Evaluation of SARS-CoV-2 neutralizing antibodies using a CPE-based colorimetric live virus micro-neutralization assay in human serum samples

The micro-neutralization assay is a fundamental test in virology, immunology, vaccine assessment, and epidemiology studies. Since the SARS-CoV-2 outbreak at the end of December 2019 in China, it has become extremely important to have well-established and validated diagnostic and serological assays for this new emerging virus. Here, we present a micro-neutralization assay with the use of SARS-CoV-2 wild type virus with two different methods of read-out. We evaluated the performance of this assay using human serum samples taken from an Italian seroepidemiological study being performed at the University of Siena, along with the human monoclonal antibody CR3022 and some iper-immune animal serum samples against Influenza and Adenovirus strains. The same panel of human samples have been previously tested in enzyme-linked immunosorbent assay (ELISA) as a pre-screening. Positive, borderline, and negative ELISA samples were evaluated in neutralization assay using two different methods of read-out: subjective (by means of an inverted optical microscope) and objective (by means of a spectrophotometer). Our findings suggest that at least 50% of positive ELISA samples are positive in neutralization as well, and that method is able to quantify different antibody concentrations in a specific manner. Taken together, our results confirm that the colorimetric cytopathic effect-based microneutralization assay could be used as a valid clinical test method for epidemiological and vaccine studies.     

The importance of anosmia,ageusia and age in community presentation of symptomatic and asymptomatic SARS-CoV-2 infection in Louisiana,USA; a cross-sectional prevalence study

ObjectiveWhile many seroprevalence studies of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been performed, few are demographically representative. This investigation focused on defining the nature and frequency of symptomatic and asymptomatic SARS-CoV-2 infection in a representative, cross-sectional sample of communities in Louisiana, USA.MethodsA sample of 4778 adults from New Orleans and Baton Rouge, Louisiana were given a survey of symptoms and co-morbidities, nasopharyngeal swab to test for active infection (PCR), and blood draw to test for past infection (IgG). Odds ratios, cluster analysis, quantification of virus and antibody, and linear modelling were used to understand whether certain symptoms were associated with a positive test, how symptoms grouped together, whether virus or antibody varied by symptom status, and whether being symptomatic was different across the age span.ResultsReported anosmia/ageusia was strongly associated with a positive test; 40.6% (93/229) tested positive versus 4.8% (218/4549) positivity in those who did not report anosmia/ageusia (OR 13.6, 95% CI 10.1–18.3). Of the people who tested positive, 47.3% (147/311) were completely asymptomatic. Symptom presentation clustered into three groups; low/no symptoms (0.4 ± 0.9, mean ± SD), highly symptomatic (7.5 ± 1.9) or moderately symptomatic (4.0 ± 1.5). Quantity of virus was lower in the asymptomatic versus symptomatic group (cycle number 23.3 ± 8.3 versus 17.3 ± 9.0; p < 0.001). Modelling the probability of symptoms showed changes with age; the highest probability of reporting symptoms was 64.6% (95% CI 50.4–76.5) at age 29 years, which decreased to a probability of 49.3% (95% CI 36.6–62.0) at age 60 years and only 25.1% (95% CI 5.0–68.1) at age 80 years.ConclusionAnosmia/ageusia can be used to differentiate SARS-CoV-2 infection from other illnesses, and, given the high ratio of asymptomatic individuals, contact tracing should include those without symptoms. Regular testing in congregant settings of those over age 60 years may help mitigate asymptomatic spread.     

