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. 2019 Oct 16;14(10):e0223920.
doi: 10.1371/journal.pone.0223920. eCollection 2019.

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

Mohammad Asadzadeh  1 , Ahlam F Alanazi  1 , Suhail Ahmad  1 , Noura Al-Sweih  1 , Ziauddin Khan  1
Affiliations

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

Mohammad Asadzadeh et al. PLoS One. .

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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. An agarose gel of amplified products obtained in mPCR using C. glabrata sensu stricto-specific (mCGLF), C. nivariensis-specific (mCNIF) and C. bracarensis-specific (mCBRF) forward and C. glabrata complex-specific (mCGCR) reverse primer with genomic DNA from reference strain of C. glabrata sensu stricto ATCC 90030 (lane CG), C. nivariensis CBS 9983 (lane CN), C. bracarensis CBS 10154 (lane CB), C. albicans ATCC 76615 (lane CA), C. dubliniensis CBS 7987 (lane CD), C. parapsilosis ATCC 22019 (lane CP) and C. tropicalis ATCC750 (lane CT).
Lane M is 100 bp DNA ladder and the position of migration of 100 bp, 300 bp and 600 bp fragments are marked.
Fig 2
Fig 2. Neighbor-joining tree based on DNA sequence data for the ITS region of rDNA from 85 C. glabrata sensu stricto isolates.
The bootstrap frequencies (>50%) on the node branches are shown. The ITS haplotype (ITSH) arbitrarily assigned for each isolate is also shown in the right hand column.
See this image and copyright information in PMC

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