key: cord-0728424-fyu420yx
authors: Herrera, Luis A.; Hidalgo-Miranda, Alfredo; Reynoso-Noverón, Nancy; Meneses-García, Abelardo A.; Mendoza-Vargas, Alfredo; Reyes-Grajeda, Juan P.; Vadillo-Ortega, Felipe; Cedro-Tanda, Alberto; Peñaloza, Fernando; Frías-Jimenez, Emmanuel; Arriaga-Canon, Cristian; Ruiz, Rosaura; Angulo, Ofelia; López-Villaseñor, Imelda; Amador-Bedolla, Carlos; Vilar-Compte, Diana; Cornejo, Patricia; Cisneros-Villanueva, Mireya; Hurtado-Cordova, Eduardo; Cendejas-Orozco, Mariana; Hernandez-Morales, Jose S.; Moreno, Bernardo; Hernández-Cruz, Irwin A.; Herrera, César A.; García, Francisco; González-Woge, Miguel A.; Munguía-Garza, Paulina; Luna-Maldonado, Fernando; Sanchez-Vizcarra, Antonia; Osnaya, Vincent G.; Medina-Molotla, Nelly; Alfaro-Mora, Yair; Caceres-Gutiérrez, Rodrigo E.; Tolentino-Garcia, Laura; Rosas-Escobar, Patricia; Román-González, Sergio A.; Escobar-Arrazola, Marco A.; Canseco-Mendez, Julio C.; Ortiz-Soriano, Diana R.; Dominguez-Ortiz, Julieta; Gonzalez-Barrera, Ana D.; Aparicio-Bautista, Diana I.; Cruz-Rangel, Armando; Alarcón-Zendejas, Ana Paula; Contreras-Espinosa, Laura; González, Rodrigo; Guerra-Calderas, Lissania; Meraz-Rodríguez, Marco A.; Montalvo-Casimiro, Michel; Montiel-Manríquez, Rogelio; Torres-Arciga, Karla; Venegas, Daniela; Juárez-González, Vasti; Guajardo-Barreto, Xiadani; Monroy-Martínez, V.; Guillén, D.; Fernández, J.; Herrera, J.; León-Rodriguez, R.; Canela-Pérez, Israel; Ruiz-Ordaz, Blanca H.; Valdez-Vazquez, Rafael; Bertin-Montoya, Jennifer; Niembro-Ortega, María; Villegas-Acosta, Liudmila; López-Castillo, Daniela; Soriano-Ríos, Andrea; Gastelum-Ramos, Michael; Zamora-Barandas, Tonatiuh; Morales-Baez, Jorge; García-Rodríguez, María; García-Martínez, Mariano; Nieto-Patlán, Erik; Quirasco-Baruch, Maricarmen; López-Martínez, Irma; Ramírez-Gonzalez, Ernesto; Olivera-Díaz, Hiram; Escobar-Escamilla, Noe
title: Saliva is a reliable and accessible source for the detection of SARS-CoV-2
date: 2021-02-11
journal: Int J Infect Dis
DOI: 10.1016/j.ijid.2021.02.009
sha: e7b852b92ed7ef734fb1d7e0c39a790153b1597e
doc_id: 728424
cord_uid: fyu420yx

Objectives Investigate the feasibility of saliva sampling as a noninvasive and safer tool to detect SARS-CoV-2 and to compare its reproducibility and sensitivity with nasopharyngeal swab samples (NPS). The use of sample pools was also investigated. Methods 2107 paired samples were collected from asymptomatic health care and office workers in Mexico City. Sixty of these samples were also analyzed in two other independent laboratories for concordance analysis. Sample processing and analysis of virus genetic material were performed according to standard protocols described elsewhere. Pooling analysis was performed by analyzing the saliva pool and the individual pool components. Results The concordance between NPS and saliva results was 95.2% (Kappa: 0.727, p = 0.0001) and 97.9% without considering inconclusive results (Kappa: 0.852, p = 0.0001). Saliva had a lower number of inconclusive results than NPS (0.9% vs 1.9%). Furthermore, saliva shows a significantly higher concentration of both total RNA and viral copies than NPS. Comparison of our results with those of the other two laboratories shows 100% and 97% concordance. Saliva samples are stable without the use of any preservative, a positive SARS-CoV-2 sample can be detected 5, 10, and 15 days after collection when the sample is stored at 4 °C. Conclusions Our results indicate that saliva is as effective as NPS for the identification of SARS-CoV-2-infected asymptomatic patients, sample pooling facilitates the analysis of a larger number of samples with the benefit of cost reduction.

