Diagnosis of infection in patients with sepsis takes days via culture and appropriate treatment of pathogens are delayed awaiting results. A faster diagnosis of the pathogen and resistance via RNA sequencing informed PCR will improve outcomes. We hypothesize that we can use RNA sequencing from patients with sepsis to identify novel targets for future nucleic acid-based tests. This cohort study of 46 sepsis patients admitted to the ICU with samples taken on days 0, 1, 3, and 7 with follow up through the hospital stay during 2021–2022. All patients had RNA sequencing, depth of > 100 million reads, conducted on days in the ICU from a single center medical intensive care unit. Patients were admitted to the ICU with a diagnosis of sepsis. Patients or surrogates were approached consecutively and those who consented were enrolled. RNA sequencing of peripheral blood was performed to identify RNA targets from pathogens. RNA sequencing data that did not map to the human genome was then aligned to resistance genes and pathogen genomes and used to design novel PCR tests. These tests were correlated with culture diagnosis and clinical outcomes. Forty-six patients (mean age 62.2, 48% female) were enrolled and samples from 87 time points were collected. These samples resulted in 8.6 billion RNA sequencing reads to identify pathogen RNA. PCR target discovery for this study focused on positive blood cultures (n = 40) due Escherichia coli (5), Staphylococcus aureus (6), and Pseudomonas aeruginosa (3) as well as identification of resistance genes. From these RNA sequencing reads, 40 targets were defined and tested by quantitative PCR. In cohort of patients (n = 9) the some of the proposed PCRs identified all cases of positive blood cultures (Pseudomonas aeruginosa and Staphylococcus aureus, Escherichia coli had no positive blood cultures in this cohort). RNA sequencing from patients with sepsis can identify RNA from pathogens causing the infection. This can be used to design PCR primers that can identify patients with positive blood cultures. Translation of these primers to clinical microbiology machines is the next step and will allow the diagnosis pathogen and resistance faster than blood culture.
