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BackgroundStudies investigating the cumulative incidence of and immune status against SARS-CoV-2 infection provide valuable information for shaping public health decision-making. MethodsThe current cross-sectional, population-based study, conducted in April 2022 in the Valencian Community (VC), recruited 935 participants of all ages. Anti-SARS-CoV-2-Receptor Binding Domain-RBD-total antibodies and anti-Nucleocapsid (N)- IgGs were measured by electrochemiluminescence assays. To account for past SARS-CoV-2 infection the VC microbiology registry (RedMiVa) was interrogated. |Quantitation of neutralizing antibodies (NtAb) against the ancestral and Omicron BA.1 and BA.2 (sub)variants by an S-pseudotyped neutralization assay and for enumeration of SARS-CoV-2-S specific-IFN{gamma}-producing CD4+ and CD8+ T cells by Intracellular Cytokine Staining assay was performed in a subset of participants (n=100 and 137, respectively). FindingsThe weighted cumulative incidence was 51{square}9% (95% CI, 48{square}7-55{square}1), and was inversely related to age. Anti-RBD total antibodies were detected in 906/931 (97{square}3%) participants, those vaccinated and SARS-CoV-2-experienced (VAC-ex;=442) displaying higher levels (P<0.001) than vaccinated/naive (VAC-n;(n=472) and non-vaccinated/experienced (UNVAC-ex; n(n=63). Antibody levels correlated inversely with the time elapsed since receipt of last vaccine dose in VAC-n (Rho, -0{square}52; 95% CI, -0{square}59 to -0{square}45; P<0.001) but not in VAC-ex. NtAbs against Omicron BA.1 were detected in 94%, 75% and 50% of VAC-ex, VAC-n and UNVAC-ex groups, respectively, while in 97%, 84% and 40%, against Omicron BA.2. SARS-CoV-2-S-reactive IFN-{gamma} T cells were detected in 73%, 75%, and 64% for VAC-ex, VAC-n, UNVAC-ex, respectively. InterpretationBy April 2022 around half of the VC population had been infected with SARS-CoV-2 and due to extensive vaccination display hybrid immunity. The large percentage of participants with detectable functional antibody and T-cell responses against SARS-CoV-2, which may be cross-reactive to some extent, points towards lower expected severity than in previous waves. FundingThis research was supported in part by the European Commission NextGenerationEU fund (CSICs Global Health Platform).
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ObjectivesIt has been suggested that rapid antigen detection assays (RADT) may perform suboptimally in terms of sensitivity for the diagnosis of SARS-CoV-2 Omicron variant infection. To address this issue, we conducted a prospective study in primary health centers to evaluate the clinical performance of the Panbio COVID-19 Ag Rapid Test Device in nasopharyngeal specimens (NP) carried out at the point of care. MethodsWe recruited 244 patients (median age, 40 years; range 2-96; 141 female) with clinical suspicion of COVID-19 (232 adults and 12 children). 228/244 patients had been fully vaccinated (two doses) with licensed COVID-19 vaccines prior to recruitment. Most patients (222/244) were SARS-CoV-2 naive prior to enrollment. Patients were tested by RT-PCR and RADT within 5 days since symptoms onset. Results126 patients (51.6%) tested positive by both RT-PCR and RADT, 90 patients (36.8%) returned negative results by both assays and 28 patients (11.4%) yielded discordant results (RT-PCR+/RADT-). No patients tested RT-PCR-/RADT+. Overall specificity and sensitivity of RADT was 100% (95% CI, 95.9-100%) and 81.8% (95% CI, 75-87.1%) respectively. The sensitivity of the assay increased from 79.6% (95% CI, 66.4-88.5) when considering specimens collected at days 0-1 after symptoms onset, to 86.4% (95% CI, 66.7-95.3) when grouping the specimens obtained on days 4-5. ConclusionThe Panbio COVID-19 Ag Rapid Test Device perform well ([≥]80% sensitivity) as a point-of-care test for early diagnosis of COVID-19 due to the Omicron variant in primary healthcare centers.
