Microfluidic qPCR for detection of 21 common respiratory viruses in children with influenza-like illness

Abstract

Multiple respiratory viruses lead to high morbidity and mortality, yet global surveillance platforms focus primarily on seasonal influenza viruses. The COVID-19 pandemic and new RSV vaccines highlight the importance of a broader approach. Upper respiratory tract swabs from children aged 24-59 months presenting with influenza-like illness in The Gambia were collected during follow-up of a live-attenuated influenza vaccine randomised controlled trial in 2017-18. A microfluidic quantitative polymerase chain reaction (qPCR) assay was established and used to detect 21 respiratory viruses. 76.6% of samples had one or more viruses detected (n = 121/158). The viruses detected most frequently were rhinovirus (n = 37/158, 23.4%) and adenovirus (n = 34/158, 21.5%), followed by parainfluenza virus 3, influenza B and human metapneumovirus B. A third of positive samples had multiple viruses detected (two n = 31/121, 25.6%; three n = 9/121, 7.4%). Our data demonstrates how microfluidic qPCR is a useful tool for high-throughput, comprehensive detection of multiple respiratory viruses in surveillance platforms. Rapidly changing epidemiology exemplifies the need for new, broader approaches to virus surveillance in low-resource settings to respond to future epidemics and to guide the need for and use of new prevention and therapeutic measures.

Keywords: Influenza-like illness; Microfluidics; Polymerase chain reaction; Respiratory virus; Surveillance.

Fig. 1

Schematic of sample processing and microfluidic assay. Created with BioRender.com.

Fig. 2

Correlation between microfluidic and NHS Laboratory PCR Cq values. The grey shaded areas represent the Cq values over the 95% LOD threshold. Datapoints are coloured based on if Cq values are available on both assays (purple), or if there was “No Cq” on at least one of the assays (black). Correlation was calculated including only the purple points. Cq Quantification cycle, PCR Polymerase Chain Reaction, RdRP RNA-Dependant RNA Polymerase, RNA Ribonucleic acid, RSV Respiratory Syncytial Virus, SARS-CoV-2 Severe Acute Respiratory Syndrome Coronavirus-2.

Fig. 3

Chord diagram showing pairs of respiratory viruses detected in co-infections (i.e. 2 or more viruses per swab) of children with ILI detected by passive surveillance in the Gambia. Colours correspond to the seasonality data shown in Fig. 4. Where more than two (one pair of) viruses were detected, each infection may be represented more than once. ILI Influenza-like illness, HMPV Human metapneumovirus, RSV Respiratory syncytial virus.

Fig. 4

Seasonal detection of respiratory viruses from upper respiratory tract swabs taken from children aged 24–59 months with influenza-like illness in the Gambia in 2017 and 2018. Rainy season months are shaded in grey. Only partial data are available for January and November, and no data for December, due to follow up windows in the parent study. HMPV Human Metapneumovirus, RSV Respiratory Syncytial Virus.