Human intestinal enteroids platform to assess the infectivity of gastroenteritis viruses in wastewater.

Fecal-orally transmitted gastroenteritis viruses, particularly human noroviruses (HuNoVs), are a public health concern. Viral transmission risk through contaminated water results underexplored as they have remained largely unculturable until recently and the robust measuring of gastroenteritis viruses infectivity in a single cell line is challenging. This study primarily aimed to test the feasibility of the human intestinal enteroids (HIE) model to demonstrate the infectivity of multiple gastroenteritis viruses in wastewater. Initially, key factors affecting viral replication in HIE model were assessed, and results demonstrated that the reagent-assisted disruption of 3D HIE represents an efficient alternative to syringe pass-through, and the filtering of HuNoV stool suspensions could be avoided. Moreover, comparable replication yields of clinical strains of HuNoV genogroup I (GI), HuNoV GII, rotavirus (RV), astrovirus (HAstV), and adenoviruses (HAdV) were obtained in single and multiple co-infections. Then, the optimized HIE model was used to demonstrate the infectivity of multiple naturally occurring gastroenteritis viruses from wastewater. Thus, a total of 28 wastewater samples were subjected to (RT)-qPCR for each virus, with subsequent testing on HIE. Among these, 16 samples (57 %) showed replication of HuNoVs (n = 3), RV (n = 5), HAstV (n = 8), and/or HAdV (n = 5). Three samples showed HuNoV replication, and sequences assigned to HuNoV GI.3[P13] and HuNoV GII.4[P16] genotypes. Concurrent replication of multiple gastroenteritis viruses occurred in 4 wastewater samples. By comparing wastewater concentrate and HIE supernatant sequences, diverse HAstV and HAdV genotypes were identified in 4 samples. In summary, we successfully employed HIE to demonstrate the presence of multiple infectious human gastroenteritis viruses, including HuNoV, in naturally contaminated wastewater samples.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and lateral flow immunochromatography for rapid identification of ß-lactamase-gene-harboring Enterobacterales in urine specimens: Performance and cost-benefit analyses.

In this single-center prospective study, we evaluated the performance to the MALDI-ToF MS based method in conjunction with lateral flow immunochromatographic (LFIC) in urine specimens for rapid diagnosis of bacterial Urinary Tract Infection (UTI) and detection of carbapenemase and/or extended-spectrum ß- lactamase (ESBL) enzymes produced by the involved bacteria, compared to standard culture, and antimicrobial susceptibility testing/genotypic resistance markers characterization performed on culture-grown colonies. In addition, a cost-benefit analysis comparing this approach against standard procedures was conducted. A total of 324 urines were included in the study, of which 288 (88.9 %) yielded concordant results by the MALDI-ToF MS and conventional culture (Kappa agreement, 0.82; P<0.001). Direct LFIC testing could be carried out in 249/324 urines. Bacterial species carrying ß-lactam genotypic resistance markers were identified in 35 urines (35 CTX-M and 2 OXA-48). Two ESBL-producing Escherichia coli were missed by LFIC (Kappa agreement with standard procedures of 0.96; P<0.001). The cost-benefit analysis indicated that our novel approach resulted in an improvement of clinical outcomes (less need of outpatient care) with a marginal incremental cost (€2.59).

Culture of Human Rotaviruses in Relevant Models Shows Differences in Culture-Adapted and Nonculture-Adapted Strains.

Rotavirus (RV) is the leading cause of acute gastroenteritis (AGE) in children under 5 years old worldwide, and several studies have demonstrated that histo-blood group antigens (HBGAs) play a role in its infection process. In the present study, human stool filtrates from patients diagnosed with RV diarrhea (genotyped as P[8]) were used to infect differentiated Caco-2 cells (dCaco-2) to determine whether such viral strains of clinical origin had the ability to replicate in cell cultures displaying HBGAs. The cell culture-adapted human RV Wa model strain (P[8] genotype) was used as a control. A time-course analysis of infection was conducted in dCaco-2 at 1, 24, 48, 72, and 96 h. The replication of two selected clinical isolates and Wa was further assayed in MA104, undifferentiated Caco-2 (uCaco-2), HT29, and HT29-M6 cells, as well as in monolayers of differentiated human intestinal enteroids (HIEs). The results showed that the culture-adapted Wa strain replicated more efficiently in MA104 cells than other utilized cell types. In contrast, clinical virus isolates replicated more efficiently in dCaco-2 cells and HIEs. Furthermore, through surface plasmon resonance analysis of the interaction between the RV spike protein (VP8*) and its glycan receptor (the H antigen), the V7 RV clinical isolate showed 45 times better affinity compared to VP8* from the Wa strain. These findings support the hypothesis that the differences in virus tropism between clinical virus isolates and RV Wa could be a consequence of the different HBGA contents on the surface of the cell lines employed. dCaco-2, HT29, and HT29M6 cells and HIEs display HBGAs on their surfaces, whereas MA104 and uCaco-2 cells do not. These results indicate the relevance of using non-cell culture-adapted human RV to investigate the replication of rotavirus in relevant infection models.

