Tracheal Aspirates in Long-term Mechanically Ventilated Patients: A Human Model of Gram-Negative Infection and Airway Inflammation

doi:10.1378/chest.108.5.1326

Chest

Volume 108, Issue 5, November 1995, Pages 1326-1332

https://doi.org/10.1378/chest.108.5.1326 Get rights and content

It is well known that patients requiring long-term mechanical ventilation and tracheostomy have nearly universal airway colonization with Gram-negative organisms. However, useful parameters to objectively describe the airway inflammation associated with airway instrumentation and colonization have not been well defined. In our respiratory care unit, patients who are medically stable except for ventilator dependence are readily available for longitudinal assessment of airway secretions and therefore provide a unique population for studying airway inflammation and infection. To quantitate production of respiratory secretions, we instituted a uniform protocol of suctioning over a 6-h period. Further, we devised a method of dilution and homogenization of tracheal aspirates that permits reproducible intrasample total cell counts (coefficient of variation, 4.6%). With these techniques, patients were then studied serially over a 4- to 7-week period. Total cell count, inflammatory cell differential, and two indices of airway inflammation, human neutrophil elastase (HLE) and soluble-intercellular adhesion molecule-1 (sICAM-1) studied in the sol phase of secretions were monitored. The mean total cell count was 42.2×10⁶ cells per gram of secretions when patients were clinically stable and not receiving antibiotics. The average differential was neutrophils 69.9%, macrophages 26.9%, and lymphocytes 2.8%. Mean active HLE was 35.6 pg/mL and mean sICAM-1 was 83 ng/mL. Six patients during the period of observation received intravenous oral or aerosolized antibiotics for tracheobronchitis. A threefold drop in volume of secretions was measured (p<0.018). The total cell count and percent neutrophils decreased from 76.4×10⁶/g of sputum to 54.9×10⁶ and 72.2 to 54.9%, respectively. While these changes were not statistically significant, the absolute number of airway neutrophils over the 6 h decreased sevenfold (p<0.014). Similarly sICAM-1 burden (micrograms per 6-h period) also decreased significantly (p<0.034). These patients provide a unique human model for future studies specifically designed to assess the effect of novel modalities of anti-inflammatory and antimicrobial agents on respiratory secretions.

Section snippets

Study Population

This investigation was a prospective 6-month study of patients in the respiratory care unit of University Medical Center at Stony Brook, NY, an 8-bed subspecialty unit for patients requiring long-term ventilatory support. Patients were enrolled in the study if they were in clinically stable condition and had no radiographic evidence of pneumonia. The patient population is described in Table 1. The protocol was approved by the Human Studies Committee and participants signed informed consent.

Microbiologic Studies

Total Cell Counts and Differentials

Determination of TCC Reproducibility: To determine the reproducibility of TCC, ten measurements of the same tracheal aspirate after dilution were performed in four patients. The coefficient of variation of multiple measurements ranged from 1.8 to 8.7% with a mean of 4.6%. Then, to determine if the tracheal aspirate as a whole was homogeneous, 14 samples of tracheal aspirate were divided into two aliquots before dilution. Samples were then diluted and total cell count was performed on each. The

Discussion

In this investigation, we describe a method for serially assessing airway secretions in mechanically ventilated patients that enabled us to monitor airway inflammation. Our purpose was to determine if this technique could provide meaningful data that would warrant future studies with precisely controlled regimens of therapeutic anti-inflammatory or antimicrobial agents. Daily access to a small group of patients provided unique longitudinal data and significant differences in secretory

Acknowledgment

The writers thank Gail Fox, MSN, ANP; April Plank, MSN, ANP; and Ann Cuccia, RT, for their assistance in obtaining specimens; and Cathy Desilvia for her assistance with cytologic examination of specimens.

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Cited by (27)