Epidemiological and clinical features of asymptomatic patients with SARS-CoV-2 infection

Few studies reported the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected patients with completely asymptomatic throughout the disease course. We investigated the epidemiological and clinical features of patients infected by SARS-CoV-2 without any symptoms. Patients with confirmed SARS-CoV-2 infection were retrospectively recruited. The demographic characteristics, clinical data, treatment, and outcomes of SARS-CoV-2 infected patients without any symptoms were analyzed. Fifteen (4.4%) of 342 SARS-CoV-2 infected patients did not develop any symptom during the course of the disease. The median time from exposure to diagnosis was 7.0 days (interquartile range [IQR]: 1.0-15.0 days). Of the 15 patients, 14 patients were diagnosed by tested positive for SARS-CoV-2 in throat swabs, while one patient was only tested positive for SARS-CoV-2 in anal swabs. During hospitalization, only 1 (6.7%) patient developed lymphopenia. Abnormalities of chest computed tomography examinations were detected in 8 (53.4%) patients on admission. As of 8 March 2020, all patients have been discharged. The median time of SARS-CoV-2 tested negative from admission was 7.0 days (IQR: 4.0-9.0 days). Patients without any symptoms but with SARS-CoV-2 exposure should be closely monitored and tested for SARS-CoV-2 both in anal and throat swabs to excluded the infection. Asymptomatic patients infected by SARS-CoV-2 have favorable outcomes.     

The first Italian outbreak of SARS-CoV-2 B.1.1.7 lineage in Corzano,Lombardy

In December 2020, Italy experienced the first case of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) B.1.1.7 lineage. In January 2021, we identified 21 cases of this variant in Corzano, defining the first outbreak of SARS-CoV-2 B.1.1.7 lineage in Italy. The high transmissibility of the B.1.1.7 variant represented an important benefit for the virus, which became rapidly dominant on the territory. Containment measures induced the epidemic curve onto a decreasing trajectory underlining the importance of appropriate control and surveillance for restraint of virus spread. Highlights The first Italian outbreak of SARS-CoV-2 B.1.1.7 lineage occurred in Lombardy in January 2021. The outbreak originated by a single introduction of the B.1.1.7 lineage. The genomic sequencing revealed, for the first time, the presence of the V551F mutation in the B.1.1.7 lineage in Italy. Surveillance, prompt sequencing and tracing efforts were fundamental to identify and to quickly contain the outbreak.     

Asymptomatic cases with SARS-CoV-2 infection

On 31 March 2020, Chinese Health Authorization announced that numbers of asymptomatic cases with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection will be made to the public daily. This was a very important step since different counties have different capacities for the detection of SARS-CoV-2 infection and control strategy for the Coronavirus Disease 2019 outbreak. We summarized the characteristics of asymptomatic SARS-CoV-2 infections and the transmission potential of asymptomatic cases. Then we provided guidelines for the management of asymptomatic cases through quarantine and nucleic acid/serology tests.     

SARS-CoV-2, the pandemic coronavirus: Molecular and structural insights

The outbreak of a novel coronavirus associated with acute respiratory disease, called COVID-19, marked the introduction of the third spillover of an animal coronavirus (CoV) to humans in the last two decades. The genome analysis with various bioinformatics tools revealed that the causative pathogen (SARS-CoV-2) belongs to the subgenus Sarbecovirus of the genus Betacoronavirus, with highly similar genome as bat coronavirus and receptor-binding domain (RBD) of spike glycoprotein as Malayan pangolin coronavirus. Based on its genetic proximity, SARS-CoV-2 is likely to have originated from bat-derived CoV and transmitted to humans via an unknown intermediate mammalian host, probably Malayan pangolin. Further, spike protein S1/S2 cleavage site of SARS-CoV-2 has acquired polybasic furin cleavage site which is absent in bat and pangolin suggesting natural selection either in an animal host before zoonotic transfer or in humans following zoonotic transfer. In the current review, we recapitulate a preliminary opinion about the disease, origin and life cycle of SARS-CoV-2, roles of virus proteins in pathogenesis, commonalities, and differences between different corona viruses. Moreover, the crystal structures of SARS-CoV-2 proteins with unique characteristics differentiating it from other CoVs are discussed. Our review also provides comprehensive information on the molecular aspects of SARS-CoV-2 including secondary structures in the genome and protein–protein interactions which can be useful to understand the aggressive spread of the SARS-CoV-2. The mutations and the haplotypes reported in the SARS-CoV-2 genome are summarized to understand the virus evolution.     

