From Delta to Omicron SARS-CoV-2 variant: switch to saliva sampling for higher detection rate

BackgroundReal-time polymerase chain reaction (RT-PCR) testing on a nasopharyngeal swab is the current standard for SARS-CoV-2 virus detection. Since collection of this sample type is experienced uncomfortable by patients, saliva- and oropharyngeal swab collections should be considered as alternative specimens. ObjectivesEvaluation of the relative performance of oropharyngeal swab, nasopharyngeal swab and saliva for the RT-PCR based SARS-CoV-2 Delta (B.1.617.2) and Omicron (B.1.1.529) variant detection. Study designNasopharyngeal swab, oropharyngeal swab and saliva were collected from 246 adult patients who presented for SARS-CoV-2 testing at the screening centre in Ypres (Belgium). RT-PCR SARS-CoV-2 detection was performed on all three sample types separately. Variant type was determined for each positive patient using whole genome sequencing or Allplex SARS-CoV-2 variants I and II Assay. Results and conclusionsSaliva is superior compared to nasopharyngeal swab for the detection of the Omicron variant. For the detection of the Delta variant, nasopharyngeal swab and saliva can be considered equivalent specimens. Oropharyngeal swab is the least sensitive sample type and shows little added value when collected in addition to a single nasopharyngeal swab. HighlightsO_LISaliva is the preferred sample type for Omicron variant (B.1.1.529) detection C_LIO_LINasopharyngeal swab and saliva are equivalent for Delta variant (B.1.617.2) detection C_LIO_LIOropharyngeal swab is the least preferred sample type for SARS-CoV-2 detection C_LI

Cov2MS: an automated matrix-independent assay for mass spectrometric detection and measurement of SARS-CoV-2 nucleocapsid protein in infectious patients

INTRODUCTIONThe pandemic readiness toolbox needs to be extended, providing diagnostic tools that target different biomolecules, using orthogonal experimental setups and fit-for-purpose specification of detection. Here we build on a previous Cov-MS effort that used liquid chromatography-mass spectrometry (LC-MS) and describe a method that allows accurate, high throughput measurement of SARS-CoV-2 nucleocapsid (N) protein. MATERIALS and METHODSWe used Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) technology to enrich and quantify proteotypic peptides of the N protein from trypsin-digested samples from COVID-19 patients. RESULTSThe Cov2MS assay was shown to be compatible with a variety of sample matrices including nasopharyngeal swabs, saliva and blood plasma and increased the sensitivity into the attomole range, up to a 1000-fold increase compared to direct detection in matrix. In addition, a strong positive correlation was observed between the SISCAPA antigen assay and qPCR detection beyond a quantification cycle (Cq) of 30-31, the level where no live virus can be cultured from patients. The automatable "addition only" sample preparation, digestion protocol, peptide enrichment and subsequent reduced dependency upon LC allow analysis of up to 500 samples per day per MS instrument. Importantly, peptide enrichment allowed detection of N protein in a pooled sample containing a single PCR positive sample mixed with 31 PCR negative samples, without loss in sensitivity. MS can easily be multiplexed and we also propose target peptides for Influenza A and B virus detection. CONCLUSIONSThe Cov2MS assay described is agnostic with respect to the sample matrix or pooling strategy used for increasing throughput and can be easily multiplexed. Additionally, the assay eliminates interferences due to protein-protein interactions including those caused by anti-virus antibodies. The assay can be adapted to test for many different pathogens and could provide a tool enabling longitudinal epidemiological monitoring of large numbers of pathogens within a population, applied as an early warning system.

Cov-MS: a community-based template assay for clinical MS-based protein detection in Sars-Cov-2 patients

