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Opportunistic Protozoans - Microbiology

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Opportunistic Protozoans--Microbiology..
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Opportunistic Protozoans - Microbiology

  1. 1. PRESENTED BY : ABHISHEK KUMARYADAV
  2. 2. Opportunistic Protozoans : Cryptosporidium Microsporidia Cyclospora Isospora
  3. 3. Cryptosporidium SPP.  Disease Cryptosporidiosis  Phylum Apicomplexa  Class Sporozoa
  4. 4. Introduction  Cryptosporidium species causes diarrhoeal disease cryptosporidiosis.  Both the parasite and the disease are commonly known as "Crypto.“  Water (drinking water and recreational water) is the most common method of transmission.  Cryptosporidium parvum and C. hominis are the most prevalent species.
  5. 5. Morphology  Morphological form detectable in faeces is oocyst  measures 4-5 m in diameter  spherical or ovoidal in shape  contains four crescentic sporozoites and amylopectin like granules (1-6 large dark granules).  Two types :  thin walled (autoinfection)  thick walled (faeces)
  6. 6. LIFE CYCLE
  7. 7. Transmission electron micrograph showing type II meront containing 4 merozoites.
  8. 8. Transmission electron micrograph of a fertilized macrogamete 4.1 µm x 2.5 µm connected to the host cell and surrounded by the parasitophorous vacuole (arrow) and the feeder organelle (arrow head).
  9. 9. Scanning electron micrograph showing numerous cryptosporidia on surface of epithelial cells: trophozoites (arrow), crater-like area (arrowhead) is a ruptured parasitophorous envelope.
  10. 10. Scanning electron micrograph showing type I meront releasing 8 merozoites
  11. 11. Transmission electron micrograph showing macrogamont with nucleus and dark bodies
  12. 12. PATHOGENESIS
  13. 13. diarrhoea • enterocyte malfunction (osmotic) • impaired absorption • enhanced secretion • inflammatory diarrhoea • mucosal invasion • leukocytes in stools • secretory diarrhoea • toxin • watery Pathogenesis • enterocytes damaged or killed •villus atrophy (blunting) • Na+ absorption • permeability • crypt cell hyperplasia • Cl- secretion • inflammation
  14. 14. Clinical Features  Incubation period : 4 to 22 days after ingestion of oocysts.  Self-limited diarrhoea lasting 5 to 14 days.  diarrhoea is cholera-like, profuse, watery, and foul smelling, with no leukocytes or blood.  Other symptoms :  Nausea and vomiting, abdominal cramps, low-grade fever, anorexia, dehydration, weight loss, weakness, myalgia, and headache.
  15. 15. Clinical Features  Patients with profound immunosuppression (AIDS) :  Fluctuation with changes in CD4 count and antiretroviral therapy.  The 4 patterns of clinical syndromes are chronic diarrhoea, cholera-like disease, transient diarrhoea, and relapsing illness.  Have a greater incidence of infection in extraintestinal sites, such as the stomach, and the biliary, pancreatic, and respiratory tracts.
  16. 16. Lab Diagnosis  Specimen  Stool  Sputum – in respiratory cryptosporidiosis  Biopsy – for histopathological examination  Blood – for detection of antibodies  Stool Examination  3 consecutive stool specimens to be examined  Direct wet mount of stool for demonstration of highly refractile, spherical oocysts.  Staining by Modified Ziehl-Neelsen Stain  Immunofluorescent Antibody test  Concentration by Sheather’s sugar concentration technique  Antigen detection in faeces by Immunochromatic test  Histopathological examination –intestinal biospy by H/E stain  Serology  Polymerase Chain Reaction (PCR)
  17. 17.  Oocysts in stool are stained by a variety of techniques:  modifed cold Kinyoun acid-fast  Ziehl-Neelsen acid-fast  Safranin-methylene blue  Giemsa  fluorescent acridine orange  auramine-rhodamine stains  Oocysts are autofluorescent.  On phase-contrast microscopy, oocysts are bright, refractile, have up to 6 black granules, and often adhere to mucus.  Differential staining :  Grocott’s methenamine silver (GMS)  Warthin-Starry silver impregnation stain (WS)  Brown-Hopps (B&H) tissue gram stain
  18. 18. Cryptosporidium parvum of calves. Fecal float with refractile unstained oocysts suspended in water
  19. 19. Cryptosporidium parvum in centrifuged human faeces stained with modified cold Kinyoun acid-fast.
  20. 20. Fecal float with unstained oocysts suspended in sugar solution and viewed with phase-contrast microscopy
  21. 21. Fecal float with oocysts stained with monocloanal antibody conjugated with fluorescent isothiocyanate.
