Browsing by Author "Mbano, Ian Maheti."

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Light forge : a microfluidic high throughput platform for rapid and affordable detection of drug resistant strains of tuberculosis.
(2015) Mbano, Ian Maheti.; Balagaddé, Frederick.
Tuberculosis is one of the most deadly infectious diseases currently plaguing the global community. Unfortunately, lack of accessible, reliable and affordable diagnostic tools in the high disease burden, and resource poor regions such as Sub-Saharan Africa has hampered efforts to eradicate the epidemic. This study documents the development of a microfluidic platform called Light Forge, which is capable of detecting genetic drug resistance signatures in M.tuberculosis DNA. The first phase of this study involved a molecular drug susceptibility assay on 7 strains of M.tuberculosis using the high resolution melt analysis at the rpoB, katG, mab-inhA and gyrA loci with the Light Cycler96 . These findings compared with phenotypic drug susceptibility testing and Sanger sequencing. The results from the preliminary tests showed that the commercial system could detect positive strains at sensitivity estimates of 86%, 17% , 0% and 100% for rpoB, katG, mab-inhA and gyrA respectively. Detection of non-synonymous mutation in gyrA region for all test strains halted further testing. The rpoB gene was selected for on chip profiling with the Light Forge system due to the higher sensitivity. The results from the Light Forge showed that the system was capable of detecting test strains with 100% sensitivity, with modest reproducibility and correspondence with the phenotypic drug susceptibility profiles and the sequencing results. A microfluidic TB assay based on the Light Forge system is on the horizon based on the findings of the study. However, more work is required to incorporate other genes and ultimately design the best-equipped device for the clinical setting.
Single cell ribonucleic acid sequencing in Tuberculosis research.
(2021) Mbano, Ian Maheti.; Leslie, Alasdair.
Tuberculosis (TB) remains a global challenge, with approximately 1,5 million deaths annually. Addressing deficits in our understanding of disease pathology and treatment is needed for the development of new treatment modalities. Despite much effort, prevalence of this disease remains high in resource limited regions, where research capacity is not sufficient to successfully combat the endemic. Research in developed countries has generally been constrained to animal models due lack of access to clinical samples from the site of TB disease, the human lung. Although these animal models have their utility, it is essential that findings from these systems be tested and validated in human tissue. In this thesis, I leveraged a relatively new technology called Seq-well, which is highly portable and low-tech single cell ribonucleic acid sequencing (scRNAseq) platform and access to TB infected lung tissue obtained from lung resections, to generate a single cell atlas of TB affected lung tissue. This involved processing the human tissue immediately post-surgery and loading unprocessed/neat cells or FACS sorted cells (tissue resident t cells) onto a microarray that allowed capture and subsequent sequencing of the cell transcriptomes. In the first part of the thesis, I identified and profiled cellular subsets from TB infected tissue, focussing on a subset of FAP+PDPN + fibroblasts associated with the organisation of tertiary lymphoid organs. I also demonstrated that this dataset can be useful in evaluating current and future TB biomarkers, by superimposing signatures from the literature onto the cellular