General Characteristics
Living trophozoites (motile feeding stage) of E. histolytica vary in size from about 12 to 60 µm in diameter. Organ isms recovered from diarrheic or dysenteric stools generally are larger than those in formed stool from an asymptomatic individual. The motility has been described as rapid and unidirectional. Although this characteristic motility is often described, amebiasis rarely is diagnosed on the basis of motility seen in a direct mount. The cytoplasm is differentiated into a clear outer ectoplasm and a more granular inner endoplasm.
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 1).
Fig1. Entamoeba histolytica trophozoite containing ingested red blood cells.
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 chroma tin 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 2). Most often, infection with E. histolytica is diagnosed on the basis of the organism’s morphology, without the presence of RBCs.
Fig2. 1, Trophozoite of Entamoeba histolytica (note ingested red blood cells). 2, Trophozoite of Entamoeba histolytica/Entamoeba dispar (morphology does not allow differentiation between the two species). 3 and 4, Early cysts of E. histolytica/E. dispar . 5 to 7, Cysts of E. histolytica/E. dispar .
Fig2. cont’d 8 and 9, Trophozoites of Entamoeba coli. 10 and 11, Early cysts of E. coli. 12 to 14, Cysts of E. coli. 15 and 16, Trophozoites of Entamoeba hartmanni. 17 and 18, Cysts of E. hartmanni. (From Garcia LS: Diagnostic medical parasitology, ed 4, Washington, DC, 2001, ASM Press. Illustrations 4 and 11 by Nobuko Kitamura.)
As part of the life cycle, the trophozoites may con dense 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 2; Figure 3), 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.
Fig3. Entamoeba histolytica/Entamoeba dispar cyst.
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 4) 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.
Fig4. Entamoeba dispar trophozoite; no ingested red blood cells are present.
Infection is acquired through the fecal-oral route from infective cysts contained in the feces. These cysts can be ingested in contaminated food or drink or contracted from fomites or various sexual practices that could include accidental ingestion of fecal organisms. Flies and cockroaches have been implicated as mechanical vectors of contaminated fecal material.
The infection occurs worldwide, particularly in areas with poor sanitation. It is estimated that E. histolytica infection kills more than 100,000 people each year.
Pathogenesis and Spectrum of Disease
The pathogenesis of E. histolytica is related to the organ ism’s ability to directly lyse host cells and cause tissue destruction. Amebic lesions show evidence of cell lysis, tissue necrosis, and damage to the extracellular matrix. Evidence indicates that E. histolytica trophozoites interact with the host through a series of steps: adhesion to the target cell, phagocytosis, and cytopathic effect. Numerous other parasite factors also play a role. From the perspective of the host, E. histolytica induces both humoral and cellular immune responses; cell-mediated immunity is the major human host defense against this complement resistant cytolytic protozoan.
The presentations of disease are seen with invasion of the intestinal mucosa or dissemination to other organs (most often the liver) or both. However, it is estimated that a small proportion (2% to 8%) of infected individuals have invasive disease beyond the lumen of the bowel. Also, organisms may be spontaneously eliminated with no disease symptoms.
Asymptomatic Infection. Individuals harboring E. histolytica may have either a negative or a weak antibody titer and negative stools for occult blood. They also may be passing cysts detectable by a routine ova and parasite (O&P) examination. However, these cysts cannot be morphologically differentiated from those of the nonpathogen, E. dispar . Although trophozoites may be identified, they will not contain any phagocytized RBCs and cannot be differentiated from E. dispar . Molecular analyses of organisms isolated from asymptomatic individuals generally indicate that the isolates belong to the nonpathogenic E. dispar . Generally, asymptomatic patients never become symptomatic and may excrete cysts for a short period. This pattern is seen in patients infected with either nonpathogenic or pathogenic organisms.
Intestinal Disease. The incubation period varies from a few days to a much longer time; in an area where E. histolytica is endemic, it is impossible to determine exactly when exposure to the organism occurred. Normally, the incubation time ranges from 1 to 4 weeks. Although the exact mode of mucosal penetration is not known, microscopic studies suggest that amebae have enzymes that lyse. The enzymes are released from lysosomes on the surface of the amebae or from enzymes in the tissue released from ruptured organisms. Amebic ulcers often develop released the cecum, appendix, or adjacent portion of the ascending colon; however, they can also be found in the sigmoid rectal area. Other lesions may occur from these primary sites. Ulcers usually are raised, with a small opening on the mucosal surface and a larger area of destruction below the surface (i.e., flask shaped). The mucosal lining may appear normal between ulcers.
