Fabíola Silveira-GomesI; Dayse Nogueira SarmentoI; Elaine Patrícia Tavares do Espírito-SantoI; Nádia de Oliveira SouzaII; Thifany Mendes PintoII; Silvia Helena Marques-da-SilvaII
IPrograma de Pós-Graduação em Biologia de Agentes Infecciosos e Parasitários, Instituto de Ciências Biológicas, Universidade Federal do Pará, Belém, PA IILaboratório de Micologia, Seção de Bacteriologia e Micologia, Instituto Evandro Chagas, Ananindeua, PA
INTRODUCTION: Opportunistic fungal infections in immunocompromised hosts are caused by Candida species, and the majority of such infections are due to Candida albicans. However, the emerging pathogen Candida dubliniensis demonstrates several phenotypic characteristics in common with C. albicans, such as production of germ tubes and chlamydospores, calling attention to the development of stable resistance to fluconazole in vitro. The aim of this study was to evaluate the performance of biochemistry identification in the differentiating between C. albicansand C. dubliniensis, by phenotyping of yeast identified as C. albicans.
METHODS: Seventy-nine isolates identified as C. albicans by the API system ID 32C were grown on Sabouraud dextrose agar at 30°C for 24-48h and then inoculated on hypertonic Sabouraud broth and tobacco agar.
RESULTS: Our results showed that 17 (21.5%) isolates were growth-inhibited on hypertonic Sabouraud broth, a phenotypic trait inconsistent with C. albicans in this medium. However, the results observed on tobacco agar showed that only 9 (11.4%) of the growth-inhibited isolates produced characteristic colonies of C. dubliniensis (rough colonies, yellowish-brown with abundant fragments of hyphae and chlamydospores).
CONCLUSIONS: The results suggest that this method is a simple tool for screening C. albicansand non-albicans yeast and for verification of automated identification.
Keywords: Biochemistry identification. Phenotypic characteristics. Candida albicans.
INTRODUÇÃO: Infecções fúngicas oportunistas em hospedeiros imunocomprometidos são causadas por espécies de Candida, cuja maioria das infecções se deve a Candida albicans. Entretanto, o patógeno emergente Candida dubliniensis demonstra várias características fenotípicas em comum com C. albicans, tais como produção de tubo germinativo e clamidósporos, solicitando atenção por desenvolver resistência in vitro estável ao fluconazol. O objetivo do presente estudo foi avaliar a performance da identificação bioquímica na diferenciação entre C. albicans e Candida dubliniensis, analisando fenotipicamente leveduras previamente identificadas como C. albicans.
MÉTODOS: Setenta e oito isolados identificados como C. albicans pelo sistema API ID 32C foram cultivados em ágar Sabouraud dextrose a 30°C por 24-48h e em seguida inoculados em caldo hipertônico Sabouraud e agar tabaco.
RESULTADOS: Nossos resultados mostraram que 17 (21,5%) isolados tiveram o crescimento inibido no caldo hipertônico Sabouraud, característica fenotípica inconsistente para C. albicansneste meio de cultura. Entretanto, os resultados observados em ágar tabaco mostraram que somente 9 (11,4%) dos isolados inibidos produziram colônias características de C. dubliniensis(colônias rugosas, marrom-amarelada com fragmentos de hifas e abundantes clamidósporos).
CONCLUSÕES: Os resultados obtidos sugerem que este é um instrumento simples para triagem entre leveduras de C. albicans e não-albicans, bem como confirmação de identificação automatizada.
Palavras-chaves: Identificação bioquímica. Características fenotípicas. Candida albicans.
The increased incidence of opportunistic fungal infections has been well documented, especially during the last two decades. The main factor contributing to this increase is growing number of immunocompromised patients1. Opportunistic fungal infections are caused above all by Candida albicans and other Candida species2.
C. albicans is a human pathogenic fungus capable of infecting both the skin and mucous membranes, often causing severe systemic infections in immunocompromised hosts3. Distinctive features of this species are morphological conversion in the body of the host yeast blastoconidia and hyphae true, and germ tube formation4. C. dubliniensis is a yeast that has been isolated in various geographical regions and is associated with superficial and systemic infections in immunocompromised individuals, especially those infected with HIV, who need of attention due to their ability to develop resistance to fluconazole in vitro5. This species has several morphological, phenotypic, biochemical and virulence characteristics that are similar to those of C. albicans, hampering the presumptive identification6.
