Project description:Mass testing is fundamental to face the pandemic caused by the coronavirus SARS-CoV-2 discovered at the end of 2019. To this aim, it is necessary to establish reliable, fast, and cheap tools to detect viral particles in biological material so to identify the people capable of spreading the infection. We demonstrate that a colorimetric biosensor based on gold nanoparticle (AuNP) interaction induced by SARS-CoV-2 lends itself as an outstanding tool for detecting viral particles in nasal and throat swabs. The extinction spectrum of a colloidal solution of multiple viral-target gold nanoparticles-AuNPs functionalized with antibodies targeting three surface proteins of SARS-CoV-2 (spike, envelope, and membrane)-is red-shifted in few minutes when mixed with a solution containing the viral particle. The optical density of the mixed solution measured at 560 nm was compared to the threshold cycle (Ct) of a real-time PCR (gold standard for detecting the presence of viruses) finding that the colorimetric method is able to detect very low viral load with a detection limit approaching that of the real-time PCR. Since the method is sensitive to the infecting viral particle rather than to its RNA, the achievements reported here open a new perspective not only in the context of the current and possible future pandemics, but also in microbiology, as the biosensor proves itself to be a powerful though simple tool for measuring the viral particle concentration.
Project description:Saliva is an attractive sample for detecting SARS-CoV-2. However, contradictory reports exist concerning the sensitivity of saliva versus nasal swabs. We followed close contacts of COVID-19 cases for up to 14 days from the last exposure and collected self-reported symptoms, midturbinate swabs (MTS), and saliva every 2 or 3 days. Ct values, viral load, and frequency of viral detection by MTS and saliva were compared. Fifty-eight contacts provided 200 saliva-MTS pairs, and 14 contacts (13 with symptoms) had one or more positive samples. Saliva and MTS had similar rates of viral detection (P = 0.78) and substantial agreement (κ = 0.83). However, sensitivity varied significantly with time since symptom onset. Early on (days -3 to 2), saliva had 12 times (95% CI: 1.2, 130) greater likelihood of viral detection and 3.2 times (95% CI: 2.8, 3.8) higher RNA copy numbers compared to MTS. After day 2 of symptoms, there was a nonsignificant trend toward greater sensitivity using MTS. Saliva and MTS demonstrated high agreement making saliva a suitable alternative to MTS for SARS-CoV-2 detection. Saliva was more sensitive early in the infection when the transmission was most likely to occur, suggesting that it may be a superior and cost-effective screening tool for COVID-19. IMPORTANCE The findings of this manuscript are increasingly important with new variants that appear to have shorter incubation periods emerging, which may be more prone to detection in saliva before detection in nasal swabs. Therefore, there is an urgent need to provide the science to support the use of a detection method that is highly sensitive and widely acceptable to the public to improve screening rates and early detection. The manuscript presents the first evidence that saliva-based RT-PCR is more sensitive than MTS-based RT-PCR in detecting SARS-CoV-2 during the presymptomatic period - the critical period for unwitting onward transmission. Considering other advantages of saliva samples, including the lower cost, greater acceptability within the general population, and less risk to health care workers, our findings further supported the use of saliva to identify presymptomatic infection and prevent transmission of the virus.
Project description:Widely available and easily accessible testing for COVID-19 is a cornerstone of pandemic containment strategies. Nasopharyngeal swabs (NPS) are the currently accepted standard for sample collection but are limited by their need for collection devices and sampling by trained healthcare professionals. The aim of this study was to compare the performance of saliva to NPS in an outpatient setting. This was a prospective study conducted at three centers, which compared the performance of saliva and NPS samples collected at the time of assessment center visit. Samples were tested by real-time reverse transcription polymerase chain reaction and sensitivity and overall agreement determined between saliva and NPS. Clinical data was abstracted by chart review for select study participants. Of the 432 paired samples, 46 were positive for SARS-CoV-2, with seven discordant observed between the two sample types (four individuals testing positive only by NPS and three by saliva only). The observed agreement was 98.4% (kappa coefficient 0.91) and a composite reference standard demonstrated sensitivity of 0.91 and 0.93 for saliva and NPS samples, respectively. On average, the Ct values obtained from saliva as compared to NPS were higher by 2.76. This study demonstrates that saliva performs comparably to NPS for the detection of SARS-CoV-2. Saliva was simple to collect, did not require transport media, and could be tested with equipment readily available at most laboratories. The use of saliva as an acceptable alternative to NPS could support the use of widespread surveillance testing for SARS-CoV-2.