The rapid spread of SARS-CoV-2 worldwide has generated the considerable demand for medical supplies to use when fighting the pandemic, among other problems, causing a shortage in nasopharyngeal swabs (NPS) and tests for the detection of SARS-CoV-2. Scarce consumables and invasive sample collection, which can expose medical personnel to biohazards, are obstacles to effective mass screening of the population to identify infected individuals. Mass screening is essential to identify and isolate infected individuals during reopening. Additionally, fast massive effective screening is essential in the event of COVID-19 resurgence and for the safe return to productive activities, an approach that has been implemented by several governments around the globe. Although this situation has been addressed using different innovative approaches, such as 3D printing of NPS (Callahan et al., 2020) , additional solutions for sample collection that are easier and less invasive, with minimal risk to health professionals, together with strategies aiming to maximize the number of samples analyzed, must be explored.

The gold standard test for the diagnosis of SARS-CoV-2 infection involves sample collection of NPS, followed by viral RNA extraction and detection by real-time polymerase chain reaction (RT-qPCR). Recent reports have indicated that saliva is a viable option for testing with several potential advantages over NPS, including that it is a less invasive procedure, making it more viable for repeated testing. Furthermore, saliva can be self-collected by the patient with minimal guidance and intervention by healthcare personnel (Azzi et al., 2020) . SARS-CoV-2 can be detected in more than 95% of saliva samples, and the virus can be cultured from saliva samples (To et al., 2020b) . Detection of the virus in saliva has also been used to monitor viral load dynamics over time, indicating that the highest viral load in saliva presents during the first week after symptom onset and then declines over time (To et al., J o u r n a l P r e -p r o o f 2020a) . Recently, the Food and Drug Administration (FDA) in the United States approved the first diagnostic test with the option for saliva sampling for SARS-CoV-2 detection (U.S.

Food and Drug Administration, 2020a)(U.S. Food and Drug Administration, 2020a). Another study found that the home-based collection method of saliva, supervised by a clinician, performed similar or even better than NPS for infection detection (Noah et al., 2020) . These findings were confirmed by recent studies, which identified that saliva is more sensitive for SARS-CoV-2 detection than NPS in patients with COVID-19 (Wyllie et al., 2020) . In another report, 229 paired samples from 95 patients also showed a high concordance and no significant temporal variation in viral load between the two sample types (Cheuk et al., 2020) .

The combination of the advantages offered by saliva and sample pooling results in an inexpensive diagnostic procedure suitable for assaying a large number of samples as they are occurring in the current pandemic (Abdalhamid et al., 2020; Yelin et al., 2020) . Sample pooling has proven its efficacy in different applications, including retrospective testing (Hogan et al., 2020) and, more importantly, in large-scale screening of asymptomatic populations (Ben-Ami et al., 2020; Lohse et al., 2020; U.S. Food and Drug Administration, 2020b) . There is work showing that pooling saliva samples for the detection of SARS-CoV-2 provides a mechanism to support testing for a greater number of individuals with substantial cost savings, especially at lower prevalence levels (Ekawat Pasomsub et al., 2020; Watkins et al., 2020) . Mirimus Clinical Labs in its Saliva Clear test already uses the pooling strategy to monitor and detect infections in groups of symptomatic and asymptomatic individuals (SalivaClear by Mirimus Clinical, 2020).

In this study, we compared the reproducibility, accuracy and feasibility of saliva sampling using NPS followed by RT-qPCR for the detection of SARS-CoV-2 in paired samples from asymptomatic clinical and laboratory personnel of two Mexico National Institutes of Health laboratories and from asymptomatic office workers (n=2107 individuals). We present evidence that saliva sample pooling is a reliable inexpensive method that allows for the screening of a large number of samples.

A cross-sectional study design was used to collect samples from personnel engaged in clinical and laboratory activities at Mexico's National Cancer Institute and National Institute of Genomic Medicine. Consecutive asymptomatic subjects were sampled after signing an informed consent form. The study was approved by the Ethics and Research Committees of both institutes (CEI/1479/20 and CEI 2020/21). Paired saliva and NPS samples were collected from 2107 asymptomatic healthcare and office workers to compare both sample sources for SARS-CoV-2 detection. Additionally, saliva samples were collected from 3983 asymptomatic office workers, 2126 asymptomatic healthcare personnel and 846 symptomatic office workers to detect SARS-CoV-2.

