Cutaneous and Visceral Leishmaniasis Caused by the Same Zymodeme of Leishmania donovani in Kerala, India

ABSTRACT.

The tribal population in and around the Western Ghats region of India is affected by both cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) with typical clinical symptoms. In this study, we recorded and analyzed seven CL and three VL cases from this emerging belt. All the cases were found as autochthonous transmission. Multiple genetic markers (minicircle kinetoplast DNA polymerase chain reaction and restriction fragment length polymorphism of 3′untranslated region heat shock protein (HSP) 70, a larger segment of HSP 70, and 6-phosphogluconate dehydrogenase [PGDH] gene sequences) were used to identify and characterize the parasite. It was found that both clinical manifestations are caused by zymodeme MON-37 of Leishmania donovani. We have investigated the detailed entomological and epidemiological aspects of disease transmission. An abundant population of the proven vector Phlebotomus argentipes was observed in the study villages.

INTRODUCTION

Leishmaniasis is a spectrum of disease manifestations caused by protozoan parasites of the genus Leishmania, which are transmitted by phlebotomine sand flies. The most prevalent form of the disease is cutaneous leishmaniasis (CL), which is endemic in the northwestern desert zone in India. Visceral leishmaniasis (VL) is the fatal form of the disease, which contributes to approximately 1 million cases globally with an annual mortality of about 20,000 to 40,000.¹ India reports about 25% of these cases, which are mainly distributed in the eastern region of the country. However, the occurrence of sporadic cases of leishmaniasis has been recorded in non-endemic states located in the southern and northern regions of the country during the last decade.²

The Western Ghats region in India was considered to be a non-endemic focus of leishmaniasis. Few sporadic cases of CL and VL have been documented in this area in recent years.^3,4 Interestingly, recent studies have reported several indigenous cases of both CL and VL in a short period, indicating the emergence of new endemic foci of leishmaniasis in India. Phlebotomus argentipes was found to be the proven vector species involved.⁵ Also, preliminary investigations identified domesticated dogs as reservoir hosts.⁶ The surveillance and natural infection assessment of vector sand fly species are inevitable for planning effective disease control measures and tracking disease transmission. In the present study, we describe the clinical investigation, genetic characterization, epidemiological findings, and entomological surveillance of patients with recently confirmed CL and VL from this emerging endemic belt of leishmaniasis.

MATERIALS AND METHODS

From February 2021 to February 2022, we carried out a cross-sectional epidemiological investigation for leishmaniasis in three districts of Kerala states (viz., Palakkad, Kollam, and Thrissur) (Figure 1A). The study was performed after obtaining ethical clearance from the Indian Council of Medical Research–Vector Control Research Center (approval no. IHEC-0119/R/M). Among the nine tribal settlements comprising 412 individuals (209 males and 203 females) from Kollam, we identified 18 suspected CL cases. All the suspected VL cases meeting the case definition criteria (prolonged fever, hepatosplenomegaly, anemia, and weight loss) as set by the WHO² were referred to us for disease confirmation by tertiary health care centers in central Kerala.

Figure 1.

Data showing the distribution and diagnosis of cutaneous and visceral leishmaniasis cases in the Western Ghats region of Kerala. (A) The map represents the distribution of visceral leishmaniasis/cutaneous leishmaniasis (VL/CL) cases in the foothills of Western Ghats, Kerala, India. (B–D) A nodular patch on the right leg of a CL patient (B) and Leishmania amastigotes in skin aspirate (C) and skin biopsy (D) samples were analyzed by histopathological examination. (E–G) The patient was diagnosed as having VL (E) and the Leishmania amastigotes in the histopathology of the liver (F) and bone marrow (G). Red arrowheads indicate the Leishmania amastigotes forms.

