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Serological assays designed to detect SARS-CoV-2 antibodies are being used in serological surveys and other specialized applications. As a result, and to ensure that the outcomes of serological testing meet high quality standards, evaluations are required to assess the performance of these assays and the proficiency of laboratories performing them.A panel of 60 plasma/serum samples from blood donors who had reverse transcriptase-polymerase chain reaction (RT-PCR) confirmed SARS-CoV-2 infections and 21 SARS-CoV-2 negative samples were secured and distributed to interested laboratories within Canada (The performance of the different assays evaluated was excellent, with the high-throughput platforms of Roche, Ortho, and Siemens demonstrating 100% sensitivity. Most other high-throughput platforms had sensitivities of93%, with the exception of the IgG assay using the Abbott ARCHITECT which had an average sensitivity of only 87%. The majority of the high-throughput platforms also demonstrated very good specificities (97%).This proficiency study demonstrates that most of the SARS-CoV-2 serological assays utilized by provincial public health or hospital laboratories in Canada have acceptable sensitivity and excellent specificity.Les dosages sérologiques conçus pour dépister les anticorps anti-SRAS-CoV-2 sont utilisés dans les études sérologiques et d’autres applications spécialisées. Par conséquent, et pour s’assurer que leurs résultats respectent des normes de qualité, il faut procéder à des évaluations de leur performance et de la compétence des laboratoires à les effectuer.Les chercheurs ont obtenu une batterie de 60 prélèvements de plasma et de sérum chez des donneurs dont l’amplification en chaîne par polymérase après transcription inverse (RT-PCR) avait confirmé des infections par le SRAS-CoV-2 et de 21 prélèvements dont les résultats étaient négatifs au SRAS-CoV-2 et les ont distribués aux laboratoires intéressés du Canada (Les divers dosages avaient une excellente exécution, les plateformes à haut débit de Roche, d’Ortho et de Siemens démontrant une sensibilité de 100 %. La plupart des autres plateformes à haut débit avaient des sensibilités de plus de 93 %, à l’exception des dosages des IgG faisant appel à l’analyseur ARCHITECT d’Abbott, dont la sensibilité moyenne était de seulement 87 %. La majorité des plateformes à haut débit avaient également une très bonne spécificité (plus de 97 %).La présente étude de compétence démontre que la plupart des dosages sérologiques du SRAS-CoV-2 évalués dans des laboratoires sanitaires provinciaux ou les laboratoires hospitaliers du Canada possèdent une sensibilité acceptable et une excellente spécificité.

Neutralization assays are important for understanding and quantifying neutralizing antibody responses toward severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 lentivirus surrogate neutralization assay (SCLSNA) can be used in biosafety level 2 (BSL-2) laboratories and has been shown to be a reliable alternative approach to the plaque reduction neutralization test (PRNT). In this study, we optimized and validated the SCLSNA to assess its ability as a comparator and prescreening method to support the PRNT. Comparability between the PRNT and SCLSNA was determined through clinical sensitivity and specificity evaluations. Clinical sensitivity and specificity assays produced acceptable results, with 100% (95% confidence interval [CI], 94% to 100%) specificity and 100% (95% CI, 94% to 100%) sensitivity against ancestral Wuhan spike-pseudotyped lentivirus. The sensitivity and specificity against B.1.1.7 spike-pseudotyped lentivirus were 88.3% (95% CI, 77.8% to 94.2%) and 100% (95% CI, 94% to 100%), respectively. Assay precision measuring intra-assay variability produced acceptable results for high (50% PRNT [PRNT

The evaluation of humoral protective immunity against SARS-CoV-2 remains crucial in understanding both natural immunity and protective immunity conferred by the several vaccines implemented in the fight against COVID-19. The reference standard for the quantification of antibodies capable of neutralizing SARS-CoV-2 is the plaque-reduction neutralization test (PRNT). However, given that it is a laboratory-developed assay, validation is crucial in order to ensure sufficient specificity and intra- and interassay precision. In addition, a multitude of other serological assays have been developed, including enzyme-linked immunosorbent assay (ELISA), flow cytometry-based assays, luciferase-based lentiviral pseudotype assays, and commercially available human ACE2 receptor-blocking antibody tests, which offer practical advantages in the evaluation of the protective humoral response against SARS-CoV-2. In this study, we validated a SARS-CoV-2 PRNT to assess both 50% and 90% neutralization of SARS-CoV-2 according to guidelines outlined by the World Health Organization. Upon validation, the reference-standard PRNT demonstrated excellent specificity and both intra- and interassay precision. Using the validated assay as a reference standard, we characterized the neutralizing antibody response in specimens from patients with laboratory-confirmed COVID-19. Finally, we conducted a small-scale multilaboratory comparison of alternate SARS-CoV-2 PRNTs and surrogate neutralization tests. These assays demonstrated substantial to perfect interrater agreement with the reference-standard PRNT and offer useful alternatives to assess humoral immunity against SARS-CoV-2. IMPORTANCE SARS-CoV-2, the causal agent of COVID-19, has infected over 246 million people and led to over 5 million deaths as of October 2021. With the approval of several efficacious COVID-19 vaccines, methods to evaluate protective immune responses will be crucial for the understanding of long-term immunity in the rapidly growing vaccinated population. The PRNT, which quantifies SARS-CoV-2-neutralizing antibodies, is used widely as a reference standard to validate new platforms but has not undergone substantial validation to ensure excellent inter- and intraassay precision and specificity. Our work is significant, as it describes the thorough validation of a PRNT, which we then used as a reference standard for the comparison of several alternative serological methods to measure SARS-CoV-2-neutralizing antibodies. These assays demonstrated excellent agreement with the reference-standard PRNT and include high-throughput platforms, which can greatly enhance capacity to assess both natural and vaccine-induced protective immunity against SARS-CoV-2.

