Journal of Medical Microbiology Papers in Press. Published December 5, 2014 as doi:10.1099/jmm.0.076695-0
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Cutaneous leishmaniasis caused by Leishmania donovani in tribal population
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of the Agasthyamala Biosphere reserve forest, Western Ghats, Kerala, India
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Short running title: Cutaneous leishmaniasis caused by Leishmania donovani
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Contents category: Original Scientific Research paper (Diagnostics, typing and
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identification)
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N. Pradeep Kumar1*, R. Srinivasan2, T. S. Anish3, G. Nandakumar3, P. Jambulingam2
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1
Vector Control Research Centre Field Station (ICMR), Kottayam, Kerala – 686002, India
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2
Vector Control Research Centre (ICMR), Indira Nagar, Puducherry – 605006, India
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3
Government Medical College, Thiruvananthapuram, Kerala – 695011, India
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*Correspondence: N. Pradeep Kumar,
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E-mail: [email protected]
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Telephone: 914812303435
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______________________________________________________________________________
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The GenBank accession numbers for 1) the Hsp70 gene sequences for the isolates from this
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study are KC884001, KF562068, KF562069, KF562070 & KF562071 and 2) the 6-PGDH
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sequences are KJ461872 & KJ461873.
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ABSTRACT
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Cutaneous leishmaniasis (CL), a neglected tropical disease infection is reported to be prevalent
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in tribal villages located in the Agasthyamala Biosphere Reserve forests of Western Ghats,
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Kerala state, India. We carried out an investigation to characterize the species of Leishmania
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parasites involved in these infections prevalent among one of the oldest human tribal populations
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in India. Skin aspirates collected from 13 clinically diagnosed cases were subjected to
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histopathological investigations, serological rapid tests using ‘rk39’ and molecular diagnostics.
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Clinical manifestations recorded among patients were hypo-pigmented erythematous nodules/
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papules on limbs and other parts of the body. Histopathological investigations of these skin
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lesions among patients showed Leishman-Donovan (LD) bodies in macrophages. None of the
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patients were found positive for ‘rk39’ tests, which detect active visceral Leishmaniasis. Using
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three different genetic markers (kinetoplast minicircle DNA, 3’ UTR region of Heat shock
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protein 70 (Hsp70) and Hsp70 gene) we identified the parasite species involved in these
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infections to be Leishmania donovani. The 6-phosphogluconate (6-PGDH) gene sequences of the
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parasite isolates from Western Ghats indicted close genetic relatedness to the L. donovani
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isolates reported from Sri Lanka, also causing CL. This could be cited as another instance of
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“local endemism” of organisms in this single “bio-geographic unit”.
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INTRODUCTION
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Leishmaniasis, is prevalent in 98 countries and about 310 million population live under the risk
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of infection of this neglected tropical disease globally. About 12 million people are infected, with
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an annual incidence of about 1.3 million cases. Cutaneous leishmaniasis (CL) is the most
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common clinical manifestation, affecting about 1 million people every year. (WHO, 2013).
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However, visceral leishmaniasis (VL) remains the serious form of infection. Also, this is the
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most important variety of leishmaniasis infection in India. Bihar, West Bengal, Jharkhand and
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Uttar Pradesh are the endemic states of VL in India (Alvar et al., 2012). In addition, sporadic
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cases have been recorded from the foothills of the Himalayas (Desjeux, 1991), Gujarat and
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Kerala (Kesavan et al., 2003; Sharma et al., 2007). Thirty three thousand one hundred and eighty
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seven and 20600 cases of VL were recorded in India during 2011 and 2012 respectively
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(NVBDCP1). Leishmania donovani is the major parasite involved in VL and Phlebotomus
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argentipes is reported as the major vector species involved in transmission (Kumar et al., 2001).
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CL is reported mainly from the Northwestern region of India bordering Pakistan (Rajasthan &
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Punjab) (Sharma et al., 1973; Aara et al., 2010) and in some other small pockets in Himachal
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Pradesh (Sharma et al., 2005). The parasite involved in the infection is L. tropica and L.
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donovani (Sharma et al., 2005; Kumar et al., 2007). Mucocutaneous forms of Leishmaniasis
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cases caused by L. donovani have been reported from Himachal Pradesh (Sharma et al., 2005).
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CL caused by L. donovani has been recorded as a major problem in the adjacent country, Sri
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Lanka (Siriwardana et al., 2007; Ranasinghe et al., 2012; Gajapathy et al., 2013).
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A recent preliminary study reported the prevalence of CL in two tribal settlements in the Western
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Ghats of Thiruvananthapuram District, Kerala State (Simi et al., 2010). They reported 12 active
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CL cases from the region based on clinical examinations. Also, histopathological examinations
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showed LD bodies in 4 cases. Based on this report, we initiated a study to elucidate the
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epidemiological factors involved in the indigenous transmission of this neglected disease in the
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tribal belt located in the protected Agasthymala, Biosphere forest region of southern India. Here,
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we report the characterization of the Leishmania species involved in the human CL infections.
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Furthermore, studies are in progress about the transmission dynamics of the CL infections in the
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region. The region is thickly forested and do not have transport accessibility. About 1500 tribal
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people live in 28 hamlets deep in the forest in this region. Most of these people dwell in thatched
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huts constructed with mud, peeled stem of bamboo, palm trees or sticks. Electricity supplies to
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the region are by installing solar cells in each hutment. One or two dogs are domesticated by
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each tribal family, especially to protect them from attack of wild animals in the region.
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METHODS
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Clinical Examination:
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Seven hundred and sixty eight persons residing in 28 hamlets were examined for the clinical
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manifestations of leishmaniasis during field visits of medical officers to these villages. The
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persons having suspected active CL lesions were advised to visit as outpatients to the Govt.
