Evaluation of the ddPCR ability to detect the SARS-CoV-2 in nasopharyngeal samples of symptomatic patients with suspected COVID-19 infection using an IgG serological assay (EUROIMMUN Anti-SARS-Cov2 ELISA Ig) as gold/reference standard (FDA validated commercial serologic test).
The Bio-Rad SARS-CoV-2 ddPCR Test is a reverse transcription (RT) droplet digital polymerase chain reaction (ddPCR) test designed to detect RNA from SARS-CoV-2 in specimens (mainly nasopharyngeal, anterior nasal, oropharyngeal and mid-turbinate swab but also nasopharyngeal wash/aspirate and nasal aspirate specimens) collected from individuals who are suspected of COVID-19 infection. This single assay multiplex test enables a one-well reaction with three sets of the oligonucleotide primers and probes which were reported by CDC. Two were selected from regions of the virus nucleocapsid (N) gene. An additional primer/probe included in the panel is set to detect the human RNase P gene (RP) in control samples and clinical specimens. RNA isolated and purified from swab specimens is added to the mastermix comprised of reverse transcriptase whereby RNA is converted into cDNA and then amplified, using the Bio-Rad One-Step RT-ddPCR Advanced Kit for Probes. Briefly, the sample and mastermix RT-ddPCR mixtures are fractionated into up to 20,000 nanoliter-sized droplets in the form of a water-in-oil emulsion. The 96-well RT-ddPCR ready plate containing droplets is sealed with foil using a plate sealer. The emulsions are then thermocycled to achieve reverse transcription to generate cDNA followed by target amplification plus probe hydrolysis in each droplet. After thermocycling is complete, the 96-well RT-ddPCR ready plate is loaded into the Droplet Reader. The Droplet Reader singulates the droplets and flows them past a two-color fluorescence detector (FAM and HEX) in order to determine which contain target (positive) and which do not (negative) for each of the targets identified with the SARS-CoV-2 ddPCR Test: N1, N2 and RP. The ddPCR system uses the QuantaSoft 1.7 and QuantaSoft Analysis Pro 1.0 for analysis software.
Study Type
INTERVENTIONAL
Allocation
NA
Purpose
DIAGNOSTIC
Masking
NONE
Enrollment
100
Nasopharyngeal and throat/oropharyngeal swabs analyzed by both RT-PCR and ddPCR
Centre Leon Berard
Lyon, France
To determine the ddPCR ability to detect the SARS-CoV-2 in nasopharyngeal samples of symptomatic patients with suspected COVID-19 infection
Using IgG serological assay (EUROIMMUN Anti-SARS-Cov2 ELISA Ig) as gold/reference standard (FDA validated commercial serologic test)
Time frame: At inclusion
To determine the RT-qPCR ability to detect the SARS-CoV-2 in nasopharyngeal samples of symptomatic patients with suspected COVID-19 infection
Using IgG serological assay (EUROIMMUN Anti-SARS-Cov2 ELISA Ig) as gold/reference standard
Time frame: Third week after inclusion
To determine the ddPCR and RT-qPCR abilities to detect the SARS-CoV-2 in oropharyngeal samples of symptomatic patients with suspected COVID-19 infection
Using IgG serological assay (EUROIMMUN Anti-SARS-Cov2 ELISA Ig) as gold/reference standard
Time frame: Third week after inclusion
To determine the ability of a clinical diagnosis based both on patients' symptoms and chest CT-scan to detect the SARS-CoV-2 in symptomatic patients with suspected COVID-19 infection
Using IgG serological assay (EUROIMMUN Anti-SARS-Cov2 ELISA Ig) as gold/reference standard
Time frame: Third week after inclusion
To determine the agreements between nasopharyngeal samples and oropharyngeal samples
Using ddPCR and RT-qPCR assays
Time frame: At inclusion
To determine the agreements between a clinical diagnosis and ddPCR and RT-qPCR assays
Using ddPCR and RT-qPCR assays
Time frame: At inclusion
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To assess the 28-day mortality rate
Rate calculated from the date of the first diagnostic procedure to the date of death of any cause
Time frame: Up to the follow-up end (28 days after inclusion)
To determine potential predictive factors of death among patients' characteristics
Demographics, type of tumor, type of anticancer, treatment, comorbidities, biological parameters
Time frame: Up to the follow-up end (28 days after inclusion)
To evaluate the over risk of death of patients COVID+ versus COVID-
After adjusting on main clinical characteristics and treatment type
Time frame: Up to the follow-up end (28 days after inclusion)