WP2 has been working on the set-up of the VALUE-Dx biobank and continuing the optimisation of the protocols in preparation for the imminent start of WP4 clinical trials.

Thanks to the expertise of LAB-Net members, the sample collection and management manuals for both trials have been developed. These manuals describe in depth how to collect samples for the microbiology sub-studies and how to process and store them until shipment to the VALUE-Dx UA/UZA biobank in Antwerp. Additionally, the preparation of kits to be sent to the sites for the sample collection has begun, starting with kits to store the leftover samples from the diagnostic intervention implemented in the paediatric arm of the ADEQUATE trial.

Regarding the optimisation of metagenomics protocols, the focus has been on the bioinformatics protocols that will be used to analyse the generated shotgun metagenomics data, as the wet lab protocols for sequencing are optimised for Illumina and PacBio. The pipeline will include tools for quality control and host reads removal, after which taxonomical classification, functional characterisation and assemblies will be performed. Furthermore, antimicrobial resistance genes and mutations will be studied with different tools, and S. pneumoniae serotypes will be identified if present in the sample. Validation of this pipeline is being done in shotgun metagenomics data from other studies, and will be further extended with a metagenome reconstruction step implemented by Bioaster.

Concerning the biomarker sub-studies, specific manuals for sample collection and processing have been developed. These manuals describe how to collect, process and store plasma (EDTA and heparin) and RNA samples prior to their shipment to the UA/UZA biobank in Antwerp. The kits required for blood collection and processing are being assembled for their subsequent use in the PRUDENCE trial. Additionally, the cytokine and chemokine analysis is being optimised, starting with the selection of the most relevant and promising biomarkers. Cytokine and chemokine panels are currently being designed. Also, we tested and recommended a double-spin plasma separation protocol to be utilised within the PRUDENCE trial, which will improve the quality of collected samples.