Cutaneous Leishmaniasis and/or Dermal Leishmanoid in Himachal Pradesh (India)


 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.


Introduction
The Leishmaniases are a group of diseases caused by the protozoan parasite Leishmania. There are three main types of leishmaniasis: i) Visceral, often known as kala-azar and the most serious form of the disease (VL); ii) Cutaneous, the most common (CL); and iii) Mucocutaneous [1].
The historical focus of CL in India was found in Bikaner, (Rajasthan) caused by L tropica [2] and transmitted by Phlebotomus salehi, P papatasi and P major (WHO, 2010). Subalpine valley along the Satluj River is known as an endemic focus of CL in Himachal Pradesh. During 1988 to 2001, a total of 38 cases of CL were reported from the Satluj valley of Himachal Pradesh and 161 CL cases were also reported between 2001-2003 where L. tropica and L. donovani were identi ed as causative parasite and Phlebotomus longiductus as the transmitting vector for CL in Himachal Pradesh. One patients was found positive with rK39 dipstick test [3, 4, 5, 6,]. Recently in 2016-2017, 337 cases of CL were registered, clinically diagnosed and treated at Mahatma Gandhi Medical Service Complex Khaneri Rampur. However, the causative parasite was not identi ed [8,9].
The present study was carried out to identify and characterize the parasite species causing CL in Himachal Pradesh.

Material Methodology
Epidemiological data and study area The present study was undertaken in Rampur (Shimla), Nirmand (Kullu) and Nichar (Kinnaur) district of Himachal Pradesh along the Sutlej river valley which stretched from 31°05′0″N and 31°42′0″N latitude and 76°52′E and 78°24′0″E latitude. As per the data recorded at IGMC Shimla from July -September 2018, CL cases were con ned in the villages along Sutlej River in Rampur (Shimla), Nirmand (Kullu) district and Nichar (Kinnaur) district. Distribution of CL cases shown in Figure 1. biopsy sample was divided into two, for smear and culture. Imprints were prepared from one portion for the detection of Leishmania amastigotes and after preparation of imprints that portion was preserved in formalin for PCR analysis, while the other portion was inoculated into culture medium (M199, RPMI supplemented with 25 mmol/L HEPES (pH 7.5) and 10% fetal bovine serum (FCS) and in NNN medium followed by incubation at 22-24°C). Cultures were examined microscopically for the growth of promastigotes up to four weeks. All the 28 patients clinically diagnosed for CL were also screened by rK39 dipstick test. The dipstick tests were performed according to the manufacturers (Inbios international, inc.seatle, wa98104) instruction.

DNA Extraction
Total DNA was extracted from the biopsy samples collected and preserved in formalin buffer using the The polymerase chain reaction was carried out on DNA isolates from punch biopsy samples .The Leishmania speci c JW11/12 primer set ampli es a 120bp fragment of KDNA of genus Leishmania [10] and the LITSR/L5.8S set ampli es a 320 bp fragment of ITS1 region of Leishmania genus-speci c [11,12] . Primers sequences and relevant PCR conditions given in Table 1. A volume of 2μl of extracted DNA from patients and L. donovani, L. tropica and L. major positive control were ampli ed for each forward and reverse primer in the presence of 2 X Kappa master mix in a nal volume of 20 μl in Bio Rad PCR Thermocycler. The ampli ed PCR products were electrophoresed on 1.

Clinical features of patients
The age of CL patients ranged from 1-72 years of which 10 were males and 18 females. Mostly females and students were affected. Duration of the lesions ranged from three weeks to two years. In most of the cases, lesions were mainly found on face. The lesions were of mucocutaneous type involving upper and lower lip and angle of mouth in three cases. The number of lesions per patient ranged from one to two and in majority of the cases single lesion was found. Size of the lesions ranged from 0.5 × 1 cm to 5 × 5 cm, the lesions were mostly erythematous, papule, and nodule-ulcerative plaques with or without crusting. None of the patients exhibited typical symptoms of VL.
Con rmation of diagnosis by smear, rK39 and culture Of 12 patients from which imprint smear was prepared, only one was found positive for Leishmania amastigotes. None of the skin biopsy samples (12) was found positive for promastigotes. Only one out of 28 patients, screened using rK39 rapid diagnostic kit, showed positivity (Fig. 2).

Analysis Of Biopsy Specimens By Pcr -r p
Of 12 biopsy samples, eight were found positive for Leishmania with JW11/12 set of primers (Table 2

Treatment Of Cl Cases
All the CL patients were treated at IGMC Shimla with Sodium stibogluconate (SSG) injected intra-lesional three injections in a month and repeated until healing was complete. Complete resolution of lesions after 3-6 months was seen in most of the patients. Intralesional SSG consistently effective without any major side effects except painful injections.

Discussion
Leishmaniasis is thought to be a disease of low altitude, with no occurrence of cases above 2000 feet (600 m). However, number of cases has been reported from subalpine valley of Himachal Pradesh. CL was not known to occur in Himachal Pradesh before 1988. L. donovani and L. tropica has been reported from cases of CL in Himachal Pradesh. In Kerala also, L. donovani identi ed from cutaneous leishmanisis cases [13]. LITSR/L5.8S set of primers were found better than JW11/12 as the former would detect leishmania in all the 12 samples. PCR-RFLP and sequencing methods were found equally good in species identi cation of Leishmania. In the present study we also identi ed L. donovani as a causative parasite from 50% of CL cases. We also found L major (16.6%) adding three species of Leishmania as causative agent of CL in Himachal Pradesh. Other countries like Kenya, Iraq, Sri Lanka, Yemen [ 14,15,16,17] have also reported L. donovani as causative parasite for CL. Looking into the information available, it evident that the CL reported from Himachal Pradesh and Kerala is not a typical CL rather it should be termed as atypical leishmaniasis. It is not known whether the encountered cases of CL in the present study area of CL, recovered VL and the parasite hiding in Dermis, or coinfection of CL and VL. Is it VL similar to China having L. donovani intriguing to note what is compelling the L. donovani not to enter viscera beyond dermis is still not clear warranting further study.
The culture did not grow due to high rate of contamination by bacteria or variable nutritional requirements of the strains as reported by Sharma at al. [18]. It is also well known that some members of the genus may be di cult to culture [ 19] and inoculation in hamsters may be required to maintain strains in the laboratory for zymodeme or genetic studies in such situations [20,21]. RFLP method was also used for detection of leishmania species, however the DNA bands in gel for some samples were faint so species identi cation was also con rmed by Sanger DNA sequencing method.

Conclusions
Detection of three species of Leishmanaia from the patients of CL from Himachal Pradesh is a matter of concern as to whether the so called CL cases are 'atypical leishmaniasis' in Himachal Pradesh. Being close to China, it is also likely that the cases of so called CL in the state of Himachal Pradesh present are Dermal Leishmaniod ( Brahmachari ,1922)  Email ID: r.c.dhiman@gmail.com