Lab 8 leishmaniasis


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  • Autochthonous أصيلة : formed or originating in the place where found. Autochthonous Leishmaniasis. Black fever ( darkening of the skin)= Kala azar
  • Members of the subfamily Phlebotominae are known outside of the United States by the name sand fly . This subfamily includes numerous genera of blood-feeding ( hematophagous ) flies. The Old World includes Africa , Asia , and Europe (collectively known as Afro-Eurasia ), plus surrounding islands. The term is in distinction from the New World , meaning the Americas and Australasia . Females Haematophagus Males sap feeders
  • Leishmaniasis, caused by 20 species of Leishmania and transmitted by 30 species of sand fly, is characterized by diversity and complexity1-4. Most of Leishmania infections are zoonotic and rodents and canids are reservoir host. Only two Leishmania species can maintain anthroponotic, human-human cycle, these species are L. donovani responsible for visceral leishmaniasis (VL) in Indian subcontinent and east Africa and L. tropica, which is responsible for cutaneous leishmaniasis (CL) in the Old World5. Female sand fly of genus Phlebotomus in the Old World and Lutzomyia in the New World are the only proven vector responsible for transmission of the disease. It occurs in 88 countries in tropical and temperate regions, 72 of them developing or least developed. An estimated 350 millions population is at risk and 10 million people are affected from this disease worldwide1. Two million cases occur annually.
  • Female sand flies have piercing mouthparts and subsist on mammalian blood, biting mostly at night.
  • Kala azar is an Indian expression means black fever.
  • The different species are morphologically indistinguishable, but they can be differentiated by isoenzyme analysis, molecular methods, or monoclonal antibodies.
  • nucleus (in red), a kinetoplast (usually perpendicular, in red to violet) and a pale blue cytoplasm.
  • The different species are morphologically indistinguishable, but they can be differentiated by isoenzyme analysis, molecular methods, or monoclonal antibodies.
  • VL: The disease is fatal when untreated, with death generally occurring 1 to 2 years after the onset of clinical manifestations. For visceral Leishmaniasis, the pathogenesis is complex. Three species of L. Donovoni cause visceral leishmaniasis: L. donovani, L. infantum, and L. chagasi. Initially, the infection is asymptomatic. If the infection spreads, the severe symptoms of kal-azar disease become increasingly apparent. Once the species causing VL parsitize reticuloendothelial cells (RE), the disease, if left untreated, may result in a 75% to 95% mortality rate within the first two years of infection (Standford refernce). In Brazil, the main vector responsible for the transmission of VL is Lutzomyia longipalpis , a small mosquito of sand-like color (sand fly) with large hairy wings directed backwards and upwards.
  • Asia and Africa (primarily Sudan, Kenya and Ethiopia) and can affect people of all ages.
  • Asia (Northeastern China, India and Iran) and Africa (primarily Sudan, Kenya and Ethiopia) and can affect people of all ages.
  • When an individual is bitten by the sandfly, promastigotes are introduced into the skin and enter small blood vessels and macrophages around the affected area. There, the parasites proflierate as amastigotes. White blood cells, called lymphocytes, then attack the immediate area using white blood cells called lymphocytes and the amastigotes are liberated from the macrophages. A lump then forms and breaks open when the blood flow to the area is compromised. Although the ulcer may become infected, the lesion usually heals as the body builds immunity against the parasite. Once an individual is infected with cutaneous leishmaniasis, it is unlikely that he or she will be re-infected by the same species.Once an individual is infected with cutaneous leishmaniasis, it is unlikely that he or she will be re-infected by the same species. (Standford Reference).
  • This rare mucosal involvement may occur if a skin lesion near the mouth or nose is not treated. Hard to confirm diagnosis as few parasites are in the lesion
  • Mucocutaneous leishmaniasis is a rare form of the disease that can occur months or years after the healing of a CL ulcer. This form of the disease can affect the nasal septum, palate and other parts of the nasopharynx. cases are focused in South America, especially in Brazil, Paraguay, Ecaudor, Bolivia, Peru, Colombia, andVenezuela. Ninety percent of the cases occur in Brazil, Bolivia, and Peru. Twenty percent of leishmaniasis patients in Brazil develop MCL.
