The value of point-of-care tests for the detection of SARS-CoV-2 RNA or antigen in bronchoalveolar lavage fluid.

BACKGROUND: Transmission of SARS-CoV-2 from donor to recipient is a clinically relevant risk for developing severe COVID-19 after lung transplantation (LTx). This risk of iatrogenic transmission can be reduced by timely detection of viral RNA or antigen in samples of bronchoalveolar lavage (BAL) fluid obtained at the time of lung procurement. We aimed to retrospectively evaluate the detection of SARS-CoV-2 RNA or antigen in BAL fluid samples using three point-of-care tests (POCTs). METHODS: BAL fluid samples came from patients hospitalized in an intensive care unit during the COVID-19 pandemic. These pandemic samples were scored as positive or negative for SARS-CoV-2 by a RT-qPCR comparator assay for orf1ab. Three commercially available POCTs were then evaluated: cobas SARS-CoV-2 & Influenza A/B assay with the cobas Liat RT-qPCR system (Roche Diagnostics), ID NOW COVID-19 and COVID-19 2.0 (Abbott), and SARS-CoV-2 Rapid Antigen Test (RAT) (Roche Diagnostics). Samples from the pre-pandemic era served as negative controls. RESULTS: We analyzed a total of 98 BAL fluid samples, each from a different patient: 58 positive pandemic samples (orf1ab Ct<38), 20 putatively negative pandemic samples (orf1ab Ct≥38), and 20 pre-pandemic samples. Univariate logistic regression shows that the probability of detection was highest for cobas Liat, followed by ID NOW, and then RAT. Of clinical relevance, cobas Liat detected SARS-CoV-2 RNA in 30 of the 31 positive pandemic samples that were collected within 10 days after RT-qPCR diagnosis of SARS-CoV-2 infection. None of the 20 pre-pandemic samples had a false-positive result for any POCT. CONCLUSIONS: POCTs enable the detection of SARS-CoV-2 RNA or antigen in BAL fluid samples and may provide additional information to decide if donor lungs are suitable for transplantation. Detection of respiratory pathogens with POCTs at the time of donor lung procurement is a potential strategy to increase safety in LTx by preventing iatrogenic transmission and severe postoperative infections.

Colorimetric immunosensing using liposome encapsulated MnO2 nanozymes for SARS-CoV-2 antigen detection.

Development of specific signal reporters with signal amplification effect are highly needed for sensitive and accurate detection of pathogen. Herein, we design a colorimetric immunosensing nanosystem based on liposome encapsulated quantum dots-sized MnO2 nanozyme (MnO2QDs@Lip) as a signal reporter for ultrasensitive and fast detection of SARS-CoV-2 antigen. The pathogenic antigens captured and separated by antibody-conjugated magnetic beads (MBs) are further connected with antibody-modified MnO2QDs@Lip to form a sandwich-like immunocomplex structure. After triggered release, MnO2 QDs efficiently catalyze colorless 3,3',5,5'-tetramethylbenzidine (TMB) to blue oxidized TMB, which can be qualitatively observed by naked eyes and quantitatively analyzed by UV-Vis spectra or smartphone platforms. By taking advantages of immuno-magnetic separation, excellent peroxidase-like catalytic activity of MnO2 QDs, and high encapsulation efficiency of MnO2QDs@Lip, ultrasensitive detection of SARS-CoV-2 antigen ranging from 0.1 pg/mL to 100 ng/mL is achieved within 20 min. The limit of detection (LOD) is calculated to be 65 fg/mL in PBS buffer. Furthermore, real clinical samples of SARS-CoV-2 antigens can be effectively identified by this immunosensing nanosystem with excellent accuracy. This proposed detection nanosystem provides a strategy for simple, rapid and ultrasensitive detection of pathogens and may shed light on the development of new POCT detection platforms for early diagnosis of pathogens and surveillance in public health.

Evaluating diagnostic accuracies of Panbio™ test and RT-PCR for the detection of SARS-CoV-2 in Addis Ababa, Ethiopia using Bayesian Latent-Class Models (BLCM).

