Abstract
Objectives
The goal of this study was to determine how frequently Microsporum canis was isolated after 1, 2 and 3 weeks of incubation on dermatophyte culture medium either from untreated cats or cats during treatment.
Methods
This was an observational retrospective study. Toothbrush fungal culture results were examined from two data pools: untreated cats with suspect skin lesions and weekly fungal cultures from cats being treated for dermatophytosis.
Results
Results from 13,772 fungal cultures were reviewed and 2876 (20.9%) were positive for M canis. Of these, 2800 were confirmed as positive within 14 days of incubation and only 76 (2.6%) required >14 days for confirmation of M canis. In pretreatment specimens, 98.2% (1057/1076) of M canis isolates were recovered within 14 days of incubation in specimens from cats not known to have received prior antifungal treatment. For cats receiving treatment, 96.8% (1743/1800) of M canis isolates were recovered within 14 days of incubation. Of the 57 cultures that required >14 days for finalization, 21 required extra incubation time because cultures were grossly abnormal, 12 had concurrent contaminant growth delaying microscopic confirmation and 24 had no growth in the first 14 days. Of these 24, 19 had 1–2 colony-forming units (cfu)/plate and the remaining five plates had 5 to >10 cfu/plate, all with abnormal morphology.
Conclusions and relevance
The findings of this study show that it is not necessary to hold pretreatment or post-treatment fungal cultures for 21 days before finalizing cultures for no growth. Growth requiring >14 days had grossly abnormal morphology.
Introduction
Feline dermatophytosis is a superficial fungal skin disease and the most commonly isolated pathogen is Microsporum canis. The disease is not life threatening and will spontaneously resolve in otherwise healthy cats and kittens. Treatment is recommended to shorten the course of the infection and to limit the spread to other animals and people.
Treatment protocols have been recently reviewed; however, key aspects include the concurrent use of an oral systemic antifungal drug, topical disinfection of the haircoat, reasonable confinement, cleaning of the environment and monitoring of treatment until mycological cure. 1 Mycological cure of cats was first described as two negative fungal cultures by Kaplan and Ajello in 1959, and is considered the standard of care. 2 It is also the standard of care in veterinary medicine to incubate cultures for up to 21 days. 3 Recommendations in human diagnostic laboratories are to incubate fungal cultures for 3–4 weeks with no distinction for various fungal pathogens.4–6 One published study focused on identification of the optimum incubation time for just dermatophytes. In that study, only 16/1128 (1.4%) of human dermatophytes (none M canis) required 17 or more days of incubation. 7
In veterinary medicine it is widely recommended to monitor dermatophyte cultures for 21 days. This recommendation is almost universal but is not based upon any prospective or retrospective studies. The goal of this descriptive retrospective study was to report on how frequently M canis was isolated after 1, 2 and 3 weeks postinoculation on dermatophyte culture medium either from untreated cats or cats during treatment.
Materials and methods
This study was conducted under approved protocols by the University of Wisconsin-Madison.
Study design
In this observational retrospective study, positive dermatophyte cultures from one of the author’s laboratories were examined. The first set of positive fungal culture data examined was from cats being screened for dermatophytosis either at a local animal shelter or during outbreak investigation. The second set of positive fungal culture data was from cats being treated for dermatophytosis at a local animal shelter with an on-site treatment program. All cats were treated using the same treatment protocol: 21 consecutive days of oral itraconazole and concurrent twice weekly lime sulfur rinses, as previously described. 8 Treatment was monitored via weekly toothbrush fungal cultures until mycological cure; ie, two negative consecutive fungal cultures.
Laboratory procedures
Toothbrush fungal cultures were inoculated onto fungal culture plates by gently stabbing the bristles onto the surface of the plate as previously described. 3 Fungal cultures were grown on BBL Mycosel agar (Becton Dickinson) modified with phenol red as a color indicator. Petri dishes (90 mm) were incubated at 25–27°C for 21 days. Plates were examined daily and fungal culture results were recorded at days 7, 14 and 21 postinoculation. Growth was recorded as no growth, contaminant growth, suspect (compatible gross morphology but not confirmed microscopically) or M canis. The number of colony-forming units (cfu) plate were recorded and plates were given a pathogen (P) score of P0 = no pathogens, P1 = 1–4 cfu/plate, P2 = 5–9 cfu/plate or P3 = ≥10 cfu/plate. Descriptive comments regarding colony characteristics (gross or microscopic) were included in the laboratory data. Confirmation of M canis was made via microscopic examination.
Data analysis
Descriptive data were calculated and reported for this observational study. The χ 2 test was used to compare the proportion of dermatophytes isolated before and after 14 days of incubation (P <0.5).
Results
Pooled fungal culture data (n = 13,772) are shown in Figure 1. When all of the positive fungal data was pooled, only 76/2876 (2.6%) fungal cultures required more than 14 days to recover to recover M canis.
