RESUMEN

Background. The COVID-19 pandemic demonstrated a need for robust SARS-CoV-2 test evaluation infrastructure to underpin biosecurity and protect the population during a pandemic health emergency.Gap statement. The first generation of rapid antigen tests was less accurate than molecular methods due to their inherent sensitivity and specificity shortfalls, compounded by the consequences of self-testing. This created a need for more accurate point-of-care SARS-CoV-2 detection methods.Aim. Here we present the lessons-learned during the COVID-19 emergency response in Western Australia including the detailed set-up, evaluation and operation of rapid antigen test in a state-run drive-through sample collection service during the COVID-19 pandemic after the strict border shutdown ended.Methods. We report a conformity assessment of a novel, second-generation rapid antigen test (Virulizer) comprising a technician-operated rapid lateral flow immunoassay with fluorescence-based detection.Results. The Virulizer rapid antigen test demonstrated up to 100% sensitivity (95% CI: 61.0-100%), 91.94% specificity (95% CI: 82.5-96.5%) and 92.65% accuracy when compared to a commercial PCR assay method. Wide confidence intervals in our series reflect the limits of small sample size. Nevertheless, the Virulizer assay performance was well-suited to point-of-care screening for SARS-CoV-2 in a drive-through clinic setting.Conclusion. The adaptive evaluation process necessary under changing pandemic conditions enabled assessment of a simple sample collection and point-of-care testing process, and showed how this system could be rapidly deployed for SARS-CoV-2 testing, including to regional and remote settings.

Asunto(s)

COVID-19 , Pruebas en el Punto de Atención , SARS-CoV-2 , Sensibilidad y Especificidad , Humanos , COVID-19/diagnóstico , SARS-CoV-2/inmunología , SARS-CoV-2/aislamiento & purificación , Inmunoensayo/métodos , Australia Occidental/epidemiología , Antígenos Virales/análisis , Prueba Serológica para COVID-19/métodos , Prueba de COVID-19/métodos , Fluorescencia , Sistemas de Atención de Punto

RESUMEN

This study comprehensively evaluated the DNA/RNA Defend Pro (DRDP) sample collection buffer, designed to inactivate and stabilize patient samples. The primary objectives were to assess DRDP's efficacy in ensuring sample stability, facilitating extraction-free polymerase chain reaction (PCR), and ensuring compatibility with rapid antigen testing (RAT). Ninety-five diagnostic nasopharyngeal swab samples tested for influenza virus (influenza A), respiratory syncytial virus (RSV A), and/or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were 10-fold diluted with DRDP and anonymized. Initial characterization and retesting of these samples using cobas Liat confirmed 88 samples as positive, validating the presence of viral targets. Results from rapid antigen testing showed lower sensitivity compared to nucleic acid amplification testing (NAAT) but maintained perfect specificity, with 40 out of 88 positive samples by cobas Liat also testing positive for RAT. Direct RT-qPCR of DRDP-diluted samples demonstrated robust compatibility, with 72 out of 88 samples positive for cobas Liat also testing positive by direct RT-qPCR. Non-concordant results could be explained by the 200-fold lower input of extraction-free NAAT. Stability testing involved incubating 31 positive samples at 4 °C, 20 °C, and 37 °C for 7 days, with extraction-free NAAT. DRDP guaranteed viral RNA stability at all temperatures for influenza A, SARS-CoV-2, and RSV A, showing stability up to 7 days at 4 °C. In conclusion, DRDP is an effective stabilizing medium compatible with direct RT-qPCR and rapid antigen testing and shows great potential for optimizing diagnostic processes, particularly in resource-limited or time-sensitive scenarios.

Asunto(s)

Nasofaringe , SARS-CoV-2 , Manejo de Especímenes , Nasofaringe/virología , Humanos , Manejo de Especímenes/métodos , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/inmunología , SARS-CoV-2/genética , ARN Viral/análisis , ARN Viral/aislamiento & purificación , ARN Viral/genética , Tampones (Química) , Virus de la Influenza A/aislamiento & purificación , Virus de la Influenza A/inmunología , Virus de la Influenza A/genética , Sensibilidad y Especificidad , COVID-19/diagnóstico , COVID-19/virología , Antígenos Virales/análisis , Gripe Humana/diagnóstico , Gripe Humana/virología

RESUMEN

This work proposed a simple and ultrasensitive nanozyme-based immunoassay on a filtration device for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (NP). Gold core porous platinum shell nanoparticles (Au@Pt NPs) were synthesized with high catalytic activity to oxidize 3,3',5,5'-tetramethylbenzidine, leading to an oblivious color change. The filtration device was designed based on the size difference of magnetic beads, filter membrane pore, and Au@Pt NPs. A simple, rapid, and consistent washing procedure can be performed with the help of a plastic syringe. This detection method could realize the quantitative detection of SARS-CoV-2 NP within 80 min for point-of-care needs. The limit of detection for the SARS-CoV-2 antigen was 0.01 ng/mL in buffer. The coefficients of variation of the assay were 1.78% for 10 ng/mL SARS-CoV-2 antigen, 2.03% for 1 ng/mL SARS-CoV-2 antigen, and 2.34% for the negative sample, respectively. The specificity of the detection platform was verified by the detection of various respiratory viruses. This simple and effective detection system was expected to promote substantial progress in the development and application of virus immunodetection technology.

