Stability of SARS-CoV-2 RNA in Nonsupplemented Saliva.

The expense of saliva collection devices designed to stabilize severe acute respiratory syndrome coronavirus 2 RNA is prohibitive to mass testing. However, virus RNA in nonsupplemented saliva is stable for extended periods and at elevated temperatures. Simple plastic tubes for saliva collection will make large-scale testing and continued surveillance easier.

Detection of pneumococcus during hospitalization for SARS-CoV-2.

Background: Infections with respiratory viruses [e.g. influenza and respiratory syncytial virus (RSV)] can increase the risk of severe pneumococcal infections. Likewise, pneumococcal coinfection is associated with poorer outcomes in viral respiratory infection. However, there are limited data describing the frequency of pneumococcus and SARS-CoV-2 coinfection and the role of coinfection in influencing COVID-19 severity. We, therefore, investigated the detection of pneumococcus in COVID-19 inpatients during the early pandemic period. Methods: The study included patients aged 18 years and older, admitted to the Yale-New Haven Hospital who were symptomatic for respiratory infection and tested positive for SARS-CoV-2 during March-August 2020. Patients were tested for pneumococcus through culture-enrichment of saliva followed by RT-qPCR (to identify carriage) and serotype-specific urine antigen detection (UAD) assays (to identify presumed lower respiratory tract pneumococcal disease). Results: Among 148 subjects, the median age was 65 years; 54.7% were male; 50.7% had an ICU stay; 64.9% received antibiotics; and 14.9% died while admitted. Pneumococcal carriage was detected in 3/96 (3.1%) individuals tested by saliva RT-qPCR. Additionally, pneumococcus was detected in 14/127 (11.0%) individuals tested by UAD, and more commonly in severe than moderate COVID-19 [OR: 2.20; 95% CI: (0.72, 7.48)]; however, the numbers were small with a high degree of uncertainty. None of the UAD-positive individuals died. Conclusions: Pneumococcal lower respiratory tract infection (LRTI), as detected by positive UAD, occurred in patients hospitalized with COVID-19. Moreover, pneumococcal LRTI was more common in those with more serious COVID-19 outcomes. Future studies should assess how pneumococcus and SARS-CoV-2 interact to influence COVID-19 severity in hospitalized patients.

The community-wide effectiveness of municipal larval control programs for West Nile virus risk reduction in Connecticut, USA.

BACKGROUND: Mosquito larval control through the use of insecticides is the most common strategy for suppressing West Nile virus (WNV) vector populations in Connecticut (CT), USA. To evaluate the ability of larval control to reduce entomological risk metrics associated with WNV, we performed WNV surveillance and assessments of municipal larvicide application programs in Milford and Stratford, CT in 2019 and 2020. Each town treated catch basins and nonbasin habitats (Milford only) with biopesticide products during both WNV transmission seasons. Adult mosquitoes were collected weekly with gravid and CO2 -baited light traps and tested for WNV; larvae and pupae were sampled weekly from basins within 500 m of trapping sites, and Culex pipiens larval mortality was determined with laboratory bioassays of catch basin water samples. RESULTS: Declines in 4th instar larvae and pupae were observed in catch basins up to 2-week post-treatment, and we detected a positive relationship between adult female C. pipiens collections in gravid traps and pupal abundance in basins. We also detected a significant difference in total light trap collections between the two towns. Despite these findings, C. pipiens adult collections and WNV mosquito infection prevalence in gravid traps were similar between towns. CONCLUSION: Larvicide applications reduced pupal abundance and the prevalence of host-seeking adults with no detectable impact on entomological risk metrics for WNV. Further research is needed to better determine the level of mosquito larval control required to reduce WNV transmission risk.

Evidence for SARS-CoV-2 Spike Protein in the Urine of COVID-19 Patients.

