Background: We have monitored influenza C virus infection using the cell culture method due to its advantage in detecting infectious virus. Application of multiplex polymerase chain reaction (PCR) improves the detection rate of multiple pathogens from the same clinical specimen, and it can be difficult to determine whether the influenza C virus is the cause of the patient´s symptoms. This study aimed to evaluate the impact of co-pathogens in children infected with the influenza C virus based on viral load in clinical specimens.

Methods: We enrolled 11 patients in whom influenza C virus and co-pathogens were isolated by cell culture. Pathogens were further detected by multiplex real-time PCR and next-generation sequencing (NGS). Viral load was determined by quantitative real-time PCR, and its association with the patients´ symptoms was investigated.

Results: Co-pathogens not isolated in cell culture, including rhinovirus, adenovirus and bocavirus, were detected in 5 of the 11 specimens by real-time PCR. Additionally, adenovirus and human metapneumovirus, which were not detected by real-time PCR, were detected by next-generation sequencing, but both had very low read numbers. Children whose specimens had influenza C virus levels of ≥104 copies/μL showed typical symptoms of influenza C virus infection, such as coughing and nasal discharge. When the viral load of a co-pathogen was greater than that of influenza C virus, atypical symptoms such as persistent fever were likely due to the co-pathogens, suggesting that co-infection contributed to symptom severity.

Conclusion: Knowledge of viral load can help in evaluating the etiologic role of the virus in influenza C-infected children with co-pathogens.