Browsing by Author "Burke, Thomas W"
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Item Open Access A blood-based host gene expression assay for early detection of respiratory viral infection: an index-cluster prospective cohort study.(The Lancet. Infectious diseases, 2020-09-24) McClain, Micah T; Constantine, Florica J; Nicholson, Bradly P; Nichols, Marshall; Burke, Thomas W; Henao, Ricardo; Jones, Daphne C; Hudson, Lori L; Jaggers, L Brett; Veldman, Timothy; Mazur, Anna; Park, Lawrence P; Suchindran, Sunil; Tsalik, Ephraim L; Ginsburg, Geoffrey S; Woods, Christopher WBACKGROUND:Early and accurate identification of individuals with viral infections is crucial for clinical management and public health interventions. We aimed to assess the ability of transcriptomic biomarkers to identify naturally acquired respiratory viral infection before typical symptoms are present. METHODS:In this index-cluster study, we prospectively recruited a cohort of undergraduate students (aged 18-25 years) at Duke University (Durham, NC, USA) over a period of 5 academic years. To identify index cases, we monitored students for the entire academic year, for the presence and severity of eight symptoms of respiratory tract infection using a daily web-based survey, with symptoms rated on a scale of 0-4. Index cases were defined as individuals who reported a 6-point increase in cumulative daily symptom score. Suspected index cases were visited by study staff to confirm the presence of reported symptoms of illness and to collect biospecimen samples. We then identified clusters of close contacts of index cases (ie, individuals who lived in close proximity to index cases, close friends, and partners) who were presumed to be at increased risk of developing symptomatic respiratory tract infection while under observation. We monitored each close contact for 5 days for symptoms and viral shedding and measured transcriptomic responses at each timepoint each day using a blood-based 36-gene RT-PCR assay. FINDINGS:Between Sept 1, 2009, and April 10, 2015, we enrolled 1465 participants. Of 264 index cases with respiratory tract infection symptoms, 150 (57%) had a viral cause confirmed by RT-PCR. Of their 555 close contacts, 106 (19%) developed symptomatic respiratory tract infection with a proven viral cause during the observation window, of whom 60 (57%) had the same virus as their associated index case. Nine viruses were detected in total. The transcriptomic assay accurately predicted viral infection at the time of maximum symptom severity (mean area under the receiver operating characteristic curve [AUROC] 0·94 [95% CI 0·92-0·96]), as well as at 1 day (0·87 [95% CI 0·84-0·90]), 2 days (0·85 [0·82-0·88]), and 3 days (0·74 [0·71-0·77]) before peak illness, when symptoms were minimal or absent and 22 (62%) of 35 individuals, 25 (69%) of 36 individuals, and 24 (82%) of 29 individuals, respectively, had no detectable viral shedding. INTERPRETATION:Transcriptional biomarkers accurately predict and diagnose infection across diverse viral causes and stages of disease and thus might prove useful for guiding the administration of early effective therapy, quarantine decisions, and other clinical and public health interventions in the setting of endemic and pandemic infectious diseases. FUNDING:US Defense Advanced Research Projects Agency.Item Open Access A comparison of host response strategies to distinguish bacterial and viral infection.(PloS one, 2021-01) Ross, Melissa; Henao, Ricardo; Burke, Thomas W; Ko, Emily R; McClain, Micah T; Ginsburg, Geoffrey S; Woods, Christopher W; Tsalik, Ephraim LObjectives
Compare three host response strategies to distinguish bacterial and viral etiologies of acute respiratory illness (ARI).Methods
In this observational cohort study, procalcitonin, a 3-protein panel (CRP, IP-10, TRAIL), and a host gene expression mRNA panel were measured in 286 subjects with ARI from four emergency departments. Multinomial logistic regression and leave-one-out cross validation were used to evaluate the protein and mRNA tests.Results
The mRNA panel performed better than alternative strategies to identify bacterial infection: AUC 0.93 vs. 0.83 for the protein panel and 0.84 for procalcitonin (P<0.02 for each comparison). This corresponded to a sensitivity and specificity of 92% and 83% for the mRNA panel, 81% and 73% for the protein panel, and 68% and 87% for procalcitonin, respectively. A model utilizing all three strategies was the same as mRNA alone. For the diagnosis of viral infection, the AUC was 0.93 for mRNA and 0.84 for the protein panel (p<0.05). This corresponded to a sensitivity and specificity of 89% and 82% for the mRNA panel, and 85% and 62% for the protein panel, respectively.Conclusions
A gene expression signature was the most accurate host response strategy for classifying subjects with bacterial, viral, or non-infectious ARI.Item Open Access A host gene expression approach for identifying triggers of asthma exacerbations.