Plasma Microbial Cell-Free DNA Sequencing in Immunocompromised Patients With Pneumonia: A Prospective Observational Study.

BACKGROUND: Pneumonia is a common cause of morbidity and mortality, yet a causative pathogen is identified in a minority of cases. Plasma microbial cell-free DNA sequencing may improve diagnostic yield in immunocompromised patients with pneumonia. METHODS: In this prospective, multicenter, observational study of immunocompromised adults undergoing bronchoscopy to establish a pneumonia etiology, plasma microbial cell-free DNA sequencing was compared to standardized usual care testing. Pneumonia etiology was adjudicated by a blinded independent committee. The primary outcome, additive diagnostic value, was assessed in the Per Protocol population (patients with complete testing results and no major protocol deviations) and defined as the percent of patients with an etiology of pneumonia exclusively identified by plasma microbial cell-free DNA sequencing. Clinical additive diagnostic value was assessed in the Per Protocol subgroup with negative usual care testing. RESULTS: Of 257 patients, 173 met Per Protocol criteria. A pneumonia etiology was identified by usual care in 52/173 (30.1%), plasma microbial cell-free DNA sequencing in 49/173 (28.3%) and the combination of both in 73/173 (42.2%) patients. Plasma microbial cell-free DNA sequencing exclusively identified an etiology of pneumonia in 21/173 patients (additive diagnostic value 12.1%, 95% confidence interval [CI], 7.7% to 18.0%, P < .001). In the Per Protocol subgroup with negative usual care testing, plasma microbial cell-free DNA sequencing identified a pneumonia etiology in 21/121 patients (clinical additive diagnostic value 17.4%, 95% CI, 11.1% to 25.3%). CONCLUSIONS: Non-invasive plasma microbial cell-free DNA sequencing significantly increased diagnostic yield in immunocompromised patients with pneumonia undergoing bronchoscopy and extensive microbiologic and molecular testing. CLINICAL TRIALS REGISTRATION: NCT04047719.

Clinically Adjudicated Reference Standards for Evaluation of Infectious Diseases Diagnostics.

Lack of a gold standard can present a challenge for evaluation of diagnostic test accuracy of some infectious diseases tests, particularly when the test's accuracy potentially exceeds that of its predecessors. This approach may measure agreement with an imperfect reference, rather than correctness, because the right answer is unknown. Solutions consist of multitest comparators, including those that involve a test under evaluation if multiple new tests are being evaluated together, using latent class modeling, and clinically adjudicated reference standards. Clinically adjudicated reference standards may be considered as comparator methods when no predefined test or composite of tests is sufficiently accurate; they emulate clinical practice in that multiple data pieces are clinically assessed together.

RADx-UP Testing Core: Access to COVID-19 Diagnostics in Community-Engaged Research with Underserved Populations.

Research on the COVID-19 pandemic revealed a disproportionate burden of COVID-19 infection and death among underserved populations and exposed low rates of SARS-CoV-2 testing in these communities. A landmark National Institutes of Health (NIH) funding initiative, the Rapid Acceleration of Diagnostics-Underserved Populations (RADx-UP) program, was developed to address the research gap in understanding the adoption of COVID-19 testing in underserved populations. This program is the single largest investment in health disparities and community-engaged research in the history of the NIH. The RADx-UP Testing Core (TC) provides community-based investigators with essential scientific expertise and guidance on COVID-19 diagnostics. This commentary describes the first 2 years of the TC's experience, highlighting the challenges faced and insights gained to safely and effectively deploy large-scale diagnostics for community-initiated research in underserved populations during a pandemic. The success of RADx-UP shows that community-based research to increase access and uptake of testing among underserved populations can be accomplished during a pandemic with tools, resources, and multidisciplinary expertise provided by a centralized testing-specific coordinating center. We developed adaptive tools to support individual testing strategies and frameworks for these diverse studies and ensured continuous monitoring of testing strategies and use of study data. In a rapidly evolving setting of tremendous uncertainty, the TC provided essential and real-time technical expertise to support safe, effective, and adaptive testing. The lessons learned go beyond this pandemic and can serve as a framework for rapid deployment of testing in response to future crises, especially when populations are affected inequitably.

Envisioning Future Urinary Tract Infection Diagnostics.

