Dataset Information

Clinical Performance of Direct RT-PCR Testing of Raw Saliva for Detection of SARS-CoV-2 in Symptomatic and Asymptomatic Individuals.

ABSTRACT: RT-PCR tests based on RNA extraction from nasopharyngeal swabs (NPS) are promoted as the "gold standard" for SARS-CoV-2 detection. However, the use of saliva samples offers noninvasive self-collection more suitable for high-throughput testing. This study evaluated performance of the TaqPath COVID-19 Fast PCR Combo kit 2.0 assay for detection of SARS-CoV-2 in raw saliva relative to a lab-developed direct RT-PCR test (SalivaDirect-based PCR, SDB-PCR) and an RT-PCR test based on RNA extraction from NPS. Saliva and NPS samples were collected from symptomatic and asymptomatic individuals (N = 615). Saliva samples were tested for SARS-CoV-2 using the TaqPath COVID-19 Fast PCR Combo kit 2.0 and the SDB-PCR, while NPS samples were tested by RT-PCR in RNA extracts according to the Irish national testing system. TaqPath COVID-19 Fast PCR Combo kit 2.0 detected SARS-CoV-2 in 52 saliva samples, of which 51 were also positive with the SDB-PCR. Compared to the NPS "gold standard" biospecimen method, 49 samples displayed concordant results, while three samples (35IMPORTANCE The scale of the COVID-19 pandemic highlighted the need for viral diagnostic systems that are accurate and could be deployed at large population scales. Large-scale diagnostic or surveillance testing of large numbers of people requires collection of infected biological samples that is easy and rapid. Here, we demonstrate that raw saliva samples can be easily collected and tested by RT-PCR assays. Indeed, we find that direct testing of raw saliva by two different RT-PCR assays is as accurate (if not more accurate) than nasal swab-based RT-PCR testing. We present a cost-effective and highly scalable system for high-throughput COVID-19 rapid-testing.

SUBMITTER: Castillo-Bravo R

PROVIDER: S-EPMC9769602 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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Clinical Performance of Direct RT-PCR Testing of Raw Saliva for Detection of SARS-CoV-2 in Symptomatic and Asymptomatic Individuals.

Castillo-Bravo Rosa R Lucca Noel N Lai Linyi L Marlborough Killian K Brychkova Galina G Sakhteh Maryam Shideh MS Lonergan Charlie C O'Grady Justin J Alikhan Nabil-Fareed NF Trotter Alexander J AJ Page Andrew J AJ Smyth Breda B McKeown Peter C PC Feenstra Jelena D M JDM Ulekleiv Camilla C Sorel Oceane O Gandhi Manoj M Spillane Charles C

Microbiology spectrum 20221121 6

RT-PCR tests based on RNA extraction from nasopharyngeal swabs (NPS) are promoted as the "gold standard" for SARS-CoV-2 detection. However, the use of saliva samples offers noninvasive self-collection more suitable for high-throughput testing. This study evaluated performance of the TaqPath COVID-19 Fast PCR Combo kit 2.0 assay for detection of SARS-CoV-2 in raw saliva relative to a lab-developed direct RT-PCR test (SalivaDirect-based PCR, SDB-PCR) and an RT-PCR test based on RNA extraction from ...[more]

PMID: 36409097

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Project description:The Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) pandemic demanded rapid diagnosis to isolate new COVID-19 cases and prevent disease transmission. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) rapidly became the gold standard for diagnosis. However, due to the high cost and delay of the results, other types of diagnosis were implemented, such as COVID-19 Ag Rapid Tests and Reverse Transcription Technique followed by Loop-Mediated isothermal Amplification (RT-LAMP). In this work, we validated the use of RT-LAMP in saliva samples rather than nasopharyngeal swabs, as the collection is more comfortable. First, we selected 5 primer sets based on the limit of detection for SARS-CoV-2 RNA, then validated their sensitivity and specificity in patient samples. A total of 117 samples were analyzed by fluorometric RT-LAMP and compared with qRT-PCR results. Our results show that the use of a high-sensitive primer ORF1-a, together with a low-sensitive primer set Gene E (time to threshold of 22.9 and 36.4 minutes, respectively, using 200 copies of viral RNA), achieved sensitivity in purified RNA from saliva samples of 95.2% (95% CI 76.1‒99.8) with 90.5% specificity (95% CI 69.6‒98.8) (n = 42).As RNA purification increases the turnaround time, we tested the outcome of RT-LAMP utilizing raw saliva samples without purification. The test achieved a sensitivity of 81.8% (95% CI 59.7‒94.8) and a specificity of 90.9% (95% CI 70.8‒98.8). As a result, the accuracy of 92.9% (95% CI 80.5‒98.5) in purified RNA-saliva samples was lowered to an acceptable level of 86.4% (95% CI 72.6‒94.8) in raw saliva. Although mass vaccination has been implemented, new strains and low vaccination progress helped to spread COVID-19. This study shows that it is feasible to track new COVID-19 cases in a large population with the use of raw saliva as sample in RT-LAMP assay which yields accurate results and offers a less invasive test.

