LITHUANIAN UNIVERSITY OF HEALTH SCIENCES
VETERINARY ACADEMY
Marija Ivaškien÷
PHARMACOLOGICAL AND CLINICAL
EVALUATION OF TOPICALLY ADMINISTERED
CREAMS E-1 AND T-1 IN THE TREATMENT OF
DERMATOPHYTOSIS
Summary of Doctoral Dissertation
Biomedical Sciences, Veterinary Medicine (12 B)
Kaunas, 2010
The doctoral dissertation was arranged in 2006–2010 in Lithuanian Veterinary Academy.
Research supervisor –
Prof. Habil. Dr. Algimantas Petras MATUSEVIČIUS (Lithuanian University of Health Sciences Veterinary Academy, Biomedical Sciences, Veterinary Medicine – 12 B).
Scientific assistant –
Prof. Dr. Jūrat÷ ŠIUGŽDAITö (Lithuanian University of Health Sciences Veterinary Academy, Biomedical Sciences, Veterinary Medicine – 12 B).
Veterinary Science Council: Chairman –
Prof. Habil. Dr. Saulius PETKEVIČIUS (Lihuanian University of Health Sciences Veterinary Academy, Biomedical Sciences, Veterinary Medicine – 12 B).
Members:
Prof. Dr. Antanas SEDEREVIČIUS (Lihuanian University of Health Sciences Veterinary Academy, Biomedical Sciences, Veterinary Medicine – 12 B);
Prof. Habil. Dr. Rimantas VELIČKA (Lithuanian University of Agriculture, Biomedical Sciences, Agronomy – 06 B);
Prof. Habil. Dr. Algimantas PAULAUSKAS (Vytautas Magnus University, Biomedical Sciences, Biology – 01 B)
Assoc prof. Dr. Algirdas ŠALOMSKAS (Lihuanian University of Health Sciences Veterinary Academy, Biomedical Sciences, Veterinary Medicine – 12 B).
Opponents:
Prof. Dr. Albina ANIULIENö (Lithuanian University of Health Sciences Veterinary Academy, Biomedical Sciences, Veterinary Medicine – 12 B);
Prof. Habil. Dr. Aniolas SRUOGA (Vytautas Magnus University, Biomedical Sciences, Biology – 01 B).
Public defence of doctoral dissertation in Veterinary Science council will take place at the Lithuanian University of Health Sciences Veterinary Academy Dr. S. Jankauskas auditorium 2 p.m. on 19th of November, 2010.
Address: Tilž÷s str. 18, LT-47181 Kaunas, Lithuania.
The summary of doctoral dissertation has been sent on 19th of October, 2010 according to the confirmed address list. The doctoral dissertation is available at the libraries of the LUHS Veterinary Academy.
LIETUVOS SVEIKATOS MOKSLŲ UNIVERSITETO
VETERINARIJOS AKADEMIJA
Marija Ivaškien÷
KREMŲ E-1 IR T-1
FARMAKOLOGINIS-KLINIKINIS ĮVERTINIMAS
ŠUNŲ IR KAČIŲ DERMATOFITIJAI GYDYTI
Daktaro disertacijos santrauka
Biomedicinos mokslai, veterinarin÷ medicina (12 B)
Kaunas, 2010
Disertacija rengta 2006–2010 metais Lietuvos veterinarijos akademijoje. Mokslinis vadovas –
Prof. habil. dr. Algimantas Petras MATUSEVIČIUS (Lietuvos sveikatos mokslų universiteto Veterinarijos akademija, biomedicinos mokslai, veterinarin÷ medicina – 12 B).
Mokslin÷ konsultant÷ –
Prof. dr. Jūrat÷ ŠIUGŽDAITö (Lietuvos sveikatos mokslų universiteto Veterinarijos akademija, biomedicinos mokslai, veterinarin÷ medicina – 12 B).
Veterinarin÷s medicinos mokslo krypties taryba: Pirmininkas –
Prof. habil. dr. Saulius PETKEVIČIUS (Lietuvos sveikatos mokslų universiteto Veterinarijos akademija, biomedicinos mokslai, veterinarin÷ medicina – 12 B).
Nariai:
Prof. dr. Antanas SEDEREVIČIUS (Lietuvos sveikatos mokslų universiteto Veterinarijos akademija, biomedicinos mokslai, veterinarin÷ medicina – 12 B);
Prof. habil. dr. Rimantas VELIČKA (Lietuvos žem÷s ūkio universitetas, biomedicinos mokslai, agronomija – 06 B);
Prof. habil. dr. Algimantas PAULAUSKAS (Vytauto Didžiojo universitetas, biomedicinos mokslai, biologija – 01 B);
Doc. dr. Algirdas ŠALOMSKAS (Lietuvos sveikatos mokslų universiteto Veterinarijos akademija, biomedicinos mokslai, veterinarin÷ medicina – 12 B).
Oponentai:
Prof. dr. Albina ANIULIENö (Lietuvos sveikatos mokslų universiteto Veterinarijos akademija, biomedicinos mokslai, veterinarin÷ medicina – 12 B);
Prof. habil. dr. Aniolas SRUOGA (Vytauto Didžiojo universitetas, biomedicinos mokslai, biologija – 01 B).
Disertacija bus ginama viešame Veterinarin÷s medicinos mokslo krypties tarybos pos÷dyje 2010 m. lapkričio 19 d. 14 val. Lietuvos sveikatos mokslų universiteto Veterinarijos akademijos dr. S. Jankausko auditorijoje. Adresas: Tilž÷s g. 18, 47181 Kaunas, Lietuva
Disertacijos santrauka išsiųsta 2010 m. spalio 19 d. pagal patvirtintą adresų sąrašą.
Disertaciją galima peržiūr÷ti LSMU Veterinarijos akademijos bibliotekoje.
INTRODUCTION
Keeping cats and dogs as pets has always been particularly popular. As a result, it is common for people to be in close contact with pets who often happen to suffer from zoonosis. There are over 250 zoonotic diseases; about 40 of them are attributed to dogs and cats. Zoonosis is defined as an infectious disease transmissible under natural conditions between vertebrate animals and human beings. Transmission of zoonotic infections occurs via bites, scratches or touch; exposure to saliva, urine or faeces; inhalation of particles or infectious aerosols; contact with a transport or intermediate host (e.g. ticks, fleas); as well as through contaminated water and soil.
Dermatophytosis is a zoonotic disease of the superficial layer of the skin, nails and hair. The disease is caused by fungi species belonging to three genera: Microsporum, Trichophyton and Epidermophyton. Based on their natural habitat dermatophytes may conveniently be classified as anthropophilic – those found mainly in humans, but very seldom in animals, zoophilic – those found in animals, but which may cause damage to human tissue, geophilic - predominantly found in soil and dangerous both to humans and animals. Zoophilic dermatophytes induce lesions which are most often quite inflammatory in humans.
Dermatophytosis is highly contagious and spreads rapidly; it often affects children and older people. Domestic pets have the potential to distribute dermatophytosis. There is a growing amount of publications focusing on the increase of superficial and invasive mycosis spread among people (Fridkin, Jarvis, 1996; Viscoli et al., 1999; Patterson et al., 2000; Marr et al., 2002). Microsporum canis, a zoophilic dermatophyte, is still the most common agent in Europe to cause tinea capitis. Children aged between 3-7 years, irrespective of gender, still remain the ones most commonly affected by tinea capitis, however, recently there has been an increase in infections in adults and the elderly (Ginter-Hanselmayer et al., 2007).
Infection amongst people and animals can come from such sources as contaminated environment and fomites, animals with sub-clinical and clinical infections, as well as animals that are merely carrying the spores on their coat (Arrese et al., 2000). Asymptomatic animal carriers of M. canis are considered a high risk factor in human dermatophytosis epidemiology. (Marchisa et al., 1995; Romano et al., 1997; Cafarchia et al., 2006). Reports of drug resistance to and the reduction in therapeutic effectiveness of currently available antifungal agents are on the rise. Many factors are likely to be involved in these trends, including immune suppression and the use of broad-spectrum antibiotics. The use of fungistatic drugs, suboptimal doses, compounded drugs, poorly absorbed drug formulations, and inadequate
tissue penetrations of antifungals also contribute to the development of acquired resistance. Because of the unique chemical complexities of the antifungal agents, drug/drug and drug/food interactions may also play a significant role in poor therapeutic outcome (Wiebe, Karriker, 2005). Dermatophytes Microsporum canis, Microsporum gypseum and Trichophyton mentagrophytes, and yeast Malassezia pachidermatis, Candida albicans, C. parapsilosis, etc. often cause dermatomycosis in humans in Lithuania (Lapinskait÷, 2007). According to Kaunas Public Health Centre, dermatophytosis incidence rate in Kaunas County over the past 9 years ranged from 5,3 to 37,3 cases/10 000 inhabitants.