(PloS one, 2019-01) Lydon, Emily C; Bullard, Charles; Aydin, Mert; Better, Olga M; Mazur, Anna; Nicholson, Bradly P; Ko, Emily R; McClain, Micah T; Ginsburg, Geoffrey S; Woods, Chris W; Burke, Thomas W; Henao, Ricardo; Tsalik, Ephraim LRATIONALE:Asthma exacerbations often occur due to infectious triggers, but determining whether infection is present and whether it is bacterial or viral remains clinically challenging. A diagnostic strategy that clarifies these uncertainties could enable personalized asthma treatment and mitigate antibiotic overuse. OBJECTIVES:To explore the performance of validated peripheral blood gene expression signatures in discriminating bacterial, viral, and noninfectious triggers in subjects with asthma exacerbations. METHODS:Subjects with suspected asthma exacerbations of various etiologies were retrospectively selected for peripheral blood gene expression analysis from a pool of subjects previously enrolled in emergency departments with acute respiratory illness. RT-PCR quantified 87 gene targets, selected from microarray-based studies, followed by logistic regression modeling to define bacterial, viral, or noninfectious class. The model-predicted class was compared to clinical adjudication and procalcitonin. RESULTS:Of 46 subjects enrolled, 7 were clinically adjudicated as bacterial, 18 as viral, and 21 as noninfectious. Model prediction was congruent with clinical adjudication in 15/18 viral and 13/21 noninfectious cases, but only 1/7 bacterial cases. None of the adjudicated bacterial cases had confirmatory microbiology; the precise etiology in this group was uncertain. Procalcitonin classified only one subject in the cohort as bacterial. 47.8% of subjects received antibiotics. CONCLUSIONS:Our model classified asthma exacerbations by the underlying bacterial, viral, and noninfectious host response. Compared to clinical adjudication, the majority of discordances occurred in the bacterial group, due to either imperfect adjudication or model misclassification. Bacterial infection was identified infrequently by all classification schemes, but nearly half of subjects were prescribed antibiotics. A gene expression-based approach may offer useful diagnostic information in this population and guide appropriate antibiotic use.Item Open Access A miRNA Host Response Signature Accurately Discriminates Acute Respiratory Infection Etiologies.(Frontiers in microbiology, 2018-01) Poore, Gregory D; Ko, Emily R; Valente, Ashlee; Henao, Ricardo; Sumner, Kelsey; Hong, Christopher; Burke, Thomas W; Nichols, Marshall; McClain, Micah T; Huang, Erich S; Ginsburg, Geoffrey S; Woods, Christopher W; Tsalik, Ephraim LBackground: Acute respiratory infections (ARIs) are the leading indication for antibacterial prescriptions despite a viral etiology in the majority of cases. The lack of available diagnostics to discriminate viral and bacterial etiologies contributes to this discordance. Recent efforts have focused on the host response as a source for novel diagnostic targets although none have explored the ability of host-derived microRNAs (miRNA) to discriminate between these etiologies. Methods: In this study, we compared host-derived miRNAs and mRNAs from human H3N2 influenza challenge subjects to those from patients with Streptococcus pneumoniae pneumonia. Sparse logistic regression models were used to generate miRNA signatures diagnostic of ARI etiologies. Generalized linear modeling of mRNAs to identify differentially expressed (DE) genes allowed analysis of potential miRNA:mRNA relationships. High likelihood miRNA:mRNA interactions were examined using binding target prediction and negative correlation to further explore potential changes in pathway regulation in response to infection. Results: The resultant miRNA signatures were highly accurate in discriminating ARI etiologies. Mean accuracy was 100% [88.8-100; 95% Confidence Interval (CI)] in discriminating the healthy state from S. pneumoniae pneumonia and 91.3% (72.0-98.9; 95% CI) in discriminating S. pneumoniae pneumonia from influenza infection. Subsequent differential mRNA gene expression analysis revealed alterations in regulatory networks consistent with known biology including immune cell activation and host response to viral infection. Negative correlation network analysis of miRNA:mRNA interactions revealed connections to pathways with known immunobiology such as interferon regulation and MAP kinase signaling. Conclusion: We have developed novel human host-response miRNA signatures for bacterial and viral ARI etiologies. miRNA host response signatures reveal accurate discrimination between S. pneumoniae pneumonia and influenza etiologies for ARI and integrated analyses of the host-pathogen interface are consistent with expected biology. These results highlight the differential miRNA host response to bacterial and viral etiologies of ARI, offering new opportunities to distinguish these entities.Item Open Access An atlas connecting shared genetic architecture of human diseases and molecular phenotypes provides insight into COVID-19 susceptibility.(Genome medicine, 2021-05) Wang, Liuyang; Balmat, Thomas J; Antonia, Alejandro L; Constantine, Florica J; Henao, Ricardo; Burke, Thomas W; Ingham, Andy; McClain, Micah T; Tsalik, Ephraim L; Ko, Emily R; Ginsburg, Geoffrey S; DeLong, Mark R; Shen, Xiling; Woods, Christopher W; Hauser, Elizabeth R; Ko, Dennis CBackground
While genome-wide associations studies (GWAS) have successfully elucidated the genetic architecture of complex human traits and diseases, understanding mechanisms that lead from genetic variation to pathophysiology remains an important challenge. Methods are needed to systematically bridge this crucial gap to facilitate experimental testing of hypotheses and translation to clinical utility.Results
Here, we leveraged cross-phenotype associations to identify traits with shared genetic architecture, using linkage disequilibrium (LD) information to accurately capture shared SNPs by proxy, and calculate significance of enrichment. This shared genetic architecture was examined across differing biological scales through incorporating data from catalogs of clinical, cellular, and molecular GWAS. We have created an interactive web database (interactive Cross-Phenotype Analysis of GWAS database (iCPAGdb)) to facilitate exploration and allow rapid analysis of user-uploaded GWAS summary statistics. This database revealed well-known relationships among phenotypes, as well as the generation of novel hypotheses to explain the pathophysiology of common diseases. Application of iCPAGdb to a recent GWAS of severe COVID-19 demonstrated unexpected overlap of GWAS signals between COVID-19 and human diseases, including with idiopathic pulmonary fibrosis driven by the DPP9 locus. Transcriptomics from peripheral blood of COVID-19 patients demonstrated that DPP9 was induced in SARS-CoV-2 compared to healthy controls or those with bacterial infection. Further investigation of cross-phenotype SNPs associated with both severe COVID-19 and other human traits demonstrated colocalization of the GWAS signal at the ABO locus with plasma protein levels of a reported receptor of SARS-CoV-2, CD209 (DC-SIGN). This finding points to a possible mechanism whereby glycosylation of CD209 by ABO may regulate COVID-19 disease severity.Conclusions
