Multiplex-PCR based method for the identification of 8 clinically relevant Candida species

Carvalho A 1, Costa-Oliveira S 2, Martins M 3, Pina-Vaz C 4, Rodrigues A 5, Rodrigues F 6

Author address: 

1 ICVS/Health Sciences School, Braga, Portugal, 2 Department of Microbiology, Faculty of Medicine, University of Porto, Porto, Portugal, 3 IHMT, University of Lisbon, Lisbon, Portugal, 4 Department of Microbiology, Faculty of Medicine, University of

Abstract: 

The purpose of the study was to identify the most relevant pathogenic Candida species by PCR. Invasive fungal infections have emerged in recent years as a public health problem of major importance. Invasive Candida infections underlie high mortality rates and thus, early detection and identification of the implicated species assumes critical relevance. Since conventional detection techniques are time-consuming, molecular approaches are expected to result in potential diagnostic methods. We describe a rapid and simple multiplex PCR-based procedure able to specifically identify 8 clinically relevant Candida species (C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. krusei, C. guilliermondii, C. lusitaniae and C. dubliniensis) based on the amplification of particular DNA fragments of the ITS1 and ITS2 regions. In fact, C. dubliniensis, a emerging and closely related species to C. albicans and whose standard diagnostic procedures often misclassify as C. albicans, was accurately identified. The method combines seven species-specific primers in a single PCR reaction yielding two amplicons of different sizes for each Candida species, except for C. glabrata. In this particular case, the amplification of the ITS1 and ITS2 regions, including the 5.8S rRNA coding region is sufficient to discriminate C. glabrata from other Candida species. Furthermore, to simplify the procedure and to shorten the time required for identification, we performed multiplex PCR directly from yeast colonies, avoiding the standard and cumbersome DNA isolation steps. No amplification was detected using bacterial DNA as template, although the method could accurately detect Candida species in the presence of coexisting bacteria without interference. In addition, the method was able to simultaneously identify coexisting Candida species in mixed cultures by the same multiplex PCR reaction. The clinical relevance of this method is enhanced by the ability to detect and identify Candida species DNA directly from blood cultures, using a simple and rapid sample preparation technique. Hence, from the time at which a blood culture becomes positive, species can be identified within 4.5 h using this method, in contrast to classical methods which can take up to several days. Thus, this assay would become clinically and epidemiologically useful in the identification of Candida up-to species level, since it has low costs and requires only standard equipment being therefore easily implemented in most clinical laboratories. * Carvalho, A. was the recipient of a fellowship from Fundaçí£o para a Ciíªncia e Tecnlogia (FCT), Lisbon, Portugal (SFRH/BD/11837/2003)
2005

abstract No: 

35

Full conference title: 

15th Annual Focus on Fungal Infections
    • FFI 15th (2005)