PRESENTATION OF DIAGNOSTIC QUIZ #55 (July, 2005)

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A 52 year old male, originally from San Francisco, presented with a three week history of diarrhea and general malaise. This patient was a geologist consultant, and he traveled throughout the world on a regular basis. These trips included Europe, Latin America, Asia, Africa, and throughout North America. He often took side trips on his travels and liked to eat many of the local dishes, since he was an excellent cook. Apparently, he had no underlying health problems. On presentation, he was complaining of intermittent diarrhea, a weight loss of approximately 10 pounds, and general malaise. He had just returned from a trip to Brazil.

Routine stool examinations for parasites (three ova and parasite examinations) revealed the following:

Orange image (x1,000), Clear image(x 400), Blue/purple image (x1,000), Green/pink image (x1,000)

 

 

QUESTIONS:

1. What organisms are suggested from the images shown above?

2. What groups of parasites might be involved, and are these typical based on his travel history?

3. What else do these objects suggest?

4. How likely is it that the patient has a mixed infection (based on the structures seen above? Why or why not?

5. What other tests might be requested before all intestinal parasites could be eliminated as possibilities?

(Scroll Down for Answers and Discussion)

 

 

 

 

 

 

 

 

 

 

ANSWER AND DISCUSSION OF DIAGNOSTIC QUIZ #55 (July, 2005)

The images presented in Diagnostic Quiz #55 are the following:

ANSWERS TO QUESTIONS (View images from left to right):

1. The orange structure on the left, photographed using the 100x oil immersion objective (x1,000), resembles an Entamoeba coli cyst. However, the "nuclei" are not the same size and the morphology is not uniform enough to be a protozoan cyst. The clear structures in the next image to the right were photographed using the 40x high dry objective (x400) and resemble helminth eggs. However, they are not uniform in size or shape. The structures in the next image were photographed using the 100x oil immersion objective (x1,000) and resemble protozoan trophozoites and cysts. The last image on the right was photographed using the 100x oil immersion objective (x1,000) and resembles Entamoeba histolytica containing red blood cells (RBCs).

2. The structures in the photographs resemble intestinal protozoa and intestinal helminth eggs. Based on the patient's travel history, any or all of these parasites are possibilities.

3. The images also suggest various artifacts and/or human cells that might be present in human fecal specimens.

4. Based on the images you have seen, it is unlikely that the patient has a mixed infection with multiple parasites. All of the structures are artifacts. However, with the presence of artifacts in the specimen, examination of the stained fecal smears should be very thorough in order to "rule out" the possibility of true parasites.

5. Because routine stool examinations do not "rule out" all the coccidia and/or the microsporidia, additional testing should be performed. Those tests would include fecal immunoassays for Cryptosporidium/Giardia or Cryptosporidium, and special staining for Cyclospora cayetanensis (modified acid-fast stains) and the microsporidia (modified trichrome stains.

COMMENTS ON THE PATIENT:

This case represents a situation in which the cause of the patient's symptoms may be something other than an infection with intestinal parasites. Another possibility is that the routine Ova and Parasite Examination (O&P) did not allow detection and identification of the particular parasite involved. Generally, the coccidia and/or microsporidia require different testing prior to identification (modified acid-fast stains, modified trichrome stains).

COMMENTS ON THE METHOD LIMITATIONS:

It is always recommended that a thorough examination be performed (minimum of two specimens for O&P examinations - three is even better) before reporting the patient as "No Parasites Seen." Also, remember that the routine O&P examination does not allow the detection of organisms like Cryptosporidium spp., Cyclospora cayetanensis, or the microsporidia. A statement listing the method limitations should be included in the report to the physician. In spite of these method limitations with some of the coccidia, the coccidian, Isospora belli, can usually be identified in the concentration sediment wet smear examination.

