Intestinal Protozoa
1. Describe the basic life cycle, distinguishing morphologic characteristics, clinical disease (if pathogenic), laboratory diagnosis, and prevention for the organisms listed in the following table.
2. Define and identify the following parasitic structures: trophozoite, cyst, oocyst, spore, pseudopodia, flagella, cilia, chromatoidal bars, karysome, central vacuole cyst form, axoneme, cytosotome, spiral groove, undulating membrane, ventral disc, shepherd’s crook, axostyle, macronucleus, micronucleus, apical complex, sporocyst, sporozoite, spore, sarcocyst, and polar tubule.
3. Define life cycle processes, including merogony, gametogony, sporogony, schizogony, and the associated organism or organisms and associated stages.
4. Correlate the parasitic life cycles with the specific diagnostic stages for the organisms listed.
The important characteristics of the intestinal protozoa are presented in Tables 48-1 to 48-7. The clinically relevant intestinal protozoa are generally considered to be Entamoeba histolytica, Blastocystis hominis, Giardia lamblia, Dientamoeba fragilis, Balantidium coli, Isospora (Cystoisospora) belli, Cryptosporidium spp., Cyclospora cayetanensis, and the microsporidia. Nonpathogenic intestinal protozoa are listed in various figures and tables but are not discussed in detail.
TABLE 48-1
Intestinal Protozoa: Trophozoites of Common Amebae
| Characteristic | Entamoeba histolytica | Entamoeba dispar | Entamoeba hartmanni | Entamoeba coli | Endolimax nana | Iodamoeba bütschlii |
| Size* (diameter or length) | 12-60 µm (usual range, 15-20 µm); invasive forms may be > 20 µm | Same size range as E. histolytica | 5-12 µm (usual range, 8-10 µm) | 15-50 µm (usual range, 20-25 µm) | 6-12 µm (usual range, 8-10 µm) | 8-20 µm (usual range, 12-15 µm) |
| Motility | Progressive, with hyaline, finger-like pseudopodia; motility may be rapid | Same motility as E. histolytica | Usually nonprogressive | Sluggish, nondirectional; blunt, granular pseudopodia | Sluggish, usually nonprogressive | Sluggish, usually nonprogressive |
| Nucleus (single) and visibility | Difficult to see in unstained preparations | Difficult to see in unstained preparations | Usually not seen in unstained preparations | Often visible in unstained preparation | Occasionally visible in unstained preparations | Usually not visible in unstained preparations |
| Peripheral chromatin (stained) | Fine granules, uniform in size and usually evenly distributed; may have beaded appearance | Fine granules, uniform in size and usually evenly distributed; may have beaded appearance | Nucleus may stain more darkly than in E. histolytica, although morphology is similar; chromatin may appear as solid ring rather than beaded (trichrome) | May be clumped and unevenly arranged on the membrane; may also appear as solid, dark ring with no beads or clumps | Usually no peripheral chromatin; nuclear chromatin may be quite variable | Usually no peripheral chromatin |
| Karyosome (stained) | Small, usually compact; centrally located but may also be eccentric | Small, usually compact; centrally located but may also be eccentric | Usually small and compact; may be centrally located or eccentric | Large, not compact; may or may not be eccentric; may be diffuse and darkly stained | Large, irregularly shaped; may appear blotlike; many nuclear variations are common; may mimic E. hartmanni or Dientamoeba fragilis | Large, may be surrounded by refractile granules that are difficult to see (“basket nucleus”) |
| Cytoplasm appearance (stained) | Finely granular, “ground glass” appearance; clear differentiation of ectoplasm and endoplasm; if present, vacuoles are usually small | Finely granular, “ground glass” appearance; clear differentiation of ectoplasm and endoplasm; if present, vacuoles are usually small | Finely granular | Granular, with little differentiation into ectoplasm and endoplasm; usually vacuolated | Granular, vacuolated | Granular, may be heavily vacuolated |
| Inclusions (stained) | Noninvasive organism may contain bacteria; presence of red blood cells (RBCs) is diagnostic; presence of RBCs is the only characteristic that allows differentiation between pathogenic E. histolytica and nonpathogenic E. dispar | Organisms usually contain bacteria; RBCs not present in cytoplasm | May contain bacteria; no RBCs | Bacteria, yeast, other debris | Bacteria | Bacteria |
*These sizes refer to wet preparation measurements. Organisms on a permanent stained smear may be 1 to 1.5 µm smaller as a result of artificial shrinkage.
