SARS-CoV-2 N-Antigen Quantification in Respiratory Tract, Plasma and Urine: Kinetics and Association with RT-qPCR Results.

Qualitative SARS-CoV-2 antigen assays based on immunochromatography are useful for mass diagnosis of COVID-19, even though their sensitivity is poor in comparison with RT-PCR assays. In addition, quantitative assays could improve antigenic test performance and allow testing with different specimens. Using quantitative assays, we tested 26 patients for viral RNA and N-antigen in respiratory samples, plasma and urine. This allowed us to compare the kinetics between the three compartments and to compare RNA and antigen concentrations in each. Our results showed the presence of N-antigen in respiratory (15/15, 100%), plasma (26/59, 44%) and urine (14/54, 28.9%) samples, whereas RNA was only detected in respiratory (15/15, 100%) and plasma (12/60, 20%) samples. We detected N-antigen in urine and plasma samples until the day 9 and day 13 post-inclusion, respectively. The antigen concentration was found to correlate with RNA levels in respiratory (p < 0.001) and plasma samples (p < 0.001). Finally, urinary antigen levels correlated with plasma levels (p < 0.001). Urine N-antigen detection could be part of the strategy for the late diagnosis and prognostic evaluation of COVID-19, given the ease and painlessness of sampling and the duration of antigen excretion in this biological compartment.

Rapid and reliable inactivation protocols for the diagnostics of emerging viruses: The example of SARS-CoV-2 and monkeypox virus.

The emergence and sustained transmission of novel pathogens are exerting an increasing demand on the diagnostics sector worldwide, as seen with the ongoing severe acute respiratory coronavirus 2 (SARS-CoV-2) pandemic and the more recent public health concern of monkeypox virus (MPXV) since May 2022. Appropriate and reliable viral inactivation measures are needed to ensure the safety of personnel handling these infectious samples. In the present study, seven commercialized diagnosis buffers, heat (56°C and 60°C), and sodium dodecyl sulfate detergent (2.0%, 1.0%, and 0.5% final concentrations) were tested against infectious SARS-CoV-2 and MPXV culture isolates on Vero cell culture. Cytopathic effects were observed up to 7 days postinoculation and viral load evolution was measured by semiquantitative polymerase chain reaction. The World Health Organization recommends an infectious titer reduction of at least 4 log10 . As such, the data show efficacious SARS-CoV-2 inactivation by all investigated methods, with >6.0 log10 reduction. MPXV inactivation was also validated with all investigated methods with 6.9 log10 reductions, although some commercial buffers required a longer incubation period to yield complete inactivation. These results are valuable for facilities, notably those without biosafety level-3 capabilities, that need to implement rapid and reliable protocols common against both SARS-CoV-2 and MPXV.

Dynamics of viral shedding during ancestral or Omicron BA.1 SARS-CoV-2 infection and enhancement of pre-existing immunity during breakthrough infections.

Omicron variant is circulating in the presence of a globally acquired immunity unlike the ancestral SARS-CoV-2 isolate. Herein, we investigated the normalized viral load dynamics and viral culture status in 44 fully vaccinated healthcare workers (HCWs) infected with the Omicron BA.1 variant. Viral load dynamics of 38 unvaccinated HCWs infected with the 20A variant during the first pandemic wave was also studied. We then explored the impact of Omicron infection on pre-existing immunity assessing anti-RBD IgG levels, neutralizing antibody titres against 19A, Delta and Omicron isolates, as well as IFN-γ release following cell stimulation with SARS-CoV-2 peptides. We reported that two weeks after diagnosis a greater proportion of HCWs infected with 20A (78.9%, 15/19) than with Omicron BA.1 (44.7%, 17/38; p = 0.02) were still positive by RT-qPCR. We found that Omicron breakthrough infections led to an overall enhancement of vaccine-induced humoral and cellular immunity as soon as a median [interquartile range] of 8 [7-9] days post symptom onset. Among samples with similar high viral loads, non-culturable samples exhibited higher neutralizing antibody titres and anti-RBD IgG levels than culturable samples. Additionally, Omicron infection led to an enhancement of antibodies neutralization capacity against other SARS-CoV-2 isolates. Taken together, the results suggest that Omicron BA.1 vaccine breakthrough infection is associated with a faster viral clearance than that of the ancestral SARS-CoV-2, in addition this new variant leads to a rapid enhancement of the humoral response against multiple SARS-CoV-2 variants, and of the cellular response.