BACKGROUND: Rapid diagnostics are vital for curving the transmission and control of the COVID-19 pandemic. Although many commercially available antigen-based rapid diagnostic tests (Ag-RDTs) for the detection of SARS-CoV-2 are recommended by the WHO, their diagnostic performance has not yet been assessed in Ethiopia. So far, the vast majority of studies assessing diagnostic accuracies of rapid antigen tests considered RT-PCR as a reference standard, which inevitably leads to bias when RT-PCR is not 100% sensitive and specific. Thus, this study aimed to evaluate the diagnostic performance of Panbio™ jointly with the RT-PCR for the detection of SARS-CoV-2. METHODS: A prospective cross-sectional study was done from July to September 2021 in Addis Ababa, Ethiopia, during the third wave of the pandemic involving two health centers and two hospitals. Diagnostic sensitivity and specificity of Panbio™ and RT-PCR were obtained using Bayesian Latent-Class Models (BLCM). RESULTS: 438 COVID-19 presumptive clients were enrolled, 239 (54.6%) were females, of whom 196 (44.7%) had a positive RT-PCR and 158 (36.1%) were Panbio™ positive. The Panbio™ and RT-PCR had a sensitivity (95% CrI) of 99.6 (98.4-100) %, 89.3 (83.2-97.6) % and specificity (95% CrI) of 93.4 (82.3-100) %, and 99.1 (97.5-100) %, respectively. Most of the study participants, 318 (72.6%) exhibited COVID-19 symptoms; the most reported was cough 191 (43.6%). CONCLUSION: As expected the RT-PCR performed very well with a near-perfect specificity and a high, but not perfect sensitivity. The diagnostic performance of Panbio™ is coherent with the WHO established criteria of having a sensitivity ≥80% for Ag-RDTs. Both tests displayed high diagnostic accuracies in patients with and without symptoms. Hence, we recommend the use of the Panbio™ for both symptomatic and asymptomatic individuals in clinical settings for screening purposes.

Pathological Studies on Hantaan Virus-Infected Mice Simulating Severe Hemorrhagic Fever with Renal Syndrome.

Hantaan virus is the causative agent of hemorrhagic fever with renal syndrome (HFRS). The Hantaan virus strain, Korean hemorrhagic fever virus clone-5 (KHF5), causes weight loss and renal hemorrhage in laboratory mice. Clone-4 (KHF4), which has a single E417K amino acid change in its glycoprotein, is an avirulent variant. In this study, KHF4 and KHF5 were compared to evaluate pathological differences in mice in vitro and in vivo. The characteristics of the two glycoproteins were not significantly different in vitro. However, the virulent KHF5 strain targeted the lungs and caused pneumonia and edema in vivo. Both strains induced high infectivity levels in the liver and caused hepatitis; however, petechial hemorrhage and glycogen storage reduction were observed in KHF5-infected mice alone. Renal hemorrhage was observed using viral antigens in the tubular region of KHF5-infected mice. In addition, an increase in white blood cell levels and neutrophilia were found in KHF5-infected mice. Microarray analysis of liver cells showed that CD8+ T cell activation, acute-phase protein production, and neutrophil activation was induced by KHF5 infection. KHF5 infectivity was significantly increased in vivo and the histological and clinicopathological findings were similar to those in patients with HFRS.

Padronização de um ELISA indireto a partir da utilização de uma proteína de superfície do vírus da anemia infecciosa equina como antígeno para o diagnóstico dessa enfermidade / Standardization of an indirect ELISA using a surface protein of the equine infectious anemia virus as an antigen for the diagnosis of this disease

