Absolute quantification of plasma mitochondrial DNA by droplet digital PCR marks COVID-19 severity over time during intensive care unit admissions.

Increased plasma mitochondrial DNA concentrations are associated with poor outcomes in multiple critical illnesses, including COVID-19. However, current methods of cell-free mitochondrial DNA quantification in plasma are time-consuming and lack reproducibility. Here, we used next-generation sequencing to characterize the size and genome location of circulating mitochondrial DNA in critically ill subjects with COVID-19 to develop a facile and optimal method of quantification by droplet digital PCR. Sequencing revealed a large percentage of small mitochondrial DNA fragments in plasma with wide variability in coverage by genome location. We identified probes for the mitochondrial DNA genes, cytochrome B and NADH dehydrogenase 1, in regions of relatively high coverage that target small sequences potentially missed by other methods. Serial assessments of absolute mitochondrial DNA concentrations were then determined in plasma from 20 critically ill subjects with COVID-19 without a DNA isolation step. Mitochondrial DNA concentrations on the day of enrollment were increased significantly in patients with moderate or severe acute respiratory distress syndrome (ARDS) compared with those with no or mild ARDS. Comparisons of mitochondrial DNA concentrations over time between patients with no/mild ARDS who survived, patients with moderate/severe ARDS who survived, and nonsurvivors showed the highest concentrations in patients with more severe disease. Absolute mitochondrial DNA quantification by droplet digital PCR is time-efficient and reproducible; thus, we provide a valuable tool and rationale for future studies evaluating mitochondrial DNA as a real-time biomarker to guide clinical decision-making in critically ill subjects with COVID-19.

Estimating dead-space fraction for secondary analyses of acute respiratory distress syndrome clinical trials.

OBJECTIVES: Pulmonary dead-space fraction is one of few lung-specific independent predictors of mortality from acute respiratory distress syndrome. However, it is not measured routinely in clinical trials and thus altogether ignored in secondary analyses that shape future research directions and clinical practice. This study sought to validate an estimate of dead-space fraction for use in secondary analyses of clinical trials. DESIGN: Analysis of patient-level data pooled from acute respiratory distress syndrome clinical trials. Four approaches to estimate dead-space fraction were evaluated: three required estimating metabolic rate; one estimated dead-space fraction directly. SETTING: U.S. academic teaching hospitals. PATIENTS: Data from 210 patients across three clinical trials were used to compare performance of estimating equations with measured dead-space fraction. A second cohort of 3,135 patients from six clinical trials without measured dead-space fraction was used to confirm whether estimates independently predicted mortality. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Dead-space fraction estimated using the unadjusted Harris-Benedict equation for energy expenditure was unbiased (mean ± SD Harris-Benedict, 0.59 ± 0.13; measured, 0.60 ± 0.12). This estimate predicted measured dead-space fraction to within ±0.10 in 70% of patients and ±0.20 in 95% of patients. Measured dead-space fraction independently predicted mortality (odds ratio, 1.36 per 0.05 increase in dead-space fraction; 95% CI, 1.10-1.68; p < 0.01). The Harris-Benedict estimate closely approximated this association with mortality in the same cohort (odds ratio, 1.55; 95% CI, 1.21-1.98; p < 0.01) and remained independently predictive of death in the larger Acute Respiratory Distress Syndrome Network cohort. Other estimates predicted measured dead-space fraction or its association with mortality less well. CONCLUSIONS: Dead-space fraction should be measured in future acute respiratory distress syndrome clinical trials to facilitate incorporation into secondary analyses. For analyses where dead-space fraction was not measured, the Harris-Benedict estimate can be used to estimate dead-space fraction and adjust for its association with mortality.

Impaired pulmonary defense against Pseudomonas aeruginosa in VEGF gene inactivated mouse lung.

