History

E. histolytica has recently been separated from E. dispar on the basis of genetic differences. Both protozoa are morphologically identical but have genetic, and functional differences; E. histolytica is invasive and causes disease such as colitis and liver abscess, and E. dispar causes a asymptomatic colonization which does not need to be medically treated (WHO/PAHO/UNESCO, 1997). In 1828, James Annesley wrote in Prevalent Diseases of India, '. . . hepatic disease seems to be induced by the disorder of the bowels, more particularly when this disorder is of a subacute or chronic kind', recognizing for the first time a link between dysentery and liver abscess. Approximately 30 years later, in 1855, Lambl described amebae in the stool of a child who had diarrhea (Stillwell, 1955). Fedor Losch described amebae in the stool of a young farmer with dysentery from his first evaluation in November 1873 until his death in April 1874. The amebae causing the ultimate demise of the farmer were described as 'round, pear-shaped or irregular form and which are in a state of almost continuous motion', and autopsy studies ultimately revealed colonic ulcerations. Koch's postulates were fulfilled when the patient's stool inoculated orally and rectally into a dog caused dysentery, with amebic ulcers (Stillwell, 1955; Kean, 1988). The first North American case of amebiases was reported in 1890 by Sir William Osler, when he described a young physician in Baltimore with dysentery. Osler wrote: 'Dr B, aged 29, resident in Panama for nearly 6 years, where he had had several attacks of dysentery, or more correctly speaking a chronic dysentery, came north in May, 1889 . . .'. Subsequently, in 1890, the patient developed tender hepatosple-nomegaly and amebae were observed in the stool and abscess fluid: 'The general character of the amoebae (found in the stool) correspond in every particular with those found in the liver' (Osler, 1890). A year later, Osler's colleagues Councilman and Lafleur (1891) proceeded through a classic investigation of 14 cases of amebic dysentery to

Principles and Practice of Clinical Parasitology

Edited by Stephen Gillespie and Richard D. Pearson © 2001 John Wiley & Sons Ltd clearly distinguish amebiasis from bacterial dysentery, and coined the terms 'amebic dysentery' and 'amebic liver abscess'.

Historically, ipecac bark was used in the treatment of dysentery. Piso introduced ipecac bark, which had been used for centuries in Peru for the treatment of dysentery, to Europe in 1658. Helvetius used ipecac to successfully treat the dysentery of King Louis XIV, and subsequently sold it as a secret remedy to the French government. Not until 1858 was the use of large doses of ipecac for the treatment of dysentery promoted by Surgeon E. S. Docker in Mauritius, who demonstrated that ipecac (60 grains, two or three times daily) decreased mortality from 10-18% to only 2%. However, large doses of ipecac by mouth was complicated by severe nausea and vomiting and necessitated the co-administration of opium, chloral hydrate or tannic acid. An alternative therapy was discovered by Leonard Rogers, Professor of Pathology in Calcutta, India, who found that emetine, the principal alkaloid in ipecac, killed amebae in the mucus of stools from patients with dysentery at dilutions as high as 1/100000. In 1912 he reported successfully treating three patients in Calcutta, who had been unable to tolerate oral ipecac, by injection of emetine (Rogers, 1912).

The cyst form of E. histolytica was implicated as the infective form of the parasite by Walker and Sellards (1913) in the Philippines, and the parasite's life-cycle was outlined by Dobell (1925). Axenic culture of E. histolytica (free of any associated microorganisms) was accomplished by Diamond (1961) at the NIH in 1961. This milestone in the history of amebiasis has enabled study of the cell biology and biochemistry of the parasite, upon which our current understanding of amebiasis is based.

DESCRIPTION OF THE ORGANISM Taxonomy

E. histolytica, a pseudopod-forming non-flagellated protozoan parasite, is the most invasive of the Entamoeba group (which includes E. dispar, E. hartmanni, E. polecki, E. coli and E. gingivalis). It is the Entamoeba that most often causes clinical disease in humans, such as amebic colitis and liver abscess. Trophozoites of this parasite contain a single nucleus, and nuclear division occurs without the formation of condensed metaphase chromosomes. No sexual forms of the parasite have been identified.

