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

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 nonviral conditions (n = 318) to develop support vector machine classifiers that rely on a parsimonious 2-gene host signature to diagnose COVID-19. We find that optimal classifiers include an interferon-stimulated gene that is strongly induced in COVID-19 compared with nonviral conditions, such as IFI6, and a second immune-response gene that is more strongly induced in other viral infections, such as GBP5. The IFI6+GBP5 classifier achieves an area under the receiver operating characteristic curve (AUC) greater than 0.9 when evaluated on an independent RNA-seq cohort (n = 553). We further provide proof-of-concept demonstration that the classifier can be implemented in a clinically relevant RT-qPCR assay. Finally, we show that its performance is robust across common SARS-CoV-2 variants and is unaffected by cross-contamination, demonstrating its utility for improved accuracy of COVID-19 diagnostics. IMPORTANCE In this work, we study upper respiratory tract gene expression to develop and validate a 2-gene host-based COVID-19 diagnostic classifier and then demonstrate its implementation in a clinically practical qPCR assay. We find that the host classifier has utility for mitigating false-negative results, for example due to SARS-CoV-2 variants harboring mutations at primer target sites, and for mitigating false-positive viral PCR results due to laboratory cross-contamination. Both types of error carry serious consequences of either unrecognized viral transmission or unnecessary isolation and contact tracing. This work is directly relevant to the ongoing COVID-19 pandemic given the continued emergence of viral variants and the continued challenges of false-positive PCR assays. It also suggests the feasibility of pan-respiratory virus host-based diagnostics that would have value in congregate settings, such as hospitals and nursing homes, where unrecognized respiratory viral transmission is of particular concern.

SARS-CoV-2 infection of airway organoids reveals conserved use of Tetraspanin-8 by Ancestral, Delta, and Omicron variants.

Ancestral SARS coronavirus-2 (SARS-CoV-2) and variants of concern (VOC) caused a global pandemic with a spectrum of disease severity. The mechanistic explaining variations related to airway epithelium are relatively understudied. Here, we biobanked airway organoids (AO) by preserving stem cell function. We optimized viral infection with H1N1/PR8 and comprehensively characterized epithelial responses to SARS-CoV-2 infection in phenotypically stable AO from 20 different subjects. We discovered Tetraspanin-8 (TSPAN8) as a facilitator of SARS-CoV-2 infection. TSPAN8 facilitates SARS-CoV-2 infection rates independently of ACE2-Spike interaction. In head-to-head comparisons with Ancestral SARS-CoV-2, Delta and Omicron VOC displayed lower overall infection rates of AO but triggered changes in epithelial response. All variants shared highest tropism for ciliated and goblet cells. TSPAN8-blocking antibodies diminish SARS-CoV-2 infection and may spur novel avenues for COVID-19 therapy.

Upper airway gene expression shows a more robust adaptive immune response to SARS-CoV-2 in children.

Unlike other respiratory viruses, SARS-CoV-2 disproportionately causes severe disease in older adults whereas disease burden in children is lower. To investigate whether differences in the upper airway immune response may contribute to this disparity, we compare nasopharyngeal gene expression in 83 children (<19-years-old; 38 with SARS-CoV-2, 11 with other respiratory viruses, 34 with no virus) and 154 older adults (>40-years-old; 45 with SARS-CoV-2, 28 with other respiratory viruses, 81 with no virus). Expression of interferon-stimulated genes is robustly activated in both children and adults with SARS-CoV-2 infection compared to the respective non-viral groups, with only subtle distinctions. Children, however, demonstrate markedly greater upregulation of pathways related to B cell and T cell activation and proinflammatory cytokine signaling, including response to TNF and production of IFNγ, IL-2 and IL-4. Cell type deconvolution confirms greater recruitment of B cells, and to a lesser degree macrophages, to the upper airway of children. Only children exhibit a decrease in proportions of ciliated cells, among the primary targets of SARS-CoV-2, upon infection. These findings demonstrate that children elicit a more robust innate and especially adaptive immune response to SARS-CoV-2 in the upper airway that likely contributes to their protection from severe disease in the lower airway.

Increased risk of severe clinical course of COVID-19 in carriers of HLA-C*04:01.

BACKGROUND: Since the beginning of the coronavirus disease 2019 (COVID-19) pandemic, there has been increasing urgency to identify pathophysiological characteristics leading to severe clinical course in patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Human leukocyte antigen alleles (HLA) have been suggested as potential genetic host factors that affect individual immune response to SARS-CoV-2. We sought to evaluate this hypothesis by conducting a multicenter study using HLA sequencing. METHODS: We analyzed the association between COVID-19 severity and HLAs in 435 individuals from Germany (n = 135), Spain (n = 133), Switzerland (n = 20) and the United States (n = 147), who had been enrolled from March 2020 to August 2020. This study included patients older than 18 years, diagnosed with COVID-19 and representing the full spectrum of the disease. Finally, we tested our results by meta-analysing data from prior genome-wide association studies (GWAS). FINDINGS: We describe a potential association of HLA-C*04:01 with severe clinical course of COVID-19. Carriers of HLA-C*04:01 had twice the risk of intubation when infected with SARS-CoV-2 (risk ratio 1.5 [95% CI 1.1-2.1], odds ratio 3.5 [95% CI 1.9-6.6], adjusted p-value = 0.0074). These findings are based on data from four countries and corroborated by independent results from GWAS. Our findings are biologically plausible, as HLA-C*04:01 has fewer predicted bindings sites for relevant SARS-CoV-2 peptides compared to other HLA alleles. INTERPRETATION: HLA-C*04:01 carrier state is associated with severe clinical course in SARS-CoV-2. Our findings suggest that HLA class I alleles have a relevant role in immune defense against SARS-CoV-2. FUNDING: Funded by Roche Sequencing Solutions, Inc.

