Evaluation of Malassezia pachydermatis antifungal susceptibility using two different methods

Avaliação da sensibilidade da Malassezia pachydermatis frente a antifúngicos através de duas técnicas

VETERINARY MICROBIOLOGY

Evaluation of Malassezia pachydermatis antifungal susceptibility using two different methods

Avaliação da sensibilidade da Malassezia pachydermatis frente a antifúngicos através de duas técnicas

Patrícia da Silva NascenteI, ^*; Márcia de Oliveira Nobre^II; Luiz Filipe Schuch^I; Thomaz Lucia-Júnior^I; Laerte Ferreiro^II; Mário Carlos Araújo Meireles^I

^IDepartamento de Veterinária Preventiva, Faculdade de Veterinária, Universidade Federal de Pelotas, Pelotas, RS, Brasil

^IIDepartamento de Patologia Clínica Veterinária, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brasil

ABSTRACT

Malassezia pachydermatis is recognized as a normal inhabitant and an opportunistic pathogen of the external ear canal and skin of dogs and cats. In especial clinical conditions, and mainly in the cases of therapeutic failure related to external otitis and dermatitis complicated by this yeast, it is recommended test susceptibility to antifungal drugs. The purpose of this work was to evaluate the susceptibility of 44 isolates of M. pachydermatis from the external ear canal and skin of dogs and cats using two different in vitro antifungal susceptibility methods: the Etest® and the broth microdilution method. Thirty-five samples were tested using the Etest®, twenty-four samples were tested using the broth microdilution method and fifteen samples were tested using both tests. The antifungal agents used were ketoconazole (KTZ), fluconazole (FLZ) and itraconazole (ITZ). In the broth microdilution method the yeast was less susceptible to ITZ while KTZ had the strongest activity. On the other hand, the Etest® showed that M. pachydermatis was more susceptible to ITZ while the less active drug was FLZ. The simultaneous evaluation using both methods identified FLZ as the antifungal drug with the highest activity (64.3%), followed by KTZ (57.1%) and ITZ (28.6%). These results showed that there is an urgent need to standardize of the values considered as parameters for growth inhibition of this yeast so a simple and efficient method can be used routinely in the laboratory practice.

Key words:Malassezia pachydermatis, Etest®, broth microdilution method, ketoconazole, itraconazole, fluconazole.

RESUMO

Malassezia pachydermatis é considerada um habitante normal e patógeno oportunista do meato acústico externo e tegumento cutâneo de cães e gatos. Em condições clínicas especiais e nos casos de fracasso terapêutico, comum em casos de otite externa ou dermatite complicadas por esta levedura, é recomendado testar a sensibilidade aos antifúngicos. O objetivo do trabalho foi avaliar a sensibilidade de 44 isolados de M. pachydermatis do meato acústico externo e do tegumento cutâneo de cães e gatos, através de duas técnicas de antifungigrama: ETEST e Microdiluição em Caldo (MC). Foram testadas 35 amostras pelo método ETEST (1994) e 24 pelo método de MC e 15 pelos dois testes. Os antifúngicos utilizados foram: cetoconazol (KTZ), fluconazol (FLZ) e itraconazol (ITZ). Através da MC, a levedura foi menos sensível ao ITZ, enquanto que o antifúngico com maior atividade foi o KTZ. M. pachydermatis foi mais sensível ao ITZ pelo ETEST, enquanto a droga menos ativa foi o FLZ. A avaliação simultanea através das duas técnicas aponta o FLZ como o antifúngico com maior concordância entre os resultados (64,3%), seguido pelo KTZ (57,1%) e ITZ (28,6%). Os resultados indicam que há uma necessidade urgente de padronização dos valores de inibição do crescimento desta levedura para que um método, simples e eficaz, possa ser amplamente utilizado na rotina laboratorial.

Palavras-chave:Malassezia pachydermatis, ETEST, antifungigrama, Microdiluição em caldo, cetoconazol, itraconazol, fluconazol.

INTRODUCTION

Malassezia pachydermatis is recognized as a normal inhabitant and an opportunistic pathogen of the external ear canal and skin of dogs and cats, also can be found in the rectum, interdigital, anal sacs and vagina (3,22,24). The identification of this yeast as a common agent of mycosis of dogs and cats (4,9,14,22) make it necessary to test the antifungal susceptibility of the isolates to the drugs currently used for its treatment (1,5,21,22) which are itraconazole, fluconazole and, mainly ketoconazole.