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Medical College and Gokulam Medical College hospitals, Thiruvananthapuram, Kerala. They
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were clinically examined and required treatment was administered. Ethical clearance for the
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study was obtained from the Institutional Ethical Committee of Vector Control Research Centre
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(Indian Council of Medical Research), Puducherry, India.
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Histopathology:
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Histopathological examination of skin biopsy specimens was carried out in the Departments of
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Pathology on 13 cases who visited the hospitals as outpatients. After identifying a relatively new
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skin lesion, 1% lignocaine was infiltrated with it and by using a surgical blade a linear incision of
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about 0.5 cm, reaching up to the level of dermis was performed. Then the blade was turned 900
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and the dermal pulp was scraped out using the blunt end of the knife taking care not to induce
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bleeding. This pulp was smeared onto a glass slide and was stained with Geimsa and observed
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under the microscope to visualize the granuloma and identify the amastigote stages of
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Leishmania parasites - Leishman-Donovan (LD) bodies.
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Molecular studies:
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Aspirates from skin nodules/lesions of all the samples were also subjected to PCR based analysis
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for Leishmania infection at the Vector Control Research Centre Field Station, Kottayam, Kerala,
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India. Parasite DNA was extracted using Genelute Mini-prep Kit (SIGMA-ALDRICH, USA),
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following the kit protocol. Briefly, the skin aspirates from all 13 cases were lysed and treated
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with RNase A. The lysate was loaded and passed through Mini-prep columns so that DNA binds
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to the column silica membranes. The DNA was eluted with de-ionized distilled water and was
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subjected to diagnostic mini-circle kinetoplast DNA (k-DNA) semi-nested PCR using DNA
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primers (Aransay et al., 2000). This PCR amplifies k-DNA in 8 species of Leishmania parasites
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and groups them into two categories, 1) major group comprising of L. major and L. aethiopica
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which yield a band of about 650 bp and 2) infantum group comprising of L. infantum, L. tropica,
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L. donovani, L. amazonensis, L. mexicana and L. braziliensis which amplify a fragment of about
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720 bp. Requena et al., (2012) proposed a PCR-RFLP analysis of the UTR region of the Heat
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shock protein 70 (Hsp70) for species characterization of Leishmania species belonging to both
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Old world and new world species. Among old world species it clearly distinguishes between L.
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tropica and L. donovani complex by restriction digestion with Hae III. Hence we carried out this
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analysis on the positives and the 750 bp fragment amplified was digested with the restriction
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enzyme and the restriction fragments were resolved in a 3.0% agarose gel to type the species
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involved. Also, a larger fragment of Hsp70 gene was amplified and sequenced to determine the
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exact species involved in the infections (Montalvo et al., 2010). Hsp 70 gene fragment amplified
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was about 1400 bp and could be used to determine the exact species either by RFLP or DNA
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sequences analyses. A partial sequence of phosphogluconate (6-PGDH) gene, a highly variable
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gene was also amplified using DNA primers already reported (Ranasinghe et al., 2012).
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Phylogenetic analyses were carried out with Hsp70 gene sequences as well as partial 6-PGDH
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gene sequences isolated in this study and those available in the GenBank from different countries
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(Ranasinghe et al., 2012; Zhang et al., 2013), using maximum likelihood analysis (bootstrapping
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with 1000 replications) to understand the genetic lineage of the parasite. Thus, four robust
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genetic markers were used in the study to confirm the species involved in the infections.
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Serological studies:
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‘rk39’ serological rapid test had been found very useful for rapid diagnosis of active
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visceral Leishmaniasis infections (Badaro et al., 1996). This rapid immunochromatographic test
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diagnoses VL cases with 97% specificity and 100% sensitivity (NVBDCP2). We carried out this
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test in all the 13 cases examined in the study following the national guidelines of Government of
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India (NVBDCP2).
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RESULTS
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Clinical manifestations:
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A total of 27 cases were recorded with suspected symptoms of CL. None reported clinical
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manifestations of VL. Among these 12 had healed dermal scars of CL lesions. Among 15 cases
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with active lesions, 13 visited hospitals for follow up and treatment. The major clinical
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manifestations recorded among these 13 patients (Table 1) were multiple hypo-pigmented to
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erythematous papules with a few showing studded erythematous nodules or ones with erosions,
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forming ulcers in the upper/lower limbs, head, ear pinna, shoulder, trunk etc. (Fig. 1).
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Histopathology:
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Skin smears examined under the microscope showed epidermis with different degrees of
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keratosis to atrophy. Dermis showed granulomas with diffuse infiltration by lymphocytes, few
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epithelioid cells and macrophages. Isolates from four patients had L-D bodies in the
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macrophages. (Fig. 2).
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Molecular studies:
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Diagnostic PCR analysis (k-DNA) showed five patients to be positive for Leishmania infection.
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Among these, 3 were positive for L-D bodies also on histopathological examination. The size of
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the k-DNA fragments amplified was about 720 bp. indicating that the species involved belonged
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to the L. infantum group (Aransay et al., 2000). 3’ UTR region of Hsp70 gene of all these
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positive samples were amplified which yielded about 750 bp fragments. On restriction digestion
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with Hae III a pattern similar to that reported to L. donovani/ L. infantum was recorded (4
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fragments of about 450 bp, 170 bp, 125 bp and 25 bp (very faint)) (Fig. 3). Phylogenetic analysis
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of Hsp70 gene sequences of Leishmania parasite isolates indicated the species to be L. donovani
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(Fig. 4).On further analysis of DNA sequences it was found that all five had a Cytosine molecule
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at 434 position of the Hsp70 gene sequences, indicating the specimens belonged to the species L.
