Periodic Reporting for period 2 - PITBUL (Point-of-Care implementation of TB testing with ultra-fast Local Heating PCR)
Berichtszeitraum: 2019-06-01 bis 2020-11-30
Tuberculosis (TB) is a wide-spread infectious disease caused by a bacterium, - Mycobacterium tuberculosis. One quarter of the world population is infected with M. tuberculosis, and 10% of infected cases develop an active TB in their lifespan, which means that patients show symptoms and are contagious. Typical symptoms are chest pain, weakness, fever, and weight loss. In 2019, an estimate of 10 million people developed an active TB. The WHO recommends the use of rapid molecular diagnostic tests as they will lead to major improvements in the early detection of TB and drug-resistant TB. A thereon linked good case-management will help to prevent the spread of TB and multi-drug resistant TB.
The overall goal of PITBUL was to bring the ultra-fast local heating PCR (LPCR) from lab-proven prototype into clinical demonstration. An automated workflow with a disposable cartridge and a point-of-care-testing (POCT) demonstrator should be developed to allow the conduction of fast and affordable tuberculosis tests at various locations (e.g. hospitals, TB centers or in-and-out clinics). A cloud-based software allowing the secured on-line data interpretation and storage should be developed for the analysis of patient data.
PCR (polymerase chain reaction) is a biochemical method for the specific, exponential amplification of DNA. The considerable amplification of the primary DNA is required for the subsequent detection of pathogens in human specimen (e.g. sputum of TB- suspects). The outstanding advantages of the LPCR technology compared to competitors are an intrinsic purification step and the drastic reduction of time.
In conventional PCR cyclers the entire reaction liquid with biochemical reaction partners in solution undergoes repetitive thermal cycles. In contrast to this, in LPCR the pathogens DNA attaches to a micro-scaled wire during the capturing process and only the reaction liquid in close proximity of this wire undergoes the repetitive cycles, while the remaining liquid is a reservoir for reaction components. Heat is conducted via electric current through these wires. The reduction of time in LPCR compared to conventional PCR can be traced back to a reduction of heating and cooling ramps. An intrinsic purification step allows the removal of inhibitory specimen after capturing the molecules of interest (e.g. pathogen DNA) prior to amplification.
The overall goal of PITBUL was to bring the ultra-fast local heating PCR (LPCR) from lab-proven prototype into clinical demonstration. An automated workflow with a disposable cartridge and a point-of-care-testing (POCT) demonstrator should be developed to allow the conduction of fast and affordable tuberculosis tests at various locations (e.g. hospitals, TB centers or in-and-out clinics). A cloud-based software allowing the secured on-line data interpretation and storage should be developed for the analysis of patient data.
PCR (polymerase chain reaction) is a biochemical method for the specific, exponential amplification of DNA. The considerable amplification of the primary DNA is required for the subsequent detection of pathogens in human specimen (e.g. sputum of TB- suspects). The outstanding advantages of the LPCR technology compared to competitors are an intrinsic purification step and the drastic reduction of time.
In conventional PCR cyclers the entire reaction liquid with biochemical reaction partners in solution undergoes repetitive thermal cycles. In contrast to this, in LPCR the pathogens DNA attaches to a micro-scaled wire during the capturing process and only the reaction liquid in close proximity of this wire undergoes the repetitive cycles, while the remaining liquid is a reservoir for reaction components. Heat is conducted via electric current through these wires. The reduction of time in LPCR compared to conventional PCR can be traced back to a reduction of heating and cooling ramps. An intrinsic purification step allows the removal of inhibitory specimen after capturing the molecules of interest (e.g. pathogen DNA) prior to amplification.
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.
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.
Within this Horizon 2020 project, a proof-of concept for LPCR in TB diagnostics was obtained with clinical samples tested in a clinical environment for the first time. All members of the consortium benefitted from the broad experience of the others and were able to develop new capacities.
The expected impact of the project targeted TB knowledge and capacity. The cooperation between clinical partners in Italy and Tanzania was deepened and allowed an intense know-how transfer to a high TB burden, low-resource country in sub-Saharan Africa. One partners expertise of guidelines for analytical processes was shared within the consortium. Another partner was able to implement a quality management system, which represents a big step in the development of the company to a certified medical device provider.
The expected impact on healthcare was the development of a solution for a reliable POC test (>95% sensitivity and <95% specificity) with a 20-30-minute turn-around time, allowing same-day time-to-treatment. Due to demanding challenges, this was not achieved to 100%. Nevertheless, within PITBUL it was shown that the technology of LPCR can fulfill these demands in the future.
The expected impact of the project targeted TB knowledge and capacity. The cooperation between clinical partners in Italy and Tanzania was deepened and allowed an intense know-how transfer to a high TB burden, low-resource country in sub-Saharan Africa. One partners expertise of guidelines for analytical processes was shared within the consortium. Another partner was able to implement a quality management system, which represents a big step in the development of the company to a certified medical device provider.
The expected impact on healthcare was the development of a solution for a reliable POC test (>95% sensitivity and <95% specificity) with a 20-30-minute turn-around time, allowing same-day time-to-treatment. Due to demanding challenges, this was not achieved to 100%. Nevertheless, within PITBUL it was shown that the technology of LPCR can fulfill these demands in the future.