The broth microdilution method, and more recently, the Etest® are recommended for testing antifungal susceptibility of yeasts like Candida sp. and Cryptococcus sp., but there are few studies dealing with susceptibility of isolates of M. pachydermatis commonly used with broth macrodilution and agar gel diffusion methods (2,6,16,17,18,25). Etest® (13) and broth microdilution methods (23) were already used for testing M. pachydermatis but there are no data comparing the performance of both methods.

The purpose of this work was to evaluate the antifungal susceptibility of 44 isolates of M. pachydermatis to ketoconazole, fluconazole and itraconazole using Etest® (13) and broth microdilution methods (23).

MATERIALS AND METHODS

The isolates were obtained from the external acoustic meatus and skin of dogs and cats and were cultivated in Sabouraud's agar-dextrose with chloramphenicol and cycloheximide. The tests were performed at the mycology laboratory of the Faculdade de Veterinária da Universidade Federal de Pelotas, Pelotas, Brazil. Antifungal susceptibility of M. pachydermatis against ketoconazole (KTZ), fluconazole (FLZ) and itraconazole (ITZ) was determinet by Etest® (35 isolates), broth microdilution (24 isolates) and both methods (15 isolates).

The strips of the Etest® have a defined and consistent gradient of the antifungal drug of 0.002 to 32.0 µg/mL to KTZ and ITZ and 0.016 to 256.0 µg/mL to FLZ allowing a quantitative reading. The culture medium used was Sabouraud dextrose agar with chloramphenicol according to the Etest® manufacturer recommendations. M. pachydermatis isolates were suspended in saline with turbidity adjusted to level 1 of the McFarland scale and cultivated by spreading the sample in the culture medium. After 15 min, the strips were distributed over the medium and incubated at 37ºC. The readings were performed at 48, 72 and 96 h and the MICs (Minimum Inhibitory Concentrations) the lowest concentration of the drug was determined by the Etest® strip pattern.

The NCCLS' broth microdilution method (23) recommendations were adapted by Eichenberg (10,11) for M. pachydermatis. Ten dilutions of each drug storage solution were prepared obtaining 10 solutions with a gradient 10 times greater than the final solution of each drug used. The ten solutions obtained this way had the drug concentration from 64 to 0.125 µg/mL to FLZ, 4 to 0.078 µg/mL to ITZ and 8 to 0.015 µg/mL to KTZ and they were put into the first 10 contiguous wells of a sterile microtiter plate. The solution containing each isolate was transferred in aliquots of 100 µL into each well of the sterile plates that already had 100 µL of the solution containing the antifungal drug tested. The wells 11 and 12 had the positive control (100 µL of Sabouraud dextrose agar and 100 µL of the half-inoculum solution) and the negative control (200 µL of the same culture medium). The plates were incubated at 37ºC for 96 h. The readings were made by visually comparing the growth of the yeast on wells one to ten with wells that had the positive control (wells 11). The lowest concentration that produced a relative significant inhibition (around 50%) of the growth of the yeast observed for the positive control was identified as the MIC of the drug (10,11).

The MICs were calculated according Coutinho (7,8) for the Etest® and Eichenberg (10,11) for the broth microdilution method. M. pachydermatis was classified as susceptible (S), intermediary susceptible (I) and resistant (R) using the criteria established by Colombo et al. (7) such as: S = MIC sample < MIC50, I = MIC50 < MIC sample < MIC90, R = MIC sample > MIC90. The results were codified and analyzed using the software Statistix 7.0 (27). The frequency distribution and the chi-square test were applied to evaluate the two in vitro antifungal susceptibility methods.

RESULTS AND DISCUSSION

The range for Etest® MICs to KTZ was 0.002 to 0.25 µg/mL and the mean MIC was 0.057 µg/mL while the broth microdilution method showed a MIC ranging from 0.03 to 8 µg/mL and the mean MIC of 1.28 µg/mL. The results of the Etest® obtained in this work for the MICs had a broader range, higher values and an average MIC higher than the values reported by Coutinho (7,8). This author found a mean MIC 0.08 µg/mL (0.015 to 0.25 µg/mL) and MIC values lower than the data obtained by Uchida et al. (28) that ranged from 0.002 µg/mL to 10 µg/mL, but similar to the values obtained by Lorenzini et al. (17), Mickelsen et al. (20).

