A High-Avidity, Thermostable, and Low-Cost Synthetic Capture for Ultrasensitive Detection and Quantification of Viral Antigens and Aerosols.

Lateral flow assays (LFAs) are currently the most popular point-of-care diagnostics, rapidly transforming disease diagnosis from expensive doctor checkups and laboratory-based tests to potential on-the-shelf commodities. Yet, their sensitive element, a monoclonal antibody, is expensive to formulate, and their long-term storage depends on refrigeration technology that cannot be met in resource-limited areas. In this work, LCB1 affibodies (antibody mimetic miniproteins) were conjugated to bovine serum albumin (BSA) to afford a high-avidity synthetic capture (LCB1-BSA) capable of detecting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and virus like particles (VLPs). Substituting the monoclonal antibody 2B04 for LCB1-BSA (stable up to 60 °C) significantly improved the thermal stability, shelf life, and affordability of plasmonic-fluor-based LFAs (p-LFAs). Furthermore, this substitution significantly improved the sensitivity of p-LFAs toward the spike protein and VLPs with precise quantitative ability over 2 and 3 orders of magnitude, respectively. LCB1-BSA sensors could detect VLPs at 100-fold lower concentrations, and this improvement, combined with their robust nature, enabled us to develop an aerosol sampling technology to detect aerosolized viral particles. Synthetic captures like LCB1-BSA can increase the ultrasensitivity, availability, sustainability, and long-term accuracy of LFAs while also decreasing their manufacturing costs.

Feasibility and Effectiveness Assessment of SARS-CoV-2 Antigenic Tests in Mass Screening of a Pediatric Population and Correlation with the Kinetics of Viral Loads.

The gold standard for diagnosis of SARS-CoV-2 infection has been nucleic acid amplification tests (NAAT). However, rapid antigen detection kits (Ag-RDTs), may offer advantages over NAAT in mass screening, generating results in minutes, both as laboratory-based test or point-of-care (POC) use for clinicians, at a lower cost. We assessed two different POC Ag-RDTs in mass screening versus NAAT for SARS-CoV-2 in a cohort of pediatric patients admitted to the Pediatric Emergency Unit of IRCCS-Polyclinic of Sant'Orsola, Bologna (from November 2020 to April 2021). All patients were screened with nasopharyngeal swabs for the detection of SARS-CoV-2-RNA and for antigen tests. Results were obtained from 1146 patients. The COVID-19 Ag FIA kit showed a baseline sensitivity of 53.8% (CI 35.4-71.4%), baseline specificity 99.7% (CI 98.4-100%) and overall accuracy of 80% (95% CI 0.68-0.91); the AFIAS COVID-19 Ag kit, baseline sensitivity of 86.4% (CI 75.0-93.9%), baseline specificity 98.3% (CI 97.1-99.1%) and overall accuracy of 95.3% (95% CI 0.92-0.99). In both tests, some samples showed very low viral load and negative Ag-RDT. This disagreement may reflect the positive inability of Ag-RDTs of detecting antigen in late phase of infection. Among all cases with positive molecular test and negative antigen test, none showed viral loads > 106 copies/mL. Finally, we found one false Ag-RDTs negative result (low cycle thresholds; 9 × 105 copies/mL). Our results suggest that both Ag-RDTs showed good performances in detection of high viral load samples, making it a feasible and effective tool for mass screening in actively infected children.

Comparison of four commercial, automated antigen tests to detect SARS-CoV-2 variants of concern.

