On-line version ISSN 1806-4841
An. Bras. Dermatol. vol.84 no.3 Rio de Janeiro July 2009
Lívia Maria Martins de AlmeidaI; Eliane Alves de Freitas SouzaII; Débora Bertoluzzi BianchinIII; Terezinha Inez Estivalet SvidzinskiIV
IUndergraduate, School of Medicine,
Universidade Estadual de Maringá (UEM) Maringá (PR), Brazil
IIUndergraduate, School of Medicine, Universidade Estadual de Maringá (UEM) Maringá (PR), Brazil
IIIBiochemical Pharmacist, Specialist, Medical Mycology - Universidade Estadual de Maringá (UEM) Maringá (PR), Brazil
IVPh.D., Associate Professor, Discipline of Medical Mycology, Universidade Estadual de Maringá (UEM) Maringá (PR), Brazil
BACKGROUND The high frequency of cutaneous mycosis justify the
need to evaluate the possible contribution of in vitro profile of susceptibility
to antifungal agents.
OBJECTIVE To evaluate whether there is variability in in vitro susceptibility by filamentous fungi, previously isolated from cutaneous mycosis, to fluconazole, ketoconazole, itraconazole and terbinafine.
METHODS - Fungi were isolated and identified by classical methods and the antifungal susceptibility test was performed using the method of broth microdilution, according to a protocol recommended by the Clinical Laboratory Standards Institute (CLSI), through M38-A document.
RESULTS Amongst the 80 filamentous fungi identified, Trichophyton genus represented 81%. The four examined drugs showed great variation for Trichophyton spp and Microsporum spp. Fusarium spp was resistant to all tested drugs. Terbinafine was the most effective drug against the majority of the isolated fungi.
CONCLUSION - There was great variability in response profiles to the tested antifungals. The definition of a reference test method will offer higher objectivity for physicians to choose the appropriate therapy.
Keywords:Antifungal Agents; Antifungal Agents/Analysis; Arthrodermataceae; Mycoses
Cutaneous mycoses are fungal infections localized on the superficial layers of skin and annexes 1, and may be caused by dermatophytes, yeasts or non-dermatophyte filamentous fungi (NDFF). Epidemiological studies have indicated that cutaneous mycosis are among the most prevalence diseases in the world 2, affecting all age ranges and leading to millions of dollars spent in treatment every year 3. The distribution and frequency of cutaneous mycosis and its etiological agents may vary according to geographical region and social economic levels of the population 2,4, whose most important predisposing factors are professional occupation and individual habits. Thus, it is important to know the epidemiology to come to controlling these infections 5.
Cutaneous mycosis vary depending on clinical forms and causal agents, but most of the authors report as the most frequent agents dermatophytes (80.0 90.0%), followed by yeasts (5.0 17.0%) and NDFF (3.0 12.0%).2 Infection by dermatophytes affects approximately 40% of the world population and represents 30% of all cutaneous mycotic infections, which most commonly affect the skin and nails.
Many patients come for dermatological offices and outpatient units owing to superficial and cutaneous mycoses 6. Cutaneous mycoses, especially onychomycosis, are sometimes difficult to treat and recurrences are frequent, which in part is due to poor compliance with treatment owing to high cost of drugs, prolonged treatment, poor use or discontinuation of the drug, inefficiency or resistance to drug, and adverse effects such as hepatotoxicity and gastrointestinal manifestations. The success of the treatment is linked to correct diagnosis and prescription, plus treatment compliance, which in many situations requires persistence and change of habits, which have to be dealt by professionals 7.
Among the cutaneous mycoses, the ones that are the most difficult to treat are those involving the nails. Monotherapy to treat onychomycosis may fail in most of the cases 8,9 and the reason for it has not been fully explained yet 8. However, some factors may be attributed to this lack of success, among which the difficulty to reach appropriate concentration of the drug in the infected site, peripheral vascular abnormalities, slow growth of nails, especially of toe nails, making them vulnerable to reinfection, onset of antifungal resistance, which according to Baran may result in fungi that do not respond to the spectrum of any drug 8.
The main groups of systemic antifungal agents normally used for treating superficial and cutaneous mycoses are imidazole (Ketoconazole), triazole (fluconazole and itraconazole) and alilamine (terbinafine). Rather than having greater variability of antifungal options, both topical and systemic agents, the therapeutic arsenal is very restricted and there is a clear need for new more effective and less toxic antifungal agents 9. Many drugs belong to the same group of pharmacological action and have the same action mechanism, however, fungi respond significantly different to these drugs 10,11. Because it takes a complex choice to select the appropriate antifungal agent, it is necessary to learn much information about the fungus itself and its interaction with antifungal drugs 12. However, treatment is normally prescribed in an empirical form, without mycological confirmation and many times it is applied without appropriate follow-up time.
