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Presentation of Quiz #39

A 24 year-old female complains of diarrhea for several days (off and on) and 2 stool specimens are submitted to the laboratory for an Ova and Parasite (O&P) examination. After examination of the concentration sediment from both specimens, nothing was seen and the results were reported as NO PARASITES SEEN. No permanent stained smears were prepared and/or examined. The patient continued to be symptomatic with GI problems and diarrhea.

Would you agree or disagree with the original diagnostic laboratory approach? Why or why not?

The patient submitted additional stool specimens and both the concentration sediment and permanent stained smears were examined. The parasites identified are seen below.

If one considers all the intestinal protozoa, what organisms might have been missed without the permanent stained smear examination? What organism identification requires the permanent stained smear for identification? Why is this true? What references might be helpful in reviewing guidelines for a complete O&P examination?

These protozoan trophozoites measure approximately 13 to 16 µm long. The one on the left is stained with iron hematoxylin; the one in the middle is stained with Wheatley's trichrome (modification of the original Gomori trichrome method for tissues). The organism on the right is B/W and contains a single nucleus; note that there appears to be some clearing within the nucleus (prior to the chromatin fragmenting). Hint: They are flagellates, although the morphology is more consistent with amebae. Note that both trophozoites contain two fragmented nuclei.

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Answer and Discussion of Quiz #39

The images presented in the Diagnostic Quiz #39 are trophozoites of Dientamoeba fragilis; thus the physician's report (NO PARASITES SEEN) was incorrect, since no permanent stains had been examined from the first stool specimens. This organism can be confused with Endolimax nana and Entamoeba hartmanni, particularly if the D. fragilis trophozoite contains only a single nucleus.  NOTE:  In the organism on the right, the clearing in the karyosome provides a clue that this organism is probably D. fragilis and the nucleus is just prior to fragmentation into ~4 pieces.  However, this particular organism could very easily be confused with Endolimax nana.

On the basis of electron microscopy studies, D. fragilis has been reclassified as an ameba-flagellate rather than an ameba and is closely related to Histomonas and Trichomonas spp. It has a cosmopolitan distribution, and past surveys provide incidence rates of from 1.4 to 19%. Much higher incidence figures have been reported for mental institution inmates, missionaries, and Indians in Arizona. D. fragilis tends to be common in some pediatric and adult populations; in some studies, incidence figures are higher for patients under 20 years of age. Trophozoites can contain one or two nuclei. The cyst form has recently been identified for this organism; however, there are very few seen in human clinical specimens (two fragmented nuclei and a distinct cyst wall).   In many laboratories, D. fragilis may be as common, or more common, than Giardia lamblia.

D. fragilis has been associated with a wide range of symptoms. Case reports of children infected with D. fragilis reveal a number of symptoms, including intermittent diarrhea, abdominal pain, nausea, anorexia, malaise, fatigue, poor weight gain, and unexplained eosinophilia. The most common symptoms in patients infected with this parasite appear to be intermittent diarrhea and fatigue; the infection is not limited to pediatric patients. In some patients, both the organism and the symptoms may persist or reappear until appropriate treatment is initiated.

Diagnosis of D. fragilis infections depends on proper collection and processing techniques (a minimum of three fecal specimens). Although survival time for this parasite has been reported as 24 to 48 h, the survival time in terms of morphology is limited, and stool specimens must be examined immediately or preserved in a suitable fixative soon after defecation. It is particularly important that permanently stained smears of stool material be examined with an oil immersion lens (100 x objective). These organisms have been recovered in formed stool; thus, a permanent stained smear must be prepared for every stool submitted for a parasite examination. If a laboratory procedure is called "Ova and Parasite Examination" - this procedure must include both the fecal concentration and permanent stained smear (CAP checklist).

If preserved specimens are submitted to the laboratory, it is more cost effective to delete the direct smear and begin the stool examination with the concentration procedure, particularly since motile protozoa will not be viable because of the prior addition of preservative. Even if parasites are seen on a direct mount of preserved stool, they would almost certainly be seen on the concentration examination as well as on the permanent stained smear (protozoa in particular). With few exceptions, intestinal protozoa should never be identified on the basis of a wet mount alone; permanent stained smears should be examined to confirm the specific identification of suspected organisms.

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:

  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.