A versatile portfolio of diagnostic tests is essential for the containment of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) pandemic. Besides nucleic acid-based test systems and point-of-care (POCT) antigen (Ag) tests, quantitative, laboratory-based nucleocapsid Ag tests for SARS-CoV-2 have recently been launched. Here, we evaluated four commercial Ag tests on automated platforms and one POCT to detect SARS-CoV-2. We evaluated PCR-positive (n = 107) and PCR-negative (n = 303) respiratory swabs from asymptomatic and symptomatic patients at the end of the second pandemic wave in Germany (February-March 2021) as well as clinical isolates EU1 (B.1.117), variant of concern (VOC) Alpha (B.1.1.7) or Beta (B.1.351), which had been expanded in a biosafety level 3 laboratory. The specificities of automated SARS-CoV-2 Ag tests ranged between 97.0 and 99.7% (Lumipulse G SARS-CoV-2 Ag (Fujirebio): 97.03%, Elecsys SARS-CoV-2 Ag (Roche Diagnostics): 97.69%; LIAISON® SARS-CoV-2 Ag (Diasorin) and SARS-CoV-2 Ag ELISA (Euroimmun): 99.67%). In this study cohort of hospitalized patients, the clinical sensitivities of tests were low, ranging from 17.76 to 52.34%, and analytical sensitivities ranged from 420,000 to 25,000,000 Geq/ml. In comparison, the detection limit of the Roche Rapid Ag Test (RAT) was 9,300,000 Geq/ml, detecting 23.58% of respiratory samples. Receiver-operating-characteristics (ROCs) and Youden's index analyses were performed to further characterize the assays' overall performance and determine optimal assay cutoffs for sensitivity and specificity. VOCs carrying up to four amino acid mutations in nucleocapsid were detected by all five assays with characteristics comparable to non-VOCs. In summary, automated, quantitative SARS-CoV-2 Ag tests show variable performance and are not necessarily superior to a standard POCT. The efficacy of any alternative testing strategies to complement nucleic acid-based assays must be carefully evaluated by independent laboratories prior to widespread implementation.

Use, Safety Assessment, and Implementation of Two Point-of-Care Tests for COVID-19 Testing.

OBJECTIVES: The Abbot ID NOW COVID-19 assay and Quidel Sofia 2 SARS Antigen FIA are point-of-care assays that offer rapid testing for severe acute respiratory syndrome coronavirus 2 viral RNA and nucleocapsid protein, respectively. Given the utility of these devices in the field, we investigated the feasibility and safety of using the ID NOW and Sofia assays in the public health response to the coronavirus disease 2019 pandemic and in future public health emergencies. METHODS: A combination of utilization and contamination testing in addition to a review of instrument workflows was conducted. RESULTS: Utilization testing demonstrated that both tests are intuitive, associated with high user test success (85%) in our study, and could be implemented by staff after minimal training. Contamination tests revealed potential biosafety concerns due to the open design of the ID NOW instrument and the transfer mechanisms with the Sofia. When comparing the workflow of the ID NOW and the Sofia, we found that the ID NOW was more user-friendly and that the transfer technology reduces the chance of contamination. CONCLUSIONS: The ID NOW, Sofia, and other emerging point-of-care tests should be used only after careful consideration of testing workflow, biosafety risk mitigations, and appropriate staff training.

Time scale performance of rapid antigen testing for SARS-CoV-2: Evaluation of 10 rapid antigen assays.

There is a great demand for more rapid tests for SARS-CoV-2 detection to reduce waiting time, boost public health strategies for combating disease, decrease costs, and prevent overwhelming laboratory capacities. This study was conducted to assess the performance of 10 lateral flow device viral antigen immunoassays for the detection of SARS-CoV-2 in nasopharyngeal swab specimens. We analyzed 231 nasopharyngeal samples collected from October 2020 to December 2020, from suspected COVID-19 cases and contacts of positive cases at Biotechnology Research Center laboratories, Tripoli, Libya. The performance of 10 COVID-19 Antigen (Ag) rapid test devices for the detection of SARS-CoV-2 antigen was compared to a quantitative reverse transcription-polymerase chain reaction (RT-qPCR). In this study, 161 cases had symptoms consistent with COVID-19. The mean duration from symptom onset was 6.6 ± 4.3 days. The median cycle threshold (Ct ) of positive samples was 25. Among the 108 positive samples detected by RT-qPCR, the COVID-19 antigen (Ag) tests detected 83 cases correctly. All rapid Ag test devices used in this study showed 100% specificity. While tests from six manufacturers had an overall sensitivity range from 75% to 100%, the remaining four tests had a sensitivity of 50%-71.43%. Sensitivity during the first 6 days of symptoms and in samples with high viral loads (Ct < 25), was 100% in all but two of the test platforms. False-negative samples had a median Ct of 34 and an average duration of onset of symptoms of 11.3 days (range = 5-20 days). Antigen test diagnosis has high sensitivity and specificity in early disease when patients present less than 7 days of symptom onset. Patients are encouraged to test as soon as they get COVID-19-related symptoms within 1 week and to seek medical advice within 24 h if they develop disturbed smell/taste. The use of rapid antigen tests is important for controlling the COVID-19 pandemic and reducing the burden on molecular diagnostic laboratories.