Thus, connecting genetically related traits across phenotypic scales links human diseases to molecular and cellular measurements that can reveal mechanisms and lead to novel biomarkers and therapeutic approaches. The iCPAGdb web portal is accessible at http://cpag.oit.duke.edu and the software code at https://github.com/tbalmat/iCPAGdb .Item Open Access Assessment of the Feasibility of Using Noninvasive Wearable Biometric Monitoring Sensors to Detect Influenza and the Common Cold Before Symptom Onset.(JAMA network open, 2021-09) Grzesiak, Emilia; Bent, Brinnae; McClain, Micah T; Woods, Christopher W; Tsalik, Ephraim L; Nicholson, Bradly P; Veldman, Timothy; Burke, Thomas W; Gardener, Zoe; Bergstrom, Emma; Turner, Ronald B; Chiu, Christopher; Doraiswamy, P Murali; Hero, Alfred; Henao, Ricardo; Ginsburg, Geoffrey S; Dunn, JessilynImportance
Currently, there are no presymptomatic screening methods to identify individuals infected with a respiratory virus to prevent disease spread and to predict their trajectory for resource allocation.Objective
To evaluate the feasibility of using noninvasive, wrist-worn wearable biometric monitoring sensors to detect presymptomatic viral infection after exposure and predict infection severity in patients exposed to H1N1 influenza or human rhinovirus.Design, setting, and participants
The cohort H1N1 viral challenge study was conducted during 2018; data were collected from September 11, 2017, to May 4, 2018. The cohort rhinovirus challenge study was conducted during 2015; data were collected from September 14 to 21, 2015. A total of 39 adult participants were recruited for the H1N1 challenge study, and 24 adult participants were recruited for the rhinovirus challenge study. Exclusion criteria for both challenges included chronic respiratory illness and high levels of serum antibodies. Participants in the H1N1 challenge study were isolated in a clinic for a minimum of 8 days after inoculation. The rhinovirus challenge took place on a college campus, and participants were not isolated.Exposures
Participants in the H1N1 challenge study were inoculated via intranasal drops of diluted influenza A/California/03/09 (H1N1) virus with a mean count of 106 using the median tissue culture infectious dose (TCID50) assay. Participants in the rhinovirus challenge study were inoculated via intranasal drops of diluted human rhinovirus strain type 16 with a count of 100 using the TCID50 assay.Main outcomes and measures
The primary outcome measures included cross-validated performance metrics of random forest models to screen for presymptomatic infection and predict infection severity, including accuracy, precision, sensitivity, specificity, F1 score, and area under the receiver operating characteristic curve (AUC).Results
A total of 31 participants with H1N1 (24 men [77.4%]; mean [SD] age, 34.7 [12.3] years) and 18 participants with rhinovirus (11 men [61.1%]; mean [SD] age, 21.7 [3.1] years) were included in the analysis after data preprocessing. Separate H1N1 and rhinovirus detection models, using only data on wearble devices as input, were able to distinguish between infection and noninfection with accuracies of up to 92% for H1N1 (90% precision, 90% sensitivity, 93% specificity, and 90% F1 score, 0.85 [95% CI, 0.70-1.00] AUC) and 88% for rhinovirus (100% precision, 78% sensitivity, 100% specificity, 88% F1 score, and 0.96 [95% CI, 0.85-1.00] AUC). The infection severity prediction model was able to distinguish between mild and moderate infection 24 hours prior to symptom onset with an accuracy of 90% for H1N1 (88% precision, 88% sensitivity, 92% specificity, 88% F1 score, and 0.88 [95% CI, 0.72-1.00] AUC) and 89% for rhinovirus (100% precision, 75% sensitivity, 100% specificity, 86% F1 score, and 0.95 [95% CI, 0.79-1.00] AUC).Conclusions and relevance
This cohort study suggests that the use of a noninvasive, wrist-worn wearable device to predict an individual's response to viral exposure prior to symptoms is feasible. Harnessing this technology would support early interventions to limit presymptomatic spread of viral respiratory infections, which is timely in the era of COVID-19.Item Open Access Blood RNA alternative splicing events as diagnostic biomarkers for infectious disease(Cell Reports Methods, 2023-01-01) Zhang, Zijun; Sauerwald, Natalie; Cappuccio, Antonio; Ramos, Irene; Nair, Venugopalan D; Nudelman, German; Zaslavsky, Elena; Ge, Yongchao; Gaitas, Angelo; Ren, Hui; Brockman, Joel; Geis, Jennifer; Ramalingam, Naveen; King, David; McClain, Micah T; Woods, Christopher W; Henao, Ricardo; Burke, Thomas W; Tsalik, Ephraim L; Goforth, Carl W; Lizewski, Rhonda A; Lizewski, Stephen E; Weir, Dawn L; Letizia, Andrew G; Sealfon, Stuart C; Troyanskaya, Olga GAssays detecting blood transcriptome changes are studied for infectious disease diagnosis. Blood-based RNA alternative splicing (AS) events, which have not been well characterized in pathogen infection, have potential normalization and assay platform stability advantages over gene expression for diagnosis. Here, we present a computational framework for developing