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Trypanosomiasis

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Leishmania and Trypanosoma
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Trypanosomiasis

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Medical presentation for undergraduates - T. Brucei, T. cruzi, African Sleeping Sickness

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  1. 1. Trypanosomiasis Mohamed Sinan Govt Medical College, Calicut
  2. 2. • Phylum : Sacromastigophora • Subphylum : Mastigophora • Class : Kinetoplastidea • Order : Trypanosomatida • Family : Trypanosomatidae
  3. 3. General Characteristics • Members of family Trypanosomatidae live in the blood and tissues of man and other vertebrate hosts and in the gut of the insect vectors. (They are called hemoflagellates) • They have a single nucleus, a kinetoplast, and a single flagellum. Kinetoplast consists of a deeply staining parabasal body and adjacent dot-like blepharoplast. • Nucleus is round or oval and is situated in the central part of the body.
  4. 4. • 4 morphological states (Based on position/arrangement of flagella) – Amastigote – Promastigote – Epimastigote and – Trypomastigote. • Each exists in 2 or more of these 4 morphologic states.
  5. 5. Staining : • For smears of body fluids, Romanowsky’s Wrights stain, Giemsa stain, and Leishman’s stain are suitable. The cytoplasm appears blue, the nucleus and flagellum appear pink, and the kinetoplast appears deep red. • For tissue section, hematoxylin eosin staining is done for demonstrating structures of the parasite.
  6. 6. T. brucei light microscopy – Giemsa stain
  7. 7. T. cruzi in blood smear – giemsa staining
  8. 8. • All members of the family have similar life cycles. They all require an insect vector as an intermediate host. • Multiplication in both the vertebrate and invertebrate host is by binary fission. No sexual cycle is known.
  9. 9. • Family Trypanosomatidae consists of 6 genera • 2 of them are pathogenic to humans – – Trypanosoma – Leishmania
  10. 10. Trypanosomes General Characters • All members of the genus Trypanosoma exist in trypomastigote stage at sometime in their life cycle. • Some trypanosomes (T. cruzi) assume amastigote forms in vertebrate hosts. • They also show polymorphism
  11. 11. • Trypanosoma pass their life cycle in 2 hosts – – vertebrate hosts (definitive hosts) – insect vectors (intermediate hosts). • During incubation period the parasite undergoes development and multiplication in the vector. • In the vector, the trypanosomes are classified into 2 groups according to mode of development :
  12. 12. • €Salivaria (anterior station): The trypanosomes migrate to mouth parts of the vectors; Infection is transmitted by their bite (inoculative transmission). (e.g. T. gambiense) • €Stercoraria (posterior station): The trypanosomes migrate to the hindgut and are passed in faeces (stercorian transmission). (e.g. T. cruzi)
  13. 13. Trypanosomes Infecting Man • Trypanosoma brucei complex, causing African trypanosomiasis (sleeping sickness) Subspecies are: - Trypanosoma brucei gambiense: causing West African sleeping sickness. - Trypanosoma brucei rhodesiense: causing East African sleeping sickness. • Trypanosoma cruzi, causing South American trypanosomiasis or Chagas’ disease. • Trypanosoma rangeli, a nonpathogenic trypanosome causing human infection in
  14. 14. Trypanosoma Brucei Gambiense (West African Trypanosomiasis) • First isolated in 1901 by Forde. • The name Trypanosoma gambiense was proposed by Dulton, in 1902 • It is endemic in West and Central Africa • Habitat: They live in man and other vertebrate hosts. They are parasites of connective tissue.
  15. 15. Morphology Vertebrate Forms • In the blood of vertebrate host, T. brucei gambiense exists as trypomastigote form, which is highly pleomorphic • It occurs as a long slender form; a stumpy short broad form with attenuated or absent flagellum; and an intermediate form. • The trypomastigotes are about 15–40 μm long and 1.5 – 3.5 μm broad.
  16. 16. Insect Forms • In insects, it occurs in 2 forms: – Epimastigotes – Metacyclic trypomastigote forms.
