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. 2020 Oct 20;12(10):1184.
doi: 10.3390/v12101184.

Frequency and Duration of SARS-CoV-2 Shedding in Oral Fluid Samples Assessed by a Modified Commercial Rapid Molecular Assay

Licia Bordi  1 , Giuseppe Sberna  1 , Eleonora Lalle  1 , Pierluca Piselli  2 , Francesca Colavita  1 , Emanuele Nicastri  3 , Andrea Antinori  3 , Evangelo Boumis  3 , Nicola Petrosillo  3 , Luisa Marchioni  3 , Giulia Minnucci  4 , Elena D'Agostini  4 , Concetta Castilletti  1 , Franco Locatelli  5 , Alimuddin Zumla  6   7 , Giuseppe Ippolito  8 , Maria Rosaria Capobianchi  1 , On Behalf Of Inmi ReCOVeRI Study Group
Affiliations

Frequency and Duration of SARS-CoV-2 Shedding in Oral Fluid Samples Assessed by a Modified Commercial Rapid Molecular Assay

Licia Bordi et al. Viruses. .

Abstract

Background: RT-PCR on nasopharyngeal (NPS)/oropharyngeal swabs is the gold standard for diagnosis of SARS-CoV-2 infection and viral load monitoring. Oral fluid (OF) is an alternate clinical sample, easy and safer to collect and could be useful for COVID-19 diagnosis, monitoring viral load and shedding.

Methods: Optimal assay conditions and analytical sensitivity were established for the commercial Simplexa™ COVID-19 Direct assay adapted to OF matrix. The assay was used to test 337 OF and NPS specimens collected in parallel from 164 hospitalized patients; 50 bronchoalveolar lavage (BAL) specimens from a subgroup of severe COVID-19 cases were also analysed.

Results: Using Simplexa™ COVID-19 Direct on OF matrix, 100% analytical detection down to 1 TCID50/mL (corresponding to 4 × 103 copies (cp)/mL) was observed. No crossreaction with other viruses transmitted through the respiratory toute was observed. Parallel testing of 337 OF and NPS samples showed highly concordant results (κ = 0.831; 95 % CI = 0.771-0.891), and high correlation of Ct values (r = 0.921; p < 0.0001). High concordance and elevated correlation was observed also between OF and BAL. Prolonged viral RNA shedding was observed up to 100 days from symptoms onset (DSO), with 32% and 29% positivity observed in OF and NPS samples, respectively, collected between 60 and 100 DSO.

Conclusions: Simplexa™ COVID-19 Direct assays on OF have high sensitivity and specificity to detect SARS-CoV-2 RNA and provide an alternative to NPS for diagnosis and monitoring SARS-CoV-2 shedding.

Keywords: SARS-CoV-2 RNA; direct assay; oral fluid; real time RT-PCR.

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Conflict of interest statement

Giulia Minnucci and Elena D’Agostino are employees of the DiaSorin S.p.A. (Gerenzano, Italy). In no way their contribution to the completion of the study influenced the study design and the analysis of the results. No additional conflict of interest or other competing relationships exist.

Figures

Figure 1
Figure 1
Probit analysis of Simplexa Covid-19 Direct Assay applied to OF samples spiked with virus preparation both for S gene (A) and ORF1ab gene (B).
Figure 2
Figure 2
(A) Correlation between Simplexa™ COVID-19 Direct assay and reference method applied to OF samples. Ct values obtained on 41 OF samples tested in parallel with Simplexa™ COVID-19 Direct assay and RT-PCR by Corman VM reference method are included in the linear regression analysis (r = 0.770; p < 0.0001). (B) Correlation between NPS and OF samples. Results obtained from 292 samples tested for the presence of SARS-CoV-2 RNA both in NPS and OF matrices are included in linear regression adjusted for cluster, sex and age (r = 0.921; p < 0.0001).
Figure 3
Figure 3
Shedding of SARS-CoV-2 RNA in OF and NPS samples based on days from symptom onset. Results obtained from 162 samples tested for the presence of SARS-CoV-2 RNA both in OF (A) and NPS (B) and expressed as Ct values vs. days from symptoms onset. Red symbols refer to samples coming from patients with severe COVID-19 disease, while black symbols refer to paucisymptomatic + asymptomatic patients.
Figure 4
Figure 4
Shedding of SARS-CoV-2 RNA in (A) OF and (B) NPS samples from individuals with repeated measures, according to days from symptoms.
Figure 5
Figure 5
Shedding of SARS-CoV-2 RNA in OF, NPS and BAL samples based on data symptoms onset and correlation analyses. Results obtained from 50 samples coming from 12 patients with severe COVID-19 disease in the Intensive Care Unit, showing more severe presentation (PaO2/FiO2 < 100) tested for the presence of SARS-CoV-2 RNA in OF ((A), round symbol), NPS ((C), triangular symbol) and BAL ((E), square symbol) and expressed as Ct values vs. days from symptoms onset. Correlation analysis from Ct values obtained from NPS vs. OF (B), BAL vs. OF (D) and NPS vs. BAL (F).
Figure 6
Figure 6
Shedding of SARS-CoV-2 RNA in OF (A), NPS (B) and BAL (C) NPS samples from individuals with repeated measures, according to days from symptoms.
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