Optimization of Saliva and Nasopharyngeal Swab-based Enzyme Immunoassays for Non-invasive Detection of Human Bocavirus 1 Antibodies

Human bocavirus 1 (HBoV1), the second human-pathogenic parvovirus after B19, causes upper and lower acute respiratory tract infections (ARTIs) in pediatric populations. Although discovered two decades ago, accurate diagnosis of HBoV1 remains challenging due to prolonged viral shedding in the respiratory tract, which leads to frequent co-detections with other respiratory pathogens. The diagnosis of HBoV1 ARTI therefore necessitates an integrated approach involving quantification of respiratory viral load and detection of viral mRNA, capsid antigen, viremia, or serology. To identify non-invasive, child-friendly alternatives to serum and to evaluate the performance of a novel HBoV1-IgA enzyme immunoassay (EIA), pediatric saliva and nasopharyngeal swab (NPS) samples were collected at the HUS New Children’s Hospital, from 34 children, and serum from 2 children with ARTI. Quantitative PCR (qPCR) of the NPS samples revealed a 27% prevalence of HBoV1 DNA. The saliva serology was optimized with saliva and serum pairs from 24 healthy adult volunteers, so the acute-marker IgM had to be omitted at this stage. The HBoV1 IgG EIA was optimized also for pediatric NPS samples. HBoV1-specific IgG and IgA were readily detectable in saliva, while HBoV1 IgG was also measurable in NPS, albeit with more varying signal intensities. Undiluted and 1:2-diluted saliva showed HBoV1-IgG levels consistent with matched serum in all adults. Of the two children who donated serum samples, one was HBoV1 NPS-PCR positive and exhibited serum IgM and IgA, but not serum or NPS IgG, indicating a very acute infection. Notably, NPS diluted at 1:5 produced distinct and diagnostically informative IgG absorbance values in all 34 pediatric samples, revealing a seroprevalence of 58%. These results support this protocol to be optimal for future sample validation with more pediatric samples, the sampling of which is ongoing. These EIAs are potentially applicable in the clinical routine. Overall, my thesis demonstrates the potential of saliva and NPS as effective, non-invasive specimens for combined qPCR and antibody EIA diagnostics of HBoV1 ARTI.