Advancing Diagnostics of Infectious Uveitis with MetagenomicAnalysis

项目总结

PROJECT SUMMARY Uveitis is a sight-threatening intraocular inflammation that affects young working-age individuals, resulting in significant socioeconomic impacts. It is most often caused by infections or immune-mediated diseases. While herpesviruses and Toxoplasma gondii are the leading causes of infectious uveitis, many other infectious agents can be also involved, including viruses, bacteria, fungi and parasites. Clinical diagnosis is often complicated by overlapping findings upon examination among cases caused by different infectious etiologies, and also with observations made for those caused by autoimmune diseases and trauma. Because of that, complementary laboratory tests are essential for the etiological diagnosis and proper clinical management. The laboratory workup relies on multiple immunological tests and pathogen-targeted singleplex polymerase chain reaction (PCR) assays. This approach increases significantly the time to diagnosis, is often prohibitive due to the limited amount of intraocular fluid that can be safely obtained, and in many cases is unrevealing. As a result, patients can be treated for weeks or months using one-size-fits-all therapeutic trials that are not tailored for an individual and do not work for everyone. The use of metagenomic next-generation sequencing (mNGS) for unbiased detection of every pathogen in a clinical specimen is an emerging diagnostic modality that has the potential to significantly improve diagnostic yields in infectious uveitis. To date, there is a lack of an approach in which the appropriate steps for analytical and clinical validations are thoroughly performed for the unique intraocular fluid matrices tested from uveitic eyes. The studies proposed here are intended to fill this void and take on the challenge of developing and systematically validating an unbiased mNGS test that can be translated into clinical application for uveitis diagnosis and identification of new etiologies that currently escape diagnosis. First, we will thoroughly establish the analytical performance characteristics of a mNGS test for detection of uveitis pathogens from vitreous specimens. This will be done by using control vitreous containing varying levels of host DNA background spiked with a reference pathogen panel that will be used to model the effects of important determinants of analytical performance. Then, we will challenge this optimized mNGS assay with patient specimens for determination of relevant clinical performance characteristics to fully validate this test for clinical use. Using a combination of specimens stored in our biorepository and additional ones that will be collected prospectively for a blinded challenge study, we will determine the diagnostic accuracy of the assay using different sample types (e.g., aqueous vs. vitreous), its quantification capabilities, and will define clinical thresholds for reporting of detected pathogens. This proposal will result in the development of a systematically validated and innovative all-in-one diagnostic assay for infectious uveitis that is expected to substantially improve diagnostic yields and help to illuminate novel infectious uveitis etiologies in cases that remain undiagnosed, with great potential to for significantly changing the management of patients suffering from this debilitating disease.