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  1. 1. Intestinal ProtozoaAmoebae and ciliatesDr. Devika IddawelaDepartment of Parasitology 2008/2009 Batch
  2. 2. OBJECTIVES1. Name the common intestinal amoebae &ciliates that infecthumans2. Of the intestinal amoebae, name the organisms that arepathogenic to human3. Outline the life cycle of Entamoeba histolytica /Balantidium coliindicating the stages that cause pathogenic effects and are ofdiagnostic importance in the above4. Identify points in Life cycle where preventive measures areapplicable5. Describe the mechanism of pathogenesis6. Describe the pathogenesis and clinical features of these stages7. Describe the mode(s) of transmission, prevention and control ofamoebiasis8. Describe the laboratory methods of diagnosis of theseorganisms
  3. 3. Intestinal protozoan• 1.Amoebae – moves by means of pseudopodia• 2. Ciliates – are propelled by rows of cilia thatbeat with a wave like motion3. Flagellates- move by long whip like flagellae4. Coccidia: lack the specialized organelles ofmotilityPhylum protozoa is classified into 4subdivisions based on methods of locomotion
  4. 4. AmoebaeUnicellular organismsCharacterized by possessingpseudopodia by which these organismsmove and engulf food particlessuch as bacteria, red blood cells
  5. 5. • Asexual reproduction – binaryfissionMost are free living
  6. 6. can exist as trophozoite (growing stage) or cyst( dormant stage)Differentiate on morphological features of eithertrophozoite or cystDifferentiating features of trophozoite:Size,Type of motility – directional or non- directionalfast or sluggishcharacter of pseudopodia,Cytoplasmic inclusion bodies : Red blood cells,food vacuoles containing bacteria, yeast
  7. 7. Differentiating features of cyst :sizeshapenumber of nuclei, structure of nucleipresence of glycogen massChromatoid body or bar - coalesced RNAwithin the cytoplasmnumber of nuclei, arrangement of peripheral chromatin,position of the karyosomeNuclear structure:Chromatin ; Nuclear DNA present as peripheralchromatinKaryosome: small condensed mass of chromatin within thenuclear spacePeripheral chromatin – chromatin adhering to nuclearmembrane
  8. 8. Genus : EntamoebaParasites of alimentary tract - man, monkeysvertebrates and invertebratesCharacteristics of this genus :Nucleus more or less sphericalNuclear membrane line with chromatingranulesSmall karyosome situated at or near thecentreTrophozoite has single nucleus
  9. 9. Endolimax andIodamoebaperipheralchromatinLarge karyozomeEntamoebakaryosomeGenus:Genus:Grouped according to the number of nuclei in the mature cyst (1,4,8)
  10. 10. Amoebae that parasitize humansIntestinal amoebae: ( inhabit the large intestine)Entamoeba histolyticaE.disparE.coliE.hartmaniEndolimax nanaIodamoeba butschliiDientamoeba fragillisOral cavity : Entamoeba gingivalis
  11. 11. There are two stages in the life cycle ofthese amoebae.1.Trophozoite:mortile and feedingstage. Multiply by binary fission2. Cyst : Inactive, non motile andinfective stageNo cyst stages in D.fragilis &E.gingivalis
  12. 12. Of several species of amoebae live in thealimentary tract of human MAJORITY arecommensals ONLY Entamoebahistolytica is pathogenic D.fragilis and I.butschlii,may cause intestinal infection
  14. 14. • cosmopolitan distribution• worldwide incidence: 0.2-50%• highest prevalence in areas withpoor sanitation• no animal reservoirs•estimated 50 million cases/year100,000 deaths/yearEntamoeba histolytica
  15. 15. Disease: amoebiasisBlood and mucous diarrhoeaPathogenic organism parasitize largeintestine of manE. dispar identical morphology but notInvasive ( non-pathogenic)
  16. 16. RBCsNucleus20-40 µm, motility-active, progressive,directionalPseudopodia- finger like, hyaline, very rapidlyextrudedInclusions- red blood cells (invasive forms)Nucleus- single, fine central kayosome,regular peripheral chromatinTrophozoite
  17. 17. Cyst – spherical, 10-20 µm (E. hartmanni <10 µm)Nuclei: 1-4, structure like in trophozoiteChromatoid bodies: thick, 1-2 stain like chromatin,disappear as cyst matures (does not stain with Iodine)E. dispar identical morphology