  22. 22. Treatment  There are several treatments for cryptosporidium enteritis.  Drugs such as nitazoxanide have been used in children and adults.  Other drugs that are sometimes used include:  Atovaquone  Metronidazole Trimethoprim-sulfamethoxazole
  23. 23. The Milwaukee Outbreak (1994) •massive cryptosporidiosis outbreak following spring thaw • >400,000 people may have been affected • based on clinical symptoms (acute watery diarrhoea) •treated water had high levels of turbidity • oocysts identified in ice made during this period • 100-fold higher prevalence of Cryptosporidium oocysts in stools • other enterics (including Giardia, bacteria, viruses) were at ~normal levels
  24. 24. ISOSPORA BELLI
  25. 25. Sporozoan of human intestine. Occurs throughout the world. Most common in patients with AIDS. First described byVirchow in 1860 & named byWenyon in 1923. It causes Isosporiasis which is a human intestinal disease.
  26. 26. Isosporiasis:- Infection causes acute, non-bloody diarrhoea with crampy abdominal pain. Can last for weeks & result in malabsorption & weight loss.
  27. 27. MORPHOLOGY:- Oocysts of Isospora belli – elongate- ovoidal. Measuring – 22 to 33 μm × 10 to 15 μm. Each oocyst is surrounded by thin, smooth, two layered cyst wall. Immature oocyst seen in faeces of patients contains two sporoblasts.
  28. 28. MORPHOLOGY:- On maturation, sporoblasts convert into sporocysts. Each sporocyst contains 4 crescent shaped sporozoites.
  29. 29. LIFE CYCLE Life cycle of Isospora belli completes in one host. Humans acquire infection by ingestion of food and water contaminated with faeces containing sporulated (mature) oocysts. 8 sporozoites are released in the small intestine & invade the epithelial cells of distal duodenum & proximal jejunum.
  30. 30. Now they undergo asexual multiplication to produce trophozoites. Trophozoites undergo sexual cycle (gametogony) to produce microgamete & macrogamete. These microgamete & macrogamete upon fertilisation form oocysts which are excreted in the stool. Usually oocysts consist of single sporoblast but soon it divides into two.
  31. 31. These oocysts mature outside the host and develop into mature oocyst. These mature oocyst is the infective stage of the parasite.
  32. 32. LIFE CYCLE:-
  33. 33. PATHOGENECITY:- Isospora belli infects both immunocompetent adults & children. It may lead to mild, self limiting diarrhoea which lasts for 6 weeks to 6 months.
  34. 34. Symptoms- 1. Vomiting 2. Headache 3. Fever 4. Malaise. Dehydration follows when diarrhoea is severe.
  35. 35. LAB DIAGNOSIS:- Sample- Stool. Oocysts of Isospora Belli in the stool establishes the diagnosis.
  36. 36. Microscopic examination- 1- Smear can be prepared by Zinc Sulphate or formalin-ether concentration methods. 2- By acid fast staining or with Auramine Rhodamine. It appears red in colour.
  37. 37. 3- Unstained oocysts are autoflourescent. They appear violet under UV light & green under green or blue-violet light.
  38. 38. Lab Diagnosis Cont….. POLYMERASE CHAIN REACTION (PCR) Highly sensitive and specific method.
  39. 39. TREATMENT:- Cotrimoxazole is usually effective.
  40. 40. Microsporodia  Phylum : Microspora  Obligate, intracellular spore forming protozoa.  There are at least 15 microsporidian species that have been identified as human pathogens.
  41. 41. Nodular Cutaneous Microsporidiosis of the leg caused by Encephalitozoon intestinalis in an HIV positive patient.
  42. 42. Morphology  Unicellular, obligate intracellular parasites.  In host cell, the parasite develops and multiply (merogony-binary fission/schizogony-multiple fission) and produces large number of spores (sporogony).  Spore is the infective stage, measures 0.5-0.2 m x 1-4 m; oval to cylindrical in shape; possess a thick double layered wall.
  43. 43. Morphology  Within cytoplasm, spore contains a coiled polar tube which uncoils and thrusts forcefully into the host cell and injects sporoplasm (infective material).  Spores :  Stained with Gram’s stain, PAS, Giemsa stain or modified trichome stain.  Gram-positive and acid fast.
  44. 44. Electron micrograph of Anncaliia (Brachiola, Nosema) connori spore in adrenal gland showing the coiled polar filament (arrow) and two nuclei.
  45. 45.  Species and Genera :  There are 15 genera which infect the humans :  Anncaliia (formerly Brachiola) algerae  A. connori  A. vesicularum  Encephalitozoon cuniculi  E. hellem  E. intestinalis  Enterocytozoon bieneusi  Microsporidium ceylonensis  M. africanum  Nosema ocularum  Pleistophora sp.  Trachipleistophora hominis  T. anthropophthera  Vittaforma corneae, and  Tubulinosema acridophagus.
