Clinical evaluation of the GSD NovaPrime® SARS-CoV-2 RTq-PCR assay.

Faced with the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), high-throughput respiratory tests are in high demand. We evaluated the clinical performance of the GSD NovaPrime® SARS-CoV-2 RTq-PCR assay, a new assay that detects 2 specific RNA sequences of the nucleocapsid (N) gene. It was assessed using 99 nasopharyngeal samples and compared in parallel with the Allplex® assay. Among those samples, 72 and 27 were included in the positive (PPA) and negative (NPA) percent agreement analyses, respectively. In case of discordance, samples were reanalyzed with another amplification technique, the Aptima® SARS-CoV-2 assay. Cross-reactivity, including specimens positive for another respiratory virus and collected before the COVID-19 outbreak, was also evaluated (n = 32). Based on the patients' clinical history, the Ct (cycle threshold) values obtained, and the results of the Aptima® assay, the clinical performances were deemed satisfactory, with the PPA reaching a minimum percentage of 87.5% and the NPA reaching 100%. No cross-reactivity with other respiratory viruses was observed.

Immunogenicity of mRNA-1273 COVID vaccine after 6 months surveillance in health care workers; a third dose is necessary.

OBJECTIVES: Scarce data are currently available on the kinetics of antibodies after vaccination with mRNA vaccines as a whole and, with mRNA-1273, in particular. We report here an ad-interim analysis of data obtained after a 6-month follow-up in a cohort of healthcare workers (HCWs) who received the mRNA-1273 vaccine. These new data provide more insight into whether and in whom a 3rd dose could be necessary. METHODS: Our study compared the anti-S antibody kinetics at 2 weeks (T1), 3 months (T3) and 6 months (T4) after the first injection, and 2 weeks after the second injection (T2). The 201 participating HCWs were stratified according to their initial serological status. The vaccine effectiveness was also assessed through a medical questionnaire. RESULTS: We report here a marked and statistically significant antibody decrease (P < 0.05) between T3 and T4, especially in naïve vaccinees. The analysis of potential confounding factors or known risk factors for severe COVID-19 disease did not reveal any influence on the drop observed. Six-month after vaccination, only one, symptomatic, infection was reported in our cohort. CONCLUSIONS: In a supply-limited environment, our results plead for reserving the 3rd dose scheme, in the upcoming months, to seronegative individuals prior to vaccination, especially when the serological status is easily accessible.

Efficacy comparison of three rapid antigen tests for SARS-CoV-2 and how viral load impact their performance.

More and more rapid antigen tests for the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) appear in the market with varying performance. The sensitivity of these tests heavily depends on the viral load, extrapolated by the threshold cycle (Ct). It is therefore essential to verify their performance before their inclusion in routine. The Coronavirus Ag Rapid Test Cassette Bio-Rad, the GSD NovaGen SARS-CoV-2 (COVID-19) Antigen Rapid Test, and the Aegle Coronavirus Ag Rapid Test Cassette were evaluated on 199 samples: 150 fresh samples from the routine and positive in quantitative reverse-transcription polymerase chain reaction (RT-qPCR), nine fresh samples negative in RT-qPCR, and 40 frozen samples, taken before the discovery of SARS-CoV-2 but positive for other respiratory viruses. Positive RT-qPCR samples were categorized according to their Ct: Ct < 20 (18.7%), ≥ 20-< 25 (27.3%), ≥ 25-< 30 (18.7%), ≥ 30-35 (17.3%), and > 35 (18.0%). Sensitivities (95% confidence interval) for Ct below 25 were 95.7% (92.4-98.9), 97.1% (94.4-99.8), and 97.1% (94.4-99.8) for GSD NovaGen, Bio-Rad, and Aegle, respectively but drastically dropped when Ct exceeded 27. Among samples with previously diagnosed viruses, seven false-positive results were found with GSD NovaGen only (specificity 85.7%). Equivalent, high sensitivities were observed with the highest viral load samples. The GSD NovaGen assay showed less specificity. Although the three kits tested in this study are inadequate for routine testing in a high throughput laboratory, they can help to quickly identify the most infectious patients and screen their close contacts in an environment where molecular tests are not readily available.

The challenge of screening SARS-CoV-2 variants of concern with RT-qPCR: One variant can hide another.

