The performance of the LightCycler SeptiFast (SF) assay was compared to that of culture methods in the detection of microorganisms in 43 purulent fluids from patients with pyogenic infections. The SF assay was more sensitive than the culture methods (86% versus 61%, respectively), irrespective of whether the infections were mono-or polymicrobial.The LightCycler SeptiFast (SF) test is a commercially available multiplex real-time PCR assay able to detect a wide range of bacterial and fungal organisms commonly involved in systemic infections (10). The SF assay has been shown to be a valuable ancillary method for the etiological diagnosis of bacteremia, sepsis, endocarditis, and more recently, periprosthetic joint infections, particularly in patients who have received antibiotics prior to diagnostic testing (1-3, 5, 6, 9, 10, 13-17). Molecular methods can allow the rapid detection of the microorganisms involved in severe pyogenic infections, thus leading to a more efficient and targeted early therapeutic intervention, which could translate into major clinical benefits for patients. To the best of our knowledge, there is no published experience on the performance characteristics of the SF assay for the detection of microorganisms in purulent fluids.A total of 43 purulent fluids obtained from 41 patients (29 male and 12 female; mean age, 64.1 years) over a period of 5 months (July to November 2010) were included in this study, as follows: 11 biliary fluid samples from patients with acute cholangitis, 7 pleural fluid samples from patients with empyema secondary to mesothelioma, lung cancer, or community-acquired and nosocomial pneumonia, 7 peritoneal fluid samples from patients with secondary peritonitis, 7 dialysis effluents from 6 patients with continuous ambulatory peritoneal dialysis (CAPD)-related peritonitis, 5 purulent liquid samples from 4 patients with intra-abdominal abscesses secondary to abdominal surgery, 4 purulent (Ͼ250 polymorphonuclear leukocytes/l) ascites fluid samples from cirrhotic patients with spontaneous bacterial peritonitis (SBP), and 2 intra-articular fluid samples from patients with acute septic arthritis. A total of 31 of the 41 patients had been treated with broad-spectrum antibiotics prior to sampling, as follows. All patients (n ϭ 11) undergoing biliary surgery, 3 patients subjected to thoracocentesis for empyema, and 3 patients undergoing laparotomy for secondary peritonitis received perioperative antimicrobial prophylaxis with amoxicillin-clavulanic. All patients with intra-abdominal abscesses and 4 patients with secondary peritonitis were empirically treated with ciprofloxacin plus metronidazole for a median of 2 days (range, 1 to 4 days) prior to specimen sampling. Four patients with secondary empyema had been treated with imipenem, imipenem plus metronidazole, or imipenem plus vancomycin for a median of 1 day prior to specimen sampling (range, 1 to 3 days). One patient with CAPD, one patient with SBP, and one patient with septic arthritis were treated with vancomycin or ceftriaxone ...