Urinary tract infections (UTIs) are among the most common bacterial infections in the United States and are a major driver of antibiotic use, both appropriate and inappropriate, across healthcare settings. Novel UTI diagnostics are a strategy that might enable better UTI treatment. Members of the Antibacterial Resistance Leadership Group Laboratory Center and the Infectious Diseases Society of America Diagnostics Committee convened to envision ideal future UTI diagnostics, with a view towards improving delivery of healthcare, patient outcomes and experiences, and antibiotic use, addressing which types of UTI diagnostics are needed and how companies might approach development of novel UTI diagnostics.

Host Gene Expression to Predict Sepsis Progression.

OBJECTIVES: Sepsis causes significant mortality. However, most patients who die of sepsis do not present with severe infection, hampering efforts to deliver early, aggressive therapy. It is also known that the host gene expression response to infection precedes clinical illness. This study seeks to develop transcriptomic models to predict progression to sepsis or shock within 72 hours of hospitalization and to validate previously identified transcriptomic signatures in the prediction of 28-day mortality. DESIGN: Retrospective differential gene expression analysis and predictive modeling using RNA sequencing data. PATIENTS: Two hundred seventy-seven patients enrolled at four large academic medical centers; all with clinically adjudicated infection were considered for inclusion in this study. MEASUREMENTS AND MAIN RESULTS: Sepsis progression was defined as an increase in Sepsis 3 category within 72 hours. Transcriptomic data were generated using RNAseq of whole blood. Least absolute shrinkage and selection operator modeling was used to identify predictive signatures for various measures of disease progression. Four previously identified gene signatures were tested for their ability to predict 28-day mortality. There were no significant differentially expressed genes in 136 subjects with worsened Sepsis 3 category compared with 141 nonprogressor controls. There were 1,178 differentially expressed genes identified when sepsis progression was defined as ICU admission or 28-day mortality. A model based on these genes predicted progression with an area under the curve of 0.71. Validation of previously identified gene signatures to predict sepsis mortality revealed area under the receiver operating characteristic values of 0.70-0.75 and no significant difference between signatures. CONCLUSIONS: Host gene expression was unable to predict sepsis progression when defined by an increase in Sepsis-3 category, suggesting this definition is not a useful framework for transcriptomic prediction methods. However, there was a differential response when progression was defined as ICU admission or death. Validation of previously described signatures predicted 28-day mortality with insufficient accuracy to offer meaningful clinical utility.

Validation of a Host Gene Expression Test for Bacterial/Viral Discrimination in Immunocompromised Hosts.

BACKGROUND: Host gene expression has emerged as a complementary strategy to pathogen detection tests for the discrimination of bacterial and viral infection. The impact of immunocompromise on host-response tests remains unknown. We evaluated a host-response test discriminating bacterial, viral, and noninfectious conditions in immunocompromised subjects. METHODS: An 81-gene signature was measured using real-time-polymerase chain reaction in subjects with immunocompromise (chemotherapy, solid-organ transplant, immunomodulatory agents, AIDS) with bacterial infection, viral infection, or noninfectious illness. A regularized logistic regression model trained in immunocompetent subjects was used to estimate the likelihood of each class in immunocompromised subjects. RESULTS: Accuracy in the 136-subject immunocompetent training cohort was 84.6% for bacterial versus nonbacterial discrimination and 80.8% for viral versus nonviral discrimination. Model validation in 134 immunocompromised subjects showed overall accuracy of 73.9% for bacterial infection (P = .04 relative to immunocompetent subjects) and 75.4% for viral infection (P = .30). A scheme reporting results by quartile improved test utility. The highest probability quartile ruled-in bacterial and viral infection with 91.4% and 84.0% specificity, respectively. The lowest probability quartile ruled-out infection with 90.1% and 96.4% sensitivity for bacterial and viral infection, respectively. Performance was independent of the type or number of immunocompromising conditions. CONCLUSIONS: A host gene expression test discriminated bacterial, viral, and noninfectious etiologies at a lower overall accuracy in immunocompromised patients compared with immunocompetent patients, although this difference was only significant for bacterial infection classification. With modified interpretive criteria, a host-response strategy may offer clinically useful diagnostic information for patients with immunocompromise.

Antibacterial Resistance Leadership Group 2.0: Back to Business.