Replication of Human Norovirus in Mice after Antibiotic-Mediated Intestinal Bacteria Depletion.

Human noroviruses (HuNoVs) are the main cause of acute gastroenteritis causing more than 50,000 deaths per year. Recent evidence shows that the gut microbiota plays a key role in enteric virus infectivity. In this context, we tested whether microbiota depletion or microbiota replacement with that of human individuals susceptible to HuNoVs infection could favor viral replication in mice. Four groups of mice (n = 5) were used, including a control group and three groups that were treated with antibiotics to eliminate the autochthonous intestinal microbiota. Two of the antibiotic-treated groups received fecal microbiota transplantation from a pool of feces from infants (age 1-3 months) or an auto-transplantation with mouse feces that obtained prior antibiotic treatment. The inoculation of the different mouse groups with a HuNoVs strain (GII.4 Sydney [P16] genotype) showed that the virus replicated more efficiently in animals only treated with antibiotics but not subject to microbiota transplantation. Viral replication in animals receiving fecal microbiota from newborn infants was intermediate, whereas virus excretion in feces from auto-transplanted mice was as low as in the control mice. The analysis of the fecal microbiota by 16S rDNA NGS showed deep variations in the composition in the different mice groups. Furthermore, differences were observed in the gene expression of relevant immunological mediators, such as IL4, CXCL15, IL13, TNFα and TLR2, at the small intestine. Our results suggest that microbiota depletion eliminates bacteria that restrict HuNoVs infectivity and that the mechanism(s) could involve immune mediators.

BD MAX Enteric Bacterial, Bacterial Plus, and Virus Panels for Diagnosis of Acute Infectious Gastroenteritis: a Cost-Benefit Analysis.

Economic assessment is required to gauge the value of implementing PCR syndromic platforms in the microbiology laboratory for the diagnosis of community-acquired acute gastroenteritis (AGE) in pediatric and adult in- and outpatients. A cost-benefit analysis was conducted from a health care system perspective using BD MAX Enteric Bacterial, Bacterial Plus, and Virus panels. Two 6-month periods were selected, in which either conventional procedures (in 2017) or BD MAX PCR multiplex panels (in 2018) were used. We retrospectively reviewed medical records of all patients with positive results and a representative sample of negative ones. A Markov model was used to represent transition probabilities between different health care states from time of stool microbiological study until completion of AGE-episode-associated health care. A total of 1,336 medical records were reviewed (829 in 2018 and 507 in 2017), showing overall a significantly higher positivity rate in 2018 than in 2017 (26% versus 6%, P < 0.001). The total cost per individual associated with health care for AGE was €314 in 2018 and €341 in 2017; when we only considered the pediatric cohort, the figures were €271 and €456, respectively. Using Tornado sensitivity analyses, we found that the three variables that most influenced the model in descending order of weight were the probability of longer hospital stays, the probability of returning to the emergency room (ER), and the probability of hospitalization from the ER. Use of BD MAX enteric PCR platforms for the diagnosis of community-acquired AGE instead of a non-PCR-based conventional approach results in an incremental benefit from a health care perspective in the general population, particularly children. IMPORTANCE The implementation of multiplex molecular panels allows microbiological laboratories to quickly, sensitively, and accurately diagnose acute infectious gastroenteritis. This methodology therefore allows faster decisions regarding treatment and infection control measures. Economic evaluations are required to gauge the value of implementing these syndromic PCR platforms in a community-based acute gastroenteritis setting. We studied the potential clinical and cost benefits, in terms of both their impact on laboratory costs and the subsequent costs of managing patients.

Recombinant Noroviruses Circulating in Spain from 2016 to 2020 and Proposal of Two Novel Genotypes within Genogroup I.