  46. 46. Spore of Enterocytozoon bieneusi demonstrating the characteristic six turns of the polar tubule, which are organized into two tiers of three turns each.
  47. 47. LIFE CYCLE
  48. 48. Pathogenesis • Microsporidia have been reported as pathogens in patients with HIV disease. • Infection is probably by ingestion, inhalation or inoculation of spores. • Microsporidia species frequently associated with AIDS are : • Enterocytozoon bieneusi • Enchepalitozoon hellem • Encephalitozoon intestinalis • Intestinal microsporidiosis – commonest infection caused mainly by E. bieneusi.
  49. 49. Clinical Features Microsporidian species Clinical manifestation Anncaliia algerae Keratoconjunctivitis, skin and deep muscle infection Enterocytozoon bieneusi* diarrhoea, acalculous cholecystitis Encephalitozoon cuniculi and Encephalitozoon hellem Keratoconjunctivitis, infection of respiratory and genitourinary tract, disseminated infection Encephalitozoon intestinalis (syn. Septata intestinalis) Infection of the GI tract causing diarrhoea, and dissemination to ocular, genitourinary and respiratory tracts Microsporidium (M. ceylonensis and M. africanum) Infection of the cornea Nosema sp. (N. ocularum), Anncaliia connori Ocular infection Pleistophora sp. Muscular infection Trachipleistophora anthropophthera Disseminated infection Trachipleistophora hominis Muscular infection, stromal keratitis, (probably disseminated infection) Tubulinosema acridophagus Disseminated infection Vittaforma corneae (syn. Nosema corneum) Ocular infection, urinary tract infection
  50. 50. Lab Diagnosis  Specimen  Small Intesinal Biopsy – for histopathological examination  Faeces  Biopsy examination  Intestinal microsporidiosis – most common type of infection  Diagnosed by microscopy of small intestinal biopsy sections.  Light microscopic and electron microscopic examinations used for demonstration of spores.  Brown-Brenn or Brown –Hopps tissue Gram stain, PAS or Giemsa stains- used for demonstration of tiny intracytoplasmic spores.  Faeces Examination  Spores detected in faeces and content of duodenum-jejunum using modifiedTrichome stain.  Culture – culture of spores.  Polymerase Chain Reaction (PCR) – Microsporidial DNA amplified and detected.
  51. 51. Treatment  For treatment of intestinal microsporidiosis :  Metronidazole  Albendazole  For treatment of microsporidial keratoconjuctivitis :  Itraconazole  Fumagillin  Albendazole
  52. 52. CYCLOSPORA CAYATENENSIS
  53. 53. It is a newly recognised protozoan parasite. Causes a disease named as Cyclosporiasis in man particularly in patients with AIDS. First human case was reported in Peru in 1995. In recent years human cyclosporiasis has emerged as an important infection.
  54. 54. MORPHOLOGY:- The morphological form found in the faeces is an oocyst. Oocyst is non- refractile, spherical. Diameter- 8 to 10 μm. Oocyst contains 2 sporocysts.
  55. 55. Each sporocyst contains 2 sporozoites. Each sporulated oocyst are passed in the faeces.
  56. 56. LIFE CYCLE:- Source- Contaminated food & water containing oocysts. Host- Single host. Has both sexual & asexual stage. Man is infected by ingestion of food & water contaminated with faeces.
  57. 57. The unsporulated oocyst in faeces sporulates outside the host. Excystation of the sporocyst releases 2 sporozoites which infect the small intestine causing diarrhoea.
  58. 58. PATHOGENESIS:- Incubation period- 1 week. Disease starts with acute watery diarrhoea with nausea, loss of appetite, abdominal pain, fever fatigue, weight loss. Diarrhoea may be prolonged & associated with muscle pain, vomiting, dehydration & substantial weight loss.
  59. 59. Illness may last six weeks before self-limiting. If the disease is left untreated, the illness may relapse.
  60. 60. LAB DIAGNOSIS:- Diagnosis- Made by stool sample.
  61. 61. To detect oocysts in faeces, conc. of faeces by floatation technique is required. Modified ZN staining is another useful method. Oocysts are acid fast & stain red in colour.
  62. 62. TAKE HOME MESSAGE AND SUMMARY  Acid fast Intestinal parasites  Persistent diarrhoea – HIV/AIDS  Cryptosporidium spp. – Oocyst  Cyclospora spp. – Oocyst  Isospora spp. – Oocyst  Microsporidium Spp. - Spores

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