Haemoparasites....

Haemoparasites....

1. HAEMOPARASITES SUNIL KUMAAR.P DEPARTMENT OF CLINICAL PATHOLOGY ST.JOHN’S MEDICAL COLLEGE BANGALORE 13 October 2018 1 SUNIL KUMAR.P

2. DEFINITION Haemoparasites are those parasites that lives within its host bloodstream. The parasites which are found in blood are Malarial parasites Filaria Leishmania Trypanosoma Babesia 13 October 2018 2 SUNIL KUMAR.P

3. MALARIA It is a protozoan disease transmitted by the bite of infected female anopheles mosquito 4 species P.vivax P.falciparum P.malariae P.ovale 13 October 2018 3 SUNIL KUMAR.P

4. EPIDEMIOLOGY World wide Death rate :1.5 – 2.7 million In India P.vivax & P.falciparum are common 13 October 2018 4 SUNIL KUMAR.P

5. LIFE CYCLE 1:-Asexual division ( schizogony ) man (intermediate host) 2:-sexual development ( sporogony ) female Anopheline mosquito(definitive host) CYCLE IN MAN COMPRISES Pre erythrocytic schizogony Erythrocytic schizogony Gametogony Exo erythrocytic schizogony 13 October 2018 5 SUNIL KUMAR.P

6. TRANSMISSION ALSO OCCUR THROUGH Blood transfusion Bone marrow transplants Transplacentaly Drug addicts 13 October 2018 6 SUNIL KUMAR.P

7. LIFE CYCLE OF MALARIA 13 October 2018 7 SUNIL KUMAR.P

8. PATHOGENICITY Infection with the plasmodium causes intermittent fever –malaria Incubation period (10-14days) in p.vivax , p.falciparum & p.ovale P.malariae28-30days 13 October 2018 8 SUNIL KUMAR.P

9. Contin … … P.vivax :-benign tertian malaria P.malariae :- quartan malaria P.ovale :-tertian malaria P.falciparum :-malignant tertian malaria &responsible for black water fever& pernicious anaemia 13 October 2018 9 SUNIL KUMAR.P

10. COMPARATIVE FEATURE OF MALARIAL PARASITES FEATURE P.VIVAX P.FALCIPARUM P.OVALE P. MALARIAE schizogony 48hrs 48hrs 48hrs 72 hrs Forms in P.S Trophozoite s schizont gametocytes Rings gametocytes Trophozoites schizont gametocytes Trophozoites schizont gametocytes Ring stage 2.5Large prominent single chromatin dot 1.25-1.5Small delicate double chromatin dots multiple rings Similar to p.vivax Similar to p.vivax trophozoite Irregular, amaeboid,vacuole present Compact form rarely amaeboid ,pigments collect into a single mass band shaped, slightly amoeboid,vacuole inconspicuous Compact ,not amoeboid, vacuole inconspicuous 13 October 2018 10 SUNIL KUMAR.P

11. p.vivax p.falciparum p.ovale p.malariae schizont 9-10 μ m almost completely fills an enlarged ery 4.5-5 μ m fills two-third of a normal RBC 6.2 μ m fills about three quarters of a slightly enlarged RBC 6.5-7 μ m almost fills a normal sized RBC merozoite 12 – 24, 18 - 24 6 - 12 6 - 12 gametocytes spherical cresentic round round Malarial pigment Yellowish- brown;fine granules Dark brown- black;coarse Dark yellowish brown;coarser than p.vivax Dark brown Infected erythrocyte Enlarged, Schuffners dots Normal size, Enlarged oval Normal size 13 October 2018 11 SUNIL KUMAR.P

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13. Clinical feature typical features - Febrile paroxysm, anaemia and spleenomegaly Febrile paroxysm comprises 3 successive days. cold stage:-20 – 60 mts hot stage:-1 – 4 hrs sweating stage:- 2 – 3 hrs high risk group pregnancy children 13 October 2018 13 SUNIL KUMAR.P

14. Anaemia- microcytic or normocytic hypochromic Splenomegaly- enlarged and palpable. -No relapses in p.falciparum infection but relapses occur in p.vivax Complications of p.falciparum infections include - Pernicious malaria - Black water fever 13 October 2018 14 SUNIL KUMAR.P