NPS were collected by a trained clinician with a flexible nylon swab that was inserted through the patient's nostrils to reach the posterior nasopharynx. It was left in place for several seconds and slowly removed while rotating. The swab was then placed in 3 mL of sterile viral transport media. Swabs from both nostrils were deposited in a single viral transport tube. Saliva samples were self-collected by the individuals without any stimulation and without rinsing the mouth before sample collection. Five milliliters of saliva was collected in a 50 mL sterile conical centrifuge tube without preservatives. Samples collection was done within the same facilities where the viral diagnosis laboratory is located. They were also collected from nearby hospitals. Because of this, swabs and the saliva samples were processed for viral RNA extraction within 5 hours after collection.

Total nucleic acid was extracted from 300 µL of viral transport media from the NPS or 300 µL of whole saliva using the MagMAX Viral/Pathogen Nucleic Acid Isolation Kit (ThermoFisher Scientific) and eluted into 75 µL of elution buffer. For SARS-CoV-2 RNA detection, 5 µL of RNA template was tested using the U.S. CDC real-time RT-qPCR primer/probe sets for 2019-nCoV_N1 and 2019-nCoV_N2 and human RNase P (RP) as an extraction control Samples were classified as positive for SARS-CoV-2 when both the N1 and N2 primer-probe sets were detected with a Ct value of less than 40 (Centers for Disease Control and Prevention, 2020). If only one of these genes was detected, the sample was labeled inconclusive. All tests were run on Thermo Fisher´s ABI QuantStudio 5 or QuantStudio 7 real-time thermal cyclers.

For validation purposes, 60 samples that were analyzed in our laboratory were also processed in two independent authorized laboratories (30 samples in each laboratory: Instituto de Investigaciones Biomédicas and Facultad de Química, Universidad Nacional Autónoma de México) using two additional RNA extraction methods and detection systems. The additional extraction methods consisted of spin-column-based RNA extraction (Total RNA Purification Kit, Jena Biosciences) and the use of a quick extraction solution from Lucigen. The two additional methods for SARS-CoV-2 detection were carried out using the GoTaq Probe 1-

Step RT-qPCR System from Promega on a 7500 ABI system and the Star Q One-step RT-qPCR from Genes 2 Life. 

The accuracy of SARS-CoV-2 saliva detection, including sensitivity, sensitivity, predictive values, and likelihood ratios, was determined using RT-qPCR in NPS as the "gold standard".

Other statistical analyses were performed using GraphPad Prism 7.0 and SPSS v24 software.

One-tailed parametric (Student's t-test) and nonparametric (Mann-Whitney U) statistical tests were used to determine the significance of the data considering a statistically significant value of p = ≤0.05. The Kappa coefficient was used to estimate the concordance between saliva and NPS results (McHugh, 2012) .

The design of this study was intended to compare the reproducibility, accuracy and feasibility of saliva sampling followed by RT-qPCR to identify SARS-CoV-2 and to evaluate the use of saliva in sample pooling strategies. It was a priori accepted that the use of NPS followed by RT-qPCR is the gold standard for identification of the virus, despite current studies showing marked variation in the accuracy of this test.

A total of 2107 paired NPS and saliva samples were included in the analysis. The distribution of the results is described in Figure 1 . Concordance between saliva and NPS results was statistically significant (Cohen´s Kappa: 0.727, standard error 0.025; p=0.0001; Table 1A ).

Concordance improved when inconclusive samples were removed from the analysis (Cohen´s Kappa: 0.852, standard error = 0.022; p = 0.0001). A total of 2006 tests out of 2050 (98%) showed the same results in both saliva and NPS (Table 1B) . Saliva had a lower number of inconclusive results than NPS (0.9% vs 1.9%) ( Table 1 and Figure 1 ).

The concordance between the RT-qPCR results from viral RNA obtained from saliva and NPS was statistically significant, indicating that saliva is at least as sensitive as NPS for SARS-CoV-2 detection. Comparison of both the Ct values and the transcript copies of RNAse P showed a significantly higher total RNA concentration in saliva than in NPS (Figures 2A and 3A) . However, when we analyzed the two viral genes in the positive samples, saliva and NPS did not show significant differences in viral load (Figures 2B-C and 3B-C). Spearman correlation analysis of viral copies confirmed that saliva and NPS are both reliable sources for SARS-CoV-2 detection (N1: r=0.4217, P=0.0001; N2: r=0.4261, P=0.0001).

