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Is sarcoidosis a persistent form of tuberculosis
Thesis (MSc)--Stellenbosch University, 2024.
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| Format: | Thesis |
| Language: | en_ZA |
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Stellenbosch : Stellenbosch University
2024
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| _version_ | 1867614137555615744 |
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| access_status_str | Open Access |
| author | Geldenhuys, Rebecca |
| author2 | Walzl, Gerhard |
| author_browse | Geldenhuys, Rebecca Walzl, Gerhard |
| author_facet | Walzl, Gerhard Geldenhuys, Rebecca |
| author_sort | Geldenhuys, Rebecca |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MSc)--Stellenbosch University, 2024. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/130913 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:47:15.645Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2024 |
| publishDateRange | 2024 |
| publishDateSort | 2024 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/130913 Is sarcoidosis a persistent form of tuberculosis Geldenhuys, Rebecca Walzl, Gerhard Beltran, Caroline G. G. Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics. Sarcoidosis Mycobacterium tuberculosis RNA-protein interactions UCTD Thesis (MSc)--Stellenbosch University, 2024. ENGLISH ABSTRACT: Sarcoidosis is an inflammatory disorder of unknown etiology, characterized by the formation of non-caseating epithelioid granulomas. Sarcoidosis and tuberculosis share several similarities in disease presentation, prompting researchers to investigate a link between sarcoidosis and Mycobacterium tuberculosis (Mtb), possibly present in an altered form. Mtb DNA and proteins have been found in sarcoidosis tissue; however, evidence of viable Mtb has not been discovered. The aim of this study was to investigate whether an altered form of Mtb could be the etiological agent of sarcoidosis in a South African setting. We utilized a combination of RNA detection and specialized culturing methods to investigate the presence of viable Mtb in sarcoidosis tissue. To detect the presence of Mtb RNA in sarcoidosis tissue, formalin-fixed paraffin-embedded (FFPE) sarcoidosis samples were sectioned and stained using custom designed Stellaris single molecule RNA FISH probes to detect Mtb 16S rRNA and Mtb heat-shock protein 70 (hsp70) mRNA, and visualized using fluorescence microscopy. To determine the limit of detection of bacterial RNA in a population of host RNA, Mycobacterium smegmatis (M. smegmatis) cells and THP-1 cells were combined at a constant total cell number with differing ratios of THP-1: M. smegmatis. Total RNA was extracted and analysed using PCR amplification with M. smegmatis rpoB gene primers to detect bacterial RNA. Fresh sarcoidosis samples were processed for Mycobacterial Growth Indicator Tube (MGIT) culture supplemented with specialised growth enhancers to enable the growth of potential mycobacteria present in these samples. Positive Mtb 16S rRNA signal was detected in 6 out of 8 archived- and 3 out of 4 fresh FFPE sarcoidosis samples. Overlapping positive Mtb hsp70 mRNA signal was observed in 2 of the archived- and 1 of the fresh FFPE sarcoidosis samples. The limit of detection was 5x105 M. smegmatis cells, meaning bacterial cells beneath 5x105 in the presence of host cells cannot be accurately identified. TiKa supplementation did not result in any growth of the sarcoidosis samples, even beyond the standard 42 days of culture. In this study, we were able to positively identify the presence of Mtb specific RNA in sarcoidosis tissue indicating the presence of viable cells. RNA FISH analysis was superior to bulk RNA analysis and enhanced culture methods for identifying rare sub-populations of Mtb in sarcoidosis tissue. Together these