1. Case report I2 劉崢偉

2. General data 29-year-old man 29-year-old man Human immunodeficiency virus (HlV)- infected Human immunodeficiency virus (HlV)- infected

3. Chief complants Severe, profuse, nonbloody, watery diarrhea Severe, profuse, nonbloody, watery diarrhea

4. Present illness Nausea Poor appetite 15-lb weight loss over the past several months

5. Lab data A very low CD4 lymphocyte count (of only 100). A very low CD4 lymphocyte count (of only 100). Stool specimens cultured for enteric bacilli were negative. Stool specimens cultured for enteric bacilli were negative.

6. Lab data(2) Fecal speci ­ mens examined for ova and parasites, which included a microscopic study of the concentrated sediment and a permanent trichrome-stained smear, were negative for intestinal parasites Fecal speci ­ mens examined for ova and parasites, which included a microscopic study of the concentrated sediment and a permanent trichrome-stained smear, were negative for intestinal parasites

7. Lab data(3) A diagnosis of intestinal parasites was used. A diagnosis of intestinal parasites was used. Microscopic examination revealed very tiny parasites, nor much larger than staphylococcal cells(1uM) Microscopic examination revealed very tiny parasites, nor much larger than staphylococcal cells(1uM)

9. 1.Which stain was used to stain the parasite shown in Fig. 5.1? A:Chromotrope stain

10. 2.Which group of obligate Intracellular protozoan parasites would you suspect of causing this infection in an AIDS patient?

11. A: 1.Cryptosporidiosis (4-6 um, round) Modified Ziehl-Neelsen carbolfuchsin stain 2.Microsporidiosis(<1um) 3.Isosporiasis (25 um, elliptic) (Modified Kinyoun stain) (Electron microscopy of biopsy - definitive diagnosis 4.Cyclospora (8-10 um) (Modified Ziehl-Neelsen carbolfuchsin stain) (trichrome)

12. 3.Which member of this group would you expect might be causing this patient's infection? Why? A: Microsporidiosis

13. Microsporidiosis E. bieneusi was found to be the cause of unexplained diarrhea in 27 to 30% of HIV- infected patients E. bieneusi was found to be the cause of unexplained diarrhea in 27 to 30% of HIV- infected patients Wasting, chronic diarrhea, and cholangiopathy Wasting, chronic diarrhea, and cholangiopathy Indistinguishable from the manifestations of isosporiasis and cryptosporidiosis in AIDS patients Indistinguishable from the manifestations of isosporiasis and cryptosporidiosis in AIDS patients Diarrheal stools are watery and are not accompanied by blood or fever Diarrheal stools are watery and are not accompanied by blood or fever Occasional hypokalemia and hypomagnesemia Occasional hypokalemia and hypomagnesemia Carbohydrate and fat malabsorption are present Carbohydrate and fat malabsorption are present

14. Cryptosporidiosis Self-limited diarrheal illness that lasts from 4 to 20 days and is associated with abdominal cramping, nausea, vomiting, low-grade fever, and anorexia Self-limited diarrheal illness that lasts from 4 to 20 days and is associated with abdominal cramping, nausea, vomiting, low-grade fever, and anorexia persistent, profuse watery diarrhea, may wax and wane, or may be asymptomatic. persistent, profuse watery diarrhea, may wax and wane, or may be asymptomatic. Stool volume - 1 to 17 L/day Stool volume - 1 to 17 L/day Frequency – Frequency – 6 to 26 bowel movements/day

15. Cyclosporiasis Watery diarrhea, abdominal cramping, flatulence, weight loss, and nausea. Watery diarrhea, abdominal cramping, flatulence, weight loss, and nausea. Typically wax and wane for several weeks and may persist for several months Typically wax and wane for several weeks and may persist for several months

16. Isosporiasis Indistinguishable from clinic presentation of Cryptosporidium infection. Indistinguishable from clinic presentation of Cryptosporidium infection. Particularly crampy abdominal pain and profuse watery diarrhea of 8 to 10 stools /day, along with weight loss, weakness, anorexia, and occasional low-grade fever Particularly crampy abdominal pain and profuse watery diarrhea of 8 to 10 stools /day, along with weight loss, weakness, anorexia, and occasional low-grade fever Progress dehydration, malnutrition, and cachexia Progress dehydration, malnutrition, and cachexia

17. 4.How do these parasites differ from other intracellular intestinal protozoans?

18. A: Enterocytozoon bieneusi Enterocytozoon bieneusi Infection of the intestinal epithelium to enterocytes covering the villi, (at the tip) Villous atrophy, cell degeneration, necrosis, and sloughing. Jejunum - preferred site of infection Jejunum - preferred site of infection Duodenum - less frequently infected Large intestine - relatively spared. Encephalitozooan intestinalis Encephalitozooan intestinalis Severe diarrhea and a granulomatous tubulointerstitial enteritis Disseminate to lungs and sinuses. Encephalitozoon cuniculi Encephalitozoon cuniculi Involve the kidneys and CNS ~continue ~continue

19. True eukaryotes - membrane-bound nucleus, an intracytoplasmic membrane system, and chromosome separation on mitotic spindles True eukaryotes - membrane-bound nucleus, an intracytoplasmic membrane system, and chromosome separation on mitotic spindles Unusual in 70S ribosomes, have no mitrochondria or peroxisomes, and have simple Golgi membranes Unusual in 70S ribosomes, have no mitrochondria or peroxisomes, and have simple Golgi membranes Microsporidial genome is smaller and less complex than those of other eukaryotes Microsporidial genome is smaller and less complex than those of other eukaryotes Chitin in the spore wall, suggest a potential link to the fungi. Chitin in the spore wall, suggest a potential link to the fungi.

