Comparison of carpet and toothbrush techniques for the detection of Microsporum cani s in cats

Abstract

Objectives

The aim of this study was to evaluate the diagnostic concordance between the toothbrush and carpet techniques for the detection of Microsporum canis in cats in a field study.

Methods

Thirty-nine Persian cats from a cattery were used. Fungal culture samples from the haircoat of each cat were collected by stroking the coat with a sterile toothbrush and a 5 × 5 cm-sized sterile carpet square (n = 78 total samples). Specimens were inoculated onto Mycosel Agar and incubated at 25°C for 21 days. Both techniques were compared using the following parameters: number of plates without fungal growth, number of plates with contaminant growth and number of plates positive for dermatophytes.

Results

The feline population in the study cattery was 39. Thirty (77%) were symptomatic and nine (23%) asymptomatic. The diagnosis was made via carpet and toothbrush methods and 78 cultures were performed. On day 21, M canis was detected in all culture plates. No contaminant molds were observed.

Conclusions and relevance

The concordance rate between the carpet and toothbrush techniques among the 78 evaluable culture plates was 100%. Both methods are equally effective for collecting material for M canis culture. Additionally, both techniques are inexpensive and easy to perform in feline clinical practice.

Introduction

Dermatophytosis is a common fungal infection in companion animals, and one of the most important infectious skin diseases that affects cats worldwide. Although other fungal species may be responsible for dermatophytosis, Microsporum canis is considered the main etiologic agent.^1,2 This fungal species remains a major concern because of its contagious and zoonotic behavior, being responsible for transmission of dermatophytosis between animals and from animals to humans.^1–4

Fungal culture has been routinely used to detect dermatophytosis, mostly because it presents high accuracy and allows the identification of dermatophytes. Fungal isolation is performed by inoculation of hair samples and/or skin scales onto Sabouraud dextrose agar or in a specific medium for dermatophytes.^5,6 In this regard, different sampling techniques have been described. One of the most widely used techniques is the Mackenzie toothbrush technique. It is considered easy to perform, reliable and a technique frequently reported in clinical studies. ¹

Even though the toothbrush method is broadly used, other sampling methods, such as hair plucking, carpet square and adhesive tape sampling have been reported. ¹ The carpet method was one of the sampling techniques first described, ⁷ but it remains an underutilized tool. Some studies report its effectiveness; however, to our knowledge, there is no study correlating the carpet method with the toothbrush technique. The carpet method can present some advantages, such as being less expensive and easier to store, and can be a potential alternative to the classic use of toothbrushes. The aim of this study was to evaluate the diagnostic concordance between the toothbrush and carpet methods for the detection of M canis in cats.

Materials and methods

Institutional approval

This study was approved by the Local Animal Care and Ethics Committee of the University of São Paulo (protocol number 854914/0217), and the owners signed written informed consent forms before enrolment of their cats into the study.

Study population

The study included 39 Persian cats from a cattery with a previous history of dermatophytosis.

Sampling procedure

Fungal culture specimen samples containing hairs and skin scales were collected from all cats by two methods: the toothbrush and the carpet method (Figure 1). For each method, the cats were gently rubbed for approximately 3 mins from the head and neck to the dorsum, trunk, ventrum, limbs and tail. Sample collection was performed first using toothbrushes, followed by the squares of carpet.

Figure 1

Toothbrush and square of carpet used for collection of hairs and skin scales. The surface area of the toothbrush was 2 cm² and the carpet area was 5 cm²

The toothbrush technique was performed according to a modified method described by Mackenzie. ⁷ In this regard, soft bristle human toothbrushes (2 × 2 cm) were used for each cat. After sample collection, the toothbrushes were placed in individual plastic bags.

The carpet technique was performed according to the modified method described by Mariat and Adam-Campos. ⁸ In brief, small squares (5 × 5 cm) of carpet, which were previously sterilized in the autoclave for 15 mins at 121°C and individually packed in aluminum foil wrappers, were used to collect the hair and skin scale samples, and then wrapped in their own pack.

Fungal cultures

For both methods, collected samples were processed within 4 h in a mycology reference laboratory. The carpet squares were pressed once onto the center of each culture plate containing a medium for dermatophyte (Mycosel Agar; Becton Dickinson). Similarly, the toothbrushes were pressed onto the surface of the agar by gently stabbing the bristles onto the surface five times. The plates were then incubated at 25°C for 21 days. Identification of dermatophytes was performed by macroscopic evaluation of fungal colonies and direct microscopic observation of the macroconidia.

Comparison of techniques

Both techniques were compared using the following parameters: number of plates without fungal growth; number of plates with contaminant growth (non-dermatophytic contaminant molds); and number of plates positive for dermatophyte. For cultures positive for M canis, colony-forming units (cfu) were counted and recorded for semi-quantitative analysis of fungal culture. Each M canis culture received a pathogen score (P-score) based on cfu/plate using the following scale: P₀ = no growth; P₁= 1–4 cfu/plate; P₂= 5–9 cfu/plate; P₃ = >10 cfu/plate). ¹

Statistical analysis

Student’s t-test was used to compare the results obtained by the two methods (toothbrush and carpet). The number of plates without fungal growth was compared with positive dermatophyte cultures. A P value of <0.05 was considered statistically significant.