Project description:The genome of the Omicron variant of concern (VOC) contains more than 50 mutations, many of which have been associated with increased transmissibility, differing disease severity, and the potential to elute immune responses acquired after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination or infection with previous VOCs. Due to a better tropism for the upper respiratory tract, it was suggested that the detection of the Omicron variant could be preferred in saliva, compared to nasopharyngeal swabs (NPS). Our objective was to compare the SARS-CoV-2 levels in saliva fluid and NPS to estimated Ct values, according to the main SARS-CoV-2 variants circulating in France since the beginning of 2021. We analyzed 1,289 positive reverse transcription-polymerase chain reaction (RT-PCR) results during the three major waves: Alpha, Delta, and Omicron. NPS and saliva sampling were performed for 909 (71%) and 380 (29%) cases, respectively. The Ct values were significantly lower in the NPS samples than in the saliva samples for the three main VOCs. Still, the difference was less pronounced with the Omicron variant than for the Alpha and Delta variants. In contrast, in the saliva samples, Ct values were significantly lower for the Omicron variant than for the Delta (difference of -2.7 Ct) and the Alpha (difference of -3.0 Ct) variants, confirming a higher viral load in saliva. To conclude, the higher viral load in saliva was evidenced for the Omicron variant, compared to the Alpha and Delta variants, suggesting that established diagnostic methods might require revalidation with the emergence of novel variants. IMPORTANCE Established methods for SARS-CoV-2 diagnostics might require revalidation with the emergence of novel variants. This is important for screening strategy programs and for the investigation of the characteristics of new variants in terms of tropism modification and increased viral burden leading to its spread. SARS-CoV-2 RT-PCR screening on saliva samples reported lower but acceptable performance, compared to nasopharyngeal samples. Due to a better tropism for the upper respiratory tract, it was suggested that the detection of the Omicron variant could be preferred in saliva, compared to nasopharyngeal swabs. Our study analyzed 1,289 positive RT-PCR results during the three major waves in France: Alpha, Delta, and Omicron. Our findings also showed a higher viral load in saliva for the Omicron variant, compared to the Alpha and Delta variants.
Project description:The gold standard method in the diagnosis of SARS-CoV-2 infection is the detection of viral RNA in the nasopharyngeal sample by RT-PCR. Recently, saliva samples have been suggested as an alternative sample. In the present study, we aimed to compare RT-PCR results in nasopharyngeal, oro-nasopharyngeal and saliva samples of COVID-19 patients. 98 of 200 patients were positive in RT-PCR analysis performed before the hospitalization. On day 0, at least one sample was positive in 67 % of 98 patients. The positivity rate was 83 % for both oro-nasopharyngeal and nasopharyngeal samples, while it was 63 % for saliva samples (p?<?0.001). On day 5, RT-PCR was performed in 59 patients, 34 % had at least one positive result. The positivity rate was 55 % for both saliva and nasopharyngeal samples, while it was 60 % for oro-nasopharyngeal samples. Our study shows that the sampling saliva does not increase the sensitivity of RT-PCR tests at the early stages of infection. However, on the 5th day, viral RNA detection rates in saliva were similar to nasopharyngeal and oro-nasopharyngeal samples. In conclusion, we suggest that, in patients receiving treatment, RT-PCR in saliva, in addition to the standard samples, is important to determine the isolation period and control transmission.
