Medical Mycology, 2014, 52, 350–355 doi: 10.1093/mmy/myt016 Advance Access Publication Date: 28 February 2014 Original Article
Original Article
In vitro and in vivo activity of a killer peptide against Malassezia pachydermatis causing otitis in dogs
1
Dipartimento di Medicina Veterinaria, Universita` degli Studi di Bari, Bari and 2 Dipartimento di Scienze Biomediche, Biotecnologiche e Traslazionali, Universita` degli Studi di Parma, Parma, Italy
*To whom correspondence should be addressed. Claudia Cafarchia, Dipartimento di Medicina Veterinaria, Universita` degli Studi di Bari, Str. prov.le per Casamassima Km 3, 70010 Valenzano, Bari, Italy. Tel: +39 080 467 9834; Fax: +39 0804679837; E-mail: [email protected] Received 9 July 2013; Revised 26 August 2013; Accepted 7 November 2013
Abstract In order to overcome the limitations inherent in current pharmacological treatments for Malassezia pachydermatis, the cause of otitis externa in dogs, the efficacy of a killer decapeptide (KP) was evaluated in vitro and in vivo. Sixteen dogs with naturally occurring M. pachydermatis otitis externa were enrolled, and the in vitro fungicidal activity of KP was evaluated using yeasts recovered from these animals. The therapeutic activity was evaluated in four groups of four animals each. The dogs were topically treated with KP (150 µl, 2 mg/ml) three times per week (group A) or every day (group B), treated with a scramble peptide every day (group C), or left untreated (group D). Assessment of clinical signs (pruritus, erythema, and lichenification and/or hyperpigmentation), expressed as mean of the total clinical index score (mTCIS), the population size of M. pachydermatis at the cytological examination (mean number of yeast cells at 40× magnification [mYC]), and culture testing (mean number of log10 CFU/swab [mCFU]), were conducted daily from the first day of treatment (T0) until two consecutive negative cultures (mCFU ≤ 2). KP showed an in vitro fungicidal effect against M. pachydermatis isolates, with an MFC90 value of 1 µg/ml. The mTCIS, mYC and mCFU were negative only in animals in group B after T8. Daily administration of KP for 8 days was safe and effective in controlling both clinical signs and the population size of M. pachydermatis causing otitis externa, thus offering an alternative to the currently available therapeutic or prophylactic protocols for recurrent cases of Malassezia otitis in dogs. Key words: Malassezia pachydermatis, otitis, dogs, killer peptide, therapy.
350
C The Author 2014. Published by Oxford University Press on behalf of The International Society for Human and Animal
Mycology. All rights reserved. For permissions, please e-mail: [email protected]
Downloaded from http://mmy.oxfordjournals.org/ at Yale University on September 30, 2014
Claudia Cafarchia1,∗ , Davide Immediato1 , Giancarlo Di Paola1 , Walter Magliani2 , Tecla Ciociola2 , Stefania Conti2 , Domenico Otranto1 and Luciano Polonelli2
Cafarchia et al.
Introduction
Materials and methods KP The previously described KP (aminoacid sequence: AKVTMTCSAS; molecular weight, 998.2) was used in this study [13]. A scramble decapeptide (SP) (aminoacid sequence: MSTAVSKCAT), previously proven to be devoid
of fungicidal activity, was included as a negative control [13].
Evaluation of in vitro antifungal activity of KP Chemically synthesized KP and SP were solubilized in sterile distilled water (2 mg/ml) the day before use. Fungicidal activity was tested on 16 Malassezia strains isolated from enrolled dogs (i.e., one for each dog) as previously described [13]. In brief, Malassezia isolates were grown on Dixon agar at 35◦ C for 4 days. Yeast cells were then transferred and suspended in sterile distilled water in order to obtain a concentration of 106 CFU/ml. Next, 10 µl of the suspension was added to 90 µl of water containing KP or SP at different concentrations (1:2 serial dilution from 1 µg/ml to 0.061 µg/ml). Yeast cells in sterile distilled water without KP or SP were used as growth control. After incubation (i.e., 6 h at 30◦ C), the suspensions were inoculated onto Dixon agar and CFU enumerated after incubation at 35◦ C for 4 days. The minimal fungicidal concentration (MFC) was assessed as the lowest KP concentration that totally inhibited the growth of yeast cells. Each assay was performed in triplicate for statistical purposes. Results are expressed as the value at which the growth of 90% (MFC90 ) of yeast cells was inhibited.
