Cutaneous Leishmaniasis Caused by an Unknown Leishmania Strain, Arizona, USA

We investigated an autochthonous case of cutaneous leishmaniasis caused by a genetically different Leishmania sp. in a patient in Arizona, USA. This parasite was classified into the subgenus Leishmania on the basis of multilocus DNA sequence and phylogenetic analyses of the rRNA locus and 11 reference genes.

In January 2018, after a third lesion erupted, we performed skin biopsies. Histologic sections showed nonnecrotizing granulomas in the papillary dermis, and tiny, basophilic, spherical inclusions within histiocyte cytoplasm resembling amastigotes, suggestive of CL. The lesions showed a limited extent and spontaneous improvement; therefore, no specific treatment was prescribed. The patient was followed for almost 2 years, and the 3 skin lesions remained nodular without ulceration, which eventually resolved by December 2019. The patient remained otherwise asymptomatic.
We tested clinical specimens from the patient following the Centers for Disease Control and Prevention (CDC)-approved protocol for using residual specimens from human subjects (use of residual diagnostic specimens from humans for laboratory methods research protocol no. 6756). We used lesion biopsy specimens submitted to CDC for DNA extraction, touch-prep smears, and in vitro culture in Roswell Park Memorial Institute medium (GIBCO-Thermo-Fisher, https://www.thermofisher.com) containing 15% fetal bovine serum at 25°C (11). Microscopic analysis of touch-prep smears identified a large number of amastigotes, whereas promastigotes with cellular shape and architecture compatible with species in the subgenus Leishmania were observed from culture ( Figure 1).
The resulting sequence (380 bp) (GenBank accession no. MT764332) had low similarity with L. , and species in the subgenus Viannia (<80.0%). We also tested DNA samples for amplicon melting temperature by using a SYBR green real-time, quantitative PCR protocol, which enables presumptive discrimination of Leishmania species (12). This analysis showed a melting temperature of 79.5°C, indicating L. (L.) infantum infection. On the basis of PCR analysis, the case-patient was identified as being infected with a Leishmania spp., without providing species-level identification.
We used DNA extracted from cultured parasites by using the MagAttract HMW DNA Kit (QIAGEN) to prepare genomic libraries by using the NEBNext Ultra II DNA Library Prep (New England Biolabs, https://www.neb.com) and subjected them to whole-genome sequencing by using the MiSeq platform (Illumina, https://www.illumina.com). MiSeq sequencing resulted in 22,808,630 Leishmania reads that had >100× coverage, 3,464 contigs of 29,491,421 bp, and a GC content of 59.68%.

Conclusions
Leishmania species associated with human clinical cases are typically prevalent in tropical and subtropical foci and classified into 2 subgenera: Viannia and Leishmania. Nonetheless, environmental changes might contribute to expansion of natural vectors, reservoirs, and emergence of novel Leishmania strains and leishmaniasis in nonendemic areas, posing a new and serious challenge to public health (5,6).
We report an autochthonous case of CL caused by a previously undescribed Leishmania parasite in a patient in Arizona. The integrated interpretation of the clinical information, travel history, parasite morphology, CDC species-specific diagnostic test results, and MLSA/phylogenetic analyses suggest that the isolate from Arizona could be a new strain or species within the subgenus Leishmania. This isolate is also genetically distinct at the internal transcribed spacer 2 locus from reported isolates for 18 previous cases of leishmaniasis from Arizona, characterized by CDC over the past 10 years, which were detected in travelers returning from disease-endemic areas. Despite these findings, we realize that classification of this parasite cannot be conclusively determined based solely on genetic evidence observed in this study. Therefore, further investigations (including multilocus enzyme electrophoresis and wholegenome sequencing with next-generation sequencing long read fragments) will be needed to confirm whether the isolate from Arizona is a new species or a new strain in the subgenus Leishmania.
Historically, human leishmaniasis in the United States has been considered an exotic, travel-acquired infection. However, this concept must be reexamined because of the expansion of sylvatic animal reservoirs and natural sand fly vectors of Leishmania spp. and reports of human and animal autochthonous cases in several states (7)(8)(9)(13)(14)(15). Considering the patient's travel history, the increased reports of zoonotic cases, and the active presence of sand fly vectors/reservoirs in southern areas of the United States, we concluded that the CL reported was probably caused by local parasite transmission. Because there is increasing evidence of likely local transmission, leishmaniasis could be emerging in the southwestern United States.