Microbial Dynamics and Pulmonary Immune Responses in COVID-19 Secondary Bacterial Pneumonia (preprint)

Secondary bacterial pneumonia (2°BP) is associated with significant morbidity following respiratory viral infection, yet mechanistically remains incompletely understood. In a prospective cohort of 112 critically ill adults intubated for COVID-19, we comparatively assessed longitudinal airway microbiome dynamics and studied the pulmonary transcriptome of patients who developed 2°BP versus controls who did not. We found that 2°BP was significantly associated with both mortality and corticosteroid treatment. The pulmonary microbiome in 2°BP was characterized by increased bacterial RNA load, dominance of culture-confirmed pathogens, and lower alpha diversity. Bacterial pathogens were detectable days prior to 2°BP clinical diagnosis, and in most cases were also present in nasal swabs. Pathogen antimicrobial resistance genes were also detectable in both the lower airway and nasal samples, and in some cases were identified prior to 2°BP clinical diagnosis. Assessment of the pulmonary transcriptome revealed suppressed TNFa signaling via NF-kB in patients who developed 2°BP, and a sub-analysis suggested that this finding was mediated by corticosteroid treatment. Within the 2°BP group, we observed a striking inverse correlation between innate and adaptive immune gene expression and bacterial RNA load. Together, our findings provide fresh insights into the microbial dynamics and host immune features of COVID-19-associated 2°BP.

Field assessment of BinaxNOW antigen tests as COVID-19 treatment entry point at a community testing site in San Francisco during evolving omicron surges (preprint)

ABSTRACT Background COVID-19 oral treatments require initiation within 5 days of symptom onset. Although antigen tests are less sensitive than RT-PCR, rapid results could facilitate entry to treatment. As SARS-CoV-2 variants and host immunity evolve, it is important to characterize the use case for rapid antigen tests for treatment entry. Methods We collected anterior nasal swabs for BinaxNOW and RT-PCR testing and clinical data at a walk-up, community site in San Francisco, California between January and June 2022. SARS-CoV-2 genomic sequences were generated from positive samples and classified according to subtype and variant. Monte Carlo simulations were conducted to estimate the expected proportion of SARS-CoV-2 infected persons who would have been diagnosed within 5 days of symptom onset using RT-PCR versus BinaxNOW testing. Results Among 25,309 persons tested with BinaxNOW, 2,952 had concomitant RT-PCR. 1321/2952 (44.7%) were SARS-CoV-2 RT-PCR positive. We identified waves of predominant omicron BA.1, BA.2, BA.2.12, BA.4, and BA.5 among 720 sequenced samples. Among 1,321 RT-PCR positive samples, 938/1321 (71%) were detected by BinaxNOW; 95% (774/817) of those with Ct value <30 were detected by BinaxNOW. BinaxNOW detection was consistent over lineages. In analyses to evaluate entry to treatment, BinaxNOW detected 82.7% (410/496, 95% CI: 79-86%) of persons with COVID-19 within 5 days of symptom onset. In comparison, RT-PCR (24-hour turnaround) detected 83.1% (412/496 95% CI: 79-86%) and RT-PCR (48-hour turnaround) detected 66.3% (329/496 95% CI: 62-70%) of persons with COVID-19 within 5 days of symptom onset. Conclusions BinaxNOW detected high viral load from anterior nasal swabs consistently across omicron sublineages emerging between January and June of 2022. Simulations support BinaxNOW as an entry point for COVID-19 treatment in a community field setting.

Computational pipeline provides mechanistic understanding of Omicron variant of concern neutralizing engineered ACE2 receptor traps (preprint)

The SARS-CoV-2 Omicron variant, with 15 mutations in Spike receptor binding domain (Spike-RBD), renders virtually all clinical monoclonal antibodies against WT SARS-CoV-2 ineffective. We recently engineered the SARS-CoV-2 host entry receptor, ACE2, to tightly bind WT-Spike-RBD and prevent viral entry into host cells (receptor traps). Here we determine cryo-EM structures of our receptor traps in complex with full length Spike. We develop a multi-model pipeline combining Rosetta protein modeling software and cryo-EM to allow interface energy calculations even at limited resolution and identify interface side chains that allow for high affinity interactions between our ACE2 receptor traps and Spike-RBD. Our structural analysis provides a mechanistic rationale for the high affinity (0.53 - 4.2nM) binding of our ACE2 receptor traps to Omicron-RBD confirmed with biolayer interferometry measurements. Finally, we show that ACE2 receptor traps potently neutralize Omicron- and Delta- pseudotyped viruses, providing alternative therapeutic routes to combat this evolving virus.

