Implementation, evaluation and use of methods to identify SARS-CoV-2 genetic variants of significance

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dc.contributor.advisorWolfgang, Preiseren_ZA
dc.contributor.advisorTongai, Mapongaen_ZA
dc.contributor.authorKarabo, Phaduen_ZA
dc.contributor.otherStellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Division of Medical Virology.en_ZA
dc.date.accessioned2023-03-02T09:43:29Z
dc.date.accessioned2023-05-18T07:16:17Z
dc.date.available2023-03-02T09:43:29Z
dc.date.available2023-05-18T07:16:17Z
dc.date.issued2023-03
dc.descriptionThesis (MSc)--Stellenbosch University, 2023. en_ZA
dc.description.abstractENGLISH SUMMARY: Background: The emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants, designated as variants of concern (VOCs) by the World Health Organization (WHO) continue to threaten measures in place to contain the spread of the virus. Consequently, efforts have been made to intensify genomic surveillance to track and monitor the evolution and spread of SARS-CoV-2 VOCs to help inform public health interventions in a timely manner. However, whole genome sequencing (WGS) is expensive, has a longer turnaround time and requires expert bioinformatics analysis, making it unfeasible for near real-time monitoring and reporting of SARS-CoV-2 variants. There is a need for affordable variant screening methods for the rapid detection and differentiation of SARS-CoV-2 VOCs. Methods: Ten SARS-CoV-2 variant screening assays targeting SARS-CoV-2 Alpha, Beta, Delta and Omicron VOCs were used. The analytical sensitivity of the assays was assessed using RNA extracted from cell culture supernatants of isolates of the Beta and Delta VOCs and the limit of detection (LOD) and linearity of the assays were determined. Furthermore, the assays were used to screen for SARS-CoV-2 VOCs in 898 patient samples diagnosed with infection between November 2020 and January 2022. Screening results were validated against WGS to determine the clinical sensitivity, and specificity of variant screening assays, and the agreement between assays (using WGS as the gold standard) was reported as the kappa value. Lastly, the TaqMan SNP genotyping panel, TaqPath COVID-19, and ModularDx assays were implemented to estimate the proportion of SARS-CoV-2 VOCs that circulated between November 2020 and January 2022. Results: The SARS-COV-2 variant screening assay had a correlation coefficient between 0.98 and 0.99, indicating good linearity between the dilution and the corresponding average cycle threshold values (Ct-values) at each standard dilution. Of the 10 screening assays, the Smartchek B.1.351 assay had the lowest clinical sensitivity (86.4%) followed by the Allplex Variants I assay (95.5%). The sensitivity of the ModularDx assay and the TaqPath COVID-19 assays was 98.6% and 99.7%, respectively. Furthermore, the TaqMan mutation panel, Allplex Variants II, IV and Master assays were 100% sensitive. All assays demonstrated 100% specificity and moderate to high concordance in comparison with WGS. The predominant circulating variant before and during May 2021 was the Beta VOC, constituting 86.7%, 100% and 79.4% of samples screened in November and December 2020 and May 2021 respectively. The Delta VOC rapidly spread, displacing the Beta VOC in May 2021 and dominating from June until October 2021. In November, December 2021, and January 2022 the Omicron VOC predominated, with the proportion of samples resulting in failure of the S gene target to amplify, SGTF rising from 87.7% in November to 99.5% in December. Conclusion: This study demonstrated that SARS-CoV-2 variant screening assays can be used as rapid and affordable tools to monitor signature mutations in SARS-CoV-2 VOCs. In addition, they can be useful tools for scaling up SARS-CoV-2 genomic surveillance. However, they need to be regularly updated and cannot replace traditional sequencing methods, but rather serve as tools to complement sequencing in monitoring circulating SARS-CoV-2 VOCs.en_ZA
