Drug resistance of yeasts isolated from oropharyngeal candidiasis in aids patients

Resistência à drogas de leveduras isoladas de candidíase orofaríngea em pacientes com Aids

DRUG RESISTANCE OF YEASTS ISOLATED FROM OROPHARYNGEAL CANDIDIASIS IN AIDS PATIENTS Maria do Rosário Rodrigues Silva1^*, Claudete Rodrigues de Paula², Soraya Cristina Silva¹, Théo Rodrigues Costa¹, Márcio Rodrigues Costa¹

¹Departamento de Microbiologia, Imunologia, Parasitologia e Imunopatologia do Instituto de Patologia Tropical da Universidade Federal de Goiás, Goiânia, Go, Brasil. ²Departamento de Microbiologia do Instituto de Ciências Biomédicas da Universidade de São Paulo, São Paulo, SP, Brasil

Submitted: September 02, 1997; Returned to authors for corrections: April 17, 1998;

Approved: September 09, 1998

Candida spp was isolated from 59 (68.60%) out of eighty six samples of oral mucosa of AIDS patients. The identification, based or the production of a germ tube and chlamydospores, and on the assimilation and fermentation of carbohydrates, revealed 52 strains (88.13%) of C. albicans, 4 (6.77%) of C. tropicalis and 3 (5.08%) of C. krusei. The susceptibility of these strains to amphotericin B, flucytosine, itraconazole, fluconazole and ketoconazole was determined using the agar dilution method. Comparing the minimum inhibitory concentration values found in the susceptibility test with the serum levels achieved by these drugs, only 8.47% and 5.08% of the yeasts strains proved to be resistant to amphotericin B and flucitosyne, respectively. A high frequency of strains resistant to azole derivatives (25.42%, to itraconazole, 45.76%, to ketoconazole and 66.10% to fluconazole) was observed.

Key words: Oropharyngeal candidiasis, yeasts, in vitro resistance, antifungal drugs.

Candida albicansC. tropicalisC.glabrataC.krusei C. parapsilosis,

The development of resistance to antifungal agents, observed in previously susceptible yeast isolates, as well as recurrence of oropharyngeal candidiasis following treatment with different antifungal drugs, are highly relevant factors which should be taken into account to determine the use of in vitro susceptibility tests in the microbiology laboratory routine (9,17).

Dilution tests in solid and liquid media, for determination of the minimum inhibitory concentration (MIC) of antifungal drugs against yeasts, have been proposed by Alves and Cury (1).

The aim of this work was to determine the resistance of 59 strains of Candida spp., isolated from the injured oral mucosa of AIDS patients to amphothericin B, flucytosine, fluconazole, ketoconazole and itraconazole.

Samples

Samples were collected from the oral mucosa of 86 AIDS patients treated at the Tropical Diseases Hospital, Goiânia, Goiás State, Brazil. Samples were maintained up to one year on Sabouraud dextrose agar kept at 25ºC.

Microorganisms

The identification of yeast isolates was carried out through the production of germ tubes and chlamydospores, as well as through carbohydrates assimilation and fermentation tests, according to Kreger van Rij (12). Fifty nine isolates were included in this study.

In vitro susceptibility tests

Each isolate was tested by the dilution method in buffered Yeast Nitrogen Base (YNB) agar, using five antifungal drugs: amphotericin B (Squibb), fnucytosine (Roche), fluconazole (Pfizer), ketoconazole (Janssen) and itraconazole (Janssen).

Dilution method in agar. Amphotericin B (AmB) and flucytosine were dissolved in 1.0 ml distilled sterilized water whereas the azoles were dissolved in 1.0 ml dimethylsulfoxide (DMSO), to a final concentration of a 25600 µg/ml. Solutions were diluted 1:20 in broth (YNB-6.7%, L-asparagine - 1.5% and phosphate buffer-0.01M-pH-7.0) and from this, two-fold dilutions were carried out in the same broth. Each dilution was mixed with YNB agar in order to get an antifungal range from 128µg/ml to 0.25 µg/ml (1,13).

Inoculum and MIC. Prior to testing, each isolate was grown at least twice on Sabouraud dextrose agar to ensure optimal growth. Colonies were suspended in 5 ml physiological solution, vortexed for 15 s, and the cell density was adjusted to a 0.5 McFarland standard, using a spectrophotometer at 530nm (16). Three µl of this suspension were transfered to plates containing the medium and the drugs in different concentrations. A control plate, free of drugs, was included. The MIC was determined as the lowest concentration in which there was no cell growth.

Determination of resistance

Candida isolates were classfied as resistant or susceptible, according to the MIC values when compared to serum levels achieved by amphotericin B, flucytosine, fluconazole, ketoconazole e itraconazole (2,19). Strains with following MIC results were considered resistant: >8.0µg/ml for ketoconazole, itraconazole and fluconazole; >64µg/ml for flucytosine and >µ2g/ml for amphotericin B.

CandidaC. albicansC. tropicalisC. krusei

The MIC

₅₀₉₀Table 1

Comparing the MIC values of each drug, it was observed that 25.42% and 45.76% of the isolates were resistant to itraconazole and ketoconazole, respectively. The highest resistance was observed for fluconazole, i.e., 66.10%. All the isolates of C. krusei were resistant to fluconazole, with MIC>128µg/ml, while 75% of C. tropicalis isolates were resistant to this drug. A high susceptibility of Candida albicans and C. krusei isolates to AmB and flucytosine was recorded, which presented MIC < 2 and MIC <64, respectively. Among the isolates of C. tropicalis, 75% were resistant to flucytosine. The frequency of yeast isolates that were resistant to the antifungal drugs tested is shown in Fig.1.

