In vitro efficacy of shampoos containing miconazole, ketoconazole, climbazole or accelerated hydrogen peroxide against Microsporum canis and Trichophyton species

Abstract

Objectives

The objective was to evaluate the antifungal efficacy of shampoo formulations of ketoconazole, miconazole or climbazole and accelerated hydrogen peroxide wash/rinse against Microsporum canis and Trichophyton species spores.

Methods

Lime sulfur (1:16)-treated control, enilconazole (1:100)-treated control, accelerated hydrogen peroxide (AHP 7%) 1:20 and a 1:10 dilution of shampoo formulations of miconazole 2%, miconazole 2%/chlorhexidine gluconate 2–2.3%, ketoconazole 1%/chlorhexidine 2%, climbazole 0.5%/chlorhexidine 3% and sterile water-untreated control were tested in three experiments. In the first, a suspension of infective spores and hair/scale fragments was incubated with a 1:10, 1:5 and 1:1 dilution of spores to test solutions for 10 mins. In the second, toothbrushes containing infected cat hair in the bristles were soaked and agitated in test solutions for 10 mins, rinsed, dried and then fungal cultured (n = 12×). In the third, a 3 min contact time combined with an AHP rinse was tested (n = 10×). Good efficacy was defined as no growth.

Results

Water controls grew >300 colony-forming units/plate and all toothbrushes were culture-positive prior to testing. For the suspension tests, all test products showed good efficacy. Miconazole 2%, ketoconazole 1% and AHP showed good efficacy after a 10 min contact time. Good efficacy was achieved with a shorter contact time (3 mins) only if combined with an AHP rinse.

Conclusions and relevance

Lime sulfur and enilconazole continued to show good efficacy. In countries or situations where these products cannot be used, shampoos containing ketoconazole, miconazole or climbazole are alternative haircoat disinfectants, with a 10 min contact time or 3 mins if combined with an AHP rinse.

Introduction

Dermatophytosis in cats is most commonly caused by Microsporum canis; however, infections with Trichophyton species can occur. ¹ Treatment involves the use of systemic antifungal drugs, which eradicate the infection within the hair follicle, and concurrent repeated use of topical antifungal therapy for disinfection of the haircoat. ² Although the value of topical therapy is well recognized, compliance with topical therapy treatment recommendations is challenging. The two most widely used whole-body rinses (enilconazole, lime sulfur) have proven efficacy; however, these products are not available and/or approved for cats in all countries. Further complicating the use of topical therapy are negative perceptions or experiences with existing products (ie, odor, time consuming, messy, irritating, stressful for the cat).

Compliance with any therapeutic recommendation is enhanced when options can be tailored for individual situations. Currently, the only alternative option supported by published studies is a 2% chlorhexidine gluconate/2% miconazole shampoo (Malaseb shampoo; DVM TEVA Animal Health).^3–5 This product is most commonly used to treat canine bacterial pyoderma and/or Malassezia species dermatitis in dogs. The importance of maintenance topical therapy for management of Malassezia species dermatitis has resulted in a number of other sole or combination products against this yeast. It is hypothesized that these single or combination products have antifungal efficacy against M canis or Trichophyton species. If so, this would increase topical treatment options for feline dermatophytosis. The goal of this in vitro study was to evaluate the antifungal efficacy of shampoo formulations of ketoconazole, miconazole, accelerated hydrogen peroxide (AHP) or climbazole using a suspension test and infected cat hair challenge model.

Materials and methods

Sources of infective material

Infected, strongly fluorescent Wood’s lamp-positive M canis-infected hairs were obtained from cats with untreated infections. Infected hairs were collected via plucking of Wood’s-positive hairs and toothbrush fungal cultures (1–3 per cat). Trichophyton species was obtained from untreated juvenile hedgehogs with spontaneous untreated infections. Infections were confirmed via fungal culture and microscopic examination.

Isolated infective spore suspension

Infective spore suspensions were prepared as previously described except the suspension was not filtered and contained hair and scale fragments. ⁶ A 100 µl inoculum of each pathogen grew >300 colony-forming units (CFU) per plate.

Fungal culture procedures

Fungal cultures were grown on BBL Mycosel agar (Becton Dickinson) modified with phenol red as a color indicator. Plates were incubated at 28–30°C for 21 days and examined daily for growth and CFU. Confirmation of M canis and Trichophyton species was made via microscopic examination.

