ISOLATION OF DERMATOPHYTES FROM INFECTED STRAY DOGS IN SELANGOR, MALAYSIA

1 Department of Pre-Clinical, International Medical School, Management & Science University, Selangor, Malaysia, pm_ridzuan@msu.edu.my 2 Department of Medical Science, International Medical School, Management & Science University, Selangor, Malaysia, drpmridzuan@gmail.com Correspondence Author: P.M. Ridzuan, pm_ridzuan@msu.edu.my / drpmridzuan@gmail.com, Department of Pre-Clinical, International Medical School, Management & Science University, University Drive, Off Persiaran Olahraga, Seksyen 13, 40100 Shah Alam, Selangor, Malaysia


INTRODUCTION
Dermatophytes are one of the most common skin diseases of pets and livestock. Transmissibility among animal communities, public health consequences, high cost of treatment, and difficulty of control measures of animal ringworm explain the importance of these diseases. Ringworm is an important skin disease because it is contagious, infectious, and can be transmitted to people. Dermatophytes in living hosts usually remain in superficial tissues such as the epidermis, hair, and nails. Serious consequences are uncommon, and infections can be self-limiting; however, the illness may be disfiguring and uncomfortable, especially when the lesions are widespread. Infrequently, dermatophytes may invade subcutaneous tissues and very rarely invade other sites, especially in immuno-compromised hosts (K. A. Moriello, Coyner, Paterson, & Mignon, 2017).
The dermatophytes will be transmitted by close direct contact with infected animals or contaminated fomites including arthropods, such as houseflies, and the hair coats of animals. Dermatophytes are subdivided into three main groups: geophilic, zoophilic, and anthropophilic.
Geophilic is a type of dermatophyte or microorganism from soil. Microsporum gypseum is a type of geophilic dermatophyte that most commonly infects dogs or cats. Microsporum gypseum is widespread in warm, humid, tropical and subtropical environments. Zoophilic dermatophytes are found in animal hosts and are rarely found in soil. This type of dermatophyte affects almost 60% dogs, compared to cats with almost 90% (Abdalla, Wisal, 2018). Anthropophilic dermatophytes are adapted to human hosts and cannot survive in soil. Species included in this type are Microsporum audouinii, Trichophyton tonsurans, Trichophyton rubrum, and Epidermophyton floccosum. This species rarely infects dogs or cats that have a history of close contact with infected humans (K. A. Moriello et al., 2017).
The disease burden of chronic-relapsing and therapy-refractory superficial dermatophytosis has dramatically increased in India within the past 5-6 years. A study by Gupta et al (2016) showed that 149 out of the 201 samples (74.13%) showed a dermatophyte-positive culture result. Out of this, 138 (92.62%) samples were identified as Trichophyton (T.) mentagrophytes and 11 (7.38%) as Trichophyton rubrum.
Tinea capitis is a lesion on the scalp that is divided into two parts, which are ectothrix, with follicular destruction, and arthroconidia formation on the outside of hair shafts. This form is often caused by zoophilic species such as M. canis, M. gypseum, T. equinum, and T. vericosum. Endothrix arthroconidia occurs within the hair shaft. This form is caused by anthropophilic species and favus a crusting form of scalp dermatophytosis caused by anthropophilic strains (Hay, 2017).
Dermatophytic infections are very common worldwide with incidence rates increasing every day because of the increasing coexistence of humans and pets. Microsporum canis is the most frequent cause of infection in domestic animals with a high risk of transmission to humans. Dermatophytosis is one of the most frequent skin diseases in dermatologic observance in México. It can affect any sex, age, and any part of the body (Rodríguez, Guzmán, García, Asz-Sigall, & Arenasa, 2018). Dermatophytosis is also the most common pet associated and occupational zoonotic disease, where in the United States almost 2 million people are infected each year due to contact with animals. Dermatophytic infections are common in pediatric and geriatric patients due to decreased host immunity. Diagnosis is easy if performing a good clinic history; a mycological examination is the gold standard because it is quick and cheap (Sahni, Singh, & Dogra, 2018).
Dermatophytosis is a contagious zoonotic disease, the most common mycosis in dogs and cats in the western hemisphere, and can be transmitted to humans (Minarikova, Hauptman, Jeklova, Knotek, & Jekl, 2015;Pasquetti, Min, Scacchetti, Dogliero, & Peano, 2017). Dermatophyte species are classified into three genera: Epidermophyton, Microsporum and Trichophyton (Hayette & Sacheli, 2015). It has been reported that the prevalence of infection varies depending on temperature, humidity, season, and geographical area (Magdum et al., 2016). Although in many countries the epidemiology of dermatophytosis from dogs is well understood, studies of the dermatophytes on stray dogs are limited, especially in Malaysia. Therefore, the aim of this study was to determine the macroscopic and microscopic identification of different types of dermatophytes present on stray dogs.