dc.description.abstractAFRIKAANSE OPSOMMING: Agtergrond: Die opkoms van akute respiratosie sindroom koronavirus (SARS-CoV-2) variante, wat deur die wereldgesondheidsorganisasie (WGO) as variante van kommer aangewys is, dreig steeds maatreels wat in plek is om die verspreiding van die virus te beperk. Gevolglik is pogings aangewend om genomiese toesig te versterk om die evolusie en verspreiding van SARS-CoV-2 variante van kommer (VOC's) op te spoor en te monitor om te help om openbare gesondheidsingrypings betyds in te lig. Heelgenoomvolgordebepaling is egter duur, het 'n langer omkeertyd en vereis kundige bioinformatika-analise en infrastruktuur, wat dit onprakties maak vir SARS-CoV-2 genomiese toesigstudies. Daar is 'n behoefte om vinnige en bekostigbare variante siftingsmetodes te ontwikkel om SARS-CoV-2 VOC's op te spoor en te onderskei en resultate betyds te rapporteer. Metodes: Tien toetse is in hierdie studie gebruik. Analitiese sensitiwiteit van die toetse is geassesseer deur gebruik te maak van ribonukleiensuur (RNS) wat uit selkultuursupernatant van variante kultuurisolate onttrek is en die limiet van opsporing en lineariteit van die toetse is bepaal. Verder is geevalueerde toetse gebruik om te sif vir die teenwoordigheid van SARS-CoV-2 VOC's in monsters wat tussen November 2020 en Januarie 2022 versamel is. Die resultate wat verkry is, is bekragtig teen heelgenoomvolgordes, die kliniese sensitiwiteit, spesifisiteit is bepaal, en die ooreenkoms tussen toetse (met heelgenoom-volgordebepaling as die goudstandaard) is bereken en as die kappa-waarde gerapporteer. Laastens is die TaqMan SNP genotiperingstoetse, TaqPath COVID-19 en ModularDx toetse gebruik om die verhouding SARS-CoV-2 VOC's in monsters wat tussen November 2020 en Januarie 2022 versamel is, te skat. Resultate: SARS-COV-2 variant siftingstoetse wat geevalueer is, het 'n korrelasiekoeffisient van 0.98 en 0.99 gehad, wat 'n goeie lineariteit tussen die verdunning en die ooreenstemmende gemiddelde siklus-drempel-waardes by elke standaardverdunning aandui. Nege siftingstoetse wat gebruik is om vir SARS-CoV-2 VOC's te sirkuleer wat tussen November 2020 en Januarie 2022 in 898 monsters gesirkuleer het. Die Smartchek B.1.351-toets het die laagste sensitiwiteit (86.4%) gevolg deur die Allplex Variants I-toets (95.5%). Die sensitiwiteit van die ModularDx- en TaqPath COVID-19-toetse was onderskeidelik 98.6% en 99.7%. Terwyl die TaqMan-mutasiepaneel, Allplex Variants II, IV en Master-toetse 100% sensitief was. Alle toetse het 100% spesifisiteit en matige tot hoe konkordansie getoon in vergelyking met heelgenoomvolgordes. Die oorheersende sirkulerende variant voor en gedurende Mei 2021 was die B.1.351 wat 86.7%, 100% en 79.4% uitmaak van monsters wat onderskeidelik in November en Desember 2020 en Mei 2021 gekeur is. Die B.1.617.2-lyn (Delta-variant) het vinnig versprei en die Beta-variant in Mei 2021 verplaas en daarna oorheers van Junie tot Oktober 2021. In November en Desember 2021 en Januarie 2022 was die omikronvariant oorheersend, met die proporsie van monsters wat daartoe gelei het dat die spike geen teiken mislukking (SGTF) van 87.7% in November 2021 tot 99.5% in Desember 2021 gestyg het. Gevolgtrekking: Hierdie studie was in staat om te demonstreer dat gevalideerde SARS-CoV-2 variant siftingstoetse gebruik kan word as vinnige en bekostigbare instrumente om handtekeningmutasie in SARS-CoV-2 VOC's te monitor. Boonop kan dit nuttige instrumente wees om SARS-CoV-2 genomiese toesig op te skaal. Hulle moet egter gereeld bygewerk word en kan nie tradisionele volgordebepalingsmetodes vervang nie, maar dien eerder as hulpmiddels om volgordebepaling aan te vul in die monitering van sirkulerende SARS-CoV-2 VOC's.af_ZA
dc.description.versionMasters
dc.format.extentxvii, pages : illustrations, includes annexures
dc.identifier.urihttp://hdl.handle.net/10019.1/127327
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch University
dc.rights.holderStellenbosch University
dc.subject.lcshCoronavirus -- geneticsen_ZA
dc.subject.lcshCoronavirus infections -- Complicationsen_ZA
dc.subject.lcshCOVID-19 (Disease) -- Diagnosisen_ZA
dc.subject.nameUCTD
dc.titleImplementation, evaluation and use of methods to identify SARS-CoV-2 genetic variants of significanceen_ZA
dc.typeThesisen_ZA
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