In December 2019, the Antibacterial Resistance Leadership Group (ARLG) was awarded funding for another 7-year cycle to support a clinical research network on antibacterial resistance. ARLG 2.0 has 3 overarching research priorities: infections caused by antibiotic-resistant (AR) gram-negative bacteria, infections caused by AR gram-positive bacteria, and diagnostic tests to optimize use of antibiotics. To support the next generation of AR researchers, the ARLG offers 3 mentoring opportunities: the ARLG Fellowship, Early Stage Investigator seed grants, and the Trialists in Training Program. The purpose of this article is to update the scientific community on the progress made in the original funding period and to encourage submission of clinical research that addresses 1 or more of the research priority areas of ARLG 2.0.

Heparin-based blood purification attenuates organ injury in baboons with Streptococcus pneumoniae pneumonia.

Bacterial pneumonia is a major cause of morbidity and mortality worldwide despite the use of antibiotics, and novel therapies are urgently needed. Building on previous work, we aimed to 1) develop a baboon model of severe pneumococcal pneumonia and sepsis with organ dysfunction and 2) test the safety and efficacy of a novel extracorporeal blood filter to remove proinflammatory molecules and improve organ function. After a dose-finding pilot study, 12 animals were inoculated with Streptococcus pneumoniae [5 × 109 colony-forming units (CFU)], given ceftriaxone at 24 h after inoculation, and randomized to extracorporeal blood purification using a filter coated with surface-immobilized heparin sulfate (n = 6) or sham treatment (n = 6) for 4 h at 30 h after inoculation. For safety analysis, four uninfected animals also underwent purification. At 48 h, necropsy was performed. Inoculated animals developed severe pneumonia and septic shock. Compared with sham-treated animals, septic animals treated with purification displayed significantly less kidney injury, metabolic acidosis, hypoglycemia, and shock (P < 0.05). Purification blocked the rise in peripheral blood S. pneumoniae DNA, attenuated bronchoalveolar lavage (BAL) CCL4, CCL2, and IL-18 levels, and reduced renal oxidative injury and classical NLRP3 inflammasome activation. Purification was safe in both uninfected and infected animals and produced no adverse effects. We demonstrate that heparin-based blood purification significantly attenuates levels of circulating S. pneumoniae DNA and BAL cytokines and is renal protective in baboons with severe pneumococcal pneumonia and septic shock. Purification was associated with less severe acute kidney injury, metabolic derangements, and shock. These results support future clinical studies in critically ill septic patients.

Discriminating Bacterial and Viral Infection Using a Rapid Host Gene Expression Test.

OBJECTIVES: 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.

Sepsis Subclasses: A Framework for Development and Interpretation.

Sepsis is defined as a dysregulated host response to infection that leads to life-threatening acute organ dysfunction. It afflicts approximately 50 million people worldwide annually and is often deadly, even when evidence-based guidelines are applied promptly. Many randomized trials tested therapies for sepsis over the past 2 decades, but most have not proven beneficial. This may be because sepsis is a heterogeneous syndrome, characterized by a vast set of clinical and biologic features. Combinations of these features, however, may identify previously unrecognized groups, or "subclasses" with different risks of outcome and response to a given treatment. As efforts to identify sepsis subclasses become more common, many unanswered questions and challenges arise. These include: 1) the semantic underpinning of sepsis subclasses, 2) the conceptual goal of subclasses, 3) considerations about study design, data sources, and statistical methods, 4) the role of emerging data types, and 5) how to determine whether subclasses represent "truth." We discuss these challenges and present a framework for the broader study of sepsis subclasses. This framework is intended to aid in the understanding and interpretation of sepsis subclasses, provide a mechanism for explaining subclasses generated by different methodologic approaches, and guide clinicians in how to consider subclasses in bedside care.

Average Weighted Accuracy: Pragmatic Analysis for a Rapid Diagnostics in Categorizing Acute Lung Infections (RADICAL) Study.

Patient management relies on diagnostic information to identify appropriate treatment. Standard evaluations of diagnostic tests consist of estimating sensitivity, specificity, positive/negative predictive values, likelihood ratios, and accuracy. Although useful, these metrics do not convey the tests' clinical value, which is critical to informing decision-making. Full appreciation of the clinical impact of a diagnostic test requires analyses that integrate sensitivity and specificity, account for the disease prevalence within the population of test application, and account for the relative importance of specificity vs sensitivity by considering the clinical implications of false-positive and false-negative results. We developed average weighted accuracy (AWA), representing a pragmatic metric of diagnostic yield or global utility of a diagnostic test. AWA can be used to compare test alternatives, even across different studies. We apply the AWA methodology to evaluate a new diagnostic test developed in the Rapid Diagnostics in Categorizing Acute Lung Infections (RADICAL) study.