Although, dermatophyte infections generally are self-limiting, treatment helps to speed the resolution of the disease and minimise the risk of spread of infected spores to the environment. Both topical and systemic therapies may be used to treat dermatophyte infection. A limited number of antifungal agents is licensed for use in animals, however, many of those available for the treatment of dermatophytosis in humans are used by veterinary practitioners. A systematic use of these drugs often has a negative impact on the animal‘s body, topical preparations available in human medicine are not suitable for use on animal skin. The use of an effective and safe antifungal therapy that shortens the time of treatment and shortens the exposure of the owners to the disease is important in veterinary medicine.
The aim of the present study is to determine the prevalence of fungi in the fur of clinically healthy dogs and cats; as well as determine the occurrence rate of dermatophytosis in cats and dogs and establish the therapeutic efficacy of E-1 and T-1 creams.
Goals of the study
1. To determine the prevalence of microscopic fungi in the fur of clinically healthy dogs and cats and in dogs and cats with clinically affected skin;
2. To determine the in vitro susceptibility of pathogenic fungi to econazole nitrate, miconazole nitrate and terbinafine hydrochloride;
3. To determine the influence of pH on the permeation of econazole nitrate and terbinafine through synthetic membrane;
4. To evaluate the effect of creams E-1 and T-1 on the healthy skin; 5. To determine the therapeutic efficacy of creams E-1 and T-1 in the treatment of experimentally induced dermatophytosis in guinea pigs;
6. To determine the therapeutic efficacy of creams E-1 and T-1 in the treatment of dermatophytosis in dogs and cats.
Novelty and practical use
We have researched the scale of the spread of pathogenic and saprophytic microscopic fungi in the fur of both clinically healthy cats and dogs and those with present skin damage. We have established that pets, especially cats, have a large potential in spreading dermatophytosis. There is a limited choice of veterinary preparations to treat dermatophytosis in animals. Safe, effective, short-term therapy that minimizes the spread of infective spores into the environment is desired. Lithuanian Veterinary Academy’s Experimental and Clinical Pharmacology Laboratory created E-1 and T-E-1 creams to treat animals infected with dermatophytosis. We have studied the therapeutic effect of these creams on small animals. We have established that creams E-1 and T-1 effectively and quickly removes signs of the disease, destroys the pathogenic agent and protects the environment from the pathogen.
MATERIALS AND METHODS
Laboratory procedures were carried out at the microbiological laboratory, Department of Infectious Diseases, Laboratory of Experimental and Clinical Pharmacology, Department of Noninfectious Diseases, Lithuanian Veterinary Academy, and at The School of Pharmacy, University of London. The experiment with laboratory animals was approved by the State Veterinary Service upon the recommendation of the Ethics Commission of Lithuania on the use of laboratory animals (permission Nr. 0185; 2009).
The prevalence of microscopic fungi in the fur of clinically healthy cats and dogs and those with skin lesions was studied during the first stage of work. The samples were taken from 153 clinically healthy animals (89 dogs and 64 cats) and 174 (129 dogs and 45 cats) animals with skin lesions. A modified Mackenzie method was used to take hair samples from clinically healthy animals (Katoh et al., 1990; Moriello, 2003 a). In animals with clinical skin lesions the damaged hair was plucked with sterile tweezers from the center of skin lesion and from the demarcation zone between healthy and damaged skin; scabs and dandruff were collected. Fungi were isolated in accordance with the procedure described (Medley, Ristic, 1992), we used a selective medium - Sabouraud dextrose agar (Liofilchem, Italy), potato dextrose agar (Liofilchem, Italy) and the commercial media “Dermatophyte Test Medium” (DTM) (E&O Laboratories Limited, Scotland). The hair sample of each test animal was divided into two groups. One sample was cultivated under aerobic conditions in a thermostat at 25°C (these conditions are favourable to the growth of moulds), the other sample
was cultivated under aerobic conditions in a thermostat at 37°C temperature. Samples were incubated for up to 14-21 days, and examined daily. Fungal identification was based on cultural morphology and microscopic examination of hyphae, microconidia and macroconidia according to the descriptions of species found in publications (Van Cutsem, Rochette, 1991; Quinn et al., 1994; 2002; Lugauskas and others, 2002). Age, sex, hair length and habitat were recorded for each animal as a potential risk factor for the incidence of fungal spores.
During the second phase of work we established the susceptibility of pathogenic fungi to antifungal agents - econazole nitrate, miconazole nitrate and terbinafine hydrochloride, using in vitro broth microdilution method according to Clinical and Laboratory Standards Institute guidelines. Stock solutions of antifungals (5 mg/ml) were prepared in 100% dimethyl sulfoxide (DMSO). Serial two-fold dilutions were prepared using RPMI 1640 medium (Sigma-Aldrich, USA). The final concentrations of the antifungal agents were 0.16-80.0 µg/ml.
During the third stage we assessed the influence of buffer pH on the penetration of econazole nitrate and terbinafine base through the synthetic polydimethylsiloxane membrane. Testing was carried out using Franz diffusion cells. We used PBS solution within the receptor cell, and saturated buffer solution in the donor cell, where the tested material was dissolved. Buffer solutions had different pH values (5, 6 and 7).
We established the effect of the creams E-1 and T-1 on healthy skin during the fourth phase. We used 10 New Zealand white rabbits and 10 Dunkin Hartley guinea-pigs for testing. The test cream was applied on rabbits’ left thigh skin, and guinea pigs’ left side of the abdominal skin, whereas the right side was free of any applications and became the control side. Creams were applied daily for 10 days in a row, by being rubbed gently into the skin. The volume of the fold of the skin, where the cream had been applied, was measured by vernier callipers during testing; skin condition was rated and the degree of irritation assessed according to a points system. At 7 and 14 days after the application of creams the condition of skin and the skin-fold volume were assessed. After 10 test days two guinea pigs from both groups were randomly selected and euthanised. Skin histopathology testing was carried out at The Centre of Pathology in Lithuanian Veterinary Academy. Pathological material was taken within an hour of the animals’ euthanasia. 1x1 cm skin samples were cut from the right and the left sides and captured with a 10% buffered formalin solution. Skin samples were embedded in paraffin. Blocks were cut with a rotary microtome in cuts 2-5 µm thick, stained with hematoxylin and eosin and examined under a fluorescence microscope (OLIMPUS BX 41, Japan).
During the fifth stage of work we evaluated the therapeutic efficacy of creams E-1 and T-1 in the treatment of small animals with dermatophytosis and compared it to that of other products. The test used 20 guinea pigs, experimentally infected with M. canis dermatophytes, and 51 dogs and cats with dermatophytosis. The hair in the 2x2 cm area on guinea pigs’ back was clipped and the skin was scarified and infected by M. canis suspension prepared beforehand. The experimental animals were divided into five groups, four animals in each group. The first group received cream T-1, the second group received cream E-1, the third group received “Imaverol” solution as a comparator every 3 days till the end of clinical changes, the fourth group received the vehicle and the fifth group was an untreated control group. Formulations were applied on the day when clinical symptoms of infection were most evident, in a volume of ~25 mg per application to the infected area, once a day and the treatment was continued till the resolution.
To evaluate the clinical and mycological efficacy the methodology described by Ghannoum et al., (2009) was used with little modification. To evaluate the clinical efficacy of different treatments, the infected area was divided into four equal quadrants. Each quadrant was scored on a scale from 0 to 5. Drug efficiency was calculated using the formula:
Effectiveness % = 100 - (T٠100 / C),
Where T - the total score of treatment and control groups; C - the total score of 20 for the unhealed lesion sections. The total score for any group denotes the average clinical score from different animals in the same group.
Laboratory animals were followed daily: skin changes were photographed, the volume of damaged skin folds was measured by vernier callipers, the changes in damaged skin were scored and registered. The test was divided into the following stages: infection, the first clinical signs of infection, significant clinical signs, lesion „cleansing“ of skin flakes and infected hair, and new hair growth in the beginning of a complete clinical recovery. Standardised mycological examination was performed on the day before the treatment and after that once a week. The hair root invasion test was used to assess mycological cure. Ten hairs were removed from each lesion, or, if the lesion was bald, hairs were removed at the edges of the lesion and placed on the Potato Dextrose Agar in Petri dishes. Mycological evaluation was based on the number of culture positive hairs obtained from each lesion. Percent efficacy of different treatments and the control group was calculated with the same formula used to determine clinical efficacy; C score of 10 was assigned for the unhealed lesion.
Dogs and cats, affected with dermatophytosis and having from one to five clearly expressed skin lesions with a diameter of less than 5 cm were
treated with creams T-1 and E-1. Skin lesions were treated with experimental creams two times a day, morning and evening, by gentle rubbing in the affected area. The animal was considered to have recovered when the hair in the damaged area had fully grown again, and the mycological test result was negative. Depending on the intensity of fungal invasion, when treating the damaged skin with creams T-1 and E-1, some animals’ coat was additionally sprayed with “Imaverol” solution every third day until the animal was clinically healed.
Statistical analysis was carried out using statistical package SPSS No. 15 Version (SPSS for Windows 9.0, SPSS Inc. Chicago, IL, USA, 1989-1995). The age, sex, hair length and habitat of each clinically healthy animal and of each animal with skin lesions, were recorded and analysed for the prevalence of microscopic fungi in the coat. Chi square test (χ2) was used to establish the link between the characteristics, when a significant link was present the correlation coefficients (φ) were calculated. Statistically significant differences between groups were defined when p<0.05 and p<0.01.
