Quench the Stench - Maximizing the Diagnostic Yield of the Faecal Examination
World Small Animal Veterinary Association World Congress Proceedings, 2013
Stanley L. Marks, BVSc, PhD, DACVIM (Internal Medicine, Oncology), DACVN
School of Veterinary Medicine, University of California-Davis, Davis, CA, USA

Parasite-associated diarrhoea in dogs and cats is one of the most common maladies facing small animal veterinarians; however, the diagnosis of these organisms can be challenging. Although immunologic and molecular assays are becoming increasingly available to aid in the diagnosis of protozoal infections, direct microscopic examination of faeces remains the mainstay of attaining a diagnosis in many animals. The techniques used most commonly include direct faecal smear (wet preparation) and a faecal floatation.

Collection and Storage of Faeces

Fresh faecal specimens (< 1 h) should be examined, whenever possible, to maximize the likelihood of identifying a parasite. At least 5 g of faeces should be placed in a clean, dry, airtight container. If samples cannot be examined immediately, they should be refrigerated (up to 1 week) to preserve the integrity of ova, oocysts and cysts. Protozoal trophozoites must be examined right away in fresh faeces, and the specimen should not be refrigerated. One can preserve the viability of trophozoites for several hours by adding 3 mL of saline to 3–5 g of faeces.

Direct Wet Preparation

Direct wet preparations are used to evaluate the smear for the presence of trophozoites, such as Giardia spp. and Tritrichomonas foetus. A small amount of faeces (peppercorn size) is placed on a warm slide and a drop of 0.9% saline is mixed with the faeces. It is important that the smear not be too thick, as trophozoites will be easily missed. After application of a coverslip, the smear is evaluated systematically for motile organisms using the 10X magnification, with confirmation at 40X magnification.

Stained Faecal Smear

The addition of iodine to the wet mount through the edge of the coverslip may aid in the visualization of internal structures of some protozoa. One must examine the direct wet preparation without any stain for motility first, because staining the preparation kills the organism. Methylene blue is useful for identifying trophozoites, particularly those of Entamoeba histolytica. Stained fecal smears are of little to no diagnostic value for the diagnosis of bacteria-associated diarrhoea.

Faecal Floatation

Faecal floatations are indicated to find cysts, oocysts, and ova in faeces. Although standing (gravitational) floatation methods are easier and quicker to perform than centrifugation floatation, the latter has clearly superior sensitivity. Solutions used in centrifugation floatation methods include zinc sulfate and Sheather's sugar.

Procedure for Single Centrifugal Floatation

1.  Prepare a faecal emulsion using 2–5 grams of faeces.

2.  Mix faeces with approximately 10 ml of floatation solution. For zinc sulfate solution, the optimal specific gravity is 1.18–1.20.

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

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

5.  Fill tube with floatation solution so that a slight positive meniscus forms.

6.  Place a coverslip on the tube, and put the tube in the centrifuge.

7.  Ensure that the centrifuge is balanced.

8.  Centrifuge at 1,200 rpm (280 x g) for 5 minutes.

9.  Remove the tube and let stand for 10 minutes.

10.  Remove the coverslip and place on a clean slide.

11.  Examine entire coverslip systematically at 100X magnification (10X objective). Use 40X objective lens to confirm diagnosis by visualizing internal structures and measuring the organism.

Parasitic Causes of Diarrhoea

Tritrichomonas foetus

Tritrichomonas foetus, the primary causative agent of bovine trichomoniasis, has recently been recognized as a protozoal pathogen in cats, although the organism has also been associated with diarrhoea in puppies. Infected cats are generally young, but have ranged in age from 3 months to 13 years (median 9 months). The prevalence of T. foetus infection at an international cat show was found to be 31% (36 out of 117 cats), with 28 out of 89 catteries affected. Co-infection by T. foetus and Giardia was common and was documented in 12% of cats. Misdiagnosis of Giardia is common in cats having T. foetus infection, and may explain why cats diagnosed with apparent Giardia infection do not respond to appropriate therapy.

