Protocols for the 3 kits were optimized and validated. The kits were produced and frozen at -20ºC. In order to have room-temperature stable kits lyophilization was tried at FASTinov and transferred to the industrial partner, Clonit (former Euroclone). However, for some of the drugs, stability was compromised. Very extensive efforts were undertaken by project partners in close collaboration with external experts to optimize lyophilization conditions. After lyophilizaion, drying down with different excipients were tried, having 6 drugs already stable for more than 2 years at room temperature. One of the stable drug was colistin that, due its importance as last line drug in critical clinical situations needs a rapid report about its susceptibility. Moreover, EUCAST invalidated most of the methods due to technical difficulties found on both manual and automated methods used on clinical labs. Considered as a strategic opportunity for FASTinov a separate kit was developed, the FASTcolistinMIC kit. The new kit was produced at Clonit, dried and validated at FASTinov and SERMAS and have been submitted to the local EU Regulatory Authority, with a dedicated software. Accelerated studies already showed a 2 years stability although, room temperature stability studys are still on going.
A pilot study with cryo-version panels produced by FASTinov team began in Oct 2018 at SERMAS. Positive Blood Culture samples were collected daily. Standard AST were performed using both MicroScan and either disk diffusion method or microdilution. Results have been interpreted using both CLSI and EUCAST breakpoints. Ninety-nine patient samples have been included in the external validation. Additionally, at FASTinov, 285 blood cultures were spiked with well-characterized bacteria and inoculated into FASTinov panels. Part of these results have been presented in different international conferences and papers are in preparation.
Meanwhile the consortium continued to look for help on finding stable panels at room temperature. A CDMO specialized in antibiotic stability drugs, was contracted to produce room temperature panels with antibiotics and probes ready to use. Several pilot lots were produced and tested with good results. In parallel, a decision was made to include in the FASTbact kits, along with the drug panels, tubes prefilled with the culture media required to resuspend the drugs and carry out the subsequent incubation. This will turn the assay much easier for the user, further standardize the method and avoid problems with flow cytometry analysis due the quality of the media used, its base fluorescence and particulate content. Optimization and manufacturing of the culture media was perfomed by Clonit. After optimization, the order of production of dried panels was made and 3 validation lots produced regarding FASTgramneg and FASTgrampos. Non-clinical performance was completed at FASTinov regarding the 3 dried kits (FASTgramneg, FASTgrampos and FASTcolistinMIC) and clinical performance took place at FASTinov (using spiked blood cultures) and at SERMAS.
Partners were trained by Profess to set-up a fail-proof QA system. QA-requirements implemented at FASTinov and μRoboptics are in line with current legislation for in vitro diagnostic tools and accompanying software packages. The software package, bioFAST, for data analysis of FAST-bact kits and routine methods has been finalized and is ready to be used by clients during the commercialization phase either as a stand-alone, integrated or web application.
Since November 2020, clinical validation with dehydrated plates was achieved at SERMAS evaluating FASTgramneg and FASTgrampos kits at the same time. A total of 180 samples have been included in the study.
The results from the clinical validation have provided the ground for commercial success subsequent to FASTbact project conclusion.
