Assessment of Multiplex Digital Droplet RT-PCR as a Diagnostic Tool for SARS-CoV-2 Detection in Nasopharyngeal Swabs and Saliva Samples

Cassinari, Kévin; Alessandri-Gradt, Elodie; Chambon, Pascal; Charbonnier, Françoise; Gracias, Ségolène; Beaussire, Ludivine; Alexandre, Kévin; Sarafan-Vasseur, Nasrin; Houdayer, Claude; Etienne, Manuel; Caron, François; Plantier, Jean Christophe; Frébourg, Thierry

doi:10.1093/clinchem/hvaa323

Cited by 34 publications

(40 citation statements)

References 24 publications

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“…It must be considered that the presence of SARS-CoV-2 RNA in saliva and negative results in nasopharyngeal samples analyzed by RT-PCR cannot be classified as false positive, but a misclassification of nasopharyngeal samples. This pivotal view is well-documented in a previous study that showed 71% of matched detection of SARS-CoV-2 RNA in saliva and nasopharyngeal swabs, 21% only in saliva, and 8% only in nasopharyngeal swabs ( 52 ). It can be related with the limitations in nasopharyngeal swab procedure and/or with low produced nasopharyngeal mucous secretion in COVID-19 patients.…”

Section: Future Directionssupporting

confidence: 63%

One-Year Update on Salivary Diagnostic of COVID-19

Caixeta

Oliveira

Cardoso-Sousa

et al. 2021

Front. Public Health

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Background: Coronavirus disease 2019 (COVID-19) is a global health problem, which is challenging healthcare worldwide. In this critical review, we discussed the advantages and limitations in the implementation of salivary diagnostic platforms of COVID-19. The diagnostic test of COVID-19 by invasive nasopharyngeal collection is uncomfortable for patients and requires specialized training of healthcare professionals in order to obtain an appropriate collection of samples. Additionally, these professionals are in close contact with infected patients or suspected cases of COVID-19, leading to an increased contamination risk for frontline healthcare workers. Although there is a colossal demand for novel diagnostic platforms with non-invasive and self-collection samples of COVID-19, the implementation of the salivary platforms has not been implemented for extensive scale testing. Up to date, several cross-section and clinical trial studies published in the last 12 months support the potential of detecting SARS-CoV-2 RNA in saliva as a biomarker for COVID-19, providing a self-collection, non-invasive, safe, and comfortable procedure. Therefore, the salivary diagnosis is suitable to protect healthcare professionals and other frontline workers and may encourage patients to get tested due to its advantages over the current invasive methods. The detection of SARS-CoV-2 in saliva was substantial also in patients with a negative nasopharyngeal swab, indicating the presence of false negative results. Furthermore, we expect that salivary diagnostic devices for COVID-19 will continue to be used with austerity without excluding traditional gold standard specimens to detect SARS-CoV-2.

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Section: Future Directionssupporting

confidence: 63%

One-Year Update on Salivary Diagnostic of COVID-19

Caixeta

Oliveira

Cardoso-Sousa

et al. 2021

Front. Public Health

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“…Similarly, Suo et al reported that 26 patients deemed negative by RT-PCR (out of 77 patients) were detected as positive by ddPCR 27 . In another study, multiplex ddPCR was used to test saliva and NP samples from 130 individuals with COVID-19 symptoms, and ddPCR was found to be superior to RT-PCR in determining positive cases 28 . Results published by several recent studies also support the use of ddPCR as a sensitive method for SARS-CoV-2 detection and quantification 29 – 31 .…”

Section: Discussionmentioning

confidence: 99%

Sensitive detection and quantification of SARS-CoV-2 in saliva

Abasıyanık

Flood

Lin

et al. 2021

Sci Rep

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Saliva has significant advantages as a test medium for detection of SARS-CoV-2 infection in patients, such as ease of collection, minimal requirement of supplies and trained personnel, and safety. Comprehensive validation in a large cohort of prospectively collected specimens with unknown SARS-CoV-2 status should be performed to evaluate the potential and limitations of saliva-based testing. We developed a saliva-based testing pipeline for detection of SARS-CoV-2 nucleic acids using real-time reverse transcription PCR (RT-PCR) and droplet digital PCR (ddPCR) readouts, and measured samples from 137 outpatients tested at a curbside testing facility and 29 inpatients hospitalized for COVID-19. These measurements were compared to the nasal swab results for each patient performed by a certified microbiology laboratory. We found that our saliva testing positively detects 100% (RT-PCR) and 93.75% (ddPCR) of curbside patients that were identified as SARS-CoV-2 positive by the Emergency Use Authorization (EUA) certified nasal swab testing assay. Quantification of viral loads by ddPCR revealed an extremely wide range, with 1 million-fold difference between individual patients. Our results demonstrate for both community screening and hospital settings that saliva testing reliability is on par with that of the nasal swabs in detecting infected cases, and has potential for higher sensitivity when combined with ddPCR in detecting low-abundance viral loads that evade traditional testing methods.

