Detection of SARS-CoV-2 RNA in Urine by RT-LAMP: A Very Rare Finding

Detection of SARS-CoV-2 is routinely performed in naso/oropharyngeal swabs samples from patients via RT-qPCR. The RT-LAMP technology has also been used for viral RNA detection in respiratory specimens with both high sensitivity and specificity. Recently, we developed a novel RT-LAMP test for SARS-CoV-2 RNA detection in nasopharyngeal swab specimens (named, N15-RT-LAMP) that can be performed as a single-tube colorimetric method, in a real-time platform, and as dry-LAMP. To date, there has been very little success in detecting SARS-CoV-2 RNA in urine by RT-qPCR, and the information regarding urine viral excretion is still scarce and not comprehensive. Here, we tested our N15-RT-LAMP on the urine of 300 patients admitted to the Hospital of Salamanca, Spain with clinical suspicion of COVID-19, who had a nasopharyngeal swab RT-qPCR-positive (n = 100), negative (n = 100), and positive with disease recovery (n = 100) result. The positive group was also tested by RT-qPCR for comparison to N15-RT-LAMP. Only a 4% positivity rate was found in the positive group via colorimetric N15-RT-LAMP and 2% via RT-qPCR. Our results are consistent with those obtained in other studies that the presence of SARS-CoV-2 RNA in urine is a very rare finding. The absence of SARS-CoV-2 RNA in urine in the recovered patients might suggest that the urinary route is very rarely used for viral particle clearance.

Analytical performance of the point-of-care BIOSYNEX COVID-19 Ag BSS for the detection of SARS‐CoV‐2 nucleocapsid protein in nasopharyngeal swabs: a prospective field evaluation during the COVID-19 third wave in France

Background The accuracy and reliability of rapid diagnostic tests are critical for monitoring and diagnosing SARS-CoV-2 infection in the general population. This study aimed to evaluate the analytical performance of the BIOSYNEX COVID-19 Ag BSS (Biosynex Swiss SA, Fribourg, Switzerland) antigen rapid diagnostic test (BIOSYNEX Ag-RDT), which targets the SARS-CoV-2 N-nucleocapsid protein for the diagnosis of COVID-19. The Ag-RDT was compared with a real-time RT-PCR (rtRT-PCR) as gold standard for performance measurement. Methods Two nasopharyngeal flocked swabs were prospectively collected simultaneously in March and April 2021 from 967 individuals aged ≥ 18 years tested for SARS-CoV-2 in two private laboratories, Paris, France. Results Overall, the Ag-RDT demonstrated high sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 81.8%, 99.6%, 96.6%, and 97.5%, respectively. The agreement (97.0%), reliability assessed using Cohen’s κ-coefficient (0.87), and accuracy evaluated using Youden index (J) (81.6%) in detecting SARS-CoV-2 were high. The analytical performance of the Ag-RDT remained high when there was significant viral shedding (i.e., N gene Ct values ≤ 33 on reference RT-PCR). The sensitivity was only 55.2% in case of low or very low viral excretion (Ct > 33). Conclusions The BIOSYNEX Ag-RDT is a promising, potentially simple diagnostic tool, especially in symptomatic COVID-19 patients with substantial viral excretion in the nasopharynx.

Analytical Performance of the Point-of-care BIOSYNEX COVID-19 Ag BSS for the Detection of SARS‐CoV‐2 Nucleocapsid Protein in Nasopharyngeal Swabs: A Prospective Field Evaluation During the COVID-19 Third Wave in France

Background: The accuracy and reliability of rapid diagnostic tests are critical for monitoring and diagnosing SARS-CoV-2 infection in the general population. This study aimed to evaluate the analytical performance of the BIOSYNEX COVID-19 Ag BSS (Biosynex Swiss SA, Fribourg, Switzerland) antigen rapid diagnostic test (Ag-RDT), which targets the SARS-CoV-2 N-nucleocapsid protein for the diagnosis of COVID-19. The Ag-RDT was compared with a real-time RT-PCR (rtRT-PCR) gold standard for performance measurement.Methods: Two nasopharyngeal flocked swabs were prospectively collected simultaneously in March and April 2021 from 967 individuals aged ≥18 years tested for SARS-CoV-2 in two private laboratories, Paris, France.Results: Overall, the Ag-RDT demonstrated high sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 81.8%, 99.6%, 96.6%, and 97.5%, respectively, as well as high or near-perfect agreement (97.0%), reliability was assessed using Cohen’s κ-coefficient (0.87), and accuracy was evaluated using Youden index (J) (81.6%) in detecting SARS-CoV-2. The analytical performance of the Ag-RDT remained high when there was significant viral shedding (i.e., N gene Ct values ≤ 33 on reference RT-PCR). The sensitivity was only 55.2% in case of low or very low viral excretion (Ct> 33).Conclusions: The BIOSYNEX COVID-19 Ag BSS Ag-RDT is a promising, potentially simple diagnostic tool, especially in symptomatic COVID -19 or proven infectiousness.