Background: SARS-CoV-2 infection has, as of April 2021, affected >133 million people worldwide, causing >2.5 million deaths. Because the large majority of individuals infected with SARS-CoV-2 are asymptomatic, major concerns have been raised about possible long-term consequences of the infection. Methods: Wedeveloped an antigen capture assay to detect SARS-CoV-2 spike protein in urine samples from patients with COVID-19whose diagnosis was confirmed by positive PCR results from nasopharyngeal swabs (NP-PCR+) forSARS-CoV-2. We used a collection of 233 urine samples from 132 participants from Yale New Haven Hospital and the Children's Hospital of Philadelphia that were obtained during the pandemic (106 NP-PCR+ and 26 NP-PCR-), and a collection of 20 urine samples from 20 individuals collected before the pandemic. Results: Our analysis identified 23 out of 91 (25%) NP-PCR+ adult participants with SARS-CoV-2 spike S1 protein in urine (Ur-S+). Interestingly, although all NP-PCR+ children were Ur-S-, one child who was NP-PCR- was found to be positive for spike protein in their urine. Of the 23 adults who were Ur-S+, only one individual showed detectable viral RNA in urine. Our analysis further showed that 24% and 21% of adults who were NP-PCR+ had high levels of albumin and cystatin C, respectively, in their urine. Among individuals with albuminuria (>0.3 mg/mg of creatinine), statistical correlation could be found between albumin and spike protein in urine. Conclusions: Together, our data showed that one of four individuals infected with SARS-CoV-2 develop renal abnormalities, such as albuminuria. Awareness about the long-term effect of these findings is warranted.

SalivaDirect: A simplified and flexible platform to enhance SARS-CoV-2 testing capacity.

BACKGROUND: Scaling SARS-CoV-2 testing to meet demands of safe reopenings continues to be plagued by assay costs and supply chain shortages. In response, we developed SalivaDirect, which received Emergency Use Authorization (EUA) from the U.S. Food and Drug Administration (FDA). METHODS: We simplified our saliva-based diagnostic test by (1) not requiring collection tubes with preservatives, (2) replacing nucleic acid extraction with a simple enzymatic and heating step, and (3) testing specimens with a dualplex qRT-PCR assay. Moreover, we validated SalivaDirect with reagents and instruments from multiple vendors to minimize supply chain issues. FINDINGS: From our hospital cohort, we show a high positive agreement (94%) between saliva tested with SalivaDirect and nasopharyngeal swabs tested with a commercial qRT-PCR kit. In partnership with the National Basketball Association (NBA) and National Basketball Players Association (NBPA), we tested 3,779 saliva specimens from healthy individuals and detected low rates of invalid (0.3%) and false-positive (<0.05%) results. CONCLUSIONS: We demonstrate that saliva is a valid alternative to swabs for SARS-CoV-2 screening and that SalivaDirect can make large-scale testing more accessible and affordable. Uniquely, we can designate other laboratories to use our sensitive, flexible, and simplified platform under our EUA (https://publichealth.yale.edu/salivadirect/). FUNDING: This study was funded by the NBA and NBPA (N.D.G.), the Huffman Family Donor Advised Fund (N.D.G.), a Fast Grant from Emergent Ventures at the Mercatus Center at George Mason University (N.D.G.), the Yale Institute for Global Health (N.D.G.), and the Beatrice Kleinberg Neuwirth Fund (A.I.K.). C.B.F.V. is supported by NWO Rubicon 019.181EN.004.

Simply saliva: stability of SARS-CoV-2 detection negates the need for expensive collection devices.

Most currently approved strategies for the collection of saliva for COVID-19 diagnostics require specialized tubes containing buffers promoted for the stabilization of SARS-CoV-2 RNA and virus inactivation. Yet many of these are expensive, in limited supply, and not necessarily validated specifically for viral RNA. While saliva is a promising sample type as it can be reliably self-collected for the sensitive detection of SARS-CoV-2, the expense and availability of these collection tubes are prohibitive to mass testing efforts. Therefore, we investigated the stability of SARS-CoV-2 RNA and infectious virus detection from saliva without supplementation. We tested RNA stability over extended periods of time (2-25 days) and at temperatures representing at-home storage and elevated temperatures which might be experienced when cold chain transport may be unavailable. We found SARS-CoV-2 RNA in saliva from infected individuals is stable at 4°C, room temperature (~19°C), and 30°C for prolonged periods and found limited evidence for viral replication in saliva. This work demonstrates that expensive saliva collection options involving RNA stabilization and virus inactivation buffers are not always needed, permitting the use of cheaper collection options. Affordable testing methods are urgently needed to meet current testing demands and for continued surveillance in reopening strategies.