Periodic Reporting for period 4 - CORONADX (Three Rapid Diagnostic tests (Point-of-Care) for COVID-19 CoronaThree Rapid Diagnostic tests (Point-of-Care) for Coronavirus, improving epidemic preparedness, public health and socio-economic benefits)
Periodo di rendicontazione: 2023-04-01 al 2023-09-30
On the 30th January 2020 the World Health Organisation (WHO) declared a global health emergency for the outbreak of the 2019 novel Coronavirus (2019-nCoV) that originated in Wuhan, China. The 2019-nCoV has spread all over the world with 772.166.517 confirmed cases and 6.981.263 deaths (22.11.2023). Diagnostic tests are essential to control such outbreaks. The first tests to receive Emergency Use Authorization (EUA) were, however, all based on methods suited only for well-equipped centralized laboratories.
CORONADX provides a fast diagnostic tool for 2019-nCoV. A Point-of-Care (POC) portable equipment that can be used by briefly trained personnel is developed for use in hospitals, primary health care units or in mobile laboratories. The POC with lab and field evaluations will allow for fast detection and surveillance of the epidemic and greatly improve the diagnosis and clinical management of patients infected with 2019-nCoV.
The development of rapid POC diagnostics is supported by clinical and molecular epidemiological studies on the characterization and spatio-temporal evolution of the 2019-nCoV virus and identify infection sources as e.g. the animal reservoir.
The social sciences research in CORONADX provides information on societal resilience in the era of social media, and the related public health preparedness and response to the epidemic.
The developed PATHPOD system has shown a sensitivity and specificity close to 100% and has a Limit of Detection (LOD) only 2.5-5 larger than RT-PCR methods, when using purified RNA samples and a specially developed sample preparation protocol based on magnetic beads. Field tests with human samples show good results, but use of animal samples with a sample matrix other than a swab, show the need for a different sample preparation method, optimized for this type of samples.
CORONADX provides a fast diagnostic tool for 2019-nCoV. A Point-of-Care (POC) portable equipment that can be used by briefly trained personnel is developed for use in hospitals, primary health care units or in mobile laboratories. The POC with lab and field evaluations will allow for fast detection and surveillance of the epidemic and greatly improve the diagnosis and clinical management of patients infected with 2019-nCoV.
The development of rapid POC diagnostics is supported by clinical and molecular epidemiological studies on the characterization and spatio-temporal evolution of the 2019-nCoV virus and identify infection sources as e.g. the animal reservoir.
The social sciences research in CORONADX provides information on societal resilience in the era of social media, and the related public health preparedness and response to the epidemic.
The developed PATHPOD system has shown a sensitivity and specificity close to 100% and has a Limit of Detection (LOD) only 2.5-5 larger than RT-PCR methods, when using purified RNA samples and a specially developed sample preparation protocol based on magnetic beads. Field tests with human samples show good results, but use of animal samples with a sample matrix other than a swab, show the need for a different sample preparation method, optimized for this type of samples.
The work in the project has been on developing the three COVID-19-tests PATHPOD, PATHLOCK and PATHAG, as well as performing the field testing of samples collected in China. Also, method development and studies of the viral diversity of human clinical samples have been carried out to give a better understanding of the variability of corona viruses. Studies on 21st century epidemics have been completed to provide a better basis for understanding the COVID-19 pandemic and its impact on the human population.
PATHAG, an antigen agglutination test, and PATHLOCK, a simple lateral flow test, were unfortunately unsuccessful and the work on these was discontinued early on in the project. The PATHPOD device, which was based on LAMP amplification of virus RNA, was successful and was developed in two versions, one based on turbidity measurements and one based on fluorescence detection.
For the PATHPOD we needed to first develop primer sets suitable for detection of SARS-CoV-2 virus causing COVID 19. These were developed based on publication and genome sequences from the web (www.gisdaidm.org) and tested for specificity and sensitivity for detecting COVID 19. When using purified RNA, the sensitivity was 94% and specificity 96.7% compared to the Altona PCR reference method.
The first design of the PATHPOD resulted in 10 PATHPOD instruments and 1000 cartridges (for 10000 tests). These instruments and cartridges were used to determine the analytic specificity (100%), analytic sensitivity (~30-50 copies) as well as clinical evaluation. This first version of the instrument had a lower sensitivity than we expected. To improve the sensitivity and LOD of the PATHPOD, a version 2 of the instrument was developed based on fluorescence detection. The PATHPOD v2 has shown 99% relative accuracy, 97,5% relative specificity, and 100% relative sensitivity to RT-qPCR, with the LOD improved to being 2.5-5X higher than RT-PCR. Due to time constraints and several delays caused by the pandemic, we were only able to test PATHPOD v1 with human, animal and environmental samples in China. The results showed that the PATHPOD performance greatly depends on the sample type and clearly works better for human samples, for which it has been validated in the laboratory.
Viral diversity and evolution are important in order to follow the development of the pandemic as well as the efficiency of tests and vaccines. Therefore, MUW established a method that does not discriminate against mutated virus sequences and is able to reliably detect also low abundant sequence variants.
We analysed and compared the epidemiological dynamics of selected epidemics of the 21st century using historical surveillance data to identify differences and similarities both in diseases' natural history and public health response measures.