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donovani. L. infantum isolates have a Thymine molecule in the position 434 of Hsp70, making
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that a restriction site of the restriction enzyme Mlu I (Montalvo et al., 2010). The GenBank
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accession numbers of Hsp70 sequences of our isolates are KC884001, KF562068-KF562071.
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GenBank BLAST analysis showed 100.0% identity for these gene sequences with L. donovani
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isolated from VL cases in India (JQ990221), China (JX021428) and African countries
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(FN395027 & FN669773). Phylogenetic analysis carried out with the 6-PGDH gene amplified
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for two positive samples in the study showed their genetic lineage to be closely related to the Sri
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Lankan strains of L. donovani (Fig. 5). The Kimura 2 Parameter (K2P) genetic distance
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calculated among “Kerala/Sri Lanka group” of CL strains was found to be zero. The 737 bp
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partial DNA sequences of the 6-PGDH gene (KJ461872 & KJ461873) exactly matched with the
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gene sequences of the Sri Lankan isolates. The K2P genetic distance between this and the “north
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Indian/Bangladesh” group (AJ88899-AJ888901) was found to be 0.003. Also, a unique transition
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event in the 976th nucleotide position (A G) resulting in a non-synonymous mutation “N326D”
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was recorded in “Kerala/Sri Lanka” group of isolates compared to the “north Indian/Bangladesh”
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isolates (VL). Isolates from China and Kenya included in the analysis belonged to the same
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genetic lineage of “Kerala/Sri Lanka” group. However, the former isolates were from VL cases.
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None of the 13 cases were found positive with rk39 kits, clearly confirming the cases included
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did not have detectable levels of antibodies in their blood.
→
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DISCUSSION
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Agasthyamala biosphere reserve (area: 3500.36 Km2) spanning both Kerala and Tamil Nadu
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states of India is located in the Western Ghats, the second largest mountainous belt in India is a
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UNESCO World Heritage site (UNESCO, 2007). It is the abode of one of the oldest surviving
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ancient tribal populations (“Kanikarans”) in the world. It is also designated as one of the world’s
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eight hotspots of biodiversity on the globe (Myers et al., 2000). The current investigation on the
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28 tribal settlements of this biosphere reserve indicated endemic transmission of L. donovani
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among the ‘Kanikaran’ tribal population, causing CL. The first investigation of the prevalence of
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this disease in this region was carried out by Simi et al. 2010. The investigators made known the
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active transmission of CL by clinical and histopathological investigations in two tribal villages of
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Thiruvananthapuram District, never recorded earlier in the literature. The region is inaccessible
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to modern developmental activities and clinical facilities and is located deep in the evergreen
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forests of Western Ghats and hence remained “isolated” for decades. These hamlets were never
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in the records of the state Department of Health services, Kerala state, for any past activity of
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vector control operations. Also, the region belongs to one of the protected bio-reserves of
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international interest, with very restricted human interventions from outside. Thus, it could be
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ascertained that the transmission of CL in this region would have remained unnoticed for many
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decades.
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L. donovani, the dominant parasite of visceral leishmaniasis in other parts of the country brings
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about cutaneous manifestations in the tribal population of this undisturbed ecosystem remains
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intriguing. None of the inhabitants clinically surveyed in these villages (n=768) exhibited the
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typical symptoms of VL. All the 13 cases included in the investigations were tested negative for
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“rk39” rapid tests for VL. The nucleotide sequences of both Hsp70 as well as 6-PGDH genes of
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L. donovani isolates from Kerala exactly matched with that of neighboring Sri Lankan isolates.
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The zymodeme classification of the strain recorded in tribal villages of Kerala may be
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“MON37”. The unique mutation recorded in the 976th position of 6-PGDH gene (responsible for
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the change from the uncharged Asparagine to negatively charged Aspartic Acid which affects the
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mobility of the 6-PGDH iso-enzyme) recorded among these L.donovani isolates was ascertained
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to the zymodeme “MON37” which causes CL in Sri Lanka (Siriwardana, et al., 2012). P.
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argentipes, the established vector species of L. donovani (Tiwary et al., 2012) was found to be
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abundant in the study area. A similar situation had been recorded from Sri Lanka also
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(Siriwardana et al., 2007). P. argentipes was found to be the vector species prevalent in this
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region (Gajapathy et al., 2013).
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Terrestrial links between ‘montane’ regions of Sri Lanka and Western Ghats existed during
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Pleistocene era (Rohling et al., 1998). These two areas (about 400 Km apart and currently
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separated by Palk Strait) are grouped as a single biodiversity hotspot by the International Union
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for Conservation of Nature (IUCN) as they form a community of species that fits together as a
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“single bio-geographic unit” (Myers, et al., 2000). Few studies have already analyzed the genetic
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similarity of different species of organisms from both regions and concluded that ‘the overall
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limited biotic interchange has left both the areas with an unexpectedly large number of
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endemics’ (Bossuyt et al., 2004; CEPF, 2007). The genetic similarity of many organisms
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recorded between the wet zone of Sri Lanka and the moist forests of the Western Ghats
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(Conservation International, 2008) may be owing to the inability of rainforest organisms to
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disperse across intervening dry lowlands (Bossuyt et al., 2004). Genetic drift between the
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organisms living in these conserved rainforest ‘montane’ regions owing to the geographical
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isolation may be negligible since these remain isolated as virgin forests without intervention
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from developmental activities. The villages included in this study are located deep inside the
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protected forests with about a 10 km inaccessible thick forested mountainous region barrier, also
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a natural reserve of wild animals. The genetic relatedness of L. donovani isolates from the
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Western Ghats region with that of Sri Lankan isolates recorded in the present study could be
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cited as another instance of local endemism of a parasite species in this ‘single bio-geographic
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unit’. The Kani tribes, from whom these parasite species were isolated, live exclusively in the
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ever green rain forested region of Western Ghats and are hesitant to migrate to the non-forested
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regions of the state, in spite of many government schemes to re-habilitate them.
