Lack of detection of Candida nivariensis and Candida bracarensis among 440 clinical Candida glabrata sensu lato isolates in Kuwait

Abstract

Occurrence of Candida nivariensis and Candida bracarensis, two species phenotypically similar to Candida glabrata sensu stricto, in human clinical samples from different geographical settings remains unknown. This study developed a low-cost multiplex PCR (mPCR) and three species-specific singleplex PCR assays. Reference strains of common Candida species were used during development and the performance of mPCR and singleplex PCR assays was evaluated with 440 clinical C. glabrata sensu lato isolates. The internal transcribed spacer (ITS) region of rDNA was also sequenced from 85 selected isolates and rDNA sequence variations were used for determining genetic relatedness among the isolates by using MEGA X software. Species-specific amplicons for C. glabrata (~360 bp), C. nivariensis (~250 bp) and C. bracarensis (~180 bp) were obtained in mPCR while no amplicon was obtained from other Candida species. The three singleplex PCR assays also yielded expected results with reference strains of Candida species. The mPCR amplified ~360 bp amplicon from all 440 C. glabrata sensu lato isolates thus identifying all clinical isolates in Kuwait as C. glabrata sensu stricto. The results of mPCR were confirmed for all 440 isolates as they yielded an amplicon only in C. glabrata sensu stricto-specific singleplex PCR assay. The rDNA sequence data identified 28 ITS haplotypes among 85 isolates with 18 isolates belonging to unique haplotypes and 67 isolates belonging to 10 cluster haplotypes. In conclusion, we have developed a simple, low-cost mPCR assay for rapid differentiation of C. glabrata sensu stricto from C. nivariensis and C. bracarensis. Our data obtained from a large collection of clinical C. glabrata sensu lato isolates show that C. nivariensis and C. bracarensis are rare pathogens in Kuwait. Considerable genetic diversity among C. glabrata sensu stricto isolates was also indicated by rDNA sequence analyses.

Antifungal drug susceptibility, molecular basis of resistance to echinocandins and molecular epidemiology of fluconazole resistance among clinical Candida glabrata isolates in Kuwait

“…PCR-sequencing of ITS region of rDNA identified 23 different haplotypes among 51 isolates including 16 isolates with unique haplotypes. The data are consistent with a recent study showing that the ITS region of rDNA varies considerably among C. glabrata strains 33 . None of the clinical isolate produced creamy white growth on CHROMagar Candida or yielded an amplicon of ~299 bp which is characteristic of C. bracarensis strains or an amplicon of ~411 bp which is characteristic of C. nivariensis strains in PCR assay 33 .…”

“…The genomic DNA from the isolates was extracted by the rapid boiling method using Chelex-100 or by using Gentra Puregene Yeast DNA extraction kit (Qiagen, Hilden Germany) used according to kit instructions and as described previously 58 . Molecular identification was performed by PCR amplification of internal transcribed spacer (ITS) region of rDNA by using mCGLF, mCNIF, mCBRF and mCGCR primers and detection of amplicons by agarose gel electrophoresis, as described previously 33 . The identity of 51 selected isolates was also confirmed by sequencing of ITS region of rDNA by using panfungal primers, as described previously 59 .…”

Identification of Cryptic Species of Four Candida Complexes in a Culture Collection

“…Twenty-four isolates of C. glabrata sensu lato were analyzed by PCR-RFLP and sequencing of the ribosomal ITS and all the strains were identified as C. glabrata sensu stricto. Several studies have investigated the presence of rare cryptic species of the glabrata complex and have not been able to prove their presence [60][61][62]. Whereas, other studies have reported C. nivariensis and C. bracarensis but with a very low prevalences [26,63,64].…”

Identification of Cryptic Species of Four Candida Complexes in a Culture Collection

“…Twenty-four isolates of C. glabrata sensu lato were analyzed by PCR-RFLP and sequencing of the ribosomal ITS and all the strains were identified as C. glabrata sensu stricto. Several studies have investigated the presence of rare cryptic species of the glabrata complex and have not been able to prove their presence [62][63][64]. However, other studies have reported C. nivariensis and C. bracarensis but with very low prevalences [28,65,66].…”