subsets and localizing them to different parenchymal, stromal and immune cell types. I also profiled tissue resident CD4 T cells from the same lung tissue, identifying canonical marker genes (ITGA1, PRF1) in one specific cluster, together with naive (CCR7, SELL), regulatory (RORA) and activated/myeloid-like T cells (LYZ, S100A9) in separate clusters. Finally, I demonstrated the applicability of this dataset in research involving other pulmonary diseases, by identifying ACE2+ TMPRSS2+ type 2 pneumocytes, a target of the SARS-CoV-2. Taken together, these findings provide new insights into the immunopathology of TB in the human lung together with the impact of HIV on specific immune subsets. It serves as a resource for cross validation of lung immune signatures generated in experimental infections of both mice and non-human primates, which is beneficial for scientists lacking access to the technology and/or tissue. Iqoqa Isifo sofuba (i-TB) silokhu siyinselelo emhlabeni jikelele, ngokufa okuhlobene naso okucishe kufike esigidini esi-1.5 njalo ngonyaka. Ukubhekana nokushoda ekuqondeni kwethu umumosakhiwo wesifo bese kuncishiswa ukufa. Ngaphandle kwemizamo emikhulu, ukudlanga kwalesi sifo kusalokhu kuphezulu ezifundeni ezintula imithombokusiza, lapho umthamokwenza wocwaningo unqindekile. Ucwaningo emazweni asethuthukile, ngakolunye uhlangothi, belwenzeka kuphela kumamodeli asebenzisa izilwane ngenxa yokuntuleka kokufinyelela amasampuleni okwelapha engxenyeni okuqubuke kuyo isifo sofuba, okuyiphaphu lomuntu. Nakuba kunamamodeli ezilwane anomsebenzi, kubalulekile ukuba okutholakele kulezo zinhlelo kuyohlolwa bese kuqinisekiswa ngesigqa somuntu ukuqinisekisa ubunjalo. Kule thesisi, ngiveze ubuchwepheshe obusha obungenayo obubizwa nge-Seq-well, iseli eyodwa e-low-tech ephathekayo ene-ribonucleic acid sequencing (scRNASeq) okuyindawo kanye nokufinyelela esicutshini sephaphu esitheleleke ngesifo sofuba esitholakale ekuhlukanisweni kabusha kwamaphaphu okukhonjwe ngokokwelapha, ukwakha iseli eyodwa yesicutshana sephaphu elitheleleke ngesifo sofuba. Lokhu kwafaka ukusebenzakuhlola isicubu somuntu ngokushesha emva kokuhlinza nokufaka amaseli ahlanzekile angasetshenziwe noma amaseli ahleliwe angama-FACS (ama-T cells asesicutshini) ohlelweni lolibofuzo olwavumela ukufaka ohlwini nokulandelanisa okulandelayo womumofuzo oqondene nezicubu. Engxenyeni yokuqala yethesisi, amaqoqwana ahlonziwe nafakwe kwiphrofayli esicubini esitheleleke ngesifo sofuba kugxilwe eqoqweni le FAP+PDPN + amafayibhroplasti ahlobene nokuhlelwa kwezingxenye zomzimba ezinkulu zamalimfoyidi kanye nemichilwana yamafayibhrodi kanye noma igranyuloma yesifo sofuba. Ngivezile ukuthi lamadathasethi angaba nomsebenzi omkhulu ekuhlaziyeni amabhayomakha amanje nawasesikhathini esizayo esifo sofuba, ngokufaka izinkombabunjalo emaqoqweni amancane nokuwabeka ezinhlotsheni ezehlukene zamaseli angamapharenikhayma nangamastroma. Ngiphinde ngachaza esizindeni sezicutshana ze-CD4 T esicutshini sephaphu elifanayo okuchaza ulibofuzo olukala amakhenoni (i-ITGA1, PRF1) eqoqweni elilodwa eliqondile, kanye namaseli angachazi lutho (CCR7, SELL), alawulayo (RORA) nama-T cell aqaliswe ukusebenza/efana ne-myeloid (LYZ, S100A9) emaqoqweni aseceleni. Okokugcina, ngiveze ukungena kwedathasethi ocwaningweni olufaka izifo zamaphaphu nokuphefumula ngokuhlonza i- ACE2+ TMPRSS2+ type 2 wama-pneumocytes, okuhlosiwe kwe-SARS-CoV- 2. Uma kuhlanganisiwe, lokhu okutholakele kuletha imibono emisha yomumobugciwane bokutheleleka ngesifo sofuba ephashini lomuntu, umthelela we-HIV kokutholakele emumwenikuphila kwephaphu ekuthelelekeni okuyilinga kwakho kokubili amagundane kanye nalokho okungebona abantu.