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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.

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