To present, laboratory diagnosis of mycoses is not commonly used. However, considering the similarities of clinical manifestations by fungal infections with other etiologies, the importance of lab characterization to provide effective treatment 2,13 is evident, which would also allow the reduction of costs generated by empirical therapy 2,13. It is increasingly important to learn about the sensitivity profile of clinical strains and the spectrum of action of antifungal agents, because the detection of resistance is extremely important when it comes to choosing a therapeutic alternative 14.
Moreover, laboratory tests may help to distinguish a reinfection by the same agent, infection by a new agent and provide evidence whether the fungus is responsible for treatment failure or whether it is a limitation of the antifungal agent 15,16. In recent years many techniques have been standardized for detection of in vitro resistance, which shows good correlation with clinical progression of patients.
Currently, the susceptibility tests for filamentous fungi may be performed in laboratories according to the document M38-A passed on by NCCLS (National Committee for Clinical Laboratory Standards), now named CLSI (Clinical and Laboratory Standards Institute), and even though it is not exclusive for dermatophytes, it standardizes AST (Antifungal Susceptibility Test) for filamentous fungi through two methodologies (macro and microdilution in broth), both with good interlaboratorial reproducibility 17,18.
The purpose of this paper was to assess whether there is variability in isolates concerning in vitro susceptibility of filamentous fungi, previously isolated from cutaneous mycosis, in response to antifungal agents such as fluconazole, ketoconazole, itraconazole and terbinafine, based on the method of microdilution in broth suggested by document M38-A of CLSI.
MATERIAL AND METHOD
This experimental study was carried out with fungal samples from the filamentous fungi bank of Sector of Medical Mycology Laboratory of Teaching and Research in Clinical Analyses (LEPAC), at Universidade Estadual de Maringa, Parana, Brazil.
All isolated filamentous fungi were analyzed (n=80) and identified as agents of cutaneous mycoses (skin, hairs and nails), according to 3-month referrals from physicians, distributed as follows Trichophyton mentagrophytes (35), Trichophyton rubrum (21), Trichophyton tonsurans (6), Trichophyton raubitscheki (3), Microsporum canis (8), Microsporum gypseum (2), Microsporum ferrugineum (1), Fusarium oxysporum (1), Fusarium solani (1), Fusarium incarnatum (1) and Fusarium verticillioides (1). Samples were maintained in sterile distilled water at 4º C until use.
Minimum inhibitory concentration (MIC) was determined for fluconazole FLC (Pfizer), ketoconazole - CTC (Janssen-Cilag), itraconazole ITC (Sporanox®-Janssen-Cilag) and terbinafine TRB (Galena and Química Farmacêutica) and the method employed was microdilution in broth according to the protocol recommended by CLSI in the document M38-A (2002). Each drug was prepared to be tested in 10 concentrations within the following limits: 0.12μg/mL to 64μg/mL for FLC, 0.06μg/mL to 32μg/mL for TRB and 0.03μg/mL to 16μg/mL for ITC and CTC. These antifungal agents were chosen because they already have test protocols, and because they are the most widely used for oral administration in clinical practice. The results may serve as a reference for possible prescriptions of topical agents.
To prepare the inoculates, the fungal samples were cultivated in Sabouraud Dextrose Agar (SDA) and incubated at 25o C for 72 hours (Fusarium) or seven days (dermatophytes). Fungal colonies were covered with 5mL of saline sterilized solution 0.85% and then gently homogenized with the Pasteur pipette. The mix of hyphae and conidia was transferred to sterilized tapered tubes and let to rest for 20 minutes for sedimentation. The supernatant was removed by aspiration, transferred to other tube and the density of the inoculate was adjusted to 68-70% of transmittance in 530nm, using spectrophotometer (Bausch Lomb). The suspension was diluted in RPMI 1640 medium, so as to obtain final concentration close to 104UFC/mL. This concentration was checked by 10ÂµL plaques of inoculate prepared with SDA and colony number counting.
Each antifungal drug was distributed in serial dilutions in columns of 1 to 10, in 96-well microplaques. For the assay, we added 100ÂµL of the obtained inoculate and the plaques were incubated in 25º C without stirring.
