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Background Clinical characteristics and control of the infection of patients with culture-negative (CN) prosthetic joint infection (PJI) have not been well assessed. Prior use of antimicrobial therapy has been speculated but not proven as a risk factor for CNPJI. Questions/purposes We therefore determined whether prior use of antimicrobial therapy, prior PJI, and postoperative wound healing complications were associated with CN PJI. Methods We performed a retrospective case-control study of 135 patients with CN PJI treated between January 1, 1985, and December 31, 2000 matched with 135 patients with culture-positive (CP) PJIs (control patients) during the study period. The time to failure of therapy compared between cases and control patients using a Kaplan-Meier analysis. Results The use of prior antimicrobial therapy and postoperative wound drainage after index arthroplasty were associated with increased odds of PJI being culture-negative (odds ratio, 4.7; 95% CI, 2.8-8.1 and odds ratio, 3.5; 95% CI, 1.5-8.1, respectively). The percent (± SE) cumulative incidence free of treatment failure at 2 years followup was similar for CN and CP PJI: 75% (± 4%) and 79% (± 4%), respectively. Conclusions Prior antimicrobial therapy and postoperative wound drainage were associated with an increased risk of negative cultures among patients with PJI. Physicians should critically evaluate the need for antimicrobial therapy before establishing a microbiologic diagnosis of PJI in patients with suspected PJI.