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1.
Sci Rep ; 12(1): 3951, 2022 03 10.
Article in English | MEDLINE | ID: covidwho-1740471

ABSTRACT

The SARS-CoV-2 pandemic has brought to light the need for expedient diagnostic testing. Cost and availability of large-scale testing capacity has led to a lag in turnaround time and hindered contact tracing efforts, resulting in a further spread of SARS-CoV-2. To increase the speed and frequency of testing, we developed a cost-effective single-tube approach for collection, denaturation, and analysis of clinical samples. The approach utilizes 1 µL microbiological inoculation loops to collect saliva, sodium dodecyl sulfate (SDS) to inactivate and release viral genomic RNA, and a diagnostic reaction mix containing polysorbate 80 (Tween 80). In the same tube, the SDS-denatured clinical samples are introduced to the mixtures containing all components for nucleic acids detection and Tween 80 micelles to absorb the SDS and allow enzymatic reactions to proceed, obviating the need for further handling of the samples. The samples can be collected by the tested individuals, further decreasing the need for trained personnel to administer the test. We validated this single-tube sample-to-assay method with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) and discovered little-to-no difference between Tween- and SDS-containing reaction mixtures, compared to control reactions. This approach reduces the logistical burden of traditional large-scale testing and provides a method of deployable point-of-care diagnostics to increase testing frequency.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , SARS-CoV-2/genetics , Saliva/virology , COVID-19 Nucleic Acid Testing/instrumentation , Humans , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , Real-Time Polymerase Chain Reaction , Specimen Handling/instrumentation , Specimen Handling/methods
2.
PLoS One ; 17(3): e0264085, 2022.
Article in English | MEDLINE | ID: covidwho-1736504

ABSTRACT

Self-collected specimens can expand access to SARS-CoV-2 testing. At a large inner-city hospital 1,082 participants self-collected saliva and anterior nasal swab (ANS) samples before healthcare workers collected nasopharyngeal swab (NPS) samples on the same day. To characterize patient preferences for self-collection, this investigation explored ability, comfort, and ease of ANS and saliva self-collection for SARS-CoV-2 testing along with associated patient characteristics, including medical history and symptoms of COVID-19. With nearly all participants successfully submitting a specimen, favorable ratings from most participants (at least >79% in ease and comfort), and equivocal preference between saliva and ANS, self-collection is a viable SARS-CoV-2 testing option.


Subject(s)
COVID-19/diagnosis , Specimen Handling/methods , Adolescent , Adult , COVID-19/virology , COVID-19 Testing , Female , Georgia , Humans , Male , Middle Aged , Nasopharynx/virology , RNA, Viral/analysis , RNA, Viral/metabolism , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Saliva/virology , Young Adult
3.
Sci Rep ; 12(1): 3706, 2022 03 08.
Article in English | MEDLINE | ID: covidwho-1735275

ABSTRACT

Scaling up SARS-CoV-2 testing and tracing continues to be plagued with the limitation of the sample collection method, which requires trained healthcare workers to perform and causes discomfort to the patients. In response, we assessed the performance and user preference of gargle specimens for qRT-PCR-based detection of SARS-CoV-2 in Indonesia. Inpatients who had recently been diagnosed with COVID-19 and outpatients who were about to perform qRT-PCR testing were asked to provide nasopharyngeal and oropharyngeal (NPOP) swabs and self-collected gargle specimens. We demonstrated that self-collected gargle specimens can be an alternative specimen to detect SARS-CoV-2 and the viral RNA remained stable for 31 days at room temperature storage. The developed method was validated for use on multiple RNA extraction kits and commercially available COVID-19 RT-PCR kits. Our developed method achieved a sensitivity of 91.38% when compared to paired NPOP swab specimens (Ct < 35), with 97.10% of patients preferring the self-collected gargle method.


Subject(s)
COVID-19/diagnosis , Saliva/virology , Specimen Handling/methods , COVID-19/virology , Humans , Mouthwashes/chemistry , Nasopharynx/virology , Oropharynx/virology , RNA, Viral/analysis , RNA, Viral/metabolism , Real-Time Polymerase Chain Reaction , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Sensitivity and Specificity
4.
Sci Rep ; 12(1): 3480, 2022 03 03.
Article in English | MEDLINE | ID: covidwho-1730307

