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Fig. 9.7 Antigen detection test for E. histolytica in stool. Stool specimens with culture-confirmed E. dispar (open circles) or E. histolytica (closed circles) infection, or stools without detectable Entamoeba detected by microscopy (crosses), were assayed using an ELISA containing mAb specific for E. histolytica sensitive, and is unable to distinguish pathogenic E. histolytica from the morphologically identical non-pathogenic E. dispar (Haque et al., 1995, 1997; Petri and Mann, 1993). Erythrophagocytic amebae are more likely to be E. histolytica than E. dispar (Gonzalez-Ruis et al., 1994), but E. dispar trophozoites have also been found to contain ingested red blood cells (Haque et al., 1995).

In patients with amebic liver abscess, microscopic examination of the stool is even less efficacious, as repeated stool examinations in patients with amebic liver abscess were only able to detect the parasite in 8-44% of cases (Katzenstein et al., 1982; Petri et al., 1990). Aspiration of the abscess is occasionally required to diagnose amebiasis, and although amebae are visualized in the pus in only the minority ofcases, antigen detection on the abscess fluid appears to be very sensitive. In pyogenic abscesses, bacteria will be seen and/or cultured from the aspirated fluid. Antibodies to E. histolytica are present in the serum of 92-97% of patients upon acute presentation with amebic liver abscess, and therefore are very useful diagnostically. Because a significant proportion of the population in developing countries is seropositive, however, antibody tests are less specific in residents or immigrants from the developed world. Identification of the parasite in aspirated pus from liver abscesses, even in the most experienced hands, is only 20% sensitive (Haque et al., 1995; Katzenstein et al., 1982).

Antigen Detection

Antigen detection is a recently developed tool which exploits the molecular differences between E. histolytica and E. dispar. A stool antigen detection test that is specific for E. histolytica is now commercially available for clinical use from TechLab Inc. (Blacksburg, VA, USA) (Figure 9.7) (Haque et al., 1993, 1995, 1997). The E. histolytica antigen test is rapid, has improved sensitivity compared to microscopy, and is only slightly less sensitive than the 'gold standard' of culture/isoenzyme analysis. The TechLab E. histolytica test is based on detection of the Gal/GalNAc lectin in stool; another E. histolytica specimen antigen test is also available (Gonzalez-Ruiz et al., 1994). A test to detect the Gal/GalNAc lectin in serum appears promising for the diagnosis of amebic liver abscess (a situation where it is more difficult than amebic colitis to demonstrate the parasite in stool), with a reported sensitivity in initial tests of 67%. Detection of Gal/GalNAc lectin in liver abscess aspirates has demonstrated to be a sensitive test in a small number of patients (R. Haque and W. Petri, unpublished).

Polymerase Chain Reaction

Polymerase chain reaction (PCR) can now be used to diagnose E. histolytica in stool samples with sensitivity comparable to antigen detection (Haque et al., 1998). Preliminary results for PCR detection of E. histolytica DNA in liver abscess pus appear encouraging. Use of PCR can be used also to distinguish amongst isolates of E. histolytica, which should prove useful for epidemiologic purposes, as well as determining the virulence characteristics of different isolates (Garfinkel et al., 1989; Tachibana et al., 1991; Tannich et al., 1991; Acuna-Solo et al., 1993).

Serology

Serology using an indirect hemagglutination (IHA) test for anti-amebic antibody is eventually reportedly 99% sensitive for amebic liver abscess and 88% sensitive for amebic colitis (Kagan, 1970). However, early in the course of amebic liver abscess the indirect hemagglutination test may be negative. The major problem limiting clinical use of the current serologic tests is that they remain positive for years after an episode of amebiasis. As a result, a substantial number (1035%) of residents of developing countries have anti-amebic antibodies detected by current sero-logic tests (Caballero-Salcedo et al., 1994; Choudhuri et al., 1991). This has resulted in a situation where it is impossible to distinguish current from past infection in individuals from countries with high prevalence of infection (Ximenez et al., 1993). In five recent series of patients in the USA with amebic liver abscess, 80-96% of patients were immigrants (Aceti et al., 1991; Agarwal et al., 1992; Irusen et al., 1992; Kelsall et al., 1994). Therefore, current serologic tests may be inadequate for the differentiation of acute from past amebiasis, even in developed nations, and one should not make the diagnosis of amebiasis in a native of a country with endemic amebiasis on the basis of a serologic test alone.

Colonoscopy

Colonoscopy is preferable to sigmoidoscopy in the diagnosis of amebic colitis because disease may be localized to the cecum or ascending colon. Cathartics or enemas interfere with the identification of the parasite and should not be used. Wet preparations of material aspirated or scraped from the base of ulcers should be examined for motile trophozoites and tested for E. histolytica antigen. The appearance of amebic colitis may resemble that of inflammatory bowel disease, with granular, friable and diffusely ulcerated mucosa, and large geographic ulcers and pseudomembranes may also be present (Chun et al., 1994). Biopsy specimens should be taken from the edge of the ulcers. The detection rate of trophozoites upon histopathologic examination of colonic biopsy specimens from patients with amebic colitis is quite variable (Yoon et al., 1991). Periodic acid-Schiff stains the parasites a magenta color (Figure 9.6), increasing the ease of detection in biopsies. E. histolytica has been shown to invade into carcinomas, causing diagnostic confusion (Mhlanga et al., 1992).

Imaging

Imaging procedures such as ultrasound, computer tomography and magnetic resonance imaging studies of the liver are equally sensitive at detecting amebic abscesses (Figure 9.8). However, no technique can differentiate an amebic from a pyogenic abscess (Sheen et al., 1989; Ravdin et al., 1988; Ahmed et al., 1990; Elizondo et al., 1987). Radiographic resolution of amebic liver abscesses is prolonged and at 6 months'

Fig. 9.8 Imaging of hepatic amebic abscess from different patients by (A) ultrasound, (B) computed tomography. (A) From Ahmed (1990) and (B) from Radin (1988), both with permission

follow-up only one-third to two-thirds of amebic liver abscesses had disappeared upon repeat ultrasonography (Sheen et al., 1989; Ahmed et al., 1990; Agarwal et al., 1992).

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