COVID-19 Impacts Highlight the Need for Holistic Evaluation of Research and in the Hydrologic Sciences

As we reckon with the effect of COVID-19 on the research enterprise in hydrologic science, it is important to acknowledge that disruptions will be persistent and that institutional-level adjustments, while helpful, are not sufficient to mitigate all impacts on hydrologic scientists. Here, we describe the breadth of research contributions in the hydrologic sciences, consider how the pandemic has impacted this portfolio of contributions, document one impact that is already being realized in publication of research, and suggest guidance to the hydrologic science community, institutions, review panels, and funding organizations in considering these impacts at various stages of hiring and promotion in our community. Acknowledging the diversity of contributions to research is particularly valuable because it provides a more objective, transparent, and holistic basis for evaluating individuals within the context of norms of the hydrologic science community. With clearly established values, it is easier to identify impacts of life events, such as those related to the COVID-19 pandemic, as they are manifested in individuals under a diversity of circumstances. This article is protected by copyright. All rights reserved.

COVID-19 Variant Detection with a High-Fidelity CRISPR-Cas12 Enzyme (preprint)

Laboratory tests for the accurate and rapid identification of SARS-CoV-2 variants can potentially guide the treatment of COVID-19 patients and inform infection control and public health surveillance efforts. Here we present the development and validation of a rapid COVID-19 variant DETECTR ® assay incorporating loop-mediated isothermal amplification (LAMP) followed by CRISPR-Cas12 based identification of single nucleotide polymorphism (SNP) mutations in the SARS-CoV-2 spike (S) gene. This assay targets the L452R, E484K/Q/A, and N501Y mutations that are associated with nearly all circulating viral lineages and identifies the two circulating variants of concern, Delta and Omicron. In a comparison of three different Cas12 enzymes, only the newly identified enzyme CasDx1 was able to accurately identify all targeted SNP mutations. An analysis pipeline for CRISPR-based SNP identification from 139 clinical samples yielded an overall SNP concordance of 98% and agreement with SARS-CoV-2 lineage classification of 138/139 compared to viral whole-genome sequencing. We also showed that detection of the single E484A mutation was necessary and sufficient to accurately identify Omicron from other major circulating variants in patient samples. These findings demonstrate the utility of CRISPR-based DETECTR ® as a faster and simpler diagnostic than sequencing for SARS-CoV-2 variant identification in clinical and public health laboratories.

Covid-19 silver linings – enhanced collaboration with hospice partners

BackgroundThe COVID-19 pandemic, whilst challenging for all, acted as a catalyst for collaboration between NHS partners and hospice colleagues to improve service delivery across all settings.The directors of care from four hospices met weekly with commissioners to provide support for one another and to work collaboratively to ensure increasing demands were met and good practice shared. This collaborative effort paid off with a successful joint bid for additional funding to enhance 24/7 care and support for care homes. Key outcomes were delivered and care enhanced through new and different ways of working.AimOur main aim is to learn from this experience to transform care for people receiving palliative and end-of-life care across our Integrated Care System. Emerging from the second wave, the four hospices were keen to learn from this experience, to optimise opportunities and improve quality of care for patients, their families and bereaved clients. A joint Hospice Collaboration Project group was set up with fortnightly meetings to develop key collaborative projects.MethodologyThe group will work collaboratively to:Identify population health needs in relation to palliative and end-of-life care.Create a model of partnership which influences and shapes best practice, that is sustainable and maximises statutory and voluntary income to meet the outcomes of the needs analysis.Meet the requirements of the national, regional and local population health agendas.Share best practice and offer joint solutions where possible demonstrating the ongoing ‘added value’ of hospices in care delivery.ResultsAt this stage the project is at the outset of shaping a sustainable way ahead, working with system partners including the Palliative and End of Life Care Delivery Group and commissioners and learning from other hospice approaches. Our methodology has backing from the ICS and regional strategic clinical network.ConclusionsIt’s early days but there’s great energy, inspiration and willingness for continued improvement to service delivery as demonstrated during the pandemic.