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The present study evinces active transmission of CL in the tribal settlements of Western Ghats
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caused by L. donovani. The genetic lineage of the parasite was found closely associated with the
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isolates from the adjacent Country Sri Lanka, which forms a single bio-geographic unit along
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with the Western Ghats. The vector species involved may be P. argentipes, as this established
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vector species for L. donovani was found abundant in the region. Further studies to confirm this
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and to determine whether any reservoir hosts are involved in the maintenance of transmission in
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the region is in progress. Chinese and African strains of L. donovani were found closely related
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genetically to the isolates from “Kerala/Sri Lanka”. Nevertheless, they cause visceral
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manifestations. To prevent the remote possibility of spread of L. donovani from its isolation in
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the deep forests, to the thickly populated lowlands of Kerala state, urgent intervention measures
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to contain and eradicate these foci of Leishmaniasis transmission is of utmost importance.
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ACKNOWLEDGEMENTS
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The authors are grateful to Dr. T. Dileep Kumar, Directorate of Health Services, Govt. of Kerala,
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for his suggestions and to Shri. P. M. Ajithlal, Vector Control Research Centre (VCRC) Field
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station, Kottayam and Shri. B. Edwin, VCRC Puducherry for the technical assistance rendered.
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Table 1: PCR and histopathology results of skin nodule aspirates from suspected Leishmania cases – tribal villages of Thiruvananthapuram Sl. No
Date of colln.
*ID. No. (OP-MCH)
Village
Clinical symptoms and Provisional Diagnosis
Histopathology
PCR (k-DNA) result (Leishmania)
1
19/10/2012
273400/12
10F
Thazheamala
No LD bodies Late lesion of CL
Negative
273398/12
38F
Thazheamala
273402/12
17F
Thazheamala
Macrophages show LD bodies, consistent with CL No significant pathology
Positive
19/10/2012
4
19/10/2012
273399/12
44F
Meleamala
No LD bodies, No features seen to suggest CL.
Positive
5
19/10/2012
273397/12
75F
Thazheamala
Positive for LD bodies. Consistent with CL
Positive
6
19/10/2012
273396/12
50M
Thazheamala
No LD bodies, Late lesions of CL
Negative
7
30/3/2013
66332/13
13F
Kombidi
Papules on exposed parts of body (limbs and upper chest) for six months. Most of the lesions are erythematous and 5-10mm in diameter. One well defined erythematous plaque of 1cmX2cm size on forehead for six months Two erythematous papules on left forearm with 0.5cm diameter for six months Two lesions, hypo-pigmented patches on left ear lobe and right side of fore head. History of diagnosis and treatment for CL one year before for the same lesions An erythematous nodule on right forearm 2X3cm in size for 8 months and another lesion on right leg, hypo-pigmented plaque of 2X2cm. Hypo-pigmented patch on right shoulder 3X4cm. History of treatment for CL one year before Two well defined hypo-pigmented nodules no surface changes on the lateral aspect of right arm. CL
2
19/10/2012
3
Macrophages with LD bodies. Consistent with CL
Positive
Age/ Sex
Positive
18
8
30/3/2013
66328/13
22M
Thazheamala
9
30/3/2013
66330/13
10M
Thazheamala
10
26/4/2013
88297/13
28M
Thazheamala
11
26/4/2013
88298/13
26M
Podium
12
26/4/2013
88299/13
25M
Thazheamala
13
26/4/2013
Gokulam Medical College, TVPM
77F
Kadackal
Asymptomatic hypo-pigmented macules and erythematous to skin colored small plaques over upper back and forearms 3 years. Suspected CL & T. versicolor Asymptomatic hypo-pigmented to reddish papules over body – 2 years. Erythematous papules on upper limbs and chest with 5-10mm in size for 6 months
Macrophages with LD bodies
Negative
No LD bodies
Negative
Epithelioid granuloma with lymphocytic infiltration. No LD bodies Single well defined hypoEpithelioid pigmented plaque on upper limbs granuloma with 1X2cm in size for one year lymphocytic infiltration. No LD bodies Erythematous papules on upper Epithelioid limbs with 5-10mm in diameter for granuloma with 3 months lymphocytic infiltration. No LD bodies Nodular skin lesions on both the Epithelioid forearms. 2-3cm in diameter for last granuloma with 6 months lymphocytes. No LD bodies seen
*outpatient identification number, Government Medical College, Thiruvananthapuram (TVPM), India
Negative
Negative
Negative
Negative
19
Legends to Figures:
Fig. 1. Photographs of typical cutaneous lesions of CL patients from the study area. Fig. 2. Histopathological examination of skin aspirates from CL patients (Fig. 2a shows the granulomatous macrophages and is at 40 X magnification; Fig 2b is a 1000 X magnification of the 2a which show amastigote stages of Leishmania parasites). Fig. 3. Restriction digest of 3’ UTR region of Hsp70 gene. (fragment with 25bp size is very faint in the agarose gel photograph). The depicted pattern indicates the species to be either L. donovani or L. infantum (infantum group). Fig. 4. Phylogenetic analysis of the Hsp70 gene of Leishmania parasite isolates from Kerala. Fig. 5. Phylogenetic analysis of the 6 PGDH gene of the Leishmania isolates from Kerala.
