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2013 PARASITOLOGY WORKSHOP Lynnegarcia2@verizon

2013 PARASITOLOGY WORKSHOP Lynnegarcia2@verizon.net. LYNNE S. GARCIA, MS, FAAM, CLS, BLM Diagnostic Medical Parasitology Workshop 2013 UPDATE – PART 2 INTESTINAL PROTOZOA SPONSORED BY MEDICAL CHEMICAL CORPORATION www.med-chem.com. Entamoeba histolytica.

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2013 PARASITOLOGY WORKSHOP Lynnegarcia2@verizon

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  1. 2013 PARASITOLOGY WORKSHOPLynnegarcia2@verizon.net LYNNE S. GARCIA, MS, FAAM, CLS, BLM Diagnostic Medical Parasitology Workshop 2013 UPDATE – PART 2 INTESTINAL PROTOZOA SPONSORED BY MEDICAL CHEMICAL CORPORATION www.med-chem.com

  2. Entamoeba histolytica • Pathogenic (E. dispar, nonpathogenic) • Morphology same (E. histolytica, E. dispar, E. moshkovskii) • Causes amebiasis (diarrhea, dysentery) • Fecal-oral transmission • Contaminated food and/or water • May disseminate from intestine to liver, skin, miscellaneous body sites 2

  3. Entamoeba histolytica Infection:Ingestion of infective cysts (4 nuclei) Trophozoites: multiply Cysts: not seen in diarrhea RBCs: seen in dysentery True amebiasis: RBCs within the trophozoites No ingested RBCs: may be E. histolytica (pathogen) or E. dispar (non-pathogen) or E. moshkovskii (may be pathogenic) 3

  4. Entamoeba histolytica • Clinical Symptoms • Intestinal: diarrhea, dysentery • Extraintestinal: right upper quadrant pain, fever • Clinical specimens • Intestinal: stool, sigmoidoscopy • Extraintestinal: liver aspirate, biopsy, serology for antibody 4

  5. Entamoeba histolytica vs E. dispar • Differentiation - morphology • Trophozoites with ingested RBCs in cytoplasm = diagnostic for E. histolytica (RBCs must be seen in the background) • Immunodetection (specific reagent) • EIA format, requires fresh, frozen, possibly Cary-Blair (check pkg insert) • Entamoeba histolytica/E. dispargroup • Entamoeba histolytica (true pathogen) 5

  6. FECAL IMMUNOASSAY FORMATS E. histolytica Antigen Detection Thermo: ProSpecT (E. histolytica) TechLab: Entamoeba histolytica II Biosite: Triage (E. histolytica/E. dispar) E. histolytica reagent designed to pick up E. histolytica ONLY – not E. dispar or E. moshkovskii. 6

  7. ENTAMOEBA HISTOLYTICAENTAMOEBA DISPAR Entamoeba dispar (non-pathogen) Note: Ingested RBCs Compact karyosome 7 Entamoeba histolytica (pathogen)

  8. ENTAMOEBA POLECKIENTAMOEBA MOSHKOVSKII Entamoeba polecki (non-pathogen) Two organisms (cyst) do NOT look alike! Trophozoites can be confused – like Entamoeba genus. Entamoeba moshkovskii (pathogenicity - ??) 8

  9. REPORTING • If cysts or no ingested RBCs (trophs) are seen or immunoassay is not available: • Entamoeba histolytica/E. dispar/E. moshkovskii NOTE:Entamoeba moshkovskiialso looks like Entamoeba histolytica/E. dispar; however, it is not that easy to differentiate, so the name is currently not added to the overall report. It tends to be more rare than the others. Some controversy per pathogenicity (Australia studies indicate some symptomatic patients) 9

  10. Entamoeba coli TrophozoiteNon Pathogen •  Ingested debris, vacuoles •  Trophs 15 – 50 µm (range 20 – 25 µm) • Peripheral chromatin clumped or uneven (may appear as solid dark ring) • Single nucleus, eccentric messy - blotlike karyosome (not compact) Ingested yeast cell 10