TABLE 48-2
Intestinal Protozoa—Cysts of Common Amebae
| Characteristic | Entamoeba Histolytica/Entamoeba dispar | Entamoeba hartmanni | Entamoeba coli | Endolimax nana | Iodamoeba bütschlii |
| Size* (diameter or length) | 10-20 µm (usual range, 12-15 µm) | 5-10 µm (usual range, 6-8 µm) | 10-35 µm (usual range, 15-25 µm) | 5-10 µm (usual range, 6-8 µm) | 5-20 µm (usual range, 10-12 µm) |
| Shape | Usually spherical | Usually spherical | Usually spherical; may be oval, triangular, or other shapes; may be distorted on permanent stained slide because of inadequate fixative penetration | Usually oval, may be round | May vary from oval to round; cyst may collapse because of large glycogen vacuole space |
| Nucleus (number and visibility) | Mature cyst: 4 nuclei Immature cyst: 1-2 nuclei; nuclear characteristics difficult to see on wet preparation |
Mature cyst: 4 nuclei; Immature cyst: 1-2 nuclei (2-nucleated cysts very common) | Mature cyst: 8 (occasionally 16 or more nuclei may be seen) Immature cysts with 2 or more nuclei are occasionally seen | Mature cyst: 4 Immature cysts: 2 Very rarely seen and may resemble cysts of Enteromonas hominis | Mature cyst: 1 |
| Peripheral chromatin (stained) | Peripheral chromatin present; fine, uniform granules, evenly distributed; nuclear characteristics may not be as clearly visible as in trophozoite | Fine granules evenly distributed on the membrane; nuclear characteristics may be difficult to see | Coarsely granular; may be clumped and unevenly arranged on membrane; nuclear characteristics not as clearly defined as in trophozoite; may resemble E. histolytica | No peripheral chromatin | No peripheral chromatin |
| Karyosome (stained) | Small, compact, usually centrally located but occasionally may be eccentric | Small, compact, usually centrally located | Large, may or may not be compact and/or eccentric; occasionally may be centrally located | Smaller than karyosome seen in trophozoites but generally larger than those of genus Entamoeba | Larger, usually eccentric refractile granules may be on one side of karyosome (“basket nucleus”) |
| Cytoplasm, chromatoidal bodies (stained) | May be present; bodies usually elongate, with blunt, rounded, smooth edges; may be round or oval | Usually present; bodies usually elongate with blunt, rounded, smooth edges; may be round or oval | May be present (less frequently than in E. histolytica); splinter shaped with rough, pointed ends | Rare chromatoidal bodies present; occasionally small granules or inclusions seen; fine linear chromatoidals may be faintly visible on well-stained smears | No chromatoidal bodies present; occasionally small granules may be present |
| Glycogen (stained with iodine) | May be diffuse or absent in mature cyst; clumped chromatin mass may be present in early cysts (stains reddish brown with iodine) | May or may not be present, as in E. histolytica | May be diffuse or absent in mature cyst; clumped mass occasionally seen in mature cysts (stains reddish brown with iodine) | Usually diffuse if present (stains reddish brown with iodine) | Large, compact, well-defined mass (stains reddish brown with iodine) |
*Wet preparation measurements; in permanent stains, organisms usually are 1 to 2 µm smaller.
