Correlation of SARS-CoV-2 RNA and nucleocapsid concentrations in samples used in INSTAND external quality assessment schemes.

OBJECTIVE: In routine clinical laboratories, severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection is determined by reverse-transcription PCR (RT-PCR). In the COVID pandemic, a wide range of antigen detection tests were also in high demand. We investigated the correlation between SARS-CoV-2 NCap antigen and N gene concentration by analyzing samples from several INSTAND external quality assessment (EQA) schemes starting in March 2021. The absolute N gene concentration was measured using reverse transcriptase digital PCR (RT-dPCR) as reference value. Moreover, the performance of five commercial ELISA tests using an EQA inactivated SARS-CoV-2 sample at different concentrations was assessed on the basis of these reference values. RESULTS: Quantitative ELISA and RT-dPCR results showed a good correlation between SARS-CoV-2 NCap antigen and RNA concentration, but this correlation varies among SARS-CoV-2 isolates. A direct correlation between SARS-CoV-2 NCap antigen concentration and genome concentration should not be generally assumed. CONCLUSION: Further correlation studies between SARS-CoV-2 RNA and NCap antigen concentrations are needed, particularly in clinical samples and for emerging SARS-CoV-2 variants, to support the monitoring and improvement of antigen testing.

Enhancing a SARS-CoV-2 nucleocapsid antigen test sensitivity with cost efficient strategy through a cotton intermembrane insertion.

Lateral flow antigen tests have been widely used in the Covid-19 pandemic, allowing faster diagnostic test results and preventing further viral spread through isolation of infected individuals. Accomplishment of this screening must be performed with tests that show satisfactory sensitivity in order to successfully detect the target protein and avoid false negatives. The aim of this study was to create a lateral flow test that could detect SARS-CoV-2 nucleocapsid protein in low concentrations that were comparable to the limits of detection claimed by existing tests from the market. To do so, several adjustments were necessary during research and development of the prototypes until they were consistent with these criteria. The proposed alternatives of increasing the test line antibody concentration and addition of an intermembrane between the conjugate pad and the nitrocellulose membrane were able to increase the sensitivity four-fold and generate a new rapid test prototype called "lateral flow intermembrane immunoassay test" (LFIIT). This prototype showed an adequate limit of detection (2.0 ng mL-1) while maintaining affordability and simplicity in manufacturing processes.

Assessment of Immunogenic and Antigenic Properties of Recombinant Nucleocapsid Proteins of Five SARS-CoV-2 Variants in a Mouse Model.

COVID-19 cases caused by new variants of highly mutable SARS-CoV-2 continue to be identified worldwide. Effective control of the spread of new variants can be achieved through targeting of conserved viral epitopes. In this regard, the SARS-CoV-2 nucleocapsid (N) protein, which is much more conserved than the evolutionarily influenced spike protein (S), is a suitable antigen. The recombinant N protein can be considered not only as a screening antigen but also as a basis for the development of next-generation COVID-19 vaccines, but little is known about induction of antibodies against the N protein via different SARS-CoV-2 variants. In addition, it is important to understand how antibodies produced against the antigen of one variant can react with the N proteins of other variants. Here, we used recombinant N proteins from five SARS-CoV-2 strains to investigate their immunogenicity and antigenicity in a mouse model and to obtain and characterize a panel of hybridoma-derived monoclonal anti-N antibodies. We also analyzed the variable epitopes of the N protein that are potentially involved in differential recognition of antiviral antibodies. These results will further deepen our knowledge of the cross-reactivity of the humoral immune response in COVID-19.

A SARS-CoV-2 nucleocapsid ELISA represents a low-cost alternative to lateral flow testing for community screening in LMI countries.

