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

doi:10.21203/rs.3.rs-50049/v1

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

https://doi.org/10.21203/rs.3.rs-50049/v1

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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.

Infectious Diseases

Leishmaniasis

Cutaneous Leishmaniasis

Himachal Pradesh

India

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 identified 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,].

A latest review of atypical leishmaniasis with emphasis on India has mapped cases of atypical leishmaniasis several parts of India like Punjab, Assam, Haryana, Delhi, Jammu & Kashmir, Kerala, and Varanasi [7].

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 identified [8, 9].

The present study was carried out to identify and characterize the parasite species causing CL in Himachal Pradesh.

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 confined in the villages along Sutlej River in Rampur (Shimla), Nirmand (Kullu) district and Nichar (Kinnaur) district. Distribution of CL cases shown in Figure 1.

Table 1: Thermocycler Polymerase chain reaction conditions.

PCR

Primer sequence

Amplification programme

KDNA genus Leishmania

specific PCR

(Rodgers et al. 1990)

JW11 (forward):

5’-CCTATTTTACACCAACCCCCAGT-3’

JW12 (reverse):

5’-GGGTAGGGGCGTTCTGCGAAA-3’

Initial denaturation at 94°C for 1 min followed by 34 cycles of denaturation at 94°C for 30s,annealing at 58°C for 30 s, and extension at 72°C for 30 s

Internal transcribed spacer 1

PCR genus Leishmania specific

(El Tai et al. 2000)

LITSR (forward):

5’-CTGGATCATTTTCCGATG-3’

L 5.8S (reverse):

5’-TGATACCACTTATCGCACTT-3’

Initial denaturation at 95°C for 2 min, followed by 34 cycles of denaturation at 95°C for 20 s,

annealing at 53°C for 30 s, and extension at

72°C for 1 min, with a final extension

of 72°C for 6 min

Patients Sample collection

A total of 28 CL patients were registered in Department of Dermatology IGMC Shimla. Out of 28 CL patients registered in 2018 at IGMC Shimla, only 12 were included in the study being fresh .The study was approved by Ethical committee of ICMR -National Institute of Malaria Research, Delhi. CL was diagnosed clinically by the appearance of plaque, erythematous nodules or ulcerated lesions by a dermatologist. Skin biopsies of 3-4 mm in diameter were taken under anesthetic and sterile conditions from the border of the lesions. Each 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 Qiagen DNA mini kit (Qiagen ,Valencia, CA), as per manufacturer’s instructions and eluted in 20ul elution buffer. DNA extracts were stored at -20 °C for further use. Genomic DNA was extracted from in-vitro cultures of L. donovani Ag83 and was used as positive control. L. tropica (MHOM/AF/87/RUP), and L. major (MHOM/SN/74/SD) positive controls were procured from BEI, Resource USA.

Polymerase Chain Reaction- Restriction Fragment Length Polymorphism (PCR –RFLP)

The polymerase chain reaction was carried out on DNA isolates from punch biopsy samples .The Leishmania specific JW11/12 primer set amplifies a 120bp fragment of KDNA of genus Leishmania[10] and the LITSR/L5.8S set amplifies a 320 bp fragment of ITS1 region of Leishmania genus-specific [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 amplified for each forward and reverse primer in the presence of 2 X Kappa master mix in a final volume of 20 μl in Bio Rad PCR Thermocycler. The amplified PCR products were electrophoresed on 1.5-2% (w/v) agarose gel stained with ethidium bromide (EtBr) (Sigma Aldrich) in 1X tris-acetate-EDTA buffer at 100 V for 45 min, Gel images were captured on a computerized gel documentation unit.

For leishmania species identification, PCR-Restriction Fragment Length Polymorphism was carried out, relevant PCR conditions given in table 1. The 10µl of LITSR/L5.8S amplified PCR products were digested with HaeIII enzyme (Thermoscientific) over night at 37°C, according to the manufacturer’s instructions and restriction fragments were analysed by gel electrophoresis in 3% metaphor gel [12].

Sequencing

Sequencing was performed commercially from Apical Scientific Selangor, Malaysia for LITSR/L5.8S amplified Leishmania positive PCR products using LITSR/L5.8S leishmania specific primers as mentioned in Table 1. The sequence generated were subjected to a homology search using NCBI’s Basic Local Alignment Tool (BLAST). The sequences were submitted to GenBank (accession no. MT423519, MT423520, MT423521, MT423522, MT423523).

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.

Confirmation 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 –rflp

Of 12 biopsy samples, eight were found positive for Leishmania with JW11/12 set of primers (Table 2 and Fig. 3) while all the 12 samples were found positive with ITS1 Leishmania specific set of primers (Fig. 4). Of twelve Leishmania positive samples, six were identified as L. donovani with band size 187 bp, 75 bp and 54 bp, 3 were identified as L. tropia with band size 200 bp and 60 bp, and two were identified as L. major with band size 210 and 140 by PCR- RFLP (Fig. 5).