Predominance of antibody-resistant SARS-CoV-2 variants in vaccine breakthrough cases from the San Francisco Bay Area, California (preprint)

Associations between vaccine breakthrough cases and infection by SARS coronavirus 2 (SARS-CoV-2) variants have remained largely unexplored. Here we analyzed SARS-CoV-2 whole-genome sequences and viral loads from 1,373 persons with COVID-19 from the San Francisco Bay Area from February 1 to June 30, 2021, of which 125 (9.1%) were vaccine breakthrough infections. Fully vaccinated were more likely than unvaccinated persons to be infected by variants carrying mutations associated with decreased antibody neutralization (L452R, L452Q, E484K, and/or F490S) (78% versus 48%, p = 1.96e-08), but not by those associated with increased infectivity (L452R and/or N501Y) (85% versus 77%, p = 0.092). Differences in viral loads were non-significant between unvaccinated and fully vaccinated persons overall (p = 0.99) and according to lineage (p = 0.09 - 0.78). Viral loads were significantly higher in symptomatic as compared to asymptomatic vaccine breakthrough cases (p < 0.0001), and symptomatic vaccine breakthrough infections had similar viral loads to unvaccinated infections (p = 0.64). In 5 cases with available longitudinal samples for serologic analyses, vaccine breakthrough infections were found to be associated with low or undetectable neutralizing antibody levels attributable to immunocompromised state or infection by an antibody-resistant lineage. These findings suggest that vaccine breakthrough cases are preferentially caused by circulating antibody-resistant SARS-CoV-2 variants, and that symptomatic breakthrough infections may potentially transmit COVID-19 as efficiently as unvaccinated infections, regardless of the infecting lineage.

Transmission, infectivity, and antibody neutralization of an emerging SARS-CoV-2 variant in California carrying a L452R spike protein mutation (preprint)

We identified a novel SARS-CoV-2 variant by viral whole-genome sequencing of 2,172 remnant nasal/nasopharyngeal swab samples from 44 counties in California. Named B.1.427/B.1.429 or 20C/L452R, the variant emerged around May 2020 and increased from 0% to >50% of sequenced cases from September 1, 2020 to January 29, 2021, exhibiting an estimated 18.6-24% increase in transmissibility relative to wild-type circulating strains. This variant is characterized by three mutations in the spike protein, including a L452R substitution in the receptor-binding domain. Our analyses revealed 2-fold increased B.1.427/B.1.429 viral shedding in vivo and increased L452R pseudovirus infection of cell cultures and lung organoids, albeit decreased relative to pseudoviruses carrying the N501Y mutation found in SARS-CoV-2 variants of concern (B.1.1.7, B.1.351, and P.1 lineages). Antibody neutralization assays showed 4.0 to 6.7-fold and 2.0-fold decreases in neutralizing titers from convalescent patients and vaccine recipients, respectively. The increased prevalence of a more transmissible variant in California associated with decreased antibody neutralization warrants further investigation.

Performance characteristics of a rapid SARS-CoV-2 antigen detection assay at a public plaza testing site in San Francisco (preprint)

We evaluated the performance of the Abbott BinaxNOWTM Covid-19 rapid antigen test to detect virus among persons, regardless of symptoms, at a public plaza site of ongoing community transmission. Titration with cultured clinical SARS-CoV-2 yielded a human observable threshold between 1.6x104-4.3x104 viral RNA copies (cycle threshold (Ct) of 30.3-28.8 in this assay). Among 878 subjects tested, 3% (26/878) were positive by RT-PCR, of which 15/26 had a Ct<30, indicating high viral load. 40% (6/15) of Ct<30 were asymptomatic. Using this Ct<30 threshold for Binax-CoV2 evaluation, the sensitivity of the Binax-CoV2 was 93.3% (14/15), 95% CI: 68.1-99.8%, and the specificity was 99.9% (862/863), 95% CI: 99.4-99.9%.

Nasopharyngeal SARS-CoV2 viral loads in young children do not differ significantly from those in older children and adults (preprint)

The role of children in the spread of the SARS-CoV2 coronavirus has become a matter of urgent debate as societies in the US and abroad consider how to safely reopen schools. Small studies have suggested higher viral loads in young children. Here we present a multicenter investigation on over five thousand SARS-CoV-2 cases confirmed by real-time reverse transcription (RT) PCR assay. Notably, we found no discernable difference in amount of viral nucleic acid among young children and adults.

Digital droplet PCR accurately quantifies SARS-CoV-2 viral load from crude lysate without nucleic acid purification (preprint)

The COVID-19 pandemic caused by the SARS-CoV-2 virus motivates diverse diagnostic approaches due to the novel causative pathogen, incompletely understood clinical sequelae, and limited availability of testing resources. Given the variability in viral load across and within patients, absolute viral load quantification directly from crude lysate is important for diagnosis and surveillance. Here, we investigate the use of digital droplet PCR (ddPCR) for SARS-CoV-2 viral load measurement directly from crude lysate without nucleic acid purification. We demonstrate ddPCR accurately quantifies SARS-CoV-2 standards from purified RNA and multiple sample matrices, including commonly utilized universal transport medium (UTM). In addition, we find ddPCR functions robustly at low input viral copy numbers on nasopharyngeal swab specimens stored in UTM without upfront RNA extraction. We also show ddPCR, but not qPCR, from crude lysate shows high concordance with viral load measurements from purified RNA. Our data suggest ddPCR offers advantages to qPCR for SARS-CoV-2 detection with higher sensitivity and robustness when using crude lysate rather than purified RNA as input. More broadly, digital droplet assays provide a potential method for nucleic acid measurement and infectious disease diagnosis with limited sample processing, underscoring the utility of such techniques in laboratory medicine.