TABLE 48-3
Intestinal Protozoa—Trophozoites of Flagellates
| Protozoa | Shape and Size | Motility | Number of Nuclei and Visibility | Number of Flagella (Usually Difficult to See) | Other Features |
| Dientamoeba fragilis | Shaped like amebae; 5-15 µm (usual range, 9-12 µm) | Usually nonprogressive; pseudopodia are angular, serrated, or broad lobed and almost transparent | Percentage may vary, but 40% of organisms have 1 nucleus and 60% have 2 nuclei; not visible in unstained preparations; no peripheral chromatin; karyosome is composed of a cluster of 4-8 granules | Internal flagella; not visible | Cytoplasm finely granular and may be vacuolated with ingested bacteria, yeasts, and other debris; may be great variation in size and shape on a single smear |
| Giardia lamblia | Pear-shaped; length 10-20 µm; width, 5-15 µm | “Falling leaf” motility may be difficult to see if organism is in mucus; slight flutter of flagella may be visible using low light (duodenal aspirate or mucus from Entero-Test capsule) | 2; not visible in unstained mounts | 4 lateral; 2 ventral, 2 caudal | Sucking disk occupies one half to three fourths of ventral surface; pear-shaped front view, spoon-shaped side view |
| Chilomastix mesnili | Pear-shaped; length 6-24 µm (usual range, 10-15 µm); width, 4-8 µm | Stiff, rotary | 1; not visible in unstained mounts | 3 anterior, 1 in cytostome | Prominent cytostome extending one third to one half the length of the body; spiral groove across ventral surface |
| Pentatrichomonas hominis | Pear-shaped; length 5-15 µm (usual range, 7-9 µm); width 7-10 µm | Jerky, rapid | 1; not visible in unstained mounts | 3-5 anterior, 1 posterior | Undulating membrane extends the length of the body; posterior flagellum extends free beyond end of body |
| Trichomonas tenax | Pear shaped; length 5-12 µm; average of 6.5-7.5 µm; width, 7-9 µm | Jerky, rapid | 1; not visible in unstained mounts | 4 anterior, 1 posterior | Seen only in preparations from mouth; axostyle (slender rod) protrudes beyond the posterior end and may be visible; posterior flagellum extends only halfway down the body; no free end |
| Enteromonas hominis | Oval; 4-10 µm (usual range, 8-9 µm); width, 5-6 µm | Jerky | 1; not visible in unstained mounts | 3 anterior, 1 posterior | One side of the body is flattened; posterior flagellum extends free posteriorly or laterally |
| Retortamonas intestinalis | Pear-shaped or oval; 4-9 µm (usual range, 6-7 µm); width, 3-4 µm | Jerky | 1; not visible in unstained mount | 1 anterior, 1 posterior | Prominent cytostome extends approximately one half the length of the body |
TABLE 48-4
Intestinal Protozoa—Cysts of Flagellates
| Protozoa | Size | Shape | Number of Nuclei | Other Features |
| Dientamoeba fragilis, Pentatrichomonas hominis, Trichomonas tenax | No cyst stage | |||
| Giardia lamblia | 8-19 µm (usual range, 11-14 µm); width, 7-10 µm | Oval, ellipsoidal, or may appear round | 4; not distinct in unstained preparations; usually located at one end | Longitudinal fibers in cysts may be visible in unstained preparations; deep staining median bodies usually lie across the longitudinal fibers. Shrinkage is common, with the cytoplasm pulling away from the cyst wall; “halo” effect may be seen around the outside of the cyst wall because of shrinkage caused by dehydrating reagents |
| Chilomastix mesnili | 6-10 µm (usual range, 7-9 µm); width, 4-6 µm | Lemon or pear shaped with anterior hyaline knob | 1; not distinct in unstained preparations | Cytostome with supporting fibrils, usually visible in stained preparation; curved fibril along side of cytostome, usually referred to as a “shepherd’s crook” |
| Enteromonas hominis | 4-10 µm (usual range, 6-8 µm); width, 4-6 µm | Elongate or oval | 1-4; usually 2 lying at opposite ends of cyst; not visible in unstained mounts | Resembles Endolimax nana cyst; fibrils or flagella usually not seen |
| Retortamonas intestinalis | 4-9 µm (usual range, 4-7 µm); width, 5 µm | Pear shaped or slightly lemon shaped | 1; not visible in unstained mounts | Resembles Chilomastix cyst; shadow outline of cytostome with supporting fibrils extends above nucleus; “bird beak” fibril arrangement |
TABLE 48-5
| Protozoa | Shape and Size | Motility | Number of Nuclei | Other Features |
| Balantidium coli trophozoite |
Ovoid with tapering anterior end; 50-100 µm long, 40-70 µm wide (usual range, 40-50 µm) | Ciliates: rotary, boring; may be rapid | 1 large kidney-shaped macronucleus; 1 small round micronucleus, which is difficult to see even in stained smear; macronucleus may be visible in unstained preparation | Body covered with cilia, which tend to be longer near cytostome; cytoplasm may be vacuolated |