1 1
Cutaneous leishmaniasis caused by Leishmania donovani in tribal population
2
of the Agasthyamala Biosphere reserve forest, Western Ghats, Kerala, India
3
Short running title: Cutaneous leishmaniasis caused by Leishmania donovani
4
Contents category: Original Scientific Research paper (Diagnostics, typing and
5
identification)
6 7
N. Pradeep Kumar1*, R. Srinivasan2, T. S. Anish3, G. Nandakumar3, P. Jambulingam2
8
1
Vector Control Research Centre Field Station (ICMR), Kottayam, Kerala – 686002, India
9
2
Vector Control Research Centre (ICMR), Indira Nagar, Puducherry – 605006, India
10
3
Government Medical College, Thiruvananthapuram, Kerala – 695011, India
11 12 13
*Correspondence: N. Pradeep Kumar,
14
E-mail: [email protected]
15
Telephone: 914812303435
16
______________________________________________________________________________
17
The GenBank accession numbers for 1) the Hsp70 gene sequences for the isolates from this
18
study are KC884001, KF562068, KF562069, KF562070 & KF562071 and 2) the 6-PGDH
19
sequences are KJ461872 & KJ461873.
20 21
2 22
ABSTRACT
23
Cutaneous leishmaniasis (CL), a neglected tropical disease infection is reported to be prevalent
24
in tribal villages located in the Agasthyamala Biosphere Reserve forests of Western Ghats,
25
Kerala state, India. We carried out an investigation to characterize the species of Leishmania
26
parasites involved in these infections prevalent among one of the oldest human tribal populations
27
in India. Skin aspirates collected from 13 clinically diagnosed cases were subjected to
28
histopathological investigations, serological rapid tests using ‘rk39’ and molecular diagnostics.
29
Clinical manifestations recorded among patients were hypo-pigmented erythematous nodules/
30
papules on limbs and other parts of the body. Histopathological investigations of these skin
31
lesions among patients showed Leishman-Donovan (LD) bodies in macrophages. None of the
32
patients were found positive for ‘rk39’ tests, which detect active visceral Leishmaniasis. Using
33
three different genetic markers (kinetoplast minicircle DNA, 3’ UTR region of Heat shock
34
protein 70 (Hsp70) and Hsp70 gene) we identified the parasite species involved in these
35
infections to be Leishmania donovani. The 6-phosphogluconate (6-PGDH) gene sequences of the
36
parasite isolates from Western Ghats indicted close genetic relatedness to the L. donovani
37
isolates reported from Sri Lanka, also causing CL. This could be cited as another instance of
38
“local endemism” of organisms in this single “bio-geographic unit”.
39 40 41 42 43
3 44
INTRODUCTION
45
Leishmaniasis, is prevalent in 98 countries and about 310 million population live under the risk
46
of infection of this neglected tropical disease globally. About 12 million people are infected, with
47
an annual incidence of about 1.3 million cases. Cutaneous leishmaniasis (CL) is the most
48
common clinical manifestation, affecting about 1 million people every year. (WHO, 2013).
49
However, visceral leishmaniasis (VL) remains the serious form of infection. Also, this is the
50
most important variety of leishmaniasis infection in India. Bihar, West Bengal, Jharkhand and
51
Uttar Pradesh are the endemic states of VL in India (Alvar et al., 2012). In addition, sporadic
52
cases have been recorded from the foothills of the Himalayas (Desjeux, 1991), Gujarat and
53
Kerala (Kesavan et al., 2003; Sharma et al., 2007). Thirty three thousand one hundred and eighty
54
seven and 20600 cases of VL were recorded in India during 2011 and 2012 respectively
55
(NVBDCP1). Leishmania donovani is the major parasite involved in VL and Phlebotomus
56
argentipes is reported as the major vector species involved in transmission (Kumar et al., 2001).
57
CL is reported mainly from the Northwestern region of India bordering Pakistan (Rajasthan &
58
Punjab) (Sharma et al., 1973; Aara et al., 2010) and in some other small pockets in Himachal
59
Pradesh (Sharma et al., 2005). The parasite involved in the infection is L. tropica and L.
60
donovani (Sharma et al., 2005; Kumar et al., 2007). Mucocutaneous forms of Leishmaniasis
61
cases caused by L. donovani have been reported from Himachal Pradesh (Sharma et al., 2005).
62
CL caused by L. donovani has been recorded as a major problem in the adjacent country, Sri
63
Lanka (Siriwardana et al., 2007; Ranasinghe et al., 2012; Gajapathy et al., 2013).
64
A recent preliminary study reported the prevalence of CL in two tribal settlements in the Western
65
Ghats of Thiruvananthapuram District, Kerala State (Simi et al., 2010). They reported 12 active
4 66
CL cases from the region based on clinical examinations. Also, histopathological examinations
67
showed LD bodies in 4 cases. Based on this report, we initiated a study to elucidate the
68
epidemiological factors involved in the indigenous transmission of this neglected disease in the
69
tribal belt located in the protected Agasthymala, Biosphere forest region of southern India. Here,
70
we report the characterization of the Leishmania species involved in the human CL infections.
71
Furthermore, studies are in progress about the transmission dynamics of the CL infections in the
72
region. The region is thickly forested and do not have transport accessibility. About 1500 tribal
73
people live in 28 hamlets deep in the forest in this region. Most of these people dwell in thatched
74
huts constructed with mud, peeled stem of bamboo, palm trees or sticks. Electricity supplies to
75
the region are by installing solar cells in each hutment. One or two dogs are domesticated by
76
each tribal family, especially to protect them from attack of wild animals in the region.
77 78
METHODS
79
Clinical Examination:
80
Seven hundred and sixty eight persons residing in 28 hamlets were examined for the clinical
81
manifestations of leishmaniasis during field visits of medical officers to these villages. The
82
persons having suspected active CL lesions were advised to visit as outpatients to the Govt.