Regarding to FLZ, the Etest® had a mean MIC of 21.24 µg/mL (0.016 to 256 µg/mL) and the broth microdilution method showed a mean MIC of 9.22 µg/mL (0.5 to 64 µg/mL). Using the Etest®, the values obtained were lower than the values of Coutinho (7,8) that found a mean MIC of 37.47 µg/mL (8 to 256 µg/mL). In addition, the broth microdilution method provided average MIC values higher than the value of 9.22 µg/mL (1 to 32 µg/mL) obtained by Eichenberg (10,11).

ITZ tested by Etest® presented mean MIC = 0.073 µg/mL (0.002 to 2 µg/mL) and using the broth microdilution method the mean MIC = 0.62 µg/mL (0.03 to 4 µg/mL). Those values were higher than average MIC reported by Coutinho (7,8) that used the Etest® and found 0.004 µg/mL (0.002 to 0.008 µg/mL). However, Eichenberg (10,11) found an average MIC of 0.05 µg/mL (0.007 to 0.125 µg/mL).

The concentrations of the antifungal drugs used were different for each of the two techniques and for this reason it was not possible to estimate whether there was a coincidence in the MICs found in each of the tests alone. It was possible to establish relations between the values just after classifying the yeast isolates in susceptible, intermediary susceptible, and resistant using the susceptibility calculation described above (Table 1).

Comparing the MICs obtained in this work with the results of Coutinho (7,8) and Eichenberg (10,11) there is little similarities among the results. At this moment, it is only possible to evaluate what drug is more efficient for an isolate in a specific method but it is not possible to compare susceptibilities (MICs) using different methods.

In the Etest®, ITZ had the highest number of susceptible isolates (82.9%) followed by KTZ (71.4%) and FLZ (60%). According to the broth microdilution test, the most efficacious drug was KTZ (70.8%), followed by FLZ (62.5%) and ITZ (41.7%). The percentage of susceptible samples to FLZ and KTZ using both methods were similar, but there was a great divergence among those results to ITZ. Using this approach, the fungus Malassezia pachydermatis was more susceptible (p<0.05) to ITZ using the Etest® while there was no difference (p>0.005) between the two different methods to FLZ and KTZ.

Also, there was no difference (p>0.005) among the activity of the three different drugs used when they were evaluated using the same method. In opposition to it, Lorenzini et al. (17) and Uchida (28), who tested respectively 5 and 10 antifungal drugs against this yeast, concluded that KTZ is the most active antifungal drug.

In the 15 isolates evaluated by both methods, it was possible to observe the percentage of results that were susceptible or resistant in the same sample (Table 2). Nonetheless, it was not possible to compare the MIC of the isolates in each test due to the difference in the pattern of the interval of the concentrations. The mean MIC to KTZ, FLZ and ITZ observed in these 15 isolates were respectively 0.059 µg/mL, 16.99 µg/mL and 0.0027 µg/mL using the Etest® and 0.70 µg/mL, 2.43 µg/mL and 0.38 µg/mL using the broth microdilution method (MC). The means MIC had different values to the same drug when comparing the two methods although there was agreement for the increasing values for MICs following the order ITZ, KTZ and FLZ. The results obtained to KTZ agreed in 57.1% (8) of the samples, 64.3% (9) to FLZ and, just 28.6% (4) to ITZ. In the reviewed literature, there no any study comparing two different in vitro antifungal susceptibility methods for Malassezia pachydermatis. However, a similar study applying the same two methods was reported by Martin-Mazuelos et al. (19) using Candida spp and testing FLZ and ITZ, whit FLZ having highest agreement (74.5% of the samples).

The differences between the methods did not allow a deep analysis of the results because the MIC test is highly dependent of factors such as the inoculum concentrations, chemical composition of the medium, pH, temperature, and incubation time (7,8,12,15,26). The results found in this work showed that it is possible to compare the degree of susceptibility to each antifungal drug but without comparing the MICs. To compare the MICs, its is necessary an agreement for the drug concentrations to be used in both methods because Etest® have a broader range for drug concentrations than the broth microdilution test.

CONCLUSION

The aspects related to the different methods indicate the necessity of standardized method that could be widely used in research and in mycology laboratories as in the same manner as in bacteriology. More studies about the MICs of M. pachydermatis are necessary to standardize the values for growth inhibition of the yeast in both methods, to allow comparisons of results obtained from various laboratories.

Submitted: October 10, 2003; Approved: February 23, 2004

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Publication Dates

• Publication in this collection
  13 Aug 2004
• Date of issue
  Dec 2003

History

• Accepted
  23 Feb 2004
• Received
  10 Oct 2003