Correspondence

Laundry Brighteners and Amebic Cysts

N Engl J Med 1994; 331:1459November 24, 1994

Article

To the Editor:

In a transplant recipient with disseminated amebic infection (July 14 issue),1 Slater et al. reported that organisms were initially overlooked on examination of the skin-biopsy specimen stained with hematoxylin and eosin. When we suspect acanthamoeba infection, we use Calcofluor white M2R, an inexpensive (about 10 cents per test), colorless compound that is widely available commercially (from American Cyanamid, Difco, Polysciences, Remel, and Sigma). By binding to polysaccharides such as cellulose and chitin, this fluorochrome highlights amebic cysts, microsporidial spores, filamentous fungi, yeasts, Pneumocystis carinii, algae, and plant-containing foreign material.

Calcofluor white (or a related toluylene derivative) is probably a proprietary ingredient of detergents containing whiteners. The agent makes fabrics appear brighter by emitting light-blue light when exposed to ultraviolet radiation. We have found a similar effect in testing for amebae: cysts shine. For this procedure we use Liquid Tide with Bleach Alternative, but any product containing a nonchlorine “optical bleach” should do as well.

We first dilute the commercial product 1:30. A few drops are used to make a wet mount under a coverslip for smears. For tissue sections, the stain is allowed to sit for approximately five minutes and is then rinsed off with water. Acanthamoeba and fungi stain apple-green or blue, depending on the filters used with fluorescent microscopy. Some organisms glow more vividly if the pH is raised before staining. Bleaching by ultraviolet excitation produces a gradual fading of fluorescent intensity.

We agree that the routine use of laboratory stains requires adequate quality assurance with control slides, but items from the local grocery store can help in the rapid diagnosis of parasitic and fungal disease.

Kirk R. Wilhelmus, M.D.
Michael S. Osato, Ph.D.
Baylor College of Medicine, Houston, TX 77030

1 References

1. 1

   Slater CA, Sickel JZ, Visvesvara GS, Pabico RC, Gaspari AA. Successful treatment of disseminated acanthamoeba infection in an immunocompromised patient. N Engl J Med 1994;331:85-87
   Full Text | Web of Science | Medline

Author/Editor Response

The authors reply:

To the Editor: We appreciate the comments by Wilhelmus and Osato and agree that amebic cysts in tissue would indeed be highlighted by the use of a Calcofluor-containing stain. However, skin-biopsy specimens from our patient contained only amebic trophozoites, which are not stained by this reagent because they lack the β-linked polysaccharide polymers contained in amebic cysts.1 Thus, this type of stain was not useful in the identification of acanthamoeba trophozoites in specimens obtained from the patient we described. Even if amebic cysts had been present in the tissue that stained with Calcofluor-containing stain, further procedures (amebic culture, electron microscopy, or immunostaining) would have been necessary to identify the species of acanthamoeba, since Calcofluor stain is nonspecific.2

Amebic trophozoites were recognized in tissue sections from our patient that were stained with hematoxylin and eosin. Because our repeated cultures for amebic organisms were negative, we used indirect immunofluorescence on tissue sections with specific antiserum to identify and speciate Acanthamoeba rhysodes. On the basis of this specific information, we planned the antimicrobial therapy that proved effective.

Calcofluor-containing dyes can be effective reagents in screening for parasitic and fungal organisms in tissue specimens, but a negative result does not rule out the presence of alternative (nonstaining) forms in dimorphic organisms such as acanthamoeba.

Anthony A. Gaspari, M.D.
Cathy A. Slater, M.D.
University of Rochester Medical Center, Rochester, NY 14642

Govinda S. Visvesvara, Ph.D.
Centers for Disease Control and Prevention, Shamblee, GA 30341

2 References

1. 1

   Wilhelmus KR, Osato MS, Font RL, Robinson NM, Jones DB. Rapid diagnosis of Acanthamoeba keratitis using calcofluor white. Arch Ophthalmol 1986;104:1309-1312
   Web of Science | Medline

2. 2

   Monheit JE, Cowan DF, Moore DG. Rapid detection of fungi in tissues using calcofluor white and fluorescence microscopy. Arch Pathol Lab Med 1984;108:616-618
   Web of Science | Medline

Citing Articles (3)

Citing Articles

1. 1

   Brian J. Harrington, PhD, MPH, George J. Hageage . PhD D(ABMM). (2003) your lab focus: Calcofluor White: A Review of its Uses and Applications in Clinical Mycology and Parasitology. Laboratory Medicine 34:5, 361-367
   CrossRef

2. 2

   George J. Hageage, Jr., PhD, D(ABMM), Brian J. Harrington, PhD, MPH. (2003) your lab focus: Calcofluor White: A Review of its Uses and Applications in Clinical Mycology and Parasitology. Laboratory Medicine 34:5, 361
   CrossRef

3. 3

   PAUL D. WORTMAN. (1996) ACANTHAMOEBA INFECTION. International Journal of Dermatology 35:1, 48-51
   CrossRef