The taxonomy of E. histolytica has changed significantly in the last decade and it has recently been reclassified into two species which are morphologically identical but genetically distinct: E. histolytica (Schaudinn, 1903), an invasive disease-causing parasite, and E. dispar (Brumpt, 1925), a non-invasive parasite. This separation was initially proposed in 1925 by Brumpt, who found that only one of the species caused disease in kittens or human volunteers, and named the non-pathogenic species E. dispar (Thompson and Glasser, 1986). However, in the absence of a means to distinguish the two morphologically identical parasites, this distinction was not generally accepted. In 1978, Sargeaunt and colleagues demonstrated that isoenzyme typing could be used to distinguish the pathogenic from the non-pathogenic species of Entamoeba and the issue was re-examined. The two species can be differentiated by isoenzyme analysis, typing by monoclonal antibodies to surface antigens, and restriction fragment length polymorphisms and PCR (Table 9.1) (Sargeaunt et al, 1978; Tannich et al., 1989; Tannich and Burchard, 1991; Diamond and Clark, 1993; Garfinkel et al., 1989; Clark and Diamond, 1993). Earlier reports that E. histolytica and E. dispar could convert in culture (Andrews et al., 1990; Mirelman et al., 1986) were recently shown to be artifactual with the apparent conversion due to contamination of laboratory strains (Clark and Diamond, 1993).

Table 9.1 Differentiation of E. histolytica and E. dispar

Biochemical characters

Discrimination by isoenzyme migration Immunological characters Monoclonal antibodies Genetic characters

Restriction fragment pattern comparisons Repetitive DNA sequences Riboprinting Clinical characters

E. dispar has not been associated with tissue invasion

From Petri (1996), with permission.

Fig. 9.1 E. histolytica trophozoite and cyst. (A) The motile and invasive trophozoite forms each contain a single nucleus and are 10 and 60 ^m in diameter, respectively. (B) The infectious cyst form is 10-15 ^m in diameter and contains four nuclei. Note that it is not possible to distinguish E. histolytica from E. dispar morphologically (photograph courtesy of Centers for Disease Control)

Fig. 9.1 E. histolytica trophozoite and cyst. (A) The motile and invasive trophozoite forms each contain a single nucleus and are 10 and 60 ^m in diameter, respectively. (B) The infectious cyst form is 10-15 ^m in diameter and contains four nuclei. Note that it is not possible to distinguish E. histolytica from E. dispar morphologically (photograph courtesy of Centers for Disease Control)

Since E. dispar infection need not be treated, an important clinical advance has been the development of antigen detection tests that differentiate infection by the two amebae (see Diagnosis).

Life-cycle

The E. histolytica life-cycle is relatively simple and consists of an infective cyst and an invasive trophozoite form. The trophozoite stage is 10-60 ^ in diameter, containing a single nucleus with a central karyosome (Figure 9.1A). The cyst is 10-15 ^ in diameter and contains four or fewer nuclei (Figure 9.1B). The quadrinucleate cyst is the infectious form of the parasite, is resistant to chlorination, gastric acidity and desiccation, and can survive in a moist environment for several weeks. Infection with E. histolytica occurs due to fecal-oral spread with fecally contaminated food or water. Excystation of the cyst occurs in the intestine where the cyst undergoes nuclear and cytoplasmic division to form eight trophozoites. The trophozoites can then colonize and/or invade the large bowel. Cysts are never found within invaded tissues. Invasion of the colonic intestinal epithelium by trophozoites leads to the formation of the classically described flask-shaped ulcers. Hepatic abscesses are thought to occur due to migration of the parasite via the portal vein. All infections with E. histolytica are not alike, and whether infection results in colonization or invasion may be influenced by the E. histolytica strain and its interaction with bacterial flora, host genetic susceptibility and factors such as malnutrition, sex, age and immunocompetence.

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