Discovery and validation of plasma-based protein biomarkers for the aetiological distinction of bacterial and non-bacterial febrile infections in African children

Background: In many low-resource settings, the clinical management of children with febrile infections is hindered by poor access to diagnostic tools to determine whether the cause of an infection is bacterial, viral or parasitic. As a result, many clinicians resort to the default prescription of antibiotics as a safety precaution, contributing to the alarming spread of antimicrobial resistance. Commonly used biomarkers for the identification of bacterial sepsis such as CRP lack aetiological specificity and are frequently elevated by non-bacterial infections including malaria. We set out to discover and validate new biomarkers for the characterization of the microbial aetiology of febrile acute infections in Kenyan children. Methods: We recruited a discovery cohort comprising of children who had been admitted to hospital with a variety of severe acute infections. Diagnostic identification of viral infections was done using a 15-target virus PCR panel, bacterial infections were identified using blood culture while malaria infections were identified by microscopy. Using mass spectrometry analysis, we identified a set of 76 plasma proteins whose abundance varied significantly by the microbial aetiology of infection and used machine learning to generate a shortlist of candidate biomarkers that had the highest diagnostic performance in distinguishing aetiologies. To validate these candidate biomarkers, we recruited a separate validation cohort where the plasma levels of the shortlisted biomarkers were assayed among children with different infectious aetiologies using a custom protein microarray. Results: In the discovery study, six candidate biomarkers whose plasma abundance was significantly different in children with bacterial and viral infections were shortlisted by random forest for cross-cohort validation (AGT, HRG, LBP, PON1, SERPINA1, SERPINA3). In the validation study, we found that of the six biomarkers, only AGT compared favourably to CRP and identified febrile bacterial infections with a sensitivity of 72.4% (95% CI 48.4% - 83.6%) compared to CRP which distinguished febrile bacterial infections with a sensitivity of 69.5% (30.8% - 88.2%). Plasma AGT was superior to CRP in distinguishing children with febrile bacterial infections from those with febrile malaria episodes, with a sensitivity of 72.5% (40% - 84.6%) for AGT and 26% (15% - 32.8%) for CRP. Conclusions: We report the discovery of AGT, as a sensitive plasma biomarker for the identification of febrile bacterial infections among African children living in a malaria-endemic setting. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was funded by the Wellcome Trust ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Scientific Ethics Review Unit of the Kenya Medical Research Institute gave ethical approval for this work. The parents and legal representatives of all children in this study provided written informed consent before recruitment into the study. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present work are contained in the manuscript