AS diagnostic biomarkers. Leveraging a large prospective cohort of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and whole-blood RNA sequencing (RNA-seq) data, we identify a major functional AS program switch upon viral infection. Using an independent cohort, we demonstrate the improved accuracy of AS biomarkers for SARS-CoV-2 diagnosis compared with six reported transcriptome signatures. We then optimize a subset of AS-based biomarkers to develop microfluidic PCR diagnostic assays. This assay achieves nearly perfect test accuracy (61/62 = 98.4%) using a naive principal component classifier, significantly more accurate than a gene expression PCR assay in the same cohort. Therefore, our RNA splicing computational framework enables a promising avenue for host-response diagnosis of infection.Item Open Access Chromatin remodeling in peripheral blood cells reflects COVID-19 symptom severity.(bioRxiv, 2020-12-05) Giroux, Nicholas S; Ding, Shengli; McClain, Micah T; Burke, Thomas W; Petzold, Elizabeth; Chung, Hong A; Palomino, Grecia R; Wang, Ergang; Xi, Rui; Bose, Shree; Rotstein, Tomer; Nicholson, Bradly P; Chen, Tianyi; Henao, Ricardo; Sempowski, Gregory D; Denny, Thomas N; Ko, Emily R; Ginsburg, Geoffrey S; Kraft, Bryan D; Tsalik, Ephraim L; Woods, Christopher W; Shen, XilingSARS-CoV-2 infection triggers highly variable host responses and causes varying degrees of illness in humans. We sought to harness the peripheral blood mononuclear cell (PBMC) response over the course of illness to provide insight into COVID-19 physiology. We analyzed PBMCs from subjects with variable symptom severity at different stages of clinical illness before and after IgG seroconversion to SARS-CoV-2. Prior to seroconversion, PBMC transcriptomes did not distinguish symptom severity. In contrast, changes in chromatin accessibility were associated with symptom severity. Furthermore, single-cell analyses revealed evolution of the chromatin accessibility landscape and transcription factor motif occupancy for individual PBMC cell types. The most extensive remodeling occurred in CD14+ monocytes where sub-populations with distinct chromatin accessibility profiles were associated with disease severity. Our findings indicate that pre-seroconversion chromatin remodeling in certain innate immune populations is associated with divergence in symptom severity, and the identified transcription factors, regulatory elements, and downstream pathways provide potential prognostic markers for COVID-19 subjects.Item Unknown COVID-19 Diagnosis and SARS-CoV-2 Strain Identification by a Rapid, Multiplexed, Point-of-Care Antibody Microarray.(Analytical chemistry, 2023-03) Heggestad, Jacob T; Britton, Rhett J; Kinnamon, David S; Liu, Jason; Anderson, Jack G; Joh, Daniel Y; Quinn, Zachary; Fontes, Cassio M; Hucknall, Angus M; Parks, Robert; Sempowski, Gregory D; Denny, Thomas N; Burke, Thomas W; Haynes, Barton F; Woods, Christopher W; Chilkoti, AshutoshAntigen tests to detect SARS-CoV-2 have emerged as a promising rapid diagnostic method for COVID-19, but they are unable to differentiate between variants of concern (VOCs). Here, we report a rapid point-of-care test (POC-T), termed CoVariant-SPOT, that uses a set of antibodies that are either tolerant or intolerant to spike protein mutations to identify the likely SARS-CoV-2 strain concurrent with COVID-19 diagnosis using antibodies targeting the nucleocapsid protein. All reagents are incorporated into a portable, multiplexed, and sensitive diagnostic platform built upon a nonfouling polymer brush. To validate CoVariant-SPOT, we tested recombinant SARS-CoV-2 proteins, inactivated viruses, and nasopharyngeal swab samples from COVID-19 positive and negative individuals and showed that CoVariant-SPOT can readily distinguish between two VOCs: Delta and Omicron. We believe that CoVariant-SPOT can serve as a valuable adjunct to next-generation sequencing to rapidly identify variants using a scalable and deployable POC-T, thereby enhancing community surveillance efforts worldwide and informing treatment selection.Item Unknown Differential chromatin accessibility in peripheral blood mononuclear cells underlies COVID-19 disease severity prior to seroconversion.(Scientific reports, 2022-07-09) Giroux, Nicholas S; Ding, Shengli; McClain, Micah T; Burke, Thomas W; Petzold, Elizabeth; Chung, Hong A; Rivera, Grecia O; Wang, Ergang; Xi, Rui; Bose, Shree; Rotstein, Tomer; Nicholson, Bradly P; Chen, Tianyi; Henao, Ricardo; Sempowski, Gregory D; Denny, Thomas N; De Ussel, Maria Iglesias; Satterwhite, Lisa L; Ko, Emily R; Ginsburg, Geoffrey S; Kraft, Bryan D; Tsalik, Ephraim L; Shen, Xiling; Woods, Christopher WSARS-CoV-2 infection triggers profound and variable immune responses in human hosts. Chromatin remodeling has been observed in individuals severely ill or convalescing with COVID-19, but chromatin remodeling early in disease prior to anti-spike protein IgG seroconversion has not been defined. We performed the Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) and RNA-seq on peripheral blood mononuclear cells (PBMCs) from outpatients with mild or moderate symptom severity at different stages of clinical illness. Early in the disease course prior to IgG seroconversion, modifications in chromatin accessibility associated with mild or moderate symptoms were already robust and included severity-associated changes in accessibility of genes in interleukin signaling, regulation of cell differentiation and cell morphology. Furthermore, single-cell analyses revealed evolution of the chromatin accessibility landscape and transcription factor motif accessibility for individual PBMC cell types over time. The most extensive remodeling occurred in CD14+ monocytes, where sub-populations with distinct chromatin accessibility profiles were observed prior to seroconversion. Mild symptom severity was marked by upregulation of classical antiviral pathways, including those regulating IRF1 and IRF7, whereas in moderate disease, these classical antiviral signals diminished, suggesting dysregulated and less effective responses. Together, these observations offer novel insight into the epigenome of early mild SARS-CoV-2 infection and suggest that detection of chromatin remodeling in early disease may offer promise for a new class of diagnostic tools for COVID-19.Item Unknown Differential chromatin accessibility in peripheral blood mononuclear cells underlies COVID-19 disease severity prior to seroconversion.