Project description:BackgroundSensitive and high throughput molecular detection assays are essential during the coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The vast majority of the SARS-CoV-2 molecular assays use nasopharyngeal swab (NPS) or oropharyngeal swab (OPS) specimens collected from suspected individuals. However, using NPS or OPS as specimens has apparent drawbacks, e.g. the collection procedures for NPS or OPS specimens can be uncomfortable to some people and may cause sneezing and coughing which in turn generate droplets and/or aerosol particles that are of risk to healthcare workers, requiring heavy use of personal protective equipment. There have been recent studies indicating that self-collected saliva specimens can be used for molecular detection of SARS-CoV-2 and provides more comfort and ease of use for the patients. Here we report the performance of QuantiVirus™ SARS-CoV-2 test using saliva as the testing specimens with or without pooling.MethodsDevelopment and validation studies were conducted following FDA-EUA and molecular assay validation guidelines. Using SeraCare Accuplex SARS-CoV-2 reference panel, the limit of detection (LOD) and clinical performance studies were performed with the QuantiVirus™ SARS-CoV-2 test. For clinical evaluation, 85 known positive and 90 known negative clinical NPS samples were tested. Additionally, twenty paired NPS and saliva samples collected from recovering COVID-19 patients were tested and the results were further compared to that of the Abbott m2000 SARS-CoV-2 PCR assay. Results of community collected 389 saliva samples for COVID-19 screening by QuantiVirus™ SARS-CoV-2 test were also obtained and analyzed. Additionally, testing of pooled saliva samples was evaluated.ResultsThe LOD for the QuantiVirus™ SARS-CoV-2 test was confirmed to be 100-200 copies/mL. The clinical performance studies using contrived saliva samples indicated that the positive percentage agreement (PPA) of the QuantiVirus™ SARS-CoV-2 test is 100% at 1xLOD, 1.5xLOD and 2.5xLOD. No cross-reactivity was observed for the QuantiVirus™ SARS-CoV-2 test with common respiratory pathogens. Testing of clinical samples showed a positive percentage agreement (PPA) of 100% (95% CI: 94.6% to 100%) and a negative percentage agreement (NPA) of 98.9% (95% CI: 93.1% to 99.9%). QuantiVirus™ SARS CoV-2 test had 80% concordance rate and no significant difference (p = 0.13) between paired saliva and NPS specimens by Wilcoxon matched pairs signed rank test. Positive test rate was 1.79% for 389 saliva specimens collected from local communities for COVID-19 screening. Preliminary data showed that saliva sample pooling up to 6 samples (1:6 pooling) for SARS-CoV-2 detection is feasible (sensitivity 94.8% and specificity 100%).ConclusionThe studies demonstrated that the QuantiVirus™ SARS-CoV-2 test has a LOD of 200 copies/mL in contrived saliva samples. The clinical performance of saliva-based testing is comparable to that of NPS-based testing. Pooling of saliva specimens for SARS-CoV-2 detection is feasible. Saliva based and high-throughput QuantiVirus™ SARS-CoV-2 test offers a highly desirable testing platform during the ongoing COVID-19 pandemic.

Project description:The coronavirus disease 2019 (COVID-19) cases could be symptomatic or asymptomatic. We (1) characterized and analyzed data collected from the first cohort of reverse transcriptase polymerase chain reaction (RT-PCR)-confirmed COVID-19 cases reported in the Emirate of Abu Dhabi, United Arab Emirates, according to the symptomatic state, and (2) identified factors associated with the symptomatic state. The association between the symptomatic state and testing positive in three subsequent RT-PCR testing rounds was also quantified. Between February 28 and April 8, 2020, 1,249 cases were reported. Sociodemographic characteristics, working status, travel history, and chronic comorbidities of 791 cases were analyzed according to the symptomatic state (symptomatic or asymptomatic). After the first confirmatory test, the results of three subsequent tests were analyzed. The mean age of the 791 cases was 35.6 ± 12.7 years (range: 1-81). Nearly 57.0% of cases were symptomatic. The two most frequent symptoms were fever (58.0%) and cough (41.0%). Symptomatic cases (mean age 36.3 ± 12.6 years) were significantly older than asymptomatic cases (mean age 34.5 ± 12.7 years). Compared with nonworking populations, working in public places (adjusted odds ratio (aOR), 1.76, 95% confidence interval (95% CI): 1.11-2.80), healthcare settings (aOR, 2.09, 95% CI: 1.01-4.31), or in the aviation and tourism sectors (aOR, 2.24, 95% CI: 1.14-4.40) was independently associated with the symptomatic state. Reporting at least one chronic comorbidity was also associated with symptomatic cases (aOR, 1.76, 95% CI: 1.03-3.01). Compared with asymptomatic cases, symptomatic cases had a prolonged duration of viral shedding and consistent odds of ?2 positive COVID-19 tests result out of the three subsequent testing rounds. A substantial proportion of the diagnosed COVID-19 cases in the Emirate of Abu Dhabi were asymptomatic. Quarantining asymptomatic cases, implementing prevention measures, and raising awareness among populations working in high-risk settings are warranted.