Saliva and paired NPS, which were previously analyzed in our laboratory (60 paired samples), were sent to two independent laboratories for extraction and SARS-CoV-2 detection and processed using different extraction and detection kits. Each laboratory processed 30 paired samples. A 100% concordance was observed in the results between our laboratory and the Instituto de Investigaciones Biomédicas (27 negatives and 3 positives both in saliva and NPS), while 96.7% of the samples sent to Facultad de Química had the same result as in our laboratory (28 negative, one positive and one discordant), this independent validation is an initial and exploratory assessment.

The accuracy of the saliva test is useful for clinical purposes. A positive likelihood ratio strongly supports its use as a reliable clinical test. We identified a statistically significant correlation and concordance of the RT-qPCR detection of the virus in the saliva samples compared to NPS, we reported a high concordance between both types of samples (Table   2A) . Given the high number of paired samples analyzed, our results show a clear indication that saliva is as good as NPS for viral detection for diagnostic of COVID-19. Our data also demonstrated that saliva is stable even without the use of any preservative during sample collection and that a positive SARS-CoV-2 sample can be detected 5, 10, and 15 days after collection when the sample is stored at 4ºC, variation in Ct values in the viral gene N was 0.88  1.92 at 5 days, -0.93  3.01 at 10 days, and -0.76  2.12 at 15 days. Other studies have also demonstrated the stability of saliva for the detection of SARS-CoV-2 out of 10 to 25 days at room temperature (Uwamino et al., 2021) without buffers or stabilizers (Ott et al., 2020) .

Positive samples were selected according to their RT-qPCR results, representing low and high Ct values, to evaluate the effect of a 1:10 pooling with negative samples in the detection capacity of the test. In the first set of saliva samples, the initial Ct values for the positive sample were 22.3 for ORF1ab, 19.6 for N and 19.8 for RNAse P. As expected, after pooling with the additional nine negative samples, the Ct values increased to 23.8 for ORF1ab, 22.4 for N and 21.6 for RNAse P, showing that pooling did not affect the detection capacity of the test. A similar situation was observed in the NPS sample pool. In the second saliva pool, the positive sample had higher Ct values (31.9 ORF1ab, 28 N and 19.1 for RNAse P). After pooling, we observed an increase in four Ct values in both viral genes. Even though this result is still within the acceptable range for detecting the positive sample in the pool ( Figure   4 ), samples with a higher Ct value might become false negatives if analyzed by pooling; for this reason, the following experiments were focused on the analysis of 5-sample pools.

We tested 130 individuals in 26 NPS pools with 5 samples each, identifying 20 positive cases (15.4%). All positive cases identified in the pools were confirmed through the analysis of the individual samples used to generate the pool. In the case of saliva, 255 individuals were grouped into 26 pools with 5 samples each. In this case, we identified 2 positive cases (7.7%), which were also confirmed through analysis of the individual samples.

Additionally, we tested asymptomatic office and healthcare personnel as well as office workers presenting mild symptoms that were suspected of being SARS-CoV-2 carriers. We used saliva only and pooled 5 samples in the first two groups. Table 2C shows the positivity among the three groups, which was increased in healthcare personnel and symptomatic office workers. A substantial reduction in direct costs for sampling compared with NPS and in the costs by testing pools instead of individual samples was observed.

J o u r n a l P r e -p r o o f considerations should be taken into account, including the use of 500 µl of saliva to generate the pool to obtain a homogeneous mixture. Dilution of 1 positive sample with 9 negative samples showed that, even though we can still obtain positive results in the pool, samples with low viral load might become difficult to detect. Therefore, we suggest pooling no more than 5 samples, even thought other reports indicate that pooling strategies of 16 and 24 samples are useful in high prevalence prevalence (≥10%) (Verwilt et al., 2020) .

Our data indicate that saliva is a reliable source for the detection of SARS-CoV-2 infection.

However, several aspects must be addressed to successfully use saliva testing: 1) Sample collection: even though saliva self-collection might be easier than NPS sampling, proper biosafety and risk evaluation protocols must be followed by medical personnel to minimize contagions due to the production of potential aerosols during saliva collection; 2) sample handling: the application of proven and standardized methods for the inactivation and handling of a saliva sample should be considered, and saliva samples must always be regarded as potentially infected. The packaging and cold-chain protocols used for NPS samples must be followed; 3) RNA extraction and RT-qPCR: it has been well documented that several components of saliva can inhibit PCR, highlighting the importance of using viral RNA extraction systems that have been tested and approved by regulatory agencies that generate pure and high-quality RNA for RT-qPCR analysis. We did not use any preservative for saliva samples and suggest that samples should be stored at 4ºC after collection and processed within 4 days post collection.