The arithmetic average (X) of and standard deviations (σ) in the size of skin fold were calculated when studying the effect of T-1 and E-1 creams on healthy skin. The arithmetic average figures of the size of tested skin fold before the application and on each day of application were compared. The significance of the means (p) was evaluated according to Student. The results were considered reliable when p<0.05.
When evaluating the therapeutic efficacy of test preparations we calculated the arithmetic average (X) of and standard deviations (σ) in skin lesion score. When comparing the effectiveness of each test preparation to another, or to self recovery, the Mann-Whitney test was applied. Statistically significant differences between the arithmetic averages of the two groups were defined when p<0.05.
GENERAL RESULTS AND DISCUSSION
Prevalence of dermatophytes in the hair coat of asymptomatic animals
Studies of clinically healthy animals have shown that the prevalence of dermatophytes in the coat of dogs is 20.2%, and in cats – 18.8%. Several types of dermatophytes were isolated in dogs – Microsporum canis, Microsporum gypseum, Trichophyton mentagrophytes, whereas only one species was isolated in cats – M. canis. We found that most common dermatophyte is M. canis, which makes up 90% of all isolated
dermatophytes. The dermatophyte in clinically healthy dog and cat coat is prevalent almost equally (16.9% and 18.8% respectively) (Table 1).
Our survey shows that more M. canis spores are isolated from dogs older than one year of age (80%, p=0.981) and from dogs kept outdoors (80%, p=0.063), but data are not statistically reliable. Sex and the hair length had no significant effect on M. canis spores spread in clinically healthy dogs’ coat. Cat age and gender had no effect on M. canis spores spread in the coat, but the hair length and storage have had.M. canis spores were isolated from short-haired cats more often than those from longhaired cats (75.0% and 25.0% respectively) (p=0.381). These results are consistent with the hypothesis, proposed by Moriello (2003 b), that it is difficult to determine if a long-haired cat is an asymptomatic carrier of spores due to the density of its fur.
Interestingly enough, slightly more dermatophytes were isolated from cats kept only indoors than from those in contact with the environment (58.3% and 41.7% respectively) (p=0.837). We believe that because M. canis spores are widespread in the environment, cats, also known as “dust collectors” can carry pathogenic fungus spores in their coat from birth.
Occurrence of dermatophytes in animals with skin lesions
We established the frequency of dermatophytosis in pets through another study. Having studied cats and dogs with clinical skin and hair damage, we found that in 27.6% of cases this damage was due to dermatophytes. Three types of dermatophytes were isolated in dogs – Microsporum canis, Microsporum gypseum, Trichophyton mentagrophytes, whereas only one species was isolated in cats – M. canis. About 85% of isolated dermatophytes were M. canis (Table 1). We discovered that cats are four times more likely than dogs to be suffering from M. canis-induced dermatophytosis, 55.6% and 12.4% respectively. As high as 68.0% of cats with dermatophytosis were younger than one year of age. This data is statistically reliable (p<0.01) and confirms other research findings that infection by dermatophytes is directly dependent on age (Cabanes et al., 1997; Paixão et al., 2001; Brilhante et al., 2003; Khosravi, Mahmoud, 2003). A higher number of cats with dermatophytosis had access to the outdoors (76%) (p<0.01). We believe that cats living outside or those able to go outside, can more frequently damage their skin, because they come into contact with the environment and other animals more often. In the coat of shorthaired cats dermatophytosis is diagnosed more often than in longhaired (76.0% and 24.0% respectively) (p=0.249). It is argued that longhaired cats (especially those of Persian breed) are predisposed to dermatophytosis because their hair may protect spores from mechanical removal; the longer
the contact with the skin, the greater the opportunity that infection may develop (Moriello, 2003 b). However, it is easier to isolate microscopic fungus spores and notice early clinical signs in shorthair cat fur, rather than in longhair one.
Having examined the dependence of the disease on the sex of cats, the data we obtained coincides with the results obtained by L. Pinter (1999) and a group of researchers, as well as A. Patel (2005) and a group of researchers, which found that more male cats suffer from dermatophytosis than female cats. The aforementioned attribute such dependence to the fact that females’ sebaceous glands secrete more fungistatic fatty acids than those of males. As a result, female cats and dogs are more resistant to dermatophytosis.
Older than one year of age and shorthaired male dogs suffered from dermatophytosis more frequently, however such data is not statistically reliable. According to some research data, the age, gender and hair length does not affect the prevalence of dermatophytosis in dogs (Paixão et al., 2001; Brilhante et al., 2003; Manciano et al., 2003). We found that more often dogs suffered from dermatophytosis caused by M. canis in autumn (43.75%) and cats – in summer (44%).
Apart from M. canis, other dermatophytes were established in dogs – M. gypseum (1.6%) and T. mentagrophytes (3.9%). During the course of our study M. gypseum dermatophyte was isolated from two dogs in winter and spring. The dogs were kept at a private estate; therefore they spent most of their day outdoors. T. mentagrophytes were isolated from five dogs at different times of the year – in autumn (n=2, 40.0%, p=0.824), winter (n=2, 40.0%, p=0.977) and spring (n=1, 20.0% p=0.703).Some scientists say that hunting dogs and other dogs, which like to dig, are more often infected with T. mentagrophytes and M. gypseum dermatophytes. These agents are distributed by wild rodents and other wild animals (Manciano et al., 2003).
During this study we focused on the prevalence of saprophytic fungal flora in animals that are asymptomatic M. canis carriers and animals which had clinical features of dermatophytosis caused by M. canis. It is argued that the natural skin saprophytic microflora inhibits the pathogen invasion (Moriello, DeBoer, 1991 b). However, when barrier properties of the skin weaken, the natural skin flora changes. When M. canis colonises the skin the dermatophyte begins to compete with the resident fungal flora for its position in the ecological niche. It was found that there are more saprophytic fungi genera in animals, whose coat contains M. canis dermatophyte and who don’t have clinical signs of dermatophytosis, than in animals affected by this infection. Saprophytic fungi in the affected skin were significantly less prevalent than in the coat of asymptomatic M. canis carriers. Our research confirms the hypothesis proposed by Moriello and DeBoer (1991
b) that when M. canis dermatophyte is established in the skin, the growth of saprophytic fungi is inhibited.
Table 1. Prevalence of dermatophytes in the hair coat of asymptomatic animals and animals with skin lesions
Age Sex Hair lenght Habitat Der ma to phyt es ≤1 year >1 year ♂ ♀ Short haired Long haired In door Out door Clinically healthy dogs
M.c % 3 (20.0) 12 (80.0) 8 (53.3) 7 (46.7) 7 (46.7) 8 (53.3) 3 (20.0) 12 (80.0) M.g % 1 (50.0) 1 (50.0) 2 (100.0) 0 0 2 (100.0) 1 (50.0) 1 (50.0) T.m % 0 1 (100.0) 1 (100.0) 0 1 (100.0) 0 0 1 (100.0) Clinically healthy cats
M.c % 6 (50.0) 6 (50.0) 6 (50.0) 6 (50.0) 9 (75.0) 3 (25.0) 7 (58.3) 5 (41.7) Dogs with suspected lesions of dermatophytosis
M.c % 4 (25.0) 12 (75.0) 11 (68.8) 5 (31.2) 9 (56.2) 7 (43.8) 16 (100.0) 0 M.g % 1 (50.0) 1 (50.0) 0 2 (100.0) 1 (50.0) 1 (50.0) 2 (100.0) 0 T.m % 1 (20.0) 4 (80.0) 4 (80.0) 1 (20.0) 2 (40.0) 3 (60.0) 4 (80.0) 1 (20.0) Cats with suspected lesions of dermatophytosis
M.c % 17** (68.0) 8 (32.0) 15 (60.0) 10 (40.0) 19 (76.0) 6 (24.0) 6 (24.0) 19** (76.0) ** p<0,01 M.c – Microsporum canis M.g – Microsporum gypseum T.m – Trichophyton mentagrophytes
During the study we collected data on the health of owners of clinically healthy animals and animals with skin lesions, related to this specific zoonose. We found that 12 pet owners had dermatophytosis: six children under the age of 14, five young women under 30 years of age, and one
woman over 50 years old. These people kept animals with a history of M. canis-induced dermatophytosis or which were asymptomatic M. canis carriers. Cats, able to go outside, made up the majority of pets. Our findings coincide with published data showing that children and older people whose immune systems are either immature or weakened are among those most susceptible to dermatophytosis (Vander Straten et al., 2003).
After finding that the incidence of dermatophytosis in dogs and cats is common in Lithuania and that the disease is transmitted to humans, the need to develop effective, non-toxic and easy to use medicines for zoonosis arose. A limited number of antifungal agents are licensed for veterinary purpose in Lithuania. “Imaverol” is the only preparation that is allowed for veterinary use. This 0.2 % enilconazole emulsion is certified for application as a topical treatment of dermatophytosis in cattle, horses and dogs. This medicine is not intended to treat cats, however our data and other publications indicate that the latter more often suffer from dermatophytosis than other animals. The use of an effective and safe antifungal therapy that shortens the time of treatment and shortens the exposure of owners to the disease is important in veterinary medicine.
