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Laboratory Diagnosis

Identification of Entamoeba histolytica is essential to the laboratory diagnosis of amebiasis. Although this process may be as simple as a microscopic examination of a single stool specimen, difficulties often occur. The standard ova and parasite investigation, with three stool specimens, should be performed as a routine on all outpatients using direct and permanent stained smears in addition to concentration methods. The permanent stained smear remains the most valuable method in the detection and identification of amebae. Iron hematoxylin or trichromic acid stains yield characteristic cytoplasmic and nuclear features necessary in differentiating pathogenic from non pathogenic organisms (see Fig.1.2). Ninety percent of infected individuals can be confirmed with this technique. It is recommended that barium, laxatives, antibiotics and soap enemas be avoided in order not to interfere with the test. Detection of amebae also depends on several factors ranging from appropriate collection and preservation of multiple fresh stool specimens (minimum of three over a 10 day interval), and concentration and staining methods, to training and experience of the microscopist. It is possible to cultivate trophozoites from fresh material and to hatch and cultivate trophozoite cysts in stools that are several days old.

Problems encountered at any step in the process may lead to false-negative reports. Likewise, inexperience in morphological recognition can lead to false-positive diagnoses with other nonpathogenic protozoa being mistaken for E. histolytica. In patients with diarrhea or dysentery, trophozoite identification should be emphasized whereas, with formed stool specimens, cysts will be more frequent and concentration methods become useful. The "steaming stool" examination or fresh biopsy material from a patient may provide a rapid inexpensive diagnosis. A warmed microscope stage and saline is ideal. The mobile trophozoites of E. histolytica can be diagnosed by two characteristic features: (1) presence of ingested red blood corpuscles (harmless amebae rarely contain red blood cells), and (2) rapidly protruding and slender pseudopodia (see Fig.1.2). Amebic cysts can survive in contact with a little moisture for many years, but the vegetative forms of E. histolytica, which have pseudopodia, are not viable outside the body.During the life cycle of this parasite in man, the trophozoite or "feeding stage" multiplies by simple binary fission in the large intestine. Eventually it rounds up and develops a wall protecting the infective form or cyst which is capable of surviving adverse environments in the host (or externally when excreted with feces). A dysenteric stool specimen with large numbers of white blood cells should alert the clinician to a superimposed bacterial colitis.

Sigmoidoscopy or colonoscopy biopsy specimens may also be helpful when several samples are obtained (five or more). The most rewarding specimens are obtained from the margins of small ulcers, but amebae in the process of migration may occasionally be found in the edematous areas between ulcers. If no ulcers are visible, biopsy should be performed where there is marked edema; even apparently normal mucosa may harbor the parasite. Despite repeated careful biopsies, there will be some patients from whom it is extremely difficult to obtain amebae, because they have few parasites. Rare false-negative biopsies can occur, even when the clinician and pathologist are experienced. Histological diagnosis is accomplished by identifying trophozoites in stained tissue sections (see Figs. 1.3, 1.4, 1.5). Hematoxylin and eosin staining demonstrates important cytological features within amebae and most often allow direct diagnosis. Periodic acid - Schiff staining is useful in locating organisms in multiple tissue sections. Genital and cutaneous lesions are examined similarly by smear and biopsy.

Amebic serology is useful where invasive or extraintestinal disease is suspected clinically. Over 60% of these patients with fever and right upper quadrant pain have no enteric symptoms and no history of dysentery. Stool examination for cysts and trophozoites will be positive in only a few of these individuals. Liver disease in particular can be confounding in situations where imaging is not available. Standard liver-function tests are of no value. Aspiration of amebic liver cysts is only likely to be needed for diagnosis if serology is not available and is instead performed occasionally for pain relief. Diagnostic aspiration often yields the characteristic "anchovy paste" fluid, which becomes thicker towards the end of drainage. This last thicker portion presumably contains debris from the wall of the cyst and with it live trophozoites. It is this smaller fraction that should be most carefully examined.

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