  17. 17. Antigenic Variation • Trypanosomes exhibit unique antigenic variation of their glycoproteins. • There is a cyclical fluctuation in the trypanosomes in the blood of infected vertebrates after every 7–10 days. • Each successive wave represents a variant antigenic type (VAT) of trypomastigote posssesing Variant Surface Specific Antigens (VSSA) or Variant Surface Glycoprotein (VSG) coat antigen. • A single trypanosome may have 1,000 or
  18. 18. Life Cycle • T. brucei gambiense passes its life cycle in 2 hosts. – Vertebrate host: Man, game animals, and other domestic animals. – Invertebrate host: Tsetse fly. • Both male and female tsetse fly of Glossina species (G. palpalis) are capable of transmitting the disease to humans. • Infective form: Metacyclic
  19. 19. • Mode of transmission: – By bite of tsetse fly – Congenital transmission has also been recorded. • Reservoirs: Man is the only reservoir host, although pigs and others domestic animals can act as chronic asymptomatic carriers of the parasite.
  20. 20. Development in Man and Other Vertebrate Hosts • Metacyclic stage (infective form) of trypomastigotes are inoculated into a man (definitive host) through skin when an infected tsetse fly takes a blood meal • The parasite transforms into slender forms that multiply asexually for 1–2 days before entering the peripheral blood and lymphatic circulation. • These become ‘stumpy’ via intermediate forms and enter the blood stream. • It invades the central nervous system in chronic infection.
  21. 21. Development in Tsetse Fly • In the midgut of the fly, short stumpy trypomastigotes develop into long, slender forms and multiply. • After 2–3 weeks, they migrate to the salivary glands, where they develop into epimastigotes, which multiply and fill the cavity of the gland and eventually transform into the infective metacyclic trypomastigotes • Development of the infective stage within the tsetse fly requires 25–50 days (extrinsic incubation period).
  22. 22. Pathogenecity and Clinical Features • T. brucei gambiense causes African trypanosomiasis (West African sleeping sickness). • The illness is chronic and can persist for many years. • There is an initial period of parasitemia, following which parasite is localized predominatly in the lymph nodes. • A painless chancre (trypanosomal chancre) appears on skin at the site of bite by tsetse fly, followed by intermittent fever, chills, rash, anemia, weight loss,
  23. 23. • Systemic trypanosomiasis without central nervous system involvement is referred to as stage I disease. • In this stage, there is hepatosplenomegaly and lymphadenopathy, particularly in the posterior cervical region (Winterbottom’s sign). • Hematological manifestations seen in stage I include anemia, moderate leucocytosis, and thrombocytopenia.
  24. 24. • Stage II disease involves invasion of central nervous system. With this, the ‘sleeping sickness’ starts. • This is marked by increasing headache, mental dullness, apathy, and day time sleepiness. • There is infiltration of the brain & spinal cord, and neuronal degeneration.
  25. 25. • Abnormalities in cerebrospinal fluid include raised intracranial pressure, pleocytosis, and raised total protein concentrations. • The patient falls into coma followed by death from asthenia.
  26. 26. Trypanosoma Brucei Rhodesiense (East African Trypanosomiasis) • It is found in Eastern and Central Africa • Discovered by Stephans and Fanthan in 1910 from the blood of a patient in Rhodesia suffering from sleeping sickness. • The principal vector is G. morisitans, G. palpalis, and G. Swynnertoni
  27. 27. Pathogenesis and Clinical Feature • T. brucei rhodesiense causes East African sleeping sickness • East African trypanosomiasis is more acute than the Gambian form and appears after an incubation period of 4 weeks. • It may end fatally within an year of onset, before the involvement of central nervous system develops. • Pathological features are similar in both diseases with some variations— Edema, myocarditis, and weakness are more prominent in East African sickness.
  28. 28. • Lymphadenitis is less prominent. • Febrile paroxysms are more frequent and severe. • There is a larger quantity of parasite in the peripheral blood. • €Central nervous system involvement occurs early. • Mania and delusions may occur but the marked somnolence, which occurs in T. brucei gambiense infection is lacking.
  29. 29. Laboratory Diagnosis The diagnosis of both types of African trypanosomiasis is similar • Nonspecific Findings – Anemia and monocytosis. – Raised ESR due to rise in gamma globulin levels. – Reversal of albumin:globulin ratio. – Increased cerebrospinal fluid (CSF) pressure and raised cell count and proteins in CSF.
