Background: Visceral Leishmaniasis (VL) is in elimination phase in India while cases of Cutaneous Leishmaniasis (CL) are spreading to new foci in different parts of the country. In Himachal Pradesh, a foci of CL have been reported along Satluj River, but the causative agent poses a dilemma. To ascertain the Leishmania species from CL cases from Shimla, Kullu and Kinnaur districts of Himachal Pradesh, the present study was undertaken.
Methods: A total of 28 CL patients registered in Department of Dermatology, Indira Gandhi Medical College (IGMC) and Hospital Shimla in 2018, were tested by rk39. Barring 16 cases undergoing treatment, 12 fresh cases were subjected to microscopic detection of Leishmania parasite, PCR and sequencing. Skin biopsies of 3-4 mm diameter were taken in culture medium and in formalin under anesthetic and sterile conditions from the border of the lesions. Imprints were prepared for the detection of Leishmania amastigotes. Biopsy samples were inoculated into different culture media (M199, RPMI 1640, and NNN) and were incubated at 22-24°C. Cultures were examined microscopically for the growth of promastigotes up to four weeks. Polymerase chain reaction (PCR) was performed to characterize leishmania parasite species.
Results: Of 28 patients, one patient was found positive for by rK39 dipstick test. One imprint was found positive for leishmania amastigotes. Twelve biopsy DNA samples were subjected to PCR for Leishmania kDNA, of which all the 12 were found positive ITS1 Leishmania specific set of primers while eight were found positive with JW11/12 lesihmania species specific set of primers. Identification of Leishmania species was confirmed by PCR-RFLP and sequencing method. Of 12 Leishmania positive samples, six were identified as L. donovani, three L. tropica, two L. major and one remained unidentified.
Conclusion: The detection of L. donovani from cutaneous leishmniasis patients is a significant finding leading towards existence of atypical leishmaniasis in Himachal Pradesh.