Validation of a simplex PCR assay enabling reliable identification of clinically relevant Candida species

Fungal bloodstream infections (BSI) may be serious and are associated with drastic rise in mortality and health care costs. Candida spp. are the predominant etiological agent of fungal sepsis. The prompt and species-level identification of Candida may influence patient outcome and survival. The aim of this study was to develop and evaluate the CanTub-simplex PCR assay coupled with T_m calling and subsequent high resolution melting (HRM) analysis to barcode seven clinically relevant Candida species.

Efficiency, coefficient of correlation and the limit of reliable detection were estimated on purified Candida EDTA-whole blood (WB) reference panels seeded with Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, Candida guilliermondii, Candida dubliniensis cells in a 6-log range. Discriminatory power was measured on EDTA-WB clinical panels on three different PCR platforms; LightCycler®96, LightCycler® Nano, LightCycler® 2.0. Inter- and intra assay consistencies were also calculated.

The limit of reliable detection proved to be 0.2-2 genomic equivalent and the method was reliable on broad concentration ranges (10⁶-10 CFU) providing distinctive melting peaks and characteristic HRM curves. The diagnostic accuracy of the discrimination proved to be the best on Roche LightCycler®2.0 platform. Repeatability was tested and proved to be % C.V.: 0.14 ± 0.06 on reference- and % C.V.: 0.14 ± 0.02 on clinical-plates accounting for a very high accuracy. Reproducibility was % C.V.: 0.11 between reference- and % C.V.: 0.12between clinical-panels which is highly acceptable.

Our assay demonstrates recent advances on T_m calling and HRM analysis for the molecular identification of relevant Candida species. This unique, simplex PCR assay may be capable to outperform conventional phenotypic methods by reducing time and providing accurate and reliable results directly from blood (2 h) or from whole blood culture bottles (12-24 h).