Trypanosome genetic diversity and differential gene expression profiles associated with Human African Trypanosomiasis (HAT) clinical phenotypes in Malawi

Abstract

Background: Clinical pathology of Rhodesian Human African trypanosomiasis (r-HAT) in sleeping sickness in endemic countries varies depending on disease foci. To gain more insight of r-HAT disease in Malawi, the current epidemiological trend of r-HAT in Malawi as well as gene expression profiles of sleeping sickness patients and T. b. rhodesiense isolates from Rumphi and Nkhotakota foci were analysed. Methodology: Data on r-HAT prevalence and demographics from epidemiological surveys carried out in Malawi from 2012 to 2020 was obtained from the ministry of health. In addition, blood samples and clinical profiles of HAT patients surveyed between 2016 to 2020 from Rumphi and Nkhotakota districts were analysed. Furthermore, gene expression profiles in blood from 23 individuals with early stage 1 and late stage 2 sleeping sickness versus 21 healthy controls as well as T. b. rhodesiense transcriptome profiles of 8 and 16 endemic isolates from Rumphi and Nkhotakota foci respectively, including one that was nonresponsive to melarsoprol treatment, were comparatively analyzed. Lastly, the genotypes of APOL-1 gene of r-HAT patients as well as the SRA gene from T. b. Rhodesiense isolates were determined. Results: A surge of r-HAT incidence occurred in Malawi from 2019 to 2020 with almost 50% of the total r-HAT presented in this study reported within 2 years of the outbreak. The r-HAT clinical phenotypes in Malawi were demonstrated to be foci dependent; cases in Nkhotakota had more of a less severe clinical phenotype, compared to Rumphi who were characterised by severe clinical phenotype. There were differences in gene expression profiles in individuals with stage 1 and stage 2 disease regardless of the focus of origin. Additionally, innate immune response transcripts were elevated in individuals with stage 1 sleeping sickness. There were indications that immune response through neutrophil activation may have a central role in human response against peripheral blood T. b. rhodesiense infections in Malawi. It was also shown that wasting in individuals with stage 2 sleeping sickness maybe due to altered lipid metabolic processes. In addition, transcripts such as ZNF354C, TCN1 and MAGI3 that in combination with other markers maybe exploited in future research for staging of sleeping sickness without the need of the invasive lumber puncture were identified. T. b. rhodesiense isolates from Nkhotakota were enriched with transcripts for cell cycle arrest and stumpy form markers, whereas isolates in Rumphi focus were enriched with transcripts for folate biosynthesis and antigenic variation pathways. The parasite focus-specific transcriptome profiles are consistent with the more virulent disease observed in Rumphi and a more silent disease in Nkhotakota associated with the non-dividing stumpy forms. PCA analysis using SNPs called from the RNA-seq data showed that T. b. rhodesiense parasites from Nkhotakota are genetically distinct from those collected in Rumphi. Interestingly, the Malawi T. b. rhodesiense isolates expressed genes enriched for reduced cell proliferation compared to the Uganda T. b. rhodesiense isolates. Transcriptome profiles of a T. b. rhodesiense isolate from a patient with recurrent r-HAT were enriched with cellular response to oxidative and chemical stress response. The isolate was also genetically different from other isolates in Nkhotakota and Rumphi foci. Lastly, genetic diversity of T. b. rhodesiense SRA gene in Malawi isolates that affected SRA protein folding on the APOL-1 binding domain was demonstrated. Interestingly, individuals with APOL-1 G2 variant had severe r-HAT disease, contrary to the current consensus that the variant may protect against T. b. rhodesiense infection. Conclusion: This thesis has added insights to the current understanding on how clinical phenotypes of r-HAT in Malawi might be associated with differences in population structure of T. b. rhodesiense circulating in Rumphi and Nkhotakota districts, as well as host and parasite gene expression profiles. A surge of r-HAT and continuing incidences of the disease in Malawi should call for a review of Malawi’s r-HAT control and elimination strategies. A one health approach with inclusion of key stakeholders such as department of parks and wildlife may also be considered in the control of Malawi’s HAT endemic foci as they characteristically occur adjacent to the national parks.