Invasive fungal infection is a matter of increasing clinical concern in immunocompromised patients. Aspergillosis and Candidiasis cause the bulk of fungal infections, but a large number of other fungi are also implicated in often-fatal infections. The existing technologies in clinical laboratories for detection and diagnosis of fungal infection are both time consuming and very insensitive. Nearly 50% of invasive fungal infections are missed with current clinical testing protocols, with devastating consequences for patient survival. In the current application, Immunetics proposes to adapt its Reverse Line Blot (RLB) Assay technology to perform molecular diagnosis of fungal infection. The strategy employed by the RLB assay is amplification of fungal or bacterial DNA sequences using primers that are pan-specific, followed by hybridization of the amplified DNA to species- specific probes. 30-50 probes can be simultaneously hybridized to each amplified clinical sample, and Immunetics'CodaXcel blotting system is used to analyze up to 8 clinical samples simultaneously. We propose to develop a series of pan-fungal amplification primers using either PCR or isothermal loop mediated amplification (LAMP) techniques, and design a series of species-specific primers for detection of multiple Aspergillus and Candida species as well as other clinically relevant fungal species. There are several advantages to the RLB assay system that make it ideal for use in fungal diagnostics: 1) with properly designed amplification primers and detection probes, the assay is extremely sensitive (1-2 CFU/ml are routinely detected by the bacterial RLB assay) and does not require any culturing of fungi (meaning that uncultivatable fungi are still detectable in the assay), 2) the assay is much quicker than diagnostic methods dependent on culture, generating a specific molecular species ID within 8 hours, 3) the assay is extremely flexible - probes can be designed for any species of interest, and panels of probes for any specific applications are easy to design and validate, 4) technical failures in the assay are rare but are easily detectable due to the existence of internal positive controls for each step of the process, generating an extremely robust assay, and 5) the assay can also be adapted for detection of other genes of interest, including antibiotic and antimicrobial resistance genes. Clinically-relevant single base polymorphisms (SNPs) are also readily detectable.
The proposed project is aimed at developing and validating a more rapid and sensitive DNA-based test for fungi which cause serious infections in humans. As fungal infections are becoming more and more frequent in seriously ill patients, there is a critical need for better diagnostic tools to help clinicians diagnose and treat these infections. The test to be developed in this project will be able to identify a wide range of fungal pathogens with same day turnaround, a singificant improvement over currently available test methods.