Sputum microRNA profiles of treatment-naïve and patients on antituberculosis therapy in Mulago National Referral Hospital
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Background: Worldwide, tuberculosis (TB) is the leading cause of death due to a single infectious agent. This is attributed to the limitations of the current diagnostic tests in terms of sensitivity and specificity. Studies have demonstrated that microRNAs (miRNA) could be better diagnostic markers based on their stability and ability to survive extremes in pH and high temperatures. miRNAs regulate gene expression by interfering with mRNA transcripts. Rationale: The miRNA expression profiles differ during different pathological conditions and are largely influenced by the environment and genetics. Owing to this, population-specific studies need to be conducted. With the limited research regarding the miRNA profiles of TB patients in Uganda, there is need for further exploration in this area. Objectives: (1) To determine the specific MTBC pathogen lineages in TB patients attending Mulago hospital. (2) To determine the expression profiles of the selected miRNAs (hsa-miR-3179, miR-155, hsa-miR-let7g, hsa-miR29a, miR-223, miR-144, and miR-48) in TB patients at baseline, and at 2 and 5 months of anti-TB therapy (3) To determine the association between the patient MTBC pathogen lineages and the expression profile of the selected miRNAs. Methods: This study was nested in the Mixed tuberculosis infections study that recruited new patients confirmed to have pulmonary TB disease and followed them through treatment. We utilized 72 processed archived sputum samples obtained from patients at baseline, months 2 and 5 of TB therapy. Mycobacterium DNA was extracted using the modified Cetyl trimethylammonium bromide (CTAB) method and the MTBC lineages were determined by using lineage-specific primers and Taqman probes. Total RNA was isolated using the conventional Trizol method and cDNA synthesis was achieved by using the microRNA cDNA synthesis kit. The expression profiles of miRNAs: hsa-miR-3179, miR155, miR-223, miR-144, hsa-miR-let7g, miR-484 and hsa-miR29a were determined by using the TaqMan® Advanced miRNA Assay Kit. Results: This study demonstrated a prevalence of: 36 (50%) for the Lineage 4UG genotype, 7(9.72%) of the Lineage 4UGI genotype, 29 (40.28%) of the Lineage 4UGII genotype, 24(33.33%) of the Lineage 3 genotype, 2(2.78% ) of the Latin American Mediterranean (LAM) genotype and 1(1.39%) of the Beijing genotypes. The study also demonstrated a prevalence of 9% for the mixed strain infections. Our study is the first to perform Real-time PCR quantification assays for the quantification of miRNAs in Uganda. However, when it came to identification of miRNAs, we did not succeed in detecting them probably due to issues related to unsuccessful polyadenylation of the miRNAs. Conclusion: We determined the MTBC lineages present in almost all the samples. Although we did not detect the miRNAs, we demonstrated that the miRNA polyadenylation step is difficult to achieve when total RNA is used as the starting template. For subsequent assays, we have highlighted the challenges experienced when optimising a miRNA real-time PCR assay.