Increased Alternative Complement Pathway Function and Improved Survival during Critical Illness.

Rationale: Complement is crucial for host defense but may also drive dysregulated inflammation. There is limited understanding of alternative complement function, which can amplify all complement activity, during critical illness.Objectives: We examined the function and key components of the alternative complement pathway in a series of critically ill patients and in a mouse pneumonia model.Methods: Total classical (CH50) and alternative complement (AH50) function were quantified in serum from 321 prospectively enrolled critically ill patients and compared with clinical outcomes. Alternative pathway (AP) regulatory factors were quantified by ELISA (n = 181) and examined via transcriptomics data from external cohorts. Wild-type, Cfb-/-, and C3-/- mice were infected intratracheally with Klebsiella pneumoniae (KP) and assessed for extrapulmonary dissemination.Measurements and Main Results: AH50 greater than or equal to median, but not CH50 greater than or equal to median, was associated with decreased 30-day mortality (adjusted odds ratio [OR], 0.53 [95% confidence interval (CI), 0.31-0.91]), independent of chronic liver disease. One-year survival was improved in patients with AH50 greater than or equal to median (adjusted hazard ratio = 0.59 [95% CI, 0.41-0.87]). Patients with elevated AH50 had increased levels of AP factors B, H, and properdin, and fewer showed a "hyperinflammatory" subphenotype (OR, 0.30 [95% CI, 0.18-0.49]). Increased expression of proximal AP genes was associated with improved survival in two external cohorts. AH50 greater than or equal to median was associated with fewer bloodstream infections (OR, 0.67 [95% CI, 0.45-0.98). Conversely, depletion of AP factors, or AH50 less than median, impaired in vitro serum control of KP that was restored by adding healthy serum. Cfb-/- mice demonstrated increased extrapulmonary dissemination and serum inflammatory markers after intratracheal KP infection compared with wild type.Conclusions: Elevated AP function is associated with improved survival during critical illness, possibly because of enhanced immune capacity.

Host-Response Subphenotypes Offer Prognostic Enrichment in Patients With or at Risk for Acute Respiratory Distress Syndrome.

OBJECTIVES: Classification of patients with acute respiratory distress syndrome into hyper- and hypoinflammatory subphenotypes using plasma biomarkers may facilitate more effective targeted therapy. We examined whether established subphenotypes are present not only in patients with acute respiratory distress syndrome but also in patients at risk for acute respiratory distress syndrome (ARFA) and then assessed the prognostic information of baseline subphenotyping on the evolution of host-response biomarkers and clinical outcomes. DESIGN: Prospective, observational cohort study. SETTING: Medical ICU at a tertiary academic medical center. PATIENTS: Mechanically ventilated patients with acute respiratory distress syndrome or ARFA. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We performed longitudinal measurements of 10 plasma biomarkers of host injury and inflammation. We applied unsupervised latent class analysis methods utilizing baseline clinical and biomarker variables and demonstrated that two-class models (hyper- vs hypoinflammatory subphenotypes) offered improved fit compared with one-class models in both patients with acute respiratory distress syndrome and ARFA. Baseline assignment to the hyperinflammatory subphenotype (39/104 [38%] acute respiratory distress syndrome and 30/108 [28%] ARFA patients) was associated with higher severity of illness by Sequential Organ Failure Assessment scores and incidence of acute kidney injury in patients with acute respiratory distress syndrome, as well as higher 30-day mortality and longer duration of mechanical ventilation in ARFA patients (p < 0.0001). Hyperinflammatory patients exhibited persistent elevation of biomarkers of innate immunity for up to 2 weeks postintubation. CONCLUSIONS: Our results suggest that two distinct subphenotypes are present not only in patients with established acute respiratory distress syndrome but also in patients at risk for its development. Hyperinflammatory classification at baseline is associated with higher severity of illness, worse clinical outcomes, and trajectories of persistently elevated biomarkers of host injury and inflammation during acute critical illness compared with hypoinflammatory patients. Our findings provide strong rationale for examining treatment effect modifications by subphenotypes in randomized clinical trials to inform precision therapeutic approaches in critical care.

Metagenomic identification of severe pneumonia pathogens in mechanically-ventilated patients: a feasibility and clinical validity study.

BACKGROUND: Metagenomic sequencing of respiratory microbial communities for pathogen identification in pneumonia may help overcome the limitations of culture-based methods. We examined the feasibility and clinical validity of rapid-turnaround metagenomics with Nanopore™ sequencing of clinical respiratory specimens. METHODS: We conducted a case-control study of mechanically-ventilated patients with pneumonia (nine culture-positive and five culture-negative) and without pneumonia (eight controls). We collected endotracheal aspirates and applied a microbial DNA enrichment method prior to metagenomic sequencing with the Oxford Nanopore MinION device. For reference, we compared Nanopore results against clinical microbiologic cultures and bacterial 16S rRNA gene sequencing. RESULTS: Human DNA depletion enabled in depth sequencing of microbial communities. In culture-positive cases, Nanopore revealed communities with high abundance of the bacterial or fungal species isolated by cultures. In four cases with resistant clinical isolates, Nanopore detected antibiotic resistance genes corresponding to the phenotypic resistance in antibiograms. In culture-negative pneumonia, Nanopore revealed probable bacterial pathogens in 1/5 cases and Candida colonization in 3/5 cases. In controls, Nanopore showed high abundance of oral bacteria in 5/8 subjects, and identified colonizing respiratory pathogens in other subjects. Nanopore and 16S sequencing showed excellent concordance for the most abundant bacterial taxa. CONCLUSIONS: We demonstrated technical feasibility and proof-of-concept clinical validity of Nanopore metagenomics for severe pneumonia diagnosis, with striking concordance with positive microbiologic cultures, and clinically actionable information obtained from sequencing in culture-negative samples. Prospective studies with real-time metagenomics are warranted to examine the impact on antimicrobial decision-making and clinical outcomes.

