Case reportCase report

I2 I2 劉崢偉劉崢偉

General dataGeneral data

29-year-old man 29-year-old man Human immunodeficiency virus (HlV)-infHuman immunodeficiency virus (HlV)-inf

ectedected

Chief complantsChief complants

Severe, profuse, nonbloody, watery diarSevere, profuse, nonbloody, watery diarrhea rhea

Present illnessPresent illness

Nausea Nausea Poor appetite Poor appetite 15-lb weight loss 15-lb weight loss over the past several months over the past several months

Lab dataLab data

A very low CD4 lymphocyte count (of A very low CD4 lymphocyte count (of only 100). only 100).

Stool specimens cultured for enteric Stool specimens cultured for enteric bacilli were negative. bacilli were negative.

Lab data(2)Lab data(2)

Fecal speci mens examined for ova and pFecal speci mens examined for ova and parasites, which included a microscopic sarasites, which included a microscopic study of the concentrated sediment and tudy of the concentrated sediment and a permanent trichrome-stained smear, a permanent trichrome-stained smear, were negative for intestinal parasites were negative for intestinal parasites

Lab data(3)Lab data(3)

A diagnosis of intestinal A diagnosis of intestinal parasites was used. parasites was used.

Microscopic examination Microscopic examination revealed very tiny parasites, nor revealed very tiny parasites, nor much larger than staphylococcal much larger than staphylococcal cells(1uM)cells(1uM)

1.Which stain was used to stain th1.Which stain was used to stain the parasite shown in Fig. 5.1?e parasite shown in Fig. 5.1?

A:Chromotrope stainA:Chromotrope stain

2.Which group of obligate2.Which group of obligate

Intracellular protozoan parasitesIntracellular protozoan parasites

would you suspect of causing thiswould you suspect of causing this

infection in an AIDS patient? infection in an AIDS patient?

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

3.Which member of this group3.Which member of this groupwould you expect might bewould you expect might becausing this patient's infection?causing this patient's infection?Why?Why?

A: MicrosporidiosisA: Microsporidiosis

Microsporidiosis Microsporidiosis E. bieneusiE. bieneusi was found to be the cause of unexplain was found to be the cause of unexplain

ed diarrhea in 27 to 30% of HIV-infected patientsed diarrhea in 27 to 30% of HIV-infected patients Wasting, chronic diarrhea, and cholangiopathy Wasting, chronic diarrhea, and cholangiopathy Indistinguishable from the manifestations of isospIndistinguishable from the manifestations of isosp

oriasis and cryptosporidiosis in AIDS patientsoriasis and cryptosporidiosis in AIDS patients Diarrheal stools are watery and are not accompaniDiarrheal stools are watery and are not accompani

ed by blood or fevered by blood or fever Occasional hypokalemia and hypomagnesemiaOccasional hypokalemia and hypomagnesemia Carbohydrate and fat malabsorption are presentCarbohydrate and fat malabsorption are present

CryptosporidiosisCryptosporidiosis

Self-limited diarrheal illness that lasts frSelf-limited diarrheal illness that lasts from 4 to 20 days and is associated with aom 4 to 20 days and is associated with abdominal cramping, nausea, vomiting, lbdominal cramping, nausea, vomiting, low-grade fever, and anorexiaow-grade fever, and anorexia

persistent, profuse watery diarrhea, may persistent, profuse watery diarrhea, may wax and wane, or may be asymptomatic.wax and wane, or may be asymptomatic.

Stool volume - 1 to 17 L/dayStool volume - 1 to 17 L/day Frequency – Frequency – 6 to 26 bowel movements/day6 to 26 bowel movements/day

Cyclosporiasis Cyclosporiasis

Watery diarrhea, abdominal Watery diarrhea, abdominal cramping, flatulence, weight loss, cramping, flatulence, weight loss, and nausea.and nausea.

Typically wax and wane for several Typically wax and wane for several weeks and may persist for several weeks and may persist for several monthsmonths

Isosporiasis Isosporiasis

Indistinguishable from clinic presentatioIndistinguishable from clinic presentation of n of CryptosporidiumCryptosporidium infection. infection.

Particularly crampy abdominal pain and Particularly crampy abdominal pain and profuse watery diarrhea of 8 to 10 stools profuse watery diarrhea of 8 to 10 stools /day, along with weight loss, weakness, /day, along with weight loss, weakness, anorexia, and occasional low-grade feveanorexia, and occasional low-grade feverr

Progress dehydration, malnutrition, and Progress dehydration, malnutrition, and cachexiacachexia

4.How do these parasites differ4.How do these parasites differfrom other intracellular intestinalfrom other intracellular intestinalprotozoans? protozoans?