Repeated bacterial and viral infections are known to contribute to worsening lung function in several respiratory diseases, including asthma, cystic fibrosis, and chronic obstructive pulmonary disease (COPD). Previous studies have reported alveolar wall cell apoptosis and parenchymal damage in adult pulmonary VEGF gene ablated mice. We hypothesized that VEGF expressed by type II cells is also necessary to provide an effective host defense against bacteria in part by maintaining surfactant homeostasis. Therefore, Pseudomonas aeruginosa (PAO1) levels were evaluated in mice following lung-targeted VEGF gene inactivation, and alterations in VEGF-dependent type II cell function were evaluated by measuring surfactant homeostasis in mouse lungs and isolated type II cells. In VEGF-deficient lungs increased PAO1 levels and pro-inflammatory cytokines, TNFα and IL-6, were detected 24 h after bacterial instillation compared to control lungs. In vivo lung-targeted VEGF gene deletion (57% decrease in total pulmonary VEGF) did not alter alveolar surfactant or tissue disaturated phosphatidylcholine (DSPC) levels. However, sphingomyelin content, choline phosphate cytidylyltransferase (CCT) mRNA, and SP-D expression were decreased. In isolated type II cells an 80% reduction of VEGF protein resulted in decreases in total phospholipids (PL), DSPC, DSPC synthesis, surfactant associated proteins (SP)-B and -D, and the lipid transporters, ABCA1 and Rab3D. TPA-induced DSPC secretion and apoptosis were elevated in VEGF-deficient type II cells. These results suggest a potential protective role for type II cell-expressed VEGF against bacterial initiated infection.

Recombinant surfactant protein C-based surfactant for patients with severe direct lung injury.

RATIONALE: Patients with acute lung injury have impaired function of the lung surfactant system. Prior clinical trials have shown that treatment with exogenous recombinant surfactant protein C (rSP-C)-based surfactant results in improvement in blood oxygenation and have suggested that treatment of patients with severe direct lung injury may decrease mortality. OBJECTIVES: Determine the clinical benefit of administering an rSP-C-based synthetic surfactant to patients with severe direct lung injury due to pneumonia or aspiration. METHODS: A prospective randomized blinded study was performed at 161 centers in 22 countries. Patients were randomly allocated to receive usual care plus up to eight doses of rSP-C surfactant administered over 96 hours (n = 419) or only usual care (n = 424). MEASUREMENTS AND MAIN RESULTS: Mortality to 28 days after treatment, the requirement for mechanical ventilation, and the number of nonpulmonary organ failure-free days were not different between study groups. In contrast to prior studies, there was no improvement in oxygenation in patients receiving surfactant compared with the usual care group. Investigation of the possible reasons underlying the lack of efficacy suggested a partial inactivation of rSP-C surfactant caused by a step of the resuspension process that was introduced with this study. CONCLUSIONS: In this study, rSP-C-based surfactant was of no clinical benefit to patients with severe direct lung injury. The unexpected lack of improvement in oxygenation, coupled with the results of in vitro tests, suggest that the administered suspension may have had insufficient surface activity to achieve clinical benefit.

Association Between Acute Kidney Injury During Invasive Mechanical Ventilation and ICU Outcomes and Respiratory System Mechanics.

Compare ICU outcomes and respiratory system mechanics in patients with and without acute kidney injury during invasive mechanical ventilation. DESIGNS: Retrospective cohort study. SETTINGS: ICUs of the University of California, San Diego, from January 1, 2014, to November 30, 2016. PATIENTS: Five groups of patients were compared based on the need for invasive mechanical ventilation, presence or absence of acute kidney injury per the Kidney Disease: Improving Global Outcomes criteria, and the temporal relationship between the development of acute kidney injury and initiation of invasive mechanical ventilation. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: A total of 9,704 patients were included and 4,484 (46%) required invasive mechanical ventilation; 2,009 patients (45%) had acute kidney injury while being treated with invasive mechanical ventilation, and the mortality rate for these patients was 22.4% compared with 5% in those treated with invasive mechanical ventilation without acute kidney injury (p < 0.01). Adjusted hazard of mortality accounting for baseline disease severity was 1.58 (95% CI, 1.22-2.03; p < 0.001]. Patients with acute kidney injury during invasive mechanical ventilation had a significant increase in total ventilator days and length of ICU stay with the same comparison (both p < 0.01). Acute kidney injury during mechanical ventilation was also associated with significantly higher plateau pressures, lower respiratory system compliance, and higher driving pressures (all p < 0.01). These differences remained significant in patients with net negative cumulative fluid balance. CONCLUSIONS: Acute kidney injury during invasive mechanical ventilation is associated with increased ICU mortality, increased ventilator days, increased length of ICU stay, and impaired respiratory system mechanics. These results emphasize the need for investigations of ventilatory strategies in the setting of acute kidney injury, as well as mechanistic studies of crosstalk between the lung and kidney in the critically ill.

Beyond mortality: future clinical research in acute lung injury.

