Performance assessment and validation of a plaque reduction neutralization test (PRNT) in support to yellow fever diagnostic and vaccine clinical trials.

Yellow fever (YF) virus is a mosquito-borne virus belonging to the Flaviviridae family that circulates in tropical and subtropical areas of Africa and South America. Despite the availability of an effective vaccine, YF remains a threat to travelers, residents of endemic areas, and unvaccinated populations. YF vaccination and natural infection both induce the production of neutralizing antibodies. Serological diagnostic methods detecting YF virus-specific antibodies demonstrate high levels of cross-reactivities with other flaviviruses. To date, the plaque reduction neutralization test (PRNT) is the most specific serological test for the differentiation of flavivirus infections and is considered the reference method for detecting YF neutralizing antibodies and assessing the protective immune response following vaccination. In this study, we developed and validated a YF PRNT. We optimized different parameters including cell concentration and virus-serum neutralization time period and then assessed the intra- and inter-assay precisions, dilutability, specificity, and lower limit of quantification (LLOQ) using international standard YF serum, sera from vaccinees and human specimens collected through YF surveillance. The YF PRNT has shown good robustness and 100% of intra-assay precision, 95.6% of inter-assay precision, 100% of specificity, 100% of LLOQ, and 95.3% of dilutability. The test is, therefore, suitable for use in the YF diagnostic as well as evaluation of the YF vaccine neutralizing antibody response and risk assessment studies.

Evaluation of eight lateral flow tests for the detection of anti-SARS-CoV-2 antibodies in a vaccinated population.

BACKGROUND: Rapid determination of an individual's antibody status can be beneficial in understanding an individual's immune response to SARS-CoV-2 and for initiation of therapies that are only deemed effective in sero-negative individuals. Antibody lateral flow tests (LFTs) have potential to address this need as a rapid, point of care test. METHODS: Here we present a proof-of-concept evaluation of eight LFT brands using sera from 95 vaccinated individuals to determine sensitivity for detecting vaccination generated antibodies. Samples were analysed on eight different brands of antibody LFT and an automated chemiluminescent microparticle immunoassay (CMIA) that identifies anti-spike antibodies which was used as our reference standard. RESULTS: All 95 (100%) participants tested positive for anti-spike antibodies by the chemiluminescent microparticle immunoassay (CMIA) reference standard post-dose two of their SARS-CoV-2 vaccine: BNT162b2 (Pfizer/BioNTech, n = 60), AZD1222 (AstraZeneca, n = 31), mRNA-1273 (Moderna, n = 2) and Undeclared Vaccine Brand (n = 2). Sensitivity increased from dose one to dose two in six out of eight LFTs with three tests achieving 100% sensitivity at dose two in detecting anti-spike antibodies. CONCLUSIONS: These tests are demonstrated to be highly sensitive to detect raised antibody levels in vaccinated individuals. RDTs are low cost and rapid alternatives to ELISA based systems.

Clinical Evaluation of Severe Acute Respiratory Syndrome Coronavirus 2 Rapid Antigen Tests During the Omicron Wave in South Africa.

We evaluated the performance of nasal and nasopharyngeal Standard Q COVID-19 [coronavirus disease 2019] Ag tests (SD Biosensor) and the Panbio COVID-19 Ag Rapid Test Device (nasal; Abbott) against the Abbott RealTime severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) assay during the Omicron (clades 21M, 21K, and 21L) wave in South Africa. Overall, all evaluated tests performed well, with high sensitivity (range, 77.78%-81.42%) and excellent specificity values (>99%). The sensitivity of rapid antigen tests increased above 90% in samples with cycle threshold <20, and all 3 tests performed best within the first week after symptom onset.

Characterization of Severe Acute Respiratory Syndrome Coronavirus 2 Omicron Variant Shedding and Predictors of Viral Culture Positivity on Vaccinated Healthcare Workers With Mild Coronavirus Disease 2019.

