#16 Chasing the Small Tail of Infectious Diseases: The Rapid, Non-invasive, Culture-independent Detection Of Rat-Bite Fever With A Plasma-based Microbial Cell-free DNA Next-generation Sequencing Test (The Karius Test)

Abstract Background Rat-bite fever (RBF) is a rare, systemic illness caused by infection with Streptobacillus moniliformis. RBF has a case-fatality risk of 7%-10% among untreated patients. Over 200 cases of RBF have been documented in the United States, but this is likely a significant under-representation because RBF is not a reportable disease. The diagnosis of these infections can be limited by: (1) Streptobacillus moniliformis fastidious nature and difficulty to culture; (2) the nonspecific manifestations of the infections and clinical overlap with a broad differential diagnosis; and (3) the unreliability of rat exposure history. We demonstrate use of unbiased microbial cell free DNA (mcfDNA) next-generation sequencing (NGS) to overcome these diagnostic limitations. Method The Karius Test (KT) was developed and validated in Karius’s CLIA certified/CAP accredited lab (Redwood City, CA) to detect and interpret mcfDNA in plasma. After mcfDNA is extracted and NGS performed, mcfDNA sequences are aligned to a curated database of > 1000 organisms. McfDNA from organisms observed above background at statistical significance are reported and quantified in molecules/µL (MPM). KT detections of Streptobacillus moniliformis were reviewed from January 2017 - June 2021; clinical information was obtained with test requisition or consultation upon result reporting. Results KT detected 7 cases of Streptobacillus moniliformis at an average of 673 MPM (35-3078; SD 1185) with an average turnaround time of 28.5 hours (SD 8.4) from sample receipt from 6 unique institutions (Table 1). Six patients were children; all were immunocompetent. Fever and rash were the most common presentation in the majority of the cases. Five of seven patients had arthritis or osteomyelitis while the remaining two patients had arthralgia. A history of rat exposure was elicited in all cases (some after microbiological diagnosis). In all patients blood cultures were negative and mcfDNA NGS was the only means of microbiological diagnosis. Conclusion Unbiased plasma-based mcfDNA NGS provides a rapid, non-invasive test to diagnose diverse clinical infections by Streptobacillus moniliformis. These cases highlight the potential of the KT to effectively identify infections caused by fastidious/unculturable pathogens with non-specific clinical manifestations and broad differential diagnoses.