  18. 18. Life cycleInfective stage• Ingestion of maturecysts• Excysts in smallintestine• Each cyst give riseimmature trophozoites• Maturation takes placein caecum•Trophozoites feed growand divide causingpathological effects
  19. 19. AmoebiasisPathogenesis - Infection withE.histolytica does notnecessarily lead to disease. Theoutcome depends on :•Host factors•Parasite factors
  20. 20. Host Factor Contributions• Physico-chemical environment of thegut influenced by bacterial flora, mucussecretion & gut motility• Degree of immunological resistance
  21. 21. Important virulence factors of E.histolytica• Adhesion molecules ( N- acetyl-D-galactosamine inhibitable lectineGal/GalNac) – adhesion to colonic mucine and hostcells• induce contact dependent cytolysis,• Channel-forming peptides(Amoebapores):Stored in cytoplasmic granules & releasefollowing target cell contact, forms ironexchanging channels in plasma membrane –lysing the target cellsParasitic factors
  22. 22. 3. Cystein protinases –Aid in penetration of host tissue by digestingextracellular matrix, cleaving collagen, elastin,fibrinoge inextracellular matrix by stimulating host cell proteolytic cascadeResistance to host response• complement resistance-inactivates theinactivates thecomplement factorscomplement factors and are thus resistantand are thus resistantto Complement mediated Complement mediated lysis.• Limit the effectiveness of humoralresponse by degradingboth IgA and IgG4. Species/strain differences; E. dispar noninvasive, Pathogenic zymodemes =E.histolytica
  23. 23. PathologyIntestinal Amoebiasis –LARGE INTESTINE
  24. 24. • Penetration of mucus layer• contact-dependent killing of epithelium• breakdown of tissues (extracellular matrix)• contact-dependent killing of neutrophils, leukocytes, etc.initially produce focal and superficial erosions in largeintestine with unaffected mucosa in betweenAdhere to colonic mucinand host clls
  25. 25. Amoebic ulcerationwith unaffected mucosa in between
  26. 26. •Trophozoites advance laterally and downwardinto the submucosa producing a flask-shapedulcer ( typical appearance of intestinalamoebiasis)Flask shaped ulcers -Base in submucosa and small opening onthe mucosal surface• Trophozoite penetrates the intestinalepithelium and then the muscularis mucosa &enter in to submucosa
  27. 27. Trophozoites penetrate the muscleand serous layers leading tointestinal perforations ,peritonitisRarely involvement of blood vessels at the base of theulcer may produce profuse bleedingAmoeboma - Amoebic granulomaAn inflammatory thickening of the intestinal wall,due to repeated invasion of colon by E histolyticacommon sites- ascending colon & caecumHaematogenous spread to other organs
  28. 28. Tissue invasion:Initial lesion – large intestine, caecum,ascending colon, sigmoidorectal region.
  29. 29. Extraintestinal Amebiasis• primarily liver (portal vein)• other sites less frequentBlood stream and lymphatic spread causeextra-intestinal amoebiasis(liver, skin, brain,heart)
  30. 30. Hepatic amoebiasisSingle abscess- Rt. Lobe (commonest)predisposing factors: alcoholSpread to other sites- direct-blood stream
  31. 31. Hepatic abscess ( common site is right lobe)
  32. 32. Pulmonary Amoebiasis• rarely primary• rupture of liver abscessthrough diaphragm• fever, cough, dyspnea,pain,Cutaneous Amoebiasis• intestinal or hepatic fistula• perianal ulcers• urogenital (eg, labia,vagina, penis)
  33. 33. Clinical featuresIntestinal diseaseMajority of infections are asymptomatic[cyst passers are infective carriers]asymptomatic cyst passer• Amoebic colitisGradual onset ( symptoms presenting over 1-2weeks)abdominal pain, tenesmus , watery or bloodydiarrhoea, anorexia, loss of weight. Fever only10- 30%Rectal bleeding without diarrhea can occur,especially in children
  34. 34. •fulminant colitis- Rare complication• abrupt onset ofprofuse bloody diarrhoea, highfever,dehydration ,wide spreadabdominal pain+ perforation (peritonitis)
  35. 35. •amoeboma (amoebic granuloma)- painfulabdominal mass• perianal ulceration