Tracheostomy and related host-patogen interaction are associated with airway inflammation as characterized by tracheal aspirate analysis
2009, Respiratory Medicine
Citation Excerpt :
Thus, the study of tracheal aspirates in tracheostomized patients could provide clues to the pathways of airway inflammation in this particular setting, information that could prove useful also for the clinical management of tracheostomized patients. In contrast with this concept, only preliminary anecdotal data on tracheal aspirate analysis is available in a limited number of patients.5 Aim of our study was to evaluate the cellular, microbiological and molecular components of tracheal aspirates and to compare these characteristics in groups of chronic trachostomized patients with different underlying diseases and conditions.
In the last years an increasing number of subjects experienced respiratory failure and underwent tracheostomy. The aim of the present study was to analyze tracheal aspirates from the inflammatory point of view. Samples were collected from 38 consecutive tracheostomized patients: 13 COPD, 6 with neurologic disorders and 19 with other different causes of respiratory failure. We analyzed cells and soluble mediators related to inflammation and/or infection. We also compared results obtained in the tracheal aspirate of COPD patients with the same determination in the sputum of another group of non-tracheostomized COPD patients (n = 41).
Regardless of the underlying diagnosis, most of the samples were Pseudomonas aeruginosa positive and cells and soluble mediator did not differ. Treatment with steroids was associated with lower amount of total cells, neutrophils and lymphocytes, whereas treatment with antibiotics was not. Tracheal aspirate neutrophils correlated with PaCO₂ values; neutrophils and eosinophils correlated with their percentages in blood. As compared with sputa obtained from another group of culture-positive non-tracheostomized COPDs, tracheal aspirates showed similar cell count, proportions of inflammatory cells, and infection/inflammatory mediators.
In conclusion tracheal aspirates presented high amounts of viable cells and soluble mediators independently to the cause of respiratory failure leading to tracheotomy and they represent suitable specimens to study infection/inflammation interactions.
Are We heading toward clarity or confusion?
2009, Chest
Assessing the effects of racemic and single-enantiomer albuterol on airway secretions in long-term intubated patients
2006, Chest
Citation Excerpt :
Compared to ICU populations, this study population, with a median duration of intubation of 21 months, was relatively stable, and only 2 of 16 patients had to be withdrawn because of the introduction of antibiotics. Systemic and inhaled antibiotics can significantly affect secretion volume1516 and would therefore be a potentially serious confounding variable in a less stable population. Before proceeding to the clinical trial, a previously described model of aerosol delivery through a mechanical ventilator was used to estimate the amount of medication that these patients would be likely to inhale.171819
data suggest that the S-enantiomer of albuterol can induce mucociliary dysfunction. This clinical study assesses the clinical significance of standard doses of the S-enantiomer on airway secretions in long-term intubated patients by comparing a racemic formulation of albuterol, an R-enantiomer formulation, and normal saline solution.
A placebo-controlled crossover study.
Fourteen stable intubated patients with a median duration of intubation of 21 months and a median age of 72 years.
Long-term ventilator unit in skilled nursing facility.
Following a 2-week washout period during which regularly scheduled β₂-agonists were discontinued, tracheal aspirates were collected for 4 h/d for a 5-day period to establish baseline values, and the patients were then randomized in crossover manner to each of three nebulized treatments: normal saline solution, racemic albuterol, and R-albuterol. Each treatment was administered three times daily for 5 days, followed by a 2-day washout.
Tracheal aspirates were analyzed for volume, sodium, chloride, bicarbonate, interleukin (IL)-8, IL-1β, soluble intercellular adhesion molecule, and tumor necrosis factor-α.
There were no consistent significant differences among the three treatment periods either in terms of volume of secretions or in the concentrations of the electrolytes or the inflammatory indexes. However, all three treatments, including saline solution, were associated with increased secretion volume after the first dose, but this effect was not apparent on subsequent doses.
There were no significant differences between racemic albuterol and R-albuterol observed in this study for any of the parameters studied, suggesting that the S-enantiomer does not adversely affect airway secretions at recommended doses. In addition, the routine administration of nebulized β₂-sympathomimetic agonists to stable patients undergoing prolonged intubation, for the sole purpose of changing the volume and composition of secretions of airway secretions, is not supported by the results of this study.
Antiprotease function of airway secretions in purulent tracheobronchitis
2005, Chest
Study objectives: Unopposed activity of the serine protease, human leukocyte elastase (HLE), is detectable in the airways of patients with purulent tracheobronchitis. The aim of this study was to assess the compartmentalization of HLE activity in the liquid sol phase and the solid gel phase of airway secretions.
Design: Seventy samples of tracheobrochial aspirates were obtained from patients who had hypersecretion and were receiving mechanical ventilation.
Methods: Samples were separated into sol and gel (“mucous pellet”) phases, and HLE activity was measured using chromogenic substrate degradation. HLE was eluted from the mucous pellet using hypertonic saline solution, 1 mol/L, or bovine pancreatic deoxyribonuclease (DNase), 16 μmol/L.