(Res Sq, 2022-04-07) Giroux, Nicholas S; Ding, Shengli; McClain, Micah T; Burke, Thomas W; Petzold, Elizabeth; Chung, Hong A; Rivera, Grecia O; Wang, Ergang; Xi, Rui; Bose, Shree; Rotstein, Tomer; Nicholson, Bradly P; Chen, Tianyi; Henao, Ricardo; Sempowski, Gregory D; Denny, Thomas N; De Ussel, Maria Iglesias; Satterwhite, Lisa L; Ko, Emily R; Ginsburg, Geoffrey S; Kraft, Bryan D; Tsalik, Ephraim L; Shen, Xiling; Woods, ChristopherSARS-CoV-2 infection triggers profound and variable immune responses in human hosts. Chromatin remodeling has been observed in individuals severely ill or convalescing with COVID-19, but chromatin remodeling early in disease prior to anti-spike protein IgG seroconversion has not been defined. We performed the Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) and RNA-seq on peripheral blood mononuclear cells (PBMCs) from outpatients with mild or moderate symptom severity at different stages of clinical illness. Early in the disease course prior to IgG seroconversion, modifications in chromatin accessibility associate with mild or moderate symptoms are already robust and include severity-associated changes in accessibility of genes in interleukin signaling, regulation of cell differentiation and cell morphology. Furthermore, single-cell analyses revealed evolution of the chromatin accessibility landscape and transcription factor motif accessibility for individual PBMC cell types over time. The most extensive remodeling occurred in CD14+ monocytes, where sub-populations with distinct chromatin accessibility profiles were observed prior to seroconversion. Mild symptom severity is marked by upregulation classical antiviral pathways including those regulating IRF1 and IRF7, whereas in moderate disease these classical antiviral signals diminish suggesting dysregulated and less effective responses. Together, these observations offer novel insight into the epigenome of early mild SARS-CoV-2 infection and suggest that detection of chromatin remodeling in early disease may offer promise for a new class of diagnostic tools for COVID-19.Item Unknown Direct-from-blood RNA sequencing identifies the cause of post-bronchoscopy fever.(BMC infectious diseases, 2019-10-28) Ko, Emily R; Philipson, Casandra W; Burke, Thomas W; Cer, Regina Z; Bishop-Lilly, Kimberly A; Voegtly, Logan J; Tsalik, Ephraim L; Woods, Christopher W; Clark, Danielle V; Schully, Kevin LBACKGROUND:Antibiotic resistance is rising at disturbing rates and contributes to the deaths of millions of people yearly. Antibiotic resistant infections disproportionately affect those with immunocompromising conditions, chronic colonization, and frequent antibiotic use such as transplant patients or those with cystic fibrosis. However, clinicians lack the diagnostic tools to confidently diagnose and treat infections, leading to widespread use of empiric broad spectrum antimicrobials, often for prolonged duration. CASE PRESENTATION:A 22 year-old Caucasian female with cystic fibrosis received a bilateral orthotopic lung transplantation 5 months prior to the index hospitalization. She underwent routine surveillance bronchoscopy and was admitted for post-procedure fever. A clear cause of infection was not identified by routine methods. Imaging and bronchoscopic lung biopsy did not identify an infectious agent or rejection. She was treated with a prolonged course of antimicrobials targeting known colonizing organisms from prior bronchoalveolar lavage cultures (Pseudomonas, Staphylococcus aureus, and Aspergillus). However, we identified Stenotrophomonas maltophilia in two independent whole blood samples using direct-pathogen sequencing, which was not identified by other methods. CONCLUSIONS:This case represents a common clinical conundrum: identification of infection in a high-risk, complex patient. Here, direct-pathogen sequencing identified a pathogen that would not otherwise have been identified by common techniques. Had results been clinically available, treatment could have been customized, avoiding a prolonged course of broad spectrum antimicrobials that would only exacerbate resistance. Direct-pathogen sequencing is poised to fill a diagnostic gap for pathogen identification, allowing early identification and customization of treatment in a culture-independent, pathogen-agnostic manner.Item Unknown Discriminating Bacterial and Viral Infection Using a Rapid Host Gene Expression Test.(Critical care medicine, 2021-10) Tsalik, Ephraim L; Henao, Ricardo; Montgomery, Jesse L; Nawrocki, Jeff W; Aydin, Mert; Lydon, Emily C; Ko, Emily R; Petzold, Elizabeth; Nicholson, Bradly P; Cairns, Charles B; Glickman, Seth W; Quackenbush, Eugenia; Kingsmore, Stephen F; Jaehne, Anja K; Rivers, Emanuel P; Langley, Raymond J; Fowler, Vance G; McClain, Micah T; Crisp, Robert J; Ginsburg, Geoffrey S; Burke, Thomas W; Hemmert, Andrew C; Woods, Christopher W; Antibacterial Resistance Leadership GroupObjectives
Host gene expression signatures discriminate bacterial and viral infection but have not been translated to a clinical test platform. This study enrolled an independent cohort of patients to describe and validate a first-in-class host response bacterial/viral test.Design