SARS-CoV-2 Omicron Variant, Lineage BA.1, Is Associated with Lower Viral Load in Nasopharyngeal Samples Compared to Delta Variant.

OBJECTIVES: High viral load in upper respiratory tract specimens observed for Delta cases might contribute to its increased infectivity compared to the other variant. However, it is not yet documented if the Omicron variant's enhanced infectivity is also related to a higher viral load. Our aim was to determine if the Omicron variant's spread is also related to higher viral loads compared to the Delta variant. METHODS: Nasopharyngeal swabs, 129 (Omicron) and 85 (Delta), from Health Care Workers were collected during December 2021 at the University Hospital of Lyon, France. Cycle threshold (Ct) for the RdRp target of cobas® 6800 SARS-CoV-2 assay was used as a proxy to evaluate SARS-CoV-2 viral load. Variant identification was performed using a screening panel and confirmed by whole genome sequencing. RESULTS: Herein, we showed that the RT-PCR Ct values in Health Care Workers sampled within 5 days after symptom onset were significantly higher for Omicron cases than Delta cases (21.7 for Delta variant and 23.8 for Omicron variant, p = 0.008). This difference was also observed regarding patient with complete vaccination. CONCLUSIONS: This result supports the studies showing that the increased transmissibility of Omicron is related to other mechanisms than higher virus excretion.

Sars-Cov-2 Inactivation by a Panel of Commercialized Buffers and by Traditional Protocols (Heat or SDS) (preprint)

Following the rapid spread of COVID-19 across the globe, the intense response that was demanded of diagnostic centers and research laboratories prompted the use of numerous products and protocols for the management of SARS-CoV-2 specimens. In these settings, proper handling of such infectious specimen is necessary to ensure the safety of personnel and to reduce the risk of active transmission. Our aim was to evaluate the inactivation efficacy of different inactivating methods, notably from commercial lysis buffers available in diagnostic kits. Heat and sodium dodecyl sulfate detergent were also included in our investigations. A cell culture-based assay was used, and supported by molecular qRT-PCR detection, to show in vitro infectivity reduction after inactivation treatment. Overall, all the investigated methods were successful in inactivating SARS-CoV-2. Ten minutes of contact with the commercial buffers completely stopped in vitro SARS-CoV-2 infectivity. Fifteen minutes at 68°C and 30 minutes at 56°C as well as one hour with sodium dodecyl sulfate detergent at 2, 1, 0.5, and 0.1% yielded the same results. These findings demonstrate the reliability of these protocols with regards to biosafety. Inactivation by heat and sodium dodecyl sulfate detergent are rather simple and can be readily available methods for rendering an infectious SARS-CoV-2 specimen inactive, especially in settings where commercial buffers are not available.

Effect of remdesivir on viral dynamics in COVID-19 hospitalized patients: a modelling analysis of the randomized, controlled, open-label DisCoVeRy trial.

BACKGROUND: The antiviral efficacy of remdesivir in COVID-19 hospitalized patients remains controversial. OBJECTIVES: To estimate the effect of remdesivir in blocking viral replication. METHODS: We analysed nasopharyngeal normalized viral loads from 665 hospitalized patients included in the DisCoVeRy trial (NCT04315948; EudraCT 2020-000936-23), randomized to either standard of care (SoC) or SoC + remdesivir. We used a mathematical model to reconstruct viral kinetic profiles and estimate the antiviral efficacy of remdesivir in blocking viral replication. Additional analyses were conducted stratified on time of treatment initiation (≤7 or >7 days since symptom onset) or viral load at randomization (< or ≥3.5 log10 copies/104 cells). RESULTS: In our model, remdesivir reduced viral production by infected cells by 2-fold on average (95% CI: 1.5-3.2-fold). Model-based simulations predict that remdesivir reduced time to viral clearance by 0.7 days compared with SoC, with large inter-individual variabilities (IQR: 0.0-1.3 days). Remdesivir had a larger impact in patients with high viral load at randomization, reducing viral production by 5-fold on average (95% CI: 2.8-25-fold) and the median time to viral clearance by 2.4 days (IQR: 0.9-4.5 days). CONCLUSIONS: Remdesivir halved viral production, leading to a median reduction of 0.7 days in the time to viral clearance compared with SoC. The efficacy was larger in patients with high viral load at randomization.

