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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 3 - FAST-bact (A novel fast test for antibiotic susceptibility testing for Gram positive and Gram negative bacteria)

Teaser

Summary

This project deals with the pressing topic of antibiotic microbial resistance (AMR). The WHOâ€™s â€œAntimicrobial resistance: global report on surveillance (2014)â€ makes clear that resistance to common bacteria has reached alarming levels in many parts of the world and that in some settings, few, if any, of the available treatment options remain effective for common infections. Current projections include 10 million annual global deaths due to resistant microorganisms by the year 2030.
Obviously, there is an urgent need for fast and accurate AST methods for proper treatment of infections from major bacterial pathogens. The novel Flow cytometry Antimicrobial Susceptibility Test (FAST-bact) delivers ultra-rapid results within 2h. The FAST-bact tests will directly impact treatment of patients contracting bacterial infections, as well as the 400.000 patients in Europe who each year contract a multi-resistant bacterial infection. By providing faster results on the antibiotic susceptibility pattern, the FAST-bact tests will improve treatment, contribute to lowering mortality and morbidity and in the process improve the quality of life for patients and reduce the pressure for AMR.
The project aims to introduce the FAST-bact tests as product to the urine AST testing market, which includes a CE-IVD software analytical package for clinical guidance as to the interpretation of the AST results according to current CLSI and EUCAST antibiotic AST breakpoint guidelines. The consortium aims for clinical validation of the 3 assay kits (FAST grampos, FAST gramneg, FAST mar), and envisions the production of the IVD AST kits on a large-scale and regulatory compliant process, addressing both present and future EU IVD-guidelines, that will be implemented in Europe in the course of the project.

Work performed

After successful analytical method validation, technology transfer from coordinator FASTinov to industrial partner Euroclone was initiated. The optimal method for conservation of the FAST-bact kits was found to be lyophilization. However, for some of the drugs, stability was compromised. To mitigate the risk of delay, FASTinov contacted an external company with extensive experience in lyophilization of antibiotics. New excipients and different lyophilization protocols were developed and transferred to Euroclone. Euroclone upgraded their freeze-drying equipment based on the new requirements and started production according to external expertâ€™s indications on formulation and protocol. The manufactured plates were functionally evaluated at FASTinov. Despite evident progress that was made compared to previous results, overall performance of the plates was still not acceptable: low activity persisted for some drugs along with a high background that was masking the effect of the activity of drugs in the flow cytometry-based assay. Several attempts were made to avoid these effects, such as modifying the support (polymer, color, transparency, surface treatment), the fill volume of individual wells, the formulation, the lyophilization protocols etc., but none of them worked.

After producing and testing more than 50 batches of plates with different conditions/parameters, Euroclone and FASTinov contacted the contracted external expert for a joint troubleshooting work, consisting in the preparation from Biofortuna of another lot of plates. Full FASTbact Gram Neg panels were used this time instead of only the 4 â€œdifficultâ€ drugs subject of the feasibility study based on which lyophilization formulation and protocol was developed. Unfortunately, performance of the produced lot was similar to that of Euroclone lots, and therefore not acceptable. The conclusion of Biofortuna lyophilization experts, who could not identify evident causes for the poor results, was that the developed process may not be reproducible.

In the meantime, FASTinov have been getting a lot of consultation on the stability of antibiotics and decided to make a plan of air-dried plates production. Different excipients were selected and the work was divided between FASTinov and Euroclone and the stability evaluation performed at FASTinov.

A consortium meeting was set up where the decision was made to go on validating in Madrid when the drugs showed a stability of at least 2 months. At this moment we are continuing that hard work having now 6 drugs already stable for more than 2 months.
Partners were trained by Profess to set-up a fail-proof QA system. QA-requirements are in line with current legislation for in vitro diagnostic tools and accompanying software packages.
The software package for data analysis of FAST-bact kits and routine methods were developed. The application can be used by clients during the commercialization phase, as a stand-alone application, as an integrated application or as web application.

As the planned pilot study began in Oct 2018 at SERMAS with freshly made plates produced by FASTinov team with all protocols in place. Results are interpreted using both CLSI and EUCAST breakpoints. Also, AST are performed with flow cytometry FAST-bact kit in direct blood culture positive samples; results are interpreted with the software tool BioFAST. Until now, 274 samples have been included in the validation and the results are being statistically analyzed.

The results of the internal validation with spiked blood culture and the clinical data were sent for presentation at ECCMID 2020 and a full paper is under revision to be published. Next year a validation on the final, stable product will take place.
The FAST-bact kit is expected to provide a significant impact on the microbiology diagnostics and subsequent treatment of severe infections.

The potential commercial impact of the FAST-bact kits is being evaluated by major industrial players under

Final results

The kits that are under development are able to produce reliable antibiotic susceptibility test results within 2h after a Positive Blood Culture. Compared to other methods, this will lead to a faster outcome on the susceptibility phenotype of the bacterial pathogen that infected a specific patient. This will allow, on-time targeted therapy, with much narrower spectrum; for example, in the case of resistant phenotypes, it will allow a more aggressive therapy in a more expedient timeframe. On the other hand, it will reduce the common empirical clinical practice of treatment of patients with wide-spectrum antibiotics and will provide a much more specific way of treating the patient immediately and efficiently using the appropriate antibiotic. The benefits of a quicker report will not only impact patient treatment and health, but also contribute to the safety of the public health system and stakeholders by allowing a timely isolation of those patients infected with AMR strains, avoiding the spread of the AMR pathogen. Technologically, the patented innovation is a technology platform that allows even further developments such as applications to determine antifungal susceptibility or even allowing the determination of mechanisms of resistance displayed by bacterial strains.