A Rapid and Sensitive (1→3)-β-D-Glucan Microfluidic Assay for Early Detection of Invasive Aspergillosis Infections in a Murine Model

R. Kapoor1 , N. Wiederhold2 , L. K. Najvar2 , T. F. Patterson2 , W. P. Chang1

Author address: 

1Wako Life Sci., Mountain Vew, CA, 2UT Hlth.Sci. Ctr., San Antonio, TX

Abstract: 

Background: Incidence of invasive aspergillosis (IA) is rising in recent decades with poor morbidity and mortality in neutropenic patients. Early detection and treatment are key to improved clinical outcomes. Timely diagnosis is suboptimal using existing tools. A fungal cell wall component, (1→3)-β-D-glucan (BDG), has been used as a surrogate marker for fungal infection. We report here the results from an automated liquid phase binding assay for the early detection of serum BDG in infected mice.Method: Immunosuppressed ICR mice were inoculated with 105 inhaled A. fumigatus conidia (Sheppard et al Antimicrob Agents Chemother 2004; 48: 1908). Serum was collected daily for 7 days post infection (5 mice for each day); kidney fungal burden and BDG levels were measured. We measured BDG level by a liquid phase binding assay (Kawabata et al Electrophoresis, 2008; 29:1399) using microfluidic electrophoresis. Here, BDG is recognized by a recombinant protein (BGRP); detection is achieved through BGRP-conjugated fluorescent dye. The BDG level is correlated to peak area of the immunocomplex (BDG-BGRP).Results: The median BDG level (10.3, integrated peak area of immunocomplex) of 5 uninfected mice is used as a cutoff. All 5 infected animals from day 1 (median = 34.8; range = 32.0 - 43.7) were positively identified. Mice from days 2-7 were also correctly identified as being infected (median = 42.9; range = 17.6 - 204.0). However, results from a commercial kit (Fungitell®, cutoff = 80pg/mL) failed to ID 2 out of 5 mice for day 1 and 1 out of 5 for day 2. Samples from later time points (days 3 - 7) remained positive in infected mice as judged by results from both assays (microfluidics and Fungitell®).Conclusion: In this study, our assay detects serum BDG as early as day 1 in the course of IA in infected mice. This assay has the potential of being a useful tool for detecting IA in at risk patient population, with several advantages over existing assays, including a shorter assay time (~ 2 min), low reagent consumption, robotic liquid and sample handling, and automated runs with a single or multiple samples. Future studies will include the assessment of the performance of this assay using a larger pool of human specimens.
2016

abstract No: 

MONDAY-228

Full conference title: 

ASM Microbe 2016
    • ASM microbe 1st (2016)