Clinical Signs

Tritrichomonas foetus infection in kittens can be associated with chronic or recurrent large intestinal diarrhoea characterized by increased mucus, tenesmus, occasional haematochezia, and increased frequency. The anus is frequently red, swollen, and painful, and faecal incontinence is not uncommon. Most animals are otherwise healthy with a normal appetite. Tritrichomonas foetus can also be cultured from the faeces of asymptomatic animals, many of whom will not develop diarrhoea.

Diagnosis

1.  Multiple direct wet preparations on diarrhoeic faecal specimens
Direct faecal smears are performed to evaluate for the presence of Tritrichomonas foetus trophozoites. The sensitivity of direct faecal smear examination for diagnosis of T. foetus is relatively low in cats with spontaneous disease (14%). T. foetus can be distinguished from Giardia based on morphology and motility. Giardia trophozoites have a concave ventral disc and motility mimicking a falling leaf, whereas trichomonads are spindle-shaped, have an undulating membrane that courses the entire length of the body, and move in a more irregular and jerky fashion. T. foetus does not have a cyst stage, underscoring the limitations of faecal floatation for diagnosing this organism.

2.  Faecal cultures performed with an InPouch TF kit
A commercially available system marketed for diagnosis of T. foetus infection in cattle and cats (InPouch TF, Biomed Diagnostics, White City, Oregon, USA) is more sensitive than the direct wet preparation. Approximately 0.05 gram (less than a peppercorn) of freshly voided faeces can be placed in the InPouch for culture, or alternatively, a saline-moistened cotton-tipped swab can be placed in the rectum and then gently agitated in the InPouch for culture. The InPouch should be incubated at 37°C for 24 h, followed by storage at room temperature in an upright position in the dark, and examined q 48 hours for up to 10 days for motile trophozoites using a 20 or 40X objective. The InPouches can also be stored at room temperature for the entire incubation period if one does not have access to an incubator; however, a brief incubation period usually results in more rapid growth of the trophozoites and a potentially earlier diagnosis. The media in the InPouch does not support the growth of Giardia or Pentatrichomonas hominis according to the manufacturer.

3.  Single-tube nested PCR of DNA extracted from faeces
A sensitive and specific single-tube nested PCR from feline or canine faeces has been described. The PCR test is more sensitive than faecal culture and tested positive in 55% of cultures that were negative for T. foetus, even when faeces were normal.

4.  Histopathology and immunohistopathology
Histopathologic changes in colonic mucosal biopsies from infected cats have revealed a lymphoplasmacytic colitis with a significant neutrophilic component. The intestinal epithelium is frequently attenuated, and immunohistochemistry can be utilized to detect the trichomonads on the surface epithelium and within crypts.

Cryptosporidium spp.

Infection with the ubiquitous protozoan parasites, Cryptosporidium parvum and C. felis, in puppies and kittens can cause a spectrum of disease, ranging from asymptomatic carrier state to mild, transient diarrhoea, or prolonged, severe, life-threatening malabsorption syndrome or inflammatory bowel disease.

Diagnosis

The laboratory detection of this ubiquitous protozoan parasite in diarrhoeic animals is difficult, predominantly because the organism is so small (average 4.6 × 4.0 µm) and difficult to find in faecal specimens via light microscopy, and because faecal shedding may be intermittent. Acid-fast staining should always be done following fecal floatation to increase the diagnostic yield of finding this parasite. Immunofluorescent (IFA) detection procedures are more sensitive and specific than acid-fast stains, and are generally the method of choice for morphological diagnosis in humans; however, this method is not practical in private hospitals. Reference laboratories can usually perform IFAs on faeces for Cryptosporidium, and faecal specimens can be transported in formalin (5%) to these labs for IFA testing. The newer enzyme immunoassays designed to detect Cryptosporidium antigens in faeces, have proven more sensitive than microscopic techniques. The ProSpecT Microplate ELISA is one of the most sensitive diagnostic tests (89% sensitivity) for Cryptosporidium spp.; however, the ProSpecT Rapid ELISA is highly insensitive (15% sensitivity) and should not be utilized.

Giardia spp.

Giardia infection in adult dogs and cats is often subclinical or associated with a transient softening of the stool early in the infection; however, acute diarrhoea tends to occur in puppies and kittens shortly after infection.

