Identification of Candida species and antifungal susceptibility in vitro: a study on 100 patients with superficial candidiasis

Abstracts

FUNDAMENTO: Leveduras do gênero Candida determinam colonização, infecções superficiais e infecções sistêmicas em imunodeprimidos. As várias apresentações da doença levam à necessidade de utilizar diferentes métodos diagnósticos e tratamentos. OBJETIVOS: Diferenciar as espécies de Candida e correlacioná-las com as regiões anatômicas. Avaliar a susceptibilidade a cetoconazol, fluconazol, itraconazol e anfotericina B. MÉTODOS: Foram avaliados 100 pacientes imunocompetentes com candidíase cutânea ou mucosa atendidos na Santa Casa de S. Paulo entre maio de 1999 e julho de 2001. Correlacionou-se a região acometida e a espécie de Candida, isolada através técnica CHROMagar Candida®. Avaliou-se a susceptibilidade das espécies a cetoconazol, fluconazol, itraconazol e anfotericina B, através do Etest®. RESULTADOS: C. albicans foi isolada em 76,0% dos materiais, C. krusei em 19% e C. tropicalis em 1%. Não houve correlação significante entre a região acometida e as espécies. A maioria das amostras mostrou susceptibilidade aos antifúngicos. CONCLUSÃO: C. albicans foi a espécie mais observada. A maioria das amostras de Candida mostrou-se susceptível aos antifúngicos.

Candida albicans; Candida tropicalis; Candidíase cutânea; Itraconazol

CLINICAL, EPIDEMIOLOGICAL, LABORATORY AND THERAPEUTIC INVESTIGATION

Identification of Candida species and antifungal susceptibility in vitro: a study on 100 patients with superficial candidiasis^*

Elisete I. Crocco^I; Lycia M. J. Mimica^II; Laura H. Muramatu^III; Cristina Garcia^IV; Valéria M. Souza^V; Ligia R. B. Ruiz^VI; Clarisse Zaitz^VII

^IM.D., received dermatology specialization from the Dermatology Clinic, Department of Medicine, Santa Casa de Misericordia de Sao Paulo Hospital

^IIAssistant Professor, Microbiology Service, Department of Pathology, Santa Casa de Misericordia de São Paulo Hospital

^IIIM.D., Dermatology Clinic, Department of Medicine, Santa Casa de Misericordia de São Paulo Hospital

^IVM.D., Microbiology Service, Department of Pathology, Santa Casa de Misericórdia de São Paulo Hospital

^VAssistant M.D., Infectious Diseases sector, Dermatology Clinic, Department of Medicine, Santa Casa de Misericordia de São Paulo Hospital

^VIVolunteer M.D., Infectious Diseases sector, Dermatology Clinic, Department of Medicine, Santa Casa de Misericordia de São Paulo Hospital

^VIIAdjunct Professor, Head of the Infectious Diseases sector of the Dermatology Clinic, Department of Medicine, Santa Casa de Misericordia de Sao Paulo Hospital

^{Correspondence}

ABSTRACT

BACKGROUND: Yeasts of the genus Candida create colonization, superficial infections and systemic infections in immunodeficient individuals. The presentations of the disease lead to the necessity of using various diagnostic methods and treatments.

OBJECTIVES: To differentiate among the Candida species, correlate them with the anatomical region involved and evaluate in vitro susceptibility to ketoconazole, fluconazole, itraconazole and amphotericin B.

METHODS: An evaluation of 100 cases of immunocompetent patients with cutaneous or mucous candidiasis attended at the Santa Casa de Sao Paulo Hospital from May 1999 to July 2001. This study attempted to correlate the site of the involvement and the Candida species, isolated using the CHROMagar Candida^® technique. The species were evaluated using the Etest^® antifungal susceptibility to ketoconazole, fluconazole, itraconazole and amphotericin B.

RESULTS: C. albicans was isolated in 76% of the material, C. krusei in 19% and C. tropicalis in 1%. It was not possible to establish a significant correlation between the involved area and the species isolated. Most of the samples proved to be susceptible to the antifungals tested.

CONCLUSIONS: C. albicans was the most commonly observed species. The majority of samples presented susceptibility to the tested antifungals.

Key-words: Candida albicans; Candida tropicalis; Candidiasis, Cutaneous; Itraconazole.

INTRODUCTION

Candidiasis expresses the variety of relations occurring between the host and autochthonous microbiota, that is, of a commensalism to fatal systemic disease.¹ Colonization by yeasts of the genus Candida are responsible for superficial fungal infections in specific immunocompetent patients, and systemic infections in immunodeficient patients. A variety of presentations of the disease leads to the necessity of using different diagnostic methods and therapeutic schemes.

