Study population
We included a selection of swabs taken from patients presenting to primary care with symptoms of ILI, willing to participate in the ALIC
4E trial, who met the inclusion criteria and gave informed consent. [
12,
13] There were no additional exclusion criteria. For the purposes of the trial, ILI was defined as a sudden onset of self-reported fever, with at least one respiratory symptom (cough, sore throat, running or congested nose) and one systemic symptom (headache, muscle ache, sweats, chills or tiredness), with symptom duration of 72 h or less. [
12,
13]
Recruitment for the ALIC4E trial was during periods of heightened influenza incidence which was determined by reviewing the data from the European Centre of Disease Prevention and Control (ECDC) in combination with local and regional alerts during the winters of 2015–2016, 2016–2017 and 2017–2018. The ALIC4E Trial team confirmed to each recruiting network when their sites could begin recruitment. Recruitment took place through recruiting sites (GP Practices, Out of Hours surgeries or Paediatric Centres); recruitment was paused when influenza activity dropped again below the epidemic threshold.
Design
This was a method comparison study where a selection of swabs taken at baseline during the ALIC4E trial from participants presenting to primary care are analysed both on the cobas® Liat® POCT with the Influenza A/B & RSV Assay, as well as the laboratory-based Fast-Track Respiratory Pathogens 21 Plus kit (FTD, Fast-Track Diagnostics). The selection of swabs was aimed to detect a representative range of test positive and test negative samples for the viral pathogens relevant to the assay under investigation, including: all RSV positive samples, and, in function of the epidemiology of the circulating strains, a more or less equal proportion of samples testing positive for influenza A H1N1, influenza A H3N2 and influenza B.
For the laboratory-based PCR test, extraction was performed on NucliSENS easyMag (bioMérieux) and amplification on Lightcycler 480 (Roche).
Sample analysis on the cobas® Liat® POCT started 12th February 2018 and ended 30th June 2018.
For the POCT, all patients aged up to 16 years recruited during the ALIC4E trial had an oropharyngeal and nasal flocked swab taken at baseline by the responsible clinician or recruiter. These two samples were placed in one 3-ml Universal Transport Medium (UTM) (Copan). Those aged 16 years or older had a nasopharyngeal swab taken which was also placed in a 3-ml UTM. Once at the local laboratory, the samples were frozen and stored at − 70 °C (if a deep-freezer was not available on site at the local laboratory, storage at − 20 °C was deemed acceptable) and then transported to a central laboratory in Antwerp, Belgium. Samples went through a freeze/thaw cycle prior to aliquoting at the central laboratory in Antwerp.
A selection of the samples collected during the three influenza seasons (2015–2016, 2016–2017, 2017–2018) of the ALIC4E trial, frozen and stored at − 70 °C, were used, to ensure a sufficient large number of positive samples of the various viral pathogens. We enriched the sample for flu positivity.
The fresh samples collected in the third influenza season in Belgium were sent to the central laboratory for analysis both before and after a freeze-thaw cycle.
All laboratory analyses were done in the central laboratory at the University of Antwerp by trained lab technicians.
Prior to testing patient samples, the 2016 Quality Control for Molecular Diagnostics (QCMD) influenza panel (proficiency testing) was tested on the cobas® Liat® POCT, containing 10 samples with dilutions of different subtypes of influenza A and influenza B, to assess the performance of the POC device.
Samples with discordant results were analysed additionally by the RespiFinder 2Smart (PathoFinder).
The participant’s date of recruitment, gender and participant trial ID number were used as identifiers for these samples. Only the laboratory and trial team had access to this information for the purposes of sample identification and tracking.
Acceptability and failure mode analysis
To assess acceptability and certain aspects of failure mode analysis, [
19] the device operators were asked to complete a survey (see electronic supplementary material S1).
Acceptability was scored on a five-point Likert scale (1 = completely disagree to 5 = completely agree), including questions regarding device start-up, cartridge handling, operating the device, duration of the test, the provided manual and presentation of error codes. The risk of misinterpretation of failure modes was scored as: low risk–medium risk–high risk–difficult to assess, considering the following errors: expired cartridge, insufficient sample volume, incorrect cartridge insertion, unexpected test result and mismatch between the error code and actual error. The detection of the failure modes was scored as: always perceivable–probably perceivable–not perceivable for the same set of potential errors.
Operators were asked to list the strengths and weaknesses of the device.
Sample size calculation and statistical analysis
We aimed to analyse a selection of swab results obtained using both platforms from a minimum of 730 participants recruited during three consecutive winters (samples selected from all stored frozen samples from season 1 and 2 and all new samples from season 3 until required sample size achieved), giving 90% power at 95% confidence to detect a maximum difference in results obtained from the two methods of 10% at a presumed sensitivity of 95%.
An expanded gold standard (EGS) approach was used to calculate sensitivities and specificities (positive by at least two tests). Results of this analysis were considered the reference standard. Sensitivity, specificity and positive and negative predictive value were determined. Wilson 95% confidence intervals were calculated with the Binom Package. All statistical analyses were performed using R 3.4.3, (
https://www.r-project.org/).