DESCRIPTION (provided by applicant): The goal of this project is to enable diagnosis of pulmonary tuberculosis at the point of care in low resource high burden countries by developing a portable, easy to use, integrated nucleic acid testing device that executes sample preparation, isothermal DNA amplification and lateral flow based detection without user intervention, providing a result in < 1.5 h sample-in-to-answer-out, at a significantly lower cost than currently
available fully integrated bench-top systems. The project builds upon ongoing efforts that involve the same team of investigators and institutions, and is strengthened by new partners. To date, we have developed a novel, rapid sample preparation process for sputum liquefaction and disinfection, pathogen lysis, and DNA extraction, and have established suitable performance of this process using clinical sputum samples. We have demonstrated that our system can accommodate different isothermal amplification methods coupled to lateral flow detection, and have developed a novel process for master-mix reagent stabilization. Going forward we will utilize an isothermal cross-priming amplification (CPA), clinically validated for TB diagnosis, which is coupled to lateral flow, and includes an internal amplification control. We have designed and built a prototype integrated cartridge and instrument, and we have demonstrated that the sample preparation and amplification/detection subunits are functional. By the beginning of the proposed project, we aim to demonstrate fully automated process execution in the integrated alpha prototype system. The proposed project will enable us to further develop and validate the system, and to obtain regulatory approval for market release as an IVD. In Aim 1, we will optimize key system components related to sample pre-processing, DNA extraction, CPA, and lateral flow detection. We will improve the thermal stability of master-mix reagents, refine on-board reagent storage, incorporate a lateral flow strip reader, and ensure overall system robustness. In Aim 2, we will refine the design for manufacturability, and perform industrial design and human factors engineering. After finalizing the design of the cartridge and instrument, we will scale up manufacturing under ISO 13485 to produce a sufficient number of cartridges and instruments to execute Aim 3, which entails analytical and clinical validation of the system. These three aims will culminate in application for CE-IVD regulatory approval, to enable market entry. In Aim 4, we will develop a docking station to which four of the portable instrument units can be attached, as an upgrade to the basic system. This docking station will provide enhanced quality control, a better user interface, plus wireless connectivity, enabling external quality assurance and electronic result transmission, two important operational aspects of disseminated TB diagnostics in low resource near patient settings. This project presents a unique opportunity to enable early diagnosis of all TB cases, which was identified as a key objective in the Global Plan to Stop TB. In the long term, the system can be applied to other pathogens relevant to bio-threat detection and infectious disease diagnosis in resource-limited settings.