Intestinal Protozoal Diseases Workup

For all protozoa-related gastroenteritis, direct observation of the parasite from stools is the confirmatory diagnostic method. Morphologic characteristics and laboratory techniques for observation of the protozoa are listed in Table 2.

Table 2. Laboratory Procedures for Identification of Intestinal Protozoa

Table. (Open Table in a new window)

See the list below:

• Amebiasis

  • Amebic colitis

    • The diagnosis of amebic infection requires examination of stools passed on 3 separate occasions. These tests include a wet preparation (must be performed within 30 min of collection) to identify motile trophozoites and a formalin-ethyl acetate concentration step to identify amebic cysts and trophozoites.

    • E histolytica is indistinguishable from the noninvasive and more prevalent E dispar, with the exception that E histolytica trophozoites may contain ingested RBCs. An ELISA test for E histolytica antigen in stools can also be used and will soon be commercially available. E histolytica can be cultured for research and susceptibility purposes (especially for emetine susceptibility) but does not contribute to diagnostic purposes. See the image below.

      This micrograph stained with chlorazol black, revealed an Entamoeba histolytica cyst.

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    • As mentioned before, techniques using both ELISA and polymerase chain reaction (PCR) have been tested to distinguish between E histolytica from E dispar (nonpathogenic); however, these tests have been used only for epidemiological purposes.

  • Amebic liver abscess: For amebic liver abscess and extraintestinal illness, serologic tests are the diagnostic hallmark. An indirect hemagglutination assay titer higher than 128 or ELISA titer higher than 40 U have more than 99% and more than 95% specificity, respectively. Abscess aspiration is no longer considered useful as a diagnostic tool because it is an invasive procedure. Usually, trophozoites are attached to the abscess wall and cannot be observed in the abscess fluid, and serologic tests are extremely sensitive and specific.

• Giardiasis

  • Use of polyclonal antisera or monoclonal antibodies against Giardia -specific antigens is improving diagnostic testing. ELISA has been used for G lamblia antigen in stool, with sensitivities of 92-98% and specificities of 87-100%. This is currently the laboratory procedure most commonly used in clinical settings.

  • No single method appears to detect every infection with G lamblia. In general, specimens should be examined within 1 hour after being collected or should be preserved in polyvinyl alcohol or 10% formalin. The following tests are generally available:

    • Fecal examination (for both trophozoites and cysts) - Direct, wet preparation in saline with/without iodine; trichrome stained slides; formalin-ethyl-acetate concentration method; ELISA

    • When giardiasis is suspected and 5 stool specimens are negative, duodenal or upper jejunum aspiration should be performed. This can be accomplished by duodenal drainage by intubation, mucus collected by the Entero-test capsule (a weighted length of nylon yarn is introduced into the duodenum in a gelatin capsule), or by endoscopy.

  • Appropriately conducted direct examination of stools establishes the diagnosis in up to 70-85% of cases after 2 examinations.

  • The main reason for failure to identify G lamblia is the use of medications such as antibiotics, antacids, and other medications that can distort protozoal morphology. Radiographic examinations with contrast material may also distort morphology. Intermittent excretion of the parasite, improper specimen collection, and inadequate training of the observer also contribute to the difficulty in identifying the organism. See the image below.

    This is a scanning electron micrograph (SEM) of an in vitro Giardia lamblia culture. This photograph contains both trophozoites and a cluster of maturing cysts (bottom right). At far left, the 2 trophozoite-staged organisms are positionally situated opposite to one another, with the farthest left G lamblia displaying its dorsal, or upper surface, and the protozoan to its immediate right, its ventral, or bottom surface.

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• Spore-forming protozoa

  • Specific diagnosis of these parasites depends on stool examination. Table 2 shows the stains recommended for adequate visualization of spores or oocysts in stool samples.

  • The modified acid-fast stain can be routinely used to visualize the oocysts of cryptosporidium, Cyclospora, and Isospora in stool or duodenal aspirate.

  • Differentiating these 3 organisms requires expertise. The sensitivity of the acid-fast stain is low, with approximately 30% sensitivity for cryptosporidiosis after 1 sample, but the diagnostic yield increases by examining multiple specimens. Enhanced sensitivity can be obtained by concentrating oocysts with various techniques, such as the Sheafer method or, for cryptosporidium, by using the monoclonal antibody-based immunofluorescent stain. See the images below.

    This photomicrograph revealed the morphologic details of Cryptosporidium parvum oocysts.

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    This is an illustration of the life cycle of Isospora belli, the causal agent of isosporiasis.

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    This photomicrograph of a fresh stool sample, which had been prepared using a 10% formalin solution, and stained with safranin, revealed the presence of 3 uniformly stained Cyclospora cayetanensis oocysts in the field of view.

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  • An ELISA test for cryptosporidium also is available.

  • The first step in diagnosing intestinal microsporidia is to examine the stool specimen using the modified trichrome stain. The spores are usually very small and can be misinterpreted as fecal debris. An available nonspecific fluorescence method may enhance speed and sensitivity.

  • Small intestine biopsy with visualization by electron microscopy is the only method that differentiates between E bieneusi and S intestinalis. For microsporidiosis, a PCR technique has been successfully used to identify a spore-shedding pattern of E bieneusi in asymptomatic children; however, this test is not available for clinical use.

• Dientamoebiasis

  • Examination of the stools by properly trained personnel is essential.