METHODS
This research was conducted from January to May 2019 in Selangor, Malaysia. Designed as a descriptive study based on laboratory research, samples were collected from an infection site (nail, hair, or skin) as determined by conducting physical examinations of stray dogs which were infected with fungi. Direct physical examination was conducted using a random sampling method.
Sabouraud Dextrose Agar (SDA) was used and prepared using 300 petri dish plates to culture 100 samples. The SDA was prepared by mixing 65 g of SDA powder into 1,000 ml of distilled water. The mixture was autoclaved and poured onto the petri dish under the flame to avoid contamination. The SDA mixtures were then kept in the chiller below 4°C for 24 hours for future use. The samples were cultured on SDA and were incubated at 26°C for two weeks to allow for sample growth. After the incubation period, the samples were stained using the Lactophenol Cotton Blue wet mount technique. Dermatophyte species are known to support gross and microscopic morphology. The macroscopic fungi identification was done by identifying the fungi colony, color, and texture. Microscopic identification was done using a light microscope to identify the fungi morphology.
The collected data were grouped and analyzed univariably. The univariable analysis was displayed in descriptive form with frequency tables. The data were then interpreted and compared with the existing theories, standards, guidelines, and provisions.

RESULTS
A total of 306 samples, which consisted of 102 samples each of nail, skin, and fur were obtained. The direct microscopy showed 63 positive sample results for Trichophyton spp., 11 positive sample results for Aspergillus spp., and 10 positive sample results for Microsporum spp. on a stray dog's hair. Almost similar prevalence was demonstrated on a stray dog's nails and skin. Other dermatophytes that were isolated from dog's skin were Absidia spp. (5%), Alternaria spp. (4%), Epidermaphyton (3%), and 1% for both Bipolaris spp. and Trichoderma. In general, there are various types of dermatophytes that present on a stray dog specimen located on their skin, nails, and fur. The most common type of dermatophyte that affects a dog is Trichophyton spp., which appeared in 198 out of 306 samples (64.7%), and the least common was Ulocladium spp., which appeared in only 1 out of 306 samples (0.3%). A summary of results for isolated dermatophytes from stray dogs from Selangor are represented in Table 2.