Asunto(s)

COVID-19 , Oro , Nanopartículas del Metal , SARS-CoV-2 , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/inmunología , Humanos , COVID-19/diagnóstico , COVID-19/virología , Oro/química , Nanopartículas del Metal/química , Filtración/instrumentación , Platino (Metal)/química , Proteínas de la Nucleocápside de Coronavirus/inmunología , Límite de Detección , Inmunoensayo/métodos , Inmunoensayo/instrumentación , Jeringas , Antígenos Virales/análisis , Antígenos Virales/inmunología , Bencidinas/química , Inmunoadsorbentes/química , Fosfoproteínas

RESUMEN

Accurate and early diagnosis of monkeypox virus (MPXV) is crucial for controlling epidemics and treating affected individuals promptly. This study aimed to assess the analytical and clinical performance of the MolecisionTM Monkeypox Virus qPCR Assay, Biorain Monkeypox Virus ddPCR Assay, and MAGLUMI® Monkeypox Virus Ag (chemiluminescence immunoassay, CLIA) Assay. Additionally, it aimed to compare the clinical application of antigen and nucleic acid assays to offer insights into using commercial monkeypox assay kits. Specimens from 117 clinical patients, serial diluted virus cell culture supernatant, and artificially created positive samples were tested to evaluate the performance of these assay kits for MPXV diagnostics. The Biorain Monkeypox Virus ddPCR Assay had a limit of detection (LoD) of 3.89 CCID50/mL, while the MolecisionTM Monkeypox Virus qPCR Assay had an LoD of 15.55 CCID50/mL. The MAGLUMI® Monkeypox Virus Ag (CLIA) Assay had an LoD of 0.500 pg/mL. The accuracy of the MolecisionTM Monkeypox Virus qPCR Assay was comparable to the Biorain Monkeypox Virus ddPCR Assay, and the MAGLUMI® Monkeypox Virus Ag (CLIA) Assay demonstrated high sensitivity. The specificity of all three MPXV diagnostic assays for clinical specimens with potential cross-reacting substances was 100%. In conclusion, this study provides valuable insights into the clinical application of monkeypox assays, supporting efforts to mitigate and control the spread of monkeypox.

Asunto(s)

Monkeypox virus , Mpox , Sensibilidad y Especificidad , Humanos , Mpox/diagnóstico , Mpox/virología , Monkeypox virus/aislamiento & purificación , Monkeypox virus/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Límite de Detección , Femenino , Juego de Reactivos para Diagnóstico/normas , Masculino , Técnicas de Diagnóstico Molecular/métodos , Antígenos Virales/análisis , Adulto , Persona de Mediana Edad

RESUMEN

BACKGROUND: Porcine deltacoronavirus (PDCoV) is a swine enteropathogenic coronavirus that affects young pigs, causing vomiting, acute diarrhea, dehydration, and even death. There is growing evidence that PDCoV can undergo cross-species as well as zoonotic transmissions. Due to the frequent outbreaks of this deadly virus, early detection is essential for effective prevention and control. Therefore, developing a more convenient and reliable method for PDCoV detection is the need of the hour. RESULTS: This study utilized a high-affinity monoclonal antibody as the capture antibody and a horseradish peroxidase labeled polyclonal antibody as the detection antibody to develop an enzyme-linked immunosorbent assay (DAS-ELSA) for PDCoV detection.Both antibodies target the PDCoV nucleocapsid (N) protein. The findings of this study revealed that DAS-ELISA was highly specific to PDCoV and did not cross-react with other viruses to cause swine diarrhea. The limit of detection of the virus titer using this method was 103 TCID50/mL of PDCoV particles. The results of a parallel analysis of 239 known pig samples revealed a coincidence rate of 97.07% (κ = 0.922) using DAS-ELISA and reverse transcriptase PCR (RT-PCR). The DAS-ELISA was used to measure the one-step growth curve of PDCoV in LLC-PK cells and the tissue distribution of PDCoV in infected piglets. The study found that the DAS-ELISA was comparable in accuracy to the TCID50 method while measuring the one-step growth curve. Furthermore, the tissue distribution measured by DAS-ELISA was also consistent with the qRT-PCR method. CONCLUSION: The developed DAS-ELISA method can be conveniently used for the early clinical detection of PDCoV infection in pigs, and it may also serve as an alternative method for laboratory testing of PDCoV.