Accuracy and cost description of rapid antigen test compared with reverse transcriptase-polymerase chain reaction for SARS-CoV-2 detection.

INTRODUCTION: Fast and accurate detection of SARS-CoV-2 is essential in limiting the COVID-19 pandemic. Rapid antigen (AG) tests provide results within minutes; however, their accuracy has been questioned. The study aims to determine the accuracy and cost of the STANDARD Q COVID-19 AG test compared with RT-PCR. METHODS: Individuals 18 years or older with an appointment for a RT-PCR test on 26-31 December 2020 at a public test centre in Copenhagen, Denmark were invited to participate. An oropharyngeal swab was collected for RT-PCR analysis, followed by a nasopharyngeal swab examined by the AG test (SD Biosensor). The diagnostic accuracy of the AG test was calculated with RT-PCR as reference. Costs were evaluated for both tests. RESULTS: A total of 4,811 paired conclusive test results were collected (median age: 45 years, female: 53%). The RT-PCR test revealed 221 (4.6%) positive tests. The overall sensitivity and specificity of the AG test were 69.7% and 99.5%, respectively. Viral cycle threshold values were significantly higher in individuals with false negative AG tests than in individuals who were true positives. The RT-PCR test and AG test costs were 67.0 DKK (10.8 USD) and 35.0 DKK (5.7 USD), respectively, per positive case detected at 100,000 daily tests. CONCLUSIONS: The AG test enables mass testing and provides immediate results, which is important in SARS-CoV-2 screening. The AG test is a good and relevant supplement to RT-PCR testing in public SARS-CoV-2 screenings. FUNDING: This project received no external funding. Copenhagen Medical A/S delivering the rapid AG tests and provided test personnel but were not otherwise involved. TRIAL REGISTRATION: Clinicaltrials.org: NCT04716088.

Longitudinal Assessment of Diagnostic Test Performance Over the Course of Acute SARS-CoV-2 Infection.

BACKGROUND: Serial screening is critical for restricting spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by facilitating timely identification of infected individuals to interrupt transmission. Variation in sensitivity of different diagnostic tests at different stages of infection has not been well documented. METHODS: In a longitudinal study of 43 adults newly infected with SARS-CoV-2, all provided daily saliva and nasal swabs for quantitative reverse transcription polymerase chain reaction (RT-qPCR), Quidel SARS Sofia antigen fluorescent immunoassay (FIA), and live virus culture. RESULTS: Both RT-qPCR and Quidel SARS Sofia antigen FIA peaked in sensitivity during the period in which live virus was detected in nasal swabs, but sensitivity of RT-qPCR tests rose more rapidly prior to this period. We also found that serial testing multiple times per week increases the sensitivity of antigen tests. CONCLUSIONS: RT-qPCR tests are more effective than antigen tests at identifying infected individuals prior to or early during the infectious period and thus for minimizing forward transmission (given timely results reporting). All tests showed >98% sensitivity for identifying infected individuals if used at least every 3 days. Daily screening using antigen tests can achieve approximately 90% sensitivity for identifying infected individuals while they are viral culture positive.

World-Wide Prevalence and Genotype Distribution of Enteroviruses.

Enteroviruses (EVs) are highly prevalent viruses world-wide, causing a wide range of diseases in both children and adults. Insight in the global prevalence of EVs is important to define their clinical significance and total disease burden, and assists in making therapeutic decisions. While many studies have been conducted to describe epidemiology of EVs in specific (sub)populations and patient cohorts, little effort has been made to aggregate the available evidence. In the current study, we conducted a search in the PubMed and Embase (Ovid) databases to identify articles reporting EV prevalence and type distribution. We summarized the findings of 153 included studies. We found that EVs are highly prevalent viruses in all continents. Enterovirus B was the most detected species worldwide, while the other species showed continent-specific differences, with Enterovirus C more detected in Africa and Enterovirus A more detected in Asia. Echovirus 30 was by far the most detected type, especially in studies conducted in Europe. EV types in species Enterovirus B-including echovirus 30-were often detected in patient groups with neurological infections and in cerebrospinal fluid, while Enterovirus C types were often found in stool samples.

