Methods: Specimens were studied for evidence of adenovirus (AdV), enterovirus (EV) and coronavirus (CoV) with panspecies gel-based nested PCR/RT-PCR assays. Gene sequences of specimens positive by panspecies assays were sequenced and studied with the NCBI Basic Local Alignment Search Tool software.
Results: There was considerable discordance between real-time and conventional molecular methods. The real-time AdV assay found a positivity of 10.4%; however, the AdV panspecies assay detected a positivity of 12.4% and the conventional AdV-Hexon assay detected a positivity of 19.6%. The CoV and EV panspecies assays similarly detected more positive specimens than the real-time assays, with a positivity of 7.8% by the CoV panspecies assay versus 4.2% by rRT-PCR, and 8.0% by the EV panspecies assay versus 1.0% by rRT-PCR. We were not able to ascertain virus viability in this setting. While most discordance was likely due to assay sensitivity for previously described human viruses, two novel, possible zoonotic AdV were detected.
Conclusions: The observed differences in the two modes of amplification suggest that where a problem with sensitivity is suspected, real-time assay results might be supplemented with panspecies conventional PCR/RT-PCR assays.