Diagnosis

Microscopic diagnosis of Giardia infection can be difficult, because cysts and trophozoites may be shed intermittently and because cysts are so delicate. Many artifacts (grass pollen, yeast, etc.) mimic to varying degrees the morphology of Giardia cysts, and care must be exercised in differentiating these from Giardia. Direct fluorescent antibody assay (DFA) is often the standard against which other tests for Giardia are measured. The Merifluor® Cryptosporidium/Giardia assay (Meridian Diagnostics, Inc., Cincinnati, OH) uses a fluorescein isothiocyanate (FITC)-labeled monoclonal antibody directed against cell wall antigens of Giardia cysts in the stool; however, a fluorescent microscope is necessary to perform this test. Enzyme immunoassays are relatively simple to perform and do not require morphological identification of the cyst or oocyst, thus saving technician time and potentially avoiding false negative interpretations. A novel SNAP® Giardia Test Kit (IDEXX Laboratories, Inc., Westbrook, ME) for detection of GCWP 1 in canine and feline faeces was recently released, and shown to have a sensitivity of 85.3% in cats when compared to the DFA test. The sensitivity improved to 97.8% when the test was used in parallel with faecal flotation, underscoring the value of this immunoassay in the clinical setting.

Quiz

1.  Giardia duodenalis is a common cause of diarrhea in dogs and cats. Which one of the following choices is not used to diagnose Giardia in dogs or cats?

a.  Faecal ELISA for Giardia

b.  Faecal fluorescent antibody for Giardia

c.  Zinc sulfate centrifugation floatation on faeces

d.  Faecal Baermann test

2.  Which one of the following diagnostic tests is not a reliable test to differentiate Giardia spp. from Tritrichomonas foetus?

a.  Faecal ELISA for Giardia

b.  PCR on faeces for T. foetus

c.  Direct wet smear on faeces

d.  Centrifugation fecal floatation

3.  Which of the following diagnostic tests can be commonly performed in veterinary hospitals for diagnosing Cryptosporidium spp?

a.  PCR on faeces

b.  Direct wet smear on faeces

c.  Fecal floatation followed by acid-fast stain

d.  Fecal culture

4.  Which one of the following statements is the most accurate regarding processing of faecal specimens?

a.  If faecal samples cannot be immediately examined, they should be frozen for up to a week

b.  Faecal samples for identification of protozoa can be refrigerated for up to 48 hours before examination

c.  One can preserve the viability of trophozoites for several hours by adding 3 mL of formalin to 3–5 g of faeces

d.  Fresh faecal specimens (< 1 hour) should be examined whenever possible

5.  Faecal flotations should best be done using which one of the following applications?

a.  Centrifugation floatation

b.  Gravitational floatation

c.  Using zinc sulfate with a specific gravity of 1.35 or greater

d.  Using sodium chloride as the floatation solution

References

1.  Dryden MW, Payne PA, Smith V. Accurate diagnosis of Giardia spp. and proper fecal examination procedures. Vet Ther. 2006;7(1):4–14.

2.  Gookin JL, Stebbins ME, et al. Prevalence of and risk factors for feline Tritrichomonas foetus and Giardia infection. J Clin Microbiol. 2004;42(6):2707–2710.

3.  Marks SL, Hanson TE, Melli AC. Comparison of direct immunofluorescence, modified acid-fast staining, and enzyme immunoassay techniques for detection of Cryptosporidium spp. in naturally exposed kittens. J Am Vet Med Assoc. 2004;225:1549–1553.

4.  Mekaru S, Marks SL, et al. Comparison of direct immunofluorescence, immunoassays, and fecal flotation for detection of Cryptosporidium spp. and Giardia spp. in naturally exposed cats in 4 Northern California animal shelters. J Vet Intern Med. 2007;21(5):959–965.

  

Speaker Information
(click the speaker's name to view other papers and abstracts submitted by this speaker)

Stanley L. Marks, BVSc, PhD, DACVIM (Internal Medicine, Oncology), DACVN
School of Veterinary Medicine
University of California-Davis
Davis, CA, USA


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