Impact of the H274Y Substitution on N1, N4, N5, and N8 Neuraminidase Enzymatic Properties and Expression in Reverse Genetic Influenza A Viruses.

The H274Y substitution (N2 numbering) in neuraminidase (NA) N1 confers oseltamivir resistance to A(H1N1) influenza viruses. This resistance has been associated with reduced N1 expression using transfected cells, but the effect of this substitution on the enzymatic properties and on the expression of other group-1-NA subtypes is unknown. The aim of the present study was to evaluate the antiviral resistance, enzymatic properties, and expression of wild-type (WT) and H274Y-substituted NA for each group-1-NA. To this end, viruses with WT or H274Y-substituted NA (N1pdm09 or avian N4, N5 or N8) were generated by reverse genetics, and for each reverse-genetic virus, antiviral susceptibility, NA affinity (Km), and maximum velocity (Vm) were measured. The enzymatic properties were coupled with NA quantification on concentrated reverse genetic viruses using mass spectrometry. The H274Y-NA substitution resulted in highly reduced inhibition by oseltamivir and normal inhibition by zanamivir and laninamivir. This resistance was associated with a reduced affinity for MUNANA substrate and a conserved Vm in all viruses. NA quantification was not significantly different between viruses carrying WT or H274Y-N1, N4 or N8, but was lower for viruses carrying H274Y-N5 compared to those carrying a WT-N5. In conclusion, the H274Y-NA substitution of different group-1-NAs systematically reduced their affinity for MUNANA substrate without a significant impact on NA Vm. The impact of the H274Y-NA substitution on viral NA expression was different according to the studied NA.

Factors Associated with Long COVID-19 in a French Multicentric Prospective Cohort Study.

(1) Background: A substantial proportion of COVID-19 patients continue to experience long-lasting effects that hamper their quality of life. The objectives of this study were (1) to report the prevalence of persistent clinical symptoms 6-12 months after the onset of COVID-19 and (2) to identify potential factors at admission associated with the occurrence of long COVID. (2) Methods: A prospective study was conducted among COVID-19 adult patients, hospitalized in four French university hospitals. Patients were invited to two ambulatory follow-up medical visits, 6-8 months (visit #1) and one year (visit #2) after the onset of their COVID-19. A multivariate logistic regression was performed to assess factors associated with long COVID. (3) Results: In total, 189 patients participated in this study (mean age of 63.4 years). BMI > 30 kg/m2 (aOR 3.52), AST levels between 31 and 42 U/L (aOR 8.68), and AST levels > 42 U/L (aOR 3.69) were associated with persistent clinical symptoms at visit #1. Anosmia (aOR 13.34), AST levels between 31 and 42 U/L (aOR 10.27), stay in ICU (aOR 5.43), pain (aOR 4.31), and longer time before hospitalization (aOR 1.14) were significantly associated with persistent clinical symptoms at visit #2. Patients with ageusia (aOR 0.17) had a lower risk of long COVID. (4) Conclusions: This study showed that some patients experienced persistent clinical symptoms one year after COVID-19 onset that were associated with some determinants at the acute phase/stage.

Patient influenza vaccination reduces the risk of hospital-acquired influenza: An incident test negative-case control study in Lyon university hospital, France (2004-2020).

BACKGROUND: Literature is limited on the impact of patient vaccination on the risk of hospital-acquired influenza (HAI). This test negative case-control study nested in a surveillance program aimed at evaluating the effectiveness of influenza vaccination in reducing the risk of HAI in hospitalized patients during 15 influenza seasons (2004-05 to 2019-20). METHODS: HAI cases were those who developed influenza like illness (ILI) symptoms at least 72 h after hospitalization and had a positive reverse transcriptase-polymerase chain reaction (RT-PCR). Controls were those with ILI symptoms and a negative RT-PCR test. A nasal swab as well as socio-demographic, clinical data and information on influenza vaccination were collected. RESULTS: Of the 296 patients included, 67 were confirmed HAI cases. Influenza vaccine coverage was significantly higher among controls compared to HAI cases (p = 0.002). The risk of HAI was reduced by almost 60 % in vaccinated patients. CONCLUSIONS: A better control of HAI can be achieved by vaccinating hospitalized patients.

Co-infection of the respiratory epithelium, scene of complex functional interactions between viral, bacterial, and human neuraminidases.

