A Real Time PCR strategy for the detection and quantification of Candida albicans in human blood

Abstract

Candidemia is a significant cause of bloodstream infections (BSI) in nosocomial settings. The identification of species can potentially improve the quality of care and decrease human mortality. Quantitative PCR (qPCR) was evaluated for Candida albicans detection using culture suspensions containing C. albicans , spiked human blood, the cloned qPCR target fragment (ITS2 region) and the results of these assays were compared. The assays showed a good detection limit: C. albicans DNA extracted from yeast (sensitivity 0.2 CFU/µL), spiked human blood (sensitivity 10 CFU/mL), and cloned fragment of ITS2 region (sensitivity 20 target copies/μL). The efficiency of ITS2 fragment-qPCR ranged from 89.67 to 97.07, and the linearity (R2) of the standard curve ranged from 0.992 to 0.999. The results showed that this ITS2-qPCR has a great potential as a molecular prototype model for the development of a test to be applied in clinical practice, greatly reducing the time of candidemia diagnosis, which is extremely important in this clinical setting.

Figure 1. Technical performance and detection range of the Candida albicans qPCR: A) Amplification curves of ten-fold dilutions of cloned targets ranging from 2 x 109 to 2 x 101 copies/μL; B) Amplification curves of ten-fold dilutions of cultures, ranging from 105 to 10-1 CFU/μL; C) Amplification curves of ten-fold dilutions of spiked blood, ranging from 107 to 101 and 5 CFU/mL of blood; D) Dissociation curves for the amplifications observed in A, B and C, where the same Melting temperature of 81.06 ± 0.19 ºC was observed. The linearity R2 value was 0.996.

Figure 2. Specificity: A) qPCR results with 5 and 0.05 ng of C. albicans . DNA samples from various microorganisms were tested: C. glabrata, C. parapsilosis, C. tropicalis, C. krusei, C. dubliniensis, Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus agalactiae, Escherichia coli, Rhodotorula sp, Cryptococcus neoformans, Histoplasma capsulatum and Fusarium sp .; B) Dissociation curves for the amplifications observed in A (melting temperature of 80.86 ºC).