DISCUSSION
Several studies on various types of dermatophytes in dogs found that some dermatophytes are the cause of ringworm, namely Microsporum and Trichophyton dermatophytes. The majority of dermatophytes found in dogs were Microsporum canis (70%), while the others were Microsporum gypseum as much as 20% and Trichophyton spp. mentagrofit as much as 10%. Trichophyton is a genus of fungi that are parasitic in nature and can cause tinea diseases including itching in the groin, water fleas, ringworm, and similar infections of the nails, beard, skin, and scalp (K. Moriello, 2019 Animal dermatophytes are communicable to humans (Bennett, Dolin, & Blaser, 2014). Fungal infections in humans because of human or animal contact are relatively uncommon, some of the most common encountered diseases of the integument are dermatomycoses. Human or animal contact may be the source of occasional candida infections, all types of tinea infections, and some other types of superficial or deep fungal infections (Baumgardner, 2017). Asymptomatic animal carriers of Microsporum canis are considered to be important factors in epidemiology. This is because 50% of people in direct contact with infected dogs or cats or arthroconidia found in the environment can contract the disease (Pin, 2017).
Another study conducted in Istanbul, Turkey showed Microsporum canis as the most frequently isolated species followed by Trichophyton spp., M. gypseum, T. mentagrophytes, M. nanum, and Microsporum spp (Sığırcı et al., 2019). Micropsorum canis is the most common pathogen causing dermatophytosis in animals, whereas Microsporum gypseum and T. mentagrophytes are other dermatophytes associated with this infection. These infections are more common during the rainy season and in contact with human patients or pet owners (Murmu et al., 2015).
A study conducted by da Cunha et al (2019) showed that among symptomatic animals, female dogs were the largest contributor to the prevalence of M. canis, namely 77.77% and up to five months of age as much as 77.90%. This suggests that younger animals have a higher susceptibility to contracting infections due to immature immune systems. Regarding sex variable, there are several other studies that show different results that there is no correlation between sex and infection, although some authors report that male dogs have more frequent dermatophyte infections (Mancianti, Nardoni, Cecchi, Corazza, & Taccini, 2002;Seker & Dogan, 2011). Regarding to the age variable, some similar epidemiological data on dermatophytosis in relation to animal age had different results, but researchers agree that young dogs and cats (especially those less than one year old) are susceptible to the development of dermatophytosis (Mancianti, Nardoni, Cecchi, Corazza, & Taccini, 2002). Seker & Dogan (2011) also stated that animal age is a risk factor that has a significant relationship to the incidence of dermatophytosis in animals. The dermatophytosis isolation rate was significantly higher in younger dogs (P <0.05). This is related to immunological immaturity, biochemical exchange of the skin, deficiency of fungistatic sebum or linoleic acid, being in the anagen phase of the hair, or its physiological situation.
Regarding the seasonal or weather variable, this study showed a relatively high isolation rate of dermatophytes in spring and winter, although, there was no correlation between seasons and prevalence of dermatophyte incidence. The results of this study are in line with other studies, which also reported the same thing that there was no conclusive evidence of seasonal variation in the incidence of dog infection, although there were some results that reported a seasonal distribution of the incidence of dermatophyte infection in dogs (Mancianti, Nardoni, Cecchi, Corazza, & Taccini, 2002;Seker & Dogan, 2011).
As mentioned earlier, dermatophyte infections are the most common human fungal infections transmitted from animals, and tinea capitis is the predominant disease of these infections. Domestic animals can transmit dermatologic fungal infections, especially tinea corporis and pedis, by direct contact. Zoophilic T. interdigitale may cause the rare acute ulcerative type of tinea pedis described earlier (Canavan & Elewski, 2015). Malassezia pachydermatis fungemia has occurred as outbreaks in neonatal intensive care units, including one case in which the employee acquired the strain from a household dog (Reiss, Shadomy, & Lyon, 2011). In general, tinea capitis due to zoophilic and geophilic species is a rare infection compared to anthropophilic species. however, they tend to cause more severe inflammatory disease than anthropophilic species. The dominant zoophilic tinea capitis species in North America is M. canis. Kerion can be caused by zoophilic Trichophyton verrucosum or T. mentagrophytes (Hay, 2017).
Anthropophilic dermatophytes isolated in pediatric patients include T. tonsurans (associated with wrestling mats) and Microsoprum audouinii (more common in kindergartens and schools). Zoophilic dermatophytes are the most common types of pathogens found in children and adolescents. This breed is generally sourced from domesticated animals, although there are some from livestock (very rare). Anthropophilic dermatophytes are more common in Germany than Austria. This is because immigration and urban density in Germany is greater than that in Austria. (Hayette & Sacheli, 2015).
According to Gupta et al (2016), the general action to reduce the risk of transmission of fungal organisms is to maintain general hygiene for pets, pet owners, and the environment where both of them (pets and pet owner) live. In addition to paying attention to general hygiene, tinea capitis sufferers are also advised to remove or wash all the combs, brushes, and headgear they use. Prevention of recurrence of onychomycosis after clinical cure can be assisted by periodic application of topical antifungals for plantar and inter-digital nails or feet, decontamination of footwear, close family care, and avoidance of public swimming pools.

Research Limitations
Limitations in this study include time constraints on the collection of data, and the highcost required to perform molecular analysis of the data.

CONCLUSION
In conclusion, this study was performed to find the most common type of dermatophytes present in the stray dog population in Selangor, Malaysia, and conduct an analysis to identify the dermatophytes' burden in stray dogs. This study can assist investigators in understanding the prevalence of dermatophytes burden in stray dogs and help prevent further complication, such as the spread of illness, especially zoonotic infection