Detection of pneumococcus during hospitalization for SARS-CoV-2

BackgroundInfections with respiratory viruses (e.g., influenza, RSV) can increase the risk of severe pneumococcal infections. Likewise, pneumococcal co-infection is associated with poorer outcomes in viral respiratory infection. However, there are limited data describing the frequency of pneumococcus and SARS-CoV-2 co-infection and the role of co-infection in influencing COVID-19 severity. MethodsThe study included patients admitted to 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). ResultsAmong 148 subjects, the median age was 65 years; 54.7% were male; 50.7% had an ICU stay; 64.9% received antibiotics; 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. ConclusionsPneumococcal 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. One Sentence SummaryPneumococcal lower respiratory tract infection, as detected by positive UAD, occurred in patients hospitalized with COVID-19 at rates similar to those reported prepandemic.

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

SARS-CoV-2 infection has so far affected over 42 million people worldwide, causing over 1.1 million deaths. With the large majority of SARS-CoV-2 infected individuals being asymptomatic, major concerns have been raised about possible long-term consequences of the infection. We developed an antigen capture assay to detect SARS-CoV-2 spike protein in urine samples from COVID-19 patients whose diagnosis was confirmed by PCR from nasopharyngeal swabs (NP-PCR+). The study used a collection of 233 urine samples from 132 participants from Yale New Haven Hospital and the Childrens Hospital of Philadelphia obtained during the pandemic (106 NP-PCR+ and 26 NP-PCR-) as well as a collection of 20 urine samples from 20 individuals collected before the pandemic. 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-, 1 NP-PCR-child was found to be positive for spike protein in urine. Of the 23 Ur-S+ adults, only 1 individual showed detectable viral RNA in urine. Our analysis further showed that 24% and 21% of NP-PCR+ adults have high levels of albumin and cystatin C in urine, respectively. Among individuals with albuminuria (>0.3 mg/mg of creatinine) statistical correlation could be found between albumin and spike protein in urine. Together, our data showe that 1 of 4 of SARS-CoV-2 infected individuals develop renal abnormalities such as albuminuria. Awareness about the long-term impact of these findings is warranted.

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{degrees}C, room temperature ([~]19{degrees}C), and 30{degrees}C for prolonged periods and found limited evidence for viral replication in stored saliva samples. 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.

SalivaDirect: Simple and sensitive molecular diagnostic test for SARS-CoV-2 surveillance

Current bottlenecks for improving accessibility and scalability of SARS-CoV-2 testing include diagnostic assay costs, complexity, and supply chain shortages. To resolve these issues, we developed SalivaDirect, which received Emergency Use Authorization (EUA) from the U.S. Food and Drug Administration on August 15th, 2020. The critical component of our approach is to use saliva instead of respiratory swabs, which enables non-invasive frequent sampling and reduces the need for trained healthcare professionals during collection. Furthermore, we simplified our diagnostic test by (1) not requiring nucleic acid preservatives at sample collection, (2) replacing nucleic acid extraction with a simple proteinase K and heat treatment step, and (3) testing specimens with a dualplex quantitative reverse transcription PCR (RT-qPCR) assay. We validated SalivaDirect with reagents and instruments from multiple vendors to minimize the risk for supply chain issues. Regardless of our tested combination of reagents and instruments from different vendors, we found that SalivaDirect is highly sensitive with a limit of detection of 6-12 SARS-CoV-2 copies/L. When comparing SalivaDirect to paired nasopharyngeal swabs using the authorized ThermoFisher Scientific TaqPath COVID-19 combo kit, we found high agreement in testing outcomes (>94%). In partnership with the National Basketball Association (NBA) and Players Association, we conducted a large-scale (n = 3,779) SalivaDirect usability study and comparison to standard nasal/oral tests for asymptomatic and presymptomatic SARS-CoV-2 detection. From this cohort of healthy NBA players, staff, and contractors, we found that 99.7% of samples were valid using our saliva collection techniques and a 89.5% positive and >99.9% negative test agreement to swabs, demonstrating that saliva is a valid and noninvasive alternative to swabs for large-scale SARS-CoV-2 testing. SalivaDirect is a flexible and inexpensive ($1.21-$4.39/sample in reagent costs) option to help improve SARS-CoV-2 testing capacity. Register to become a designated laboratory to use SalivaDirect under our FDA EUA on our website: publichealth.yale.edu/salivadirect/.