The Matrix Effect in the RT-PCR Detection of SARS-CoV-2 Using Saliva without RNA Extraction

Morais, Orlando Miguel; Alves, M. Rui; Ramos, Carla; Ferreira, Fernando; Fernandes, Paulo

doi:10.3390/diagnostics12071547

Cited by 12 publications

(13 citation statements)

References 32 publications

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“…As saliva is easy to collect from a large number of people, pooling strategies are thus a natural extension to surveillance programs 17 . While pooling saliva does impact assay sensitivity and potentially decrease virus detection, as also observed by others 14 , 18 – 20 , we found that the actual impact appears to be minimal, likely due to the dilution of possible contaminants in certain saliva samples that can have an inhibitory effect on the PCR reaction 21 .…”

Section: Discussionsupporting

confidence: 76%

Pooled RNA-extraction-free testing of saliva for the detection of SARS-CoV-2

Allicock

Yolda-Carr

Todd³

et al. 2023

Sci Rep

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The key to limiting SARS-CoV-2 spread is to identify virus-infected individuals (both symptomatic and asymptomatic) and isolate them from the general population. Hence, routine weekly testing for SARS-CoV-2 in all asymptomatic (capturing both infected and non-infected) individuals is considered critical in situations where a large number of individuals co-congregate such as schools, prisons, aged care facilities and industrial workplaces. Such testing is hampered by operational issues such as cost, test availability, access to healthcare workers and throughput. We developed the SalivaDirect RT-qPCR assay to increase access to SARS-CoV-2 testing via a low-cost, streamlined protocol using self-collected saliva. To expand the single sample testing protocol, we explored multiple extraction-free pooled saliva testing workflows prior to testing with the SalivaDirect RT-qPCR assay. A pool size of five, with or without heat inactivation at 65 °C for 15 min prior to testing resulted in a positive agreement of 98% and 89%, respectively, and an increased Ct value shift of 1.37 and 1.99 as compared to individual testing of the positive clinical saliva specimens. Applying this shift in Ct value to 316 individual, sequentially collected, SARS-CoV-2 positive saliva specimen results reported from six clinical laboratories using the original SalivaDirect assay, 100% of the samples would have been detected (Ct value < 45) had they been tested in the 1:5 pool strategy. The availability of multiple pooled testing workflows for laboratories can increase test turnaround time, permitting results in a more actionable time frame while minimizing testing costs and changes to laboratory operational flow.

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Section: Discussionsupporting

confidence: 76%

Pooled RNA-extraction-free testing of saliva for the detection of SARS-CoV-2

Allicock

Yolda-Carr

Todd³

et al. 2023

Sci Rep

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“…Positive saliva and negative nasopharyngeal aspirate have also been observed in the detection of other respiratory viruses [ 51 ]. In addition, high between-person variability and false negative rates have been obtained with minimally processed saliva [ 56 , 57 ], highlighting the need to optimize collection and processing before saliva could be applied as a standardized specimen for nucleic acid testing and large-scale screening of viral infections.…”

Section: Discussionmentioning

confidence: 99%

“…First, the saliva collection and processing methods have varied among different studies, which might directly influence the testing results. Although several studies have shown that SARS-CoV-2 RNA could be detected in unprocessed saliva [ 64 , 65 ], other studies found a high false negative rate and between-person variability using minimally processed saliva [ 56 , 57 ]. In addition, the types of saliva (unstimulated, stimulated, unforced saliva, and forced deep throat saliva), the part of saliva (cell or cell-free), and behaviors before testing (eating and mouth washing) also interfere with SARS-CoV-2 detection [ 66 – 68 ].…”

Section: Discussionmentioning

confidence: 99%

Saliva-based detection of SARS-CoV-2: a bibliometric analysis of global research

et al. 2023

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Saliva has emerged as a promising noninvasive biofluid for the diagnosis of oral and systemic diseases, including viral infections. During the coronavirus disease 2019 (COVID-19) pandemic, a growing number of studies focused on saliva-based detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Taking advantage of the WoS core collection (WoSCC) and CiteSpace, we retrieved 1021 articles related to saliva-based detection of SARS-CoV-2 and conducted a comprehensive bibliometric analysis. We analyzed countries, institutions, authors, cited authors, and cited journals to summarize their contribution and influence and analyzed keywords to explore research hotspots and trends. From 2020 to 2021, research focused on viral transmission via saliva and verification of saliva as a reliable specimen, whereas from 2021 to the present, the focus of research has switched to saliva-based biosensors for SARS-CoV-2 detection. By far, saliva has been verified as a reliable specimen for SARS-CoV-2 detection, although a standardized procedure for saliva sampling and processing is needed. Studies on saliva-based detection of SARS-CoV-2 will promote the development of saliva-based diagnostics and biosensors for viral detection. Collectively, our findings could provide valuable information to help scientists perceive the basic knowledge landscapes on saliva-based detection of SARS-CoV-2, the past and current research hotspots, and future opportunities.

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“…On the other hand, saliva as specimen poses challenges for sampling handling and processing in laboratories due to its inherent properties such as viscosity and heterogeneity [10]. Furthermore, saliva contains PCR inhibitors which may affect the effectiveness for PCR-based diagnostics especially if saliva is directly used in the PCR reaction [11].…”

Section: Introductionmentioning

confidence: 99%

Comparison of the performance of SARS-CoV-2 RNA qRT-PCR testing based on expectorated and drooled saliva samples

Kay

Futschik²,

Turek

et al. 2023

Preprint

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Purpose Saliva has been considered a suitable sample material for SARS-CoV-2 testing but uncertainty remained regarding the sensitivity and reliability of different saliva collection methods for community mass testing. This study aimed to investigate the potential utility of expectorated saliva (ES) and drooled saliva (DS) through a large cohort study. Methods ES and DS samples were collected in a two-stage non-randomized prospective cohort study. Their utility for SARS-CoV-2 RNA qRT-PCR testing was assessed by comparison with results for combined throat and nose (CTN) swabs. A total of 2,878 subjects were recruited, from which 2,747 were evaluable for statistical analyses. Results Using CTN swab-based results as reference,DS- and ES-based tests showed the same high level of concordance (98% vs 98%) or specificity (99% vs 99%). Sensitivity seemed to be higher for DS than for ES (93% vs 80%) but not significantly once viral concentration was taken into account. Multivariable analysis indicated however an inferior sensitivity of saliva-based testing for female compared to male subjects with low viral burden. Assuming no false positive qRT-PCR results, an unbiased comparison showed no significant difference in sensitivity between saliva- and swab-based testing. Conclusion SARS-CoV-2 RNA testing based on saliva showed high diagnostic accuracy and can be considered an alternative where swabbing may not be tolerated or operationally feasible. Drooled saliva yielded the same diagnostic performance compared to expectorated saliva and may present a preferred option with reduced aerosol risk and increased compliance. Observed sex-specific difference in detection performance however warrant further investigations.

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The Matrix Effect in the RT-PCR Detection of SARS-CoV-2 Using Saliva without RNA Extraction

Cited by 12 publications

References 32 publications

Pooled RNA-extraction-free testing of saliva for the detection of SARS-CoV-2

Pooled RNA-extraction-free testing of saliva for the detection of SARS-CoV-2

Saliva-based detection of SARS-CoV-2: a bibliometric analysis of global research

Comparison of the performance of SARS-CoV-2 RNA qRT-PCR testing based on expectorated and drooled saliva samples

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