The Laboratory of Experimental and Clinical pharmacology in Lithuanian Veterinary Academy has developed two antifungal cream formulations for the treatment of dermatophytosis in dogs and cats – T-1 and E-1. T-1 formulation contains 1% terbinafine hydrochloride, E-1 formulation contains 1% econazole nitrate.
The production of creams requires several conditions to be fulfilled: the cream should be produced on the basis of homogeneous oil-in-water emulsions. Evaporation of the aqueous phase gives a cooling effect after application (Williams, 2003). Creams should be spread easily and dry rapidly on animal skin, leaving no detectable residue and adhering to the treated area without being tacky, having optimal pH and being non-irritating to the skin, as well as not having an objectionable texture or odour, having keratolitic and moisturising effect on skin. Oil-in-water emulsions intensively hydrate the skin by donating water. Increased skin hydration opens the structure of the superficial layers of the skin leading to an increase in penetration of active agents (Benson, 2005).
Econazole belongs to the family of imidazoles, exerts fungistatic effect against dermatophytes, is relatively cheap, widely used in the topical formulations for the treatment of dermatomycosis in human. Terbinafine hydrochloride, a synthetic antimycotic agent that belongs to the family of compounds known as the allylamines, exerts a fungicidal effect against dermatophytes and is widely used in the topical formulations for the treatment of dermatophytosis in human medicine, it is also more expensive
than econazole. Azoles, and allylamines possess anti-inflammatory activity (Rosen, 1997; Camera et al., 1999).
The influence of pH on the diffusion of antifungal agents through membrane
Econazole nitrate and terbinafine hydrochloride are weak bases with pKa 6.6 and 7.1, respectively, and log P – 5.4 and 5.9. It was established that lipophilic compounds are those which log P>4. Both these substances are highly lipophilic and therefore soluble in skin lipids, and are poorly soluble in aqueous media. Ionised membranes of the body, including skin, allow unionized substances to penetrate well (Williams, 2003 a). It is known that the pH value of the drug affects the penetration of the active substance through biological membranes.Increasing the pH of formulation, econazole nitrate and terbinafine hydrochloride become less ionised, and should go through the stratum corneum easier. The study was carried out using Franz diffusion cells to determine whether changing the solution pH can affect penetration of terbinafine and econazole through the membrane. We used a synthetic polydimethylsiloxane membrane as the skin replacement. This test revealed that the diffusion of terbinafine and econazole nitrate through silicone membrane was slow and the pH did not affect faster diffusion of these substances. After 24 hrs econazole nitrate flux from the pH 5 solution was equal to 6.78±0.58 µg/cm²/hr and the flux from solution pH 7 was equal to 7.34±1.18 µg/cm²/h. After 24 hrs terbinafine flux from the pH 5 solution was equal to 1.38±0.32 µg/cm²/h, and the flux from solution pH 6 – 1.34±0.29 µg/cm²/h (Figure 1).
After conducting the experiment on the transmembrane penetration of creams, the chromatographic system didn’t detect terbinafine or econazole in the receptor phase. Knowing that these substances are lipophilic and poorly soluble in aqueous solutions, we believe that terbinafine and econazole could accumulate in the polydimethylsiloxane membrane.
Determining the efficacy of creams T-1 and E-1 in the topical treatment of dermatophytosis in animals
The therapeutic effect of creams T-1 and E-1 was evaluated after testing on animals was carried out. During the first test guinea pigs, experimentally infected with M. canis dermatophyte, were treated with creams T-1 and E-1. During the second test small domestic pets affected with dermatophytosis were treated with creams T-1 and E-1. These tests revealed that creams T-1 and E-1 are effective in the treatment of this zoonosis.
When treating guinea pigs affected with experimentally induced dermatophytosis, we found that the two creams used once per day,
effectively eliminate the disease agent and the clinical signs of disease. When using cream T-1 clinical signs were removed in 30.3±3.4 days and the agent was eliminated after two weeks of treatment. When treating the animals with cream E-1 clinical symptoms disappeared in 44.3±3.5 days, and we could not find the agent after three weeks of treatment. “Imaverol” solution eliminated clinical signs of the disease in 42.3±3.4 days and the agent could not be found after four weeks of treatment.
0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 24 Time, h F lu x , µ g /c m 2 /h
Econazole nitrate pH 5 Terbinafine pH 5 Econazole nitrate pH 7 Terbinafine pH 6
Figure 1. Permeation of econazole nitrate and terbinafine through membrane
The therapeutic efficiency of “Imaverol” was achieved after 13.5 applications every third day. “Imaverol” did not have an adverse effect on the skin or general health of the animal. We believe that the duration of treatment period with cream E-1 coincides with that of “Imaverol” because the preparations have similar antifungal substances, according to their structure and mechanism of action – imidazoles. E-1 cream contains econazole, whereas “Imaverol” solution contains enilconazole.
During the test, when dermatophytosis was experimentally induced, we treated animals with the vehicle of experimental creams containing no active therapeutic substances. We found that vehicle had a positive therapeutic effect in 46.0±3.5 days. The disease agent in this group of animals was removed after five weeks. Figure 2 and figure 3 shows comparative clinical and mycological efficacy of each tested formulation.
0 20 40 60 80 100 0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 Days C li n ic al e ff ec ti v en es s % .
T-1 E-1 Imaverol Vehicle Control
Figure 2. Clinical effectiveness of the formulations
0 20 40 60 80 100 0 1 2 3 4 5 6 7 8 Weeks M y co lo g ic al e ff ec ti v en es s % .
T-1 E-1 Imaverol Vehicle Control Figure 3. Mycological effectiveness of the formulations
When testing laboratory animals we confirmed the statement that dermatophytosis is a self-limiting disease (DeBoer, Moriello, 1994; Colombo et al., 2001; Scott et al., 2001). Healthy animals, kept in good conditions and good hygiene, with well-developed immune system recover
spontaneously. The clinical symptoms in our control guinea pigs resolved spontaneously within 64.3±6.1 days after infection, but the disease agent was only removed after 10 weeks.
During the test in which dogs and cats with dermatophytosis were treated with the experimental creams twice a day, we found that cats, suffering from the most widely M. canis-induced dermatophytosis, when treated with T-1 cream recovered in 23.6±7.22 days; whereas when treated with E-1 cream they recovered in 30.2±10.85 days. Dogs with M. canis-induced dermatophytosis when treated with T-1 cream recovered in 26.9±6.17 days, while when treated with E-1 cream they recovered in 26.3±5.03 days. During this test a group of cats with localised skin lesions and a significant invasion of dermatophyte spores in their coat, were treated with cream T-1 and also with “Imaverol”. Such a combined treatment had a positive therapeutic effect in 25.5±9.28 days (Table 2).
Table 2. Efficacy of creams T-1 and E-1 in the topical treatment of dermatophytosis in dogs and cats
Dogs Cats Dogs Cats Cream E-1 Cream T-1 Cream T-1+ „Imaverol“ Dermatophytes Treatment days (X±SD) M. canis 26.3±5.03 30.2±10.85 26.9±6.17 23.6±7.22 25.5±9.28 M. gypseum 26.0±6.36 - 17 - - T. mentagro-phytes 21.0±1.41 - 11 - - After performing testing with small animals infected with dermatophytosis we can say that a successful recovery is influenced not only by the nature of the treatment, but also by the species of animals, physiological characteristics of an individual, pathogen species and the extent of damage caused, as well as the environmental conditions under which animals are treated.
During testing cream T-1 exhibited a greater therapeutic efficacy and fewer side effects on the skin than cream E-1. When treating with cream T-1, those animals who had lesions on the ear, displayed reddened skin. Most animals treated with E-1 cream displayed reddening and irritation of skin at the site of application. Animals would begin to scratch the patch of skin which the cream was applied to immediately after application; however the irritation would disappear after an hour. It is known that azoles and
allylamines used in topical formulations rarely cause side effects such as skin itching, redness and burning sensation (Katzung, 2007).
Animal skin irritation test
A study with clinically healthy laboratory animals was carried out to determine whether the reddening of the skin is due to the skin‘s reaction to the components of creams or inflammatory reaction to the pathogen. By studying the effects of creams on a healthy rabbit and guinea pig skin we found that rabbits’ skin remained red for quite some time after the application of cream E-1, whereas guinea pig skin did not become red. When using cream T-1, rabbits’ skin reddened for a short while, but after an hour the redness would disappear. When using the same cream on guinea pigs, skin redness was not observed. Histological testing of guinea pig skin samples did not show obvious characteristics of spongiosis or edema, which are characteristic features of contact dermatitis, however we have found limited eosinophilic infiltrate in the dermis. We also found that the epidermis of the skin rubbed with creams was thicker than that of the skin which wasn’t rubbed with creams. Knowing that the vehicle of the experimental creams consists 90% of water, we believe that creams had a moisturizing effect on the skin. We believe that the onset of skin redness and discomfort in rabbits and animals, treated with creams, may depend on the individual physiological characteristics of the organism, as well as the application area. Rabbit skin redness after the application can be explained by the fact that their skin is thinner than that of guinea pigs.