Test products

Sterile water was used as an untreated control. Lime sulfur 1:16 (LimePlus Dip; Decra Veterinary Products) and enilconazole 1:100 (Imaverol; Janssen Animal Health) were used as treatment controls. Test shampoos included chlorhexidine 3%/climbazole 0.5% shampoo (Douxo Shampoo Chlorhexidine PS with Climbazole; Sogeval Laboratories), AHP shampoo/rinse 7% (Pure Oxygen Derma Wash; Ogena Solutions), three chlorhexidine gluconate 2–2.3%/ketoconazole 1% shampoos (Equishield Shampoo [Kinetic Lexington]; Ketochlor Shampoo (Virbac Animal Health); Mal-A-Ket Shampoo [Dechra Animal Health]), two chlorhexidine gluconate 2%/miconazole 2% shampoos (Michonahex+Triz shampoo; Dechra Veterinary Products; and Malaseb Shampoo; DVM TEVA Animal Health) and a miconazole 2% shampoo (Miconazole Shampoo; Davis Manufacturing). Similar shampoo formulations by different manufacturers were tested to evaluate different proprietary formulations. Shampoo products were used a 1:10 dilution.

Experimental protocols

Experiment 1: suspension test

Spore suspensions of Microsporum canis and Trichophyton species were tested at a 1:10, 1:5 and 1:1 dilution of spores to product with a contact time of 10 mins. Test solutions were vortexed during the contact time to ensure adequate exposure of spores to product. Aliquots (100 µl) of each test suspension were inoculated on fungal culture plates by spreading the suspension evenly on the surface with a sterile loop. Shampoo products were diluted 1:10 dilution for this study. All testing was repeated six times.

Experiment 2: Microsporum canis-infected cat hair challenge (10 min contact time)

Whole cat hair was tested as follows. Toothbrushes containing hair in the bristles from culture positive and Wood’s lamp-positive cats were used. Each toothbrush head acted as its own control; immediately before testing two impressions from each toothbrush were inoculated onto a fungal culture plate. Immediately afterward, the entire toothbrush was placed in a sterile 50 ml test tube with 40 ml test compound and agitated for 10 mins. The toothbrush was rinsed in sterile water and allowed to dry for 24 h before repeat fungal culture. Shampoos were tested as a group and prepared just prior to testing. This was repeated 12 times; 132 toothbrushes were used in this experiment. Data from toothbrushes without a positive pretreatment fungal culture was not used. Trichophyton species toothbrush samples were not available for testing.

Experiment 3: infected cat hair challenge with a 3 min contact time

Experiment 2 was repeated except that the contact time was 3 mins and immediately after the second culturing all of the toothbrushes were thoroughly wetted with a 1:20 AHP solution, allowed to dry and then cultured again. (Each toothbrush had three cultures: pretreatment, post-treatment with a 3 min contact time and a post-AHP rinse treatment.) Tests were repeated 10 times; 110 toothbrushes were used in this experiment.

Data analysis

Descriptive data were collected. The number of CFUs per plate was counted. For the purposes of this study good efficacy was defined as no growth. Fungal culture growth was considered delayed if it was noted between days 14 and 21 of culture.

This study had the appropriate institutional approval.

Results

Experiment 1

Untreated controls grew >300 CFU/plate and the treated controls (enilconazole and lime sulfur) inhibited all growth (Table 1). All products were considered to have good efficacy.

Table 1.

Number of culture-positive plates at each testing dilution

		Spore to test dilution concentration
Compound	Commercial product	1:10	1:5	1:1
Lime sulfur 1:16	LimePlus Dip-treated control	0	0	0
Enilconazole 1:100	Imaverol-treated control	0	0	0
Sterile water	Untreated control	6	6	6
Climbazole 0.5%/chlorhexidine 3%	Douxo Shampoo Chlorhexidine PS with Climbazole	0	0	0
Acclerated hydrogen peroxide 1:20 (7%)	Pure Oxygen Derma Wash	0	0	0
Miconazole nitrate 2%	Miconazole Shampoo	0	0	0
Miconazole nitrate 2%, chlorhexidine gluconate 2%	Malaseb Shampoo	0	0	0
Miconazole nitrate 2%, chlorhexidine gluconate 2%, TrizEDTA	Michonahex+Triz	0	0	0
Ketoconazole 1%, chlorhexidine gluconate 2%	Equishield Shampoo	0	0	0
Ketoconazole 1%, chlorhexidine gluconate 2.3%	Ketochlor Shampoo	0	0	0
Ketoconazole 1%, chlorhexidine gluconate 2%, 2% acetic acid	Mal-A-Ket Shampoo	0	0	0