Emergence of E484K Mutation Following Bamlanivimab Monotherapy among High-Risk Patients Infected with the Alpha Variant of SARS-CoV-2

An Emergency Use Authorization was issued in the United States and in Europe for a monoclonal antibody monotherapy to prevent severe COVID-19 in high-risk patients. This study aimed to assess the risk of emergence of mutations following treatment with a single monoclonal antibody. Bamlanivimab was administered at a single dose of 700 mg in a one-hour IV injection in a referral center for the management of COVID-19 in France. Patients were closely monitored clinically and virologically with nasopharyngeal RT-PCR and viral whole genome sequencing. Six patients were treated for a nosocomial SARS-CoV-2 infection, all males, with a median age of 65 years and multiple comorbidities. All patients were infected with a B.1.1.7 variant, which was the most frequent variant in France at the time, and no patients had E484 mutations at baseline. Bamlanivimab was infused in the six patients within 4 days of the COVID-19 diagnosis. Four patients had a favorable outcome, one died of complications unrelated to COVID-19 or bamlanivimab, and one kidney transplant patient treated with belatacept died from severe COVID-19 more than 40 days after bamlanivimab administration. Virologically, four patients cleared nasopharyngeal viral shedding within one month after infusion, while two presented prolonged viral excretion for more than 40 days. The emergence of E484K mutants was observed in five out of six patients, and the last patient presented a Q496R mutation potentially associated with resistance. CONCLUSIONS: These results show a high risk of emergence of resistance mutants in COVID-19 patients treated with monoclonal antibody monotherapy.

Immunogenicity of an Inactivated Novel Duck Parvovirus Vaccine for Short Beak and Dwarf Syndrome in Cherry Valley Ducks

Duck short beak and dwarf syndrome (SBDS) is a viral infectious disease caused by novel duck parvovirus (NDPV). It has brought serious economic losses to the Chinese duck industry in recent years. Currently, there exists no effective vaccine against this disease. In this study, we developed an inactivated virus vaccine based on NDPV-DS15 for SBDS. Immune efficacy was evaluated in 112 ducks, which were randomly divided into vaccination, challenge control, vaccination-challenge, and blank control groups (n = 28 each). Clinical characteristics, antibodies, viral excretion, viremia, and pathological changes were monitored and analyzed. No morbidity or death was observed in the immunized ducks, which showed normal weight and good mental state. High levels of serum antibodies (OD450 nm: ~0.63) were detected in ducks immunized with inactivated vaccine at 7 days post-vaccination (dpv), and the amount of virus neutralizing antibodies increased from 1:23 to 1:28.5 from 7 dpv to 42 dpv. The anal swab, serum, and tissue viral load tests showed that vaccination could significantly inhibit the replication of NDPV in immunized ducks. Moreover, NDPV could not be isolated from the spleens of immunized or vaccination-challenged ducks. Our results show that the developed inactivated NDPV vaccine, administered in an oil emulsion adjuvant, possesses good immunogenicity and represents a potentially powerful tool for SBDS prevention and control.

Analytical Performances of the Point-of-Care BIOSYNEX COVID-19 Ag BSS for the Detection of SARS‐CoV‐2 Nucleocapsid Protein in Nasopharyngeal Swabs: A Prospective Field Evaluation During the COVID-19 Third Wave in France

Background: Evaluating the accuracy and reliability of rapid diagnostic testing kits is crucial for surveillance and diagnosis of SARS-CoV-2 infections in general population. The aim of the study was to assess the analytical performances of the antigen-rapid diagnosis test (Ag-RDT) BIOSYNEX COVID-19 Ag BSS (Biosynex Swiss SA, Freiburg, Switzerland), targeting the SARS-CoV-2 N nucleocapsid protein, for the diagnosis of COVID-19, by reference to real-time RT-PCR (rtRT-PCR).Methods: A total 967 adults living in Paris region were prospectively included during the third wave of the COVID-19 epidemic in France. Paired nasopharyngeal flocked swabs were collected at the same timepoint from persons aged ≥18 years receiving testing for SARS-CoV-2, at two private laboratories.Results: Overall, the Ag-RDT showed high sensitivity, specificity, PPV and NPV of 81.8%, 99.6%, 96.6% and 97.5%, respectively, as well as high or almost perfect agreement (97.0%), reliability assessed by Cohen’s κ coefficient (0.87), and accuracy assessed by Youden’s J index (81.6%) to detect SARS-CoV-2. The analytical performances of the Ag-RDT remained high in the event of significant viral excretion (i.e., N gene Ct values ≤ 33 by reference rtRT-PCR), while the sensitivity of the Ag-RDT dropped to 55.2% with low or very low viral shedding (Ct> 33).Conclusions: The Ag-RDT BIOSYNEX COVID-19 Ag BSS showed high specificity and sufficient sensitivity for the detection of SARS-CoV-2. This test is a promising potential easy diagnostic tool, especially in situations of symptomatic COVID-19 and/or proven contagiousness.