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ObjectivesThere is scarce information as to the durability of immune responses elicited by the Comirnaty(R) COVID-19 vaccine in nursing home residents. Here, we assessed SARS-CoV-2-Spike (S)-targeted antibody and functional T cell responses at around 6 months after complete vaccination. MethodsThe sample comprised 46 residents (34 females; age, 60-100 years), of whom 10 had COVID-19 prior to vaccination. Baseline (median of 17.5 days after vaccination) and follow-up (median, 195 days) plasma specimens were available for quantitation of SARS-CoV-2-S antibodies and enumeration of SARS-CoV-2-S-reactive IFN-{gamma} CD4+ and CD8+ T cells by flow cytometry. ResultsIn total, 44/45 participants had detectable SARS-CoV-2-S antibodies at follow-up. Overall, antibody levels were found to decrease (median, 4.8 fold). Antibodies waning was more frequent (P<0.001) in SARS-CoV-2 naive (29/35) than in recovered (1/10) residents. SARS-CoV-2-S IFN-{gamma} CD8+ T cells were detected in 33/46 and 24/46 at baseline and follow-up, respectively. The figures for CD4+ T cell counterparts were 12/46 and 30/46. Detectable SARS-CoV-2 IFN-{gamma} CD8+ and CD4+ T cell responses at follow-up were more common in recovered (8/10 and 7/10, respectively) than in naive residents (9/36 and 25/36, respectively). For those with detectable responses at both time points, SARS-CoV-2-S IFN-{gamma} CD8+ T cell frequencies decreased significantly (P=0.001) over time whereas the opposite (P=0.01) was observed in CD4+ T cells. ConclusionAlmost all residents displayed detectable SARS-CoV-2-S-reactive antibodies and T cell responses, respectively, by around 6 months after complete vaccination with Comirnaty(R) COVID-19 vaccine, albeit generally waning in magnitude over time.
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Immunosenescence may impact the functionality and breadth of vaccine-elicited humoral immune responses. The ability of sera to neutralize the SARS-CoV-2 spike protein (S) from Beta, Gamma, Delta, and Epsilon variants of concern (VOCs) relative to the ancestral Wuhan-Hu-1 strain was compared in Comirnaty(R) COVID-19-vaccinated elderly nursing home residents (n=30) or younger individuals (n=18) and non-vaccinated individuals who recovered from severe COVID-19 (n=19). In all groups, some participants lacked NtAb against one or more VOCs, mainly the Beta variant (15-20%). Serum NtAb titers were lowest against the Beta variant followed by Gamma, Epsilon, and Delta variants. Fold change reduction in NtAb titers relative to the ancestral strain was greatest for the Beta variant (6.7-18.8) followed by Gamma (3.6-6.2), Epsilon (2.9-5.8), and Delta (3.5-4.3) variants, regardless of the study group considered. In summary, older age, frailty, and concurrence of co-morbidities had no impact on the serum NtAb activity profile against SARS-CoV-2 VOCs.
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ObjectivesThe immunogenicity of the BNT162b2 COVID-19 vaccine is understudied in elderly people with comorbidities. We assessed SARS-CoV-2-S-targeted antibody and T cell responses following full vaccination in nursing home residents (NHR). MethodsWe recruited 60 NHR (44 female; median age, 87.5 years), of whom 10 had previously had COVID-19, and 18 healthy controls (15 female; median age, 48.5 years). Pre- and post-vaccination blood specimens were available for quantitation of total antibodies binding RBD and enumeration of SARS-CoV-2-S-reactive IFN-{gamma} CD4+ and CD8+ T cells by flow cytometry. ResultsThe seroconversion rate in presumably SARS-CoV-2 naive NHR (95.3%), either with or without comorbidities, was similar to controls (94.4%). A robust booster effect was documented in NHR with prior COVID-19. Plasma antibody levels were higher in convalescent NHR than in individuals across the other two groups. A large percentage of NHR had SARS-CoV-2 S-reactive IFN-{gamma} CD8+ and/or CD4+ T cells at baseline, in contrast to healthy controls. Either CD8+ and/or CD4+ T-cell responses were documented in all control subjects after vaccination. Contrariwise, the percentage of NHR exhibiting detectable SARS-CoV-2 IFN-{gamma} CD8+ or CD4+ T-cell responses (or both), irrespective of their baseline SARS-CoV-2 infection status, dropped consistently after vaccination. Overall, SARS-CoV-2 IFN-{gamma} CD8+ and CD4+ T-cell responses in NHR decreased in post-vaccination specimens. ConclusionThe BNT162b2 COVID-19 vaccine elicits robust SARS-CoV-2-S antibody responses in NHR. Nevertheless, the frequency and magnitude of detectable SARS-CoV-2 IFN-{gamma} T-cell responses after vaccination was lower in NHR compared to controls.