Impact of original, B.1.1.7, and B.1.351/P.1 SARS-CoV-2 lineages on vaccine effectiveness of two doses of COVID-19 mRNA vaccines: Results from a nationwide case-control study in France.

BACKGROUND: We aimed to assess the effectiveness of two doses of mRNA COVID-19 vaccines against COVID-19 with the original virus and other lineages circulating in France. METHODS: In this nationwide case-control study, cases were SARS-CoV-2 infected adults with onset of symptoms between 14 February and 3 May 2021. Controls were non-infected adults from a national representative panel matched to cases by age, sex, region, population density and calendar week. Participants completed an online questionnaire on recent activity-related exposures and vaccination history. Information about the infecting virus was based on a screening RT-PCR for either B.1.1.7 or B.1.351/P.1 variants. FINDINGS: Included in our analysis were 7 288 adults infected with the original SARS-CoV-2 virus, 31 313 with the B.1.1.7 lineage, 2 550 with B.1.351/P1 lineages, and 3 644 controls. In multivariable analysis, the vaccine effectiveness (95% confidence interval) seven days after the second dose of mRNA vaccine was estimated at 88% (81-92), 86% (81-90) and 77% (63-86) against COVID-19 with the original virus, the B.1.1.7 lineage, and the B.1.351/P.1 lineages, respectively. Recent (2 to 6 months) history of virologically confirmed SARS-CoV-2 infection was found to be 83% (76-88), 88% (85-91) and 83% (71-90) protective against COVID-19 with the original virus, the B.1.1.7 lineage, and the B.1.351/P.1 lineages, respectively; and more distant (> 6 months) infections were 76% (54-87), 84% (75-90), and 74% (41-89) protective against COVID-19 with the original virus, the B.1.1.7 lineage, and the B.1.351/P.1 lineages, respectively. INTERPRETATION: In real-life settings, two doses of mRNA vaccines proved to be effective against COVID-19 with the original virus, B.1.1.7 lineage and B.1.351/P.1 lineages. FUNDING: Institut Pasteur, Research & Action Emerging Infectious Diseases (REACTing), Fondation de France (Alliance "Tous unis contre le virus").

Co-infection with Legionella and SARS-CoV-2, France, March 2020.

We describe a March 2020 co-occurrence of Legionnaires' disease (LD) and coronavirus disease in France. Severe acute respiratory syndrome coronavirus 2 co-infections were identified in 7 of 49 patients from LD case notifications. Most were elderly men with underlying conditions who had contracted severe pneumonia, illustrating the relevance of co-infection screening.

Characteristics of the delayed respiratory syncytial virus epidemic, 2020/2021, Rhône Loire, France.

The Rhône-Loire metropolitan areas' 2020/21 respiratory syncytial virus (RSV) epidemic was delayed following the implementation of non-pharmaceutical interventions (NPI), compared with previous seasons. Very severe lower respiratory tract infection incidence among infants ≤ 3 months decreased twofold, the proportion of cases among children aged > 3 months to 5 years increased, and cases among adults > 65 years were markedly reduced. NPI appeared to reduce the RSV burden among at-risk groups, and should be promoted to minimise impact of future RSV outbreaks.

Clinical and laboratory characteristics of symptomatic healthcare workers with suspected COVID-19: a prospective cohort study.

