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Visceral Leishmaniasis  Learning Objectives (LOs) By the end of the lecture the students should be able to: Identify classification, geographical distribution, morphology, and life cycle of Leishmania spp. causing visceral leishmaniasis. Illustrate pathogenesis, and management...

Visceral Leishmaniasis  Learning Objectives (LOs) By the end of the lecture the students should be able to: Identify classification, geographical distribution, morphology, and life cycle of Leishmania spp. causing visceral leishmaniasis. Illustrate pathogenesis, and management of visceral leishmaniasis. 2  Case scenario A 20-year-old Indian male was admitted to a hospital because of recurrent fever with chills for six months, progressive generalised weakness for five months, loss of appetite and body rashes for one month. He took local symptomatic treatment (paracetamol and multivitamins) for 5 days but did not get any relief, meanwhile he started developing petechial haemorrhages. On general examination the patient had moderate pallor, no icterus or lymphadenopathy. On abdominal examination, the liver and spleen were palpable. Routine haematological investigations revealed pancytopenia with haemoglobin level of 7g/dl. Morphological structure of red blood cells was normocytic hypochromic with thrombocytopaenia (platelet count 63000/ml). Bone marrow smears revealed abundance of mononucleated rounded bodies about 3µ in size both intracellularly within the macrophages as well as extracellularly. 3  Leishmania The second deadliest parasitic disease in the world following malaria. A flagellated insect-transmitted protozoa that infect blood & tissues of human and reservoir hosts. Different spp. infect man with same morphology, life cycle in insect. They differ in geographical distribution, vector, manifestations, antigenic structure, biochemical characters, DNA analysis and culture characteristics. 4  They cause 3 different diseases: 1-Visceral leishmaniasis: Leishmania donovani complex. 2-Cutaneous leishmaniasis: Leishmania tropica complex and Leishmania mexicana complex. 3-Mucocutaneous leishmaniasis: Leishmania braziliensis complex. 5  Visceral leishmaniasis (Kala azar, black fever or Dum-dum fever) L. donovani complex Old world visceral New world visceral leishmaniasis leishmaniasis L. donovani L. infantum L. chagasi N.B : -Old world refers to Africa, Asia, Europe. 6 -New world refers to Americas.  Geographical distribution India, Pakistan, Indonesia, Thailand, L. donovani Ethiopia, East and Central Africa. Mediterranean areas, Middle East L. infantum and China. L. chagasi Central and South America. 7 Visceral leishmaniasis distribution. 8  Morphology Amastigote form Promastigote form Human Reservoir Insect Culture body hosts vector 9  Morphology 1- Amastigote form (Leishman Donovan; LD body) in: Reticuloendothelial cells (RECs) all over the human body & reservoir hosts (intracellular in macrophages). 10  Leishmania (Leishman-Donovan A macrophage filled with bodies) lying in macrophage cells from liver (Giemsa ×12000). Leishmania amastigotes. 11 2- Promastigote form: In insect vector and culture. 12  Life cycle Habitat: In RECs of viscera: Littoral cells of spleen, Kupffer cells of liver, bone marrow, peyer's patches & mesenteric lymph nodes. In endothelial cells of kidney (urine), and suprarenal capsules. In macrophages of intestinal wall (faeces), blood, heart, lungs, meninges, CSF & nasal secretions. 13  Life cycle Definitive Man host Reservoir Dogs, rodents and wild & host domestic animals Insect vector Female sand flies; Phelobotomus Lutzomyia Phelobotomus or Lutzomyia Promastigotes Infective Promastigotes in buccal cavity stage of sand fly 14 Mode of infection: 1. Bite of infected vector. 2. Blood transfusion or organ transplantation. 3. Direct in epidemics by nasal secretions. 4. Congenital. 5. Accidental in laboratory. 15  16 Video link: https://www.youtube.com/watch?v=gW9Nj84IBmo  Life cycle in human and reservoir host -Promastigotes in mouth of blocked sand fly are inoculated in skin during blood meal. -Ingested by macrophages amastigotes, and reproduce by binary fission. -Parasitized cells rupture, amastigotes are free & phagocytosed by other macrophages. -Circulating macrophages phagocytose the parasites and carried to viscera generalized infection of RECs. 17  Life cycle in sand fly insect - Transmission is biological of cyclo-propagative type. - Amastigotes in blood meal are taken by sand fly, transformed to promastigotes in its gut. -They multiply by binary fission, migrate to the pharynx which is blocked by the parasites, then parasites go to the buccal cavity. -When sand fly attempts another blood meal, some of promastigotes are regurgitated and introduced into the skin bite by their own motility. 