Within the course of PITBUL a method for the safe processing of the contagious sputum samples was developed. The workflow covered the self-collection of the sputum samples by the patient in a closed cup in a well-ventilated area, the inactivation of the mycobacteria within the sample and the liquefaction of lumpy specimen within a closed and disinfected cup and the transfer to the integrated cartridge. Within the cartridge, the bacteria are lysed with ultrasound, the released bacterial DNA is captured by pathogen specific molecules, the remaining inhibitory specimen is removed, and the M. tuberculosis DNA is amplified and detected. Assays for the sensitive detection of TB per se and for the detection of mutations causing resistances against the common first-line antibiotics Rifampicin and Isoniazid were developed. For the detection of the minor changes in DNA sequence causing resistances, new technologies were introduced. The cartridge that was developed. It allowed the automated conduction of necessary steps for sample processing (e.g. pumping of the sample to the reaction chamber for conduction of the thermal cycles) and was tested with a POCT demonstrator. Actuators, addressed by a software, allowed to perform the conduction of the test in a closed system. A cloud-based software capable of complex analyses was developed.
Nevertheless, the development of the cartridge was challenging and not finalized within the project. Even though first tests proved the conduction of the complete workflow within the cartridge, an automatization was not possible yet and will need further improvement of assembly processes and material of the cartridge.
For this reason, the LPCR technology was tested with a manual workflow and a prototype (capable of LPCR, but not of preparative steps) in clinical trials in Latvia and Italy. This served as proof of concept for the technology and the developed molecular assays. More than 150 patient samples were tested with this prototype in a laboratory environment. A high sensitivity and specificity for the detection of tuberculosis was revealed. First TB samples were diagnosed within less than 3 minutes of LPCR. Assuming that a complete automatization of the process in the cartridge will be possible, a diagnosis of TB can be given within less than 20 minutes. The detection of resistances in the genome of M. tuberculosis is more time-consuming and will need further optimization. Intense dissemination and communication activities were prevented by the SARS-CoV-2 outbreak. Nevertheless, the consortium shared videos to create awareness for TB and arise interest for the project.
A transfer of the knowledge gained within the tuberculosis project to SARS-CoV-2 detection was possible. Within the 6-month cost-neutral extension of the PITBUL project, the successful detection of the virus in different sample types (e.g. oropharyngeal swab, nasopharyngeal swab or saliva) was shown.
