Fecal PCR test for Tritrichomonas foetus

General information

Tritrichomonas foetus is an important cause of diarrhea in cats. This assay amplifies the DNA of T. foetus in feces and has been shown to detect as few as 10 organisms per 1 gram of feces. All PCR reactions are performed with positive and negative controls and a restriction enzyme digest is performed on positive samples to ensure the amplified DNA sequence is specific to T. foetus.

For this test we will require up to 1 gram of fresh feces. To increase the probability of detecting infection, the cat should be sampled when it is having diarrhea. The fecal sample can be collected from a spontaneously voided stool, using a fecal loop, or using the colonic saline flush technique (see below). The sample must be free from cat litter as some components of certain cat litters may inhibit PCR reactions. Collect the fecal sample as promptly as possible. However, once the fecal sample has been collected it can be stored in a refrigerator for up to one week prior to shipping. Please ship cooled samples overnight with a gel ice pack (the samples do not have to be frozen but these precautions will help prevent the samples getting overheated during transit). The turnaround time for this assay is 1-2 business days after receipt of the sample at the Gastrointestinal Laboratory.

General information about Tritrichomonas foetus

When evaluating feline patients with diarrhea it is essential to include infectious etiologies on the list of differential diagnoses. One such infectious organism is Tritrichomonas foetus, a flagellated protozoal parasite that is usually associated with venereal trichomoniasis in cattle. Recently, T. foetus has also been identified as an intestinal pathogen in cats. Diarrhea has been reported in cats after both experimental and natural infection. Although the true prevalence of T. foetus infection in cats is unknown, it is suspected to be relatively high. In one study, 31% of 117 cats examined at an international cat show were infected with T. foetus.

While cats of any age, breed, or sex can be infected, young cats that are densely housed (e.g., cats in catteries, animal shelters, or multi-cat households) seem to be at increased risk. Infection is most commonly seen in young cats (i.e., those that are less than 12 months of age), but older cats may also be infected. T. foetus primarily colonizes the surface of the colonic mucosa, leading to chronic large bowel diarrhea. Without appropriate treatment cats usually remain persistently infected. While diarrhea may spontaneously resolve, cats will often experience recurrent bouts of diarrhea after being exposed to stress. Cats infected with T. foetus generally appear healthy but show an increased frequency of defecation with loose to liquid stools, which may contain blood and/or mucus. Fecal incontinence is also frequently observed. The anal region often appears edematous and may become painful with severe diarrhea. A rectal prolapse may occur in some cases.

Who should be tested?

In general, kittens and young cats from multi-cat environments with chronic diarrhea should be tested for T. foetus infection. However, it should be noted that cats in single cat households and older cats can also get the disease, so cats with clinical signs that do not have another obvious cause for their diarrhea should also be tested. Furthermore, cats that are unresponsive to treatment for suspected Giardia spp. infection should be tested, as the two parasites are sometimes confused on routine fecal smear evaluation.

Diagnosis

Diagnosis of a T. foetus infection can be made by identification of tritrichomonads on a direct fecal smear examination, fecal culture, PCR analysis of fecal material, or by colonic mucosal biopsy. Disadvantages of direct fecal smear examination include a low sensitivity (14%), low specificity (T. foetus can be misdiagnosed as Giardia spp. or the nonpathogenic Pentatrichomonas hominis), and the fact that only fresh fecal samples can be used.

Tritrichomonas foetus can also be cultured in-house using the commercially available culture system In Pouch™ TF (Biomed Diagnostics, San Jose, CA). Pouches should be inoculated with less than 0.1 g of freshly voided feces and then incubated at 25°C. Pouches need to be evaluated under a microscope every couple of days. Results are usually obtained between 1 and 11 days of setting up a pouch. Although fecal culture is more sensitive than direct fecal smear examination, difficulties in interpretation of results, the necessity of using freshly voided feces, and the fact that results might not be available for up to 11 days, are important disadvantages of this method.