Data analyses were performed on mortality data (COVID-19 mortality and excess mortality for all causes) and data on estimated prevalence of the infection, at the national and European level. A systematic listing of containment models and measures and their impact in different European settings was completed in this last period.
The work has been described in several peer-reviewed publications and has resulted in 1 patent. Moreover, the consortium has produced a flyer, set up and managed the CORONADX online communication channels (website and social media), produced and distributed public communication contents aimed at raising awareness and visibility around CORONADX (such as press and news release and video teasers), created a series of Fact or Myth? posts aimed at clarifying common misunderstandings and providing useful, science-based recommendations related to the topic towards the general public.
PATHAG, an antigen agglutination test, and PATHLOCK, a simple lateral flow test, were unfortunately unsuccessful and the work on these was discontinued early on in the project. The PATHPOD device, which was based on LAMP amplification of virus RNA, was successful and was developed in two versions, one based on turbidity measurements and one based on fluorescence detection.
For the PATHPOD we needed to first develop primer sets suitable for detection of SARS-CoV-2 virus causing COVID 19. These were developed based on publication and genome sequences from the web (www.gisdaidm.org) and tested for specificity and sensitivity for detecting COVID 19. When using purified RNA, the sensitivity was 94% and specificity 96.7% compared to the Altona PCR reference method.
The first design of the PATHPOD resulted in 10 PATHPOD instruments and 1000 cartridges (for 10000 tests). These instruments and cartridges were used to determine the analytic specificity (100%), analytic sensitivity (~30-50 copies) as well as clinical evaluation. This first version of the instrument had a lower sensitivity than we expected. To improve the sensitivity and LOD of the PATHPOD, a version 2 of the instrument was developed based on fluorescence detection. The PATHPOD v2 has shown 99% relative accuracy, 97,5% relative specificity, and 100% relative sensitivity to RT-qPCR, with the LOD improved to being 2.5-5X higher than RT-PCR. Due to time constraints and several delays caused by the pandemic, we were only able to test PATHPOD v1 with human, animal and environmental samples in China. The results showed that the PATHPOD performance greatly depends on the sample type and clearly works better for human samples, for which it has been validated in the laboratory.
Viral diversity and evolution are important in order to follow the development of the pandemic as well as the efficiency of tests and vaccines. Therefore, MUW established a method that does not discriminate against mutated virus sequences and is able to reliably detect also low abundant sequence variants.
We analysed and compared the epidemiological dynamics of selected epidemics of the 21st century using historical surveillance data to identify differences and similarities both in diseases' natural history and public health response measures.
Data analyses were performed on mortality data (COVID-19 mortality and excess mortality for all causes) and data on estimated prevalence of the infection, at the national and European level. A systematic listing of containment models and measures and their impact in different European settings was completed in this last period.
The work has been described in several peer-reviewed publications and has resulted in 1 patent. Moreover, the consortium has produced a flyer, set up and managed the CORONADX online communication channels (website and social media), produced and distributed public communication contents aimed at raising awareness and visibility around CORONADX (such as press and news release and video teasers), created a series of Fact or Myth? posts aimed at clarifying common misunderstandings and providing useful, science-based recommendations related to the topic towards the general public.
Two versions of the PATHPOD system have successfully been developed and tested for specificity, sensitivity and initial clinical evaluation. Applications for Emergency Use Authorization (EUA) have been filed by September 2022. The system could however not receive approval. In the end the biggest challenge of all proved to be that the PATHPOD is a complete system consisting of instrument (with electronic components), software and a detection cartridge loaded with the test reagents – each of which will have their own challenges from a regulatory point of view.
The typical EUA test approved by the regulatory authorities would be a set of test reagents (primers, probes, nucleotides and enzymes) that could be loaded onto any standardized multi-well PCR plate format and incubated/read in a regulatorily approved instrument – thus eliminating the separate instrument/software validation and approval issue that the PATHPOD is facing.
The PATHPOD v1 was used to test several human, animal and environmental samples in China, and the tests highlighted the importance of sample preparation methods suitable for the sample used.
Public Health response and socio-economic impacts. Findings from: i) epidemiological analysis of previous and current viral epidemic, ii) mathematical modelling on the impact of rapid diagnostic implementation, iii) socio-economic evaluations of the current epidemic and analysis of population resilience patterns will enable the development and consolidation of solid methods and models for public health preparedness in the era of digitalization and innovative means of communication targeting both the general populations, healthcare and public health professionals and decision-makers.
The typical EUA test approved by the regulatory authorities would be a set of test reagents (primers, probes, nucleotides and enzymes) that could be loaded onto any standardized multi-well PCR plate format and incubated/read in a regulatorily approved instrument – thus eliminating the separate instrument/software validation and approval issue that the PATHPOD is facing.
The PATHPOD v1 was used to test several human, animal and environmental samples in China, and the tests highlighted the importance of sample preparation methods suitable for the sample used.
Public Health response and socio-economic impacts. Findings from: i) epidemiological analysis of previous and current viral epidemic, ii) mathematical modelling on the impact of rapid diagnostic implementation, iii) socio-economic evaluations of the current epidemic and analysis of population resilience patterns will enable the development and consolidation of solid methods and models for public health preparedness in the era of digitalization and innovative means of communication targeting both the general populations, healthcare and public health professionals and decision-makers.