| Cyst | Spherical or oval; 50-70 µm (usual range, 50-55 µm) | 1 large macronucleus visible in unstained preparation; micronucleus difficult to see | Macronucleus and contractile vacuole are visible in young cysts; in older cysts, internal structure appears granular; cilia difficult to see in cyst wall |
TABLE 48-6
Morphologic Criteria Used to Identify Intestinal Protozoa (Coccidia, Blastocystis hominis)
| Protozoa | Shape and Size | Other Features |
| Cryptosporidium spp. C. parvum (humans and animals) C. hominis (humans) |
Oocyst generally round, 4-6 µm; each mature oocyst contains four sporozoites | Oocyst, diagnostic stage in stool, sporozoites occasionally visible within oocyst wall; acid-fast positive using modified acid-fast stains; various other stages in life cycle can be seen in biopsy specimens taken from gastrointestinal tract (brush border of epithelial cells) and other tissues; disseminated infection well documented in compromised host; oocysts immediately infective (in both formed and/or watery specimens); nosocomial infections documented; use enteric precautions for inpatients. |
| Cyclospora cayetanensis | Oocyst generally round, 8-10 µm; oocysts are not mature, no visible internal structure; oocysts may appear wrinkled | Oocyst, diagnostic stage in stool; acid-fast variable using modified acid-fast stains; color range from clear to deep purple (tremendous variation); best results obtained with decolorizing solution consisting of 1% acid, 3% maximum; oocysts may appear wrinkled (like crumpled cellophane); mimic Cryptosporidium oocysts but are twice as large. |
| Isospora (Cystoisospora) belli | Ellipsoidal oocyst; range 20-30 µm long, 10-19 µm wide; sporocysts rarely seen broken out of oocysts but measure 9-11µm | Mature oocyst contains two sporocysts with four sporozoites each; usual diagnostic stage in feces is immature oocyst containing spherical mass of protoplasm (intestinal tract). Oocysts are modified acid-fast positive. Whole oocyst may stain pink, but just the internal sporocysts stain if the oocyst is mature. |
| Sarcocystis hominis S. suihominis S. bovihominis |
Oocyst thin-walled and contains two mature sporocysts, each containing four sporozoites; frequently thin oocyst wall ruptures; ovoid sporocysts each measure 10-16 µm long and 7.5-12 µm wide | Thin-walled oocyst or ovoid sporocysts occur in stool (intestinal tract) |
| S. “lindemanni” | Shapes and sizes of skeletal and cardiac muscle sarcocysts vary considerably | Sarcocysts contain several hundred to several thousand trophozoites, each measuring 12-16µm long and 4-9µm wide. Sarcocysts may also be divided into compartments by septa, which are not seen in Toxoplasma cysts (tissue/muscle). |
| Blastocystis hominis | Organisms are generally round, measure approximately 6-40 µm, and are usually characterized by a large, central body (looks like a large vacuole); this stage has been called the central body form | The more amebic form can be seen in diarrheal fluid but is difficult to identify. The central body forms vary tremendously in size, even on a single fecal smear; this is the most common form seen. Routine fecal examinations may indicate a positive rate much higher than other protozoa; some laboratories report figures of 20% and higher. |
TABLE 48-7
Microsporidia That Cause Human Infection
| Microsporidia | Immunocompromised Patient | Immunocompetent Patient | Comments |
| Common | |||
| Enterocytozoon bieneusi | Chronic diarrhea; wasting syndrome, cholangitis, acalculous cholecystitis, chronic sinusitis, chronic cough, pneumonitis; cause of diarrhea in organ transplant recipients | Self-limiting diarrhea in adults and children; traveler’s diarrhea; asymptomatic carriers | Short-term culture only; three strains identified but not named; AIDS patients with chronic diarrhea (present in 5% to 30% of patients when CD4 lymphocyte counts are very low); pigs, nonhuman primates |
| Encephalitozoon hellem | Disseminated infection; keratoconjunctivitis; sinusitis, bronchitis, pneumonia, nephritis, ureteritis, cystitis, prostatitis, urethritis | Possibly diarrhea | Cultured in vitro; detected in people with traveler’s diarrhea and co-infection with E. bieneusi; pathogenicity unclear; spores not reported yet from stool; psittacine birds |
| Encephalitozoon intestinalis | Chronic diarrhea, cholangiopathy; sinusitis, bronchitis, pneumonitis; nephritis, bone infection, nodular cutaneous lesions | Self-limiting diarrhea; asymptomatic carriers | Cultured in vitro; formerly Septata intestinalis; AIDS patients with chronic diarrhea; dogs, donkeys, pigs, cows, goats |
| Encephalitozoon cuniculi | Disseminated infection; keratoconjunctivitis, sinusitis, bronchitis, pneumonia; nephritis; hepatitis, peritonitis, symptomatic and asymptomatic intestinal infection; encephalitis | Not described. Two HIV-serologically negative children with seizure disorder (suspect E. cuniculi infection) presumably were immunocompromised | Cultured in vitro; wide mammalian host range |