Evaluation of in vivo therapeutic activity of KP Sixteen client-owned dogs with M. pachydermatis otitis externa were enrolled in the study after receiving written consent from the animals’ owners. Individual data (i.e., sex, age, breed) together with medical history were collected for each dog. The 4 females and 12 males were crossbred, aged 3–5 years, and had pendulous ears. Dogs were included in the study if they had no previous history of skin and underlying metabolic diseases. Dogs with infectious otitis were recruited based on clinical signs compatible with the disease, including head shaking, pruritus, pain, erythema, and swelling. The presence of otitis externa was confirmed by otoscopic exam. Systemic and alternative topical antimicrobial and antiinflammatory treatments were not used for 3 weeks prior to and during the study. All dogs were fed the same commercial food (Hill’s Science Diet Canine Adult Maintenance, Rome, Italy) for 3 weeks prior to and during the study. Dogs were clinically and microbiologically evaluated to ascertain the diagnosis of Malassezia infections during their first clinical presentation at day -7 (T-7) and later on day 0 (T0) before treatment and daily throughout the study (see below). Animals were randomly enrolled into four groups, four animals each, according to the baseline of the mean of log10 CFU (mCFU) value at T-7 and KP treatment regimen as follows: group A, treated with KP
Downloaded from http://mmy.oxfordjournals.org/ at Yale University on September 30, 2014
Malassezia pachydermatis is a commensal yeast of the skin of animals; it may become pathogenic under the influence of predisposing factors [1–3]. Clinical manifestations in domestic animals and nosocomial infections in humans include otitis externa/media and various forms of dermatitis [3–6]. Otitis externa caused by M. pachydermatis is one of the most common diseases of dogs, and treatment of severe infections may require systemic therapy with high doses of antifungal agents (e.g., ketoconazole and itraconazole) for prolonged periods [6,7]. Primary underlying diseases involved in Malassezia overgrowth include allergic dermatitis, intertrigo, endocrinopathies, primary keratinization defects and pyoderma. Additionally, pendulous ears are considered a predisposing factor for otitis externa [3–6]. Since otitis externa related to M. pachydermatis is secondary to underlying conditions, the best therapeutic response is usually achieved when topical therapy including either azole antifungal drugs (principally miconazole or clotrimazole) or nystatin is used in combination with antibiotics and glucocorticoid to control concurrent bacterial infection and reduce inflammation [6,7]. However, this clinical condition may be unresponsive to the antifungal therapy and remain unresolved for long periods [6,7]. Additionally, the overuse of polypharmaceutical products that contain antibiotics and antifungal agents might result in development of resistance to one or the other or both. The threat of such resistance has spurred interest in the scientific community to discover new, effective antifungal drugs [8–10]. Recently, chelating agents have been found to be useful in increasing the efficacy of antifungal drugs in treating Malassezia-causing otitis externa [11]. A killer decapeptide (KP) engineered from the variable region of a single-chain recombinant antiidiotypic antibody and representing the internal image of a yeast killer toxin (KT) characterized by wide-spectrum activity against fungi that present specific β-glucan KT cell-wall receptors displayed in vitro and in vivo antifungal effects [12,13]. The aims of this study were to evaluate, for the first time, the in vitro antifungal activity of KP against M. pachydermatis isolates and the in vivo therapeutic activity vs. Malassezia otitis externa in naturally infected dogs.
