Application of a High-Resolution Melt Assay for Monitoring SARS-CoV-2 Variants in Burkina Faso and Kenya

The rapid emergence and global dissemination of SARS-CoV-2 highlighted a need for robust, adaptable surveillance systems. However, financial and infrastructure requirements for whole genome sequencing (WGS) mean most surveillance data have come from higher-resource geographies, despite unprecedented investment in sequencing in low-middle income countries (LMICs) throughout the SARS-CoV-2 pandemic. Consequently, the molecular epidemiology of SARS-CoV-2 in some LMICs is limited, and there is a need for more cost-accessible technologies to help close data gaps for surveillance of SARS-CoV-2 variants. To address this, we have developed two high-resolution melt curve (HRM) assays that target key variant-defining mutations in the SARS-CoV-2 genome, which give unique signature profiles that define different SARS-CoV-2 variants of concern (VOCs). Extracted RNA from SARS-CoV-2 positive samples collected from 205 participants (112 in Burkina Faso, 93 in Kenya) on the day of enrolment in the MALCOV study (Malaria as a Risk Factor for COVID-19) between February 2021 and February 2022 were analysed using our optimised HRM assays and compared to Next Generation Sequencing (NGS) on Oxford Nanopore MinION . With NGS as a reference, two HRM assays, HRM-VOC-1 and HRM-VOC-2, demonstrated sensitivity/specificity of 100%/99.29% and 92.86/99.39%, respectively, for detecting Alpha, 90.08%/100% and 92.31%/100% for Delta and 93.75%/100% and 100%/99.38% for Omicron. The assays described here provide a lower-cost approach (<$1 per sample) to conducting molecular epidemiology, capable of high-throughput testing. We successfully scaled up the HRM-VOC-2 assay to screen a total of 506 samples from which we were able to show the replacement of Alpha with the introduction of Delta and the replacement of Delta by the Omicron variant in this community in Kisumu, Kenya. These assays are readily adaptable and can focus on local epidemiological surveillance questions or be updated quickly to accommodate the emergence of a novel variant or adapt to novel and emerging pathogens ### Competing Interest Statement The authors have declared no competing interest. ### Clinical Protocols ### Funding Statement This work was partially funded by UK Aid from the Department of Health and Social Care (https://www.gov.uk/government/collections/official-development-assistance-oda--2) via the UK Public Health Rapid Support Team Research Programme (Grant No. RST6\_00\_03, EPIDZK3828). The funder had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. This work was also supported by the National Environment Research Council (grant number NE/V010441/1). The MALCOV study was funded by the Bill and Melinda Gates Foundation. This study received funding from the UK Research Council through a PhD scholarship from the MRC Doctoral Training Partnership to CGB. ERA is supported by the U.S. Food and Drug Administration Medical Countermeasures Initiative contract 75F40120C00085 and by the National Institute for Health Research Health Protection Research Unit (HPRU) in Emerging and Zoonotic Infections (NIHR200907) at the University of Liverpool in partnership with Public Health England (PHE), in collaboration with Liverpool School of Tropical Medicine and the University of Oxford. This publication is based on research funded by (or in part by) the Bill & Melinda Gates Foundation (INV-017985 and INV-019400). The findings and conclusions contained within are those of the authors and do not necessarily reflect positions or policies of the Bill & Melinda Gates Foundation ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The ethics committee of Kenya Medical Research Institute (KEMRI) Scientific and Ethics Review Unit (SERU) gave ethical approval for this work (Reference 4097) The ethics committee of Kenya Pharmacy and Poisons Board gave ethical approval for this work The ethics committee of The Health Research Ethics Committee (CERS) and Technical Committee for Clinical Trials (Comite Technique dExamen des d emandes d autorisation d Essais Cliniques [CTEC]) of the Ministry of Health (Burkina Faso) gave ethical approval for this work The Ethics Committee of Liverpool School of Tropical Medicine (LSTM UK) gave ethical approval for this work (Ref 20-063) The Ethics Committee of the London School of Hygiene and Tropical Medicine (LSHTM UK) gave ethical approval for this work(Ref 22599) I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All sequencing data is deposited in BioProjects : PRJNA1095865 and PRJNA1096688 All other data produced in the present work can be made available upon reasonable request to the authors.