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.

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.

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.

Launching a saliva-based SARS-CoV-2 surveillance testing program on a university campus.

Regular surveillance testing of asymptomatic individuals for SARS-CoV-2 has been center to SARS-CoV-2 outbreak prevention on college and university campuses. Here we describe the voluntary saliva testing program instituted at the University of California, Berkeley during an early period of the SARS-CoV-2 pandemic in 2020. The program was administered as a research study ahead of clinical implementation, enabling us to launch surveillance testing while continuing to optimize the assay. Results of both the testing protocol itself and the study participants' experience show how the program succeeded in providing routine, robust testing capable of contributing to outbreak prevention within a campus community and offer strategies for encouraging participation and a sense of civic responsibility.

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.