In this prospective cohort of 30 vaccinated healthcare workers with mild Omicron variant infection, we evaluated viral culture, rapid antigen test (RAT), and real-time reverse-transcription polymerase chain reaction (RT-PCR) of respiratory samples at days 5, 7, 10, and 14. Viral culture was positive in 46% (11/24) and 20% (6/30) of samples at days 5 and 7, respectively. RAT and RT-PCR (Ct ≤35) showed 100% negative predictive value (NPV), with positive predictive values (PPVs) of 32% and 17%, respectively, for predicting viral culture positivity. A lower RT-PCR threshold (Ct ≤24) improved culture prediction (PPV = 39%; NPV = 100%). Vaccinated persons with mild Omicron infection are potentially transmissible up to day 7. RAT and RT-PCR might be useful tools for shortening the isolation period.

Head-to-head performance comparison of self-collected nasal versus professional-collected nasopharyngeal swab for a WHO-listed SARS-CoV-2 antigen-detecting rapid diagnostic test.

In 2020, the World Health Organization (WHO) recommended two SARS-CoV-2 lateral flow antigen-detecting rapid diagnostics tests (Ag-RDTs), both initially with nasopharyngeal (NP) sample collection. Independent head-to-head studies are necessary for SARS-CoV-2 Ag-RDT nasal sampling to demonstrate comparability of performance with nasopharyngeal (NP) sampling. We conducted a head-to-head comparison study of a supervised, self-collected nasal mid-turbinate (NMT) swab and a professional-collected NP swab, using the Panbio™ Ag-RDT (distributed by Abbott). We calculated positive and negative percent agreement between the sampling methods as well as sensitivity and specificity for both sampling techniques compared to the reference standard reverse transcription polymerase chain reaction (RT-PCR). A SARS-CoV-2 infection could be diagnosed by RT-PCR in 45 of 290 participants (15.5%). Comparing the NMT and NP sampling the positive percent agreement of the Ag-RDT was 88.1% (37/42 PCR positives detected; CI 75.0-94.8%). The negative percent agreement was 98.8% (245/248; CI 96.5-99.6%). The overall sensitivity of Panbio with NMT sampling was 84.4% (38/45; CI 71.2-92.3%) and 88.9% (40/45; CI 76.5-95.5%) with NP sampling. Specificity was 99.2% (243/245; CI 97.1-99.8%) for both, NP and NMT sampling. The sensitivity of the Panbio test in participants with high viral load (> 7 log10 SARS-CoV-2 RNA copies/mL) was 96.3% (CI 81.7-99.8%) for both, NMT and NP sampling. For the Panbio supervised NMT self-sampling yields comparable results to NP sampling. This suggests that nasal self-sampling could be used for to enable scaled-up population testing.Clinical Trial DRKS00021220.

Risk of Zika virus transmission in the Euro-Mediterranean area and the added value of building preparedness to arboviral threats from a One Health perspective.

In the alarming context of risk of Zika virus (ZIKV) transmission in the Euro-Mediterranean area, there is a need to examine whether capacities to detect, diagnose and notify ZIKV infections in the region are in place and whether ongoing capacity-building initiatives are filling existing gaps.The MediLabSecure network, created in 2014, comprises 55 laboratories of virology and medical entomology and 19 public health institutions in 19 countries in the Balkans, North-Africa, the Middle-East and the Black Sea regions. It aims to set up awareness, risk assessment, monitoring and control of emerging and re-emerging vector-borne viruses. We here examine the actions and strategies that MediLabSecure has been implementing and how they will contribute to the prevention and control of the ZIKV threat in the Euro-Mediterranean area.Capacity-building for arbovirus diagnostics is a major objective of the project and follows a methodological rather than disease-driven approach. This enables the implementation of laboratory trainings on techniques that are common to several arboviruses, including ZIKV, and putting into action appropriate diagnostic tools in the target region.Moreover, by its One Health approach and the interaction of its four sub-networks in human virology, animal virology, medical entomology and public health, MediLabSecure is fostering intersectoral collaboration, expertise and sharing of information. The resulting exchanges (methodological, communication and operational) across disciplines and across countries, dedicated research on intersectoral collaboration and increasing diagnostic capacities are providing new paths and tools to public health professionals to face emerging viral threats such as a ZIKV epidemic in the Euro-Mediterranean region.

Diagnostic performance of GENEDIA W and ActiveXpress+ COVID-19 antigens tests among symptomatic individuals in Peru and The United Kingdom.

