment strategy for the patient. Conclusions: An increased detection of the potential causative agents by sonication of retrieved implants may improve the current diagnostics of BAI. In addition, determination of MBEC for clinically relevant antimicrobial agents has the potential to optimize the treatment choices for patients.