A novel anterior nasal swab to detect respiratory viruses: a prospective study of diagnostic accuracy.

Detection of respiratory viruses requires testing of the upper respiratory tract to obtain specimens for analysis. However, nasal and throat swabs can cause discomfort and procedural anxiety in children. Respiratory sampling methods which are accurate and less invasive are needed. We aim to determine the positive and negative percentage agreement of a novel anterior nasal swab (ANS) compared with the combined throat and anterior nasal swab (CTN), the reference standard, for detection of respiratory viruses. Children 5 - 18 years of age presenting to a tertiary paediatric hospital with respiratory symptoms were tested with both swabs in randomised order. Respiratory samples were tested on a multiplex RT-PCR panel. Viral detections, RT-PCR cycle-threshold values and child/parent/clinician experience of the swab were recorded. There were 157 viral detections from 249 participant CTN swabs. In comparison with the CTN, the overall positive and negative percentage agreement of ANS for detection of respiratory viruses was 96.2% (95% CI, 91.8-98.3%) and 99.8% (95% CI, 99.6-99.9%), respectively. The ANS was "extremely comfortable", or only a "little uncomfortable" for 90% of children compared with 48% for CTN. 202 children (84%) rated the ANS as the preferred swab, and 208 (87%) indicated they would prefer ANS for future testing. The ANS required additional laboratory handling processes compared to the CTN. The ANS has high positive percentage agreement and is comparable to the current standard of care. The high acceptability from the less invasive ANS provides a more comfortable method for respiratory virus testing in children.Trial registrationClinicalTrials.gov ID NCT05043623.

A simplified, amplicon-based method for whole genome sequencing of human respiratory syncytial viruses.

BACKGROUND: Human Respiratory Syncytial Virus (RSV) infections pose a significant risk to human health worldwide, especially for young children. Whole genome sequencing (WGS) provides a useful tool for global surveillance to better understand the evolution and epidemiology of RSV and provide essential information that may impact on antibody treatments, antiviral drug sensitivity and vaccine effectiveness. OBJECTIVES: Here we report the development of a rapid and simplified amplicon-based one-step multiplex reverse-transcription polymerase chain reaction (mRT-PCR) for WGS of both human RSV-A and RSV-B viruses. STUDY DESIGN: Two mRT-PCR reactions for each sample were designed to generate amplicons for RSV WGS. This new method was tested and evaluated by sequencing 206 RSV positive clinical samples collected in Australia in 2020 and 2021 with RSV Ct values between 10 and 32. RESULTS: In silico analysis and laboratory testing revealed that the primers used in the new method covered most of the currently circulating RSV-A and RSV-B. Amplicons generated were suitable for both Illumina and Oxford Nanopore Technologies (ONT) NGS platforms. A success rate of 83.5% with a full coverage for the genome of 98 RSV-A and 74 RSV-B was achieved from all clinical samples tested. CONCLUSIONS: This assay is simple to set up, robust, easily scalable in sample preparation and relatively inexpensive, and as such, provides a valuable addition to existing NGS RSV WGS methods.

Factors associated with severe respiratory syncytial virus disease in hospitalised children: a retrospective analysis.

BACKGROUND: Early recognition of children at risk of severe respiratory syncytial virus (RSV) lower respiratory tract infection is important as it informs management decisions. We aimed to evaluate factors associated with severe disease among young children hospitalised with RSV infection. METHODS: We conducted a retrospective cohort study of all children <2 years of age hospitalised for RSV lower respiratory tract infection at a single tertiary paediatric hospital over three RSV seasons (January 2017-December 2019). We classified children as having 'moderate' or 'severe' disease based on the level of respiratory intervention and used univariable and multivariable regression models to determine factors associated with severe disease. RESULTS: Of 970 hospitalised children, 386 (40%) were classified as having 'severe' and 584 (60%) as having 'moderate' RSV disease. On multivariable analyses, age <2 months (OR: 2.3, 95% CI 1.6 to 3.3, p<0.0001), prematurity (OR: 1.6, 95% CI 1.1 to 2.4, p=0.02) and RSV-parainfluenza virus type 3 (PIV3) codetection (OR: 2.6, 95% CI 1.05 to 6.5, p=0.04) were independently associated with severe disease. CONCLUSION: Younger age, prematurity and PIV3 codetection were associated with severe RSV disease in children <2 years of age hospitalised with RSV infection. The association between PIV3 and severe RSV disease is a novel finding and warrants further investigation.

Rapid detection of human respiratory syncytial virus A and B by duplex real-time RT-PCR.

Respiratory syncytial virus (RSV) is a common cause of acute respiratory disease worldwide, especially in young children. The World Health Organization (WHO) has initiated an RSV Surveillance Pilot program that aims to perform worldwide RSV surveillance, requiring the development of reliable and rapid molecular methods to detect and identify RSV. A duplex real-time RT-PCR assay developed for simultaneous detection of both A and B subtypes of RSV was included as part of this program. This duplex assay targeted a conserved region of the RSV polymerase gene and was validated for analytical sensitivity, specificity, reproducibility and clinical performance with a wide range of respiratory specimens. The assay was highly specific for RSV and did not react with non-RSV respiratory pathogens, including the SARS-CoV-2 virus.