COMMENTS ON THE IMAGES:

Wheatley's trichrome stain (permanent stained smear) (x1,000)

Although this structure is the same size as an E. coli cyst, the "nuclei" are quite different sizes, a finding that makes the structure unlikely to be a protozoan cyst. Also, E. coli cysts do not tend to stain well on the permanent stained smear (fixative does not penetrate the cyst wall very well, even using mercury-based fixatives), but their color tends to be more blue or purple (normal cyst); if the cyst is very shrunk, it may even appear pink to red to purple in color (Wheatley's trichrome stain), rather than being this color orange.

Saline wet mount (x400)

Although these structures (wet mount) tend to resemble helminth eggs, notice that the sizes are somewhat different and the overall shape/size are not consistent. Also, it is unusual to find groups of eggs clustered together. These structures are probably plant cells of some type.

Wheatley's trichrome stain (permanent stained smear) (x1,000)

Although these structures (permanent stained smear) tend to resemble protozoan trophozoites and cysts, notice that the cell nuclei tend to take up a very large part of the cell; this large ratio of nuclear material to cytoplasm (about 1:2 to 1:4) is much more typical of WBCs, not intestinal protozoa. The cell in the center and upper left of the image are probably macrophages with a single nucleus; these can tend to mimic Entamoeba histolytica/E. dispar trophozoites. However, actual protozoan trophozoites tend to have a ratio of nucleus to cytoplasm of about 1:8 to 1:12. The cells in the lower right section of the image are PMNs; they can mimic cysts of Entamoeba histolytica/E. dispar. The lobed nucleus of the PMN tends to look normal (as it would on a routine hematology smear) if the cells are fresh in the stool; however, if the PMNs have been in the gut for a while, the nuclei tend to come apart and the resulting structure can mimic a protozoan cyst. Another example of WBCs in a fecal specimen can be seen below.

Note: the pink cell is an eosinophil. There are a number of PMNs in this field; note the variations in the cell nuclei (also the nucleus/cytoplasm ratio). While PMNs tend to measure about 14 microns on a routine stained hematology blood film, the ones in stool tend to be a bit smaller and measure from 10 to 12 microns. So, although the staining characteristics are identical to intestinal protozoa, the morphology of the cells will differ considerably.

Wheatley's trichrome stain (permanent stained smear) (x1,000)

Although this structure (permanent stained smear) tends to resemble a trophozoite of Entamoeba histolytica containing RBCs, it is an artifact. There is no nucleus visible and this structure appears to be a cluster of objects. This type of artifact can be very confusing; however, if it is a true organism, the nucleus should be visible.

Here are some examples of Entamoeba histolytica/E. dispar trophozoites and cysts, as well as some examples of E. coli cysts.

The image on the left is an E. histolytica/E. dispar trophozoite (note the ratio of the nucleus to cytoplasm is about 1:9 and the next two images are E. histolytica/E. disparcysts (note the nuclei are about the same size). Note: the magnification for these organisms is somewhat larger than the images of the WBCs above.

The image on the left is an E. coli cyst (Wheatley's trichrome stain - permanent stained smear); note the nuclei are about the same size. The image in the middle is also an E. coli cyst (shrunk, Wheatley's trichrome stain). The image on the right is an E. coli cyst on an iodine wet mount; again, note the consistency of the nuclear size. Depending on the amount of iodine added to the wet mount, the cyst can appear to be yellow to gold to orange to almost reddish brown.

The image above is an E. histolytica trophozoite (Wheatley's trichrome stain - permanent stained smear); note the nucleus is visible (central karyosome with evenly arranged nuclear chromatin). The cytoplasm contains ingested RBCs; this finding confirms the presence of the true pathogen, E. histolytica.

 

REFERENCES

Garcia, L.S. 2001. Diagnostic Medical Parasitology, 4th Ed., ASM Press, Washington, D.C.

Leber, A.L. and S.M. Novak. 2003. Intestinal and Urogenital Amebae, Flagellates, and Ciliates In: Murray, P.R., E.J. Baron, J.H. Jorgensen, M.A. Pfaller, and R.H. Yolken (eds). Manual of Clinical Microbiology, 8th ed, vol 2, ASM Press, Washington, D.C.