Invasive intestinal amebiasis has four clinical forms, all of which are generally acute: dysentery (bloody diarrhea), fulminating colitis, amebic appendicitis, and ameboma of the colon. Dysentery and diarrhea account for 90% of cases of invasive intestinal amebiasis. The severity of symptoms can range from asymptomatic to severe symptoms that mimic ulcerative colitis. Patients with colicky abdominal pain, frequent bowel movements, and tenesmus (a persistent feeling of needing to pass stool) may present with a gradual onset of disease. With the onset of dysentery, bowel movements are frequent (up to 10 per day). Although dysentery may last for months, it varies from severe to mild and may lead to weight loss and prostration. In severe cases, symptoms may begin very suddenly and include profuse diarrhea, fever, and dehydration with electrolyte imbalances.
Hepatic Disease. Blood flow from the mesenteric veins surrounding the intestine returns blood, via the portal vein, to the liver, most commonly the upper right lobe. Amebae in the submucosa can be carried by the blood stream to the liver. The onset of symptoms may be gradual or sudden; upper right abdominal pain and fever (38° to 39°C) are the most consistent findings. Although the liver may be enlarged and tender, liver function tests may be normal or slightly abnormal (jaundice is rare). The abscess can be visualized radiologically, sonically, or by radionuclear scan; most patients have a single abscess in the right lobe of the liver. The most common complication is rupture of the abscess into the pleural space. An abscess also can extend into the peritoneum and through the skin. Hematogenous spread to the brain, lung, pericardium, and other sites is possible.
Pyogenic and amebic liver abscesses are the two most common hepatic abscesses. The severity of a pyogenic abscess depends on the bacterial source and the patient’s underlying condition. An amebic abscess tends to be more prevalent in those with suppressed cell-mediated immunity, men, and younger individuals. E. histolytica cysts and trophozoites are found in the stools of only a few patients with liver abscess. About 60% of these patients have no intestinal symptoms or any history of dysentery.
Laboratory Diagnosis
Routine Methods. The standard O&P examination is the recommended procedure for recovery and identification of E. histolytica in stool specimens. Microscopic examination of a direct saline wet mount may reveal motile trophozoites, which may contain RBCs. However, trophozoites with RBCs are found only in a limited number of cases. In many patients who do not present with acute dysentery, trophozoites may be present but do not contain RBCs, and the organisms may be pathogenic E. histolytica or nonpathogenic E. dispar . An asymptomatic individual may have few trophozoites and possibly only cysts in the stool. Although the concentration technique is helpful for demonstrating cysts, the most important technique for the recovery and identification of protozoan organisms is the permanent stained smear (normally stained with trichrome or iron-hematoxylin). A minimum of three specimens collected over not more than 10 days may be required for identification.
Sigmoidoscopy specimens may be very helpful for identifying organisms. At least six areas of the mucosa should be sampled. Smears from these areas should be examined after permanent staining. However, these specimens are not considered a substitute for the recommended minimum of three stool specimens submitted for O&P examination (direct, concentration, and permanent stained smear).
Liver aspirate material is rarely examined, and often the specimen was not collected properly. Aspirated mate rial must be aliquoted into several different containers as it is removed from the abscess; amebae may be found only in the last portion of the aspirated material, theoretically material from the abscess wall, not necrotic debris from the abscess center.
Antigen Detection. A number of enzyme immunoassay reagents are commercially available, and their specificity and sensitivity provide excellent options for the clinical laboratory. These tests can differentiate the E. histolytica/ E. dispar group from the rest of the Entamoeba species, such as nonpathogenic Entamoeba coli or Entamoeba hartmanni. Other test reagents can distinguish between E. histolytica and E. dispar (Entamoeba histolytica II test, TechLab, Blacksburg, VA or Entamoeba CELESA path, Cellabs, Brookvale, Australia). These kits require fresh or frozen stool; fecal preservatives have been found to inter fere with the Entamoeba spp. reagents. Because of the specificity of the immunoassay reagents, the laboratory can inform the physician whether the E. histolytica/ E. dispar organisms seen in the stool specimen are pathogenic E. histolytica or nonpathogenic E. dispar . Without the use of these reagents, the only way to identify true pathogenic E. histolytica morphologically is to detect the rare presence of trophozoites containing ingested RBCs. If the laboratory does not use these reagents, the presence of E. histolytica/E. dispar should be reported to the physician, accompanied by commentary related to the newer information on pathogenicity. Depending on each state’s requirements, pathogenic E. histolytica generally is reported to the public health facility (county).