The most accurate method for identifying of C. dubliniensis and discriminating it from C. albicans is based on PCR7. Carbohydrate assimilation for yeast identification is widely used in laboratories due to the speed. However, systems such as API 20C AUX and Vitek YBC have not been entirely specific in differentiating of C. albicans and C. dubliniensis and thus require additional phenotypic tests8.
Several phenotypic tests differentiating to differentiate between the two species have been described, such as discrimination by color on CHROMagar, growth at 42 and 45°C9, and produce fluorescence in Sabouraud methylene blue (for isolates of C. albicans) and abundant production of chlamydospores on cornmeal agar with Tween-80 (by isolates of C. dubliniensis)10.
Hypertonic Sabouraud broth11 and tobacco agar12 culture media have been described to distinguish between these species with high reproducibility and simplicity at low cost, results using this media agreed with those of molecular identification. In hypertonic Sabouraud broth (Sabouraud dextrose broth with 6.5% NaCl), C. dubliniensis displays no growth, and its micromorphological aspects on tobacco agar is distinguishable from that of C. albicans.
In this study, we evaluated the performance of carbohydrate assimilation (API ID 32C) and phenotypic identifications in the differentiation between C. albicans and C. dubliniensis of yeasts previously identified as C. albicans.
A total of 79 yeast isolates identified as C. albicans by the API ID 32C system (bio Merieux, France) were studied. The analyzed isolates belong to the culture collection of the Mycology Laboratory of the Instituto Evandro Chagas. The yeasts were stored in Malte Extrat broth medium and 15% glycerol at -70°C, and were grown on Sabouraud dextrose agar (Difco, Detroit, Mich.) and maintained at 30°C for 24-48h for testing. All were isolates from culture of wide spectrum of clinical presentation of patients (hospital/outpatient) suspected of fungal infection (skin, sputum, bronchoalveolar lavage and hemoculture), in the period of 2005 to 2009. C. albicans (ATCC 90028) and C. dubliniensis (was obtained from the Escola Paulista de Medicina Yeast Collection, Mycology Laboratory, São Paulo, Brazil.) strains were used as control both for hypertonic Sabouraud broth and tobacco agar.
API ID 32C system
According to the instructions of the manufacturer (bio Merieux, France), 250µL of the yeast suspension was inoculated and homogenizated in an ampule of API C medium. An aliquot (135µL) of this suspension was dispensed into each well of the strip and incubated at 28°C for 24-48h. Strip was read using themini API® instruments and identification was obtained using the database (V2.0). After automatic reading the results transmitted to the computer was interpreted by the identification software.
Hypertonic Sabouraud broth
Hypertonic Sabouraud broth was produced as described by Alves et al11. Briefly, isolates from a 48h culture on Sabouraud agar plates were suspended in sterile distilled water (0.5 Mc Farland). Aliquots (20µL) of each suspension were added to tubes containing 1ml of sterile Sabouraud broth supplemented with 6.5% NaCl and then statically incubated for 96h. The cultures were then visually examined for the detection of fungal growth at 24h intervals. A positive test result was defined as the absence of any visible colony growth of in the test tube after 96h of incubation. In this case, C. albicans but not C. dubliniensis is able to grow in an excess of NaCl.
Tobacco was produced with agar as described previously12. Briefly, 50g of tobacco from commercially available tobacco (Fumo Extra Forte LTDA, Brazilian Industry, Manaus, State of Amazonas) was mixed with 1L of distilled water. The mixture was boiled for 30min and then filtered through several layers of gauze. To this filtrate, 20g of agar was added, and the volume was adjusted made up to 1L. The pH of the medium at this point was 5.4. It was autoclaved at 121°C for 15min and poured into each Petri plates. Yeast isolates were freshly subcultured on Sabouraud dextrose agar (Difco, Detroit, Mich.), and the tobacco agar plates were streaked with a small amount of inoculum from the isolated colonies. The culture plates were incubated at 28°C, and the macro and micromorphological characteristics were observed daily up to 96h. The isolates were observed under a light microscope (Carl Zeiss) at lower (10X) and higher magnification (40X) to identify fragments of hyphae and chlamydospores produced by C. dubliniensis.
