Prolonged silent carriage, genomic virulence potential and transmission between staff and patients characterize a neonatal intensive care unit (NICU) outbreak of methicillin-resistant Staphylococcus aureus (MRSA).

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen in neonatal intensive care units (NICU) that confers significant morbidity and mortality. OBJECTIVE: Improving our understanding of MRSA transmission dynamics, especially among high-risk patients, is an infection prevention priority. METHODS: We investigated a cluster of clinical MRSA cases in the NICU using a combination of epidemiologic review and whole-genome sequencing (WGS) of isolates from clinical and surveillance cultures obtained from patients and healthcare personnel (HCP). RESULTS: Phylogenetic analysis identified 2 genetically distinct phylogenetic clades and revealed multiple silent-transmission events between HCP and infants. The predominant outbreak strain harbored multiple virulence factors. Epidemiologic investigation and genomic analysis identified a HCP colonized with the dominant MRSA outbreak strain who cared for most NICU patients who were infected or colonized with the same strain, including 1 NICU patient with severe infection 7 months before the described outbreak. These results guided implementation of infection prevention interventions that prevented further transmission events. CONCLUSIONS: Silent transmission of MRSA between HCP and NICU patients likely contributed to a NICU outbreak involving a virulent MRSA strain. WGS enabled data-driven decision making to inform implementation of infection control policies that mitigated the outbreak. Prospective WGS coupled with epidemiologic analysis can be used to detect transmission events and prompt early implementation of control strategies.

Integrated host-microbe plasma metagenomics for sepsis diagnosis in a prospective cohort of critically ill adults.

We carried out integrated host and pathogen metagenomic RNA and DNA next generation sequencing (mNGS) of whole blood (n = 221) and plasma (n = 138) from critically ill patients following hospital admission. We assigned patients into sepsis groups on the basis of clinical and microbiological criteria. From whole-blood gene expression data, we distinguished patients with sepsis from patients with non-infectious systemic inflammatory conditions using a trained bagged support vector machine (bSVM) classifier (area under the receiver operating characteristic curve (AUC) = 0.81 in the training set; AUC = 0.82 in a held-out validation set). Plasma RNA also yielded a transcriptional signature of sepsis with several genes previously reported as sepsis biomarkers, and a bSVM sepsis diagnostic classifier (AUC = 0.97 training set; AUC = 0.77 validation set). Pathogen detection performance of plasma mNGS varied on the basis of pathogen and site of infection. To improve detection of virus, we developed a secondary transcriptomic classifier (AUC = 0.94 training set; AUC = 0.96 validation set). We combined host and microbial features to develop an integrated sepsis diagnostic model that identified 99% of microbiologically confirmed sepsis cases, and predicted sepsis in 74% of suspected and 89% of indeterminate sepsis cases. In summary, we suggest that integrating host transcriptional profiling and broad-range metagenomic pathogen detection from nucleic acid is a promising tool for sepsis diagnosis.

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.

Physical Activity Associates With Lower Systemic Inflammatory Gene Expression in Rheumatoid Arthritis.

OBJECTIVE: While general population studies have shown inverse associations between physical activity and common inflammatory biomarkers, the effects of physical activity on inflammatory gene expression and signaling pathways in rheumatoid arthritis (RA) remain unknown. We aimed to determine whether physical activity independently associates with expression of inflammatory genes among people with RA. METHODS: This was a prospective observational study of adults with RA. Physical activity was measured by quantitative actigraphy over 7 consecutive days, and peripheral blood collected during the same time period was used for RNA sequencing followed by differential gene expression, pathway, and network analyses. RESULTS: Actigraphy and RNA sequencing data were evaluated in 35 patients. The cohort had a mean age of 56 (SD 12) years, and was 91% female, 31% White, 9% Black, 9% Asian, and 40% Hispanic. We found 767 genes differentially expressed (adjusted P < 0.1) between patients in the greatest vs lowest physical activity tertiles, after adjusting for sex, age, race, and ethnicity. The most active patients exhibited dose-dependent downregulation of several immune signaling pathways implicated in RA pathogenesis. These included CD40, STAT3, TREM-1, interleukin (IL)-17A, IL-8, Toll-like receptor, and interferon (IFN) signaling pathways. Upstream cytokine activation state analysis predicted reduced activation of tumor necrosis factor-α and IFN in the most active group. In sensitivity analyses, we adjusted for RA disease activity and physical function and found consistent results. CONCLUSION: Patients with RA who were more physically active had lower expression of immune signaling pathways implicated in RA pathogenesis, even after adjusting for disease activity, suggesting that physical activity may confer a protective effect in RA.

