[Updated March 2007]
P R E A N A L Y T I C A L C O N S I D E R A T I O N S The diagnosis of intestinal
microspori-diosis (Enterocytozoon bieneusi, Ence-phalitozoon intestinalis) has depended on the use of invasive procedures and sub-sequent examination of biopsy specimens, often by electron microscopy methods.
However, the need for a practical method for the routine clinical laboratory has
stim-ulated some work in the development of additional methods. Slides prepared from fresh or formalin-fixed stool specimens can be stained using calcofluor white (CFW) (optical brightening agent) and can be examined using fluorescence micros-copy. This staining method is based on the
fact that routine stain penetration of the microsporidial spore is very difficult; thus, the use of CFW enhances the ability of the spores to be seen. The spore coat will stain with CFW, but the stain is nonspecific and may also stain other structures within the specimen (yeast, etc.).
I. PRINCIPLE
Acanthamoeba, a free-living ameba found in soil, sewage, and fresh, salt, or brackish water, can cause painful keratitis that may lead to eye removal or loss of eye function and has also has been implicated in gran-ulomatous amebic encephalitis (1) (see also procedure 9.9.2). A key to successful treatment of Acanthamoeba-caused
infec-tion is the rapid detecinfec-tion of the organisms in patient samples. Cultures may require several days, delaying diagnosis and treat-ment.
CFW can be used for direct detection of Acanthamoeba cysts from clinical speci-mens (4). The active ingredient is the di-sodium salt of 4,4
⬘-bis-(4-anilino-bis-di-ethylamino-5-triazin-2-ylamino)-2,2 ⬘-stil-bene disulfonic acid, which is a nonspe-cific fluorescent dye binding to the poly-saccharide polymers of amebic cysts and microsporidial spores (3). It is very simple to use and provides a rapid and reliable means of demonstrating these organisms.
II. SPECIMEN With the exception of stool, collect all specimens aseptically, and hold them at room temperature (24 to 28C). Do not freeze or refrigerate specimens. Use sterile containers and solutions where indicated; any remaining specimen may be used to inoculate culture media.
A. Stool
1. The specimen can be fresh stool or stool that has been preserved in 5 or 10%
formalin, sodium acetate-acetic acid-formalin (SAF), or some of the newer single-vial system fixatives.
2. Any specimen other than tissue thought to contain microsporidia could be stained by these methods.
3. Polyvinyl alcohol-preserved fecal material is not recommended.
B. CSF
1. Centrifuge at 250⳯ g for 10 min.
2. Remove and place into another sterile tube all except 0.5 ml of the super-natant fluid.
3. Suspend the sediment with remaining supernatant fluid for examination.
Observe standard precautions.
Calcofluor White 9.3.8.2
C. Tissue
Triturate a small portion in sterile water or saline.
1. Brain
2. Corneal scrapings or biopsy specimen 3. Lung
4. Skin lesion
D. Swab of conjunctiva or corneal ulcer
1. Place swab in 2 ml of sterile water or saline in a tube.
2. Vigorously shake the cotton-tipped portion of the swab in the liquid to sus-pend specimen.
3. Remove swab from tube, and return to original holder.
4. The sterile water can be examined directly or concentrated by centrifugation if a larger volume is used.
E. Contact lens paraphernalia 1. Contact lens solutions
Solutions must be from opened containers already used by patient. If volume of solution is greater than 2 ml, then centrifuge prior to examination (250
⳯ g for 10 min).
2. Contact lenses
Submit in 2 ml of sterile water or saline. Examine a small portion of lens and the fluid containing the lens.
F. Water samples
1. Collect at least 100 ml of water in a sterile container.
2. Concentrate specimen by filtration or centrifugation prior to examination (2).
G. Slide prepared by physician Submit at least two slides.
1. Place specimen in the center of the slide, covering no more than a dime-sized area.
2. Circle the material with a wax pencil or magic marker to denote location of the specimen.
3. Air dry slides thoroughly.
4. Place the slide in a slide holder or envelope for staining in the laboratory.
III. MATERIALS A. Reagents (see Appendix 9.3.8–1) B. Supplies
1. Microscope slides (1 by 3 in.), one ring (15 mm), frosted
2. Microscope slides (1 by 3 in.), two rings (12 mm), frosted
6. Sterile wooden applicator sticks 7. Sterile swabs
8. Disposable latex gloves
9. Biohazard container for disposal of contaminated supplies and pa-tient specimens.
10. Positive fecal specimen (micro-sporidial spores)
11. Acanthamoeba castellanii ATCC 30010 stock culture
12. Escherichia coli ATCC 25922 stock culture
13. Wax pencil or magic marker C. Equipment
1. Epifluorescence microscope equipped with an exciter filter that transmits the 250- to 400-nm group of intense mercury spectral emis-sion lines (Zeiss UGI or G365).
