A Large Cohort Study of SARS-CoV-2 Detection in Saliva: A Non-Invasive Alternative Diagnostic Test for Patients with Bleeding Disorders

Diagnosis of SARS-CoV-2 infections is mostly based on the nasopharyngeal swabs (NPS). However, this collection is invasive and uncomfortable, especially for children and patients with coagulopathies, whose NPS collection often causes bleeding. Thus, the aim of this study was to evaluate the usefulness and accuracy of saliva for the diagnosis of COVID-19 in patients presenting bleeding disorders. Samples of NPS, oropharyngeal swabs (OPS), and saliva were collected simultaneously from 1159 hospitalized patients with hematological diseases and from 524 healthcare workers, both symptomatic and asymptomatic for SARS-CoV-2. All samples were evaluated for SARS-CoV-2 by qRT-PCR. SARS-CoV-2 was detected in NPS, OPS and saliva from 16.9%, 14.4% and 15.6% individuals, respectively. Tests in saliva showed sensitivity, specificity, and overall agreement of 73.3%, 96.9% and 92.7% (=0.74), respectively. Salivary tests had good accuracy (AUC = 0.7) for discriminating negative and positive qRT-PCR for SARS-CoV-2. Higher sensitivity was observed in symptomatic than in non-symptomatic patients, as well as in healthy subjects than in patients with hematological disease, in both OPS and saliva. The mean viral load in NPS was significantly higher than in OPS and in saliva samples (p < 0.001). Saliva is a good diagnostic tool to detect SARS-CoV-2, especially among patients symptomatic for COVID-19, and is a valuable specimen for mass screening of hospitalized patients with hematological diseases, especially for those that with bleeding disorders.     

Performance of Self-Collected Saliva Testing Compared with Nasopharyngeal Swab Testing for the Detection of SARS-CoV-2

The aim of this study was to determine whether self-collected pure saliva (SCPS) is comparable to nasopharyngeal (NP) swabs in the quantitative detection of SARS-CoV-2 by RT-PCR in asymptomatic, mild patients with confirmed COVID-19. Thirty-one patients aged from 18 to 85 years were included between 9 June and 11 December 2020. A SCPS sample and a NP sample were taken for each patient. Quantitative PCR was performed to detect SARS-CoV-2 viral load. Results of SCPS vs. NP samples testing were compared. Statistical analyses were performed. Viral load was significantly correlated (r = 0.72). The concordance probability was estimated at 73.3%. In symptomatic adults, SCPS performance was similar to that of NP swabs (Percent Agreement = 74.1%; p = 0.11). Thus, the salivary test based on pure oral saliva samples easily obtained by noninvasive techniques has a fair agreement with the nasopharyngeal one in asymptomatic, mild patients with a confirmed diagnosis of COVID-19.     

SARS-CoV-2 Transmission in Belgian French-Speaking Primary Schools: An Epidemiological Pilot Study

Schools have been a point of attention during the pandemic, and their closure one of the mitigating measures taken. A better understanding of the dynamics of the transmission of SARS-CoV-2 in elementary education is essential to advise decisionmakers. We conducted an uncontrolled non-interventional prospective study in Belgian French-speaking schools to describe the role of attending asymptomatic children and school staff in the spread of COVID-19 and to estimate the transmission to others. Each participant from selected schools was tested for SARS-CoV-2 using a polymerase chain reaction (PCR) analysis on saliva sample, on a weekly basis, during six consecutive visits. In accordance with recommendations in force at the time, symptomatic individuals were excluded from school, but per the study protocol, being that participants were blinded to PCR results, asymptomatic participants were maintained at school. Among 11 selected schools, 932 pupils and 242 school staff were included between January and May 2021. Overall, 6449 saliva samples were collected, of which 44 came back positive. Most positive samples came from isolated cases. We observed that asymptomatic positive children remaining at school did not lead to increasing numbers of cases or clusters. However, we conducted our study during a period of low prevalence in Belgium. It would be interesting to conduct the same analysis during a high prevalence period.     