A anemia infecciosa equina é uma importante enfermidade que acomete os equídeos em todo o mundo, se apresentando de forma aguda, crônica e assintomática causando grandes prejuízos para a economia tanto para criadores que vivem do trabalho desses animais quantos aos criadores que investem no melhoramento das raças, impedindo o acesso ao mercado tanto nacional quanto internacional. O Ministério da Agricultura, Pecuária e Abastecimento considera o IDGA como teste oficial para diagnóstico dessa enfermidade, porém essa técnica é demorada e muita vez acaba sendo subjetiva, dependendo da experiencia particular de cada Laboratorista. Além de não conseguir detectar animais no início da infecção. Logo, a necessidade de se buscar novas técnicas como o ELISA indireto que aperfeiçoem o tempo de análise dos resultados, facilita a automação e obtém resultados confiáveis. O estudo realizado teve como objetivo padronizar uma técnica de ELISA indireto utilizando uma proteína de envelope viral GP90 como antígeno para diagnóstico da anemia infecciosa equina. Avaliando o desempenho do teste a partir da sensibilidade, especificidade e valores preditivos positivo e negativo. Os valores obtidos foram: 91,11%, 93,33%, 91,11% e 93,33% respectivamente. Concluiu-se que o teste apresenta bom desempenho, além da possibilidade de detectar amimais positivos no início da infecção.

Equine infectious anemia is an important disease that affects horses all over the world, presenting in an acute, chronic and asymptomatic way, causing great damage to the economy, both for breeders who live off the work of these animals and for breeders who invest in the improvement of breeds, preventing access to both national and international markets. The Ministry of Agriculture, Livestock and Food Supply considers AGID to be the official test for diagnosing this disease, but this technique takes time and often ends up being subjective, depending on the particular experience of each laboratory worker. In addition to not being able to detect animals at the beginning of the infection. Therefore, the need to seek new techniques such as indirect ELISA that improve the time of analysis of results, facilitate automation and obtain reliable results. The aim of this study was to standardize an indirect ELISA technique using a GP90 viral envelope protein as an antigen for the diagnosis of equine infectious anemia. Evaluating test performance based on sensitivity, specificity and positive and negative predictive values. The values obtained were 91.11%, 93.33%, 91.11 and 93.33 respectively. It was concluded that the test performs well, in addition to the possibility of detecting positive animals at the beginning of the infection.

Padronização de um teste ELISA indireto para diagnóstico da cinomose canina / Standardization of an indirect ELISA test for canine diagnosis diagnosis

A cinomose é uma enfermidade causada pelo vírus Canine Distemper Virus (CDV). Essa doença afeta principalmente cães, mas também acomete outras espécies domésticas e selvagens. A imunidade do animal está relacionada ao grau que a esse patógeno vai atingir o organismo do indivíduo. Ela afeta a respiração do animal, pode causar vômito, diarreia, convulsões, podendo levar o animal à óbito. O objetivo do presente trabalho foi padronizar um teste ELISA indireto com antígeno de superfície para o diagnostico cinomose utilizando amostras de soro canino. Para padronização da técnica, fez-se necessário o estudo da diluição do antígeno para identificar a melhor concentração para sensibilização da placa. O teste foi aplicado primeiramente com diferentes diluições do antígeno para detecção do melhor desempenho do antígeno. Feito isso, foi testado em um banco de soro de 45 animais comprovadamente positivos no teste ELISA comercial e em soro de 45 animais comprovadamente negativos no teste ELISA comercial, posteriormente foi calculado o ponto de corte, especificidade e sensibilidade do teste. O teste ELISA indireto se mostrou com excelência como um teste de diagnóstico para a cinomose canina, obtendo-se ponto de corte de densidade óptica de 0,229, sensibilidade de 95,5% e especificidade de 84,4%.

Distemper is a disease or the disease by the CDV virus, Distemper Virus. This disease mainly affects dogs, but also affects other domestic and wild species. The animal's immunity is related to the degree to which it will reach the individual's organism. It affects the animal's breathing, can cause vomiting, diarrhea, convulsions, and can lead to death. The aim of the present work test was to standardize an indirect ELISA for distemper diagnosis in experiments using a surface antigen. For the study of technical identification, it was necessary to specify the antigen for the best concentration of plaque sensitization. The test was initially applied with different dilutions of the antigen to detect the best performance of the antigen. This was tested in a serum bank of 45 animals proven positive in the commercial ELISA test and in the serum of 45 animals proven negative in the commercial ELISA test, later it was tested on the cut-off point, specificity and sensitivity of the test. The indirect ELISA test proved to be excellent as a diagnostic test for canine distemper, with an optical density cut-off of 0.229, sensitivity of 95.5% and specificity of 84.4% being obtained.