In treating animals suffering from dermatophytosis, the high therapeutic efficacy of cream T-1 can be attributed to the characteristics of the active substance pharmacokinetics. Terbinafine, exerts fungicidal activity against dermatophytes. Its minimum inhibitory concentration ranged from 0.001 to 0.05 µg/ml. Thus, terbinafine eliminates fungi at concentrations that are several times below those found with azole derivatives, which exert fungistatic activity, the minimum inhibitory concentration range mainly spreading from 0.1 to 10 µg/ml (Schäfer-Körting et al., 2008). We have found in our studies that the dermatophytes M. canis, M. gypseum and T. mentagrophytes are susceptible to azoles – econazole nitrate and miconazole nitrate, as well as to allylamine terbinafine hydrochloride. We found that dermatophytes were especially susceptible to terbinafine hydrochloride. The average MIC of econazole nitrate ranges from 1.3 to 10.0 µg/ml, miconazole nitrate – from 2.5 to 10.0 µg/ml, and terbinafine hydrochloride – from 0.65 to 1.3 µg/ml.
Terbinafine provides continued increase in cure rates for days to weeks after the discontinuation of treatment. The studies conducted on animals
(Hosseini-Yeganeh, McLachlan, 2001; 2002; Foust et al., 2007) and humans (Finlay, 1992; Hill et al., 1992; Faergemann et al., 1991; 1993; Kienzler et al., 2007) showed that terbinafine accumulates in the skin either when applied externally or orally. High concentrations of it remain after discontinuation for up to several weeks.
Terbinafine is a lipophilic drug, it tends to accumulate in stratum corneum and hair follicles and persists there at concentrations above the MIC for several weeks after a short-term therapy (Uchida, Yamaguchi, 1993; Funke et al., 2002).
Orally administered terbinafine accumulates in nails, hair, dermis, and adipose tissue (Faergemann et al., 1991; Finlay, 1992). When used locally, the long-term retention of terbinafine in skin is demonstrated by its minimal transdermal permeation into the systemic circulation (Dyke et al., 1990; Hill et al., 1992; Tanum et al., 2000; Kienzler et al., 2007).
Azoles also tend to accumulate in the horny layer of the skin and as with terbinafine, systemic absorption of topically applied azoles is low (Pershing et al., 1994; Kawada et al., 2000; Sobue et al., 2004; Monti et al., 2005; Degreef et al., 2006). However, they require a longer duration of treatment because of their fungistatic mode of action.
Summarising the results we can say that clinically healthy dogs and cats are asymptomatic carriers of dermatophytes. Cats are four times more prone to be suffering from dermatophytosis than dogs are. The newly prepared topical formulations T-1 and E-1 are effective in treating small animals affected with dermatophytosis. Cream T-1 is more effective and less irritating to skin than cream E-1.Cream E-1 causes mild contact dermatitis, which occurs in the first days of treatment. We believe that correctly selected antifungal agent and type of formulation resulted in significant therapeutic effect of cream T-1. The fungicidal effect of terbinafine and its property to accumulate in the skin’s horny layer, the moisturising effect of the cream and its slightly alkaline pH (6.2) all contributed to a faster healing of damaged skin in comparison to “Imaverol” solution.
CONCLUSIONS
1. M. canis is the most common species isolated from clinically healthy dogs and cats. M. canis prevalence in dog coat is 16.9%, and in cat coat – 18.8%.
2. M. canis was isolated from 12.4% of dogs with suspected dermatophytosis and 55.6% from cats, which is four times as high. Younger than 1 year old cats showed twice higher prevalence of M. canis infection than older cats (p<0.01); cats having access to the outdoor were affected with M. canis three times more often than when kept indoor (p<0.01).
3. Dermatophytes are most susceptible to terbinafine hydrochloride; the MIC values range from 0.65 to 1.3 µg/ml.
4. The pH of the buffer solution did not have a significant impact on the penetration of econazole nitrate and terbinafine through synthetic membrane. The flux of econazole nitrate from pH 5 and pH 7 buffers through membrane within 24 hours was similar, 6.78±0.58 µg/cm²/h and 7.34±1.18 µg/cm²/h respectively. The flux of terbinafine from pH 5 and pH 6 buffers through membrane within 24 hours was also similar, 1.38±0.32 µg/cm²/h and 1.34±0.29 µg/cm²/h respectively.
5. E-1 cream caused mild contact dermatitis, which occurred on first days of the treatment.
6. Experimental creams E-1 and T-1 are effective in the treatment of experimentally induced dermatophytosis in guinea pigs. Treatment with E-1 cream eliminated the pathogen in 21 days and a complete resolution of clinical signs of infection was achieved on approximately 44.3±3.5 day. Treatment with T-1 cream eliminated the pathogen in 14 days and the clinical symptoms disappeared in 30.3±3.4 days.
7. Experimental creams T-1 and E-1 are effective in treating of dermatophytosis in dogs and cats. Dogs affected with the most common dermatophytosis caused by M. canis and treated with T-1 cream recovered in 26.9±6.17 days, while those treated with E-1 cream – in 26.3±5.03 days. Cats affected with dermatophytosis caused by M. canis and treated with T-1 cream, recovered in 23.6±7.22 days, while those treated with E-1 cream – in 30.2±10.85 days.
LIST OF PUBLICATIONS
1. Algimantas Matusevičius, Marija Ivaškien÷, Vytautas Špakauskas. Vaistai nuo mikroskopinių grybų. I dalis. Mikroskopinių grybų ląstel÷s struktūra, funkcija ir vaistų veikimo taikiniai. Literatūros apžvalga. 2008. 43 (65). P. 3–13.
2. Algimantas Matusevičius, Marija Ivaškien÷, Vytautas Špakauskas, Gintaras Daunoras. Vaistai nuo mikroskopinių grybų. II dalis. Vaistin÷s medžiagos ir preparatai nuo grybų. Literatūros apžvalga. Veterinarija ir zootechnika. 2008. 44 (66). P. 3–22.
3. Marija Ivaškien÷, Jūrat÷ Šiugždait÷, Algimantas Matusevičius, Aidas Grigonis, Gintaras Zamokas, Vytautas Špakauskas. Mikroskopinių grybų išskyrimas iš kliniškai sveikų šunų ir kačių plaukų. Veterinarija ir zootechnika. 2009. 45 (67). P. 13–19.
ACKNOWLEDGEMENT
I would like to thank my supervisor, Prof. habil. dr. Algimantas Matusevičius and scientific assistant – Prof. dr. Jūrat÷ Šiugždait÷, for the patient guidance and advice they have provided. I had the pleasure of working with dr. Aidas Grigonis and assoc. prof. dr. Gintaras Zamokas and would like to thank them for their help and encouragement. We sincerely thank professor of School of Pharmacy University of London Jonathan Hadgraft and dr. Majella Lane for hospitality and introduction to the work at their laboratory. We are also grateful to the staff of the Pathology center LUHS Veterinary Academy and Prof. dr. D. Pangonyt÷ from LUHS Institute of Cardiology for their input. I wish to thank my family and best friends who made this thesis possible.
REZIUMö
Laikyti namuose šunis, kates ir kitus smulkiuosius žinduolius ypač populiaru. Žmonių fizinis kontaktas su naminiais gyvūn÷liais, kurie neretai serga zoonoz÷mis, dažnai yra labai glaudus. Pasaulyje žinoma apie 250 zoonozinių susirgimų, apie 40 jų priskiriami šunims ir kat÷ms. Zoonoz÷s – gyvūnų ir žmonių užkrečiamosios ligos, kuriomis susergama įkandus, įdr÷skus arba prisilietus, įkv÷pus užkr÷stų aerozolio dalelių, per seiles, šlapimą arba išmatas, per ektoparazitus (erkes, blusas), per užterštą vandenį, žemę.
Dermatofitija yra zoonozin÷ paviršinių odos sluoksnių ir odos darinių liga. Susirgimą sukelia mikroskopiniai grybai – dermatofitai, priklausantys trims gentims – Microsporum, Trichophyton ir Epidermophyton. Šie grybai skirstomi į antropofilinius, zoofilinius ir geofilinius, nelygu jų natūrali aplinka žmogaus, gyvūno audiniuose ar žem÷je. Antropofilin÷s mikroskopinių grybų rūšys pavojingos žmon÷ms, rečiau – gyvūnams. Zoofilin÷s rūšys dažniausiai yra gyvūnų patogenai, bet gali pažeisti ir žmogaus audinius. Geofilin÷s rūšys pavojingos ir žmon÷ms, ir gyvūnams. Žmonių odos pažeidimai, sukelti zoofilinių arba geofilinių dermatofitų yra sunkesni, nei sukelti antropofilinių.
Dermatofitija greitai plinta ir yra lengvai užkrečiama, ja dažniau serga vaikai ir vyresnio amžiaus žmon÷s. Naminiai augintiniai turi didelį potencialą platinti dermatofitiją.
