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The role of underdiagnosed viruses in neonatal sepsis
Thesis (MMed) -- Stellenbosch University, 2022.
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Stellenbosch : Stellenbosch University
2022
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| _version_ | 1867613948426059776 |
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| access_status_str | Open Access |
| author | Mahlangu, Tshegofatso Perseverance |
| author2 | Van Zyl, Gert Uves |
| author_browse | Mahlangu, Tshegofatso Perseverance Van Zyl, Gert Uves |
| author_facet | Van Zyl, Gert Uves Mahlangu, Tshegofatso Perseverance |
| author_sort | Mahlangu, Tshegofatso Perseverance |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MMed) -- Stellenbosch University, 2022. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/126028 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:44:15.221Z |
| 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:10019.1/126028 The role of underdiagnosed viruses in neonatal sepsis Mahlangu, Tshegofatso Perseverance Van Zyl, Gert Uves Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Division of Medical Virology. Septicemia in children Antibiotics -- Therapeutic use Septicemia -- Molecular aspects Third generation sequencing Newborn infants -- Mortality Neonatal infections UCTD Thesis (MMed) -- Stellenbosch University, 2022. ENGLISH ABSTRACT: Background Neonatal sepsis is a clinical syndrome that is difficult to diagnose due to the unspecific symptoms that patients present with. Several different diagnostic techniques are used to diagnose neonatal sepsis; but currently, blood cultures are regarded as the gold standard for diagnosis of bacterial sepsis. Broad spectrum antibiotics are administered to neonates with suspected sepsis and the therapy is adjusted accordingly after diagnosis. This is, however, inappropriate therapy if sepsis is caused by viral and not bacterial agents. Numerous studies have found certain viruses to cause sepsis or a sepsis-like syndrome, but because viruses require virus specific assays, they may be underdiagnosed. Although the incidence of viral neonatal sepsis has been low, the effect of its underdiagnosis has detrimental effects. Therefore, in this study, a rapid detection multiplex polymerase chain reaction (PCR) was developed to potentially assist with the diagnosis of viral neonatal sepsis. A third-generation sequencing (TGS) technology was used for species specific identification of the agents detected by the multiplex PCR. TGS technology is increasingly utilised in medical research and appears promising for future rapid infectious agent diagnosis due to its real-time and long read sequencing. Methods Various sample types were included: residual patient laboratory samples with known positive diagnoses (none of which were from neonatal sepsis patients), external quality assurance panel samples and culture isolates from known agents. These samples were used to first optimise a singleplex assay followed by the multiplex assay. The assay was developed to have two rounds of PCR to ensure increased sensitivity. Two different extraction methods were compared to determine the method best suited for the assay. In addition, a simple sensitivity test was conducted using serially diluted samples for both the singleplex, and multiplex assays. As a result of the presence of an unknown fragment in the second-round multiplex PCR non-template controls (NTCs), several troubleshooting experiments were performed. Products with the incorrect band size or present in NTCs, which were observed during assay optimisation, were initially investigated with Sanger sequencing. Thereafter Oxford Nanopore Technology (ONT) sequencing was used to confirm the PCR amplified products. In addition, ONT sequencing was used to attempt to identify the unknown fragment in the NTCs. Results The singleplex assay was successfully optimised, however, during the multiplex optimisation an unknown fragment was discovered in the second round PCR NTCs. Troubleshooting excluded all other sources and we concluded that the “contaminant” was possibly an impurity of bacterial origin carried over from recombinant technology used in the production of the enzyme system. A new enzyme system was introduced to continue the multiplex optimisation, but the sensitivity was lower than with the previous enzyme system. As a result, the previous enzyme system was utilised for all targets except bacterial targets, for which a separate reaction including the 16s rRNA primers were used. Thereafter, the multiplex assay was further optimised. ONT sequencing confirmed that the correct agents were amplified, rendering the entire workflow successful. The unknown fragment of bacterial origin discovered in the NTCs was sequenced with ONT as well as Sanger sequencing. However, both platforms failed to confirm the specific bacterial species present. Conclusion The multiplex assay developed in this study can successfully amplify the intended agents but only at high concentrations. The comparison of sensitivity of the singleplex versus the multiplex versions of the assay revealed that the multiplex assay was significantly less sensitive. ONT sequencing could rapidly confirm all the amplified agents, and this is beneficial for diagnostic purposes. Currently, this study serves as a proof of concept because the multiplex assay, in its current form, does not appear to be suitable for primary (uncultured) diagnostic samples due to inadequate sensitivity