Subjects were recruited from 2006 to 2016. Enrollment blood samples were collected in an RNA preservative and banked for later testing. The reference standard was an expert panel clinical adjudication, which was blinded to gene expression and procalcitonin results.Setting
Four U.S. emergency departments.Patients
Six-hundred twenty-three subjects with acute respiratory illness or suspected sepsis.Interventions
Forty-five-transcript signature measured on the BioFire FilmArray System (BioFire Diagnostics, Salt Lake City, UT) in ~45 minutes.Measurements and main results
Host response bacterial/viral test performance characteristics were evaluated in 623 participants (mean age 46 yr; 45% male) with bacterial infection, viral infection, coinfection, or noninfectious illness. Performance of the host response bacterial/viral test was compared with procalcitonin. The test provided independent probabilities of bacterial and viral infection in ~45 minutes. In the 213-subject training cohort, the host response bacterial/viral test had an area under the curve for bacterial infection of 0.90 (95% CI, 0.84-0.94) and 0.92 (95% CI, 0.87-0.95) for viral infection. Independent validation in 209 subjects revealed similar performance with an area under the curve of 0.85 (95% CI, 0.78-0.90) for bacterial infection and 0.91 (95% CI, 0.85-0.94) for viral infection. The test had 80.1% (95% CI, 73.7-85.4%) average weighted accuracy for bacterial infection and 86.8% (95% CI, 81.8-90.8%) for viral infection in this validation cohort. This was significantly better than 68.7% (95% CI, 62.4-75.4%) observed for procalcitonin (p < 0.001). An additional cohort of 201 subjects with indeterminate phenotypes (coinfection or microbiology-negative infections) revealed similar performance.Conclusions
The host response bacterial/viral measured using the BioFire System rapidly and accurately discriminated bacterial and viral infection better than procalcitonin, which can help support more appropriate antibiotic use.Item Unknown Dysregulated transcriptional responses to SARS-CoV-2 in the periphery support novel diagnostic approaches.(medRxiv, 2020-07-26) McClain, Micah T; Constantine, Florica J; Henao, Ricardo; Liu, Yiling; Tsalik, Ephraim L; Burke, Thomas W; Steinbrink, Julie M; Petzold, Elizabeth; Nicholson, Bradly P; Rolfe, Robert; Kraft, Bryan D; Kelly, Matthew S; Sempowski, Gregory D; Denny, Thomas N; Ginsburg, Geoffrey S; Woods, Christopher WIn order to elucidate novel aspects of the host response to SARS-CoV-2 we performed RNA sequencing on peripheral blood samples across 77 timepoints from 46 subjects with COVID-19 and compared them to subjects with seasonal coronavirus, influenza, bacterial pneumonia, and healthy controls. Early SARS-CoV-2 infection triggers a conserved transcriptomic response in peripheral blood that is heavily interferon-driven but also marked by indicators of early B-cell activation and antibody production. Interferon responses during SARS-CoV-2 infection demonstrate unique patterns of dysregulated expression compared to other infectious and healthy states. Heterogeneous activation of coagulation and fibrinolytic pathways are present in early COVID-19, as are IL1 and JAK/STAT signaling pathways, that persist into late disease. Classifiers based on differentially expressed genes accurately distinguished SARS-CoV-2 infection from other acute illnesses (auROC 0.95). The transcriptome in peripheral blood reveals unique aspects of the immune response in COVID-19 and provides for novel biomarker-based approaches to diagnosis.Item Unknown Dysregulated transcriptional responses to SARS-CoV-2 in the periphery.(Nature communications, 2021-02-17) McClain, Micah T; Constantine, Florica J; Henao, Ricardo; Liu, Yiling; Tsalik, Ephraim L; Burke, Thomas W; Steinbrink, Julie M; Petzold, Elizabeth; Nicholson, Bradly P; Rolfe, Robert; Kraft, Bryan D; Kelly, Matthew S; Saban, Daniel R; Yu, Chen; Shen, Xiling; Ko, Emily M; Sempowski, Gregory D; Denny, Thomas N; Ginsburg, Geoffrey S; Woods, Christopher WSARS-CoV-2 infection has been shown to trigger a wide spectrum of immune responses and clinical manifestations in human hosts. Here, we sought to elucidate novel aspects of the host response to SARS-CoV-2 infection through RNA sequencing of peripheral blood samples from 46 subjects with COVID-19 and directly comparing them to subjects with seasonal coronavirus, influenza, bacterial pneumonia, and healthy controls. Early SARS-CoV-2 infection triggers a powerful transcriptomic response in peripheral blood with conserved components that are heavily interferon-driven but also marked by indicators of early B-cell activation and antibody production. Interferon responses during SARS-CoV-2 infection demonstrate unique patterns of dysregulated expression compared to other infectious and healthy states. Heterogeneous activation of coagulation and fibrinolytic pathways are present in early COVID-19, as are IL1 and JAK/STAT signaling pathways, which persist into late disease. Classifiers based on differentially expressed genes accurately distinguished SARS-CoV-2 infection from other acute illnesses (auROC 0.95 [95% CI 0.92-0.98]). The transcriptome in peripheral blood reveals both diverse and conserved components of the immune response in COVID-19 and provides for potential biomarker-based approaches to diagnosis.Item Unknown Mucosal Associated Invariant T (MAIT) Cell Responses Differ by Sex in COVID-19.