During 2014–2018, an increase in CL cases occurred in Himachal Pradesh; case reports came from different tehsils (i.e., townships) in Kinnaur, Shimla, and Kullu and the previously nonendemic districts of Mandi and Solan (Appendix Table 1, Figure 1). We confirmed 60 CL cases indigenous to the state with detailed patient information, demonstration of the presence of Leishman-Donovan bodies and CL-specific histopathologic changes in skin lesional specimens, and PCR detection of parasitic infection (Appendix).

We conducted PCR and restriction fragment-length polymorphism (RFLP) analysis of parasite species–specific internal transcribed spacer 1 (ITS1) sequences by using appropriate standard controls. We detected the expected ≈320-bp product with a HaeIII RFLP pattern specific to L. donovani complex in all patient biopsy specimens, indicating L. donovani, L. infantum, or both as the causative agent of infection (Appendix Figure 4) (10).

Figure 1

Figure 1. ITS1-based molecular analysis of clinical isolates from cutaneous leishmaniasis (CL) patients, Himachal Pradesh, India, 2014–2018. A) Multiple sequence alignment of ITS1 microsatellite repeat sequences of representative parasite isolates from CL patients...

BLAST analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi) of 44 ITS1 test sequences showed all the samples to be closest to L. donovani, having maximum identity to L. donovani isolates from Bhutan (GenBank accession nos. JQ730001–2) and possibly L. infantum. None of the CL cases were consistent with L. tropica infection, unlike in a previous report (8). To distinguish whether HP isolates were L. donovani, L. infantum, or both and to infer genetic and geographic relatedness between these isolates and standard reference strains, we performed ITS1 microsatellite repeat analysis and phylogenetic classification (11–13). The 4 ITS1 polymorphic microsatellite repeat analysis indicate HP isolates different from L. infantum and closest to the L. donovani isolates from Bhutan (Table 1; Figure 1, panel A). We detected a polymorphism in the third poly (TA) microsatellite locus with 5 repeats and an atypical insert of TAA and the fourth poly (A) microsatellite tract with 8 repeats; these polymorphisms were identical to the VL-causing L. donovani isolates from Bhutan. An L. donovani Chandigarh isolate originally from HP is reported to be closest to the Bhutan isolates and matched with HP isolates at the third poly (TA) stretch (12). However, Himachal Pradesh isolates were distinct at the first poly C and the second poly A microsatellite tracts and had heterogeneous base sequences. Thus, these isolates represent L. donovani genetic variants; none showed the ITS1 sequence type previously assigned to the referred L. donovani isolates by Kuhls et al. (13). Our phylogenetic analysis of 44 ITS1 test sequences and ITS1 reference sequences placed all the CL-causing L. donovani isolates from Himachal Pradesh into a discrete cluster different from the VL-causing L. donovani from India and elsewhere and the CL-causing L. donovani isolates from Sri Lanka. The Himachal Pradesh CL isolates within the cluster exhibited considerable heterogeneity (Table 1; Figure 1, panel B; Appendix Table 4).

Figure 2

Figure 2. 6PGDH-based molecular analysis of clinical isolates from cutaneous leishmaniasis (CL) patients, Himachal Pradesh, India, 2014–2018. A) Sequence alignment of partial 6PGDH amino acid of CL isolates exhibit replacement of asparagine (N)...

Sequences of the 6-phosphogluconate dehydrogenase gene (6PGDH) exhibit a high degree of polymorphism and have been used to identify Leishmania species and differentiate region-specific zymodemes (14). We performed multiple sequence alignment of the representative partial 6PGDH amino acid sequences from Himachal Pradesh isolates by using the homologous 6PGDH protein sequences of the reference Leishmania isolates to determine their genetic and geographic relatedness (Table 2; Figure 2, panel A; Appendix Table 4, Figure 5). Himachal Pradesh isolates exhibited a 6PGDH sequence specific to Mon-37 and different from Mon-2 (having aspartic acid in place of asparagine) at position 326 (Figure 2, panel A). Thus, CL-causing L. donovani from Himachal Pradesh were distinct from the most common VL-causing India Mon-2 L. donovani and the Bangladesh L. donovani isolate, whereas they were similar to the CL-causing L. donovani isolate from Kerala and CL- and VL-causing Mon-37 isolates from Sri Lanka and the isolates from Kenya, Brazil, and China.

Phylogenetic analysis of 6PGDH amino acid sequences of CL isolates grouped them into a heterogeneous cluster; variants were closer to a viserotropic L. donovani isolate from Sri Lanka and distinct from the VL-causing L. donovani isolates from India and Bangladesh and CL-causing isolates from Kerala and Sri Lanka (Figure 2, panel B). However, the HPCL55 isolate (GenBank accession no. MH208450) grouped differently. The HPCL49 isolate (GenBank accession no. MH208446) showed relatedness to the standard L. infantum strain, although ITS1 analysis using BLAST and microsatellite repeat sequences showed regions of similarity with L. donovani. ITS1 and 6PGDH sequence analysis suggest that Himachal Pradesh isolates from CL patients consist of heterogenous L. donovani variants and possibly represent hybrid genotypes.

None of the CL patients had VL-specific symptoms or VL history. Ten of 43 patient blood samples tested positive for rK39 antibody, and 37 of 51 samples were positive for the circulating parasite DNA with L. donovani–specific ITS1 (Appendix Figure 6, panel A, B). The result suggests asymptomatic systemic L. donovani infection in a fraction of CL patients.

The presence of leishmaniasis in Himachal Pradesh is not yet well known in India and globally (15). Our epidemiologic study shows newer CL pockets during 2014–2018; thus, the state needs to be recognized as leishmaniasis-endemic by public health authorities (Appendix Figure 1). We conclude that CL cases in Himachal Pradesh are caused by L. donovani variants distinct from the viscerotropic L. donovani strain from northeast India. The CL isolates in Himachal Pradesh exhibit considerable heterogeneity and indicate the possible existence of genetic hybrids. The scenario appears somewhat similar to Sri Lanka and Kerala, where L. donovani parasites cause cutaneous disease, albeit with differences in the region-specific L. donovani variants. In lieu of the coexistence of VL and CL in Himachal Pradesh, parasite isolates from VL patients also need to be characterized. To understand the biology of atypical L. donovani variants with cutaneous manifestations and to genetically differentiate the dermotropic versus viscerotropic potential of L. donovani variants, comparison of CL- and VL-causing isolates in Himachal Pradesh using whole-genome sequence analysis is required.

L. donovani parasites in the blood of some CL patients represent human reservoirs similar to asymptomatic VL carriers, and the parasite variants have the potential to cause full-blown VL manifestations. An elaborate surveillance program dedicated to the Himachal Pradesh region is urgently required for better diagnosis, treatment, prediction of parasite variants in different afflicted pockets, and prevention of transmission of the disease to other regions.