Higher Relative Viral Load Excretion Determined by Normalised Threshold Crossing Value in Acute Cases infected with the B.1.1.7 Lineage VOC 202012/01 (Using S gene target failure as a Proxy) When Compared to other Circulating Lineages in Wales

Since the emergence of SARS-CoV-2, global monitoring of the virus using whole genome sequencing has identified mutations occurring across the viral genome. Whilst the majority have little impact on the virus, they are used effectively to monitor the movement of the virus globally and to inform locally on transmission chains. In late 2020, a variant of SARS-CoV-2 (B.1.1.7 - VOC 202012/01) was identified in the UK with a distinct constellation of mutations, including in the spike gene that increased transmissibility. A deletion in spike also affected one of the screening qPCR tests being used in the UK outside of Wales, causing a failure to detect the target. This quickly became a surrogate marker for the variant to allow rapid monitoring of the virus as it seeded into new regions of the UK. A screening study using this assay as a proxy marker, was undertaken to understand the prevalence of the variant in Wales. Secondary analysis of a screening qPCR targeting N and ORF and also included an endogenous control, was also performed to understand viral load excretion in those infected with the variant compared to other circulating lineages. Using a combination of analytical methods based on the Ct values of two gene targets normalised against the endogenous control, there was a difference in the excreted viral load. Those with the variant excreting more virus than those not infected with the variant. Supporting not only increased infectivity but offering a plausible reason why increased transmission was associated with this particular variant. Whilst there are limitations in this study, the method using Ct as a proxy for viral load can be used at the population level to determine differences in viral excretion kinetics associated with different variants.

A laboratory-based study to explore the use of honey-impregnated cards to detect chikungunya virus in mosquito saliva

Mosquito control is implemented when arboviruses are detected in patients or in field-collected mosquitoes. However, mass screening of mosquitoes is usually laborious and expensive, requiring specialized expertise and equipment. Detection of virus in mosquito saliva using honey-impregnated filter papers seems to be a promising method as it is non-destructive and allows monitoring the viral excretion dynamics over time from the same mosquito. Here we test the use of filter papers to detect chikungunya virus in mosquito saliva in laboratory conditions, before proposing this method in large-scale mosquito surveillance programs. We found that 0.9 cm2 cards impregnated with a 50% honey solution could replace the forced salivation technique as they offered a viral RNA detection until 7 days after oral infection of Aedes aegypti and Aedes albopictus mosquitoes with CHIKV.

Experimental Lagos bat virus infection in straw-colored fruit bats: A suitable model for bat rabies in a natural reservoir species

Rabies is a fatal neurologic disease caused by lyssavirus infection. Bats are important natural reservoir hosts of various lyssaviruses that can be transmitted to people. The epidemiology and pathogenesis of rabies in bats are poorly understood, making it difficult to prevent zoonotic transmission. To further our understanding of lyssavirus pathogenesis in a natural bat host, an experimental model using straw-colored fruit bats (Eidolon helvum) and Lagos bat virus, an endemic lyssavirus in this species, was developed. To determine the lowest viral dose resulting in 100% productive infection, bats in five groups (four bats per group) were inoculated intramuscularly with one of five doses, ranging from 100.1 to 104.1 median tissue culture infectious dose (TCID50). More bats died due to the development of rabies after the middle dose (102.1 TCID50, 4/4 bats) than after lower (101.1, 2/4; 101.1, 2/4) or higher (103.1, 2/4; 104.1, 2/4) doses of virus. In the two highest dose groups, 4/8 bats developed rabies. Of those bats that remained healthy 3/4 bats seroconverted, suggesting that high antigen loads can trigger a strong immune response that abrogates a productive infection. In contrast, in the two lowest dose groups, 3/8 bats developed rabies, 1/8 remained healthy and seroconverted and 4/8 bats remained healthy and did not seroconvert, suggesting these doses are too low to reliably induce infection. The main lesion in all clinically affected bats was meningoencephalitis associated with lyssavirus-positive neurons. Lyssavirus antigen was detected in tongue epithelium (5/11 infected bats) rather than in salivary gland epithelium (0/11), suggesting viral excretion via the tongue. Thus, intramuscular inoculation of 102.1 TCID50 of Lagos bat virus into straw-colored fruit bats is a suitable model for lyssavirus associated bat rabies in a natural reservoir host, and can help with the investigation of lyssavirus infection dynamics in bats.

Pilot Study: Long-Term Shedding of SARS-CoV-2 in Urine: A Threat for Dispersal in Wastewater

Only 4 months after the beginning of SARS-CoV-2 epidemic, the world is facing a global pandemic due to a complex and insidious virus that today constantly poses new challenges. In this study, we highlight a persistent shedding of SARS-CoV-2 RNA into the urine, even in patients with a negative nasopharyngeal swab and in patients considered recovered. What does it mean? Besides the fact that the kidney is a probable site of viral replication, the prolonged viral excretion is a matter of great concern for our drainage system contamination.