  11. Entamoeba coli CystNon Pathogen •  Splintered ends on chromatoidal bars •  Mature cyst = 8 nuclei(range 15 – 20 µm) • Cytoplasm may be clean • Nuclei may vary in morphology 11

  12. Entamoeba hartmanni TrophozoiteNon Pathogen •  NO ingested RBCs •  Single nucleus, central karyosome, even chromatin(5 – 12 µm) • Cytoplasm may be clean • Nucleus looks like “bull’s eye” • Compact karyosome 12

  13. Entamoeba hartmanni CystNon Pathogen •  Rounded ends on chromatoidal bars (many) •  Mature cyst = 4 nuclei (range 5 – 10 µm) • Cytoplasm may be clean • Cyst often stops with 2 nuclei • Remember “halo” effect measurement Note “halo” around cyst wall 13

  14. Endolimax nana Trophozoite, CystNon Pathogen Karyosome very Pleomorphic in trophozoite  Troph: 1 nucleus, usually no peripheral chromatin, clean cytoplasm, some debris  Cyst: four nuclei, no chromatoidals 14

  15. Iodamoeba bütschlii Troph, CystNon Pathogen  Troph: 1 nucleus, large karyosome, ± chromatin, dirty cytoplasm, some debris  Cyst: 1 nucleus, glycogen vacuole 15

  16. Blastocystis Life Cycle Vacuolar form most commonly seen.

  17. Blastocystis Classification, Forms An analysis of gene sequences was performed in 1996, which placed it into the group Stramenopiles.Other Stramenopiles include brown algae, mildew, diatoms, the organism that caused the Irish potato famine, and the organism responsible for Sudden oak death disease. However, the position of Blastocystis within the stramenopiles remains enigmatic.

  18. Blastocystis spp. Pathogenic • Central body form, large size range; peripheral nuclei • Irritable bowel syndrome; urticaria; infective arthritis • Multiple subtypes, some pathogenic, all look alike • Undergoing reclassification, quantitate • Rare dissemination, immunocompromised • Paromymycin best; metronidazole 22%; long term therapy required in immunocompromised hosts; reinfection common

  19. Blastocystis spp.Pathogenesis • Relationships between Blastocystis and intestinal obstruction and perhaps even infective arthritis have been suggested. In patients with other underlying conditions, the symptoms are pronounced and require longer treatment. • Infection with AIDS (ST 3), megacolon, cancer (ST 3), IBS/IBD, anemia, renal disease • Immunocompromised Infection Combinations: Cryptosporidium / Blastocystis OR • Microsporidia / Blastocystis • Cancer patients undergoing therapy should be tested repeatedly for intestinal parasites, particularly Blastocystis, • Cryptosporidium, and Microsporidia

  20. Giardia lambliaTrophozoitePathogen •  Teardrop shape, spoon •  Two nuclei, stain pale • Curved median bodies • Linear axonemes • Pathogen, 19,733 in 2005 • Water, food borne • Typical motility, but caught up in mucus 20

  21. Giardia lamblia CystPathogen 21 •  Oval to round •  Four nuclei • Curved median bodies • Linear axonemes • Pathogenic • Water, food borne • Sporadic shedding • Fecal immunoassays need 2 stools for NEG

  22. Dientamoeba fragilis: Pathogen 22 •  Very pleomorphic, 1 or 2 nuclei •  Nuclei fragmented chromatin or solid • Pathogenic, transmitted via helminth eggs • No cyst stage, permanent stained smear • As common or more common than Giardia

  23. Pentatrichomonas hominis (NP)Trichomonas vaginalis (P) 23 T. vaginalis P P. hominis NP •  No cyst forms •  Note different position of undulating membrane • Nonpathogen (GI tract) and pathogen (urinary/genital tract) (possible urine contamination with stool)

  24. Chilomastix mesniliNon Pathogen 24 •  Nonpathogenic flagellate •  Trophozoite often looks rounded up (like ameba) • Note the curved fibril in the cyst (Shepherd’s Crook) • Difficult to see without the permanent stained smear.