Tritrichomonas foetus DNA can be amplified from fecal samples by PCR. PCR has been shown to be the most sensitive method for detecting T. foetus in fecal samples, and is ideally suited for direct diagnosis of T. foetus infection. Advantages of PCR testing compared to culture include a higher sensitivity, faster turnaround time, and easier handling and storing of samples because DNA is relatively stable under various temperature conditions. Using the colonic saline flush technique (http://www.ncsu.edu/project/cvm_gookin/buttflush.mov ) or a fecal loop to obtain a fecal sample directly may help increase the probability of detecting T. foetus. The sediment of the sample of fluid obtained from a colonic saline flush should be separated by centrifugation or allowing it to settle. This material is then submitted for PCR. Ideally, fecal samples should be collected when the cat is having diarrhea.

Therapy

Until recently, a successful treatment strategy for T. foetus infections in cats was not available. However, studies have shown that administration of ronidazole may be effective in both resolving diarrhea and eradicating T. foetus. Based on a study of the pharmacokinetics of ronidazole in cats the current treatment recommendation is a dose of 30 mg/kg by mouth once daily for 14 days. Retesting may be performed 2 weeks after the last dose of ronidazole. Apparent treatment failure may be due to lack of patient or owner compliance, administration of an inappropriate dose or duration of ronidazole, reinfection from the environment or from other cats, or infection with a ronidazole resistant strain of T. foetus. Ronidazole can cause neurotoxicosis in cats so patients should be monitored carefully during treatment and treatment should be discontinued if any neurological or other clinical signs develop. Some infected cats do not have diarrhea so testing non-diarrheic cats in the same household is warranted. Where possible, in multi-cat households separation of infected and non-infected cats is advised. Infection is via the fecal-oral route and environmental contamination may be important in the epidemiology of T. foetus. A recent study documented trophozoite survival in water, urine, and cat food but not cat litter. Further information on T. foetus for cat owners is available from Dr. Jody Gookin's website ( http://www.cvm.ncsu.edu/docs/personnel/gookin_jody.html ).

Several compounding pharmacies compound ronidazole. Some examples are Diamondback Drugs (phone: 1-866-646-2223; http://www.diamondbackdrugs.com ), Pet Health Pharmacy (phone: 1-800-742-0516), Abrams Royal Pharmacy (phone: 1-800-458-0804), Westlab Pharmacy (phone: 1-800-493-7852), and Creative Compounding (phone: 1-800-672-2177), but other compounding pharmacies may also carry ronidazole.

References

1. Foster DM, Gookin JL, Poore MF, et al. Outcome of cats with diarrhea and Tritrichomonas foetus infection. JAVMA 2004; 225:888-892.
2. Gookin JL, Stebbins ME, et al. Prevalence of and risk factors for feline Tritrichomonas foetus and giardia infection. J Clin Microbiol 2004; 42:2707-2710.
3. Gookin JL, Birkenheuer AJ, et al. Single-tube nested PCR for detection of Tritrichomonas foetus in feline feces. J Clin Microbiol 2002; 40:4126-4130.
4. Gookin JL, Copple CN, Papich MG, et al. Efficacy of ronidazole for treatment of feline Tritrichomonas foetus infection. JVIM 2006; 20:536-543.
5. LeVine DN, Papich MG, Gookin JL, et al. Ronidazole pharmacokinetics after intravenous and oral intermediate-release capsule administration in healthy cats. JFMS 2011; 12:244-250.
6. Gookin JL, Stauffer SH, Dybas D, Cannon DH. Documentation of in vivo and in vitro aerobic resistance of feline Tritrichomonas foetus isolates to ronidazole. JVIM 2010; 24:1003-1007.
7. Rosado TW, Specht A, Marks SL. Neurotoxicosis in 4 cats receiving ronidazole. JVIM 2007; 21:328-321.
8. Rosypal AC, Ripley A, Stockdale Walden HD, et al. Survival of Tritrichomonas foetus in water, cat urine, cat food, and cat litter. Vet Parasit 2012; 185:279-281.

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