Asunto(s)

Deltacoronavirus , Ensayo de Inmunoadsorción Enzimática , Enfermedades de los Porcinos , Animales , Ensayo de Inmunoadsorción Enzimática/veterinaria , Ensayo de Inmunoadsorción Enzimática/métodos , Porcinos , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/diagnóstico , Enfermedades de los Porcinos/inmunología , Deltacoronavirus/aislamiento & purificación , Infecciones por Coronavirus/veterinaria , Infecciones por Coronavirus/diagnóstico , Infecciones por Coronavirus/virología , Infecciones por Coronavirus/inmunología , Anticuerpos Monoclonales/inmunología , Sensibilidad y Especificidad , Antígenos Virales/análisis , Antígenos Virales/inmunología , Anticuerpos Antivirales/sangre

RESUMEN

African swine fever virus (ASFV) has spread through many countries and regions worldwide, causing significant losses. Timely detection of ASFV-infected pigs is crucial for disease control. In this study, we assessed the performance of two pen-side tests: a portable real-time PCR (qPCR) test for detecting viral genomic DNA and a lateral flow immunoassay (LFIA) for detecting viral antigens. To determine the time from infection to the earliest detection, 10 ASFV-seronegative pigs were inoculated intramuscularly with 104.0 hemadsorption dose 50 of a highly virulent ASFV strain. Whole blood and oral swab samples were alternately collected from each group of five pigs daily until all succumbed to the infection. Samples were promptly subjected to the two pen-side tests upon collection, and a subset was transported to a veterinary diagnostic laboratory for analysis using a reference qPCR assay. Viral genomic DNA was consistently detected by the reference qPCR assay in all blood samples from 2 days postinfection (dpi), preceding the onset of clinical signs, and in oral swabs from 4 dpi onwards. The portable qPCR test demonstrated comparable performance to the reference qPCR assay for both whole blood and oral swab samples. The LFIA exhibited 100% specificity when testing with whole blood samples but showed reduced sensitivity, particularly for blood samples collected early or late after infection. The antigen test did not perform well with oral swabs.

Asunto(s)

Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Reacción en Cadena en Tiempo Real de la Polimerasa , Sensibilidad y Especificidad , Animales , Virus de la Fiebre Porcina Africana/aislamiento & purificación , Virus de la Fiebre Porcina Africana/genética , Fiebre Porcina Africana/diagnóstico , Fiebre Porcina Africana/virología , Porcinos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , ADN Viral/genética , Inmunoensayo/métodos , Antígenos Virales/análisis

RESUMEN

An enhanced lateral flow assay (LFA) is presented for rapid and highly sensitive detection of acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antigens with gold nanoflowers (Au NFs) as signaling markers and gold enhancement to amplify the signal intensities. First, the effect of the morphology of gold nanomaterials on the sensitivity of LFA detection was investigated. The results showed that Au NFs prepared by the seed growth method showed a 5-fold higher detection sensitivity than gold nanoparticles (Au NPs) of the same particle size, which may benefit from the higher extinction coefficient and larger specific surface area of Au NFs. Under the optimized experimental conditions, the Au NFs-based LFA exhibited a detection limit (LOD) of 25 pg mL-1 for N protein using 135 nm Au NFs as the signaling probes. The signal was further amplified by using a gold enhancement strategy, and the LOD for the detection of N protein achieved was 5 pg mL-1. The established LFA also exhibited good repeatability and stability and showed applicability in the diagnosis of SARS-CoV-2 infection.

Asunto(s)

Antígenos Virales , Proteínas de la Nucleocápside de Coronavirus , Oro , Límite de Detección , Nanopartículas del Metal , SARS-CoV-2 , Oro/química , SARS-CoV-2/inmunología , Nanopartículas del Metal/química , Humanos , Antígenos Virales/análisis , Antígenos Virales/inmunología , Proteínas de la Nucleocápside de Coronavirus/inmunología , Proteínas de la Nucleocápside de Coronavirus/análisis , Fosfoproteínas/inmunología , Fosfoproteínas/análisis , Fosfoproteínas/química , COVID-19/diagnóstico , COVID-19/virología , Inmunoensayo/métodos , Prueba Serológica para COVID-19/métodos

