Browsing by Author "Ndou, Sedzani"

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    Cytokine gene expression assay as a diagnostic tool for detection of Mycobacterium bovis infection in warthogs (Phacochoerus africanus)
    (Nature Research (part of Springer Nature), 2019) Roos, Eduard O .; Scott, Leere A.; Ndou, Sedzani; Olea-Popelka, Francisco; Buss, Peter E.; De Klerk-Lorist, Lin-Mari; Warren, Robin M.; Van Helden, Paul D.; Sylvester, Tashnica T .; Miller, Michele A.; Parsons, Sven D. C.
    ENGLISH ABSTRACT: Mycobacterium bovis infection has been described in many wildlife species across Africa. However, diagnostic tests are lacking for many of these, including warthogs (Phacochoerus africanus). Most literature on suids has focused on using serological tools, with few studies investigating the use of cell-mediated immune response (CMI) assays. A recent study showed that warthogs develop measurable CMI responses, which suggests that cytokine gene expression assays (GEAs) may be valuable for detecting M. bovis-infection, as shown in numerous African wildlife species. Therefore, the aim of the study was to develop GEAs capable of distinguishing between M. bovis-infected and uninfected warthogs. Whole blood was stimulated using the QuantiFERON-TB Gold (In-Tube) system, using ESAT-6 and CFP-10 peptides, before determining the relative gene expression of five reference (B2M, H3F3A, LDHA, PPIA and YWHAZ) and five target (CXCL9, CXCL10, CXCL11, IFNG and TNFA) genes through qPCR. The reference gene H3F3A was the most stably expressed, while all target genes were significantly upregulated in M. bovis-infected warthogs with the greatest upregulation observed for CXCL10. Consequently, the CXCL10 GEA shows promise as an ante-mortem diagnostic tool for the detection of M. bovis-infected warthogs.
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    Evaluation of host transcriptional biomarkers in the diagnosis of TB disease and optimization of multi-colour immunophenotyping assay from the QuantiFERON® TB GOLD Plus cell sediments
    (2020-11) Ndou, Sedzani; Chegou, Novel N. ; Gutschmidt, Andrea; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
    ENGLISH ABSTRACT: Background QuantiFERON-TB Gold-Plus (QFT-Plus) test are designed for the diagnosis of Mycobacterium tuberculosis (M. tb) sensitization, hence it’s low specificity in the diagnosis of active TB. It remains unknown if the cell sediments left-over after performing the QFT-Plus ELISA can be useful for the diagnosis of active TB. Objectives To assess the expression of selected genes that have been shown in the literature as TB diagnostic or prognostic candidate biomarkers, in the cell sediments left-over after performance of the QFT-Plus test, and ascertain whether such genes when detected in QFT-Plus sediments, have any potential in the diagnosis of active TB. Methods We analysed the expression level of 13 genes namely: STAT1, GBP1, GBP5, TRAFD1, SERPING1, DUSP3, BATF2, TAP1, ETV7, SCARF1, FCGR1B, KLF2 and, GBP2, from the QFT-Plus left-over sediments using Taqman-probe qRT-PCR arrays in a total of 44 patients who were enrolled after presenting with the signs and symptoms of TB. The diagnostic accuracy of genes was evaluated using receiver operator characteristics (ROC) curves analysis whereas multi-biomarker analysis techniques were used to assess the usefulness of combinations between different genes in the diagnosis of TB. Some of the sediments from the QFT-Plus were fixed and stored for Flow Cytometry analysis in the future. The Flow Cytomery panel was designed to include the T cell subsets as well as other immune biomarkers such as IL-1B, CX3CR1, CD154 and CD56 which have been previously shown to have potential in the diagnosis of TB disease using Luminex platform Results Of the 13 individual genes evaluated in this study, the expression of 5 genes namely, BATF2, KLF2, DUSP3, GBP1 and GBP5 was significantly different (P<0.05) between the TB patients and individuals with other respiratory diseases (ORD). Individually, these genes diagnosed TB with area under the ROC curve (AUC) ≥0.89 in both the unstimulated and M. tb antigen-stimulated samples. When the genes detected in the unstimulated (Nil) sediments were fitted into general discriminant analysis (GDA) models, a 3-marker biosignature consisting of BATF2, GBP1 and GBP5 was found to be the most optimal in diagnosing TB and performed with an AUC of 0.99. However, when the Nil-derived 3-marker signature was applied on TB1 stimulated samples, the AUC reduced slightly to 0.98 and when applied to TB2 stimulated samples, the AUC remained the same as what was observed for unstimulated samples. Overall, stimulation of blood with different antigens led to different expression patterns for different genes in the specimens. Conclusion In conclusion, the results presented in this thesis show that sediments left-over after supernatants are harvested for performance of the QFT-Plus test, and which are often discarded, may be valuable samples for evaluation of both unspecific and M. tb antigen-specific gene expression. We furthermore showed that the detection of such genes may be a valuable method for the diagnosis of active TB and for future implementation purposes especially where limited blood volumes are available. Our sample size was small; hence these findings require further validation in larger studies.