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Background Children affected by infectious diseases may not always have a detectable infectious etiology. Diagnostic uncertainty can lead to prolonged hospitalizations, inappropriately broad or extended courses of antibiotics, invasive diagnostic procedures, and difficulty predicting the clinical course and outcome. Cell-free plasma next-generation sequencing (cfNGS) can identify viral, bacterial, and fungal infections by detecting pathogen DNA in peripheral blood. This testing modality offers the ability to test for many organisms at once in a shotgun metagenomic approach with a rapid turnaround time. We sought to compare the results of cfNGS to conventional diagnostic test results and describe the impact of cfNGS on clinical care in a diverse pediatric population at a large academic children’s hospital. Methods We performed a retrospective chart review of hospitalized subjects at a tertiary pediatric hospital to determine the diagnostic yield of cfNGS and its impact on clinical care. Results We describe the clinical application of results from 142 cfNGS tests in the management of 110 subjects over an 8-month study period. In comparison to conventional testing as a reference standard, cfNGS was found to have a positive percent agreement of 89.6% and negative percent agreement of 52.3%. Furthermore, 32.4% of cfNGS results were directly applied to make a clinical change in management. Conclusions We demonstrate the clinically utility of cfNGS in the management of acutely ill children. Future studies, both retrospective and prospective, are needed to clarify the optimal indications for testing.

Secretory immunoglobulins (SIg) are a first line of mucosal defense by the host. They are secreted into the gut lumen via the polymeric immunoglobulin receptor (pIgR) where they bind to antigen and are transported back across the FAE via M cells. Noroviruses are highly prevalent, enteric pathogens that cause significant morbidity, mortality and economic losses worldwide. Murine norovirus (MNV) exploits microfold (M) cells to cross the lymphoid follicle-associated epithelium (FAE) and infect the underlying population of immune cells. However, whether natural, innate SIg can protect against norovirus infection remains unknown. To investigate the role of natural SIg during murine norovirus pathogenesis, we used pIgR-deficient animals, which lack SIg in the intestinal lumen. Contrary to other enteric pathogens, acute MNV replication was significantly reduced in the gastrointestinal tract of pIgR-deficient animals compared to controls, despite increased numbers of dendritic cells, macrophages, and B cells in the Peyers patch, established MNV target cell types. Also, natural SIg did not alter MNV FAE binding or FAE crossing into the lymphoid follicle. Instead, further analysis revealed enhanced baseline levels of the antiviral molecules interferon gamma (IFN-gamma) and inducible nitric oxide synthase (iNOS) in the small intestine of naive pIgR-deficient animals compared to controls. Removing the microbiota equalized IFN-gamma and iNOS transcript levels as well as MNV viral loads in germ-free pIgR KO mice compared to germ-free controls. These data are consistent with a model whereby SIg sensing reduces pro-inflammatory, antiviral molecules, which facilitates intestinal homeostasis but thereby promotes MNV infection. In conclusion, these findings demonstrate that natural SIg are not protective during norovirus infection in mice and represent another example of indirect modulation of enteric virus pathogenesis by the microbiota.