Project description:Accurate and early diagnoses are prerequisites for prompt treatment. For coronavirus disease 2019 (COVID-19), it is even more crucial. Currently, choice of methods include rapid diagnostic tests and reverse transcription polymerase chain reaction (RT-PCR) using samples mostly of respiratory origin and sometimes saliva. We evaluated two rapid diagnostic tests with three specimen types using viral transport medium (VTM) containing naso-oropharyngeal (NOP) swabs, direct nasal and direct nasopharyngeal (NP) samples from 428 prospective patients. We also performed RT-PCR for 428 NOP VTM and 316 saliva samples to compare results. The sensitivity of the SD Biosensor Standard Q COVID-19 antigen (Ag) test kit drastically raised from an average of 65.55% (NOP VTM) to 85.25% (direct nasal samples), while RT-PCR was the gold standard. For the CareStart kit, the sensitivity was almost similar for direct NP swabs; the average was 84.57%. The specificities were ≥95% for both SD Biosensor Standard Q and CareStart COVID-19 Ag tests in all platforms. The kits were also able to detect patients with different variants as well. Alternatively, RT-PCR results from saliva and NOP VTM samples showed high sensitivities of 96.45% and 95.48% with respect to each other as standard. The overall results demonstrated high performance of the rapid tests, indicating the suitability for regular surveillance at clinical facilities when using direct nasal or direct NP samples rather than NOP VTM. Additionally, the analysis also signifies not showed that RT-PCR of saliva can be used as an choice of method to RT-PCR of NOP VTM, providing an easier, non-invasive sample collection method. IMPORTANCE There are several methods for the diagnosis of coronavirus disease 2019 (COVID-19), and the choice of methods depends mostly on the resources and level of sensitivity required by the user and health care providers. Still, reverse transcription polymerase chain reaction (RT-PCR) has been chosen as the best method using direct naso-oropharyngeal swabs. There are also other methods of fast detection, such as rapid diagnostic tests (RDTs), which offer result within 15 to 20 min and have become quite popular for self-testing and in the clinical setting. The major drawback of the currently used RT-PCR method is compliance, as it may cause irritation, and patients often refuse to test in such a way. RDTs, although inexpensive, suffer from low sensitivity due to technical issues. In this article, we propose saliva as a noninvasive source for RT-PCR samples and evaluate various specimen types at different times after infection for the best possible output from COVID-19 rapid tests.

Project description:Abstract Background Anterior nasal swabs (ANS) are established specimen collection methods for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection detection. While saliva (SA) specimens provide an alternative, few studies have comprehensively characterized the performance of SA specimens in longitudinal studies. Methods We compared SARS-CoV-2 detections between paired self-collected ANS and SA specimens from a household transmission study. Participants recorded symptoms and paired ANS and SA specimens daily for 14 days. Specimens were tested using RT-PCR. We calculated the proportion of detections identified by each specimen type among the detections from both types combined. We computed percent agreement and Kappa statistics to assess concordance in detections. We also computed estimates stratified by presence of symptoms and examined the influence of traditional and inactivating transport media on the performance of ANS. Results We examined 2535 self-collected paired specimens from 216 participants. Among 1238 (49%) paired specimens with detections by either specimen type, ANS identified 77.1% (954; 95% CI, 74.6% to 79.3%) and SA 81.9% (1014; 95% CI, 79.7% to 84.0%), with a difference of 4.9% (95% CI, 1.4% to 8.5%). Overall agreement was 80.0%, and Kappa was 0.6 (95% CI, 0.5 to 0.6). Nevertheless, the difference in the proportion of detections identified by ANS and SA using traditional and inactivating transport media was 32.5% (95% CI, 26.8% to 38.0%) and –9.5% (95% CI, −13.7% to –5.2%), respectively. Among participants who remained asymptomatic, the difference in detections between SA and ANS was 51.2% (95% CI, 31.8% to 66.0%) and 26.1% (95% CI, 0% to 48.5%) using traditional and inactivating media, respectively. Conclusions Self-collected saliva specimens provide a noninvasive alternative to nasal swabs, especially to those collected in traditional transport media, for longitudinal field studies that aim to detect both symptomatic and asymptomatic SARS-CoV-2 infections.

Dataset Information

Clinical Performance of Direct RT-PCR Testing of Raw Saliva for Detection of SARS-CoV-2 in Symptomatic and Asymptomatic Individuals.

Publications

Clinical Performance of Direct RT-PCR Testing of Raw Saliva for Detection of SARS-CoV-2 in Symptomatic and Asymptomatic Individuals.

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