The activity of sialic acids, known to play critical roles in biology and many pathological processes, is finely regulated by a class of enzymes called sialidases, also known as neuraminidases. These are present in mammals and many other biological systems, such as viruses and bacteria. This review focuses on the very particular situation of co-infections of the respiratory epithelium, the scene of complex functional interactions between viral, bacterial, and human neuraminidases. This intrinsically multidisciplinary topic combining structural biology, biochemistry, physiology, and the study of host-pathogen interactions, opens up exciting research perspectives that could lead to a better understanding of the mechanisms underlying virus-bacteria co-infections and their contribution to the aggravation of respiratory pathology, notably in the context of pre-existing pathological contexts. Strategies that mimic or inhibit the activity of the neuraminidases could constitute interesting treatment options for viral and bacterial infections.

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.

Evaluation of commercial Anti-SARS-CoV-2 neutralizing antibody assays in seropositive subjects.

The virus neutralization test (VNT) is the reference for the assessment of the functional ability of neutralizing antibodies (NAb) to block SARS-CoV-2 entry into cells. New competitive immunoassays measuring antibodies preventing interaction between the spike protein and its cellular receptor are proposed as surrogate VNT (sVNT). We tested three commercial sVNT (a qualitative immunochromatographic test and two quantitative immunoassays named YHLO and TECO) together with a conventional anti-spike IgG assay (bioMérieux) in comparison with an in-house plaque reduction neutralization test (PRNT50) using the original 19A strain and different variants of concern (VOC), on a panel of 306 sera from naturally-infected or vaccinated patients. The qualitative test was rapidly discarded because of poor sensitivity and specificity. Areas under the curve of YHLO and TECO assays were, respectively, 85.83 and 84.07 (p-value >0.05) using a positivity threshold of 20 for PRNT50, and 95.63 and 90.35 (p-value =0.02) using a threshold of 80. However, the performances of YHLO and bioMérieux were very close for both thresholds, demonstrating the absence of added value of sVNT compared to a conventional assay for the evaluation of the presence of NAb in seropositive subjects. In addition, the PRNT50 assay showed a reduction of NAb titers towards different VOC in comparison to the 19A strain that could not be appreciated by the commercial tests. Despite the good correlation between the anti-spike antibody titer and the titer of NAb by PRNT50, our results highlight the difficulty to distinguish true NAb among the anti-RBD antibodies with commercial user-friendly immunoassays.

Performance of the QIAprep& Viral RNA UM Kit assay (Qiagen), an automatable method for RT-qPCR detection of SARS-CoV-2 without RNA extraction.

We evaluated the performance of the QIAprep& Viral RNA UM Kit (Qiagen) for SARS-CoV-2 detection. It displayed specificity and sensitivity required for SARS-CoV-2 RNA detection from swab transport media without RNA extraction. This method identifies accurately patients at risk of transmission while saving time and cost of extraction.

Baseline clinical features of COVID-19 patients, delay of hospital admission and clinical outcome: A complex relationship.

INTRODUCTION: Delay between symptom onset and access to care is essential to prevent clinical worsening for different infectious diseases. For COVID-19, this delay might be associated with the clinical prognosis, but also with the different characteristics of patients. The objective was to describe characteristics and symptoms of community-acquired (CA) COVID-19 patients at hospital admission according to the delay between symptom onset and hospital admission, and to identify determinants associated with delay of admission. METHODS: The present work was based on prospective NOSO-COR cohort data, and restricted to patients with laboratory confirmed CA SARS-CoV-2 infection admitted to Lyon hospitals between February 8 and June 30, 2020. Long delay of hospital admission was defined as ≥6 days between symptom onset and hospital admission. Determinants of the delay between symptom onset and hospital admission were identified by univariate and multiple logistic regression analysis. RESULTS: Data from 827 patients were analysed. Patients with a long delay between symptom onset and hospital admission were younger (p<0.01), had higher body mass index (p<0.01), and were more frequently admitted to intensive care unit (p<0.01). Their plasma levels of C-reactive protein were also significantly higher (p<0.01). The crude in-hospital fatality rate was lower in this group (13.3% versus 27.6%), p<0.01. Multiple analysis with correction for multiple testing showed that age ≥75 years was associated with a short delay between symptom onset and hospital admission (≤5 days) (aOR: 0.47 95% CI (0.34-0.66)) and CRP>100 mg/L at admission was associated with a long delay (aOR: 1.84 95% CI (1.32-2.55)). DISCUSSION: Delay between symptom onset and hospital admission is a major issue regarding prognosis of COVID-19 but can be related to multiple factors such as individual characteristics, organization of care and severe pathogenic processes. Age seems to play a key role in the delay of access to care and the disease prognosis.

Evaluation of Commercial Anti-SARS-CoV-2 Antibody Assays and Comparison of Standardized Titers in Vaccinated Health Care Workers.