  30. 30. • Specific Findings – Definitive diagnosis of sleeping sickness is established by the demonstration of trypanosomes in peripheral blood, bone marrow, lymphnode, CSF, and chancre fluid.
  31. 31. Microscopy • Wet mount preparation of lymph node aspirates and chancre fluid are used as a rapid method for demonstration of trypanosomes. • These specimen are also examined for parasites after fixing and staining with Giemsa stain. • Examination of Giemsa stained thick peripheral blood smears reveals the presence of trypomastigotes
  32. 32. • If parasitemia is low, then examination of concentrated blood smear is a highly sensitive method. • Different concentration techniques employed are buffy coat examination, differential centrifugation, membrane filtration, and ion exchange column chromatography. • Examination of wet mount and stained smear of the CSF may also show trypanosomes
  33. 33. Culture • The organisms are difficult to grow, hence culture is not routinely used. However, it can be cultivated in Weinman’s or Tobie’s medium. Animal Inoculation • Inoculation of specimens from suspected cases to white rat or white mice is a highly sensitive procedure for detecting T. brucei rhodesiense infection.
  34. 34. Serodiagnosis Antibody detection • Almost all infected have very high levels of total serum IgM & CSF IgM • Various serological methods to detect these antibodies are: – Indirect hemagglutination (IHA) – Indirect immunofluroscence (IIF) – Enzymelinked immunosorbent assay (ELISA) – Card agglutination trypanosomiasis test (CATT) – Complement fixation test (CFT) • Specific antibodies are detected by these tests in serum within 2–3 weeks of infection. • Specific antibodies in CSF - demonstrated by
  35. 35. Antigen detection • Antigens from serum and CSF can be detected by ELISA.
  36. 36. Imaging • CT scan of the brain shows cerebral edema and MRI shows white matter enhancement in patients with late stage CNS involvement
  37. 37. Treatment • In stage I (i.e. No CNS involvement), pentamidine is the drug of choice for gambiense HAT and suramin is the drug of choice for rhodesiense HAT. • Dose: – Pentamidine: 3 - 4 mg/kg body weight, IM daily for 7–10 days. – Suramin: 20 mg/kgbody weight in a course of 5 injections intravenously, at an interval of 5– 7 days.
  38. 38. • In stage II, melarsoprol (MelB) is the drug of choice, as it can cross the blood brain barrier. • Dose: 2–3 mg/kg/day(max. 40 mg) for 3–4 days
  39. 39. Prophylaxis • Control - based on early diagnosis and treatment of cases to reduce the reservoir of infection. • Control of tsetse fly population (most important preventive measure) by wide spraying of insecticides, traps, and baits impregnated with insecticides. • No vaccine is available.
  40. 40. Trypanosoma Cruzi (Chagas’ Disease) • It is the causative organism of chagas’s disease or South American trypanosomiasis. History and Distribution: • Carlos Chagas, investigating malaria, accidently discovered T. cruzi • Zoonotic disease; limited to south and central America
  41. 41. Habitat • In humans – exist in Amastigote and trypomastigote forms. Amastigotes - Intracellular parasites - Found in muscular tissue,nervous tissue and RE system. Trypomastigotes - Found in peripheral blood
  42. 42. • In reduviid bugs – Amastigotes found in midgut – Trypomastigote present in hindgut and feces
  43. 43. Morphology Amastigote Oval body{2-4 micro mtr dia}.it has a nucleus and kinetoplast. • Flagellum is absent • Resembles amastigote of Leishmania spp, so it is called leishmanial form. • Multiplication occurs in this stage
  44. 44. Trypomastigote • Non muliplying form. • In blood, they appear either as long,thin flagellates{20 μm long} or short stumpy form {15μm long} • Posterior end is wedge-shaped • In stained blood smears, shaped like ‘C’, ’U’ or ‘S’, having a free flagellum of 1/3 length of body
  45. 45. Epimastigote Form Found in insect vector, reduviid bug and in culture • It has a kinetoplast adjascent to nucleus. • An undulating membrane runs along anterior half of parasite. • Divide by binary fission in hindgut of vector
  46. 46. Life Cycle T.Cruzi passes its life cycle in 2 hosts Definitive host :Man Intermediate host(vector): Reduviid bug or triatomine bugs Infective form: Metacyclic trypomastigotes , found in reduviid bugs Reservoir host: Armadillo, cat, dog and pigs.
