....Amebiasis

Causal Agent:

Several protozoan species in the genus Entamoeba infect 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. The other species are important because they may be confused with E. histolytica in diagnostic investigations

Life Cycle:

Cysts and trophozoites are passed in feces . Cysts are typically found in formed stool, whereas trophozoites are typically found in diarrheal stool. Infection by Entamoeba histolytica occurs by ingestion of mature cysts in fecally contaminated food, water, or hands. Excystation occurs in the small intestine and trophozoites are released, which migrate to the large intestine. The trophozoites multiply by binary fission and produce cysts , and both stages are passed in the feces . Because of the protection conferred by their walls, the cysts can survive days to weeks in the external environment and are responsible for transmission. Trophozoites passed in the stool are rapidly destroyed once outside the body, and if ingested would not survive exposure to the gastric environment. In many cases, the trophozoites remain confined to the intestinal lumen (: noninvasive infection) of individuals who are asymptomatic carriers, passing cysts in their stool. In some patients the trophozoites invade the intestinal mucosa (: intestinal disease), or, through the bloodstream, extraintestinal sites such as the liver, brain, and lungs (: extraintestinal disease), with resultant pathologic manifestations. It has been established that the invasive and noninvasive forms represent two separate species, respectively E. histolytica and E. dispar. These two species are morphologically indistinguishable unless E. histolytica is observed with ingested red blood cells (erythrophagocystosis). Transmission can also occur through exposure to fecal matter during sexual contact (in which case not only cysts, but also trophozoites could prove infective).

Geographic Distribution:

Worldwide, with higher incidence of amebiasis in developing countries. In industrialized countries, risk groups include male homosexuals, travelers and recent immigrants, and institutionalized populations.

Clinical Features:

A wide spectrum, from asymptomatic infection ("luminal amebiasis"), to invasive intestinal amebiasis (dysentery, colitis, appendicitis, toxic megacolon, amebomas), to invasive extraintestinal amebiasis (liver abscess, peritonitis, pleuropulmonary abscess, cutaneous and genital amebic lesions).

Laboratory Diagnosis:

Entamoeba histolytica must be differentiated from other intestinal protozoa including: E. coli, E. hartmanni, E. gingivalis, Endolimax nana, and Iodamoeba buetschlii (the nonpathogenic amebas); Dientamoeba fragilis (which is a flagellate not an ameba); and the possibly pathogenic Entamoeba polecki. Differentiation is possible, but not always easy, based on morphologic characteristics of the cysts and trophozoites. The nonpathogenic Entamoeba dispar, however, is morphologically identical to E. histolytica, and differentiation must be based on isoenzymatic or immunologic analysis. Molecular methods are also useful in distinguishing between E. histolytica and E. dispar and can also be used to identify E. polecki. Microscopic identification of cysts and trophozoites in the stool is the common method for diagnosing E. histolytica. 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). Concentration procedures, however, are not useful for demonstrating trophozoites.
In addition, E. histolytica trophozoites can also be identified in aspirates or biopsy samples obtained during colonoscopy or surgery.

Diagnostic findings:

1-Microscopy

Cysts

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.

Trophozoites

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.

Erythrophagocytosis (ingestion of red blood cells by the parasite) is the only morphologic characteristic that can be used to differentiate E. histolytica from the nonpathogenic E. dispar. However, erthrophagocytosis is not typically observed on stained smears of E. histolytica

• Immunodiagnosis

Antibody Detection

Enzyme immunoassay (EIA) kits for Entomoeba 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. Antigen detection may be useful as an adjunct to microscopic diagnosis in detecting parasites and can distinguish between pathogenic and nonpathogenic infections.

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. Specificity is 95% or higher: false-positive reactions rarely occur. Although the immunodiffusion test is as specific, it is slightly less sensitive than the IHA and EIA and requires a minimum of 24 hours to obtain a result, in contrast to 2 hours required for the IHA or EIA tests. However, the simplicity of the procedure makes it ideal for the laboratory that has only an occasional specimen to test. The IHA and EIA tests are more suitable for laboratories that have frequent requests for amebiasis serology. 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.

Antigen Detection
Antigen detection may be useful as an adjunct to microscopic diagnosis in detecting parasites and to distinguish between pathogenic and nonpathogenic infections. 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.

In reference diagnosis laboratories, molecular analysis by PCR-based assays is the method of choice for discriminating between the pathogenic species (E. histolytica) and the nonpathogenic species (E. dispar).

A: Agarose gel (2%) analysis of a PCR diagnostic test for differentiation between E. histolytica and E. dispar.

Lanes 1 - 4: Amplification with the Psp3/Psp51 PCR primer pair specific for E. histolytica. Diagnostic band size - 876 bp.
Lanes 6 - 9: Amplification with the NPsp3/NPsp51 PCR primer pair specific for E. dispar. Diagnostic band size - 876 bp.
Lanes 1 and 6: E. histolytica 200:NIH, zymodeme II (positive control for E. histolytica).
Lanes 2 and 7: E. dispar 351:IMMiT, zymodeme I (positive control for E. dispar).
Lanes 3 and 8: Specimen from a patient with a liver abscess (positive with E. histolytica primers and negative with E. dispar primers). E. histolytica 333:IMMiT, zymodeme XIV.
Lanes 4 and 9: Specimen from an asymptomatic patient (positive with E. dispar primers and negative with E. histolytica primers). E. dispar 389:IMMiT, zymodeme I.
Lane 5: Molecular base pair standard, 100-bp ladder (from 600 to 1,000 bp).
Figure A contributed by Assist. Prof. Przemyslaw Myjak, Ph.D., Institute of Maritime and Tropical Medicine, Gdynia, Poland.

Real-Time PCR

A TaqMan real-time PCR approach has been validated at CDC and is used for differential laboratory diagnosis of amebiasis.2 The assay targets the 18S rRNA gene with species-specific TaqMan probes in a duplex format, making it possible to detect both species in the same reaction vessel

Treatment:

For asymptomatic infections, iodoquinol, paromomycin, or diloxanide furoate (not commercially available in the U.S.) are the drugs of choice. For symptomatic intestinal disease, or extraintestinal, infections (e.g., hepatic abscess), the drugs of choice are metronidazole or tinidazole, immediately followed by treatment with iodoquinol, paromomycin, or diloxanide furoate. For additional information, see the recommendations in The Medical Letter (Drugs for Parasitic Infections).