An evaluation of the use of ronidazole for the treatment of Tritrichomonas foetus in cats

PICO question 
In cats infected with Tritrichomonas foetus, does treatment with oral ronidazole compared to an alternative antiprotozoal treatment or placebo result in successful resolution of clinical signs and eradication of disease? 
Clinical bottom line 
Ronidazole use appears to be efficacious in eradicating infection with Tritrichomonas foetus and resolving diarrhoea associated with infection. A dose range of 30–50 mg/kg 12–24 hourly has been suggested, with evidence suggesting that a dose of 30 mg/kg 24 hourly for 14 days may be effective. However, some cats may require higher doses and some may not respond to treatment, and relapse may occur during a protracted period following completion of the treatment course. Neurological side effects appear to be uncommon but may occur with doses of 30 mg/kg and above. 
A total of six studies are reviewed: Three randomised, controlled studies, one cohort study and two case series (one retrospective). Findings indicate efficacy of ronidazole treatment in eradicating infection and resolving diarrhoea, however many studies involved small sample sizes and limited follow-up. Therefore, evidence to support the use of ronidazole in Tritrichomonas foetus infected cats remains relatively limited. 
  


• Treatment group received ronidazole at 30 mg/kg once daily for 14 days in guar gum coated capsule formulation, placebo received capsule without ronidazole.

Study design: Randomised, controlled, double-blinded study
Outcome studied: • Repeat rectal swab undertaken after 14 days treatment, repeat PCR undertaken.
• Cats deemed still infected if PCR detected T. foetus presence (objective assessment).
• Clinical examination undertaken and presence of adverse effects recorded (subjective assessment).
Limitations: • Adverse effects of treatment are mentioned in the introduction, and the authors have stated that any 'adverse effects' were recorded; however, they do not describe which specific adverse effects were being screened for in methods. • Dose of ronidazole administered is alluded to in the introduction, but there is no explanation in the method section as to why the specific dose/dosing schedule used in this study was selected. • Final PCR undertaken at end of 14 day treatment course, no longer-term follow-up once treatment stopped. • 4/25 (16%) of treated cats were still infected at the end of the study, however cats appear to have not been kept separately during the treatment phase and were in a multi-cat scenario. Therefore it is difficult to know whether these cats were still infected due to treatment failure, resistance of T. foetus to ronidazole or reinfection. • Infection with T. foetus confirmed in adult cat via direct microscopy, faecal culture and PCR on faecal sample. Organisms obtained to use in in vitro susceptibility study.
• Adult cat treated with 10 mg/kg ronidazole q 24 hours for 10 days.
• Specific-pathogen free kittens randomly split into two equal groups (n=5). Health determined during initial 3 week acclimatisation period via clinical examination, biochemistry, haematology and urinalysis. Faeces examined for enteric pathogens via faecal flotation, direct microscopy, microbial culture, antigen testing for Giardia spp. and PCR for detection of T. foetus ribonucleic acid (RNA) • Kittens experimentally infected via orogastric intubation with T. foetus in specifically prepared medium containing approximately 3 x 10 6 live T. foetus.
• Treatment initiated 4 weeks after experimentally induced infection. Kittens received either ronidazole (10 mg/kg) or dextrose (placebo) twice daily q 12 hours via colour coded gel capsules for 14 days.
Study design: Randomised, controlled study (specific-pathogen free kittens) Outcome studied: • PCR undertaken on faeces weekly for 6 weeks to determine presence of T. foetus (objective).
• Cats with residual T. foetus still present given treatment with higher dose ronidazole 30 or 50 mg/kg q 12 hours for 14 days. Weekly PCR undertaken on faeces for minimum of 20 weeks.
• Presence of adverse effects (subjective). • Kitten placebo group: all remained positive.
• All relapsed and placebo-treated cats were re-treated with either 30 or 50 mg/kg ronidazole q 12 hours for 14 days. Resolution of infection documented. No relapse identified in follow-up period of 30 and 21-23 weeks respectively. • No adverse events reported during or after treatment in all groups. Subjectively softer faeces were reported in cats while receiving 50 mg/kg ronidazole.
Limitations: • Unclear why the single naturally infected cat was included in the study. • Small sample size of 10 specific-pathogen free kittens, no power calculation included. • Study not fully blinded as colour coded capsules used to differentiate those containing ronidazole from placebo. • Unclear why a low dose of ronidazole was initially utilised, then two different higher doses (30 mg/kg or 50 mg/kg) were used when relapse occurred or to treat infected kittens originally administered with the placebo.

Sample size: 11 cats
Intervention details: • 8/11 cats in a single cattery with diarrhoea and positive diagnosis of T. foetus on faecal culture.

Study design: Case series
Outcome studied: • Resolution of diarrhoea (subjective) • Faecal culture for presence of T. foetus (objective) • In vitro susceptibility to ronidazole in aerobic and anaerobic conditions (objective).