Table 2

Details of different diagnostic methods for detection of Leishmania species
S. No	Smear	rK39	Culture	Leishmania positivity by PCR with JW 11/12 Lesihmania specific set of primers	Leishmania positivity by PCR with Leishmania specific (LITSR/L5.8S ) set of primers	Leishmania species -specific identification by PCR- RFLP	Leishmania species -specific identification by sequencing method
1	Negative	Positive	Negative	Positive	Positive	L. donovani	L. donovani
2	Negative	Negative	Negative	Positive	Positive	L. donovani	L. donovani
3	Positive	Negative	Negative	Positive	Positive	L. donovani	L. donovani
4	Negative	Negative	Negative	Positive	Positive	L. major	L. major
5	Negative	Negative	Negative	Positive	Positive	L. major	L. major
6	Negative	Negative	Negative	Negative	Positive	L. donovani	L. donovani
7	Negative	Negative	Negative	Positive	Positive	L. Tropica	L. tropica
8	Negative	Negative	Negative	Negative	Positive	L. donovani	L. donovani
9	Negative	Negative	Negative	Positive	Positive	Not identified	Not identified
10	Negative	Negative	Negative	Positive	Positive	L. tropica	L. tropica
11	Negative	Negative	Negative	Negative	Positive	L. donovani	L. donovani
12	Negative	Negative	Negative	Negative	Positive	L. tropica	L. tropica

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.

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 identified 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 identification of Leishmania. In the present study we also identified 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 difficult 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 identification was also confirmed by Sanger DNA sequencing method.

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) a form of VL wherein Leishmania parasite remain confined to skin after treatment of VL) transmitted by P. longiductus or both VL and CL cases are coexisting. The Dermal Leishmanoid termed by Brahmachari in 1922 is Post kala-azar Dermal Leishmaniasis today (Desjeux et al. 2012). While India is close to elimination of VL, the occurrence of CL cases in HP, Kerala or elsewhere should not be ignored considering them just cases of CL alone. The goal of elimination of VL will be considered over only when the atypical leishmaniasis foci of VL are also taken into consideration.

PCR Polymerase Chain Reaction

NIMR National Institute of Malaria Research

ICMR Indian Council of Medical Research

WHO World Health Organization

SSG Sodium stibogluconate

RFLP Restriction Fragment Length Polymorphism

IGMC Indira Gandhi Medical College

Ethical approval: The study was approved by Ethical committee of ICMR -National Institute of Malaria Research, Delhi (committee’s reference number NIMR/ECR/2015/369)

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Consent of participants: Consent form was filled and signed by the patients prior taking the samples.

Competing interest: The authors declare that they have no competing interests.

Financial support: This work was supported by the Indian Council of Medical Research New Delhi, India.

Authors contribution

Suman Lata Collected patients samples, performed the experiment and drafted the manuscript ,Sandhya Chauhan collected patients samples , Ram Das Reviewed the manuscript, Shweta pasi reviewed the manuscript, Ramesh C Dhiman designed the study, wrote and edited the manuscript

Acknowledgments

We acknowledge the Indian Council of Medical Research for funding the project. Thanks, due to the State Program Officer Himachal Pradesh. IGMC Dermatology staff medical staff.

Authors information

1 Suman Lata

Senior Research Fellow,

Environmental Epidemiology Division

ICMR-National Institute of Malaria Research

Dwarka sector 8, New Delhi-11007

Email ID: [email protected]

2 Dr Sandhya Chauhan

Senior Residence Dermatology

Indira Gandhi Medical College and Hospital

Himachal Pradesh Shimla –171001

Email ID-[email protected]

Dr Ram Das

Scientist D

ICMR-National Institute of Malaria Research

Dwarka sector 8, New Delhi-11007

Email: [email protected]

Dr. Shweta pasi

Scientist B

ICMR-National Institute of Malaria Research

Dwarka sector 8, New Delhi-11007

Email: [email protected]

Dr. Ramesh C. Dhiman, (corresponding)

Former Scientist ‘G’ and Senior Consultant

Environmental Epidemiology Division

ICMR-National Institute of Malaria Research

Dwarka sector 8, New Delhi-11007

Email ID: [email protected]

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

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Abstract

Figures

Introduction

Material Methodology

Results

Clinical features of patients

Analysis Of Biopsy Specimens By Pcr –rflp

Treatment Of Cl Cases

Discussion

Conclusions

Abbreviations

Declarations

Availability of data and materials

References

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