(Med (New York, N.Y.), 2021-04-13) Yu, Chen; Littleton, Sejiro; Giroux, Nicholas S; Mathew, Rose; Ding, Shengli; Kalnitsky, Joan; Yang, Yuchen; Petzold, Elizabeth; Chung, Hong A; Rivera, Grecia O; Rotstein, Tomer; Xi, Rui; Ko, Emily R; Tsalik, Ephraim L; Sempowski, Gregory D; Denny, Thomas N; Burke, Thomas W; McClain, Micah T; Woods, Christopher W; Shen, Xiling; Saban, Daniel RSexual dimorphisms in immune responses contribute to coronavirus disease 2019 (COVID-19) outcomes, yet the mechanisms governing this disparity remain incompletely understood. We carried out sex-balanced sampling of peripheral blood mononuclear cells from confirmed COVID-19 inpatients and outpatients, uninfected close contacts, and healthy controls for 36-color flow cytometry and single cell RNA-sequencing. Our results revealed a pronounced reduction of circulating mucosal associated invariant T (MAIT) cells in infected females. Integration of published COVID-19 airway tissue datasets implicate that this reduction represented a major wave of MAIT cell extravasation during early infection in females. Moreover, female MAIT cells possessed an immunologically active gene signature, whereas male counterparts were pro-apoptotic. Collectively, our findings uncover a female-specific protective MAIT profile, potentially shedding light on reduced COVID-19 susceptibility in females.Item Unknown Nasopharyngeal Protein Biomarkers of Acute Respiratory Virus Infection.(EBioMedicine, 2017-03) Burke, Thomas W; Henao, Ricardo; Soderblom, Erik; Tsalik, Ephraim L; Thompson, J Will; McClain, Micah T; Nichols, Marshall; Nicholson, Bradly P; Veldman, Timothy; Lucas, Joseph E; Moseley, M Arthur; Turner, Ronald B; Lambkin-Williams, Robert; Hero, Alfred O; Woods, Christopher W; Ginsburg, Geoffrey SInfection of respiratory mucosa with viral pathogens triggers complex immunologic events in the affected host. We sought to characterize this response through proteomic analysis of nasopharyngeal lavage in human subjects experimentally challenged with influenza A/H3N2 or human rhinovirus, and to develop targeted assays measuring peptides involved in this host response allowing classification of acute respiratory virus infection. Unbiased proteomic discovery analysis identified 3285 peptides corresponding to 438 unique proteins, and revealed that infection with H3N2 induces significant alterations in protein expression. These include proteins involved in acute inflammatory response, innate immune response, and the complement cascade. These data provide insights into the nature of the biological response to viral infection of the upper respiratory tract, and the proteins that are dysregulated by viral infection form the basis of signature that accurately classifies the infected state. Verification of this signature using targeted mass spectrometry in independent cohorts of subjects challenged with influenza or rhinovirus demonstrates that it performs with high accuracy (0.8623 AUROC, 75% TPR, 97.46% TNR). With further development as a clinical diagnostic, this signature may have utility in rapid screening for emerging infections, avoidance of inappropriate antibacterial therapy, and more rapid implementation of appropriate therapeutic and public health strategies.Item Unknown Previously Derived Host Gene Expression Classifiers Identify Bacterial and Viral Etiologies of Acute Febrile Respiratory Illness in a South Asian Population.(Open forum infectious diseases, 2020-06) Tillekeratne, L Gayani; Suchindran, Sunil; Ko, Emily R; Petzold, Elizabeth A; Bodinayake, Champica K; Nagahawatte, Ajith; Devasiri, Vasantha; Kurukulasooriya, Ruvini; Nicholson, Bradly P; McClain, Micah T; Burke, Thomas W; Tsalik, Ephraim L; Henao, Ricardo; Ginsburg, Geoffrey S; Reller, Megan E; Woods, Christopher WBackground:Pathogen-based diagnostics for acute respiratory infection (ARI) have limited ability to detect etiology of illness. We previously showed that peripheral blood-based host gene expression classifiers accurately identify bacterial and viral ARI in cohorts of European and African descent. We determined classifier performance in a South Asian cohort. Methods:Patients ≥15 years with fever and respiratory symptoms were enrolled in Sri Lanka. Comprehensive pathogen-based testing was performed. Peripheral blood ribonucleic acid was sequenced and previously developed signatures were applied: a pan-viral classifier (viral vs nonviral) and an ARI classifier (bacterial vs viral vs noninfectious). Results:Ribonucleic acid sequencing was performed in 79 subjects: 58 viral infections (36 influenza, 22 dengue) and 21 bacterial infections (10 leptospirosis, 11 scrub typhus). The pan-viral classifier had an overall classification accuracy of 95%. The ARI classifier had an overall classification accuracy of 94%, with sensitivity and specificity of 91% and 95%, respectively, for bacterial infection. The sensitivity and specificity of C-reactive protein (>10 mg/L) and procalcitonin (>0.25 ng/mL) for bacterial infection were 100% and 34%, and 100% and 41%, respectively. Conclusions:Previously derived gene expression classifiers had high predictive accuracy at distinguishing viral and bacterial infection in South Asian patients with ARI caused by typical and atypical pathogens.Item Unknown Prospective Validation of a Rapid Host Gene Expression Test to Discriminate Bacterial From Viral Respiratory Infection.(JAMA network open, 2022-04) Ko, Emily R; Henao, Ricardo; Frankey, Katherine; Petzold, Elizabeth A; Isner, Pamela D; Jaehne, Anja K; Allen, Nakia; Gardner-Gray, Jayna; Hurst, Gina; Pflaum-Carlson, Jacqueline; Jayaprakash, Namita; Rivers, Emanuel P; Wang, Henry; Ugalde, Irma; Amanullah, Siraj; Mercurio, Laura; Chun, Thomas H; May, Larissa; Hickey, Robert W; Lazarus, Jacob E; Gunaratne, Shauna H; Pallin, Daniel J; Jambaulikar, Guruprasad; Huckins, David S; Ampofo, Krow; Jhaveri, Ravi; Jiang, Yunyun; Komarow, Lauren; Evans, Scott R; Ginsburg, Geoffrey S; Tillekeratne, L Gayani; McClain, Micah T; Burke, Thomas W; Woods, Christopher W; Tsalik, Ephraim L; Antibacterial Resistance Leadership GroupImportance