15. Pernicious malaria Life threatning occur in acute falciparum malaria Due to heavy parasitization Various manifestations of PM Cerebral malaria: characterised by hyperpyrexia,coma and paralysis.Brain is congested Algid malaria:cold clammy skin leading to peripheral circulatory failure. Septicaemic malaria:high continuous fever , involvement of various organs 13 October 2018 15 SUNIL KUMAR.P

16. Black water fever It is the manifestation of repeated infections Pl. falciparum , which were inadequately treated with quinine Clinical condition: Intravascular haemolysis High fever vomiting haemoglobinuria 13 October 2018 16 SUNIL KUMAR.P

17. IMMUNITY Species-specific, Stage –specific and lasts only till malarial parasite infection remains active. ( premunition immunity) 13 October 2018 17 SUNIL KUMAR.P

18. DIAGNOSIS Specimen: blood - febrile paroxysm -Before starting treatment. METHODS OF EXAMINATION 1)Light microscopy 2)Fluorescence microscopy 3) QBC 13 October 2018 18 SUNIL KUMAR.P

19. Light microscopy conventional light microscopy of stained blood smear is the gold standard for confirmation of malaria Ring forms and gametocytes are most commonly seen in the PBS THICK & THIN smears are prepared from the capillary blood Stained with Giemsa or Leishman stain Examined under oil immersion lens 13 October 2018 19 SUNIL KUMAR.P

20. Collection of Blood Smears 5. Touch the drop of blood to the slide from below. 4. Slide must always be grasped by its edges. 2. Puncture at the side of the ball of the finger. 3. Gently squeeze toward the puncture site. 1. The second or third finger is usually selected and cleaned. 13 October 2018 20 SUNIL KUMAR.P

21. Preparing thick and thin films 1. Touch one drop of blood to a clean slide. 2. Spread the first drop to make a 1 cm circle. 3. Touch a fresh drop of blood to the edge of another slide. 6. Wait for both to dry before fixing and staining. 5. Pull the drop of blood across the first slide in one motion. 4. Carry the drop of blood to the first slide and hold at 45 degree angle . 13 October 2018 21 SUNIL KUMAR.P

22. THICK SMEAR Thick smear is based on the principle that during preparation of the smear, conc red blood cells are lysed with distilled water, showing intact parasites. The smear is dried thoroughly and stained. USES(THICK SMEAR) Detecting parasites quantitating parasitemia and Demonstrating malarial pigment not used for species diagnosis Not used for Species diagnosis 13 October 2018 22 SUNIL KUMAR.P

23. Quantitation of parasitaemia is of prognostic value determine whether parasitaemia is increasing or decreasing during antimalarial treatment At least 100-200 fields, each containing 20WBCs should be examined before a thick smear is reported as negative for malaria. 13 October 2018 23 SUNIL KUMAR.P

24. THIN SMEAR Detecting parasites and for determining the species of the infecting parasite The major diagnostic feature s, which suggest P.falciparum in a stained blood smear are Occurrence of ring forms alone or along with gametocytes the tendency for multiple rings in an individual RBC with ‘ accole ’ forms. 13 October 2018 24 SUNIL KUMAR.P

25. Presence of maurer’s clefts in the RBC’s containing large rings, and Banana –shaped gametocytes The diagnosis of malaria is ruled out by obtaining negative thick blood smears on at least 3 different occasions 13 October 2018 25 SUNIL KUMAR.P

26. Plasmodium falciparum Rings: double chromatin dots; appliqué forms; multiple infections in same red cell Gametocytes: mature (M)and immature (I) forms (I is rarely seen in peripheral blood) Trophozoites: compact (rarely seen in peripheral blood) Schizonts: 8-24 merozoites (rarely seen in peripheral blood) Infected erythrocytes: normal size M I 13 October 2018 26 SUNIL KUMAR.P

27. Plasmodium vivax Trophozoites: ameboid; deforms the erythrocyte Gametocytes: round-oval Schizonts: 12-24 merozoites Rings Infected erythrocytes: enlarged up to 2X; deformed; (Schüffner’s dots) 13 October 2018 27 SUNIL KUMAR.P

