Amebiasis

[Entamoeba histolytica]

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Causal Agents

Several protozoan species in the genus Entamoeba colonize humans, but not all of them are associated with disease. Entamoeba histolytica is well recognized as a pathogenic ameba, associated with intestinal and extraintestinal infections. Other morphologically-identical Entamoeba spp., including E. dispar, E. moshkovskii, and E. bangladeshi, are generally not associated with disease although investigations into pathogenic potential are ongoing.

While the discussed species are morphologically-identical, E. histolytica may be observed with ingested red blood cells (erythrophagocytosis); E. dispar may occasionally be seen with ingested erythrocytes as well, although its capacity for erythrophagocytosis is much less than that of E. histolytica. Non-pathogenic amebae (e.g. Endolimax nana, Iodamoeba buetschlii, other Entamoeba species) are important because they may be confused with E. histolytica in diagnostic investigations.

Life Cycle

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Cysts and trophozoites are passed in feces . Cysts are typically found in formed stool, whereas trophozoites are typically found in diarrheal stool. Infection with Entamoeba histolytica (and E.dispar) occurs via ingestion of mature cysts from fecally contaminated food, water, or hands. Exposure to infectious cysts and trophozoites in fecal matter during sexual contact may also occur. Excystation occurs in the small intestine and trophozoites are released, which migrate to the large intestine. Trophozoites may remain confined to the intestinal lumen (A: noninvasive infection) with individuals continuing to pass cysts in their stool (asymptomatic carriers). Trophozoites can invade the intestinal mucosa (B: intestinal disease), or blood vessels, reaching extraintestinal sites such as the liver, brain, and lungs (C: extraintestinal disease). Trophozoites multiply by binary fission and produce cysts , and both stages are passed in the feces . Cysts can survive days to weeks in the external environment and remain infectious in the environment due to the protection conferred by their walls. Trophozoites passed in the stool are rapidly destroyed once outside the body, and if ingested would not survive exposure to the gastric environment.

Geographic Distribution

Pathogenic Entamoeba species occur worldwide and are frequently recovered from fresh water contaminated with human feces. The majority of amebiasis cases occur in developing countries.

In industrialized countries, risk groups include men who have sex with men, travelers, recent immigrants, immunocompromised persons, and institutionalized populations.

Clinical Presentation

The majority of infections restricted to the lumen of the intestine (“luminal amebiasis”) are asymptomatic. Amebic colitis, or invasive intestinal amebiasis, occurs when the mucosa is invaded. Symptoms include severe dysentery and associated complications. Severe chronic infections may lead to further complications such as peritonitis, perforations, and the formation of amebic granulomas (ameboma).

Amebic liver abscesses are the most common manifestation of extraintestinal amebiasis. Pleuropulmonary abscess, brain abscess, and necrotic lesions on the perianal skin and genitalia have also been observed.

E. histolytica/E. dispar cysts in concentrated wet mounts.

Mature Entamoeba histolytica/Entamoeba dispar cysts have 4 nuclei that characteristically have centrally-located karyosomes and fine, uniformly distributed peripheral chromatin. Cysts usually measure 12 to 15 µm.

Figure A: Cyst of E. histolytica/E. dispar in an unstained concentrated wet mount of stool. Notice the chromatoid bodies with blunt, rounded ends (arrow).

Figure B: Cyst of E. histolytica/E. dispar in an unstained concentrated wet mount of stool. Notice the chromatoid bodies with blunt, rounded ends (arrow).

Figure C: Cyst of E. histolytica/E. dispar in a concentrated wet mount stained with iodine.

Figure D: Cyst of E. histolytica/E. dispar in a concentrated wet mount stained with iodine. Notice the chromatoid body with blunt, rounded ends (arrow).

E. histolytica/E. dispar cysts stained with trichrome.

Figure A: Cyst of E. histolytica/E. dispar stained with trichrome. Note the chromatoid body with blunt ends (red arrow).

Figure B: Cyst of E. histolytica/E. dispar stained with trichrome.

Figure C: Cyst of E. histolytica/E. dispar stained with trichrome. Three nuclei are visible in the focal plane (black arrows), and the cyst contains a chromatoid body with typically blunted ends (red arrow). The chromatoid body in this image is particularly well demonstrated.

Figure D: Cyst of E. histolytica/E. dispar stained with trichrome. Two nuclei are visible in the focal plane (black arrows), and the cyst contains a chromatoid body with typically blunted ends (red arrow).

E. histolytica/E. dispar immature cysts stained with trichrome.

Mature Entamoeba histolytica/Entamoeba dispar cysts have 4 nuclei that characteristically have centrally located karyosomes and fine, uniformly distributed peripheral chromatin. Cysts usually measure 12 to 15 µm.

Figure A: Immature cyst of E. histolytica. The specimen was preserved in poly-vinyl alcohol (PVA) and stained with trichrome. PCR was performed on this specimen to differentiate between E. histolytica and E. dispar

Figure B: Immature cyst of E. histolytica/E. dispar stained with trichrome. Image taken at 1000x oil magnification and contributed by the Kansas Department of Health and Environment.