Rising population density and global mobility are among the reasons why pathogens such as SARS-CoV-2, the virus that causes COVID-19, spread so rapidly across the globe. The policy response to such pandemics will always have to include accurate monitoring of the spread, as this provides one of the few alternatives to total lockdown. However, COVID-19 diagnosis is currently performed almost exclusively by Reverse Transcription Polymerase Chain Reaction (RT-PCR). Although this is efficient, automatable and acceptably cheap, reliance on one type of technology comes with serious caveats, as illustrated by recurring reagent and test shortages. We therefore developed an alternative diagnostic test that detects proteolytically digested SARS-CoV-2 proteins using Mass Spectrometry (MS). We established the Cov-MS consortium, consisting of fifteen academic labs and several industrial partners to increase applicability, accessibility, sensitivity and robustness of this kind of SARS-CoV-2 detection. This in turn gave rise to the Cov-MS Digital Incubator that allows other labs to join the effort, navigate and share their optimizations, and translate the assay into their clinic. As this test relies on viral proteins instead of RNA, it provides an orthogonal and complementary approach to RT-PCR, using other reagents that are relatively inexpensive and widely available, as well as orthogonally skilled personnel and different instruments. Data are available via ProteomeXchange with identifier PXD022550.

Kinetics of the humoral immune response to SARS-CoV-2: comparative analytical performance of seven commercial serology tests

BackgroundSARS-CoV-2 serology tests are clinically useful to document a prior SARS-CoV-2 infection in patients with no or inconclusive PCR results and suspected COVID-19 disease or sequelae. Data are urgently needed to select the assays with optimal sensitivity at acceptable specificity. MethodsA comparative analysis of analytical sensitivity was performed of seven commercial SARS-CoV-2 serology assays on 171 sera from 135 subjects with PCR-confirmed SARS-CoV-2 infection, composed of 71 patients hospitalized for COVID-19 pneumonia and 64 healthcare workers with paucisymptomatic infections. The kinetics of IgA/IgM/IgG seroconversion to viral N-and S-protein epitopes were studied from 0 to 54 days after symptom onset. Specificity was verified on 57 pre-pandemic samples. ResultsWantai SARS-COV-2 Ab ELISA and Orient Gene COVID-19 IgG/IgM Rapid Test achieved a superior overall sensitivity. Elecsys Anti-SARS-CoV-2 assay and EUROIMMUN Anti-SARS-CoV-2 combined IgG/IgA also showed acceptable sensitivity (>95%) versus the consensus result of all assays from 10 days post symptom onset. Optimal specificity (>98%) was achieved only by Wantai SARS-COV-2 Ab ELISA, Elecsys Anti-SARS-CoV-2 assay and Innovita 2019-nCoV Ab rapid test. LIAISON SARS-CoV-2 S1/S2 IgG showed a significantly lower sensitivity as compared to all other assays. Lack of seroconversion by any test was seen in 1.4% of hospitalized and 4.7% of paucisymptomatic infections. Within 10 days from symptom onset, only the Wantai SARS-COV-2 Ab ELISA has acceptable sensitivity. ConclusionsWantai SARS-COV-2 Ab ELISA and Elecsys Anti-SARS-CoV-2 assays are suitable for sensitive and specific screening of a SARS-CoV-2 infection from 10 days after symptom onset. Brief summaryThere is an urgent need for SARS-CoV-2 serology tests for the sensitive and specific detection of prior SARS-CoV-2 infection as a complementary diagnostic tool to molecular testing. Various commercial assays are becoming available but comparison of their relative performance is difficult unless they are head-to-head evaluated. Here we compared seven commercial assays on sera equally composed of mild and severe PCR-confirmed SARS-CoV-2 infections. Our analysis indicates a superior performance of the Wantai SARS-COV-2 ELISA for total antibodies to the S-RBD domain. Also, the Elecsys Anti-SARS-CoV-2 assay for total antibodies to the N-protein shows good performance for high-throughput screening.

Vitamin D deficiency as risk factor for severe COVID-19: a convergence of two pandemics