RESUMEN

Diagnostics employing multiple modalities have been essential for controlling and managing COVID-19, caused by SARS-CoV-2. However, scaling up Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR), the gold standard for SARS-CoV-2 detection, remains challenging in low and middle-income countries. Cost-effective and high-throughput alternatives like enzyme-linked immunosorbent assay (ELISA) could address this issue. We developed an in-house SARS-CoV-2 nucleocapsid capture ELISA, and validated on 271 nasopharyngeal swab samples from humans (n = 252), bovines (n = 10), and dogs (n = 9). This ELISA has a detection limit of 195 pg/100 µL of nucleocapsid protein and does not cross-react with related coronaviruses, ensuring high specificity to SARS-CoV-2. Diagnostic performance was evaluated using receiver operating characteristic curve analysis, showing a diagnostic sensitivity of 67.78 % and specificity of 100 %. Sensitivity improved to 74.32 % when excluding positive clinical samples with RT-qPCR Ct values > 25. Furthermore, inter-rater reliability analysis demonstrated substantial agreement (κ values = 0.73-0.80) with the VIRALDTECT II Multiplex RT-qPCR kit and perfect agreement with the CoVeasy™ COVID-19 rapid antigen self-test (κ values = 0.89-0.93). Our findings demonstrated that the in-house nucleocapsid capture ELISA is suitable for SARS-CoV-2 testing in humans and animals, meeting the necessary sensitivity and specificity thresholds for cost-effective, large-scale screening.

Asunto(s)

COVID-19 , Ensayo de Inmunoadsorción Enzimática , SARS-CoV-2 , Sensibilidad y Especificidad , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/economía , Humanos , SARS-CoV-2/inmunología , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/genética , Animales , COVID-19/diagnóstico , Bovinos , Perros , Prueba Serológica para COVID-19/métodos , Prueba Serológica para COVID-19/economía , Análisis Costo-Beneficio , Antígenos Virales/análisis , Antígenos Virales/inmunología , Ensayos Analíticos de Alto Rendimiento/economía , Ensayos Analíticos de Alto Rendimiento/métodos , Nasofaringe/virología , Proteínas de la Nucleocápside de Coronavirus/inmunología , Fosfoproteínas/inmunología

RESUMEN

BACKGROUND/OBJECTIVES: We investigated if performing two lateral flow device (LFD) tests, LFD2 immediately after LFD1, could improve diagnostic sensitivity or specificity for detecting severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) antigen. STUDY DESIGN: Individuals aged ≥16 years attending UK community testing sites (February-May 2021) performed two successive LFD tests and provided a nose-and-throat sample for a polymerase chain reaction (PCR) test. Using the PCR result as the reference diagnosis, we assessed whether improvements could be achieved in sensitivity (by counting a positive result in either LFD as a positive overall test result) or specificity (by using LFD2 as confirmatory test). RESULTS: Overall, 2231 participants were included with 159 (7 %) having a positive PCR test. Of 2223 participants who completed both LFD tests, LFD results were highly concordant both with each other and with PCR tests (>97 %). The proportion of discord LFD results decreased significantly over the study period. Combined LFD usage achieved a sensitivity of 68.6 %, versus 67.1 % for either LFD individually. The specificity increased from 99.5 % to 99.8 % when using LFD2 as confirmatory test. Observed increases in sensitivity and specificity were not statistically significant. Void results were recorded for 31 (1.4 %) LFD1s, 19 (0.9 %) LFD2s and 6 (0.3 %) combined LFD tests. CONCLUSIONS: LFD tests were highly reproducible even when they were performed by untrained users following only written instructions and without supervision. While performing two LFD tests of the same type in quick succession marginally increased sensitivity or specificity, statistically significant improvements were not detected in our study.

Asunto(s)

Antígenos Virales , Prueba Serológica para COVID-19 , COVID-19 , SARS-CoV-2 , Sensibilidad y Especificidad , Humanos , COVID-19/diagnóstico , Adulto , SARS-CoV-2/inmunología , SARS-CoV-2/genética , SARS-CoV-2/aislamiento & purificación , Masculino , Femenino , Persona de Mediana Edad , Prueba Serológica para COVID-19/métodos , Antígenos Virales/análisis , Adulto Joven , Anciano , Adolescente , Reino Unido