Identification of a polymorphism in the N gene of SARS-CoV-2 that adversely impacts detection by a widely-used RT-PCR assay (preprint)

We identify a mutation in the N gene of SARS-CoV-2 that adversely affects annealing of a commonly used RT-PCR primer; epidemiologic evidence suggests the virus retains pathogenicity and competence for spread. This reinforces the importance of using multiple targets, preferably in at least 2 genes, for robust SARS-CoV-2 detection. Article Summary LineA SARS-CoV-2 variant that occurs worldwide and has spread in California significantly affects diagnostic sensitivity of an N gene assay, highlighting the need to employ multiple viral targets for detection.

The SARS-CoV-2 Spike mutation D614G increases entry fitness across a range of ACE2 levels, directly outcompetes the wild type, and is preferentially incorporated into trimers (preprint)

Early in the current pandemic, the D614G mutation arose in the Spike protein of SARS-CoV-2 and quickly became the dominant variant globally. Mounting evidence suggests D614G enhances viral entry. Here we use a direct competition assay with single-cycle viruses to show that D614G outcompetes the wildtype. We developed a cell line with inducible ACE2 expression to confirm that D614G more efficiently enters cells with ACE2 levels spanning the different primary cells targeted by SARS-CoV-2. Using a new assay for crosslinking and directly extracting Spike trimers from the pseudovirus surface, we found an increase in trimerization efficiency and viral incorporation of D614G protomers. Our findings suggest that D614G increases infection of cells expressing a wide range of ACE2, and informs the mechanism underlying enhanced entry. The tools developed here can be broadly applied to study other Spike variants and SARS-CoV-2 entry, to inform functional studies of viral evolution and vaccine development.

Epitopes targeted by T cells in convalescent COVID-19 patients (preprint)

There is growing evidence pointing to the protective role of T cells against COVID-19. Vaccines eliciting targeted T cell responses have the potential to provide robust, long-lasting immunity. However, their design requires knowledge of the SARS-CoV-2-specific epitopes that can elicit a T cell response and confer protection across a wide population. Here, we provide a unified description of emerging data of SARS-CoV-2 T cell epitopes compiled from results of 8 independent studies of convalescent COVID-19 patients. We describe features of these epitopes relevant for vaccine design, while indicating knowledge gaps that can, in part, be augmented using prior immunological data from SARS-CoV. The landscape of SARS-CoV-2 T cell epitopes that we describe can help guide SARS-CoV-2 vaccine development as well as future immunological studies. A web-based platform has also been developed to complement these efforts.

AI aided design of epitope-based vaccine for the induction of cellular immune responses against SARS-CoV-2 (preprint)

The heavy burden imposed by the COVID-19 pandemic on our society triggered the race towards the development of therapies or preventive strategies. Among these, antibodies and vaccines are particularly attractive because of their high specificity, low probability of drug-drug interaction, and potentially long-standing protective effects. While the threat at hand justifies the pace of research, the implementation of therapeutic strategies cannot be exempted from safety considerations. There are several potential adverse events reported after the vaccination or antibody therapy, but two are of utmost importance: antibody-dependent enhancement (ADE) and cytokine storm syndrome (CSS). On the other hand, the depletion or exhaustion of T-cells has been reported to be associated with worse prognosis in COVID-19 patients. This observation suggests a potential role of vaccines eliciting cellular immunity, which might simultaneously limit the risk of ADE and CSS. Such risk was proposed to be associated with FcR-induced activation of proinflammatory macrophages (M1) by Fu et al. 2020 and Iwasaki et al. 2020. All aspects of the newly developed vaccine (including the route of administration, delivery system, and adjuvant selection) may affect its effectiveness and safety. In this work we use a novel in silico approach (based on AI and bioinformatics methods) developed to support the design of epitope-based vaccines. We evaluated the capabilities of our method for predicting the immunogenicity of epitopes. Next, the results of our approach were compared with other vaccine-design strategies reported in the literature. The risk of immuno-toxicity was also assessed. The analysis of epitope conservation among other Coronaviridae was carried out in order to facilitate the selection of peptides shared across different SARS-CoV-2 strains and which might be conserved in emerging zootic coronavirus strains. Finally, the potential applicability of the selected epitopes for the development of a vaccine eliciting cellular immunity for COVID-19 was discussed, highlighting the benefits and challenges of such an approach.