| Uncommon | |||
| Pleistophora sp. | Myositis (skeletal muscle) | Not described | Tend to infect fish |
| Pleistophora ronneafiei | Myositis | Not described | |
| Trachipleistophora hominis | Myositis; myocarditis keratoconjunctivitis; sinusitis | Keratitis | Cultured in vitro; AIDS patients |
| Trachipleistophora anthropophthera | Disseminated infection; keratitis | Not described | AIDS patients |
| Anncaliia connori | Disseminated infection | Not described | (Formerly Nosema connori); often infects insects; disseminated in infant with SCID |
| Anncaliia vesicularum | Myositis | Not described | Formerly Brachiola vesicularum |
| Anncaliia algerae | Myositis; nodular cutaneous lesions | Keratitis | (Formerly Nosema algerae or Brachiola algerae); cultured in vitro; skin nodules in boy with acute lymphocytic leukemia; found in arthropods |
| Nosema ocularum | Not described | Keratitis | HIV–serologically negative individual |
| Vittaforma corneae | Disseminated infection; urinary tract infection | Keratitis | (Formerly Nosema corneum); cultured in vitro; non-HIV patient |
| Microsporidium ceylonensis* | Not described | Corneal ulcer, keratitis | HIV–serologically negative individual, autopsy |
| Microsporidium africanum* | Not described | Corneal ulcer, keratitis | HIV–serologically negative individual, autopsy |
| Microsporidia (not classified) | Keratoconjunctivitis in a contact lens wearer |
AIDS, Acquired immunodeficiency syndrome; HIV, human immunodeficiency virus; SCID, severe combined immunodeficiency
*Microsporidium is a collective generic name for microsporidia that cannot be classified
Amebae
The class Sarcodina, or Amebae, includes the organisms capable of movement by means of cytoplasmic protrusions called pseudopodia. This group includes free-living organisms, in addition to nonpathogenic and pathogenic organisms found in the intestinal tract and other areas of the body (see Tables 48-1 and 48-2). Occasionally, when fresh stool material is examined as a direct wet mount, motile trophozoites may be seen, as well as other, nonparasitic structures (Figure 48-1).
Entamoeba histolytica
General Characteristics
E. histolytica has directional and progressive motility, whereas the other amebae tend to move more slowly and at random. However, motility is rarely seen even in a fresh wet mount from a patient with diarrhea or dysentery. The cytoplasm is generally more finely granular, and the presence of red blood cells (RBCs) in the cytoplasm is considered diagnostic for E. histolytica (Figure 48-2).
Permanent stained smears demonstrate accurate morphology compared with other techniques. When the organism is examined on a permanent stained smear (trichrome or iron-hematoxylin stain), the morphologic characteristics of E. histolytica/E. dispar are readily seen. The nucleus is characterized by evenly arranged chromatin on the nuclear membrane and a small, compact, centrally located karyosome (condensed chromatin). As mentioned, the cytoplasm usually is described as finely granular, with few ingested bacteria and scant debris in vacuoles. As stated previously, in organisms isolated from a patient with dysentery, RBCs may be visible in the cytoplasm, a feature diagnostic for E. histolytica (Figure 48-3). Most often, infection with E. histolytica is diagnosed on the basis of the organism’s morphology, without the presence of RBCs.
As part of the life cycle, the trophozoites may condense into a round mass (precyst), and a thin wall is secreted around the immature cyst. Two types of inclusions may be found in this immature cyst: a glycogen mass and highly refractile chromatoidal bars (refractile chromatin structure) with smooth, rounded edges. As the cyst matures (metacyst) (see Figure 48-3; Figure 48-4), nuclear division occurs, with the production of four nuclei. Often chromatoidals may be absent in the mature cyst. Cyst morphology does not differentiate E. histolytica from E. dispar. Cyst formation occurs only in the intestinal tract; once the stool has left the body, cyst formation does not occur. The one-, two-, and four-nucleated cysts are infective and represent the mode of transmission from one host to another.
Epidemiology
Amebiasis is caused by infection with the true pathogen, Entamoeba histolytica. Recent evidence from molecular studies confirms the differentiation of pathogenic E. histolytica and nonpathogenic E. dispar (Figure 48-5) as two distinct species. E. histolytica is considered the etiologic agent of amebic colitis and extraintestinal abscesses (amebic liver abscess), whereas nonpathogenic E. dispar produces no intestinal symptoms and is not invasive in humans.
Entamoeba coli
General Characteristics
E. coli trophozoites are somewhat larger than those of E. histolytica and E. dispar
