The clinical role of codetection of HBoV and other viruses in NPAs has been questioned. It is not easy to determine whether such co-infections are sequential infections or simultaneous viral infections. Serologic analysis is a more precise approach for diagnosis of HBoV infections. When compared with PCR-positive results in serum and NPAs, diagnostic sensitivity and specificity of our antibody EIAs were as high as 97% and 99.5%, respectively; positive predictive value was 97%. We showed by using EIAs that among wheezing children, >60% of co-infections in children with HBoV NPA PCR-positive results, particularly in children with a high HBoV DNA load, are acute primary HBoV infections and should be considered in the diagnosis of respiratory disease. No differences in occurrence of co-infections were observed in children with a serologically confirmed diagnosis of infection with HBoV compared with children positive for HBoV by only PCR of NPAs.