Background Diagnosis of periprosthetic infection (PPI) is crucial for management of bone and joint infection. The preoperative gold-standard is joint aspiration, providing results after 2-14 days’ culture, with non-negligible false negative rates due to the fragility of certain micro-organisms and/or prior antibiotic treatment. The Synovasure™ alpha-defensin lateral flow test (Zimmer, Warsaw, IN, USA) contributes within minutes to joint fluid diagnosis of almost all infectious agents, including in case of concomitant antibiotic therapy. Validity remains controversial, notably in complex microbiological situations: multi-operated patients, diagnostic doubt despite iterative sterile culture, long-course antibiotic therapy. We extended a prospective study reported in 2018, to determine whether the test maintained diagnostic value in a larger population, assessing 1) negative (NPV) and positive (PPV) predictive value, and 2) sensitivity and specificity. Hypothesis Synovasure™ maintains NPV above 95% in a broader population of microbiologically complex suspected PPI. Material and methods Synovasure™’s performance was assessed between October 2015 and October 2019 in 106 patients (112 tests) in complex diagnostic situations: 37 discordant cultures (discordant findings between 2 samples), 65 cases with clinically or biologically suspected infection but iterative sterile culture, 10 emergencies (requiring surgery, precluding antibiotic window, or mechanical failure in suspected infection), including 5 with ongoing antibiotic therapy for infection in another organ. Six tests were repeated in the same patient and same joint at >6 months’ interval for strong clinical suspicion of infection. The main endpoint was the MSIS score (MusculoSkeletal Infection Society, 2018). Results NPV was 98.8%, PPV 72.4%, sensitivity 95.5% and specificity 91%. Prevalence of infection was 19.6%. Only 1 of the 22 infected patients had negative Synovasure™ tests, compared to 81 of the 84 non-infected patients. Conclusion Synovasure™ is a reliable novel diagnostic test, contributing mainly to ruling out infection thanks to its strong NPV. The cost imposes sparing use, but medico-economic assessment would be worthwhile.