A comprehensive clinical and microbiological assessments of COVID-19 in front-line healthcare workers (HCWs) is needed. Between April 10th and May 28th, 2020, 319 HCWs with acute illness were reviewed. In addition to SARS-CoV-2 RT-PCR screening, a multiplex molecular panel was used for testing other respiratory pathogens. For SARS-CoV-2 positive HCWs, the normalized viral load, viral culture, and virus neutralization assays were performed weekly. For SARS-CoV-2 negative HCWs, SARS-CoV-2 serological testing was performed one month after inclusion. Among the 319 HCWs included, 67 (21.0%) were tested positive for SARS-CoV-2; 65/67 (97.0%) developed mild form of COVID-19. Other respiratory pathogens were found in 6/66 (9.1%) SARS-CoV-2 positive and 47/241 (19.5%) SARS-Cov-2 negative HCWs (p = 0.07). The proportion of HCWs with a viral load > 5.0 log10 cp/mL (Ct value < 25) was less than 15% at 8 days after symptom onset; 12% of HCWs were positive after 40 days (Ct > 37). More than 90% of cultivable virus had a viral load > 4.5 log10 cp/mL (Ct < 26) and were collected within 10 days after symptom onset. Among negative HCWs, 6/190 (3.2%) seroconverted. Our data suggest that the determination of viral load can be used for appreciating the infectiousness of infected HCWs. These data could be helpful for facilitating their return to work.

Early assessment of diffusion and possible expansion of SARS-CoV-2 Lineage 20I/501Y.V1 (B.1.1.7, variant of concern 202012/01) in France, January to March 2021.

The emergence of SARS-CoV-2 variant 20I/501Y.V1 (VOC-202012/1 or GR/501Y.V1) is concerning given its increased transmissibility. We reanalysed 11,916 PCR-positive tests (41% of all positive tests) performed on 7-8 January 2021 in France. The prevalence of 20I/501Y.V1 was 3.3% among positive tests nationwide and 6.9% in the Paris region. Analysing the recent rise in the prevalence of 20I/501Y.V1, we estimate that, in the French context, 20I/501Y.V1 is 52-69% more transmissible than the previously circulating lineages, depending on modelling assumptions.

Modeling SARS-CoV-2 viral kinetics and association with mortality in hospitalized patients from the French COVID cohort.

The characterization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral kinetics in hospitalized patients and its association with mortality is unknown. We analyzed death and nasopharyngeal viral kinetics in 655 hospitalized patients from the prospective French COVID cohort. The model predicted a median peak viral load that coincided with symptom onset. Patients with age ≥65 y had a smaller loss rate of infected cells, leading to a delayed median time to viral clearance occurring 16 d after symptom onset as compared to 13 d in younger patients (P < 10-4). In multivariate analysis, the risk factors associated with mortality were age ≥65 y, male gender, and presence of chronic pulmonary disease (hazard ratio [HR] > 2.0). Using a joint model, viral dynamics after hospital admission was an independent predictor of mortality (HR = 1.31, P < 10-3). Finally, we used our model to simulate the effects of effective pharmacological interventions on time to viral clearance and mortality. A treatment able to reduce viral production by 90% upon hospital admission would shorten the time to viral clearance by 2.0 and 2.9 d in patients of age <65 y and ≥65 y, respectively. Assuming that the association between viral dynamics and mortality would remain similar to that observed in our population, this could translate into a reduction of mortality from 19 to 14% in patients of age ≥65 y with risk factors. Our results show that viral dynamics is associated with mortality in hospitalized patients. Strategies aiming to reduce viral load could have an effect on mortality rate in this population.

Two-step strategy for the identification of SARS-CoV-2 variant of concern 202012/01 and other variants with spike deletion H69-V70, France, August to December 2020.

We report the strategy leading to the first detection of variant of concern 202012/01 (VOC) in France (21 December 2020). First, the spike (S) deletion H69-V70 (ΔH69/ΔV70), identified in certain SARS-CoV-2 variants including VOC, is screened for. This deletion is associated with a S-gene target failure (SGTF) in the three-target RT-PCR assay (TaqPath kit). Subsequently, SGTF samples are whole genome sequenced. This approach revealed mutations co-occurring with ΔH69/ΔV70 including S:N501Y in the VOC.

Cluster of Coronavirus Disease 2019 (COVID-19) in the French Alps, February 2020.