In this study, 79 yeasts previously identified as C. albicans by the API ID 32C system were tested for salt tolerance (hypertonic Sabouraud broth) and growth on tobacco agar. Seventeen isolates (17/79; 21.5%) were growth inhibited in hypertonic Sabouraud broth, initially suggesting that they represented C. dubliniensis as described by Alves et al in 200211. However, eight of the growth-inhibited isolates (8/79; 10.1%) showed morphotype and micromorphology on tobacco agar typical of C. albicans. Smooth colonies of a whitish-cream color developed, and microscopy revealed globose yeasts with or without one bud, eliminating the possibility of identification as C. dubliniensis.
Colonial and micromorphological aspects suggestive of C. dubliniensis on tobacco agar were observed for nine isolates (9/79; 11.4%) inhibited in hypertonic Sabouraud broth. This presented rough/brownish-yellow colonies constituted of pseudohyphae and hypae with chlamydospores.Table 1 summarizes the results obtained on tobacco agar for isolates inhibited in hypertonic Sabouraud broth, and the values indicated the percentage of identified as C. albicans by API ID 32C.
Hypertonic Sabouraud broth and tobacco agar were developed to discriminate between C. albicansand C. dubliniensis, based on the different phenotypic characteristics of these two species. In these studies11,12, the carbohydrate assimilation was used as a test standard to identify and correctly differentiate between the two species. However, our results suggest that there may be a bias in species identification during the discrimination by API ID 32C system. One reason for this is that the carbohydrate assimilation profile, which is foundation of the API ID 32C identification system, shows significant variations among strains of the same species; some authors have suggested that such tests have low discriminatory power 8,13. Other trials have been conducted that identify C. dubliniensis by the absence of growth at 42°C and the activity of α-D-glucosidase, but false positives can be obtained because of atypical strains of C. albicans are capable of displaying these phenotypic aspects14.
The isolates inhibited in hypertonic Sabouraud broth, with typical aspects of C. albicans (colonies white/smooth with single yeasts) on tobacco agar, may represent non-osmotolerant, non-albicansspecies (e.g., C. glabrata) that are mistakenly identified by carbohydrate assimilation15,16. It is known that C. albicans isolates after long-term storage can produce both morphological and enzymatic changes17, by phenomenon known as phenotypic switching. This event is spontaneous, reversible and observed at high frequencies18. At despite of morphotyping on stored isolates is not recommended due to occurrence of switching, in our laboratory was not observed change of colony in culture medium of routine, such as Sabouraud dextrose agar, during this experiment. Only were obtained colonies smooth/white before of the proceeding of salt tolerance and submission at tobacco agar. Nevertheless, we cannot to exclude the hypothesis of that the cause from isolates inhibited in hypertonic Sabouraud broth and constituted of pseudohyphaes (n = 5) is the switching. Additionally, this study is one of few relates of that salt tolerance can be affected by phenotypic switching in isolates of C. albicans storage for long-term, but the inclusion of others parameters, such as molecular identification, is critical for to corroborate this result.
Tobacco agar was developed for the presumptive identification of Cryptococcus neoformans19 and later to discriminate between isolates of C. albicans and C. dubliniensis with 100% accuracy12, displays a rough colony on tobacco agar, although isolates of C. krusei, C. tropicalis, and C. parapsilosis also produce fragments of hyphae after 48h of incubation. This leads us to believe that the three isolates inhibited in hypertonic Sabouraud broth and with morphotyping in tobacco agar suggestive of C. dubliniensis need of confirmation using molecular tools based in species-specific primers.
Based on these results, the carbohydrate assimilation and phenotyping gave the same result in 78.5% of isolates for identifying C. albicans. However, the results of 21.5% of the isolates were outliers for biochemistry identification, suggesting a low discriminatory power between the two closely related species C. albicans and C. dubliniensis. Therefore, the low cost and minimal infrastructure necessary to carry out the methodology in our study make the application of hypertonic Sabouraud broth and tobacco agar an attractive strategy to inquire concerning identification for carbohydrate assimilation in the discrimination between these two species.
We expressed our gratitude to American Journal Expert for proofreading this manuscript.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.