Figure C: Immature cyst of E. histolytica/E. dispar stained with trichrome. The cyst has large vacuoles and the chromatin around the nucleus is clumpy. Image taken at 1000x oil magnification and contributed by the Kansas Department of Health and Environment.

Figure D: Immature cyst of E. histolytica/E. dispar stained with trichrome. The cyst has large vacuoles and the chromatin around the nucleus is clumpy. These cysts range in size from 10.3 to 15.7 µm. Image taken at 1000x oil magnification and contributed by the Kansas Department of Health and Environment.

E. histolytica/E. dispar trophozoites in direct wet mounts.

Pathogenic Entamoeba trophozoites have a single nucleus, which have a centrally placed karyosome and uniformly distributed peripheral chromatin. The cytoplasm has a granular or “ground-glass” appearance. Trophozoites usually measure 15 to 20 µm (range 10 to 60 µm), tending to be more elongated in diarrheal stool.

Figure A: Trophozoite of E. histolytica/E. dispar in a direct wet mount stained with iodine.

Figure B: Trophozoite of E. histolytica/E. dispar in a direct wet mount stained with iodine.

Entamoeba histolytica/Entamoeba dispar trophozoites stained with trichrome.

Entamoeba histolytica/Entamoeba dispar trophozoites have a single nucleus, which have a centrally placed karyosome and uniformly distributed peripheral chromatin. This typical appearance of the nucleus is not always observed as some trophozoites can have nuclei with an eccentric karyosome and unevenly distributed peripheral chromatin. The cytoplasm has a granular or “ground-glass” appearance. E. histolytica/E. dispar trophozoites usually measure 15 to 20 µm (range 10 to 60 µm), tending to be more elongated in diarrheal stool.

Figure A: Trophozoite of E. histolytica/E. dispar stained with trichrome.

Figure B: Trophozoite of E. histolytica/E. dispar stained with trichrome.

Figure C: Trophozoite of E. histolytica. The specimen was preserved in poly-vinyl alcohol (PVA) and stained with trichrome. PCR was performed on this specimen to differentiate between E. histolytica and E. dispar.

Figure D: Trophozoite of E. histolytica. The specimen was preserved in poly-vinyl alcohol (PVA) and stained with trichrome. The vacuolated cytoplasm seen in this image may be the result of less than optimal preservation. PCR was performed on this specimen to differentiate between E. histolytica and E. dispar.

Figure E: Trophozoite of E. histolytica/E. dispar, measuring approximately 16.7 µm, stained with trichrome. The image was taken at 1000× magnification and contributed by the Kansas Department of Health and Environment.

Trophozoites of E. histolytica with ingested erythrocytes.

Pathogenic Entamoeba trophozoites have a single nucleus, which have a centrally placed karyosome and uniformly distributed peripheral chromatin. This typical appearance of the nucleus is not always observed as some trophozoites can have nuclei with an eccentric karyosome and unevenly distributed peripheral chromatin. The cytoplasm has a granular or “ground-glass” appearance. Trophozoites usually measure 15 to 20 µm (range 10 to 60 µm), tending to be more elongated in diarrheal stool.

Erythrophagocytosis (ingestion of red blood cells by the parasite) is classically associated with E. histolytica but may rarely occur with E. dispar, thus this is not a reliable criterion by which to distinguish these species. Note that erythrophagocytosis is rarely observed on stained smears.

Figure A: Trophozoite of E. histolytica with ingested erythrocytes stained with trichrome. The ingested erythrocytes appear as dark inclusions. The parasite above shows nuclei that have the typical small, centrally located karyosome, and thin, uniform peripheral chromatin.

Figure B: Trophozoites of E. histolytica with ingested erythrocytes stained with trichrome. The ingested erythrocytes appear as dark inclusions. The parasites above show nuclei that have the typical small, centrally located karyosome, and thin, uniform peripheral chromatin.

Figure C: Trophozoite of E. histolytica with ingested erythrocytes stained with trichrome.

Figure D: Trophozoite of E. histolytica with ingested erythrocytes stained with trichrome.

Figure E: Trophozoite of E. histolytica ingested erythrocytes stained with trichrome.

Figure F: Trophozoite of E. histolytica with ingested erythrocytes (arrow), under differential interference contrast (DIC) microscopy.

E. histolytica trophozoites in tissue stained with hematoxylin and eosin (H&E).

Figure A: Numerous E. histolytica trophozoites in a rectal biopsy, H&E stained. Arrows indicate examples of trophozoites with visible nuclei.

Figure B: Closer view of E. histolytica trophozoites in the rectal biopsy shown in Figure A. Arrows indicate examples of trophozoites with visible nuclei. Note the typical small, central karyosomes, fine peripheral chromatin, and “ground-glass” cytoplasm.