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BackgroundLittle is known about the comparative kinetics of SARS-CoV-RNA load in the lower respiratory tract and in blood compartment in patients admitted to the intensive care unit, and how these relate to biomarkers of COVID-19 severity. MethodsSeventy-three consecutive critically ill COVID-19 patients (median age, 65 years) were recruited. Serial lower respiratory tract (n=165) and plasma (n=340) specimens were collected. RT-PCR and lateral flow immunochromatography assay were used for SARS-CoV-2 RNA quantitation and N protein detection in plasma, respectively. Serum levels of inflammatory and tissue-damage biomarkers in paired specimens were analyzed. ResultsSARS-CoV-RNA was detected in the lower respiratory tract of most patients (92%). Viral RNAemia and N-antigenemia were documented in 35.6% and 40.1% of patients, respectively. Viral RNAemia and N-antigenemia cleared at a faster rate than SARS-CoV-2 RNA in tracheal aspirates (TA). SARS-CoV-2 RNA load was higher (P<0.001) in TA than in plasma, and correlated significantly (Rho, 0.41; P<0.001). A modest correlation was found between SARS-CoV-2 RNA load in TA and plasma and levels of ferritin and lactose dehydrogenase (Rho[≤]0.3; P[≤]0.008) in paired serum specimens. Neither the dynamics of SARS-CoV-2 RNA load in TA and plasma, nor N-antigenemia detection rate differed between surviving and deceased patients. Yet, a trend towards a higher mortality was seen in patients with viral RNAemia (OR; 2.82; 95% CI, 0.94-8.47; P=0.06). ConclusionNeither SARS-CoV-2 replication rate in the lower respiratory tract nor its presence in the blood appeared to critically impact on survival in ICU COVID-19 patients. SUMMARYSARS-CoV-2 RNA load in the lower respiratory tract and plasma and N-antigenemia followed different kinetics, correlated modestly with serum levels of inflammatory and tissue-damage biomarkers and lymphopenia and did not appear to increase overall mortality risk in critically ill adult COVID-19 patients.
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The performance of a laboratory-developed quantitative IgG/IgA flow cytometry-based immunoassay (FCI) using Jurkat T cells stably expressing full-length native S protein was compared against Elecsys(R) electrochemiluminiscent (ECLIA) Anti-SARS-CoV-2 S (Roche Diagnostics, Pleasanton, CA, USA), and LIAISON(R) SARS-CoV-2 TrimericS IgG chemiluminiscent assay (CLIA) (Diasorin S.p.a, Saluggia, IT) for detection and quantitation of SARS-CoV-2-specific antibodies. A total of 225 serum/plasma specimens from 120 acute or convalescent COVID-19 individuals were included. Overall, IgG/IgA-FCI yielded the highest number of positives (n=179), followed by IgA-FCI (n=177), Roche ECLIA (n=175), IgG-FCI (n=172) and Diasorin CLIA (n=154). Positive percent agreement between FCI and compared immunoassays was highest for Roche ECLIA, ranging from 96.1% (IgG/IgA-FCI) to 97.7% (IgG-FCI), whereas negative percent agreement was higher between FCI and Diasosin CLIA, regardless of antibody isotype. A strong correlation (Rho:0.6-0.8) was found between IgG-FCI or IgA-FCI levels and antibodies quantified by Roche ECLIA and Diasorin CLIA. The trajectory of antibody levels delineated by the different immunoassays in 22 of patients with sequential specimens ([≥]3) was frequently discordant, with the exception of IgG and IgA determined by FCI assay and to a lesser extent antibodies quantified by Roche ECLIA and Diasorin CLIA. The data suggest that FCI may outperform Roche ECLIA and Diasorin CLIA in terms of clinical sensitivity for serological diagnosis of SARS-CoV-2 infection.