INTRODUCTION: Following the emergence of SARS-CoV-2 variants of concern (VOCs) worldwide, it is important to monitor local epidemiology to better understand the occurrence of clusters, reinfections, or infection after vaccination. Detecting mutations by specific RT-qPCR is a rapid and affordable alternative to sequencing. However, care must be taken to ensure that the techniques used are up-to-date and adapted to the variants circulating in the studied population. MATERIAL AND METHODS: All samples tested positive for SARS-CoV-2 were screened for detection of mutations of the spike protein using the Novaplex™ SARS-CoV-2 Variants I Assay from week 11 of 2021. Target sought were deletion H69/V70 and mutations N501Y and E484K. From week 18 we used in addition the new Novaplex™ SARS-CoV-2 Variants II Assay for samples with no targets found with the Variants I assay or with the mutation E484K alone, in order to screen the mutations L452R, K417N/T and W152C. RESULTS: Between weeks 11 and 25, 2239 positive samples out of 54,317 were tested with the Variants I Assay. Between weeks 18 and 25, 94 samples met the criteria for being tested with the Variants II Assay. Of these, 47 had the L452R mutation without the W152C mutation, typical in the B.1.617 variant. At week 25, this profile was found in 45.5 % of the samples and was the most frequent. CONCLUSION: According to our observations, variant B.1.617 has become predominant in our institution and most probably in our region. In the absence of the use of the Variants II Assay, they would have been considered wild.

Development and implementation of a RT-qPCR extraction-free protocol for the detection of SARS-CoV-2 and impact on the turn-around-time.

The occurrence of the COVID-19 second-wave outbreak in Europe has pushed laboratories performing molecular SARS-CoV-2 tests to increase their throughput and decrease the result rendering time. In this evaluation, we tested for the first time a new, extraction-free, protocol with the Allplex SARS-CoV-2 Assay RT-qPCR kit on a Nimbus platform. Ninety-one samples, of which 71 previously tested positive with RT-qPCR with extraction were immediately analyzed without extraction, using only a dilution and thermal shock protocol. The positive and negative percentage agreements were respectively 97.2% (95% confidence interval [CI]: 0.90-0.99) and 95.0% (95% CI: 0.76-0.99). The two false negatives observed were very weakly positive with the comparison method. Moderate variations in Ct of the targeted genes were observed (median ± 95% CI): E gene, +2.49 ± 0.44; N gene, +0.98 ± 0.54; RdRP/S genes, +2.64 ± 0.48. On the other hand, the number of tests performed within 24 h raised from 86.4% to 97.8%, the turn-around time decreased from 19:18 to 09:03 (p < .0001), and the number of tests that can be performed per day doubled since this technique was introduced routinely in our laboratory.

Analytical and clinical validation of an ELISA for specific SARS-CoV-2 IgG, IgA, and IgM antibodies.

The development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serological tests is massive. The external validation of their performance is needed before use in clinical routine practice. Our study aims at assessing the analytical and clinical performance of two enzyme-linked immunosorbent assay tests detecting antibodies directed against the virus nucleocapsid protein: The NovaLisa SARS-CoV-2 immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin M (IgM) test (NovaTec) allowing a separate detection of each antibody and the Platelia SARS-CoV-2 Total Ab test (Bio-Rad) detecting total antibodies (IgM, IgA, and IgG). Two-hundred and eight coronavirus disease 2019 samples from 48 quantitative reverse transcription-polymerase chain reaction (RT-qPCR) confirmed patients were used to perform the sensitivity analysis. Non-SARS-CoV-2 sera (n = 79) with a potential cross-reaction to SARS-CoV-2 immunoassays were included in the specificity analysis. In addition, using receiver operator characteristic curves, adapted cut-off for improvement of the performances were proposed. The kinetics of these antibodies was also assessed over 8 weeks. Two weeks after the RT-qPCR positive detection, the NovaLisa test shows a sensitivity and specificity of 94.9% (95% confidence interval [CI]: 83.1%-98.6%) and 96.2% (95% CI: 89.4%-98.7%) for IgG, of 89.7% (95% CI: 76.4%-95.9%) and 98.7% (95% CI: 93.2%-98.8%) for IgA, and of 48.7% (95% CI: 33.9%-63.8%) and 98.7% (95% CI: 93.2%-99.8%) for IgM. With the Platelia system, the specificity and sensitivity were 97.4% (95% CI: 92.1%-99.7%) and 94.9% (95% CI: 87.7%-98.0%) for total antibodies using the adapted cut-offs. The NovaLisa and the Platelia tests have satisfactory analytical performances. The clinical performances are excellent for IgG, IgA, and total antibodies especially if the cut-off is optimized.