351
352
Microbiological diagnosis Malassezia infection was evaluated using both cytological examination and fungal culture. Briefly, samples were collected from each animal from the right and left ear canal using two sterile cotton swabs introduced into both external ear canals at the furthest accessible level of the auditory canal. Samples were stained with May-Grunwald Giemsa ¨ for microscopic examination [15,16]. The number of yeasts in five random fields at 40× magnification was recorded and a mean yeast count (mYC) calculated for each animal group. Swab samples were also immersed into 1 ml of sterile distilled water and gently vortexed. Three serial dilutions (1:10 v/v) were prepared and 0.1 ml of each dilution was inoculated onto modified Dixon agar and incubated at 32◦ C for 4 days. The colonies were counted and the results reported as number of CFU per swab. The CFU for each dog
was expressed as log10 CFU and then averaged for each treatment group (mCFU). Samples were considered positive when numbers exceeding 10 Malassezia cells were present in five fields at 40× magnification on cytological examination or when numbers in excess of 100 CFU (mCFU >2) were counted on Dixon agar plates [15,16]. Four colonies from each positive sample at T0 were subcultured on modified Dixon agar and identified physiologically and phenotypically by their macro- and microscopic morphology [17].
Statistical analysis Statistical calculations were performed using mTCIS, mYC, and mCFU/swab values. The homogeneity of the groups was checked with respect to the CFU counts obtained on T-7 by using a two-sample t test. Differences in mTCIS, mYC, and mCFU/swab between T0 and different sampling times were compared using the Mann-Whitney test for two independent samples for ordinal data (mTCIS scores) and the Student t test for continuous data (mYC and mCFU). A P < 0.05 was considered significant.
Results All Malassezia isolates recovered were physiologically and morphologically identified as M. pachydermatis. All were found to be susceptible to KP, and the MFC90 value was 1 µg/ml. SP had no effect at any concentration. KP therapeutic activity on animals in groups A and B and SP activity on animals in group C during the study period are reported as mTCIS, mYC, and mCFU values (Tables 1–3). Briefly, SP had no effect on animals, confirming the specificity of KP activity (Tables 1–3). mTCIS decreased significantly at 4 days post treatment (T4) in group A (mTCIS from 9 to 6.5) and in group B (mTCIS from 5.8 to 2.5), reaching a negative value (mTCIS 0.05) of mYC was observed in samples from groups A, C, and D throughout the study period (Table 2). The mCFU decreased significantly at T2 in group B (mCFU from 7.9 to 4.9) and at T4 in group A (mCFU from 7.5 to 4.1; Table 3). At T8, the values of mCFU were
Downloaded from http://mmy.oxfordjournals.org/ at Yale University on September 30, 2014
three times per week; group B, treated with KP every day; group C, treated with SP every day; and group D, untreated controls. On day 0 (T0), KP solution (150 µl, 2 mg/ml) was gently rubbed (i.e., filling the ear canal with liquid and then massaging the ears) into ears of animals in groups A and B; ear hairs were cut and ear cerumen removed with a sterile swab. The same amount of SP solution (2 mg/ml) was used for animals in group C and sterile distilled water (i.e., the peptide solvent) was used for animals in group D. Treatment was continued until two consecutive cultures were negative (mCFU ≤ 2) in one of the KP-treated groups (i.e., groups A and B; see below). Animals were observed daily throughout the study for any adverse reactions and were evaluated clinically and microbiologically for 2 months after the last treatment. A clinical index score (CIS) was assigned to each dog, and lesions (i.e., presence of erythema and greasy exudates and lichenification and/or hyperpigmentation) were evaluated by three examiners using Canine Atopic Dermatitis Extent and Severity Index - CADESI-03 scores with a severity scale of 0 to 5 [14]. The severity of pruritic manifestations was evaluated using the pruritus visual analog scale (PVAS) graded from 0 to 3, with the left most corner underlined by the statement “not itchy, no scratching, chewing, rubbing, or licking observed” and the right most end with the statement “extremely itchy, scratching, chewing, rubbing, or licking constantly” [14]. Total CIS (TCIS = CIS pruritus + CIS erythema + CIS lichenification and/or hyperpigmentation) was calculated for each dog and then averaged for each treatment group (mean of total clinical index score [mTCIS]).
Medical Mycology, 2014, Vol. 52, No. 4
Cafarchia et al.