COVID-19 symptoms and duration of direct antigen test positivity at a community testing and surveillance site, January 2021-2022 (preprint)

Importance: Characterizing clinical symptoms and evolution of community- based SARS Co-V-2 infections can inform health practitioners and public health officials in a rapidly changing landscape of population immunity and viral variants. Objective: To characterize COVID-19 symptoms during the Omicron period compared to pre-Delta and Delta variant periods and assess the duration of COVID-19 BinaxNOW rapid antigen test positivity during the Omicron variant surge. Design, Setting, and Participants: This public health surveillance study was undertaken between January 2021- January 2022, at a walk-up community COVID-19 testing site in San Francisco, California. Testing with BinaxNOW rapid antigen tests was available regardless of age, vaccine status, or symptoms throughout. Main Outcomes and Measures: We characterized the prevalence of specific symptoms for people with a positive BinaxNOW test during the Omicron period and compared it to the pre-Delta and Delta periods. During the Omicron period, we examined differences in symptoms by age and vaccine status. Among people returning for repeat testing during Omicron period, we estimated the proportion with a positive BinaxNOW antigen test between 4-14 days from symptom onset or since first positive test if asymptomatic. Results: Of 63,277 persons tested, 18,301 (30%) reported symptoms and 4,568 (25%) tested positive for COVID-19. During the Omicron period, 41.6% (3032/7283) of symptomatic testers tested positive, and the proportion reporting cough (67.4%) and sore throat (43.4%) was higher than during Delta and pre-Delta periods. Congestion was higher during Omicron (38.8%) than during the pre-Delta period and loss of taste/smell (5.3%) and fever (30.4%) were less common. Fevers and myalgias were less common among persons who had received boosters compared to unvaccinated people or those who received the primary series. Five days after symptom onset, 31.1% of people with COVID-19 stated their symptoms were similar or worsening. An estimated 80.2% of symptomatic re-testers remained positive five days after symptom onset and 60.5% after ten days. Conclusions and Relevance: COVID-19 upper respiratory tract symptoms were more commonly reported during the Omicron period compared to pre-Delta and Delta periods, with differences by vaccination status. Antigen test positivity remained high after 5 days, supporting guidelines requiring a negative test to shorten the isolation period.

A 2-Gene Host Signature for Improved Accuracy of COVID-19 Diagnosis Agnostic to Viral Variants (preprint)

The continued emergence of SARS-CoV-2 variants is one of several factors that may cause false negative viral PCR test results. Such tests are also susceptible to false positive results due to trace contamination from high viral titer samples. Host immune response markers provide an orthogonal indication of infection that can mitigate these concerns when combined with direct viral detection. Here, we leverage nasopharyngeal swab RNA-seq data from patients with COVID-19, other viral acute respiratory illnesses and non-viral conditions (n=318) to develop support vector machine classifiers that rely on a parsimonious 2-gene host signature to predict COVID-19. Optimal classifiers achieve an area under the receiver operating characteristic curve (AUC) greater than 0.9 when evaluated on an independent RNA-seq cohort (n=553). We show that a classifier relying on a single interferon-stimulated gene, such as IFI6 or IFI44, measured in RT-qPCR assays (n=144) achieves AUC values as high as 0.88. Addition of a second gene, such as GBP5, significantly improves the specificity compared to other respiratory viruses. The performance of a clinically practical 2-gene RT-qPCR classifier is robust across common SARS-CoV-2 variants, including Omicron, and is unaffected by cross-contamination, demonstrating its utility for improving accuracy of COVID-19 diagnostics.

