#	Pagina
attuale pagina	/open-h2020/projects/193272/results.html

Report

Teaser, summary, work performed and final results

Periodic Reporting for period 3 - FAPIC (Fast Assay for Pathogen Identification and Characterisation)

Teaser

Summary

The need for fast diagnostic tests characterising pathogens is high and only those avoiding the time-consuming bacteria growing step can meet the customersâ€™ requirements. A few multiplex assays analysing patient samples directly are already available on the market (e.g. SeptiFastÂ®, Roche). However, they require laborious handling and are considerably limited regarding the number of target genes (<20), because they are based only on molecular biology techniques such as RT- or QC-PCR. The laborious handling can be avoided if the protocols are automated. The challenge in the automatization process lies in the successful implementation of a wide range of different sample materials. Blood, swabs or urine require a specialised sample processing due to the varying physical and biological parameters.
In the project FAPIC, will be developed two very fast, automated and cost-effective diagnostic systems for the identification and characterisation of infectious diseases pathogens. The two systems are based on an innovative nucleotide probe concept and a novel highly sensitive DNA assay. The systems will significantly improve the diagnosis of infectious diseases, the subsequent patientâ€™s antibiotic treatment and the monitoring of multidrug-resistant pathogens including their dissemination paths. The two testing systems
i) a low-cost point-of-care testing (POCT) device (PathoDoc) for a limited range of pathogens but fast and reliable diagnostics including resistance patterns for doctorâ€™s offices and local ambulatories for a variety of sample types
ii) a high-throughput automated test system (PathoRobot) for centralised clinical laboratories with the main advantage of broad spectrum and quantitative determination of pathogens from a variety of sample types

Work performed

During this period of the FAPIC project, the following hardware, finalized during the previous period, were validated in spiked samples according to reproducibility and robustness:
- A low-cost point-of-care testing (POCT) device named PathoDoc with a fully automated approach both for sample treatment and assay analysis.
- A high-throughput PathoRobot and AXOBot automated test system for routine use.
Then, at the scientific level:
- A unique pathogen strains collection was gather, characterized and banked.
- An integrated PCR cycler was developed specifically for the PathoDoc environment.
- Ultraplex assays were developed and partially validated.
- A sample analysis using three different microarrays integrated within one single product was finalized and pre-validated.

Detailed descriptions of these achievements are presented below.

PathoDoc
The PathoDoc is a bench-top apparatus with the capacity of handling blood sample, run a DNA extraction from it, amplify the genes of interest and identify them. Three functional machines were produced during this period and their functions validated by the different partners.

High-throughput system
The high-throughput system is dedicated to the routine hospital laboratories dealing with large number of samples per day. One run can handle 96 samples at a time and performed DNA extraction, amplification of the genes of interest and identification, in a fully automated manner. Two functional machines were produced during this period and its functions validated.

Ultraplex assay
During this period, the molecular biology reagents designed using the reagent selection/design tool, which is an in silico design program, were used to generate microarrays and multiplex PCRs. The obtained ultraplex assays are composed of 3 different microarrays (50, 24 and 46 targets), useful for identification and characterisation of pathogens related to sepsis. The validation of these arrays is now half way to be completed.

Extraction of DNA
DNA is the core part of the sample to be analysed in the FAPIC project, since pathogens targets, resistance and virulence are coded by genes, and that these genes are the target the system is detecting. DNA isolation from sample is then one particularly important step in the development of FAPIC. During this period, extraction protocols were fully validated on the PathoDoc system.

Analytical validation prior clinical evaluation
In order to fulfil the clinical and ethical requirements necessary for clearance of the clinical trial, large series of analytical validation (spiked blood samples) and pre-clinical validation (banked real samples) were performed. The achieved performances were hindered by reproducibility issue and les to a reschedule of the clinical trial.

Final results

This period mainly covered the again the tentative validation of the tools necessary to the implementation of the project clinical trial in the two hospital centres. Thus, in term of progress beyond the state of the art, the main achievements were obtained from the validation of non-existing tools before the project.
These tools are, as listed above: i) a software package useful for multiplex reagents determination and choices, ii) a benchtop fully automated apparatus able to handle extraction, amplification and microarray based assay and iii) ultraplex assays.
Also, and this is of upmost importance, arrays targeting 120 different parameters were developed but only partially validated, delaying the implementation of the clinical trial. This clinical trial will take place during the next period and is expected to have strong impact, both at socio-economical and societal level. Indeed, the content of these arrays are perfectly fitting the clinician expectations but also goes well beyond the state of the art in the field of pathogen detection and characterisation. Indeed, a very wide range of pathogens, resistance and virulence genes is targeted.