Dr. sanjay s negi leishmaniasis
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  • The lesion generally commences as a small papulopustular swelling of the skin localised in the region of nostrils, mouth or eyes or widespread on the face, ears, elbows and knees. <br /> After a variable period of time the Mucosal surface of the mouth and nose are involved causing destructive and mutilating erosions. <br /> Some time the whole of the nasal septum are involved. <br /> Nasal mucous membrane, pharynx, larynx and upper lips are involved. Sometimes whole of the nasal septum is destroyed. Granulomas develop at mucocutaneous junction followed by gross destruction of soft tissue and cartilage causing disfigurement of nose and mouth. Death may occur from severe respiratory infection due to respiratory acute obstruction. <br />
  • A classical lesion shows a nodule at the site of inoculation followed by a formation of a central crust. The crust may fall away exposing a wet type of ulcer. <br /> A depressed scar and altered pigment develops on healing of that nodule at the edge of lesion are characteristic features. <br /> Or <br /> CL may be presented with papulonodular lesion covered by sperficial scales(dry type of ulcer). <br /> Whole of the nasal septum is destroyed producing the typical tapir or camel nose. <br /> Nasal mucous membrane, pharynx, larynx and upper lips are involved. Sometimes whole of the nasal septum is destroyed. Granulomas develop at mucocutaneous junction followed by gross destruction of soft tissue and cartilage causing disfigurement of nose and mouth. Death may occur from severe respiratory infection due to respiratory acute obstruction. <br />
  • Profile view of a teenage boy suffering from visceral leishmaniasis. The boy exhibits splenomegaly, distended abdomen and severe muscle wasting.  <br /> Pyrexia: Must say One of the early symptom <br /> Splenic enlargement: One of the most striking features <br />
  • The lesion commences as a papulopustular swelling of the skin localised in the region of nostrils, mouth or eyes or widespread on the face, ears, elbows and knees. <br /> After a variable time the mucosal surface of the mouth and nose are involved causing destructive and mutilating erosions. Sometimes the whole of nasal septum is destroyed. <br />
  • Amastigote stage is present in man only <br /> Promastigote stage is present in Sandflies as well as cultures. <br />
  • The cells of the R.E. system of the affected organs proliferate and become heavily parasitised, accompained by an increase in the IgG fraction of the serum gamma globulin(hypergammaglobulinaemia) which however is not protective. <br /> The increase in gamma globulin causes a reversal of albumin-globulin ration. <br />
  • In variably cause infection once bite any susceptible person <br />
  • Must talk of Leishmanin tests. <br />
  • Drawback with the culture: Slow and takes a long time(about one month) <br /> L.Donovani can be cultured in a medium composed of two parts of salt agar and one part of defibrinated rabbit’s blood. <br /> The medium was first introduced by Novy, MacNeal and later modified by Nicolle. <br />
  • Average total count is 3000 to 7-8000 per mm3 and there is progressive diminution during the course of the disease, the count falling to 1000 per mm3. <br /> Must talk about drawback of Aldehyde test. <br /> There is a reversal of albumin:globulin ratio <br />

Dr. sanjay s negi leishmaniasis Dr. sanjay s negi leishmaniasis Presentation Transcript

  • LEISMANIASIS DR. SANJAY SINGH NEGI ASSISTANT PROFESSOR, DEPARTMENT OF MICROBIOLOGY, AIIMS, RAIPUR
  • SYNONYMS kala azar, black fever, sandfly disease, Dum-Dum fever and espundia. 1903 – Sir William Leishman discovered L. donovani in spleen smears of a soldier who died of fever at Dum-Dum, India. The disease was known locally as Dum-Dum fever or kala-azar. 1903 – Charles Donovan found same parasite in a spleen biopsy. •Cultured by Rogers(1904) •Amastigote and promastigote forms were described by Batton(1907) •Phlebotomus argentipes identified as vector.