18  Pathogenicity 1) Visceral leishmaniasis is an opportunistic disease. Risk of severe form of the disease is expected in: Immunocompromised status Low protein diet Deficient iron, vitamin A and zinc diet The parasitized macrophages are present in small numbers in blood, but are numerous in RECs. 19  Pathogenicity 2) Amastigotes multiply in fixed macrophages causing marked hyperplasia & destruction of RECs in organs. 3) Multiplication of amastigotes in RECs of liver, spleen and lymph nodes leads to hepato- splenomegaly & lymphadenopathy. 4) Bone marrow is heavily infiltrated with parasitized macrophages leading to pancytopenia. 20  Pathogenicity 5) Lymphoid macrophages in intestinal mucosa & submucosa are packed with parasites causing ulceration with LD bodies in faeces. 6) Urinary tract infiltrated with parasitized macrophages leads to break down of mucosa with LD bodies in urine. 7) Naso-pharyngeal affection leading to mucopurulent discharge containing leishmanial bodies. 21 8) Types and causes and of anemia in Kala-azar Type of Normocytic normochromic Macrocytic Microcytic Anaemia Causes Increased destruction of RBCs by spleen. Reticulo- Lack of iron Decreased erythropoiesis. endothelial absorption from hyperplasia. intestine Auto-antibodies to RBCs. Hemorrhage. Fatty Alterations in RBCs membrane infiltration of permeability. liver leading Production of haemolysin by parasites. to deficient storage of vitamin B12. 22  Clinical picture Incubation period: 2-6 ms. A primary lesion at site of infection in infants in Africa in legs & arms (leishmanioma). The most important sign is fever: remittent with a twice- daily rise, intermittent, or less often continuous. 23  Fever is due to: -Release of pyrogens by infected macrophages due to: Phagocytosis of amastigotes. Uptake of cellular debris from ruptured parasitized macrophages. 24  Clinical picture Hepatosplenomegaly. Weakness, weight loss, and emaciation. Lymphadenopathy in African patients. Diarrhea and/or dysentry. Epistaxis and bleeding gums. Skin is dry, thin, rough, and darkly pigmented. A butterfly distribution over the nose. 25  Enlarged spleen and liver in an autopsy of an infant dying of visceral leishmaniasis. Kala Azar - Visceral Leishmaniasis 26  Complications The most fatal complications of VL is disseminated intravascular coagulation. Thrombocytopenia spontaneous bleeding from nasal and oral mucosa. Pancytopenia due to suppression of hematopoiesis, splenic sequestration & hemolysis. Intestinal invasion diarrhea or dysentery, malabsorption &hypoalbuminemia. Hepatic failure may develop with jaundice and ascites. Renal impairment may occur due to immune complex formation. 27  Post-Kala azar dermal leishmaniasis (PKDL) - Chronic, progressive, granulomatous, non-ulcerating, hypopigmented nodular cutaneous lesion, 6 ms-5 ys after spontenous or drug cure (Pentostam). - Parasites migrate and localized mainly in face, resembling Lupromatous leprosy or disseminated cutaneous leishmaniasis. 28 Post Kala-Azar dermal leishmaniasis Dermal lesions may contain parasites in great numbers. 29  Para-Kala azar dermal leishmaniasis (Para-KDL) Para-kala azar dermal leishmaniasis (Para-KDL) is a condition when PKDL occurs with VL and is characterized by fever, splenomegaly, hepatomegaly, lymphadenopathy, and poor nutritional status It can occur while a patient is still undergoing treatment for VL, affecting approximately 50% of patients. It is documented mainly in East Africa and South Asia. 30  Diagnosis A) Clinical: - In endemic areas, kala azar may be suspected in a patient specially children, with persistent, irregular or remittent fever (double daily peak). - Hepatosplenomegaly, anaemia, leucopenia & emaciation. 31 B) Laboratory: I. Direct: 1. Microscopy: - Detection of amastigotes in smears made from: Peripheral blood by thick film or buffy coat smears. Bone marrow puncture (sternal or iliac crest). Splenic puncture (spleen pulp) & liver puncture. Enlarged lymph node aspirate or puncture. Nasopharyngeal secretions, stool and urine. Nodular lesions in PKDL. 32  The smears of body fluids are stained with Leishman, Giemsa or Wrights stain. H& E stain is used for tissue sections. Amastigotes can be seen inside the macrophages in large numbers and little extracellular form can also be seen. 33 2.Culture on Novy-MacNeal-Nicolle (NNN) medium: shows promastigotes in rosette grouping, after 1-4 weeks. 34 3. Animal inoculation: - Intra-peritoneal inoculation of hamster. - In positive cases, amastigotes can be seen in smears from ulcers or nodules at site of inoculation or from the spleen, weeks post infection. 