Applicability and performance of EUCAST's rapid antimicrobial susceptibility testing (RAST) on primarily sterile body fluids in blood culture bottles in laboratory routine with total lab automation

Abstract

Optimisation of microbiological diagnostics in primarily sterile body fluids is required. Our objective was to apply EUCAST's RAST on primarily sterile body fluids in blood culture bottles with total lab automation (TLA) and to compare results to our reference method Vitek2 in order to report susceptibility results earlier. Positive blood culture bottles (BACTEC™ Aerobic/Anaerobic/PEDS) inoculated with primarily sterile body fluids were semi-automatically subcultured onto Columbia 5% SB agar, chocolate agar, MacConkey agar, Schaedler/KV agar and Mueller-Hinton agar. On latter, cefoxitin, ampicillin, vancomycin, piperacillin/tazobactam, meropenem and ciprofloxacin were added. After 6 h, subcultures and RAST were imaged and MALDI-TOF MS was performed. Zone sizes were digitally measured and interpreted following RAST breakpoints for blood cultures. MIC values were determined using Vitek2 panels. During a 1-year period, 197 Staphylococcus aureus, 91 Enterococcus spp., 38 Escherichia coli, 11 Klebsiella pneumoniae and 8 Pseudomonas aeruginosa were found. Categorical agreement between RAST and MIC was 96.5%. Comparison showed no very major errors, 2/7 (28.6%) and 1/7 (14.3%) of major errors for P. aeruginosa and meropenem and ciprofloxacin, 1/9 (11.1%) for K. pneumoniae and ciprofloxacin, 4/69 (7.0%) and 3/43 (5.8%) for Enterococcus spp. and vancomycin and ampicillin, respectively. Minor errors for P. aeruginosa and meropenem (1/8; 12.8%) and for E. coli and ciprofloxacin (2/29; 6.5%) were found. 30/550 RAST measurements were within area of technical uncertainty. RAST is applicable and performs well for primarily sterile body fluids in blood culture bottles, partially better than blood-based RAST. Official EUCAST evaluation is needed.

Keywords: Blood cultures; EUCAST; Primarily sterile body fluids; RAST; TLA.

Fig. 1

RAST image of Staphylococcus aureus isolated from joint fluid after 6 h on Mueller-Hinton agar with visible zone diameters taken by a total lab automation (TLA) at the Department for Infectious Diseases at the University Hospital Heidelberg, Germany. As soon as a blood culture bottle inoculated with primarily sterile body fluid flagged as positive i.a. rapid antimicrobial susceptibility testing (RAST) was prepared on a Mueller-Hinton agar as established by EUCAST for blood-based RAST. After 6 h, automatic imaging was done by TLA. Images were digitally viewed by a technician and zone diameters were measured (not measured here) (CIP, ciprofloxacin; TZP, piperacillin/tazobactam; FOX, cefoxitin; VA, vancomycin; MEM, meropenem; AM, ampicillin)

Fig. 2

RAST image of bla_OXA-48 carbapenemase-producing K. pneumoniae in drainage fluid inoculated in blood culture bottle at the Department for Infectious Diseases at the University Hospital Heidelberg, Germany. With a zone diameter of 19 mm, meropenem is susceptible according to the clinical breakpoints for RAST (version 1.1). MIC value of 1 mg/L obtained from Vitek2 confirmed the susceptible RAST result. bla_OXA-48 carbapenemase was detected by PCR. The growth-free area outside the zone diameter of meropenem was due to manually correction of the antimicrobial plate after stamping the disks (CIP, ciprofloxacin; TZP, piperacillin/tazobactam, FOX, cefoxitin; VA, vancomycin; MEM, meropenem; AM, ampicillin)