  47. 47. Mode of transmission • Man is infected : ˉ By fecal matter of reduviid bug being rubbed into bite wound . ˉ Through contamination of conjunctiva and other mucous membrane surfaces. ˉ Congenitally ˉ By blood transfusion
  48. 48. Pathogenicity and Clinical Features • Incubation period - 1-2 weeks in man • Disease manifest in acute and chronic form.
  49. 49. Acute Chagas’ Disease • Occurs soon after infection. Last for 1-4 months. • Often seen in children under 2 yrs of age • First sign appears with in a week after invasion. • Inoculation of parasite in conjunctiva causes unilateral, painless oedema of
  50. 50. • In few patients, there may be generalised infection with fever, lymphadenopathy and hepatosplenomegaly • Patient may die of acute myocarditis and meningoencephalitis
  51. 51. Chronic Chagas’ Disease Found in adults and older children • It become apparent,years or even decades after the initial infection • It produces inflammatory response,cellular destruction,and fibrosis of muscles and nerves,that control tone of hollow organs like heart,oesophagus,colon etc. • Thus,it can lead to cardiac myopathy and megaoesophagus and megacolon
  52. 52. Congenital Infection Congenital transmission is possible in both acute and chronic phase ,causing myocardial and neurological damage in the foetus
  53. 53. Laboratory Diagnosis Diagnosis is done by demonstration of T.cruzi in blood or tissues or by serology Microscopy • Microscopic examination of fresh anticoagulated blood or buffy coat is the simplest way to see motile organisms • In wet mount,Trypomastigotes are fainly visible but their snake- like motion against RBC’s makes their presents apparent
  54. 54. • Trypanomastigotes can also be seen in thick and thin peripheral blood smear, stained with Giemsa stain • Microhematocrit containing acridine orange as a stain can also be used
  55. 55. Culture Novy, Neal and Nicolle (NNN) medium or its modifications are used • Medium is inoculated with blood and other specimens and incubated at 22-24 deg C • Fluid from culture is examined microscopically by 4 th day and then every week for 6 weeks • Epimastigotes and Trypomastigotes are found in culture
  56. 56. Animal Inoculation Blood or CSF is inoculated intraperitoneally into mice or guinea pigs
  57. 57. • Xenodiagnosis ˉ Is the method of choice in suspected chagas disease,if other examinations are negative,especially during the early phase of disease onset. ˉ The reduviid bugs are reared in a trypanosome-free laboratory and starved for 2 wks, then fed on patients blood.if trypomastigotes are ingested ,they will multiply and develope into epimastigotes and trypomastigotes,which can be found in
  58. 58. • Histopathology ˉ Biopsy examination of lymphnodes and skeletal muscles and aspirate from chagoma may reveal amastigotes of T.cruzi
  59. 59. Serology Consits of antigen detection and antibody detection Antigen detection ˉ ELISA has been developed for detection of antigen. ˉ Detected in urine and sera in patients
  60. 60. • Antibody detection Antibodies(IgG)may be detected by the following tests. ˉ IHA ˉ CFT(Machado-Guerreiro test) ˉ ELISA ˉ IIF ˉ Direct agglutination test(DAT) ˉ Chagas Radio Immune Precipitation Assay(RIPA)-Highly specific and sensitive test.
  61. 61. • Intradermal test ˉ Antigen cruzin is used to detect delayed hypersensitivity • Molecular diagnosis ˉ PCR is used to detect trypomastigotes in blood.
  62. 62. Treatment • No effective specific treatment is available • Nifutrimox and benznidazole- used with some success in both acute and chronic chaga’s disease,kills only extracellular form • Dose- nifutrimox 8-10 mg/kg adult 15mg/kg children benznidazole 5-10 mg/kg orally for 60 days
  63. 63. Prophylaxis • Application of insectiside to control the vector bug. • Personal protection using insect repellent and mosquito net. • Improvement in housing and environment to eliminate breeding places of bugs .