83
Medical College and Gokulam Medical College hospitals, Thiruvananthapuram, Kerala. They
84
were clinically examined and required treatment was administered. Ethical clearance for the
85
study was obtained from the Institutional Ethical Committee of Vector Control Research Centre
86
(Indian Council of Medical Research), Puducherry, India.
87
Histopathology:
5 88
Histopathological examination of skin biopsy specimens was carried out in the Departments of
89
Pathology on 13 cases who visited the hospitals as outpatients. After identifying a relatively new
90
skin lesion, 1% lignocaine was infiltrated with it and by using a surgical blade a linear incision of
91
about 0.5 cm, reaching up to the level of dermis was performed. Then the blade was turned 900
92
and the dermal pulp was scraped out using the blunt end of the knife taking care not to induce
93
bleeding. This pulp was smeared onto a glass slide and was stained with Geimsa and observed
94
under the microscope to visualize the granuloma and identify the amastigote stages of
95
Leishmania parasites - Leishman-Donovan (LD) bodies.
96
Molecular studies:
97
Aspirates from skin nodules/lesions of all the samples were also subjected to PCR based analysis
98
for Leishmania infection at the Vector Control Research Centre Field Station, Kottayam, Kerala,
99
India. Parasite DNA was extracted using Genelute Mini-prep Kit (SIGMA-ALDRICH, USA),
100
following the kit protocol. Briefly, the skin aspirates from all 13 cases were lysed and treated
101
with RNase A. The lysate was loaded and passed through Mini-prep columns so that DNA binds
102
to the column silica membranes. The DNA was eluted with de-ionized distilled water and was
103
subjected to diagnostic mini-circle kinetoplast DNA (k-DNA) semi-nested PCR using DNA
104
primers (Aransay et al., 2000). This PCR amplifies k-DNA in 8 species of Leishmania parasites
105
and groups them into two categories, 1) major group comprising of L. major and L. aethiopica
106
which yield a band of about 650 bp and 2) infantum group comprising of L. infantum, L. tropica,
107
L. donovani, L. amazonensis, L. mexicana and L. braziliensis which amplify a fragment of about
108
720 bp. Requena et al., (2012) proposed a PCR-RFLP analysis of the UTR region of the Heat
109
shock protein 70 (Hsp70) for species characterization of Leishmania species belonging to both
110
Old world and new world species. Among old world species it clearly distinguishes between L.
6 111
tropica and L. donovani complex by restriction digestion with Hae III. Hence we carried out this
112
analysis on the positives and the 750 bp fragment amplified was digested with the restriction
113
enzyme and the restriction fragments were resolved in a 3.0% agarose gel to type the species
114
involved. Also, a larger fragment of Hsp70 gene was amplified and sequenced to determine the
115
exact species involved in the infections (Montalvo et al., 2010). Hsp 70 gene fragment amplified
116
was about 1400 bp and could be used to determine the exact species either by RFLP or DNA
117
sequences analyses. A partial sequence of phosphogluconate (6-PGDH) gene, a highly variable
118
gene was also amplified using DNA primers already reported (Ranasinghe et al., 2012).
119
Phylogenetic analyses were carried out with Hsp70 gene sequences as well as partial 6-PGDH
120
gene sequences isolated in this study and those available in the GenBank from different countries
121
(Ranasinghe et al., 2012; Zhang et al., 2013), using maximum likelihood analysis (bootstrapping
122
with 1000 replications) to understand the genetic lineage of the parasite. Thus, four robust
123
genetic markers were used in the study to confirm the species involved in the infections.
124
Serological studies:
125
‘rk39’ serological rapid test had been found very useful for rapid diagnosis of active
126
visceral Leishmaniasis infections (Badaro et al., 1996). This rapid immunochromatographic test
127
diagnoses VL cases with 97% specificity and 100% sensitivity (NVBDCP2). We carried out this
128
test in all the 13 cases examined in the study following the national guidelines of Government of
129
India (NVBDCP2).
130 131
RESULTS
132
Clinical manifestations:
7 133
A total of 27 cases were recorded with suspected symptoms of CL. None reported clinical
134
manifestations of VL. Among these 12 had healed dermal scars of CL lesions. Among 15 cases
135
with active lesions, 13 visited hospitals for follow up and treatment. The major clinical
136
manifestations recorded among these 13 patients (Table 1) were multiple hypo-pigmented to
137
erythematous papules with a few showing studded erythematous nodules or ones with erosions,
138
forming ulcers in the upper/lower limbs, head, ear pinna, shoulder, trunk etc. (Fig. 1).
139
Histopathology:
140
Skin smears examined under the microscope showed epidermis with different degrees of
141
keratosis to atrophy. Dermis showed granulomas with diffuse infiltration by lymphocytes, few
142
epithelioid cells and macrophages. Isolates from four patients had L-D bodies in the
143
macrophages. (Fig. 2).
144
Molecular studies:
145
Diagnostic PCR analysis (k-DNA) showed five patients to be positive for Leishmania infection.
146
Among these, 3 were positive for L-D bodies also on histopathological examination. The size of
147
the k-DNA fragments amplified was about 720 bp. indicating that the species involved belonged
148
to the L. infantum group (Aransay et al., 2000). 3’ UTR region of Hsp70 gene of all these
149
positive samples were amplified which yielded about 750 bp fragments. On restriction digestion
150
with Hae III a pattern similar to that reported to L. donovani/ L. infantum was recorded (4
151
fragments of about 450 bp, 170 bp, 125 bp and 25 bp (very faint)) (Fig. 3). Phylogenetic analysis
152
of Hsp70 gene sequences of Leishmania parasite isolates indicated the species to be L. donovani
153
(Fig. 4).On further analysis of DNA sequences it was found that all five had a Cytosine molecule
8 154
at 434 position of the Hsp70 gene sequences, indicating the specimens belonged to the species L.