353
Table 1. Evolution of total clinical index score, reported as mean values (mTCIS) with standard deviation in brackets, during 10 days of treatment with killer decapeptide. mTCIS (standard deviation) at T0
T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
Group A
9 (0)
9 (0)
9 (0)
9 (0)
6.5∗ (0.6)
6.5∗ (0.6)
6.5∗ (0.6)
6.5∗ (0.6)
6.5∗ (0.6)
6.5∗ (0.6)
5∗ (1.2)
Group B
5.8 (0.5)
5.8 (0.5)
5 (0.8)
5 (0.8)
2.5∗ (1.3)
2.3∗ (1.3)
1.5∗ (1.7)
1.5∗ (1.7)
0.8∗ (0.5)
0.8∗ (0.5)
0.3∗ (0.5)
Group C
6.5 (1.0)
6.5 (1.0)
6.5 (1.0)
6.7 (1.0)
6.5 (1.0)
6.3 (1.3)
6.3 (1.3)
6.3 (1.3)
6.3 (1.3)
6.3 (1.3)
6.3 (1.3)
Group D
8 (1.2)
8 (1.2)
7.8 (1)
7.8 (1)
7.8 (1)
7.8 (1)
7.8 (1)
7.8 (1)
7.8 (1)
7.8 (1)
7.8 (1)
Table 2. Evolution of the mean number of Malassezia pachydermatis yeast cells (mYC), with standard deviation in brackets, on cytological examination at 40× magnification during 10 days of treatment with killer decapeptide. mYC (standard deviation) at T0
T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
Group A
16 (9.6)
16 (9.6)
13 (9.6)
13 (9.6)
7.5 (3)
5.3 (1.5)
4.5∗ (1.7)
7.5 (5.2)
10 (5.2)
8.3 (4.5)
6 (0)
Group B
51 (27)
49 (28)
33 (17)
33 (17)
25 (17)
19∗ (5)
17∗ (6.8)
16∗ (9.7)
10∗ (3.4)
8∗ (2.3)
8∗ (2.3)
Group C
28 (4)
28 (4)
31 (26)
30 (25)
30 (25)
31 (26)
33 (25)
33 (25)
28 (11)
28 (11)
33 (12)
Group D
45 (17)
45 (16)
38 (20)
36 (16)
33 (18)
32 (18)
41 (40)
42 (40)
44 (22)
41 (26)
26 (6.7)
Statistical significant differences (P < 0.05) vs. T0 of the same group (∗ ).
negative (mCFU
Original Article
In vitro and in vivo activity of a killer peptide against Malassezia pachydermatis causing otitis in dogs
1
Dipartimento di Medicina Veterinaria, Universita` degli Studi di Bari, Bari and 2 Dipartimento di Scienze Biomediche, Biotecnologiche e Traslazionali, Universita` degli Studi di Parma, Parma, Italy
*To whom correspondence should be addressed. Claudia Cafarchia, Dipartimento di Medicina Veterinaria, Universita` degli Studi di Bari, Str. prov.le per Casamassima Km 3, 70010 Valenzano, Bari, Italy. Tel: +39 080 467 9834; Fax: +39 0804679837; E-mail: [email protected] Received 9 July 2013; Revised 26 August 2013; Accepted 7 November 2013
Abstract In order to overcome the limitations inherent in current pharmacological treatments for Malassezia pachydermatis, the cause of otitis externa in dogs, the efficacy of a killer decapeptide (KP) was evaluated in vitro and in vivo. Sixteen dogs with naturally occurring M. pachydermatis otitis externa were enrolled, and the in vitro fungicidal activity of KP was evaluated using yeasts recovered from these animals. The therapeutic activity was evaluated in four groups of four animals each. The dogs were topically treated with KP (150 µl, 2 mg/ml) three times per week (group A) or every day (group B), treated with a scramble peptide every day (group C), or left untreated (group D). Assessment of clinical signs (pruritus, erythema, and lichenification and/or hyperpigmentation), expressed as mean of the total clinical index score (mTCIS), the population size of M. pachydermatis at the cytological examination (mean number of yeast cells at 40× magnification [mYC]), and culture testing (mean number of log10 CFU/swab [mCFU]), were conducted daily from the first day of treatment (T0) until two consecutive negative cultures (mCFU ≤ 2). KP showed an in vitro fungicidal effect against M. pachydermatis isolates, with an MFC90 value of 1 µg/ml. The mTCIS, mYC and mCFU were negative only in animals in group B after T8. Daily administration of KP for 8 days was safe and effective in controlling both clinical signs and the population size of M. pachydermatis causing otitis externa, thus offering an alternative to the currently available therapeutic or prophylactic protocols for recurrent cases of Malassezia otitis in dogs. Key words: Malassezia pachydermatis, otitis, dogs, killer peptide, therapy.
