Each specimen was first screened for RSV by means of the Resp-4-Plex kit (Abbott Molecular Inc., Des Plaines, IL, USA) used with the fully automated Alinity m System (Abbott Molecular Inc., Des Plaines, IL, USA) and according to the manufacturer’s instructions. This kit is a multiplex real-time reverse transcription polymerase chain reaction (RT-PCR) for the qualitative detection and differentiation of RNA from SARS-CoV-2, RSV, influenza A and B viruses. According to the manufacturer, the limit of detection is 0.300 and 0.100 median tissue culture infectious dose (TCID
50)/ml for RSV-A and RSV-B, respectively [30 ].
To discern RSV subgroup and detect the presence of other respiratory pathogens, samples positive for RSV were further tested by means of the Allplex Respiratory Panel (RP) assays (Seegene Inc.; Seoul, Republic of Korea) according to the manufacturer’s instructions. Briefly, nucleic acids were first extracted using the STARMag Universal Cartridge Kit (Seegene Inc.; Seoul, Republic of Korea) on the automated Nimbus IVD (Seegene Inc.; Seoul, Republic of Korea) platform. For this purpose, 200
µl of each specimen was extracted and eluted with 100
µl of elution buffer and set up for RT-PCR. RT-PCR was then performed on a CFX96 instrument (Bio-Rad Laboratories, Inc; Hercules, CA, USA) with the Allplex RPs 1–4 kits. These four panels are able to detect the most common respiratory pathogens - both viruses [RP 1: RSV-A, RSV-B, influenza viruses A, A(H1N1), A(H1N1)pdm09, A(H3N2) and B; RP 2: adenovirus (AdV), enterovirus (EV), metapneumovirus (MPV), parainfluenza (PIV) viruses 1–4; RP 3: bocaviruses (BoV) 1–4, coronaviruses (CoV) 229E, NL63, OC43, rhinovirus (RV)] and bacteria [RP 4:
Streptococcus pneumoniae (SP),
Bordetella parapertussis (BPP),
Bordetella pertussis (BP),
Chlamydophila pneumoniae (CP),
Haemophilus influenzae (HI),
Legionella pneumophila (LP),
Mycoplasma pneumoniae (MP)]. For each RT-PCR, 8
µl of the extracted nucleic acid in a final volume of 25
µl was used. The diagnostic accuracy of this assay in detecting RSV-A and RSV-B is 100% [31 (
link)].
Samples showing cycle threshold (Ct) values < 40 in at least one assay were deemed positive. Ct values were used as a proxy measure of viral load: lower Ct values indicate higher viral load.
Panatto D., Domnich A., Lai P.L., Ogliastro M., Bruzzone B., Galli C., Stefanelli F., Pariani E., Orsi A, & Icardi G. (2023). Epidemiology and molecular characteristics of respiratory syncytial virus (RSV) among italian community-dwelling adults, 2021/22 season. BMC Infectious Diseases, 23, 134.