OBJECTIVES: In order to generate independent performance data regarding accuracy of COVID-19 antigen-based rapid diagnostic tests (Ag-RDTs), prospective diagnostic evaluation studies across multiple sites are required to evaluate their performance in different clinical settings. This report describes the clinical evaluation the GENEDIA W COVID-19 Ag Device (Green Cross Medical Science Corp., Chungbuk, Korea) and the ActiveXpress+ COVID-19 Complete Testing Kit (Edinburgh Genetics Ltd, UK), in two testing sites Peru and the United Kingdom. METHODS: Nasopharyngeal swabs collected from 456 symptomatic patients at primary points of care in Lima, Peru and 610 symptomatic participants at a COVID-19 Drive-Through testing site in Liverpool, England were analyzed by Ag-RDT and compared to RT-PCR. Analytical evaluation of both Ag-RDTs was assessed using serial dilutions of direct culture supernatant of a clinical SARS-CoV-2 isolate from the B.1.1.7 lineage. RESULTS: For GENEDIA brand, the values of overall sensitivity and specificity were 60.4% [95% CI 52.4-67.9%], and 99.2% [95% CI 97.6-99.7%] respectively; and for Active Xpress+ the overall values of sensitivity and specificity were 66.2% [95% CI 54.0-76.5%], and 99.6% [95% CI 97.9-99.9%] respectively. The analytical limit of detection was determined at 5.0 x 102 pfu/ml what equals to approximately 1.0 x 104 gcn/ml for both Ag-RDTs. The UK cohort had lower median Ct values compared to that of Peru during both evaluations. When split by Ct, both Ag-RDTs had optimum sensitivities at Ct<20 (in Peru; 95% [95% CI 76.4-99.1%] and 100.0% [95% CI 74.1-100.0%] and in the UK; 59.2% [95% CI 44.2-73.0%] and 100.0% [95% CI 15.8-100.0%], for the GENDIA and the ActiveXpress+, respectively). CONCLUSIONS: Whilst the overall clinical sensitivity of the Genedia did not meet WHO minimum performance requirements for rapid immunoassays in either cohort, the ActiveXpress+ did so for the small UK cohort. This study illustrates comparative performance of Ag-RDTs across two global settings and considers the different approaches in evaluation methods.

Head-to-head comparison of nasal and nasopharyngeal sampling using SARS-CoV-2 rapid antigen testing in Lesotho.

OBJECTIVES: To assess the real-world diagnostic performance of nasal and nasopharyngeal swabs for SD Biosensor STANDARD Q COVID-19 Antigen Rapid Diagnostic Test (Ag-RDT). METHODS: Individuals ≥5 years with COVID-19 compatible symptoms or history of exposure to SARS-CoV-2 presenting at hospitals in Lesotho received two nasopharyngeal and one nasal swab. Ag-RDT from nasal and nasopharyngeal swabs were performed as point-of-care on site, the second nasopharyngeal swab used for polymerase chain reaction (PCR) as the reference standard. RESULTS: Out of 2198 participants enrolled, 2131 had a valid PCR result (61% female, median age 41 years, 8% children), 84.5% were symptomatic. Overall PCR positivity rate was 5.8%. The sensitivity for nasopharyngeal, nasal, and combined nasal and nasopharyngeal Ag-RDT result was 70.2% (95%CI: 61.3-78.0), 67.3% (57.3-76.3) and 74.4% (65.5-82.0), respectively. The respective specificity was 97.9% (97.1-98.4), 97.9% (97.2-98.5) and 97.5% (96.7-98.2). For both sampling modalities, sensitivity was higher in participants with symptom duration ≤ 3days versus ≤ 7days. Agreement between nasal and nasopharyngeal Ag-RDT was 99.4%. CONCLUSIONS: The STANDARD Q Ag-RDT showed high specificity. Sensitivity was, however, below the WHO recommended minimum requirement of ≥ 80%. The high agreement between nasal and nasopharyngeal sampling suggests that for Ag-RDT nasal sampling is a good alternative to nasopharyngeal sampling.

Standardized evaluation of Zika nucleic acid tests used in clinical settings and blood screening.