ABSTRACT

The COVID-19 pandemic has resulted in significant diversion of human and material resources to COVID-19 diagnostics, to the extent that influenza viruses and co-infection in COVID-19 patients remains undocumented and pose serious public-health consequences. We optimized and validated a highly sensitive RT-PCR based multiplex-assay for the detection of SARS-CoV-2, influenza A and B viruses in a single-test. This study evaluated clinical specimens (n = 1411), 1019 saliva and 392 nasopharyngeal swab (NPS), tested using two-assays: FDA-EUA approved SARS-CoV-2 assay that targets N and ORF1ab gene, and the PKamp-RT-PCR based assay that targets SARS-CoV-2, influenza viruses A and B. Of the 1019 saliva samples, 17.0% (174/1019) tested positive for SARS-CoV-2 using either assay. The detection rate for SARS-CoV-2 was higher with the multiplex assay compared to SARS-specific assay [91.9% (160/174) vs. 87.9% (153/174)], respectively. Of the 392 NPS samples, 10.4% (41/392) tested positive for SARS-CoV-2 using either assay. The detection rate for SARS-CoV-2 was higher with the multiplex assay compared to SARS-specific assay [97.5% (40/41) vs. 92.1% (39/41)], respectively. This study presents clinical validation of a multiplex-PCR assay for testing SARS-CoV-2, influenza A and B viruses, using NPS and saliva samples, and demonstrates the feasibility of implementing the assay without disrupting the existing laboratory workflow.


Subject(s)
Influenza A virus/isolation & purification , Influenza B virus/isolation & purification , Multiplex Polymerase Chain Reaction/methods , Nasopharynx/virology , SARS-CoV-2/isolation & purification , Saliva/virology , Humans , Limit of Detection , Reproducibility of Results
5.
J Med Microbiol ; 71(2)2022 Feb.
Article in English | MEDLINE | ID: covidwho-1709252

ABSTRACT

Introduction. The importance of human saliva in aerosol-based transmission of SARS-CoV-2 is now widely recognized. However, little is known about the efficacy of virucidal mouthwash formulations against emergent SARS-CoV-2 variants of concern and in the presence of saliva.Hypothesis. Mouthwashes containing virucidal actives will have similar inactivation effects against multiple SARS-CoV-2 variants of concern and will retain efficacy in the presence of human saliva.Aim. To examine in vitro efficacy of mouthwash formulations to inactivate SARS-CoV-2 variants.Methodology. Inactivation of SARS-CoV-2 variants by mouthwash formulations in the presence or absence of human saliva was assayed using ASTM International Standard E1052-20 methodology.Results. Appropriately formulated mouthwashes containing 0.07 % cetylpyridinium chloride but not 0.2 % chlorhexidine completely inactivated SARS-CoV-2 (USA-WA1/2020, Alpha, Beta, Gamma, Delta) up to the limit of detection in suspension assays. Tests using USA-WA1/2020 indicates that efficacy is maintained in the presence of human saliva.Conclusions. Together these data suggest cetylpyridinium chloride-based mouthwashes are effective at inactivating SARS-CoV-2 variants. This indicates potential to reduce viral load in the oral cavity and mitigate transmission via salivary aerosols.


Subject(s)
Cetylpyridinium , Mouthwashes , SARS-CoV-2 , Saliva , COVID-19 , Cetylpyridinium/pharmacology , Humans , Mouthwashes/pharmacology , SARS-CoV-2/drug effects , Saliva/virology
6.
Sci Rep ; 12(1): 2356, 2022 02 18.
Article in English | MEDLINE | ID: covidwho-1706307

ABSTRACT

Effective testing is essential to control the coronavirus disease 2019 (COVID-19) transmission. Here we report a-proof-of-concept study on hyperspectral image analysis in the visible and near-infrared range for primary screening at the point-of-care of SARS-CoV-2. We apply spectral feature descriptors, partial least square-discriminant analysis, and artificial intelligence to extract information from optical diffuse reflectance measurements from 5 µL fluid samples at pixel, droplet, and patient levels. We discern preparations of engineered lentiviral particles pseudotyped with the spike protein of the SARS-CoV-2 from those with the G protein of the vesicular stomatitis virus in saline solution and artificial saliva. We report a quantitative analysis of 72 samples of nasopharyngeal exudate in a range of SARS-CoV-2 viral loads, and a descriptive study of another 32 fresh human saliva samples. Sensitivity for classification of exudates was 100% with peak specificity of 87.5% for discernment from PCR-negative but symptomatic cases. Proposed technology is reagent-free, fast, and scalable, and could substantially reduce the number of molecular tests currently required for COVID-19 mass screening strategies even in resource-limited settings.


