European Journal of Clinical Microbiology & Infectious Diseases

RT-qPCR utilising upper respiratory swabs are the diagnostic gold standard for SARS-CoV-2 despite reported low sensitivity and limited scale up due to global shortages. Saliva is a non-invasive, equipment independent alternative to swabs. We collected 145 paired saliva and nasal/throat (NT) swabs at diagnosis (day 0) and repeated on day 2 and day 7 dependent on inpatient care and day 28 for study follow up. Laboratory cultured virus was used to determine the analytical sensitivity of spiked saliva and swabs containing amies preservation media. Self-collected saliva samples were found to be consistent, and in some cases superior when compared to healthcare worker collected NT swabs from COVID-19 suspected participants. We report for the first time the analytical limit of detection of 10-2and 100 pfu/ml for saliva and swabs respectively. Saliva is a easily self-collected, highly sensitive specimen for the detection of SARS-CoV-2.

The accepted gold standard for diagnosing coronavirus disease (COVID-19) is the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA from nasopharyngeal swabs (NPS). However, shortage of reagents has made NPS collection challenging, and alternative samples need to be explored. Due to its non-invasive nature, saliva has considerable diagnostic potential. Therefore, to guide diagnostic laboratories globally, we conducted a systematic review to determine the utility of saliva for the detection of SARS-CoV-2. A systematic search of major databases (PubMed, ISI Web of Science, Scopus, and Google Scholar) was performed to identify published studies in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. There was a total of 10 publications that fit the criteria for review. Most studies collected drooled whole saliva from hospitalized patients or pipetted saliva from intubated patients. Saliva was positive in 31-92% of patients depending on the cohort and length of hospitalization. Viral loads in saliva are comparable to those in NPS and ranged from 9.9 x 102 to 1.2 x 108 copies/mL during the first week of symptoms and decrease over time. Saliva can be positive up to 20 days post-symptom onset with viral loads correlating with symptom severity and degree of tissue damage. Based on these findings, we made suggestions to guide the clinical laboratory and suggest the need for diagnostic accuracy studies for the detection of SARS-CoV-2 from saliva.

ObjectivesThe number of COVID-19 cases is increasing globally and there is an urgency for a simple non-invasive method for the detection of SARS-CoV-2. Our study aimed to demonstrate that saliva can be used as a specimen for SARS-CoV-2 detection notably for the screening of extensive population groups via pooling. MethodsTo demonstrate that saliva is an appropriate specimen for SARS-CoV-2 detection a field study including 3,660 participants was performed between September 29 and October 1, 2020. We collected paired nasopharyngeal/oropharyngeal swabs (NPS) and saliva specimens and processed them within 24 hours of collection. We performed 36 serial measurements of 8 SARS-CoV-2 positive saliva samples to confirm the stability of the specimen and completed 37 pools of saliva samples by adding one positive specimen per pool. ResultsSaliva specimens were stable for testing for up to 24 hours. Overall, 44 salival samples (1.2%) tested positive for SARS-CoV-2 during the field study. The results of saliva samples were consistent with those obtained from NPS from the same patient with 90% sensitivity (95% CI 68.3%-98.7%) and 100% specificity during the first two weeks after the onset of symptoms. Using pooling strategy 796 RT-PCR tests were performed. All pools showed 100% positivity in different pooling proportions. ConclusionsOur findings demonstrate that saliva is an appropriate specimen for pooling and SARS-CoV-2 screening with accurate diagnostic performance. Patient-performed simple specimen collection allows testing an extensive number of people rapidly, obtaining results of the spread of SARS-CoV-2 and allowing authorities to take timely measures.

In the context of an unprecedented shortage of nasopharyngeal swabs (NPS) or sample transport media during the coronavirus disease 2019 (COVID-19) crisis, alternative methods for sample collection are needed. To address this need, we validated a cell culture medium as a viral transport medium, and compared the analytical sensitivity of SARS-CoV-2 real-time RT-PCR in nasal wash (NW), oropharyngeal swab (OPS) and NPS specimens. Both the clinical and analytical sensitivity were comparable in these three sample types. OPS and NW specimens may therefore represent suitable alternatives to NPS for SARS-CoV-2 detection.