Early detection of Zika virus (ZIKV) transmission within geographic regions informs implementation of community mitigation measures such as vector reduction strategies, travel advisories, enhanced surveillance among pregnant women, and possible implementation of blood and organ donor screening or deferral. Standardized, comparative assessments of ZIKV assay and testing lab performance are important to develop optimal approaches to ZIKV diagnostic testing and surveillance. We conducted an expanded blinded panel study to characterize and compare the analytical performance of fifteen diagnostic and blood screening ZIKV NAT assays, including detection among single- and multiplex assays detecting ZIKV, dengue virus (DENV) and chikungunya virus (CHIKV). A 300 member blinded panel was constructed, consisting of 11 serial half-log dilutions ranging from ~104 to 10-1 genome equivalents/mL in 25 replicates each of the Tahitian Asian ZIKV isolate in ZIKV-negative human serum. Additionally, clinical samples from individuals with DENV-like syndrome or suspected ZIKV infection in Brazil were evaluated. The majority of assays demonstrated good specificity. Analytical sensitivities varied 1-2 logs, with a substantially higher limit of detection (LOD) in one outlier. Similar analytical sensitivity for ZIKV RNA detection in singleplex and multiplex assays of the Grifols and ThermoFisher tests were observed. Coefficient of Assay Efficiency (CE), calculated to characterize assays' RNA extraction and amplification efficiency, ranged from 0.13 for the Certest VIASURE multiplex and 0.75 for the Grifols multiplex assays. In general, assays using transcription mediated amplification (TMA) technology had greater CE compared to assays using conventional PCR technology. Donor screening NAT assays were significantly more sensitive than diagnostic RT-qPCR assays, primarily attributable to higher sample input volumes. However, ideal assays to maximize sensitivity and throughput may not be a viable option in all contexts, with other factors such as cost, instrumentation, and regulatory approval status influencing assay availability and selection, particularly in resource constrained settings.

Correction: Two-test algorithms for infectious disease diagnosis: Implications for COVID-19.

[This corrects the article DOI: 10.1371/journal.pgph.0000293.].

Performance of rapid antigen tests in identifying Omicron BA.4 and BA.5 infections in South Africa.

BACKGROUND: Concerns around accuracy and performance of rapid antigen tests continue to be raised with the emergence of new SARS-CoV-2 variants. OBJECTIVE: To evaluate the performance of two widely used SARS-CoV-2 rapid antigen tests during BA.4/BA.5 SARS-CoV-2 wave in South Africa (May - June 2022). STUDY DESIGN: A prospective field evaluation compared the SARS-CoV-2 Antigen Rapid test from Hangzhou AllTest Biotech (nasal swab) and the Standard Q COVID-19 Rapid Antigen test from SD Biosensor (nasopharyngeal swab) to the Abbott RealTime SARS-CoV-2 assay (nasopharyngeal swab) on samples collected from 540 study participants. RESULTS: Overall 28.52% (154/540) were SARS-CoV-2 RT-PCR positive with median cycle number value of 12.30 (IQR 9.30-19.40). Out of the 99 successfully sequenced SARS-CoV-2 positive samples, 18 were classified as BA.4 and 56 were classified as BA.5. The overall sensitivities of the AllTest SARS-CoV-2 Ag test and Standard Q COVID-19 Ag test were 73.38% (95% CI 65.89-79.73) and 74.03% (95% CI 66.58-80.31) and their specificities were 97.41% (95% CI 95.30-98.59) and 99.22% (95% CI 97.74-99.74) respectively. Sensitivity was >90% when the cycle number value was <20. The sensitivity of both rapid tests was >90% in samples infected with Omicron sub-lineage BA.4 and BA.5. CONCLUSION: Accuracy of tested rapid antigen tests that target the nucleocapsid SARS-CoV-2 protein, were not adversely affected by BA.4 and BA.5 Omicron sub-variants.

Multicenter Diagnostic Evaluation of OnSite COVID-19 Rapid Test (CTK Biotech) among Symptomatic Individuals in Brazil and the United Kingdom.