Mortality in National Heart, Lung and Blood Institute-sponsored clinical trials of treatments for acute lung injury (ALI) has decreased dramatically during the past two decades. As a consequence, design of such trials based on a mortality outcome requires ever-increasing numbers of patients. Recognizing that advances in clinical trial design might be applicable to these trials and might allow trials with fewer patients, the National Heart, Lung and Blood Institute convened a workshop of extramural experts from several disciplines. The workshop assessed the current state of clinical research addressing ALI, identified research needs, and recommended: (1) continued performance of trials evaluating treatments of patients with ALI; (2) development of strategies to perform ALI prevention trials; (3) observational studies of patients without ALI undergoing prolonged mechanical ventilation; and (4) development of a standardized format for reporting methods, endpoints, and results of ALI trials.

A Search for subgroups of patients with ARDS who may benefit from surfactant replacement therapy: a pooled analysis of five studies with recombinant surfactant protein-C surfactant (Venticute).

BACKGROUND: Studies to date have shown no survival benefit for the use of exogenous surfactant to treat patients with the ARDS. To identify specific patient subgroups for future study, we performed an exploratory post hoc analysis of clinical trials of recombinant surfactant protein-C (rSP-C) surfactant (Venticute; Nycomed GmbH; Konstanz, Germany). METHODS: We performed a pooled analysis of all five multicenter studies in which patients with ARDS due to various predisposing events were treated with rSP-C surfactant. Patients received either usual care (n = 266) or usual care plus up to four intratracheal doses (50 mg/kg) of rSP-C surfactant (n = 266). Factors influencing the study end points were analyzed using descriptive statistics, analysis of covariance, and logistic regression models. RESULTS: ARDS was most often associated with pneumonia or aspiration, sepsis, and trauma or surgery. For the overall patient population, treatment with rSP-C surfactant significantly improved oxygenation (p = 0.002) but had no effect on mortality (32.6%). Multivariate analysis showed age and acute physiology and chronic health evaluation (APACHE) II score to be the strongest predictors of mortality. In the subgroup of patients with severe ARDS due to pneumonia or aspiration, surfactant treatment was associated with markedly improved oxygenation (p = 0.0008) and improved survival (p = 0.018). CONCLUSIONS: rSP-C surfactant improved oxygenation in patients with ARDS irrespective of the predisposition. Post hoc evidence of reduced mortality associated with surfactant treatment was obtained in patients with severe respiratory insufficiency due to pneumonia or aspiration. Those patients are the focus of a current randomized, blinded, clinical trial with rSP-C surfactant.

Effect of recombinant surfactant protein C-based surfactant on the acute respiratory distress syndrome.

BACKGROUND: Preclinical studies suggest that exogenous surfactant may be of value in the treatment of the acute respiratory distress syndrome (ARDS), and two phase 2 clinical trials have shown a trend toward benefit. We conducted two phase 3 studies of a protein-containing surfactant in adults with ARDS. METHODS: In two multicenter, randomized, double-blind trials involving 448 patients with ARDS from various causes, we compared standard therapy alone with standard therapy plus up to four intratracheal doses of a recombinant surfactant protein C-based surfactant given within a period of 24 hours. RESULTS: The overall survival rate was 66 percent 28 days after treatment, and the median number of ventilator-free days was 0 (68 percent range, 0 to 26); there was no significant difference between the groups in terms of mortality or the need for mechanical ventilation. Patients receiving surfactant had a significantly greater improvement in blood oxygenation during the initial 24 hours of treatment than patients receiving standard therapy, according to both univariate and multivariate analyses. CONCLUSIONS: The use of exogenous surfactant in a heterogeneous population of patients with ARDS did not improve survival. Patients who received surfactant had a greater improvement in gas exchange during the 24-hour treatment period than patients who received standard therapy alone, suggesting the potential benefit of a longer treatment course.

Quantifying unintended exposure to high tidal volumes from breath stacking dyssynchrony in ARDS: the BREATHE criteria.