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ObjectivesThere is limited information comparing SARS-CoV-2 RNA load in the upper respiratory tract (URT) between children and adults, either presenting with COVID-19 or asymptomatic. Here we conducted a retrospective, single center study involving a large cohort of SARS-CoV-2 infected individuals to address this issue. Patients and MethodsA total of 1,184 consecutive subjects (256 children and 928 adults) testing positive for SARS-COV-2 RNA in nasopharyngeal exudates (NP) were included, of whom 424 (121 children and 303 adults) had COVID-19 not requiring hospitalization and 760 (135 children and 625 adults) were asymptomatic close contacts of COVID-19 patients. SARS-CoV-2 RNA testing was carried out using the TaqPath COVID-19 Combo Kit (Thermo Fisher Scientific, MS, USA). The AMPLIRUN(R) TOTAL SARS-CoV-2 RNA Control (Vircell SA, Granada, Spain) was used for estimating SARS-CoV-2 RNA loads (in copies/mL). ResultsMedian SARS-COV-2 RNA loads were comparable between adults and children with COVID-19 (7.14 log10 copies/ml vs. 6.98 log10 copies/ml; P=0.094). Median SARS-CoV-2 RNA load in asymptomatic children and adults was similar (6.20 log10 copies/ml vs. 6.48 log10 copies/ml; P=0.97). Children with COVID-19 symptoms displayed SARS-CoV-2 RNA loads comparable to their asymptomatic counterparts (P=0.61). Meanwhile in adults, median SARS-CoV-2 RNA load was significantly higher in symptomatic than in asymptomatic subjects (P=<0.001), yet comparable (P=0.61) when the analysis excluded patients sampled within 48 h after symptoms onset. ConclusionsThe data suggest that children may be drivers of SARS-CoV-2 transmission in the general population at the same level as adults.
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Rapid antigen assays (RAD) based on lateral flow immunochromatography (LFIC) technology have emerged as a valuable tool for the control of COVID-19 pandemic. Manufacturer{square}independent, real{square}world evaluation of these assays is crucial given the considerable heterogeneity reported in their clinical and analytical performances. Here, we report for the first time on the point-of-care performance of the CLINITEST(R) Rapid COVID-19 Antigen Test (Siemens, Healthineers, Erlangen, Germany) to detect SARS-CoV-2 infection in presumptive COVID-19 cases or asymptomatic close contacts of COVID-19 patients. When compared to RT-PCR, the overall sensitivity of the assay was 80.2 (95% CI, 70.9-87.1) for symptomatic patients sampled (nasopharyngeal specimens) within five days after the onset of symptoms and 60% (95% CI, 40.7-76.6%) for asymptomatic participants. The overall specificity was 100% in both population groups.
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ObjectivesNear-source tracking of SARS-CoV-2 RNA in the sewage drains serving particular buildings may allow rapid identification of SARS-CoV-2 infection cases or local outbreaks. In this pilot study, we investigated whether this was the case for nursing homes (NH). MethodsThe study involved five NH (from A to E) affiliated to the Clinico-Malvarrosa Health Department, Valencia (Spain). These were nursing or mixed nursing/care homes of different sizes, altogether providing care for 472 residents attended by a staff of 309. Near-source sewage samples were screened for presence of SARS-CoV-2 RNA by RT-qPCR at least 5 days per week during the study period. SARS-CoV-2 RNA testing in nasopharyngeal swabs from residents and staff was performed with the TaqPath COVID-19 Combo Kit (Thermo Fisher Scientific, Massachusetts, USA). ResultsSARS-CoV-2 RNA was detected in wastewater samples from four of the five NH. SARS-CoV-2 infection cases were documented in three of these four NH. Of the two NH without SARS-CoV-2 infection cases, no SARS-CoV-2 RNA was detected in sewer samples from one facility, while it was repeatedly detected in samples from the other. Presence of SARS-CoV-2 RNA in sewage preceded identification of isolated cases among residents or staff or outbreak declaration in two NH, with lag times ranging from 5 to 19 days. ConclusionOur study demonstrated that intermittent or persistent detection of SARS-CoV-2 RNA in NH sewers can provide an early warning of subsequent individual cases or outbreaks in these facilities.