Bacterial and viral causes of acute respiratory illness (ARI) are difficult to clinically distinguish, resulting in the inappropriate use of antibacterial therapy. The use of a host gene expression-based test that is able to discriminate bacterial from viral infection in less than 1 hour may improve care and antimicrobial stewardship.Objective
To validate the host response bacterial/viral (HR-B/V) test and assess its ability to accurately differentiate bacterial from viral infection among patients with ARI.Design, setting, and participants
This prospective multicenter diagnostic study enrolled 755 children and adults with febrile ARI of 7 or fewer days' duration from 10 US emergency departments. Participants were enrolled from October 3, 2014, to September 1, 2019, followed by additional enrollment of patients with COVID-19 from March 20 to December 3, 2020. Clinical adjudication of enrolled participants identified 616 individuals as having bacterial or viral infection. The primary analysis cohort included 334 participants with high-confidence reference adjudications (based on adjudicator concordance and the presence of an identified pathogen confirmed by microbiological testing). A secondary analysis of the entire cohort of 616 participants included cases with low-confidence reference adjudications (based on adjudicator discordance or the absence of an identified pathogen in microbiological testing). Thirty-three participants with COVID-19 were included post hoc.Interventions
The HR-B/V test quantified the expression of 45 host messenger RNAs in approximately 45 minutes to derive a probability of bacterial infection.Main outcomes and measures
Performance characteristics for the HR-B/V test compared with clinical adjudication were reported as either bacterial or viral infection or categorized into 4 likelihood groups (viral very likely [probability score <0.19], viral likely [probability score of 0.19-0.40], bacterial likely [probability score of 0.41-0.73], and bacterial very likely [probability score >0.73]) and compared with procalcitonin measurement.Results
Among 755 enrolled participants, the median age was 26 years (IQR, 16-52 years); 360 participants (47.7%) were female, and 395 (52.3%) were male. A total of 13 participants (1.7%) were American Indian, 13 (1.7%) were Asian, 368 (48.7%) were Black, 131 (17.4%) were Hispanic, 3 (0.4%) were Native Hawaiian or Pacific Islander, 297 (39.3%) were White, and 60 (7.9%) were of unspecified race and/or ethnicity. In the primary analysis involving 334 participants, the HR-B/V test had sensitivity of 89.8% (95% CI, 77.8%-96.2%), specificity of 82.1% (95% CI, 77.4%-86.6%), and a negative predictive value (NPV) of 97.9% (95% CI, 95.3%-99.1%) for bacterial infection. In comparison, the sensitivity of procalcitonin measurement was 28.6% (95% CI, 16.2%-40.9%; P < .001), the specificity was 87.0% (95% CI, 82.7%-90.7%; P = .006), and the NPV was 87.6% (95% CI, 85.5%-89.5%; P < .001). When stratified into likelihood groups, the HR-B/V test had an NPV of 98.9% (95% CI, 96.1%-100%) for bacterial infection in the viral very likely group and a positive predictive value of 63.4% (95% CI, 47.2%-77.9%) for bacterial infection in the bacterial very likely group. The HR-B/V test correctly identified 30 of 33 participants (90.9%) with acute COVID-19 as having a viral infection.Conclusions and relevance
In this study, the HR-B/V test accurately discriminated bacterial from viral infection among patients with febrile ARI and was superior to procalcitonin measurement. The findings suggest that an accurate point-of-need host response test with high NPV may offer an opportunity to improve antibiotic stewardship and patient outcomes.Item Unknown Rapid, Sample-to-Answer Host Gene Expression Test to Diagnose Viral Infection(Open Forum Infectious Diseases, 2019-11-01) Tsalik, Ephraim L; Khine, Ayeaye; Talebpour, Abdossamad; Samiei, Alaleh; Parmar, Vilcy; Burke, Thomas W; Mcclain, Micah T; Ginsburg, Geoffrey S; Woods, Christopher W; Henao, Ricardo; Alavie, TinoAbstract Background Distinguishing bacterial, viral, or other etiologies of acute illness is diagnostically challenging with significant implications for appropriate antimicrobial use. Host gene-expression offers a promising approach although no clinically useful tests have yet been developed to accomplish this. Here, Qvella’s FAST™ HR process was developed to quantify previously identified host gene-expression signatures in whole blood in <45 minutes. Methods Whole blood was collected from 128 human subjects (mean age 47, range 18-88) with clinically adjudicated, microbiologically confirmed viral infection, bacterial infection, non-infectious illness, or healthy controls. Stabilized mRNA was released from cleaned and stabilized RNA-surfactant complexes using e-lysisTM, an electrical process providing a qRT-PCR-ready sample. Threshold cycle values (CT) for 10 host response targets were normalized to HPRT1 expression, a control mRNA. The transcripts in the signature were specifically chosen to discriminate viral from non-viral infection (bacterial, non-infectious illness, or healthy). Classification accuracy was determined using cross-validated sparse logistic regression. Results Reproducibility of mRNA quantification was within 1 cycle as compared to the difference seen between subjects with viral vs. non-viral infection (up to 5.0 normalized CT difference). Classification of 128 subjects into viral or non-viral etiologies demonstrated 90.6% overall accuracy compared to 82.0% for procalcitonin (p=0.06). FASTTM HR achieved rapid and accurate measurement of the host response to viral infection in less than 45 minutes. Conclusions These results demonstrate the ability to translate host gene expression signatures to clinical platforms for use in patients with suspected infection.