Clinical diagnostic parasitology: The inefficiency of common techniques (Proceedings)

To ensure the health and well-being of pet dogs and cats, coprologic examinations for parasite eggs, oocysts, and cysts are an important part of the daily routine for most veterinary practices. Many different procedures and techniques are used, each with its own advantages and limitations. Direct fecal smears are useful for detecting motile protozoa, and sedimentation examinations are useful for recovering heavy (e.g., Physaloptera spp) or operculated (e.g., fluke) eggs that do not float well because of the hypertonic effects exerted by the flotation solution. The methods most frequently used to recover parasite eggs, oocysts, and cysts are flotation techniques that rely on the differences in the specific gravity (SG) of the egg(s), fecal debris, and flotation solution.

The SG of most parasite eggs is between 1.05 and 1.23.1 For parasite eggs to float, the SG of the flotation solution must be greater than that of the eggs. Ideally, all helminth eggs and protozoan cysts and oocysts would float and still maintain their morphologic integrity while fecal debris would sink in the chosen flotation solution. Flotation solutions are made by adding a measured amount of salt or sugar to a specific amount of water to produce a solution with the desired SG. Common flotation solutions include saturated sodium chloride (NaCl; SG 1.18), sugar (Sheather's solution; SG 1.27 to 1.33), sodium nitrate (NaNO3; SG 1.18 to 1.2), magnesium sulfate (MgSO4; SG 1.2), and zinc sulfate (ZnSO4; SG 1.2). These solutions are effective, easy to make or commercially available, and relatively inexpensive.

Flotation procedures vary from the simple to the complex. The simplest procedure involves mixing a small amount of feces with flotation solution in a cylinder (shell vial or centrifuge tube) and adding solution until the cylinder is nearly full. The preparation is then allowed to stand until the eggs float to the top, and a sample from the top is removed to a microscope slide using a tool such as a wire loop, straw, needle hub, or glass rod. A refinement of this method involves filling the cylinder until a slight positive meniscus is formed and placing a glass coverslip over it. Again, the cylinder is allowed to stand until the eggs have had time to float to the top, and the coverslip is then removed to a microscope slide and examined. Several commercial apparatuses that use a screen to retain debris from floating to the top are variations of the simple shell vial technique.

A further refinement of the flotation technique involves centrifugation to spin down the debris and allow the eggs to float to the surface of the solution where they can be recovered. If a fixed-angle centrifuge head is used, the centrifuge tubes cannot be filled completely and thus should be removed from the centrifuge after spinning and placed vertically in a test tube rack. If a swing-head centrifuge is used, the tubes can be filled to a slight positive meniscus and covered with 18- or 22-mm2 coverslips before centrifuging. When tubes are spun with coverslips in place, care should be taken not to open the centrifuge before it stops spinning, or the coverslips can shift and ruin the preparation. Veterinary hospitals usually use one or more of these methods based on cost, ease of use, availability of hardware, or simply tradition.

The Ovassay method with 1.1-SG ZnSO4 solution readily floats, hookworm (A. caninum) eggs (SG 1.05591 ); however, ascarid (T. canis) eggs (SG 1.0900) may not be recovered and whipworm (T. vulpis) eggs (SG 1.1453) are virtually impossible to float with such a solution.2 This points out the necessity for using care in weighing the salts and measuring water when preparing flotation solutions and for assuring proper SG by testing the solution with an SG hydrometer. When the SG of the salt solution (ZnSO4) is raised to 1.2, T. vulpis, and T. canis eggs are recovered in the Ovassay but in fewer numbers than with a centrifugation method using either ZnSO4 or sugar. A centrifugation method will recover significantly higher fecal counts compared with the Ovassay method.

For A. caninum, a centrifugation method using 1.2-SG NaNO3 solution results in significantly higher fecal egg counts than the simple flotation method, which is allowed to stand for 5 or 10 minutes.2 A 15- or 20-minute simple flotation method recovers significantly similar fecal counts as compared with the centrifugation method. With low numbers of T. vulpis eggs the 5' and 10' simple floats can miss eggs in 2 out of 3 samples.

