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Revista de Odontologia da Universidade de São Paulo

Print version ISSN 0103-0663

Rev Odontol Univ São Paulo vol.13 n.3 São Paulo July/Sept. 1999

https://doi.org/10.1590/S0103-06631999000300003 

Microbiologia

 

Antifungal susceptibility of Candida albicans isolated from oral mucosa of patients with cancer

Suscetibilidade de Candida albicans a antifúngicos: cepas isoladas da mucosa bucal de pacientes com câncer

 

Lino João da COSTA*
Esther Goldenberg BIRMAN**
Sidney Hartz ALVES***
Arlete Emily CURY****

 

 


COSTA, L. J.; BIRMAN, E. G.; ALVES, S. H.; CURY, A. E. Antifungal susceptibility of Candida albicans isolated from oral mucosa of patients with cancer. Rev Odontol Univ São Paulo, v. 13, n. 3, p. 219-223, jul./set. 1999.

The increasing number of oral infections due to opportunistic fungi in immunocompromised patients, needs a new evaluation of the drugs in use. The susceptibility in vitro of Candida albicans strains from the oral mucosa of cancer patients to amphotericin B, ketoconazole, miconazole, fluconazole and itraconazole were evaluated. A dilution technique in YNB agar or subculture on Sabouraud agar was utilised for MIC or MFC determinations. With this methodology, the best fungicidal drug for C.albicans collected from the oral mucosa of 40 cancer patients, divided in two groups: one treated by radiotherapy and the other not, the best results were obtained with amphotericin B, presenting low values of MIC compared to azoles and MFC values. However it is important to take into consideration the utilisation in vivo of this polyenic antibiotic and the possible toxic levels necessary to achieve good results. The coexistence of other fungi and the local conditions must also be pondered with these patients, who are mostly undergoing radiotherapy. In order to achieve better results without undesirable consequences, higher levels of MIC are expected with the daily clinical use of new drugs.

UNITERMS: Candida albicans; Opportunistic infections; Antifungal agents; Neoplasms.


 

 

INTRODUCTION

Radiotherapy is considered an effective tool to control and cure neoplasms of the head and neck19. Undesirable consequences, like xerostomia can occur, leading to colonisation or oral infection by Candida species, mainly C. albicans7,11,13,17,19. To avoid these complications, prophylactic measures have been utilised, including the use of topic and systemic antifungal drugs2,5,10,11,14. The prophylactic use of antifungal drugs and even their efficacy in some cases, is still open to discussion13, since the treatment of candidiasis in patients submitted to radiotherapy, is empirical in many cases. Our aim was to analyse the possible influence of radiotherapy in strains of C. albicans isolated from oral lesions of patients with treated and non treated head and neck cancer, and the role of the most used drugs for candidiasis treatment.

 

MATERIAL AND METHODS

Using conventional techniques8, we isolated and identified 40 strains of C. albicans from lesions of the oral mucosa of patients with head and neck cancer. Twenty of them had not been submitted to antineoplastic therapy and the other 20, were undergoing radiotherapy, both groups not being submitted to antifungal treatment. The strains were recovered on Sabouraud's agar (SA) plus chloramphenicol, and identified by germ tube test and chlamydospore production. The isolates were maintained on SA at 30oC for 15 days. Replications for susceptibility testing were performed each 24 hours. S. cerevisae ATCC 9763, C. pseudotropicalis "Carshalton" and C. parapsilosis ATCC 22019 were used as control strains.

Inoculum

At the moment of MIC testing, a suspension of each yeast containing 106 cells/mL utilising a Newbauer chamber was prepared in sterile saline with Tween 80. The suspension was diluted 1:50 in yeast nitrogen base (Difco), prepared in phosphate buffer (0.01 M, pH 7.0), and supplemented with 0.15% l-asparagine and 1% glucose18. This broth was named YNBG.

Susceptibility test

Stock solutions of amphotericin B (Squibb), ketoconazole, miconazole (Johnson & Johnson) or itraconazole (Janssen) were prepared in dimethyl sulfoxide, while fluconazole (Pfizer) was prepared with distilled water. The solutions were stored at -20°C and used within a week of their preparation. MIC was determined by the modified agar dilution method18. From a stock solution of antifungal agent, two serial fold dilutions were prepared in YNBG. Then, one mL of each dilution was homogenised with 19 mL of YNBG with 1% agar in a tube kept at 45°C and plated. The final concentrations of amphotericin B ranged from 0.06 to 8.0 ug/mL and of the azoles from 0.12 to 64.0 ug/mL. Five microliters of each suspension were inoculated on the agar medium and the plates were kept at 30°C until the growth of the control on the medium without drug. MIC was regarded as the lowest drug concentration that avoided visible growth of the yeast. From the test with amphotericin B, the inoculum that did not present growth was subcultured for 48 hours in SA incubated at 30°C. MFC was considered as the lowest drug concentration that avoided visible growth of the yeast on the subculture. The tests were performed three times and in triplicate at each time. The final results were obtained by geometric mean of the final values of each test. Considering that now we still do not have a consensus to define sensibility or resistance of C. albicans in face of the different antifungical drugs, we utilised the values suggested by some authors14 to fluconazole (£ 32 ug/mL) and to itraconazole sensibilities (£ 0.5 ug/mL). For amphotericin B (£ 2 ug/mL), ketoconazole (£ 5.9 ug/mL) and miconazole (£ 8.0 ug/mL), we adopted the values of the concentrations viable to be reached at serum16.