Recent insights into SARS-CoV-2 omicron variant

The SARS-CoV-2 omicron variant (B.1.1.529) was first identified in Botswana and South Africa, and its emergence has been associated with a steep increase in the number of SARS-CoV-2 infections. The omicron variant has subsequently spread very rapidly across the world, resulting in the World Health Organization classification as a variant of concern on 26 November 2021. Since its emergence, great efforts have been made by research groups around the world that have rapidly responded to fill our gaps in knowledge for this novel variant. A growing body of data has demonstrated that the omicron variant shows high transmissibility, robust binding to human angiotensin-converting enzyme 2 receptor, attenuated viral replication, and causes less severe disease in COVID-19 patients. Further, the variant has high environmental stability, high resistance against most therapeutic antibodies, and partial escape neutralisation by antibodies from convalescent patients or vaccinated individuals. With the pandemic ongoing, there is a need for the distillation of literature from primary research into an accessible format for the community. In this review, we summarise the key discoveries related to the SARS-CoV-2 omicron variant, highlighting the gaps in knowledge that guide the field's ongoing and future work.     

COVID-19 vaccinations and rates of infections,hospitalizations, ICU admissions,and deaths in Europe during SARS-CoV-2 Omicron wave in the first quarter of 2022

The vaccination campaigns brought hope to minimizing the coronavirus disease 2019 (COVID-19) burden. However, the emergence of novel, highly transmissible Omicron lineage of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the waning of neutralizing antibodies a few months after vaccination has brought concerns over the vaccine efficacy. The present work analyzed the relationships between COVID-19 vaccine coverage (completion of primary course and booster dose intake) in the European Economic Area and rates of infection, hospitalizations, admissions to intensive care units (ICU), and deaths during the Omicron wave in the first quarter of 2022 (January–April). As demonstrated, infection rates were not correlated to vaccine coverage in any considered month. For January and February, the rates of hospitalizations, intensive care unit (ICU) admissions, and death due to COVID-19 were strongly negatively correlated (r =− 0.54 to −0.82) with the percentage of individuals who completed initial vaccination protocol and the percentage of those who received a booster dose. However, in March and April, the percentage of the population with primary vaccination course correlated negatively only with ICU admissions (r = −0.77 and −0.46, respectively). The uptake of boosters in March still remained in significant negative correlation with hospitalizations (r = −0.45), ICU admissions (r = −0.70) and deaths due to COVID-19 (r = −0.37), although in April these relationships were no longer observed. The percentage of individuals with confirmed SARS-CoV-2 infection did not correlate with the pandemic indices for any considered month. The study indicates that COVID-19 vaccination, including booster administration, was beneficial in decreasing the overwhelming of healthcare systems during the Omicron wave, but novel vaccine strategies may be required in the long term to enhance the effectiveness and durability of vaccine-induced protection during future waves of SARS-CoV-2 infections.     

Estimating the effects of asymptomatic and imported patients on COVID-19 epidemic using mathematical modeling

The epidemic of Coronavirus Disease 2019 has been a serious threat to public health worldwide. Data from 23 January to 31 March at Jiangsu and Anhui provinces in China were collected. We developed an adjusted model with two novel features: the asymptomatic population and threshold behavior in recovery. Unbiased parameter estimation identified faithful model fitting. Our model predicted that the epidemic for asymptomatic patients (ASP) was similar in both provinces. The latent periods and outbreak sizes are extremely sensitive to strongly controlled interventions such as isolation and quarantine for both asymptomatic and imported cases. We predicted that ASP serve as a more severe factor with faster outbreaks and larger outbreak sizes compared with imported patients. Therefore, we argued that the currently strict interventions should be continuously implemented, and unraveling the asymptomatic pool is critically important before preventive strategy such as vaccines.     

