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.

Author summary
Visceral leishmaniasis (VL), or kala-azar, is a deadly disease if untreated. In Kenya, outbreaks of the disease commonly occur in Garissa, Isiolo, Marsabit, Turkana, and Wajir. During the 2019–2022 outbreak in Garissa, we collected samples from patients reporting at Garissa County Referral Hospital to study the genetic structure of the parasites’ population. To avoid biases from lab-grown parasites, we used advanced genetic methods on parasites directly obtained from patients’ blood. We identified two parasite groups: one related to a previously known group 5 (Ldon5) from Kenya and Ethiopia, and a new group with mixed ancestry from Sudan, Ethiopia (Ldon3), and Iraq (Ldon4). Notably, we found mutations that might be linked to drug resistance. These findings are crucial because they help in understanding how the parasites evolve and spread. As caveat, the presence of drug resistance genes should be interpreted with caution until their functional implication are clarified in future studies.