Given the abovementioned situations, the use of saliva represents a viable option for SARS-CoV-2 detection. Thus, saliva and the pooling strategy presented here are effective options for the analyses of samples in well-controlled cohorts, which provide a cost-effective screening tool in asymptomatic populations, cost reduction was calculated considering the number of tests necessary to identify the positive individuals in the positive pool. This is particularly suitable, for example, in office workers, faculty or other groups where testing is necessary on a periodic basis to identify and isolate infected individuals. Implementation of testing for SARS-CoV-2 infection by RT-qPCR using saliva as a source for viral RNA constitutes an easy, noninvasive, inexpensive and less risky option compared to NPS, without compromising the accuracy of the test. The combination of saliva sampling and pooling represents a viable and useful method for population-based studies that will be necessary for a safe return to economic activities.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 

Assessment of Specimen Pooling to Conserve SARS CoV-2 Testing Resources

Saliva is a reliable tool to detect SARS-CoV-2

Largescale implementation of pooled RNA extraction and RT-PCR for SARS-CoV-2 detection

Open development and clinical validation of multiple 3D-printed sample-collection swabs: Rapid resolution of a critical COVID-19 testing bottleneck

CDC 2019-Novel Coronavirus Panel, Real-Time RT-PCR Diagnostic 2020

Evaluating the use of posterior oropharyngeal saliva in a point-of-care assay for the detection of SARS-CoV-2

Posterior oropharyngeal saliva for the detection of SARS-CoV-2

Retrospective Screening for SARS-CoV-2 RNA in California

Spot Posterior Oropharyngeal Saliva for Diagnosis of SARS-CoV-2 Infection: Implication of Timing of Specimen Collection for Community-Wide Screening

Clinical evaluation of self-collected saliva by RT-qPCR, direct RT-qPCR, RT-LAMP, and a rapid antigen test to diagnose COVID-19

Comparison of SARS-CoV-2 detection in nasopharyngeal swab and saliva

Sensitivity of nasopharyngeal swabs and saliva for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

Deep throat saliva as an alternative diagnostic specimen type for the detection of SARS-CoV-2

Pooling of samples for testing for SARS-CoV-2 in asymptomatic people

Interrater reliability: The kappa statistic

Validation of a Self-administrable, Saliva-based RT-qPCR Test Detecting SARS-CoV-2

Saliva sampling and its direct lysis, an excellent option to increase the number of SARS-CoV-2 diagnostic tests in settings with supply shortages

Self-collected oral fluid and nasal swabs demonstrate comparable sensitivity to clinician collected nasopharyngeal swabs for Covid-19 detection

Simply saliva: Stability of SARS-CoV-2 detection negates the need for expensive collection devices

Saliva sample as a non-invasive specimen for the diagnosis of coronavirus disease 2019: a cross-sectional study

Saliva sample pooling for the detection of SARS-CoV-2

SalivaClear by Mirimus Clinical. The SalivaClear Solution

Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study

Consistent detection of 2019 novel coronavirus in saliva

First Diagnostic Test Using At-Home Collection of Saliva Specimens

Food and Drug Administration. Coronavirus (COVID-19) Update: FDA Authorizes First Diagnostic Test for Screening of People Without Known or Suspected

Infection Authorization is also second to allow testing of pooled samples 2020b

Accuracy and stability of saliva as a sample for reverse transcription PCR detection of SARS-CoV-2

Evaluation of efficiency and sensitivity of 1D and 2D sample pooling strategies for diagnostic screening purposes

Pooling saliva to increase SARS-CoV-2 testing capacity

Saliva as a noninvasive specimen for detection of sars-cov-2

Saliva is more sensitive for SARS-CoV-2 detection in COVID-19 patients than nasopharyngeal swabs

Evaluation of COVID-19 RT-qPCR Test in Multi sample Pools

Viral dynamics of SARS-CoV-2 in saliva from infected

The authors acknowledge M. C. Isabel Gracia Mora for providing BSL2 facilities at UNAM and Facultad de Química-UNAM, Patronato de la Facultad de Química-UNAM for providing all materials used in RT-qPCR analyses, and CONACyT 314298 project funding.

This work was funded by the Secretaría de Educación, Ciencia, Tecnología e Innovación de la Ciudad de México (SECTEI).The authors do not have an association that might pose a conflict of interest J o u r n a l P r e -p r o o f