350
C The Author 2014. Published by Oxford University Press on behalf of The International Society for Human and Animal
Mycology. All rights reserved. For permissions, please e-mail: [email protected]
Downloaded from http://mmy.oxfordjournals.org/ at Yale University on September 30, 2014
Claudia Cafarchia1,∗ , Davide Immediato1 , Giancarlo Di Paola1 , Walter Magliani2 , Tecla Ciociola2 , Stefania Conti2 , Domenico Otranto1 and Luciano Polonelli2
Cafarchia et al.
Introduction
Materials and methods KP The previously described KP (aminoacid sequence: AKVTMTCSAS; molecular weight, 998.2) was used in this study [13]. A scramble decapeptide (SP) (aminoacid sequence: MSTAVSKCAT), previously proven to be devoid
of fungicidal activity, was included as a negative control [13].
Evaluation of in vitro antifungal activity of KP Chemically synthesized KP and SP were solubilized in sterile distilled water (2 mg/ml) the day before use. Fungicidal activity was tested on 16 Malassezia strains isolated from enrolled dogs (i.e., one for each dog) as previously described [13]. In brief, Malassezia isolates were grown on Dixon agar at 35◦ C for 4 days. Yeast cells were then transferred and suspended in sterile distilled water in order to obtain a concentration of 106 CFU/ml. Next, 10 µl of the suspension was added to 90 µl of water containing KP or SP at different concentrations (1:2 serial dilution from 1 µg/ml to 0.061 µg/ml). Yeast cells in sterile distilled water without KP or SP were used as growth control. After incubation (i.e., 6 h at 30◦ C), the suspensions were inoculated onto Dixon agar and CFU enumerated after incubation at 35◦ C for 4 days. The minimal fungicidal concentration (MFC) was assessed as the lowest KP concentration that totally inhibited the growth of yeast cells. Each assay was performed in triplicate for statistical purposes. Results are expressed as the value at which the growth of 90% (MFC90 ) of yeast cells was inhibited.
Evaluation of in vivo therapeutic activity of KP Sixteen client-owned dogs with M. pachydermatis otitis externa were enrolled in the study after receiving written consent from the animals’ owners. Individual data (i.e., sex, age, breed) together with medical history were collected for each dog. The 4 females and 12 males were crossbred, aged 3–5 years, and had pendulous ears. Dogs were included in the study if they had no previous history of skin and underlying metabolic diseases. Dogs with infectious otitis were recruited based on clinical signs compatible with the disease, including head shaking, pruritus, pain, erythema, and swelling. The presence of otitis externa was confirmed by otoscopic exam. Systemic and alternative topical antimicrobial and antiinflammatory treatments were not used for 3 weeks prior to and during the study. All dogs were fed the same commercial food (Hill’s Science Diet Canine Adult Maintenance, Rome, Italy) for 3 weeks prior to and during the study. Dogs were clinically and microbiologically evaluated to ascertain the diagnosis of Malassezia infections during their first clinical presentation at day -7 (T-7) and later on day 0 (T0) before treatment and daily throughout the study (see below). Animals were randomly enrolled into four groups, four animals each, according to the baseline of the mean of log10 CFU (mCFU) value at T-7 and KP treatment regimen as follows: group A, treated with KP
Downloaded from http://mmy.oxfordjournals.org/ at Yale University on September 30, 2014