Summary of incubation culture data from 13,772 cultures
Results from 8911 screening dermatophyte cultures were available for review from seven animal shelters. Of these, 1076 (12.1%) were culture positive for M canis (Table 1). Six hundred and seventy-four (62.6%) were recovered within the first 7 days, 383 (35.6%) between days 7 and 14, and 19 (1.8%) between days 14 and 21. Suspect growth was reported by day 14 for all 19 specimens requiring an additional 7 days for microscopic confirmation. Overall, 98.2% of M canis isolates were recovered within 14 days of incubation in specimens from cats not known to have received prior antifungal treatment.
Summary of incubation culture data from untreated and treated cats
Data are n (%)
Results from 4861 dermatophyte fungal cultures from treated cats were available for review from November 2006 to December 2016. Of these, 1800 (37.0%) were culture positive for M canis (Table 1). Eight hundred and thirty-six (46.4%) were recovered within the first 7 days, 907 (50.4%) between days 7 and 14 and 57 (3.2%) between days 14 and 21. The difference was significant compared with the untreated group (P <0.02). Overall, 96.8% of M canis isolates were recovered within 14 days of incubation from specimens from cats being monitored for treatment.
In 21/57 post-treatment dermatophyte cultures identified on days 14–21, all of the cultures had gross morphology consistent with M canis but an additional week of incubation was needed before the culture results were finalized. In 24/57 cultures, no growth was present in the first 2 weeks. Nineteen of these dermatophyte cultures had only 1–2 cfu/plate and five had 5 to >10 cfu/plate but all had abnormal gross morphology. In 12/57 dermatophyte cultures, concurrent growth of contaminants delayed microscopic confirmation; one plate grew >10 cfu/plate.
Discussion
The findings of this study show that the incubation times shorter than 21 days are adequate to isolate M canis from screening pretreatment and post-treatment toothbrush fungal cultures (Figure 1). This pathogen was isolated within 14 days of incubation within 98.2%, 96.8% and 97.4% of untreated, treated and pooled data, respectively.
In veterinary medicine the general recommendation is to hold fungal cultures for 3–4 weeks before confirming negative growth, although the authors are not aware of any scientific studies to support this recommendation.3,9 In human medicine, fungal cultures are routinely held for 4 weeks, although some published evidence has demonstrated few clinically relevant results are obtained beyond the third week of culture.4–6 In most cases this recommendation is based upon the need to rule out dimorphic fungi from a clinical specimen. Most recently, a human study showed that only 1.4% (n = 16/1128) of dermatophyte cultures samples were isolated after 17 days of incubation. Furthermore, the strains isolated beyond day 17 were limited to Trichophyton rubrum, Trichophyton mentagrophytes complex and Epidermophyton floccosum. These species rarely cause clinically significant infections in feline patients.1,3 Our study findings are similar to those reported by these authors.
There are several weaknesses of the study. First, this was a retrospective study and, although the toothbrush fungal culture procedure is simple, it is possible that there may have been sampling errors. Second, cultures were performed in a research laboratory routinely performing dermatophyte cultures. A valid concern is whether or not findings would be similar in a point-of-care setting. A recent study comparing point-of-care dermatophyte test medium and mycology laboratory culture results found that when medium was stored, inoculated, incubated and colonies examined properly for gross and microscopic growth there was only a 3% chance of error. 10 However, when gross and microscopic examination was not performed properly there was a significant chance of error (19.4%). Third, with regard to treatment data, the treatment protocol remained consistent with twice-weekly application of lime sulfur solution and a 21 day course of oral itraconazole applied to singly-housed and isolated cats in a shelter environment. The results presented may not be generalizable to alternative treatment protocols.
From a clinical perspective the results of this study are most applicable to finalizing cultures with no growth after 14 days of incubation. In samples collected to rule in or rule out a suspect lesion, only 1.8% (n = 19) required >14 days to finalize fungal culture results. It is important to note that in all cases specimens had evidence of growth by day 14, so if samples are examined routinely false negatives would be unlikely. For post-treatment samples, only 24/1800 fungal cultures first showed growth after day 14 and all had abnormal gross morphology as expected as a result of antifungal therapy. Changes in gross morphology included delayed or absent formation of macroconidia, and slow-growing, highly fragmented hyphae. The clinical relevance of this growth can only be interpreted in conjunction with Wood’s lamp and clinical examination.
The results of this study have a direct impact on a very important aspect of this disease – quality of life. Because feline dermatophytosis is most commonly spread by direct contact, some degree of confinement during treatment is used to minimize spread to other animals or people. Isolation makes provision of behavioral care and enrichment challenging, particularly in a shelter environment. Reducing the isolation period by up to a week would have important positive consequences for cats undergoing treatment, as well as pet owners or animal shelters. Benefits to shelters, which include lower cost of treatment and shorter average time of treatment help save resources that can be directly translated into measurable improvements to animal welfare and life-saving.
Conclusions
The findings of this study show that for toothbrush fungal cultures, fungal cultures can be finalized for M canis if there is no growth after 14 days.
Footnotes
Conflict of interest
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
This work was supported by an unrestricted gift from Maddie’s Fund.