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Investigating HIV-1 drug resistance testing using Oxford Nanopore sequencing on dried blood spots (DBS) and plasma samples
Thesis (MSc)--Stellenbosch University, 2022.
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Stellenbosch : Stellenbosch University
2022
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| _version_ | 1867613974132948992 |
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| access_status_str | Open Access |
| author | Nyakubaya, Tafadzwa Dylan |
| author2 | Van Zyl, Gert Uves
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| author_browse | Nyakubaya, Tafadzwa Dylan Van Zyl, Gert Uves |
| author_facet | Van Zyl, Gert Uves
Nyakubaya, Tafadzwa Dylan |
| author_sort | Nyakubaya, Tafadzwa Dylan |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MSc)--Stellenbosch University, 2022. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/126336 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:44:39.798Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2022 |
| publishDateRange | 2022 |
| publishDateSort | 2022 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/126336 Investigating HIV-1 drug resistance testing using Oxford Nanopore sequencing on dried blood spots (DBS) and plasma samples Nyakubaya, Tafadzwa Dylan Van Zyl, Gert Uves Preiser, Wolfgang Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology. Drug resistance -- South Africa Nanopores -- South Africa HIV (Viruses) -- Alternative treatment -- South Africa Dried blood spots Plasma samples UCTD Thesis (MSc)--Stellenbosch University, 2022. ENGLISH ABSTRACT: Introduction: The expansion of antiretroviral therapy (ART) in South Africa has been linked to the emergence of human immunodeficiency virus type 1 (HIV-1) drug resistance in HIV-1 infected people. In patients with virologic failure due to drug resistance, one expects lower treatment effectiveness and the risk of transmitting these resistant variants to others. Although Sanger sequencing is routinely being used for HIV-1 genotypic resistance testing on plasma samples, this may not be ideal for resource-limited settings that lack laboratory infrastructure and cold-chain facilities. Oxford Nanopore Technologies (ONT) sequencing offers options in terms of device size, with equipment that is portable and requires a far lower start-up cost than other sequencing platforms. It can thus be utilized for near-patient diagnostics in resource-limited settings. HIV-1 drug resistance testing on dried blood spots (DBS) as an alternative to plasma would increase the accessibility of HIV drug resistance testing in resource-limited settings. Limitations of bulk sequencing (Sanger sequencing) and inadequate inclusion of at-risk populations in HIV-1 drug resistance surveillance may lead to an underestimation of the actual burden of HIV-1 drug resistance. Improved ONT base calling accuracy, in addition to using DBS as sample type, would increase the utility of ONT for HIV sequencing and facilitate the expansion of HIV-1 drug resistance surveillance to populations from understudied resource-limited settings. Methods: In this retrospective cross-sectional study, we used a sensitive extraction method to isolate ribonucleic acid (RNA) from 25 matching plasma and DBS samples that were obtained from HIV-1 infected individuals with virologic failure. These individuals had a median (interquartile range (IQR) of 101 080 (8751-21600) viral RNA copies per ml of plasma. Samples were amplified using a one step reverse transcriptase polymerase chain reaction (RT-PCR) protocol and touchdown conditions obtained from collaborators at the University of Pittsburgh, Virology Laboratory, Virology and Pharmacodynamics Core. Modifications to the protocol were made for the nested primers, and a nested PCR was performed on both plasma and DBS. Nested PCR products were purified and ligated to ONT sequencing adapters. The sequence library was then sequenced on the ONT GridION sequencing platform, and the data was processed using the Nano Recall bioinformatic pipeline that had been in development by our collaborator, Dr. Richard Harrigan, at the time of writing this thesis. Drug resistance mutations detected by each of three different ONT base calling algorithms were compared to drug resistance mutations detected by Sanger as a reference. Findings: Overall, ONT high accuracy basecalling (HAC) had the highest agreement with Sanger followed by ONT super accuracy basecalling (SUPER) and ONT fast basecalling (FAST). ONT FAST’s lower basecalling accuracy may have significantly contributed to the overcalling of some mutations, resulting in a lower agreement with the reference method. Additionally, sequencing results of DBS samples (n=12) derived from ONT HAC were compared to the corresponding plasma samples. Among the DBS-plasma pairs, 93% had identical drug-resistance mutations. Despite the low number of DBS samples sequenced, we managed to efficiently detect HIV-1 drug resistance mutations via ONT using DBS. Findings from this study may assist with wider implementation of national HIV-1 drug resistance surveillance programs in resource-limited settings. HIV-1 drug resistance surveillance is pivotal in identifying the emergence of drug resistance in different populations, to provide effective ART to maintain a high proportion of undetectable HIV viral loads in the population, as per the United Nations (UN) 95-95-95 goals to be achieved by 2025. AFRIKAANS OPSOMMING: Inleiding: Die uitbreiding van antiretrovirale terapie (ART) in Suid-Afrika gaan gepaard met die toename van menslike immuniteitsgebreksvirus tipe 1 (MIV-1)-middelweerstandigheid in