28. Species Differentiation on Thin Films 13 October 2018 28 SUNIL KUMAR.P

29. GRADING 1 – 10 PARASITES/100 FIELD- + 11 – 100 PARASITES/100 FIELD + + 1 – 10 PARASITES /FIELD + + + >10 PARASITES /FIELD + + + + 13 October 2018 29 SUNIL KUMAR.P

30. QBC PRINCIPLE Ability of acridine orange to stain nucleic acid containing parasites. blood is collected in a capillary tube coated with fluorescent dye . After centrifugation the buffy coat in the centrifuged capillary tube is examined directly under the fluorescent microscope. Acridine orange stained malaria parasites appear brillant green. 13 October 2018 30 SUNIL KUMAR.P

31. Reagents Each QBC capillary blood tube is internally coated with Acridine Orange , Potassium oxalate, Sodium Heparin and K2EDTA. Prepare and centrifuge blood tube Fill the QBC capillary blood tube, from end nearest the two blue lines, directly from a finger(or heel)puncture or a collection tube of well-mixed venous blood - fill the tube by capillary action to a level between the two blue lines . -wipe off any blood on the outside of the tube 13 October 2018 31 SUNIL KUMAR.P

32. 2. Keep the tube nearly horizontal and roll between the fingers several times to mix the blood with the anticoagulant coating. 3.Turn the tube around and tilt, allowing the blood to flow to the end with the orange-coated stain.roll the tube between the fingers 5 times to mix the blood with the staining agent. 13 October 2018 32 SUNIL KUMAR.P

33. 4.Tilt the tube slightly so that blood flows away from the orange –coated end by at least ¼ to allow space for installing the closure;then place the index finger over the end of the tube nearest the blue fill lines. 5.Press a plastic closure onto the unsealed end of the tube. Then manually twist and press the closure to form a tight seal. 6.With a clean forceps, pick up a float and insert it into the unsealed end of the tube.Tap with the forceps until the float is inside the tube. 13 October 2018 33 SUNIL KUMAR.P

34. QBC MICROSCOPY 13 October 2018 34 SUNIL KUMAR.P

35. FLUORESCENCE MICROSCOPY Kawamoto technique blood smears are stained with acridine orange This result in a differential staining of the malarial parasites. Nuclear DNA is green & cytoplasmic RNA is red The stained slide is examined with a flourescent microscope Sensitivity 90% 13 October 2018 35 SUNIL KUMAR.P

36. SEROLOGICAL DIAGNOSIS IHA IFA ELISA -To identify the infected donors incase of transfusion malaria. -To confirm past malaria in patients. -Epidemiological survey in malaria. 13 October 2018 36 SUNIL KUMAR.P

37. Rapid diagnostic tests (DIPSTICK METHOD) Principle Enzyme Immunoassay. Which detects HRP-2 Dipstick containing monoclonal antibodies directed against the parasitic antigens ,is used Takes 5mnts Test has High sensitivity & specificity. Useful in detecting only P.falciparum 13 October 2018 37 SUNIL KUMAR.P

38. Molecular diagnosis DNA probe: high sensitive & specific. Detect <10/µl of blood. PCR TREATMENT: Chloroquine –acute malaria Mefloquine active against chloroquine resistant strains Artemisinin & its derivatives newer drugs 13 October 2018 38 SUNIL KUMAR.P

39. Filarial nematodes Nematodes which infect the diff tissues are called tissue or somatic nematodes. The Filarial nematodes are the major group of tissue nematodes Family filarioidea 13 October 2018 39 SUNIL KUMAR.P

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41. FILARIASIS PARASITE ADULT MICROFILARIA CHARACTERISTICS 1.LYMPHATIC FILARIASIS Wu.bancrofti lymphatics blood sheathed Brugia malayi lymphatics blood sheathed Subcutaneous Loa loa Connective tissue blood sheathed Onchocerca volvulus subcutaneous skin unsheathed Serous cavity Mansonella ozardi Body cavities blood unsheathed Mansonella perstans Body cavities blood 13 October 2018 41 SUNIL KUMAR.P

42. morphology Adult worms:- Whitish, thread &smooth surface Lives in lympatics,connective tissue, &muscle in males 4 – 6cm,in females 8 – 10 cm Microfilaria:- Found in PB, hydrocele fluid,& chylous urine Covered by hyaline sheath 13 October 2018 42 SUNIL KUMAR.P

