In Vitro Susceptibility Testing of Different Antimycotics and Disinfectants against Microsporum canis Isolated from Clinical Cases of Feline Dermatophytosis

Pets carry many diseases which spread readily to other species including humans, hence are of zoonotic importance. Dermatophytosis is among the readily spread conditions to human population which is caused by fungal species living on the superficial layers of the skin causing ringworm in animals whereas tinea and onycomycosis in human. As the species invade the non-living skin layers (stratum corneum), hair and adnexa superficially, clinical signs involving erythema, excoriation, encrustation, hyperkeratosis and alopecia leading to bacterial dermatitis are common (Fehr 2015; Carlotti et al., 2010). M. canis is main causative agent of clinical dermatophytosis in humans, cats and dogs with prevalence of>50%, >80% and >75% respectively. The condition is spread by clinically affected as well as asymptomatic cats and fomite carriage of spores (Cafarchia et al., 2006).

Many antimycotic agents are available commercially for the treatment of the said condition but due to concurrent infections and inadequate response towards these agents resistance has developed (Nweze et al., 2010; Singh et al., 2007; Galuppi et al., 2010; Agarwal et al., 2015). Although different methods have been developed to evaluate the efficacy of anti-mycotics for yeast and mould testing but no specific method has yet been devised for dermatophytes (Agarwal et al., 2015; Fothergill, 2012). Reports suggest that Agar Based disk diffusion methods can be a reliable cost-effective method for selection of target antimycotic agents (Nweze et al., 2010; Agarwal et al., 2015; Khadka et al., 2017). Furthermore, due to fomite carriage from affected environment the condition persists and for effective control of the condition it is necessary to treat patient and its environment simultaneously. Keeping the need of time in consideration and lack of any available data in Pakistan for the resistance and efficacy of antimycotics and disinfectants respectively, this study was designed to check in vitro efficacy of commonly used antimycotic agents and disinfectants in the treatment and disinfection of M. canis in cats.

Sample collection
 
A total of 30 feline (Felis silvestris catus) patients were randomly selected for this study, from the Pet Centre clinic, UVAS, Lahore. Cats having typical clinical lesions for dermatophytosis and green fluorescence on UV (ultra-violet) light exposure were selected (Fig 1).



Samples were collected by mechanical debridement method (skin scrapping) by sterile surgical blades from lesions treated with 70% alcohol to avoid any chances of bacterial contamination. All samples were labeled, treated and processed individually as and when isolated, as mentioned by Nweze et al., (2010).
 
Laboratory processing
 
The skin scraping samples were inoculated on Sabouraud dextrose agar (SDA)1 in petri plates and incubated at 30°C for 07 days. After incubation the colonies growing in the petri plates were evaluated on the basis of their visual appearance and characteristics (roughly wooly colonies with feathery texture) (Fig 2). M. canis colonies were picked from the culture and shifted to another clean SDA petri plate and incubated again for purification (07 days at 30°C). A 01 mL of sterile physiological saline (0.9% NaCl)2 was poured on the purified culture colonies within the petri plate and probed with the tip of sterile Pasteur pipette to harvest a mixture of conidia and hyphae (mycelium). The conidial suspension was adjusted at 1.0x106 conidia/mL by hemocytometer (Fig 3).




 
Testing for antimycotics
 
This suspension was then used for streaking onto the Mueller Hinton Agar (MHA)3 in 100x15 mm petri plates by sterile swabs dipped in the solution and streaking the plates in 3 rotations to cover all area of petri plates. Then the petri plates were left ajar and turned upside down for 3 minutes till all the moisture was absorbed by the agar. The drug-impregnated-disks3 Itraconazole (10 µg), Fluconazole (25 µg), Ketoconazole (30 µg), Clotrimazole (10 µg), Miconazole (10 µg) were applied on the agar by red-heated sterile forceps, followed by incubation at 30°C for 05 days to allow growth of dermatophytes. Sterile disks were used as negative control. All steps were processed in biosafety cabinets class II and the samples were run in duplicates. Inhibition zone diameters were measured from one end to the other end through the disk by the help of measuring scale; the results were recorded in millimeters (Fig 4).


 
Testing of disinfectants
 
A 75 µL of same conidial suspension was suspended with an equal volume (75 µL) of each of following disinfectant to get a final volume of 150 µL. Each sample was tested for every disinfectant individually on separate petri plates:
 
• 5% aqueous solution of phenol
• Over the counter Hydrogen peroxide (6%)4 diluted with equal volumes of sterile water to get the final percentage of 3%.
• Sterile water5. 
• Sodium Hypochlorite or household bleach6 was used in 1:10 dilution.
• Physiological Saline was used as a negative control.