 

RESULTS

The employed technique demonstrated reproducible results in different occasions. The geometric mean of all three tests performed in triplicate, demonstrated that the control strains S. cerevisae ATCC 9763 and C. parapsilosis ATCC 22019, were sensible to amphotericin B (Am B) in MICs of 0.3 and 0.2 ug/mL respectively, being both strains sensible to MFC of 0.5 ug/mL of this polienic. C. parapsilopsis ATCC 22019 was sensible to the azoles studied responding to MIC of 0.2 ug/mL for ketoconazole or itraconazole and of 0.15 ug/mL of fluconazole. C. pseudotropicalis "Carshalton", was sensible to miconazole MIC of 0.4 ug/mL.

Independently of the patients groups, all studied strains were inhibited by AmB represented by MICs £ 0.06 ug/mL. Differences among them were observed considering the polienic response in relation to MFCs (Table I), as also the MICs of the studied azoles (Table II). The majority of the yeasts isolated from patients under neoplastic treatment, responded to more elevated concentrations when compared to those isolated from patients without treatment.

 

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Having in mind the sensibility related values, it was observed that all strains of both groups were sensible to the inhibitory and lethal activity of Am B in low concentration levels, reaching the maximum value of 0.4 ug/mL (table I). In relation to azoles, three isolates of patients without treatment were resistant to miconazole (MIC = 10.0 ug/mL), one of them presenting the same results for itraconazole (MIC = 1.0 ug/mL). Among the yeast from patients under treatment, seven demonstrated resistance to only one of the azoles. Additionaly, one strain was not sensible to miconazole (MIC = 10.0 ug/mL) and to ketoconazole (MIC - 8.0 ug/mL), and another one did not respond to any of the azoles (Table II).

 

DISCUSSION

Antifungigrams are still in improvement and we can say that a worldwide accepted pattern test is not yet available, although some of them are already used, with reproducible results. Our employed technique presented also reproducible characteristics since the results obtained with the control strain C. parapsilopsis ATCC 22019 were compatible with the data obtained by other authors utilising a different technique. This compatibility was obtained by using a lower concentrated inoculum than that indicated to antifungigrams in broth medium12,15. In agar medium, as employed in the present study, this test was easier to perform, the break-point was clear to read and up to 23 strains, only one could not be evaluated against each drug.

We could confirm that the majority of strains evaluated either from treated or non treated cancer patients were sensible to the evaluated antifungal drugs. All samples were isolated from oral lesions representing possible cases of infection and not only a fungal colonisation.

So, we can consider that all patients studied, presented the same basic disease predisposing them to candidiasis, with the possibility that those submitted to radiotherapy have additional factors which could improve infection, as observed in other studies with patients in similar conditions10,13,17.

The patients major predisposition to infections, does not explain the strains isolated with the tendency to answer to higher concentrations of the drugs as well as a major number of resistant yeasts to one or more azoles, differently from those isolated from non treated patients. However, it is reasonable to consider that the fungal cells, like the human ones, can suffer some radiation induced degree of morphological and physiologic modifications. These alterations on Candida spp, have been studied in relation to antineoplastic chemotherapy, confirming an interference on the susceptibility of the yeast to fungicide drugs1,3,6.

At a first glance, the occurrence of strains presenting crossed resistance among azoles seems not to be associated to radioactive effects. This opinion is based on the observations of a particular resistance profile presented by each one of these strains isolated from patients with or without treatment. These results, associated to patients histories without reports of antifungal treatment, call our attention to a primary crossed resistance among azoles in strains of C. albicans isolated from cancer patients. Additionaly, the resistance to miconazole and itraconazole observed with the strain of a patient not submitted to radiotherapy, conflicts with the opinion of some researchers9 that the resistance to fluconazole is the first manifestation and it can be used to evaluate the resistance of the strain to other azoles.

 

CONCLUSION

The obtained results allows us to consider that the confirmation of our data as well as an amplification of this study is important, due to the difficulties observed in relation to prophylaxis4,13 and cure5 of candidiasis mainly in cancer patients submitted to radiotherapy, since they present different responses compared to those not submitted to the same treatment.

 

 


COSTA, L. J.; BIRMAN, E. G.; ALVES, S. H.; CURY, A. E. Suscetibilidade de Candida albicans a antifúngicos: cepas isoladas da mucosa bucal de pacientes com câncer. Rev Odontol Univ São Paulo, v. 13, n. 3, p. 219-223, jul./set. 1999.