A 2-Gene Host Signature for Improved Accuracy of COVID-19 Diagnosis Agnostic to Viral Variants (preprint)

The continued emergence of SARS-CoV-2 variants is one of several factors that may cause false negative viral PCR test results. Such tests are also susceptible to false positive results due to trace contamination from high viral titer samples. Host immune response markers provide an orthogonal indication of infection that can mitigate these concerns when combined with direct viral detection. Here, we leverage nasopharyngeal swab RNA-seq data from patients with COVID-19, other viral acute respiratory illnesses and non-viral conditions (n=318) to develop support vector machine classifiers that rely on a parsimonious 2-gene host signature to predict COVID-19. Optimal classifiers achieve an area under the receiver operating characteristic curve (AUC) greater than 0.9 when evaluated on an independent RNA-seq cohort (n=553). We show that a classifier relying on a single interferon-stimulated gene, such as IFI6 or IFI44, measured in RT-qPCR assays (n=144) achieves AUC values as high as 0.88. Addition of a second gene, such as GBP5, significantly improves the specificity compared to other respiratory viruses. The performance of a clinically practical 2-gene RT-qPCR classifier is robust across common SARS-CoV-2 variants, including Omicron, and is unaffected by cross-contamination, demonstrating its utility for improving accuracy of COVID-19 diagnostics.

No Significant Difference in Viral Load Between Vaccinated and Unvaccinated, Asymptomatic and Symptomatic Groups Infected with SARS-CoV-2 Delta Variant (preprint)

We found no significant difference in cycle threshold values between vaccinated and unvaccinated, asymptomatic and symptomatic groups infected with SARS-CoV-2 Delta. Given the substantial proportion of asymptomatic vaccine breakthrough cases with high viral levels, interventions, including masking and testing, should be considered for all in settings with elevated COVID-19 transmission.

Exposures to different SARS-CoV-2 spike variants elicit neutralizing antibody responses with differential specificity towards established and emerging strains (preprint)

The wide spectrum of SARS-CoV-2 variants with phenotypes impacting transmission and antibody sensitivity necessitates investigation of the immune response to different spike protein versions. Here, we compare the neutralization of variants of concern, including B.1.617.2 (Delta) in sera from individuals exposed to variant infection, vaccination, or both. We demonstrate that neutralizing antibody responses are strongest against variants sharing one or more spike mutations with the immunizing exposure. We also observe that exposure to multiple spike variants increases the breadth of variant cross-neutralization. These findings contribute to understanding the relationship between exposures and antibody responses and may inform booster vaccination strategies.

High parental vaccine motivation at a neighborhood-based vaccine and testing site serving a predominantly Latinx community (preprint)

Purpose. To understand vaccine attitudes of Latinx parents highly impacted by COVID-19. Methods. In April 2021, we surveyed parents about their attitudes for COVID-19 vaccination of their children at a community-based outdoor testing/vaccination site serving predominantly low-income, Latinx persons in San Francisco. Results. Among 1,033 parents (75% Latinx), 92% would 'definitely' or 'probably' vaccinate their children. Vaccine hesitancy was higher for younger children; concerns included side effects and impacts on fertility. Doctors and community organizations were noted as trusted sources of information, including among vaccine-hesitant parents. Conclusion. Latinx parents accessing neighborhood-based COVID-19 testing/vaccination services are highly motivated to vaccinate their children for COVID-19.

A multi-component, community-based strategy to facilitate COVID-19 vaccine uptake among Latinx populations: from theory to practice (preprint)