Vis dažniau literatūroje rašoma apie padid÷jusį paviršinių ir invazinių mikozių išplitimą tarp žmonių (Fridkin, Jarvis, 1996; Viscoli et al., 1999; Patterson et al., 2000; Marr et al., 2002). Microsporum canis yra dažnai išskiriamas iš dermatomikoze sergančių žmonių ir vis dar yra pagrindinis vaikų galvos dermatofitijos suk÷l÷jas Europoje (Ginter-Hanselmayer et al., 2007). Šia zoonoze dažniausiai užsikrečiama nuo gyvūnų, turinčių odos pažeidimų, arba nuo gyvūnų – besimptomių suk÷l÷jo nešiotojų, arba aplinkoje (Arrese et al., 2000). Žmonių dermatofitijos epidemiologijoje besimptomiai M. canis gyvūnai nešiotojai laikomi dideliu rizikos veiksniu (Marchisio et al., 1995; Romano et al., 1997; Cafarchia et al., 2006).
Pranešimų apie atsparumą vaistin÷ms medžiagoms ir šiuo metu esančių vaistų nuo grybų terapinio efektyvumo maž÷jimą taip pat daug÷ja. Šios problemos priežastimi tapo daugelis veiksnių – imunin÷ supresija, fungistatinių vaistų naudojimas, suboptimalių dozių parinkimas, sumaišytų (kelių veikliųjų medžiagų), silpnai absorbuojamų vaistų naudojimas, vaistų, nepakankamai prasiskverbiančių į audinį, naudojimas. Silpnam terapiniam veikimui taip pat turi įtakos vaisto/vaisto ir vaisto/maisto sąveika (Wiebe, Karriker, 2005).
Lietuvoje žmonių ir gyvūnų dermatomikozių priežastimi dažniausiai tampa dermatofitai Microsporum canis, Microsporum gypseum ir Trichophyton mentagrophytes, taip pat mieliagrybiai Malassezia pachidermatis, Candida albicans, C. parapsilosis etc. (Lapinskait÷, 2007). Kauno visuomen÷s sveikatos centro duomenimis, per pastaruosius 9 metus Kauno apskrities gyventojų sergamumo dermatofitija rodiklis svyravo nuo 5,3 iki 37,3 susirgimų 10 000 gyventojų.
Nors gyvūnams su stipria imunine sistema dermatofitija gali praeiti savaime, šį susirgimą būtina gydyti. Taip galima sutrumpinti ir sušvelninti ligos eigą, apsaugoti aplinką nuo patogeno. Dermatofitija gydoma vietiškai arba sistemiškai.
Veterinarinei medicinai skirtų vaistų nuo dermatofitijos pasiūla yra ribota. Daugelis medicinoje vartojamų vaistų nuo mikroskopinių grybų naudojami veterinarin÷je medicinoje, tačiau sisteminis jų poveikis dažnai daro neigiamą įtaką gyvūno organizmui, o išorinio naudojimo preparatai yra mažai efektyvūs ir n÷ra skirti gyvūno odai. Efektyvios ir saugios terapijos nuo mikroskopinių grybų poreikis veterinarijoje did÷ja.
Darbo tikslas – nustatyti mikroskopinių grybų paplitimą šunų ir kačių kailyje; jų sergamumą dermatofitija ir įvertinti kremų E-1 ir T-1 gydomąjį poveikį šunims ir kat÷ms, sergantiems dermatofitija.
Darbo uždaviniai:
1. Nustatyti mikroskopinių grybų paplitimą sveikų ir sergančių odos ligomis šunų ir kačių kailyje;
2. Ištirti patogeninių mikroskopinių grybų jautrumą vaistin÷ms medžiagoms – ekonazolio nitratui, mikonazolio nitratui ir terbinafino hidrochloridui;
3. Nustatyti tirpalo pH reikšmę transmembraniniam ekonazolio nitrato ir terbinafino skverbimuisi;
4. Nustatyti kremų E-1 ir T-1 poveikį sveikai odai;
5. Įvertinti kremų E-1 ir T-1 terapinį efektyvumą jūrų kiaulyt÷ms, sergančioms eksperimentiškai sukelta dermatofitija;
6. Įvertinti kremų E-1 ir T-1 terapinį efektyvumą šunims ir kat÷ms, sergantiems dermatofitija.
Mokslinis darbo naujumas, mokslin÷ ir praktin÷ vert÷
Nustatytas patogeninių ir saprofitinių mikroskopinių grybų paplitimo mastas kliniškai sveikų ir odos pažeidimų turinčių šunų ir kačių kailyje. Nustatyta, kad naminiai augintiniai, ypač kat÷s, turi didelį potencialą platinti dermatofitiją. Kat÷s dermatofitija serga keturis kartus dažniau nei šunys.
Lietuvos veterinarijos akademijos Eksperimentin÷s ir klinikin÷s farmakologijos laboratorijoje sukurti kremai E-1 ir T-1 gyvūnams, sergantiems dermatofitija, gydyti. Ištirtas terapinis šių kremų poveikis smulkiesiems gyvūnams. Nustatyta, kad kremai E-1 ir T-1 greitai ir efektyviai pašalina ligos požymius ir sunaikina suk÷l÷ją, saugo aplinką nuo patogeno.
MEDŽIAGOS IR METODAI
Pirmame darbo etape ištirtas mikroskopinių grybų paplitimas kliniškai sveikų ir odos pažeidimų turinčių šunų ir kačių kailyje. M÷giniai imti iš 153 kliniškai sveikų gyvūnų (89 šunų ir 64 kačių) ir 174 (129 šunų ir 45 kačių) odos pažeidimų turinčių gyvūnų. Iš kliniškai sveikų gyvūnų plaukų m÷giniai imti modifikuotu Makenzio metodu (Katoh et al., 1990; Moriello, 2003 a). Iš gyvūnų turinčių klinikinių odos pažeidimų steriliu pincetu plaukai išpešti iš odos pažeidimo centro ir demarkacin÷s zonos tarp sveikos ir pažeistos odos, surinkti šašai ir pleiskanos. M÷giniai s÷ti į selektyvias terpes. Ant terp÷s išaugę mikroskopiniai grybai identifikuoti analizuojant jų morfologines ir kultūrines savybes pagal literatūroje pateiktus mikromicetų rūšių aprašymus (Van Cutsem, Rochette, 1991; Quinn et al., 1994; 2002; Lugauskas ir kt., 2002).
Antrame darbo etape in vitro sultinio mikroskiedimo metodu pagal Klinikinių ir laboratorinių standartų instituto (CLSI – Clinical and Laboratory Standards Institute) nurodymus nustatytas patogeninių mikroskopinių grybų jautrumas vaistams nuo grybų – ekonazolio nitratui, mikonazolio nitratui ir terbinafino hidrochloridui. Darbiniai 0,5 proc. (5 mg/ml) tirpalai buvo paruošti 100 proc. dimetilsulfokside (DMSO). Serijiniai dukartiniai vaistinių medžiagų skiediniai paruošti naudojant RPMI 1640 terpę (Sigma-Aldrich, JAV).
Trečiame darbo etape įvertinta buferinio tirpalo pH reikšm÷ ekonazolio nitrato ir terbinafino skverbimuisi per sintetinę polidimetilsiloksano membraną. Tyrimai atlikti naudojant Franco difuzijos kameras. Receptorin÷je kameros dalyje panaudojome PBS tirpalą, donoro – prisotintą buferinį tirpalą, kuriame ištirpinta tiriama vaistin÷ medžiaga. Buferiniai tirpalai buvo skirtingų pH reikšmių (5, 6 ir 7).
Ketvirtame darbo etape nustatytas kremų E-1 ir T-1 poveikis sveikai odai. Bandyme panaudota 10 Naujosios Zelandijos baltųjų triušių ir 10 Dunkin Hartley veisl÷s jūrų kiaulyčių. Triušiams kair÷s šlaunies oda, o jūrų kiaulyčių kairiojo pilvo šono oda buvo tepta bandomuoju kremu, o dešin÷s pus÷s oda nebuvo tepta kremais ir buvo kontrolin÷. Kremai tepti kasdien 10 dienų iš eil÷s. Tyrimo metu slankmačiu matuota teptos odos raukšl÷s
apimtis, vertinta odos būkl÷, o sudirginimo laipsnis vertintas balais. Po tepimo pra÷jus 7 ir 14 dienų buvo vertinama odos būkl÷ tepimo vietose ir odos raukšl÷s apimtis. Po 10 bandymo dienų iš abiejų jūrų kiaulyčių grupių atsitiktiniu būdu parinktos dvi ir eutanazuotos, atlikti odos histopatologiniai tyrimai.