A:A: Enterocytozoon bieneusiEnterocytozoon bieneusi Infection of the intestinal epithelium to enterocytes covering the villInfection of the intestinal epithelium to enterocytes covering the vill

i, (at the tip)i, (at the tip)Villous atrophy, cell degeneration, necrosis, and sloughing. Villous atrophy, cell degeneration, necrosis, and sloughing. Jejunum - preferred site of infectionJejunum - preferred site of infectionDuodenum - less frequently infectedDuodenum - less frequently infectedLarge intestine - relatively spared.Large intestine - relatively spared. Encephalitozooan intestinalisEncephalitozooan intestinalis Severe diarrhea and a granulomatous tubulointerstitial enteritis Severe diarrhea and a granulomatous tubulointerstitial enteritis Disseminate to lungs and sinuses.Disseminate to lungs and sinuses. Encephalitozoon cuniculiEncephalitozoon cuniculi Involve the kidneys and CNSInvolve the kidneys and CNS ~continue~continue

True eukaryotes - membrane-bound nucleus, True eukaryotes - membrane-bound nucleus, an intracytoplasmic membrane system, and can intracytoplasmic membrane system, and chromosome separation on mitotic spindleshromosome separation on mitotic spindles

Unusual in 70S ribosomes, have no mitrochonUnusual in 70S ribosomes, have no mitrochondria or peroxisomes, and have simple Golgi medria or peroxisomes, and have simple Golgi membranes mbranes

Microsporidial genome is smaller and less comMicrosporidial genome is smaller and less complex than those of other eukaryotesplex than those of other eukaryotes

Chitin in the spore wall, suggest a potential linChitin in the spore wall, suggest a potential link to the fungi. k to the fungi.

5.How do these parasites multiply? 5.How do these parasites multiply? A:A: Spore-forming protists with no active metabolSpore-forming protists with no active metabol

ic stages outside of the host cell. ic stages outside of the host cell. The life cycle involves a proliferative merogoniThe life cycle involves a proliferative merogoni

c stage followed by sporogony, which results ic stage followed by sporogony, which results in spores containing a tubular extrusion apparn spores containing a tubular extrusion apparatus (polar tubule) for injecting infective spore atus (polar tubule) for injecting infective spore contents into the host cell contents into the host cell

6.How is infection with this6.How is infection with thisparasite transmitted?parasite transmitted?

In soil,food, water, In soil,food, water, Contaminated with infected human or Contaminated with infected human or

animal feces animal feces

7.How is the laboratory diagnosis7.How is the laboratory diagnosis

of infection with this parasiteof infection with this parasite

made?made?

TechniqueTechnique UseUse CommentsComments

Light Light microscopymicroscopy

Stool specimensStool specimens

Modified trichroModified trichromemebb

++++ Reliable, available; light infections difficult to Reliable, available; light infections difficult to identifyidentify

GiemsaGiemsa −− Not recommended for routine use, hard to readNot recommended for routine use, hard to read

ChemofluoresceChemofluorescencence

++++ Calcofluor, Fungifluor, Unitex 2B; sensitive but Calcofluor, Fungifluor, Unitex 2B; sensitive but nonspecificnonspecific

ImmunofluorescImmunofluorescenceence

(+(+++))

Commercial availability limits use; products in Commercial availability limits use; products in developmentdevelopment

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

Cytology testingc

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

Chemofluorescence

++ Calcofluor, Fungifluor, Unitex 2B; sensitive but nonspecific

Immunofluorescence

(++)

Commercial availability limits use; products in development

Plastic-embedded sections

Toluidine blue ++

Recommended; sensitive method

Methylene blue-azure II-basic fuchsin

++

Recommended as alternative to toluidine blue

Electron microscopy

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)

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 patients

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 specimens

Weber-Green modified trichrome stWeber-Green modified trichrome staining of microsporidial spores. Spoaining of microsporidial spores. Spo

res in stool specimen res in stool specimen

Ryan-Blue modified trichrome staining of microsporidialRyan-Blue modified trichrome staining of microsporidialspores. (A) Spores in stool specimen; (B) spores in intestinspores. (A) Spores in stool specimen; (B) spores in intestin

al tract tissueal tract tissue. .

Single smear stained by an acid-fast trichrome stain methoSingle smear stained by an acid-fast trichrome stain method showing both an d showing both an Isospora belliIsospora belli oocyst (modified acid-fast oocyst (modified acid-fast positive stain) and microsporidial spores (modified trichrompositive stain) and microsporidial spores (modified trichrom

e stain).e stain).

Giemsa staining of microsporidial spores in iGiemsa staining of microsporidial spores in intestinal tract cells. The images show the dentestinal tract cells. The images show the de

velopment of the sporesvelopment of the spores

Calcofluor white staining of microsporidCalcofluor white staining of microsporidial spores in urine sedimential spores in urine sediment

EncephalitozoonEncephalitozoon spp. detected with im spp. detected with immunofluorescent reagent. (A) Urine sedmunofluorescent reagent. (A) Urine sed

iment; (B) positive control sporesiment; (B) positive control spores

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

Hematoxylin-eosin staining of eye tiHematoxylin-eosin staining of eye tissue (note clear spores)ssue (note clear spores)

PAS staining of eye tissue (note PAS-positivPAS staining of eye tissue (note PAS-positiv

e granule at the end of each spore).e granule at the end of each spore).

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

Transmission electron micrograph of a jejunal biopsy demonstrating nuTransmission electron micrograph of a jejunal biopsy demonstrating numerous septated parasitophorous vacuoles of merous septated parasitophorous vacuoles of Encephalitozoon intestinEncephalitozoon intestinalisalis, which are located in the Golgi-rich supranuclear cytoplasm, which are located in the Golgi-rich supranuclear cytoplasm

ReferenceReference

Journal of Clinical Microbiology, June 20Journal of Clinical Microbiology, June 2002, p. 1892-1901, Vol. 40, No. 602, p. 1892-1901, Vol. 40, No. 6

HIV InSite Knowledge Base ChapterHIV InSite Knowledge Base ChapterNovember 1998 Carolyn Petersen, MD, UNovember 1998 Carolyn Petersen, MD, University of California San Francisco niversity of California San Francisco