Respiratory infections remain a significant global threat to child health, contributing to a substantial proportion of pediatric mortality (1–3). Respiratory infections in early childhood have a significant impact on the broncho-alveolar and vascular development of the lungs which cause long-term effects on the health of children (4). Viruses and bacteria are the primary causative agents of these infections, making the accurate identification of pathogens essential for effective treatment (5).

Currently, the differentiation between viral and bacterial respiratory infections in pediatric patients relies largely on clinical symptoms, leukocyte count and classification, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and other laboratory indicators (6–8). However, these diagnostic tools lack specificity and often show limited sensitivity, complicating the accurate diagnosis of the infection type. While current biomarkers can estimate the likelihood of bacterial infections, there remains a critical gap in reliable biomarkers for detecting viral infections. This diagnostic gap underscores the urgent need for novel biomarkers, especially given the strict antibiotic stewardship regulations in many countries that necessitate accurate differential diagnoses to avoid unnecessary antibiotic prescriptions (9, 10).

Recent advancements in basic research have led to the discovery and development of novel biomarkers for infectious diseases. Notably, in the context of respiratory viral infections, the host immune response triggers the production of interferons, which subsequently induce the synthesis of Myxovirus resistance protein A (MxA). MxA, primarily activated by type I interferons (IFN-α/β), has gained significant attention due to its potential as a diagnostic marker for viral infections (11, 12). Studies have demonstrated that MxA holds diagnostic value in distinguishing viral infections from bacterial infections in both pediatric and adult populations. Furthermore, integrating MxA with specific bacterial infection markers has been explored to improve the management of children presenting with respiratory infection symptoms (13, 14). Despite these promising findings, more comprehensive research is needed to fully leverage the diagnostic potential of MxA and other emerging biomarkers in clinical practice (15).

This study aims to provide a systematic evaluation of blood MxA levels among children with viral, bacterial, and mixed respiratory infections compared to healthy controls in Xiamen, a coastal city in southeastern China. The findings highlight the diagnostic potential of MxA for distinguishing viral from bacterial infections. Additionally, we propose a novel diagnostic strategy that combines MxA with CRP to enhance the accuracy of differentiating between viral and bacterial respiratory infections in clinical settings in China.

This study enrolled a total of 314 pediatric patients diagnosed with respiratory tract infections, alongside a healthy control group consisting of 89 children. Among the 314 patients, 205 were diagnosed with viral infections, 21 with bacterial infections, and 88 with mixed infections involving both viral and bacterial pathogens. Detailed clinical characteristics and specific diagnoses for these patient groups are summarized in Table 1 . Notably, the most frequently observed conditions were acute upper respiratory tract infections and bronchopneumonia.

In this study, we demonstrated that MxA has good diagnostic performance in distinguishing viral from bacterial respiratory tract infections in pediatric patients, with an AUC of 0.8019 (95% CI: 0.6989–0.9049). Furthermore, combining MxA with CRP further enhanced the diagnostic accuracy, as the MxA/CRP ratio achieved an AUC of 0.8713 (95% CI: 0.7916 to 0.9510), highlighting its potential as a powerful diagnostic tool in this context. However, both MxA alone and the MxA/CRP ratio showed poor performance in differentiating viral infections from mixed infections, with AUCs of 0.5161 and 0.5429, respectively. These findings suggest that while MxA and its combination with CRP are effective for distinguishing viral and bacterial infections, they are insufficient for mixed infections. To address this limitation, integrating additional biomarkers or clinical indicators may be necessary to improve the diagnostic accuracy for mixed infections.

Pediatric patients are particularly susceptible to respiratory tract infections due to the vulnerability of their developing respiratory systems. Without timely and accurate diagnosis, infections can progress to severe conditions like pneumonia, posing significant health risks (16). Currently, the early clinical differentiation between viral and bacterial infections largely relies on symptomatology, WBC counts, and markers like CRP and PCT (17, 18). Upon diagnosis of a bacterial infection, antibiotics are usually prescribed. To date, viral infections can be determined by molecular methods, which are expensive, time-consuming and complicated. Clinically, children with viral infections often present with a range of symptoms, such as respiratory symptoms, rash, fever, and meningitis. Once such suspicious clinical symptoms appear, antibiotics are frequently administered as a precautionary measure during the diagnostic process, leading to the overutilization of antibiotics (9, 19). Up to now, there has been a lack of commercially available immunoassay reagents capable of accurately distinguishing between various viral infectious diseases.

