Rapid Identification of Fungal Pathogens in Blood from Hospitalized Patients by Semi-automated Reverse Line Blot Assay

A. Jain, K. Hennessey, S. Furniss, S. Shekar, D. Snydman, S. Doron, N. Krueger, A. Levin


Background: The incidence of Invasive Fungal Disease (IFD) has increased significantly in recent decades with ~7.2 new episodes of Candidemia and ~2.2 episodes of Aspergillosis reported per 100,000 inhabitants each year. The healthcare costs are estimated at $30,000/case for Candidemia and $60,000/patient for Aspergillosis. Current diagnostic methods are inadequate due to slow turn around time and lack of comprehensive species detection. New molecular diagnostic approaches dramatically reduce the time to detection of IFD compared to conventional culture methods, potentially allowing better patient outcomes. Methods: We have developed a reverse line blot (RLB) assay for rapid and sensitive detection of fungal pathogens. The assay utilizes a flow-through CodaXcel™ device, allowing simultaneous hybridization of multiple PCR amplicons to 30+ species-specific probes immobilized on a membrane. For testing of clinical blood samples, DNA was extracted from 29 blinded blood samples obtained from 23 hospitalized patients suspected of IFD. For spiking studies, total DNA was extracted from yeast culture (Candida, and Cryptococcus) or spore suspension (Aspergillus, Rhizopus, Mucor, Absidia and Fusarium) and whole blood was spiked with fungal culture. DNA was amplified with pan-fungal primers targeting 25-28S fungal rDNA genes and processed with the RLB assay. Results: In blood spiking studies, the assay was able to distinguish 19 fungal species including 9 Candida sp., 5 Aspergillus sp., Cryptococcus sp., Fusarium solani, Rhizopus oryzae, Mucor indicus and Absidia corymbifera. Detection limits for these species were 1-10 CFU/ml of blood. For clinical sample testing, RLB results correlated 100% with results obtained using traditional culture methods. In patient samples, the RLB assay detected C. albicans (n=4), C. glabrata (n=2), C. tropicalis (n=1), C. glabrata (n=2), C. parapsilosis (n=4) and C. krusei (n=2), all with 100% concordance to microbiological results. Overall, 15 Candida sp. were identified in 29 clinical samples with 100% concordance. Conclusions: The rapid and sensitive RLB assay detects IFD-causing species from blood in <8 hrs with very high concordance to microbiology culture. This assay holds promise as a method to enable faster diagnosis of IFD, resulting in improved patient outcomes


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Full conference title: 

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