In each test plaque a positive control was included, which represented growth of each isolate in the absence of the drug (column 11), plus a negative control, which corresponded to absence of drug and fungus (column 12). Moreover, a standard microorganism Candida parapsilosis ATCC 22019 was tested in all plaques, with all dilutions of each drug to ensure reproducibility and accuracy of the test.
MIC values were set after 72 hours of incubation for genus Fusarium and 7 days for dermatophytes by visual observation of inhibition of growth in each well compared to growth observed in the positive control well. MIC for FLC and CTC were defined as concentration in which there was 50% of the reduction of fungal growth. For ITC and TRB MIC values were the lowest concentration of the drug that resulted in 100% of inhibition of microorganism growth.
MIC90 was defined as the MIC value capable of inhibiting 90% of the isolates of the same species.
Out of 80 tested samples, genus Trichophyton represented 81%, Microsporum 14% and Fusarium 5%. It was possible to demonstrate broad variability concerning in vitro response of these agents in view of the most widely used antifungal agents to treat cutaneous mycoses. Species of Trichophyton (except for T. raubitscheki) showed great variation of MIC to the four tested drugs, as well as samples of M. canis (except for TRB). Fusarium species did not show variability, because they presented high MIC to all analyzed drugs.
MIC of ITC was low for most fungal isolates (about 0.25 0.5μg/mL), but in view of the eight samples of T. mentagrophytes (23%) and 11 of T. rubrum (52%), MIC was elevated =16μg/mL (Graph 1).
The trend of the samples for CTC was to present from medium to high MIC (1 - 2μg/mL), but two samples of M. gypseum presented high MIC - 8 μg/mL (Graph 2).
Fungal samples presented significant variation of MIC to FLC, and most had responses of about 4- 8μg/mL (Graph 3).
TRB was the drug that had the lowest MIC (0.06μg/mL) for most samples, but 48% of the isolates of T. rubrum revealed high MIC =32μg/mL (Graph 4).
Fusarium SP did not show in vitro response to none of the tested antifungal agents, given that all samples grew even in the presence of concentrations higher than recommended for the test.
By analyzing genus Trichophyton, T. mentagrophytes and T. tonsurans had elevated MIC90 to all tested drugs, T. rubrum also presented high MIC90 to most antifungal agents, and the best response was for FLC (CIM90 = 16μg/mL). Conversely, T. raubitscheki responded well to the tested drugs.
As to genus Microsporum sp, M. canis responded well only to TRB (CIM90 = 1 μg/mL), and the other species presented good responses to TRB and ITC.
Medical mycology has been through a number of changes. Antifungal prophylaxis and empirical therapy have contributed to epidemiological changes, such as onset of strains with secondary resistance to antifungal agents and replacement of some sensitive species by others with intrinsic resistance 11,14. Within this context, sensitivity tests in mycology are highly important 19, even though the correlation between in vitro tests and in vivo therapeutic response has not been totally defined yet, given that treatment depends on variables linked to the clinical presentation of the host 14,16.
In vitro tests to assess antimicrobial susceptibility are essential for appropriate antibiotic therapy because they contribute to choosing the best alternative of treatment, reducing the possibility of therapeutic failure. However, differently from what happens with bacteria and yeasts, there is no reference standardized method to measure in vitro activity of dermatophyte filamentous fungi to antifungals 5,5, 15,18,20. The Document M38A by CLSI, directed to filamentous fungus, does not determine the cut-off point (sensitive, intermediate or resistance), nor includes protocols to dermatophytes. This is one of the reasons why treatment is still empirical, given that AST is not standardized for dermatophytes.
In addition, it is important to learn about the biological characteristics of the main agents, because the identification may guide the treatment- for example, Fusarium, which showed high MIC to all assessed drugs, confirmed the results by Guillermetti at. al, 200721 and Azor et al, 2007.22 A significant problem that hinders the treatment of invasive infections by Fusarium is the high rate of generalized resistance to antifungals 23,24. Apparently, resistance is an intrinsic characteristic of this genus, which hinders the treatment of cutaneous infections, requiring new options of antifungal treatment.
Fusarium belongs to the group of NDFF, which is widely distributed in the soil, plants, air and water 22. The species that have most frequently caused pathogenicity to man are F. solani, considered to be the most resistant to treatment, followed by F. oxysporum.23 Infections by Fusarium are frequently reported in immunocompromised patients, but in the study by Paz et al., 2004, 76% of the infections were in immunocompetent subjects, and Guilhermetti et al, 2007, reported that all isolates of Fusarium were obtained from immunocompetent subjects 21,25.
