Search

Summary: Mechanisms of infection and pathogenesis have predominantly been studied based on differential gene or protein expression. Less is known about posttranslational modifications, which are essential for protein functional diversity. We applied an innovative glycoproteomics method to study the systemic proteome-wide glycosylation in response to infection. The protein site-specific glycosylation was characterized in plasma derived from well-defined controls and patients. We found 3862 unique features, of which we identified 463 distinct intact glycopeptides, that could be mapped to more than 30 different proteins. Statistical analyses were used to derive a glycopeptide signature that enabled significant differentiation between patients with a bacterial or viral infection. Furthermore, supported by a machine learning algorithm, we demonstrated the ability to identify the causative pathogens based on the distinctive host blood plasma glycopeptide signatures. These results illustrate that glycoproteomics holds enormous potential as an innovative approach to improve the interpretation of relevant biological changes in response to infection.

Background: The complement system is a central component of the innate immune system. Constitutive biosynthesis of complement proteins is essential for homeostasis. Dysregulation as a consequence of genetic or environmental cues can lead to inflammatory syndromes or increased susceptibility to infection. However, very little is known about steady state levels in children or its kinetics during infection. Methods: With a newly developed multiplex mass spectrometry-based method we analyzed the levels of 32 complement proteins in healthy individuals and in a group of pediatric patients infected with bacterial or viral pathogens. Findings: In plasma from young infants we found reduced levels of C4BP, ficolin-3, factor B, classical pathway components C1QA, C1QB, C1QC, C1R, and terminal pathway components C5, C8, C9, as compared to healthy adults; whereas the majority of complement regulating (inhibitory) proteins reach adult levels at very young age. Both viral and bacterial infections in children generally lead to a slight overall increase in complement levels, with some exceptions. The kinetics of complement levels during invasive bacterial infections only showed minor changes, except for a significant increase and decrease of CRP and clusterin, respectively. Interpretation: The combination of lower levels of activating and higher levels of regulating complement proteins, would potentially raise the threshold of activation, which might lead to suppressed complement activation in the first phase of life. There is hardly any measurable complement consumption during bacterial or viral infection. Altogether, expression of the complement proteins appears surprisingly stable, which suggests that the system is continuously replenished. Fund: European Union's Horizon 2020, project PERFORM, grant agreement No. 668303. Keywords: Targeted mass spectrometry, Multiple reaction monitoring (MRM), Complement system, Infectious disease, C-reactive protein (CRP), Clusterin

Summary: Background: Emergency admissions for infection often lack initial diagnostic certainty. COVID-19 has highlighted a need for novel diagnostic approaches to indicate likelihood of viral infection in a pandemic setting. We aimed to derive and validate a blood transcriptional signature to detect viral infections, including COVID-19, among adults with suspected infection who presented to the emergency department. Methods: Individuals (aged ≥18 years) presenting with suspected infection to an emergency department at a major teaching hospital in the UK were prospectively recruited as part of the Bioresource in Adult Infectious Diseases (BioAID) discovery cohort. Whole-blood RNA sequencing was done on samples from participants with subsequently confirmed viral, bacterial, or no infection diagnoses. Differentially expressed host genes that met additional filtering criteria were subjected to feature selection to derive the most parsimonious discriminating signature. We validated the signature via RT-qPCR in a prospective validation cohort of participants who presented to an emergency department with undifferentiated fever, and a second case-control validation cohort of emergency department participants with PCR-positive COVID-19 or bacterial infection. We assessed signature performance by calculating the area under receiver operating characteristic curves (AUROCs), sensitivities, and specificities. Findings: A three-gene transcript signature, comprising HERC6, IGF1R, and NAGK, was derived from the discovery cohort of 56 participants with bacterial infections and 27 with viral infections. In the validation cohort of 200 participants, the signature differentiated bacterial from viral infections with an AUROC of 0·976 (95% CI 0·919−1·000), sensitivity of 97·3% (85·8−99·9), and specificity of 100% (63·1−100). The AUROC for C-reactive protein (CRP) was 0·833 (0·694−0·944) and for leukocyte count was 0·938 (0·840−0·986). The signature achieved higher net benefit in decision curve analysis than either CRP or leukocyte count for discriminating viral infections from all other infections. In the second validation analysis, which included SARS-CoV-2-positive participants, the signature discriminated 35 bacterial infections from 34 SARS-CoV-2-positive COVID-19 infections with AUROC of 0·953 (0·893−0·992), sensitivity 88·6%, and specificity of 94·1%. Interpretation: This novel three-gene signature discriminates viral infections, including COVID-19, from other emergency infection presentations in adults, outperforming both leukocyte count and CRP, thus potentially providing substantial clinical utility in managing acute presentations with infection. Funding: National Institute for Health Research, Medical Research Council, Wellcome Trust, and EU-FP7.