Mixture Of Yeast Cells Research Articles

Multiplex Tandem PCR: a Novel Platform for Rapid Detection and Identification of Fungal Pathogens from Blood Culture Specimens

We describe the first development and evaluation of a rapid multiplex tandem PCR (MT-PCR) assay for the detection and identification of fungi directly from blood culture specimens that have been flagged as positive. The assay uses a short-cycle multiplex amplification, followed by 12 simultaneous PCRs which target the fungal internal transcribed spacer 1 (ITS1) and ITS2 region, elongation factor 1-alpha (EF1-alpha), and beta-tubulin genes to identify 11 fungal pathogens: Candida albicans, Candida dubliniensis, Candida glabrata, Candida guilliermondii, Candida krusei, Candida parapsilosis complex, Candida tropicalis, Cryptococcus neoformans complex, Fusarium solani, Fusarium species, and Scedosporium prolificans. The presence or absence of a fungal target was confirmed by melting curve analysis. Identification by MT-PCR correlated with culture-based identification for 44 (100%) patients. No cross-reactivity was detected in 200 blood culture specimens that contained bacteria or in 30 blood cultures without microorganisms. Fungi were correctly identified in five specimens with bacterial coinfection and in blood culture samples that were seeded with a mixture of yeast cells. The MT-PCR assay was able to provide rapid (<2 h), sensitive, and specific simultaneous detection and identification of fungal pathogens directly from blood culture specimens.

Antigenicity of Cell Wall Mannans of Candida albicans and Candida stellatoidea Cultured at High Temperatures in BACTEC Medium

The study of the antigenicity of pathogenic Candida albicans and Candida stellatoidea cells grown in BACTEC fungal medium (BFM) is useful for clinical analysis so as accurately to diagnose candidiasis. When C. albicans NIH A-207 was grown in BFM and fetal bovine serum-added BFM at the high temperatures of 36 and 40 degrees C, the cell density increased, with a mixture of yeast cells, pseudohyphae, and hyphae and with full hyphal development in the cultures compared with cultivation (mostly cells in yeast form) at 27 degrees C in both media. The mannans produced when cells were grown at these high temperatures were less reactive by enzyme-linked immunosorbent assay with factor sera 4, 5, and 6 in the commercially available kit 'Candida Check' than were the mannans obtained following growth at 27 degrees C. Based on 1H-nuclear magnetic resonance analysis, the mannans from cells grown at high temperatures had lost a phosphate group and a beta-1,2-linked mannopyranose unit, and had increased the number of non-reducing terminal alpha-1,3-linked mannopyranose units. We obtained similar results for mannans produced by C. albicans J-1012, C. albicans NIH B-792, C. albicans JCM 9061, C. stellatoidea ATCC 20408, and C. stellatoidea ATCC 36232 strains cultivated in BFM at 36 degrees C. These results suggest that both C. albicans and C. stellatoidea cells cultured at high temperatures, irrespective of the medium and shape of the cells, alter their antigenicity and chemical structure of cell wall mannans.