From 2000 to 2004, students at KSU evaluated 206 fecal samples known to contain hookworm (A. caninum) eggs.2 When all hookworm data were combined, the direct smear technique failed to detect hookworm eggs 72.82% of the time. The Ovassay and centrifugation techniques yielded false-negative results 4.85% and 0.97% of the time, respectively, and recovered more than 50 eggs/slide 36.41% and 74.76% of the time, respectively.2

Students evaluated 171 fecal samples known to contain ascarid (T. canis or T. cati) eggs. When all ascarid data were combined, the direct smear technique failed to detect ascarid eggs 85.38% of the time. The Ovassay and centrifugation techniques yielded false-negative results 25.88% and 10.53% of the time, respectively, and recovered more than 50 eggs/slide 1.18% and 42.69% of the time, respectively.2

Students evaluated 203 fecal samples known to contain whipworm (T. vulpis) eggs. When all whipworm data was combined, the direct smear technique failed to detect whipworm eggs 92.61% of the time. The Ovassay and centrifugation techniques yielded false-negative results 32.02% and 4.93% of the time, respectively, and recovered more than 50 eggs/slide 2.96% and 23.65% of the time, respectively.2

Students also evaluated 53 fecal samples known to contain tapeworm (Taenia sp) oocysts and 26 samples known to contain coccidia (Isospora sp) oocysts. The direct smear technique failed to detect tapeworm eggs 96.15% of the time. The Ovassay and centrifugation techniques yielded false-negative results 76.92% and 11.54% of the time, respectively. When the two sets of coccidia data were combined, the direct smear technique failed to detect coccidia oocysts 94.34% of the time. The Ovassay and centrifugation techniques yielded false-negative results 50.94% and 5.66% of the time, respectively.2

Evaluations of centrifugation fecal techniques and IDEXX SNAP® Giardia fecal antigen test kits of puppy fecal samples by 2nd year veterinary students showed that almost half (56/116) of the fecal samples were recorded as positive for Giardia. The direct smear technique detected the fewest number of positives with students recording only 4 positive samples. This data may be artificially low since the fecals were collected several hours prior to laboratory and trophozoites may have been dead at time of examination. Students recorded that the SNAP® Giardia fecal antigen test identified 55 of 116 samples as Giardia positive and ZnSO4 centrifugation technique recorded 45 of 116 samples as positive.

At a wet lab conducted at the Central Veterinary Conference in 2005 twenty-seven (27) participants returned completed fecal data forms. When a centrifugation fecal flotation technique was compared to passive flotation technique the data demonstrated that centrifugation with either 1.18 sp. gr. ZNSO4 or 1.27 sp. gr. Sheather's sugar solution routinely recovers more eggs and oocysts than the passive Ovassay technique. Not only did the centrifugation technique recover more eggs and oocysts in addition the participants recorded many more samples as positive with the centrifugation technique. Strikingly only once (T. canis – Ovassay - ZNSO4) did the Ovassay technique recover all parasites in all samples, while only once did the centrifugation technique fail to recover all parasites in all samples. In the group that used 1.18 sp. gr. ZNSO4 solution only 2 of 14 participants recovered Taenia sp. eggs. While in the group using 1.27 sp. gr. Sheather's sugar solution all 13 participants recovered Taenia sp. eggs using.

Even though the participants knew the samples were positive for Giardia recovery and identification of Giardia sp. oocysts was problematic for the 27 participants irregardless of technique. Only 6 of the 27 participants were able to recover and identify Giardia sp. oocysts from a known positive sample. One participant each using the Centrifugation with ZNSO4, Ovassay with ZNSO4 and Ovassay with Sugar was able to recover and identify Giardia sp. cysts. Three participants using the Centrifugation with Sugar were able to recover and identify Giardia sp. cysts. All 27 participants had a positive SNAP® Giardia fecal antigen test on the mixed sample.