Unless otherwise indicated, products were tested at a 1:10 dilution

Experiments 2 and 3

All pretreatment toothbrushes were culture-positive for M canis within less than 7 days of incubation; no data were discarded. Excluding the 12 untreated controls, after a 10 min contact time 11/120 treated toothbrushes were culture positive (Table 2). The range of CFU/plate was 1–7. Three products had no growth on all toothbrushes post-treatment and three additional products had one positive toothbrush with 1 CFU/plate each at the site of bristle impressions. Given the robust challenge of this model and that enilconazole had one positive post-treatment toothbrush with 1 CFU/plate, six products were considered to have good efficacy when used alone (Table 2). After a 3 min contact time, excluding the 10 untreated controls, 33/100 treated toothbrushes were culture positive (Table 2). With the exception of lime sulfur and enilconazole, all test products had at least two post-treatment culture-positive toothbrushes. Miconazole shampoo had 7/10 culture-positive toothbrushes. Three products had post-treatment cultures with >10 CFU/plate (Malaket Plus [n = 3], Ketochlor [n = 1] and Miconazole [n = 1]) (Table 2). All positive fungal cultures showed delayed growth. In addition, abnormal gross and microscopic colony morphology was noted. In experiment 3, all toothbrushes, including the untreated controls, were culture negative post-AHP rinse. A 3 min contact time combined with a leave-on AHP rinse had good efficacy.

Table 2.

Cat hair challenge

Compound	Commercial product	10 mins	3 mins	3 min/AHP rinse
Lime sulfur 1:16	LimePlus Dip-treated control	0	0	0
Enilconazole 1:100	Imaverol-treated control	1 (1 CFU/plate)	0	0
Sterile water	Untreated control	12	10	0
Climbazole 0.5%/chlorhexidine 3%	Douxo Shampoo Chlorhexidine PS with Climbazole	4 (1–4 CFU/plate)	5 (1–4 CFU/plate)	0
AHP 1:20 (7%) †	Pure Oxygen Derma Wash	1 (1 CFU/plate)	2 (2–5 CFU/plate)	0
Miconazole nitrate 2%	Miconazole Shampoo	1 (1 CFU/plate)	7 (1–20 CFU/plate)*	0
Miconazole nitrate 2%, chlorhexidine gluconate 2%	Malaseb Shampoo	0	4 (1–4 CFU/plate)	0
Miconazole nitrate 2%, chlorhexidine gluconate 2%, TrizEDTA	Michonahex+Triz	0	3 (1–10 CFU/plate)	0
Ketoconazole 1%, chlorhexidine gluconate 2%	Equishield Shampoo	1 (1 CFU/plate)	4 (1–10 CFU/plate)	0
Ketoconazole 1%, chlorhexidine gluconate 2.3%	Ketochlor Shampoo	3 (1–7 CFU/plate)	4 (1 to >10 CFU/plate)*	0
Ketoconazole 1%, chlorhexidine gluconate 2%, 2% acetic acid	Mal-A-Ket Shampoo	0	4 (2 to >10 CFU/plate)*	0

Data show the number of culture-positive plates after 10 mins, 3 mins and a 3 min/AHP rinse

^*Products that had post-treatment cultures with >10 CFU/plate (Malaket Plus [n = 3], Ketochlor [n = 1] and Miconazole [n = 1])

^†Product concentration is currently 3.5%

AHP = accelerated hydrogen peroxide; CFU = colony-forming unit

Discussion

The findings in this study showed that antifungal shampoos or rinses containing chlorhexidine combined with either miconazole, ketoconazole or climbazole, miconazole alone or 7% AHP diluted 1:20 had antifungal efficacy against Trichophyton species or M canis and may be suitable choices as haircoat disinfectants. The spore suspension test was a robust challenge because it contained infective spores, hair/scale fragments and organic debris. In addition, the challenge increased from a dilution of spores to test product from 1:10 to 1:5 to 1:1. When tested in a dilute solution of 1:10, all of the products showed good efficacy with a 10 min contact time against both M canis and Trichophyton species. Using the toothbrush culture cat hair challenge, which more closely simulated a ‘real cat’, products formulated as shampoos containing miconazole 2% and ketoconazole 1% had similar efficacy to enilconazole and lime sulfur when used with a 10 min contact time. None of the products had good efficacy as defined by study parameters when tested with a 3 min contact time. It is interesting to note that fungal colony growth was delayed and that both the gross and microscopic growth were abnormal. This suggests that even though the spores grew they were less viable as a result of direct contact with the active ingredients, even for short periods of time.

A shorter contact time (3 mins) was tested for several reasons. Firstly, topical rinses and shampoo are inherently stressful for both cat and owner. A 3 min contact time seemed an achievable bath time for most pet cats. Secondly, a shorter time would most likely decrease an owner’s direct exposure to untreated infective hairs, thus helping minimize disease transmission. Thirdly, a shorter contact time minimized the softening hairs making them easier to macerate. Soaking of infective hairs is a critical part of preparing an infective spore suspension. The greater the length of time infected hairs are soaked in water, the more friable the hairs become and the easier it is to macerate them with simple mechanical manipulation (ie, crushing with an instrument) thus releasing spores into the water suspension. Moisture and microtrauma are important parts of the pathogenesis of dermatophytosis. It is conceivable that the combination of bathing and lathering with subsequent release of spores from friable hairs may be a risk factor for development of new lesions elsewhere on the cat. It could be argued that shampooing should be avoided for this reason and topical antifungal rinses used instead.

