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  1. 1. Leishmania Dr N P Singh ProfessorDeptt. Of Microbiology
  2. 2. Leishmania Phylum Sarcomastigophora Order Kinetoplastida Family Trypanosomatidae Genus Leishmania• Transmitted to the mammalian hosts by the bite of infected sandflies, Phlebotomus and Lutzomyia
  3. 3. • Currently, leishmaniasis occurs in 4 continents and is considered to be endemic in 88 countries, 72 of which are developing countries:  90% of all VL: Bangladesh, Brazil, India, Nepal and Sudan  90% of all MCL: Bolivia, Brazil and Peru  90% of all CL : Afghanistan, Brazil, Iran, Peru, Saudi Arabia and Syria• Annual incidence: 1- 1.5 million cases of CL : 500,000 cases of VL• Prevalence: 12 million people• Population at risk: 350 million (WHO, 2010)
  4. 4. SITUATION IN INDIA • 40-50% of global burden (Bora 1999, Natl Med J India) • Surveillance being done by NVBDCP • INDIA: 15538 cases and 47 deaths by VL (2010) • Endemic states in Eastern India: Bihar, Jharkhand, West Bengal, Uttar Pradesh • Estimated 165.4 million population at risk in 4 states (NVBDCP, 2010)
  5. 5. • On the basis of development, divided in two genera: Subgenus Leishmania: in the anterior part of alimentary tract of sandfly Subgenus Viannia: midgut and hindgut of sandflyLeishmania L. major complex (L. major) Old L. tropica complex (L. tropica, L. killicki) World L. aethiopica complex (L. aethiopica) L. donovani complex (L. donovani, L. infantum) L. donovani complex (L. chagasi) New L. mexicana complex World (L. mexicana, L. venezuelensis, L. garnhami, L. amazonensis, L.pifanoi)Viannia L. braziliensis complex (L. braziliensis, L. peruviana, L. columbiensis, L. lainsoni) L. guyanensis complex (L. guyanensis, L. panamensis)
  6. 6. Leishmania Parasites and Diseases SPECIES Disease Leishmania tropica Leishmania major Cutaneous leishmaniasisLeishmania aethiopica (CL)Leishmania mexicanaLeishmania braziliensis Mucocutaneous leishmaniasis (MCL)Leishmania donovaniLeishmania infantum Visceral leishmaniasis Leishmania chagasi (VL)
  7. 7. Digenetic Life CyclePromastigote Amastigote– Insect – Mammalian stage– Motile – Non-motile– Midgut – Intracellular
  8. 8. INFECTION Sub-clinical or inapparent infection Recovery DeathImmune to reinfection Concurrent infection PKDL
  9. 9. LEISHMANIASIS• Vector borne disease• A wide clinical spectrum  VISCERAL  PKDL  DIFFUSE CUTANEOUS  CUTANEOUS  MUCOCUTANEOUS
  10. 10. VL - Clinical Manifestation• Variable - Incubation 3-100+ weeks• Lowgrade fever• Hepato-splenomegaly• Bone marrow hyperplasia• Anemia, Leucopenia & Cachexia• Hypergammaglobulinnemia• Epistaxis , Proteinuria, Hematuria
  11. 11. • Most severe form of the disease, may be fatal if left untreated• Usually associated with fever, weight loss, and an enlarged spleen and liver• Anemia (low RBC), leukopenia (low WBC), and thrombocytopenia (low platelets) are common• Lymphadenopathy may be present• Visceral disease from the Middle East is usually milder with less specific findings than visceral leishmaniasis from other areas of the world
  12. 12. Post Kala Azar Dermal Leishmaniasis• Normally develops <2 years after recovery• Recrudescence• Restricted to skin• Rare but varies geographically
  13. 13. Cutaneous Leishmaniasis• Most common form• Characterized by one or more sores, papules or nodules on the skin• Sores can change in size and appearance over time• Often described as looking somewhat like a volcano with a raised edge and central crater• Sores are usually painless but can become painful if secondarily infected• Swollen lymph nodes may be present near the sores (under the arm if the sores are on the arm or hand…)
  14. 14. Cutaneous Leishmaniasis• Most sores develop within a few weeks of the sandfly bite, however they can appear up to months later• Skin sores of cutaneous leishmaniasis can heal on their own, but this can take months or even years• Sores can leave significant scars and be disfiguring if they occur on the face• If infection is from L. tropica it can spread to contiguous mucous membranes (upper lip to nose)
  15. 15. Mucocutaneous Leishmaniasis• Occurs with Leishmania species from Central and South America• Very rarely associated with L. tropica which is found in the Middle East - This type occurs if a cutaneous lesion on the face spreads to involve the nose or mouth - This rare mucosal involvement may occur if a skin lesion near the mouth or nose is not treated• May occur months to years after original skin lesion• Hard to confirm diagnosis as few parasites are in the lesion• Lesions can be very disfiguring