  • Permanent stained slides from freshly passed feces or from specimens preserved in polyvinyl alcohol are essential for the diagnosis because this parasite does not have a cystic phase.

  • Examination of 3 specimens has a diagnostic yield of 70-85%, but the diagnostic yield increases to 90-100% after 10 specimen collections.

• Balantidiasis

  • B coli is shed irregularly, and repeated examinations of stools are necessary for identification.

  • As in amebiasis, a wet preparation using saline can be useful for the identification of trophozoites if the examination is performed immediately after stool sample collection.

  • Concentration techniques are useful for identification, but the parasite does not stain well on permanently stained smears.

• Blastocystosis: This organism remains intact after concentration of stool for examination by wet mount or by using trichrome or iron hematoxylin permanent-stained smears.

In general, imaging studies do not contribute to the etiologic diagnosis of intestinal protozoal infections, but these studies may be helpful in some situations:

• Amebic colitis: In cases of necrotizing colitis, plain abdominal radiography can reveal air-fluid levels in the intestine, radiologic signs of mucosal edema, and/or evidence of perforation.
• Ameboma: It typically appears as single or multiple apple core lesions on barium enema findings.
• Amebic liver abscess: Chest radiography reveals an elevation of the right hemidiaphragm, with or without atelectasis and pleural effusion, in 70-80% of cases. In complicated cases, patients may have lung infiltrates, pericardial effusions, or both. Ultrasonography, CT scanning, or MRI are equally sensitive in revealing liver abscesses, but ultrasonography is the imaging study of choice because of a much lower cost. Amebic liver abscesses disappeared on ultrasonographic findings in one to two thirds of cases following 6 months of therapy.

For the other intestinal protozoa, imaging studies are not usually necessary for diagnosis. For some parasites, such as G lamblia, contrast-based studies are contraindicated because of potential morphologic disruption for microscopic analysis.

In cases of severe balantidiasis, plain abdominal radiography can be useful to rule out the possibility of other causes of acute and severe abdominal pain. In some cases of giardiasis, edema and segmentation of the small intestine can be observed.

For some parasites, axenic cultures can be obtained for research purposes. They are not needed for diagnosis.

Serologic tests are used only for epidemiological surveys, with the exception of invasive amebiasis.

For most spore-forming protozoals, molecular assays, such as reverse transcriptase (RT)-PCR, have not shown a significant superiority when compared with staining methods as standard diagnostic tools.

Endoscopic evaluation is useful in patients with chronic and/or severe giardiasis and spore-forming parasitosis.

Macroscopic inspection and histologic evaluation are necessary for diagnosis.

Colonoscopy is useful for the diagnosis of chronic colitis due to either E histolytica or B coli. It is also helpful for the diagnosis of an ameboma. The colonic mucosa usually appears hemorrhagic, with discrete shallow-based ulcers that have raised edges.

Endoscopic studies are also useful to differentiate these diseases from other pathologic conditions, such as pseudomembranous colitis, Crohn disease, and many others.

Histologic evaluation is not indicated routinely. Usually, biopsy specimens are collected only when the parasite has not been observed by routine laboratory techniques or when the presence of other pathologic conditions needs to be ruled out.

• Amebiasis: Biopsy specimens from the margin of colonic ulcerations are the most rewarding. Special stains, such as periodic acid-Schiff, are used to visualize E histolytica in the biopsy specimen.

• Giardiasis

  • Duodenal biopsy represents the optimal method for diagnosing giardiasis and should be considered in patients with characteristic clinical symptoms, negative stool and duodenal specimens, and one of the following: abnormal radiographic findings (eg, edema and segmentation of the small intestine), abnormal lactose tolerance test, absent secretory IgA, hypogammaglobulinemia, or achlorhydria.

  • When a small-bowel biopsy is obtained, a touch preparation stained with Giemsa should be performed in addition to routine histology. The pathology can be assessed to detect spruelike lesions that may occur with G lamblia or with other conditions, such as gluten-sensitive enteropathy. Searching for trophozoites is necessary, mainly because they may be difficult to recognize. Commonly, a disruption of the brush border of the epithelium is observed in association with giardiasis.

• Spore-forming protozoa

  • Both cryptosporidia and Isospora are easily observed in intestinal biopsy specimens with routine light microscopy, but many patients whose stools test positive for these organisms may have duodenal biopsy specimens that test negative.

  • Cyclospora have not been observed with light microscopy in duodenal biopsy specimens, but, in one study, electron microscopy showed the organisms. Small-bowel biopsy may be more sensitive than stool examination for the diagnosis of intestinal microsporidiosis. Hematoxylin and eosin stains are adequate in most cases, but other stains, such as Warthin-Starry, Giemsa, toluidine blue, and Gram, can be used.

  • The histologic spectra of spore-forming protozoal infection can vary from normal to villus shortening or flattening, crypt hyperplasia, and increased number of leukocytes in the lamina propria and epithelium. Invasion and ulceration do not occur, with the exception of the microsporidia S intestinalis. Microsporidia typing is done by the morphologic appearance on electronic microscopy of biopsy specimens.

• Other protozoa: Indications for intestinal biopsy or pathognomonic pathologic lesions are not described for dientamoebiasis and blastocystosis. In balantidiasis with chronic colitis, colonoscopy with biopsy can reveal similar histologic findings as in amebiasis, and identification of the parasite is diagnostic.