  25. Balantidium coliPathogen 25 •  Pathogenic ciliate, can penetrate mucosa •  Trophozoite and cysts are quite large (90+ microns) • Covered with cilia, large bean-shaped macronucleus • Can penetrate GI tract, diarrhea similar to cholera • Uncommon in US (pigs), proficiency testing specimens

  26. Cryptosporidium spp. – Clinical10,500 Cases Reported in 2010 • Immunocompetent – GI tract • Self-limiting, profuse watery diarrhea • Cramping pain, nausea, anorexia • Immunocompromised - Disseminated • Severe diarrhea (3-6 liters/day), weeks • HIV patients, CD4 cell count marker • 180-200 cells/mm3 or higher, good 26

  27. Cryptosporidium EpidemiologyPathogen • Associated with traveling, farm animal exposure, person-to-person (day care, nosocomial) – may not be coccidian • Self-limiting, profuse watery diarrhea • Cramping pain, nausea, anorexia • Waterborne outbreaks • Milwaukee and Georgia, >400,000 cases • C. hominis, C. parvum, C. cuniculus (rabbit), others 27

  28. Cryptosporidium DiagnosisC. hominis – C. parvum look alike • Examination for oocysts (4-6 µm) • C. hominis (humans), C. parvum (also animals) • Wet mount very poor, high numbers only • Routine stains don’t work (O&P stains) • Modified acid-fast stains, 1% decolorizer recommended, thin preps, histology • Auramine-rhodamine OK, but nonspecific • Fecal immunoassays available, EM 28

  29. CRYPTOSPORIDIUM SPP.C. hominis, C. parvum 29 Modified acid-fast: stool specimen; note sporozoites, 4-6 µm Regular trichrome (O&P) note lack of staining SEM intestinal surface; Intracellular, but extra-cytoplasmic

  30. GIARDIA, CRYPTOSPORIDIUMCombination FA Test Reagent Giardia lamblia cyst Cryptosporidium spp. oocysts Immunofluorescence Method (FA scope); water testing, not specific (algae, etc.) Two filters: FITC plus counterstain (FITC only OK) 30

  31. Cyclospora cayetanensis (Lab confirmed) 1,110 Cases (1997-2008)* • Immunocompetent – GI tract • Malaise, fever, watery diarrhea • Fatigue, anorexia, vomiting, weight loss • Immunocompromised – May disseminate • Relapses for many weeks – in sputum • Up to 12 weeks, biliary disease – AIDS • TMP-SMX effective • *Does not include year of big outbreaks, 1996 – U.S. 31

  32. CYCLOSPORA CAYETANENSIS(Suspected Food Borne Outbreaks) 32 Modified acid-fast stain Autofluorescence Acid-fast variable Often 1+ to 3+ CPT: 87015 + 87207 CPT: 87015 + 87210

  33. North American Cyclospora cayetanensis Outbreaks • 1990 Tap water • 1995 Fresh Guatemalan raspberries • 1996 Fresh Guatemalan raspberries • 1996 Basil-Pesto pasta salad • 1996 Fresh basil • 1997 US, Canada raspberries, mesclun • 1997 Import voluntarily suspended • 1998 US, Canada, fruit salad, raspberries • 1999 US, chicken pasta, tomato basil salad • 2000 Imported raspberries • 2001 Canada, Thai basil • 2004 Guatemalan snow peas • 2005 US, fresh basil • 2009 Cruise ship – multiple countries • 2010 Canada, salad blends, leafy greens 33

  34. Cyclospora DiagnosisOne US outbreak in 1990 - water • Detection of oocysts (8 - 10 µm) (7.7 – 9.9 µm) • Oocysts do not stain well with Giemsa, trichrome, hematoxylin • Autofluorescence used to confirm • Autofluorescence may be only 1+ to 3+ • Modified acid-fast stain recommended • Modified acid-fast stain – 1% acid decolorizer only – stain variable 34