With the availability of vaccines, commercial assays detecting anti-severe acute respiratory syndrome coronavirus-2 antibodies (Ab) evolved toward quantitative assays directed to the spike glycoprotein or its receptor binding domain (RBD). The main objective of the present study was to compare the Ab titers obtained with quantitative commercial binding Ab assays, after one dose (convalescent individuals) or two doses (naive individuals) of vaccine, in health care workers (HCW). Antibody titers were measured in 255 sera (from 150 HCW) with five quantitative immunoassays (Abbott RBD IgG II quant, bioMérieux RBD IgG, DiaSorin Trimeric spike IgG, Siemens Healthineers RBD IgG, Wantai RBD IgG). One qualitative total antibody anti-RBD detection assay (Wantai) was used to detect previous infection before vaccination. The results are presented in binding Ab units (BAU)/mL after application, when possible, of a conversion factor provided by the manufacturers and established from a World Health Organization internal standard. There was a 100% seroconversion with all assays evaluated after two doses of vaccine. With assays allowing BAU/mL correction, Ab titers were correlated (Pearson correlation coefficient, ρ, range: 0.85-0.94). The titer differences varied by a mean of 10.6% between Siemens and bioMérieux assays to 60.9% between Abbott and DiaSorin assays. These results underline the importance of BAU conversion for the comparison of Ab titer obtained with the different quantitative assays. However, significant differences persist, notably, between kits detecting Ab against the different antigens. A true standardization of the assays would be to include the International Standard in the calibration of each assay to express the results in IU/mL.

Evaluation of High-Throughput SARS-CoV-2 Serological Assays in a Longitudinal Cohort of Patients with Mild COVID-19: Clinical Sensitivity, Specificity, and Association with Virus Neutralization Test.

BACKGROUND: The association between SARS-CoV-2 commercial serological assays and virus neutralization test (VNT) has been poorly explored in mild patients with COVID-19. METHODS: 439 serum specimens were longitudinally collected from 76 healthcare workers with RT-PCR-confirmed COVID-19. The clinical sensitivity (determined weekly) of 9 commercial serological assays were evaluated. Clinical specificity was assessed using 69 pre-pandemic sera. Correlation, agreement, and concordance with the VNT were also assessed on a subset of 170 samples. Area under the ROC curve (AUC) was estimated at 2 neutralizing antibody titers. RESULTS: The Wantai Total Ab assay targeting the receptor binding domain (RBD) within the S protein presented the best sensitivity at different times during the course of disease. The clinical specificity was greater than 95% for all tests except for the Euroimmun IgA assay. The overall agreement with the presence of neutralizing antibodies ranged from 62.2% (95%CI; 56.0-68.1) for bioMérieux IgM to 91.2% (87.0-94.2) for Siemens. The lowest negative percent agreement (NPA) was found with the Wantai Total Ab assay (NPA 33% (21.1-48.3)). The NPA for other total Ab or IgG assays targeting the S or the RBD was 80.7% (66.7-89.7), 90.3% (78.1-96.1), and 96.8% (86.8-99.3) for Siemens, bioMérieux IgG, and DiaSorin, respectively. None of the commercial assays have sufficient performance to detect a neutralizing titer of 80 (AUC < 0.76). CONCLUSIONS: Although some assays show a better agreement with VNT than others, the present findings emphasize that commercialized serological tests, including those targeting the RBD, cannot substitute a VNT for the assessment of functional antibody response.

Factors associated with admission to intensive care units in COVID-19 patients in Lyon-France.

INTRODUCTION: A new respiratory virus, SARS-CoV-2, has emerged and spread worldwide since late 2019. This study aims at analysing clinical presentation on admission and the determinants associated with admission in intensive care units (ICUs) in hospitalized COVID-19 patients. PATIENTS AND METHODS: In this prospective hospital-based study, socio-demographic, clinical and biological characteristics, on admission, of adult COVID-19 hospitalized patients presenting from the community for their first admission were prospectively collected and analysed. Characteristics of patients hospitalized in medical ward to those admitted in ICU were compared using Mann-Whitney and Chi-square or Fisher exact test when appropriate. Univariate logistic regression was first used to identify variables on admission that were associated with the outcome i.e. admission to an ICU versus total hospital stay in a medical ward. Forward selection was then applied beginning with sex, age and temperature in the multivariable logistic regression model. RESULTS: Of the 412 patients included, 325 were discharged and 87 died in hospital. Multivariable regression showed increasing odds of ICU hospitalization with temperature (OR, 1.56 [95% CI, 1.06-2.28] per degree Celsius increase), oxygen saturation <90% (OR, 12.45 [95% CI, 5.27-29.4]), abnormal lung auscultation on admission (OR, 3.58 [95% CI, 1.58-8.11]), elevated level of CRP (OR, 2.7 [95% CI, 1.29-5.66for CRP>100mg/L vs CRP<10mg/L). and monocytopenia (OR, 3.28 [95% CI, 1.4-7.68]) were also associated with increasing odds of ICU hospitalization. Older patients were less likely to be hospitalized in ICU (OR, 0.17 [95%CI, 0.05-0.51]. CONCLUSIONS: Age and delay between onset of symptoms and hospital admission were associated with the risk of hospitalisation in ICU. Age being a fixed variable, interventions that shorten this delay would improve the prognosis of Covid-19 patients.