Candida albicans is the most common pathogen among cutaneous candidiasis and of the oropharynx. However, non albicans species have increased in number and importance in vaginal and systemic candidiasis. ²

The behavior variability of the different Candida species prompted the need to develop fast and easy methods for identifying them. The CHROMagar Candida^® technique proved to be a sensitive method in addition to an accurate one for presumptive identification of more commonly isolated yeast species of the genus Candida.^3,4,5

Many of the most commonly isolated non albicans species are less susceptible to azolic derivatives, which makes treating these infections harder. While susceptibility of yeasts of the genus Candida to available antifungals is variable and predictable, a specifically determined isolated sample does not always end up following the general pattern. This is one of the reasons for the growing importance of susceptibility testing. The effort to standardize these tests has culminated in the elaboration of M27-A methodology by the National Committee for Clinical Laboratory Standards (NCCLS).^2,6

Etest^® is used to gauge in vitro susceptibility to antifungals. It is comparable as a method to the NCCLS M27-A. However, it is simpler and easier to incorporate into the laboratory routine.^4,7,8,9

MATERIAL AND METHODS

One hundred patients with cutaneous or mucous infections caused by yeasts of the genus Candida were assessed and attended to at the Ambulatory Clinic of Dermatology, or hospitalized at the Medicine Infirmaries of the Irmandade da Santa Casa de Misericordia de São Paulo Hospital, from May 1999 to July 2001.

Patients with yeasts of the genus Candida traced in their lesions, from either of the sexes, and from all races and all ages, were included. Patients were not included if they were immunodeficient in any way or had mixed infections spotted in the direct examination. The patients included in the study were grouped together according to the anatomical region of lesions involved.

The diagnosis of candidiasis was confirmed by means of a direct examination clarified with potassium hydroxide (20%) in aqueous DMSO solution, either by visualizing the yeasts and/or at least two growths, that is two sowings of the same material, in cultures done in Sabouraud agar dextrose to which chloramphenicol was added.^1,10

To identify the yeast species of the isolated genus Candida, chromogenic media plates were used — Chromagar Candida^®, (Probac of Brasil^®, São Paulo). After sowing the strain in this medium the colony's color changed as a function of the Candida species isolated. After forty-eight hours of incubation at 30-37 ºC, it is possible to identify: Candida albicans, which turns green, Candida krusei, turning pink, Candida tropicalis, blue-grey, as well as other species changing to a pink-white color (Figure 1).¹⁰

The isolated species were grouped together in an attempt to correlate them with the anatomical region involved. To evaluate in vitro susceptibility of the isolated species, the Etest^® method (AB Biodisk, Solna, Sweden^®) was used. This method has a predefined concentration gradient for ketoconazole, itraconazole, fluconazole and amphotericin B, determining the minimum inhibitory concentration (MIC) in mg/ml for each antifungal. With this method, the colonies are grown in RPMI^® (Probac do Brasil^®, São Paulo), a medium consisting of the RPMI 1640, MOPS, and 2% agar glucose (Figure 2).¹¹ The plates were incubated in a humid chamber at 35ºC for 48 hours, at which time a reading was carried out to evaluate the intersection point between the formed halo and the Etest^® strip. According to the NCCLS criteria,⁶ the species sensitivity profile to antifungals must be classified as sensitive, intermediate or resistant, in accordance with table 1.

For the statistical analysis of the estimated frequency of the different yeast species of genus Candida in the group studied, percentages were calculated with their respective trust interval. To correlate clinical variants/regions involved with the isolated Candida species, the Fisher bimodal test was used. A descriptive study was carried out to demonstrate the in vitro susceptibility of these species to the antifungals ketoconazole, fluconazole, itraconazole and amphotericin B.

The protocol was approved by the medical ethics committee of the Irmandade da Santa Casa de Misericordia de São Paulo.

RESULTS

Only six of the 100 patients assessed were hospitalized. The sample was constituted for the most part by Caucasian women between the ages of 41 and 60 years. The demographic characteristics of the group studied are found in table 2.

Results relative to the identification of yeast species of the genus Candida by means of the Chromagar Candida® may be observed in table 3.

It was not possible to ascertain a significant correlation between the regions involved and the species isolated through the Fisher bimodal test. The results found may be seen in table 4.

Table 5 shows cutaneous involvement by different yeast species of the genus Candida, illustrating a distinct predominance of fingernail involvement.

