One vs two negative fungal cultures to confirm mycological cure in shelter cats treated for Microsporum canis dermatophytosis: a retrospective study

Abstract

Objectives

The aim of this study was to determine how often one negative fungal culture (FC) was indicative of mycological cure (MC) when compared with two negative consecutive FCs in cats treated for Microsporum canis dermatophytosis.

Methods

In this retrospective study, weekly FC data from shelter cats treated for M canis dermatophytosis were reviewed.

Results

Complete records from 371 cats were reviewed. The first negative FC was indicative of MC in 335 (90.3%) cats. In this group, all cats were otherwise healthy and either had obvious lesions (n = 237) or no lesions or evidence of resolving lesions (n = 98). In the 36 cats in which the first negative culture was not indicative of MC, there were two clinical subgroups. The first consisted of healthy but lesional cats (n = 19) that had one negative FC within the first 3 weeks of treatment followed by one or more positive FCs. The most likely explanation was sampling error. These cats went on to cure and the next negative FC was indicative of MC. In the second clinical group, cats were lesional but had concurrent medical problems (n = 17). These cats showed an initial good response to treatment (lesion resolution and an initial negative FC). However, this negative FC was followed by at least one strongly positive FC (>10 colony-forming units/plate) before proceeding to cure. These cats took the longest time to cure (mean 11 weeks; range 8–28 weeks). MC occurred after resolution of the concurrent health issues. There was very good agreement between using one negative FC vs two negative FCs for the determination of MC in healthy cats (kappa = 0.903)

Conclusions and relevance

In cats where there has been high compliance with environmental cleaning, as well as topical and systemic treatment recommendations, two consecutive negative FCs may not be necessary to determine MC. The first negative FC in an otherwise healthy cat is likely indicative of MC. Good sampling technique is needed to avoid false-negative FC results.

Introduction

Feline dermatophytosis is a superficial fungal skin disease and the most commonly isolated pathogen is Microsporum canis. Current treatment recommendations include the use of oral non-compounded itraconazole, ¹ topical antifungal therapy to disinfect hairs, ² routine cleaning of the premises to minimize fomite contamination, which can interfere with determination of mycological cure (MC), ² limited confinement and treatment monitoring. ² Toothbrush fungal culture is the most commonly used test for treatment monitoring and when performed properly, this point-of-care diagnostic test has less than a 3% error rate vs a reference laboratory. ³

The endpoint of treatment of ‘mycological cure’ was introduced into the veterinary literature from a 1959 study describing the use of griseofulvin in the treatment of Persian cats (n = 22) with chronic dermatophytosis. ⁴ In that study MC was defined as ‘two successive negative cultures taken at 2 weeks apart’. To our knowledge, this recommendation has not been examined by any recent study. Much has changed since 1959 in the treatment of feline dermatophytosis (eg, oral itraconazole and topical therapy). The objective of this study was to determine how often the first negative fungal culture (FC) was indicative of MC when compared with two negative consecutive FCs in cats treated for M canis dermatophytosis.

Materials and methods

This study was conducted under approved protocols by the University of Wisconsin–Madison and Dane County Humane Society, Madison, WI, USA.

Study design

In this observational retrospective study, clinical and FC data from cats in a dermatophyte treatment program at an animal shelter were reviewed (2006–2016). All cats in the treatment program were treated with at least 21 consecutive days of oral itraconazole and concurrent twice-weekly lime sulfur rinses, as previously described. ⁵ Cats were housed in large cages and/or in cages that connected. Litters of kittens were not separated and not all cats were housed individually. MC was defined as two negative consecutive FCs obtained at weekly intervals, with FC finalized at day 21 post inoculation. During this waiting period, weekly FCs continued to be taken and inoculated. By the time of the second negative FC at day 42, each cat had four additional FCs in progress, which were all held and finalized at day 21.

Laboratory procedures

FCs were processed in the laboratory of one of the authors (KAM). All FCs were inoculated onto BBL Mycosel agar (Becton Dickinson) modified with phenol red as a color indicator, incubated at 25–27°C for 21 days, examined daily and final weekly data recorded on days 7, 14 and 21, as previously described.

Data analysis

Descriptive statistics were calculated and reported for this study, including the mean and range of the number of weeks of treatment for each of the clinical groups. Agreement between two testing methods (one vs two negative FCs for MC) was evaluated using the kappa (κ) test. The χ ² test was used to compare the proportion of healthy cats vs sick cats where MC was identified by one negative FC using a 2 × 2 contingency table (P <0.05). Statistical analysis of data was performed with Prism (GraphPad Software).

Results

During the 10-year study period, data were available for 570 cats. Data from 199 cats were discarded for one or more of the following reasons: cats were uninfected FC-negative littermates, there was incomplete admission or treatment data, there was isolation of a non-M canis pathogen, cats were transferred to foster care to complete treatment or cats were admitted for completion of treatment started elsewhere. Complete data for 371 cats were reviewed. All 371 cats had M canis-positive FCs (⩾5 colony-forming units [cfu]/plate) at the time of admission to the treatment facility. A summary of response to treatment data is shown in Table 1. Four clinical patterns of treatment response were noted, referred here to as ‘groups’.