Subject(s)
Exudates and Transudates/virology , Mass Screening/methods , SARS-CoV-2/isolation & purification , Saliva/virology , Spectroscopy, Near-Infrared , Humans , Point-of-Care Testing , Proof of Concept Study
7.
PLoS One ; 17(2): e0264130, 2022.
Article in English | MEDLINE | ID: covidwho-1704633

ABSTRACT

The global COVID-19 pandemic has highlighted the need for rapid, accurate and accessible nucleic acid tests to enable timely identification of infected individuals. We optimized a sample-to-answer nucleic acid test for SARS-CoV-2 that provides results in <1 hour using inexpensive and readily available reagents. The test workflow includes a simple lysis and viral inactivation protocol followed by direct isothermal amplification of viral RNA using RT-LAMP. The assay was validated using two different instruments, a portable isothermal fluorimeter and a standard thermocycler. Results of the RT-LAMP assay were compared to traditional RT-qPCR for nasopharyngeal swabs, nasal swabs, and saliva collected from a cohort of patients hospitalized due to COVID-19. For all three sample types, positive agreement with RT-LAMP performed using the isothermal fluorimeter was 100% for samples with Ct <30 and 69-91% for samples with Ct <40. Following validation, the test was successfully scaled to test the saliva of up to 400 asymptomatic individuals per day as part of the campus surveillance program at Rice University. Successful development, validation, and scaling of this sample-to-answer, extraction-free real-time RT-LAMP test for SARS-CoV-2 adds a highly adaptable tool to efforts to control the COVID-19 pandemic, and can inform test development strategies for future infectious disease threats.


Subject(s)
COVID-19 Testing , COVID-19/diagnosis , Nasopharynx/virology , Nose/virology , Population Surveillance/methods , SARS-CoV-2/isolation & purification , Saliva/virology , COVID-19/virology , Humans , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , RNA, Viral/genetics , Sensitivity and Specificity
8.
Sci Rep ; 12(1): 2843, 2022 02 18.
Article in English | MEDLINE | ID: covidwho-1701343

ABSTRACT

In the context of social events reopening and economic relaunch, sanitary surveillance of SARS-CoV-2 infection is still required. Here, we evaluated the diagnostic performances of a rapid, extraction-free and connected reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay on saliva. Nasopharyngeal (NP) swabs and saliva from 443 outpatients were collected simultaneously and tested by reverse-transcription quantitative PCR (RT-qPCR) as reference standard test. Seventy-one individuals (16.0%) were positive by NP and/or salivary RT-qPCR. Sensitivity and specificity of salivary RT-LAMP were 85.9% (95%CI 77.8-94.0%) and 99.5% (98.7-100%), respectively. Performances were similar for symptomatic and asymptomatic participants. Moreover, SARS-CoV-2 genetic variants were analyzed and no dominant mutation in RT-LAMP primer region was observed during the period of the study. We demonstrated that this RT-LAMP test on self-collected saliva is reliable for SARS-CoV-2 detection. This simple connected test with optional automatic results transfer to health authorities is unique and opens the way to secure professional and social events in actual context of economics restart.


Subject(s)
COVID-19 Nucleic Acid Testing/statistics & numerical data , Molecular Diagnostic Techniques/statistics & numerical data , Nucleic Acid Amplification Techniques/statistics & numerical data , SARS-CoV-2/isolation & purification , Saliva/virology , Adult , Asymptomatic Infections , Female , Humans , Male , Mass Screening , Middle Aged , Viral Load , Young Adult
9.
Sci Rep ; 12(1): 2806, 2022 02 18.
Article in English | MEDLINE | ID: covidwho-1699734

ABSTRACT

Saliva is an attractive sample for coronavirus disease 2019 testing due its ease of collection and amenability to detect viral RNA with minimal processing. Using a direct-to-RT-PCR method with saliva self-collected from confirmed COVID-19 positive volunteers, we observed 32% false negative results. Confirmed negative and healthy volunteer samples spiked with 106 genome copies/mL of heat-inactivated severe acute respiratory syndrome coronavirus 2 showed false negative results of 10% and 13%, respectively. Additional sample heating or dilution of the false negative samples conferred only modest improvements. These results highlight the potential to significantly underdiagnose COVID-19 infections when testing directly from minimally processed heterogeneous saliva samples.


Subject(s)
COVID-19 Nucleic Acid Testing , SARS-CoV-2/isolation & purification , Saliva/virology , False Negative Reactions , Healthy Volunteers , Humans , Point-of-Care Testing
10.
Microbiol Spectr ; 10(1): e0059121, 2022 02 23.
Article in English | MEDLINE | ID: covidwho-1691413

ABSTRACT

Coronavirus disease 2019 (COVID-19) is a mild to severe respiratory illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The diagnostic accuracy of the Centers for Disease Control and Prevention (CDC)- or World Health Organization (WHO)-recommended real-time PCR (RT-qPCR) primers in clinical practice remains unproven. We conducted a prospective study on the accuracy of RT-qPCR using an in-house-designed primer set (iNP) targeting the nucleocapsid protein as well as various recommended and commercial primers. The accuracy was assessed by culturing or seroconversion. We enrolled 12 confirmed COVID-19 patients with a total of 590 clinical samples. When a cutoff value of the cycle threshold (Ct) was set to 35, RT-qPCRs with WHO RdRp primers and CDC N1, N2, and N3 primers showed sensitivity of 42.1% to 63.2% and specificity of 90.5% to 100% in sputum, and sensitivity of 65.2% to 69.6% and specificity of 65.2% to 69.6% in nasopharyngeal samples. The sensitivity and specificity of iNP RT-qPCR in sputum and nasopharyngeal samples were 94.8%/100% and 69.6%/100%, respectively. Sputum testing had the highest sensitivity, followed by nasopharyngeal testing (P = 0.0193); self-collected saliva samples yielded better characteristics than oropharyngeal samples (P = 0.0032). Our results suggest that iNP RT-qPCR has better sensitivity and specificity than RT-PCR with WHO (P < 0.0001) or CDC (N1: P = 0.0012, N2: P = 0.0013, N3: P = 0.0012) primers. Sputum RT-qPCR analysis has the highest sensitivity, followed by nasopharyngeal, saliva, and oropharyngeal assays. Our study suggests that considerable improvement is needed for the RT-qPCR WHO and CDC primer sets for detecting SARS-CoV-2. IMPORTANCE Numerous research campaigns have addressed the vast majority of clinical and diagnostic specificity and sensitivity of various primer sets of SARS-CoV2 viral detection. Despite the impressive progress made to resolve the pandemic, there is still a need for continuous and active improvement of primers used for diagnosis in clinical practice. Our study significantly exceeds the scale of previously published research on the specificity and sensitivity of different primers comparing with different specimens and is the most comprehensive to date in terms of constant monitoring of primer sets of current usage. Henceforth, our results suggest that sputum samples sensitivity is the highest, followed by nasopharyngeal, saliva, and oropharyngeal samples. The CDC recommends the use of oropharyngeal specimens, leading to certain discrepancy between the guidelines set forth by the CDC and IDSA. We proved that the oropharyngeal samples demonstrated the lowest sensitivity for the detection of SARS-CoV-2.


Subject(s)
COVID-19/diagnosis , Real-Time Polymerase Chain Reaction/standards , SARS-CoV-2/isolation & purification , Adult , Aged , COVID-19/virology , Cross Reactions , Female , Humans , Male , Middle Aged , Nasopharynx/virology , Oropharynx/virology , SARS-CoV-2/genetics , Saliva/virology , Sensitivity and Specificity , Sputum/virology , Viral Load , Young Adult
11.
Microbiol Spectr ; 10(1): e0161421, 2022 02 23.
Article in English | MEDLINE | ID: covidwho-1691406

ABSTRACT

The antigen-based rapid diagnostic test (Ag-RDT) using saliva specimens is fast, noninvasive, and suitable for SARS-CoV-2 self-testing, unlike nasopharyngeal swab (NPS) testing. We evaluated a novel Beanguard gargle (BG)-based virus collection method that can be applied to Ag-RDT as an alternative to the current RT-PCR with an NPS for early diagnosis of COVID-19. This clinical trial comprised 102 COVID-19-positive patients hospitalized after a governmental screening process and 100 healthy individuals. Paired NPS and BG-based saliva specimens from COVID-19 patients and healthy individuals were analyzed using NPS-RT-PCR, BG-RT-PCR, and BG-Ag-RDTs, whose diagnostic performance for detecting SARS-CoV-2 was compared. BG-Ag-RDTs showed high sensitivity (97.8%) and specificity (100%) in 45 patients within 6 days of illness and detected all cases of SARS-CoV-2 Alpha and Delta variants. In 11 asymptomatic active COVID-19 cases, both BG-Ag-RDTs and BG-RT-PCR showed sensitivities and specificities of 100%. Sensitivities of BG-Ag-RDT and BG-RT-PCR toward salivary viral detection were highly concordant, with no discrimination between symptomatic (97.0%), asymptomatic (100%), or SARS-CoV-2 variant (100%) cases. The intermolecular interactions between SARS-CoV-2 spike proteins and truncated canavalin, an active ingredient from the bean extract (BE), were observed in terms of physicochemical properties. The detachment of the SARS-CoV-2 receptor-binding domain from hACE2 increased as the BE concentration increased, allowing the release of the virus from hACE2 for early diagnosis. Using BG-based saliva specimens remarkably enhances the Ag-RDT diagnostic performance as an alternative to NPS and enables noninvasive, rapid, and accurate COVID-19 self-testing and mass screening, supporting efficient COVID-19 management. IMPORTANCE An Ag-RDT is less likely to be accepted as an initial test method for early diagnosis owing to its low sensitivity. However, our self-collection method, Ag-RDT using BG-based saliva specimens, showed significantly enhanced detection sensitivity and specificity toward SARS-CoV-2 including the Alpha and Delta variants in all patients tested within 6 days of illness. The method represents an attractive alternative to nasopharyngeal swabs for the early diagnosis of symptomatic and asymptomatic COVID-19 cases. The evidence suggests that the method could have a potential for mass screening and monitoring of COVID-19 cases.


Subject(s)
COVID-19 Serological Testing/methods , COVID-19/diagnosis , SARS-CoV-2/isolation & purification , Saliva/virology , Adult , Aged , Aged, 80 and over , COVID-19/virology , COVID-19 Nucleic Acid Testing , COVID-19 Serological Testing/instrumentation , Female , Humans , Male , Middle Aged , Nasopharynx/virology , Republic of Korea , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Sensitivity and Specificity , Young Adult
12.
PLoS One ; 17(2): e0263341, 2022.
Article in English | MEDLINE | ID: covidwho-1690730

ABSTRACT

Rapid and accurate detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for the successful control of the current global COVID-19 pandemic. The real-time reverse transcription polymerase chain reaction (Real-time RT-PCR) is the most widely used detection technique. This research describes the development of two novel multiplex real-time RT-PCR kits, AccuPower® COVID-19 Multiplex Real-Time RT-PCR Kit (NCVM) specifically designed for use with the ExiStation™48 system (comprised of ExiPrep™48 Dx and Exicycler™96 by BIONEER, Korea) for sample RNA extraction and PCR detection, and AccuPower® SARS-CoV-2 Multiplex Real-Time RT-PCR Kit (SCVM) designed to be compatible with manufacturers' on-market PCR instruments. The limit of detection (LoD) of NCVM was 120 copies/mL and the LoD of the SCVM was 2 copies/µL for both the Pan-sarbecovirus gene and the SARS-CoV-2 gene. The AccuPower® kits demonstrated high precision with no cross reactivity to other respiratory-related microorganisms. The clinical performance of AccuPower® kits was evaluated using the following clinical samples: sputum and nasopharyngeal/oropharyngeal swab (NPS/OPS) samples. Overall agreement of the AccuPower® kits with a Food and Drug Administration (FDA) approved emergency use authorized commercial kit (STANDARD™ M nCoV Real-Time Detection kit, SD BIOSENSOR, Korea) was above 95% (Cohen's kappa coefficient ≥ 0.95), with a sensitivity of over 95%. The NPS/OPS specimen pooling experiment was conducted to verify the usability of AccuPower® kits on pooled samples and the results showed greater than 90% agreement with individual NPS/OPS samples. The clinical performance of AccuPower® kits with saliva samples was also compared with NPS/OPS samples and demonstrated over 95% agreement (Cohen's kappa coefficient > 0.95). This study shows the BIONEER NCVM and SCVM assays are comparable with the current standard confirmation assay and are suitable for effective clinical management and control of SARS-CoV-2.


Subject(s)
COVID-19/virology , Multiplex Polymerase Chain Reaction , Nasopharynx/virology , Oropharynx/virology , Real-Time Polymerase Chain Reaction , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Saliva/virology , Sputum/virology , Cross Reactions , Humans , Limit of Detection , Sensitivity and Specificity
13.
PLoS One ; 17(1): e0259886, 2022.
Article in English | MEDLINE | ID: covidwho-1666744

ABSTRACT

COVID-19 has exposed stark inequalities between resource-rich and resource-poor countries. International UN- and WHO-led efforts, such as COVAX, have provided SARS-CoV-2 vaccines but half of African countries have less than 2% vaccinated in their population, and only 15 have reached 10% by October 2021, further disadvantaging local economic recovery. Key for this implementation and preventing further mutation and spread is the frequency of voluntary [asymptomatic] testing. It is limited by expensive PCR and LAMP tests, uncomfortable probes deep in the throat or nose, and the availability of hardware to administer in remote locations. There is an urgent need for an inexpensive "end-to-end" system to deliver sensitive and reliable, non-invasive tests in resource-poor and field-test conditions. We introduce a non-invasive saliva-based LAMP colorimetric test kit and a $51 lab-in-a-backpack system that detects as few as 4 viral RNA copies per µL. It consists of eight chemicals, a thermometer, a thermos bottle, two micropipettes and a 1000-4000 rcf electronically operated centrifuge made from recycled computer hard drives (CentriDrive). The centrifuge includes a 3D-printed rotor and a 12 V rechargeable Li-ion battery, and its 12 V standard also allows wiring directly to automobile batteries, to enable field-use of this and other tests in low infrastructure settings. The test takes 90 minutes to process 6 samples and has reagent costs of $3.5 per sample. The non-invasive nature of saliva testing would allow higher penetration of testing and wider adoption of the test across cultures and settings (including refugee camps and disaster zones). The attached graphical procedure would make the test suitable for self-testing at home, performing it in the field, or in mobile testing centers by minimally trained staff.


Subject(s)
COVID-19/diagnosis , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , RNA, Viral/analysis , COVID-19/virology , COVID-19 Nucleic Acid Testing/economics , COVID-19 Nucleic Acid Testing/methods , Colorimetry , Humans , Molecular Diagnostic Techniques/economics , Molecular Diagnostic Techniques/instrumentation , Nucleic Acid Amplification Techniques/economics , Nucleic Acid Amplification Techniques/instrumentation , Point-of-Care Systems , RNA, Viral/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Saliva/virology
14.
Viruses ; 14(2)2022 02 02.
Article in English | MEDLINE | ID: covidwho-1667351

ABSTRACT

Efficient, wide-scale testing for SARS-CoV-2 is crucial for monitoring the incidence of the infection in the community. The gold standard for COVID-19 diagnosis is the molecular analysis of epithelial secretions from the upper respiratory system captured by nasopharyngeal (NP) or oropharyngeal swabs. Given the ease of collection, saliva has been proposed as a possible substitute to support testing at the population level. Here, we used a novel saliva collection device designed to favour the safe and correct acquisition of the sample, as well as the processivity of the downstream molecular analysis. We tested 1003 nasopharyngeal swabs and paired saliva samples self-collected by individuals recruited at a public drive-through testing facility. An overall moderate concordance (68%) between the two tests was found, with evidence that neither system can diagnose the infection in 100% of the cases. While the two methods performed equally well in symptomatic individuals, their discordance was mainly restricted to samples from convalescent subjects. The saliva test was at least as effective as NP swabs in asymptomatic individuals recruited for contact tracing. Our study describes a testing strategy of self-collected saliva samples, which is reliable for wide-scale COVID-19 screening in the community and is particularly effective for contact tracing.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19 Nucleic Acid Testing/standards , RNA, Viral/analysis , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Saliva/virology , COVID-19/diagnosis , COVID-19/virology , Female , Humans , Male , Mass Screening , Nasopharynx/virology , RNA, Viral/genetics , SARS-CoV-2/isolation & purification , Specimen Handling/methods
15.
Microbiol Spectr ; 10(1): e0122021, 2022 02 23.
Article in English | MEDLINE | ID: covidwho-1636464

ABSTRACT

Accurate tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been critical in efforts to control its spread. The accuracy of tests for SARS-CoV-2 has been assessed numerous times, usually in reference to a gold standard diagnosis. One major disadvantage of that approach is the possibility of error due to inaccuracy of the gold standard, which is especially problematic for evaluating testing in a real-world surveillance context. We used an alternative approach known as Bayesian latent class modeling (BLCM), which circumvents the need to designate a gold standard by simultaneously estimating the accuracy of multiple tests. We applied this technique to a collection of 1,716 tests of three types applied to 853 individuals on a university campus during a 1-week period in October 2020. We found that reverse transcriptase PCR (RT-PCR) testing of saliva samples performed at a campus facility had higher sensitivity (median, 92.3%; 95% credible interval [CrI], 73.2 to 99.6%) than RT-PCR testing of nasal samples performed at a commercial facility (median, 85.9%; 95% CrI, 54.7 to 99.4%). The reverse was true for specificity, although the specificity of saliva testing was still very high (median, 99.3%; 95% CrI, 98.3 to 99.9%). An antigen test was less sensitive and specific than both of the RT-PCR tests, although the sample sizes with this test were small and the statistical uncertainty was high. These results suggest that RT-PCR testing of saliva samples at a campus facility can be an effective basis for surveillance screening to prevent SARS-CoV-2 transmission in a university setting. IMPORTANCE Testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been vitally important during the COVID-19 pandemic. There are a variety of methods for testing for this virus, and it is important to understand their accuracy in choosing which one might be best suited for a given application. To estimate the accuracy of three different testing methods, we used a data set collected at a university that involved testing the same samples with multiple tests. Unlike most other estimates of test accuracy, we did not assume that one test was perfect but instead allowed for some degree of inaccuracy in all testing methods. We found that molecular tests performed on saliva samples at a university facility were similarly accurate as molecular tests performed on nasal samples at a commercial facility. An antigen test appeared somewhat less accurate than the molecular tests, but there was high uncertainty about that.


Subject(s)
Antigens, Viral/analysis , COVID-19/diagnosis , SARS Virus/immunology , SARS-CoV-2/isolation & purification , Saliva/virology , Antigens, Viral/blood , Bayes Theorem , COVID-19/epidemiology , COVID-19/virology , COVID-19 Nucleic Acid Testing , Humans , Predictive Value of Tests , Prevalence , Reproducibility of Results , SARS-CoV-2/immunology , Sensitivity and Specificity , Universities , Young Adult
16.
PLoS One ; 16(12): e0260894, 2021.
Article in English | MEDLINE | ID: covidwho-1623649

ABSTRACT

BACKGROUND: Performance of the SD Biosensor saliva antigen rapid test was evaluated at a large designated testing site in non-hospitalized patients, with or without symptoms. METHOD: All eligible people over 18 years of age presenting for a booked appointment at the designated SARS-CoV-2 testing site were approached for inclusion and enrolled following verbal informed consent. One nasopharyngeal swab was taken to carry out the default antigen rapid test from which the results were reported back to the patient and one saliva sample was self-taken according to verbal instruction on site. This was used for the saliva antigen rapid test, the RT-PCR and for virus culture. Sensitivity of the saliva antigen rapid test was analyzed in two ways: i, compared to saliva RT-PCR; and ii, compared to virus culture of the saliva samples. Study participants were also asked to fill in a short questionnaire stating age, sex, date of symptom onset. Recommended time of ≥30mins since last meal, drink or cigarette if applicable was also recorded. The study was carried out in February-March 2021 for 4 weeks. RESULTS: We could include 789 people with complete records and results. Compared to saliva RT-PCR, overall sensitivity and specificity of the saliva antigen rapid test was 66.1% and 99.6% which increased to 88.6% with Ct ≤30 cutoff. Analysis by days post onset did not result in higher sensitivities because the large majority of people were in the very early phase of disease ie <3 days post onset. When breaking down the data for symptomatic and asymptomatic individuals, sensitivity ranged from 69.2% to 50% respectively, however the total number of RT-PCR positive asymptomatic participants was very low (n = 5). Importantly, almost all culture positive samples were detected by the rapid test. CONCLUSION: Overall, the potential benefits of saliva antigen rapid test, could outweigh the lower sensitivity compared to nasopharyngeal antigen rapid test in a comprehensive testing strategy, especially for home/self-testing and in vulnerable populations like elderly, disabled or children where in intrusive testing is either not possible or causes unnecessary stress.


Subject(s)
Biosensing Techniques/methods , COVID-19 Serological Testing/methods , Saliva/virology , Adolescent , Adult , Aged , COVID-19/diagnosis , COVID-19/etiology , Carrier State/virology , Female , Hospitalization , Humans , Male , Middle Aged , Nasopharynx/virology , Sensitivity and Specificity , Young Adult
17.
Dis Markers ; 2022: 6478434, 2022.
Article in English | MEDLINE | ID: covidwho-1622116

ABSTRACT

Background: Since the beginning of the pandemic, clinicians and researchers have been searching for alternative tests to improve the screening and diagnosis of the SARS-CoV-2 infection. Currently, the gold standard for virus identification is the nasopharyngeal (NP) swab. Saliva samples, however, offer clear, practical, and logistical advantages but due to a lack of collection, transport, and storage solutions, high-throughput saliva-based laboratory tests are difficult to scale up as a screening or diagnostic tool. With this study, we aimed to validate an intralaboratory 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. Methods: In 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. Results: The 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. Conclusions: RT-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.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , Saliva/virology , Adult , Aged , Case-Control Studies , Humans , Middle Aged , Nasopharynx/virology , Predictive Value of Tests , RNA, Viral/isolation & purification , SARS-CoV-2/genetics , Specimen Handling/methods
18.
J Nanobiotechnology ; 20(1): 6, 2022 Jan 04.
Article in English | MEDLINE | ID: covidwho-1608546

ABSTRACT

BACKGROUND: Gold nanoparticles (AuNPs) have been widely used in local surface plasmon resonance (LSPR) immunoassays for biomolecule sensing, which is primarily based on two conventional methods: absorption spectra analysis and colorimetry. The low figure of merit (FoM) of the LSPR and high-concentration AuNP requirement restrict their limit of detection (LOD), which is approximately ng to µg mL-1 in antibody detection if there is no other signal or analyte amplification. Improvements in sensitivity have been slow in recent for a long time, and pushing the boundary of the current LOD is a great challenge of current LSPR immunoassays in biosensing. RESULTS: In this work, we developed spectral image contrast-based flow digital nanoplasmon-metry (Flow DiNM) to push the LOD boundary. Comparing the scattering image brightness of AuNPs in two neighboring wavelength bands near the LSPR peak, the peak shift signal is strongly amplified and quickly detected. Introducing digital analysis, the Flow DiNM provides an ultrahigh signal-to-noise ratio and has a lower sample volume requirement. Compared to the conventional analog LSPR immunoassay, Flow DiNM for anti-BSA detection in pure samples has an LOD as low as 1 pg mL-1 within only a 15-min detection time and 500 µL sample volume. Antibody assays against spike proteins of SARS-CoV-2 in artificial saliva that contained various proteins were also conducted to validate the detection of Flow DiNM in complicated samples. Flow DiNM shows significant discrimination in detection with an LOD of 10 pg mL-1 and a broad dynamic detection range of five orders of magnitude. CONCLUSION: Together with the quick readout time and simple operation, this work clearly demonstrated the high sensitivity and selectivity of the developed Flow DiNM in rapid antibody detection. Spectral image contrast and digital analysis further provide a new generation of LSPR immunoassay with AuNPs.


