Comparison of four DNA-based methods for strain delineation of Candida lusitaniae

Abstract

Four methods for the accurate delineation of epidemiologically related and unrelated strains of Candida lusitaniae were compared. Three pulsed-field electrophoretic methods, including two contour-clamped homogeneous field gel electrophoresis methods (EKP-1 and EKP-2) yielding electrophoretic karyotype patterns of intact chromosomal DNA and a method in which the chromosomal DNA was macrodigested with the endonuclease SfiI prior to pulsed-field electrophoresis (MDP), and a random amplified polymorphic DNA (RAPD) assay were evaluated. A selected panel of 21 well-characterized isolates representing 13 strains of C. lusitaniae, including 7 epidemiologically related isolates of one strain (group I-A), 3 epidemiologically related isolates of another strain (group I-B), and 11 epidemiologically unrelated isolates (group II), were tested. All isolates were coded and tested in a blinded manner. All seven group I-A isolates were confirmed to be a single strain by the EKP-1 and MDP methods, and the three group I-B isolates were shown to be a single strain by the EKP-1, EKP-2, MDP, and RAPD methods. Subtle differences were noted with two of the group I-A isolates by the EKP-2 method, whereas three of these isolates were different by the RAPD method. Each group II isolate had distinct patterns by all four methods. These data support the fact that the three pulsed-field electrophoretic methods and the RAPD method can be used to delineate strains of C. lusitaniae. The EKP-1, EKP-2, and MDP gave results that correlated with the epidemiologic characteristics of the isolates tested in the study, whereas the RAPD method was perhaps too sensitive in detecting DNA changes for epidemiologic studies.

Molecular epidemiology ofCandidaspecies isolated from clinical specimens of intensive care unit patients

“…Some authors used the primer with highest discriminative power, 21,30 while others used more than one primer and combined the results for analysis. 11,22,[32][33][34][35] We preferred this latter method, using several primers.…”

“…12,24 As the use of genotypical methods has increased in recent years, exogenous nosocomial infections have also begun to attract attention, but are still less prevalent than endogenous infections. 23,[30][31][32][33][34][35][36] There are numerous studies investigating the genetic diversity of Candida isolates recovered from ICU patients, and the results about the clonal relationship of the isolates have been varied. Marco et al 23 found crosscontamination between C. albicans isolates recovered from blood cultures of ICU patients, and Arif et al 36 also reported cross-contamination between C. glabrata isolates of ICU patients.…”

Phenotypic and genotypic characterization of oral yeasts from Finland and the United States

“…Previous studies used purified DNA as template for PCR typing of Candida species (4, 11,19,23,35,39,42,43,45). Our use of yeast cell lysate makes the PCR protocol easy and fast to perform.…”

“…PCR seems to be a promising technique for yeast genotyping. However, since previous PCR studies have been performed using purified yeast DNA as template, the PCR-based genotyping methods applied so far are still relatively laborious (4,11,19,23,35,36,39,42,43,45). PCR performed using crude yeast cell lysates would greatly facilitate the yeast genotyping procedure.…”

Molecular typing for fungi—a critical review of the possibilities and limitations of currently and future methods

“…In another inter-laboratory study, EK was used effectively to compare strains of C. albicans, C. lusitaniae, C. parapsilosis, C. tropicalis, and C. glabrata [49]. Additionally, this technique has been used to biotype C. parapsilosis, C. dubliniensis, C. lusitaniae, and C. neoformans, by several groups [27,47,[50][51][52][53][54]. However, it cannot be used for epidemiologic investigations of Malassezia spp., since the karyotype of this fungus does not vary intraspecifically [34].…”