Simultaneous Detection and Differentiation of Medically Important Fungal Pathogens - Candida, Aspergillus, and Cryptococcus spp. - on ICEPlex System

K. Madanahally Divakar, J. Riley, L. Kong

Abstract: 

Background: In recent years, opportunistic fungal infections are in rise. Majority of these fungal pathogens belong to the genera Candida, Aspergillus, and Cryptococcus. Invasive infection by Aspergillus spp is the prominent cause of morbidity and mortality. Cryptococcus causes cryptococcosis and meningitides. Candida causes candidaemia, which has become fourth main cause of blood stream infections in USA. Current technologies are limited to detection of only few microbial pathogens in a single reaction. Here, we developed a multiplexed assay that can simultaneously detect and discriminate fungal pathogens, Candida albicans, C. glabrata, C. kruisi, C. parapsilosis, C. tropicalis, Cryptococcus neoformans, Aspergillus flavus, and A. fumigatus in a single reaction on the ICEPlex System. Material and Methods: A unique region of the genome for each target in the assay was identified by bioinformatics analyses using BLAST program from the NCBI. ClustalW analyses were carried out to identify conserved regions, and primers were designed using Primer3 program. In silico primer-primer interaction analyses were determined using ThermoBlast . Fungal and other microbial genomic DNAs used in the study were obtained from ATCC whereas human genomic DNA was obtained from Roche Applied Science. Sensitivity control was custom synthesized by Integrated DNA Technologies. Calibrator control is a product from PrimeraDx, Inc. Results:Our results demonstrated simultaneous detection and discrimination of fungal pathogens, C. albicans, C. glabrata, C. kruisi, C. parapsilosis, C. tropicalis, Cryptococcus neoformans, Aspergillus flavus, and A. fumigatus in a single reaction on ICEPlexer System. Analytical sensitivity studies indicated a detection limit of a single fungal cell per milliliter of whole blood, and analytical specificity studies indicated no cross-reactivity with human genomic DNA and other microbial DNA. The assay demonstrated high concordance with the gold standard method. Conclusion:ICEPlex Fungal Assay shows rapid and accurate detection of majority fungal pathogens in a single reaction. Simultaneous detection reduces the cost and facilitates physicians to initiate appropriate treatment in a timely fashion. *ICEPlex and the fungal panel are for research use only. Not for clinical diagnostic use.
2013

abstract No: 

2350

Full conference title: 

American Society for Microbiology General Meeting
    • ASM 113th (2013)