Laboratory Diagnosis

Differential diagnosis among other amebae

Pathogenic Entamoeba species must be differentiated from other intestinal protozoa such as the nonpathogenic amebae (Entamoeba coli, E. hartmanni, E. gingivalis, Endolimax nana, Iodamoeba buetschlii) and the flagellate Dientamoeba fragilis. Morphologic differentiation among these is possible, but potentially complicated, based on morphologic characteristics of the cysts and trophozoites.

In culture, differential growth characteristics of E. moshkovskii may aid in distinguishing it from other species, but culture methods have important limitations (missing mixed infections, contamination, labor-intensive, limited availability). Historically, differentiation of E. dispar and E. histolytica was based on isoenzymatic or immunologic analysis, but these are no longer favored with the availability of effective molecular methods and are seldom performed. Molecular methods are currently recommended for distinguishing pathogenic Entamoeba species.

Microscopic Detection

Microscopic identification of cysts and trophozoites in the stool is the common method for diagnosing pathogenic Entamoeba species. This can be accomplished using:

Fresh stool: wet mounts and permanently stained preparations (e.g., trichrome).
Concentrates from fresh stool: wet mounts, with or without iodine stain, and permanently stained preparations (e.g., trichrome). While useful for cysts, concentration methods may not be useful for demonstrating trophozoites.
Microscopy also has a low sensitivity if only one stool sample is analyzed, and requires personnel trained in morphological diagnosis. Collection and analysis of three consecutive stool samples within ten days improves the chances for detection. Also, E. dispar, E. histolytica, and E. moshkovskii are not distinguishable based on morphology.

Trophozoites can also be identified in aspirates or biopsy samples obtained during colonoscopy or surgery.

Immunodiagnosis

Enzyme immunoassay (EIA) kits for Entamoeba histolytica antibody detection as well as EIA kits for antigen detection are commercially available in the United States. Antibody detection is most useful in patients with extraintestinal disease (i.e., amebic liver abscess) when organisms are not generally found on stool examination. Antibody detection is of limited diagnostic value on patients from highly endemic areas that are likely to have prior exposure and seroconversion, but may be of more use on patients from areas where pathogenic Entamoeba spp. are rare. Antigen detection during active infections may be useful as an adjunct to microscopic diagnosis in detecting parasites and can distinguish between pathogenic and nonpathogenic infections.

Antibody detection

The indirect hemagglutination (IHA) test has been replaced by commercially available EIA test kits for routine serodiagnosis of amebiasis. Antigen consists of a crude soluble extract of axenically cultured organisms. The EIA test detects antibody specific for E. histolytica in approximately 95% of patients with extraintestinal amebiasis, 70% of patients with active intestinal infection, and 10% of asymptomatic persons who are passing cysts of E. histolytica. If antibodies are not detectable in patients with an acute presentation of suspected amebic liver abscess, a second specimen should be drawn 7-10 days later. If the second specimen does not show seroconversion, other agents should be considered. Detectable E. histolytica-specific antibodies may persist for years after successful treatment, so the presence of antibodies does not necessarily indicate acute or current infection. Also, patients who have lived in highly endemic areas are likely to be seropositive due to past exposures. Specificity is 95% or higher: false-positive reactions rarely occur.

Although detection of IgM antibodies specific for E. histolytica has been reported, sensitivity is only about 64% in patients with current invasive disease. Several commercial EIA kits for antibody detection are available in the United States. No commercial antibody detection kits exist for E. dispar or E. moshkovskii or E. bangladeshi.

Antigen Detection

Antigen detection may be useful as an adjunct to microscopic diagnosis in detecting parasites and to distinguish between pathogenic and nonpathogenic infections. However, utility is limited for frozen or fixed specimens and for post-treatment specimens. Recent studies indicate improved sensitivity and specificity of fecal antigen assays with the use of monoclonal antibodies which can distinguish between E. histolytica and E. dispar infections. At least one commercial kit is available which detects only pathogenic E. histolytica infection in stool; several kits are available which detect E. histolytica antigens in stool but do not exclude E. dispar infections.

Molecular Diagnosis

Conventional PCR

In reference diagnosis laboratories, molecular analysis by conventional PCR-based assays is the method of choice for discriminating between E. histolytica and E. dispar. Some assays also can distinguish E. moshkovskii.

Real-Time PCR

A TaqMan real-time PCR approach has been validated at CDC and is used for differential laboratory diagnosis of amebiasis. The assay targets the 18S rRNA gene with species-specific TaqMan probes in a duplex format, making it possible to detect both E. histolyrica and E. dispar in the same reaction vessel.

References:
Qvarnstrom Y, James C, Xayavong M, Holloway B, Moura I, Visvesvara GS, et al. Comparison of real-time PCR rationales for differential laboratory diagnosis of amebiasis. J Clin Microbiol 2005;43:5491-5497.

Laboratory Safety

Cysts in unfixed stool samples are potentially infective. Observe standard precautions that apply to stool specimens: https://www.cdc.gov/dpdx/diagnosticprocedures/stool/safety.html.