Hematological investigations, ultrasonography, rK39 immunochromatographic dipstick test, and histopathological examination of bone marrow biopsy samples were performed for the diagnosis of individuals with suspected VL. Histopathological analysis was carried out for suspected CL cases using skin biopsy and skin aspirate samples. In addition, all patient samples (both CL and VL) were processed for molecular diagnosis using multiple genetic markers.³ As an initial step of diagnosis, minicircle kinetoplast DNA (kDNA) was amplified using gDNA, as it is specific for the class Kinetoplastida to which Leishmania belongs and has a large copy number.⁷ After this, restriction fragment length polymorphism (RFLP) analysis of 3′untranslated region (UTR) of heat shock protein (HSP) 70 using Hae III was carried out to ascertain the Leishmania species complex.⁸ The larger segment of HSP 70 gene sequence was amplified and sequenced for parasite species identification.⁹ The strain characterization of the parasite was carried out by the analysis of the 6-Phosphogluconate dehydrogenase (6-phosphogluconate dehydrogenase [PGDH]) gene.¹⁰

The phylogenetic analysis of the HSP-70 and 6-PGDH gene sequences was carried out using maximum likelihood methods based on the Tamura-Nei model.¹¹ The consensus of 1,197 nucleotide sequences of HSP 70 and 737 nucleotides of 6-PGDH was used for evolutionary analysis using MEGA 7.0.¹² A bootstrap analysis of 1,000 simulations was carried out to generate the phylogenetic trees of CL and VL clinical isolates. HSP 70 and 6-PGDH gene sequences obtained in this study were deposited in the National Center for Biotechnology Information Genbank. Clinically diagnosed CL cases were treated as per WHO standard treatment protocol. Patients with VL were administered intravenous infusion of liposomal amphotericin B (10 mg/kg of body weight).

After leishmaniasis diagnosis, in-depth entomological surveillance was conducted across a 0.5-km radius around the patients’ residences. Indoor and outdoor habitats were screened for the collection of sand flies using CDC-modified light traps and mechanical aspirators. Field-collected specimens were preserved in 70% ethanol and used for taxonomical identification and molecular detection of Leishmania.^5,13 Vector control operations by indoor residual spraying (IRS) of lambda-cyhalothrin 10% wettable powder formulation was carried out soon after case detection and entomological surveillance.

RESULTS

The clinical presentation of CL among the confirmed cases showed marked phenotypic variations, from asymptomatic nodules to severe itching of ulcerated lesions, irrespective of age (Table 1, Figure 1B). Histopathological examination of skin biopsy samples revealed the presence of Leishmania amastigotes (Figure 1C and D). Of the 18 suspected cases, seven were diagnosed to be CL by the diagnostic kDNA polymerase chain reaction (PCR) method. The remaining eight suspected CL cases were negative for the diagnostic kDNA PCR. All three VL cases had typical symptoms, such as moderate to high-grade fever, significant weight loss, and generalized malaise (Figure 1E). Hematological investigations revealed that patients had anemia and pancytopenia. Ultrasonography reports diagnosed two patients as having hepatosplenomegaly. An rK39 immunochromatographic dipstick test and histopathological examination of bone marrow biopsy confirmed the diagnosis of VL (Figure 1F and G).

Table 1.