  • AMASTIGOTES PROMASTIGOTES
  • Leishmania Phylum Sarcomastigophora Order Kinetoplastida Family Trypanosomatidae Genus Leishmania • Transmitted to the mammalian hosts by the bite of infected sandflies, Phlebotomus and Lutzomyia In India: Phlebotomus argentipus
  • • 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, India(Central & Western India) • 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)
  • Leishmania distribution (all species)
  • SITUATION IN INDIA • 40-50% of global burden (Bora 1999, Natl Med J India) • • INDIA: 15538 cases and 47 deaths by VL (2010) • Endemic states in Eastern India: Bihar, Jharkhand, West Bengal, Assam, Orissa, Tamil Nadu, Uttar Pradesh • (NVBDCP, 2010) Surveillance being done by NVBDCP Estimated 165.4 million population at risk in 4 states
  • Vector: Phlebotomus
  • procyclics and metacyclics (detached) (attached) • Infected macrophages are taken up with the blood meal and amastigotes are released by digestion, transform into procyclic promastigotes and attach to the midgut epithelium • Attached promastigotes divide rapidly (procyclics are not infective to mammals) • Metacyclic (infective) promastigotes cease replication, detach and pass forward into the pharynx from where they are regurgitated into the bite site
  • TYPES OF LEISMANIASIS • VISCERAL LEISHMANIASIS or Kala-azar ( Middle east, Africa, Bangladesh, Brazil, India,China, South America, Europe, Nepal and Sudan) • Post kala azar dermal leishmaniasis (Endemic to India and the Sudan) • India(Bihar, West bengal, Orissa, Assam, Tamil Nadu, Gujarat, Punjab & Jammu) • Species responsible: L.donovani • Vector: P.argentipes • Resvoir: Man
  • Other species causing Visceral Leishmaniasis Leishmania infantum: Cause Zoonotic visceral leishmaniasis (ZVL) in Mediterranean areas, Middle east, and China Reservoir: Dogs, foxes and jackals Leishmania chagasi: Zoonotic visceral leishmaniasis(ZVL) in New World. Reservoir: Dogs and foxes
  • Pathogenicity of Leishmania donovani(Visceral leishmaniasis • • • • • • • • • • • Weeks to months incubation period. May exceed one and sometimes two years. Clinical features. High fever. Fever often oscillates with a peak every second day The lead symptom is abdominal swelling due to hepato- and splenomegaly Lymphadenopathy Progressive drastic weight loss (kachexia). Epistaxix(presenting symptom) In fully developed cases, emaciation and anaemia become noticeable. Darkening of the skin Mortality of untreated disease 75-95%
  • Hepatosplenomegaly and emaciation.
  • • Enlarged spleen and liver in an autopsy of an infant dying of visceral leishmaniasis.
  • Post Kala Azar Dermal Leishmaniasis • • • • • • • • • Non ulcerative cutaneous lesion prevalent in endemic areas of kala azar in India. In 10 % treated cases of Kala azar, Normally develops <2 years after recovery(When Visceral infection disappears but skin infection persists). Clinical feature: Depigmented macules: earliest lesion, trunk, extremities and face Erythematous patches: Nose, cheeks and chin Yellowish pink nodules: Nodules mostly on face & are soft, painless granulomatous growth of varying sizes(Absences of ulceration is a noticeable feature) Do not heal spontaneously. Recrudescence Restricted to skin Extensive nodular lesion, resembling lepromatous leprosy. A very resistant case cured after long continued treatment
  • CUTANEOUS LEISHMANIASIS (Oriental Sore /Old World Cutaneous Leishmaniasis PKDL): (Middle east, Mediterranean areas, N.Africa, N.W. India and Pakistan) Cause dry type of cutaneous lesion(non-ulcerating type) Urban distribution Incubation period (2 months to > year) Lesion usually facial. Ulcer starts as small itching papule covered with fine whitish scale which subsequently becomes thick, dark and finally falls off. The lesion may be found on the face, feet, legs and arms. Children are usually affected. Reservoir: man, domestic dog VL in exceptional cases Species: Leismania tropica Leishmania major: Cause a moist type local cutaneous lesion(Ulcerating type). Ulcer found on extremities with regional lymphadenitis. Incubation period 2-6 weeks
  • Leishmania tropica (Cutaneous Leishmaniasis or Oriental Sore or Tropical Sore) Life Cycle: Same as L.donovani except amastigote form resides in large mononuclear cells of the skin and not in the viscera. Reservoir: Dogs Clinical features: Cutaneous lesion begins as a raised nodule, its ulcerates, heal spontaneously taking about 6 months or more by Ulcer filled up by granaluation tissues and a depressed white scar is often left. Laboratory Diagnosis: Smear made from specimen obtained by puncture of indurated edges of the sore and stained by Leishman method. Leishmanin reaction Oriental Sore on the face)
  • DIFFUSE CUTANEOUS LEISHMANIASIS / MUCO CUTANEOUS LEISHMANIASIS / New world Cutaneous Leishmaniasis OR ESPUNDIA Geographical Distribution: Central and South America (Bolivia, Brazil,Argentina, Columbia,Mexico, panama, Paraguay, Venezuela and Peru. Species: Leishmania braziliensis Clinical feature: Two stages primary cutaneous lesion followed by secondary mucosal involvement which occurs after a variable time of latency of primary cutaneous lesion Nasal mucous membrane, pharynx, larynx & upper lip are involved. mouth and throat cavities Develops in 5 % patients suffering from primary cutaneous lesion. Diagnosis by demonstrating amastigote forms of L.brazilensis in skin and mucocutaneous lesions
  • Leishmania donovani Morphology • Promastigote – – – Insect Motile Midgut Digenetic Life Cycle Flagella Kinetoplast Golgi Nucleus Cytoskeleton • Amastigote – Mammalian stage – Non-motile – Intracellular
  • • Leishmania (Leishman-Donovan or LD bodies). Lying in macrophage cells from liver. Giemsa. ×12000. Enlarged by 9.6.