Due to global shortages of flocked nasopharyngeal swabs and appropriate viral transport media during the COVID-19 pandemic, alternate diagnostic specimens for SARS-CoV-2 detection are sought. The accuracy and feasibility of saliva samples collected and transported without specialized collection devices or media were evaluated. Saliva demonstrated good concordance with paired nasopharyngeal swabs for SARS-CoV-2 detection in 67/74 cases (90.5%), though barriers to saliva collection were observed in long-term care residents and outbreak settings. SARS-CoV-2 RNA was stable in human saliva at room temperature for up to 48 hours after initial specimen collection, informing appropriate transport time and conditions.

COVID-19 is diagnosed by detecting SARS-CoV-2 by nasopharyngeal swab (NPS) using real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Emerging evidences have shown the utility of saliva, although conflicting results have been reported regarding viral loads between NPS and saliva. We conducted a study to compare the viral loads in 42 patients with COVID-19. Both NPS and saliva specimens were simultaneously obtained at a median of 6 days (range, 1-12) after symptom onset. SARS-CoV-2 was detected in 34 (81%) using NPS (median Ct value [IQR]=27.4 [21.3, 35.6]) and 38 (90%) using saliva (median Ct value [IQR]= 28.9 [23.1, 33.6]). There was no significance difference between them (Wilcoxon signed rank test: P=0.79) and Kendalls W was 0.82, showing a high degree of agreement, indicating equivalent viral loads in NPS and saliva. After symptom onset, the Ct values of both NPS and saliva continued to increase over time, with no substantial difference. Self-collected saliva has a detection sensitivity comparable to that of NPS and is a useful diagnostic tool with mitigating uncomfortable process and the risk of aerosol transmission to healthcare workers.

With surging global demand for increased SARS-CoV-2 testing capacity, clinical laboratories seek automated, high-throughput molecular solutions, particularly for specimen types which do not rely upon supply of specialized collection devices or viral transport media (VTM). Saliva was evaluated as a diagnostic specimen for SARS-CoV-2 using the cobas(R) SARS-CoV-2 Test on the cobas(R) 6800 instrument. Saliva specimens submitted from various patient populations under investigation for COVID-19 from March-July 2020 were processed in the laboratory with sterile phosphate-buffered saline in a 1:2 dilution and vortexed with glass beads. The processed saliva samples were tested using a commercial assay for detection of the SARS-CoV-2 E gene (LightMix(R)) in comparison to the cobas(R) SARS-CoV-2 Test. 22/64 (34.4%) of the saliva samples were positive for SARS-CoV-2. Positive and negative concordance between the LightMix(R) and cobas(R) assays were 100%. There was no cross-contamination of samples observed on the cobas(R) 6800. The overall invalid rate for saliva on the cobas(R) 6800 (1/128, 0.78%) was similar to the baseline invalid rate observed for nasopharyngeal swabs/VTM and plasma samples. Saliva is a feasible specimen type for SARS-CoV-2 testing on the cobas(R) 6800, with potential to improve turnaround time and enhance testing capacity.

BackgroundThe adoption of saliva as a specimen type for SARS-CoV-2 mass surveillance can significantly increase population compliance with decreased exposure risk for healthcare workers. However, studies evaluating the clinical performance of saliva compared to nasopharyngeal swab (NPS) samples have demonstrated conflicting results regardless of the collection being in healthcare or community settings. Further, pooled testing with saliva remains a challenge owing to the ambiguous sensitivity, limit of detection (LoD), and processing challenges. To overcome these limitations, SalivaAll protocol was developed and validated as a cost-effective measure that must be used on saliva collected in health care or community settings with pooling utility for SARS-CoV-2 mass surveillance. MethodsThe study evaluated 429 matched NPS and saliva samples collected from 344 individuals in either healthcare or community setting. In phase I (protocol U), 240 matched NPS, and saliva samples were tested for SARS-CoV-2 detection by RT-PCR. In phase II (SalivaAll protocol), 189 matched NPS and saliva samples were tested, with an additional sample homogenization step for saliva before RNA extraction, followed by RT-PCR. Eighty-five saliva samples were evaluated with both protocols (U and SalivaAll). Subsequently, adopting SalivaAll protocol, a five-sample pooling strategy was evaluated for saliva samples based on FDA recommendations. ResultsIn phase I, 28.3% (68/240) samples tested positive for SARS-CoV-2 from either saliva, NPS, or both. The detection rate was lower in saliva compared to NPS samples (50.0% vs. 89.7%). In phase II, 50.2% (95/189) samples tested positive for SARS-CoV-2 from either saliva, NPS, or both. The detection rate for SARS-CoV-2 was higher in saliva compared to NPS testing (97.8% vs. 78.9%). Of the 85 saliva samples evaluated by both protocols, 57.6% (49) tested positive for SARS-CoV-2 with either protocol U, SalivaAll, or both. The detection rate was 100% for samples tested with SalivaAll, whereas it was 36.7% with protocol U. Also, the LoD with SalivaAll protocol was 20 copies/ml. The pooled testing approach demonstrated a 95% positive and 100% negative percent agreement. ConclusionThis single-site study demonstrated the variability of results reported in the literature for saliva samples, and found that the discrepancies are explained by processing challenges associated with saliva samples. We have optimized a protocol for saliva samples that results in higher sensitivity compared to NPS samples and also breaks the barrier to using pooled saliva testing for SARS-CoV-2. SummarySalivaAll is a very sensitive (LoD 20 copies/ml) cost-effective test validated on saliva collected in health care and community settings with pooling utility and submitted for FDA Emergency Use Authorization.