RESUMEN

Polymerase chain reaction (PCR) and antigen test (AgT) are frequently used in the diagnosis of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Routine PCR tests that detect the virus genome cannot determine whether the virus is infectious or not. However, detection of subgenomic RNA (sgRNA) produced during the replication period may indicate active viral infection. Active virus detection can offer various health and economic benefits from isolation time to treatment. Antigen tests are also considered as indicators of infectiousness since they can detect viruses above a certain load amount. The aim of this study was to use two different subgenomic RNAs and antigen test instead of genomic RNA to examine the relationship with each other and the clinic in terms of infectiousness. Evaluating the antigen test together with subgenomic RNA as an indicator of infectiousness may show the importance of this test. SARS-CoV-2 PCR positive 109 naso/oropharyngeal swab samples stored at -80 °C were included in the study. In order to confirm the PCR positivity of these samples, E gene PCR was performed and AgT, and E and N sgRNA quantitative real-time reverse transcription-PCR (RT-qPCR) detection was performed. Of the 109 SARSCoV-2 PCR positive samples, 83 (76.14%) had antigen test positivity, 88 (80.73%) had E gene sgRNA, 96 (88.07%) had N gene sgRNA and 97 (89%) had at least one sgRNA positivity.The antigen test was found positive in 77.3% of the samples in which at least one sgRNA was detected and in 66.7% of the negative samples and this difference was not statistically significant (p= 0.475). The difference between E sgRNA and AgT positivity was significant (p= 0.023). N sgRNA was positive in 98.9% of E sgRNA positive samples and 42.9% of the negative samples and this difference was statistically significant (p= 0.0001). The AgT positivity rate was found to be 98.15% (53/54) for cycle threshold (Ct) value ≤ 25, 57.14% (12/21) for Ct 25-30, and 52.94% (18/34) for Ct ≥ 30. The difference in antigen test positivity between E gRNA Ct value ≤ 25 and > 25, ≤ 29 and > 29, < 30 and ≥ 30 was statistically significant (p= 0.0001). Antigen test positivity appears to be associated with viral load and infectivity, as expected. In our study, it has been shown that sgRNAs and AgT which are indicators of infectiousness can be detected at least 10 days after the symptom period. Using these two tests together could detect infective individuals with higher accuracy and shorten the duration of hospital stay and isolation.

Asunto(s)

Antígenos Virales , COVID-19 , ARN Viral , SARS-CoV-2 , Humanos , ARN Viral/análisis , COVID-19/diagnóstico , COVID-19/virología , SARS-CoV-2/genética , SARS-CoV-2/inmunología , SARS-CoV-2/aislamiento & purificación , Antígenos Virales/análisis , Antígenos Virales/inmunología , Prueba de Ácido Nucleico para COVID-19/métodos , Masculino , Prueba Serológica para COVID-19/métodos , Persona de Mediana Edad , Femenino , Adulto , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas de la Envoltura de Coronavirus/genética , Anciano , Proteínas de la Nucleocápside de Coronavirus/inmunología , Proteínas de la Nucleocápside de Coronavirus/genética , Genoma Viral

RESUMEN

BACKGROUND: The COVID-19 pandemic underscored the need for rapid and accurate diagnostic tools. In August 2020, the Abbott BinaxNOW COVID-19 Antigen Card test became available as a timely and affordable alternative for SARS-CoV-2 molecular testing, but its performance may vary due to factors including timing and symptomatology. This study evaluates BinaxNOW diagnostic performance in diverse epidemiological contexts. METHODS: Using RT-PCR as reference, we assessed performance of the BinaxNOW COVID-19 test for SARS-CoV-2 detection in anterior nasal swabs from participants of two studies in Puerto Rico from December 2020 to May 2023. Test performance was assessed by days post symptom onset, collection strategy, vaccination status, symptomatology, repeated testing, and RT-PCR cycle threshold (Ct) values. RESULTS: BinaxNOW demonstrated an overall sensitivity of 84.1% and specificity of 98.8%. Sensitivity peaked within 1-6 days after symptom onset (93.2%) and was higher for symptomatic (86.3%) than asymptomatic (67.3%) participants. Sensitivity declined over the course of infection, dropping from 96.3% in the initial test to 48.4% in testing performed 7-14 days later. BinaxNOW showed 99.5% sensitivity in participants with low Ct values (≤ 25) but lower sensitivity (18.2%) for participants with higher Cts (36-40). CONCLUSIONS: BinaxNOW demonstrated high sensitivity and specificity, particularly in early-stage infections and symptomatic participants. In situations where test sensitivity is crucial for clinical decision-making, nucleic acid amplification tests are preferred. These findings highlight the importance of considering clinical and epidemiological context when interpreting test results and emphasize the need for ongoing research to adapt testing strategies to emerging SARS-CoV-2 variants.