BACKGROUND: On 7 February 2020, French Health authorities were informed of a confirmed case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in an Englishman infected in Singapore who had recently stayed in a chalet in the French Alps. We conducted an investigation to identify secondary cases and interrupt transmission. METHODS: We defined as a confirmed case a person linked to the chalet with a positive reverse-transcription polymerase chain reaction sample for SARS-CoV-2. RESULTS: The index case stayed 4 days in the chalet with 10 English tourists and a family of 5 French residents; SARS-CoV-2 was detected in 5 individuals in France, 6 in England (including the index case), and 1 in Spain (overall attack rate in the chalet: 75%). One pediatric case, with picornavirus and influenza A coinfection, visited 3 different schools while symptomatic. One case was asymptomatic, with similar viral load as that of a symptomatic case. Seven days after the first cases were diagnosed, 1 tertiary case was detected in a symptomatic patient with from the chalet a positive endotracheal aspirate; all previous and concurrent nasopharyngeal specimens were negative. Additionally, 172 contacts were monitored; all contacts tested for SARS-CoV-2 (N = 73) were negative. CONCLUSIONS: The occurrence in this cluster of 1 asymptomatic case with similar viral load as a symptomatic patient suggests transmission potential of asymptomatic individuals. The fact that an infected child did not transmit the disease despite close interactions within schools suggests potential different transmission dynamics in children. Finally, the dissociation between upper and lower respiratory tract results underscores the need for close monitoring of the clinical evolution of suspected cases of coronavirus disease 2019.

Chest CT for rapid triage of patients in multiple emergency departments during COVID-19 epidemic: experience report from a large French university hospital.

OBJECTIVES: To assess the diagnostic performances of chest CT for triage of patients in multiple emergency departments during COVID-19 epidemic, in comparison with reverse transcription polymerase chain reaction (RT-PCR) test. METHOD: From March 3 to April 4, 2020, 694 consecutive patients from three emergency departments of a large university hospital, for which a hospitalization was planned whatever the reasons, i.e., COVID- or non-COVID-related, underwent a chest CT and one or several RT-PCR tests. Chest CTs were rated as "Surely COVID+," "Possible COVID+," or "COVID-" by experienced radiologists. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated using the final RT-PCR test as standard of reference. The delays for CT reports and RT-PCR results were recorded and compared. RESULTS: Among the 694 patients, 287 were positive on the final RT-PCR exam. Concerning the 694 chest CT, 308 were rated as "Surely COVID+", 34 as "Possible COVID+," and 352 as "COVID-." When considering only the "Surely COVID+" CT as positive, accuracy, sensitivity, specificity, PPV, and NPV reached 88.9%, 90.2%, 88%, 84.1%, and 92.7%, respectively, with respect to final RT-PCR test. The mean delay for CT reports was three times shorter than for RT-PCR results (187 ± 148 min versus 573 ± 327 min, p < 0.0001). CONCLUSION: During COVID-19 epidemic phase, chest CT is a rapid and most probably an adequately reliable tool to refer patients requiring hospitalization to the COVID+ or COVID- hospital units, when response times for virological tests are too long. KEY POINTS: • In a large university hospital in Lyon, France, the accuracy, sensitivity, specificity, PPV, and NPV of chest CT for COVID-19 reached 88.9%, 90.2%, 88%, 84.1%, and 92.7%, respectively, using RT-PCR as standard of reference. • The mean delay for CT reports was three times shorter than for RT-PCR results (187 ± 148 min versus 573 ± 327 min, p < 0.0001). • Due to high accuracy of chest CT for COVID-19 and shorter time for CT reports than RT-PCR results, chest CT can be used to orient patients suspected to be positive towards the COVID+ unit to decrease congestion in the emergency departments.

Characterization and Treatment of SARS-CoV-2 in Nasal and Bronchial Human Airway Epithelia.