  36. 36. Extraintestinal Disease _ sings &symptoms depend on the organ affectedliver abscess –Frequently affect adults than children,Male>female60-70% of patients with amebic liver abscess donot have concomitant colitis, a history of dysenterywithin the previous yearhepatomegaly, liver tenderness, pain in theupper abdomen, High fever and anorexia,Weight loss, vomiting, fatigue
  37. 37. Diagnosis of Amoebiasis
  38. 38. TrophozoitesDirect wet faecal smears in saline candemonstrate motile trophozoite. Fresh sample offaeces ( preferably with in 30 min) should beexamine to visualize live trophozoite.confirmed on a permanently stained smear toidentify morphological features of nucleusEg; Trichrome or Iron haematoxylin• Biochemical Methods: Culture andIsoenzyme analysis to differentiateE.dispar from E.histolytica
  39. 39. Entamoeba histolytica
  40. 40. Sigmoidoscopy:Visualize characteristic ulcersLook for trophozoites in mucosal aspirateBiopsy can be taken from the edge of ulcerstained with H &E
  41. 41. CystWet faecal smear ( saline or iodine)If cysts are few to be present in directsmear, cysts can be concentratedeither by floatation ( Zinc sulphatecentrifugal floatation) or bysedimentation ( Formal-Ether )Faecal concentration methods
  42. 42. Trophozoite4.Mature cyst with 4nucleiImmature cystImmature cyst
  43. 43. E.Coli cystSize – 10 -20 µm, >4nucleiNucleus ; eccentric karyosome withirregular coarse chromatinChromatoid bodies infrequent ,needleshape when presentDifferentiation of E.Hislolytica from other non-pathogenic intestinal protozoa is very important
  44. 44. Iodamoeba butshclii cyst7 -15 µm, , glycogen mass is large, darkbrown with iodine
  45. 45. Acute dysentery- predominant formtrophozoitessaline, stained smear, cultureColitis – cysts - saline, iodine, concentrationmethodsFaecal examination: minimum of 3 samples in7 dayswet/permanent/culture
  46. 46. Diagnosis – Intestinal amoebiasisDefinitive diagnosis[GOLD STANDARD]– demonstrate parasite instools/rectal smearsSTOOLFULL REPORT= SFRTrophozoiteswith ingestedred blood cells indicateinvasive amoebiasisPresence of cysts doesnot indicate activedisease but infectivecarriers(cysts are infective)Without the specificpresenceof ingested RBCs in thecytoplasmthe pathogen, E. histolytica& the non pathogen, E.disparAre morphologicalyidentical BUTBiochemically different
  47. 47. Detection of E.histolytic specific antibodiesBy Enzyme linked immunosorbent assy(ELISA)Useful in non-endemic areas where E. histolyticainfection is not commonAntigen Detection in stool• Antigen-based ELISA sAdvantagesDifferentiate E. histolytica from E. dispar; (ii) they haveexcellent sensitivity and specificity;Immunodiagnosis
  48. 48. Emerging methods in Diagnosis• These are considered themost useful tests fordetecting E. histolytica.They test directly for theparasite itself by exposingsome stool to a strip ofpaper coated withantibodies. The parasiteswill stick to the antibodieson the paper. The testdistinguishes E.histolytica from otherparasites.• Disadvantage : costly
  49. 49. Molecular Biology-Based DiagnosticTests - PCR• Detection of parasite DNA in faeces by PCR• Provide high sensitivity and specificity for thediagnosis of intestinal amoebiasis
  50. 50. •WBC/DC –leucocytosis >10,000/mm3• immunodiagnosis :•serology - Serum antibody detection –ELISA•Serum antigen detection by ELISAExtraintestinal -Hepatic
  51. 51. •abscess aspirationonly selected casesreddish brown liquidtrophozoites at the abscess wall•imagingX –ray, CT, MRI,ultrasound•Abscess fluid Ag detection (ELISA)
  52. 52. Typical aspirate- chocolate syrupTrophozoitesare found on marginal wallCommonly found in the last portions ofaspirated material
  53. 53. Peters & Gilles. Atlas of Tropical Medicine and Parasitology- 4thEd. Mosby-Wolfe 1995CT scan of abscess in R lobeX ray showing fluid level
  54. 54. amoebic hepatic abscesscausing a raised rightdiaphragm with pleural effusionNormal chest X ray
  55. 55. TestColitis Liver abscessSensitivity Specificity SensitivityMicroscopy(stool)<60% 10-50% <10%Microscopy(abscess fluid)NAbNA <25%Stool antigendetection(ELISA)>95% >95% Usually negativeSerum antigendetection(ELISA)65% (early) >90% ∼75% (late),100% (first 3∼days)Abscessantigendetection(ELISA)NA NA ∼100% (beforetreatment)PCR (stool) >70% >90% Not doneSerumantibodydetection(ELISA)>90% >85% 70-80% (acute),>90%(convalescent)sensitivity and specificity of tests ofdiagnosis for amoebiasisa