Malassezia pachydermatis is a commensal yeast of the skin of animals; it may become pathogenic under the influence of predisposing factors [1–3]. Clinical manifestations in domestic animals and nosocomial infections in humans include otitis externa/media and various forms of dermatitis [3–6]. Otitis externa caused by M. pachydermatis is one of the most common diseases of dogs, and treatment of severe infections may require systemic therapy with high doses of antifungal agents (e.g., ketoconazole and itraconazole) for prolonged periods [6,7]. Primary underlying diseases involved in Malassezia overgrowth include allergic dermatitis, intertrigo, endocrinopathies, primary keratinization defects and pyoderma. Additionally, pendulous ears are considered a predisposing factor for otitis externa [3–6]. Since otitis externa related to M. pachydermatis is secondary to underlying conditions, the best therapeutic response is usually achieved when topical therapy including either azole antifungal drugs (principally miconazole or clotrimazole) or nystatin is used in combination with antibiotics and glucocorticoid to control concurrent bacterial infection and reduce inflammation [6,7]. However, this clinical condition may be unresponsive to the antifungal therapy and remain unresolved for long periods [6,7]. Additionally, the overuse of polypharmaceutical products that contain antibiotics and antifungal agents might result in development of resistance to one or the other or both. The threat of such resistance has spurred interest in the scientific community to discover new, effective antifungal drugs [8–10]. Recently, chelating agents have been found to be useful in increasing the efficacy of antifungal drugs in treating Malassezia-causing otitis externa [11]. A killer decapeptide (KP) engineered from the variable region of a single-chain recombinant antiidiotypic antibody and representing the internal image of a yeast killer toxin (KT) characterized by wide-spectrum activity against fungi that present specific β-glucan KT cell-wall receptors displayed in vitro and in vivo antifungal effects [12,13]. The aims of this study were to evaluate, for the first time, the in vitro antifungal activity of KP against M. pachydermatis isolates and the in vivo therapeutic activity vs. Malassezia otitis externa in naturally infected dogs.
351
352
Microbiological diagnosis Malassezia infection was evaluated using both cytological examination and fungal culture. Briefly, samples were collected from each animal from the right and left ear canal using two sterile cotton swabs introduced into both external ear canals at the furthest accessible level of the auditory canal. Samples were stained with May-Grunwald Giemsa ¨ for microscopic examination [15,16]. The number of yeasts in five random fields at 40× magnification was recorded and a mean yeast count (mYC) calculated for each animal group. Swab samples were also immersed into 1 ml of sterile distilled water and gently vortexed. Three serial dilutions (1:10 v/v) were prepared and 0.1 ml of each dilution was inoculated onto modified Dixon agar and incubated at 32◦ C for 4 days. The colonies were counted and the results reported as number of CFU per swab. The CFU for each dog
was expressed as log10 CFU and then averaged for each treatment group (mCFU). Samples were considered positive when numbers exceeding 10 Malassezia cells were present in five fields at 40× magnification on cytological examination or when numbers in excess of 100 CFU (mCFU >2) were counted on Dixon agar plates [15,16]. Four colonies from each positive sample at T0 were subcultured on modified Dixon agar and identified physiologically and phenotypically by their macro- and microscopic morphology [17].
Statistical analysis Statistical calculations were performed using mTCIS, mYC, and mCFU/swab values. The homogeneity of the groups was checked with respect to the CFU counts obtained on T-7 by using a two-sample t test. Differences in mTCIS, mYC, and mCFU/swab between T0 and different sampling times were compared using the Mann-Whitney test for two independent samples for ordinal data (mTCIS scores) and the Student t test for continuous data (mYC and mCFU). A P < 0.05 was considered significant.