A cross-sectional study of SARS-CoV-2 seroprevalence among asymptomatic healthcare workers in a tertiary healthcare centre: Assessing the impact of PPE guidelines

PurposeThe present study estimates the seroprevalence of SARS-COV-2 among asymptomatic HCWs and assess the impact of various categories of PPE.MethodsA cross-sectional study of asymptomatic HCW using different levels of PPE as per their risk profile was undertaken between 18th and 24th September 2020. Participant demographics and other relevant details including the levels of PPE used were recorded using a customized questionnaire. IgG antibodies against SARS-COV-2 were detected by chemiluminescence method & used as a surrogate marker for incomplete protection.ResultsOut of 1033 HCWs tested, overall SARS-COV-2 sero-prevalence was 25.8%. Univariate and multivariate analysis both demonstrated that ancillary workers including security staff (OR 5.589, P ​< ​0.001) and sanitary workers (OR 3.946, P ​< ​0.001) were at significantly higher risk of seropositivity irrespective of the PPE used as per guidelines, whereas doctors were at significantly lower risk of seropositivity (OR 0.307, P ​= ​0.005). Staff working in office areas was associated with reduced risk of seropositivity (OR 0.21, P ​= ​0.045).ConclusionsWe document high seroprevalence of SARS-COV-2 antibodies in asymptomatic HCWs. Doctors who are at the highest risk had the lowest seropositivity and seroprevalence among office staff having a risk level comparable to the general community was lower than that reported in general population, supporting the efficacy of PPE practices as per guidelines in these groups. In contrast, much higher rates of seropositivity were seen among ancillary workers despite the availability of adequate PPE. Active screening, proper PPE use as per guidelines, and regular infection control trainings including Covid appropriate behaviour are therefore essential to contain COVID-19 spread among HCW & preventing them to transfer infection to the patients.     

Understanding evolution of SARS-CoV-2: A perspective from analysis of genetic diversity of RdRp gene

Coronavirus disease 2019 emerged as the first example of “Disease X”, a hypothetical disease of humans caused by an unknown infectious agent that was named as novel coronavirus and subsequently designated as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The origin of the outbreak at the animal market in Wuhan, China implies it as a case of zoonotic spillover. The study was designed to understand evolution of Betacoronaviruses and in particular diversification of SARS-CoV-2 using RNA dependent RNA polymerase (RdRp) gene, a stable genetic marker. Phylogenetic and population stratification analyses were carried out using maximum likelihood and Bayesian methods, respectively. Molecular phylogeny using RdRp showed that SARS-CoV-2 isolates cluster together. Bat-CoV isolate RaTG13 and Pangolin-CoVs are observed to branch off prior to SARS-CoV-2 cluster. While SARS-CoV form a single cluster, Bat-CoVs form multiple clusters. Population-based analyses revealed that both SARS-CoV-2 and SARS-CoV form separate clusters with no admixture. Bat-CoVs were found to have single and mixed ancestry and clustered as four sub-populations. Population-based analyses of Betacoronaviruses using RdRp revealed that SARS-CoV-2 is a homogeneous population. SARS-CoV-2 appears to have evolved from Bat-CoV isolate RaTG13, which diversified from a common ancestor from which Pangolin-CoVs have also evolved. The admixed Bat-CoV sub-populations indicate that bats serve as reservoirs harboring virus ensembles that are responsible for zoonotic spillovers such as SARS-CoV and SARS-CoV-2. The extent of admixed isolates of Bat-CoVs observed in population diversification studies underline the need for periodic surveillance of bats and other animal reservoirs for potential spillovers as a measure towards preparedness for emergence of zoonosis.     