Table 1

Treatment related data from 371 cats

Patterns of response to treatment	Mean time to cure (weeks)	Range	Comments
Group 1 (n = 237)	4.3	3–12	Otherwise healthy but lesional – simple infections
Group 2 (n = 98)	2.0	2	Otherwise healthy, no apparent lesions – fomite or had suspect lesions or occasional Wood’s lamp-positive hairs on the face
All 335 cats	3.6	2–12
Group 3 (n = 19)	7.3	4–13	Otherwise healthy, lesional, but had one negative fungal culture early in treatment
Group 4 (n = 17)	11.9	8–28	Dermatophytosis and concurrent systemic illness, had intermittent negative cultures followed by positive cultures, cure occurred only after resolution of medical illness
All 36 cats	9.4	4–28
All 371 cats	4.2	2–28

In 335/371 (90.3%) cats, the first negative FC was indicative of MC and all subsequent fungal cultures remained negative (n = 5; ie, all follow-up cultures were negative). Examination of admission and FC data revealed two subgroups. Group 1 consisted of 237 highly suspect (lesional, positive Wood’s lamp fluorescence) but otherwise healthy cats. Group 2 consisted of 98 cats that were FC negative after 1 week. Intake notes revealed them to be otherwise healthy but considered high risk because they were either admitted with or exposed to another infected cat (n = 62/98) or had suspect skin lesions on the nose, ears and/or face (n = 36/98). In 18/36 cats with skin lesions, fluorescent hairs were noted.

In 36/371 cats (9.7%), the first negative FC was not indicative of MC. These cats had one or more positive FCs, with at least one FC having >10 cfu/plate. Again, two subgroups were noted. Group 3 consisted of 19 cats that had a single negative FC within the first 3 weeks of treatment, followed by one or more FCs having >10 cfu/plate. Excluding this initial early negative FC, group 3 cats were cured but required longer treatment times (Table 1), for unknown reasons. Group 4 consisted of 17 cats that showed an initial good response to treatment (decreasing cfu/plate along with clinical recovery) but during treatment had at least one positive culture with >10 cfu/plate following the initial negative FC. These cats took the longest to cure (Table 1). Intake notes described these cats as being ‘unthrifty’, having concurrent upper respiratory infection, poor body condition or otherwise physiologically stressed at the time of admission. Once their underlying medical problems resolved, cats achieved MC and all follow-up FCs remained negative.

Identification of MC by one negative FC was significantly more common in cats that were otherwise healthy than in cats with illnesses (P = 0.001). When calculating agreement between using one negative FC or two negative FCs to determinate MC, there was very good agreement (κ = 0.903 ± 0.016 SEM; 95% confidence interval 0.872–0.934).

Discussion

One of the most interesting and unexpected findings was the large number of cats (n = 335/371; 90.3%) in which the first negative FC was sufficient to predict MC. These cats were otherwise healthy and responded quickly to treatment and had ‘simple infections’. ⁶ A subgroup of these cats (group 2; n = 98) most likely consisted of either ‘fomite carriers’ ⁶ or cats with resolving infections at the time of admission, as their first post-treatment FCs were negative. A resolving infection would explain why 18 of these cats had small foci of positive Wood’s lamp fluorescence; residual positive fluorescence on hair tips is common in cats cured of M canis infections.

In the 36/371 (9.7%) cats where the first negative FC was followed by a positive MC, the findings were equally interesting. Human error is a likely explanation for the single outlier negative FC (n = 19 cats). One possibility is that an error was made in inoculation of the toothbrush FC. Another is that the haircoat was inadequately sampled as this negative FC occurred within the first few weeks of treatment, before clinical cure. These cats went on to be cured without event and the next negative FC was indicative of MC as in groups 1 and 2. In this study, there were 17 cats with concurrent medical illness and dermatophytosis. These cats had more prolonged treatment periods and waxing and waning numbers of cfu/plate with intermittent negative FCs. These cats did not achieve MC until their physiological health was normal. Infected cats with concurrent illnesses have been described as having ‘complicated infections’. ⁶

The question of when to FC, how frequently and how many FCs are needed will not be easily solved, even though there was good agreement between using one vs two negative FCs to identify MC. A recent consensus statement suggests FCs should begin once cats have achieved clinical cure (ie, are lesion-free with no fluorescing hair shafts). ² The current label recommendations for oral itraconazole recommend a 6-week treatment protocol, ⁷ and these study findings suggest the following cost-effective approaches for shelter cats.

1. For otherwise healthy but lesional cats: obtain the first post-treatment FC 1 week after completing treatment unless clinical findings suggest active infection. If FC is negative, assume the cat has achieved MC. If FC is positive, continue treatment and obtain the next FC 4–6 weeks later.

2. For lesion-free FC-positive cats or cats exposed to high-risk cats, obtain the first post-treatment FC after 1 week of treatment to determine if the original positive FC was due to fomite carriage.

3. In cats that are ill at the time of diagnosis or otherwise unthrifty, the first post-treatment FC is best delayed until there is both clinical cure and resolution of underlying medical problems.

The data were collected over a long period of time. A major strength of the study is the large number of cats, that the same treatment protocol was used, and that all FCs were processed in the same laboratory by the same investigator. It is difficult to extrapolate individual animal medicine to shelters and vice versa. One explanation for the results in this study is the fact it was conducted in a shelter where there was good compliance with current recommended treatment protocols. Whether or not these results are representative of home-treated cats is unknown, but with good client education likely.

Conclusions

In otherwise healthy cats where there has been high compliance with treatment recommendations, the findings in this study found that the first negative FC was indicative of MC in 90% of cats undergoing treatment for M canis dermatophytosis.