Accurate evaluation of fecal samples critical to patient

In many veterinary clinics, the routine fecal analysis has been passed down to the newest member of the staff, with very little instruction or emphasis on the importance of the task.

However, the accurate evaluation of fecal samples is critical and should be taken seriously by all members of the veterinary health care team.

The public has become more aware of zoonotic diseases that may be transmitted to them by their pets including, visceral and cutaneous larval migrans, intestinal hookworm disease, giardiasis and toxoplasmosis. The diagnostic stages of these parasites, Toxocara sp., Ancylostoma caninum, Giardia sp. and Toxoplasma gondii, may be found in fecal samples from dogs and cats. Veterinarians are the first line of defense to help prevent the spread of these diseases. Client education, including the true versus perceived dangers of disease transmission, and the use of the most accurate diagnostic methods available are both imperatives in veterinary medicine today.

From the collection of an adequate sample to the entry into the medical record, fecal analysis should be done in a professional, routine manner with emphasis on personal protection, good record keeping, standardized procedures, sanitation and recognition and reporting of the usual as well as the unusual findings.

To spin or not to spin?

The data supports flotation with centrifugation. If you are a recent Kansas State University (KSU) graduate or if you have hired one of these fine young veterinarians, they participated in gathering the following data.

One of the first sessions in the parasitology laboratory is a technique comparison. Students are provided with positive fecal samples and are instructed to use the same sample to compare results from a direct smear, a standing fecal (See Photo 1) and a fecal flotation with centrifugation. The results are remarkable. In 2000, the sample that was positive for Ancylostoma caninum, 16.7 percent of the students classified the direct smear results to be moderate to heavy, 84.4 percent of the standing fecal results were similar as were 98.7 percent of the centrifugation results.

Photo 1: Typical standing fecal technique.

A. caninum ova were found in most of the standing fecals, but the numbers were not considered significant and therefore not life threatening. The number of ova found with the centrifugation technique by almost all of the students was indicative of a severe infection (See Photo 2).

In 2001, the positive Toxocara cati sample was found by 70 percent of the students to be negative in the direct smear, 38 percent negative by the standing technique; however 100 percent of the students reported positive results when they used flotation with centrifugation technique. Because the detection of Toxocara sp. ova is very important to families with puppies or kittens and young children, a 38 percent false negative result should be unacceptable in practice.

Photo 2: Ancylostoma caninum -- severe infection.

Mixed fecal sample

A mixed fecal sample was provided to the students in 2002. The direct smear results included 100 percent false negative results for Trichuris vulpis, 90 percent false negative results for Toxocara canis and 70 percent for Ancylostoma caninum. Standing fecal results included 20 percent false negative for T. vulpis and 50 percent false negative for T. canis. All of the centrifuged samples were found to be positive for all three egg types. A missed diagnosis of T. vulpis often leads to extensive and unnecessary diagnostic tests for gastrointestinal signs caused by the presence of these parasites.

Table 1.

Why do eggs, oocysts, cysts float?

The principal involved in fecal flotation depends on differences in specific gravities of the parasite and the flotation solution. (Tables 1 and 2). Specific gravity (sp. gr.) is defined as weight per unit volume compared with water. The hydrometer is the device used to measure specific gravity. It consists of a weighted, sealed, long-necked glass bulb that is immersed in the liquid being measured; the depth of flotation gives an indication of liquid density. (See Photo 3). Hydrometers are readily available from laboratory supply companies and cost about $20. Hydrometers are essential to assure the necessary specific gravity of flotation solutions. If the specific gravity of the flotation solution is less than the specific gravity of the parasitic ova they will not float. If the only positive fecal result being reported in your clinic is Ancylostoma caninum (sp. gr. 1.06) now is a good time to check the specific gravity of the flotation solution used in your laboratory.

Photo 3: Specific gravity hydrometer.

Commercial solutions and dry granular salts that water is added to up to a fill line should all be checked with a hydrometer for specific gravity before use and water or salt added until the reading is correct. A specific gravity that is too low will not float parasitic ova and one that is too high (from evaporation, etc.) will cause ova and cysts to rupture.

Microscope and micrometer

The purchase of a good quality microscope with a calibrated micrometer will pay for itself by enabling you and your technician to accurately diagnose parasitic infections accurately.

Table 2.

If the equipment works well, your technicians will want to use it and will spend more time examining slides. Some student microscopes are available for less than $2,000 and are rugged and equipped with good quality optics.

The ability to measure ova or cysts is extremely important. Many veterinarians are confident in their ability to compare relative sizes. This ability is dependant on the presence of a well-known egg such as Trichuris vulpis and one that may be rather rare such as Physoloptera sp. in the sample field. (See Photo 4, p. 10). However, if faced with a small oocyst, it is very difficult to rule a diagnosis of toxoplasma in or out in feline fecal samples (See Table 3, p. 9).