Evaluation of an Automated High-Throughput Liquid-Based RNA Extraction Platform on Pooled Nasopharyngeal or Saliva Specimens for SARS-CoV-2 RT-PCR

SARS-CoV-2 RT-PCR with pooled specimens has been implemented during the COVID-19 pandemic as a cost- and manpower-saving strategy for large-scale testing. However, there is a paucity of data on the efficiency of different nucleic acid extraction platforms on pooled specimens. This study compared a novel automated high-throughput liquid-based RNA extraction (LRE) platform (PHASIFY™) with a widely used magnetic bead-based total nucleic acid extraction (MBTE) platform (NucliSENS^® easyMAG^®). A total of 60 pools of nasopharyngeal swab and 60 pools of posterior oropharyngeal saliva specimens, each consisting of 1 SARS-CoV-2 positive and 9 SARS-CoV-2 negative specimens, were included for the comparison. Real-time RT-PCR targeting the SARS-CoV-2 RdRp/Hel gene was performed, and GAPDH RT-PCR was used to detect RT-PCR inhibitors. No significant differences were observed in the Ct values and overall RT-PCR positive rates between LRE and MBTE platforms (92.5% (111/120] vs. 90% (108/120]), but there was a slightly higher positive rate for LRE (88.3% (53/60]) than MBTE (81.7% (49/60]) among pooled saliva. The automated LRE method is comparable to a standard MBTE method for the detection of SAR-CoV-2 in pooled specimens, providing a suitable alternative automated extraction platform. Furthermore, LRE may be better suited for pooled saliva specimens due to more efficient removal of RT-PCR inhibitors.     

A Novel Method for the Detection of SARS-CoV-2 Based on Graphene-Impedimetric Immunosensor

Due to the SARS-CoV-2 pandemic, there has been an increase in the search for affordable healthcare devices for mass testing and rapid diagnosis. In this context, this work described a new methodology for SARS-CoV-2 detection based on an impedimetric immunosensor developed using the advantageous immobilization of antibodies in the reduced graphene oxide (rGO). The rGO was obtained by chemical synthesis from the commercial graphene oxide (GO), and the materials were morphologically, electrochemically and visually characterized. The cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques were used to evaluate the fabrication steps of the immunosensor. The electrochemical immunoassay was considered for SARS-CoV-2 spike protein RBD detection using a impedimetric immunosensor and redox couple ([(Fe(CN)₆)]^3−/4−) as a probe. The immunosensor was effectively developed and applied in the detection of SARS-CoV-2 spike protein RBD in saliva samples.     

SARS-CoV-2 and Arthropods: A Review

The emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that led to the unprecedented COVID-19 pandemic exemplifies how a lack of understanding and preparedness for emerging viruses can result in consequences on a global scale. Statements that SARS-CoV-2 could not be transmitted by arthropod vectors were made without experimental support. Here we review laboratory-based research, field studies, and environmental studies to evaluate the potential for the virus to be transmitted either biologically or mechanically by arthropods. Based on these data, we conclude that transmission by arthropods is highly unlikely to play a significant epidemiological role in the transmission of SARS-CoV-2.     

COVID-19: A year later: Practical guidance for clinical laboratories for SARS-CoV-2 serology testing

The landscape of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostic testing is rapidly evolving. While serology testing has limited diagnostic capacity for acute infection, its role in providing population-based information on positivity rates and informing evidence-based decision making for public health recommendations is increasing. With the global availability of vaccines, there is increasing pressure on clinical laboratories to provide antibody screening and result interpretation for vaccinated and non-vaccinated individuals. Here we present the most up-to-date data on SARS-CoV-2 antibody timelines, including the longevity of antibodies, and the production and detection of neutralizing antibodies. Additionally, we provide practical guidance for clinical microbiology laboratories to both verify commercial serology assays and choose appropriate testing algorithms for their local populations.     

A Novel Immunofluorescence Assay for the Rapid Serological Detection of SARS-CoV-2 Infection

As of April 2021, the COVID-19 pandemic has swept through 213 countries and infected more than 132 million individuals globally, posing an unprecedented threat to human health. There are currently no specific antiviral treatments for COVID-19 and vaccination programmes, whilst promising, remain in their infancy. A key to restricting the pandemic is the ability to minimize human–human transmission and to predict the infection status of the population in the face of emerging SARS-CoV-2 variants. Success in this area is dependent on the rapid detection of COVID-19 positive individuals with current/previous SARS-CoV-2 infection status. In this regard, the ability to detect antibodies directed against the SARS-CoV-Spike protein in patient sera represents a powerful biomarker for confirmation of infection. Here, we report the design of a proof-of-concept cell–based fluorescent serology assay (termed C19-S-I-IFA) to detect SARS-CoV-2 infection. The assay is based on the capture of IgG antibodies in the serum of COVID-19-positive patients using cells exogenously expressing SARS-CoV-2-Spike and their subsequent fluorescent detection. We validate the assay in 30 blood samples collected in Oxford, UK, in 2020 during the height of the pandemic. Importantly, the assay can be modified to express emerging Spike-variants to permit assessments of the cross-reactivity of patient sera to emerging SARS-CoV-2 strains.     

