Direct sequencing of Leishmania donovani from patients in Garissa County, Northern Kenya, reveals a newly emerging intra-specific hybrid genotype

INTRODUCTION: Leishmaniasis is endemic in many countries, and in Kenya outbreaks of visceral leishmaniasis (VL) commonly occur in Garissa, Isiolo, Marsabit, Turkana, and Wajir. Despite the rising frequency of VL outbreaks, there is limited data on the genetic structure and epidemiology of Leishmania parasites from these regions. This study used molecular methods to characterize Leishmania parasites collected at Garissa County Referral Hospital during the 2019-2022 VL outbreak. METHODS: 286 blood samples, collected from patients suspected of having VL at Garissa County Referral Hospital between 2019 and 2022 were used. Leishmania parasites were screened at genus level by a quantitative real-time PCR assay targeting the arginine permease gene AAP3 (AAP3-qRT-PCR). Species identification and targeted gene sequencing were made on Illumina MiSeq using PCR amplicons of Hsp70 gene and ITS regions. Whole genome sequencing (WGS) was performed directly on eight selected blood samples using a target enrichment method after which data was analyzed using phylogenomic tools. RESULTS: By AAP3-qRT-PCR, 128/286 (45%) blood specimens were determined to have Leishmania parasites. We obtained 86 Hsp70 and 79 ITS sequences that phylogenetically clustered with the L. donovani species complex. By WGS, the eight selected samples had L. donovani s.s., and clustered in two separate groups: one similar to the previously reported L. donovani group 5 and the other constituted a new and intra-specific hybrid genetic variant not reported previously. In all the 8 Kenya samples, we found SNPs in genes previously shown to be involved in L. donovani resistance to Antimony, Amphotericin B and Miltefosine. CONCLUSION: This pilot study reveals the complex nature of Leishmania genetic structure in Kenya and sheds light on the genomic polymorphism of L. donovani in this region, which in turn, may explain the evolving threat of VL in the region. As caveat, the genomic signatures of drug resistance genes that were identified should be interpreted with caution until their functional implication is clarified in future phenotypic studies.