Asunto(s)

COVID-19 , SARS-CoV-2 , Sensibilidad y Especificidad , Humanos , COVID-19/diagnóstico , Puerto Rico/epidemiología , SARS-CoV-2/inmunología , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/genética , Masculino , Adulto , Femenino , Persona de Mediana Edad , Antígenos Virales/análisis , Adulto Joven , Adolescente , Prueba Serológica para COVID-19/métodos , Anciano , Prueba de COVID-19/métodos

RESUMEN

Quantitative immunoassays, such as the traditional enzyme-linked immunosorbent assay (ELISA), are used to determine concentrations of an antigen in a matrix of unknown antigen concentration. Magnetic immunoassays, such as the Luminex xMAP technology, allow for the simultaneous detection of multiple analytes and offer heightened sensitivity, specificity, low sample volume requirements, and high-throughput capabilities. Here, we describe a quantitative immunoassay using the Luminex MAGPIX® System to determine the antigen concentration from liquid samples with unknown concentrations. In detail, we describe a newly developed assay for determining production yields of Drosophila S2-produced Marburg virus (MARV) glycoprotein in insect-cell-culture-derived supernatant. The potential applications of this assay could extend to the quantification of viral antigens in fluids derived from both in vitro and in vivo models infected with live MARV, thereby providing additional applications for virological research.

Asunto(s)

Antígenos Virales , Microesferas , Animales , Inmunoensayo/métodos , Antígenos Virales/inmunología , Antígenos Virales/análisis , Marburgvirus/inmunología , Marburgvirus/aislamiento & purificación , Drosophila , Técnicas de Cultivo de Célula/métodos , Línea Celular , Ensayo de Inmunoadsorción Enzimática/métodos

RESUMEN

Lateral flow immunoassays (LFIAs) are recognized for their practicality in homecare and point-of-care testing, owing to their simplicity, cost-efficiency, and rapid visual readouts. Despite these advantages, LFIAs typically fall short in sensitivity, particularly in detecting viruses such as SARS-CoV-2, thus limiting their broader application. In response to this challenge, we have innovated an approach to substantially enhance LFIA sensitivity. This involves the integration of a water-soluble dextran-methacrylate polymer wall with a 15% grafting degree positioned between the test and control lines on the LFIA strip. This novel modification significantly improved the sensitivity of the assay, achieving detection limits as low as 50 pg mL-1 and enhancing the sensitivity by 5-20-fold relative to existing LFIA kits available on the market. Furthermore, our developed LFIA kit (WSPW-LFIA) demonstrated exceptional specificity for SARS-CoV-2. Coupled with a straightforward fabrication process and robust stability, the WSPW-LFIA represents a promising advancement for real-time in vitro diagnosis across a spectrum of diseases.

Asunto(s)

COVID-19 , Polímeros , SARS-CoV-2 , SARS-CoV-2/inmunología , Humanos , COVID-19/diagnóstico , Inmunoensayo/métodos , Polímeros/química , Técnicas Biosensibles , Antígenos Virales/análisis , Agua , Sensibilidad y Especificidad , Límite de Detección , Prueba Serológica para COVID-19/métodos , Dextranos

RESUMEN

The Panbio COVID-19/Flu A&B Panel (Abbott) is an in vitro diagnostic rapid test designed for the qualitative detection of nucleocapsid proteins SARS-CoV-2 and nucleoprotein influenza A and B antigens in nasal mid-turbinate (NMT) swab specimens from symptomatic individuals meeting COVID-19 and influenza clinical and/or epidemiological criteria. This study, the largest global one to date using fresh samples, aimed to assess the diagnostic sensitivity and specificity of the Panbio COVID-19/Flu A&B Panel in freshly collected NMT swab specimens from individuals suspected of respiratory viral infection consistent with COVID-19 and/or influenza within the first 5 days of symptom onset compared with results obtained with the cobas SARS-CoV-2 and influenza A/B qualitative assay (cobas 6800/8800 systems), which were tested using nasopharyngeal swab samples. A total of 512 evaluable subjects were enrolled in the COVID-19 cohort across 18 sites, and 1,148 evaluable subjects were enrolled in the influenza cohort across 22 sites in the Asia-Pacific, Europe, and the USA. The Panbio COVID-19/Flu A&B Panel demonstrated a sensitivity of 80.4% and a specificity of 99.7% for COVID-19. For influenza A, the sensitivity and specificity rates were 80.6% and 99.3%, respectively. Likewise, for influenza B, the sensitivity and specificity rates were 80.8% and 99.4%, respectively. In conclusion, the Panbio COVID-19/Flu A&B Panel emerges as a suitable rapid test for detecting COVID-19 and influenza in symptomatic subjects across diverse global populations, exhibiting high sensitivity. The assay achieved a sensitivity of 94.4% in samples with Ct ≤24 for COVID-19 and 92.6% in samples with Ct ≤30 for influenza A and B. IMPORTANCE: The Panbio COVID-19/Flu A&B Panel is a suitable rapid test for detecting COVID-19 and influenza in symptomatic subjects across diverse global populations, exhibiting high sensitivity. The assay achieved a sensitivity of 94.0% in samples with Ct ≤24 for COVID-19 and 92.6% in samples with Ct ≤30 for influenza A and B.