Proposed plan for fecal analysis

Clients should be instructed to provide a fresh (within 24 hours), adequate sample of feces (2- 5 grams, at least the size of a walnut). Zip lock bags or a latex glove turned inside out and placed into a zip lock bag are readily available to most clients. This is an excellent time to mention routine removal of feces from the yard, litter box or methods of "scooping poop" on daily walks to prevent environmental contamination.

Table 3.

Once the sample is in the clinic, the record or laboratory slip should be generated, feces examined immediately or refrigerated until time is available for analysis. The technician should be encouraged or required to wear gloves and there should be absolutely no food or drink in the laboratory space.

Photo 4: Trichuris vulpis, Physoloptera spp.

Step one: Gross examination of feces: record consistency, color, and presence of blood, mucus or whole parasites. The examiner should be on the lookout for tapeworm segments and know how to place them on a slide with saline, crush and determine egg type for identification. Eggs of Dipylidium sp. and Taenia sp. may not be in the float regardless of technique.

Step two: Direct smear: The direct smear, when done properly, may be useful for the diagnosis of protozoal parasites which have motile trophozoite stages that are passed in the feces such as giardia and trichomonads. In order to be diagnostic, direct smears must be performed using very fresh feces (body temperature, less than five or 10 minutes old. Saline, as opposed to tap water, will help to preserve the integrity of the organisms. The addition of one drop of fresh Lugol's iodine, before adding the coverslip, will stain giardia trophozoites and cysts.

Step three: Flotation with centrifugation. The technique of choice in small animal practice for routine fecal analysis is flotation with centrifugation with very few exceptions.

Standard Centrifugation Fecal Exam (See Photo 5)

Photo 5: The tube holders swing out from the head of the centrifuge.

1. Weigh out (or guestimate) 2 to 5 grams of feces.

2. Mix feces with approximately 10 ml of flotation solution (Sheather's Sugar, sp. gr. 2.27, is the flotation solution of choice in our diagnostic laboratory).

3. Pour mixture through a tea strainer into a beaker or fecal cup.

4. Pour strained solution into a 15 ml centrifuge tube.

Photo 6: Giardia cysts in zinc sulfate, stained with Lugol's iodine.

5. Fill tube with flotation solution to a slight positive meniscus. Do not overfill the tube. There should be a small bubble under the coverslip if the correct amount of flotation solution was added.

6. Place a coverslip on the tube and place in a centrifuge.

7. Make sure the centrifuge is balanced.

8. Centrifuge at 1200 rpm (280XG) for five minutes.

9. Remove the tube and let stand 10 minutes.

10. Remove the coverslip and place on a glass slide. Systematically examine the entire area under the coverslip 10X magnification. Careful examination of the entire slide is imperative. Use the 40X objective lens to confirm your diagnosis and make measurements.

Adjustments in procedure for fixed head centrifuges.

If the centrifuge has a fixed angle head, the tube is not filled to the top (about 3/4 full) before centrifugation. The tube is removed after the five minutes spin time and then filled to a slight positive meniscus and placement of a coverslip. A small bubble under the coverslip means that the right amount of flotation solution was added. The tube is then allowed to stand for an additional 10 minutes before removing the coverslip and examining the preparation.

Step four: Record results, including negative results.

Step five: Empty and wash centrifuge tubes with soap and water. Clean centrifuge frequently to prevent sugar or salt accumulation. Clean laboratory area with disinfectant. Dispose of waste materials appropriately. Wash hands!

1. Giardia trophozoites are rarely passed in solid or semi solid stools. The diagnostician has a much greater chance of finding these cysts using the flotation with centrifugation technique in zinc sulfate, sp. gr. 1.18. One drop of Lugol's iodine on the slide before adding the cover slip (See Photo 6, 6A) will make these delicate cysts much easier to see. The slide must be examined immediately because the cysts will disintegrate and the salt will crystallize across the slide rapidly.

Photo 6A: Giardia cysts 40X.

2. Larval recovery: The larvae of lung worms (Aelurostrongylus abstrusus, Filaroides osleri) may be recovered with a Baermann exam. We have downsized the standard Baermann stand and funnel used in large animal diagnostic parasitology to a plastic champagne glass for the smaller samples from dogs and cats. The fecal sample must be very fresh and not refrigerated to recover larvae. The diagnostic stage of Strongyloides stercoralis is a larvated egg, but the larvae may be recovered from dog feces using the Baermann technique.

3. Sedimentation is the technique of choice to recover fluke ova (Alaria canis, Nanophyetus salmincola, Paragonimus kellicotti, Platynosum fastosum) because they are very heavy and may not float in typical solutions.

Conclusions

Equipment and time may be perceived drawbacks to the use of the most accurate diagnostic methods available for parasitic infections.

Suggested Reading

Equipment costs (centrifuge, hydrometer and a good quality microscope with micrometer) and the compensation of trained technicians are not that great. The accurate diagnosis of clinically important parasites is actually priceless to the veterinarian, the pet, and most importantly, to your clients and their families.