Laboratory and field evaluation of the STANDARD Q and Panbio™ SARS-CoV-2 antigen rapid test in Namibia using nasopharyngeal samples.

BACKGROUND: As new SARS-CoV-2 variants of concern emerge, there is a need to scale up testing to minimize transmission of the Coronavirus disease 2019 (COVID-19). Many countries especially those in the developing world continue to struggle with scaling up reverse transcriptase polymerase reaction (RT-PCR) to detect SARS-CoV-2 due to scarcity of resources. Alternatives such as antigen rapid diagnostics tests (Ag-RDTs) may provide a solution to enable countries scale up testing. METHODS: In this study, we evaluated the Panbio™ and STANDARD Q Ag-RDTs in the laboratory using 80 COVID-19 RT-PCR confirmed and 80 negative nasopharyngeal swabs. The STANDARD Q was further evaluated in the field on 112 symptomatic and 61 asymptomatic participants. RESULTS: For the laboratory evaluation, both tests had a sensitivity above 80% (Panbio™ = 86% vs STANDARD Q = 88%). The specificity of the Panbio™ was 100%, while that of the STANDARD Q was 99%. When evaluated in the field, the STANDARD Q maintained a high specificity of 99%, however the sensitivity was reduced to 56%. CONCLUSION: Using Ag-RDTs in low resource settings will be helpful in scaling-up SARS-CoV-2 testing, however, negative results should be confirmed by RT-PCR where possible to rule out COVID-19 infection.

Validity of at-home rapid antigen lateral flow assay and artificial intelligence read to detect SARS-CoV-2.

BACKGROUND: The gold standard for COVID-19 diagnosis-reverse-transcriptase polymerase chain reaction (RT-PCR)- is expensive and often slow to yield results whereas lateral flow tests can lack sensitivity. METHODS: We tested a rapid, lateral flow antigen (LFA) assay with artificial intelligence read (LFAIR) in subjects from COVID-19 treatment trials (N = 37; daily tests for 5 days) and from a population-based study (N = 88; single test). LFAIR was compared to RT-PCR from same-day samples. RESULTS: Using each participant's first sample, LFAIR showed 86.2% sensitivity (95% CI 73.6%-98.8) and 94.3% specificity (88.8%-99.7%) compared to RT-PCR. Adjusting for days since symptom onset and repeat testing, sensitivity was 97.8% (89.9%-99.5%) on the first symptomatic day and decreased with each additional day. Sensitivity improved with artificial intelligence (AI) read (86.2%) compared to the human eye (71.4%). CONCLUSION: LFAIR showed improved accuracy compared to LFA alone. particularly early in infection.

Performance evaluation of antigen detection rapid diagnostic test (Ag-RDT) for COVID-19 diagnosis in a primary healthcare center during the Shanghai COVID-19 quarantine period.

BACKGROUND: Rapid and accurate detection of SARS-CoV-2 infection is the cornerstone of prompt patient care. However, the reliability of the antigen rapid diagnostic test (Ag-RDT) in the diagnosis of SARS-CoV-2 infection remains inconclusive. METHODS: We conducted a field evaluation of Ag-RDT performance during the Shanghai Coronavirus disease 2019 (COVID-19) quarantine and screened 7225 individuals visiting our Emergency Department. 83 asymptomatic SARS-CoV-2 (+) individuals were enrolled in the current study. Simultaneously, Ag-RDT was performed to evaluate its testing performance. RESULTS: For the Ag-RDT(-) cases, the average cycle threshold (Ct) values of the N gene were 27.26 ± 4.59, which were significantly higher than the Ct value (21.9 ± 4.73) of the Ag-RDT(+) individuals (p < 0.0001). The overall sensitivity of Ag-RDT versus that of RT-PCR was 43.37%. The Ag-RDT(+) individuals regarding the N gene's Ct value were 16 cases in the < 20 range, 12 in 20-25, 5 in 25-30, and 3 in 30-35. The corresponding sensitivity was 84.21%, 52.17%, 21.74% and 16.67%, respectively. Meanwhile, sampling had a straight specificity of 100% regardless of the Ct value. CONCLUSIONS: The Ag-RDT were extremely sensitive in asymptomatic COVID-19 individuals with a Ct value < 20.

