An integrated centrifugation-washing-lysis method significantly improves microbiological diagnosis of spontaneous bacterial peritonitis

Culture-negative spontaneous bacterial peritonitis (SBP) remains a major diagnostic challenge in patients with cirrhotic ascites, largely due to prior antibiotic exposure, low bacterial burden, and phagocytosis of bacteria. To address these factors and reduce false-negative results, we developed an integrated centrifugation-washing-lysis method and evaluated its impact on diagnostic turnaround time and antimicrobial stewardship. Ascitic fluid was concentrated and washed, followed by selective leukocyte lysis with 0.5% Triton X-100 (optimized to lyse host cells while preserving bacterial viability during 1-min incubation). The resulting pellet was inoculated onto solid media (chocolate and blood agar). This method was prospectively evaluated against standard bedside inoculation into aerobic and anaerobic blood culture bottles using 160 ascitic fluid samples from patients with clinically suspected SBP. The modified culture method demonstrated a significantly higher positivity rate (30.6% [49/160] vs 13.8% [22/160]; <i>P</i> < 0.001) and a 2.82-fold increase in total bacterial isolates (62 vs 22), revealing previously undetected monomicrobial and polymicrobial infections. Time to detection was reduced by 56.5% (1.0 day vs 2.3 days; <i>P</i> < 0.001) , with visible colony growth on solid media after a mean time of 24 h, enabling same-day organism identification and antimicrobial susceptibility testing. By releasing viable phagocytosed bacteria, the integrated centrifugation-washing-lysis method significantly improves the microbiological diagnosis of SBP by increasing culture yield, shortening time to diagnosis, and enhancing detection of polymicrobial and multidrug-resistant infections. This modified cultrue method may support earlier targeted antimicrobial therapy and improved stewardship in patients with cirrhosis.IMPORTANCEOur research addresses the critical limitation of low positivity rates in ascitic fluid cultures for spontaneous bacterial peritonitis (SBP) diagnosis. By developing a novel ascitic fluid culture technique, we increases SBP culture positivity from 13.8% to 30.6%. This rapid and cost-effective approach not only cuts diagnosis time from 2.3 to 1 day but also reveals previously missed polymicrobial infections, enabling timely targeted therapy and reducing unnecessary antibiotic use in high-risk patients.