Asunto(s)

Antígenos Virales , COVID-19 , Virus de la Influenza A , Virus de la Influenza B , Gripe Humana , SARS-CoV-2 , Sensibilidad y Especificidad , Humanos , COVID-19/diagnóstico , Gripe Humana/diagnóstico , Gripe Humana/virología , Virus de la Influenza B/aislamiento & purificación , Virus de la Influenza B/inmunología , SARS-CoV-2/inmunología , SARS-CoV-2/aislamiento & purificación , Adulto , Persona de Mediana Edad , Femenino , Masculino , Antígenos Virales/análisis , Antígenos Virales/inmunología , Adulto Joven , Adolescente , Anciano , Virus de la Influenza A/aislamiento & purificación , Virus de la Influenza A/inmunología , Niño , Preescolar , Nasofaringe/virología , Prueba de COVID-19/métodos , Lactante , Anciano de 80 o más Años

RESUMEN

The global spread of monkeypox has become a worldwide public healthcare issue. Therefore, there is an urgent need for accurate and sensitive detection methods to effectively control its spreading. Herein, we screened by phage display two peptides M4 (sequence: DPCGERICSIAL) and M6 (sequence: SCSSFLCSLKVG) with good affinity and specificity to monkeypox virus (MPXV) B21R protein. To simulate the state of the peptide in the phage and to avoid spatial obstacles of the peptide, GGGSK was added at the C terminus of M4 and named as M4a. Molecular docking shows that peptide M4a and peptide M6 are bound to different epitopes of B21R by hydrogen bonds and salt-bridge interactions, respectively. Then, peptide M4a was selected as the capture probe, phage M6 as the detection probe, and carbonized polymer dots (CPDs) as the fluorescent probe, and a colorimetric and fluorescent double-signal capture peptide/antigen/signal peptide-displayed phage sandwich ELISA triggered by horseradish peroxidase (HRP) through a simple internal filtration effect (IFE) was constructed. HRP catalyzes H2O2 to oxidize 3,3',5,5'-tetramethylbenzidine (TMB) to generate blue oxidized TMB, which can further quench the fluorescence of CPDs through IFE, enabling to detect MPXV B21R in colorimetric and fluorescent modes. The proposed simple immunoassay platform shows good sensitivity and reliability in MPXV B21R detection. The limit of detection for colorimetric and fluorescent modes was 27.8 and 9.14 pg/mL MPXV B21R, respectively. Thus, the established double-peptide sandwich-based dual-signal immunoassay provides guidance for the development of reliable and sensitive antigen detection capable of mutual confirmation, which also has great potential for exploring various analytical strategies for other respiratory virus surveillance.

Asunto(s)

Ensayo de Inmunoadsorción Enzimática , Péptidos , Ensayo de Inmunoadsorción Enzimática/métodos , Péptidos/química , Antígenos Virales/inmunología , Antígenos Virales/análisis , Antígenos Virales/química , Simulación del Acoplamiento Molecular , Peroxidasa de Rábano Silvestre/química , Peroxidasa de Rábano Silvestre/metabolismo , Límite de Detección , Colorantes Fluorescentes/química , Biblioteca de Péptidos , Bencidinas/química , Colorimetría/métodos

RESUMEN

So far, the 2019 novel coronavirus (COVID-19) is spreading widely worldwide. The early diagnosis of infection by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is essential to provide timely treatment and prevent its further spread. Lateral flow assays (LFAs) have the advantages of rapid detection, simple operation, low cost, ease of mass production, and no need for special devices and professional operators, which make them suitable for self-testing at home. This review focuses on the early diagnosis of SARS-CoV-2 infection based on optical LFAs including colorimetric, fluorescent (FL), chemiluminescent (CL), and surface-enhanced Raman scattering (SERS) LFAs for the detection of SARS-CoV-2 antigens and nucleic acids. The types of recognition components, detection modes used for antigen detection, labels employed in different optical LFAs, and strategies to improve the detection sensitivity of LFAs were reviewed. Meanwhile, LFAs coupled with different nucleic acid amplification techniques and CRISPR-Cas systems for the detection of SARS-CoV-2 nucleic acids were summarized. We hope this review provides research mentalities for developing highly sensitive LFAs that can be used in home self-testing for the early diagnosis of SARS-CoV-2 infection.