Background. Molecular assays on nasopharyngeal swabs remain the cornerstone of COVID-19 diagnostic. Despite massive worldwide efforts, the high technicalities of nasopharyngeal sampling and molecular assays, as well as scarce resources of reagents, limit our testing capabilities. Several strategies failed, to date, to fully alleviate this testing process (e.g. saliva sampling or antigen testing on nasopharyngeal samples). We assessed the performances of a new ELISA microplate assay quantifying SARS-CoV-2 nucleocapsid antigen (N-antigen) in serum or plasma. Methods. The specificity of the assay, determined on 63 non-COVID patients, was 98.4% (95% confidence interval [CI], 85.3 to 100). Performances were determined on 227 serum samples from 165 patients with RT-PCR confirmed SARS-CoV-2 infection included in the French COVID and CoV-CONTACT cohorts. Findings. Sensitivity was 132/142, 93.0% (95% CI, 84.7 to 100), within the first two weeks after symptoms onset. A subset of 73 COVID-19 patients had a serum collected within 24 hours following or preceding a positive nasopharyngeal swab. Among patients with high nasopharyngeal viral loads, Ct value below 30 and 33, only 1/50 and 4/67 tested negative for N-antigenemia, respectively. Among patients with a negative nasopharyngeal RT-PCR, 8/12 presented positive N-antigenemia. The lower respiratory tract was explored for 6/8 patients, showing positive PCR in 5 cases. Interpretation. This is the first demonstration of the N-antigen antigenemia during COVID-19. Its detection presented a robust sensitivity, especially within the first 14 days after symptoms onset and high nasopharyngeal viral loads. These findings have to be confirmed with higher representation of outpatients. This approach could provide a valuable new option for COVID-19 diagnosis, only requiring a blood draw and easily scalable in all clinical laboratories.

Reducing procedural discomfort for children requiring respiratory testing for SARS-CoV-2 is important in supporting testing strategies for case identification. Alternative sampling methods to nose and throat swabs, which can be self-collected, may reduce laboratory-based testing requirements and provide rapid results for clearance to attend school or hospital settings. The aim of this study was to compare preference and diagnostic sensitivity of a novel anterior nasal swab (ANS), and saliva, with a standard combined nose and throat (CTN) swab. The three samples were self-collected by children aged 5-18 years who had COVID-19 or were a household close contact. Samples were analysed by reverse transcription polymerase chain reaction (RT-PCR) on the Allplex SARS-CoV-2 Assay. Most children and parents preferred the ANS and saliva swab over the CTN swab for future testing. The ANS was highly sensitive (sensitivity 1.000 (95% Confidence Interval (CI) 0.920, 1.000)) for SARS-CoV-2 detection, compared to saliva (sensitivity 0.886, 95% CI 0.754, 0.962). We conclude the novel ANS is a highly sensitive and more comfortable method for SARS-CoV-2 detection when compared to CTN swab.

