Clinical sensitivity and specificity of a high-throughput microfluidic nano-immunoassay combined with capillary blood microsampling for the identification of anti-SARS-CoV-2 Spike IgG serostatus

BackgroundWe evaluate the diagnostic performance of dried blood microsampling combined with a high-throughput microfluidic nano-immunoassay (NIA) for the identification of anti-SARS-CoV-2 Spike IgG seropositivity. MethodsWe conducted a serological study among 192 individuals with documented prior SARS-CoV-2 infection and 44 SARS-CoV-2 negative individuals. Participants with prior SARS-CoV-2 infection had a long interval of 11 months since their qRT-PCR positive test. Serum was obtained after venipuncture and tested with an automated electrochemiluminescence anti-SARS-CoV-2 S total Ig reference assay, a commercial ELISA anti-S1 IgG assay, and the index test NIA. 109 participants from the positive cohort and 44 participants from the negative cohort also participated in capillary blood collection using three microsampling devices: Mitra, repurposed glucose test strips, and HemaXis. Samples were dried, shipped by regular mail, extracted, and measured with NIA. FindingsUsing serum samples, we achieve a clinical sensitivity of 98{middle dot}33% and specificity of 97{middle dot}62% on NIA, affirming the high performance of NIA in participants 11 months post infection. Combining microsampling with NIA, we obtain a clinical sensitivity of 95{middle dot}05% using Mitra, 61{middle dot}11% using glucose test strips, 83{middle dot}16% using HemaXis, and 91{middle dot}49% for HemaXis after automated extraction, without any drop in specificity. InterpretationHigh sensitivity and specificity was demonstrated when testing micro-volume capillary dried blood samples using NIA, which is expected to facilitate its use in large-scale studies using home-based sampling or samples collected in the field. FundingSwiss National Science Foundation NRP 78 Covid-19 grant 198412 and Private Foundation of the Geneva University Hospital. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSSerological surveillance is of importance to better understand the evolution and spread of SARS-CoV-2 and adapt public health measures. We identified multiple studies conducting such serological surveys using decentralized collection of capillary blood, facilitating the logistics and reducing burden on participants and healthcare facilities. To perform the detection of anti-SARS-CoV-2 antibodies with a high-throughput and at low-cost, a microfluidic nano-immunoassay (NIA) was developed which requires ultra-low sample volumes and minimizes reagent consumption. Added value of this studyIn this study we showed the possibility of combining capillary microsampling with NIA. We validated the use of NIA in serum samples obtained 11 months after infection and show the good clinical performance of the assay in samples with waning antibody titers. Using three different microsampling device, namely Mitra, repurposed glucose test strips, and HemaXis, we implemented a protocol using dried blood sample collection, shipping, extraction, and testing on the microfluidic assay. The sensitivity and specificity were measured and are presented when using the different microsampling devices. Implications of all the available evidenceWe show that the performance of NIA is good when using serum samples, but also in combination with microsampling. Facilitated logistics and increased convenience of microsampling, together with high-throughput and low-cost testing on a microfluidic assay should facilitate the conduction of serological surveys.

Neutralization of ancestral SARS-CoV-2 and variants Alpha, Beta, Gamma, Delta, Zeta and Omicron by mRNA vaccination and infection-derived immunity through homologous and heterologous variants