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ObjectivesThere is an imperative need to determine the durability of adaptive immunity to SARS-CoV-2. We enumerated SARS-CoV-2-reactive CD4+ and CD8+ T cells targeting S1 and M proteins and measured RBD-specific serum IgG over a period of 2-6 months after symptoms onset in a cohort of subjects who had recovered from severe clinical forms of COVID-19. MethodsWe recruited 58 patients (38 males and 20 females; median age, 62.5 years), who had been hospitalized with bilateral pneumonia, 60% with one or more comorbidities. IgG antibodies binding to SARS-CoV-2 RBD were measured by ELISA. SARS-CoV-2-reactive CD69+-expressing-IFN{gamma}-producing-CD4+ and CD8+ T cells were enumerated in heparinized whole blood by flow cytometry for ICS. ResultsDetectable SARS-CoV-2-S1/M-reactive CD69+-IFN-{gamma} CD4+ and CD8+ T cells were displayed in 17 (29.3%) and 6 (10.3%) subjects respectively, at a median of 84 days after onset of symptoms (range, 58-191 days). Concurrent comorbidities increased the risk (OR, 3.15; 95% CI, 1.03-9.61; P=0.04) of undetectable T-cell responses in models adjusted for age, sex and hospitalization ward. Twenty-one out of the 35 patients (60%) had detectable RBD-specific serum IgGs at a median of 118 days (range, 60 to 145 days) after symptoms onset. SARS-CoV-2 RBD-specific IgG serum levels were found to drop significantly over time. ConclusionA relatively limited number of subjects who developed severe forms of COVID-19 had detectable SARS-CoV-2-S1/M IFN{gamma} CD4+ and CD8+ T cells at midterm after clinical diagnosis. Our data also indicated that serum levels of RBD-specific IgGs decline over time, becoming undetectable in some patients.
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The COVID-19 pandemic has shaken the world since the beginning of 2020. Spain is among the European countries with the highest incidence of the disease during the first pandemic wave. We established a multidisciplinar consortium to monitor and study the evolution of the epidemic, with the aim of contributing to decision making and stopping rapid spreading across the country. We present the results for 2170 sequences from the first wave of the SARS-Cov-2 epidemic in Spain and representing 12% of diagnosed cases until 14th March. This effort allows us to document at least 500 initial introductions, between early February-March from multiple international sources. Importantly, we document the early raise of two dominant genetic variants in Spain (Spanish Epidemic Clades), named SEC7 and SEC8, likely amplified by superspreading events. In sharp contrast to other non-Asian countries those two variants were closely related to the initial variants of SARS-CoV-2 described in Asia and represented 40% of the genome sequences analyzed. The two dominant SECs were widely spread across the country compared to other genetic variants with SEC8 reaching a 60% prevalence just before the lockdown. Employing Bayesian phylodynamic analysis, we inferred a reduction in the effective reproductive number of these two SECs from around 2.5 to below 0.5 after the implementation of strict public-health interventions in mid March. The effects of lockdown on the genetic variants of the virus are reflected in the general replacement of preexisting SECs by a new variant at the beginning of the summer season. Our results reveal a significant difference in the genetic makeup of the epidemic in Spain and support the effectiveness of lockdown measures in controlling virus spread even for the most successful genetic variants. Finally, earlier control of SEC7 and particularly SEC8 might have reduced the incidence and impact of COVID-19 in our country.
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ObjectivesThere is limited information on the performance of rapid antigen detection (RAD) tests to identify SARS-CoV-2-infected asymptomatic individuals. In this field study, we evaluated the Panbio COVID-19 Ag Rapid Test Device (Abbott Diagnostics, Jena, Germany) for the purpose. MethodsA total of 634 individuals (355 female; median age, 37 years; range, 9-87) were enrolled. Household (n=338) contacts were tested at a median of 2 days (range, 1-7) after diagnosis of the index case and non-household contacts (n=296) at a median of 6 days (range, 1-7) after exposure. RAD testing was carried out at the point of care. The RT-PCR test used was the TaqPath COVID-19 Combo Kit (Thermo Fisher Scientific, Massachusetts, USA). ResultsIn total, 79 individuals (12.4%) tested positive by RT-PCR, of whom 38 (48.1%) yielded positive RAD results. The overall sensitivity and specificity of the RAD test was 48.1% (95% CI: 37.4-58.9) and 100% (95% CI: 99.3-100), respectively. Sensitivity was higher in household (50.8%; 95% CI: 38.9-62.5) than in non-household (35.7%; 95% CI:16.3-61.2%) contacts. Individuals testing positive by RAD test were more likely (P<0.001) to become symptomatic than their negative counterparts. ConclusionThe Panbio test displays low sensitivity in asymptomatic close contacts of COVID-19 patients, particularly in non-household contacts. Nonetheless, establishing the optimal timing for upper respiratory tract collection in this group seems imperative to pinpoint test sensitivity.