O crescente número de infecções fúngicas bucais em pacientes imunocomprometidos tem estimulado a pesquisa de drogas mais eficientes. Procuramos, assim, avaliar a suscetibilidade in vitro de cepas de Candida albicans de pacientes com câncer submetidos ou não a radioterapia frente às seguintes drogas: anfotericina B, cetoconazol, miconazol e itraconazol. A técnica de diluição em ágar YNB e o subcultivo em AS foram respectivamente utilizados para determinação da CIM (Concentração Inibitória Mínima) e da CFM (Concentração Fungicida Mínima). Com esta metodologia, a droga que mostrou melhor atividade antifúngica para cepas de C. albicans isoladas de qualquer dos grupos de pacientes foi a anfotericina B, apresentando baixos valores de CIM em comparação aos azóis, e de CFM. Contudo, é necessário lembrar que o tratamento com antibióticos poliênicos pode causar efeitos tóxicos, mesmo em doses necessárias para se atingir bons resultados com os antifúngicos. A coexistência de outras espécies nesses pacientes, principalmente entre os submetidos à radioterapia, as condições locais, bem como o uso de outras drogas, devem ser também considerados. Acredita-se, porém, que o uso adequado dos antifúngicos atuais e a disponibilidade de novas drogas que não produzam conseqüências indesejáveis possam conduzir a melhores resultados na clínica diária.

UNITERMOS: Candida albicans; Infecções oportunistas; Antimicóticos; Neoplasias.


 

 

BIBLIOGRAPHIC REFERENCES

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4. EPSTEIN, J. B. More on head and neck radiation therapy. J Can Dent Assoc, v. 55, p. 96, 1989.         [ Links ]

5. FINLAY, P. M.; RICHARDSON, M. D.; ROBERTSON, A. G. A comparative study of the efficacy of fluconazole and amphotericin B in the treatment of oropharyngeal candidosis in patients undergoing radiotherapy for head and neck tumors. Br J Oral Maxillofac Surg, v. 34, p. 23-25, 1996.         [ Links ]

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8. KREGER- VAN RIJ, N. J. W. The yeast: a taxonomic study. Amsterdam : Elsevier, 1984.1082 p.         [ Links ]

9. MARTINEZ-SUAREZ, J. V.; RODRIGUEZ-TUDELA, J. L. Patterns of in vitro activity of itraconazole and imidazole antifungal agents against Candida albicans with decreased susceptibility to fluconazole from Spain. Antimicrob Agents chemoter, v. 39, p. 1512-1526, 1995.         [ Links ]

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11. PAU, H. W.; STREHLER-POHL, H. J.; EXNER, M. Yeast fungus flora in tumor irradiation of the upper aerodigestive tract. HNO, v. 33, p. 485-488, 1985.         [ Links ]

12. FALLER, M. A; BALE, M.; BUSCHELMAN, B. et al. Quality control guidelines for National Comittee for Clinical Laboratory Standards recommended broth macrodilution testin of amphotericin B, fluconazole and flucytosine. J Clin Microbiol, v. 33, p. 1104-1107, 1995.         [ Links ]

13. RAMIREZ-AMADOR, V.; SILVERMAN, JUNIOR, s.; MAYER, P. et al. Candidal colonization and oral candidiasis in patients undergoing oral and pharyngeal radiation therapy. Oral Surg Oral Med Oral Pathol Oral Radiol Endod, v. 84, p. 149-153, 1997.         [ Links ]

14. REX, J. H.; PFALLER, M. A.; GALGIANI, J. N., et al. Development of interpretative breakpoints for antifungal susceptibility testing: conceptual framework and analysis of in vitro - in vivo correlation for fluconazole, itraconazole and Candida infection. Clin Infect Dis, v. 24, p. 235-247, 1997.

15. REX, J. H.; PFALLER MA.; LANCASTER, M. et al. Quality control guidelines for National comitee for clinical Laboratory standards-recomended broth macrodilution testing of ketoconazole and itraconazole. J Clin Micro-biol, v. 34, p. 816-817, 1996.         [ Links ]

16. RICHARDSON, M. D.; WARNOCK, D. W. Fungal Infection: diagnosis and management. Oxford: Blackwell Scientific, 1993 p. 17-42.         [ Links ]

17. ROSSIE, K. M.; TAYLOR, J.; BECK, F. M. et al. Influence of radiation therapy on oral Candida albicans colonizations: a quantitative assessment. Oral Surg Oral Med Oral Pathol, v. 64, p. 698-701, 1987.

18. SHADOMY, S.; ESPINEL-INGROFF, A.; CARTWRIGHT, R. Laboratory studies with antifungal agents :susceptibility test and bioassay. In: LenNette, E. H.; Ballows, A.; HauSlers, W. J.; shadomy, H. J. (eds.) Manual of clinical microbiology, 4 ed. Washington : American society of Microbiology, 1985. p. 991-999.

19. WHITMYER, C. C.; ESPOSITO, S. J.; TEREZHALMY, G. T. Radiotherapy for head and neck neoplasms. Gen Dent, v. 45, p. 363-370, 1997.         [ Links ]

 

Recebido para publicação em 09/04/99
Aceito para publicação em 21/07/99

 

 

* PhD Federal University of Paraíba, João Pessoa - PB, Brazil.
*** PhD, ** and **** Professors and chair School of Dentistry and School of Pharmaceutical Sciences of the University of São Paulo - USP, Brazil.

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