PRESENTATION OF DIAGNOSTIC QUIZ #4 (April, 2001)

A patient is a 48 year-old male from Japan who has traveled throughout the world as a professional photographer (Europe, Asia, Central and South America, Australia, New Zealand, etc). He was diagnosed as having diarrhea, cough, and general malaise. He had a history of vague health problems over the past few years, but was never sick enough to see a physician. Three stool specimens were submitted to the laboratory. After examination of the concentration sediment and permanent stained smears from all three specimens, the following objects were seen. Please comment on the identification of the structures seen.

1. 60 x 40 µm

2. 30 x 15 µm

3. 140 x 70 µm

4. 90 x 50 µm

5. 360 x 20 µm

6. 18 µm

 

 

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ANSWER AND DISCUSSION OF DIAGNOSTIC QUIZ #4 (April, 2001)

The images presented in Diagnostic Quiz #4 are the following:

1. Hookworm egg
2. Isospora belli oocyst
3. Fasciolopsis buski or Fasciola hepatica egg
4. Paragonimus spp. egg
5. Artifact: root hair
6. Entamoeba coli cyst

Comments on the patient:

The patient may have experienced vague abdominal symptoms from several of the parasites found, including Isospora, hookworm or Fasciolopsis buski/Fasciola hepatica. The cough is probably related to the Paragonimus spp. infection, often causing symptoms on a random basis (particularly when egg production leads to cough and the production of sputum containing the egg packets). When Paragonimus spp. eggs are found in the stool, the sputum can also be checked; however, specimens may be negative unless the patient is symptomatic at the time. Isospora belli infections are not that common and are usually associated with symptoms in the immunocompromised patient; in this case, it may be an incidental finding. Symptoms with either F. buski or F. hepatica are definitely related to parasite burden; a light infection will generally cause no symptoms. Entamoeba coli is a nonpathogen and would merely be an indication that the patient had ingested something contaminated with fecal material.

Hookworm:
1. Eggs are normally passed in the stool in the unembryonated state (usually about an 8 to 32 ball cell stage of development). There is typically a thin shell and clear space between the developing embryo and the shell.
2. If the stool remains unpreserved for over 24 h, the eggs may continue to develop and the larvae may hatch. These larvae must be differentiated from those of S. stercoralis, since therapies for the two infections are different.
3. The eggs will normally be distorted on the permanent stained smear, and morphology is more easily seen in wet preparations.

Isospora belli:
1. The oocysts are more easily recovered and identified when wet preparations are examined (direct smear, concentration smear).
2. Oocysts recovered in a concentrate sediment from PVA-preserved stool may be very difficult to detect (oocyst wall is very difficult to see).
3. A biopsy can be positive while no organisms are seen in the stool. This is not necessarily due to poor?quality laboratory work but may reflect normal findings.
4. Although these organisms can be stained with auramine-rhodamine stains, they should be confirmed by wet smears or acid-fast stains.

Fasciolopsis buski:
1. The eggs of the intestinal trematodes are found in the stool. Infrequently, adult worms may also be detected.
2. Eggs of F. buski, Echinostoma ilocanum, and F. hepatica are similar in size and shape and cannot be easily differentiated. When shed, these eggs contain an undifferentiated embryo.
3. The formalin-ether sedimentation technique is the recommended method for concentrating the eggs from stool.

Fasciola hepatica:
1. Multiple stool specimens may be required in order to find the trematodes eggs; in a very light infection, eggs may not be recovered; however, the patient might be asymptomatic.
2. The sedimentation concentration method should be used; because the eggs are operculated, they will not float using the zinc sulfate flotation concentration method.
3. When adding iodine to the wet preparation, don't add too much or the eggs will stain very darkly and will resemble debris.
4. The wet preparation can be examined using the 10X (low power) objective, these eggs are large enough that they can usually be seen using this magnification.
5. Eggs of F. hepatica can resemble those Fasciola gigantica (liver fluke) and Fasciolopsis buski (intestinal fluke). 6. Do not make the wet preparation too thick, if so, the eggs can be obscured by normal stool debris.
7. If available, antigen or antibody detection may be helpful in confirming the infection

Paragonimus spp.:
1. Paragonimus spp. eggs may be confused with Diphyllobothrium latum eggs because of similarities in size and shape. However, Paragonimus eggs have opercular shoulders and a marked thickening at the abopercular end, features not found in D. latum eggs.
2. The sedimentation concentration method should be used; because the eggs are operculated, they will not float using the zinc sulfate flotation concentration method.
3. When adding iodine to the wet preparation, don't add too much or the eggs will stain very darkly and will resemble debris.
4. The wet preparation can be examined using the 10X (low power) objective; these eggs are large enough that they can usually be seen using this magnification.
5. Do not make the wet preparation too thick, if so, the eggs can be obscured by normal stool debris.
6. When looking at sputum specimens for eggs, carefully examine any blood-tinged flecks for eggs; the egg clusters have been described as looking like "iron filings."
7. In light infections, multiple stool and sputum specimens may be necessary to find the eggs.

Artifact - Root hair:
Plant or root hairs, such as the fuzz on peaches, may resemble nematode larvae. The root hairs tend to be clear and refractile while the larvae pick up stain (iodine), which will reveal internal structures. It is important to recognize this potential error when examining formalin-fixed specimens submitted as proficiency testing specimens. In cases of diarrhea, partially digested plant material, examples being bean sprouts or other vegetable material, can mimic adult nematodes or tapeworm proglottids. Also, all stages of free-living nematodes can occur in feces or as contaminants of water used in making fecal suspensions.

Entamoeba coli:
With the exception of the mature cyst, the morphologies of E. histolytica and E. coli are similar in most of the stages. Therefore, it is very important to examine permanent stained smears, even if a tentative identification has been made from a wet preparation examination. Specific treatment is not recommended for this nonpathogen. Consequently, the correct differentiation between the two species is critical to good patient care. Since the two species are acquired in the same way, it is also important to remember that both can be found in the same patient. If few E. histolytica are present among many E. coli organisms, additional searching may be necessary to correctly identify both. Remember: Entamoeba histolytica can be diagnosed using morphologic criteria ONLY if RBCs are seen within the cytoplasm of the trophozoite. Otherwise, the organisms should be identified as E. histolytica/E. dispar.

Review (Consistent with CAP Inspection Checklist):
Every stool submitted for an O&P examination must be examined using the concentration and permanent stained smear procedures.
O&P Exam (Fresh Stool Specimen/liquid and/or very soft): Direct wet smear, concentration, permanent stained smear.
O&P Exam (Fresh Stool Specimen/formed stool): Concentration, permanent stained smear. O&P Exam (Preserved Stool Specimen): Concentration, permanent stained smear. Remember that all intestinal protozoan infections can be missed if the concentration ONLY is performed. The permanent stained smear is much more sensitive than the concentration alone.
To date, there are no commercial immunoassay products available for the confirmation of infection with D. fragilis.

Additional Reading:

Garcia, LS, 2001. Diagnostic Medical Parasitology, 4th Ed., ASM Press, Washington, DC.

Garcia, LS, 1999. Practical Guide to Diagnostic Parasitology, ASM Press, Washington, DC

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.