Evaluation of the Boson rapid Ag test vs RT-PCR for use as a self-testing platform.

The gold standard test available for detecting COVID-19 patients is Real Time RT-PCR. However, this method is expensive, needing special equipment and skilled laboratory staff. Recently, less expensive antigen tests have become available, that could easily and rapidly identify new COVID-19 cases. Our objective was to evaluate the Boson Rapid Antigen Test Card versus the RT-rtPCR, using samples taken both by laymen (self-testing) and professionals. The sensitivity, specificity and accuracy rates were, 98.18%, 100.00%, and 99.28%, respectively. The positive and negative predictive values were 100.00% and 98.82%, respectively. The detection rate for asymptomatic patients was 90.48%, and detection rate for Ct values ≥30 was 91.67%. Our results indicate a high coincidence rate between the Boson and the referencing RT-rtPCR method, meeting the performance standards recommended by the WHO. Therefore, this test could facilitate a fast self-testing screening method, for the detection of infected individuals.

High Sensitivity and NPV for BinaxNOW Rapid Antigen Test in Children at a Mass Testing Site during Prevalent Delta Variant Period.

SARS-CoV-2 continues to develop new, increasingly infectious variants including delta and omicron. We evaluated the efficacy of the Abbott BinaxNOW Rapid Antigen Test against Reverse Transcription PCR (RT-PCR) in 1,054 pediatric participants presenting to a high-volume Coronavirus Disease 2019 (COVID-19) testing site while the delta variant was predominant. Both tests utilized anterior nares swabs. Participants were grouped by COVID-19 exposure and symptom status. 5.2% of samples tested positive by RT-PCR for SARS-CoV-2. For all participants, sensitivity of the BinaxNOW was 92.7% (95% CI 82.4%-98.0%), and specificity was 98.0% (95% CI 97.0%-98.8%). For symptomatic participants, positive predictive value (PPV) was 72.7% (95% CI 54.5%-86.7%) and negative predictive value (NPV) was 99.2% (95% CI 98.2%-100%). Among asymptomatic participants, PPV was 71.4% (95% CI 53.7%-85.4%) and NPV was 99.7% (95% CI 99.0%-100%). Our reported sensitivity and NPV are higher than other pediatric studies, potentially because of higher viral load from the delta variant, but specificity and PPV are lower. IMPORTANCE The BinaxNOW rapid antigen COVID-19 test had a sensitivity of nearly 92% in both symptomatic and asymptomatic children when performed at a high-throughput setting during the more transmissible delta variant dominant period. The test may play an invaluable role in asymptomatic screening and keeping children safe in school.

Preparation of the luciferase-labeled antibody for improving the detection sensitivity of viral antigen.

