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Untitled Document

Presentation of Quiz #6

A patient is a 24 year-old male from the northwest part of the United States who has just returned from a hiking trip throughout Oregon, Washington, and California. On his return, he felt fine, but tired. About a week after he returned, he began complaining of diarrhea, a slight fever, some abdominal pain, and general malaise. The examination of stool using routine Ova and Parasite examinations (3 specimens) revealed nothing significant. He began to feel better, but again complained of diarrhea the following week. He also complained of multiple bouts of indigestion and antacids didn't seem to help eliminate the problem. During a discussion of his trip, he indicated he carried a water filter, but didn't always use it for his drinking water. He then submitted stools for examination and stool immunoassay procedures were performed. The following routine trichrome stain results are seen in images #1 and #2. Stool immunoassay results can be seen in images #3 and #4. Please comment on the possible diagnosis.

1. Trichrome Stain

2. Trichrome Stain

3. FA Immunoassay

4. Immunochromatographic Immunoassay (cartridge format)

Note: Giardia cysts and Cryptosporidium oocysts are seen in Image #3.

The Immunochromatographic immunoassay cartridge is positive for both Giardia and Cryptosporidium.

NOTE:

IT IS NOT RECOMMENDED THAT YOU PERFORM MULTIPLE IMMUNOASSAY METHODS; TWO METHODS ARE SHOWN ABOVE JUST TO SHOW YOU WHAT THE RESULTS LOOK LIKE.  HOWEVER, WHEN TESTING FOR GIARDIA, IF THE FIRST STOOL IS NEGATIVE (USING ANY OF THE IAs), REQUEST A SECOND STOOL FOR TESTING.  THIS IS NOT NECESSARY IN A SUSPECT CASE OF CRYPTOSPORIDIOSIS.

ALSO, REMEMBER THAT IT IS NOT APPROPRIATE TO USE THE O&P EXAMINATION TO TRY AND CONFIRM IMMUNOASSAY RESULTS. THE IMMUNOASSAY METHODS ARE MORE SENSITIVE THAN THE O&P METHODS.

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Answer and Discussion of Quiz #6

The images presented in Diagnostic Quiz #6 are the following:

  1. Stool stained with Trichrome stain; note the image contains no parasites.
  2. Stool stained with Trichrome stain; note the image contains no parasites.
  3. Combination Giardia/Cryptosporidium FA immunoassay; note that both a Giardia lamblia cyst and Cryptosporidium spp. oocysts are visible in the field.
  4. Combination Giardia/Cryptosporidium immunochromatographic immunoassay; note that both the Giardia and Cryptosporidium lines are positive, as is the positive control line.

Comments on the patient:

This case is somewhat unusual in that the patient acquired infections with both Giardia and Cryptosporidium. With a history of hiking and water consumption of stream and/or river water, it is very likely that both infections were acquired by drinking contaminated water. The patient was treated for giardiasis and recovered with no problems; as an immunocompetent patient, the Cryptosporidium infection resolved via the patient's own immune system. With this type of history, the most appropriate test(s) to order should have been a Giardia/Cryptosporidium immunoassay, rather than the O&P examinations.

Comments on Giardia lamblia:

The incubation time for giardiasis ranges from approximately 12-20 days. Because the acute stage usually lasts only a few days, giardiasis may not be recognized as the cause, but may mimic acute viral enteritis, bacillary dysentery, bacterial or other food poisonings, acute intestinal amebiasis or "traveler's diarrhea" (toxigenic E. coli). However, the type of diarrhea plus the lack of blood, mucus and cellular exudate is consistent with giardiasis.

The acute phase is often followed by a subacute or chronic phase. Symptoms in these patients include recurrent, brief episodes of loose, foul stools; there may be increased distention and foul flatus. Between mushy stools, the patient may have normal stools or may be constipated. Abdominal discomfort continues to include marked distention and belching with a rotten egg taste. Chronic disease must be differentiated from amebiasis and other intestinal parasites such as Dientamoeba fragilis, Cryptosporidium spp., Cyclospora cayetanensis, Isospora belli, Strongyloides stercoralis and from inflammatory bowel disease and irritable colon. Based on symptoms such as upper intestinal discomfort, heartburn and belching, giardiasis must also be differentiated from duodenal ulcer, hiatal hernia, and gallbladder and pancreatic disease.

Routine stool examinations are normally recommended for the recovery and identification of intestinal protozoa. However, in the case of G. lamblia, because the organisms are attached so securely to the mucosa by means of the sucking disc, a series of even five or six stools may be examined without recovering the organisms. Many of the newer immunoassay methods are being recommended for patients suspected of having giardiasis or for those who may be involved in an outbreak situation.

