Metagenomic next-generation sequencing (mNGS) for pan-pathogen detection has been successfully tested in proof-of-concept case studies in patients with acute illness of unknown etiology but to date has been largely confined to research settings. Here, we developed and validated a clinical mNGS assay for diagnosis of infectious causes of meningitis and encephalitis from cerebrospinal fluid (CSF) in a licensed microbiology laboratory. A customized bioinformatics pipeline, SURPI+, was developed to rapidly analyze mNGS data, generate an automated summary of detected pathogens, and provide a graphical user interface for evaluating and interpreting results. We established quality metrics, threshold values, and limits of detection of 0.2-313 genomic copies or colony forming units per milliliter for each representative organism type. Gross hemolysis and excess host nucleic acid reduced assay sensitivity; however, spiked phages used as internal controls were reliable indicators of sensitivity loss. Diagnostic test accuracy was evaluated by blinded mNGS testing of 95 patient samples, revealing 73% sensitivity and 99% specificity compared to original clinical test results, and 81% positive percent agreement and 99% negative percent agreement after discrepancy analysis. Subsequent mNGS challenge testing of 20 positive CSF samples prospectively collected from a cohort of pediatric patients hospitalized with meningitis, encephalitis, and/or myelitis showed 92% sensitivity and 96% specificity relative to conventional microbiological testing of CSF in identifying the causative pathogen. These results demonstrate the analytic performance of a laboratory-validated mNGS assay for panpathogen detection, to be used clinically for diagnosis of neurological infections from CSF.

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6Metagenomic next-generation sequencing (mNGS) for pan-pathogen detection has been 3 7 successfully tested in proof-of-concept case studies in patients with acute illness of unknown 3 8 etiology, but to date has been largely confined to research settings. Here we developed and 3 9 validated an mNGS assay for diagnosis of infectious causes of meningitis and encephalitis from 4 0 cerebrospinal fluid (CSF) in a licensed clinical laboratory. A clinical bioinformatics pipeline, 4 1 SURPI+, was developed to rapidly analyze mNGS data, automatically report detected 4 2 pathogens, and provide a graphical user interface for evaluating and interpreting results. We 4 3 established quality metrics, threshold values, and limits of detection of between 0.16 -313 4 4 genomic copies or colony forming units per milliliter for each representative organism type. 4 5 Gross hemolysis and excess host nucleic acid reduced assay sensitivity; however, a spiked 4 6 phage used as an internal control was a reliable indicator of sensitivity loss. Diagnostic test 4 7 accuracy was evaluated by blinded mNGS testing of 95 patient samples, revealing 73% 4 8 sensitivity and 99% specificity compared to original clinical test results, with 81% positive 4 9

A number of "suicide genes" have been developed as safety switches for gene therapy vectors or as potential inducible cytotoxic agents for hyperproliferative disorders, such as cancer or restenosis. However, most of these approaches have relied on foreign proteins, such as HSV thymidine kinase, that primarily target rapidly dividing cells. In contrast, novel artificial death switches based on chemical inducers of dimerization (CIDs) and endogenous proapoptotic molecules function efficiently in both dividing and nondividing cells. In this approach, lipid-permeable, nontoxic CIDs are used to conditionally cross-link target proteins that are fused to CID-binding domains (CBDs), thus activating signaling cascades leading to apoptosis. In previous reports, CID-regulated Fas and caspases 1, 3, 8, and 9 were described. Since the maximum efficacy of these artificial death switches requires low basal and high specific activity, we have optimized these death switches for three parameters: (1) extent of oligomerization, (2) spacing between CBDs and target proteins, and (3) intracellular localization. We describe improved conditional Fas and caspase 1, 3, 8, and 9 alleles that function at subnanomolar levels of the CID AP1903 to trigger apoptosis. Further, we demonstrate for the first time that oligomerization of the death effector domain of the Fas-associated protein, FADD, is sufficient to trigger apoptosis, suggesting that the primary function of FADD, like that of Apaf-1, is oligomerization of associated caspases. Finally, we demonstrate that nuclear-targeted caspases 1, 3, and 8 can trigger apoptosis efficiently, implying that the cleavage of nuclear targets is sufficient for apoptosis.

A diagnosis of brucellosis can be difficult because routine culture and serological methods exhibit variable sensitivity and specificity. We present the use of a metagenomic next- generation sequencing assay to diagnose a case of neurobrucellosis from cerebrospinal fluid, resulting in the institution of appropriate antibiotic treatment and a favorable clinical outcome.