Field evaluation of COVID-19 antigen tests versus RNA based detection: Potential lower sensitivity compensated by immediate results, technical simplicity, and low cost.

One year into the coronavirus disease 2019 (COVID-19) pandemic, diagnostic strategies, although central for contact tracing and other preventive measures, are still limited. To meet the global demand, lower cost and faster antigen tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection are a convenient alternative to the gold standard reverse transcription-polymerase chain reaction (RT-PCR) assay. We tested laboratory-based RT-PCR RNA detection and two rapid antigen detection (RAD) tests, based on the immunochromatography test for nucleocapsid protein of SARS-CoV-2 (COVID-19 Ag ECO Test, ECO Diagnóstica, and Panbio COVID-19 Ag Rapid Test Abbott). Paired collection and testing were done in a small prospective open study in three clinical services in São Paulo, constituted of mostly symptomatic volunteers at collection (97%, 109/112) for a median of 4 days (interquartile range: 3-6), ranging from 1 to 30. Among the 108 paired RT-PCR/RAD tests, results were concordant in 96.4% (101/108). The test's performance was comparable, with an overall sensitivity of 87% and a specificity of 96%. These observations add to other data that suggest that antigen tests may provide reasonable sensitivity and specificity and deserve a role to improve testing strategies, especially in resource-limited settings.

Clinical assessment of the Roche SARS-CoV-2 rapid antigen test.

OBJECTIVES: Novel point-of-care antigen assays present a promising opportunity for rapid screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. The purpose of this study was the clinical assessment of the new Roche SARS-CoV-2 Rapid Antigen Test. METHODS: The clinical performance of Roche SARS-CoV-2 Rapid Antigen Test was evaluated vs. a reverse transcription polymerase chain reaction (RT-PCR) laboratory-based assay (Seegene AllplexTM2019-nCoV) in nasopharyngeal swabs collected from a series of consecutive patients referred for SARS-CoV-2 diagnostics to the Pederzoli Hospital (Peschiera del Garda, Verona, Italy) over a 2-week period. RESULTS: The final study population consisted of 321 consecutive patients (mean age, 46 years and IQR, 32-56 years; 181 women, 56.4%), with 149/321 (46.4%) positive for SARS-CoV-2 RNA via the Seegene AllplexTM2019-nCoV Assay, and 109/321 (34.0%) positive with Roche SARS-CoV-2 Rapid Antigen Test, respectively. The overall accuracy of Roche SARS-CoV-2 Rapid Antigen Test compared to molecular testing was 86.9%, with 72.5% sensitivity and 99.4% specificity. Progressive decline in performance was observed as cycle threshold (Ct) values of different SARS-CoV-2 gene targets increased. The sensitivity was found to range between 97-100% in clinical samples with Ct values <25, between 50-81% in those with Ct values between 25 and <30, but low as 12-18% in samples with Ct values between 30 and <37. CONCLUSIONS: The clinical performance of Roche SARS-CoV-2 Rapid Antigen Test is excellent in nasopharyngeal swabs with Ct values <25, which makes it a reliable screening test in patients with high viral load. However, mass community screening would require the use of more sensitive techniques.

The Sensitivity and Costs of Testing for SARS-CoV-2 Infection With Saliva Versus Nasopharyngeal Swabs : A Systematic Review and Meta-analysis.

