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Untitled Document

Presentation of Quiz #10

A 35-year-old male with AIDS presented to his physician with a continuing history of symptoms, including chronic intractable diarrhea, fever, malaise, and weight loss. He reported having four to six watery, nonbloody stools per day, and often felt nauseated. The patient had also been diagnosed with nephritis. Three ova and parasite examinations had been performed and were reported as negative. The following laboratory tests were then ordered: modified acid-fast stains for the coccidia and modified trichrome stains for the microsporidia.

Please comment on the possible diagnosis related to the history of AIDS, the patient's clinical symptoms and the laboratory test results to date. Examination of the modified acid-fast stained smears and the modified trichrome stained smears revealed the following.

1. Modified acid-fast stain, modified trichrome stain- stool

2. Modified trichrome stain (Ryan Blue) - stool

3. Modified trichrome stain (Ryan Blue) - urine

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Answer and Discussion of Quiz #10

The images presented in Diagnostic Quiz #10 are the following:

  1. Isospora (Cystoisospora) belli oocyst and microsporidial spores in stool (combination stain: modified acid-fast stain + modified trichrome stain)
  2. Microsporidial spores in stool (modified trichrome stain - Ryan Blue)
  3. Microsporidial spores in urine (modified trichrome stain - Ryan Blue)

Comment: This was a case of microsporidiosis in an immunocompromised patient with AIDS. The two organisms that are found in the intestinal tract are Enterocytozoon bieneusi and Encephalitozoon (Septata) intestinalis. The spores seen in the stool and urine measure approximately 1-2 microns, and the morphology does not allow differentiation between the two genera. Both can also disseminate to the kidneys and elsewhere, so they could both be found in urine. The patient also had an infection with Isospora belli; these oocysts stained using the modified acid-fast stain, as seen above. The first photograph represents a combination stain, using both the modified acid-fast and modified trichrome staining formulas. Organisms that would stain using this method include: Cryptosporidium parvum, Cyclospora cayetanensis, Isospora belli and the microsporidia.

Encephalitozoon spp. (probably intestinalis) (Monoclonal reagent)

Microsporidian spores in urine (Calcofluor white)

Spores in enterocytes (Giemsa stain)

Comments:

Using an experimental monoclonal reagent directed against Encephalitozoon spp., the spores were found to fluoresce, thus confirming spores in this genus were present. Smears were also stained using the optical brightening agent, calcofluor white. Although the spores did stain, this approach is non-specific and results cannot differentiate among the different genera of microsporidia. Unfortunately, commercial immunoassay reagents are not yet available for the detection and/or differentiation of the microsporidia. One can also see the spores within the enterocytes within the intestine; this preparation was stained with Giemsa stain.

Microsporidiosis is an important emerging opportunistic infection in HIV-infected patients and appears to have an ever-expanding clinicopathologic spectrum. This infection has also been identified in transplant patients. Although the majority of information is available regarding the immunocompromised host, it is likely that the immunocompetent host can also acquire these infections; there may be many more infections seen, but not recognized, in normal individuals with diarrhea.

The spore is the only life cycle stage able to survive outside of the host cell and is the infective stage. The spore normally reaches the new host through ingestion, although other routes of infection have been identified including: inhalation, direct inoculation, and sexual transmission. Currently, there are many genera and species of microsporidia that have been implicated in human infections. There are a number of methods used for the recovery and identification of microsporidia in clinical specimens. Tissue Gram stains, PAS, silver stains, Giemsa stain or modified trichrome stains are available. Differentiation to the genus level often requires specific experimental reagents or electron microscopy. A positive infection should be reported as: Microsporidian spores seen; unable to identify to the genus level.

Key Points - Laboratory Diagnosis

  1. The modified trichrome staining procedure for stool may be difficult to interpret without positive controls to review (see product information within this web site).
  2. Make sure the material on the slides is very thin, the smear is stained for the recommended time frame, and the smear is examined under oil immersion (total magnification of at least x1,000).
  3. When using various stains, optical brightening agents, or experimental immunoassay reagents where diagnosis is based on seeing the actual spores, it is highly recommended that the fecal specimen be concentrated and centrifuged at 500 x g for 10 min.
  4. The optical brightening agents (calcofluor, FungiFluor, Uvitex 2B) provide a sensitive screening method, but the results are nonspecific. False positives have been reported as a result of fluorescent artifact material.
  5. Touch preparations can be methanol fixed and stained with Giemsa.

References:

  1. Garcia, LS, 2016. Diagnostic Medical Parasitology, 6th Ed., ASM Press, Washington, DC.
  2. Wittner, M. and L.M. Weiss (eds). 1999. The Microsporidia and Microsporidiosis. ASM Press, Washington, D.C.