of the Specimen
Specimens should be obtained in the morning before the patient bathes or goes to the bathroom.
1. Prepare a mixture of 4 parts petrolatum to 1 part paraffin, and heat until liquid (just melted).
2. Dip the end of the cotton swab into the mixture, remove the swab, and allow it to cool. If the cotton is not thoroughly coated, dip it again.
3. Store the coated swab in a 100- by 13-mm tube, and plug the end of the tube with cotton. These tubes may be stored for long periods, preferably under refrigeration.
4. Rub the swab gently over the perianal surface and into the folds. Insert the swab into the anal opening about 1/4 in. and then replace it in the tube.
Examination
1. Fill the tube containing the swab half full of xylene or xylene substitute, and let it stand for 3 to 5 min.
2. Remove the swab, and centrifuge the tube at 500 × g for 1 min.
3. Remove the supernatant fluid with a pipette (do not pour it off).
4. Place the sediment on a slide, and examine the material for eggs. The fluid can be examined under a coverslip, in a depression slide, or in a wax pencil circle drawn on the slide (to prevent the fluid from spreading).
Material obtained from sigmoidoscopy can be helpful in the diagnosis of amebiasis that has not been detected by routine fecal examinations; however, a series of at least three routine stool examinations for parasites should be performed on each patient before sigmoidoscopy examination is done. Another option would be to use the immunoassay kits that are designed to detect either the Entamoeba histolytica/E. dispar group/complex or specifically pathogenic E. histolytica; fresh or frozen stools are required for these kits.
Material from the mucosal surface should be aspirated or scraped and should not be obtained with cotton-tipped swabs. If swabs are the only method available, a small amount of cotton should be used on the end of the stick and should be wound tightly to prevent absorption of the sigmoidoscopy material into the cotton. At least six representative areas of the mucosa should be sampled and examined (six samples, six slides) (8, 13).
The specimen should be processed immediately. Usually, the amount of material is limited and should be handled properly to ensure the best examination possible. Various methods of examination are available (direct mount, several options for permanent stains). All are recommended; however, depending on the availability of trained personnel, proper fixation fluids, or the amount of specimen obtained, one or two procedures may be used. If the amount of material limits the examination to one procedure, the use of fixative containing polyvinyl alcohol (PVA) is highly recommended.
If the material is to be examined by any of the new immunoassay detection kits (for Cryptosporidium spp. or Giardia lamblia [G. duodenalis, G. intestinalis]), 5 or 10% formalin or sodium acetate-acetic acid-formalin (SAF) or the Universal Fixative is recommended. However, as mentioned above, if immunoassay kits are to be used for detection of the E. histolytica/E. dispar group/complex or specifically pathogenic E. histolytica, fresh or frozen stools are required. It is always important to read the package insert to verify collection recommendations. Many physicians performing sigmoidoscopy procedures do not realize the importance of selecting the proper fixative for material to be examined for parasites. For this reason, it is recommended that a parasitology specimen tray (containing Schaudinn’s liquid fixative containing PVA, the Universal Fixative, and 5 or 10% formalin) be provided or that a trained technologist be available at the time of sigmoidoscopy to prepare the slides. Even the most thorough examination will be meaningless if the specimen has been improperly prepared.
If there is no lag time after collection and a microscope is available in the immediate vicinity, some of the material should be examined as a direct saline mount for the presence of motile trophozoites. A drop of material is mixed with a drop of 0.85% sodium chloride and examined under low light intensity for the characteristic movement of amebae. It may take time for the organisms to become acclimated to this type of preparation; therefore, motility may not be obvious for several minutes. There will be epithelial cells, macrophages, and possibly polymorphonuclear leukocytes and red blood cells (RBCs), which will require a careful examination to reveal amebae (Fig. 5.4).
Figure 5.4 Entamoeba histolytica trophozoites stained with trichrome stain. Note the clearly defined internal details (nuclear and cytoplasmic characteristics, plus the ingested red blood cells). doi:10.1128/9781555819002.ch5.f4
Note Since specific identification of protozoan organisms can be difficult when only the direct saline mount is used, this technique should be used only when sufficient material is left to prepare permanent stained smears.
Morphologic differentiation between the E. histolytica/E. dispar group/complex and Entamoeba coli can be difficult, in addition to the problem of differentiating human cells from protozoa. Also, unless trophozoites containing ingested RBCs are seen, it is impossible to differentiate organisms in the E. histolytica/E. dispar group/complex from the actual pathogen, E. histolytica.
Schaudinn’s Fixative
Most of the material obtained at sigmoidoscopy can be smeared (gently) onto a slide and immediately immersed in Schaudinn’s fixative. These slides can then be stained with trichrome stain and examined for specific cell morphology, either protozoan or otherwise. The procedure and staining times are identical to those for routine fecal smears.
PVA Fixative
If the material is bloody, contains a lot of mucus, or is a “wet” specimen, a few (no more than 2 or 3) drops of fixative containing PVA can be mixed with 1 or 2 drops of specimen directly on the slide, which is allowed to air dry (a 37°C incubator can be used) for at least 2 h before being stained. If time permits, the PVA smears should be allowed to dry overnight; they can be routinely stained with trichrome stain and examined as a permanent mount.
SAF Fixative
Material obtained at sigmoidoscopy can be placed in small amounts of SAF. After fixation for 30 min, the specimen can be centrifuged for 10 min at 500 × g, and smears from the small amount of sediment can be prepared for permanent staining with iron hematoxylin (trichrome stain would be the second choice). One of the organisms most strongly suspected when sigmoidoscopy is performed is E. histolytica, whose morphology is normally seen from the permanent stained smear; however, this identification assumes that RBCs are seen within the cytoplasm of the trophozoites (Fig. 5.4). If RBCs are not seen in the trophozoite cytoplasm, the report should indicate that organisms in the E. histolytica/E. dispar group/complex have been seen and identified. However, if SAF is used, both the permanent stained smear and a fecal immunoassay kit can be used. If enough material for only a single procedure is available, the permanent stained smear is recommended, particularly if the iron hematoxylin stain (incorporating the carbol fuchsin step) is used (34).
Universal Fixative
Material obtained at sigmoidoscopy can be placed in small amounts of the Universal Fixative (TOTAL-FIX). After fixation for 30 min, the specimen can be centrifuged for 10 min at 500 × g, and smears from the small amount of sediment can be prepared for permanent staining with trichrome or iron hematoxylin. The sediment should be smeared onto a slide and allowed to dry 30 min at 37°C