  16. 16. DIAGNOSIS
  17. 17. Direct evidence: Demonstration of LeishmaniaSpecimens that may be collected• Splenic aspirate and biopsy• Liver biopsy• Bone marrow (Sternum or iliac crest)• FNAC and biopsy• Blood buffy coat• Tegumantary leishmaniasis- dermal scrapings, sections from skin biopsy DEMONSTRATION OF Leishmania AMASTIGOTES/ L.D. BODIES
  18. 18. MICROSCOPY
  19. 19. CULTURECulture media for axenic culture• SOLID MEDIUM  NNN medium  Evan’s modified Tobie’s medium• LIQUID MEDIA  Schneider’s Drosophila medium  Grace’s insect tissue culture mediumDEMONSTRATION OF Leishmania PROMASTIGOTESAnimal inoculation• Golden hamsters inoculated intraperitoneally
  20. 20. Promastigotes as seen in artificial culture medium
  21. 21. IMMUNOLOGICAL METHODS• Aldehyde (formol gel) Test (Napier)• Antimony test (Chopra)• Complement fixation test with W.K.K Ag
  22. 22. Indirect Fluorescent Antibody test• Detection of anti-leishmanial antibody using fixed promastigotes• Demonstrated in the very early stages of infection and undetectable six to nine months after cure• Titers >1:20 are significant and above 1:128 are diagnostic• Cross reaction with trypanosomal sera (overcome by using Leishmania amastigotes as the antigen instead of the promastigotes)
  23. 23. Direct Agglutination Test• Use of whole, stained • Relative long incubation time promastigotes either as a of 18 hours suspension or in a freeze-dried • Need for serial dilutions of form. serum• The freeze-dried form is heat • No prognostic value stable • Remain positive for several• Utilized for field purposes years after cure
  24. 24. Modifications of DAT• Fast Agglutination Screening Test (Schoone et al, 2001)  Need of only 1 serum dilution  Rapid: results available in less than 3 hours• EasyDAT method (Gomez-Ochoa et al, 2003, Clin Diagn Lab Immunol)
  26. 26. Many antigens have been explored for the diagnosis of leishmaniasis:• Whole soluble antigens (Ld-ESM—Excretory, secretory and metabolic antigen by L.donovani)• Purified antigens such as fucose- mannose• Defined, synthetic peptides• Recombinant antigens  rGBP (L.major protein encoding a hydrophilic protein)  rORFF (L. infantum)  gp63  rK39  rK26, rK9  rKE16
  27. 27. rK39• Rapid dipstick test• Based on the recombinant k39 protein, a 39-amino acid cloned in Escherichia coli, from the C terminus of the kinesin protein of Leishmania major in India
  28. 28. • Case definition for enrolling a subject “A case presenting to a clinician with a fever of more than two weeks duration, with splenomegaly and not responding to the full course of anti-malarials”• Sensitivity-100%, Specificity-97% (NVBDCP, 2010)• Not to be used in the following cases:  Kala-azar relapses  In cases of kala-azar re-infection  Kala-azar and HIV co-infection• This test has been incorporated in NVBDCP for the diagnosis of VL in India.
  29. 29. Prevention• Suppress the reservoir: dogs, rats, gerbils, other small mammals and rodents• Suppress the vector: Sandfly • Critical to preventing disease in stationary troop populations• Prevent sandfly bites: Personal Protective Measures • Most important at night • Sleeves down • Insect repellent w/ DEET • Permethrin treated uniforms • Permethrin treated bed nets
  30. 30. Treatment Cutaneous and Mucocutaneous• Antimony (Pentostam®, Sodium stibogluconate) is the drug of choice • Given under an experimental protocol at Walter Reed Army Medical Center (WRAMC) • 20 days of intravenous therapy • Available at WRAMC for all branches of the military • Requires patient to come to WRAMC• Fluconazole may decrease healing time in L. major infection • Biopsy and culture to determine species is required • Six weeks of therapy is needed
  31. 31. Visceral Leishmaniasis• Liposomal amphotericin-B (AmBisome®) is the drug of choice • 3 mg/kg per day on days 1-5, day 14 and day 21• Pentostam® is an alternative therapy • 28 days of therapy is required• Although AmBisome® is widely available, the difficulty of accurate diagnosis and the potential severity of visceral infection suggest possible patients be referred to the Leishmania Treatment Center at WRAMC for maximal diagnostic efficiency
  32. 32. Vaccine• There is as yet no effective vaccine for prevention of any form of leishmaniasis.• first generation vaccine was prepared using whole killed parasites combined or not with BCG.• Live: including new genetically modified constructs• 1st generation vaccines: whole killed parasite with/without adjuvants or fractions of the parasite• 2nd generation vaccines: recombinant proteins, DNA vaccines & combinations