155
donovani. L. infantum isolates have a Thymine molecule in the position 434 of Hsp70, making
156
that a restriction site of the restriction enzyme Mlu I (Montalvo et al., 2010). The GenBank
157
accession numbers of Hsp70 sequences of our isolates are KC884001, KF562068-KF562071.
158
GenBank BLAST analysis showed 100.0% identity for these gene sequences with L. donovani
159
isolated from VL cases in India (JQ990221), China (JX021428) and African countries
160
(FN395027 & FN669773). Phylogenetic analysis carried out with the 6-PGDH gene amplified
161
for two positive samples in the study showed their genetic lineage to be closely related to the Sri
162
Lankan strains of L. donovani (Fig. 5). The Kimura 2 Parameter (K2P) genetic distance
163
calculated among “Kerala/Sri Lanka group” of CL strains was found to be zero. The 737 bp
164
partial DNA sequences of the 6-PGDH gene (KJ461872 & KJ461873) exactly matched with the
165
gene sequences of the Sri Lankan isolates. The K2P genetic distance between this and the “north
166
Indian/Bangladesh” group (AJ88899-AJ888901) was found to be 0.003. Also, a unique transition
167
event in the 976th nucleotide position (A G) resulting in a non-synonymous mutation “N326D”
168
was recorded in “Kerala/Sri Lanka” group of isolates compared to the “north Indian/Bangladesh”
169
isolates (VL). Isolates from China and Kenya included in the analysis belonged to the same
170
genetic lineage of “Kerala/Sri Lanka” group. However, the former isolates were from VL cases.
171
None of the 13 cases were found positive with rk39 kits, clearly confirming the cases included
172
did not have detectable levels of antibodies in their blood.
→
173 174
DISCUSSION
9 175
Agasthyamala biosphere reserve (area: 3500.36 Km2) spanning both Kerala and Tamil Nadu
176
states of India is located in the Western Ghats, the second largest mountainous belt in India is a
177
UNESCO World Heritage site (UNESCO, 2007). It is the abode of one of the oldest surviving
178
ancient tribal populations (“Kanikarans”) in the world. It is also designated as one of the world’s
179
eight hotspots of biodiversity on the globe (Myers et al., 2000). The current investigation on the
180
28 tribal settlements of this biosphere reserve indicated endemic transmission of L. donovani
181
among the ‘Kanikaran’ tribal population, causing CL. The first investigation of the prevalence of
182
this disease in this region was carried out by Simi et al. 2010. The investigators made known the
183
active transmission of CL by clinical and histopathological investigations in two tribal villages of
184
Thiruvananthapuram District, never recorded earlier in the literature. The region is inaccessible
185
to modern developmental activities and clinical facilities and is located deep in the evergreen
186
forests of Western Ghats and hence remained “isolated” for decades. These hamlets were never
187
in the records of the state Department of Health services, Kerala state, for any past activity of
188
vector control operations. Also, the region belongs to one of the protected bio-reserves of
189
international interest, with very restricted human interventions from outside. Thus, it could be
190
ascertained that the transmission of CL in this region would have remained unnoticed for many
191
decades.
192
L. donovani, the dominant parasite of visceral leishmaniasis in other parts of the country brings
193
about cutaneous manifestations in the tribal population of this undisturbed ecosystem remains
194
intriguing. None of the inhabitants clinically surveyed in these villages (n=768) exhibited the
195
typical symptoms of VL. All the 13 cases included in the investigations were tested negative for
196
“rk39” rapid tests for VL. The nucleotide sequences of both Hsp70 as well as 6-PGDH genes of
197
L. donovani isolates from Kerala exactly matched with that of neighboring Sri Lankan isolates.
10 198
The zymodeme classification of the strain recorded in tribal villages of Kerala may be
199
“MON37”. The unique mutation recorded in the 976th position of 6-PGDH gene (responsible for
200
the change from the uncharged Asparagine to negatively charged Aspartic Acid which affects the
201
mobility of the 6-PGDH iso-enzyme) recorded among these L.donovani isolates was ascertained
202
to the zymodeme “MON37” which causes CL in Sri Lanka (Siriwardana, et al., 2012). P.
203
argentipes, the established vector species of L. donovani (Tiwary et al., 2012) was found to be
204
abundant in the study area. A similar situation had been recorded from Sri Lanka also
205
(Siriwardana et al., 2007). P. argentipes was found to be the vector species prevalent in this
206
region (Gajapathy et al., 2013).
207
Terrestrial links between ‘montane’ regions of Sri Lanka and Western Ghats existed during
208
Pleistocene era (Rohling et al., 1998). These two areas (about 400 Km apart and currently
209
separated by Palk Strait) are grouped as a single biodiversity hotspot by the International Union
210
for Conservation of Nature (IUCN) as they form a community of species that fits together as a
211
“single bio-geographic unit” (Myers, et al., 2000). Few studies have already analyzed the genetic
212
similarity of different species of organisms from both regions and concluded that ‘the overall
213
limited biotic interchange has left both the areas with an unexpectedly large number of
214
endemics’ (Bossuyt et al., 2004; CEPF, 2007). The genetic similarity of many organisms
215
recorded between the wet zone of Sri Lanka and the moist forests of the Western Ghats
216
(Conservation International, 2008) may be owing to the inability of rainforest organisms to
217
disperse across intervening dry lowlands (Bossuyt et al., 2004). Genetic drift between the
218
organisms living in these conserved rainforest ‘montane’ regions owing to the geographical
219
isolation may be negligible since these remain isolated as virgin forests without intervention
220
from developmental activities. The villages included in this study are located deep inside the
11 221
protected forests with about a 10 km inaccessible thick forested mountainous region barrier, also
222
a natural reserve of wild animals. The genetic relatedness of L. donovani isolates from the
223
Western Ghats region with that of Sri Lankan isolates recorded in the present study could be
224
cited as another instance of local endemism of a parasite species in this ‘single bio-geographic
225
unit’. The Kani tribes, from whom these parasite species were isolated, live exclusively in the
226
ever green rain forested region of Western Ghats and are hesitant to migrate to the non-forested
227
regions of the state, in spite of many government schemes to re-habilitate them.