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We evaluated the Panbio COVID-19 AG Rapid Test Device (RAD) for the diagnosis of COVID-19 in symptomatic patients attended in primary healthcare centers (n=412). Overall specificity and sensitivity of RAD was 100% and 79.6%, respectively, taking RT-PCR as the reference. SARS-CoV-2 could not be cultured from specimens yielding RT-PCR+/RAD- results.
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BackgroundWhether antibody levels measured by commercially-available enzyme or chemiluminescent immunoassays targeting the SARS-CoV-2 spike (S) protein can act as a proxy for serum neutralizing activity remains to be established for many of these assays. ObjectivesTo evaluate the degree of correlation between neutralizing antibodies (NtAb) binding the SARS-CoV-2 Spike (S) protein and SARS-CoV-2-S-IgG levels measured by four commercial immunoassays in sera drawn from hospitalized COVID-19 patients. Patients and MethodsNinety sera from 51 hospitalized COVID-19 patients were assayed by a pseudotyped virus neutralization assay, the LIAISON SARS-CoV-2 S1/S2 IgG, the Euroimmun SARS-CoV-2 IgG ELISA, the MAGLUMI 2019-nCoV IgG and the COVID-19 ELISA IgG assays. ResultsOverall, the results obtained with the COVID-19 ELISA IgG test showed the highest agreement with the NtAb assay ({kappa}, 0.85; 95% CI, 0.63-1). The most sensitive tests were the pseudotyped virus NtAb assay and the COVID-19 ELISA IgG assay (92.2% for both). Overall, the degree correlation between antibody titers resulting in 50% virus neutralization (NtAb50) in the pseudotyped virus assay and SARS-CoV-2 IgG levels was strong for the Euroimmun SARS-CoV-2 IgG ELISA (Rho = 0.73) and moderate for the remaining assays (Rho = 0.48 to 0.59). The kinetic profile of serum NtAb50 titers could not be reliably predicted by any of the SARS-CoV-2 IgG immunoassays. ConclusionsThe suitability of SARS-CoV-2-S-IgG commercial immunoassays for inferring neutralizing activity of sera from hospitalized COVID-19 patients varies widely across tests and is influenced by the time of sera collection after the onset of symptoms.
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BackgroundThe involvement of SARS-CoV-2 antibodies in mediating immunopathogenetic events in COVID-19 patients has been suggested. By using several experimental approaches, we investigated the potential association between SARS-CoV-2 IgGs recognizing the spike (S) protein receptor-binding domain (RBD), neutralizing antibodies (NtAb) targeting S, and COVID-19 severity. Patients and MethodsThis unicenter, retrospective, observational study included 51 hospitalized patients (24 at the intensive care unit; ICU). A total of 93 sera from these patients collected at different time points from the onset of symptoms were analyzed. SARS-CoV-2 RBD IgGs were quantitated by ELISA and NtAb50 titers were measured in a GFP reporter-based pseudotyped virus platform. Demographic and clinical data, complete blood counts, as well as serum levels of ferritin, Dimer-D, C reactive protein (CRP), lactose dehydrogenase (LDH), and interleukin-6 (IL-6) were retrieved from clinical charts. ResultsThe overall correlation between levels of both antibody measurements was good (Rho=0.79; P=0<0.001). SARS-CoV-2 RBD IgG and NtAb50 levels in sera collected up to day 30 after the onset of symptoms were comparable between ICU and non-ICU patients (P=>0.1). The percentage of patients who exhibited high NtAb50 titers ([≥] 160) was similar (P=0.20) in ICU (79%) and non-ICU (60%) patients. Four ICU patients died; two of these achieved NtAb50 titers [≥] 1/160 while the other two exhibited a 1/80 titer. Very weak (Rho=>0.0-<0.2) or weak (Rho=>0.2-<0.4) correlations were observed between anti-RBD IgGs, NtAb50, and serum levels pro-inflammatory biomarkers. ConclusionsThe data presented herein do not support an association between SARS-CoV-2 RBD IgG or NtAb50 levels and COVID-19 severity.
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Qualitative assessment of SARS-CoV-2-specific antibody avidity was conducted using an urea (6M) dissociation test performed on a lateral flow immunochromatographic IgG/IgM device. We included a total of 76 serum specimens collected from 57 COVID-19 patients, of which 39 tested positive for both IgG and IgM and 37 only for IgG. Sera losing IgG reactivity after urea treatment (n=28) were drawn significantly earlier (P=0.04) after onset of symptoms than those which preserved it (n=48). This assay may be helpful to estimate the time of acquisition of infection in patients with mild to severe COVID-19.