BACKGROUND: Nasopharyngeal swabs are the primary sampling method used for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but they require a trained health care professional and extensive personal protective equipment. PURPOSE: To determine the difference in sensitivity for SARS-CoV-2 detection between nasopharyngeal swabs and saliva and estimate the incremental cost per additional SARS-CoV-2 infection detected with nasopharyngeal swabs. DATA SOURCES: Embase, Medline, medRxiv, and bioRxiv were searched from 1 January to 1 November 2020. Cost inputs were from nationally representative sources in Canada and were converted to 2020 U.S. dollars. STUDY SELECTION: Studies including at least 5 paired nasopharyngeal swab and saliva samples and reporting diagnostic accuracy for SARS-CoV-2 detection. DATA EXTRACTION: Data were independently extracted using standardized forms, and study quality was assessed using QUADAS-2 (Quality Assessment of Diagnostic Accuracy Studies 2). DATA SYNTHESIS: Thirty-seven studies with 7332 paired samples were included. Against a reference standard of a positive result on either sample, the sensitivity of saliva was 3.4 percentage points lower (95% CI, 9.9 percentage points lower to 3.1 percentage points higher) than that of nasopharyngeal swabs. Among persons with previously confirmed SARS-CoV-2 infection, saliva's sensitivity was 1.5 percentage points higher (CI, 7.3 percentage points lower to 10.3 percentage points higher) than that of nasopharyngeal swabs. Among persons without a previous SARS-CoV-2 diagnosis, saliva was 7.9 percentage points less (CI, 14.7 percentage points less to 0.8 percentage point more) sensitive. In this subgroup, if testing 100 000 persons with a SARS-CoV-2 prevalence of 1%, nasopharyngeal swabs would detect 79 more (95% uncertainty interval, 5 fewer to 166 more) persons with SARS-CoV-2 than saliva, but with an incremental cost per additional infection detected of $8093. LIMITATION: The reference standard was imperfect, and saliva collection procedures varied. CONCLUSION: Saliva sampling seems to be a similarly sensitive and less costly alternative that could replace nasopharyngeal swabs for collection of clinical samples for SARS-CoV-2 testing. PRIMARY FUNDING SOURCE: McGill Interdisciplinary Initiative in Infection and Immunity. (PROSPERO: CRD42020203415).

COVID-19 diagnostics in context.

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the need for different types of diagnostics, comparative validation of new tests, faster approval by federal agencies, and rapid production of test kits to meet global demands. In this Perspective, we discuss the utility and challenges of current diagnostics for COVID-19.

Streamlining the screening cascade for active Hepatitis C in Russia: A cost-effectiveness analysis.

OBJECTIVE: Screening for hepatitis C in Russia is a complex process that involves several visits and stepwise testing, limiting adherence and substantially reducing the yield in the identification of active infections. We aimed to evaluate the cost-effectiveness of different screening algorithms from a health system perspective. METHODS: A decision analytic model was applied to a hypothetical adult population eligible to participate in a general screening program for hepatitis C in Russia. The standard pathway (I: Screen for anti-HCV antibodies followed by a nucleic acid test for HCV RNA on antibody positives) was compared to three alternatives (II: Screen for antibodies, a reflexed test for HCV antigen on antibody positives, and RNA on antigen negatives; III: Screen for antibodies, a reflexed test for HCV antigen on antibody positives; IV: Screen for antigen). Each strategy considered a cascade of events (referral, adherence, testing, diagnosis) that must occur for screening to be effective. The primary measure of effectiveness was the number of diagnosed active infections. Calculations followed a health system perspective with costs derived from 2017 reimbursement rates and a willingness-to-pay of 2,000RUB ($82) per diagnosed active infection. Model was tested with deterministic and probabilistic sensitivity analyses. RESULTS: Non-adherence to screening stages reduced the capture rate of active infections in Strategy I from 79.0% to 40.6%. Strategies II, III, and IV were less affected and identified 69%, 67%, and 104% more infections. Average costs per diagnosed infection were decreased by 41% from 89,599RUB ($3,681) for I to 53,072RUB ($2,180), 53,004RUB ($2,177), and 59,633RUB ($2,450) for II, III, and IV, respectively. With a probability of 97%, Strategy III was most cost-effective with an incremental cost-effectiveness ratio vs. I of -1,373RUB (CI: -5,011RUB to -2,033RUB; $-56; CI: -$206 to -$84). Below a willingness-to-pay of 91,000RUB ($3,738), Strategy IV was not cost-effective. Sensitivity analyses confirmed the robustness of results. CONCLUSIONS: Testing strategies for hepatitis C with HCV antigen on HCV antibody positive cases offer a streamlining opportunity for population screening programs. Those shall increase the chances for detecting active infections and are cost-effective over current practice in Russia.