Description

Medical presentation for undergraduates - T. Brucei, T. cruzi, African Sleeping Sickness

Transcript

  1. 1. Trypanosomiasis Mohamed Sinan Govt Medical College, Calicut
  2. 2. • Phylum : Sacromastigophora • Subphylum : Mastigophora • Class : Kinetoplastidea • Order : Trypanosomatida • Family : Trypanosomatidae
  3. 3. General Characteristics • Members of family Trypanosomatidae live in the blood and tissues of man and other vertebrate hosts and in the gut of the insect vectors. (They are called hemoflagellates) • They have a single nucleus, a kinetoplast, and a single flagellum. Kinetoplast consists of a deeply staining parabasal body and adjacent dot-like blepharoplast. • Nucleus is round or oval and is situated in the central part of the body.
  4. 4. • 4 morphological states (Based on position/arrangement of flagella) – Amastigote – Promastigote – Epimastigote and – Trypomastigote. • Each exists in 2 or more of these 4 morphologic states.
  5. 5. Staining : • For smears of body fluids, Romanowsky’s Wrights stain, Giemsa stain, and Leishman’s stain are suitable. The cytoplasm appears blue, the nucleus and flagellum appear pink, and the kinetoplast appears deep red. • For tissue section, hematoxylin eosin staining is done for demonstrating structures of the parasite.
  6. 6. T. brucei light microscopy – Giemsa stain
  7. 7. T. cruzi in blood smear – giemsa staining
  8. 8. • All members of the family have similar life cycles. They all require an insect vector as an intermediate host. • Multiplication in both the vertebrate and invertebrate host is by binary fission. No sexual cycle is known.
  9. 9. • Family Trypanosomatidae consists of 6 genera • 2 of them are pathogenic to humans – – Trypanosoma – Leishmania
  10. 10. Trypanosomes General Characters • All members of the genus Trypanosoma exist in trypomastigote stage at sometime in their life cycle. • Some trypanosomes (T. cruzi) assume amastigote forms in vertebrate hosts. • They also show polymorphism
  11. 11. • Trypanosoma pass their life cycle in 2 hosts – – vertebrate hosts (definitive hosts) – insect vectors (intermediate hosts). • During incubation period the parasite undergoes development and multiplication in the vector. • In the vector, the trypanosomes are classified into 2 groups according to mode of development :
  12. 12. • €Salivaria (anterior station): The trypanosomes migrate to mouth parts of the vectors; Infection is transmitted by their bite (inoculative transmission). (e.g. T. gambiense) • €Stercoraria (posterior station): The trypanosomes migrate to the hindgut and are passed in faeces (stercorian transmission). (e.g. T. cruzi)
  13. 13. Trypanosomes Infecting Man • Trypanosoma brucei complex, causing African trypanosomiasis (sleeping sickness) Subspecies are: - Trypanosoma brucei gambiense: causing West African sleeping sickness. - Trypanosoma brucei rhodesiense: causing East African sleeping sickness. • Trypanosoma cruzi, causing South American trypanosomiasis or Chagas’ disease. • Trypanosoma rangeli, a nonpathogenic trypanosome causing human infection in
  14. 14. Trypanosoma Brucei Gambiense (West African Trypanosomiasis) • First isolated in 1901 by Forde. • The name Trypanosoma gambiense was proposed by Dulton, in 1902 • It is endemic in West and Central Africa • Habitat: They live in man and other vertebrate hosts. They are parasites of connective tissue.
  15. 15. Morphology Vertebrate Forms • In the blood of vertebrate host, T. brucei gambiense exists as trypomastigote form, which is highly pleomorphic • It occurs as a long slender form; a stumpy short broad form with attenuated or absent flagellum; and an intermediate form. • The trypomastigotes are about 15–40 μm long and 1.5 – 3.5 μm broad.
  16. 16. Insect Forms • In insects, it occurs in 2 forms: – Epimastigotes – Metacyclic trypomastigote forms.
