Informational Tables

4.1. Protozoa of the intestinal tract and urogenital system: key characteristics^a

Organism

Trophozoite or tissue stage

Cyst or other stage in specimen

Comments

Amebae

Entamoeba histolytica (pathogenic)

Cytoplasm clean; presence of RBCs is diagnostic, but cytoplasm may also contain some ingested bacteria; peripheral nuclear chromatin evenly distributed, with central, compact karyosome

Mature cyst contains four nuclei; chromatoidal bars have smooth, rounded ends; immature cyst usually contains one enlarged nucleus (precyst)

Considered pathogenic; report to public health department; trophozoites can be confused with macrophages and cysts can be confused with WBCs in the stool; unless ingested RBCs are seen within the trophozoite cytoplasm, the organism cannot be identified morphologically as the true pathogen, Entamoeba histolytica; trophozoites with no ingested RBCs, precysts, and/or cysts would be identified as Entamoeba histolytica/E. dispar group/complex

Entamoeba dispar (nonpathogenic)

Morphology identical to that of E. histolytica (confirmed by presence of RBCs in cytoplasm). If no RBCs are present, NAAT or immunoassay specific for the true pathogen is necessary to confirm species designation.

Trophozoites with no ingested RBCs, precysts, and mature cysts have morphology identical to that of E. histolytica; correct identification would be Entamoeba histolytica/E. dispar group/complex

Nonpathogenic: morphology resembles that of E. histolytica; these organisms will continue to be signed out as Entamoeba histolytica/E. dispar and reported to the public health department (optional without identification of the true pathogen, Entamoeba histolytica).

Immunoassay reagents are now available to differentiate pathogenic E. histolytica and nonpathogenic E. dispar; some laboratories may decide to use these reagents on a routine basis, depending on positivity rate and cost.

Entamoeba histolytica/E. dispar group/complex

Correct way to report, unless NAAT or immunoassay is used to identify E. histolytica or trophozoites are seen with ingested RBCs (E. histolytica). Although Entamoeba moshkovskii and Entamoeba bangladeshi also resemble the other members of the Entamoeba histolytica/E. dispar group, they are generally not reported (rare and very difficult to differentiate from the other organisms) without the use of NAAT.

Entamoeba hartmanni (nonpathogenic)

Looks identical to E. histolytica but smaller (<12 μm); RBCs will not be ingested

Mature cyst contains four nuclei but often stops at two; chromatoidal bars often present and look like those in E. histolytica (size, <10 μm); immature cyst (precyst) may mimic E. histolytica but will be <10 μm

Shrinkage occurs on permanent stain (especially in cyst form). E. histolytica may actually be below the 12- and 10-μm cutoff limits; it could be as much as 1.5 μm below the limits quoted for wet preparation measurements. Entire space (including shrinkage) must be measured from organisms seen on the permanent stained smear.

Entamoeba coli (nonpathogenic)

Cytoplasm dirty, may contain ingested bacteria or debris; peripheral nuclear chromatin unevenly distributed, with a large, eccentric karyosome

Mature cyst contains eight nuclei, may see more; chromatoidal bars (if present) tend to have sharp, pointed ends; immature cyst (precyst) usually contains two enlarged nuclei, one at either side of the precyst

If a smear is too thick or thin and if stain is too dark or light, then E. histolytica/E. dispar and E. coli are often confused; there is much overlap in morphology. Assess smear thickness before identifying organisms to the species level.

Endolimax nana (nonpathogenic)

Cytoplasm clean, not diagnostic, great deal of nuclear variation, may even be some peripheral nuclear chromatin. Normally, only karyosomes are visible.

Cyst is round to oval, with four nuclear karyosomes visible; there may be a large vacuole at one end of the cyst (can cause confusion with Iodamoeba.

More nuclear variation in these trophozoites than in other protozoa; can be confused with Dientamoeba fragilis and/or E. hartmanni

Iodamoeba buetschlii (nonpathogenic)

Cytoplasm contains much debris; organisms usually larger than E. nana but may look similar; large karyosome

Cyst contains single nucleus (may be “basket nucleus”) with bits of nuclear chromatin arranged on nuclear membrane (karyosome is the basket, bits of chromatin are the handle); large glycogen vacuole

Glycogen vacuole stains brown with addition of iodine in wet preparation; “basket nucleus” more common in cyst but can be seen in trophozoite; vacuole may be so large that the cyst collapses on itself

Stramenopile
Blastocystis spp.

The central body form is the most common and recognizable form seen in human fecal specimens; although the vacuolar form may be present (especially in acute infection), it is much more difficult to identify.

Other stages occur, but identification is difficult from microscopic examinations

Of 17 known subtypes, approximately half are pathogenic; pathogenicity cannot be determined from microscopic morphology. In many studies, this is the most common organism identified in human stool (worldwide). Giardia and Dientamoeba often rotate between 2nd and 3rd most common. Pathogenic subtypes have been linked to irritable bowel syndrome and urticaria.