  56. 56. TransmissionThrough cystsSources of infection:Food and water contaminated with infectedfaeces.Food handlers excreting cysts are animportant source of contamination of foodsHouseflies also act as a mechanical vectorscontaminating foodSexual transmission• Direct – hand to mouth• Indirect- contamination of food/water
  57. 57. Man is only reservoir hostBecause of the protection conferred bycyst wall , cyst can survive days andweeks in external environmentCyst Can be killed:Boiling- Above 68 ° CIodine (200 ppm)/acetic acid 5-10%Remove from water by sand filtrationOrdinary chlorination does not kill cysts
  58. 58. EpidemiologyAmoebiasis is cosmopolitan but no correlationbetween infection and diseaseGenerally in developed countries asymptomaticIn tropics/low socio-economic standardsHigh pathogenicityHigh risk groups: travelers, institutional inmateshomosexuals,immunocompromised individuals, children in daycare centers
  59. 59. PreventionReduce environmental contamination:detecting and treating infected personsImprove environmental sanitationAvoid ingestion of infected cyst bypersonal protection
  60. 60. Food safety• Thoroughly cook all raw foods.• * Thoroughly wash rawvegetables and fruits beforeeating.• * Reheat food until the internaltemperature of the foodreaches at least 167º.• Wash your hands beforepreparing food, before eating,after going to the toilet orchanging diapers
  61. 61. CiliatesCiliates
  62. 62. What are ciliates ?Protozoa with ciliaCilia -Hair like structures used for locomotionand feeding.Shorter than flagella and more innumber
  63. 63. • Use cilia for movement or feeding• Can have more than one nucleus(macronucleus, micronucleus)• Feed through a “mouth” like structure (oralgroove,Ciliophora – ciliates
  64. 64. Generally larger than other protozoaReproduce by binary fissionONLY ciliate that is known to parasitizeman is Balantidium coli
  65. 65. Balantidium coliLargest protozoan parasite of manA common parasite of pigsPig the main reservoirHuman infection is less frequentParasitize distal ileum and colonInvade the mucosa and causes bloodand mucous diarrheoaIt is a zoonotic infection
  66. 66. C/f similar to amoebic dysenterybut no extra-intestinal spreadPathogenic to man as it invade theintestinal tissue
  67. 67. Two morphological formsTrophozoiteCyst
  68. 68. Trophozoit Cyst
  69. 69. EM viewCilia
  70. 70. Life cycle
  71. 71. DiagnosisDetection of cysts and trophozoits infaecal smears.
  72. 72. Regarding E. histolyticaA. Cyst is the infective stageB. Does not attach to intestinal mucosaC. Inhabits the human large intestineD. Extaintestinal spread is possibleE. Nucleus has a central karyosomeRegarding amoebaA. E. gingivalis has cyst stage in their life cycleB. Can differentiated by their characteristic movementsC. E. dispar is a human pathogenD. E. coli and E. histolytica are morphologically identical
  73. 73. Regarding Balantidium coliA. It is not pathogenic to humanB. Trophozoite has only one nucleusC. It is a zoonotic parasiteD. Cyst is covered with ciliaE. Trophozoite is the infective stage to humanTrue /false E.histolyticaInhabits human large intestineE. Histolytica cyst is a infective stage to humanTransmitted by faeco-oral routeE.Histolytica trophozoite is morphologically identical to E. dispar
  74. 74. True or falseGenus Entamoeba has large katyosome in side the nucleusE. Histolytica trophozoite moves sluggishlyE. Histolytica trophozoite has single nucleus with centrally placed karyosomeE. Gingivalis has trophozoite and cyst in their life cycleAcute amoebic dysentery, predominant form is cyst in stoolsFlask shaped ulcers are typical lesion in intestinal amoebiasisTrophozoites in faecal samples is a commonly associated with hepatic amoebiasisIn amoebic colitis, predominant form in the faeces is trophozoiteE histolytica and E dispar cysts cannot differentiate microscopicallyFever is a common clinical feature of amoebic colitis.Abscess fluid microscopy is useful in the diagnosis of amoebic liver abscess