  • • A macrophage filled with Leishmania amastigotes.
  • • Amastigotes (*) of Leishmania donovani in the cells of a spleen. The individual amastigotes measure approximately 1 µm in diameter.
  • Susceptible Animal: Dog naturally infected with L.donovani Common laboratory animals mice, rats and guinea pigs not susceptible. Hamster very susceptible.
  • Leishmania infects and thrives in macrophages • • • • • Macrophages are important “microbe killers”, however several pathogens have found ways to escape killing Trypansoma cruzi -- induces phagocytosis but then escapes into the cytoplasm Toxoplasma -- active invasion, parasitophorous vacuole is never part of the endocytic pathway Mycobacterium tuberculosis -induce phagocytosis and block lysosomal maturation Leishmania ...
  • Leishmania parasites exist Amastigote & Pro mastigote) • The parasite lives in the digestive tract of sandflies as extracellular promastigote • In the mammalian parasites multiply intracellular amastiogotes host as
  • LIFE CYCLE
  • 1. Leishmaniasis is transmitted by the bite of female phlebotomine sandflies. The sandflies inject the infective stage, promastigotes, during blood meals. 2. Promastigotes that reach the puncture wound are phagocytized by macrophages. 3.They transform into amastigotes. 4. Amastigotes multiply in infected cells and affect different tissues. 5. Sandflies become infected during blood meals on an infected host when they ingest macrophages infected with amastigotes. 6. In the sandfly's midgut, the parasites differentiate into promastigotes. 7. They multiply and migrate to the proboscis.
  • Other method of transmission Congenital infection of a child in utero Transmission by blood transfusion Transmission by inoculation of cultures of L.donovani Possibly transmission during coitus.
  • DIAGNOSIS
  • Direct evidence: Demonstration of Leishmania Specimens that may be collected • Splenic aspirate and biopsy • Bone marrow (Sternum or iliac crest) • Blood buffy coat • Liver biopsy • FNAC and biopsy • Tegumantary leishmaniasis- dermal scrapings, sections from skin biopsy DEMONSTRATION OF Leishmania AMASTIGOTES/ L.D. BODIES
  • MICROSCOPY
  • CULTURE Culture media for axenic culture • SOLID MEDIUM  NNN medium (Novy, MacNeal & Nicolle) (2ml of patient blood + 10ml of Citrated saline kept at 22 0 C overnight and deposit inoculated into the water of condensation of NNN medium and incubated at 220 C for 1 to 4 wks  Evan’s modified Tobie’s medium • LIQUID MEDIA  Schneider’s Drosophila medium  Grace’s insect tissue culture medium DEMONSTRATION OF Leishmania PROMASTIGOTES Animal inoculation • Golden hamsters inoculated intraperitoneally
  • Promastigotes as seen in artificial culture medium
  • IMMUNOLOGICAL METHODS (Indirect Evidence) • Leishmanin test: 0.1 to 0.2 ml of a suspension(having 6 to 10 million promastigotes/ml) injected intradermally. Positive reaction after 72 hours in cured kala azar cases 6 to 8 wks after recovery. • Blood Count: Leucopenia(Neutropenia) • Aldehyde (formol gel) Test (Napier)(To test the rise of gamma globulin): 1 to 2 ml of a serum + one or two drop of 40% formalin: Jellification of milk white opacity • Complement fixation test with W.K.K Ag.(Not used nowadays). • Other Serological tests: CIEP, IHA, IFA(Most Commonly Used), ELISA, Direct agglutination test and Latex particle agglutination test) • Molecular(PCR)
  • 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)
  • Direct Agglutination Test • • • Use of whole, stained promastigotes either as a suspension or in a freeze-dried form. The freeze-dried form is heat stable Utilized for field purposes • • • • Relative long incubation time of 18 hours Need for serial dilutions of serum No prognostic value Remain positive for several years after cure
  • 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)
  • ELISA BASED ASSAYS
  • 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
  • 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
  • GOAL OF NATIONAL HEALTH POLICY (INDIA) 2002 ELIMINATION OF KALA AZAR 2010
  • TREATMENT • SODIUM ANTIMONY COMPOUND: SODIUM ANTIMONY GLUCONATE(SAG 600mg daily for 6-10 days IV route) • PENTAMIDINE ISTHIONATE • AMPHOTERICIN-B • Phase III Trials with a first-generation vaccine (killed Leishmania organism mixed with a low concentration of BCG as an adjuvant) have also yielded promising results • Leishmania major mixed with BCG have been successful in preventing infection with Leishmania donovani.
  • 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