Emerging SARS-CoV-2 variants of concern/interest (VOC/VOI) raise questions about effectiveness of neutralizing antibodies derived from infection or vaccination. As the population immunity to SARS-CoV-2 has become more complex due to prior infection and/or vaccination, understanding the antigenic relationship between variants is needed. Here, we have assessed in total 104 blood specimens from convalescent individuals after infection with early-pandemic SARS-CoV-2 (pre-VOC) or with Alpha, Beta, Gamma or Delta, post-vaccination after double-dose mRNA-vaccination and break through infections due to Delta or Omicron. Neutralization against seven authentic SARS-CoV-2 isolates (B.1, Alpha, Beta, Gamma, Delta, Zeta, Omicron) was assessed by plaque-reduction neutralization assay. We found highest neutralization titers against the homologous (previously infecting) variant, with lower neutralization efficiency against heterologous variants. Significant loss of neutralization for Omicron was observed but to a varying degree depending on previously infecting variant (23.0-fold in Beta-convalescence up to 56.1-fold in Alpha-convalescence), suggesting that infection-derived immunity varies, but independent of the infecting variant is only poorly protective against Omicron. Of note, Zeta VOI showed also pronounced escape from neutralization of up to 28.2-fold in Alpha convalescent samples. Antigenic mapping reveals both Zeta and Omicron as separate antigenic clusters. Double dose vaccination showed robust neutralization for Alpha, Beta, Gamma, Delta and Zeta, with fold-change reduction of only 2.8 (for Alpha) up to 6.9 (for Beta). Escape from neutralization for Zeta was largely restored in vaccinated individuals, while Omicron still showed a loss of neutralization of 85.7-fold compared to pre-VOC SARS-CoV-2. Combined immunity from infection followed by vaccination or vaccine breakthrough infection showed highest titers and most robust neutralization for heterologous variants. Breakthrough infection with Delta showed only 12.5-fold reduced neutralization for Omicron, while breakthrough infection with Omicron showed only a 1.5-fold loss for Delta, suggests that infection with antigenically different variants can boost immunity for antigens closer to the vaccine strain. Antigenic cartography showed also a tendency towards broader neutralizing capacity for heterologous variants. We conclude that the complexity of background immunity needs to be taken into account when assessing new VOCs. Development towards separate serotypes such as Zeta was already observed before Omicron emergence, thus other factors than just immune escape must contribute to Omicrons rapid dominance. However, combined infection/vaccination immunity could ultimately lead to broad neutralizing capacity also against non-homologous variants.

Diagnostic accuracy of SARS-CoV-2 rapid antigen detection testing in symptomatic and asymptomatic children in the clinical setting

ImportanceAntigen-based rapid diagnostic tests (RDTs) have shown good sensitivity for SARS-CoV-2 detection in adults and are used in children despite the lack data from children. ObjectiveWe evaluated the diagnostic performance of the Panbio-COVID-19 Ag Rapid Test Device (P-RDT) in symptomatic and asymptomatic children against reverse-transcription polymerase chain reaction (RT-PCR) on nasopharyngeal swabs (NPS). DesignProspective diagnostic study from 11.2020 to 03.2021. SettingSingle-center. ParticipantsConsecutive symptomatic and asymptomatic participants 0-16yo. InterventionTwo NPS for both RT-PCR and P-RDT. Main outcomeP-RDT sensitivity and specificity. ResultsEight-hundred and twenty-two participants completed the study, of which 533 (64.9%) were symptomatic. Among the 119 (14.5%) RT-PCR positive patients, the overall P-RDT sensitivity was 0.66 (95%CI 0.57-0.74). Mean viral load (VL) was higher among P-RDT positive than negative ones (p<0.001). Sensitivity was 0.87 in specimens with VL>1.0E6 copies/mL (95%CI 0.87-1.00), which is the accepted cut-off for the presence of infectious virus, and decreased to 0.67 (95%CI 0.59-0.76) for specimens >1.0E3 copies/mL. Among symptomatic participants, the P-RDT displayed a sensitivity of 0.73 (95%CI 0.64-0.82), which peaked at 1.00 at 2 days post onset of symptoms (DPOS; 95%CI 1.00-1.00), then decreased to 0.56 (95%CI 0.23-0.88) at 5 DPOS. There was a trend towards lower P-RDT sensitivity in symptomatic children <12 years (0.62 [95%CI 0.45-0.78]) versus [≥]12 years (0.80 [95%CI 0.69-0.91]; p=0.09). VL which was significantly lower in asymptomatic participants than in symptomatic ones (p<0.001). The P-RDT displayed a sensitivity of 0.43 (95%CI 0.26-0.61). Specificity was 1.00 in symptomatic and asymptomatic children (95%CI 0.99-1.00). Conclusion and relevanceThe overall respective 73% and 43% sensitivities of P-RDT in symptomatic and asymptomatic children was below the 80% cut-off recommended by the World Health Organization. These findings are likely explained by lower VLs in children at the time of diagnosis. As expected, we observed a direct correlation between VL and P-RDT sensitivity as well as variation of sensitivity according to DPOS, a major determinant of VL. These data highlight the limitations of RDTs both in symptomatic and asymptomatic children, with the potential exception in early symptomatic children [≥]12yrs where sensitivity reached 80%.