35 II. Indirect: 1. Immunological diagnosis: a. Serological tests: - Specific leishmanial antigens prepared from cultures are used to detect anti- leishmanial antibodies using: IFA, IHA, ELISA. Complement fixation test (CFT). Immunofluoresence of Leishmania Direct agglutination test (DAT) Rapid immunchromatographic dipstick (ICT). ELISA 36  Rapid immunchromatographic dipstick ICT is considered +ve with the appearance of two red lines (one in the control area and the other in the test area. 37 B. Leishmanin skin test (Montenegro test): It is a delayed hypersensitivity skin test. Used to detect exposure and immunity to Leishmania Intradermal 0.1ml of killed promastigotes of L. donovani. Positive result: induration and erythema of ≥ 5 mm after 48-72 hrs. Positive test: past infection with Leishmania (positive 6-8 weeks after cure). The test is negative in active infection and in PKDL due to marked depression of cellular immune response. 38 2. Molecular diagnosis: It helps in species identification of Leishmania. 3. Blood picture: Complete blood count shows: anemia, leucopenia and thrombocytopenia (Pancytopenia or Aplastic anemia). Hypergammaglobulinemia (IgG)& low albumin level. 39  Treatment I. Supportive: -Diet rich in vitamins, iron and liver therapy. -Proper antibiotics. -Blood transfusion. II. Specific: a. Systemic therapy (parenteral, IV) - Pentavalant antimony compounds: Pentostam (Sodium stibogluconate). -Amphotrocin B. -Interferron gamma + Pentostam in case of relapse. b. Systemic therapy (oral) - Miltefosine: The first oral drug approved for ttt of leishmaniasis. III. In Para-Kala azar cases: The patient is treated with two cycles of Amphotericin B with Miltefosine in between cycles for 12 weeks to obtain full recovery 40  Prevention and control 1. Control of sand flies: destruction of their breeding grounds by use of residual chlorinated hydrocarbon, DDT (dichloro-diphenyl-trichloroethane). 2. Control of reservoir hosts. 3. Treatment of infected persons. 4. Humans can be protected by using: bed nets, window mesh screen (40 meshes / inch2), repellents as dimethyl phthalate, and spraying of insecticides. 41  Summary Visceral Leishmaniasis is a vector-borne disease caused by L. donovani complex. Transmitted by bite of sand flies, promastigote is the infective stage. Clinical picture includes: fever, lymphadenopathy, hepatosplenomegaly, anaemia, diarrhea or dysentry, weight loss and emaciation. Diagnosed by detection of amastigotes in smears and treated with pentostam. Control of sand flies and reservoir hosts besides personal protection from vector bites are important measures for disease prevention and control. 42 43  Case scenario A 20-year-old Indian male was admitted to a hospital because of recurrent fever with chills for six months, progressive generalised weakness for five months, loss of appetite and body rashes for one month. He took local symptomatic treatment (paracetamol and multivitamins) for 5 days but did not get any relief, meanwhile he started developing petechial haemorrhages. On general examination the patient had moderate pallor, no icterus or lymphadenopathy. On abdominal examination, the liver and spleen were palpable. Routine haematological investigations revealed pancytopenia with haemoglobin level of 7g/dl. Morphological structure of red blood cells was normocytic hypochromic with thrombocytopaenia (platelet count 63000/ml). Bone marrow smears revealed abundance of mononucleated rounded bodies about 3µ in size both intracellularly within the macrophages as well as extracellularly. 44  Case scenario Questions: 1. What is the provisional diagnosis of this case and the causative parasite? 2. Identify the mode of infection. 3. Explain the mechanisms of anemia in this case. 4. What is the prognosis of this disease? 5. Propose other diagnostic procedures to confirm your diagnosis. 6. Predict two other complications that can occur with this infection. 45  References Garcia, L.S., 2016. Diagnostic Medical Parasitology, 6th ed., ASM press, Washington, DC. Paniker, C.K.J, Ghosh, H., Chander, J., 2018. Paniker's Textbook of Medical Parasitology, 8th ed., Jaypee Brothers Medical Publishers (P) Ltd. Gunn, A. and Pitt, S.J., 2021. Parasitology: An Integrated Approach. Wiley, The New York Academy of Science. Ghosh, S., 2021. Paniker's Textbook Of Medical Parasitology As Per The Competency Based Medical Education Curriculum. 9th edition, ISBN-13: 978-8194802884. Jaypee Brothers Medical Publishers. Mehlhorn, H., 2023. Human Parasites: Diagnosis, Treatment, Prevention. 2nd edition. Springer International Publishing Switzerland. https://www.cdc.gov/parasites/leishmaniasis/ 46 47