  35. CYCLOSPORA CAYETANENSISPathogen Modified acid-fast stain; Modified acid-fast stain; Safranin stain no internal structure; 8-10 µm, note variability “wrinkled cellophane” of stain retention Attempts to infect animals with Cyclospora oocysts unsuccessful. No solid information on reservoirs or mechanical transmission. In 2005, 16% of vegetable supply imported into US; more rare now. 35

  36. CYCLOSPORA CAYETANENSISPathogen Calcofluor white filters Autofluorescence; Autofluorescence; no internal structure; 8-10 µm, color depends on filter Nomarski image selection 36

  37. CYCLOSPORA CAYETANENSISCRYPTOSPORIDIUM SPP. AND ARTIFACT Cyclospora Artifact Cyclospora Cryptosporidium Cryptosporidium Artifact 37 Modified acid-fast stain, 1% acid decolorizer

  38. Cystoisospora (Isospora) belli – Clinical - Pathogen • Immunocompetent – GI tract • Self-limiting, profuse watery diarrhea • Cramping pain, malabsorption • Immunocompromised - Disseminated • Severe diarrhea (3-6 liters/day), months • Compromised, infants, children, AIDS • Extraintestinal dissemination rare 38

  39. Cystoisospora belli Diagnosis • Examination for oocysts (15-25 µm) • Wet mount good, typical shape • Routine stains don’t work (O&P stains) • Modified acid-fast stains, 1% decolorizer recommended, thin preps, histology • Auramine-rhodamine OK, but nonspecific • Autofluorescence/shape confirmatory 39

  40. Cystoisospora belli Oocysts 40

  41. MICROSPORIDIAPathogen (now Fungi) • Group of obligate intracellular, spores protozoa/fungi: 10 cases up to 1985 • Term for phylum Microspora, 100 genera • Genera (7), 14 species = human pathogens • Possibilities include person-to-person and animal-to-person – Insects??? (water & foodborne; widespread antibodies) • Questions remain (reservoir hosts, congenital infections) 41

  42. MICROSPORIDIA, SPORE 42

  43. MICROSPORIDIA – ClinicalPathogen • Immunocompetent • Self-limiting, profuse watery diarrhea • Symptoms like coccidian infections • Eye infections; confusing Gram stains • Immunocompromised - Disseminated • Severe diarrhea (3-6 liters/day), weeks • HIV patients, CD4 cell count marker • 180-200 cells/mm3 or higher, good 43

  44. MicrosporidiaDiagnosis • Modified trichrome stains (chromotrope) • 10X amount of chromotrope 2R, dye in routine Wheatley’s trichrome (O&P) • Tissue Gram stains recommended • PAS, silver stains acceptable, H&E NO • Calcofluor, but non specific (stool) • Fecal immunoassays under development 44

  45. Microsporidia – Polar Tubule 45

  46. MICROSPORIDIATest Stool and Urine 46 Intestinal Tissue Urine: Calcofluor White

  47. MICROSPORIDIA O O O O Ryan Blue Trichrome Weber Green Trichrome Note: horizontal “stripes” (polar tubule) in some spores 47

  48. MICROSPORIDIARemember eye infections Gram Stain, Cytology Electron Microscopy Look like bacteria or ??? Cell ready to slough off 48

  49. MICROSPORIDIA in GI TRACT(Enterocytozoon, Encephalitozoon) 49 Intestinal Tissue Urine: Calcofluor White FA Immunoassay CPT Codes: 87015 + 87206 Spores of E. intestinalis

  50. MICROSPORIDIA 50 Cryptosporidium oocysts Cystoisospora belli oocyst MicrosporidialsporesMicrosporidialspores DAPI DNA stain Modified acid-fast FA Immunoassay Modified trichrome Encephalitozoon

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