Antibody Detection. Serologic testing for intestinal disease is rarely recommended unless the patient has true dysentery; even in these cases, the titer (e.g., indirect hemagglutination) may be low and thus difficult to interpret. A definitive diagnosis of intestinal amebiasis should not be made without demonstrating the presence of the organisms. In patients suspected of having extraintestinal disease, serologic tests are diagnostically more effective. Indirect hemagglutination and indirect fluorescent anti body tests have been reported positive with titers greater than or equal to 1 : 256 and greater than or equal to 1 : 200, respectively, in almost 100% of cases of amebic liver abscess. In the absence of STAT serologic tests for amebiasis (tests with very short turnaround times for results), the decision on diagnosis must be made on clinical grounds and on the basis of results of other diagnostic tests, such as scans.
Histology. A histologic diagnosis of amebiasis can be made when the trophozoites in the tissue are identified. Organisms must be differentiated from host cells, particularly histiocytes and ganglion cells. Periodic acid-Schiff (PAS) staining often is used to help locate the organisms, which appear bright pink with a green-blue background (depending on the counterstain used). Hematoxylin and eosin staining also allows visualization of the typical morphology, thus allowing accurate identification. As a result of sectioning, some organisms exhibit the evenly arranged nuclear chromatin with the central karyosome, and some no longer contain the nucleus.
Nucleic Acid-Based Techniques
Nucleic acid-based amplification methods, including polymerase chain reaction (real-time and multiplex assays), have been developed for the identification of E. histolytica. Stool specimens, however, may contain inhibitors that would prevent accurate detection using amplification methods. These tests are not widely used, because they require more technical expertise and currently have not proven to be more sensitive than antigen-based immunoassays.
Therapy
Two classes of drugs are used in the treatment of amebic infections: luminal amebicides, such as iodoquinol or diloxanide furoate, and tissue amebicides, such as metronidazole, chloroquine, or dehydroemetine. Because of the differences in drug efficacy, it is important that the laboratory report indicates whether cysts, trophozoites, or both are present in the stool specimen.
Asymptomatic Infection. Patients found to have true E. histolytica in the intestinal tract, even if asymptomatic, should be treated to eliminate the organisms. Both diloxanide furoate and iodoquinol or paromomycin are available for treatment of patients who have cysts in the lumen of the gut. In general, these treatments are ineffective against extraintestinal disease. If the patient is passing both trophozoites and cysts, the recommended treatment is metronidazole plus iodoquinol.
Mild to Moderate Disease. In patients with mild to moderate disease, metronidazole (Flagyl) should be used when tissue invasion occurs, regardless of the tissue involved. Drugs directed against the lumen organisms should also be used in these cases.
Severe Intestinal Disease. Metronidazole plus one of the luminal drugs should be used for therapy.
Hepatic Disease. Metronidazole plus one of the luminal drugs should be used to treat hepatic disease. Some other combinations also can be used; some contain emetine, in which case the patient must be monitored very carefully for possible cardiotoxicity. The importance of using both luminal and tissue amebicides is emphasized in patients with amebic liver abscesses. Asymptomatic colonization may be present with the true pathogen, E. histolytica. In patients treated with metronidazole (tissue amebicide), generally a 100% clinical response to the hepatic lesions is seen; however, failure to eliminate the organism from the bowel can lead to second bouts with invasive disease and intestinal colonization. Also, these carriers constitute a public health hazard because of continued shedding of infective cysts.
Prevention
Humans are the reservoir host for E. histolytica, and infection can be transmitted to other humans, primates, dogs, cats, and possibly pigs. Accidental consumption of sewage-contaminated water provides another route of infection. Amebiasis is considered a zoonotic waterborne infection. The cyst stages are resistant to environmental conditions and can remain viable in the soil for 8 days at 28° to 34°C, for 40 days at 2° to 6°C, and for 60 days at 0°C. Cysts normally are removed by sand filtration or destroyed by 200 ppm of iodine, 5% to 10% acetic acid, or boiling. However, an asymptomatic carrier who is a food handler generally is thought to play the most important role in transmission. Proper disposal of contaminated feces is considered the most important preventive measure. Although vaccines have been discussed as a possibility for eliminating human disease, nothing currently is available.
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