MxA is one of the antiviral proteins, distributed in the cytoplasm, which exhibits a broad spectrum of antiviral activities and exerts inhibitory effects on numerous RNA viruses and select DNA viruses, including influenza virus, enterovirus, and respiratory syncytial virus., etc (20, 21). Studies have shown that even a minimal viral load can stimulate the production of MxA protein in cells, whereas cells infected with bacteria, parasites, and other microorganisms do not exhibit induction of MxA protein expression (22–24). Therefore, the MxA protein expression levels in cells have been proposed as a potential indicator of viral infection, facilitating differential diagnosis from other microbial infections, notably bacterial (25). In recent years, several studies have demonstrated the potential diagnostic value of MxA protein levels in identifying viral infections; however, its integration into routine clinical practice remains limited (26, 27). This study provides new evidence to support the utilization of MxA as a diagnostic indicator for respiratory viral infections in clinical settings, and the combination of CRP with MxA improves the diagnostic value of MxA.

However, it is noteworthy that our study found slightly elevated MxA levels in the bacterial infection group compared to the control group. This observation may be due to challenges in differentiating between bacterial infections and viral-bacterial co-infections, as some bacterial cases may involve undetected viral components, leading to misclassification as purely bacterial infections. It is already commonly known that CRP is less specific than PCT in bacterial infections. CRP is an acute-phase protein produced by the liver in response to increases in proinflammatory cytokines such as IL-1β and IL-6, while PCT is produced by various cell types in response to bacterial cell wall components such as lipopolysaccharide, peptidoglycan, and lipoteichoic acid, as well as proinflammatory cytokines like IL-1β, IL-6, and TNF-α. However, in our study, CRP outperformed PCT in distinguishing viral from bacterial infections (AUC: 0.7893 vs. 0.6088). Similarly, another study has also demonstrated superior performance of CRP over PCT for this purpose (28). We hypothesize that this finding may be due to differences in the patient cohort or the underlying inflammatory responses in pediatric respiratory infections. Furthermore, viral-bacterial co-infections might modulate the levels of these biomarkers differently, potentially affecting their relative diagnostic utility. We also acknowledge that the relatively small sample size in our study might have influenced the results, and we emphasize the need for further research to validate these findings.

Theoretically, CRP is expected to perform well in distinguishing between viral infections and viral-bacterial co-infections. However, the results of this study revealed that CRP had limited effectiveness in differentiating between viral infections and viral-bacterial co-infections, while showing better performance in distinguishing bacterial infections from bacterial-viral co-infections. We hypothesize that this phenomenon may be attributed to several factors. In the viral-bacterial co-infection group, although bacteria were detected, some of the bacteria might have been colonizing rather than serving as primary pathogens, as colonizing bacteria typically do not significantly induce elevated CRP levels in the host. Additionally, viral infections might interfere with the CRP levels induced by bacterial infections through immune system regulation. Furthermore, the relatively small sample size in the bacterial infection group may have affected the robustness of the statistical analysis. The reasons also contribute to the inability of MxA/CRP to effectively distinguish between the viral infection group and the viral-bacterial co-infection group. In this study, MxA and the combined use of MxA/CRP can effectively differentiate viral infections from bacterial infections, but they fail to show significant differences between viral infections and mixed infections. This suggests that when MxA or MxA/CRP levels are elevated, it is not sufficient to distinguish between isolated viral infections and mixed infections. Therefore, decisions on antibiotic use cannot rely solely on MxA or MxA/CRP levels but must integrate other clinical markers, such as white blood cell count (WBC), C-reactive protein (CRP), and procalcitonin (PCT), as well as patient history and clinical symptoms.

The cohort of this study was relatively small, comprising 314 children with respiratory infections and 89 healthy controls, which may limit the generalizability of our findings to all respiratory infections. Additionally, all participants were recruited from Xiamen Maternal and Child Health Hospital, introducing potential regional and selection biases. In this study, the majority of patients in the infection group were children under 2 years of age, while the healthy control group consisted primarily of children aged 3-15 years. This discrepancy is a limitation of our retrospective analysis. It is primarily due to the fact that respiratory infections are more common in children under 2 years, whereas younger children undergoing routine health examinations rarely have blood samples collected, or only very small amounts of peripheral blood are obtained. As a result, it was challenging to recruit enough age-matched healthy controls for this study. Future studies should aim to expand the sample size, optimize the criteria for infection grouping, and incorporate MxA and other biomarkers to enhance the diagnostic accuracy for viral and bacterial infections.

To date, limited research has been conducted on the utilization of blood MxA protein level for the differential diagnosis of respiratory tract infections in Chinese pediatric populations. By using an enzymatic immunoassay to measure MxA, this study aims to promote more rational and targeted clinical management of pediatric respiratory infections, ultimately reducing unnecessary antibiotic use.