results provide further evidence to support the hypothesis that Mtb may be the causative agent of sarcoidosis. AFRIKAANSE OPSOMMING: Sarkoïdoseis 'n inflammatoriese versteuring van onbekende etiologie, wat gekenmerk word deur die vorming van nie-kaseerende epithelioïde granulome. Sarkoïdoseen tuberkulose deel verskeie ooreenkomste in siektevoorstelling, wat navorsers aangespoor het om 'n verband tussen sarkoïdose en Mycobacterium tuberculosis(Mtb) te ondersoek, wat moontlik in 'n veranderde vorm voorkom. Mtb DNA en proteïene is in sarkoïdoseweefsel gevind; bewyse van lewensvatbare Mtb is egter nie ontdek nie. Die doel van hierdie studie was om te ondersoek of 'n veranderde vorm van Mtb die etiologiese agent van sarkoïdose in 'n Suid-Afrikaanse omgewing kan wees.Ons het 'n kombinasie van RNA-opsporing en gespesialiseerde kweekmetodes gebruik om die teenwoordigheid van lewensvatbare Mtb in sarkoïdoseweefsel te ondersoek. Om die teenwoordigheid van Mtb RNA in sarkoïdoseweefsel op te spoor, is FFPE sarkoïdose monsters gesny en gekleur met gebruik van pasgemaakte Stellaris enkelmolekule RNA FISH probes om Mtb 16S rRNA en Mtb heat-shock protein70(hsp70) mRNAop te spoor, en gevisualiseer met behulp van fluoressensiemikroskopie. Om die limiet van opsporing van bakteriële RNA in 'n populasie van gasheer-RNA te bepaal, is Mycobacterium smegmatis(M. smegmatis)-selle en THP-1-selle gekombineer teen 'n konstante totale selgetal met verskillende verhoudings van THP-1: M. smegmatis. Totale RNA is onttrek en ontleed deur gebruik te maak van PCR-amplifikasie met M. smegmatisrpoBgeeninleiders om bakteriële RNA op te spoor. Varssarkoïdosemonsters is verwerk vir Mycobacterial Growth Indicator Tube (MGIT) kultuur en aangevul met gespesialisieërde groei bevorderings mediaom die groei van potensiële mikobakterieë wat in hierdiemonsters teenwoordig is, te verbeter. Positiewe Mtb 16S rRNA sein is opgespoor in 6 uit 8 geargiveerde-en 3 uit 4 vars FFPE sarkoïdose monsters. Oorvleuelende positiewe Mtb hsp70 mRNA sein is waargeneem in 2 van die geargiveerde-en 1 van die vars FFPE sarkoïdose monsters. Die limiet van opsporing was 5x105M. smegmatis-selle, wat beteken dat bakteriese selle onder 5x105in die teenwoordigheid van gasheerselle nie akkuraat geïdentifiseer kan word nie.TiKa supplementasie het nie enige groei van sarkoïdose monsters bevorder nie, selfs na die 42dae van standaard kultuur. In hierdie studie kon ons the teenwoordigheid van Mtb spesifieke RNA in sarkoïdose weefsel indentifiseer wat dui op die teenwoordigheid van lewensvatbare selle.RNA FISH analiese was beter as grootskaalse RNA analiese en bevorderde kultuur metodes vir die indentifiseëring van rare sub-populaises van Mtb in sarkoïdose weefsel. Saamgevat verskaf hierdie resultate verdere bywyse om die hipotese dat Mtb moontlik ‘n veroorsakende agent van sarkoïdose is. Masters 2024-02-14T08:39:54Z 2024-05-08T15:10:17Z 2024-02-14T08:39:54Z 2024-05-08T15:10:17Z 2024-02 Thesis https://scholar.sun.ac.za/handle/10019.1/130913 en_ZA Stellenbosch University 105 pages : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Sarcoidosis Mycobacterium tuberculosis RNA-protein interactions UCTD Geldenhuys, Rebecca Is sarcoidosis a persistent form of tuberculosis |
| title | Is sarcoidosis a persistent form of tuberculosis |
| title_full | Is sarcoidosis a persistent form of tuberculosis |
| title_fullStr | Is sarcoidosis a persistent form of tuberculosis |
| title_full_unstemmed | Is sarcoidosis a persistent form of tuberculosis |
| title_short | Is sarcoidosis a persistent form of tuberculosis |
| title_sort | is sarcoidosis a persistent form of tuberculosis |
| topic | Sarcoidosis Mycobacterium tuberculosis RNA-protein interactions UCTD |
| url | https://scholar.sun.ac.za/handle/10019.1/130913 |
| work_keys_str_mv | AT geldenhuysrebecca issarcoidosisapersistentformoftuberculosis |