Organisation and quality monitoring for point-of-care testing (POCT) in Belgium: proposal for an expansion of the legal framework for POCT into primary health care.

BACKGROUND: There is a trend towards decentralisation of laboratory tests by means of Point-of-Care testing (POCT). Within hospitals, Belgian law requires a POCT policy, coordinated by the clinical laboratory. There is however no legal framework for POCT performed outside the hospital: no reimbursement, no compulsory quality monitoring and no limits nor control on the prices charged to the patient. Uncontrolled use of POCT can have negative consequences for individual and public health. PROPOSAL: We propose that POCT outside hospitals would only be reimbursed for tests carried out within a legal framework, requiring evidence-based testing and collaboration with a clinical laboratory, because clinical laboratories have procedures for test validation and quality monitoring, are equipped for electronic data transfer, are familiar with logistical processes, can provide support when technical issues arise and can organise and certify training. Under these conditions the government investment will be offset by health benefits, e.g. fall in antibiotic consumption with POCT for CRP in primary care, quick response to SARS-CoV2-positive cases in COVID-19 triage centres. PRIORITIES: 1° extension of the Belgian decree on certification of clinical laboratories to decentralised tests in primary care; 2° introduction of a separate reimbursement category for POCT; 3° introduction of reimbursement for a limited number of specified POCT; 4° setup of a Multidisciplinary POCT Advisory Council, the purpose of which is to draw up a model for reimbursement of POCT, to select tests eligible for reimbursement and to make proposals to the National Institute for Health and Disability Insurance (RIZIV/INAMI).

Implementation of rapid SARS-CoV-2 antigenic testing in a laboratory without access to molecular methods: Experiences of a general hospital.

BACKGROUND: The COVID-19 Ag (Antigen) Respi-Strip assay is a new immunochromatographic diagnostic tool recently available for antigenic diagnosis of SARS-CoV-2. The proposed sensitivity is not higher than 60 %, but its high specificity allows both quick decisions for the management of patients and confirmation by molecular diagnosis for only negative tests. However, from the first tests performed, we suspected that the sensitivity observed with routine use was much lower than that announced by the manufacturer. MATERIALS AND METHODS: Over a period of one month, we compared the negative results obtained with the COVID-19 Ag Respi-Strip kit with those obtained from qRT-PCR performed in a laboratory qualified for the molecular diagnosis of SARS-CoV-2. All samples tested were naso-pharyngeal smears from UTM-RT medium. RESULTS: Of 774 patients tested, 714 negative samples were sent for confirmation, and 159 were found to be positive by qRT-PCR. The median positive percentage agreement was 23.9 % (95 % CI: 14.2 %-38.2 %). The Cohen's kappa score was 0.35. CONCLUSION: Using this immunochromatographic assay as a triage test did not significantly reduce the number of samples outsourced for COVID-19 confirmation by qRT-PCR. In addition, even if the turn-around time is short, the assay is completely manual, which is not suitable for large volumes of routine samples. The sensitivity of this rapid test is poor, and improvements are needed to enhance its performance.

Validation of a chemiluminescent assay for specific SARS-CoV-2 antibody.

Objectives Faced with the COVID-19 pandemic and its impact on the availability and quality of both therapeutic and diagnostic methods, the Belgian authorities have decided to launch a procedure for additional evaluation of the performance of serological tests offered for sale on the national territory. This has been proposed with a double aim: (1) an in-depth verification of the analytical and clinical performances presented by the manufacturer and (2) an economy of scale in terms of centralized validation for all the laboratories using the tests subject to evaluation. Methods A retrospective validation study was conducted including the serum of 125 patients in order to determine the analytical and clinical performances of the LIAISON®SARS-CoV-2 from DiaSorin® detecting anti-SARS-CoV-2 IgG and to compare its clinical performance with the enzyme-linked immunosorbent assay (ELISA) test from Euroimmun®, one of the first commercially available tests allowing the detection of anti-SARS-CoV-2 IgA and IgG. Results The performances of the LIAISON®SARS-CoV-2 satisfied all the acceptance criteria and provided "real world" analytical and clinical performances very close to the ones reported by the manufacturer in its insert kit. Comparison between the LIAISON®SARS-CoV-2 and the ELISA method did not reveal any difference between the two techniques in terms of sensitivities and specificities regarding the determination of the IgG. Conclusions This study reports the validation of the LIAISON®SARS-CoV-2 allowing to detect IgG antibodies specifically directed against SARS-CoV-2. The analytical and clinical performances are excellent, and the automation of the test offers important rates, ideal for absorbing an extension of testing.