Nasopharyngeal swab sampling remains the gold standard for influenza A diagnosis but has limitations, such as dependence on medical staff, invasiveness, nosocomial transmission, and occupational exposure risk. This study aimed to investigate whether saliva and nasal vestibular swabs are suitable non-invasive alternatives to nasopharyngeal swabs for influenza A detection in severe acute respiratory infection (SARI) cases. Paired saliva and nasal vestibular swabs were collected on the same day from 16 cases diagnosed with influenza. Saliva samples demonstrated a higher sensitivity (87.5%) than did nasal vestibular swabs (31.3%) in RT-qPCR, when compared with the diagnostic results obtained from nasopharyngeal swabs. While nasal vestibular swabs showed inconsistent results, saliva samples consistently tested positive, particularly within 7 days of symptom onset (100% positive agreement). In addition, diagnosis with RT-qPCR is often delayed because it requires trained laboratory technicians and facilities with appropriate laboratory settings. Therefore, the GenPad(R), a rapid diagnostic device, was evaluated using saliva samples and showed promising performance (92.9%) compared with the efficiency of RT-qPCR. Factors such as the location of infection (upper vs. lower respiratory tract infections), sample collection timing, pre-collection instructions, and nucleic acid extraction possibly contributed to the detection efficiency. Despite the small sample size and lack of influenza-negative controls, our findings support saliva as a viable self-collected sample for influenza A diagnosis and surveillance programs. Non-invasive sampling mitigates discomfort, minimizes infection risk for healthcare workers, and improves testing capacity, particularly under frequent staff shortages during pandemics.

Children have been disproportionately affected during the COVID-19 pandemic. We aimed to assess a saliva-based algorithm for SARS-CoV-2 testing to be used in schools and childcare institutions under pandemic conditions. A weekly SARS-CoV-2 sentinel study in primary schools, kindergartens and childcare facilities was conducted over a 12-week-period. In a sub-study covering 7 weeks, 1895 paired oropharyngeal and saliva samples were processed for SARS-CoV-2 rRT-PCR testing in both asymptomatic children (n=1243) and staff (n=652). Forty-nine additional concurrent swab and saliva samples were collected from SARS-CoV-2 infected patients (patient cohort). The Salivette(R) system was used for saliva collection and assessed for feasibility and diagnostic performance. For children a mean of 1.18 ml saliva could be obtained. Based on results from both cohorts, the Salivette(R) testing algorithm demonstrated specificity of 100% (95% CI 99.7 - 100) and sensitivity of 94.9% (95% CI 81.4 - 99.1) with oropharyngeal swabs as reference. Agreement between sampling systems was 100% for moderate to high viral load situations (defined as Ct-values < 33 from oropharyngeal swabs). Comparative analysis of Ct-values derived from saliva vs. oropharyngeal swabs demonstrated a significant difference (mean 4.23; 95% CI 2.48-6.00). In conclusion, the Salivette(R) system proved to be an easy-to-use, safe and feasible saliva collection method and a more pleasant alternative to oropharyngeal swabs for SARS-CoV-2 testing in children aged 3 years and above.

In children, the gold standard for the detection of pneumococcal carriage is conventional culture of a nasopharyngeal swab. Saliva, however, has a history as one of the most sensitive methods for surveillances on pneumococcal colonisation and has recently been shown to improve carriage detection in older age groups. Here, we compared the sensitivity of nasopharyngeal and saliva samples from PCV7-vaccinated 24-month-old children for pneumococcal carriage detection using conventional and molecular diagnostic methods. Nasopharyngeal and saliva samples were collected from 288 24-month-old children during the autumn/winter, 2012/2013. All samples were first processed by conventional diagnostic techniques. Next, DNA extracted from all plate growth was tested by qPCR for the presence of pneumococcal genes piaB and lytA and a subset of serotypes. By culture, 164/288 (57%) nasopharyngeal swabs tested positive for pneumococcus, but detection was not possible from saliva due to abundant polymicrobial growth on culture-plates. Molecular methods increased the number of children pneumococci-positive to 172/288 (60%) when testing culture-enriched saliva and to 212/288 (73%) when testing nasopharyngeal samples. Altogether, by molecular methods 239/288 (83%) infants were positive, with qPCR-based carriage detection of culture-enriched nasopharyngeal swabs significantly detecting more carriers compared to either conventional culture (p<0.001) or qPCR-detection of saliva (p<0.001). However, 27/240 (11%) carriers were positive only in saliva, significantly contributing to the overall number of carriers detected (p<0.01). While testing nasopharyngeal swabs with qPCR proved most sensitive for pneumococcal detection in infants, saliva sampling could be considered as complementary to provide additional information on carriage and serotypes which may not be detected in the nasopharynx and may be particularly useful in longitudinal studies, requiring repeated sampling events.