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There is limited information on SARS-CoV-2 T-cell immune responses in patients with Covid-19. Both CD4+ and CD8+ T cells may be instrumental in the resolution of and protection from SARS-CoV-2 infection. Here, we tested 25 hospitalized patients with either microbiologically documented Covid-19 (n=19) or highly suspected of having the disease (n=6) for the presence of SARS-CoV-2-reactive-CD69+-expressing interferon--producing-(IFN-) CD8+ T cells by a flow-cytometry for intracelular cytokine staining assay. Two sets of overlapping peptides encompassing the SARS-CoV-2 Spike glycoprotein N-terminal 1-643 amino acid sequence and the entire sequence of SARS-CoV-2 M protein were used simultaneously as antigenic stimulus. Ten patients (40%) had detectable responses, displaying frequencies ranging from 0.15 to 2.7% (median of 0.57 cells/L; range, 0.43-9.98 cells/L). The detection rate of SARS-CoV-2-reactive IFN-{gamma} CD8+ T cells in patients admitted to intensive care was comparable (P=0.28) to that in patients hospitalized in other medical wards. No correlation was found between SARS-CoV-2-reactive IFN-{gamma} CD8+ T-cell counts and SARS-CoV-2 S-specific antibody levels. Likewise, no correlation was observed between either SARS-CoV-2-reactive IFN-{gamma} CD8+ T cells or S-specific IgG-antibody titers and blood cell count or levels of inflammatory biomarkers. In summary, in this descriptive, preliminary study we showed that SARS-CoV-2-reactive IFN-{gamma} CD8+ T cells can be detected in a non-negligible percentage of patients with moderate to severe forms of Covid-19. Further studies are warranted to determine whether quantitation of these T-cell subsets may provide prognostic information on the clinical course of Covid-19.
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Real-time reverse transcription polymerase-chain reaction (RT-PCR) is the mainstay of Covid-19 diagnosis. False-negative RT-PCR results may hamper clinical management of patients and hinder the adoption of epidemiological measures to control the pandemic. The current study was aimed at assessing whether amplification of {beta}-glucoronidase (GUSB) gene would help estimate the accuracy of SARS-CoV-2 RT-PCR negative results in upper respiratory tract (URT) specimens. URT specimens that tested negative by SARS-CoV-2 RT-PCR displayed higher GUSB RT-PCR cycle thresholds (CT) (P=0.070) than those testing positive (median, 30.7; range, 27.0-40.0, and median 29.7; range 25.5-36.8, respectively), this reflecting poorer cellularity. Receiver operating characteristic (roc) curve analysis indicated that a CT threshold of 31.2 discriminated best between positive and negative SARS CoV-2 RT-PCRs (area under a curve, 0.66; 95% CI, 0.50-0.81; P=0.08). This cut-off yielded a true negative ratio of 89% and accuracy of 70%. The data suggested that amplification of the GUSB gene by RT-PCR may help to appraise the accuracy of negative SARS-CoV-2 RT-PCR results in patients in whom Covid-19 is eventually diagnosed.
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Systematic testing of large population groups by RT-PCR is mandatory to Covid-19 case identification and contact tracing in order to minimize the likelyhood of resurgence in contagion. Sample pooling for RT-PCR has been effectively used to detect community transmission of SARS CoV-2. Nevertheless, this procedure may decrease the sensitivity of RT-PCR assays due to specimen dilution. We evaluated the efficacy of this strategy for diagnosis of Covid-19 using a sensitive commercially-available RT-PCR targeting SARS CoV-2 E and RdRp genes in a single reaction. A total of 20 mini-pools containing either 5 (n=10) or 10 (n=10) nasopharyngeal exudates collected in universal transport medium were made, each of which including a unique positive NP specimen. Positive specimens yielding CT <32 for the E gene (6 out of 10) or <35.2 for the RdRP gene (7 out of 10) were detected in mini-pools of both sizes. In contrast, most NP samples displaying CTs > 35.8 for the E gene or 35.7 for the RdRP gene remained undetected in mini-pools of 5 specimens (3/4 and 2/3, respectively) or in mini-pools of 10 samples (4/4 and 3/3, respectively.