  17. 17. Antigenic Variation • Trypanosomes exhibit unique antigenic variation of their glycoproteins. • There is a cyclical fluctuation in the trypanosomes in the blood of infected vertebrates after every 7–10 days. • Each successive wave represents a variant antigenic type (VAT) of trypomastigote posssesing Variant Surface Specific Antigens (VSSA) or Variant Surface Glycoprotein (VSG) coat antigen. • A single trypanosome may have 1,000 or
  18. 18. Life Cycle • T. brucei gambiense passes its life cycle in 2 hosts. – Vertebrate host: Man, game animals, and other domestic animals. – Invertebrate host: Tsetse fly. • Both male and female tsetse fly of Glossina species (G. palpalis) are capable of transmitting the disease to humans. • Infective form: Metacyclic
  19. 19. • Mode of transmission: – By bite of tsetse fly – Congenital transmission has also been recorded. • Reservoirs: Man is the only reservoir host, although pigs and others domestic animals can act as chronic asymptomatic carriers of the parasite.
  20. 20. Development in Man and Other Vertebrate Hosts • Metacyclic stage (infective form) of trypomastigotes are inoculated into a man (definitive host) through skin when an infected tsetse fly takes a blood meal • The parasite transforms into slender forms that multiply asexually for 1–2 days before entering the peripheral blood and lymphatic circulation. • These become ‘stumpy’ via intermediate forms and enter the blood stream. • It invades the central nervous system in chronic infection.
  21. 21. Development in Tsetse Fly • In the midgut of the fly, short stumpy trypomastigotes develop into long, slender forms and multiply. • After 2–3 weeks, they migrate to the salivary glands, where they develop into epimastigotes, which multiply and fill the cavity of the gland and eventually transform into the infective metacyclic trypomastigotes • Development of the infective stage within the tsetse fly requires 25–50 days (extrinsic incubation period).
  22. 22. Pathogenecity and Clinical Features • T. brucei gambiense causes African trypanosomiasis (West African sleeping sickness). • The illness is chronic and can persist for many years. • There is an initial period of parasitemia, following which parasite is localized predominatly in the lymph nodes. • A painless chancre (trypanosomal chancre) appears on skin at the site of bite by tsetse fly, followed by intermittent fever, chills, rash, anemia, weight loss,
  23. 23. • Systemic trypanosomiasis without central nervous system involvement is referred to as stage I disease. • In this stage, there is hepatosplenomegaly and lymphadenopathy, particularly in the posterior cervical region (Winterbottom’s sign). • Hematological manifestations seen in stage I include anemia, moderate leucocytosis, and thrombocytopenia.
  24. 24. • Stage II disease involves invasion of central nervous system. With this, the ‘sleeping sickness’ starts. • This is marked by increasing headache, mental dullness, apathy, and day time sleepiness. • There is infiltration of the brain & spinal cord, and neuronal degeneration.
  25. 25. • Abnormalities in cerebrospinal fluid include raised intracranial pressure, pleocytosis, and raised total protein concentrations. • The patient falls into coma followed by death from asthenia.
  26. 26. Trypanosoma Brucei Rhodesiense (East African Trypanosomiasis) • It is found in Eastern and Central Africa • Discovered by Stephans and Fanthan in 1910 from the blood of a patient in Rhodesia suffering from sleeping sickness. • The principal vector is G. morisitans, G. palpalis, and G. Swynnertoni
  27. 27. Pathogenesis and Clinical Feature • T. brucei rhodesiense causes East African sleeping sickness • East African trypanosomiasis is more acute than the Gambian form and appears after an incubation period of 4 weeks. • It may end fatally within an year of onset, before the involvement of central nervous system develops. • Pathological features are similar in both diseases with some variations— Edema, myocarditis, and weakness are more prominent in East African sickness.
  28. 28. • Lymphadenitis is less prominent. • Febrile paroxysms are more frequent and severe. • There is a larger quantity of parasite in the peripheral blood. • €Central nervous system involvement occurs early. • Mania and delusions may occur but the marked somnolence, which occurs in T. brucei gambiense infection is lacking.
  29. 29. Laboratory Diagnosis The diagnosis of both types of African trypanosomiasis is similar • Nonspecific Findings – Anemia and monocytosis. – Raised ESR due to rise in gamma globulin levels. – Reversal of albumin:globulin ratio. – Increased cerebrospinal fluid (CSF) pressure and raised cell count and proteins in CSF.