Epidemiology and economic losses of rotavirus infection associated with hospitalization of Armenian children.

INTRODUCTION: Rotavirus (RV) is the leading cause of severe diarrhea-associated morbidity and mortality among children worldwide. Limited data exist on the epidemiology and burden of rotavirus gastroenteritis in Armenia. The purpose of this study is to estimate the economic losses and describe the epidemiological characteristics of rotavirus infections in hospitalized children in Armenia. METHODOLOGY: A retrospective chart review was performed of all children (aged < 5 years) with Rotavirus infection admitted to the "Nork" Republican Infection Clinical Hospital in Yerevan, the capital of Armenia, between January and July 2014. Criteria of inclusion were age under 5 years old and presence of RV antigen in stool by enzyme linked immunosorbent assay. RESULTS: The total number of patients was 126; average age was 28.7 ± 13.3 months; 54.8% were male. The highest number of cases (31.8%) was observed in April. Most of the patients (71.4%) were hospitalized in the first three days, demonstrating an acute onset of the disease. In total 19% of the patients had received RV vaccine and, despite this, were infected with RV. Based on bacteriological examination of stool, 18.3% of patients had RV infection combined with pathogenic or conditional pathogenic microflora. All patients spent 817 days in total in the hospital. Economic losses associated with hospitalization were 16340000 AMD (≈ 33346 USD). CONCLUSION: Rotavirus carries significant morbidity and economic losses. Comprehensive estimates of the disease characteristics and introduction of a national immunization program against RV initiated in 2012 may decrease this burden. Further studies to evaluate the feasibility and cost-effectiveness of such a program are warranted.

Pediatric hospitalizations attributable to rotavirus gastroenteritis among Cambodian children: Seven years of active surveillance, 2010-2016.

BACKGROUND: Each year, approximately 1,066 Cambodian children under five years old die of diarrhea, and 51% of these deaths are due to rotavirus gastroenteritis. Quantifying childhood hospitalizations caused by severe rotavirus infections is also important in demonstrating disease burden caused by this virus. The objective of this study is to update and confirm the current burden of pediatric hospitalizations attributable to rotavirus gastroenteritis among Cambodian children using seven years of continuous active, prospective surveillance from 2010 to 2016. We also characterize the circulating rotavirus genotypic strains during this period. METHODS: Active surveillance for rotavirus gastroenteritis was conducted from January 2010 through December 2016 at a national hospital in Phnom Penh, Cambodia. Children <60 months of age who were hospitalized for acute gastroenteritis (AGE) were consented and enrolled. Information on gender, age, clinical characteristics, and month of onset were collected. Stool specimens were collected and tested by enzyme immunoassay for the presence of rotavirus antigen, and genotyping was performed on rotavirus test-positive specimens to characterize predominant rotavirus strains during the surveillance period. RESULTS: Of 7007 children enrolled with AGE and having specimens collected, 3473 (50%) were attributed to rotavirus gastroenteritis. The majority of rotavirus hospitalizations occurred in children younger than two years old (92%). Year-round rotavirus transmission was observed, with seasonal peaks during the cooler, dry months between November and May. Genotypic trends in rotavirus were observed over the surveillance period; the predominant rotavirus strains changed from G1P[8] (2010-2012), to G2P[4] (2013-2014), the emergence of genotype G8P[8] in 2015, and G3P[8] in 2016. CONCLUSIONS: Rotavirus is the leading cause of severe acute gastroenteritis hospitalizations in Cambodian children under five years old, with 50% of such hospitalizations attributable to rotavirus. Over 90% of rotavirus hospitalizations occurred in children under 2 years of age. Changes in the predominant rotavirus strains occurred over time among these unvaccinated children. This information is important to understand and prioritize the current potential impacts upon child health that could be achieved through the introduction of rotavirus vaccines in Cambodia.