Conclusions

In today's litigious society, failure to detect a light infection in a pet, regardless of whether treatment was initiated, could be significant from a legal standpoint. Although lawsuits resulting from OLM have usually revolved around failure to initiate appropriate deworming procedures, inappropriate diagnostic methodology could be an issue. Practitioners have told me that the reasons they use commercial fecal kits or a simple flotation method instead of centrifugation are that the former cost less to run and take less time. However, these data show that centrifugation consistently recovered more eggs than either of the other techniques, even when comparing a 5-minute centrifugation with a 20-minute simple floatation. Also, examining the coverslip before allowing the sample to stand for 15 minutes when using the simple flotation technique and a solution with an appropriate SG could result in a missed diagnosis of T. vulpis.

Failure to ensure that a prepared flotation solution has the proper SG could result in a missed diagnosis of either T. vulpis or T. canis, both of which are pathogenic parasites in dogs. Solutions should be properly prepared following standard formulas when using bulk sugar or salts or specific label directions when hydrating commercial salt solutions. After the solution has been prepared, it is recommended that the SG be checked with a hydrometer.

While the sugar solution was very effective in the centrifugation method, it consistently recovered fewer parasite eggs than did NaNO3 when the simple flotation method was used. The increased viscosity of the sugar solution might impede egg recovery in a simple flotation. Examining the coverslip before all the eggs in the sample have had a chance to rise to the surface might result in a missed diagnosis or alter a clinical impression if far fewer eggs are recovered. Veterinarians might be well advised to reevaluate their fecal examination protocols or, at the very least test, to be sure their flotation solutions are formulated to attain a SG heavy enough to allow T. vulpis eggs to float. Spirurid (e.g. Physaloptera sp; SG 1.23761 ) and tapeworm (e.g., Taenia sp; SG 1.2251) eggs are even heavier and require a SG of 1.22 or greater to effectively recover eggs from fecal samples.

ZnSO4 has been shown to be the most efficient flotation solution for recovery of Giardia cysts and is often used in veterinary practices. The wet lab conducted at the CVC highlighted a potential problem in using 1.18 sp. gr. ZnSO4 even in a centrifugation procedure. Only 2 of 14 (14.29%) participants that used the 1.18 sp. gr. ZnSO4 centrifugation procedure correctly recorded that sample as positive for Taenia sp. eggs. While 100% of the participants using the 1.27 sp. gr. sugar solution recovered Taenia sp. eggs from the same sample. This result was not completely unexpected since Taenia sp. eggs have an average sp. gr. of 1.2251. This indicates that veterinary practices using 1.18 sp. gr. ZnSO4 as their flotation solution are likely failing to identify some dogs infected with Taenia sp. tapeworms and possibly other parasites that shed heavy eggs such as Physaloptera sp. which has eggs with an average sp. gr. of 1.2376.

If Giardiasis is on the differential list of a dog (or cat) with diarrhea the data suggests that conducting both ZnSO4 centrifugation fecal examination and a SNAP Giardia sp. fecal antigen test may increase the chances of recording a positive finding. However, it must also be remembered that a single negative examination, even if both tests are conducted simultaneously, does not necessarily rule out Giardiasis.

The major question is what procedure or procedures should be conducted for routine fecal examinations. Data from this current study and another study previously published by this author would suggest that swing-head centrifugation technique using 1.27 sp. gr. Sheather's sugar solution is the most efficient in recovering many commonly encountered parasite eggs and oocysts. While the sugar solution is effective for many eggs and oocysts it will distort and/or destroy most Giardia sp. cysts making them often unrecognizable to most veterinarians and technicians. Many practices therefore use ZnSO4 as their flotation solution. However, as demonstrated in this investigation a ZnSO4 flotation solution may not be able to float some heavy parasite eggs. In addition, there are the previously mentioned problems in many practices of correctly identifying Giardia sp. cysts even using ZnSO4. In this investigation registered veterinary technicians and veterinarians had great difficulty in identifying cysts even when informed the samples were positive. While proper training of veterinarians and clinical staff in correctly identifying Giardia sp. cysts is important and would likely greatly improve correct diagnoses, it may be difficult on a large scale basis.

