Molecular Identification of The Parasites

Causing Indian Kala-Azar

Madhumita MannaAssociate Professor

Dept. of ZoologyBethune College

Govt. of West BengalKolkata, India

THE PARASITE Leishmaniasis is a neglected tropical disease Leishmania sp is a kinetoplast protozoa, causing the disease

There are two forms –• Amastigote (infective stage in human)• Promastigote (insect form)

The disease is transmitted by female Sand flies

•PKDL is a sequel of Kala-azar after apparent cure in 10-20% cases.

•Cutaneous Leishmaniasis by L.tropica complex

•Mucocutaneous Leishmaniasis by L.mexicana complex, L.braziliensis

•Visceral Leishmaniasis or Kala-azar caused by Leishmania donovani complex

THE DISCOVERERS

Charles Donovan

William Boog LeishmanSir Ronald Ross named the parasite

THE SAVI0UR

Lives of hundreds of thousands were saved in British India using U.N. Brahmachari’s Urea Stibamine

Urea stibamate

Life Cycle of the parasite

•The Parasite shows digenetic life cycle

•Amastigote or aflagellated infective stage in Vertebrate macrophage (Humans are accidental host)

•Promastigote or flagellated stage present in the gut of the vector [the female sand fly]

• Once thought eradicated, the disease came back in Indian subcontinent with full vengeance

• Approx. 147 million people at risk with an estimated 100 000 new cases each year

• More than 90% of VL cases occur in five countries (Bangladesh, Brazil, India, Nepal and Sudan)

• VL is reported from 96 districts bordering Bangladesh, India and Nepal

CURRENT SCENARIO

KALA-AZAR IN INDIA

Presently, in India, 33 districts endemic in Bihar, 11 districts in West Bengal, 4 districts each in Jharkhand & UP.

THE PROBLEM

• Over 60% patients in India are not responding to the prevailing regimen of pentavalent antimonials, the first line drug against Kala-azar

• Amphotericin B and Pentamidine , the second line drugs are highly toxic

• Miltefosine, the oral drug is not within the reach of patients at least in West Bengal, India

9th August, 2012

• Historically it is known that Kala-azar in India is caused by Leishmania donovani

But

Time to time, there are reports on the

occurrence of different types of isolates causing Kala-azar

• Sacks et al (1995) claimed that Indian Kala-azar can be caused by L.tropica, a species related to cutaneous leishmaniasis

• The peculiar occurrence of both L. donovani and L.tropica in the Localized Cutaneous Leishmaniasis (LCL) patients from the northern part of India have also been reported by Sharma et al(2005)

• Khanra et al (2011) reported the association of L.tropica with the disease by constructing the RAPD profiles of recent isolates (2006-2010)

• Srivastava et al (2010) have claimed the association of Leptomonas sp. with Kala-azar

Identification of any parasite is a must For

Epidemiology and Taxonomy Proper drug regimen

Phylogenetic relationship analysis

Techniques employed for such studies are many Isozyme analysis: ‘Gold standard’

Random Amplified Polymorphic DNA Analysis

Ribosomal Internal Transcribed Spacer [ITS]-PCR

RFLPs of amplified targets [ITS, ITS1, hsp70 ]

• Earlier we have performed RAPD-PCR with 8 random primers taking nine clinical isolates of recent time collected from Bihar, West Bengal of India and Bangladesh.

Amplified genomic DNA of clinical isolates with primers OPA8 & OPA3

Lanes;1, MW ;2,DD8; 3,S2;4,S4;5,P1;6,T2;7,T5;8,K27.DD8 &K27 are WHO reference strains for L.donovani & L.tropica

[Khanra, S., Bandopadhyay, S.K., Chakraborty, P., Datta, S., Mondal, D., Chatterjee, M., Naskar, K., Roy, S., Manna, M., 2011. Characterization of the recent clinical isolates of Indian kala-azar patients by RAPD-PCR method. . J. Parasit. Dis. 35, 116-122].

We have collected KA and PKDL isolates from India and Bangladesh

•Indian clinical isolates from bone marrow aspirates of patients admitted in the Calcutta National Medical College, Kolkata. Patients were majority from Bihar and also from West Bengal.