Incidence of hospital-acquired influenza in adults: A prospective surveillance study from 2004 to 2017 in a French tertiary care hospital.

BACKGROUND: Hospital-acquired influenza potentially leads to significant morbidity and mortality in already vulnerable patients, but its overall burden is not fully understood. We undertook this study to estimate the incidence and trends of hospital-acquired laboratory-confirmed influenza among adults, and to compare clinical characteristics between hospital-acquired and community-acquired influenza cases. METHODS: This was a prospective surveillance study over 11 years of adults with influenza-like-illness (ILI) hospitalized in surgery, medicine and geriatric wards in a tertiary acute-care hospital in Lyon, France. Nasal swabs were systematically collected from those with ILI and tested for influenza by reverse transcriptase-polymerase chain reaction at the national influenza reference laboratory (Lyon, France). RESULTS: Influenza was laboratory confirmed at a rate of 1 in 13 patients who developed ILI during their hospitalization. Having an underlying disease was an important characteristic of hospital-acquired ILI cases. Cardiovascular disease was the most frequent underlying condition in both influenza-positive and influenza-negative patients. Complications were more frequent for influenza-positive than influenza-negative patients. The influenza incidence rate was highest in the geriatric ward and increased over the study period. CONCLUSIONS: Hospital-acquired influenza poses a significant risk to already vulnerable patients. Longitudinal surveillance data are essential to support better recognition and monitoring of viral infections in hospitals.

Protocol for a prospective, observational, hospital-based multicentre study of nosocomial SARS-CoV-2 transmission: NOSO-COR Project.

INTRODUCTION: The newly identified SARS-CoV-2 can cause serious acute respiratory infections such as pneumonia. In France, mortality rate in the general population was approximately 10% and could reach higher levels at the hospital. In the current context of high incidence rates of SARS-CoV-2 in the community, a significant increase in the rate of nosocomial transmission is expected. The risk of nosocomial transmission could even be higher in low-income countries that have fragile healthcare systems. This protocol is intended to estimate the prevalence and incidence of suspected or confirmed cases of nosocomial SARS-CoV-2 infection, the clinical spectrum and the determinants (risk factors/protective) at participating hospitals. METHODS AND ANALYSIS: This will be an international multicentre prospective, observational, hospital-based study in adults and children. It will include volunteer patients and healthcare professionals in France and hospitals affiliated with the GABRIEL network. Demographic and clinical data will be collected using case report forms designed especially for the purpose of the project. A nasopharyngeal swab will be collected and tested for SARS-CoV-2 by reverse-transcriptase PCR. Characteristics of the study participants, the proportion of confirmed nosocomial SARS-CoV-2 infections relative to all patients with syndromes suggestive of SARS-CoV-2 infection, will be analysed. Appropriate multivariate modelling will be used to identify the determinants associated with nosocomial onset. ETHICS AND DISSEMINATION: This study was approved by the clinical research and committee of all participating countries. The findings will be submitted to peer-reviewed journal for publication and shared with national health authorities. TRIAL REGISTRATION NUMBER: NCT04290780.

Comparison of eight commercial, high-throughput, automated or ELISA assays detecting SARS-CoV-2 IgG or total antibody.

BACKGROUND: Many commercial assays, of different designs, detecting SARS-CoV-2-specific antibodies exist but with little experience with them. OBJECTIVES: The aim of this study was to compare the performance of assays detecting IgG or total antibodies to N or S antigens, validated for routine use in France, with samples from subjects with more or less severe SARS-CoV-2 infection. METHODS: Eight assays were used: Abbott Architect, DiaSorin Liaison®, bioMérieux Vidas®, Roche Elecsys Cobas®, Siemens Atellica®, BioRad Platelia ELISA, Epitope Diagnostics ELISA, and Wantai ELISA. The tested population included 86 samples from 40 hospitalized subjects and 28 outpatients at different time from symptom onset. RESULTS: The positivity rate varied depending on the assay but was greater for all assays in hospitalized than non-hospitalized patients. Despite a good correlation between the assays, discrepancies occurred, without a systematic origin, even for samples taken more than 20 days after symptom onset. These discrepancies were linked to low antibody levels in pauci-symptomatic patients. CONCLUSION: Whichever assay is chosen, a false negative result may need to be ruled out with another test in a risk situation.