Tracheal aspirate RNA sequencing identifies distinct immunological features of COVID-19 ARDS.

The immunological features that distinguish COVID-19-associated acute respiratory distress syndrome (ARDS) from other causes of ARDS are incompletely understood. Here, we report the results of comparative lower respiratory tract transcriptional profiling of tracheal aspirate from 52 critically ill patients with ARDS from COVID-19 or from other etiologies, as well as controls without ARDS. In contrast to a "cytokine storm," we observe reduced proinflammatory gene expression in COVID-19 ARDS when compared to ARDS due to other causes. COVID-19 ARDS is characterized by a dysregulated host response with increased PTEN signaling and elevated expression of genes with non-canonical roles in inflammation and immunity. In silico analysis of gene expression identifies several candidate drugs that may modulate gene expression in COVID-19 ARDS, including dexamethasone and granulocyte colony stimulating factor. Compared to ARDS due to other types of viral pneumonia, COVID-19 is characterized by impaired interferon-stimulated gene (ISG) expression. The relationship between SARS-CoV-2 viral load and expression of ISGs is 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 reveals distinct immunological features of COVID-19 ARDS.

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

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.

Upper airway gene expression reveals suppressed immune responses to SARS-CoV-2 compared with other respiratory viruses.

SARS-CoV-2 infection is characterized by peak viral load in the upper airway prior to or at the time of symptom onset, an unusual feature that has enabled widespread transmission of the virus and precipitated a global pandemic. How SARS-CoV-2 is able to achieve high titer in the absence of symptoms remains unclear. Here, we examine the upper airway host transcriptional response in patients with COVID-19 (n = 93), other viral (n = 41) or non-viral (n = 100) acute respiratory illnesses (ARIs). Compared with other viral ARIs, COVID-19 is characterized by a pronounced interferon response but attenuated activation of other innate immune pathways, including toll-like receptor, interleukin and chemokine signaling. The IL-1 and NLRP3 inflammasome pathways are markedly less responsive to SARS-CoV-2, commensurate with a signature of diminished neutrophil and macrophage recruitment. This pattern resembles previously described distinctions between symptomatic and asymptomatic viral infections and may partly explain the propensity for pre-symptomatic transmission in COVID-19. We further use machine learning to build 27-, 10- and 3-gene classifiers that differentiate COVID-19 from other ARIs with AUROCs of 0.981, 0.954 and 0.885, respectively. Classifier performance is stable across a wide range of viral load, suggesting utility in mitigating false positive or false negative results of direct SARS-CoV-2 tests.

Clinical features, diagnostics, and outcomes of patients presenting with acute respiratory illness: A retrospective cohort study of patients with and without COVID-19.

BACKGROUND: Most data on the clinical presentation, diagnostics, and outcomes of patients with COVID-19 have been presented as case series without comparison to patients with other acute respiratory illnesses. METHODS: We examined emergency department patients between February 3 and March 31, 2020 with an acute respiratory illness who were tested for SARS-CoV-2. We determined COVID-19 status by PCR and metagenomic next generation sequencing (mNGS). We compared clinical presentation, diagnostics, treatment, and outcomes. FINDINGS: Among 316 patients, 33 tested positive for SARS-CoV-2; 31 without COVID-19 tested positive for another respiratory virus. Among patients with additional viral testing (27/33), no SARS-CoV-2 co-infections were identified. Compared to those who tested negative, patients with COVID-19 reported longer symptoms duration (median 7d vs. 3d, p < 0.001). Patients with COVID-19 were more often hospitalized (79% vs. 56%, p = 0.014). When hospitalized, patients with COVID-19 had longer hospitalizations (median 10.7d vs. 4.7d, p < 0.001) and more often developed ARDS (23% vs. 3%, p < 0.001). Most comorbidities, medications, symptoms, vital signs, laboratories, treatments, and outcomes did not differ by COVID-19 status. INTERPRETATION: While we found differences in clinical features of COVID-19 compared to other acute respiratory illnesses, there was significant overlap in presentation and comorbidities. Patients with COVID-19 were more likely to be admitted to the hospital, have longer hospitalizations and develop ARDS, and were unlikely to have co-existent viral infections. FUNDING: National Center for Advancing Translational Sciences, National Heart Lung Blood Institute, National Institute of Allergy and Infectious Diseases, Chan Zuckerberg Biohub, Chan Zuckerberg Initiative.