  30. 30. • Specific Findings – Definitive diagnosis of sleeping sickness is established by the demonstration of trypanosomes in peripheral blood, bone marrow, lymphnode, CSF, and chancre fluid.
  31. 31. Microscopy • Wet mount preparation of lymph node aspirates and chancre fluid are used as a rapid method for demonstration of trypanosomes. • These specimen are also examined for parasites after fixing and staining with Giemsa stain. • Examination of Giemsa stained thick peripheral blood smears reveals the presence of trypomastigotes
  32. 32. • If parasitemia is low, then examination of concentrated blood smear is a highly sensitive method. • Different concentration techniques employed are buffy coat examination, differential centrifugation, membrane filtration, and ion exchange column chromatography. • Examination of wet mount and stained smear of the CSF may also show trypanosomes
  33. 33. Culture • The organisms are difficult to grow, hence culture is not routinely used. However, it can be cultivated in Weinman’s or Tobie’s medium. Animal Inoculation • Inoculation of specimens from suspected cases to white rat or white mice is a highly sensitive procedure for detecting T. brucei rhodesiense infection.
  34. 34. Serodiagnosis Antibody detection • Almost all infected have very high levels of total serum IgM & CSF IgM • Various serological methods to detect these antibodies are: – Indirect hemagglutination (IHA) – Indirect immunofluroscence (IIF) – Enzymelinked immunosorbent assay (ELISA) – Card agglutination trypanosomiasis test (CATT) – Complement fixation test (CFT) • Specific antibodies are detected by these tests in serum within 2–3 weeks of infection. • Specific antibodies in CSF - demonstrated by
  35. 35. Antigen detection • Antigens from serum and CSF can be detected by ELISA.
  36. 36. Imaging • CT scan of the brain shows cerebral edema and MRI shows white matter enhancement in patients with late stage CNS involvement
  37. 37. Treatment • In stage I (i.e. No CNS involvement), pentamidine is the drug of choice for gambiense HAT and suramin is the drug of choice for rhodesiense HAT. • Dose: – Pentamidine: 3 - 4 mg/kg body weight, IM daily for 7–10 days. – Suramin: 20 mg/kgbody weight in a course of 5 injections intravenously, at an interval of 5– 7 days.
  38. 38. • In stage II, melarsoprol (MelB) is the drug of choice, as it can cross the blood brain barrier. • Dose: 2–3 mg/kg/day(max. 40 mg) for 3–4 days
  39. 39. Prophylaxis • Control - based on early diagnosis and treatment of cases to reduce the reservoir of infection. • Control of tsetse fly population (most important preventive measure) by wide spraying of insecticides, traps, and baits impregnated with insecticides. • No vaccine is available.
  40. 40. Trypanosoma Cruzi (Chagas’ Disease) • It is the causative organism of chagas’s disease or South American trypanosomiasis. History and Distribution: • Carlos Chagas, investigating malaria, accidently discovered T. cruzi • Zoonotic disease; limited to south and central America
  41. 41. Habitat • In humans – exist in Amastigote and trypomastigote forms. Amastigotes - Intracellular parasites - Found in muscular tissue,nervous tissue and RE system. Trypomastigotes - Found in peripheral blood
  42. 42. • In reduviid bugs – Amastigotes found in midgut – Trypomastigote present in hindgut and feces
  43. 43. Morphology Amastigote Oval body{2-4 micro mtr dia}.it has a nucleus and kinetoplast. • Flagellum is absent • Resembles amastigote of Leishmania spp, so it is called leishmanial form. • Multiplication occurs in this stage
  44. 44. Trypomastigote • Non muliplying form. • In blood, they appear either as long,thin flagellates{20 μm long} or short stumpy form {15μm long} • Posterior end is wedge-shaped • In stained blood smears, shaped like ‘C’, ’U’ or ‘S’, having a free flagellum of 1/3 length of body
  45. 45. Epimastigote Form Found in insect vector, reduviid bug and in culture • It has a kinetoplast adjascent to nucleus. • An undulating membrane runs along anterior half of parasite. • Divide by binary fission in hindgut of vector
  46. 46. Life Cycle T.Cruzi passes its life cycle in 2 hosts Definitive host :Man Intermediate host(vector): Reduviid bug or triatomine bugs Infective form: Metacyclic trypomastigotes , found in reduviid bugs Reservoir host: Armadillo, cat, dog and pigs.
