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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - PITBUL (Point-of-Care implementation of TB testing with ultra-fast Local Heating PCR)

Teaser

Summary

The aim of this project is to research and develop the ultrafast Local Heating Polymerase Chain Reaction (LPCR) technology and its laboratory demonstrator from a non-integrated into an integrated system, for clinical proof-of-concept testing in tuberculosis (TB) centers in Italy, Latvia, and Tanzania. This will imply a fully automated workflow in the demonstrator, all necessary reagents stored onboard of the prototype cartridge, and a cloud-based analysis software. An ultrafast point-of-care test (POCT) for TB and emerging TB multi-drug resistant markers (MDR) will allow patients with active TB to be rapidly and accurately diagnosed and adequately treated.

Work performed

At the projectâ€™s kick-off meeting, specifications for the POCT system and workflow were firstly defined by all partners, in particular the technical and user requirements based on ease of use, turn-around-time, sensitivity, specificity, safety, and costs. This established the basis for a development plan of the complete workflow, namely sample processing, instrument, cartridge and software.
In favor of the systemâ€™s portability, cost of goods, and sample purification abilities, the project consortium made the decision early on to focus exclusively on the Local Heating PCR (LPCR) variant that can be employed in a compact and portable system.
A complete manual workflow concept from sample to result was successfully demonstrated, establishing: an easy-to-use and user-safe sputum liquefaction, Mycobacteria lysis protocols, sample purification, and a TB assay with LPCR. This workflow was first demonstrated at GNA using surrogate sputum and manual pipetting on a prototype chip. The same was later successfully demonstrated by partners with TB-negative patient sputum spiked with TB from culture. This served as a proof of concept for the subsequent integration of the separate steps into one single, disposable cartridge, namely: lysis, target DNA capture plus sample purification, and LPCR (amplification and detection).

Final results

The workflow concept can allow for TB testing near the patient at an unprecedented speed. A safe sample handling and transfer process have been designed, beyond the current state of art. Furthermore, assays for highly relevant, but largely neglected mutations responsible for drug-resistance in TB will be designed and tested. These aspects can contribute to a broad implementation of the future test system.