Project description:Nasopharyngeal (NP) swabs are considered the highest-yield sample for diagnostic testing for respiratory viruses, including SARS-CoV-2. The need to increase capacity for SARS-CoV-2 testing in a variety of settings, combined with shortages of sample collection supplies, have motivated a search for alternative sample types with high sensitivity. We systematically reviewed the literature to understand the performance of alternative sample types compared to NP swabs. We systematically searched PubMed, Google Scholar, medRxiv, and bioRxiv (last retrieval 1 October 2020) for comparative studies of alternative specimen types (saliva, oropharyngeal [OP], and nasal [NS] swabs) versus NP swabs for SARS-CoV-2 diagnosis using nucleic acid amplification testing (NAAT). A logistic-normal random-effects meta-analysis was performed to calculate % positive alternative-specimen, % positive NP, and % dual positives overall and in subgroups. The QUADAS 2 tool was used to assess bias. From 1,253 unique citations, we identified 25 saliva, 11 NS, 6 OP, and 4 OP/NS studies meeting inclusion criteria. Three specimen types captured lower % positives (NS [82%, 95% CI: 73 to 90%], OP [84%, 95% CI: 57 to 100%], and saliva [88%, 95% CI: 81 to 93%]) than NP swabs, while combined OP/NS matched NP performance (97%, 95% CI: 90 to 100%). Absence of RNA extraction (saliva) and utilization of a more sensitive NAAT (NS) substantially decreased alternative-specimen yield of positive samples. NP swabs remain the gold standard for diagnosis of SARS-CoV-2, although alternative specimens are promising. Much remains unknown about the impact of variations in specimen collection, processing protocols, and population (pediatric versus adult, late versus early in disease course), such that head-to head studies of sampling strategies are urgently needed.
Project description:The processing of swabs for respiratory virus detection involves vortexing while still in the viral transport medium (VTM). The effect of not vortexing swabs prior to analysis has not been studied extensively for SARS-CoV-2 detection, and presents an opportunity to improve pre-analytic laboratory workflow. We aimed to assess the impact of not vortexing nasopharyngeal/throat swabs submitted in VTM for SARS-CoV-2 testing. To assess the impact of not vortexing swabs, 277 swab samples were tested for SARS-CoV-2 RNA in paired vortexed and non-vortexed aliquots using eight routine nucleic acid amplification assays. We compared the qualitative (positive/negative) and semi-quantitative (cycle threshold, Ct) results. Following discordant analysis, all but one non-vortexed sample had the same qualitative result as the vortexed sample. 27.4 % of samples were SARS-CoV-2 positive. Comparison of Ct values revealed an apparent reduction in human cellular nucleic acid in the non-vortexed samples (mean Ct values of 24.0 and 26.5 for vortexed and non-vortexed samples, respectively, p < 0.0001) and increased Ct values for non-vortexed samples using a laboratory-developed SARS-CoV-2 assay (mean Ct values of 4.1 and 4.2 for vortexed and non-vortexed samples, respectively; p < 0.0001), but this was not observed for a more automated commercial SARS-CoV-2 assay (mean Ct values of 15.2 for both vortexed and non-vortexed samples, respectively; p = 0.68). While vortexing swabs appears to improve the recovery of cellular material, it does not have an appreciable impact on the qualitative sensitivity of SARS-CoV-2 nucleic acid tests, which may support omission of this step and simplification of front-end sample processing.
Project description:BackgroundNasal swabs and saliva samples are being considered alternatives to nasopharyngeal swabs (NPSs) for detecting severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2); however, few studies have compared the usefulness of nasal swabs, NPSs, and saliva samples for detecting SARS-CoV-2 and other respiratory virus infections. We compared the positivity rates and concentrations of viruses detected in nasal swabs, NPSs, and saliva samples using cycle threshold (Ct) values from real-time PCR tests for respiratory viruses.MethodsIn total, 236 samples (48 five-rub and 10 10-rub nasal swabs, 96 NPSs collected using two different products, 48 saliva swabs, and 34 undiluted saliva samples) from 48 patients (34 patients with SARS-CoV-2 and 14 with other respiratory virus infections) and 40 samples from eight healthy controls were obtained. The PCR positivity and Ct values were compared using Allplex Respiratory Panels 1/2/3 and Allplex SARS-CoV-2 real-time PCR.ResultsNPSs showed the lowest Ct values (indicating the highest virus concentrations); however, nasal and saliva samples yielded positive results for SARS-CoV-2 and other respiratory viruses. The median Ct value for SARS-CoV-2 E gene PCR using nasal swab samples collected with 10 rubs was significantly different from that obtained using nasal swabs collected with five rubs (Ct=24.3 vs. 28.9; P=0.002), but not from that obtained using NPSs.ConclusionsOur results confirm that the NPS is the best sample type for detecting respiratory viruses, but nasal swabs and saliva samples can be alternatives to NPSs. Vigorously and sufficiently rubbed nasal swabs can provide SARS-CoV-2 concentrations similar to those obtained with NPSs.