In the current COVID-19 pandemic context, proposing and validating effective treatments represents a major challenge. However, the scarcity of biologically relevant pre-clinical models of SARS-CoV-2 infection imposes a significant barrier for scientific and medical progress, including the rapid transition of potentially effective treatments to the clinical setting. We use reconstituted human airway epithelia to isolate and then characterize the viral infection kinetics, tissue-level remodeling of the cellular ultrastructure, and transcriptional early immune signatures induced by SARS-CoV-2 in a physiologically relevant model. Our results emphasize distinctive transcriptional immune signatures between nasal and bronchial HAE, both in terms of kinetics and intensity, hence suggesting putative intrinsic differences in the early response to SARS-CoV-2 infection. Most important, we provide evidence in human-derived tissues on the antiviral efficacy of remdesivir monotherapy and explore the potential of the remdesivir-diltiazem combination as an option worthy of further investigation to respond to the still-unmet COVID-19 medical need.

Performance Assessment of SARS-CoV-2 PCR Assays Developed by WHO Referral Laboratories.

A reliable diagnostic assay is crucial to early detect new COVID-19 cases and limit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission. Since the onset of the COVID-19 pandemic, the World Health Organization has published several diagnostic molecular approaches developed by referral laboratories, including Charité (Germany), HKU (Hong Kong), China CDC (China), US CDC (United States), and Institut Pasteur, Paris (France). We aimed to compare the sensitivity and specificity of these different RT-PCR assays using SARS-CoV-2 cell culture supernatants and clinical respiratory samples. Overall, the different RT-PCR assays performed well for SARS-CoV-2 detection and were all specific except the N Charité (Germany), and N2 US CDC (United States) assays. RdRp Institut Pasteur (IP2, IP4), N China CDC, and N1 US CDC were found to be the most sensitive assays. The data presented herein are of prime importance to facilitate the equipment choice of diagnostic laboratories, as well as for the development of marketed tests.

Clinical and virological data of the first cases of COVID-19 in Europe: a case series.

BACKGROUND: On Dec 31, 2019, China reported a cluster of cases of pneumonia in people at Wuhan, Hubei Province. The responsible pathogen is a novel coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We report the relevant features of the first cases in Europe of confirmed infection, named coronavirus disease 2019 (COVID-19), with the first patient diagnosed with the disease on Jan 24, 2020. METHODS: In this case series, we followed five patients admitted to Bichat-Claude Bernard University Hospital (Paris, France) and Pellegrin University Hospital (Bordeaux, France) and diagnosed with COVID-19 by semi-quantitative RT-PCR on nasopharyngeal swabs. We assessed patterns of clinical disease and viral load from different samples (nasopharyngeal and blood, urine, and stool samples), which were obtained once daily for 3 days from hospital admission, and once every 2 or 3 days until patient discharge. All samples were refrigerated and shipped to laboratories in the National Reference Center for Respiratory Viruses (The Institut Pasteur, Paris, and Hospices Civils de Lyon, Lyon, France), where RNA extraction, real-time RT-PCR, and virus isolation and titration procedures were done. FINDINGS: The patients were three men (aged 31 years, 48 years, and 80 years) and two women (aged 30 years and 46 years), all of Chinese origin, who had travelled to France from China around mid-January, 2020. Three different clinical evolutions are described: (1) two paucisymptomatic women diagnosed within a day of exhibiting symptoms, with high nasopharyngeal titres of SARS-CoV-2 within the first 24 h of the illness onset (5·2 and 7·4 log10 copies per 1000 cells, respectively) and viral RNA detection in stools; (2) a two-step disease progression in two young men, with a secondary worsening around 10 days after disease onset despite a decreasing viral load in nasopharyngeal samples; and (3) an 80-year-old man with a rapid evolution towards multiple organ failure and a persistent high viral load in lower and upper respiratory tract with systemic virus dissemination and virus detection in plasma. The 80-year-old patient died on day 14 of illness (Feb 14, 2020); all other patients had recovered and been discharged by Feb 19, 2020. INTERPRETATION: We illustrated three different clinical and biological types of evolution in five patients infected with SARS-CoV-2 with detailed and comprehensive viral sampling strategy. We believe that these findings will contribute to a better understanding of the natural history of the disease and will contribute to advances in the implementation of more efficient infection control strategies. FUNDING: REACTing (Research & Action Emerging Infectious Diseases).