Simultaneous Evaluation of Diagnostic Assays for Pharyngeal and Rectal Neisseria gonorrhoeae and Chlamydia trachomatis Using a Master Protocol.

BACKGROUND: Pharyngeal and rectal Neisseria gonorrhoeae and Chlamydia trachomatis play important roles in infection and antibacterial resistance transmission, but no US Food and Drug Administration (FDA)-cleared assays for detection at these sites existed prior to this study. The objective was to estimate performance of assays to detect those infections in pharyngeal and rectal specimens to support regulatory submission. METHODS: We performed a cross-sectional, single-visit study of adults seeking sexually transmitted infection testing at 9 clinics in 7 states. We collected pharyngeal and rectal swabs from participants. The primary outcome was positive and negative percent agreement for detection of N. gonorrhoeae and C. trachomatis for 3 investigational assays compared to a composite reference. Secondary outcomes included positivity as well as positive and negative predictive values and likelihood ratios. Subgroup analyses included outcomes by symptom status and sex. RESULTS: A total of 2598 participants (79% male) underwent testing. We observed N. gonorrhoeae positivity of 8.1% in the pharynx and 7.9% in the rectum and C. trachomatis positivity of 2.0% in the pharynx and 8.7% in the rectum. Positive percent agreement ranged from 84.8% to 96.5% for different anatomic site infection combinations, whereas negative percent agreement was 98.8% to 99.6%. CONCLUSIONS: This study utilized a Master Protocol to generate diagnostic performance data for multiple assays from different manufacturers in a single study population, which ultimately supported first-in-class FDA clearance for extragenital assays. We observed very good positive percent agreement when compared to a composite reference method for the detection of both pharyngeal and rectal N. gonorrhoeae and C. trachomatis. CLINICAL TRIALS REGISTRATION: NCT02870101.

New Molecular Diagnostic Approaches to Bacterial Infections and Antibacterial Resistance.

Recent advances in the field of infectious disease diagnostics have given rise to a number of host- and pathogen-centered diagnostic approaches. Most diagnostic approaches in contemporary infectious disease focus on pathogen detection and characterization. Host-focused diagnostics have recently emerged and are based on detecting the activation of biological pathways that are highly specific to the type of infecting pathogen (e.g., viral, bacterial, protozoan, fungal). Although this progress is encouraging, it is unlikely that any single diagnostic platform will fully address the clinician's need for actionable data with short turnaround times in all settings.

Direct-from-blood RNA sequencing identifies the cause of post-bronchoscopy fever.

BACKGROUND: 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.

Future Research Directions in Pneumonia. NHLBI Working Group Report.

Pneumonia is a complex pulmonary disease in need of new clinical approaches. Although triggered by a pathogen, pneumonia often results from dysregulations of host defense that likely precede infection. The coordinated activities of immune resistance and tissue resilience then dictate whether and how pneumonia progresses or resolves. Inadequate or inappropriate host responses lead to more severe outcomes such as acute respiratory distress syndrome and to organ dysfunction beyond the lungs and over extended time frames after pathogen clearance, some of which increase the risk for subsequent pneumonia. Improved understanding of such host responses will guide the development of novel approaches for preventing and curing pneumonia and for mitigating the subsequent pulmonary and extrapulmonary complications of pneumonia. The NHLBI assembled a working group of extramural investigators to prioritize avenues of host-directed pneumonia research that should yield novel approaches for interrupting the cycle of unhealthy decline caused by pneumonia. This report summarizes the working group's specific recommendations in the areas of pneumonia susceptibility, host response, and consequences. Overarching goals include the development of more host-focused clinical approaches for preventing and treating pneumonia, the generation of predictive tools (for pneumonia occurrence, severity, and outcome), and the elucidation of mechanisms mediating immune resistance and tissue resilience in the lung. Specific areas of research are highlighted as especially promising for making advances against pneumonia.

Unsupervised Analysis of Transcriptomics in Bacterial Sepsis Across Multiple Datasets Reveals Three Robust Clusters.