  47. 47. Mode of transmission • Man is infected : ˉ By fecal matter of reduviid bug being rubbed into bite wound . ˉ Through contamination of conjunctiva and other mucous membrane surfaces. ˉ Congenitally ˉ By blood transfusion
  48. 48. Pathogenicity and Clinical Features • Incubation period - 1-2 weeks in man • Disease manifest in acute and chronic form.
  49. 49. Acute Chagas’ Disease • Occurs soon after infection. Last for 1-4 months. • Often seen in children under 2 yrs of age • First sign appears with in a week after invasion. • Inoculation of parasite in conjunctiva causes unilateral, painless oedema of
  50. 50. • In few patients, there may be generalised infection with fever, lymphadenopathy and hepatosplenomegaly • Patient may die of acute myocarditis and meningoencephalitis
  51. 51. Chronic Chagas’ Disease Found in adults and older children • It become apparent,years or even decades after the initial infection • It produces inflammatory response,cellular destruction,and fibrosis of muscles and nerves,that control tone of hollow organs like heart,oesophagus,colon etc. • Thus,it can lead to cardiac myopathy and megaoesophagus and megacolon
  52. 52. Congenital Infection Congenital transmission is possible in both acute and chronic phase ,causing myocardial and neurological damage in the foetus
  53. 53. Laboratory Diagnosis Diagnosis is done by demonstration of T.cruzi in blood or tissues or by serology Microscopy • Microscopic examination of fresh anticoagulated blood or buffy coat is the simplest way to see motile organisms • In wet mount,Trypomastigotes are fainly visible but their snake- like motion against RBC’s makes their presents apparent
  54. 54. • Trypanomastigotes can also be seen in thick and thin peripheral blood smear, stained with Giemsa stain • Microhematocrit containing acridine orange as a stain can also be used
  55. 55. Culture Novy, Neal and Nicolle (NNN) medium or its modifications are used • Medium is inoculated with blood and other specimens and incubated at 22-24 deg C • Fluid from culture is examined microscopically by 4 th day and then every week for 6 weeks • Epimastigotes and Trypomastigotes are found in culture
  56. 56. Animal Inoculation Blood or CSF is inoculated intraperitoneally into mice or guinea pigs
  57. 57. • Xenodiagnosis ˉ Is the method of choice in suspected chagas disease,if other examinations are negative,especially during the early phase of disease onset. ˉ The reduviid bugs are reared in a trypanosome-free laboratory and starved for 2 wks, then fed on patients blood.if trypomastigotes are ingested ,they will multiply and develope into epimastigotes and trypomastigotes,which can be found in
  58. 58. • Histopathology ˉ Biopsy examination of lymphnodes and skeletal muscles and aspirate from chagoma may reveal amastigotes of T.cruzi
  59. 59. Serology Consits of antigen detection and antibody detection Antigen detection ˉ ELISA has been developed for detection of antigen. ˉ Detected in urine and sera in patients
  60. 60. • Antibody detection Antibodies(IgG)may be detected by the following tests. ˉ IHA ˉ CFT(Machado-Guerreiro test) ˉ ELISA ˉ IIF ˉ Direct agglutination test(DAT) ˉ Chagas Radio Immune Precipitation Assay(RIPA)-Highly specific and sensitive test.
  61. 61. • Intradermal test ˉ Antigen cruzin is used to detect delayed hypersensitivity • Molecular diagnosis ˉ PCR is used to detect trypomastigotes in blood.
  62. 62. Treatment • No effective specific treatment is available • Nifutrimox and benznidazole- used with some success in both acute and chronic chaga’s disease,kills only extracellular form • Dose- nifutrimox 8-10 mg/kg adult 15mg/kg children benznidazole 5-10 mg/kg orally for 60 days
  63. 63. Prophylaxis • Application of insectiside to control the vector bug. • Personal protection using insect repellent and mosquito net. • Improvement in housing and environment to eliminate breeding places of bugs .

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