The evolution of radiographic edema in ARDS and its association with clinical outcomes: A prospective cohort study in adult patients.

PURPOSE: To assess the longitudinal evolution of radiographic edema using chest X-rays (CXR) in patients with Acute Respiratory Distress Syndrome (ARDS) and to examine its association with prognostic biomarkers, ARDS subphenotypes and outcomes. MATERIALS AND METHODS: We quantified radiographic edema on CXRs from patients with ARDS or cardiogenic pulmonary edema (controls) using the Radiographic Assessment of Lung Edema (RALE) score on day of intubation and up to 10 days after. We measured baseline plasma biomarkers and recorded clinical variables. RESULTS: The RALE score had good inter-rater agreement (r = 0.83, p < 0.0001) applied on 488 CXRs from 129 patients, with higher RALE scores in patients with ARDS (n = 108) compared to controls (n = 21, p = 0.01). Baseline RALE scores were positively correlated with levels of the receptor for end-glycation end products (RAGE) in ARDS patients (p < 0.05). Baseline RALE scores were not predictive of 30- or 90-day survival. Persistently elevated RALE scores were associated with prolonged need for mechanical ventilation (p = 0.002). CONCLUSIONS: The RALE score is easily implementable with high inter-rater reliability. Longitudinal RALE scoring appears to be a reproducible approach to track the evolution of radiographic edema in patients with ARDS and can potentially predict prolonged need for mechanical ventilation.

Improved Detection of Culprit Pathogens by Bacterial DNA Sequencing Affects Antibiotic Management Decisions in Severe Pneumonia.

BACKGROUND Severe pneumonia requiring admission to an intensive care unit carries high morbidity and mortality. Evidence-based management includes early administration of empiric antibiotics against plausible bacterial pathogens while awaiting results of microbiologic cultures. However, in over 60% of pneumonia cases, no causative pathogen is identified with conventional diagnostic techniques. In this case report, we demonstrate how direct-from-sample sequencing of bacterial DNA could have identified the multiple culprit pathogens early in the disease course to guide appropriate antibiotic management. CASE REPORT A previously healthy, 21-year-old man presented with neck pain and fever and rapidly developed acute respiratory distress syndrome (ARDS) requiring mechanical ventilation. He was started on broad-spectrum antibiotics and was found to have septic thrombophlebitis of the left internal jugular vein (Lemierre syndrome), with blood cultures growing Fusobacterium necrophorum. While his antibiotics were narrowed to piperacillin-tazobactam monotherapy, his clinical condition worsened, but repeated efforts to define an additional/alternative respiratory pathogen resulted in negative cultures. He eventually developed bilateral empyemas growing Mycoplasma hominis. Once azithromycin was added to the patient's regimen, he improved dramatically. Retrospective sequencing of consecutive endotracheal aspirates showed Fusobacterium as the dominant pathogen early in the course, but with significant and increasing Mycoplasma abundance several days prior to clinical detection. CONCLUSIONS Had sequencing information been available to the treating clinicians, the causative pathogens could have been detected earlier, guiding appropriate antibiotic therapy and perhaps preventing his clinical complications. Real-time bacterial DNA sequencing has the potential to shift the diagnostic paradigm in severe pneumonia.

Respiratory Microbiome Profiling for Etiologic Diagnosis of Pneumonia in Mechanically Ventilated Patients.

Etiologic diagnosis of bacterial pneumonia relies on identification of causative pathogens by cultures, which require extended incubation periods and have limited sensitivity. Next-generation sequencing of microbial DNA directly from patient samples may improve diagnostic accuracy for guiding antibiotic prescriptions. In this study, we hypothesized that enhanced pathogen detection using sequencing can improve upon culture-based diagnosis and that certain sequencing profiles correlate with host response. We prospectively collected endotracheal aspirates and plasma within 72 h of intubation from patients with acute respiratory failure. We performed 16S rRNA gene sequencing to determine pathogen abundance in lung samples and measured plasma biomarkers to assess host responses to detected pathogens. Among 56 patients, 12 patients (21%) had positive respiratory cultures. Sequencing revealed lung communities with low diversity (p < 0.02) dominated by taxa (>50% relative abundance) corresponding to clinically isolated pathogens (concordance p = 0.009). Importantly, sequencing detected dominant pathogens in 20% of the culture-negative patients exposed to broad-spectrum empiric antibiotics. Regardless of culture results, pathogen dominance correlated with increased plasma markers of host injury (receptor of advanced glycation end-products-RAGE) and inflammation (interleukin-6, tumor necrosis factor receptor 1-TNFR1) (p < 0.05), compared to subjects without dominant pathogens in their lung communities. Machine-learning algorithms identified pathogen abundance by sequencing as the most informative predictor of culture positivity. Thus, enhanced detection of pathogenic bacteria by sequencing improves etiologic diagnosis of pneumonia, correlates with host responses, and offers substantial opportunity for individualized therapeutic targeting and antimicrobial stewardship. Clinical translation will require validation with rapid whole meta-genome sequencing approaches to guide real-time antibiotic prescriptions.