for a low target concentration input. AFRIKAANS OPSOMMING: Agtergrond Neonatale sepsis is `n kliniese sindroom wat moeilik is om te diagnoseer omdat pasiënte nie-spesifieke simptome toon. `n Aantal verskillende tegnieke word gebruik om neonatale sepsis te diagnoseer, alhoewel bloedkulture as die goudstandaard vir die diagnose van bakterieë beskou word. Neonate met vermoedelike sepsis word aanvanklik behandel met breëspektrum antibiotika, wat ná diagnose dienooreenkomstig aangepas word. Dit is egter onvanpaste terapie wanneer die sepsis sindroom veroorsaak word deur virusse in plaas van bakterieë. Virusse is oënskynlik die mins algemene oorsaak van neonatale sepsis; nietemin dui etlike studies daarop dat sekere virusse wel sepsis of `n sepsis-agtige sindroom veroorsaak. Neonatale sepsis veroorsaak deur virusse vereis virus-spesifieke toetse wat min gebruik word vir diagnose en wat dus kan lei tot onderdiagnose. Daarom was die doel van hierdie studie om `n multipleks-polimerase kettingreaksie (PKR) te ontwikkel vir die snelle opsporing en diagnose van virale neonatale sepsis. Derde generasie volgordebepalingstegnologie is gebruik vir die spesiebevestiging van die agense wat deur die multipleks-PKR opgespoor is. Dié tegnologie word al hoe meer vir mediese navorsing ingespan, en omdat dit intydse en langlesing-volgordebepaling bied, hou dit groot belofte in vir toekomstige diagnostiek. Metodes Verskeie monstertipes is ingesluit: Oorblywende laboratorium monsters met bekende positiewe diagnoses (waarvan geen van neonatale sepsis pasiënte was nie), gehalte-beheer paneelmonsters en kultuur isolate. Hierdie monsters is gebruik om eers `n enkelpleks-toets te optimiseer, waarna die multipleks-toets ontwikkel is. Die toets is ontwikkel om twee rondtes van PKRs in te sluit om maksimum sensitiwiteit te verseker. Verder is `n sensitiwiteits toets met reeksverdunde monsters is vir beide die enkelpleks- en die multipleks-toetse onderneem. Weens die teenwoordigheid van `n onbekende fragment in die nul templaat kontroles (NTK`s) is `n aantal foutopsporings eksperimente uitgevoer. Produkte met die verkeerde band-grootte of teenwoordig in die NTKs, wat waargeneem is tydens optimisering-eksperimente is aanvanklik met Sanger-volgordebepaling ondersoek. Daarna is Oxford Nanopore Tegnologie (ONT)- volgordebepaling gebruik om die PKR-geamplifiseerde produkte te bevestig. Verder is ONT-volgordebepaling ook gebruik om die onbekende fragment in die NTK`s te probeer identifiseer. Resultate Die enkelpleks-toets is suksesvol geoptimiseer. Gedurende optimisering van die multipleks-toets is `n onbekende fragment in die NTK`s van die tweede rondte PKR opgemerk. Foutopsporing het alle ander bronne van kontaminasie uitgeskakel en daar derhalwe bepaal dat die “kontaminant” `n onsuiwerheid van bakteriese oorsprong was, oorgedra vanaf rekombinate tegnologie, in die produksie van die meestermengsel wat gebruik is. `n Nuwe ensiemstelsel is gevolglik gebruik om met die multipleks-optimisering voort te gaan. Hierdie ensiemstelsel was minder sensitief as die vorige ensiemstelsel. Dus is daar weer op die vorige ensiemstelsel teruggeval, vir alle agense behalwe bakterieë, waarvoor `n aparate reaksie met 16s-rRNA inleiers gebruik is. Die multipleks-toets is daarna verder geoptimiseer. ONT-volgordebepaling het bevestig dat die korrekte agense geamplifiseer is, en dus was die oorhoofse werkvloei suksesvol. Die onbekende bakteriese fragment in die NTK`s is ook aan ONT-volgordebepaling onderwerp, maar die data kon nie die spesifieke bakterieële spesie bevestig nie. Gevolgtrekking Die multipleks-toets wat in hierdie studie ontwikkel is, kan die beoogde agense amplifiseer, maar slegs vanaf hoë konsentrasies. `n Vergelyking van die enkelpleks- en multipleks-weergawes van die toets het getoon dat die multipleks-toets aansienlik minder sensitief is. ONT-volgordebepaling het die geamplifiseerde spesies in `n kits bevestig, wat voordelig is vir diagnostiese doeleindes. Hierdie studie dien tans as `n konsepbewys aangesien die multipleks toets nie geskik blyk te wees vir primêre (ongekultuurde) diagnostiese monsterns nie, weens die onvoldoende sensitiwiteit vir `n lae teikenkonsentrasie inset. Masters 2022-11-21T06:44:53Z 2023-01-16T12:46:28Z 2022-11-21T06:44:53Z 2023-01-16T12:46:28Z 2022-12 Thesis http://hdl.handle.net/10019.1/126028 en_ZA Stellenbosch University xviii, 86 pages : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Septicemia in children Antibiotics -- Therapeutic use Septicemia -- Molecular aspects Third generation sequencing Newborn infants -- Mortality Neonatal infections UCTD Mahlangu, Tshegofatso Perseverance The role of underdiagnosed viruses in neonatal sepsis |
| title | The role of underdiagnosed viruses in neonatal sepsis |
| title_full | The role of underdiagnosed viruses in neonatal sepsis |
| title_fullStr | The role of underdiagnosed viruses in neonatal sepsis |
| title_full_unstemmed | The role of underdiagnosed viruses in neonatal sepsis |
| title_short | The role of underdiagnosed viruses in neonatal sepsis |
| title_sort | role of underdiagnosed viruses in neonatal sepsis |
| topic | Septicemia in children Antibiotics -- Therapeutic use Septicemia -- Molecular aspects Third generation sequencing Newborn infants -- Mortality Neonatal infections UCTD |
| url | http://hdl.handle.net/10019.1/126028 |
| work_keys_str_mv | AT mahlangutshegofatsoperseverance theroleofunderdiagnosedvirusesinneonatalsepsis AT mahlangutshegofatsoperseverance roleofunderdiagnosedvirusesinneonatalsepsis |