Utility of two PCR‐RFLP‐based techniques for identification of Candida parapsilosis complex blood isolates

Abstract

Background: Candida parapsilosis is the second or third most frequently isolatedCandida species related to nosocomial infections, even overtaking Candida albicans in some hospitals. C. parapsilosis constitutes a complex of closely related species:Candida parapsilosis sensu stricto, Candida orthopsilosis and Candida metapsilosis.Accurate detection of these species is of importance, as the incidence of C. orthopsilosis has been reported to surpass that of Candida krusei.Objective: To evaluate the diagnostic utility of two PCR-RFLP methods targeting the SADH and FKS1 genes and to determine the prevalence of cryptic species in 96 bloodstream isolates of C. parapsilosis from 93 patients. Methods:Restriction patterns of the SADH and FKS1 genes were analysed, and sequencing of the D1/D2 regions of the ribosomal RNA was used to evaluate the reliability of both PCR-RFLP methods.Results: In our study, 77 C. parapsilosis sensu stricto, 13 C. orthopsilosis and five C. metapsilosis were identified by sequencing. Both PCR-RFLP methods demonstrated strong agreement with D1/D2 sequencing in the identification of C. parapsilosis and C. orthopsilosis, while both methods were unable to identify the C. metapsilosis isolates. Moreover, unexpected restriction patterns were observed for two isolates on SADH PCR-RFLP and for four isolates on FKS1 PCR-RFLP. Mixed bloodstream infections of C. parapsilosis sensu stricto and C. orthopsilosis were detected for three patients, for which differential growth characteristics were observed. Conclusion:The molecular method chosen for identification could have an impact on determination of the real prevalence of C. metapsilosis in candidaemia, and mixed fungaemias can remain undetected. K E Y W O R D SCandida metapsilosis, Candida orthopsilosis, D1/D2 large subunit sequencing, PCR-RFLP, SADH and FKS1 genes

Species distribution, azole resistance and related molecular mechanisms in invasive Candida parapsilosis complex isolates: Increase in fluconazole resistance in 21 years

“…8 Differentiation up to species level mostly relies on molecular methods, although promising results were achieved using matrix-assisted laser desorption-time-of-flight mass spectrometry (MALDI-TOF MS). [8][9][10][11] Prevalence, virulence factors and antifungal resistance may vary among these species. [12][13][14][15][16] C. parapsilosis sensu stricto is the most common member of the complex causing human infections.…”

“…Candida parapsilosis complex consists of C. parapsilosis sensu stricto , Candida orthopsilosis and Candida metapsilosis 8 . Differentiation up to species level mostly relies on molecular methods, although promising results were achieved using matrix‐assisted laser desorption‐time‐of‐flight mass spectrometry (MALDI‐TOF MS) 8–11 . Prevalence, virulence factors and antifungal resistance may vary among these species 12–16 .…”

Candida duobushaemulonii: An Old But Unreported Pathogen

“…Currently, only matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and molecular techniques, such as sequencing of genetic loci of the ribosomal DNAi ncluding the D1/D2 domain, internal transcribed spacer (ITS) regions, and RPB1 and RPB2, have been demonstrated to provide reliable identification of these species [16,18]. However, these techniques are not available in most routine microbiology laboratories, and there may be issues when testing mixed clinical samples, as they could be not able to distinguish between the species involved [26]. Aiming to achieve simple and easily accessible techniques for accurate identification of C. auris, various species-specific conventional or real-time PCR-based assays have been developed.…”