Background COVID-19 vaccine coverage in the Latinx community depends on delivery systems that overcome barriers such as institutional distrust, misinformation, and access to care. We hypothesized that a community-centered vaccination strategy that included mobilization, vaccination, and "activation" components could successfully reach an underserved Latinx population, utilizing its social networks to boost vaccination coverage. Methods and Findings Our community-academic-public health partnership, "Unidos en Salud," utilized a theory-informed approach to design our "Motivate, Vaccinate, and Activate" COVID-19 vaccination strategy. Our strategy's design was guided by the PRECEDE Model and sought to address and overcome predisposing, enabling, and reinforcing barriers to COVID-19 vaccination faced by Latinx individuals in San Francisco. We evaluated our prototype outdoor, "neighborhood" vaccination program located in a central commercial and transport hub in the Mission District in San Francisco, using the Reach, Effectiveness, Adoption, Implementation and Maintenance (RE-AIM) framework during a 16-week period from February 1, 2021 to May 19, 2021. Programmatic data, city-wide COVID-19 surveillance data, and a survey conducted between May 2, 2021 and May 19, 2021 among 997 vaccinated clients at least 16 years old were used in the evaluation. There were 20,792 COVID-19 vaccinations administered at the neighborhood site during the 16-week evaluation period. Vaccine recipients had a median age of 43 (IQR 32-56) years, 53.9% were male and 70.5% were Latinx, 14.1% white, 7.7% Asian, 2.4% Black, and 5.3% other. Latinx vaccinated clients were substantially more likely than non-Latinx clients to have an annual household income of less than $50,000 a year (76.1% vs. 33.5%), be a first-generation immigrant (60.2% vs. 30.1%), not have health insurance (47.3% vs. 16.0%), and not have access to primary care provider (62.4% vs. 36.2%). The most frequently reported reasons for choosing vaccination at the site were its neighborhood location (28.6%), easy and convenient scheduling (26.9%) and recommendation by someone they trusted (18.1%); approximately 99% reported having an overall positive experience, regardless of ethnicity. Notably, 58.3% of clients reported that they were able to get vaccinated earlier because of the neighborhood vaccination site, 98.4% of clients completed both vaccine doses, and 90.7% said that they were more likely to recommend COVID-19 vaccination to family and friends after their experience; these findings did not substantially differ according to ethnicity. There were 40.3% of vaccinated clients who said they still knew at least one unvaccinated person (64.6% knew 3 or more). Among clients who received both vaccine doses (n=729), 91.0% said that after their vaccination experience, they had personally reached out to at least one unvaccinated person they knew (61.6% reached out to 3 or more) to recommend getting vaccinated; 83.0% of clients reported that one or more friends, and/or family members got vaccinated as a result of their outreach, including 18.9% who reported 6 or more persons got vaccinated as a result of their influence. Conclusions A multi-component, "Motivate, Vaccinate, and Activate" community-based strategy addressing barriers to COVID-19 vaccination for the Latinx population reached the intended population, and vaccinated individuals served as ambassadors to recruit other friends and family members to get vaccinated.

Impaired immune signaling and changes in the lung microbiome precede secondary bacterial pneumonia in COVID-19 (preprint)

Secondary bacterial infections, including ventilator-associated pneumonia (VAP), lead to worse clinical outcomes and increased mortality following viral respiratory infections including in patients with coronavirus disease 2019 (COVID-19). Using a combination of tracheal aspirate bulk and single-cell RNA sequencing (scRNA-seq) we assessed lower respiratory tract immune responses and microbiome dynamics in 28 COVID-19 patients, 15 of whom developed VAP, and eight critically ill uninfected controls. Two days before VAP onset we observed a transcriptional signature of bacterial infection. Two weeks prior to VAP onset, following intubation, we observed a striking impairment in immune signaling in COVID-19 patients who developed VAP. Longitudinal metatranscriptomic analysis revealed disruption of lung microbiome community composition in patients with VAP, providing a connection between dysregulated immune signaling and outgrowth of opportunistic pathogens. These findings suggest that COVID-19 patients who develop VAP have impaired antibacterial immune defense detectable weeks before secondary infection onset.

The COVID-19 Epidemic in Madagascar: clinical description and laboratory results of the first wave, March-September 2020 (preprint)

