PROJECT SUMMARY Peritonitis is a common complication in patients with end stage renal disease on peritoneal dialysis (PD) and is associated with significant morbidity and mortality in this population. Diagnosing the etiology for peritonitis can be challenging with approximately 25% of peritonitis cases being culture-negative. Currently, conventional cell culture is the gold standard for diagnosing the etiology, but this technique is limited to the detection of culturable organisms. Culture-independent methods based on metagenomic sequencing are promising techniques to screen infection in biofluids. However, the specificity of metagenomic sequencing is limited by two factors. First, contamination with microbial DNA introduced during sample preparations leads to background noise that can be significant for samples with an inherent low biomass. Second, conventional metagenomic sequencing assays do not inform about the host’s response to infection. The overall objective of this application is to apply precision medicine approaches to monitor PD patients with culture-negative peritonitis and culture-positive peritonitis. We will develop and apply a metagenomic cell-free DNA sequencing assay that is insensitive to environmental contamination and that informs the host’s response to infection. This is achieved by sequencing of cell-free DNA in peritoneal fluid after bisulfite conversion of unmethylated cytosines to uracils. Bisulfite conversion will be performed directly on the biofluid prior to DNA isolation, thereby tagging any microbial and human cell-free DNA that is intrinsic to the sample. Any contaminating environmental DNA introduced in the sample in downstream steps will not be bisulfite converted, making it straightforward to bio-informatically identify and remove any contaminant sequences. In addition to making this assay robust against contamination, bisulfite conversion of the host DNA will enable quantification of the host injury response to infection through genome-wide profiling of methylation marks in cfDNA. In this study, we will recruit PD patients at the time of suspected peritonitis: 50 PD patients with culture-positive peritonitis and 25 PD patients with culture-negative peritonitis. We will also recruit 40 PD patients with no evidence of clinical peritonitis. In Aim 1, we will determine the utility of whole genome bisulfite sequencing for monitoring PD patients with suspected peritonitis. In Aim 2, we will investigate the host-pathogen response during culture-positive peritonitis and culture-negative peritonitis. Our study will lead to the development of a metagenomic assay that is insensitive to environmental microbial contamination and informs the host’s response to infection, which can be more broadly applicable to profiling of other low biomass specimens. In addition, our study will lead to new avenues for personalized assessment and management of suspected peritonitis which cause significant morbidity and mortality in PD patie...