Carbohydrate assimilation profiles of Brazilian Candida dubliniensis isolates based on ID 32C system

Identificação de Candida dubliniensis isoladas no Brasil, através do método comercial ID 32C

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Carbohydrate assimilation profiles of Brazilian Candida dubliniensis isolates based on ID 32C system

Identificação de Candida dubliniensis isoladas no Brasil, através do método comercial ID 32C

Sydney Hartz Alves^I; Jorge A. Horta^II; Eveline P. Milán^III; Liliane A. Scheid^I; Marilene H. Vainstein^II; Janio M. Santurio^I; Arnaldo L. Colombo^IV

^IDepartamento de Microbiologia e Parasitologia; Centro de Ciências da Saúde, Universidade Federal de Santa Maria. Santa Maria, RS, Brasil, phone/fax [phone], [e-mail] ^IIUniversidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brasil

^IIIUniversidade Federal do Rio Grande do Norte, Natal, RN, Brasil

^IVUniversidade Federal de São Paulo, São Paulo, SP, Brasil

^{Correspondence}

SUMMARY

The purpose of the present study was to evaluate the identification of 19 Brazilian C. dubliniensis based on the biochemical profile exhibited when tested by the commercial identification kit ID 32C (bioMerieux). Thirteen of the isolates were rigorously identified as C. dubliniensis and the remaining isolates (six) were considered as having a doubtful profile but the software also suggested that there was 83.6% of chances for them to be C. dubliniensis. As well as pointed by the literature the identification obtained by phenotypic tests should be considered presumptive for C. dubliniensis due to variability of this new species.

Keywords: Candida dubliniensis; ID 32C; Phenotypic identification.

RESUMO

Dezenove culturas de C. dubliniensis isoladas no Brasil, previamente identificadas através de métodos genotípicos, foram avaliadas pelo kit comercial ID 32C (bioMerieux). Treze culturas foram identificadas como C. dubliniensis, mas as demais (seis) evidenciaram perfil duvidoso, embora o software do sistema sugerisse 83,6% de chances das mesmas pertencerem à espécie C. dubliniensis. A literatura tem registrado grande variabilidade fenotípica com esta espécie e, por isto, as identificações obtidas com este sistema deverão ser consideradas como presuntivas.

INTRODUCTION

Candida dubliniensis is a newly described species of Candida, which was first reported by Sullivan et al. in 1995²¹. Despite C. dubliniensis has been recovered from several body sites in many human populations, it is most often isolated from the oral cavities of patients infected with the Human Immunodeficiency Virus. This new yeast is found all over the world^{2,4,10,15,16,18,22} and is similar to C. albicans in a number of ways including morphology and metabolism, being considered as an opportunistic yeast pathogen but also recognized as a minor constituent of the normal human oral microbial flora^11,15.

A multicenter surveillance study conducted in Brazil by Milán et al.¹⁰ showed a 2.8% prevalence rate of HIV-infected/AIDS adult patients harboring C. dubliniensis in their oral cavities. In contrast with patients from the north-hemisphere countries where this particular species may be recovered from 27% of asymptomatic HIV-positive patients and 32% of AIDS patients with oral candidiasis²¹, different studies from South America suggest that C. dubliniensis is more rarely found^2,10,16,18.

C. dubliniensis is phylogenetically related to C. albicans and the distinction between C. dubliniensis and C. albicans remains a challenge for clinical microbiology laboratories. The importance of the correct identification seems to be meaningful for epidemiological proposals and therapeutic interventions. The majority of C. dubliniensis isolates show susceptibility to currently used antifungal drugs¹², but it has been demonstrated that they may rapidly develop a stable resistance to fluconazole upon in vitro exposure¹¹.