Noroviruses are the leading cause of sporadic cases and outbreaks of viral gastroenteritis. For more than 20 years, most norovirus infections have been caused by the pandemic genotype GII.4, yet recent studies have reported the emergence of recombinant strains in many countries. In the present study, 4,950 stool samples collected between January 2016 and April 2020 in Valencia, Spain, from patients with acute gastroenteritis were analyzed to investigate the etiological agent. Norovirus was the most frequently detected enteric virus, with a positivity rate of 9.5% (471/4,950). Among 224 norovirus strains characterized, 175 belonged to genogroup II (GII) and 49 belonged to GI. Using dual genotyping based on sequencing of the open reading frame 1 (ORF1)/ORF2 junction region, we detected 25 different capsid-polymerase-type associations. The most common GII capsid genotype was GII.4 Sydney 2012, followed by GII.2, GII.3, GII.6, and GII.17. A high prevalence of recombinant strains (90.4%) was observed among GII infections between 2018 and 2020. GII.4 Sydney[P16] was the predominant genotype from 2019 to 2020. In addition, GII.P16 polymerase was found harbored within six different capsid genes. GI.4 and GI.3 were the predominant genotypes in genogroup I, in which recombinant strains were also found, such as GI.3[P10], GI.3[P13], and GI.5[P4]. Interestingly, applying the criterion of 2 times the standard deviation, we found that 12 sequences initially classified as GI.3 may represent two new tentative genotypes in genogroup I, designated GI.10 and GI.11. This study shows the extensive diversity of recombinant noroviruses circulating in Spain and highlights the role of recombination events in the spread of noroviruses. IMPORTANCE Human noroviruses are the most common cause of viral diarrhea. There are no approved vaccines to prevent their infections yet, which would be very useful to protect infants, small children, and the elderly in residential institutions. These viruses are extremely contagious and can be transmitted by contaminated food and water as well as directly from person to person. Molecular surveillance and epidemiology of norovirus infections allow the identification of the most common viral strains in different geographical areas over time. Noroviruses show wide genetic variability due to a high rate of mutations but also due to genomic recombinations, as we demonstrate in this study. We have detected 25 different viral capsid-polymerase gene associations among 224 norovirus strains characterized in Spain between January 2016 and April 2020, including two tentative new capsid genotypes in genogroup I.

The Role of Host Glycobiology and Gut Microbiota in Rotavirus and Norovirus Infection, an Update.

Rotavirus (RV) and norovirus (NoV) are the leading causes of acute gastroenteritis (AGE) worldwide. Several studies have demonstrated that histo-blood group antigens (HBGAs) have a role in NoV and RV infections since their presence on the gut epithelial surfaces is essential for the susceptibility to many NoV and RV genotypes. Polymorphisms in genes that code for enzymes required for HBGAs synthesis lead to secretor or non-secretor and Lewis positive or Lewis negative individuals. While secretor individuals appear to be more susceptible to RV infections, regarding NoVs infections, there are too many discrepancies that prevent the ability to draw conclusions. A second factor that influences enteric viral infections is the gut microbiota of the host. In vitro and animal studies have determined that the gut microbiota limits, but in some cases enhances enteric viral infection. The ways that microbiota can enhance NoV or RV infection include virion stabilization and promotion of virus attachment to host cells, whereas experiments with microbiota-depleted and germ-free animals point to immunoregulation as the mechanism by which the microbiota restrict infection. Human trials with live, attenuated RV vaccines and analysis of the microbiota in responder and non-responder individuals also allowed the identification of bacterial taxa linked to vaccine efficacy. As more information is gained on the complex relationships that are established between the host (glycobiology and immune system), the gut microbiota and intestinal viruses, new avenues will open for the development of novel anti-NoV and anti-RV therapies.

Adaptive immune responses to SARS-CoV-2 in recovered severe COVID-19 patients.

BACKGROUND: There 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. PATIENTS AND METHODS: We 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-IFNγ-producing-CD4+ and CD8+ T cells were enumerated in heparinized whole blood by flow cytometry for ICS. RESULTS: Detectable SARS-CoV-2-S1/M-reactive CD69+-IFN-γ 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, 60-145 days) after symptoms onset. SARS-CoV-2 RBD-specific IgG serum levels were found to drop significantly over time. CONCLUSION: A relatively limited number of subjects who developed severe forms of COVID-19 had detectable SARS-CoV-2-S1/M IFNγ 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.