OBJECTIVES: To find and validate generalizable sepsis subtypes using data-driven clustering. DESIGN: We used advanced informatics techniques to pool data from 14 bacterial sepsis transcriptomic datasets from eight different countries (n = 700). SETTING: Retrospective analysis. SUBJECTS: Persons admitted to the hospital with bacterial sepsis. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: A unified clustering analysis across 14 discovery datasets revealed three subtypes, which, based on functional analysis, we termed "Inflammopathic, Adaptive, and Coagulopathic." We then validated these subtypes in nine independent datasets from five different countries (n = 600). In both discovery and validation data, the Adaptive subtype is associated with a lower clinical severity and lower mortality rate, and the Coagulopathic subtype is associated with higher mortality and clinical coagulopathy. Further, these clusters are statistically associated with clusters derived by others in independent single sepsis cohorts. CONCLUSIONS: The three sepsis subtypes may represent a unifying framework for understanding the molecular heterogeneity of the sepsis syndrome. Further study could potentially enable a precision medicine approach of matching novel immunomodulatory therapies with septic patients most likely to benefit.

MASTERMIND: Bringing Microbial Diagnostics to the Clinic.

New diagnostics are urgently needed to address emerging antimicrobial resistance. The Antibacterial Resistance Leadership Group proposes a strategy called MASTERMIND (Master Protocol for Evaluating Multiple Infection Diagnostics) for advancement of infectious diseases diagnostics. The goal of this strategy is to generate the data necessary to support US Food and Drug Administration clearance of new diagnostic tests by promoting research that might not have otherwise been feasible with conventional trial designs. MASTERMIND uses a single subject's sample(s) to evaluate multiple diagnostic tests at the same time, providing efficiencies of specimen collection and characterization. MASTERMIND also offers central trial organization, standardization of methods and definitions, and common comparators.

Advancing Diagnostics to Address Antibacterial Resistance: The Diagnostics and Devices Committee of the Antibacterial Resistance Leadership Group.

Diagnostics are a cornerstone of the practice of infectious diseases. However, various limitations frequently lead to unmet clinical needs. In most other domains, diagnostics focus on narrowly defined questions, provide readily interpretable answers, and use true gold standards for development. In contrast, infectious diseases diagnostics must contend with scores of potential pathogens, dozens of clinical syndromes, emerging pathogens, rapid evolution of existing pathogens and their associated resistance mechanisms, and the absence of gold standards in many situations. In spite of these challenges, the importance and value of diagnostics cannot be underestimated. Therefore, the Antibacterial Resistance Leadership Group has identified diagnostics as 1 of 4 major areas of emphasis. Herein, we provide an overview of that development, highlighting several examples where innovation in study design, content, and execution is advancing the field of infectious diseases diagnostics.

Host-Based Peripheral Blood Gene Expression Analysis for Diagnosis of Infectious Diseases.

Emerging pandemic infectious threats, inappropriate antibacterial use contributing to multidrug resistance, and increased morbidity and mortality from diagnostic delays all contribute to a need for improved diagnostics in the field of infectious diseases. Historically, diagnosis of infectious diseases has relied on pathogen detection; however, a novel concept to improve diagnostics in infectious diseases relies instead on the detection of changes in patterns of gene expression in circulating white blood cells in response to infection. Alterations in peripheral blood gene expression in the infected state are robust and reproducible, yielding diagnostic and prognostic information to help facilitate patient treatment decisions.

Transcriptomic Analysis of the Host Response and Innate Resilience to Enterotoxigenic Escherichia coli Infection in Humans.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is a globally prevalent cause of diarrhea. Though usually self-limited, it can be severe and debilitating. Little is known about the host transcriptional response to infection. We report the first gene expression analysis of the human host response to experimental challenge with ETEC. METHODS: We challenged 30 healthy adults with an unattenuated ETEC strain, and collected serial blood samples shortly after inoculation and daily for 8 days. We performed gene expression analysis on whole peripheral blood RNA samples from subjects in whom severe symptoms developed (n = 6) and a subset of those who remained asymptomatic (n = 6) despite shedding. RESULTS: Compared with baseline, symptomatic subjects demonstrated significantly different expression of 406 genes highlighting increased immune response and decreased protein synthesis. Compared with asymptomatic subjects, symptomatic subjects differentially expressed 254 genes primarily associated with immune response. This comparison also revealed 29 genes differentially expressed between groups at baseline, suggesting innate resilience to infection. Drug repositioning analysis identified several drug classes with potential utility in augmenting immune response or mitigating symptoms. CONCLUSIONS: There are statistically significant and biologically plausible differences in host gene expression induced by ETEC infection. Differential baseline expression of some genes may indicate resilience to infection.