Clinical profiles of cutaneous and visceral leishmaniasis patients

Sl no	Date of collection	Age/sex	Village/district	Site of lesion	Clinical symptoms	Duration	PCR kDNA result	Parasite sp.	Clinical manifestation
1	March 1, 2021	23 years/M	Kulathupuzha, Kollam	Lateral aspect of the right upper arm, upper chest	Multiple lesions of 1–1.5 cm in diameter with crust formation; 2–2.5 cm in diameter, ulcerated lesion with surrounding erythema and severe itching	> 1 year	Positive	L. donovani	CL
2	February 22, 2021	17 years/F	Kulathupuzha, Kollam	Left and right elbows	2–2.5 cm in diameter, well-circumscribed ulcerated lesion with surrounding hyperpigmentation; 1.5–2 cm in diameter ill-defined lesion with scaling	> 6 months	Positive	L. donovani	CL
3	February 22, 2021	20 years/F	Kulathupuzha, Kollam	Right cheek	1.5–2 cm in diameter, nodule with central ulceration and raised margins	> 1 year	Positive	L. donovani	CL
4	February 22, 2021	40 years /F	Kulathupuzha, Kollam	Right upper arm and wrist joint	1–1.5 cm in diameter, raised lesion with surrounding hypopigmentation; 1–1.5 cm in diameter, nodule with central scab formation with surrounding hyperpigmentation	> 1 year	Positive	L. donovani	CL
5	March 1, 2021	18 years /M	Kulathupuzha, Kollam	Right and left calves	2–2.5 cm in diameter, ulcerated lesion with surrounding hyperpigmentation; multiple healed hyperpigmented lesions of 1.5–2 cm	> 1 year	Positive	L. donovani	CL
6	March 1, 2021	18 years /F	Kulathupuzha, Kollam	Dorsal aspect of right and left ankle joints	0.5–1 cm in diameter, crusted lesion with surrounding hyperpigmentation; 0.5 cm in diameter, multiple healed hyperpigmented lesions	> 1 year	Positive	L. donovani	CL
7	March 23, 2021	28 years /M	Kulathupuzha, Kollam	Dorsal aspect of left shoulder	0.5–1 cm in diameter, nodule	> 1.5 years	Positive	L. donovani	CL
8	March 15, 2021	35 years /M	Kulathupuzha, Kollam	Right pinna, lower elbow of right arm	0.5–1 cm in diameter, multiple healed lesions with hyperpigmentation	> 1 year	Negative	Negative	–
9	March 15, 2021	5 years /F	Kulathupuzha, Kollam	Lateral aspect of the left buttock	0.5 cm in diameter, multiple lesions healed with hyperpigmentation	> 4 months	Negative	Negative	–
10	March 15, 2021	40 years /M	Kulathupuzha, Kollam	Umbilical region of the abdomen	2–2.5 cm in diameter, hyperpigmented papule with diffused margins	> 3 months	Negative	Negative	–
11	March 15, 2021	6 years /F	Kulathupuzha, Kollam	Right knee and calf	2.5–3 cm in diameter, ulcerated nodular lesion with scaling of surrounding skin	> 3 months	Negative	Negative	–
12	November 3, 2021	42 years /M	Achenkovil, Kollam	Left lumbar and iliac region	Multiple healed pinpoint lesions with surrounding hyperpigmentation	> 2 months	Negative	Negative	–
13	September 22, 2021	6 years /M	Anapantham, Thrissur	Posterior aspect of the left middle arm	2–2.5 cm in diameter, ulcerated lesion with central scale formation	> 3 months	Negative	Negative	–
14	September 22, 2021	10 years /M	Anapantham, Thrissur	Dorsal aspect of the left ankle region	0.5–1 cm in diameter, multiple lesions with central scaling	> 6 months	Negative	Negative	–
15	September 22, 2021	8 years /M	Anapantham, Thrissur	Dorsal aspect of the right ankle region	1.5–2 cm in diameter, central ulcerated lesion with surrounding scale formation	> 3 months	Negative	Negative	–
16	December 2, 2021	13 years /F	Kumaramputhur, Palakkad	–	Hepatospleno-megaly; significant weight loss, pancytopenia, fever	> 2 years	Positive	L. donovani	VL
17	January 25, 2022	60 years /M	Chelakkara, Thrissur	–	Hepatospleno-megaly, significant weight loss, fever, anemia	> 2 years	Positive	L. donovani	VL
18	February 7, 2022	62 years /M	Manalithara, Thrissur	–	Hepatospleno-megaly, weight loss, mild fever	> 5 months	Positive	L. donovani	VL

CL = cutaneous leishmaniasis; F = female; kDNA = kinetoplast DNA; L. donovani = Leishmania donovani; M = male; PCR = polymerase chain reaction; Sl no = serial number; VL = visceral leishmaniasis.

The primary diagnostic PCR involving the kDNA region gave positive results for all the VL patients and for seven CL patients (Table 1), for whom Leishmania amastigotes were also positive. The kDNA PCR yielded an amplicon of 720-bp size, and confirmatory findings were obtained from RFLP analysis of 3′UTR HSP 70 and larger segments of HSP 70 gene sequences. The 3′UTR of HSP 70 PCR amplified a 750-bp fragment that, on Hae III digestion, showed an L. donovani–specific pattern of four fragments of ≈450, 170, 125, and 25 bp (Figure 2A). HSP 70 sequence analysis confirmed that the infection was L. donovani. A unique nonsynonymous single nucleotide polymorphism at the 976th position, causing an amino acid change from asparagine to aspartic acid, was recorded in the 6-PGDH gene sequence analysis, confirming that the parasite belonged to the zymodeme MON-37 strain (Figure 2B, Supplemental Figure 1).

Figure 2.

