Evaluation of ultra-rapid susceptibility testing of ceftolozane-tazobactam by a flow cytometry assay directly from posit
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ORIGINAL ARTICLE
Evaluation of ultra-rapid susceptibility testing of ceftolozane-tazobactam by a flow cytometry assay directly from positive blood cultures Inês Martins-Oliveira 1,2 & Blanca Pérez-Viso 3 & Sofia Quintas 2 & Ana Silva-Dias 1,4 & Rosário Gomes 1 & Acácio G Rodrigues 1,2,4 & Rafael Cantón 3 & Cidalia Pina-Vaz 1,2,4 Received: 11 February 2020 / Accepted: 6 May 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract The urgent need for rapid antimicrobial susceptibility is broadly apparent from government reports to the lay press. Accordingly, we developed a flow-cytometry assay (FCM) for evaluating ceftolozane-tazobactam (C/T) susceptibility directly on blood cultures (BC) requiring < 2 h from flag positivity to report. The protocol was optimized with C/T-susceptible and C/Tresistant gram-negative bacilli inoculated in BC aerobic bottles (Becton-Dickinson, USA), and afterward optimized for different C/T concentrations (1/4, 2/4, 4/4, and 8/4 mg/L) for 1 h incubation (37 °C), followed by FCM and software analysis. Fluorescent membrane permeability and membrane potential dyes were comparatively used to detect early cell lesions using the CytoFLEX cytometer (Beckman-Coulter, USA). Repeatability, reproducibility, and stability of the assay up to 48 h after BC positivity were determined. Internal validation was performed in spiked BC bottles with 130 Enterobacterales and 32 Pseudomonas aeruginosa isolates from Porto University (Portugal), including 13 ATCC isolates. Additionally, 64 gram-negative bacilli recovered from positive BC at Ramon y Cajal Hospital (Madrid, Spain) were tested. Categorical agreement (CA) and analytical errors were calculated comparing FCM with broth microdilution results. Only the membrane potential dyes clearly distinguished CTsusceptible and CT-resistant isolates. Excellent repeatability, reproducibility, and inter-method concordance was observed. Overall, CA was 99.1% using EUCAST criteria with 2 major errors and 98.7% with CLSI criteria with 2 major and 1 minor errors. A new, accurate, and ultra-rapid FCM (< 2 h) for testing C/T susceptibility gave accurate results and would expand current FCM antimicrobial susceptibility assay. Keywords Flow cytometry . Ceftolozane-tazobactam . Antimicrobial susceptibility testing . Blood culture . Anti-microbial resistance . Microbiology diagnostics
Introduction Antimicrobial resistance is increasing and becoming a major global problem for antimicrobial therapy [1]. According to the * Cidalia Pina-Vaz [email protected] 1
FASTinov, S.A., Matosinhos, Portugal
2
Division of Microbiology, Department of Pathology, Faculty of Medicine, University of Porto, Porto, Portugal
3
Servicio de Microbiología, Hospital Universitario Ramón y Cajal, Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain
4
Faculty of Medicine, University of Porto, CINTESIS - Center for Health Technology and Services Research, Porto, Portugal
World Health organization (WHO), the world is headed for a post-antibiotic; common inf
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