Table 6 shows the behavior of Candida albicans species isolated from the lesions of patients studied. Most of them proved to be susceptible to the antifungals tested.

Table 7 shows the behavior of Candida krusei species isolated from the lesions of the patients studied. The species of Candida krusei showed greater susceptibility (94.7%) to amphotericin B than to other antifungals.

DISCUSSION

Most patients assessed were attended to at the Dermatology Clinic of the Santa Casa de Misericordia de São Paulo Hospital. The Clinic's clientele consists predominantly of women. The demographic characteristics of the group studied may be partly related to the profile of patients frequenting the Clinic.

Yeasts of the genus Candida are usually found on the nails. Candida albicans is the most common pathogen. Candida tropicalis, Candida krusei, Candida parapsilosis and Candida guilliermondii are found in lower frequency.¹² Yeasts of the genus Candida may behave like a primary pathogen. They tend to invade the normal nail mainly in patients with chronic mucocutaneous candidiasis as well as in Aids patients. Indeed, in immunocompetent patients, they are often secondary pathogens, and invade a nail only after previously being altered by trauma, hyperhydratation or irritation from contact with chemical substances. These cases clinically show as onycholysis or paronychia. They may also act as secondary pathogens in nails affected by psoriasis and lichen planus, and by other skin diseases.¹²

That 60% of patients studied presented with nail involvement was something to be expected just as much by the fact that yeasts of the genus Candida in nails of immunocompetent patients were secondary to trauma, hyperhydratation (continued exposure to water) or irritation from contact with chemical substances (household cleaning products) — all of which are most common to women —, as by the profile of the group studied: predominantly female.

Candida albicans is known to be the most common pathogen in cutaneous candidiasis and oropharynx.² On the other hand, publications studying the species of Candida, its correlation with the regions involved and the susceptibility of these species to antifungals have been especially rare with respect to immunocompetent groups. As for immunodeficient patients, mainly with Aids groups, studies are quite common.

A multicenter Brazilian study isolated Candida albicans in 91% of oropharynx samples taken from Aids patients. The non albicans species isolated in decreasing frequency were: C. glabrata, C. tropicalis, C. parapsilosis and C. krusei.¹³

For the isolation of yeast species of the genus Candida the use of Chromagar Candida, was selected because it is a susceptible and specific method. Candida albicans was isolated in 76% of the material, Candida krusei in 19%, and Candida tropicalis in 1% of patients assessed, amounting to 96% of the isolated cases. All of the other species corresponded to only 4% of those isolated. A predominance of isolated Candida albicans is what can be observed in the literature in either immunocompetent or immunodeficient patients.^2,13,14 The frequency of isolation and prevalence of non albicans species isolated in different studies are variable.^13,14

It was not possible to ascertain a significant correlation between the regions involved and the main species isolated. Moreover, there were no studies found in the literature which correlated the isolated species and region involved.

The National Committee for Clinical Laboratory Standards (NCCLS) Subcommittee on Antifungal Susceptibility Testing developed and standardized a methodology through broth dilution in order to determine the susceptibility of yeasts.⁶ The broth macro- and microdilution method was published in document NCCLS M27-A, and updated in 2002 (M27-A2).

The albicans, tropicalis and parapsilosis of yeast species of the genus Candida are susceptible to antifungals amphotericin B, itraconazole and fluconazole in accordance with the general pattern of susceptibility (method M27-A NCCLS).² By the same method, Candida lusitaniae is susceptible to itraconazole and fluconazole, which may vary or be resistant to amphotericin B. Many isolated species of Candida krusei and Candida glabrata are resistant to these antifungals.²

The Etest^® method was assessed and proved to be an acceptable alternative to the reference methods.^4,7,8,9 In the present study, the authors made use of Etest^®. Most of the isolated species of Candida albicans from the lesions of patients studied showed susceptibility to tested antifungals. These results agree with the literature as far as the evaluation of immunocompetent patients is concerned.² Cases were reported of Candida albicans resistance to azolic derivatives in HIV-infected (human immunodeficiency virus) patients and in patients with invasive candidiasis.²

As for the behavior of isolated Candida krusei species from the lesions of patients studied by Etest^®, greater susceptibility to amphotericin B than to other funguses was observed. Candida krusei is known to have intrinsic resistance particularly to fluconazole and to azolic derivatives in general.^2,15

In studies of candidiasis on the oropharynx of Aids patients, the susceptibility ranking of the azolic derivatives of yeast species of the genus Candida, in accordance with the NCCLS-approved method of microdilution, was: Candida albicans > C. tropicalis > C. glabrata > C. krusei.¹³

CONCLUSION

In conclusion, the following features were observed in the group of predominantly ambulatory immunocompetent patients with superficial candidiasis caused by yeasts of the genus Candida, studied from May 1999 to July 2001:

- 60% of cases involved the nails, where the yeast probably behaves like a secondary pathogen;

- isolation of Candida albicans in 76% of material, Candida krusei in 19%, and Candida tropicalis in 1% of samples;

- it was not possible to ascertain a correlation between the anatomical regions involved and the species isolated;

- the majority of the isolated species tested by Etest^® were susceptible to antifungals.