•Bone marrow aspirates were confirmed with parasites by Giemsa stain and culture in media.

• Nine isolates were from Bangladesh.

•Two reference strains for L.donovani and L.tropica were taken for comparison

•RFLPs of ITS, ITS1 and hsp-70 amplicons were performed followed by ITS1 sequence alignment

THE PRESENT STUDY

RFLPs of ITS & ITS1

Upper panel: ITS-RFLP with Alu1 Lower panel: ITS-RFLP with Msp1 Lane1: Marker; lane 2, DD8; lanes 3-10, Clinical

isolates of KA & PKDL; lane 11, T5; lane 12, K27

5.8s RNA LSU RNAITS1 ITS2

LITSR L5.8SR

L5.8S

LITSV

Schematic representation of the internal transcribed spacer (ITS) in the ribosomal operon with primers amplifying different parts of the spacer.SSU = small subunit rRNA geneLSU = large subunit rRNA gene

Schematic representation of the internal transcribed spacer (ITS) in the ribosomal operon with primers amplifying different parts of the spacer.SSU = small subunit rRNA geneLSU = large subunit rRNA gene

300-350 bp 700-750 bp

Target sequences : ITS, ITS1Target sequences : ITS, ITS1

ITS1 ITS2

SSUrRNA

ITS1 –RFLP with HaeIII & Rsa 1

• The amplified ITS 1region of nuclear DNA of different clinical isolates of KA along with DD8 and K27 were digested with enzymes Hae III & Rsa 1

Lane1, MW marker (100 bp); lane 2, DD8; lanes 3-10, clinical isolates of KA and PKDL from India & Bangladesh,lane 11,T5; lane 12, K27

Hae III RFLP of hsp70

Hae III-RFLP of hsp 70 of one clinical isolate of Kala-azar (lane 11) showed profile similar to Leishmania tropica (lane 12) suggesting its association with the disease.Lane1, MW marker (100 bp); lane 2, DD8; lane 3, T2; lane 4, T3; lane 5, T-085; lane 6, T4; lane 7, T7; lane 8, Raj04; lane 9, Raj 05; lane 10, Raj07; lane 11,T5; lane 12, K27

The sequences of ITS1 of K27, T5 and DD8 K27 were subjected to CLUSTAL W (2.1) multiple sequence alignment analysis for the homology search

T5 (Genbank Accession no. JQ229828) was clearly grouped with L. tropica WHO strain, K27 (Genbank Accession no. JQ517279) and far apart from the other clinical isolates which were shown to be closely related to L. donovani WHO strain, DD8 (Genbank Accession no. AJ000292)

Phylogram based on ITS1 sequence alignment

Molecular typing of recent clinical isolates of Kala azar from India and Bangladesh confirms the association of L. tropica with the disease. Supriya Khanra, Sanchita Datta, Dinesh Mondal, Partha Saha, Subir K Bandopadhyay, Syamal Roy and Madhumita Manna (accepted in Acta Tropica)

This study showed that one of fifteen clinical isolates studied here, was grouped with L.tropica while rest were with L.donovani.

This finding put credence to the earlier reports that L.tropica causes Indian Kala-azar.

Systematic parasite typing is lacking in India assuming that all are L. donovani

•The molecular epidemiology of Indian Kala-azar should be taken into serious consideration as it has direct bearing with the suitable drug schedule to be referred to the patients, especially for drug unresponsive cases and

This is, in turn, related to design effective control programmes.

THE OBSERVATION

My Laboratory

1. SUPRIYA KHANRA, UGC fellow

2. SANCHITA DATTA, CSIR SRF

3. SANGITA LAHIRY, UGC DAE fellow

4. TULIKA JANA, summer trainee

5. AMRITA HALDER, summer trainee

ACKNOWLEDGEMENT

• The authors sincerely acknowledge the financial help from University Grant Commission, New Delhi.

• The authors are thankful to the Organisers of 3rd International Conference on Neglected Tropical Diseases, Dhaka, Bangladesh