This is a prospective study which was conducted between July 2022 and February 2023 in six departments of internal medicine located at public hospitals in Greece. The study was a sub-study part of the clinical trials ACCESS and ImmunoSep, which were licensed by the National Ethics Committee of Greece (approvals 122/20 and 2/21) and the National Organization for Medicines of Greece (approvals IS113/20 and IS008/21) (ClinicalTrials.gov registrations NCT04724044 and NCT04990232). Written informed consent was provided by the patients or their legal representative. Patients who were screened for eligibility for both trials participated in the present study. The study was performed in accordance with the Helsinki Declaration of 1964 and its later amendments.

Enrolled patients should meet both the following inclusion criteria: (1) male or female adults (age 18 years or more), and (2) any of the following clinical signs of infection: fever, cough, dyspnea, sore throat, headache, nasal congestion, or diarrhea. Exclusion criteria were: age less than 18 years; chronic oral or intravenous intake of corticosteroids at a dose more than 0.4 mg/kg of equivalent prednisone daily; known infection by the human immunodeficiency virus; neutropenia; any chronic anti-cytokine treatment; and pregnancy or lactation.

Forty patients who did not meet any of the exclusion criteria and who did not present any sign of active infection were enrolled as comparators.

All the study participants were subject to an intense work-out, including present and past medical history and thorough physical examination; laboratory evaluation including complete blood cell counting, biochemistry, blood gas and procalcitonin; chest X-ray, and, when required, high-resolution chest computed tomography; nasopharyngeal swab collection for real-time PCR testing for SARS-CoV-2 and, when needed, by the BioFire® FilmArray® panel of the upper or lower respiratory tract (Respiratory Panel 2.1 plus or Pneumonia plus Panel respectively) (bioMérieux, Lyon France); urine detection of the antigens of Streptococcus pneumoniae and Legionella by the BinaxNOW assay (Abbott Point of care); glutamate dehydrogenase and toxin A/B immunosorbent assays for Clostridioides difficile in the stool; and blood, sputum, or urine culture. The SOFA (sequential organ failure assessment) score and the Charlson’s comorbidity index (CCI) were calculated. Patients were followed-up for 28 days for survival.

In parallel to routine blood sampling, another 4 mL of venous whole blood was collected into one EDTA-coated tube (Vacutainer; Becton Dickinson, Cockeysville MD, USA) and analyzed using the AFIAS MxA/CRP test.

AFIAS MxA/CRP is a fluorescent lateral flow immunoassay test for the quantitative determination of MxA and CRP in human blood, consisting of an all-in-one cartridge with a detector part, a diluent part, and the test strip with anti-human MxA, anti-human CRP, and CRP antigen in the test line and chicken IgY in the control line. The test was run on an AFIAS-10 automated immunoassay analyzer by Boditech Med (Chuncheon, Republic of Korea). The lower limit of detection was 10 ng/mL for MxA and 1 mg/L for CRP.

Patients were classified into seven categories following adjudication by two experts, who were given access to all clinical and laboratory information for the patients 30 days after completion of the follow-up. The only non-accessible information was the result of AFIAS MxA/CRP. Adjudicators were told to classify patients into one the following seven categories using their clinical judgment and available laboratory and microbiology results. In case the two experts did not agree, a third expert served as an arbiter: (1) no infection: patients without infection; (2) definitive viral infection: infection definitively caused by an isolated virus and where no bacterial pathogen was detected; (3) definitive bacterial infection: infection definitively caused by an isolated bacterial species and where no viral pathogen was detected; (4) definitive viral/bacterial co-infection: infection definitively caused by both viral and bacterial species; (5) high probability for viral infection: infection most probably of viral etiology without any viral pathogen detected, (6) high probability of bacterial infection: infection most probably of bacterial etiology without any bacterial pathogen detected, and (7) definitive viral infection and high probability for bacterial co-infection: infection definitively caused by an isolated virus with high probability of bacterial co-infection without, however, any bacterial pathogen detected.

The study primary endpoint was to develop an algorithm which uses blood levels of MxA, CRP, and their ratio to classify patients into viral and bacterial infections or co-infection.

The study's secondary endpoint was to develop cut-offs of MxA, CRP, and their ratio for the early prognosis of a 28-day unfavorable outcome.

Qualitative variables were expressed as frequencies and 95% confidence intervals (CIs) and qualitative variables as medians and 95% CIs. Comparisons of qualitative variables between groups were carried out by Fisher’s exact test. Comparisons of quantitative variables between groups were carried out by the Mann–Whitney U test after Bonferroni correction for multiple testing. Patients with definitive viral and bacterial infections were the discovery cohort. In these patients, two analyses were carried out: (1) the diagnostic performance of the combination of the suggested diagnostic cut-offs for MxA (> 15 ng/mL) and for CRP (> 10 mg/L) were determined; and (2) logistic regression analysis was performed to provide an equation which can define the probability of a patient for bacterial or viral infection. Then, a receiver operator characteristic (ROC) curve analysis was carried out to define a cut-off of the probability with the best trade-off using the Youden index. The area under the curve (AUC) and the 95% CIs were calculated. In parallel, the ratio of MxA to CRP was calculated and plotted as the ROC curve, which provides the best trade-off for the diagnosis of viral infection. Then, the two cut-offs, the cut-off of probability for bacterial infection and the cut-off of the MxA/CRP ratio, were combined to a final diagnostic rule for the discrimination between bacterial and viral infections. This rule was validated at the validation cohort. Finally, these cut-offs were applied for patients with definitive viral/bacterial co-infection and for patients with definitive viral and highly probable bacterial co-infection. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated. Any p value below 0.05 was considered statistically significant.