Multiplex tandem PCR: a novel platform for rapid detection and identification of fungal pathogens from blood culture specimens

Abstract

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.

FIG. 1.

Cycling curve produced from blood samples seeded with serial dilutions of C. albicans, showing the detection limit of MT-PCR (CFU/ml). The positive control (+ve) indicates that the assay was not inhibited.

FIG. 2.

Melting curves generated from a mixture of 49 different DNA templates (Table 1) illustrating the melting peaks obtained from the 12 MT-PCR targets: C. albicans (Ca), C. dubliniensis (Cd), C. glabrata (Cg), C. guilliermondii (Cgu), C. krusei (Ck), C. parapsilosis (Cp), C. tropicalis (Ct), C. neoformans (Cn), F. solani (Fs), Fusarium sp. (Fsp), S. prolificans (Sp), and a positive control (+ve). deg, degrees; dF/dT, derivative of fluorescence with respect to temperature.

FIG. 3.

Melting curves generated from blood cultures seeded with C. albicans and C. glabrata (a) and with C. parapsilosis and C. krusei (b). The melting temperatures determined were compared to the expected melting temperatures (Table 2) by automated analysis software to determine the presence or absence of an organism. +ve, positive control; deg, degrees; dF/dT, derivative of fluorescence with respect to temperature.