SARS-CoV-2 seroprevalence and neutralizing activity in donor and patient blood from the San Francisco Bay Area (preprint)

We report very low SARS-CoV-2 seroprevalence in two San Francisco Bay Area populations. Seropositivity was 0.26% in 387 hospitalized patients admitted for non-respiratory indications and 0.1% in 1,000 blood donors. We additionally describe the longitudinal dynamics of immunoglobulin-G, immunoglobulin-M, and in vitro neutralizing antibody titers in COVID-19 patients. Neutralizing antibodies rise in tandem with immunoglobulin levels following symptom onset, exhibiting median time to seroconversion within one day of each other, and there is >93% positive percent agreement between detection of immunoglobulin-G and neutralizing titers.

Test performance evaluation of SARS-CoV-2 serological assays (preprint)

Background Serological tests are crucial tools for assessments of SARS-CoV-2 exposure, infection and potential immunity. Their appropriate use and interpretation require accurate assay performance data. Method We conducted an evaluation of 10 lateral flow assays (LFAs) and two ELISAs to detect anti-SARS-CoV-2 antibodies. The specimen set comprised 128 plasma or serum samples from 79 symptomatic SARS-CoV-2 RT-PCR-positive individuals; 108 pre-COVID-19 negative controls; and 52 recent samples from individuals who underwent respiratory viral testing but were not diagnosed with Coronavirus Disease 2019 (COVID-19). Samples were blinded and LFA results were interpreted by two independent readers, using a standardized intensity scoring system. Results Among specimens from SARS-CoV-2 RT-PCR-positive individuals, the percent seropositive increased with time interval, peaking at 81.8-100.0% in samples taken >20 days after symptom onset. Test specificity ranged from 84.3-100.0% in pre-COVID-19 specimens. Specificity was higher when weak LFA bands were considered negative, but this decreased sensitivity. IgM detection was more variable than IgG, and detection was highest when IgM and IgG results were combined. Agreement between ELISAs and LFAs ranged from 75.7-94.8%. No consistent cross-reactivity was observed. Conclusion Our evaluation showed heterogeneous assay performance. Reader training is key to reliable LFA performance, and can be tailored for survey goals. Informed use of serology will require evaluations covering the full spectrum of SARS-CoV-2 infections, from asymptomatic and mild infection to severe disease, and later convalescence. Well-designed studies to elucidate the mechanisms and serological correlates of protective immunity will be crucial to guide rational clinical and public health policies.

Association of COVID-19 Infections in San Francisco in Early March 2020 with Travel to New York and Europe (preprint)

Real-time dissemination of epidemiological survey data from positive COVID-19 cases is critical to support efforts to contain or reduce spread of viral infection in the community. Here we detected a significant association between domestic travel or travel to Europe and the identification of new cases in San Francisco, California, USA. These findings suggest that domestic and European travelers may need to be prioritized for evaluation of acute infection from COVID-19 in the setting of limited testing capacity.

A Genomic Survey of SARS-CoV-2 Reveals Multiple Introductions into Northern California without a Predominant Lineage (preprint)

The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 has spread globally, resulting in >300,000 reported cases worldwide as of March 21st, 2020. Here we investigate the genetic diversity and genomic epidemiology of SARS-CoV-2 in Northern California using samples from returning travelers, cruise ship passengers, and cases of community transmission with unclear infection sources. Virus genomes were sampled from 29 patients diagnosed with COVID-19 infection from Feb 3rd through Mar 15th. Phylogenetic analyses revealed at least 8 different SARS-CoV-2 lineages, suggesting multiple independent introductions of the virus into the state. Virus genomes from passengers on two consecutive excursions of the Grand Princess cruise ship clustered with those from an established epidemic in Washington State, including the WA1 genome representing the first reported case in the United States on January 19th. We also detected evidence for presumptive transmission of SARS-CoV-2 lineages from one community to another. These findings suggest that cryptic transmission of SARS-CoV-2 in Northern California to date is characterized by multiple transmission chains that originate via distinct introductions from international and interstate travel, rather than widespread community transmission of a single predominant lineage. Rapid testing and contact tracing, social distancing, and travel restrictions are measures that will help to slow SARS-CoV-2 spread in California and other regions of the USA.