Study setting
A prospective evaluation was conducted directly from blood cultures, between January and May 2018 in the microbiology laboratory of South West London Pathology in London. The diagnostic laboratory processes approximately 3500 positive blood cultures per year. All blood culture samples were initially incubated on the BD BacTec FX400 (Becton Dickinson, USA). Blood culture bottles, including adult and paediatric samples, that flagged positive were then tested in parallel for antimicrobial susceptibility by Alfred 60 AST and by routine testing on the BD Phoenix™, after Gram staining performance to determine a Gram-positive or Gram-negative antibiotic panel required and exclude the polymicrobial samples.
Bacterial species were identified by Bruker Biotyper matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS, Germany), this technique was performed, as per routine laboratory practice, from isolated colonies obtained following 4 h agar plate subculture, in order to fully interpret the results of the Alfred 60AST with species ID. This meant the species of the samples were identified before the Alfred 60AST result was available. (Fig. 2).
Alfred 60 AST system
The Alfred 60 ST was performed according to the manufacturer’s instructions on blood culture bottle that had flagged positive on the BD BacTec FX system, this including running weekly controls with Pseudomonas aeruginosa 27,853; E.coli 25,922; E.coli 35,218; S.aureus 29,213; E.faecalis 29,212; E. faecalis 51,299. 30 μL of the sample was inoculated into an enrichment broth and loaded onto the instrument. Each vial of antibiotic contains 45 mg of a preparation of lyophilized antibiotic that required to be dissolved in 2 mL of regenerating solution, as per manufacturer’s specifications. Some antibiotics, including amikacin, piperacillin-tazobactam and meropenem, required different specific antibiotic vials for testing Enterobacteriaceae and Pseudomonas Spp. As the identity of organism is unknown when the Alfred 60 AST system was set up, a combined panel of antibiotics with both concentrations was used. When the identity of the organism was known the appropriate antibiotic result was reported.
Antimicrobial agents
The following antibiotic panel was performed on Alfred 60AST™ for all Gram-negative organisms (Enterobacteriaceae and Pseudomonas spp).: ampicillin, amikacin, ciprofloxacin, gentamicin, meropenem, and piperacillin-tazobactam. Ceftriaxone was included on the Enterobacteriaceae panel later in March 2018 and tested in 78 isolates. For Gram-positive organisms the antibiotic panel included: ampicillin, cefoxitin, clindamycin, teicoplanin and vancomycin. Purity plates were set up both on CLED agar and blood agar plates and examined the next day to confirm that the inoculum was not mixed.
Comparator AST technique
The BD Phoenix™ automated susceptibility testing system CE marked IVD (BD Diagnostics, Sparks, MD, USA) (software version 5.02H/4.11B) was used as the routine laboratory method according to the manufacturer instructions, including running weekly controls with Pseudomonas aeruginosa 27,853; E.coli 25,922; E.coli 35,218; S.aureus 29,213; E.faecalis 29,212; E. faecalis 51,299 using cartridge PMIC-96 for Gram-positive and NMIC-417 for Gram-negative isolates.
Interpretation and comparison of results
Isolates were classified as susceptible, intermediate or resistant (S/I/R) according to interpreted reports provided by Alfred 60AST™ and BD Phoenix™. The final report interpretation of susceptibility results provided by Alfred 60 AST™ are based on bacterial growth curve analysis Fig. 1.
Each sample result by Alfred 60 AST system was compared against BD Phoenix™. Categorical agreement (CA) was defined as agreement of test results interpreted within the same susceptibility category(S/I/R).
Discordant results were categorized as very major error (VME, reported susceptible on Alfred AST60™ when reported resistant Phoenix™), major error (ME, reported resistant when susceptible) or minor error (mE, reported intermediate when susceptible or resistant). The criteria for antimicrobial susceptibility testing proposed by Jorgensen has been used [26] [27].
Time to AST results
The time to AST was defined as the time between the culture bottle flagging positive on the BacTec and the availability of the AST result by Alfred 60 AST system or BD Phoenix™. Wilcoxon test was performed for comparison of median AST time analyses, considering p < 0.05 as a significant value.
SPSS (Statistical Package for the Social Sciences, IBM, USA) version 25.0 statistics software was used for all analyses.