Study design and sample collection
The diagnostic accuracy of the Hyris Kit was evaluated by a retrospective and a prospective analysis on SARS-CoV-2 samples previously assessed (and validated positive and negative) by an FDA “authorized for the emergency use - EUA” reference method. The study was conducted in the Department of Laboratory Medicine of the National Institute for The Study and Treatment of Cancer IRCCS "Fondazione G Pascale" (IRCCS Pascale) of Naples (Italy).
For the retrospective analysis, SARS CoV-2-positive samples were taken during the pandemic peak period (from March to June 2020) and were provided by the Azienda Ospedaliera Universitaria Federico II (Federico II University Hospital) and the Azienda Ospedaliera dei Colli – Ospedale Domenico Cotugno (Cotugno Hospital) of Naples (Italy). SARS-CoV-2-negative samples were collected in July 2020 by the IRCCS Pascale.
For the prospective analysis, new positive samples were prepared by Federico II University Hospital starting from pools of one or two positive samples, diluted in a negative sample (pooled samples).
All participant patients were aged ≥18 years; they understood and signed the informed consent form. The study protocol was approved by the Ethics Committee of IRCCS Pascale.
Reference analysis methods
The reference methods, used to verify the positivity or negativity of collected samples for the retrospective analysis were: 1) Abbott Kit: RealTime SARS-CoV-2 (#09N77-095), EUA by the FDA, where a complete analysis run lasts for 6 hours on average; and 2) Roche Kit: LightMix®Modular SARS-Cov-2 (COVID19) RdRP, where a complete analysis run lasts for 3 hours on average. Using the Roche Kit, samples are rated negative if they have a late amplification beyond 35 threshold cycle (Ct, Kit cut-off) or no amplification for the target gene.
Moreover, positive samples by the Federico II University Hospital and negative samples by IRCCS Pascale were analyzed with the Abbott Kit. Positive samples by Cotugno Hospital were analyzed with the Roche Kit.
The Roche Kit, which needs 200 μL of sample, was used in place of the Abbott Kit if the sample volume was not enough for the specifications of the Abbott Kit, which requires 800 μL.
The prospective analysis on pooled samples was first carried out with the Hyris Kit, and then with the Abbott kit.
All samples were analyzed in compatible soil (universal transport media/viral transport medium) and then stored at -80°C.
Hyris Kit point-of-care test
The Hyris Kit is intended for use on the bCUBE instrument, a miniaturized device for the in vitro qualitative detection of SARS-CoV-2 nucleic acid in nasopharyngeal swabs or nasal swabs specimens (Figure 1). No sample extraction is needed prior to the PCR amplification step. To allow a quick detection of the disease, without necessarily going through an analysis laboratory, all the solutions required for the analysis process are premixed within the kit. A small aliquot of the swab transport medium (5 μL), which contains the collected specimen, can be directly added to 15 μL of the reaction mix, previously loaded into the bCUBE cartridge. The sample will be amplified as per the amplification kit procedure. During the RT step of the RT-PCR thermal protocol, the viral RNA will be released into the qPCR master mix. The direct amplification RT-PCR assay is performed in the bCUBE. The bCUBE is designed to be fully portable and to work in network and the embedded automatic results interpretation delivers a test output readable by non-experts. At the end of the analysis, a “detected” or “not detected” result can be assigned to each sample for positive and negative samples, respectively. The Hyris Kit automatic interpretation algorithm classifies all those samples that do not show amplification for viral targets and very late amplification for endogenous control, namely the human gene RPP30 (i.e., the ubiquitous gene that encodes for RNase P), as "indeterminate". Late amplification of the RPP30 gene may depend on a poorly performed sample, this can alter the outcome of the analysis in the case of a patient with a viral load very close to the limit of the detection of the method, causing cases of "false negativity". Therefore, for the present study, samples with an "indeterminate" outcome are excluded from the trial.
Samples analyzed with the Hyris Kit that have the amplification of only one of the two targets of the SARS-CoV-2 virus are classified as "inconclusive". Samples with "inconclusive" results are excluded from the trial for the present study.
In case of positive samples that showed a no amplification signal or showed partial amplification during retrospective analysis with the Hyris kit, analyses were repeat with the Roche Kit.
Determination of the lower limit of detection of the Hyris kit
Two positive samples provided by the Federico II, analyzed with the Hyris Kit and evaluated positive, were diluted and tested in technical duplication in order to identify a concentration of analyte beyond the limit of detection of the method of analysis. All dilutions where one or more replicated were not properly amplified (both targets for both replicated) have been discarded. Having defined the dilution to be tested, the samples were processed with the Roche Kit to identify the correlation between the Ct obtained with Hyris Kit, and the Roche Kit, in order to exclude all samples that have Ct values greater than the threshold value identified, which corresponds to 33 Ct.
To perform a blinded analysis with Hyris kit, a selection of positive and negative samples was prepared and by IRCCS Pascale staff and provided anonymously for analysis to Hyris kit staff, who carried out the analysis. At the end of the tests, IRCCS Pascale's staff disclosed all the information for data analysis.
Descriptive statistics were used for the present study.