228
The present study evinces active transmission of CL in the tribal settlements of Western Ghats
229
caused by L. donovani. The genetic lineage of the parasite was found closely associated with the
230
isolates from the adjacent Country Sri Lanka, which forms a single bio-geographic unit along
231
with the Western Ghats. The vector species involved may be P. argentipes, as this established
232
vector species for L. donovani was found abundant in the region. Further studies to confirm this
233
and to determine whether any reservoir hosts are involved in the maintenance of transmission in
234
the region is in progress. Chinese and African strains of L. donovani were found closely related
235
genetically to the isolates from “Kerala/Sri Lanka”. Nevertheless, they cause visceral
236
manifestations. To prevent the remote possibility of spread of L. donovani from its isolation in
237
the deep forests, to the thickly populated lowlands of Kerala state, urgent intervention measures
238
to contain and eradicate these foci of Leishmaniasis transmission is of utmost importance.
239 240
ACKNOWLEDGEMENTS
12 241
The authors are grateful to Dr. T. Dileep Kumar, Directorate of Health Services, Govt. of Kerala,
242
for his suggestions and to Shri. P. M. Ajithlal, Vector Control Research Centre (VCRC) Field
243
station, Kottayam and Shri. B. Edwin, VCRC Puducherry for the technical assistance rendered.
244
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Table 1: PCR and histopathology results of skin nodule aspirates from suspected Leishmania cases – tribal villages of Thiruvananthapuram Sl. No
Date of colln.
*ID. No. (OP-MCH)
Village
Clinical symptoms and Provisional Diagnosis
Histopathology
PCR (k-DNA) result (Leishmania)
1
19/10/2012
273400/12
10F
Thazheamala
No LD bodies Late lesion of CL
Negative
273398/12
38F
Thazheamala
273402/12
17F
Thazheamala
Macrophages show LD bodies, consistent with CL No significant pathology
Positive
19/10/2012
4
19/10/2012
273399/12
44F
Meleamala
No LD bodies, No features seen to suggest CL.
Positive
5
19/10/2012
273397/12
75F
Thazheamala
Positive for LD bodies. Consistent with CL
Positive
6
19/10/2012
273396/12
50M
Thazheamala
No LD bodies, Late lesions of CL
Negative
7
30/3/2013
66332/13
13F
Kombidi
Papules on exposed parts of body (limbs and upper chest) for six months. Most of the lesions are erythematous and 5-10mm in diameter. One well defined erythematous plaque of 1cmX2cm size on forehead for six months Two erythematous papules on left forearm with 0.5cm diameter for six months Two lesions, hypo-pigmented patches on left ear lobe and right side of fore head. History of diagnosis and treatment for CL one year before for the same lesions An erythematous nodule on right forearm 2X3cm in size for 8 months and another lesion on right leg, hypo-pigmented plaque of 2X2cm. Hypo-pigmented patch on right shoulder 3X4cm. History of treatment for CL one year before Two well defined hypo-pigmented nodules no surface changes on the lateral aspect of right arm. CL
2
19/10/2012
3
Macrophages with LD bodies. Consistent with CL
Positive
Age/ Sex
Positive
18
8
30/3/2013
66328/13
22M
Thazheamala
9
30/3/2013
66330/13
10M
Thazheamala
10
26/4/2013
88297/13
28M
Thazheamala
11
26/4/2013
88298/13
26M
Podium
12
26/4/2013
88299/13
25M
Thazheamala
13
26/4/2013
Gokulam Medical College, TVPM
77F
Kadackal
Asymptomatic hypo-pigmented macules and erythematous to skin colored small plaques over upper back and forearms 3 years. Suspected CL & T. versicolor Asymptomatic hypo-pigmented to reddish papules over body – 2 years. Erythematous papules on upper limbs and chest with 5-10mm in size for 6 months
Macrophages with LD bodies
Negative
No LD bodies
Negative
Epithelioid granuloma with lymphocytic infiltration. No LD bodies Single well defined hypoEpithelioid pigmented plaque on upper limbs granuloma with 1X2cm in size for one year lymphocytic infiltration. No LD bodies Erythematous papules on upper Epithelioid limbs with 5-10mm in diameter for granuloma with 3 months lymphocytic infiltration. No LD bodies Nodular skin lesions on both the Epithelioid forearms. 2-3cm in diameter for last granuloma with 6 months lymphocytes. No LD bodies seen
*outpatient identification number, Government Medical College, Thiruvananthapuram (TVPM), India
Negative
Negative
Negative
Negative
19
Legends to Figures:
Fig. 1. Photographs of typical cutaneous lesions of CL patients from the study area. Fig. 2. Histopathological examination of skin aspirates from CL patients (Fig. 2a shows the granulomatous macrophages and is at 40 X magnification; Fig 2b is a 1000 X magnification of the 2a which show amastigote stages of Leishmania parasites). Fig. 3. Restriction digest of 3’ UTR region of Hsp70 gene. (fragment with 25bp size is very faint in the agarose gel photograph). The depicted pattern indicates the species to be either L. donovani or L. infantum (infantum group). Fig. 4. Phylogenetic analysis of the Hsp70 gene of Leishmania parasite isolates from Kerala. Fig. 5. Phylogenetic analysis of the 6 PGDH gene of the Leishmania isolates from Kerala.