43. Life cycle Definitive host:-human Intermediate host:-female culex anopheles & aedes mosquito Infective form:-third stage larva (mosquito) 13 October 2018 43 SUNIL KUMAR.P

44. Clinical presentation Fever Lymphangitis Hydrocele Chyluria elephantasis 13 October 2018 44 SUNIL KUMAR.P

45. Lab diagnosis Blood collection:- Mid night ( nocturnaly ) Other specimens- chylous urine,hydrocele fluid. 15 -20 mts after administration of DEC drug Detection wet preparation Thick & thin smear examination Microfilaria differentiated - sheath pattern - nuclei distribution &size 13 October 2018 45 SUNIL KUMAR.P

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47. Concentration method Knott method:- 1 ml blood + 9 ml formalin centrifuge at 2000 rpm for 20 mts Survey work:- Counter chamber technique Membrane filter conc. technique 13 October 2018 47 SUNIL KUMAR.P

48. DEC provocation test 2-8mg/ kgBW , DEC orally administrated After 30 mts capillary blood collected By wet mount & stained smear QBC TEST Urine microscopy SEROLOGICAL TEST IHA; IFA ELISA 13 October 2018 48 SUNIL KUMAR.P

49. Molecular methods:- pcr PCR –detect as low as 1 pg of filarial DNA. TREATMENT DEC 13 October 2018 49 SUNIL KUMAR.P

50. LESHMANIA World wide Only the visceral form( kala azar )is associated with organism in haemopoeitic tissue Indian visceral leishmaniasis is caused by L.donovani 13 October 2018 50 SUNIL KUMAR.P

51. MORPHOLOGICAL FORMS Promasitogte Spindle shaped Flagellar Insect(sand flies) Motile Amastigote Round or oaval Vertebrate stage Non-motile Intracellular Aflagellar stage In macrophage Digenetic Life Cycle 13 October 2018 51 SUNIL KUMAR.P

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53. Vector-phlebotomus fly(Sand fly) 13 October 2018 53 SUNIL KUMAR.P

54. LIFE CYCLE 13 October 2018 54 SUNIL KUMAR.P

55. Clinical features produces KALA AZAR(black disease) Visceral leishmaniasis is a serious & pottentially fatal systemic disease caused by L . donovani Incubation period:-3 – 6 months high fever Pyrexia Spleenomegaly Hepatomegaly Lymphadenopathy 13 October 2018 55 SUNIL KUMAR.P

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57. Haematological Anaemia Leucopenia Thrombocytopenia LAB DIAGNOSIS Specimen :- -Blood -B.M aspiration & splenic puncture 13 October 2018 57 SUNIL KUMAR.P

58. cs LYMPH NODE PUNCTURE BONE MARROW PUNCTURE SPLEEN PUNCTURE 13 October 2018 58 SUNIL KUMAR.P

59. Peripheral Blood smear Thick blood film-demonstration of amastigote form LD bodies –monocytes and neutrophils in stained PBS Smears stained by leishman , giemsa or wright stain. 13 October 2018 59 SUNIL KUMAR.P

60. CULTURE N.N.N medium is used for culture Incubated at 22-24c. Promastigote forms can be demonstrated 13 October 2018 60 SUNIL KUMAR.P

61. PROMASTIGOTES 13 October 2018 61 SUNIL KUMAR.P

62. culture N.Nnmedium GRADING >100 PARASITES/FIELD 6+ 10 -100 PARASITES/FIELD 5+ 1 -10 PARASITES/ FIELD 4+ 1 – 10 PARASITES/10 FIELD 3+ 1 -10PARASITES/100FIELD 2+ 1- 10 PARASITES/1000 FIELD 1+ OPARASITES/1000FIELD 0 13 October 2018 62 SUNIL KUMAR.P

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64. BONE MARROW ASPIRATION From sternum / iliac crest Amastigote forms demonstrated in macrophages and monocytes Promastigote –demonstrated in N.N.N medium 13 October 2018 64 SUNIL KUMAR.P

