Entamoeba histolytica

Cosmopolitan amoeba. In man, the main reservoir of infection, this species inhabits the lumen of colon and caecum. Extraintestinal localizations – in liver, lung, brain, skin and other tissues – are always secondary to an invasive amoebic colitis.

Infection with Entamoeba histolytica is acquired by indirect fecal-oral route or by sexual intercourse. Tetranucleate cysts eliminated with feces are infective, can contaminate drinking water or water used for irrigation (especially for cultivating vegetables), and can be carried by insects (e.g. flies). Once ingested, four “metacystic” trophozoites are released into the duodenum where they multiply until they assume their typical morphology in the caecum and ascending colon. In the terminal part of the large bowel, due to micro-environmental changes, trophozoites slow down, become round (precystic forms) and subsequently encyst. The cysts eliminated with feces may be mono-, bi- or tetranucleate. In amoebic dysentery, hematophagous trophozoites of Entamoeba histolytica are frequently found.


Size: 10-60 μm; usual range, 10-20 μm (>20 μm in the hematophagous form).

Motility: movements can be seen only in fresh, not fixed, specimens. The trophozoite forms a small, clear hyaline pseudopod which is filled with endoplasm and then disappears, while the amoeba takes a definite direction crawling fluidly like a snail (videoclip ). To study trophozoitic motility, slides should be sealed to prevent dehydration and stored at a proper temperature (about 37 °C). In addition, the type of motility observed can vary considerably according to hydration and temperature conditions of the specimen under the microscope.

Cytoplasm: usually clean or finely granular, it may contain small, hard-to-see vacuoles as well as Sphaerita.  In degenerating trophozoites, it is possible to see larger vacuoles containing bacteria or yeast.  In mucopurulent specimens, the cytoplasm of degenerating trophozoites takes on a granular appearance.  In these samples, the trophozoites are often surrounded by a mass of degenerating leukocytes.

In hematophagous trophozoites, it is possible to find 1-40 ingested red blood cells that, while still red in color, appear more or less digested and of variable size. One should always compare the color of ingested erythrocytes with that of free ones in the same microscopic field, to avoid confusion between RBCs and small, clear vacuoles in the amoebic cytoplasm.

The presence of hematophagous trophozoites is generally sufficient to diagnose Entamoeba histolytica, although Entamoeba coli can also ingest erythrocytes under particular circumstances. Recent studies suggested that also Entamoeba dispar can phagocytize red blood cells, both in vitro and in vivo. Moreover, in bacillary dysentery and in hemorrhagic rectocolitis, erythrocytes can also be phagocytized by macrophages.

Nucleus: not visible in fresh specimens, but only in degenerating trophozoites or in fixed specimens; its diameter ranges from 3 to 8μm.

-     Karyosome: in unstained or temporary stained wet mounts of formalin-fixed (or, better, SAF-fixed) specimens, the karyosome is generally small, compact and centrally or eccentrically located. With permanent staining, the karyosome is clearly visible and is surrounded by a clear, achromatic halo; there may be refractile achromatic granules (chromosomes) inside (depending on the stage of division of the trophozoite).

-     Peripheral chromatin: a layer of chromatin, in the form of small, evenly distributed granules lining the thin nuclear membrane. Sometimes chromatin is coarse, irregular, in the form of long aggregates along the nuclear membrane.

This description is purely indicative because, even in the same sample, different forms with different appearances may be present.  For example, the karyosome can be eccentric and diffuse, and the peripheral chromatin can be heterogeneous. In addition, in improperly fixed specimens, the cytoplasm may appear vacuolated and consequently these trophozoites can be mistaken for those of Entamoeba coli.  A single specimen may also contain trophozoites of different species: in fact, co-infection with Entamoeba coli is not rare.  For this reason, many trophozoites should be examined in order to identify all the species present in a sample. Stool specimens may also contain other cells (neutrophils, macrophages, intestinal epithelial cells) that can be confused with amoebic cysts or trophozoites.


Size: from 10 to 20 μm (usual range, 12-14 μm).  Immature cysts are generally larger.

Shape and features: generally round but sometimes oval, although the shape is less variable than that of Entamoeba coli.  By wet mount examination, cysts are surrounded by a double (split) wall that is more difficult to see than that in Entamoeba coli.

Nucleus/i: One to four nuclei are present, depending on the stage of maturation of the cyst.  On rare occasions, more than 4 nuclei can be observed (supernucleate cysts).

-     Cysts with one nucleus: the nucleus is large, with a diameter that can reach half the diameter of the cyst; it is round, oval, elliptical or tapered; a glycogen vacuole is generally present. The karyosome is often diffuse and comprises coarse, rather dispersed chromatin (easy to see on permanent stained smears), which is a sign of intense karyokinesis. In contrast, a nucleus at rest is small, almost always spherical, with a delicate nuclear membrane and small, compact karyosome. Be careful not to mistake mononucleate vacuolated cysts with cysts of Iodamoeba buetschlii, whose nucleus has a completely different structure. Chromatoid bodies, when present, are found on the periphery of the vacuole; they are colorless and refractile in fresh specimens, with rounded ends.

-     Cysts with two nuclei: the nuclei are generally paired and not at opposite ends as in Entamoeba coli; a glycogen vacuole may be present.

-     Cysts with four nuclei: these are the mature forms. Chromatoid bodies and vacuoles are no longer present. Very rarely it is possible to observe cysts with more than 4 nuclei.

A tetranucleate cyst may cause a problem of differential diagnosis between a mature cyst of Entamoeba histolytica and an immature cyst of Entamoeba coli.  To avoid confusion, it is useful to remember that:

-     The diameter of Entamoeba histolytica cysts is rarely greater than 15μm;

-     The four spherical nuclei of Entamoeba histolytica are all of the same size and have thin peripheral chromatin and a prominent, dot-like karyosome;

-     If the four nuclei are of different sizes, or if one or more of them display a karyosome with evident karyokinesis, mitotic division is probably still incomplete and the cyst is likely an immature Entamoeba coli cyst. 

During the microscopic examination, correct focusing on the sample is important.  Since the nuclei are rarely at the same focusing level, it is necessary to focus up and down through the cyst, in a sort of tomographic examination.  This permits an accurate count of all the nuclei present in the cyst.

videoclip 1

videoclip 2