Structured abstractO_ST_ABSContextC_ST_ABSThrough its immunological functions, vitamin D attenuates inflammatory responses to respiratory viruses. Vitamin D deficiency might be a highly prevalent risk factor for severe SARS-CoV-2 infections. ObjectiveTo investigate the level of vitamin D deficiency in West Flanders, Belgium and its correlation to severity of COVID-19 as staged by CT DesignRetrospective observational study Setting. Central network hospital Participants186 SARS-CoV-2-infected patients hospitalized from March 1, 2020 to April 7, 2020 Main outcome measureAnalysis of 25(OH)D in COVID-19 patients versus season/age/sex-matched diseased controls ResultsThe rate of vitamin D deficiency (25(OH)D<20 ng/mL) in West Flanders varies with age, sex and season but is overall very high (39.9%) based on analysis of 16274 control samples. We measured 25(OH)D levels in 186 COVID-19 patients (109 males (median age 68 years, IQR 53-79) and 77 females (median age 71 years, IQR 65-74)) and 2717 age/season-matched controls (999 males (median age 69 years, IQR 53-81) and 1718 females (median age 68 years, IQR 43-83)). COVID-19 patients showed lower median 25(OH)D (18.6 ng/mL, IQR 12.6-25.3, versus 21.5 ng/mL, IQR 13.9-30.8; P=0.0016) and higher vitamin D deficiency rates (58.6% versus 45.2%, P=0.0005). Surprisingly, this difference was restricted to male COVID-19 patients who had markedly higher deficiency rates than male controls (67.0% versus 49.2%, P=0.0006) that increased with advancing radiological stage and were not confounded vitamin D-impacted comorbidities. Conclusionsvitamin D deficiency is a prevalent risk factor for severe COVID-19. Vitamin D supplementation might be an inexpensive and safe mitigation for the SARS-CoV-2 pandemic. O_TEXTBOXSUMMARY BOXO_ST_ABSWhat is already known on this topicC_ST_ABSVitamin D deficiency increases the incidence and severity of respiratory viral infections by exacerbation of pro-inflammatory immune responses. Lung damage in COVID-19 involves excessive inflammation and cytokine release. With more than 1 billion people worldwide affected by vitamin D deficiency the world might now face a convergence of two pandemics. Data are emerging that variations in vitamin D deficiency rates across ethnic and demographic subgroups are correlated to severity of SARS-CoV-2 infections. What this study addsOur data reveal that more than 40% of the adult population in a wealthy European country is vitamin D deficient. We show that vitamin D deficiency is correlated with the risk for hospitalization for COVID-19 pneumonia and predisposes to more advanced radiological disease stages. Specifically, men were at risk. This correlation was not confounded by vitamin D-impacted morbidities such as coronary artery disease, diabetes and chronic lung disease. Our findings support a causal relation between vitamin D deficiency and severe COVID-19 and call for vitamin D supplementation as safe, widely available and inexpensive mitigation strategy. C_TEXTBOX

Diagnostic power of chest CT for COVID-19: to screen or not to screen

Structured abstractO_ST_ABSBackgroundC_ST_ABSchest CT is increasingly used for COVID-19 screening in healthcare systems with limited SARS-CoV-2 PCR capacity. Its diagnostic value was supported by studies with methodological concerns and its use is controversial. Here we investigated its potential to diagnose COVID-19 in symptomatic patients and to screen asymptomatic patients in a prospective study with minimal selection bias. MethodsFrom March 19, 2020 to April 20, 2020 we performed parallel SARS-CoV-2 PCR and CT with categorization of COVID-19 suspicion by CO-RADS, in 859 patients with COVID-19 symptoms and 1138 controls admitted to the hospital for COVID-19 unrelated medical urgencies. CT-CORADS was categorized on a 5-point scale from 1 (very low suspicion) to 5 (very high suspicion). AUC under ROC curve were calculated in symptomatic versus asymptomatic patients to predict positive SARS-CoV-2 positive PCR and likelihood ratios for each CO-RADS score were used for rational selection of diagnostic thresholds. FindingsCT-CORADS had significant (P<0.0001) diagnostic power in both symptomatic (AUC=0.891) and asymptomatic (AUC=0.700) patients hospitalized during SARS-CoV-2 peak prevalence. In symptomatic patients (41.7% PCR+), CO-RADS [≥] 3 detected positive PCR with high sensitivity (89.1%) and 72.5% specificity. In asymptomatic patients (5.3% PCR+), a CO-RADS score [≥] 3 detected SARS-CoV-2 infection with low sensitivity (45.0%) but high specificity (88.8%). InterpretationCT-CORADS has meaningful diagnostic power in symptomatic patients, supporting its application for time-sensitive triage. Sensitivity in asymptomatic patients is insufficient to justify its use as screening approach. Incidental detection of CO-RADS [≥] 3 in asymptomatic patients should trigger reflex testing for respiratory pathogens.