ACKNOWLEDGEMENTS

The authors should like to thank the Fundação de Amparo à Pesquisa de São Paulo (FAPESP: The State of São Paulo Research Foundation) and the Medical Sciences Faculty of the Santa Casa de Misericordia de Sao Paulo Hospital for their support.

REFERENCES

Received on Jannuary 27, 2004

Approved by the Consultive Council and accepted for publication on October 30, 2004

Project financed by FAPESP. No. 99/06792-7 — October 1999 to September 2001

References

• 1. Paula CR. Candidíases. In: Zaitz C, Campbell I, Marques AS, et al Compêndio de Micologia Médica. Rio de Janeiro: Medsi; 1998. p. 99-107.
• 2. Rex JH, Walsh TJ, Sobel JD, et al Practice Guidelines for the treatment of candidiasis. J Infect Dis. 2000; 30: 662-678.
• 3. Houang ETS, Chu KC, Koehler AP, Cheng AFB. Use of Chromagar Candida for genital specimens in the diagnostic laboratory. J Clin Pathol. 1997; 50: 563-565.
• 4. Ambler JE, Kerawala M, Yaneza A, Drabu YJ. Evaluation of Chromagar Candida for rapid idenfication and Etest for antifungal susceptibility testing in a district general hospital laboratory. J Clin Pathol. 2001; 54: 158-159.
• 5. Yocesoy M, Marol S. Performance of Chromagar Candida and BIGGY Agar for presumptive identification of yeasts. Clin Microbiol Infect. 2003; 9: 253.
• ⁶ 6. National Committee for Clinical Laboratory Standards. Reference method for broth dilution antifungal susceptibility testing of yeasts; approved standard M27-A. Wayne, Pa, USA: National Committee for Clinical Laboratory Standards. 1995.
• 7. Li J, Nong H, Nong D, Cheng L. Study on susceptibility test of pathogenic fungi from otorhinolaryngology by Etest method. Lin Chuang Er Bi Yan Hou Ke Za Zhi. 2001; 15: 77-79.
• 8. Matar M, Ostrosky-Zeichner L, Paetznick VL, et al Correlation between Etest, disk diffusion, and microdilution methods for antifungal susceptibility testing of fluconazole and voriconazole. Antimicrob Agents Chemother. 2003; 47: 1647-1951.
• 9. Morace G, Amato G, Bistoni F, et al Multicenter comparative evaluation of six commercial systems and the National Committee for clinical laboratory standards M27-A broth microdilution method for fluconazole susceptibility testing of Candida species. J Clin Microbiol. 2002; 40: 2953-2958.
• 10. Lacaz CS, Porto E, Marins JEC, et al Tratado de Micologia Médica Lacaz. São Paulo: Sarvier; 2002. p. 918-990.
• 11. Lacaz CS, Porto E, Marins JEC, et al Tratado de Micologia Médica Lacaz. São Paulo: Sarvier; 2002. p. 998-1067.
• 12. Daniel CRI, Gupta AK, Daniel MP, et al. Candida infection of the nail: role of Candida as a primary or secondary pathogen. Dermatology. 1998; 37: 904-907.
• 13. Sant' Ana PL, Milan EP, Martinez R, et al Multicenter Brazilian study of oral Candida species isolated from Aids patients. Mem Inst Oswaldo Cruz. 2002; 97: 253-257.
• 14. Dorko E, Jautová J. Candida species isolated in skin infections. J Eur Acad Dermatol Venereol. 2002; 16: 234.
• 15. Coleman DC, Rinaldi MG, Haynes KA, et al Importance of Candida species other than Candida albicans as opportunistic pathogens. Med Mycol. 1998; 36: 156-165.

*

Work performed at the Irmandade da Santa Casa de Misericordia de São Paulo Hospital

Publication Dates

• Publication in this collection
  30 Mar 2005
• Date of issue
  Dec 2004

History

• Received
  27 Jan 2004
• Accepted
  30 Oct 2004