The COVID-19 pandemic has given rise to numerous commercially available antigen rapid diagnostic tests (Ag-RDTs). To generate and to share accurate and independent data with the global community requires multisite prospective diagnostic evaluations of Ag-RDTs. This report describes the clinical evaluation of the OnSite COVID-19 rapid test (CTK Biotech, CA, USA) in Brazil and the United Kingdom. A total of 496 paired nasopharyngeal (NP) swabs were collected from symptomatic health care workers at Hospital das Clínicas in São Paulo, Brazil, and 211 NP swabs were collected from symptomatic participants at a COVID-19 drive-through testing site in Liverpool, United Kingdom. Swabs were analyzed by Ag-RDT, and results were compared to quantitative reverse transcriptase PCR (RT-qPCR). The clinical sensitivity of the OnSite COVID-19 rapid test in Brazil was 90.3% (95% confidence interval [CI], 75.1 to 96.7%) and in the United Kingdom was 75.3% (95% CI, 64.6 to 83.6%). The clinical specificity in Brazil was 99.4% (95% CI, 98.1 to 99.8%) and in the United Kingdom was 95.5% (95% CI, 90.6 to 97.9%). Concurrently, analytical evaluation of the Ag-RDT was assessed using direct culture supernatant of SARS-CoV-2 strains from wild-type (WT), Alpha, Delta, Gamma, and Omicron lineages. This study provides comparative performance of an Ag-RDT across two different settings, geographical areas, and populations. Overall, the OnSite Ag-RDT demonstrated a lower clinical sensitivity than claimed by the manufacturer. The sensitivity and specificity from the Brazil study fulfilled the performance criteria determined by the World Health Organization, but the performance obtained from the UK study failed to do. Further evaluation of Ag-RDTs should include harmonized protocols between laboratories to facilitate comparison between settings. IMPORTANCE Evaluating rapid diagnostic tests in diverse populations is essential to improving diagnostic responses as it gives an indication of the accuracy in real-world scenarios. In the case of rapid diagnostic testing within this pandemic, lateral flow tests that meet the minimum requirements for sensitivity and specificity can play a key role in increasing testing capacity, allowing timely clinical management of those infected, and protecting health care systems. This is particularly valuable in settings where access to the test gold standard is often restricted.

Analytical evaluation of thirty-two severe acute respiratory syndrome 2 lateral flow antigen tests demonstrates sensitivity remains with the SARS-CoV-2 Gamma lineage.

BACKGROUND: The emergence of variants of concern (VOCs) requires an ongoing assessment of the performance of antigen lateral flow tests (Ag-RDTs). The limit of detection (LOD) of 32 Ag-RDTs was evaluated using the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Gamma variant. METHODS: Ag-RDTs were performed according to the manufacturer's instructions with a clinical isolate of the Gamma variant. RESULTS: Twenty-eight of the 32 Ag-RDTs exceeded the World Health Organization criteria. CONCLUSIONS: This comprehensive analytical evaluation of Ag-RDTs demonstrated that the test performance was maintained with Gamma VOC.

Two-test algorithms for infectious disease diagnosis: Implications for COVID-19.

Diagnostic assays for various infectious diseases, including COVID-19, have been challenged for their utility as standalone point-of-care diagnostic tests due to suboptimal accuracy, complexity, high cost or long turnaround times for results. It is therefore critical to optimise their use to meet the needs of users. We used a simulation approach to estimate diagnostic outcomes, number of tests required and average turnaround time of using two-test algorithms compared with singular testing; the two tests were reverse transcription polymerase chain reaction (RT-PCR) and an antigen-based rapid diagnostic test (Ag-RDT). A web-based application of the model was developed to visualise and compare diagnostic outcomes for different disease prevalence and test performance characteristics (sensitivity and specificity). We tested the model using hypothetical prevalence data for COVID-19, representing low- and high-prevalence contexts and performance characteristics of RT-PCR and Ag-RDTs. The two-test algorithm when RT-PCR was applied to samples negative by Ag-RDT predicted gains in sensitivity of 27% and 7%, respectively, compared with Ag-RDT and RT-PCR alone. Similarly, when RT-PCR was applied to samples positive by Ag-RDT, specificity gains of 2.9% and 1.9%, respectively, were predicted. The algorithm using Ag-RDT followed by RT-PCR as a confirmatory test for positive patients limited the requirement of RT-PCR testing resources to 16,400 and 3,034 tests when testing a population of 100,000 with an infection prevalence of 20% and 0.05%, respectively. A two-test algorithm comprising a rapid screening test followed by confirmatory laboratory testing can reduce false positive rate, produce rapid results and conserve laboratory resources, but can lead to large number of missed cases in high prevalence setting. The web application of the model can identify the best testing strategies, tailored to specific use cases and we also present some examples how it was used as part of the Access to Covid-19 Tools (ACT) Accelerator Diagnostics Pillar.

Analytical Sensitivity of Eight Different SARS-CoV-2 Antigen-Detecting Rapid Tests for Omicron-BA.1 Variant.

