Direct sequencing ofLeishmania donovanifrom patients in Garissa County, Northern Kenya, reveals a newly emerging intra-specific hybrid genotype

Introduction Leishmaniasis is endemic in many countries, including Kenya. Despite the rising frequency of visceral leishmaniasis (VL) outbreaks in Garissa County, Northeastern Kenya, there is limited data on the genetic structure and epidemiology of Leishmania parasites from this region. This study used molecular methods to characterize Leishmania parasites collected at Garissa County Referral Hospital during the 2019 - 2022 VL outbreak.

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.

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, Amphotericine B and Miltefosine.

This study reveals the complex nature of Leishmania genetic structure in Kenya and sheds light on the genomic polymorphism of L. donovani in the 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 studies. Non-technical Summary Visceral leishmaniasis (VL), or kala-azar, is a deadly disease if untreated. In Kenya, it is common in Garissa, Isiolo, Marsabit, Turkana, and Wajir. During the 2019-2022 outbreak, we collected samples from patients at Garissa County Referral Hospital to study the genetic structure of the parasites. To avoid biases from lab-grown parasites, we used advanced genetic methods on parasites directly from patients’ blood. We identified two parasite groups: one related to a known group 5 (Ld5) from Kenya and Ethiopia, and a new group with mixed ancestry from Sudan, Ethiopia (Ld3), and Iraq (Ld4). Notably, we found genes 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. Competing Interest Statement The authors have declared no competing interest. Funding Statement Yes Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This study was conducted as part of a public health response following a request from the Ministry of Health, Garissa County to support a Leishmania outbreak investigation. The WRAIR Human Subject Protection Branch determined that the outbreak response did not meet the definition of research and does not require IRB review. WGS of eight samples using Leishmania target enrichment (SureSelect Sequencing, SuSL-seq) was approved by the IRB of the Institute of Tropical Medicine, Antwerp (code 45/2024). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data availability All sequencing data are available at http://www.ncbi.nlm.nih.gov/bioproject/1251555 (PRJNA1251555) for whole genome sequences. Hsp70 and ITS loci are available under GenBank accession numbers PQ700286–PQ700371 and PV557379–PV557457, respectively. All other data are available in the main text or supplementary material.