Anthropophilic fungi, such as T. rubrum and T. tonsurans, have greater adaptation to human beings and we expected higher MICs, which was a fact. Some important data observed in this study were the responses of T. rubrum to terbinafine - 48% of the sample responded to lower MIC (0.06 μg/mL), but 48% were highly resistant, because they were not inhibited even with the highest assessed concentration, that is, MIC =32 μg/mL. It means that in addition to identification, in the near future, the use of effective methods to test routine antifungal sensitivity will be mandatory, facilitating the selection of therapy.
Among the 35 assessed samples of T. mentagrophytes we observed great variation concerning susceptibility to antifungals, with a trend to respond to intermediate MIC; however, eight samples were inhibited only by the highest MIC of itraconazole and other four exhibited this behavior to all other drugs, suggesting they were resistant to all tested antifungals. This intense variability can be related with the species varieties, because apparently zoophilic responded better than anthropophilic varieties. However, it is important to remember that in vitro behavior to antifungals does not have direct implications to therapeutic response in vivo. Even so, it is interesting to learn about the inherit characteristics of microorganisms of not responding homogeneously to antifungals.
Fluconazole is characterized by good availability, low binding to proteins and long half-life 19. However, it was the antifungal that presented the greatest variability, whose MICs ranged from 0.25 to =64 μg/mL in all 80 assessed samples. In practice, treatment with fluconazole is associated with good cure indices, despite some limitations 26.
Terbinafine is one of the newest antifungal agents, it has in vitro activity directed to a broad range of dermatophytes and NDFF, even though it has low activity against yeast 19. In this study, it was the antifungal that had the best performance among the tested drugs, and 67% of the samples were inhibited at the lowest MIC (00.6 μg/mL). However, it is important to highlight that 24% of the analyzed fungi were sensitive to only the highest concentration (MIC = 32 μg/mL). Some cases of resistance to terbinafine in dermatophyte have been reported 27, but in general the response is good. It is one of the most recommended antifungal agents for treatment, especially in onychomycosis, both for topical and oral use, including pulse therapy 28.
Thus, the present study was exclusively directed to microorganisms, but it confirmed the assumptions of the authors confirmed by clinical efficacy of systemic treatment with itraconazole, terbinafine and fluconazole, considered drugs of choice in managing skin and nail fungi infections 29. Despite that, some authors have pointed out the need to approach special populations such as elderly, children, pregnant women and immunocompromised subjects 30.
The present study has shown that there is major diversity in relation to susceptibility to antifungal drugs to isolated filamentous fungi in cutaneous infections. Even though the results of in vitro tests are not necessarily correspondent to in vivo response, it is impossible to deny the broad variability of fungi responses to different antifungal agents. This fact, associated with the identification of the fungus responsible for the cutaneous mycosis, enables clinicians to objectively choose the appropriate therapy, facilitating the individualization of treatment and changes in dosing or choice of drugs.
Considering the in vitro confirmation of intrinsic resistance of some fungi species to specific antifungal agents, the need to have a reference method that is standardized and validated to enable the use of susceptibility tests to antifungals in clinical practice becomes evident. The method of microdilution in broth, even though labor-intensive, is feasible and has presented good interlaboratorial reproducibility. It is evident that in practice therapeutic success depends on a set of variables that combine information about the agent, clinical aspects - type, location and duration of lesion, genetic predisposition, discipline and compliance to treatment, which play a relevant role and should be taken into account. Thus, clinicians and mycologists should act in synchrony to adapt to new requirements of modern medicine, aiming at improving the approaches to enhance the chances of therapeutic success and patient satisfaction.
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Mailing Address: Conflict of interest:
Profa. Dra. Terezinha Inez Estivalet Svidzinski
Universidade Estadual de Maringá
Departamento de Análises Clínicas
Programa de Pós-Graduação em Análises Clínicas
Av. Colombo, 5.790, bl J90, sala 11, zona 7
87020 900 Maringá Paraná
Tel./fax: 44 3261 4809 3261 4860
Financial funding: Este estudo recebeu auxílio financeiro da Fundação Araucária e do Laboratório de Ensino e Pesquisa em Análises
Clínicas Universidade Estadual de Maringá (Lepac/UEM) Maringá (PR), Brasil.
How to cite this article: Almeida LMM, Souza EAF, Bianchin DB, Svidzinski TIE. Resposta in vitro de fungos agentes de micoses cutâneas frente aos antifúngicos sistêmicos mais utilizados na dermatologia. An Bras Dermatol. 2009;84(3):249-55.
Conflict of interest: