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1.
Sci Rep ; 12(1): 3480, 2022 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-35241679

RESUMO

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.


Assuntos
Vírus da Influenza A/isolamento & purificação , Vírus da Influenza B/isolamento & purificação , Reação em Cadeia da Polimerase Multiplex/métodos , Nasofaringe/virologia , SARS-CoV-2/isolamento & purificação , Saliva/virologia , Humanos , Limite de Detecção , Reprodutibilidade dos Testes
2.
Sci Rep ; 12(1): 3706, 2022 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-35260654

RESUMO

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.


Assuntos
COVID-19/diagnóstico , Saliva/virologia , Manejo de Espécimes/métodos , COVID-19/virologia , Humanos , Antissépticos Bucais/química , Nasofaringe/virologia , Orofaringe/virologia , RNA Viral/análise , RNA Viral/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Sensibilidade e Especificidade
3.
PLoS One ; 17(3): e0264085, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35263342

RESUMO

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.


Assuntos
COVID-19/diagnóstico , Manejo de Espécimes/métodos , Adolescente , Adulto , COVID-19/virologia , Teste para COVID-19 , Feminino , Georgia , Humanos , Masculino , Pessoa de Meia-Idade , Nasofaringe/virologia , RNA Viral/análise , RNA Viral/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Saliva/virologia , Adulto Jovem
4.
Sci Rep ; 12(1): 3951, 2022 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-35273232

RESUMO

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.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , SARS-CoV-2/genética , Saliva/virologia , Teste de Ácido Nucleico para COVID-19/instrumentação , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Reação em Cadeia da Polimerase em Tempo Real , Manejo de Espécimes/instrumentação , Manejo de Espécimes/métodos
5.
PLoS One ; 17(2): e0264130, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35213596

RESUMO

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.


Assuntos
Teste para COVID-19 , COVID-19/diagnóstico , Nasofaringe/virologia , Nariz/virologia , Vigilância da População/métodos , SARS-CoV-2/isolamento & purificação , Saliva/virologia , COVID-19/virologia , Humanos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral/genética , Sensibilidade e Especificidade
6.
J Med Microbiol ; 71(2)2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-35180046

RESUMO

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.


Assuntos
Cetilpiridínio , Antissépticos Bucais , SARS-CoV-2 , Saliva , COVID-19 , Cetilpiridínio/farmacologia , Humanos , Antissépticos Bucais/farmacologia , SARS-CoV-2/efeitos dos fármacos , Saliva/virologia
7.
PLoS Negl Trop Dis ; 16(2): e0010206, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35139066

RESUMO

The tiger mosquito was introduced to the Eastern region of the Mediterranean basin more than twenty years ago. In Lebanon, it was first observed in 2002 in a limited number of locations mainly from the coastal area of the country. In the absence of national entomological control program, this invasive mosquito became an established species and is now considered in many localities, a source of nuisance because of its human biting behavior. Several entomological surveys were conducted to monitor the geographic spread and the seasonal dynamics of Aedes albopictus by collecting adult stages and by monitoring oviposition activity. Moreover, its susceptibility to the common groups of insecticides was assessed using WHO standard bioassays. Previous vector competence studies revealed that local strains were able to transmit Chikungunya and Dengue viruses. Due to the increased risk of Zika virus introduction in the country, we determined the competence of local populations to transmit this virus. Mapping results showed that Ae. albopictus is mainly spread in the relatively humid western versant of the Mount Lebanon chain reaching 1000m altitude, while it is absent from arid and semi-arid inland areas. Besides, this mosquito is active during 32 weeks from spring till the end of autumn. Local strains of the tiger mosquito are susceptible to pyrethroids and carbamates but resistant to organophosphates and organochlorines. They showed ability to transmit Zika virus; however, only 9% of females were capable to excrete the virus in their saliva at day 28 post infection. Current and previous observations highlight the need to establish a surveillance system in order to control this mosquito and monitor the potential introduction of related diseases.


Assuntos
Aedes/fisiologia , Espécies Introduzidas/estatística & dados numéricos , Mosquitos Vetores/fisiologia , Aedes/efeitos dos fármacos , Aedes/virologia , Distribuição Animal , Animais , Vírus da Dengue/genética , Vírus da Dengue/isolamento & purificação , Feminino , Inseticidas/farmacologia , Líbano , Masculino , Mosquitos Vetores/efeitos dos fármacos , Mosquitos Vetores/virologia , Piretrinas/farmacologia , Saliva/virologia , Estações do Ano , Zika virus/genética , Zika virus/isolamento & purificação
8.
Microbiol Spectr ; 10(1): e0059121, 2022 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-35170995

RESUMO

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.


Assuntos
COVID-19/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/normas , SARS-CoV-2/isolamento & purificação , Adulto , Idoso , COVID-19/virologia , Reações Cruzadas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Nasofaringe/virologia , Orofaringe/virologia , SARS-CoV-2/genética , Saliva/virologia , Sensibilidade e Especificidade , Escarro/virologia , Carga Viral , Adulto Jovem
9.
Viruses ; 14(2)2022 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-35215902

RESUMO

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.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , Teste de Ácido Nucleico para COVID-19/normas , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , SARS-CoV-2/genética , Saliva/virologia , COVID-19/diagnóstico , COVID-19/virologia , Feminino , Humanos , Masculino , Programas de Rastreamento , Nasofaringe/virologia , RNA Viral/genética , SARS-CoV-2/isolamento & purificação , Manejo de Espécimes/métodos
10.
Sci Rep ; 12(1): 2806, 2022 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-35181678

RESUMO

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.


Assuntos
Teste de Ácido Nucleico para COVID-19 , SARS-CoV-2/isolamento & purificação , Saliva/virologia , Reações Falso-Negativas , Voluntários Saudáveis , Humanos , Testes Imediatos
11.
Sci Rep ; 12(1): 2843, 2022 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-35181680

RESUMO

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.


Assuntos
Teste de Ácido Nucleico para COVID-19/estatística & dados numéricos , Técnicas de Diagnóstico Molecular/estatística & dados numéricos , Técnicas de Amplificação de Ácido Nucleico/estatística & dados numéricos , SARS-CoV-2/isolamento & purificação , Saliva/virologia , Adulto , Infecções Assintomáticas , Feminino , Humanos , Masculino , Programas de Rastreamento , Pessoa de Meia-Idade , Carga Viral , Adulto Jovem
12.
Sci Rep ; 12(1): 2356, 2022 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-35181702

RESUMO

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.


Assuntos
Exsudatos e Transudatos/virologia , Programas de Rastreamento/métodos , SARS-CoV-2/isolamento & purificação , Saliva/virologia , Espectroscopia de Luz Próxima ao Infravermelho , Humanos , Testes Imediatos , Estudo de Prova de Conceito
13.
PLoS One ; 17(2): e0263341, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35143538

RESUMO

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.


Assuntos
COVID-19/virologia , Reação em Cadeia da Polimerase Multiplex , Nasofaringe/virologia , Orofaringe/virologia , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Saliva/virologia , Escarro/virologia , Reações Cruzadas , Humanos , Limite de Detecção , Sensibilidade e Especificidade
14.
Microbiol Spectr ; 10(1): e0161421, 2022 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-35171037

RESUMO

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.


Assuntos
Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , SARS-CoV-2/isolamento & purificação , Saliva/virologia , Adulto , Idoso , Idoso de 80 Anos ou mais , COVID-19/virologia , Teste de Ácido Nucleico para COVID-19 , Teste Sorológico para COVID-19/instrumentação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Nasofaringe/virologia , República da Coreia , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Sensibilidade e Especificidade , Adulto Jovem
15.
Dis Markers ; 2022: 6478434, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35035611

RESUMO

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.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , Saliva/virologia , Adulto , Idoso , Estudos de Casos e Controles , Humanos , Pessoa de Meia-Idade , Nasofaringe/virologia , Valor Preditivo dos Testes , RNA Viral/isolamento & purificação , SARS-CoV-2/genética , Manejo de Espécimes/métodos
16.
PLoS One ; 17(1): e0263114, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35077513

RESUMO

In many countries a second wave of infections caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has occurred, triggering a shortage of reagents needed for diagnosis and compromising the capacity of laboratory testing. There is an urgent need to develop methods to accelerate the diagnostic procedures. Pooling samples represents a strategy to overcome the shortage of reagents, since several samples can be tested using one reaction, significantly increasing the number and speed with which tests can be carried out. We have reported the feasibility to use a direct lysis procedure of saliva as source for RNA to SARS-CoV-2 genome detection by reverse transcription quantitative-PCR (RT-qPCR). Here, we show that the direct lysis of saliva pools, of either five or ten samples, does not compromise the detection of viral RNA. In addition, it is a sensitive, fast, and inexpensive method that can be used for massive screening, especially considering the proximity of the reincorporation of activities in universities, offices, and schools.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Saliva/virologia , COVID-19/epidemiologia , COVID-19/prevenção & controle , Teste de Ácido Nucleico para COVID-19/normas , Humanos , Programas de Rastreamento/métodos , Programas de Rastreamento/normas , Quarentena/normas , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/patogenicidade , Sensibilidade e Especificidade
17.
PLoS One ; 17(1): e0263033, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35089942

RESUMO

SARS-CoV-2 pandemic has forced frequent testing of populations. It is necessary to identify the most cost-effective strategies for the detection of COVID-19 outbreaks. Nasopharyngeal samples have been used for SARS-CoV-2 detection but require a healthcare professional to collect the sample and cause discomfort and pain to the individual. Saliva has been suggested as an appropriate fluid for the diagnosis of COVID-19. We have investigated the possibility of using pools of saliva samples to detect SARS-CoV-2 in symptomatic and asymptomatic patients. Two hundred and seventy-nine saliva samples were analyzed through RT-PCR of Envelope, Nucleocapsid and Open Reading Frame 1ab genes. Reproducibility assays showed an almost perfect agreement as well as high sensitivity (96.6%), specificity (96.8%), positive predicted value (96.6%), and negative predicted value (96.8%). The average Cycle Threshold of the genes detected was 29.7. No significant differences (p > 0.05) were detected when comparing the cycle threshold average of two consecutive reactions on the same positive saliva samples. Saliva samples have a higher median viral load (32.6) than in nasopharyngeal samples (28.9), although no significant differences were detected (p > 0.05). Saliva-pool samples allowed effective SARS-CoV-2 screening, with a higher sensibility (96.9%) on 10-sample pools than in 20-sample pools (87.5%). Regardless of pools size specificity was high (99.9%) and an almost perfect agreement was observed. Our strategy was successfully applied in population wide testing of more than 2000 individuals, showing that it is possible to use pooled saliva as diagnostic fluid for SARS-CoV-2 infection.


Assuntos
Teste para COVID-19/métodos , SARS-CoV-2/isolamento & purificação , Saliva/virologia , Manejo de Espécimes/métodos , COVID-19/diagnóstico , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
18.
J Nanobiotechnology ; 20(1): 6, 2022 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-34983543

RESUMO

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.


Assuntos
Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , SARS-CoV-2/isolamento & purificação , Ressonância de Plasmônio de Superfície/métodos , Anticorpos Antivirais/imunologia , COVID-19/imunologia , Teste Sorológico para COVID-19/instrumentação , Desenho de Equipamento , Ouro/química , Humanos , Imunoensaio/instrumentação , Imunoensaio/métodos , Nanopartículas Metálicas/química , SARS-CoV-2/imunologia , Saliva/virologia , Glicoproteína da Espícula de Coronavírus/imunologia , Ressonância de Plasmônio de Superfície/instrumentação
19.
PLoS One ; 17(1): e0259886, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35081119

RESUMO

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.


Assuntos
COVID-19/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral/análise , COVID-19/virologia , Teste de Ácido Nucleico para COVID-19/economia , Teste de Ácido Nucleico para COVID-19/métodos , Colorimetria , Humanos , Técnicas de Diagnóstico Molecular/economia , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Amplificação de Ácido Nucleico/economia , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Sistemas Automatizados de Assistência Junto ao Leito , RNA Viral/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Saliva/virologia
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