  • Although the pathogenesis of visceral and cutaneous leishmaniasis are well understood, the pathogenesis of mucotaneous leishmaniasis (MCL) is still unclear. However, it is believed that host genetic factors are important in the advancement of the disease. MCL development is similar to that of cutaneous leishmaniasis, and the two infections can occur simultaneously. MCL occurs when cutaneous lesions expand to the mucosal region or through metastasis. Moreover, it is not uncommon for MCL to develop many years after the recovery of an initial lesion. The result is a gradual and progressive development of destructive lesions. (standford reference).
  • Leishmaniasis is transmitted by the bite of infected female phlebotomine sandflies. The sandflies inject the infective stage (i.e., promastigotes) from their proboscis during blood meals (1). Promastigotes that reach the puncture wound are phagocytized by macrophages (2) and other types of mononuclear phagocytic cells. Progmastigotes transform in these cells into the tissue stage of the parasite (i.e., amastigotes)(3) , which multiply by simple division and proceed to infect other mononuclear phagocytic cells (4) . Parasite, host, and other factors affect whether the infection becomes symptomatic and whether cutaneous or visceral leishmaniasis results. Sandflies become infected by ingesting infected cells during blood meals (5,6). In sandflies, amastigotes transform into promastigotes, develop in the gut (7)(in the hindgut for leishmanial organisms in the Viannia subgenus; in the midgut for organisms in the Leishmania subgenus), and migrate to the proboscis(8) . VL : Hematogenic dissemination to other tissues rich in cells of the mononuclear phagocyte system, such as lymph nodes, liver, spleen and bone marrow is observed
  • Complete identification requires the isolation of parasites in culture. Slit skin smears were taken from the most prominent lesions. After cleaning with alcohol, the area was grasped between the thumb and forefinger of the non-dominant hand until the site was blanched. A 5-mm long and 3-mm deep incision was made with Bard Parker No. 15 blade and the sides of the incision scraped to obtain tissue fluid and pulp. A smear was made on a clean glass slide, stained with Leishman stain, and examined under the oil-immersion objective of the microscope.
  • Complete identification requires the isolation of parasites in culture.
  • They possess a large nucleus, a prominent kinetoplast
  • Lab 8 leishmaniasis

    1. 1. Hemoflagellates : Leishmania spp. University of Sulaimani College of Science Department of Biology Practical Parasitology 2 nd stage Lab 8 : Leishmaniasis
    2. 2. <ul><li>Objectives: Students should be able to: </li></ul><ul><li>List human pathogenic species of Leishmania </li></ul><ul><li>Identify amastigote stage. </li></ul><ul><li>Describe methods of diagnosis of Leishmania s pp. </li></ul>Leishmania spp.
    3. 3. <ul><li>The leishmaniasis is a group of vector-borne diseases caused by protozoan haemoflagelates of the genus Leishmania </li></ul><ul><li>It is spread through females of Sandflies of the genus Phlebotomus spp. in the Old World , and of the genus Lutzomyia in the New World. </li></ul><ul><li>Leishmaniasis is a zoonotic deadly parasitic disease that affects over 12 million people worldwide, with 350 millions are at risk. </li></ul><ul><li>With more than 2 million new cases reported every year. </li></ul>Leishmania spp.