Microbiota Depletion Promotes Human Rotavirus Replication in an Adult Mouse Model.

Intestinal microbiota-virus-host interaction has emerged as a key factor in mediating enteric virus pathogenicity. With the aim of analyzing whether human gut bacteria improve the inefficient replication of human rotavirus in mice, we performed fecal microbiota transplant (FMT) with healthy infants as donors in antibiotic-treated mice. We showed that a simple antibiotic treatment, irrespective of FMT, resulted in viral shedding for 6 days after challenge with the human rotavirus G1P[8] genotype Wa strain (RVwa). Rotavirus titers in feces were also significantly higher in antibiotic-treated animals with or without FMT but they were decreased in animals subject to self-FMT, where a partial re-establishment of specific bacterial taxons was evidenced. Microbial composition analysis revealed profound changes in the intestinal microbiota of antibiotic-treated animals, whereas some bacterial groups, including members of Lactobacillus, Bilophila, Mucispirillum, and Oscillospira, recovered after self-FMT. In antibiotic-treated and FMT animals where the virus replicated more efficiently, differences were observed in gene expression of immune mediators, such as IL1ß and CXCL15, as well as in the fucosyltransferase FUT2, responsible for H-type antigen synthesis in the small intestine. Collectively, our results suggest that antibiotic-induced microbiota depletion eradicates the microbial taxa that restrict human rotavirus infectivity in mice.

The Rotavirus Vaccine Landscape, an Update.

Rotavirus is the leading cause of severe acute childhood gastroenteritis, responsible for more than 128,500 deaths per year, mainly in low-income countries. Although the mortality rate has dropped significantly since the introduction of the first vaccines around 2006, an estimated 83,158 deaths are still preventable. The two main vaccines currently deployed, Rotarix and RotaTeq, both live oral vaccines, have been shown to be less effective in developing countries. In addition, they have been associated with a slight risk of intussusception, and the need for cold chain maintenance limits the accessibility of these vaccines to certain areas, leaving 65% of children worldwide unvaccinated and therefore unprotected. Against this backdrop, here we review the main vaccines under development and the state of the art on potential alternatives.

Global Trends in Norovirus Genotype Distribution among Children with Acute Gastroenteritis.

Noroviruses are a leading cause of acute gastroenteritis (AGE) among adults and children worldwide. NoroSurv is a global network for norovirus strain surveillance among children <5 years of age with AGE. Participants in 16 countries across 6 continents used standardized protocols for dual typing (genotype and polymerase type) and uploaded 1,325 dual-typed sequences to the NoroSurv web portal during 2016-2020. More than 50% of submitted sequences were GII.4 Sydney[P16] or GII.4 Sydney[P31] strains. Other common strains included GII.2[P16], GII.3[P12], GII.6[P7], and GI.3[P3] viruses. In total, 22 genotypes and 36 dual types, including GII.3 and GII.20 viruses with rarely reported polymerase types, were detected, reflecting high strain diversity. Surveillance data captured in NoroSurv enables the monitoring of trends in norovirus strains associated childhood AGE throughout the world on a near real-time basis.

Epidemiological and Genetic Characterization of Sapovirus in Patients with Acute Gastroenteritis in Valencia (Spain).

Sapovirus is a common cause of acute gastroenteritis in all age groups. Sapovirus infections are seldom investigated in Spain, and its epidemiology in the country is not well known. The use of molecular diagnostic procedures has allowed a more frequent detection of sapoviruses in patients with diarrhea. A total of 2545 stool samples from patients with acute gastroenteritis attended from June 2018 to February 2020 at the Clinic University Hospital in Valencia, Spain, were analyzed by reverse transcription (RT) and real-time multiplex PCR (RT-PCR) to investigate the etiology of enteric infections. Sapovirus was the second enteric virus detected with a positive rate of 8%, behind norovirus (12.2%) and ahead of rotavirus (7.1%), astrovirus (4.9%) and enteric adenoviruses (2.9%). Most sapovirus infections occurred in infants and young children under 3 years of age (74%) with the highest prevalence in autumn and early winter. Coinfections were found in 25% of the patients with sapovirus diarrhea, mainly with other enteric viruses. Genotyping demonstrated the circulation of seven different genotypes during the study period, with a predominance of genotypes GI.1, GI.2, and GII.1. Phylogenetic analysis showed that genogroup GII strains form a cluster separated from genogroup GI and GV, being genotype GV.1 strains related to genotype GI.1 and GI.2 strains.