Penktame darbo etape įvertintas kremų E-1 ir T-1 terapinis efektyvumas gydant smulkius gyvūnus, sergančius dermatofitija bei palygintas su kitais preparatais. Bandymui naudota 20 jūrų kiaulyčių, eksperimentiškai užkr÷stų M. canis dermatofitu, bei 51 šuo ir kat÷, sergantys dermatofitija. Jūrų kiaulyt÷ms nugaros srityje 2x2 cm plote nukirpti plaukai ir skarifikuota oda, užkr÷sta paruošta M. canis suspensija. Jūrų kiaulyt÷s suskirstytos į 5 grupes. I bandomųjų gyvūnų grup÷ buvo gydoma kremu T-1 tepant pažeistą vietą 1xd. kasdien, iki gyvūnas pasveiko kliniškai ir mikologiškai, II grup÷ buvo gydoma kremu E-1, III grup÷ buvo gydoma Lietuvoje registruotu vaistu nuo grybų „Imaverol“ pagal aprašymo nuorodas (pažeista vieta 0,2 proc. emulsija purkšta kas 3 dienos), IV grup÷ gydyta eksperimentinius kremus sudarančiu pagrindu be veikliųjų medžiagų, V grup÷ buvo kontrolin÷ ir nebuvo gydyta jokiais preparatais. Vertindami kiekvieno bandomojo preparato klinikinį efektyvumą, kiekvienos jūrų kiaulyt÷s pažeistos odos plotą dalijome į keturias dalis. Kremų E-1 ir T-1, šių kremų pagrindo ir „Imaverol“ klinikinis efektyvumas nustatytas įvertinus kiekvienos dalies pažeidimo mastą balais pagal M. A. Ghannoum ir kitų mokslininkų (2009) metodiką. Vaistų efektyvumas apskaičiuotas pagal formulę:
efektyvumo % = 100 – (T٠100/C),
čia: T – gydomos pažeistos odos dalių bendra balų suma; C – negyjančios pažeistos odos dalių bendra balų suma – 20.
Tiriamų gydomųjų preparatų ir negydomų odos pažeidimų savaiminio gijimo antimikotinis efektyvumas nustatytas atliekant plauko šaknies invazijos tyrimą pagal M. A. Ghannoum ir kitų mokslininkų (2009) aprašytą metodiką. Antimikotinio efektyvumo procentin÷ išraiška apskaičiuota pagal tą pačią formulę, kaip ir vertinant klinikinį efektyvumą (efektyvumo % = 100 – (T٠100/C)), tik C skaičiuota 10 balų.
Šunys ir kat÷s, kuriems nustatytas sergamumas dermatofitija, turintys nuo vieno iki penkių aiškiai išreikštų odos pažeidimų, kurių skersmuo ne didesnis kaip 5 cm, buvo gydomi kremais T-1 ir E-1. Odos pažeidimai eksperimentiniais kremais tepti du kartus per dieną, ryte ir vakare, švelniai įtrinant į pažeistą odą. Gyvūnas buvo laikomas pasveikęs, kai pažeistos odos plaukai visiškai ataugo, o mikologinio tyrimo rezultatas buvo neigiamas.
Gydant pažeistą odą kremais T-1 ir E-1, atsižvelgiant į grybin÷s invazijos intensyvumą, kai kurių gyvūnų kailis papildomai buvo purškiamas „Imaverol“ tirpalu kas 3 dienas, iki gyvūnas kliniškai pasveiko. Statistin÷
tyrimų duomenų analiz÷ atlikta SPSS statistiniu paketu Nr. 15 versija (SPSS for Windows 9.0, SPSS Inc., Chicago, IL, USA, 1989-1995). Analizuota kačių ir šunų amžiaus, lyties, plauko ilgio ir laikymo sąlygų įtaka mikroskopinių grybų paplitimui kliniškai sveikų ir odos pažeidimų turinčių gyvūnų kailyje. Kokybinių požymių ryšiui nustatyti naudotas chi kvadratas (χ2), esant patikimam ryšiui apskaičiuoti koreliacijos koeficientai (φ). Statistiškai reikšmingi skirtumai tarp grupių apibr÷žti, kai p<0,05 ir p<0,01. Atliekant kremų T-1 ir E-1 poveikį sveikai odai apskaičiuoti odos raukšl÷s apimties aritmetiniai vidurkiai (X), vidutiniai kvadratiniai nuokrypiai (σ). Buvo lyginti bandomųjų odos raukšlių apimčių aritmetiniai vidurkiai prieš tepimą kremu ir kiekvieną tepimo dieną. Aritmetinių vidurkių skirtumo patikimumas (p) nustatytas pagal Stjudentą. Rezultatai laikyti patikimais, kai p<0,05. Atlikdami bandomųjų preparatų terapinio efektyvumo vertinimą apskaičiavome odos pažeidimų balų aritmetinius vidurkius (X), vidutinius kvadratinius nuokrypius (σ). Lygindami bandomųjų preparatų efektyvumą tarpusavyje ar su savaiminiu sveikimu, panaudojome Mano-Vitnio testą, statistiškai patikimi aritmetinių vidurkių skirtumai tarp dviejų grupių nustatyti kai p<0,05.
TYRIMŲ REZULTATAI IR APTARIMAS
Tyrimai su kliniškai sveikais gyvūnais parod÷, kad šunų kailyje dermatofitai paplitę 20,2 proc., o kačių – 18,8 proc. Iš šunų išskirti kelių rūšių dermatofitai − Microsporum canis, Microsporum gypseum, Trichophyton mentagrophytes, o iš kačių tik vienos rūšies – M. canis. Nustat÷me, kad dažniausiai paplitęs dermatofitas yra M. canis, šunų ir kačių kailyje jis paplitęs atitinkamai 16,9 proc. ir 18,8 proc. Daugiau M. canis sporų išskirta iš vyresnių nei vieneri metai šunų (80 proc.; p=0,981) ir šunų, laikomų lauke (80 proc.; p=0,063). Lytis ir plauko ilgis netur÷jo reikšmingos įtakos M. canis sporų paplitimui kliniškai sveikų šunų kailyje. Kačių amžius ir lytis netur÷jo įtakos M. canis sporų paplitimui kailyje, tačiau plauko ilgis ir laikymo sąlygos − tur÷jo. Iš trumpaplaukių kačių M. canis sporos išskirtos dažniau nei iš ilgaplaukių (atitinkamai 75,0 proc. ir 25,0 proc.) (p=0,381). Įdomu tai, kad iš kačių, laikomų tik patalpose, išskirta šiek tiek daugiau dermatofitų nei iš kačių, kontaktuojančių su aplinka (atitinkamai 58,3 proc. ir 41,7 proc.) (p=0,837).
Ištyrę šunis ir kates, turinčius klinikinių odos ir plauko pažeidimų, nustat÷me, kad 27,6 proc. šių pažeidimų priežastis buvo dermatofitai. Iš šunų išskirti trijų rūšių dermatofitai, o iš kačių − tik vienos. Apie 85 proc. visų išskirtų dermatofitų sudar÷ M. canis. Nustat÷me, kad kat÷s keturis kartus dažniau nei šunys serga M. canis sukelta dermatofitija, atitinkamai
55,6 proc. ir 12,4 proc. Net 68,0 proc. mikrosporija sergančių kačių buvo jaunesn÷s nei vieneri metai. Šie duomenys statistiškai patikimi (p<0,01) ir patvirtina kitų mokslininkų tyrimų rezultatus: gyvūnų sergamumas dermatofitija priklauso nuo amžiaus (Cabanes et al., 1997; Paixão et al., 2001; Brilhante et al., 2003; Khosravi, Mahmoudi, 2003). Didesn÷ dalis kačių, sergančių mikrosporija, gal÷jo išeiti į lauką (76 proc.) (p<0,01). Kaip ir kliniškai sveikoms, taip ir odos pažeidimų turinčioms trumpaplauk÷ms kat÷ms mikrosporija diagnozuota dažniau nei ilgaplauk÷ms (atitinkamai 76,0 proc. ir 24,0 proc.) (p=0,249).
Ištyrus sergamumo priklausomybę nuo kačių lyties, mūsų gauti duomenys sutapo su L. Pinter (1999) ir grup÷s tyr÷jų, taip pat A. Patel (2005) ir grup÷s mokslininkų gautais rezultatais, kai nustatyta, jog dažniau dermatofitija serga katinai, o ne kat÷s.
Dermatofitija dažniau sirgo vyresni nei vienerių metų šunys, trumpo plauko patinai. Nustat÷me, kad šunys M. canis sukelta dermatofitija dažniau serga rudenį (43,75 proc.), o kat÷s − vasarą (44 proc.).
Be M. canis dermatofito, nustatyti ir kiti šunų dermatofitijos suk÷l÷jai − M. gypseum (1,6 proc.) ir T. mentagrophytes (3,9 proc.). Mūsų tyrimo metu dermatofitas M. gypseum išskirtas iš dviejų šunų žiemą ir pavasarį. Šunys buvo laikomi privačioje valdoje, tod÷l didžiąją dienos dalį praleisdavo lauke. T. mentagrophytes išskirtas iš penkių šunų skirtingais metų laikais – rudenį (n=2, 40,0 proc.; p=0,824), žiemą (n=2, 40,0 proc.; p=0,977) ir pavasarį (n=1, 20,0 proc.; p=0,703).