(A) Polymerase chain reaction‒RFLP pattern of 3′UTR of the HSP 70 gene using Hae III. (The lowest band of 25 bp is very faint in the agarose gel). (B) Single nucleotide polymorphism showing the nucleotide change (A→G) at the 976th position of the 6-PGDH gene. (C) Phylogenetic analysis of the HSP 70 gene. (D) Phylogenetic analysis of 6-PGDH gene sequences from dermatropic and viscerotropic clinical manifestations of Leishmania donovani. RFLP = restriction fragment length polymorphism; UTR = untranslated region.

The genetic analysis of the nucleotide sequences of HSP 70 and 6-PGDH genes among the CL/VL strain in Kerala revealed they were genetically identical (Figure 2C and D). All the CL/VL sequences analyzed branched to a single clade in the phylogenetic analysis of HSP 70 and 6-PGDH gene sequences, indicating that the clinical isolates of the parasites were 100% identical and that the parasite strain was zymodeme MON-37 of L. donovani. The Genbank accession numbers of HSP 70 sequences are OM471994, OM471995, OM471996, OM948860, and OM948861; for 6-PGDH they are OM948862, OM948863, OM471992, and OM471993.

Patients showed an excellent prognosis after the treatments. One of the VL cases (12/female) was an immunocompromised child who presented with intermittent fever and had comorbidities such as type 1 diabetes mellitus, hypothyroidism, and congenital bilateral hydronephrosis. The child expired soon after diagnosis as a result of the comorbidities.

The entomological collections yielded an abundant population of sand flies from the epidemiologically active areas. A total of 1,530 sand fly specimens belonging to two genera and 12 species were collected from the study areas after leishmaniasis confirmation. Phlebotomus argentipes was the predominant species (52.68%), followed by Sergentomyia zeylanica (13.46) and Sergentomyia babu (12.42%) (Supplemental Figure 2). Of the 806 P. argentipes caught, 537 were females and none of the specimens were positive for Leishmania infection. A drastic reduction in the sand fly population was observed in case-reported areas after IRS with 10% lambda-cyhalothrin.

DISCUSSION

Circulation of the Leishmania parasite was reported in the Western Ghats region of Kerala state.^3,4 However, our observations on MON-37 of L. donovani causing different types of manifestations (CL/VL) in different ecological situations are intriguing and warrant a detailed investigation of the determinants. The case history records of the patients indicated the occurrence of the infection was indigenous, as none of the patients had traveled to endemic states in the eastern part of India or had been in contact with a vulnerable population. The active entomological surveys conducted in and around a 0.5-km radius of the patients’ residences indicated a conducive ecological setup for leishmaniasis transmission. Of the 12 sand fly species collected during the study period, a high abundance of vector species, P. argentipes, was recorded from these areas. However, Leishmania infection was not detected in sand flies, which may be due to the small sample size and the large incubation period of Leishmania parasites. Sergentomyia zeylanica, S. babu, Phlebotomus colabaensis, etc., were also present in the study areas. Active surveillance of parasites in reservoir hosts is imminent to validate the zoonotic transmission, as reservoir hosts such as dogs and goats have been observed in the region.^6,14 The environment was found to be ideal for parasite circulation. Atypical reports of CL caused by L. donovani zymodeme MON-37 have also been documented in Sri Lanka, which forms a single biogeographical unit with the Western Ghats.¹⁰

These case incidences reveal a unique observation that there is a marked difference in the topographical distribution of CL and VL within the Western Ghats belt (Figure 1A). Reports of CL were mostly confined to tribal people residing in the deep forest zones of the Western Ghats.^3,15 Because the peripheral population residing in the foothill regions of the Western Ghats relies on forest-based products for their livelihood, their contact with these indigenous tribal populations and the forest habitat is frequent. Cases of VL were mostly reported in the population living at the outskirts of the Western Ghats.⁴ Further investigation regarding interactions among hosts, vectors, and parasites is necessary to validate these observations.

CONCLUSION

Leishmaniasis is not yet a notified disease in the state of Kerala. At present, India is a signatory to the global VL elimination program with a target of 2023. Recent observation and description of an emerging focus of CL and VL in the Western Ghats is of high epidemiological significance. We propose the initiation of a systematic surveillance program for leishmaniasis in this endemic area for the successful achievement of this elimination goal.

Note: Supplemental material appears at www.ajtmh.org.