PURPOSE: Breath stacking dyssynchrony generates higher tidal volumes than intended, potentially increasing lung injury risk in acute respiratory distress syndrome (ARDS). Lack of validated criteria to quantify breath stacking dyssynchrony contributes to its under-recognition. This study evaluates performance of novel, objective criteria for quantifying breath stacking dyssynchrony (BREATHE criteria) compared to existing definitions and tests if neuromuscular blockade eliminates high-volume breath stacking dyssynchrony in ARDS. METHODS: Airway flow and pressure were recorded continuously for up to 72 h in 33 patients with ARDS receiving volume-preset assist-control ventilation. The flow-time waveform was integrated to calculate tidal volume breath-by-breath. The BREATHE criteria considered five domains in evaluating for breath stacking dyssynchrony: ventilator cycling, interval expiratory volume, cumulative inspiratory volume, expiratory time, and inspiratory time. RESULTS: The observed tidal volume of BREATHE stacked breaths was 11.3 (9.7-13.3) mL/kg predicted body weight, significantly higher than the preset volume [6.3 (6.0-6.8) mL/kg; p < 0.001]. BREATHE identified more high-volume breaths (≥2 mL/kg above intended volume) than the other existing objective criteria for breath stacking [27 (7-59) vs 19 (5-46) breaths/h; p < 0.001]. Agreement between BREATHE and visual waveform inspection was high (raw agreement 96.4-98.1 %; phi 0.80-0.92). Breath stacking dyssynchrony was near-completely eliminated during neuromuscular blockade [0 (0-1) breaths/h; p < 0.001]. CONCLUSIONS: The BREATHE criteria provide an objective definition of breath stacking dyssynchrony emphasizing occult exposure to high tidal volumes. BREATHE identified high-volume breaths missed by other methods for quantifying this dyssynchrony. Neuromuscular blockade prevented breath stacking dyssynchrony, assuring provision of the intended lung-protective strategy.

Lung surfactant in subacute pulmonary disease.

Pulmonary surfactant is a surface active material composed of both lipids and proteins that is produced by alveolar type II pneumocytes. Abnormalities of surfactant in the immature lung or in the acutely inflamed mature lung are well described. However, in a variety of subacute diseases of the mature lung, abnormalities of lung surfactant may also be of importance. These diseases include chronic obstructive pulmonary disease, asthma, cystic fibrosis, interstitial lung disease, pneumonia, and alveolar proteinosis. Understanding of the mechanisms that disturb the lung surfactant system may lead to novel rational therapies for these diseases.

Surfactant from diving aquatic mammals.

Diving mammals that descend to depths of 50-70 m or greater fully collapse the gas exchanging portions of their lungs and then reexpand these areas with ascent. To investigate whether these animals may have evolved a uniquely developed surfactant system to facilitate repetitive alveolar collapse and expansion, we have analyzed surfactant in bronchoalveolar lavage fluid (BAL) obtained from nine pinnipeds and from pigs and humans. In contrast to BAL from terrestrial mammals, BAL from pinnipeds has a higher concentration of phospholipid and relatively more fluidic phosphatidylcholine molecular species, perhaps to facilitate rapid spreading during alveolar reexpansion. Normalized concentrations of hydrophobic surfactant proteins B and C were not significantly different among pinnipeds and terrestrial mammals by immunologic assay, but separation of proteins by gel electrophoresis indicated a greater content of surfactant protein B in elephant seal surfactant than in human surfactant. Remarkably, surfactant from the deepest diving pinnipeds produced moderately elevated in vitro minimum surface tension measurements, a finding not explained by the presence of protein or neutral lipid inhibitors. Further study of the composition and function of pinniped surfactants may contribute to the design of optimized therapeutic surfactants.

Efficacy of methylprednisolone in preventing lung injury following pulmonary thromboendarterectomy.

BACKGROUND: We sought to determine the efficacy and safety of perioperative treatment with methylprednisolone on the development of lung injury after pulmonary thromboendarterectomy. METHODS: This was a randomized, prospective, double-blind, placebo-controlled study of 98 adult patients with chronic thromboembolic pulmonary hypertension who were undergoing pulmonary thromboendarterectomy at a single institution. The patients received either placebo (n = 47) or methylprednisolone (n = 51) (30 mg/kg in the cardiopulmonary bypass prime, 500 mg IV bolus following the final circulatory arrest, and 250 mg IV bolus 36 h after surgery). The primary end point was the presence of lung injury as determined by two independent, blinded physicians using prospectively defined criteria. The secondary end points included ventilator-free, ICU-free, and hospital-free days and selected levels of cytokines in the blood and in BAL fluid. RESULTS: The incidence of lung injury was similar in both treatment groups (45% placebo, 41% steroid; P = .72). There were no statistical differences in the secondary clinical end points between treatment groups. Treatment with methylprednisolone, compared with placebo, was associated with a statistically significant reduction in plasma IL-6 and IL-8, a significant increase in plasma IL-10, and a significant reduction in postoperative IL-1ra and IL-6, but not IL-8 in BAL fluid obtained 1 day after surgery. CONCLUSIONS: Perioperative methylprednisolone does not reduce the incidence of lung injury following pulmonary thromboendarterectomy surgery despite having an antiinflammatory effect on plasma and lavage cytokine levels.

