Leishmania donovani Infection with Atypical Cutaneous Manifestations, Himachal Pradesh, India, 2014–2018

We conducted a molecular study of parasite sequences from a cohort of cutaneous leishmaniasis patients in Himachal Pradesh, India. Results revealed atypical cutaneous disease caused by Leishmania donovani parasites. L. donovani variants causing cutaneous manifestations in this region are different from those causing visceral leishmaniasis in northeastern India.

A part of the lesional biopsy from the CL cases was processed for examining CL specific histopathological changes. The samples were processed in 10% NBF, embedded in paraffin and processed to 4 to 5 µm thick tissue sections. Tissue sections were stained with H&E and examined for epidermal and dermal histopathological changes specific to cutaneous lesions (2).

Serum Isolation and rK39 Strip Assay
Blood samples collected from 50/60 patients were used to isolate sera. The rK39 immunochromatographic rapid diagnostic test was performed to determine the seroprevalence of rK39 antibody as per manufacturer instructions (InBios International, Inc. Seattle, WA 98104).
Briefly, 20 µl of serum was loaded on to the strip followed with addition of chase buffer solution. Samples were read after 10 minutes and considered positive for the presence of anti-K39 IgG with two distinct red lines corresponding to the, test region and the control region.

Molecular Analysis
Skin lesion specimens from 57/60 patients and laboratory-grown L. donovani and L. major cultures (used as controls) were used to isolate Genomic DNA (gDNA) as described previously (3). gDNA from patients' blood samples was also isolated to analyze presence of circulating parasite. gDNA from the test and control samples was used to perform speciesspecific ribosomal internal transcribed spacer 1 region (ITS1) PCR-RFLP assay and 6phosphogluconate dehydrogenase (6PGDH) gene amplification (Appendix Figure 4, 5). Both ITS1 and 6PGDH amplification products were sequenced for species identification and to decipher genetic relatedness of the HP isolates with other region-specific VL and CL causing Leishmania isolates (4-8).

ITS1 PCR-RFLP
Parasite specific ITS1PCR amplification was done for all the samples with primers LITSR (5′-CTGGATCATTTTCCGATG-3′) and L5.8S (5′-TGATACCACTTATCGCACTT-3′) as described previously (3,4). Briefly 50-100 ng of gDNA was used as template and amplified with10 pmol of each primer using Go Taq Green Master mix, 1X (Promega, Cat # M7122) in a reaction volume of 25 µl. Reaction conditions comprised an initial denaturation at 95°C for 2 min, 34 cycles of denaturation at 95°C for 20 sec, annealing at 53°C for 30 sec and extension at 72°C for 1 min with the final extension at 72°C for 6 min. The PCR product of ~ 320 bp size was subjected to HaeIII RFLP with overnight HaeIII digestion at 37°C and run on 2.5% agarose gel to identify the Leishmania species.

6PGDH PCR
6PGDH amplification was done with primers 6PGDH-F: AATCGAGCAGCTCAAGGAAG and 6PGDH-R: GAGCTTGGCGAGAATCTGAC as described previously (7,8). 50-100 ng of gDNA was amplified with 10 pmol of each primer using Go Taq Green Master mix, 1X (Promega, Cat # M7122) in a reaction volume of 25 µl. The reaction conditions comprised denaturation at 95°C for 5 min, 30 cycles of denaturation at 95°C for 1 min, annealing at 56°C for 30 secs and extension at 72°C for 1 min with the final extension at 72°C for 10 min. The PCR product was run on 1.8% agarose gel.

