Stanley L. Marks, BVSc, PhD, Dip. ACVIM (Internal Medicine, Oncology), Dip. ACVN
There are many well-established techniques for recovering parasite stages from feces, ranging from those designed to recover specific diagnostic stages (the Baermann recovers only larvae), to concentration techniques designed to recover as many organisms as possible (flotation and sedimentation). There is, however, no single method that is 100% efficient for all parasite stages, and best results will be obtained by combining two or more techniques.
As the zoonotic potential and public health significance of several organisms that commonly parasitize companion animals become more clearly appreciated, a strong argument must be made for reevaluating current veterinary methodologies in an effort to increase test sensitivity. A few relatively simple improvements to protocols for routine ova and parasite examinations will bring most private practices (and reference labs) up to a more acceptable standard.
1. Always use fresh feces
Older feces may contain eggs, oocysts, and larvae that have developed beyond their diagnostic stage. Because descriptions and photographs are of parasite diagnostic stages as they appear in fresh feces, matching what you see in old feces with these descriptions can result in an incorrect identification. It is recommended that a calibrated ocular micrometer be part of your microscopic set-up, because size and morphology are the two key elements for correct identification of intestinal parasites.
2. Understand the indications and limitations of the tests you choose to run
If you suspect lungworm infection in a cat, recall that some lungworms shed ova as their diagnostic stage (e.g., Capillaria aerophila) while another (Aelurostrongylus abstrusus) has a first stage larva as its diagnostic stage. If the animal is from the Midwest, you should consider Paragonimus, a fluke that sheds an egg that is usually too heavy to float. A sedimentation technique will recover all of these stages, and is the technique of choice to recover fluke ova. Capillaria may be hard to find in the fecal debris of a sedimentation, so a flotation might be a better choice for these eggs; however, Aelurostrongylus often will not be present in sufficient numbers to show up on a flotation, or will be distorted, so Baermann and Walters techniques are the method of choice for detecting Aelurostrongylus. These techniques will eliminate distortion, which can interfere with larval identification and allow the use of a much larger fecal specimen, thereby increasing the likelihood of larval recovery. However, sedimentation techniques also result in more fecal debris, making it difficult to find very small organisms such as coccidia oocysts and cysts of Giardia, both of which are detected more easily using a flotation technique.
3. Direct Smears
Direct smears are indicated for recovery of parasites whose diagnostic stage is a trophozoite. They must be done with saline (not water) and performed using fresh feces (body temperature, less than 1 hour old). Trophozoites in older specimens will lose their motility and degenerate, becoming unrecognizable. The main limitation of direct smears is sample size, with the result that negative smears are not uncommon with low parasite levels. Trophozoites of Giardia and intestinal trichomonads can be detected by direct smear only (trichomonads have no cyst stage).
Procedure for Direct Smear
1. Place a small drop of saline (water may rupture trophozoites) on a microscope slide.
2. Add a very small amount of well-mixed fresh feces and mix carefully with saline to form a very thin slurry. The final preparation should be thin enough to read newsprint through it.
3. Place a coverslip over the drop.
4. Adjust the light intensity on the microscope to yield maximum contrast and examine at 10x. For closer examination or to confirm a diagnosis, use the 40x objective.
Lugol's or D'Antoni's iodine may be added to help visualize internal structures of trophozoites and cysts. Because stains will kill motile trophozoites, it is recommended that a saline smear and an iodine smear be prepared separately.
4. Fecal flotations
Flotations are excellent for recovering common nematode ova, oocysts of coccidia (including Cryptosporidium spp.), and Giardia cysts. The main limitation of flotations is their inability to float organisms whose diagnostic stage has a specific gravity higher than that of the flotation medium. Most commonly these are the heavy ova of trematodes and acanthocephalans (thorny-headed worms). Fragile cysts and larvae, though recovered, may be too distorted to identify. It is always preferable to use a centrifugation flotation technique. This is probably the single most important change you can make to improve routine recovery of parasite stages by flotation. Flotation methods not utilizing a centrifugation technique are often not sensitive enough to recover small numbers of organisms in the feces.
Always use a coverslip rather than a loop or glass rod to transfer the meniscus to a slide. If your centrifuge has free-swinging buckets, use a coverslip on the final centrifugation to recover parasites. If your centrifuge has a fixed-angle, carefully add more flotation medium after the final spin to create a meniscus and set the coverslip on top for several minutes before lifting it off for examination. If at this point you are still determined to do standing flotations, use a coverslip to transfer the meniscus. Coverglass improves the optics of any microscope.
Check the specific gravity of flotation medium. If possible, switch to zinc sulphate (specific gravity of 1.18 or 1.2) to improve detection and morphology of Giardia spp. Remember that all eggs do not float regardless of the flotation solution. Choice of flotation solution is less important than the actual method of flotation. Examine preparations as soon as possible after preparing them. Delay will result in distortion of some parasite stages and result in incorrect or missed diagnoses, especially with delicate cysts and oocysts.
Procedure for Centrifugal Flotation
(Centrifuge must have free-swinging buckets)
1. Prepare a fecal emulsion using 2-5 grams of feces and 30 ml of flotation solution.
2. Strain the emulsion through a tea straineror cheesecloth into a 15-ml conical centrifuge tube. Suspending a funnel over the tube facilitates filling the tube.
3. Fill the tube with flotation medium to create a positive meniscus.
4. Place a coverslip on top of the tube.
5. Create a balance tube of equal weight, containing another sample or water.
6. Place the tubes in the centrifuge buckets and weigh them on a balance. Water may be added to the buckets to make them equal weights.
7. Centrifuge the tubes for 10 minutes at 400-600 gravities (approximately 1500 rpm).
8. Carefully remove the coverslips from the tubes by lifting straight up and place them on a slide.
9. Examine the slide within 10 minutes. Examine entire coverslip at 10X. Use 40X to confirm identification by visualizing internal structures and measuring the organism.
If your centrifuge is fixed-angle, proceed as above but fill centrifuge tube to within an inch or so of the top, and do not add a coverslip for the final spin. When final centrifugation step is complete, carefully set the tube upright in a test tube rack. Use a pipette to gently run additional flotation solution down the side of the tube while disturbing the contents as little possible. Create a positive meniscus and set a coverslip on top. Let stand for 5 minutes only. Remove the coverslip to a slide and examine as described in step 9.
5. Technical Expertise
Have one technician become very skilled at parasite identification. This will improve detection and identification of parasites once the preparation is complete. The utilization of immunoassay techniques will improve detection of Giardia spp. and Cryptosporidium spp. Both organisms are frequently missed or misidentified in fecal flotations.
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