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A real-time reverse transcription polymerase chain reaction for the sensitive detection of hepatitis a virus in various clinical and environmental samples
Thesis (MSc)--Stellenbosch University, 2023.
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| Format: | Thesis |
| Language: | en_ZA |
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Stellenbosch : Stellenbosch University
2023
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| _version_ | 1867613937023844352 |
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| access_status_str | Open Access |
| author | Mpazi, Nothukela |
| author2 | Nkosi, Nokwazi |
| author_browse | Mpazi, Nothukela Nkosi, Nokwazi |
| author_facet | Nkosi, Nokwazi Mpazi, Nothukela |
| author_sort | Mpazi, Nothukela |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MSc)--Stellenbosch University, 2023. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/126977 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:44:04.029Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2023 |
| publishDateRange | 2023 |
| publishDateSort | 2023 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/126977 A real-time reverse transcription polymerase chain reaction for the sensitive detection of hepatitis a virus in various clinical and environmental samples Mpazi, Nothukela Nkosi, Nokwazi De Beer, Corena Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Division of Medical Virology. Hepatitis A virus Virus diseases Hepatitis viruses UCTD Thesis (MSc)--Stellenbosch University, 2023. ENGLISH SUMMARY: Hepatitis A virus (HAV) is a growing public health concern worldwide due to its contribution to acute viral hepatitis. In South Africa, HAV data has been accounted for in clinical and environmental studies separately. Simultaneous detection of the HAV in clinical and environmental samples in the Western Cape province has not yet been explored. The current serological method of HAV detection has been linked to cross-reactivity between antibodies resulting in false positive results. Supplemental use of real-time reverse transcription (RT) polymerase chain reaction (PCR) may have the benefit of limiting cross-reactivity and serving as an early detection method in outbreak settings as it detects HAV ribonucleic acid (RNA) two weeks prior to seroconversion. The aim was to assess the presence of HAV (RNA) in various clinical and environmental wastewater samples using an in-house established real-time RT-PCR assay and to describe circulating HAV genotype(s). The objectives were to identify serologically tested HAV residual serum, plasma and stool samples referred to the National Health Laboratory Service (NHLS) Medical Virology Laboratories at Tygerberg Hospital (TBH), Groote Schuur Hospitals (GSH); and Western Cape Blood Service (WCBS); to screen untreated wastewater environmental samples for HAV presence from the South African Medical Research Council (SAMRC); to analyse data by age, sex, liver function enzyme parameters and location of retrieval using Microsoft Excel and to assess circulating genotype (s) through Sanger sequencing. Two different primers and probes were evaluated for HAV RNA detection in 353 samples comprising of 179 clinical (serum, stool, and plasma) and 174 environmental (untreated wastewater RNA eluates) samples obtained through convenience sampling from TBH; GSH NHLS serology laboratories; WCBS and SAMRC (from five Stellenbosch University residences and two Cape Town areas). BioEdit software; Genome detectives Krisp genotyping tool version 2.43 and Mega 11 software were used to assess HAV genotype(s) on selected clinical samples only. HAV RNA was positive in 43.6% (78/179) clinical samples and 32.7% (57/174) environmental samples. In the ages younger 15 years HAV RNA positive samples were higher in males in compared to females. In contrast age groups >16 years HAV RNA positive samples were higher in females compared to males. The City of Cape Town areas had a higher detection of HAV RNA positive environmental samples (>40%), while a 28% detection was recorded from the Stellenbosch University student residence samples. The identified circulating HAV genotype was HAV IB. Using the two primer and probe sets this assay successfully detected HAV RNA in various clinical and environmental untreated wastewater samples offering a 92% and 100% sensitivity for the first and second primer set, respectively. Specificity was 100% and 75% respectively. Performance characteristics of second primer set evaluated limited by sample number evaluated. Using assays with 100% specificity might be used in diagnostic settings as a supplemental confirmatory technique. This study was semi-qualitative in nature, future studies may assess the quantitative aspect and cost related to the implementation of the assay in diagnostic settings. AFRIKAANSE OPSOMMING: Hepatitis A-virus (HAV) is 'n groeiende openbare gesondheidsorg entiteit wereldwyd as gevolg van sy bydrae tot akute virale hepatitis. In Suid-Afrika is HAV-data afsonderlik in kliniese en omgewingstudies verreken. Gelyktydige opsporing van die HAV in kliniese