65. INDIRECT EVIDENCE Blood examination- pancytopenia , mainly neutropenia ↓erythrocyte count leucopenia, thrombocytopenia. A:G ratio reversed 13 October 2018 65 SUNIL KUMAR.P

66. IMMUNOLOGICAL TEST NON SPECIFIC TEST Aldehyde test Antimony test CFT with WKKantigen (Ag prepared from TB by witebsky,kleingenstein&kuhn SPECIFIC TEST DAT,IHA IFAT ELISA 13 October 2018 66 SUNIL KUMAR.P

67. TREATMENT PENTAVALENT ANTIMONIAL SODIUM STIBOGLUCONATE 13 October 2018 67 SUNIL KUMAR.P

68. BABESIA Named after Babes – in blood of cattle & sheep Tick borne disease;( USA&Europe ) 3 species B.bovis , B.divergens , B microti . Habitat :-inside RBC MORPHOLOGY INFECTIVE FORM: sporozoite (ticks) Trophozoites Oval & spindle shaped size 1 – 5 µm Small chromatin dot and scanty cytoplasm 13 October 2018 68 SUNIL KUMAR.P

69. Merozoite : -oval or round present as pyriform bodies arranged in pairs or multiples of 2 LIFE CYCLE Definitive host:-ticks of ixodes,boophilus Intermediate host:-man & domestic animals 13 October 2018 69 SUNIL KUMAR.P

70. Severe complications Acute tubular necrosis Pulmonary oedema Respiratory failure 13 October 2018 70 SUNIL KUMAR.P

71. Clinical features Incubation period :-1 – 4 weeks Fever Mild splenomegaly Anaemia Jaundice haemoglobinuria 13 October 2018 71 SUNIL KUMAR.P

72. haematological Haemolytic anaemia WBC count;-normal or decreased Reticulocyte count:-increased Thrombocytopenia ESR:-increased 13 October 2018 72 SUNIL KUMAR.P

73. Lab diagnosis Thin & thick smear Stain:- leishman,wrights,giemsa Only ring form seen Feature:- Haemozoin & gametocyte are absent Merozoite arranged in tetrads or maltese cross form 13 October 2018 73 SUNIL KUMAR.P

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75. Serodiagnosis - IFA MOLECULAR DIAGNOSIS;--PCR OTHER TES T:- Serum protein electrophoresis:-poly clonalgammapathy Urine:- haemoglobinuria & proteinuria Haptoglobin :-decreased 13 October 2018 75 SUNIL KUMAR.P

76. treatment Combination of clindamicin (600mg 3times daily) & oral quinine20mg/kg body wt daily for children; 650mg 3-4times daily for adults) Period 7-10 days. 13 October 2018 76 SUNIL KUMAR.P

77. TRYPANOSOMA These are haemoflagellates that live in blood & tissue of their hosts. They cause two important diseases. 1:-sleeping diseases 2:- chagas diseases. CHAGAS DISEASES (American trypanosomiasis ) it is caused by protozoan parasites T.cruzi . Indurated inflammatory lesion called chagoma 13 October 2018 77 SUNIL KUMAR.P

78. Life cycle Definitive host:-man Intermediate host:- reduviid bugs Clinical features Malaise Fever Anorexia Oedema of face Hepatosplenomegaly lymphadenopathy Development of subcutaneous inflammatory nodule 13 October 2018 78 SUNIL KUMAR.P

79. Laboratory diagnosis Wet,thin &thick blood film Conc .method Serodiagnosis -IHA -IFA -ELISA Molecular diagnosis Blood culture- NNN MEDIUM -LIT MEDIUM Animal inoculation(mice) 13 October 2018 79 SUNIL KUMAR.P

80. Sleeping sickness( african trypanosomiasis ) Caused by - T.brucei Clinical features Head ache Arthralgia Malaise Wtg loss Oedema Hepatosplenomegaly 13 October 2018 80 SUNIL KUMAR.P

81. Life cycle Defenitive host -man Intermediate host- tse tse fly Hematological abnormalities Anemia Luekocytosis Thrombocytopenia Treatment Pentamidine & Arsenical 13 October 2018 81 SUNIL KUMAR.P

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83. THANK YOU…… 13 October 2018 83 SUNIL KUMAR.P