An investigation of the potential association between gastrointestinal viral and bacterial infection and development of intestinal acute graft versus host disease following allogeneic hematopoietic stem cell transplantation.

It is uncertain whether gastrointestinal (GI) infection caused by viral and bacterial pathogens may predispose to gastrointestinal acute Graft-versus-host disease (aGvHD-GI) in allogeneic hematopoietic stem cell transplant recipients (allo-HSCT). We investigated the potential association between detection of enteropathogenic viruses or bacteria in stools and subsequent occurrence of aGvHD-GI in a cohort of 121 allo-HSCT patients. Eighty-six out of 121 patients (71%) had acute diarrhea and underwent screening for primary GI pathogens by molecular diagnostic methods. One or more GI pathogens were detected in 27 out of the 86 patients with diarrhea (31.3%). Specifically, Clostridioides difficile was found in 16 patients (18.6%), enteropathogenic viruses in 11 patients (12.7%) (Astrovirus, n = 4; Norovirus, n = 2; Sapovirus, n = 2; Adenovirus, n = 2; and Rotavirus, n = 1), and Campylobacter spp. in two patients (2.3%). Thirty patients were diagnosed with all grade aGvHD-GI by histopathology. Detection of primary GI pathogens was achieved in 12 out of 30 patients (Clostridium difficile, n = 5; enteric viruses, n = 8; Campylobacter spp., n = 1) who either subsequently developed (n = 9) or previously had (n = 3) grade I-IV IaGvHD (n = 9). Neither the detection of these microorganisms (all combined), enteric viruses, nor C. difficile was significantly associated with subsequent aGvHD-GI development in Cox models (hazard ratio [HR] = 1.11, p = .80; HR = 1.64, p = .62; HR = 0.75, p = .64, respectively). Analogous results were obtained when grade II-IV aGvHD-GI was selected as the clinical outcome. In summary, data in the current study did not support an association between GI infection and subsequent occurrence of aGvHD-GI in an unselected cohort of allo-HSCT recipients.

Interaction of Intestinal Bacteria with Human Rotavirus during Infection in Children.

The gut microbiota has emerged as a key factor in the pathogenesis of intestinal viruses, including enteroviruses, noroviruses and rotaviruses (RVs), where stimulatory and inhibitory effects on infectivity have been reported. With the aim of determining whether members of the microbiota interact with RVs during infection, a combination of anti-RV antibody labeling, fluorescence-activated cell sorting and 16S rRNA amplicon sequencing was used to characterize the interaction between specific bacteria and RV in stool samples of children suffering from diarrhea produced by G1P[8] RV. The genera Ruminococcus and Oxalobacter were identified as RV binders in stools, displaying enrichments between 4.8- and 5.4-fold compared to samples nonlabeled with anti-RV antibodies. In vitro binding of the G1P[8] Wa human RV strain to two Ruminococcus gauvreauii human isolates was confirmed by fluorescence microscopy. Analysis in R. gauvreauii with antibodies directed to several histo-blood group antigens (HBGAs) indicated that these bacteria express HBGA-like substances on their surfaces, which can be the target for RV binding. Furthermore, in vitro infection of the Wa strain in differentiated Caco-2 cells was significantly reduced by incubation with R. gauvreauii. These data, together with previous findings showing a negative correlation between Ruminococcus levels and antibody titers to RV in healthy individuals, suggest a pivotal interaction between this bacterial group and human RV. These results reveal likely mechanisms of how specific bacterial taxa of the intestinal microbiota could negatively affect RV infection and open new possibilities for antiviral strategies.

Inference of SARS-CoV-2 spike-binding neutralizing antibody titers in sera from hospitalized COVID-19 patients by using commercial enzyme and chemiluminescent immunoassays.

Whether 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. We evaluated 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. Ninety sera from 51 hospitalized COVID-19 patients were tested 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. Overall, the results obtained with the COVID-19 ELISA IgG test showed the highest agreement with the NtAb assay (κ, 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. The 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.

Recommendations for the introduction of metagenomic high-throughput sequencing in clinical virology, part I: Wet lab procedure.

