BackgroundSeptic and aseptic nonunion require different therapeutic strategies. However, differential diagnosis is challenging, as low-grade infections and biofilm-bound bacteria often remain undetected. Therefore, the examination of biofilm on implants by sonication and the evaluation of its value for differentiating between femoral or tibial shaft septic and aseptic nonunion in comparison to tissue culture and histopathology was the focus of this study.Materials and methodsOsteosynthesis material for sonication and tissue samples for long-term culture and histopathologic examination from 53 patients with aseptic nonunion, 42 with septic nonunion and 32 with regular healed fractures were obtained during surgery. Sonication fluid was concentrated by membrane filtration and colony-forming units (CFU) were quantified after aerobic and anaerobic incubation. CFU cut-off values for differentiating between septic and aseptic nonunion or regular healers were determined by receiver operating characteristic analysis. The performances of the different diagnostic methods were calculated using cross-tabulation.ResultsThe cut-off value for differentiating between septic and aseptic nonunion was ≥ 13.6 CFU/10 ml sonication fluid. With a sensitivity of 52% and a specificity of 93%, the diagnostic performance of membrane filtration was lower than that of tissue culture (69%, 96%) but higher than that of histopathology (14%, 87%). Considering two criteria for infection diagnosis, the sensitivity was similar for one tissue culture with the same pathogen in broth-cultured sonication fluid and two positive tissue cultures (55%). The combination of tissue culture and membrane-filtrated sonication fluid had a sensitivity of 50%, which increased up to 62% when using a lower CFU cut-off determined from regular healers. Furthermore, membrane filtration demonstrated a significantly higher polymicrobial detection rate compared to tissue culture and sonication fluid broth culture.ConclusionsOur findings support a multimodal approach for the differential diagnosis of nonunion, with sonication demonstrating substantial usefulness.Level of Evidence: Level 2Trial registrationDRKS00014657 (date of registration: 2018/04/26)