Flagellates

Giardia duodenalis (G. lamblia, G. intestinalis) (pathogenic)

Trophozoites teardrop shaped from front, like a curved spoon from side; contain nuclei, linear axonemes, and curved median bodies

Cyst is round to oval; contains multiple nuclei, axonemes, and median bodies

Organisms live in duodenum, and multiple stools may be negative; may have to use additional techniques (immunoassay, aspirate, Entero-Test). Organism numbers may have little relevance to presence of symptoms. Note: two negative immunoassay tests (same test, does not require two different test formats) required before patient is reported negative (problems with intermittent shedding).

Chilomastix mesnili (nonpathogenic)

Trophozoites teardrop shaped; cytostome must be visible for identification

Cyst is lemon shaped, with one nucleus and curved fibril called shepherd’s crook

Cyst can be identified much easier than trophozoite form. Trophozoite will look like other small flagellates.

Dientamoeba fragilis (symptomatic adults and children seen)

Cytoplasm contains debris; may contain one or two nuclei (chromatin often fragmented into four dots)

Cyst identified; however, very rare in clinical specimens

Tremendous size and shape range on a single smear; trophozoites with one nucleus can resemble E. nana; often found as frequently as Giardia

Trichomonas vaginalis (pathogenic)

Supporting rod (axostyle) present; undulating membrane comes halfway down the organism; small dots may be seen in cytoplasm along axostyle

No known cyst form

Recovered from genitourinary system; often diagnosed at bedside with wet preparation (motility)

Pentatrichomonas hominis (nonpathogenic)

Supporting rod (axostyle) present; undulating membrane comes all the way down the organism; small dots may be seen in cytoplasm along axostyle

No known cyst form

Recovered in stool; trophozoites may resemble other small flagellate trophozoites; risk management issue if misidentified as pathogen T. vaginalis from urine contaminated with stool (especially in pediatric patients)

Ciliates

Balantioides coli (pathogenic)

Very large trophozoites (50–100 μm long) covered with cilia; large bean-shaped nucleus

Morphology not significant except for large, bean-shaped nucleus

Rarely seen in the United States; causes severe diarrhea with large fluid loss; may be seen in proficiency testing specimens

Apicomplexa
>Includes Cryptosporidium spp. and the coccidia

Cryptosporidium spp. (pathogenic) Apicomplexan

Seen in intestinal mucosa (edge of brush border), gallbladder, and lung biopsy specimens (less common in lung).

Oocysts seen in stool and/or sputum; organisms are modified acid-fast positive, 4–6 μm; hard to find if few in number

Chronic infection in compromised host (internal autoinfective cycle), self-cure in immunocompetent host; numbers of oocysts correlate with stool consistency; can cause severe, watery diarrhea; oocysts immediately infective when passed

Cyclospora cayetanensis (pathogenic) coccidian

Experience with this organism is not extensive; organism may be difficult to identify in tissue; since patients are immunocompetent, biopsy specimens will probably rarely be required or requested

Oocysts seen in stool; approximately 8–10 μm; are unsporulated and thus difficult to recognize as coccidia; will mimic C. parvum (4–6 μm) on modified acid-fast-stained smears; hot safranin stain; autofluorescence helpful in determining presumptive positives

Oocysts have been seen in stool but were originally not recognized as coccidia. To date, most infections are associated with immunocompetent individuals, but they may also be seen in immunosuppressed patients (more severe disease); organism may be associated with traveler’s diarrhea and has been linked to the ingestion of imported food (raspberries, mesclun, basil) with periodic outbreaks seen in the U.S.

Cystoisospora belli (pathogenic) coccidian

Seen in intestinal mucosal cells and in biopsy specimens; less common than Cryptosporidium parvum

Oocysts seen in stool; organisms acid fast; best technique is concentration, not permanent stained smear

Thought to be the only Cystoisospora sp. that infects humans; oocysts not immediately infective when passed; oocysts will be missed if preserved specimens containing PVA are concentrated

Microsporidia

Brachiola spp.
Nosema spp.
Encephalitozoon spp.
Pleistophora spp.
Trachipleistophora spp.
Anncaliia spp.
Enterocytozoon bieneusi
Microsporidium spp.
Vittaforma corneae

Developing stages sometimes difficult to identify; spores can be identified (microsporidian spores) by size, shape, and presence of polar tubules; routine microscopy cannot be used to identify the spores to the genus and/or species levels

Depending on the genus involved, spores could be identified in stool or urine using NAAT or the modified trichrome stain, calcofluor, or immunoassay reagents (experimental).

Spores are generally quite small (1–2.0 μm for Enterocytozoon spp.) and can easily be confused with other organisms or artifacts (particularly in stool). Infections tend to be present in immunosuppressed patients.

It is unclear whether all genera listed here can disseminate to all parts of the body (gastrointestinal tract, urogenital system); however, the list is complete in terms of all body sites.