en omgewingsmonsters in die Wes-Kaap provinsie is nog nie ondersoek nie. Die huidige serologiese metode van HAV-opsporing is gekoppel aan kruisreaktiwiteit tussen teenliggaampies wat lei tot vals positiewe resultate met 'n toetsherhalingsperiode van twee weke. Aanvullende gebruik van intydse omgekeerde transkripsie polimerase kettingreaksie (RT-PCR) kan die ernstig siektes bevoordeel deur die herhalingsperiode van twee weke te beperk, kruisreaktiwiteit te beperk en te dien as 'n vroee opsporingsmetode in uitbraak-instellings aangesien dit HAV RNA twee weke voor opspoor tot seroomskakeling. Die doel was om die teenwoordigheid van HAV-ribonukleiensuur (RNA) in verskeie kliniese en omgewingsafvalwatermonsters te bepaal deur gebruik te maak van 'n in-huis gevestigde intydse RT-PCR-toets en om sirkulerende HAV-genotipe(s) te beskryf. Die doelwitte was om serologies-getoetste HAV-serum-, plasma- en stoelmonsters te identifiseer wat verwys is na die Nasionale Gesondheids Laboratorium Dienste (NGLD) Mediese Virologie Laboratoria by Tygerberg-hospitaal (TBH), Groote Schuur-hospitaal (GSH); en Wes-Kaapse Bloeddiens (WKB); om onbehandelde afvalwater-omgewingsmonsters vir HAV teenwoordigheid van die Suid-Afrikaanse Mediese Navorsingsraad (SAMNR) te verkry en te sift; om data volgens ouderdom, geslag, lewerfunksie-ensiemparameters en ligging van herwinning te ontleed met behulp van Microsoft Excel en om sirkulerende genotipe(s) deur middel van Sanger-volgordebepaling te assesseer. In-huis-intydse RT-PCR-toetse vir HAV-RNA-opsporing is opgestel vir 353 monsters wat bestaan uit 179 kliniese (serum, stoelgang en plasma) en 174 omgewings- (onbehandelde afvalwater-RNA eluate) monsters wat verkry is deur middel van geriefsmonsters van TBH; GSH NHLS serologie laboratoriums; WKBS en SAMNR (vanaf vyf Universiteit Stellenbosch-koshuise en twee Kaapstad areas). Om genotipe(s) en volgordebelynings te assesseer Sanger-volgordebepaling, BioEdit sagteware; Genoom Speurders Krisp genotipering instrument weergawe 2.43 en Mega 11 sagteware is slegs op geselekteerde kliniese monsters gebruik. HAV RNA was positief in 43.6% (78/179) kliniese monsters en 32.73% (57/174) omgewingsmonsters. HAV RNA positiewe monsters was hoer by mans in ouderdomsgroep 1 tot 15 jaar in vergelyking met vroue; in ouderdomsgroepe >16 jaar was hoer vroulik in vergelyking met mans. Verspreiding van positiewe monsters volgens ouderdom, geslag en lewerfunksie-ensieme is nie vir die omgewingsmonsters geassesseer nie, maar die meeste HAV RNA-positiewe monsters was van die Kaapstad-gebiede (>40%) in vergelyking met die Universiteit Stellenbosch-koshuise (28%). Die geidentifiseerde genotipe was die HAV IB genotipe wat slegs op kliniese monsters geassesseer is. Die toets het 100% spesifisiteit gebied. Hierdie studie was in staat om HAV RNA in verskeie kliniese en omgewings onbehandelde afvalwatermonsters op te spoor deur gebruik te maak van 'n in-huis gevestigde intydse RT-PCR-toets. Sensitiwiteit was 92% en 100% tussen die twee reaksies, onderskeidelik. Die spesifiteit was 100% en 75% onderskeidelik. Prestasie eienskappe van die tweede reaksie geevalueer beperk deur monster beskikbaarheid.Hierdie toets was egter kwalitatief van aard, toekomstige studies kan die kwantitatiewe aspek assesseer. In diagnostiese laboratorium implementering het hoe kliniese spesifisiteit geskiktheid vir gebruik en aanvulling tot bestaande serologiese tegnieke aangedui. Masters 2023-02-23T04:29:00Z 2023-05-18T06:58:27Z 2023-02-23T04:29:00Z 2023-05-18T06:58:27Z 2023-03 Thesis http://hdl.handle.net/10019.1/126977 en_ZA Stellenbosch University xiv, 97 pages : illustrations, maps, includes annexures application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Hepatitis A virus Virus diseases Hepatitis viruses UCTD Mpazi, Nothukela A real-time reverse transcription polymerase chain reaction for the sensitive detection of hepatitis a virus in various clinical and environmental samples |
| title | A real-time reverse transcription polymerase chain reaction for the sensitive detection of hepatitis a virus in various clinical and environmental samples |
| title_full | A real-time reverse transcription polymerase chain reaction for the sensitive detection of hepatitis a virus in various clinical and environmental samples |
| title_fullStr | A real-time reverse transcription polymerase chain reaction for the sensitive detection of hepatitis a virus in various clinical and environmental samples |
| title_full_unstemmed | A real-time reverse transcription polymerase chain reaction for the sensitive detection of hepatitis a virus in various clinical and environmental samples |
| title_short | A real-time reverse transcription polymerase chain reaction for the sensitive detection of hepatitis a virus in various clinical and environmental samples |
| title_sort | real time reverse transcription polymerase chain reaction for the sensitive detection of hepatitis a virus in various clinical and environmental samples |
| topic | Hepatitis A virus Virus diseases Hepatitis viruses UCTD |
| url | http://hdl.handle.net/10019.1/126977 |
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