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“…Experimental setups, such as specimen type and primer–probe sets used, can impact test accuracy, which ranges from 47% to 100% in various PCR studies. 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 LAMP also differs in reported accuracy (61–100%) depending on the viral content of the sample and reference test used. 12 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 This large variation in reported accuracies of PCR and LAMP is due to the absence of a standardised measure for evaluating diagnostic test accuracy and the experimental setups that vary across studies.…”

Section: Introductionmentioning

confidence: 99%

“…Even though many studies have assessed the accuracy of the PCR and LAMP methods separately, their experimental setups differ greatly, leading to test result variations and disagreement in conclusions on which nucleic acid test is the most accurate. 6 , 7 , 8 , 9 , 10 , 11 , 13 , 14 , 15 , 17 , 20 , 21 , 24 , 25 , 26 , 28 , 29 , 35 , 36 , 37 , 38 , 39 For example, of the three studies that previously reviewed diagnostic tests, including chest CT, antigen tests, isothermal amplification, and qPCR, a small number of trials and limited subgroups were analysed with large experimental variations. 4 , 40 , 41 …”

Section: Introductionmentioning

confidence: 99%

Diagnostic performances of common nucleic acid tests for SARS-CoV-2 in hospitals and clinics: a systematic review and meta-analysis

Cheung

2021

The Lancet Microbe

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Background An optimised standard experimental setup across different hospitals is urgently needed to ensure consistency in nucleic acid test results for SARS-CoV-2 detection. A standard comparison across different nucleic acid tests and their optimal experimental setups is not present. We assessed the performance of three common nucleic acid tests, namely digital PCR (dPCR), quantitative PCR (qPCR), and loop-mediated isothermal amplification (LAMP), to detect SARS-CoV-2 in clinical settings. Methods In this systematic review and meta-analysis we compared sensitivity and specificity of qPCR, dPCR, and LAMP and their performances when different experimental setups (namely specimen type used, use of RNA extraction, primer–probe sets, and RNA extraction methods) are applied. We searched PubMed, BioRxiv, MedRxiv, SciFinder, and ScienceDirect for studies and preprints published between Feb 29 and Dec 15, 2020. Included dPCR, qPCR, and LAMP studies using any type of human specimens should report the number of true-positive, true-negative, false-positive, and false-negative cases with Emergency Use Authorization (EUA)-approved PCR assays as the comparator. Studies with a sample size of less than ten, descriptive studies, case studies, reviews, and duplicated studies were excluded. Pooled sensitivity and specificity were computed from the true and false positive and negative cases using Reitsma's bivariate random-effects and bivariate latent class models. Test performance reported in area under the curve (AUC) of the three nucleic acid tests was further compared by pooling studies with similar experimental setups (eg, tests that used RNA extracted pharyngeal swabs but with either the open reading frame 1ab or the N primer). Heterogeneity was assessed and reported in I 2 and τ 2 . Findings Our search identified 1277 studies of which we included 66 studies (11 dPCR, 32 qPCR, and 23 LAMP) with 15 017 clinical samples in total in our systematic review and 52 studies in our meta-analysis. dPCR had the highest pooled diagnostic sensitivity (94·1%, 95% CI 88·9–96·6, by Reitsma's model and 95·8%, 54·9–100·0, by latent class model), followed by qPCR (92·7%, 88·3–95·6, and 93·4%, 60·9–99·9) and LAMP (83·3%, 76·9–88·2, and 86·2%, 20·7–99·9), using EUA-approved PCR kits as the reference standard. LAMP was the most specific with a pooled estimate of 96·3% (93·8–97·8) by Reitsma's model and 94·3% (49·1–100·0) by latent class model, followed by qPCR (92·9%, 87·2–96·2, and 93·1%, 47·1–100·0) and dPCR (78·5%, 57·4–90·8, and 73·8%, 0·9–100·0). The overall heterogeneity was I 2 0·5% (τ 2 2·79) for dPCR studies, 0% (4·60) for qPCR studies, and 0% (3·96) for LAMP studies. AUCs of the three nucleic acid tests were the highest and differed the least between tests (ie, AUC>0·98 for all tests) when performed with RNA extracted pharynge...

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Assessment of Multiplex Digital Droplet RT-PCR as a Diagnostic Tool for SARS-CoV-2 Detection in Nasopharyngeal Swabs and Saliva Samples

Cited by 34 publications

References 24 publications

One-Year Update on Salivary Diagnostic of COVID-19

One-Year Update on Salivary Diagnostic of COVID-19

Sensitive detection and quantification of SARS-CoV-2 in saliva

Diagnostic performances of common nucleic acid tests for SARS-CoV-2 in hospitals and clinics: a systematic review and meta-analysis

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