These conidial-disinfectant suspensions were incubated at room temperature for 5 minutes (Moriello 2015; Moriello et al., 2013) and were used for streaking on the Dermatophyte Test Medium (DTM)3 petri plates, in 3 directions to cover all area. The plates were monitored daily for the growth of any colony until 12 days of incubation at 28°C. All samples were run in duplicates. 

Antimycotic testing
 
The results were interpreted on the basis of inhibition zone diameters (IZD) measured in millimeters according to Keyvan et al., (2009) and Nweze et al., (2010). The tabulated results of the IZDs at day 05 of incubation are mentioned in the Table 1.



The mean IZD of Itraconazole measuring 31.70±2.21 mm showed significant efficacy (P<0.05) against M. canis. These results were well in accordance with Perea et al., (2001). But contrary to this, Agarwal et al., (2015) documented resistance of M. canis against Itraconazole as well as Fluconazole. On the other hand non-significant results were recorded for Fluconazole having IZD mean 4.76±1.17 mm (P>0.05) which indicated lack of in vitro efficacy. The smaller IZDs were contributed by resistance against the species. The same results were reported by (Galuppi et al., 2010; Singh et al., 2007; Murmu et al., 2017). Multiple studies carried out in different regions suggest overall resistance of the species against Fluconazole. But significant results (P<0.05) were obtained for Ketoconazole with mean IZD 7.30±1.5 mm. The same findings have been reported earlier by Khadka et al., (2017) and Keyvan et al., (2009) in contrary to findings of Agarwal et al., (2015). The change in the opinions among the authors can be validated on the basis of geographical regions, use of specific drugs in those regions and developed resistance by the strain of dermatophyte. M. canis was also susceptible to Miconazole validated on the basis of significant efficacy (P<0.05) with mean IZD of 12.10±1.51 mm that was in accordance with the findings of Nweze et al., (2010). Khadka et al., (2017). Significant results of Clotrimazole IZDs were obtained measuring 10.03±1.63 mm which is in line with the findings of Keyvan et al., (2009) (P<0.05). The data suggested that efficacies of the antimycotics vary in the real time clinical practice as Itraconazole had maximum antimycotic effect followed by Miconazole, Clotrimazole and Ketoconazole, whereas resistance was recorded against Fluconazole. The sterile disks were used as negative controls and did not inhibit the growth of colonies (Fig 5).



In the study overall percentage share of efficacy for Itraconazole was 48%, 19% for Miconazole, 15% for Clotrimazole, 11% for Ketoconazole whereas for Fluconazole only 7% was recorded (Fig 6).



This study also showed that Agar Based Disk Diffusion method can be utilized to demonstrate the antimycotic susceptibility testing which must be performed prior to start of treatment to avoid treatment failures and persistence of the condition on account of developed resistance.
 
Disinfectant testing
 
In disinfectant testing, the numbers of colony forming units (CFUs) per plate were counted. The disinfectant in the petri plates showing too many colony units to count was considered ineffective whereas disinfectant in plates containing 1-10 colonies per plate was considered effective (Moriello 2015; Moriello and Hondzo 2014). Significant results were recorded (P<0.05) as all the samples suspended with 5% Phenol, 3% Hydrogen Peroxide and 1:10 Sodium hypochlorite bleach solution did not grow any colony on DTM. The mean number of colonies 27.86 ± 5.19 was observed for sterile water which showed non-significant efficacy towards M. canis (P>0.05). These findings were in consistence with the finding reported earlier by Moriello et al., (2004) and Moriello (2015). Physiological saline was used as negative control. The results indicate that 5% Phenol solution, 3% Hydrogen Peroxide and 1:10 Bleach solution are effective against M. canis conidia whereas sterile water without any antimycotic agent has no inhibitory effect on the growth of M. canis conidia (Fig 7).

It can be concluded that the Agar based disk diffusion method can be cost effective and easily available test for determination of in vitro efficacy against M. canis. Furthermore, the treatment should be initiated on the basis of results of antimycotic sensitivity testing. Both the treatment of patient and disinfection of environment should be performed in order to control the further spread of the condition.

The study was partially supported by Pet Centre, University of Veterinary and Animal Sciences, Lahore.     

This study confers no conflict of interest.