A new SARS-CoV-2 variant with high lethality poorly detected by RT-PCR on nasopharyngeal samples: an observational study

ObjectivesIn early January 2021 an outbreak of nosocomial cases of coronavirus disease 2019 (COVID-19) emerged in Western France; RT-PCR tests were repeatedly negative on nasopharyngeal samples but positive on lower respiratory tract samples. Whole-genome sequencing (WGS) revealed a new variant, currently defining a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineage B.1.616. In March, the WHO classified this as a ‘variant under investigation’ (VUI). We analysed the characteristics and outcomes of COVID-19 cases related to this new variant.MethodsClinical, virological, and radiological data were retrospectively collected from medical charts in the two hospitals involved. We enrolled those inpatients with: (a) positive SARS-CoV-2 RT-PCR on a respiratory sample, (b) seroconversion with anti-SARS-CoV-2 IgG/IgM, or (c) suggestive symptoms and typical features of COVID-19 on a chest CT scan. Cases were categorized as B.1.616, a variant of concern (VOC), or unknown.ResultsFrom 1st January to 24th March 2021, 114 patients fulfilled the inclusion criteria: B.1.616 (n = 39), VOC (n = 32), and unknown (n = 43). B.1.616-related cases were older than VOC-related cases (81 years, interquartile range (IQR) 73–88 versus 73 years, IQR 67–82, p < 0.05) and their first RT-PCR tests were rarely positive (6/39, 15% versus 31/32, 97%, p < 0.05). The B.1.616 variant was independently associated with severe disease (multivariable Cox model HR 4.0, 95%CI 1.5–10.9) and increased lethality (28-day mortality 18/39 (46%) for B.1.616 versus 5/32 (16%) for VOC, p = 0.006).ConclusionWe report a nosocomial outbreak of COVID-19 cases related to a new variant, B.1.616, which is poorly detected by RT-PCR on nasopharyngeal samples and is associated with high lethality.     

The acceptance of SARS-CoV-2 rapid antigen self-testing: A cross-sectional study in China

Compared with the nucleic acid amplification test (NATT), the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapid antigen self-testing (RAST) has advantages in speed and convenience. However, little is known about people's acceptance and influencing factors for SARS-CoV-2 RAST. A cross-sectional study was conducted from April 21 to 30, 2022 in China. The χ² test and multivariate logistic regressions were used to identify the influencing factors. The structural equation model was used to test the extended protective motivation theory (PMT) model hypotheses. Among the total of 5107 participants, 62.5% were willing to accept the SARS-CoV-2 RAST. There were significant differences in acceptance among different residences (p < 0.001), educational level (p < 0.001), occupation (p < 0.001), monthly income (p < 0.001), travel frequency (p < 0.05), and feelings about NATT (p < 0.001). Response efficacy (β = 0.05; p = 0.025) and self-efficacy (β = 0.84; p < 0.001) had a positive effect, while response cost showed a negative effect (β = −0.07; p < 0.001). The public's major concerns about SARS-CoV-2 RAST are its reliability, testing method, price, and authority. Overall, a moderate intention to use SARS-CoV-2 RAST was found among the Chinese population. The extended PMT can be used for the prediction of intention to accept the RAST. We need to take measures to increase people's acceptance of SARS-CoV-2 RAST.     

Magnetic resonance imaging study of cross-sectional area of the cervical extensor musculature in an asymptomatic cohort