There remains an urgent need for assays to quantify humoral protective immunity to SARS-CoV-2 to understand the immune responses of COVID-19 patients, evaluate efficacy of vaccine candidates in clinical trials, and conduct large-scale epidemiological studies. The plaque-reduction neutralization test (PRNT) is the reference-standard for quantifying antibodies capable of neutralizing SARS-CoV-2. However, the PRNT is logistically demanding, time-consuming, and requires containment level-3 facilities to safely work with live virus. In contrast, a surrogate virus neutralization test (sVNT) manufactured by Genscript is a quick and simple assay that detects antibodies that inhibit the RBD-ACE2 interaction, crucial for virus entry into host cells. In this study, we evaluate the sensitivity, specificity, and cross-reactivity of the sVNT compared with the PRNT using both 50% and 90% SARS-CoV-2 neutralization as a reference-standard. We found that the sVNT provides a high-throughput screening tool prior to confirmatory PRNT testing for the evaluation of SARS-CoV-2 neutralizing antibodies.

Because of the global spread of Zika virus, accurate and high-throughput diagnostic immunoassays are needed. We compared the sensitivity and specificity of 5 commercially available Zika virus serologic assays to the recommended protocol of Zika virus IgM-capture ELISA and plaque-reduction neutralization tests. Most commercial immunoassays showed low sensitivity, which can be increased.

Mass spectrometry-based phenotypic H-antigen typing (MS-H) combined with whole-genome-sequencing-based genetic identification of H antigens, O antigens, and toxins (WGS-HOT) was used to type 60 clinical Escherichia coli isolates, 43 of which were previously identified as nonmotile, H type undetermined, or O rough by serotyping or having shown discordant MS-H and serotyping results. Whole-genome sequencing confirmed that MS-H was able to provide more accurate data regarding H antigen expression than serotyping. Further, enhanced and more confident O antigen identification resulted from gene cluster based typing in combination with conventional typing based on the gene pair comprising wzx and wzy and that comprising wzm and wzt . The O antigen was identified in 94.6% of the isolates when the two genetic O typing approaches (gene pair and gene cluster) were used in conjunction, in comparison to 78.6% when the gene pair database was used alone. In addition, 98.2% of the isolates showed the existence of genes for various toxins and/or virulence factors, among which verotoxins (Shiga toxin 1 and/or Shiga toxin 2) were 100% concordant with conventional PCR based testing results. With more applications of mass spectrometry and whole-genome sequencing in clinical microbiology laboratories, this combined phenotypic and genetic typing platform (MS-H plus WGS-HOT) should be ideal for pathogenic E. coli typing.

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has gained popularity in recent years for rapid bacterial identification, mostly at the genus or species level. In this study, a rapid method to identify the Escherichia coli flagellar antigen (H antigen) at the subspecies level was developed using a MALDI-TOF MS platform with high specificity and sensitivity. Flagella were trapped on a filter membrane, and on-filter trypsin digestion was performed. The tryptic digests of each flagellin then were collected and analyzed by MALDI-TOF MS through peptide mass fingerprinting. Sixty-one reference strains containing all 53 H types and 85 clinical strains were tested and compared to serotyping designations. Whole-genome sequencing was used to resolve conflicting results between the two methods. It was found that DHB (2,5-dihydroxybenzoic acid) worked better than CHCA (α-cyano-4-hydroxycinnamic acid) as the matrix for MALDI-TOF MS, with higher confidence during protein identification. After method optimization, reference strains representing all 53 E. coli H types were identified correctly by MALDI-TOF MS. A custom E. coli flagellar/H antigen database was crucial for clearly identifying the E. coli H antigens. Of 85 clinical isolates tested by MALDI-TOF MS-H, 75 identified MS-H types (88.2%) matched results obtained from traditional serotyping. Among 10 isolates where the results of MALDI-TOF MS-H and serotyping did not agree, 60% of H types characterized by whole-genome sequencing agreed with those identified by MALDI-TOF MS-H, compared to only 20% by serotyping. This MALDI-TOF MS-H platform can be used for rapid and cost-effective E. coli H antigen identification, especially during E. coli outbreaks.