BACKGROUND: Viral antigen detection test is the most common method used to detect viruses in the field rapidly. However, due to the low sensitivity, it can only be used as an auxiliary diagnosis method for virus infection. Improving sensitivity is crucial for developing more accurate viral antigen tests. Nano luciferase (Nluc) is a sensitive reporter that has not been used in virus detection. RESULTS: In this study, we produced an intracellularly Nluc labeled detection antibody (Nluc-ch2C5) and evaluated its ability to improve the detection sensitivity of respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens. Compared with the traditional horse-radish peroxidase (HRP) labeled antibody (HRP-ch2C5), Nluc-ch2C5 was 41 times more sensitive for inactivated SARS-CoV-2 virus by sandwich chemiluminescence ELISA. Then we applied Nluc-ch2C5 to establish an automatic magnet chemiluminescence immune assay (AMCA) for the SARS-CoV-2 viral spike protein, the limit of detection was 68 pfu/reaction. The clinical sensitivity and specificity reached 75% (24/32) and 100% (48/48) using 32 PCR-positive and 48 PCR-negative swab samples for clinical evaluation, which is more sensitive than the commercial ELSA kit and colloid gold strip kit. CONCLUSIONS: Here, monoclonal antibody ch2C5 served as a model antibody and the SARS-CoV-2 served as a model pathogen. The Nluc labeled detecting antibody (Nluc-ch2C5) significantly improved the detection sensitivity of SARS-CoV-2 antigen. This labeling principle applies to other viral infections, so this labeling and test format could be expected to play an important role in detecting other virus antigens.

Detecting Sources of Immune Activation and Viral Rebound in HIV Infection.

Anti-retroviral therapy (ART) generally suppresses HIV replication to undetectable levels in peripheral blood, but immune activation associated with increased morbidity and mortality is sustained during ART, and infection rebounds when treatment is interrupted. To identify drivers of immune activation and potential sources of viral rebound, we modified RNAscope in situ hybridization to visualize HIV-producing cells as a standard against which to compare the following assays of potential sources of immune activation and virus rebound following treatment interruption: (i) envelope detection by induced transcription-based sequencing (EDITS) assay; (ii) HIV-Flow; (iii) Flow-FISH assays that can scan tissues and cell suspensions to detect rare cells expressing env mRNA, gag mRNA/Gag protein and p24; and (iv) an ultrasensitive immunoassay that detects p24 in cell/tissue lysates at subfemtomolar levels. We show that the sensitivities of these assays are sufficient to detect one rare HIV-producing/env mRNA+/p24+ cell in one million uninfected cells. These high-throughput technologies provide contemporary tools to detect and characterize rare cells producing virus and viral antigens as potential sources of immune activation and viral rebound. IMPORTANCE Anti-retroviral therapy (ART) has greatly improved the quality and length of life for people living with HIV, but immune activation does not normalize during ART, and persistent immune activation has been linked to increased morbidity and mortality. We report a comparison of assays of two potential sources of immune activation during ART: rare cells producing HIV and the virus' major viral protein, p24, benchmarked on a cell model of active and latent infections and a method to visualize HIV-producing cells. We show that assays of HIV envelope mRNA (EDITS assay), gag mRNA, and p24 (Flow-FISH, HIV-Flow. and ultrasensitive p24 immunoassay) detect HIV-producing cells and p24 at sensitivities of one infected cell in a million uninfected cells, thereby providing validated tools to explore sources of immune activation during ART in the lymphoid and other tissue reservoirs.

An all-in-one point-of-care testing device for multiplexed detection of respiratory infections.

The impact of the COVID-19 pandemic has reinforced the need for rapid, cost-effective, and reliable point-of-care testing (POCT) devices for massive population screening. The co-circulation of SARS-CoV-2 with several seasonal respiratory viruses highlights the need for multiplexed biosensing approaches. Herein, we present a fast and robust all-in-one POCT device for parallel viral antigen and serological analysis. The biosensing approach consists of a functionalized polycarbonate disc-shaped surface with microfluidic structures, where specific bioreagents are immobilized in microarray format, and a portable optoelectronic analyzer. The biosensor quantifies the concentration of viral antigens and specific immunoglobulins G and M for SARS-CoV-2, influenza A/B, adenovirus, and respiratory syncytial virus, using 30 µL of a sample. The semi-automated analysis of 6 samples is performed in 30 min. Validation studies performed with 135 serum samples and 147 nasopharyngeal specimens reveal high diagnostic sensitivity (98-100%) and specificity (84-98%), achieving an excellent agreement (κ = 0.937) with commercial immunoassays, which complies with the World Health Organization criteria for POC COVID-19 diagnostic tests. The versatility of the POCT device paves the way for the detection of other pathogens and analytes in the incoming post-pandemic world, integrating specific bioreagents against different variants of concerns and interests.

