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Review 1: "Validation of a Saliva-Based Test for the Molecular Diagnosis of SARS-CoV-2 Infection"

This study finds that saliva samples can be used for population screening at a high rate of sensitivity and specificity, supporting other studies already showing that saliva tests can be comparable to NP swabs for COVID-19 testing. While reliable, there are some study flaws.

Published onOct 21, 2021
Review 1: "Validation of a Saliva-Based Test for the Molecular Diagnosis of SARS-CoV-2 Infection"
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ABSTRACTBackgroundSince the beginning of the pandemic, clinicians and researchers have been searching for alternative tests to improve screening and diagnosis of SARS-CoV-2 infection (Y. Yang et al., medRxiv 2020; W. Wang et al., 2020.3786; A Senok et al., Infect Drug Resist 2020). Currently, the gold standard for virus identification is the nasopharyngeal (NP) swab (N. Sethuraman et al., JAMA 2020; A.J. Jamal et al Clinical Infect Disease 2021). Saliva samples, however, offer clear practical and logistical advantages (K.K.W To et al, Clinical Microb and Infect; A.L. Wylle et al. N Engl J Med 2020; N. Matic et al, Eur J Clin 2021) but due to lack of collection, transport, and storage solutions, high-throughput saliva-based laboratory tests are difficult to scale up as a screening or diagnostic tool (D. Esser et al., Biomark Insights 2008; E. Kaufman et al., Crit Rev Oral Biol Med2002). With this study, we aimed to validate an intra-laboratory molecular detection method for SARS-CoV-2 on saliva samples collected in a new storage saline solution, comparing the results to NP swabs to determine the difference in sensitivity between the two tests.MethodsIn this study, 156 patients (cases) and 1005 asymptomatic subjects (controls) were enrolled and tested simultaneously for the detection of the SARS-CoV-2 viral genome by RT-PCR on both NP swab and saliva samples. Saliva samples were collected in a preservative and inhibiting saline solution (Biofarma Srl). Internal method validation was performed to standardize the entire workflow for saliva samples.ResultsThe identification of SARS-CoV-2 conducted on saliva samples showed a clinical sensitivity of 95.1% and specificity of 97.8% compared to NP swabs. The positive predictive value (PPV) was 81% while the negative predictive value (NPV) was 99.5%. Test concordance was 97.6% (Cohen’s Kappa=0.86; 95% CI 0.81-0.91). The LoD of the test was 5 viral copies for both samples.ConclusionsRT-PCR assays conducted on a stored saliva sample achieved similar performance to those on NP swabs and this may provide a very effective tool for population screening and diagnosis. Collection of saliva in a stabilizing solution makes the test more convenient and widely available; furthermore, the denaturing properties of the solution reduce the infective risks belonging to sample manipulation.

RR:C19 Evidence Scale rating by reviewer:

  • Reliable. The main study claims are generally justified by its methods and data. The results and conclusions are likely to be similar to the hypothetical ideal study. There are some minor caveats or limitations, but they would/do not change the major claims of the study. The study provides sufficient strength of evidence on its own that its main claims should be considered actionable, with some room for future revision.


Review: The manuscript shows a useful saliva-based validation study with comparison data from both saliva and the golden standard samples - NP swab samples. It shows the potential use of the saliva samples in population screening since the saliva samples are more accessible. They have very good sensitivity and specificity, and decent reproducibility due to the feature of their stability. In my opinion, it can be considered as publishable if the authors solve some of my questions as below:

1. The authors collected samples from previously tested patients but did not mention the previous testing methods for the patients. Are those diagnostic tests and screening tests certain PCR-based tests on NP swabs? The authors’ testing methods in this study, including RNA extraction and PCR test on NP swabs, should be both validated and confirmed to have the similar accuracy to the previous tools, or the authors should quote a reference as a bridge study to show their test’s accuracy. Otherwise, the authors should at least calculate their current test’s sensitivity and specificity, by using the data of the cases that they used in the study to compare to the previous testing results.

2. Since there are Ct value related viral load evaluations in the study, it is necessary to briefly mention the volume of saliva collected and the input volume for the RNA extraction. This is also helpful for understanding the settings of the LoD in the assay and the association between viral load and severity, and comparing these datasets to the ones in other studies.

3. What are the sampling criteria for the diagnosed and symptomatic cases? Is there a sampling window according to the onset of symptom, hospital admission or ICU? It is important to describe the sampling time since the viral loads change along with the course of disease.

4. It is interesting that the saliva in stabilizing saline has higher viral load than the fresh saliva does. Since the authors used 24h and 48h time points in the sample storage study, the data of Ct values should be displayed. Is there any growing trend along with time? This data may reveal if the virus possibly replicates in the cells in the stabilizing saline, in the sense of when the conserving saline does not deactivate the virus or lyse the cells.

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