An informatics strategy to assure enrollment criteria compliance in studies of the critically ill.

Up to 18% of acutely ill patients randomized into multicenter clinical trials may not satisfy inclusion/exclusion criteria. To improve compliance with such criteria in an ICU-based multicenter international drug trial, we established a novel Internet/telephone-based strategy for providing rapid case approval or disapproval by centralized panels of critical care physicians. We assessed whether these panels could acquire and record accurate patient information, and whether this approach would minimize enrollment of ineligible patients and could be accomplished in a timely fashion. Analysis of the first 1000 submitted patients showed accurate data capture for 98.7% of enrolled and randomized patients. Median response time from case submission to panel member decision was 34.7 min. Over 99% of enrolled patients met critical study criteria. We conclude that, an Internet-based communications strategy appears to be a valuable adjunct to multicenter clinical trials in acutely ill patients when rapid assurance of eligibility is required.

The future of surfactant therapy for patients with acute lung injury - new requirements and new surfactants.

New requirements must be considered when designing trials of new lung surfactants for patients with acute lung injury (ALI). Radiographic inclusion criteria must be carefully applied if they are to generate reproducible patient groups. Strategies for ventilation are now known to significantly affect outcome and also must be clearly defined and applied. Similar mortality rates in patients with different degrees of gas exchange and radiographic abnormalities suggest that prior clinical distinctions should be re-examined. Current trials of surfactant therapy for ALI are examining the efficacy of a natural surfactant, a surfactant containing recombinant SP-C and a surfactant based on an SP-B-like peptide.

Acute lung injury in 2003.

During the past several decades, clinical investigators world-wide have continued to study the causes, pathophysiology, and treatment strategies for acute lung injury (ALI). This syndrome, which is characterized by nonhydrostatic pulmonary edema and hypoxemia associated with a variety of etiologies, is slowly becoming better understood as a result of these efforts.

Effect of CTP:phosphocholine cytidylyltransferase overexpression on the mouse lung surfactant system.

CTP:phosphocholine cytidylyltransferase (CT) is the rate-limiting enzyme in the biosynthesis by type II pneumocytes of phosphatidylcholine (PC), the predominant phospholipid in lung surfactant. Augmentation of endogenous CT activity might therefore result in enhanced surfactant PC production. To test this hypothesis, transgenic mice were created in which rat CT (rCT) was expressed under control of the human surfactant protein C (SP-C) promoter. Transgenic mice were identified by tail-clip PCR analysis and studies of four founder lines were initiated. Lung CT gene expression was enhanced in two transgenic founder lines relative to wild-type controls. These two transgenic lines also exhibited significantly higher levels of immunoreactive CT protein and CT activity in whole-lung homogenates and in cultured type II cell extracts. Disaturated PC (DSPC) content in whole-lung homogenates and the rate of DSPC synthesis in cultured type II cells were significantly increased in one transgenic line. However, neither the incorporation of radiolabeled precursors (choline and palmitate) into DSPC in vivo nor the cellular metabolism of DSPC differed significantly between transgenic and control mice. This transgenic model provides opportunity for further study of factors controlling surfactant phospholipid production in vivo.

Treatment of acute respiratory distress syndrome with recombinant surfactant protein C surfactant.

We performed a phase I/II trial in North America of a recombinant surfactant protein C-based surfactant (Venticute) as treatment for the acute respiratory distress syndrome. Patients were prospectively randomized to receive either standard therapy or standard therapy plus one of two doses of exogenous surfactant given four times over 24 hours. Surfactant administration was well tolerated. No significant treatment benefit was associated with surfactant treatment. Bronchoalveolar lavage of treated patients at 48 hours reflected the presence of exogenous surfactant components, did not show evidence of improved surface tension lowering function, and had interleukin-6 concentrations that were significantly lower than control group values, consistent with an antiinflammatory treatment effect. The presence of exogenous surfactant was not detected in lavage fluid obtained at 120 hours. Future studies might rationally employ larger surfactant doses and a more prolonged dosing schedule.