1. Hung CC, Yang YL, Lauderdale TL, Clifford McDonald L, Hsiao C, Cheng H, et al. Colonization of human immunodeficiency virus-infected outpatients in Taiwan with Candida species. J Clin Microbiol 2005; 43:1600-1603. [ Links ]
2. Pfaller MA. Nosocomial candidiasis: Emerging species, reservoirs, and modes of transmission. Clin Infect Dis 1996; 22 (suppl 2):89-94. [ Links ]
3. Calderone RA. Candida and candidiasis. 1st ed. Washington (DC): ASM Press; 2002. [ Links ]
4. Cutler JE. Putative virulence factors of Candida albicans. Annu Rev Microbiol 1991; 45:187-218. [ Links ]
5. Sullivan D, Coleman D. Candida dubliniensis: Characteristics and Identification. J Clin Microbiol 1998; 36:329-334. [ Links ]
6. Sullivan DJ, Moran G, Donnelly S, Gee S, Pinjon E, McCartan B, et al. Candida dubliniensis: An update. Rev Iberoam Micol 1999; 16:72-76. [ Links ]
7. Selvarangan R, Limaye AP, Cookson BT. Rapid identification and differentiation of Candida albicans and Candida dubliniensis by capillary-based amplification and fluorescent probe hybridization. J Clin Microbiol 2002; 40:4308-4312. [ Links ]
8. Gales AC, Pfaller MA, Houston AK, Joly S, Sullivan DJ, Coleman DC, et al. Identification of Candida dubliniensis Based on Temperature and Utilization of Xylose and a-Methyl-D-Glucoside as Determined with the API 20C AUX and Vitek YBC Systems. J Clin Microbiol 1999; 37:3804-3808. [ Links ]
9. Kirkpatrick WR, Revankar SG, Mcatee RK, Lopez-Ribot JL, Fothergill AW, McCarthy DI, et al. Detection of Candida dubliniensis in oropharyngeal samples from human immunodeficiency virus-infected patients in North America by primary CHROMagar candida screening and susceptibility testing of isolates. J Clin Microbiol 1998; 36:3007-3012. [ Links ]
10. Schoofs A, Odds FC, Colebunders R, Ieven M, Goossens H. Use of specialised isolation media for recognition and identification of Candida dubliniensis isolates from HIV-infected patients. Eur J Clin Microbiol Infect Dis 1997; 16:296-300. [ Links ]
11. Alves SH, Milan EP, Sant’ana PL, Oliveira LO, Santurio JM, Colombo AL. Hypertonic sabouraud broth as a simple and powerful test for Candida dubliniensis screening. Diagnostic Microbiol Infect Dis 2002; 43:85-86. [ Links ]
12. Khan ZU, Ahmad S, Mokaddas E, Chandy R. Tobacco Agar, a New Medium for Differentiating Candida dubliniensis from Candida albicans. J Clin Microbiol 2004; 42: 4796-4798. [ Links ]
13. Salkin IF, Pruitt WR, Padhye AA, Sullivan D, Coleman D, Pincus DH. Distinctive carbohydrate assimilation profiles used to identify the first clinical isolates of Candida dubliniensis recovered in the United States. J Clin Microbiol 1998; 36:1467. [ Links ]
14. Tintelnot K, Haase G, Seibold M, Bergmann F, Staemmler M, Franz T, et al. Evaluation of phenotypic markers for selection and identification of C. dubliniensis. J Clin Microbiol 2000; 38:1599-1608. [ Links ]
15. Akgül Ö, ÇerikçioĞlu N. Hypertonic Sabouraud Dextrose Agar as a Substrate for Differentiation of Candida dubliniensis. Mycopathologia 2009; 167:357-359. [ Links ]
16. Krauke Y, Sychrova H. Four Pathogenic Candida Species Differ in Salt Tolerance. Curr Microbiol 2010; 61:335-339. [ Links ]
17. Bacelo KL, Costa KRC, Ferreira JC, Candido RC. Biotype stability of Candida albicans isolates after culture storage determined by randomly amplified polymorphic DNA and phenotypical methods. Mycoses 2009; 53:468-474. [ Links ]
18. Soll DR. High frequency switching in Candida albicans. Clin Microbiol Rev 1992; 5:183-203. [ Links ]
19. Tendolkar U, Taniwala S, Jog S, Mathur M. Use of a new médium – tobacco agar, for pigment production of Cryptococcus neoformans. Indian J Med Microbiol 2003; 21:277-279. [ Links ]
Dra. Silvia Helena Marques da Silva
Rodovia BR 316, Km 07 s/nº, Levilândia
67030-000 Ananindeua, PA, Brasil.
Phone: 55 91 3214-2120; Fax: 55 91 3214-2129.
Received in 22/02/2011
Accepted in 28/04/2011