The identification and differentiation of the Candida parapsilosis complex species by polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer region of the rDNA

Abstract

Currently, it is accepted that there are three species that were formerly grouped under Candida parapsilosis: C. para- psilosis sensu stricto, Candida orthopsilosis, andCandida metapsilosis. In fact, the antifungal susceptibility profiles and distinct virulence attributes demonstrate the differences in these nosocomial pathogens. An accurate, fast, and economical identification of fungal species has been the main goal in mycology. In the present study, we searched sequences that were available in the GenBank database in order to identify the complete sequence for the internal transcribed spacer (ITS)1-5.8S-ITS2 region, which is comprised of the forward and reverse primers ITS1 and ITS4. Subsequently, an in silico polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to differentiate the C. parapsilosis complex species. Ninety-eight clinical isolates from patients with fungaemia were submitted for analysis, where 59 isolates were identified as C. parapsilosis sensu stricto, 37 were identified as C. orthopsilosis, and two were identified as C. metapsilosis. PCR-RFLP quickly and accurately identified C. parapsilosis complex species, making this method an alternative and routine identification system for use in clinical mycology laboratories.

Comparison of four molecular approaches to identify Candida parapsilosis complex species

“…Figure illustrates the species-specific PCR of the reference strains and five representative isolates. These results agree with published sequences of the D1/D2 region of the LSU 28S rDNA gene, PCR-RFLP patterns of the ITS1-5.8S-ITS2 region of the rDNA gene, and microsatellite typing of C. parapsilosis sensu stricto ( Barbedo et al 2015 , 2016 ) ( ).…”

“…Fungaemia caused by Candida parapsilosis is commonly associated with the provision of parenteral nutrition and use of a central venous catheter. C. parapsilosis is also the main species that causes bloodstream infections in premature newborns in neonatal intensive care units ( Barbedo et al 2016 ).…”

“…Therefore, the current work was carried out to compare four different molecular methods. The species-specific uniplex PCR was performed using specific primer pairs CPAF-CPAR, CORF-CORR, and CMEF-CMER, described by Asadzadeh et al (2009) , to discriminate between C. parapsilosis complex species and to compare to the partial D1/D2 region of LSU 28S rDNA gene sequences, PCR-RFLP patterns of the ITS1-5.8S-ITS2 region of the rDNA gene, and microsatellite typing of C. parapsilosis sensu stricto ( Barbedo et al 2015 , 2016 ).…”

“…Ninety-eight clinical isolates of C. parapsilosis sensu lato obtained from the bloodstream and from catheter tips were included in this study. Information on all isolates, such as origin, biochemical characteristics, and DNA extraction, has been published previously ( Barbedo et al 2015 , 2016 ). C. parapsilosis ATCC 22019, C. orthopsilosis ATCC 96141, and C. metapsilosis ATCC 96143 were used as reference strains.…”

Estudo comparativo entre a produção de fosfolipases extracelulares e proteinases do gênero Candida isoladas a partir de infecções de cavidade oral

“…Amplificação da sequência ITS Para a reação de PCR, foram utilizados primers específicos para seis espécies, que amplificam os genes ITS1 e ITS2 do RNAr, e o gene da topoimerase II (Tabela 1) [11][12][13] . A reação de PCR foi preparada em um volume de 50 µL, composto por solução tampão 10X (20 mM Tris-HCl, pH 8,4, 50 mM KCl), 1,5 mM Mg Cl 2 , 0,5 µM de primer (Invitrogen, EUA), 2U de Taq DNA polimerase (Promega, EUA) e 50 ng de DNA previamente extraído.…”