Metagenomic high-throughput sequencing (mHTS) is a hypothesis-free, universal pathogen detection technique for determination of the DNA/RNA sequences in a variety of sample types and infectious syndromes. mHTS is still in its early stages of translating into clinical application. To support the development, implementation and standardization of mHTS procedures for virus diagnostics, the European Society for Clinical Virology (ESCV) Network on Next-Generation Sequencing (ENNGS) has been established. The aim of ENNGS is to bring together professionals involved in mHTS for viral diagnostics to share methodologies and experiences, and to develop application recommendations. This manuscript aims to provide practical recommendations for the wet lab procedures necessary for implementation of mHTS for virus diagnostics and to give recommendations for development and validation of laboratory methods, including mHTS quality assurance, control and quality assessment protocols.

Suitability of two rapid lateral flow immunochromatographic assays for predicting SARS-CoV-2 neutralizing activity of sera.

Assessment of commercial severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoassays for their capacity to provide reliable information on sera neutralizing activity is an emerging need. We evaluated the performance of two commercially available lateral flow immunochromatographic assays (LFIC; Wondfo SARS-CoV-2 Antibody test and the INNOVITA 2019-nCoV Ab test) in comparison with a SARS-CoV-2 neutralization pseudotyped assay for coronavirus disease 2019 (COVID-19) diagnosis in hospitalized patients and investigate whether the intensity of the test band in LFIC associates with neutralizing antibody (NtAb) titers. Ninety sera were included from 51 patients with moderate to severe COVID-19. A green fluorescent protein (GFP) reporter-based pseudotyped neutralization assay (vesicular stomatitis virus coated with SARS-CoV-2 spike protein) was used. Test line intensity was scored using a 4-level scale (0 to 3+). The overall sensitivity of LFIC assays was 91.1% for the Wondfo SARS-CoV-2 Antibody test, 72.2% for the INNOVITA 2019-nCoV IgG, 85.6% for the INNOVITA 2019-nCoV IgM, and 92.2% for the NtAb assay. Sensitivity increased for all assays in sera collected beyond day 14 after symptoms onset (93.9%, 79.6%, 93.9%, and 93.9%, respectively). Reactivities equal to or more intense than the positive control line (≥2+) in the Wondfo assay had a negative predictive value of 100% and a positive predictive value of 96.4% for high NtAb50 titers (≥1/160). Our findings support the use of LFIC assays evaluated herein, particularly the Wondfo test, for COVID-19 diagnosis. We also find evidence that these rapid immunoassays can be used to predict high SARS-CoV-2-S NtAb50 titers.

SARS-CoV-2 antibodies, serum inflammatory biomarkers and clinical severity of hospitalized COVID-19 patients.

BACKGROUND: The 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 METHODS: This 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 reporterbased 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. RESULTS: The overall correlation between levels of both antibody measurements was good (Rho = 0.82; 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). 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. CONCLUSIONS: The data presented herein do not support an association between SARS-CoV-2 RBD IgG or NtAb50 levels and COVID-19 severity.

Sero-epidemiological study of the rotavirus VP8* protein from different P genotypes in Valencia, Spain.

The aims of the present work were to determine the prevalence and titer of serum antibodies against several rotavirus VP8* proteins from different P genotypes in children and adults in Valencia, Spain; and to determine the role of the secretor status (FUT2G428A polymorphism) in the antibody response. The VP8* protein from the P[4], P[6], P[8], P[9], P[11], P[14] and P[25] genotypes were produced in E. coli. These proteins were tested with 88 serum samples from children (n = 41, 3.5 years old in average) and from adults (n = 47, 58 years old in average) by ELISA. A subset of 55 samples were genotyped for the FUT2G428A polymorphism and the antibody titers compared. The same subset of samples was also analysed by ELISA using whole rotavirus Wa particles (G1P[8]) as antigen. Ninety-three per cent of the samples were positive for at least one of the VP8* antigens. Differences in the IgG seroprevalence were found between children and adults for the P[4], P[8] and P[11] genotypes. Similarly, significant differences were found between adults and children in their antibody titers against the P[4], P[8], and P[11] VP8* genotypes, having the children higher antibody titers than adults. Interestingly, positive samples against rare genotypes such as P[11] (only in children), P[14] and P[25] were found. While no statistical differences in the antibody titers between secretors and non-secretors were found for any of the tested P genotypes studied, a higher statistic significant prevalence for the P[25] genotype was found in secretors compared to non-secretors. Significant differences in the antibody titers between secretors and non-secretors were found when the whole viral particles from the Wa rotavirus strain (G1P[8]) were used as the antigen.