The emergence of each novel SARS-CoV-2 variant of concern (VOC) requires investigation of its potential impact on the performance of diagnostic tests in use, including antigen-detecting rapid diagnostic tests (Ag-RDTs). Although anecdotal reports have been circulating that the newly emerged Omicron-BA.1 variant is in principle detectable by Ag-RDTs, few data on sensitivity are available. We have performed (i) analytical sensitivity testing with cultured virus in eight Ag-RDTs and (ii) retrospective testing in duplicates with clinical samples from vaccinated individuals with Omicron-BA.1 (n = 59) or Delta (n = 54) breakthrough infection on seven Ag-RDTs. Overall, in our analytical study we have found heterogenicity between Ag-RDTs for detecting Omicron-BA.1. When using cultured virus, we observed a trend toward lower endpoint sensitivity for Omicron-BA.1 detection than for earlier circulating SARS-CoV-2 and the other VOCs. In our retrospective study, the detection of Delta and Omicron-BA.1 was assessed in a comparable set of stored clinical samples using seven Ag-RDTs. Four hundred ninety-seven of all 826 tests (60.17%) performed on Omicron-BA.1 samples were positive, compared to 489/756 (64.68%) for Delta samples. In the analytical study, the sensitivity for both Omicron-BA.1 and Delta between the Ag-RDTs was variable. All seven Ag-RDTs showed comparable sensitivities to detect Omicron-BA.1 and Delta in the retrospective study. IMPORTANCE Sensitivity for detecting Omicron-BA.1 shows high heterogenicity between Ag-RDTs, necessitating a careful consideration when using these tests to guide infection prevention measures. Analytical and retrospective testing is a proxy and timely solution to generate rapid performance data, but it is not a replacement for clinical evaluations, which are urgently needed. Biological and technical reasons for detection failure by some Ag-RDTs need to be further investigated.

Multicenter Diagnostic Evaluation of a Novel Coronavirus Antigen Lateral Flow Test among Symptomatic Individuals in Brazil and the United Kingdom.

The COVID-19 pandemic has led to the commercialization of many antigen-based rapid diagnostic tests (Ag-RDTs), requiring independent evaluations. This report describes the clinical evaluation of the Novel Coronavirus 2019-nCoV Antigen Test (Colloidal Gold) (Beijing Hotgen Biotech Co., Ltd.), at two sites within Brazil and one in the United Kingdom. The collected samples (446 nasal swabs from Brazil and 246 nasopharyngeal samples from the UK) were analyzed by the Ag-RDT and compared to reverse transcription-quantitative PCR (RT-qPCR). Analytical evaluation of the Ag-RDT was performed using direct culture supernatants of SARS-CoV-2 strains from the wild-type (B.1), Alpha (B.1.1.7), Delta (B.1.617.2), Gamma (P.1), and Omicron (B.1.1.529) lineages. An overall sensitivity and specificity of 88.2% (95% confidence interval [CI], 81.3 to 93.3) and 100.0% (95% CI, 99.1 to 100.0), respectively, were obtained for the Brazilian and UK cohorts. The analytical limit of detection was determined as 1.0 × 103 PFU/mL (Alpha), 2.5 × 102 PFU/mL (Delta), 2.5 × 103 PFU/mL (Gamma), and 1.0 × 103 PFU/mL (Omicron), giving a viral copy equivalent of approximately 2.1 × 104 copies/mL, 9.0 × 105 copies/mL, 1.7 × 106 copies/mL, and 1.8 × 105 copies/mL for the Ag-RDT, respectively. Overall, while a higher sensitivity was claimed by the manufacturers than that found in this study, this evaluation finds that the Ag-RDT meets the WHO minimum performance requirements for sensitivity and specificity of COVID-19 Ag-RDTs. This study illustrates the comparative performance of the Hotgen Ag-RDT across two global settings and considers the different approaches in evaluation methods. IMPORTANCE Since the beginning of the SARS-CoV-2 pandemic, we have witnessed growing numbers of antigen rapid diagnostic tests (Ag-RDTs) being brought to market. In the United Kingdom, this was somewhat controlled indirectly as the government offered free tests from a small number of companies. However, as this has now ceased, individuals are responsible for their own acquisition of test kits. Similarly in Brazil, as of January 2022, pharmacies and other health care retailers are permitted to sell Ag-RDTs directly to the community. Many of these Ag-RDTs have not been externally evaluated, and results are not readily available to the public. Thus, there is now a need for a transparent evaluation of Ag-RDTs with both analytical and clinical evaluation. We present an independent review of the Novel Coronavirus 2019-nCoV Antigen Test (Colloidal Gold) (Beijing Hotgen Biotech Co., Ltd.), at two sites within Brazil and one in the United Kingdom.

