A simple and reliable PCR-restriction fragment length polymorphism assay to identify Candida albicans and its closely related Candida dubliniensis

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Abstract

Candida dubliniensis is an emerging pathogen capable of causing superficial as well as systemic infections. Due to its close similarity to C. albcians, conventional methods based on phenotypic traits are not always reliable in identification of C. dubliniensis. In this study, we developed a PCR-restriction fragment length polymorphism (RFLP) assay to identify and discriminate between the two closely related species. The D1/D2 region of 28S rDNA was amplified by PCR and enzymatically digested by ApaI and BsiEI respectively. PCR products of both species were digested into two fragments by ApaI, but those of other yeast species were undigested. BsiEI cut the PCR products of C. albicans into two fragments but not those of C. dubliniensis. Thus two species were differentiated. We evaluated 10 reference strains representing 10 yeast species, among which C. albicans and C. dubliniensis were successfully identified. A total of 56 phenotypically characterized clinical isolates (42 C. albicans isolates and 14 C. dubliniensis isolates) were also investigated for intra-species variability. All tested isolates produced identical RFLP patterns to their respective reference strains except one initially misidentified isolate. Our method offers a simple, rapid and reliable molecular method for the identification of C. albicans and C. dubliniensis.

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Most cited references 63

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Epidemiology of invasive candidiasis: a persistent public health problem.

M Pfaller, D J Diekema (2007)

Invasive candidiasis (IC) is a leading cause of mycosis-associated mortality in the United States. We examined data from the National Center for Health Statistics and reviewed recent literature in order to update the epidemiology of IC. IC-associated mortality has remained stable, at approximately 0.4 deaths per 100,000 population, since 1997, while mortality associated with invasive aspergillosis has continued to decline. Candida albicans remains the predominant cause of IC, accounting for over half of all cases, but Candida glabrata has emerged as the second most common cause of IC in the United States, and several less common Candida species may be emerging, some of which can exhibit resistance to triazoles and/or amphotericin B. Crude and attributable rates of mortality due to IC remain unacceptably high and unchanged for the past 2 decades. Nonpharmacologic preventive strategies should be emphasized, including hand hygiene; appropriate use, placement, and care of central venous catheters; and prudent use of antimicrobial therapy. Given that delays in appropriate antifungal therapy are associated with increased mortality, improved use of early empirical, preemptive, and prophylactic therapies should also help reduce IC-associated mortality. Several studies have now identified important variables that can be used to predict risk of IC and to help guide preventive strategies such as antifungal prophylaxis and early empirical therapy. However, improved non-culture-based diagnostics are needed to expand the potential for preemptive (or early directed) therapy. Further research to improve diagnostic, preventive, and therapeutic strategies is necessary to reduce the considerable morbidity and mortality associated with IC.

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Nosocomial bloodstream infections in United States hospitals: a three-year analysis.

M B Edmond, S. Wallace, D K McClish … (1999)

Nosocomial bloodstream infections are important causes of morbidity and mortality. In this study, concurrent surveillance for nosocomial bloodstream infections at 49 hospitals over a 3-year period detected >10,000 infections. Gram-positive organisms accounted for 64% of cases, gram-negative organisms accounted for 27%, and 8% were caused by fungi. The most common organisms were coagulase-negative staphylococci (32%), Staphylococcus aureus (16%), and enterococci (11%). Enterobacter, Serratia, coagulase-negative staphylococci, and Candida were more likely to cause infections in patients in critical care units. In patients with neutropenia, viridans streptococci were significantly more common. Coagulase-negative staphylococci were the most common pathogens on all clinical services except obstetrics, where Escherichia coli was most common. Methicillin resistance was detected in 29% of S. aureus isolates and 80% of coagulase-negative staphylococci. Vancomycin resistance in enterococci was species-dependent--3% of Enterococcus faecalis strains and 50% of Enterococcus faecium isolates displayed resistance. These data may allow clinicians to better target empirical therapy for hospital-acquired cases of bacteremia.

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Candida dubliniensis sp. nov.: phenotypic and molecular characterization of a novel species associated with oral candidosis in HIV-infected individuals.

Mark Bennett, T Westerneng, David Sullivan … (1995)

Atypical oral Candida isolates were recovered from 60 HIV-infected and three HIV-negative individuals. These organisms were germ-tube-positive and produced abundant chlamydospores which were frequently arranged in triplets or in contiguous pairs. They belonged to C. albicans serotype A and had atypical carbohydrate assimilation profiles. Fingerprinting the genomic DNA of a selection of these organisms with the C. albicans-specific probe 27A and five separate oligonucleotides, homologous to eukaryotic microsatellite repeat sequences, demonstrated that they had a very distinct genomic organization compared to C. albicans and C. stellatoidea. This was further established by random amplified polymorphic DNA (RAPD) and karyotype analysis. Comparison of 500 bp of the V3 variable region of the large ribosomal subunit genes from nine atypical isolates and the corresponding sequences determined from C. albicans, C. stellatoidea, C. tropicalis, C. parapsilosis, C. glabrata, C. kefyr and C. krusei showed that they atypical organisms formed a homogeneous cluster (100% similarity) that was significantly different from the other Candida species analysed, but was most closely related to C. albicans and C. stellatoidea. These genetic data combined with the phenotypic characteristics of these atypical organisms strongly suggest that they constitute a novel species within the genus Candida for which the name Candida dubliniensis is proposed.

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Author and article information

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SciELO Brazil ID:: S1517-83822012000300004

DOI:: 10.1590/S1517-83822012000300004

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http://creativecommons.org/licenses/by/4.0/

ScienceOpen disciplines: Microbiology & Virology

Keywords: Candida albicans,Candida dubliniensis,PCR-restriction fragment length polymorphism,identification,differentiation

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ScienceOpen disciplines: Microbiology & Virology

Keywords: Candida albicans, Candida dubliniensis, PCR-restriction fragment length polymorphism, identification, differentiation

A simple and reliable PCR-restriction fragment length polymorphism assay to identify Candida albicans and its closely related Candida dubliniensis

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SciELO Brazil

Most cited references 63

Epidemiology of invasive candidiasis: a persistent public health problem.

Nosocomial bloodstream infections in United States hospitals: a three-year analysis.

Candida dubliniensis sp. nov.: phenotypic and molecular characterization of a novel species associated with oral candidosis in HIV-infected individuals.

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