Periodic Reporting for period 2 - KRONO (Evaluation of a production ready portable, Point-of-Need Platform (instrument and reagents), direct from nasal swab test for the molecular diagnostic detection of COVID-19 infection.)
Periodo di rendicontazione: 2022-01-01 al 2022-06-30
The KRONO consortium is focused on providing simple, rapid and accurate low-cost molecular diagnostics, ensuring that Europe is better prepared for response to future emerging viral pathogens. The requirement for lab bases testing has been shown to present issues during the current COVID pandemic, including with scaling, logistical difficulties of obtaining test components and delays resulting from shipping to remote laboratories. The KRONO team are developing a platform called the QuRapID XF1, able to perform molecular diagnostic testing portably without requirement for expert laboratory users or time consuming nucleic acid extraction. The reagents do not require refrigeration and so the new platform technology could be swiftly deployed to outbreaks of emerging disease in remote settings. This technology could therefore be used for screening travelers at the point of entry, testing in the community, supporting the military and healthcare systems to rapidly scale testing capability, amongst other uses. Outside of human diseases the same platform technology could be used to prevent the economic losses and food supply insecurity caused by veterinary pathogens. The consortium are developing a duoplex test that can detect SARS CoV-2 directly from a nasal swab or saliva sample and validating it using archived patient samples collected during the pandemic. KRONO will develop the novel direct from crude sample reagent, the portable platform and the assay itself, demonstrating that the platform will support future response to outbreaks of emerging diseases. The consortium are also developing a direct from blood assay that can detect yellow fever infections in the field. This will demonstrate that the technology can provide a solution for the detection of a number of neglected tropical diseases which have severe health and economic impacts in the developing world. The ultimate goal is to be able to provide tests with the low cost and ease of use of lateral flow tests with the sensitivity of a molecular approach. The project partners are INMI (Rome), AMU (Marseilles), UCPP (Corsica) and the commercial partner BioGene. KRONO will generate new intellectual property, academic publications and data to be used for future regulatory approvals. The system will be made commercially available and new assay panels will be developed that will assist in detection of important pathogens.
This project covers a number of technical areas; design and development of the novel instrument and reaction vessel, development of the reagent including the enzyme, the buffer system and it’s lyophilization, assay development and validation, finally a performance evaluation for the complete platform. The commercial partner has developed the instrument and reaction vessel from concept to Alpha prototype entirely within the duration of the KRONO project. A new injection mould has been developed making the reaction vessels for the assays, including development of sealing means to ensure that the lyophilized reagents can be stored correctly and a barcoding system, such that the instrument can be programmed by scanning the vessel. In terms of the instrument this has involved the complete design of the electronics, mechanics and hardware of the system from the ground up. BG research have taken on additional staff to complete and have developed new software, firmware, electronics and the prototype instrument within the duration of the programme of work. The current status is that a set of Alpha prototype instruments have been built and are currently undergoing final PCR testing prior to shipment to partner labs in early February. User manuals and training manuals for these have been developed. Using the prototype BioGene demonstrated direct detection of a SARS CoV-2 at below 5,000 virions/ml direct from a contrived nasal swab sample using a lyophilized version of the reagent, passing the originally stated aim of detection at below 10,000 virions/ml of nasal swab eluate. In terms of the reagent, a method of producing the enzyme at scale has been developed and validated. A final version of the buffer was developed and validated using live SARS CoV-2 virus and archived patient samples. The reagent was lyophilized and demonstrated identical sensitivity to the frozen reagent, indicating that the project goal of portable diagnostics could be supported. The novel reagent was tested with saliva and it was possible to detect infection from unprocessed saliva. With regard to the assay itself, initial studies were performed with frozen reagents and a singleplex N gene assay. As new variants emerged the team moved to an N gene and RdRp duoplex assay, demonstrating a lower limit of detection below 5 virions per reaction using a surrogate virus. An internal positive control was developed and shown to not negatively impact the target lower limit of detection for the assay. To support future use in emerging outbreaks an assay was developed to detect “disease X”, in this case the direct from blood detection of yellow fever. An assay was developed and the migration to direct detection from blood is underway and this assay will be validated shortly. In terms of SARS CoV-2 INMI have performed live viral culture testing and showed that the virus is lysed and rendered amplifiable by the reagent and that the assay detects the pathogen to below 40 actual virions per reaction, the assays have been validated at the addition of 10µl of crude sample per reaction and meet the originally outlined performance criteria. The WHO have developed a target product profile for COVID stating that simple, low-cost tests must be able to detect to below 10,000 virions/ml and as such this technology can easily meet this requirement.
A number of important innovations have been developed during the project. A stretch goal was the use of alternate crude sample matrices and the consortium have been able to demonstrate direct detection from unprocessed saliva at below the targeted 10,000 virions/ml level and shown that breath condensates are a potential input sample for future investigation. A direct from unprocessed whole blood version of the reagent has been developed, capable of detection of viral pathogens at below 5,000 virions/ml - supporting WHO R&D blueprint goals of the development of assays for important viral hemorrhagic fevers such as CCHF, Ebola and others. The portable platform would support detection direct from minimally invasive samples such as saliva and finger prick blood in a field setting and could have significant impact in resource poor environments and for a number of economically important diseases. KRONO will have demonstrated cold-chain free direct detection from unprocessed nasal swabs and saliva shortly and so generated proof of concept for portable, simplified, low-cost molecular testing. New IP filings have been made and academic publications will follow shortly. Between now and project end the academic research partners will be tested the lyophilized reagents on the prototype platform using archived patient samples. This will enable a performance evaluation to be performed that will support the publications and provide the basis for future regulatory approvals. The impact of the project will include further ongoing research collaborations, publications and following the expected commercial release in Q3 2022, benefits arising from the use of the system itself as a diagnostic platform.