Rapid sample-to-answer tests for detection of SARS-CoV-2 are emerging and data on their relative performance is urgently needed. We evaluated the analytical performance of two rapid nucleic acid tests, Cepheid Xpert(R) Xpress SARS-CoV-2 and Mobidiag Novodiag(R) Covid-19, in comparison to a combination reference of three large-scale PCR tests. Moreover, utility of the Novodiag(R) test in tertiary care emergency departments was assessed. In the preliminary evaluation, analysis of 90 respiratory samples resulted in 100% specificity and sensitivity for Xpert(R), whereas analysis of 107 samples resulted in 93.4% sensitivity and 100% specificity for Novodiag(R). Rapid SARS-CoV-2 testing with Novodiag(R) was made available for four tertiary care emergency departments in Helsinki, Finland between 18 and 31 May, coinciding with a rapidly declining epidemic phase. Altogether 361 respiratory specimens, together with relevant clinical data, were analyzed with Novodiag(R) and reference tests: 355/361 of the specimens were negative with both methods, and 1/361 was positive in Novodiag(R) and negative by the reference method. Of the 5 remaining specimens, two were negative with Novodiag(R), but positive with the reference method with late Ct values. On average, a test result using Novodiag(R) was available nearly 8 hours earlier than that obtained with the large-scale PCR tests. While the performance of novel sample-to-answer PCR tests need to be carefully evaluated, they may provide timely and reliable results in detection of SARS-CoV-2 and thus facilitate patient management including effective cohorting.

Identifying SARS-CoV-2 infections through aggressive diagnostic testing remains critical in tracking and curbing the spread of the COVID-19 pandemic. Collection of nasopharyngeal swabs (NPS), the preferred sample type for SARS-CoV-2 detection, has become difficult due to the dramatic increase in testing and consequential supply strain. Therefore, alternative specimen types have been investigated, that provide similar detection sensitivity with reduced health care exposure and potential for self-collection. In this study, the detection sensitivity of SARS-CoV-2 in nasal swabs (NS) and saliva was compared to that of NPS, using matched specimens from two outpatient cohorts in New York State (total n = 463). The first cohort showed only a 5.4% positivity but the second cohort (n=227) had a positivity rate of 41%, with sensitivity in NPS, NS and saliva of 97.9%, 87.1%, and 87.1%, respectively. Whether the reduced sensitivity of NS or saliva is acceptable must be assessed in the settings where they are used. However, we sought to improve on it by validating a method to mix the two sample types, as the combination of nasal swab and saliva resulted in 94.6% SARS-CoV-2 detection sensitivity. Spiking experiments showed that combining them did not adversely affect the detection sensitivity in either. Virus stability in saliva was also investigated, with and without the addition of commercially available stabilizing solutions. The virus was stable in saliva at both 4{degrees}C and room temperature for up to 7 days. The addition of stabilizing solutions did not enhance stability and in some situations reduced detectable virus levels.

ObjectivesAmid the increasing number of global pandemic coronavirus disease 2019 (COVID-19) cases, there is a need for a quick and easy method to obtain a non-invasive sample for the detection of this novel coronavirus 2019 (SARS-CoV-2). We aimed to investigate the potential use of saliva samples as a non-invasive tool for the diagnosis of COVID-19. MethodsFrom 27 March to 4 April, 2020, we prospectively collected saliva samples and a standard nasopharyngeal and throat swab in persons seeking care at an acute respiratory infection clinic in a university hospital during the outbreak of COVID-19. Real-time polymerase chain reaction (RT-PCR) was performed, and the results of the two specimens were compared. ResultsTwo-hundred pairs of the samples were collected. Sixty-nine (34.5%) patients were male, and the median (interquartile) age was 36 (28-48) years. Using nasopharyngeal and throat swab RT-PCR as the reference standard, the prevalence of COVID-19 diagnosed by nasopharyngeal and throat swab RT-PCR was 9.5%. The sensitivity and specificity of the saliva sample RT-PCR were 84.2% [95% confidence interval (CI) 79.2%-89.3%], and 98.9% (95% CI 97.5-100.3%), respectively. An analysis of the agreement between the two specimens demonstrated 97.5% observed agreement (kappa coefficient 0.851, 95% CI 0.723-0.979; p <0.001). ConclusionsSaliva specimens can be used for the diagnosis of COVID-19. The collection method is non-invasive, and non-aerosol generating. Using a saliva sample as a specimen for the detection of SARS-CoV-2 could facilitate the diagnosis of the disease, which is one of the strategies that helps in controlling the epidemic.

