The development and evaluation of a novel rapid lateral flow diagnostic test for Crimean-Congo Haemorrhagic Fever Virus

Thompson, Caitlin (2024) The development and evaluation of a novel rapid lateral flow diagnostic test for Crimean-Congo Haemorrhagic Fever Virus, Thesis (Doctoral), Liverpool School of Tropical Medicine.

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Abstract

Following the COVID-19 pandemic, G7 leaders called for a 100-day mission to respond to future pandemic threats by developing vaccines, therapeutics and diagnostics within 100 days. Crimean-Congo Haemorrhagic Fever (CCHF) is the most widespread tick-borne viral disease globally and can cause a potentially fatal viral infection in humans. It is deemed a high priority pathogen by the World Health Organisation (WHO) due to its epidemic and pandemic potential. There is an urgent unmet need to develop rapid diagnostic tests as there are no CCHF rapid diagnostic test available for research or commercial use. Lateral flow tests (LFTs) are rapid, inexpensive, easy-to-use diagnostic platforms that require no specialised training, laboratory facility or equipment and can be used directly at the point-of-care.

The aim of this PhD project was to develop a robust lateral flow pipeline, that produced a highly sensitive and specific lateral flow test for the WHO priority pathogen, Crimean-Congo Haemorrhagic Fever (CCHF) Virus. The prototype LFT was designed and developed with the intent for commercialisation, therefore, this pipeline incorporated regulatory procedures such as a design history file and quality management system.

CCHF target antigens were identified and recombinantly expressed in HEK293 cells. Successful recombinant expression of the CCHF nucleoprotein provided the primary antigen for monoclonal antibody development. Forty-seven anti-CCHF nucleoprotein monoclonal antibodies (mAbs) were produced using a mouse model and, in addition to a commercially produced anti-CCHF nucleoprotein mAb, were evaluated for their binding affinity, kinetics and pairing capabilities using an IBIS MX96 surface plasmon resonance imager. Following evaluation of 2304 potential pairings, 15 mAbs were transferred onto a lateral flow platform for initial development and optimisation. Six versions of the prototype lateral flow assay were taken to the Ministry of Health CCHF reference laboratory in Ankara, Türkiye (a country endemic for CCHF) in July 2022 to test on a small number of prospective clinical samples for proof-of-concept evaluation. The top two performing lateral flow test prototypes had a sensitivity of 75.0% [95% CI 57.9 – 86.8%] and specificity of 90.6% [95% CI 75.8 – 96.8%] (prototype 1) and a sensitivity of 71.9% [95% CI 54.6 – 84.4%] and specificity of 96.8% [95% CI 83.8 – 99.8%] (prototype 2). These prototypes met the minimum requirement for the WHO CCHF lateral flow test target product profile of a specificity of ≥90%, however did not meet the sensitivity requirement of ≥80%.
Extensive optimisation of the component parts of the lateral flow prototypes including nitrocellulose membrane, conjugation pad, nanoparticle size and sample incubation time took place to increase lateral flow test sensitivity. Following this, a final multisite clinical evaluation in six regional clinical facilities in Türkiye on prospective clinical samples took place between July and October 2023 and a retrospective clinical evaluation on stored samples took place in Baghdad, Iraq. The optimal performing optimised prototype lateral flow test had a sensitivity of 90.4% [95% Cl 81.5-95.3%] and a specificity of 96.2% [95% Cl 87.0-99.3%] compared to RT-qPCR in prospective Turkish samples. In retrospective stored samples in Iraq, selected to represent a range of cycle threshold (Ct) values (Ct 14-39), the test demonstrated a sensitivity of 82.2% [95% Cl 68.7-90.7%] (n=45) in RT-qPCR samples Ct≤30 and a sensitivity of 71.7% [95% Cl 59.2-81.5%] (n=60) in all samples (Ct ≤40) with a specificity of 92.5% [95% Cl 80.1-97.8%] (n=40) compared to RT-qPCR.

The culmination of this PhD project was the development of a highly sensitive and specific prototype antigen targeting CCHF lateral flow test for potential use in regional clinical facilities, to provide diagnostic results for a suspected CCHF infection within 30 minutes, and the development and evaluation of a diagnostic pipeline for lateral flow development for a WHO priority pathogen. This project highlighted the critical challenges to producing an LFT within 100 days, specifically the generation of monoclonal antibodies and access to prospective samples.

Item Type:	Thesis (Doctoral)
Subjects:	QY Clinical Pathology > QY 4 General works WC Communicable Diseases > WC 20 Research (General) WC Communicable Diseases > Virus Diseases > Viral Hemorrhagic Fevers. Other Virus Diseases > WC 534 Viral hemorrhagic fevers
Faculty: Department:	Clinical Sciences & International Health > Clinical Sciences Department
Depositing User:	Lynn Roberts-Maloney
Date Deposited:	20 Nov 2024 11:38
Last Modified:	20 Nov 2024 11:43
URI:	https://archive.lstmed.ac.uk/id/eprint/25649

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