Molecular Identification of The Parasites Causing Indian Kala-Azar

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Molecular Identification of The Parasites
Causing Indian Kala-Azar

• Madhumita Manna
• Associate Professor
• Dept. of Zoology
• Bethune College
• Govt. of West Bengal
• Kolkata, India

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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

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• Cutaneous Leishmaniasis by L.tropica complex

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

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

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

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THE DISCOVERERS
Charles Donovan
Sir Ronald Ross named the parasite
William Boog Leishman
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THE SAVI0UR
Lives of hundreds of thousands were saved in
British India using U.N. Brahmacharis Urea
Stibamine
Urea stibamate
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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

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CURRENT SCENARIO

• 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

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KALA-AZAR IN INDIA
Presently, in India, 33 districts endemic in
Bihar, 11 districts in West Bengal, 4 districts
each in Jharkhand UP.
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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

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9th August, 2012
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• 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

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• 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

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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
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• 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.

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.
Amplified genomic DNA of clinical isolates with
primers OPA8 OPA3 Lanes1, MW 2,DD8
3,S24,S45,P16,T27,T58,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.
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THE PRESENT STUDY

• 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

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RFLPs of ITS ITS1
LITSR
L5.8SR
ITS1
ITS2
SSUrRNA
5.8s RNA
LSU RNA
LITSV
L5.8S
700-750 bp
300-350 bp
ITS1
ITS2
Target sequences ITS, ITS1
Schematic representation of the internal
transcribed spacer (ITS) in the ribosomal operon
with primers amplifying different parts of the
spacer. SSU small subunit rRNA gene LSU large
subunit rRNA gene

• 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

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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

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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
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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
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Phylogram based on ITS1 sequence alignment
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)
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)
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THE OBSERVATION

• 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.

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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

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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

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THANKYOU