Results

A total of 39 Persian cats were examined and 30 cats were symptomatic for dermatophytosis (ranging from focal to generalized hair loss, associated with scaling and erythema) and nine cats had no clinical signs. A total of 78 cultures were performed; the results are summarized in Table 1. There was no growth of contaminant molds on any plate. M canis was identified in all 78 samples: 39 from the carpet method and 39 from the toothbrush method. Additionally, M canis was the only dermatophyte species isolated, and no differences were observed regarding the identification of M canis colonies in both methods. On day 21, all 78 fungal cultures had more than 10 cfu/plate (P-score 3).

Table 1

Fungal cultures obtained using toothbrush and carpet methods

Fungal culture result	Toothbrush method	Carpet method
Number of plates with NDM growth	0	0
Number of plates positive for Microsporum canis	39	39
P-score 0	0	0
P-score 1	0	0
P-score 2	0	0
P-score 3	39	39

P-score 0 = no growth; P-score 1 = 1–4 cfu/plate; P-score 2 = 5–9 cfu/plate; P-score 3 = >10 cfu/plate

NDM = non-dermatophytic mold

In some cultures, more abundant colonies of M canis were observed with the carpet method than with the toothbrush method (Figure 2). However, since we performed a semi-quantitative analysis, it was not possible to quantify the difference with a high degree of accuracy.

Figure 2

Comparison of fungal culture of Microsporum canis obtained by the carpet and toothbrush methods. (a) Animal 01 – toothbrush method; (b) animal 01 – carpet method; (c) animal 02 – toothbrush method; (d) animal 02 – carpet method

Discussion

In this study, we compared the toothbrush and carpet methods for sampling dermatophytosis in cats. Our results showed that both methods are equally effective for the detection of M canis in culture plates. Indeed, there is no difference in the ability to isolate dermatophytes or the rate of isolation of contaminating fungi (non-dermatophytic mold). Although the toothbrush method was performed before the carpet method, the order of collection did not influence the dermatophyte growth. In fact, since the cats were heavily contaminated with a high fungal load (>10 cfu/plate), the amount of infective material was enough for both methods. Furthermore, no differences were observed between the fungal cultures of the two groups of cats. Cats with and without clinical signs presented a similar number of colonies of M canis. Cats from the latter group were classified as asymptomatic because they did not present clinical signs and also presented culture plates with confluent fungal growth and >10 cfu/plate.

Although both techniques have been individually used in several studies in cats with dermatophytosis, a full study comparing agreement between the two methods has not been previously reported. Recently, different protocols for sampling and identifying M canis have been investigated to improve fungal isolation rates. ⁹ Indeed, the toothbrush remains the most commonly used method by sampling dermatophytosis in companion animals. This technique has been broadly used for small animal clinicians in the USA, Canada and in some European countries.^1,4,9 In 1967, the carpet method was reported by Mariat and Adam-Campos, when these authors used small fragments of sterile carpets to collect hair and skin scales from children with tinea capitis. ⁸ Subsequently, this method was adapted to veterinary medicine, and then it was used as the preferred method by clinicians from France and Brazil.^2,10,11 In Brazil, the carpet technique has been widely used for more than three decades as a rapid and inexpensive method with suitable fungal isolation rates.^2,3 In a recent study, the carpet method was used to identify dermatophytosis in 70 Persian cats, with 100% of cultures being positive for M canis. ¹²

In this study, M canis was properly isolated using both methods, which reinforces that the toothbrush and carpet methods could be useful for sampling dermatophytosis. Both methods are easy and safe to perform on cats, but have particular aspects that affect their ease of use. In this regard, while toothbrushes are commercially available, carpets require a sterilization process. However, carpets can present some advantages, such as being a less expensive method and are easier to store. Another possible advantage of carpets is that they can encompass a larger area and could trap more arthrospores. A previous study reported the highest rates of M canis isolation in children with dermatophytosis using a hairbrush method when compared with a toothbrush method. ¹³ The authors suggested that the larger surface area of the hairbrush might be responsible for these findings. ¹³ The limitation of our study is the absence of quantitative analysis, which could provide a better understanding regarding the correlation between surface area and the ability to trap arthrospores.

Conclusions

This study provides evidence that both carpet and toothbrush techniques are effective for sampling hairs and skin scales to use for fungal culture of dermatophytes. There was 100% concordance between the carpet and toothbrush techniques. Indeed, this study revealed toothbrush and carpet technique as suitable and comparable sampling methods for dermatophytosis. Moreover, these methods are easy to perform, and the carpet method can be a reliable alternative for sample collection. Further studies should compare these techniques for sampling animals with low fungal load, such as asymptomatic or mechanical carriers living solitarily in household environments.