Background Controlling the spread of SARS-CoV-2 is problematic because of transmission driven by asymptomatic and pre-symptomatic individuals. Community screening can help identify these individuals but is often too expensive for countries with limited health care resources. Low-cost ELISA assays may address this problem, but their use has not yet been widely reported. Methods We developed a SARS-CoV-2 nucleocapsid ELISA and assessed its diagnostic performance on nose and throat swab samples from UK hospitalised patients and sputum samples from patients in Ghana. Results The ELISA had a limit of detection of 8.4 pg/ml antigen and 16 pfu/ml virus. When tested on UK samples (128 positive and 10 negative patients), sensitivity was 58.6% (49.6-67.2) rising to 78.3% (66.7-87.3) if real-time PCR Ct values > 30 were excluded, while specificity was 100% (69.2-100). In a second trial using the Ghanaian samples (121 positive, 96 negative), sensitivity was 52% (42.8-61.2) rising to 72.6% (61.8-81.2) when a > 30 Ct cut-off was applied, while specificity was 100% (96.2-100). Conclusions: Our data show that nucleocapsid ELISAs can test a variety of patient sample types while achieving levels of sensitivity and specificity required for effective community screening. Further investigations into the opportunities that this provides are warranted.

Longitudinal Assessment of SARS-CoV-2 Antinucleocapsid and Antispike-1-RBD Antibody Testing Following PCR-Detected SARS-CoV-2 Infection.

BACKGROUND: SARS-CoV-2 serologic assays are becoming increasingly available and may serve as a diagnostic aid in a multitude of settings relating to past infection status. However, there is limited literature detailing the longitudinal performance of EUA-cleared serologic assays in US populations, particularly in cohorts with a remote history of PCR-confirmed SARS-CoV-2 infection (e.g., >2 months). METHODS: We evaluated the diagnostic sensitivities and specificities of the Elecsys® Anti-SARS-CoV-2 (anti-N) and Elecsys Anti-SARS-CoV-2 S (anti-S1-RBD) assays, using 174 residual clinical samples up to 267 days post-PCR diagnosis of SARS-CoV-2 infection (n = 154) and a subset of samples obtained prior to the COVID-19 pandemic as negative controls (n = 20). RESULTS: The calculated diagnostic sensitivities for the anti-N and anti-S1-RBD assays were 89% and 93%, respectively. Of the 154 samples in the SARS-CoV-2-positive cohort, there were 6 discrepant results between the anti-N and anti-S1-RBD assays, 5 of which were specimens collected ≥200 days post-PCR positivity and only had detectable levels of anti-S1-RBD antibodies. When only considering specimens collected ≥100 days post-PCR positivity (n = 41), the sensitivities for the anti-N and anti-S1-RBD assays were 85% and 98%, respectively. CONCLUSIONS: The anti-S1-RBD assay demonstrated superior sensitivity at time points more remote to the PCR detection date, with 6 more specimens from the SARS-CoV-2-positive cohort detected, 5 of which were collected more than 200 days post-PCR positivity. While analytical differences and reagent lot-to-lot variability are possible, this may indicate that, in some instances, anti-S1-RBD antibodies may persist longer in vivo and may be a better target for detecting remote SARS-CoV-2 infection.

Quantitative Assessment of SARS-CoV-2 Virus in Nasopharyngeal Swabs Stored in Transport Medium by a Straightforward LC-MS/MS Assay Targeting Nucleocapsid, Membrane, and Spike Proteins.

Alternative methods to RT-PCR for SARS-CoV-2 detection are investigated to provide complementary data on viral proteins, increase the number of tests performed, or identify false positive/negative results. Here, we have developed a simple mass spectrometry assay for SARS-CoV-2 in nasopharyngeal swab samples using common laboratory reagents. The method employs high sensitivity and selectivity targeted mass spectrometry detection, monitoring nine constitutive peptides representative of the three main viral proteins and a straightforward pellet digestion protocol for convenient routine applications. Absolute quantification of N, M, and S proteins was achieved by addition of isotope-labeled versions of best peptides. Limit of detection, recovery, precision, and linearity were thoroughly evaluated in four representative viral transport media (VTM) containing distinct total protein content. The protocol was sensitive in all swab media with limit of detection determined at 2 × 103 pfu/mL, corresponding to as low as 30 pfu injected into the LC-MS/MS system. When tested on VTM-stored nasopharyngeal swab samples from positive and control patients, sensitivity was similar to or better than rapid immunoassay dipsticks, revealing a corresponding RT-PCR detection threshold at Ct ∼ 24. The study represents the first thorough evaluation of sensitivity and robustness of targeted mass spectrometry in nasal swabs, constituting a promising SARS-CoV-2 antigen assay for the first-line diagnosis of COVID-19 and compatible with the constraints of clinical settings. The raw files generated in this study can be found on PASSEL (Peptide Atlas) under data set identifier PASS01646.