Twelve lateral flow immunoassays (LFAs) to detect SARS-CoV-2 antibodies.

BACKGROUND: There are an abundance of commercially available lateral flow assays (LFAs) that detect antibodies to SARS-CoV-2. Whilst these are usually evaluated by the manufacturer, externally performed diagnostic accuracy studies to assess performance are essential. Herein we present an evaluation of 12 LFAs. METHODS: Sera from 100 SARS-CoV-2 reverse-transcriptase polymerase chain reaction (RT-PCR) positive participants were recruited through the FASTER study. A total of 105 pre-pandemic sera from participants with other infections were included as negative samples. RESULTS: At presentation sensitivity against RT-PCR ranged from 37.4 to 79% for IgM/IgG, 30.3-74% for IgG, and 21.2-67% for IgM. Sensitivity for IgM/IgG improved ≥ 21 days post symptom onset for 10/12 tests. Specificity ranged from 74.3 to 99.1% for IgM/IgG, 82.9-100% for IgG, and 75.2-98% for IgM. Compared to the EuroImmun IgG enzyme-linked immunosorbent assay (ELISA), sensitivity and specificity ranged from 44.6 to 95.4% and 85.4-100%, respectively. CONCLUSION: There are many LFAs available with varied sensitivity and specificity. Understanding the diagnostic accuracy of these tests will be vital as we come to rely more on the antibody status of a person moving forward, and as such manufacturer-independent evaluations are crucial.

A Multicenter Clinical Diagnostic Accuracy Study of SureStatus, an Affordable, WHO Emergency Use-Listed, Rapid, Point-Of-Care Antigen-Detecting Diagnostic Test for SARS-CoV-2.

Access to reverse transcription-PCR (RT-PCR) testing, the gold standard for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection, is limited throughout the world, due to restricted resources, available infrastructure, and high costs. Antigen-detecting rapid diagnostic tests (Ag-RDTs) overcome some of these barriers, but independent clinical validations in settings of intended use are scarce. To inform the World Health Organization's (WHO) emergency use listing (EUL) procedure and ensure affordable, high-quality Ag-RDTs, we assessed the performance and ease of use of the SureStatus for SARS-CoV-2. For this prospective, multicenter diagnostic accuracy study, we recruited unvaccinated participants with presumed SARS-CoV-2 infection in India and Germany from December 2020 to March 2021, when the Alpha (B.1.1.7) variant was predominantly circulating. Paired swabs were performed for (i) routine clinical RT-PCR testing (sampling was either nasopharyngeal [NP] or combined NP and oropharyngeal [NP/OP]) and (ii) Ag-RDT (sampling was NP). Performance of the Ag-RDT was compared to RT-PCR overall and by predefined subgroups, e.g., cycle threshold (CT) value, symptoms, and days from symptom onset. To understand the usability, a system usability scale (SUS) questionnaire and ease-of-use (EoU) assessment were performed. A total of 1,119 participants were included in the analysis, of whom 205 (18.3%) were RT-PCR positive. SureStatus detected 169 out of 205 RT-PCR-positive participants, reporting a sensitivity of 82.4% (95% confidence interval [CI]: 76.6% to 87.1%) and a specificity of 98.5% (95% CI: 97.4% to 99.1%). In the first 7 days post-symptom onset, the sensitivity was 90.7% (95% CI: 83.5% to 94.9%), when CT values were low and viral loads were high. The test was characterized as easy to use (SUS, 85/100) and considered suitable for point-of-care settings, although quality concerns were raised due to visibly contaminated packaging of swabs included in the test kits. The SureStatus diagnostic test can be considered a reliable test during the first week of SARS-CoV-2 infection, with high sensitivity in combination with excellent usability. IMPORTANCE Our manufacturer-independent, prospective diagnostic accuracy study assessed clinical performance in participants presumed to have a SARS-CoV-2 infection at three study sites in two countries. We assessed the accuracy overall and in predefined subgroups (CT values and symptom duration). SureStatus performed with high sensitivity. Its sensitivity was particularly high in the first 3 days after symptom onset and when CT values were low (i.e., the viral load was high). The system usability and ease-of-use assessment complements the accuracy assessment of the test and highlights critical factors to facilitate the widespread use of SureStatus in point-of-care settings. The high sensitivity demonstrated by the evaluated Ag-RDT within the first days of symptoms, when most transmission occurs, supports the role of Ag-RDTs for public health-relevant screening. Evidence from this study was used to inform the World Health Organization Emergency Use Listing procedure.