The AHP product is not formulated as a shampoo but as a rinse. The product does not lather as ‘richly’ as products formulated for shampoo use; however, it is adequate for cleansing purposes. Two previous studies have shown that this product has good efficacy against dermatophyte spores when tested in a suspension model.^7,8 In the current study, the product had good efficacy when tested as a ‘shampoo’ with a 10 min contact time and showed similar trends to other products when tested with a 3 min contact time. It showed good efficacy when used as a leave-on rinse; the water-treated controls were all culture negative in experiment 3. The use of this product as a rinse alternative to lime sulfur or enilconazole needs to take into account that AHP breaks down to water vapor and oxygen when drying and it does not have residual activity. (Note: since conducting this AHP product has been reformulated from a 7% to 3.5% concentrate and a comparable dilution would be 1:10.)

There are several weaknesses in this study. Firstly, in vitro findings may or may not reflect what happens in vivo. Contact time and thoroughness of application were controlled in this study and are two important variables when used in a clinical setting. Secondly, the testing model of the cat hair challenge may have over- or underestimated the efficacy of some products in spite of efforts to minimize this. For this trial, efforts were made to keep the toothbrush challenges for a particular treatment trial similar. All of the pretreatment toothbrushes were examined for the amount of hair in the toothbrushes, the presence of positive Wood’s lamp hairs, source (a litter vs single cats) and severity of the infection. After collecting this information toothbrushes were divided so that shampoos were all tested in a similar treatment trial against a similar challenge, for example large amounts of hair, easily found Wood’s lamp-positive hairs and so on. This may account for some of the culture-positive differences noted between the 10 and 3 min contact time. Another possible explanation for these differences was the 1:10 dilution of products. This dilution was selected because it rinsed easily from the toothbrushes, thus minimizing concern about residual shampoo being deposited onto the surface of fungal culture plates, inhibiting culture growth and hence increasing the chance of false negatives. Thirdly, post-treatment sampling methods for the cat hair challenge may have influenced the results. Toothbrush fungal cultures are inoculated by pressing the bristles into the plate with the assumption that the results are representative of the entire toothbrush. It is possible that culture-positive hairs were trapped in the bristles but not detected. Because of these anticipated weaknesses, each shampoo was tested 10–12 times. Experiments 2 and 3 were repeated only 10 times because of a lack of test toothbrushes.

It is important to remember the pathogenesis of dermatophytosis when selecting a topical antifungal treatment and to provide thorough information on its use and expectations. Systemic therapy targets the infection within the hair follicle which is the source of the infective spores. Until the infection is eradicated within hair follicles, the haircoat will continually be reseeded with infective spores. In addition, the haircoat will remain culture positive long after the infection is eradicated from hair follicles because of infected extrafollicular hairs. Topical antifungal treatments are used concurrently with a systemic antifungal drug, applied several times weekly, and need to be continued for at least several weeks after completion of the systemic antifungal therapy. Use of reasonable barrier protection (ie, gloves) is recommended with all products. Simple combing of the haircoat to remove easily shed hairs is recommended prior to any topical therapy to enhance penetration of the product on the haircoat. These easily shed hairs are most fragile and their removal will help minimize contact with infective material. Products should be thoroughly applied and the coat wetted for the appropriate time (leave-on) or shampoo contact time, but efforts should be made to avoid excess ‘scrubbing’ of the haircoat to prevent maceration of hairs. Because shampoo products are rinsed from the haircoat, it is not expected that these products will have residual activity. AHP has good antifungal efficacy but it does not have residual activity. This needs to be considered when selecting haircoat disinfectant rinses.

In my opinion the therapeutic goal is to find a topical therapy protocol that minimizes the risk of disease transmission. The ‘best’ product is one that an owner will use and the cat can tolerate. The goal of this study was not to prove which product was ‘best’ but to identify options for more flexible topical therapy options. For example, it is reasonable to consider recommending a combination protocol that uses enilconazole or lime sulfur in the early stages of treatment and, when clinical cure is apparent, use a topical shampoo formulated for use against Malassezia species until mycological cure is reached. The use of a shampoo formulation as a first-choice topical therapy option is supported not only by the findings in this study, but also by field studies that have shown that miconazole based shampoo therapy are efficacious as haircoat disinfectants.^3,9 The use of an AHP rinse post-shampoo or use of this product as an alternative rinse alone is yet another treatment option.

Conclusions

Lime sulfur and enilconazole continued to show good efficacy. In countries or situations where these products cannot be used, shampoos containing ketoconazole, miconazole or climbazole are alternative hair coat disinfectants when used with a 10 min contact time, or a 3 min contact time if combined with an AHP rinse. The use of AHP alone is a possible hair coat rinse alternative.