MIV-1- geïnfekteerde mense. In pasiënte met verhoogde virusladings weens middelweerstandigheid, verwag mens ‘n laer terapie behandelingsdoeltreffendheid en die risiko om hierdie weerstandige variante aan ander oor te dra. Alhoewel Sanger-volgordebepaling vir MIV-1 genotipiese toetsing op plasma monsters meestal gebruik word, is dit dalk nie ideaal vir hulpbronbeperkte instellings wat ʼn gebrek het aan laboratorium infrastruktuur en koueketting fasiliteite nie. ONT-volgordebepaling bied opsies in terme van toestelgrootte, met toerusting wat draagbaar is wat 'n baie laer aanvangskoste vereis in vergelyking met ander volgordebepalingplatforms. Dit kan ook gebruik word vir punt-van-sorg diagnostiek in hulpbronbeperkte instellings. Wanneer middelweerstandstoetse op gedroogde bloedvlekke (GBV) uitgevoer word as 'n alternatief vir plasma, sal dit die toeganklikheid van MIV-middelweerstandstoetsing in hulpbronbeperkte instellings verhoog. Beperkings van grootmaat-volgordebepaling (Sanger-volgordebepaling) en onvoldoende insluiting van risiko-bevolkings by MIV-1-middelweerstand-toesig programme kan lei tot 'n onderskatting in die werklike las van MIV-1-middelweerstandigheid. Verbeterde ONT-basis-identifikasie, benewens the gebruik van GBV as monster tipe sal die nut van ONT vir MIV-volgordebepaling verhoog. Dit fasiliteer die uitbreiding van MIV-1-middelweerstandtoetsingprogramme in onderbestudeerde populasies in hulpbronbeperkte omgewings. Metodes: In hierdie retrospektiewe deursneestudie het ons 'n sensitiewe ekstraksiemetode gebruik om ribonukleïensuur (RNS) te isoleer van 25 plasma- en GBV-monsters wat verkry is van MIV-1- geïnfekteerde individue met verhoogde virusladings. Monsters is geamplifiseer deur gebruik te maak van 'n een-stap tru-transkriptase polimerase kettingreaksie (TT-PKR) protokol en “afwentel” PKR kondisies wat van medewerkers van die Universiteit van Pittsburgh, Virologie Laboratorium, Virologie en Farmakodinamika Kern verkry is. Modifikasies aan die protokol is gemaak vir die tweede rondte PKR inleiers; en twee rondtes PKR is op beide plasma en GBV uitgevoer. Tweede rondte PKR-produkte is versuiwer en aan ONT-volgordebepalings aansluiters geligeer. Die volgorde van die volgorde-biblioteek is toe bepaal deur die ONT-volgordebepalingsplatform, en die data is verwerk met behulp van die NanoRecall bioinformatiese pyplyn wat tydens die skrywe van hierdie tesis ontwikkel is. Middelweerstands-mutasies wat onderskeidelik deur die verskillende ONT-basis-oproep algoritmes opgespoor is, is elk vergelyk met middelweerstands-mutasies wat deur Sanger as verwysing opgespoor is. https://scholar.sun.ac.za vi Bevindinge: In samevatting, ONT hoë akkuraatheid basis-identifikasie (HAC) het die beste met Sanger ooreengekom, gevolg deur ONT super akkuraatheid basis-identifikasie (SUPER) en ONT vinnige basis-identifikasie (FAST). ONT FAST se laer basis-identifikasie akkuraatheid kon dalk aansienlik bygedra het tot die oorraportering van sommige mutasies, wat gelei het tot 'n laer ooreenstemming met die verwysing Sanger. Additioneel, is volgordebepaling resultate van GBV monsters (n=12) afgelei van ONT HAC vergelyk met die ooreenstemmende plasma monsters. Van die GBV-plasmapare het 93% identiese middelweerstands-mutasies gehad. Ten spyte van die lae aantal GBV-monsters wat se volgorde bepaal is, het ons daarin geslaag om MIV-1- middelweerstands-mutasies doeltreffend op te spoor met ONT deur gebruik te maak van GBV. Bevindinge van hierdie studie kan ook beleidmakers help om nasionale MIV-1-middelweerstandtoesigprogramme meer suksesvol in hulpbronbeperkte omgewings te implementeer. MIV1-middelweerstand-toesigprogramme is noodsaaklik in die identifisering van ontluikende middelweerstandigheid in verskillende bevolkings, om effektiewe ART te voorsien ten einde 'n hoë voorkoms van onopspoorbare MIV-virusladings in die bevolking te handhaaf, wat die laaste 95% van die Verenigde Nasies (VN) 95-95- 95 doelwitte teen 2025, is. Masters 2022-11-23T09:45:58Z 2023-01-23T06:52:50Z 2023-05-23T03:00:10Z 2022-12 Thesis http://hdl.handle.net/10019.1/126336 en_ZA Stellenbosch University xv, 61 pages : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Drug resistance -- South Africa Nanopores -- South Africa HIV (Viruses) -- Alternative treatment -- South Africa Dried blood spots Plasma samples UCTD Nyakubaya, Tafadzwa Dylan Investigating HIV-1 drug resistance testing using Oxford Nanopore sequencing on dried blood spots (DBS) and plasma samples |
| title | Investigating HIV-1 drug resistance testing using Oxford Nanopore sequencing on dried blood spots (DBS) and plasma samples |
| title_full | Investigating HIV-1 drug resistance testing using Oxford Nanopore sequencing on dried blood spots (DBS) and plasma samples |
| title_fullStr | Investigating HIV-1 drug resistance testing using Oxford Nanopore sequencing on dried blood spots (DBS) and plasma samples |
| title_full_unstemmed | Investigating HIV-1 drug resistance testing using Oxford Nanopore sequencing on dried blood spots (DBS) and plasma samples |
| title_short | Investigating HIV-1 drug resistance testing using Oxford Nanopore sequencing on dried blood spots (DBS) and plasma samples |
| title_sort | investigating hiv 1 drug resistance testing using oxford nanopore sequencing on dried blood spots dbs and plasma samples |
| topic | Drug resistance -- South Africa Nanopores -- South Africa HIV (Viruses) -- Alternative treatment -- South Africa Dried blood spots Plasma samples UCTD |
| url | http://hdl.handle.net/10019.1/126336 |
| work_keys_str_mv | AT nyakubayatafadzwadylan investigatinghiv1drugresistancetestingusingoxfordnanoporesequencingondriedbloodspotsdbsandplasmasamples |