View through a barrier filter (Zeiss 41 or LP420), which removes UV while transmitting visible blue light and longer wavelengths (1, 3).
2. Biological safety cabinet II. SPECIMEN (continued)
A N A L Y T I C A L C O N S I D E R A T I O N S
A. A QC slide must be run with each batch of specimens stained with CFW.
B. Unfortunately, the only way to perform acceptable QC procedures for this method is to use actual microsporidial spores as the control organisms (Medical Chemical Corporation; http://www.med-chem.com). Obtaining these positive controls may be somewhat difficult. It is particularly important to use the actual organisms because the spores are difficult to stain and the size is very small (1 to 2.5 lm). Prepare control slides for microsporidia as follows:
1. Using a 10-ll aliquot of concentrated (formalin-ethyl acetate sedimentation concentration; centrifugation at 500⳯ g for 10 min), preserved liquid stool (5 or 10% formalin or SAF), prepare the smear by spreading the material over an area of 45 by 25 mm.
2. Allow the smear to air dry.
C. Prepare control slides for amebae as follows.
1. Make a suspension of A. castellanii from stock culture with sterile water or Page ameba saline.
2. Make a suspension of E. coli from stock culture with sterile water or Page ameba saline.
3. Add 1 drop of the A. castellanii suspension to one of the rings of a two-ring slide and 1 drop of the E. coli suspension to the other ring.
4. Allow the smear to air dry.
5. Fix the smear in absolute methanol for 3 to 5 min.
6. Store at room temperature. The smears are stable for 1 year.
D. Positive control
1. Acanthamoeba cysts are doubled walled (10 to 25 lm), and outer wall is wrinkled. The cysts will fluoresce.
2. The spores will be ovoid and refractile, and the spore wall will fluoresce.
Occasionally, the polar tube can be seen either as a stripe or as a diagonal line across the spore; however, the internal spore contents will normally not be visible.
E. Negative control
1. E. coli will not fluoresce.
2. Most of the bacteria and other debris will not fluoresce. However, there will still be some yeast and debris that may also fluoresce.
F. Perform all scheduled maintenance on all equipment.
G. Record all QC results, including a description of QC specimens tested.
H. Known positive microscope slides, projection slides (2 by 2 in.), photographs, and reference books should be available at the workstation.
A. Slide preparation of clinical specimens
1. Using a sterile swab, stick, or pipette, thinly spread the specimen evenly over the area circumscribed by the ring.
䊓 NOTE: Do not apply excessive specimen on the slide, because the smear may be too thick to visualize any organisms present.
2. Allow the smear to air dry.
3. Fix the smear in absolute methanol for 3 to 5 min.
4. Air dry.
B. Stain procedure
1. Add 3 or 4 drops of CFW and 3 or 4 drops of Evan’s blue into a tube (12 by 75 mm) and mix well.
2. Add several drops of this mixture to the specimen and allow to stand for 5 min.
IV. QUALITY CONTROL
V. PROCEDURE
Calcofluor White 9.3.8.4
3. Turn slide on its side and allow excess stain to run off.
4. Add coverslip, blot excess stain from slide, and examine immediately.
VI. RESULTS A. Microsporidial spores will fluoresce. The outer wall (1 to 3 lm) will fluoresce, with the interior being clear, or perhaps the horizontal or diagonal stripe will fluoresce.
B. Acanthamoeba cysts will fluoresce. Cysts are double walled (10 to 25 lm), and the outer cyst wall is wrinkled (hexacanth cyst). Although more rare than Acan-thamoeba, the cysts of Balamuthia mandrillaris are usually spherical, appear to have two walls (outer irregular wall and inner round wall), and measure 6 to 30 lm in diameter. Naegleria cysts can be confirmed from culture plates but are not seen in clinical specimens. They tend to measure from 7 to 15 lm and have a thick double wall.