Application of cation exchange chromatography in bind and elute and flowthrough mode for the purification of enteroviruses.

Members of the enterovirus genus are promising oncolytic agents. Their morphogenesis involves the generation of both genome-packed infectious capsids and empty capsids. The latter are typically considered as an impurity in need of removal from the final product. The separation of empty and full capsids can take place with centrifugation methods, which are of low throughput and poorly scalable, or scalable chromatographic processes, which typically require peak cutting and a significant trade-off between purity and yield. Here we demonstrate the application of packed bed cation exchange (CEX) column chromatography for the separation of empty capsids from infectious virions for a prototype strain of Coxsackievirus A21. This separation was developed using high throughput chromatography techniques and scaled up as a bind and elute polishing step. The separation was robust over a wide range of operating conditions and returned highly resolved empty and full capsids. The CEX step could be operated in bind and elute or flowthrough mode with similar selectivity and returned yields greater than 70% for full mature virus particles. Similar performance was also achieved using a selection of other bead based CEX chromatography media, demonstrating general applicability of this type of chromatography for Coxsackievirus A21 purification. These results highlight the wide applicability and excellent performance of CEX chromatography for the purification of enteroviruses, such as Coxsackievirus A21.

Performance differences among commercially available antigen rapid tests for COVID-19 in Brazil.

A rapid and accurate diagnosis is a crucial strategy for containing the coronavirus disease (COVID-19) pandemic. Considering the obstacles to upscaling the use of RT-qPCR, rapid tests based on antigen detection (Ag-RDT) have become an alternative to enhance mass testing, reducing the time for a prompt diagnosis and virus spreading. However, the performances of several commercially available Ag-RDTs have not yet been evaluated in several countries. Here, we evaluate the performance of eight Ag-RDTs available in Brazil to diagnose COVID-19. Patients admitted to tertiary hospitals with moderate or mild COVID-19 symptoms and presenting risk factors for severe disease were included. The tests were performed using a masked protocol, strictly following the manufacturer's recommendations and were compared with RT-qPCR. The overall sensitivity of the tests ranged from 9.8 to 81.1%, and specificity greater than 83% was observed for all the evaluated tests. Overall, slight or fair agreement was observed between Ag-RDTs and RT-PCR, except for the Ag-RDT COVID-19 (Acro Biotech), in which moderate agreement was observed. Lower sensitivity of Ag-RDTs was observed for patients with cycle threshold > 25, indicating that the sensitivity was directly affected by viral load, whereas the effect of the disease duration was unclear. Despite the lower sensitivity of Ag-RDTs compared with RT-qPCR, its easy fulfillment and promptness still justify its use, even at hospital admission. However, the main advantage of Ag-RDTs seems to be the possibility of increasing access to the diagnosis of COVID-19 in patients with a high viral load, allowing immediate clinical management and reduction of infectivity and community transmission.

Field evaluation of the performance of seven Antigen Rapid diagnostic tests for the diagnosis of SARs-CoV-2 virus infection in Uganda.

OBJECTIVE: The objective of this study was to evaluate the performance of seven antigen rapid diagnostic tests (Ag RDTs) in a clinical setting to identify those that could be recommended for use in the diagnosis of SARS-CoV-2 infection in Uganda. METHODS: This was a cross-sectional prospective study. Nasopharyngeal swabs were collected consecutively from COVID-19 PCR positive and COVID-19 PCR negative participants at isolation centers and points of entry, and tested with the SARS-CoV-2 Ag RDTs. Test sensitivity and specificity were generated by comparing results against qRT-PCR results (Berlin Protocol) at a cycle threshold (Ct) cut-off of ≤39. Sensitivity was also calculated at Ct cut-offs ≤29 and ≤33. RESULTS: None of the Ag RDTs had a sensitivity of ≥80% at Ct cut-off values ≤33 and ≤39. Two kits, Panbio™ COVID-19 Ag and VivaDiag™ SARS-CoV-2 Ag had a sensitivity of ≥80% at a Ct cut-off value of ≤29. Four kits: BIOCREDIT COVID -19 Ag, COVID-19 Ag Respi-Strip, MEDsan® SARS-CoV-2 Antigen Rapid Test and Panbio™ COVID-19 Ag Rapid Test had a specificity of ≥97%. CONCLUSIONS: This evaluation identified one Ag RDT, Panbio™ COVID-19 Ag with a performance at high viral load (Ct value ≤29) reaching that recommended by WHO. This kit was recommended for screening of patients with COVID -19-like symptoms presenting at health facilities.