Main findings: (relevant to PICO question):
• Treatment with one (seven cats) or two (one cat) courses of ronidazole 35-45 mg/kg q 12 hourly for 14 days resulted in repeatedly negative faecal cultures for T. foetus in six cats and resolution of diarrhoea. • Infection was still present in 2/8 cats. Repeat courses of metronidazole (20-32 mg/kg q 24 hours, unknown duration), tinidazole (50 mg/kg q 24 hours for 14 days) and ronidazole (35-60 mg/kg q 12 hours for 14 days) were administered to these cats over the course of 1 year. • Multiple courses of metronidazole, tinidazole and ronidazole did not clear T. foetus infection suggesting in vivo resistance. • After 1 year of treatment, infection was still not eradicated in 2/8 cats. Faeces from these two cats was collected and in vitro susceptibility to ronidazole tested. • In vitro resistance of T. foetus to ronidazole demonstrated in aerobic conditions. • No weight loss, inappetence or neurological signs observed. One cat vomited three times in first 24 hours of treatment although unclear which drug was being administered at this point.

Limitations:
• In vivo part of study not controlled or blinded.
• Small sample size.
• Resistance of T. foetus to ronidazole in vivo assumed from nonresponse to treatment, however small cohort examined (n=2). • Cats included in study were blanket treated with different drug courses/combinations rather than a defined treatment regimen. • Susceptibility of T. foetus to ronidazole in aerobic conditions suggested in this study, however this was only demonstrated in one cat. Further studies required to substantiate this claim.

Koster et al. (2015)
Population: Medical records of domestic cats presented to two veterinary clinics in Hong Kong over a 5 year period.
Only cases with complete medical record and confirmed T. foetus diagnosis (history, clinical examination and details of diagnostic testing) included.

Sample size: 29 cats
Intervention details: • Diagnosis of T. foetus made by combination of faecal smear microscopy and faecal PCR. • All cats treated with 30 mg/kg ronidazole q 24 hourly for 14 days.
• Response to ronidazole treatment at recommended dosing schedule (objective). • Presence of co-infection with Giardia spp. (objective).
Limitations: • Retrospective study of medical records, therefore lack of control group and not, randomised or blinded. • Bias in signalment may be present as owned cats presenting to hospital/clinic reflect overrepresentation of popular breeds in Hong Kong.
• No discussion of any side effects of treatment.
• Only the resolution of clinical signs was used as a measure of ronidazole treatment success, no further faecal testing to ensure resolution of T. foetus (or Giardia spp.) infection appears to have been undertaken. Co-infection with Giardia spp. cannot be ruled out as a contributing factor for apparent treatment failure. • Kittens divided into two groups (n=3), sedated and administered media containing T. foetus trophozoites via feeding tube.
• Infection with T. foetus confimed via faecal examination of trophozoites by day 20 post inoculation. • Faecal smear, culture from rectal swab and PCR undertaken once weekly for following 4 weeks.

Study design: Randomised controlled, non-blinded study
Outcome studied: • Presence of diarrhoea following experimental infection (subjective). • Presence of T. foetus in faeces during treatment period and 4 week follow-up (objective).

Main findings: (relevant to PICO question):
• No diarrhoea was observed in any cat following experimental infection. • Treatment group: All cats negative for T. foetus during treatment period and 4 week follow-up period. • Placebo group: All cats remained positive for T. foetus throughout the study.
• Blinding difficult to achieve as empty capsules used for placebo.
• No discussion of why higher dose/dosing schedule than in previous studies was used in this study. • No discussion of any side effects that may or may not have been seen with higher dose of ronidazole administered. • Short-term follow-up period of 4 weeks, treatment appears effective during the immediate post-treatment period, however it cannot be determined whether ronidazole administration at this dose is effective at preventing relapse over longer periods.

Reinert et al. (2016)
Population: Abyssinian cats. Two male, five female cats with intermittent large bowel diarrhoea despite treatment with fenbendazole.

Sample size: Seven cats
Intervention details: • T. foetus diagnosed via faecal PCR.
• Five remaining cats treated with 30 mg/kg ronidazole q 24 hourly for 14 days.
• Cats kept separately in isolation and given physical examinations and neurological examinations daily.
• Repeat PCR undertaken on faeces over period of 345-800 days.
• Two cats lost through euthanasia as result of other conditions. Cats euthanised following T. foetus diagnosis but prior to starting treatment with ronidazole.

Study design: Cohort study
Outcome studied: • Presence of T. foetus in faeces via PCR in follow-up period (objective).
• Presence of side effects of treatment (subjective).

Main findings: (relevant to PICO question):
• Neurological side effects seen in two cats, treatment paused for one day then restarted. • No evidence of T. foetus (via faecal PCR) in any cat during up to 800 day follow-up, infection deemed eliminated. • 2/7 cats initially diagnosed with T. foetus lost to follow up due to euthanasia.

