Department of Biomedical Sciences

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Browsing Department of Biomedical Sciences by browse.metadata.advisor "Beltran, Caroline G. G."

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    Is sarcoidosis a persistent form of tuberculosis
    (Stellenbosch : Stellenbosch University, 2024-02) 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.
    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.