Metagenomic prediction of antimicrobial resistance in critically ill patients with lower respiratory tract infections.

BACKGROUND: Antimicrobial resistance (AMR) is rising at an alarming rate and complicating the management of infectious diseases including lower respiratory tract infections (LRTI). Metagenomic next-generation sequencing (mNGS) is a recently established method for culture-independent LRTI diagnosis, but its utility for predicting AMR has remained unclear. We aimed to assess the performance of mNGS for AMR prediction in bacterial LRTI and demonstrate proof of concept for epidemiological AMR surveillance and rapid AMR gene detection using Cas9 enrichment and nanopore sequencing. METHODS: We studied 88 patients with acute respiratory failure between 07/2013 and 9/2018, enrolled through a previous observational study of LRTI. Inclusion criteria were age ≥ 18, need for mechanical ventilation, and respiratory specimen collection within 72 h of intubation. Exclusion criteria were decline of study participation, unclear LRTI status, or no matched RNA and DNA mNGS data from a respiratory specimen. Patients with LRTI were identified by clinical adjudication. mNGS was performed on lower respiratory tract specimens. The primary outcome was mNGS performance for predicting phenotypic antimicrobial susceptibility and was assessed in patients with LRTI from culture-confirmed bacterial pathogens with clinical antimicrobial susceptibility testing (n = 27 patients, n = 32 pathogens). Secondary outcomes included the association between hospital exposure and AMR gene burden in the respiratory microbiome (n = 88 patients), and AMR gene detection using Cas9 targeted enrichment and nanopore sequencing (n = 10 patients). RESULTS: Compared to clinical antimicrobial susceptibility testing, the performance of respiratory mNGS for predicting AMR varied by pathogen, antimicrobial, and nucleic acid type sequenced. For gram-positive bacteria, a combination of RNA + DNA mNGS achieved a sensitivity of 70% (95% confidence interval (CI) 47-87%) and specificity of 95% (CI 85-99%). For gram-negative bacteria, sensitivity was 100% (CI 87-100%) and specificity 64% (CI 48-78%). Patients with hospital-onset LRTI had a greater AMR gene burden in their respiratory microbiome versus those with community-onset LRTI (p = 0.00030), or those without LRTI (p = 0.0024). We found that Cas9 targeted sequencing could enrich for low abundance AMR genes by > 2500-fold and enabled their rapid detection using a nanopore platform. CONCLUSIONS: mNGS has utility for the detection and surveillance of resistant bacterial LRTI pathogens.

Functional Transcriptomic Studies of Immune Responses and Endotoxin Tolerance in Early Human Sepsis.

BACKGROUND: Limited studies have functionally evaluated the heterogeneity in early ex vivo immune responses during sepsis. Our aim was to characterize early sepsis ex vivo functional immune response heterogeneity by studying whole blood endotoxin responses and derive a transcriptional metric of ex vivo endotoxin response. METHODS: Blood collected within 24 h of hospital presentation from 40 septic patients was divided into two fractions and incubated with media (unstimulated) or endotoxin. Supernatants and cells were isolated, and responses measured using: supernatant cytokines, lung endothelial permeability after supernatant exposure, and RNA expression. A transcriptomic signature was derived in unstimulated cells to predict the ex vivo endotoxin response. The signature was tested in a separate cohort of 191 septic patients to evaluate for association with clinical outcome. Plasma biomarkers were quantified to measure in vivo host inflammation. RESULTS: Ex vivo response to endotoxin varied and was unrelated to immunosuppression, white blood cell count, or the causative pathogen. Thirty-five percent of patients demonstrated a minimal response to endotoxin, suggesting early immunosuppression. High ex vivo cytokine production by stimulated blood cells correlated with increased in vitro pulmonary endothelial cell permeability and was associated with attenuated in vivo host inflammation. A four-gene signature of endotoxin response detectable without the need for a functional assay was identified. When tested in a separate cohort of septic patients, its expression was inversely associated with hospital mortality. CONCLUSIONS: An attenuated ex vivo endotoxin response in early sepsis is associated with greater host in vivo inflammation and a worse clinical outcome.

Upper airway gene expression reveals a more robust innate and adaptive immune response to SARS-CoV-2 in children compared with older adults.