Detection of Torquetenovirus and Redondovirus DNA in Saliva Samples from SARS-CoV-2-Positive and -Negative Subjects

Objectives: Torquetenovirus (TTV) and Redondovirus (ReDoV) are the most prevalent viruses found in the human respiratory virome in viral metagenomics studies. A large-scale epidemiological study was performed to investigate their prevalence and loads in saliva samples according to SARS-CoV-2 status. Methods: Saliva samples from 448 individuals (73% SARS-CoV-2 negative and 27% SARS-CoV-2 positive) aged 23–88 years were tested. SARS-CoV-2 and TTV were determined in saliva by specific qualitative and quantitative real-time PCRs, respectively. A sub-cohort of 377 subjects was additionally tested for the presence and load of ReDoV in saliva, and a different sub-cohort of 120 subjects for which paired saliva and plasma samples were available was tested for TTV and ReDoV viremia at the same timepoints as saliva. Results: TTV in saliva was 72% prevalent in the entire cohort, at a mean DNA load of 4.6 log copies/mL, with no difference regardless of SARS-CoV-2 status. ReDoV was found in saliva from 61% of the entire cohort and was more prevalent in the SARS-CoV-2-negative subgroup (65% vs. 52%, respectively). In saliva, the total mean load of ReDoV was very similar to the one of TTV, with a value of 4.4 log copies/mL. The mean viral loads in subjects infected with a single virus, namely, those infected with TTV or ReDoV alone, was lower than in dually infected samples, and Tukey’s multiple-comparison test showed that ReDoV single-infected samples resulted in the only true outlier (p = 0.004). Differently from TTV, ReDoV was not detected in any blood samples. Conclusions: This study establishes the prevalence and mean value of TTV and ReDoV in saliva samples and demonstrates the existence of differences between these two components of the human virome.     

Fast,Reliable, and Simple Point-of-Care-like Adaptation of RT-qPCR for the Detection of SARS-CoV-2 for Use in Hospital Emergency Departments

During COVID-19 pandemics, the availability of testing has often been a limiting factor during patient admissions into the hospital. To circumvent this problem, we adapted an existing diagnostic assay, Seegene Allplex SARS-CoV-2, into a point-of-care-style direct qPCR (POC dqPCR) assay and implemented it in the Emergency Department of Clinical Hospital Center Rijeka, Croatia. In a 4-month analysis, we tested over 10,000 patients and demonstrated that POC-dqPCR is robust and reliable and can be successfully implemented in emergency departments and similar near-patient settings and can be performed by medical personnel with little prior experience in qPCR.     

Modeling Within-Host Dynamics of SARS-CoV-2 Infection: A Case Study in Ferrets

The pre-clinical development of antiviral agents involves experimental trials in animals and ferrets as an animal model for the study of SARS-CoV-2. Here, we used mathematical models and experimental data to characterize the within-host infection dynamics of SARS-CoV-2 in ferrets. We also performed a global sensitivity analysis of model parameters impacting the characteristics of the viral infection. We provide estimates of the viral dynamic parameters in ferrets, such as the infection rate, the virus production rate, the infectious virus proportion, the infected cell death rate, the virus clearance rate, as well as other related characteristics, including the basic reproduction number, pre-peak infectious viral growth rate, post-peak infectious viral decay rate, pre-peak infectious viral doubling time, post-peak infectious virus half-life, and the target cell loss in the respiratory tract. These parameters and indices are not significantly different between animals infected with viral strains isolated from the environment and isolated from human hosts, indicating a potential for transmission from fomites. While the infection period in ferrets is relatively short, the similarity observed between our results and previous results in humans supports that ferrets can be an appropriate animal model for SARS-CoV-2 dynamics-related studies, and our estimates provide helpful information for such studies.