C. Yeast cells, pseudohyphae, hyphae, and other fungal elements will stain with CFW (2).
D. Pneumocystis carinii can be detected with CFW (3).
E. Bacteria will not fluoresce.
F. Epithelial cells and blood cells will stain red by Evan’s blue counterstain.
G. Cotton fibers will fluoresce strongly and can be distinguished as artifacts.
P O S T A N A L Y T I C A L C O N S I D E R A T I O N S
A. Positive
Report as microsporidial spores or Acanthamoeba cysts seen. Notify physician immediately.
B. Negative
No organisms seen.
A. Collagen, elastin, and keratin also fluoresce.
B. Other microorganisms (e.g., yeast cells, fungi) will fluoresce.
C. Although bacteria will not fluoresce, microsporidial spores (approximately the same size as some small yeasts and bacteria [1 to 2 lm]) will fluoresce. These organisms have been implicated as a cause of eye disease and have been found in other body tissues. The use of modified trichrome stains would be helpful in differentiating microsporidial spores from other organisms.
D. One drop of 10% KOH can be added to the CFW reagent, and a wet mount can be made of the specimens which require clearing or teasing (e.g., skin scrapings, hair, or viscous specimens).
E. Various concentrations of CFW and Evan’s blue are commercially available.
Some work better than others.
F. Confirm findings (positive or negative) with culture.
G. Depending on what filter combination is used, the cysts will fluoresce either blue-white or apple green.
H. If Naegleria cysts are present, they may be seen using calcofluor.
1. Garcia, L. S. 2001. Diagnostic Medical Par-asitology, 4th ed., p. 723. ASM Press, Wash-ington, D.C.
2. Green, L. K., and D. G. Moore. 1987. Fluo-rescent compounds that nonspecifically stain fungi. Lab. Med. 18:456–458.
3. Polysciences, Inc. April 1990. Data sheet 316.
4. Wilhelmus, K. R., M. S. Osato, R. L. Font, N. M. Robinson, and D. B. Jones. 1986.
Rapid diagnosis of Acanthamoeba keratitis us-ing calcofluor white. Arch. Ophthalmol.
104:1309–1312.
V. PROCEDURE (continued)
VII. REPORTING RESULTS
VIII. PROCEDURE NOTES
REFERENCES
SUPPLEMENTAL READING Baselski, V. S., and M. K. Robinson. 1989. A staining kit for detection of opportunistic patho-gens in bronchoalveolar lavage specimens. Am.
Clin. Lab. 8:36–37.
Polysciences, Inc. 1991. Material safety data sheet for Fungi-Fluor kit, catalog no. 17442. Poly-sciences, Inc., Washington, Pa.
Ryan, N. J., G. Sutherland, K. Coughlan, M.
Globan, J. Doultree, J. Marshall, R. W. Baird, J. Pedersen, and B. Dwyer. 1993. A new tri-chrome-blue stain for detection of microsporidial species in urine, stool, and nasopharyngeal spec-imens, J. Clin. Microbiol. 31:3264–3269.
Weber, R., R. T. Bryan, R. L. Owen, C. M. Wil-cox, L. Gorelkin, and G. S. Visvesvara. 1992.
Improved light-microscopical detection of micro-sporidia spores in stool and duodenal aspirates. N.
Engl. J. Med. 326:161–166.
APPENDIX 9.3.8–1 Reagents
A. Commercially available solution of CFW with an Evan’s blue counterstain (such as Fungi-Fluor kit, catalog no. 17442; Polysciences, Inc., Warrington, Pa.) or solution prepared as follows:
1. 0.1% CFW
CFW M2R, purified ...0.1 g distilled water ... 99.9 ml
Mix. Filter, and store in dark container. The mixture is stable at room temperature for 1 year.
2. 0.5% Evan’s blue
Evan’s blue ...0.5 g distilled water ... 99.5 ml Mix. The mixture is stable at room temperature for 1 year.
B. Page’s ameba saline (1ⴒ)
NaCl ... 6 mg MgSO4•7H2O ...0.2 mg CaCl2•2H2O ...0.2 mg Na2HPO4 ...7.1 mg KH2PO4...6.8 mg distilled water ... 500 ml
Autoclave at 121C for 15 min. Store refrigerated in a glass bottle. The mixture is stable for 3 months.
C. Absolute methanol D. Sterile distilled water Include QC information on
reagent container and in QC records.
9.4.1.1