Subject(s)
COVID-19 Serological Testing/methods , COVID-19/diagnosis , SARS-CoV-2/isolation & purification , Surface Plasmon Resonance/methods , Antibodies, Viral/immunology , COVID-19/immunology , COVID-19 Serological Testing/instrumentation , Equipment Design , Gold/chemistry , Humans , Immunoassay/instrumentation , Immunoassay/methods , Metal Nanoparticles/chemistry , SARS-CoV-2/immunology , Saliva/virology , Spike Glycoprotein, Coronavirus/immunology , Surface Plasmon Resonance/instrumentation
19.
Front Immunol ; 12: 759688, 2021.
Article in English | MEDLINE | ID: covidwho-1605844

ABSTRACT

Antibodies (Abs) are essential for the host immune response against SARS-CoV-2, and all the vaccines developed so far have been designed to induce Abs targeting the SARS-CoV-2 spike. Many studies have examined Ab responses in the blood from vaccinated and infected individuals. However, since SARS-CoV-2 is a respiratory virus, it is also critical to understand the mucosal Ab responses at the sites of initial virus exposure. Here, we examined plasma versus saliva Ab responses in vaccinated and convalescent patients. Although saliva levels were significantly lower, a strong correlation was observed between plasma and saliva total Ig levels against all SARS-CoV-2 antigens tested. Virus-specific IgG1 responses predominated in both saliva and plasma, while a lower prevalence of IgM and IgA1 Abs was observed in saliva. Antiviral activities of plasma Abs were also studied. Neutralization titers against the initial WA1 (D614G), B.1.1.7 (alpha) and B.1.617.2 (delta) strains were similar but lower against the B.1.351 (beta) strain. Spike-specific antibody-dependent cellular phagocytosis (ADCP) activities were also detected and the levels correlated with spike-binding Ig titers. Interestingly, while neutralization and ADCP potencies of vaccinated and convalescent groups were comparable, enhanced complement deposition to spike-specific Abs was noted in vaccinated versus convalescent groups and corresponded with higher levels of IgG1 plus IgG3 among the vaccinated individuals. Altogether, this study demonstrates the detection of Ab responses after vaccination or infection in plasma and saliva that correlate significantly, although Ig isotypic differences were noted. The induced plasma Abs displayed Fab-mediated and Fc-dependent functions with comparable neutralization and ADCP potencies, but a greater capacity to activate complement was elicited upon vaccination.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19 Vaccines/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Saliva/immunology , Spike Glycoprotein, Coronavirus/immunology , Adult , Aged , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Antibody Formation/immunology , COVID-19/blood , COVID-19/virology , Female , Humans , Immunoglobulin G/immunology , Male , Middle Aged , SARS-CoV-2/metabolism , SARS-CoV-2/physiology , Saliva/virology , Vaccination
20.
Microbiol Spectr ; 9(3): e0099621, 2021 12 22.
Article in English | MEDLINE | ID: covidwho-1594122

ABSTRACT

Due to increased demand for testing, as well as restricted supply chain resources, testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection continues to face many hurdles. Pooling several samples has been proposed as an alternative approach to address these issues. We investigated the feasibility of pooling nasopharyngeal swab (NPS) or saliva samples for SARS-CoV-2 testing with a commercial assay (Idylla SARS-CoV-2 test; Biocartis). We evaluated the 10-pool and 20-pool approaches for 149 subjects, with 30 positive samples and 119 negative samples. The 10-pool approach had sensitivity of 78.95% (95% confidence interval [CI], 54.43% to 93.95%) and specificity of 100% (95% CI, 71.51% to 100%), whereas the 20-pool approach had sensitivity of 55.56% (95% CI, 21.20% to 86.30%) and specificity of 100% (95% CI, 25% to 100%). No significant difference was observed between the results obtained with pooled NPS and saliva samples. Given the rapidity, full automation, and practical advantages of the Idylla SARS-CoV-2 assay, pooling of 10 samples has the potential to significantly increase testing capacity for both NPS and saliva samples, with good sensitivity. IMPORTANCE To control outbreaks of coronavirus disease 2019 (COVID-19) and to avoid reagent shortages, testing strategies must be adapted and maintained for the foreseeable future. We analyzed the feasibility of pooling NPS and saliva samples for SARS-CoV-2 testing with the Idylla SARS-CoV-2 test, and we found that sensitivity was dependent on the pool size. The SARS-CoV-2 testing capacity with both NPS and saliva samples could be significantly expanded by pooling 10 samples; however, pooling 20 samples resulted in lower sensitivity.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , Nasopharynx/virology , SARS-CoV-2/isolation & purification , Saliva/virology , Specimen Handling/methods , Adult , Diagnostic Tests, Routine , Female , Humans , Male , Middle Aged , Retrospective Studies
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