Results All Malassezia isolates recovered were physiologically and morphologically identified as M. pachydermatis. All were found to be susceptible to KP, and the MFC90 value was 1 µg/ml. SP had no effect at any concentration. KP therapeutic activity on animals in groups A and B and SP activity on animals in group C during the study period are reported as mTCIS, mYC, and mCFU values (Tables 1–3). Briefly, SP had no effect on animals, confirming the specificity of KP activity (Tables 1–3). mTCIS decreased significantly at 4 days post treatment (T4) in group A (mTCIS from 9 to 6.5) and in group B (mTCIS from 5.8 to 2.5), reaching a negative value (mTCIS 0.05) of mYC was observed in samples from groups A, C, and D throughout the study period (Table 2). The mCFU decreased significantly at T2 in group B (mCFU from 7.9 to 4.9) and at T4 in group A (mCFU from 7.5 to 4.1; Table 3). At T8, the values of mCFU were
Downloaded from http://mmy.oxfordjournals.org/ at Yale University on September 30, 2014
three times per week; group B, treated with KP every day; group C, treated with SP every day; and group D, untreated controls. On day 0 (T0), KP solution (150 µl, 2 mg/ml) was gently rubbed (i.e., filling the ear canal with liquid and then massaging the ears) into ears of animals in groups A and B; ear hairs were cut and ear cerumen removed with a sterile swab. The same amount of SP solution (2 mg/ml) was used for animals in group C and sterile distilled water (i.e., the peptide solvent) was used for animals in group D. Treatment was continued until two consecutive cultures were negative (mCFU ≤ 2) in one of the KP-treated groups (i.e., groups A and B; see below). Animals were observed daily throughout the study for any adverse reactions and were evaluated clinically and microbiologically for 2 months after the last treatment. A clinical index score (CIS) was assigned to each dog, and lesions (i.e., presence of erythema and greasy exudates and lichenification and/or hyperpigmentation) were evaluated by three examiners using Canine Atopic Dermatitis Extent and Severity Index - CADESI-03 scores with a severity scale of 0 to 5 [14]. The severity of pruritic manifestations was evaluated using the pruritus visual analog scale (PVAS) graded from 0 to 3, with the left most corner underlined by the statement “not itchy, no scratching, chewing, rubbing, or licking observed” and the right most end with the statement “extremely itchy, scratching, chewing, rubbing, or licking constantly” [14]. Total CIS (TCIS = CIS pruritus + CIS erythema + CIS lichenification and/or hyperpigmentation) was calculated for each dog and then averaged for each treatment group (mean of total clinical index score [mTCIS]).
Medical Mycology, 2014, Vol. 52, No. 4
Cafarchia et al.
353
Table 1. Evolution of total clinical index score, reported as mean values (mTCIS) with standard deviation in brackets, during 10 days of treatment with killer decapeptide. mTCIS (standard deviation) at T0
T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
Group A
9 (0)
9 (0)
9 (0)
9 (0)
6.5∗ (0.6)
6.5∗ (0.6)
6.5∗ (0.6)
6.5∗ (0.6)
6.5∗ (0.6)
6.5∗ (0.6)
5∗ (1.2)
Group B
5.8 (0.5)
5.8 (0.5)
5 (0.8)
5 (0.8)
2.5∗ (1.3)
2.3∗ (1.3)
1.5∗ (1.7)
1.5∗ (1.7)
0.8∗ (0.5)
0.8∗ (0.5)
0.3∗ (0.5)
Group C
6.5 (1.0)
6.5 (1.0)
6.5 (1.0)
6.7 (1.0)
6.5 (1.0)
6.3 (1.3)
6.3 (1.3)
6.3 (1.3)
6.3 (1.3)
6.3 (1.3)
6.3 (1.3)
Group D
8 (1.2)
8 (1.2)
7.8 (1)
7.8 (1)
7.8 (1)
7.8 (1)
7.8 (1)
7.8 (1)
7.8 (1)
7.8 (1)
7.8 (1)
Table 2. Evolution of the mean number of Malassezia pachydermatis yeast cells (mYC), with standard deviation in brackets, on cytological examination at 40× magnification during 10 days of treatment with killer decapeptide. mYC (standard deviation) at T0
T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
Group A
16 (9.6)
16 (9.6)
13 (9.6)
13 (9.6)
7.5 (3)
5.3 (1.5)
4.5∗ (1.7)
7.5 (5.2)
10 (5.2)
8.3 (4.5)
6 (0)
Group B
51 (27)
49 (28)
33 (17)
33 (17)
25 (17)
19∗ (5)
17∗ (6.8)
16∗ (9.7)
10∗ (3.4)
8∗ (2.3)
8∗ (2.3)
Group C
28 (4)
28 (4)
31 (26)
30 (25)
30 (25)
31 (26)
33 (25)
33 (25)
28 (11)
28 (11)
33 (12)
Group D
45 (17)
45 (16)
38 (20)
36 (16)
33 (18)
32 (18)
41 (40)
42 (40)
44 (22)
41 (26)
26 (6.7)
Statistical significant differences (P < 0.05) vs. T0 of the same group (∗ ).
negative (mCFU