    4. 4. Leishmania spp. Phlebotomus
    5. 5. Leishmania spp. <ul><li>In 1901, professor William B. Leishman ( Scotish Pathologist ), identified certain organisms in smears taken from the spleen of a patient who had died from “Dum-Dum fever&quot;. </li></ul><ul><li>This disease was similar to what Indian physicians called kala-azar (black fever). </li></ul><ul><li>Initially, these organisms were considered to be trypanosomes. </li></ul>
    6. 6. Leishmania spp. <ul><li>In 1903, Captain Charles Donovan described them as being new. </li></ul><ul><li>These new microorganisms were given the name ‘Leishman-Donovan bodies’ </li></ul><ul><li>After that taxonomically designated Leishmania donovani . </li></ul>
    7. 7. Amastigote . (Leishmanial form) <ul><li>Spherical or Oval in shape </li></ul><ul><li>2-3 μm in diameter. </li></ul><ul><li>non-motile </li></ul><ul><li>Large eccentrically located </li></ul><ul><li>nucleus </li></ul><ul><li>Rode shape Kinetoplast </li></ul><ul><li>Usually found in the </li></ul><ul><li>vertebrate host. </li></ul>Leishmania spp.
    8. 8. <ul><li>A macrophage filled with Leishmania amastigotes. </li></ul>Leishmania spp.
    9. 9. Promastigote : (Leptomonad stage) <ul><li>Spindle shaped with 14-20 by 1.5 - 4 μm in diameter </li></ul><ul><li>Free anterior flagellum </li></ul><ul><li>Kinetoplast at the anterior end of the body. </li></ul><ul><li>There is no undulating membrane. </li></ul><ul><li>Usually promastigote found in female Sandflies and Culture </li></ul>Leishmania spp.
    10. 10. Leishmania spp. <ul><li>In humans, There are different forms of this disease : </li></ul><ul><li>Visceral leishmaniasis: involving liver, spleen, and bone marrow </li></ul><ul><li>Cutaneous leishmaniasis: involving the skin at the site of a sandfly bite </li></ul><ul><li>mucocutaneous leishmaniasis: involving mucous membranes of the mouth and nose after spread from a nearby cutaneous lesion (very rare). </li></ul>Different species of Leishmania cause different forms of disease
    11. 11. <ul><li>Most severe form of the disease, usually fatal if left untreated </li></ul><ul><li>Usually associated with fever, weight loss, and an enlarged spleen and liver. </li></ul>Leishmania donovani Visceral leishmaniasis (VL)
    12. 12. <ul><li>G.D. Asia, Africa and south America and can affect people of all ages </li></ul><ul><li>90% of all visceral leishmaniasis occurs in Bangladesh, Brazil, India, Iraq and the Sudan </li></ul><ul><ul><li>2893 cases were reported in Iraq in 2001 </li></ul></ul><ul><ul><li>12 visceral leishmania cases were reported in Americans in Desert Storm. </li></ul></ul>Leishmania donovani Visceral leishmaniasis (VL)
    13. 13. Habitat:- internal organ (liver, lymph nodes, spleen ,bone marrow) Disease Visceral leishmaniasis, Kala azar, Dum Dum fever Transmission VL. is transmitted chiefly by female Sandflies ( Phlebotomus spp.) Leishmania donovani Visceral leishmaniasis (VL)
    14. 14. Leishmania tropica <ul><li>Most common form </li></ul><ul><li>Characterized by one or more sores, or nodules on the skin </li></ul><ul><li>Sores can change in size and appearance over time </li></ul><ul><li>Often described as looking somewhat like a volcano with a raised edge and central crater </li></ul><ul><li>Sores are painless or painful. </li></ul>Cutaneous leishmaniasis (CL)
    15. 15. <ul><li>Most sores develop within a few weeks of the sandfly bite, however they can appear up to months later </li></ul><ul><li>Skin sores of cutaneous leishmaniasis can heal on their own, but this can take months or even years </li></ul><ul><li>Sores can leave significant scars and be disfiguring if they occur on the face </li></ul>Leishmania tropica Cutaneous leishmaniasis (CL)