Unlike other respiratory viruses, SARS-CoV-2 disproportionately causes severe disease in older adults and only rarely in children. To investigate whether differences in the upper airway immune response could contribute to this disparity, we compared 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 adults (>40-years-old; 45 with SARS-CoV-2, 28 with other respiratory viruses, 81 with no virus). Expression of interferon-stimulated genes (ISGs) was robustly activated in both children and adults with SARS-CoV-2 compared to the respective non-viral groups, with only relatively subtle distinctions. Children, however, demonstrated markedly greater upregulation of pathways related to B cell and T cell activation and proinflammatory cytokine signaling, including TNF, IFNγ, IL-2 and IL-4 production. Cell type deconvolution confirmed greater recruitment of B cells, and to a lesser degree macrophages, to the upper airway of children. Only children exhibited a decrease in proportions of ciliated cells, the primary target of SARS-CoV-2, upon infection with the virus. These findings demonstrate that children elicit a more robust innate and adaptive immune response to SARS-CoV-2 infection 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.

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.

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 we assessed lower respiratory tract immune responses and microbiome dynamics in 23 COVID-19 patients, 10 of whom developed VAP, and eight critically ill uninfected controls. At a median of three days (range: 2-4 days) before VAP onset we observed a transcriptional signature of bacterial infection. At a median of 15 days prior to VAP onset (range: 8-38 days), 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.

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

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.

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.

Draft Genome Sequence of an Extensively Drug-Resistant Salmonella enterica Serovar Typhi Strain from a Returned Traveler from Pakistan.

We report a draft genome sequence of extensively drug-resistant (XDR) Salmonella enterica serotype Typhi isolated from a returned traveler from Pakistan who developed sepsis. Whole-genome sequencing revealed relatedness to a previously reported outbreak in Pakistan and identified the bla CTX-M-15 and qnrS resistance genes.

Upper airway gene expression differentiates COVID-19 from other acute respiratory illnesses and reveals suppression of innate immune responses by SARS-CoV-2.

We studied the host transcriptional response to SARS-CoV-2 by performing metagenomic sequencing of upper airway samples in 238 patients with COVID-19, other viral or non-viral acute respiratory illnesses (ARIs). Compared to other viral ARIs, COVID-19 was characterized by a diminished innate immune response, with reduced expression of genes involved in toll-like receptor and interleukin signaling, chemokine binding, neutrophil degranulation and interactions with lymphoid cells. Patients with COVID-19 also exhibited significantly reduced proportions of neutrophils and macrophages, and increased proportions of goblet, dendritic and B-cells, compared to other viral ARIs. Using machine learning, we built 26-, 10- and 3-gene classifiers that differentiated COVID-19 from other acute respiratory illnesses with AUCs of 0.980, 0.950 and 0.871, respectively. Classifier performance was stable at low viral loads, suggesting utility in settings where direct detection of viral nucleic acid may be unsuccessful. Taken together, our results illuminate unique aspects of the host transcriptional response to SARS-CoV-2 in comparison to other respiratory viruses and demonstrate the feasibility of COVID-19 diagnostics based on patient gene expression.

Investigating Transfusion-related Sepsis Using Culture-Independent Metagenomic Sequencing.

BACKGROUND: Transfusion-related sepsis remains an important hospital infection control challenge. Investigation of septic transfusion events is often restricted by the limitations of bacterial culture in terms of time requirements and low yield in the setting of prior antibiotic administration. METHODS: In 3 gram-negative septic transfusion cases, we performed metagenomic next-generation sequencing (mNGS) of direct clinical blood specimens in addition to standard culture-based approaches utilized for infection control investigations. Pathogen detection leveraged IDSeq, a new open-access microbial bioinformatics portal. Phylogenetic analysis was performed to assess microbial genetic relatedness and understand transmission events. RESULTS: mNGS of direct clinical blood specimens afforded precision detection of pathogens responsible for each case of transfusion-related sepsis and enabled discovery of a novel Acinetobacter species in a platelet product that had become contaminated despite photochemical pathogen reduction. In each case, longitudinal assessment of pathogen burden elucidated the temporal sequence of events associated with each transfusion-transmitted infection. We found that informative data could be obtained from culture-independent mNGS of residual platelet products and leftover blood specimens that were either unsuitable or unavailable for culture or that failed to grow due to prior antibiotic administration. We additionally developed methods to enhance accuracy for detecting transfusion-associated pathogens that share taxonomic similarity to contaminants commonly found in mNGS library preparations. CONCLUSIONS: Culture-independent mNGS of blood products afforded rapid and precise assessment of pathogen identity, abundance, and genetic relatedness. Together, these challenging cases demonstrated the potential for metagenomics to advance existing methods for investigating transfusion-transmitted infections.

Sentinel Case of Candida auris in the Western United States Following Prolonged Occult Colonization in a Returned Traveler from India.

Candida auris is an emerging multidrug-resistant yeast with high mortality. We report the sentinel C. auris case on the United States West Coast in a patient who relocated from India. We identified close phylogenetic relatedness to the South Asia clade and ERG11 Y132F and FKS1 S639Y mutations potentially explaining antifungal resistance.