Tyrimo metu kreiptas d÷mesys į saprofitin÷s grybin÷s mikrofloros paplitimą sveikame kailyje ir pažeistoje odoje tų gyvūnų, iš kurių išskirtas patogeninis dermatofitas M. canis. Nustatyta, kad klinikinių odos pažeidimų neturinčiuose gyvūnuose, kurių kailyje rastas M. canis dermatofitas, randama daugiau saprofitinių mikroskopinių grybų genčių nei kailyje gyvūnų, sergančių šio patogeno sukelta dermatomikoze. Saprofitiniai grybai pažeistoje gyvūnų odoje buvo paplitę ženkliai mažiau nei sveikų gyvūnų kailyje.
Atlikdami tyrimus su kliniškai sveikais ir odos pažeidimų turinčiais gyvūnais, rinkome duomenis apie jų savininkų sveikatos būklę, susijusią su šia zoonoze. Tyrimų metu nustat÷me, kad dvylika gyvūnų savininkų sirgo dermatofitija, iš jų − šeši vaikai iki 14 metų, penkios jaunos moterys iki 30 metų ir viena vyresn÷ nei 50 metų moteris. Šie žmon÷s laik÷ gyvūnus, kurie sirgo M. canis sukelta dermatofitija arba buvo besimptomiai M. canis nešiotojai. Didesnę laikomų gyvūnų dalį sudar÷ kat÷s, galinčios išeiti į lauką.
Lietuvos veterinarijos akademijos Eksperimentin÷s ir klinikin÷s farmakologijos laboratorijoje pagamintos dvi kremų pavidalo vaistin÷s
formos – T-1 ir E-1, skirtos šunų ir kačių, sergančių dermatofitija, paviršiniam gydymui. Į kremo T-1 sud÷tį įeina alilaminas terbinafinas (1 proc. terbinafino hidrochloridas), o į kremo E-1 – azolis ekonazolis (1 proc. ekonazolio nitratas).
Panaudojus Franco difuzijos kameras ištirta tirpalo pH įtaka ekonazolio nitrato ir terbinafino skverbimuisi per membraną. Nustatyta, kad terbinafino ir ekonazolio nitrato difuzija per silikoninę membraną buvo l÷ta ir pH reikšm÷ netur÷jo įtakos spartesnei šių medžiagų difuzijai. Po 24 val. ekonazolio nitrato srautas iš pH 5 tirpalo buvo lygus 6,78±0,58 µg/cm²/val., o srautas iš pH 7 tirpalo buvo lygus 7,34±1,18 µg/cm²/val. Po 24 val. terbinafino srautas iš pH 5 tirpalo buvo lygus 1,38±0,32 µg/cm²/val., o srautas iš pH 6 tirpalo – 1,34±0,29 µg/cm²/val. Atlikus eksperimentinių kremų transmembraninio praeinamumo bandymą, chromatografin÷ sistema receptorin÷je faz÷je neaptiko veikliųjų medžiagų terbinafino ir ekonazolio.
Kremų T-1 ir E-1 gydomasis poveikis įvertintas atlikus bandymus su gyvūnais. Gydydami jūrų kiaulytes, sergančias eksperimentiškai sukelta dermatofitija, nustat÷me, kad abu kremai, naudojami vieną kartą per dieną, efektyviai pašalino ligos suk÷l÷ją ir klinikinius ligos požymius. Kremas T-1 klinikinius ligos požymius pašalino per 30,3±3,4 dienos, o suk÷l÷jas buvo likviduotas po dviejų gydymo savaičių. Gydant kremu E-1 klinikiniai ligos požymiai išnyko per 44,3±3,5 dienos, o suk÷l÷jo nebeišskyr÷me po trijų gydymo savaičių. „Imaverol“ tirpalas klinikinius ligos požymius pašalino per 42,3±3,4 dienos, o suk÷l÷jo neišskirta po keturių gydymo savaičių. Bandymo metu su eksperimentiškai sukelta dermatofitija gyvūnus gyd÷me eksperimentinių kremų pagrindu, kurio sud÷tyje n÷ra veikliųjų ar pagalbinių gydomųjų medžiagų, pasižyminčių fungistatin÷mis arba keratolitin÷mis savyb÷mis. Nustat÷me, kad kremų pagrindas teigiamą terapinį poveikį padar÷ per 46,0±3,5 gydymo dienas. Šioje gyvūnų grup÷je ligos suk÷l÷jas buvo pašalintas po penkių savaičių. Kontrolin÷s grup÷s jūrų kiaulyt÷ms klinikiniai ligos požymiai savaime išnyko per 64,3±6,1 dienos nuo užkr÷timo, bet ligos suk÷l÷jas buvo pašalintas tik po 10 savaičių.
Bandymu, kurio metu šunys ir kat÷s, sergantys dermatofitija, buvo gydomi eksperimentiniais kremais du kartus per dieną, nustat÷me, kad kat÷s, sergančios labiausiai paplitusia M. canis sukelta dermatofitija ir gydomos kremu T-1, pasveiko per 23,6±7,22 dienos, o gydomos kremu E-1 – per 30,2±10,85 dienos. Šunys, sergantys M. canis sukelta dermatofitija ir gydyti kremu T-1, pasveiko per 26,9±6,17 dienos, o gydyti kremu E-1 – per 26,3±5,03 dienos. Šio bandymo metu grupei kačių, turinčių lokalinių odos pažeidimų ir didelę dermatofitų sporų invaziją kailyje, be gydymo kremu T-1, buvo taikomas papildomas gydymas „Imaverol“ tirpalu. Toks sinergetinis gydymas teigiamą terapinį efektą dav÷ per 25,5±9,28 dienos.
Mūsų bandymų metu kremas T-1 pasižym÷jo didesniu terapiniu efektyvumu ir mažesniu šalutiniu poveikiu odai nei kremas E-1. Gydant kremu T-1, oda buvo paraudusi tų gyvūnų, kurie tur÷jo pažeidimų ant ausies kaušelio. Didesnei daliai gyvūnų, gydytų kremu E-1, oda paraudo ir buvo dirgli kremo tepimo vietoje. Gyvūnai prad÷davo kasyti pateptą vietą tuoj pat užtepus kremo, bet dirginimas išnykdavo po valandos. Žinoma, kad azoliai ir alilaminai, naudojami kaip išviršiniai vaistai, sukelia odos niež÷jimą, paraudimą, deginimo pojūtį (Katzung, 2007). Nor÷dami išsiaiškinti, ar odos paraudimas, atsiradęs nuo kremų, yra reakcija į kremo sud÷tines dalis, ar yra odos uždegimin÷ reakcija į patogeną, atlikome tyrimą su kliniškai sveikais laboratoriniais gyvūnais. Tirdami kremų poveikį sveikai triušių ir jūrų kiaulyčių odai nustat÷me, kad nuo kremo E-1 triušių oda ilgai išliko paraudusi, o jūrų kiaulyčių oda neparaudo. Nuo kremo T-1 trumpam parausdavo triušių oda, bet po valandos paraudimas išnykdavo. Nuo to paties kremo jūrų kiaulyčių oda neparausdavo. Histologiškai ištyrę jūrų kiaulyčių odos m÷ginius, ryškių spongioz÷s ar edemos požymių, būdingų alerginiam kontaktiniam dermatitui, nenustat÷me, tačiau nustat÷me nedidelę eozinofilų infiltraciją dermoje. Taip pat nustatyta, kad kremais teptos odos epidermis yra šiek tiek storesnis nei neteptos.
Tyrimais nustat÷me, kad dermatofitai M. canis, M. gypseum ir T. mentagrophytes yra jautrūs azoliams − ekonazolio nitratui ir mikonazolio nitratui, taip pat alilaminui terbinafino hidrochloridui. Nustat÷me, kad dermatofitai labiausiai jautrūs terbinafino hidrochloridui. Vidutin÷ minimali ekonazolio nitrato koncentracija, slopinanti dermatofitus, svyruoja nuo 1,3 iki 10,0 µg/ml, mikonazolio nitrato – nuo 2,5 iki 10,0 µg/ml, o terbinafino hidrochlorido – nuo 0,65 iki 1,3 µg/ml.
Apibendrindami tyrimų rezultatus galime teigti, kad kliniškai sveiki šunys ir kat÷s yra besimptomiai dermatofitų nešiotojai. Kat÷s dermatofitija serga keturis kartus dažniau nei šunys. Naujai paruoštos išorinio naudojimo T-1 ir E-1 vaistin÷s formos yra efektyvios gydant smulkiuosius gyvūnus, sergančius dermatofitija. Kremas T-1 yra efektyvesnis ir mažiau dirgina odą nei kremas E-1. Pastarasis sukelia nestiprų kontaktinį dermatitą, kuris pasireiškia pirmosiomis gydymo dienomis. Manome, kad tikslingai parinkta vaistin÷ medžiaga ir vaistin÷ forma l÷m÷ didelį kremo T-1 gydomąjį poveikį. Fungicidinis terbinafino poveikis dermatofitams, jo savyb÷ kauptis odos raginiame sluoksnyje, kremo savyb÷ dr÷kinti odą ir silpnai šarmin÷ pH (6,2) tur÷jo įtakos spartesniam pažeistos odos gijimui palyginti su Lietuvoje registruotu „Imaverol“ tirpalu.
