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Comparative Analyses of Classical Phenotypic Method and Ribosomal RNA Gene Sequencing for Identification of Medically Relevant Candida Species

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Comparative Analyses of Classical Phenotypic Method and Ribosomal RNA Gene Sequencing for Identification of Medically Relevant Candida Species 178 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 108(2): 178-185, April 2013 Comparative analyses of classical phenotypic method and ribosomal RNA gene sequencing for identification of medically relevant Candida species Constanza Giselle Taverna1/+, María Eugenia Bosco-Borgeat1, Omar Alejandro Murisengo1, Graciela Davel1, Mariana Côrtes Boité2, Elisa Cupolillo2, Cristina Elena Canteros1 1Departamento Micología, Instituto Nacional de Enfermedades Infecciosas Dr Carlos G Malbrán, Buenos Aires, Argentina 2Laboratório de Pesquisa em Leishmaniose, Instituto Oswaldo Cruz-Fiocruz, Rio de Janeiro, RJ, Brasil As the distribution of Candida species and their susceptibility to antifungal agents have changed, a new means of accurately and rapidly identifying these species is necessary for the successful early resolution of infection and the subsequent reduction of morbidity and mortality. The current work aimed to evaluate ribosomal RNA gene sequenc- ing for the identification of medically relevant Candida species in comparison with a standard phenotypic method. Eighteen reference strains (RSs), 69 phenotypically identified isolates and 20 inconclusively identified isolates were examined. Internal transcribed spaces (ITSs) and D1/D2 of the 26S ribosomal RNA gene regions were used as tar- gets for sequencing. Additionally, the sequences of the ITS regions were used to establish evolutionary relationships. The sequencing of the ITS regions was successful for 88% (94/107) of the RS and isolates, whereas 100% of the remaining 12% (13/107) of the samples were successfully analysed by sequencing the D1/D2 region. Similarly, geno- typic analysis identified all of the RS and isolates, including the 20 isolates that were not phenotypically identified. Phenotypic analysis, however, misidentified 10% (7/69) of the isolates. Phylogenetic analysis allowed the confirma- tion of the relationships between evolutionarily close species. Currently, the use of genotypic methods is necessary for the correct identification of Candida species. Key words: Candida - ribosomal DNA - DNA sequence analysis Candida species are fungal pathogens that can cause tropicalis (15.4%) and Candida glabrata (4.3%) are fre- a wide range of superficial and deep mycoses, collective- quently isolated species (Cordoba et al. 2011). ly known as candidiasis, that are commonly observed in The proper identification of Candida species is in- immunocompromised patients. Candidaemia is the most creasingly necessary, not only because the distribution clinically important Candida infection, both because is of Candida species has changed, but also because these the most frequent yeast infection in hospitalised patients species differ in their susceptibility to antifungal agents and because this infection results in significant mortal- (Pfaller & Diekema 2002, Ellepola & Morrison 2005). ity. The incidence has risen over the past decades as the Accurate and rapid identification can facilitate the suc- number of immunocompromised patients has increased cessful early resolution of infections and the subsequent (Pfaller & Diekema 2002, Falagas et al. 2010) and cur- reduction of morbidity and mortality (Pincus et al. 2007). rently, candidaemia is the fourth most common noso- The classical identification of fungi has been based on comial bloodstream infection in the United States of the morphological and physiological features of the sex- America (USA) (Pfaller et al. 1998, Edmond et al. 1999, ual and/or asexual state. Because most medically impor- Wisplinghoff et al. 2004). tant yeast species lack a sexual state and/or distinctive Although Candida albicans remains the most fre- asexual morphological features, the correct identification quently isolated species in human infection, more than of these species is often difficult and inconclusive when 50% of candidaemias are due to infection with other based solely on physiological traits. These morphological Candida species (Price et al. 1994, Nguyen et al. 1996, and physiological characteristics are often unstable, vari- Abi-Said et al. 1997, Trick et al. 2002, Hajjeh et al. 2004, able and subjective (Latouche et al. 1997). Furthermore, Wisplinghoff et al. 2004). For example, in Argentina, C. genetically diverse yeast species can yield similar pheno- albicans (38.4%), Candida parapsilosis (26%), Candida typic profiles, resulting in poor discrimination between unrelated yeast species (Sullivan et al. 1996). In this con- text, genotypic identification may be preferable as this method is faster and more accurate (Pincus et al. 2007). The ribosomal RNA (rDNA) gene complex is large- ly used as a