Clinical Validation of a Rapid Variant-Proof RT-RPA Assay for the Detection of SARS-CoV-2

The COVID-19 pandemic has unveiled a pressing need to expand the diagnostic landscape to permit high-volume testing in peak demand. Rapid nucleic acid testing based on isothermal amplification is a viable alternative to real-time reverse transcription polymerase chain reaction (RT-PCR) and can help close this gap. With the emergence of SARS-CoV-2 variants of concern, clinical validation of rapid molecular tests needs to demonstrate their ability to detect known variants, an essential requirement for a robust pan-SARS-CoV-2 assay. To date, there has been no clinical validation of reverse transcription recombinase polymerase amplification (RT-RPA) assays for SARS-CoV-2 variants. We performed a clinical validation of a one-pot multi-gene RT-RPA assay with the E and RdRP genes of SARS-CoV-2 as targets. The assay was validated with 91 nasopharyngeal samples, with a full range of viral loads, collected at University College London Hospitals. Moreover, the assay was tested with previously sequenced clinical samples, including eleven lineages of SARS-CoV-2. The rapid (20 min) RT-RPA assay showed high sensitivity and specificity, equal to 96% and 97%, respectively, compared to gold standard real-time RT-PCR. The assay did not show cross-reactivity with the panel of respiratory pathogens tested. We also report on a semi-quantitative analysis of the RT-RPA results with correlation to viral load equivalents. Furthermore, the assay could detect all eleven SARS-CoV-2 lineages tested, including four variants of concern (Alpha, Beta, Delta, and Omicron). This variant-proof SARS-CoV-2 assay offers a significantly faster and simpler alternative to RT-PCR, delivering sensitive and specific results with clinical samples

Lateral flow test engineering and lessons learned from COVID-19

The acceptability and feasibility of large-scale testing with lateral flow tests (LFTs) for clinical and public health purposes has been demonstrated during the COVID-19 pandemic. LFTs can detect analytes in a variety of samples, providing a rapid read-out, which allows self-testing and decentralized diagnosis. In this Review, we examine the changing LFT landscape with a focus on lessons learned from COVID-19. We discuss the implications of LFTs for decentralized testing of infectious diseases, including diseases of epidemic potential, the ‘silent pandemic’ of antimicrobial resistance, and other acute and chronic infections. Bioengineering approaches will play a key part in increasing the sensitivity and specificity of LFTs, improving sample preparation, incorporating nucleic acid amplification and detection, and enabling multiplexing, digital connection and green manufacturing, with the aim of creating the next generation of high-accuracy, easy-to-use, affordable and digitally connected LFTs. We conclude with recommendations, including the building of a global network of LFT research and development hubs to facilitate and strengthen future diagnostic resilience

Harnessing state-of-the-art diagnostic technologies for point-of-care testing of emerging and neglected tropical diseases

The emergence of epidemic-prone infectious diseases has been identified as a major global health issue of the twenty-first century. Global strategies have been initiated for the development of drugs, vaccines and diagnostics to fight pandemic threats. Recent breakthroughs in biosensing technologies are strengthening the diagnostic landscape. Stopping pandemics requires collaboration fair and wide access to these state-of-the-art diagnostic technologies. Decentralisation of laboratory testing and adaptation of these technologies to point-of-care testing is one strategy to widen access. Moreover, harnessing state-of-the-art diagnostic tools for rapid, simple and cost-effective decentralised testing can bolster the fight against neglected tropical diseases, which still affect over 1 billion people across the world. In this PhD thesis, I present the development and evaluation of three new in vitro diagnostics targeting topical infectious diseases – Ebola virus disease, COVID-19 and schistosomiasis – adapting a range of state-of-the art biosensing technologies to meet the needs of real-world settings. The first diagnostic developed is a proof-of-concept dual-species antigen test which detects and differentiates the glycoproteins of Zaire and Sudan ebolavirus. Twelve monoclonal antibodies were characterised and functionalised to select species-specific antibody pairs. Finally, two species-specific dipstick components were evaluated in buffer spiked with Ebola recombinant glycoproteins. The second diagnostic presented herein is a rapid multi-gene molecular assay able to simultaneously detect two genes of SARS-CoV-2 using reverse transcription recombinase polymerase amplification. The one-pot assay offered two alternative readouts – dipstick or real-time fluorescence – and a clinical validation using the fluorescence readout was conducted with 91 clinical samples. Last, the first CRISPR-based test for urogenital schistosomiasis was developed and evaluated with parasitic DNA and eggs. An in-house CRISPR-compatible protocol for urine sample extraction was proposed and the assay was lyophilised to facilitate transport and field-testing. To conclude, the diagnostic technology research presented in this thesis is a step towards closing the gap of high-performance tests adapted to infectious diseases and endemic settings. In addition, innovation in access is needed to bring these diagnostic technologies to patients who need them and to achieve universal health coverage

Clinical Validation of a Rapid Variant-Proof RT-RPA Assay for the Detection of SARS-CoV-2

The COVID-19 pandemic has unveiled a pressing need to expand the diagnostic landscape to permit high-volume testing in peak demand. Rapid nucleic acid testing based on isothermal amplification is a viable alternative to real-time reverse transcription polymerase chain reaction (RT-PCR) and can help close this gap. With the emergence of SARS-CoV-2 variants of concern, clinical validation of rapid molecular tests needs to demonstrate their ability to detect known variants, an essential requirement for a robust pan-SARS-CoV-2 assay. To date, there has been no clinical validation of reverse transcription recombinase polymerase amplification (RT-RPA) assays for SARS-CoV-2 variants. We performed a clinical validation of a one-pot multi-gene RT-RPA assay with the E and RdRP genes of SARS-CoV-2 as targets. The assay was validated with 91 nasopharyngeal samples, with a full range of viral loads, collected at University College London Hospitals. Moreover, the assay was tested with previously sequenced clinical samples, including eleven lineages of SARS-CoV-2. The rapid (20 min) RT-RPA assay showed high sensitivity and specificity, equal to 96% and 97%, respectively, compared to gold standard real-time RT-PCR. The assay did not show cross-reactivity with the panel of respiratory pathogens tested. We also report on a semi-quantitative analysis of the RT-RPA results with correlation to viral load equivalents. Furthermore, the assay could detect all eleven SARS-CoV-2 lineages tested, including four variants of concern (Alpha, Beta, Delta, and Omicron). This variant-proof SARS-CoV-2 assay offers a significantly faster and simpler alternative to RT-PCR, delivering sensitive and specific results with clinical samples

Synthesis of amidoximated polyacrylonitrile fibers and its use as adsorbent for Cr (VI) ions removal from aqueous solutions

International audienc

CRISPR-assisted test for Schistosoma haematobium

Abstract Schistosomiasis is a major neglected tropical disease targeted for elimination as a public health issue by 2030, however there is an urgent need for more sensitive and specific diagnostic tests suitable to resource-limited settings. Here we developed CATSH, a CRISPR-assisted diagnostic test for Schistosoma haematobium, utilising recombinase polymerase amplification, Cas12a-targeted cleavage and portable real-time fluorescence detection. CATSH showed high analytical sensitivity, consistent detection of a single parasitic egg and specificity for urogenital Schistosoma species. Thanks to a novel CRISPR-compatible sample preparation developed using simulated urine samples containing parasitic eggs, CATSH had a sample-to-result within 2 h. The components of CATSH can be lyophilised, reducing cold chain dependence and widening access to lower and middle-income countries. This work presents a new application of CRISPR diagnostics for highly sensitive and specific detection of parasitic pathogens in remote areas and could have a significant impact on the elimination of neglected tropical diseases