Restriction Enzyme Analysis of Ribosomal DNA Shows that Candida inconspicua Clinical Isolates Can Be Misidentified as Candida norvegensis with Traditional Diagnostic Procedures

Abstract

We identified 29 yeast isolates from 22 patients using the API ID32C panel. Twenty-eight of these isolates were Candida norvegensis and one was C. inconspicua. Although C. norvegensis is considered a pseudohyphaproducing species, only one isolate produced pseudohyphae. Restriction enzyme analysis of PCR-amplified ribosomal DNA with four different enzymes proved that all isolates were C. inconspicua.The frequency of human infections caused by yeasts is increasing. Candida albicans is still the opportunistic pathogenic yeast most frequently isolated from humans with fungal diseases; however, several non-C. albicans Candida species are also cultured more and more frequently from different body sites (3,15). Preliminary identification of non-C. albicans Candida species has been incorporated into the majority of routine laboratory algorithms (18). In doubtful cases, additional techniques are required to obtain a precise identification of the isolated species, e.g., the determination of different biochemical patterns with the API 20C and API ID32C systems (API/ ATB package inserts, BioMerieux, 1996). Recently, molecular biology techniques have been introduced for the more precise identification of Candida isolates (21,22). This urgent need is based on at least two factors: the spread of Candida species resistant to antifungal agents (6) and the threat of nosocomial Candida outbreaks (4). An additional advantage of these techniques is the ability to differentiate between closely related species or previously misidentified yeasts.C. inconspicua and C. norvegensis are rarely isolated opportunistic pathogens (17,20). During the past 5 years, the number of yeast isolates identified in the Laboratory for Bacteriological Diagnostics, University of Debrecen, as C. inconspicua or C. norvegensis increased from 3 to 4 per year to 29 per year (in 2001), while the overall increase in the rate of yeast isolation was only threefold (9). We could not reliably distinguish the two species by traditional identification methods (with the API ID32C system and detection of pseudohypha production) because the species identities obtained by different methods were contradictory. To precisely identify our isolates we applied ribosomal DNA (rDNA) analysis.The genetic background of the method chosen can be summarized as follows. The tandemly repeated rDNA genes of yeasts contain highly conservative and less conservative regions. Sequences coding for the 18S, 5.8S, and 25S rRNAs evolved slowly during the phylogenesis of fungi; thus, they are suitable for use as tools for discrimination and identification at the interspecies or higher level. On the contrary, the internal transcribed spacer region 1 (ITS1) and ITS2 rDNA regions are more variable; thus, they can serve as a tool in the discrimination of closely related yeast species (21). Restriction enzyme analysis of PCR-amplified rDNA is a superior typing method suitable for correct identification, even with a large number of yeast isolates. In addition, this method can correct misidentification r...

Results from the ARTEMIS DISK Global Antifungal Surveillance Study, 1997 to 2007: a 10.5-Year Analysis of Susceptibilities of Candida Species to Fluconazole and Voriconazole as Determined by CLSI Standardized Disk Diffusion

“…The majority of C. inconspicua isolates reported in the literature are from the respiratory tract; however, wound, blood, and genital isolates have also been obtained (3,11,(33)(34)(35). Whereas the previous reports concerning C. inconspicua contained no more than 50 clinical isolates, the present data set is considerably more robust and shows C. inconspicua isolated from numerous body sites, including blood and NSBF.…”

Evaluation of the New Micronaut- Candida System Compared to the API ID32C Method for Yeast Identification

“…Rare species with proven decreased susceptibility to certain antifungals (2,5,8,10) were identified correctly by MicronautCandida with (C. inconspicua) or without extra tests (C. kefyr, C. guilliermondii, and C. lusitaniae). Thus, this new system seems to be a reliable and useful method for identification of medically relevant yeasts in routine mycology laboratories.…”

“…Conventional methods are time-consuming, while DNA-based methods are not available to all laboratories. Relatively simple tests based on assimilation reactions (API ID32C, API 20C, API Candida) are commercially available, though they may sometimes yield equivocal results or even lead to misidentification (1,3,5,11).…”

“…If these were in accord, we accepted them as final. Otherwise or when additional tests (microscopic morphology, esculin hydrolysis, growth at 45°C, nitrate assimilation) yielded equivocal results, PCR ribotyping was performed in each case and ribotyping results were accepted as valid identification (5).…”

Identification of Medically Important Candida and Non- Candida Yeast Species by an Oligonucleotide Array

“…These limitations are probably attributable, in part, to the databases currently employed in the profile indexes. Misidentifications of some species by commercial kits have been reported (7,11,28,29,40), and even the well-known and medically important yeast Candida glabrata has been misidentified by phenotypic methods (7).…”