Trichrome Stain, Stool (Trophozoite)

Trichrome Stain, Mucus from Duodenal Aspirate (Trophozoites)

Comments on Cryptosporidium spp.:

In humans, age and immune status at the time of primary exposure to Cryptosporidium do not appear to be primary factors influencing susceptibility to infection. Symptomatic intestinal and respiratory cryptosporidiosis has been seen in both immunocompetent and immunodeficient patients of all ages. However, once the primary infection has been established, the immune status of the host plays a very important role in determining the length and severity of the illness. People who are immunocompetent usually develop a short-term, self-limited diarrhea lasting approximately 2 weeks.

Clinical symptoms include nausea, low-grade fever, abdominal cramps, anorexia, and 5 to 10 watery, frothy bowel movements per day, which may be followed by constipation. Some patients may present with diarrhea as described above, and others may have relatively few symptoms, particularly later in the course of the infection. In patients with the typical watery diarrhea, the stool specimen contains very little fecal material, mainly water and mucous flecks. Often the organisms are entrapped in the mucus, and diagnostic procedures are performed accordingly. Generally a patient with a normal immune system will have a self-limited infection; however, patients who are compromised may have a chronic infection with a wide range of symptoms (asymptomatic to severe).

Either fresh or preserved specimens can be examined using the routine stool formalin-ethyl acetate concentration and one of the modified acid-fast stains or the newer stool immunoassays. Fluorescence (FA), enzyme-linked immunosorbent assay (ELISA), and immunochromatographic methods are currently available. It is recommended that the stool specimen be centrifuged at 500Xg for 10 minutes prior to performance of the FA (Image #3) or modified acid-fast stains (visual identification of the organism itself); centrifugation at this speed and time ensures maximum recovery of the oocysts. However, centrifugation is not required for performance of the ELISA or immunochromatographic cartridge methods (Image #4) (detection of antigen).

The more normal the stool (semiformed, formed), the fewer the organisms and the more artifact material will be present. The normal permanent stains (trichrome, iron hematoxylin) do not adequately stain Cryptosporidium spp., although occasionally oocysts may be seen in these preparations, especially in heavy infections. Although the organisms can be stained with auramine-rhodamine stains, the identification should be confirmed by using acid-fast stains or the immunoassay methods (most sensitive). This is particularly true if the stool contains other cells or a lot of artifact material (more normal stool consistency).

Studies have shown that calves, and perhaps other animals, serve as potential sources of human infections. Contact with these animals may be an unrecognized cause of gastroenteritis in humans, both in a rural and in an urban setting. Direct person-to-person transmission is also likely and may occur through direct or indirect contact with stool material. Direct transmission may occur during sexual practices involving oral-anal contact. Indirect transmission may occur through exposure to positive specimens in a laboratory setting or from contaminated surfaces or food or water.

The epidemiologic considerations for cryptosporidiosis emphasize transmission by environmentally resistant oocysts, numerous potential reservoir hosts for zoonotic transmission, documentation of person-to-person transmission in day-care centers, nosocomial transmission within the health care setting, occurrence of asymptomatic infections (infective, carrier state), widespread environmental distribution resulting in the probability of waterborne transmission, and the link between cryptosporidiosis and severe, life-threatening disease in individuals with impaired immune function.

Modified Acid-Fast Stain (Cryptosporidium oocysts)

Routine Trichrome Stain (Cryptosporidium oocysts)

Note:

Cryptosporidium oocysts do not stain using routine Trichrome or Iron-hematoxylin stains. Also remember that the decolorizer used in the modified acid-fast stain should be no more than a 1% sulfuric acid. The use of a stronger decolorizer will tend to decolorize Cyclospora cayetanensis (very acid-fast variable). The use of the 1% decolorizer solution is recommended for all modified acid-fast stains for the coccidia (Cryptosporidium spp, Cyclospora cayetanensis, and Isospora (Cystoisospora) belli).

Additional Reading:

  1. Garcia, LS, 2016. Diagnostic Medical Parasitology, 6th Ed., ASM Press, Washington, DC.
  2. Garcia, LS, 2009. Practical Guide to Diagnostic Parasitology, 2nd Ed., ASM Press, Washington, DC
  3. NCCLS, 1997, Procedures for the recovery and identification of parasites from the intestinal tract, Approved Guideline, M28-A, National Committee for Clinical Laboratory Standards, Villanova, PA.