Sequencing and Phylogenetic Analysis
DNA sequencing of ITS1 and 6PGDH PCR products was done for identification of  Table 4).
The homologous gene sequences for ITS1 and 6PGDH of standard WHO Leishmania species specific isolates and region specific L. donovani isolates were retrieved from the NCBI-GenBank database. ITS1 nt query sequences from 44 specimens were analyzed using BLAST and multiple alignment software, MUSCLE using default parameters (9). The final alignment was made using BioEdit sequence alignment editor (version 7.0.5.3). The maximum likelihood tree from the aligned sequences was obtained with 5000 bootstraps with default parameters using the dnaml program of the phylip package (10). The final tree was plotted using FigTree software (version 1.4.3). To analyze 6PGDH protein sequences, gene sequences from 28 CL samples were translated into the corresponding homologous protein sequences using translate tool at ExPASy.
The representative protein sequences of the seven clusters obtained from the 28 test 6PGDH protein sequences were analyzed in relation to the homologous 6PGDH protein sequences of the reference Leishmania strains using the methods explained previously. The partial 6PGDH sequence alignment was made using Jalview multiple alignment editor version 2.10.4b1 (11).
The maximum likelihood tree was obtained with 5000 bootstraps using the proml program of the phylip package with default parameters (10).

Results Section Disease Epidemiology
During the period from 2014-2018, an increase in CL cases in the routine skin OPD was recorded in IGMC, Shimla and MGMSC, Khaneri, Rampur. A detailed record of patients suspected with CL was taken at the time of diagnosis (Appendix Table 2). In our study on 60 CL patients, there was an almost equivalent frequency of females (51.6%) and males (48.3%) ranging in age from 4 years to 70 years. The majority of the patients belonged to the indigenous students and the farming community. All the patients had localized cutaneous skin lesions predominantly on exposed body parts with involvement of the face in majority of the cases. The time gap between the appearance of lesions and disease diagnostics on the day of sample collection, ranged from 10 days to 2.6 years with most cases reporting within 3-4 months of disease occurrence, indicating a lack of awareness among local population. Most of the patients had one or two raised and itchy lesions presented as plaques, nodules and/or papules, often ulcerated unlike those present in post kala azar dermal leishmaniasis. Around 26% of cases had mucocutaneous like lesions extending to the inner nose consistent with previous reports (12, 13; Appendix Table 2).

Clinical Diagnostics
Clinical confirmation of the CL cases was performed by microscopic examination of Giemsa stained lesion touch smears, H & E stained biopsy sections for the presence of LD bodies and by parasite-specific ITS1 and 6PGDH PCR analysis (Appendix Figure 2, panels A-C; Appendix Table 2). Giemsa stained tissue smears were LD positive for 50% (23/46) of the samples processed with non-availability of the smears for 14 patients. 38% (19/50) H&E stained biopsy sections were positive for LD bodies. Only 11 patients demonstrated LD positivity in both the tissue smears and the histologic sections. Patients negative for LD bodies were, however, positive for the infection based on the PCR based parasite detection. Also, the CL lesion-specific histopathological analysis of biopsy sections performed for 50 patients exhibited characteristic dermal and epidermal changes to variable extents (Appendix Figure 3, panels A-F; Appendix Table 3). The characteristic CL lesion-specific epidermal changes with acanthosis and papillomatosis along with varying degree of keratosis accompanied granulomatous inflammation as accessed by a trained pathologist. These findings confirmed that all the 60 CL cases were positive for Leishmania infection and displayed varying degrees of CL specific lesional pathologies.  HPCL50  MG982979  MH208447  HPCL51  MG982980  HPCL52  MG982981  MH208448  HPCL54  MG982982  MH208449  HPCL55  MG982983  MH208450  HPCL57  MG982984 *ITS1, internal transcribed spacer 1. Disease endemicity in state districts is indicated by different colors: yucca yellow shading for previously reported endemic districts and tzavorite green shading for newly emerging endemic districts. Regions with blue and red dots indicate previously reported and newly emerging endemic Blocks/Tehsils in the endemic districts of the state, respectively. The map was created using ArcGIS 10.3 software. The map

Appendix
showing regional distribution of disease was geo-referenced with UTM projection taking WGS84 datum.
The unit of measurement for the scale bar is Kilometers.