Limitations:
• No discussion about presence/improvement of diarrhoea over follow-up period. • No discussion as to why ronidazole dose chosen was used.
• Small sample size.

Appraisal, application and reflection
There are few papers examining the in vivo efficacy of oral ronidazole treatment in cats infected with Tritrichomonas foetus. Of these papers, half were randomised, controlled, blinded studies comparing the use of oral ronidazole with placebo, however the remaining half were case series or cohort studies. Therefore, there is a significant amount of lower quality evidence present. One major problem present in most of the papers is that a small sample size was utilised. Although many results were significant, this may mean that the results are not representative of a wider population and conclusions may not be robust.
Infection with T. foetus was induced experimentally via orogastric intubation in two of the randomised, controlled studies (Gookin et al. 2006 andLim et al. 2012), therefore the results of these studies may not be truly reflective of naturally occurring infection.
There is significant variation in the treatment protocols described in the studies. All three randomised, controlled, blinded studies utilised different doses and dosing schedules of ronidazole. Grellet  Koster et al. (2015) presented a retrospective case series also describing the use of 30 mg/kg ronidazole q 24 hours for 14 days in cats presented to a hospital environment with T. foetus confirmed via faecal smear microscopy and PCR. This dosing schedule was found to successfully resolve diarrhoea in 24/29 cats, however confirmation of resolution of infection via faecal PCR was not undertaken and there was no follow-up to determine whether relapse occurred.
The use of 30 mg/kg ronidazole q 24 hours for 14 days was also documented by Reinert et al. (2016) in a small cohort study and found to be successful in eradicating infection (demonstrated via faecal PCR) and preventing relapse up to 800 days following treatment completion. Presence of diarrhoea was not described. However, this was a small cohort study of five treated cats with no control or comparator group to substantiate the result.
A confounding factor for both the Koster et al. (2015) case series and Grellet et al. (2017) randomised, controlled study is that treated cats which continued to live in a multi-cat household environment were included. In both studies, a proportion of cats did not respond to treatment/remained infected, however it is impossible to know why treatment failure occurred as this could be a demonstration of resistance of T. foetus to ronidazole therapy, or simply reinfection. Therefore, the use of 30 mg/kg ronidazole q 24 hours for 14 days may be a useful initial treatment regimen in eradicating infection with T. foetus and resolution of diarrhoea, with a successful result documented in the randomised, controlled study by Grellet et al. (2017) and supported by the cohort study by Reinert et al. (2016) and case series by Koster et al. (2015). However further case controlled, analytical studies should be undertaken to substantiate this. The case series and cohort studies are subject to bias and the result of the randomised, controlled study is complicated by the test and placebo cats not being housed separately during treatment, leading to possible reinfection. Ronidazole appeared to be superior in efficacy against T. foetus compared to metronidazole and tinidazole (Gookin et al. 2006 andGookin et al. 2010) and was effective in eradicating infection in cats previously treated unsuccessfully with fenbendazole in the small cohort study by Reinert et al. (2016). However, two cats did not respond to repeated ronidazole administration in the case series by Gookin et al. (2010). Resistance in vivo was suggested, and in vitro resistance was demonstrated in aerobic conditions in one cat, although further studies on larger numbers of cats are required to validate this finding.
Ronidazole is suggested to have a narrow therapeutic range and neurological side effects are documented to occur with treatment (Gookin et al., 2017), however these were infrequently evidenced in this knowledge summary, with only Reinert et al. (2016) observing effects in 2 cats which resolved on pausing treatment. Lim et al. (2012) specifically described the use of high dose 50 mg/kg ronidazole 12 hourly for 14 days. This was found to be successful in eradicating infection on faecal analysis, however the presence of any neurological side effects was unfortunately not discussed. Gookin et al. (2006) also administered doses up to 50 mg/kg twice daily and no adverse effects were seen.

Conclusions
There is a small amount of good quality evidence for the use of ronidazole to treat cats with diarrhoea as a result of Tritrichomonas foetus infection, and a range of therapeutic protocols have been described. A dose of 30-50 mg/kg administered once to twice daily for 14 days appears to be successful in eradicating infection and may successfully prevent relapse of infection for up to 800 days, although the evidence for this is limited. Neurological side effects may be observed in cats treated with doses of 30 mg/kg and above. Resistance of T. foetus to ronidazole has been suggested (Gookin et al., 2010), however additional work is needed to substantiate this further.
The small body of evidence currently available would suggest that the use of ronidazole is efficacious in treating T. foetus in cats and does resolve the associated diarrhoea, supporting the use of ronidazole in clinical practice in cats with diarrhoea and T. foetus confirmed via PCR/faecal culture. Based on this, the use of ronidazole as an off-license medication is therefore justified in the absence of a licensed alternative. The studies by Importantly it appears that some cats may not respond to treatment and relapse may occur in a protracted period post-treatment, therefore patients should be monitored for this occurring.