Due to the inability of 1.18 sp gr. ZnSO4 flotation solution to consistently recover heavier parasite eggs these authors recommend that for routine fecal examinations 1.27 sp. gr. Sheather's sugar solution should be used in a swing-head centrifugation technique. In addition, if Giardiasis is encountered in your practice area then the fecal examination should be accompanied by an efficient Giardia sp. fecal antigen snap test. Due to the difficulty of identifying Giardia sp. cysts, the in-clinic soluble Giardia spp. fecal antigen snap test likely will improve a clinic's ability to arrive at a correct diagnosis.

References

David ED, Lindquist WD: Determination of the specific gravity of certain helminth eggs using sucrose density gradient centrifugation. J Parasitol 68:916–919, 1982.

Dryden MW, Payne PA, Ridley R, Smith V. Comparison of common fecal flotation techniques for the recovery of parasite eggs and oocysts. Vet Therapeutics 6(1), 14 – 28, 2005.

Appendix 1. Flotation Solutions for Helminth Ova a

Flotation Solutions –

• Sugar: 454g / 355ml water ≈1.27 sp. gr. (water must be heated to get sugar into solution). add 2ml 37% formaldehyde (10%formalin)) – as a preservative

o Veterinary Lab Supply: 315 E. Madison, Winterset, Iowa 50273 USA; Toll-Free (800) 325-3144, FAX (515) 462-9207 www.veterinarylabsupply.com

• Fecal Floatation Sheather's Sugar, 1 gallon 4/case $51.00

o Jorgensen Laboratory www.jorvet.com

• Sheather's Sugar Floatation Solution, J1028G 1 gallon $15.60

• Sodium Chloride: 400g / 1000ml water ≈ 1.2 sp. gr.

• Magnesium sulfate: 400g / 1000ml water ≈ 1.2 sp. gr.

• Zinc sulfate: 371g / 1000ml water ≈ 1.18 – 1.2 sp. gr.

• Sodium nitrate: 400g / 1000ml water ≈ 1.18 – 1.2 sp. gr.

1. Swinging Head Centrifuge

Standard Qualitative Fecal:

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

2. Mix with 10ml of sugar solution.

3. Pour through tea strainer into a beaker/fecal cup.

4. Pour solution from beaker/fecal cup into 12ml or 15ml centrifuge tube. (depending on the size the centrifuges uses).

5. Place tube into the centrifuge.

6. Fill tube with sugar solution to a slight positive meniscus and cover with a coverslip. There should be a small bubble under the coverslip if correct amount of flotation solution was added.

7. Centrifuge at 1200rpm for 5 minutes. Make sure the centrifuge is balanced.

8. Let stand for 10 minutes.

9. Remove coverslip from tube and place on slide labeled with the animal name or number.

10. Examine entire coverslip at 10X. Use 40X to identify parasites or eggs.

11. Record results.

2. Fixed Head Centrifuge

Standard Qualitative Fecal:

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

2. Mix with 10ml of sugar solution.

3. Pour through tea strainer into a beaker/fecal cup.

4. Pour solution from beaker/fecal cup into 12ml or 15ml centrifuge tube. (depending on the size the centrifuges uses).

5. Place tube into the centrifuge.

6. Fill tube with sugar solution about 1 inch from the top of the tube. DO NOT place a coverslip on the tube.

7. Centrifuge at 1200rpm for 5 minutes. Make sure the centrifuge is balanced.

8. Remove the test tube from the centrifuge and fill to the top with sugar solution.

9. Place a coverslip on the tube. There should be a small bubble under the coverslip if the correct amount of flotation solution was added.

10. Let Stand for 10 minutes.

11. Remove coverslip from tube and place on slide labeled with the animal name or number.

12. Examine entire coverslip at 10X. Use 40X to identify parasites or eggs.

13. Record results.