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.

Impact of Pregnancy on Intra-Host Genetic Diversity of Influenza A Viruses in Hospitalised Women: A Retrospective Cohort Study.

The authors wish to make the following corrections to this paper [...].

Impact of Pregnancy on Intra-Host Genetic Diversity of Influenza A Viruses in Hospitalised Women: A Retrospective Cohort Study.

Characterising dynamics of Influenza A Viruses (IAV) within-host evolution is an active field of research which may lead to a better understanding of viral pathogenesis. Using a pregnant mouse model, a study has recently suggested that immune modulation during pregnancy could promote the emergence of IAV quasispecies with increased virulence. Herein, we assess the clinical relevance of these findings in humans. We studied IAV intra-host diversity (ihD) in pregnant (n = 36) and non-pregnant (n = 23) women hospitalized in Lyon for IAV infection (01/2015-05/2018). Whole IAV genomes present in nasopharyngeal samples were sequenced in duplicate to analyze reproducible intra-host single nucleotide variants (ihSNV). Counts, relative frequencies and locations of ihSNV were used as indicators of ihD. The median ihSNV/kb counts per segment were between 0 and 1.3. There was >81% ihSNV at relative frequencies between 1-5% for H1N1 and >51% for H3N2 IAV. No significant difference was noted between pregnant and non-pregnant women when considering all or only non-synonymous ihSNV. Seven convergent non-synonymous ihSNV were found; none were significantly associated with pregnancy. These results suggest that modulation of the immune system during pregnancy in humans does not impact IAV ihD, in contrast to mice.

Human herpesvirus 6 infection after autologous stem cell transplantation: A multicenter prospective study in adult patients.

OBJECTIVES: to prospectively evaluate the incidence and the clinical relevance on hematopoietic reconstitution of HHV-6 infection in autologous hematopoietic stem cell transplantation (ASCT) recipients. METHODS: HHV-6 DNA load was measured in whole blood specimens once during the 7 days before stem cell re-infusion and once a week after transplantation until hematopoietic recovery. Active HHV-6 infection was defined by 2 consecutive positive DNA loads. RESULTS: from July 2012 to February 2015, 196 adult patients undergoing ASCT were enrolled. Twenty-two (11.2%) patients developed active HHV-6 infection with a cumulative incidence of 19% at 40 days after transplantation. The onset of active HHV-6 infection occurred with a median of 13 days after stem cell re-infusion. HHV-6 infection was associated with an increased frequency of non-infectious complications (OR = 5.05; 95%CI 1.78-14.32; P < 0.001). Moreover, the severity of these non-infectious complications was higher in recipients exhibiting HHV-6 infection (OR = 4.62; 95%CI 1.32-16.2; p < 0.01). Delayed neutrophils 10 (IQR: 8-14) vs 8 (IQR: 6-11) days and platelets recoveries 15 (IQR: 11.8-18.5) vs 8 (IQR: 4-14) days were observed in patients with active HHV-6 infection compared to non-infected ones. CONCLUSIONS: in this study, 11.2% ASCT recipients presented active HHV-6 infection associated with significantly delayed hematologic reconstitution.

[Do liquid wastes from automated instruments in medical laboratories have their proper microbicide effect?] / Les rejets liquides des automates de biologie médicale ont-ils un effet microbicide propre qui permettrait de s'affranchir de leur traitement pour risque infectieux ?

Liquid wastes from clinical biology automated systems are currently evacuated in the urban network after chemical treatment to eliminate a possible risk of infection. Since these wastes are ecotoxic because of the presence of numerous chemical reagents, we studied their intrinsic microbicidal power towards a selection of infectious agents widely found in clinical specimens. The objective was to determine if an additional anti-infectious treatment before elimination is necessary. Thus, we evaluated the bactericidal effect of liquid wastes of several automated systems towards four bacterial species (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Enterococcus faecalis) and their virucidal activity against a non-enveloped virus, resistant in the environment (adenovirus). This effect was determined for different exposure times. Our results showed that the antibacterial activity was highly variable depending on the waste-bacteria pair considered (varying from no activity to complete sterilization of a strong bacterial inoculum). The liquid wastes were on the other hand globally inactive towards adenovirus.