Rapid Antigen Test LumiraDxTM vs. Real Time Polymerase Chain Reaction for the Diagnosis of SARS-CoV-2 Infection: A Retrospective Cohort Study.

Background: Real time reverse transcription polymerase chain reaction (real time RT-PCR) testing is the gold standard for the diagnosis of SARS-CoV-2 infections. However, to expand the testing capacity, new SARS-CoV-2 rapid antigen tests (Ag-RDTs) have been implemented. Ag-RDTs are more rapid, but less reliable in terms of sensitivity, and real-life data on their performance in comparison with the real time RT-PCR test are lacking. Methods: We aimed at assessing the diagnostic performance of the third-generation antigenic swab LumiraDx™ compared with real time RT-PCR in a retrospective cohort study at the Infectious Diseases Unit of Padua. All of the patients who were consecutively tested for SARS-CoV-2 in our centre (by both real time RT-PCR and Ag-RTD LumiraDxTM) from 19 January to 30 May 2021, were included. Cycle-threshold (Ct) values of positive real time RT-PCR were recorded as well as the number of days from symptoms' onset to testing. Results: Among the 282 patients included, 80.9% (N = 228) tested positive to real time RT-PCR, and among these, 174 tested positive also to LumiraDx™. Compared with real time RT-PCR, which is considered as the gold standard for the assessment of the presence/absence of SARS-CoV-2 infection, LumiraDx™ showed an overall sensitivity of 76.3% and specificity of 94.4%. Sensitivity increased to 91% when testing was performed <10 days from symptoms' onset, and to 95% when considering Ct < 25. Multivariable binomial logistic regression showed that false negative LumiraDx™ results were significantly associated with high Ct values, and with further testing from symptoms' onset. Conclusions: The results of our study suggested that the LumiraDx™ SARS-CoV-2 antigen assay may be appropriate for the detection of SARS-CoV-2 infection, especially in its early phase when the test largely meets the performance requirements of the European Centre for Disease Prevention and Control (ECDC).

Clinical validation of two immunochromatographic SARS-CoV-2 antigen tests in near hospital facilities.

INTRODUCTION: Rapid antigen tests to detect SARS-CoV-2 virus need to be validated. The purpose of clinical validation is to place the test into the everyday working process in health care institutions. METHODOLOGY: The clinical validation of Alltest Covid19 antigen test (Alltest, China) and Vivadiag Pro SARS- CoV-2 antigen tests (Vivacheck, China) started in four Slovenian health care institutions in December as a point-of-care test. Institutions compared the results of antigen tests to Seegene Allplex™ 2019-nCoV rt-PCR assay (SeeGene, South Korea) and Cobas 6800 SARS CoV-2 rt-PCR (Roche, USA). RESULTS: Sensitivity (90.6%, 95% CI = 84.94%-94.36%) and specificity (100%, 95% CI = 99.41%-100%) of Vivadiag Pro SARS CoV-2 Ag test were observed. While validating Alltest Covid19 Ag assay we got similar results (sensitivity 94.37%, 95% CI = 89.20% - 97.54%), specificity 100% (95% CI = 98.83% - 100%). CONCLUSIONS: Vivadiag Pro SARS CoV-2 Ag test and Alltest Covid19 test proved to be a good screening tool to detect SARS-CoV-2. The accurate information about the patient's status was available almost immediately and there was no need to wait for rt-PCR results. We could prevent further spread of the SARS-CoV-2 in primary care and hospital settings.