Developing a high-throughput panel for rapid detection of pathogens causing infectious keratitis

PROJECT SUMMARY Infectious keratitis, also known as corneal infection, is frequently caused by bacteria, fungi, viruses, or parasites. Each pathogen needs a unique treatment approach. Hence, rapid determination of the underlying pathogen causing the disease is of utmost importance. However, current gold standard still includes culture of corneal samples, which not only can take days to identify the pathogen, but also still leads to false negative results in many cases. The delay in identifying the appropriate treatment strategy causes significant complications and unfavorable clinical outcomes, including blindness, which is why infectious keratitis is the 5th leading cause of vision impairment and blindness and the main cause of corneal blindness worldwide. Together, more rapid methods to determine pathogens are urgently needed, and would present a significant improvement in patient care. Here, we describe the development of a novel TaqMan-based assay, termed TLLPS (Thermal Lysis Low Input Pathogen Screen) that can rapidly distinguish between the most common pathogens causing infectious keratitis, with several advantages over other approaches: First, we designed our system to work with genomic DNA, which allows avoiding RNA-extraction, reverse transcription steps, and the significant challenge of heterogeneity emerging from isolating small amounts of heavily degraded RNA from the eye surface. Second, our assay allows using 1 µl of raw and un-processed biological fluids, which are submitted directly into a thermal lysis and genomic pre-amplification step, avoiding any purification steps, which would introduce variability and cause sample loss of the small amounts extracted from the ocular surface. Here we propose to expand and prospectively validate the assay. Further, we will test the use of ocular swabs for more rapid detection. With the completion of this proposal, we envision to have developed a clinically feasible, quick, and reliable way to identify the most common pathogens causing keratitis, and significantly improve patient outcomes through rapid identification of the correct treatment strategies. 1