Asunto(s)

COVID-19 , Diagnóstico Precoz , SARS-CoV-2 , COVID-19/diagnóstico , COVID-19/virología , Humanos , SARS-CoV-2/aislamiento & purificación , Técnicas de Amplificación de Ácido Nucleico/métodos , Espectrometría Raman/métodos , Colorimetría/métodos , Antígenos Virales/análisis , Mediciones Luminiscentes/métodos , Prueba de COVID-19/métodos

Asunto(s)

Prueba Serológica para COVID-19 , COVID-19 , Humanos , Reacciones Falso Positivas , Prueba Serológica para COVID-19/métodos , COVID-19/diagnóstico , SARS-CoV-2/inmunología , Antígenos Virales/análisis , Antígenos Virales/sangre

RESUMEN

SARS-CoV-2 is the causative agent of COVID-19. Timely and accurate diagnostic testing is vital to contain the spread of infection, reduce delays in treatment and care, and inform patient management. Optimal specimen type (e.g. nasal swabs or saliva), timing of sampling, viral marker assayed (RNA or antigen), and correlation with viral infectivity and COVID-19 symptoms severity remain incompletely defined. We conducted a field study to evaluate SARS-CoV-2 viral marker kinetics starting from very early times after infection. We measured RNA and antigen levels in nasal swabs and saliva, virus outgrowth in cell culture from nasal swabs, and antibody levels in blood in a cohort of 30 households. Nine household contacts (HHC) became infected with SARS-CoV-2 during the study. Viral RNA was detected in saliva specimens approximately 1-2 days before nasal swabs in six HHC. Detection of RNA was more sensitive than of antigen, but antigen detection was better correlated with culture positivity, a proxy for contagiousness. Anti-nucleocapsid antibodies peaked one to three weeks post-infection. Viral RNA and antigen levels were higher in specimens yielding replication competent virus in cell culture. This study provides important data that can inform how to optimally interpret SARS-CoV-2 diagnostic test results.

Asunto(s)

Anticuerpos Antivirales , Biomarcadores , COVID-19 , Composición Familiar , ARN Viral , SARS-CoV-2 , Saliva , Humanos , COVID-19/diagnóstico , COVID-19/transmisión , COVID-19/virología , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/inmunología , Saliva/virología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Femenino , Antígenos Virales/análisis , Antígenos Virales/inmunología , Cinética , Masculino , Adulto , Persona de Mediana Edad

RESUMEN

African swine fever (ASF) is a lethal disease caused by ASF virus (ASFV), severely impacting the global swine industry. Though nuclear acid-based detection methods are reliable, they are laboratory-dependent. In this study, we developed a device-independent, user friendly and cost-effective quantum dots based immunochromatographic strip (QDs-ICS) with high specificity and sensitivity for the rapid and on-site detection of ASFV antigen. For the preparation of the QDs-ICS, we generated a monoclonal antibody (mAb) mAb-8G8 and polyclonal antibody (pAb) against ASFV-p72 protein. The pAb was labelled with QDs to be used as the detection probe and the mAb-8G8 was coated on the nitrocellulose membrane as the test line. Our results proved that the strip displayed no cross-reactivity with other swine viruses and detection limit of the QDs-ICS was down to 1 ng/mL for the ASFV-p72 protein with great reproducibility. The strip also exhibited high stability with a storage period up to 12 months under room temperature. Twenty blind samples and one hundred clinical samples were examined by the QDs-ICS, conventional PCR and real-time PCR method, respectively. Results showed that the agreement rate between the QDs-ICS and PCR method was 100%, and the agreement rate between the strip and real-time PCR was 94%. The novel QDs-ICS developed here would be an effective tool for on-site detection of ASFV.

Asunto(s)

Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Anticuerpos Monoclonales , Anticuerpos Antivirales , Antígenos Virales , Cromatografía de Afinidad , Puntos Cuánticos , Sensibilidad y Especificidad , Virus de la Fiebre Porcina Africana/aislamiento & purificación , Virus de la Fiebre Porcina Africana/inmunología , Virus de la Fiebre Porcina Africana/genética , Animales , Fiebre Porcina Africana/diagnóstico , Fiebre Porcina Africana/virología , Fiebre Porcina Africana/inmunología , Porcinos , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Cromatografía de Afinidad/métodos , Antígenos Virales/análisis , Antígenos Virales/inmunología , Reproducibilidad de los Resultados , Tiras Reactivas

Asunto(s)

Prueba Serológica para COVID-19 , COVID-19 , Humanos , Reacciones Falso Positivas , COVID-19/diagnóstico , Prueba Serológica para COVID-19/métodos , SARS-CoV-2/inmunología , Antígenos Virales/análisis , Antígenos Virales/sangre