Background: Following the first detection of SARS-CoV-2 in passengers arriving from Europe on 19 March 2020, Madagascar took several mitigation measures to limit the spread of the virus in the country. Methods: Nasopharyngeal and/or oropharyngeal swabs were collected from travellers to Madagascar, suspected SARS-CoV-2 cases, and contact of confirmed cases. Swabs were tested at the national reference laboratory using real-time RT-PVR. Data collected from patients were entered in an electronic database for subsequent statistical analysis. All distribution of laboratory confirmed cases were mapped and six genomes of viruses were fully sequenced. Results: Overall, 26,415 individuals were tested for SARS-CoV-2 between 18 March and 18 September 2020, of whom 21.0% (5,553/26,145) returned positive. Among laboratory-confirmed SARS-CoV-2 positive patients, the median age was 39 years (CI95%: 28-52), and 56.6% (3,311/5,553) were asymptomatic at the time of sampling. The probability of testing positive increased with age with the highest adjusted odds ratio of 2.2 [95% CI: 1.9-2.5] for individuals aged 49 years and more. Viral strains sequenced belong to clades 19A, 20A, and 20B in favour of several independent introduction of viruses. Conclusions. Our study describes the first wave of the COVID-19 in Madagascar. Despite early strategies in place Madagascar could not avoid the introduction and spread of the virus. More studies are needed to estimate the true burden of disease and make public health recommendations for a better preparation to another wave.

COVID-19 ARDS is characterized by a dysregulated host response that differs from cytokine storm and is modified by dexamethasone (preprint)

We performed comparative lower respiratory tract transcriptional profiling of 52 critically ill patients with the acute respiratory distress syndrome (ARDS) from COVID-19 or from other etiologies, as well as controls without ARDS. In contrast to a cytokine storm, we observed reduced proinflammatory gene expression in COVID-19 ARDS when compared to ARDS due to other causes. COVID-19 ARDS was characterized by a dysregulated host response with increased PTEN signaling and elevated expression of genes with non-canonical roles in inflammation and immunity that were predicted to be modulated by dexamethasone and granulocyte colony stimulating factor. Compared to ARDS due to other types of viral pneumonia, COVID-19 was characterized by impaired interferon-stimulated gene expression (ISG). We found that the relationship between SARS-CoV-2 viral load and expression of ISGs was decoupled in patients with COVID-19 ARDS when compared to patients with mild COVID-19. In summary, assessment of host gene expression in the lower airways of patients with COVID-19 ARDS did not demonstrate cytokine storm but instead revealed a unique and dysregulated host response predicted to be modified by dexamethasone.

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%.

IDseq – An Open Source Cloud-based Pipeline and Analysis Service for Metagenomic Pathogen Detection and Monitoring (preprint)

ABSTRACT Background Metagenomic next generation sequencing (mNGS) has enabled the rapid, unbiased detection and identification of microbes without pathogen-specific reagents, culturing, or a priori knowledge of the microbial landscape. mNGS data analysis requires a series of computationally intensive processing steps to accurately determine the microbial composition of a sample. Existing mNGS data analysis tools typically require bioinformatics expertise and access to local server-class hardware resources. For many research laboratories, this presents an obstacle, especially in resource limited environments. Findings We present IDseq, an open source cloud-based metagenomics pipeline and service for global pathogen detection and monitoring ( https://idseq.net ). The IDseq Portal accepts raw mNGS data, performs host and quality filtration steps, then executes an assembly-based alignment pipeline which results in the assignment of reads and contigs to taxonomic categories. The taxonomic relative abundances are reported and visualized in an easy-to-use web application to facilitate data interpretation and hypothesis generation. Furthermore, IDseq supports environmental background model generation and automatic internal spike-in control recognition, providing statistics which are critical for data interpretation. IDseq was designed with the specific intent of detecting novel pathogens. Here, we benchmark novel virus detection capability using both synthetically evolved viral sequences, and real-world samples, including IDseq analysis of a nasopharyngeal swab sample acquired and processed locally in Cambodia from a tourist from Wuhan, China, infected with the recently emergent SARS-CoV-2. Conclusion The IDseq Portal reduces the barrier to entry for mNGS data analysis and enables bench scientists, clinicians, and bioinformaticians to gain insight from mNGS datasets for both known and novel pathogens.

Rapid metagenomic characterization of a case of imported COVID-19 in Cambodia (preprint)

Rapid production and publication of pathogen genome sequences during emerging disease outbreaks provide crucial public health information. In resource-limited settings, especially near an outbreak epicenter, conventional deep sequencing or bioinformatics are often challenging. Here we successfully used metagenomic next generation sequencing on an iSeq100 Illumina platform paired with an open-source bioinformatics pipeline to quickly characterize Cambodias first case of COVID-2019.