Figure 1. Resistance of Candida spp isolated from the oral mucosa of AIDS patients to antifungal agents.

DISCUSSION

Identification of 52 Candida albicans isolates (88.13%), out of the 59 yeasts isolated from the oral mucosa in AIDS patients, showed the prevalence of this species among Candida sp. C. albicans can be often found either in healthy individuals or in HIV positive asymptomatic patients (4).

Oropharyngeal candidiasis, diagnosed in more than 75% of AIDS patients, leads to some discomfort in food chewing, causing a stress in the immunological state of the host, due to bad nutrition (7). Diagnosis, followed by efficient treatment is necessary and the in vitro susceptibility tests are important for a correct therapy. Oropharyngeal candidiasis in AIDS patients has proved to be difficult to treat (9).

In this study, most of the C. albicans and C. krusei isolates presented low MIC values for AmB and flucytosine however, 75% C. tropicalis isolates were found to be resistant to flucytosine. Rodero et al (18) noted that 82% yeasts, isolated from the oropharyngeal mucosa in HIV positive patients, were susceptible to flucytosine, when a resistance level higher than 12.5µg/ml was considered. Bonifácio e Souza et al (5) observed inhibition of growth of C. albicans isolated from clinical materials by 2 µg/ml AmB. The situation seems to be different in AIDS patients. An increased frequency of C. albiccns serotype B in this type of patients was observed and this may correlate with a higher incidence of resistant strains (8). The increased resistance of C. tropicalis isolates to flucytosine observed in this study can be related to strains less sensitive to the drug during AIDS.

Azole derivatives, however, presented different results. High resistance was observed to fluconazole and ketoconazole, while only 25.42 % of the isolates were resistant to itraconazole. All C. krusei isolates were resistant to fluconazole, and 75% of C. tropicalis were resistant to this drug and to ketoconazole. Gallagher et al. (9)observed that 54% of the C. albicans isolates from HIV+ patients exhibited lower susceptibility to ketoconazole. Resistance to fluconazole in C. albicans appear to be a less common phenomenon than in other Candida species. Comparative in vitro studies have consistently shown that C. krusei strains show notably higher fluconazole MICs than C. albicans (15). C. krusei exhibits innate resistance to fluconazole, and fluconazole prophylaxis has been associated with an increased incidence of C. krusei infection in some centers (20).

Studies on the genotypic characterization of C. albicans have demonstrated that the recurrent oral candidiasis, mainly in AIDS patients, is caused by the emergence of fluconazole resistant strains (3). Recurrences after treatment with azole derivatives, mainly fluconazole, in HIV patients have been explained by the persistence of Candida colonies in the lesion area after the clinical cure, or by a fungistatic but not fungicidal action of the drug (10,14).

The mechanism of resistance to azoles is related to their mode of action (11,14). Azoles are inhibitors of P450 cytochrome dependent on C14 demethylase, an important enzyme which synthesizes the ergosterol of the fungal cell. Resistance of yeasts to these drugs may be due to alterations in the C14 demethylase or to a lower capacity of the azoles to bind to P450 cytochrome oxidase.

Considering the emergence of isolates resistant to antifungal drugs, particularly to fluconazole, in vitro susceptibility tests, based on the MIC values, are necessary for the choice of the appropriate therapy.

RESUMO

Resistência à drogas de leveduras isoladas de candidíase orofaríngea em pacientes com Aids

Entre oitenta e seis amostras da mucosa oral de pacientes com AIDS, 59 (68,60%) foram positivas para leveduras do gênero Candida. A identificação, feita pela produção de tubo germinativo e clamidósporos e através de assimilação e fermentação de hidratos de carbono, revelou 52 cepas (88,13%) de C.albicans, 4 (6,77%) de C. tropicalis e 3 (5,08%) de C.krusei. Avaliação destas leveduras para susceptibilidade in vitro frente a anfotericina B, flucitosina, itraconazol, fluconazol e cetoconazol, foi realizada pelo método de diluição em ágar. Comparando-se os valores de concentração inibitória mínima encontrados com os níveis séricos alcançados por estes antifúngicos verificou-se que apenas 8,47% e 5,08% das 59 leveduras foram resistentes a anfotericina B e flucitosina, respectivamente. Foi registrada uma percentagem de cepas resistentes aos derivados azólicos, sendo 25,42% ao itraconazol, 45,76% ao cetoconazol e 66,10% ao fluconazol.

Palavras-chave: candidíase orofaríngea, leveduras, resistência in vitro, antifúngicos.

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*

Corresponding author. Mailing Address: Departamento de Microbiologia, Imunologia, Parasitologia e Patologia do Instituto de Patologia Tropical e Saúde Pública da Universidade Federal Goiás, Av. Delenda de Rezende S/N Esq. com 1ª Avenida. Setor Universitário. CEP 74605-050. Goiânia, Go, Brasil.

Publication Dates

• Publication in this collection
  27 May 1999
• Date of issue
  Oct 1998

History

• Accepted
  09 Sept 1998
• Reviewed
  17 Apr 1998
• Received
  02 Sept 1997