    16. 16. Leishmania tropica <ul><li>G.D. in Asia, Africa, and Mediterranean </li></ul><ul><li>90% of cutaneous leishmaniasis occurs in Afghanistan, Iran, Iraq, Saudi Arabia, Syria, Brazil and Peru </li></ul><ul><ul><li>8,779 cases were reported in Iraq in 1992 </li></ul></ul><ul><ul><li>At least 20 cases of cutaneous leishmaniasis were reported in Americans from Desert Storm </li></ul></ul>Cutaneous leishmaniasis (CL)
    17. 17. Leishmania tropica Habitat:- Skin Disease Cutaneous leishmaniasis, Oriental sore, Delhi or Baghdad boils Transmission female Sandflies ( Phlebotomus sp.) Cutaneous leishmaniasis (CL)
    18. 18. Leishmania tropica Multiple lesions on arm with a variety of appearances. Cutaneous leishmaniasis (CL)
    19. 19. Note the raised border and wet appearance of the sore on the back of the hand. Both lesions are leishmaniasis. Leishmania tropica Cutaneous leishmaniasis (CL)
    20. 20. Back of hand. Note raised border and wet appearance. Patient has bacitracin ointment applied to lesion. Leishmania tropica Cutaneous leishmaniasis (CL)
    21. 21. Leishmania tropica Small, raised lesion on trunk. Cutaneous leishmaniasis (CL)
    22. 22. Leishmania tropica Upper Eyelid. Note the dry, crusted appearance which is different than previous sores shown. Cutaneous leishmaniasis (CL)
    23. 23. Leishmania tropica Three lesions on face. Raised and dry. Another different presentation. Cutaneous leishmaniasis (CL)
    24. 24. Mucocutaneous Leishmaniasis (MCL) <ul><li>This type occurs if a cutaneous lesion on the face spreads to involve the nose or mouth </li></ul><ul><li>May occur months to years after original skin lesion </li></ul><ul><li>Lesions can be very disfiguring </li></ul>Leishmania braziliensis
    25. 25. Mucocutaneous Leishmaniasis (MCL) Leishmania braziliensis G.D. In South and Central America (Brazil, Bolivia, and Peru)   Habitat:- Skin and mucosa Disease mucocutaneous leishmaniasis, espundia, Uta, American leishmaniasis Transmission : female Sandflies Lutzomyia
    26. 26. Mucocutaneous Leishmaniasis (MCL) Leishmania braziliensis
    27. 27. Life Cycle
    28. 28. Laboratory Diagnosis: <ul><li>Microscopy: </li></ul><ul><li>(slit-skin smear, splenic aspirate, liver biopsy or bone marrow biopsy). Examination of Giemsa and Leishman stained slides of the relevant tissue is still the technique most commonly used to detect the parasite. </li></ul><ul><li>2. Culture: </li></ul><ul><li>The aspirates can be cultured in NNN. In culture the amastigote stage converts to the promastigote stage. However, this is not a rapid technique, as the parasites may take from 10 - 21 days to grow. </li></ul>
    29. 29. 3. Serodiagnosis: VL produces large amounts of specific IgG which can be used for diagnosis. Currently the most used serodiagnostic tests Enzyme Linked Immunosorbent Assay (ELISA). 4. Molecular techniques: PCR, such technique, however, are not readily available in general diagnostic laboratories. Laboratory Diagnosis:
    30. 30. Promastigotes from culture. Amastigotes from blood.
    31. 31. References <ul><li>Schmidt GD, & Roberts LS. (2005). Foundations of Parasitology. 7 th ed. McGraw Hill. Boston. </li></ul><ul><li>Gillespie S, & Pearson RD. (2001). Principles and Practice of Clinical Parasitology. John Wiley & Sons Ltd. Chichester. </li></ul><ul><li>Singh RK, Pandey HP, Sundar S. ( 2006 ). Visceral leishmaniasis (kala-azar): challenges ahead. Indian J Med Res. 123(3):331-44. </li></ul><ul><li>Soto J`, et al. (2001). Treatment of American cutaneous leishmaniasis with miltefosine, an oral agent. Clin Infect Dis. 33(7):57-61. </li></ul><ul><li> </li></ul><ul><li> </li></ul><ul><li> </li></ul>
    32. 32. Next Lab Apicomplexa: Tissue Apicomplexa Toxoplasma gondii