Magnetic Resonance Imaging (MRI) can be regarded as the gold standard for muscle imaging; however there is little knowledge about in vivo morphometric features of neck extensor muscles in healthy subjects and how muscle size alters across vertebral segments. It is not known how body size and activity levels may influence neck muscle cross-sectional area (CSA) or if the muscles differ from left and right. The purpose of this study was to establish relative CSA (rCSA) data for the cervical extensor musculature with a reliable MRI measure in asymptomatic females within a defined age range and to determine if side-side and vertebral level differences exist. MRI of the cervical spine was performed on 42 asymptomatic female subjects within the age range of 18-45. The rCSA values for the cervical extensor muscles were measured from axial T1-weighted images. We found significant side-side rCSA differences for the rectus capitis posterior minor, major (P < 0.001), multifidus (P = 0.002), and the semispinalis cervicis/capitis (P = 0.001, P < 0.001). There were significant vertebral level differences in rCSA of the semispinalis cervicis/capitis, multifidus, splenius capitis, and upper trapezius (P < 0.001). Activity levels were shown to impact on the size of semispinalis cervicis (P = 0.027), semispinalis capitis (P = 0.003), and the splenius capitis (P = 0.004). In conclusion, measuring differences in neck extensor muscle rCSA with MRI in an asymptomatic population provides the basis for future study investigating relationships between muscular atrophy and symptoms in patients suffering from persistent neck pain. Clin.     

Prevalence and risk factor assessment of Tropheryma whipplei in a rural community in Gabon: a community-based cross-sectional study

Tropheryma whipplei is the causative agent of Whipple's disease and has been detected in stools of asymptomatic carriers. Colonization has been associated with precarious hygienic conditions. There is a lack of knowledge about the epidemiology and transmission characteristics on a population level, so the aim of this study was to determine the overall and age-specific prevalence of T. whipplei and to identify risk factors for colonization. This molecular epidemiological survey was designed as a cross-sectional study in a rural community in Central African Gabon and inhabitants of the entire community were invited to participate. Overall prevalence assessed by real-time PCR and sequencing was 19.6% (95% CI 16–23.2%, n = 91) in 465 stool samples provided by the study participants. Younger age groups showed a significantly higher prevalence of T. whipplei colonization ranging from 40.0% (95% CI 27.8–52.2) among the 0–4 year olds to 36.4% (95% CI 26.1–46.6) among children aged 5–10 years. Prevalence decreased in older age groups (p <0.001) from 12.6% (95% CI 5.8–19.4%; 11–20 years) to 9.7% (95% CI 5.7–13.6) among those older than 20. Risk factor analysis revealed young age, male sex, and number of people sharing a bed as factors associated with an increased risk for T. whipplei carriage. These results demonstrate that T. whipplei carriage is highly prevalent in this part of Africa. The high prevalence in early life and the analysis of risk factors suggest that transmission may peak during childhood facilitated through close person-to-person contacts.     

A compendium answering 150 questions on COVID-19 and SARS-CoV-2

In December 2019, China reported the first cases of the coronavirus disease 2019 (COVID-19). This disease, caused by the severe acute respiratory syndrome–related coronavirus 2 (SARS-CoV-2), has developed into a pandemic. To date, it has resulted in ~9 million confirmed cases and caused almost 500 000 related deaths worldwide. Unequivocally, the COVID-19 pandemic is the gravest health and socioeconomic crisis of our time. In this context, numerous questions have emerged in demand of basic scientific information and evidence-based medical advice on SARS-CoV-2 and COVID-19. Although the majority of the patients show a very mild, self-limiting viral respiratory disease, many clinical manifestations in severe patients are unique to COVID-19, such as severe lymphopenia and eosinopenia, extensive pneumonia, a “cytokine storm” leading to acute respiratory distress syndrome, endothelitis, thromboembolic complications, and multiorgan failure. The epidemiologic features of COVID-19 are distinctive and have changed throughout the pandemic. Vaccine and drug development studies and clinical trials are rapidly growing at an unprecedented speed. However, basic and clinical research on COVID-19–related topics should be based on more coordinated high-quality studies. This paper answers pressing questions, formulated by young clinicians and scientists, on SARS-CoV-2, COVID-19, and allergy, focusing on the following topics: virology, immunology, diagnosis, management of patients with allergic disease and asthma, treatment, clinical trials, drug discovery, vaccine development, and epidemiology. A total of 150 questions were answered by experts in the field providing a comprehensive and practical overview of COVID-19 and allergic disease.