Development and clinical application of a rapid SARS-CoV-2 antibody test strip: A multi-center assessment across China.

BACKGROUND: The ongoing coronavirus disease 19 (COVID-19) is posing a threat to the public health globally. Serological test for SARS-CoV-2 antibody can improve early diagnosis of COVID-19 and serves as a valuable supplement to RNA detection. METHOD: A SARS-CoV-2 IgG/IgM combined antibody test strip based on colloidal gold immunochromatography assay was developed, with both spike protein and nucleocapsid protein of SARS-CoV-2 antigen used for antibody detection. From 3 medical institutions across China, serum or plasma of 170 patients with confirmed COVID-19 diagnosis and 300 normal controls were collected and tested with the strip. Sensitivity, specificity, kappa coefficient, receiver operating characteristic (ROC) curve, and area under the curve (AUC) were analyzed. Positive rates in different medical centers, age group, gender, and different disease course were compared. RESULTS: 158 out 170 samples from confirmed COVID-19 patients had positive results from the test, and 296 out of 300 samples from normal controls had negative results. The kit was 92.9% sensitive and 98.7% specific. The positive rate was 77.3% during the first week after disease onset, but reached 100% since day 9. AUC and kappa coefficient were 0.958 and 0.926, respectively, which showed the consistency of the test results with the standard diagnosis. Age or gender caused little variations in the kit sensitivity. CONCLUSION: The rapid, easy-to-use SARS-CoV-2 IgG/IgM combined antibody test kit has a superior performance, which can help with accurate diagnosis and thus timely treatment and isolation of COVID-19 patients, that contributes to the better control of the global pandemic.

Detection of severe human metapneumovirus infection by real-time polymerase chain reaction and histopathological assessment.

BACKGROUND: Infections with common respiratory tract viruses can cause high mortality, especially in immunocompromised hosts, but the impact of human metapneumovirus (hMPV) in this setting was previously unknown. METHODS: We evaluated consecutive bronchoalveolar lavage and bronchial wash fluid samples from 688 patients--72% were immunocompromised and were predominantly lung transplant recipients--for hMPV by use of quantitative real-time polymerase chain reaction (PCR), and positive results were correlated with clinical outcome and results of viral cultures, in situ hybridization, and lung histopathological assessment. RESULTS: Six cases of hMPV infection were identified, and they had a similar frequency and occurred in a similar age range as other paramyxoviral infections. Four of 6 infections occurred in immunocompromised patients. Infection was confirmed by in situ hybridization for the viral nucleocapsid gene. Histopathological assessment of lung tissue samples showed acute and organizing injury, and smudge cell formation was distinct from findings in infections with other paramyxoviruses. Each patient with high titers of hMPV exhibited a complicated clinical course requiring prolonged hospitalization. CONCLUSIONS: Our results provide in situ evidence of hMPV infection in humans and suggest that hMPV is a cause of clinically severe lower respiratory tract infection that can be detected during bronchoscopy by use of real-time PCR and routine histopathological assessment.

Assessment of high-affinity hybridization, RNase H cleavage, and covalent linkage in translation arrest by antisense oligonucleotides.

Antisense oligonucleotides (ONs) are designed to hybridize target mRNA in a sequence-specific manner and inhibit gene expression by preventing translation, either by activation of RNase H or steric blockage of the ribosome complex. Second-generation ONs, which possess greater binding affinity for target RNA relative to the isosequential phosphodiester (PO) ONs, have been developed and include, among others, peptide nucleic acids (PNA) and N3' P5' phosphoramidate oligonucleotides (npONs). In the present study, PNA and npON derivatives were targeted to the coding portion of the complementary mRNA of the N protein of the vesicular stomatitis virus (VSV) in order to evaluate their ability to arrest translation in an in vitro rabbit reticulocyte lysate system. High-affinity hybridization of ONs lacking RNase H activity was not sufficient to block translation in this test system. Only antisense ONs acting via an RNase H mechanism or by steric hindrance through covalent attachment (via transplatin modification) to the target mRNA were found to definitively arrest translation in this study.