Respiratory exacerbations are a frequent cause of hospitalisation in children with severe neurodisability (ND). Direct aspiration of food/saliva, reflux aspiration of gastric contents or a combination of both is thought to be a common cause of respiratory symptoms and disease, particularly when this occurs silently. A number of aspiration biomarkers, including bile acids and pepsin, have been proposed, however, no gold-standard diagnostic tests are currently available. In children with severe ND at high risk of both direct and reflux aspiration, we analysed lower airway samples for saliva- and/or gastric-specific proteins with biomarker potential.

The performance of Oracol collection device (Oracol) for oral fluid sampling for SARS-CoV-2 molecular detection was compared to nasopharyngeal swabs (NPS). Samples (n = 128) were collected from symptomatic and asymptomatic adults after PCR confirmed COVID-19 on NPS. All samples were tested by 3 real-time PCRs (RT-qPCR) for E, N and RdPR genes. Accuracy of Oracol compared to NPS was 38.28 % for E-gene, 47.66 % for RdRp-gene and 83.59 % for N-gene. A clear correlation between the N-gene Ct values on NPS and on Oracols was observed (p < 0.001). The sensitivity of the Oracol compared to the NPS increased in participants with an N-gene Ct value <30 on NPS (89.08%,) reaching 93.2 % with a N-gene Ct value <25. Oracol can be considered an alternative to NPS to detect SARS-CoV-2, especially in populations where NPS is not well tolerated.

BackgroundNasopharyngeal sampling has been the standard collection method for COVID-19 testing. Due to its invasive nature and risk of contamination for health care workers who collect the sample, non-invasive and safe sampling methods like saliva, can be used alternatively. MethodsA rapid systematic search was performed in PubMed and medRxiv, with the last retrieval on June 6th, 2020. Studies were included if they compared saliva with nasopharyngeal sampling for the detection of SARS-CoV-2 RNA using the same RT-qPCR applied on both types of samples. The primary outcome of interest was the relative sensitivity of SARS-CoV-2 testing on saliva versus nasopharyngeal samples (used as the comparator test). A secondary outcome was the proportion of nasopharyngeal-positive patients that tested also positive on a saliva sample. ResultsEight studies were included comprising 1070 saliva-nasopharyngeal sample pairs allowing assessment of the first outcome. The relative sensitivity of SARS-CoV-2 testing on saliva versus nasopharyngeal samples was 0.97 (95% CI=0.92-1.02). The second outcome incorporated patient data (n=257) from four other studies (n=97 patients) pooled with four studies from the first outcome (n=160 patients). This resulted in a pooled proportion of nasopharyngeal positive cases that was also positive on saliva of 86% (95% CI=77-93%). DiscussionSaliva could potentially be considered as an alternative sampling method when compared to nasopharyngeal swabs. However, studies included in this review often were small and involved inclusion of subjects with insufficient information on clinical covariates. Most studies included patients who were symptomatic (78%, 911/1167). Therefore, additional and larger studies should be performed to verify the relative performance of saliva in the context of screening of asymptomatic populations and contact-tracing.

RT-qPCR on nasopharyngeal swabs is currently the reference COVID-19 diagnosis method. We developed a multiplex RT-ddPCR assay, targeting six SARS-CoV-2 genomic regions, and evaluated it on nasopharyngeal swabs and saliva samples collected from 130 COVID-19 positive or negative ambulatory individuals, who presented symptoms suggestive of mild or moderate Sars-CoV2 infection. The 6-plex RT-ddPCR assay was shown to have 100% sensitivity on nasopharyngeal swabs and a higher sensibility than RT-qPCR on saliva (85% versus 62%). Saliva samples from 2 individuals with negative results on nasopharyngeal swabs were found to be positive. These results show that multiplex RT-ddPCR should represent an alternative and complementary tool for the diagnosis of COVID-19, in particular to control RT-qPCR ambiguous results, and its application to saliva an appropriate strategy for repetitive sampling and testing individuals for whom nasopharyngeal swabbing is not possible.