Various phenotype screening tests have been used to discriminate those organisms, including the colony color on CHROMagar Candida, growth temperature test at 42/45 °C¹⁴, sugar assimilation tests and, b-glucosidase activity^17,23. In addition, C. dubliniensis strains may be recognized by their ability to produce abundant chlamydospores often observed in triplets or in contiguous pairs²¹, to coaggregate in vitro with Fusobacterium nucleatum⁸, to produce rough colonies and chlamydospores on Staib agar²⁰ and also by its intolerance to 6.5% sodium chloride broth³ and inability to produce an opacity halo at Tween 80 agar¹⁹. It is important to highlight that no single phenotype test has proven to be highly effective and the use of genotypic tests may be necessary for definitive identification purposes^7,9,22. Considering that molecular methods are relatively time consuming and expensive there is a need for clinical laboratories to have phenotype tests as reliable as the carbohydrate assimilation profiles.

The present study was undertaken to evaluate the biochemical profile exhibited by nineteen Brazilian C. dubliniensis isolates formerly identified by genotyping methods, when tested by the commercial identification kit ID 32C (bioMerieux). This kit provides an evaluation for the assimilation of 30 carbon sources and for the growth of yeasts in the presence of cyclohexemide. The assays were performed according to the manufacturer's instructions. It consists of 32 cupules, each containing a dehydrated carbohydrate substrate. A semi-solid, chemically defined, minimal medium was inoculated with a suspension of the yeast organism to be tested. After 24-48 hours of incubation, growth in each cupule was detected by visual reading. Identification was obtained using the identification software (bioMerieux).

After 48 h of incubation, the ID 32 C system was able to rigorously identify and classify great part of the isolates as positive (13) for C. dubliniensis at three different levels: excellent (seven), very good (five) and as good (one). Almost all the remaining isolates (six) were considered as having a doubtful profile but the software also suggest that there is 83.6% of chances for them to be C. dubliniensis. It should be pointed here that in laboratory routine the percentage of 83.6% would be an important indication of the species and should be taken into account.

Some assimilation profiles were not consistent among the strains of C. dubliniensis tested once they exhibited variation of results among different isolates tested. The lower agreement rate was found with results generated by palatinose, 2-keto-gluconate, N-acetylglucosamine, lactate, trehalose, galactose and sorbitol (Table 1). Best discrimination and consistence of results were obtained with a-methyl-D-glucoside (MDG) and xylose (XYL) that were not assimilated by 100% of the isolates tested. Lactate (LAT) and trehalose (TRE) were assimilated by one isolate. Our results were slightly different from those reported by Pincus et al.¹³ in which 30% of the isolates assimilated trehalose (TRE) and no one of the isolates assimilated lactate (LAT). When Sullivan et al.²¹ described C. dubliniensis as a new species, they reported that this organism was not able to assimilate xylose (XYL) and a-methyl-D-glucoside (MDG). However, some positive reactions with this subtract were described previously by testing some strains of C. dubliniensis with commercial kits as the Vitek system^5,13.

The results obtained in the present study are in accordance with the results reported by other authors^6,13. The major identification problem related to the ID32 system was the discrepancy of results generated by some reactions causing misidentification of C. dubliniensis. It has been reported that variations in the density of the inoculum may produce false-positive or false-negative results requiring the adjustment of the inoculum to the proper density⁵. Otherwise, one could speculate that different strains of C. dubliniensis may exhibit some differences in their metabolic pathways like that involving trehalose and N-acetylglucosamine²⁴. The variant phenotypes could also arise from the process of high-frequency switching to adapt to the host environment, like almost observed with C. albicans¹.

In conclusion, despite the promising results obtained with the ID 32C system, this method exhibited some limitations in the identification of C. dubliniensis. Since variability has been reported in the literature, the results obtained by phenotypic tests should be considered presumptive for C. dubliniensis and one or more confirmatory tests should be employed. Therefore, we do not recommend the use of ID 32C system as the only screening test for the identification of C. dubliniensis.

Received: 10 September 2004

Accepted: 25 November 2004

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Correspondence to

Sydney Hartz Alves

Rua Venancio Aires 2766/403

97010-004 Santa Maria, RS, Brazil

Publication Dates

• Publication in this collection
  04 May 2005
• Date of issue
  Apr 2005

History

• Accepted
  25 Nov 2004
• Received
  10 Sept 2004