20. 5.How do these parasites multiply? A: Spore-forming protists with no active metabolic stages outside of the host cell. Spore-forming protists with no active metabolic stages outside of the host cell. The life cycle involves a proliferative merogonic stage followed by sporogony, which results in spores containing a tubular extrusion apparatus (polar tubule) for injecting infective spore contents into the host cell The life cycle involves a proliferative merogonic stage followed by sporogony, which results in spores containing a tubular extrusion apparatus (polar tubule) for injecting infective spore contents into the host cell

22. 6.How is infection with this parasite transmitted? In soil,food, water, In soil,food, water, Contaminated with infected human or animal feces Contaminated with infected human or animal feces

23. 7.How is the laboratory diagnosis of infection with this parasite made?

24. TechniqueUseComments Light microscopy Stool specimens Modified trichrome b b ++ Reliable, available; light infections difficult to identify Giemsa− Not recommended for routine use, hard to read Chemofluoresce nce ++ Calcofluor, Fungifluor, Unitex 2B; sensitive but nonspecific Immunofluoresc ence (++) Commercial availability limits use; products in development

25. Other bodily fluids Modified trichrome++Reliable, available; light infections difficult to identify Giemsa+Urine, conjunctival swab, BAL, CSF, duodenal aspirate Chemofluorescence++Calcofluor, Fungifluor, Unitex 2B; sensitive but nonspecific Immunofluorescence(++)Commercial availability limits use; products in development

26. Cytology testing c c Modified trichrome ++Reliable, available; light infections difficult to identify Giemsa+Urine, conjunctival swab, BAL, CSF, duodenal aspirate Gram+Recommended, especially for specimens with little debris Chemofluoresc ence ++Calcofluor, Fungifluor, Unitex 2B; sensitive but nonspecific Immunofluores cence (++)Commercial availability limits use; products in development

27. Plastic-embedded sections Toluidine blue+ Recommended; sensitive method Methylene blue-azure II- basic fuchsin+ Recommended as alternative to toluidine blue Electron microscopy

28. Plastic- embedded sections Toluidine blue++Recommended; sensitive method Methylene blue-azure II-basic fuchsin ++Recommended as alternative to toluidine blue Electron microscopy Body fluids+Specific, sensitivity unknown; used for identification to species level (some exceptions) Tissue sections++Gold standard for confirmation, but sensitivity lower than for detection of spores in stool or urine; used for identification to species level (some exceptions)

29. Molecular testing −Availability limited to research laboratories; studies ongoing and appear promising; molecular identification to the species level possible Serologic testing (serum) −Reagents not commercially available; preliminary results controversial; have been reported for Encephalitozoon; not available for Enterocytozoon; not relevant for immunocompromised patientsEncephalitozoonEnterocytozoon Culture−Generally used in the research setting; continued advances in culture and organism survival and growth; Encephalitozoon, Nosema, Trachipleistophora, Vittaforma can be isolated; delivery to specialty lab within 2 to 3 days recommended; use Universal Precautions when handling specimensEncephalitozoon NosemaTrachipleistophoraVittaforma

30. Weber-Green modified trichrome staining of microsporidial spores. Spores in stool specimen

31. Ryan-Blue modified trichrome staining of microsporidial spores. (A) Spores in stool specimen; (B) spores in intestinal tract tissue.

32. Single smear stained by an acid-fast trichrome stain method showing both an Isospora belli oocyst (modified acid-fast positive stain) and microsporidial spores (modified trichrome stain).

33. Giemsa staining of microsporidial spores in intestinal tract cells. The images show the development of the spores

34. Calcofluor white staining of microsporidial spores in urine sediment

35. Encephalitozoon spp. detected with immunofluorescent reagent. (A) Urine sediment; (B) positive control spores

36. Cytospin preparation of bronchoalveolar lavage fluid from a patient with AIDS and intestinal E. bieneusi infection, showing intracellular gram- positive microsporidial spores (Gram stain)

37. Hematoxylin-eosin staining of eye tissue (note clear spores)

38. PAS staining of eye tissue (note PAS- positive granule at the end of each spore).

39. Warthin-Starry silver staining of eye tissue (note dark spores)

40. Transmission electron micrograph of a jejunal biopsy demonstrating numerous septated parasitophorous vacuoles of Encephalitozoon intestinalis, which are located in the Golgi-rich supranuclear cytoplasm

41. Reference Journal of Clinical Microbiology, June 2002, p. 1892-1901, Vol. 40, No. 6 Journal of Clinical Microbiology, June 2002, p. 1892-1901, Vol. 40, No. 6 HIV InSite Knowledge Base Chapter November 1998 Carolyn Petersen, MD, University of California San Francisco HIV InSite Knowledge Base Chapter November 1998 Carolyn Petersen, MD, University of California San Francisco