Browsing by Author "Tshivhula, Happy"
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- ItemExtract from used Xpert MTB/ RIF Ultra cartridges is useful for accurate second-line drug-resistant tuberculosis diagnosis with minimal rpoB-amplicon cross-contamination risk(Nature Research (part of Springer Nature), 2020) Venter, Rouxjeane; Minnies, Stephanie; Derendinger, Brigitta; Tshivhula, Happy; De Vos, Margaretha; Dolby, Tania; Ruiters, Ashley; Warren, Robin M.; Theron, GrantXpert MTB/RIF Ultra (Ultra) detects Mycobacterium tuberculosis and rifampicin resistance. Follow-on drug susceptibility testing (DST) requires additional sputum. Extract from the diamond-shaped chamber of the cartridge (dCE) of Ultra’s predecessor, Xpert MTB/RIF (Xpert), is useful for MTBDRsl-based DST but this is unexplored with Ultra. Furthermore, whether CE from non-diamond compartments is useful, the performance of FluoroType MTBDR (FT) on CE, and rpoB cross-contamination risk associated with the extraction procedure are unknown. We tested MTBDRsl, MTBDRplus, and FT on CEs from chambers from cartridges (Ultra, Xpert) tested on bacilli dilution series. MTBDRsl on Ultra dCE on TB-positive sputa (n = 40) was also evaluated and, separately, rpoB amplicon cross-contamination risk . MTBDRsl on Ultra dCE from dilutions ≥103 CFU/ml (CTmin <25, >“low semi-quantitation”) detected fluoroquinolone (FQ) and second-line injectable (SLID) susceptibility and resistance correctly (some SLIDs-indeterminate). At the same threshold (at which ~85% of Ultra-positives in our setting would be eligible), 35/35 (100%) FQ and 34/35 (97%) SLID results from Ultra dCE were concordant with sputa results. Tests on other chambers were unfeasible. No tubes open during 20 batched extractions had FT-detected rpoB cross-contamination. False-positive Ultra rpoB results was observed when dCE dilutions ≤10−3 were re-tested. MTBDRsl on Ultra dCE is concordant with isolate results. rpoB amplicon cross-contamination is unlikely. These data mitigate additional specimen collection for second-line DST and cross-contamination concerns.
- ItemIdentification of distinct bio-signatures in whole blood and in-tube quantiferon pellets of Tuberculosis (TB) close contacts (CCs) and TB patients with and without type 2 diabetes (T2D)(Stellenbosch : Stellenbosch University, 2019-12) Tshivhula, Happy; Ronacher, Katharina; Kleynhans, Leanie; Stellenbosch University. Faculty of Medicine and Health Science. Dept. of Biomedical Sciences: Molecular Biology and Human GeneticsENGLISH ABSTRACT: Tuberculosis (TB) remains difficult to control despite effective treatment and the presence of more sensitive diagnostic tools. Immunity against Mycobacterium tuberculosis (Mtb) infection can further be impacted by other concomitant infections (like human immunodeficiency virus) and non-communicable diseases (like Type 2 diabetes (T2D)) rendering individuals susceptible to TB. T2D patients are three times more likely to progress from latent to active TB.Therefore, it is crucial to better understand why people with T2D are at increased risk for TB progression. We hypothesize that distinct bio-signatures exist in whole blood of TB patients and close contacts (CCs) of TB patients with latent TB infection (LTBI) with T2D compared to those without T2D and that these bio-signatures correlate with impaired immune responses to Mtb in peripheral blood mononuclear cells (PBMCs) and monocytes (MNs) in CCs. RNA was extracted from whole blood stimulated with Mtb antigens using the Quantiferon TB Gold in-tube assay and NanoString analysis was performed to determine whether specific transcription signatures exist between CCs with LTBI with and without T2D. We characterized serum cytokines and endocrine signatures by luminex and enzyme-linked immunosorbent assay. PBMCs and MNs from the same individuals were isolated and stimulated with live H37Rv Mtb to determine Mtb uptake and killing. Bacterial uptake and killing was correlated with HbA1c to determine whether there was an association with glycaemic control. RNA from TB patients, TB patients with transient hyperglycaemia (TB-THG) and TB patients with T2D (TB-T2D) was also extracted from whole blood stimulated with Mtb antigens and analysed using NanoString. Interleukin-22 was also measured in serum from these patients. PBMCs from TB patients were further used to determine the frequency of mucosal associated invariant T (MAIT) cells before, during and after TB treatment. Fifteen gene transcripts were downregulated in TB-T2D compared to TB and TB-THG patients. We identified a gene signature that was able to differentiate between TB-THG and TB-T2D. We further identified a gene signature unique to the CCs with LTBI and T2D, which could be associated with an increased risk of developing active TB. Differences in cytokines, hormones, lipids, differential blood counts and Mtb uptake was observed when comparing CCs with and without T2D. Differential expression of IL-6, IL-18 and IL-22 in CCs with and without T2D highlights the differential regulation of the immune response during T2D. We showed that the frequency of MAIT cells in the periphery of TB-T2D was significantly lower compared to TB-THG at baseline, suggesting the MAIT cells in TB-T2D have redistributed to either the lung or adipose tissue. The increase of MAIT cells in the periphery at month 2 compared to baseline could mean that MAIT cells population was restored in the blood due to TB treatment. Our gene expression results suggests that downregulated gene transcripts may be involved in pathways that favour immunopathology in TB-T2D. In addition, gene signatures differentiating THG from T2D could be useful in preventing the unnecessary diagnosis of patients with THG as having T2D. Differential gene expression in CCs with LTBI with and without T2D shows potential to be involved in the mechanisms leading to susceptibility to active TB. Cytokine and hormone data confirms that during T2D, the immune response is differentially regulated which may influence the response to infections. Lastly, MAIT cell frequency could be useful for monitoring treatment responses in TB-T2D patients.