Distinguishing bacterial versus non-bacterial causes of febrile illness - A systematic review of host biomarkers.

BACKGROUND: Acute febrile illnesses (AFIs) represent a major disease burden globally; however, the paucity of reliable, rapid point-of-care testing makes their diagnosis difficult. A simple tool for distinguishing bacterial versus non-bacterial infections would radically improve patient management and reduce indiscriminate antibiotic use. Diagnostic tests based on host biomarkers can play an important role here, and a target product profile (TPP) was developed to guide development. OBJECTIVES: To qualitatively evaluate host biomarkers that can distinguish bacterial from non-bacterial causes of AFI. DATA SOURCES: The PubMed database was systematically searched for relevant studies published between 2015 and 2019. STUDY ELIGIBILITY CRITERIA: Studies comparing diagnostic performances of host biomarkers in patients with bacterial versus non-bacterial infections were included. PARTICIPANTS: Studies involving human participants and/or human samples were included. METHODS: We collected information following PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. A risk of bias assessment was performed, based on a modified QUADAS-2 (Quality Assessment of Diagnostic Accuracy Score 2). RESULTS: We identified 1107 publications. Following screening, 55 publications were included, with 265 biomarker entries. Entries mostly comprised protein biomarkers (58.9%), followed by haematological, RNA, and metabolite biomarkers (15.5%, 8.7%, 12.5%). Sensitivity/specificity was reported for 45.7% of biomarker entries. We assessed a high overall risk of bias for most entries (75.8%). In studies with low/medium risk of bias, four biomarker entries tested in blood samples had sensitivity/specificity of more than 0.90/0.80. Only 12 additional biomarker entries were identified with sensitivity/specificity of more than 0.65/0.65. CONCLUSIONS: Most recently assessed biomarkers represent well-known biomarkers, e.g. C-reactive protein and procalcitonin. Some protein biomarkers with the highest reported performances include a combined biomarker signature (CRP, IP-10, and TRAIL) and human neutrophil lipocalin (HNL). Few new biomarkers are in the pipeline; however, some RNA signatures show promise. Further high-quality studies are needed to confirm these findings.

Limit of detection in different matrices of 19 commercially available rapid antigen tests for the detection of SARS-CoV-2.

In the context of the coronavirus disease 2019 (COVID-19) pandemic there has been an increase of the use of antigen-detection rapid diagnostic tests (Ag-RDT). The performance of Ag-RDT vary greatly between manufacturers and evaluating their analytical limit of detection (LOD) has become high priority. Here we describe a manufacturer-independent evaluation of the LOD of 19 marketed Ag-RDT using live SARS-CoV-2 spiked in different matrices: direct culture supernatant, a dry swab, and a swab in Amies. Additionally, the LOD using dry swab was investigated after 7 days' storage at - 80 °C of the SARS-CoV-2 serial dilutions. An LOD of ≈ 5.0 × 102 pfu/ml (1.0 × 106 genome copies/ml) in culture media is defined as acceptable by the World Health Organization. Fourteen of 19 Ag-RDTs (ActiveXpress, Espline, Excalibur, Innova, Joysbio, Mologic, NowCheck, Orient, PanBio, RespiStrip, Roche, Standard-F, Standard-Q and Sure-Status) exceeded this performance criteria using direct culture supernatant applied to the Ag-RDT. Six Ag-RDT were not compatible with Amies media and a decreased sensitivity of 2 to 20-fold was observed for eleven tests on the stored dilutions at - 80 °C for 7 days. Here, we provide analytical sensitivity data to guide appropriate test and sample type selection for use and for future Ag-RDT evaluations.