target in many polymerase chain reaction (PCR)-based assays because the complex is present This study was part of the CGT thesis for the Master in Science degree/ in all microorganisms, occurs as tandem repeats of as FIOCRUZ in collaboration with the ANLIS Dr Carlos G Malbrán. + Corresponding author: [email protected] many as 100-200 copies and contains highly conserved Received 6 September 2012 domains separated by variable domains, thus enabling Accepted 12 December 2012 the design of universal PCR primers for fungi (White et online | memorias.ioc.fiocruz.br Identification of Candida species • Constanza Giselle Taverna et al. 179 al. 1990, Kurtzman & Fell 1998, Iwen et al. 2002, Pincus ITS amplification - The ITS1 (5′-TCCGTAGGTGAA- et al. 2007). Moreover, nucleotide-sequence heterogene- CCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGAT- ity within this complex may be used to phylogenetically ATGC-3′) primers were used (White et al. 1990). The classify microorganisms, including yeast species (Kurtz- reactions were performed in a volume of 100 µL con- man & Robnett 1998, Fell et al. 2000, Iwen et al. 2002). taining 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 2 mM The taxonomy of yeast and other fungal species has Mg2Cl2, 5.2% DMSO, 0.2 mM each of dATP, dCTP, profoundly changed since the advent of DNA sequencing dGTP and dTTP (Fermentas International, Inc), 0.1 µM for the classification of microorganisms. Single gene se- each of the primers ITS1 and ITS4, 1 U Taq DNA poly- quences, such as those from the D1/D2 region of 26S rDNA merase (Invitrogen-Life Technologies, Brazil) and 30 ng or from the internal transcribed space (ITS) regions, are of DNA. All of the amplifications were performed in an commonly used to identify yeast species (Kurtzman 2006, iCycler (Bio-Rad Laboratories, Inc) using the follow- 2010). Recently, the sequence of the ITS region has been ing parameters: 95ºC for 7 min, followed by 40 cycles proposed to be a primary fungal barcode marker by the at 95ºC for 1 min, 54ºC for 2 min, 72ºC for 1 min and a Consortium for the Barcode of Life (Schoch et al. 2012). final extension at 72ºC for 10 min. At present, the rDNA sequences of nearly all clini- Amplification of D1/D2 region of 26S rDNA - The NL1 cally relevant yeast species are available in public data- (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL4 bases, such as GenBank (ncbi.nlm.nih.gov/genbank/) or (5′-GGTCCGTGTTTCAAGACGG-3′) primers were used the Centraalbureau voor Schimmelcultures (CBS) Yeast (White et al. 1990). The reactions were performed in a Database (cbs.knaw.nl), making it possible to genetical- volume of 100 µL containing 20 mM Tris-HCl (pH 8.4), ly identify one unknown yeast isolate by comparing its 50 mM KCl, 2.5 mM Mg2Cl, 5% DMSO, 0.2 mM each of rDNA sequences with those sequences in the database. dATP, dCTP, dGTP and dTTP, 0.1 µM each of the prim- The aim of the current study was to compare a clas- ers NL1 and NL4, 1 U Taq DNA polymerase and 30 ng sical phenotypic method with rDNA sequencing for the of DNA. All of the amplifications were performed in an identification of medically relevant Candida species. iCycler using the following parameters: 95ºC for 7 min, ITS sequences were also used to evaluate the genetic re- followed by 40 cycles at 95ºC for 1 min, 53ºC for 2 min, lationships between the samples analysed. 72ºC for 1 min and a final extension at 72ºC for 10 min. MATERIALS AND METHODS Agarose gel electrophoresis - The PCR products Reference strains (RSs) and isolates - Eighteen RSs, were electrophoresed on 1.5% agarose gels in 40 mM which our laboratory commonly uses for identification Tris-Acetate and 1 mM EDTA buffer (1X TAE) for 1 h and/or susceptibility testing, and 89 additional isolates at 100 V, stained with ethidium bromide (10 µg/mL) and (Supplementary data) were included in the study. The RS then visualised under ultraviolet (UV) light and photo- were obtained from the American Type Culture Collec- documented using an LAS 3000 version 2.1 (Fuji Photo Film Co, Ltd). A GeneRuler 100 bp DNA Ladder (Fer- tion, USA, the culture collection of the Carlos III Institute mentas Internationa, Inc) was used. of Health, Spain, and the culture collection of the De- partment of Mycology of the National Institute of Infec- Purification of PCR products - The PCR products tious Diseases Dr Carlos G Malbrán, Argentina. Based were purified using a PureLink PCR Purification Kit on their phenotypic identification, isolates were selected (Invitrogen). The products were then electrophoresed on from those received at the yeast identification laboratory 1.5% agarose gels in 1X TAE for 1 h at 100 V, stained from January 2009-December 2010. A total of 69 isolates with ethidium bromide (10 µg/mL) and visualised under of Candida species frequently isolated from patient in UV light. A ready-to-use MassRuler Express DNA Lad- Argentina were included in the study (Rodero et al. 2005, der, LR Reverse (Fermentas),
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