Results: HLE activity partitioned between the sol and gel phases of the secretions, with most of the activity present in the gel phase (32:1 ratio of gel to sol HLE activity). The activity of HLE was 95% inhibited when bound to the gel phase, but activity appeared to be largely restored after elution from the gel phase. The gel phase was capable of binding additional exogenous HLE, and its binding capacity for exogenous HLE was not saturated by concentrations that exceeded the highest clinically relevant HLE levels (1.1 mg/mL). Hypertonic saline solution and DNase I efficiently liberated endogenous and exogenous gel phase-bound HLE activity, suggesting that electrostatic bonds and DNA, respectively, play important roles in binding HLE to the gel phase.
Conclusions: The solid phase of airway secretions is a more important modulator of elastase-antielastase balance than has been previously recognized.
Sputum matrix metalloproteinase-9, tissue inhibitor of metalloprotinease-1, and their molar ratio in patients with chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis and healthy subjects
2003, Respiratory Medicine
Background: Matrix-metalloproteinases (MMPs) and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), are involved in the turnover of extracellular matrix. Chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) are inflammatory diseases characterized by excessive matrix degradation and tissue fibrosis. We have compared sputum concentrations of MMP-9, TIMP-1 and the controlling cytokine tumor necrosis factor (TNF)-alpha in patients with COPD, IPF and healthy subjects. Methods: In a cross-sectional analysis, 12 patients with stable COPD, 15 patients with IPF and 14 healthy subjects underwent sputum induction. Induced sputum cells were counted and concentrations of MMP-9, TIMP-1 and TNF were measured by enzyme immunoassays. Results: Sputum neutrophils were markedly elevated in COPD and IPF patients compared with controls (P<0.001, both comparisons). Concentrations of MMP-9 and the MMP-9:TIMP-1 ratio were increased in COPD (P<0.001 vs. IPF and controls), whereas sputum TIMP-1 levels were both elevated in COPD and IPF (P<0.01 vs. controls, both comparisons). TNF levels were similar in all three groups (P>0.2, all comparisons). MMP-9 concentrations were negatively correlated with airway obstruction (FEV₁%FVC) in COPD (rho=−0.62, P=0.03), but not with diffusion capacity or vital capacity (% predicted) in IPF (rho=−0.06, P=0.85, and rho=−0.3, P=0.29, respectively). MMP-9 was positively correlated with sputum neutrophils in all patients (rho=0.68, P<0.0001), and with TNF in COPD patients (rho=0.76, P=0.004). Conclusions: These data underline the significance of protease/antiprotease imbalance for the pathogenesis of inflammatory lung diseases. Despite similar cellular inflammatory patterns both in COPD and IPF sputa, marked differences were observed with regard to MMP-9:TIMP-1 balance.
Long-term repeatability of induced sputum cells and inflammatory markers in stable, moderately severe COPD
2003, Chest
Neutrophilic inflammation is a major feature of COPD. Induced sputum is increasingly used to monitor inflammatory airway diseases. Although short-term repeatability of selected sputum markers has been extensively studied in several populations, data on the long-term repeatability of induced sputum markers in stable COPD are scant.
Sputum supernatant of 12 patients with stable COPD was analyzed on three separate occasions with 4-weekly intervals. Sputum cells and inflammatory markers interleukin (IL)-8 and soluble intercellular adhesion molecule (sICAM)-1 were measured in supernatant using enzyme-linked immunosorbent assay. Repeatability of sputum markers was expressed by intraclass correlation coefficients (Ri).
Sputum induction was safe in all patients. None of the sputum parameters analyzed changed significantly throughout the study. The repeatability for cell differential counts in stable COPD was as follows: total cells, Ri = 0.07; neutrophils, Ri = 0.66; macrophages, Ri = 0.47; eosinophils, Ri = 0.49; and lymphocytes, Ri = 0.58. The repeatability of soluble markers was as follows: IL-8, Ri = 0.50; and sICAM, Ri = 0.58. Sputum neutrophils were negatively correlated with lung function on each separate occasion, whereas soluble markers were not correlated with sputum cells (p > 0.16, all correlations) or lung function (p > 0.24, all correlations).
Clinically stable, moderate COPD is associated with equally stable sputum inflammatory markers. Repeatability of induced-sputum markers of neutrophilic inflammation in stable COPD is satisfactory, even over extended periods of time. These data support the usefulness of serial monitoring of induced-sputum inflammatory markers in COPD.

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: Supported by operational grant 371318 of University Medical Center, Stony Brook, NY.

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Chest

Clinical Investigations in Critical CareTracheal Aspirates in Long-term Mechanically Ventilated Patients: A Human Model of Gram-Negative Infection and Airway Inflammation

Section snippets

Study Population

Microbiologic Studies

Total Cell Counts and Differentials

Discussion

Acknowledgment

Chest

Chest

Chest

Chest

Changing pharyngeal flora of hospitalized patients

N Engl J Med

Nosocomial respiratory infections with Gram-negative bacilli: the significance of colonization of the respiratory tract

Ann Intern Med

Bacterial colonization profile with tracheal intubation and mechanical ventilation

Arch Surg

Sources of Gram-negative bacilli colonizing the trachea of intubated patients

J Infect Dis

Gastric and pharyngeal flora in nosocomial pneumonia acquired during mechanical ventilation

Am Rev Respir Dis

Role of gastric colonization in nosocomial infections and endotoxemia: a prospective study in neurosurgical patients on mechanical ventilation

J Infect Dis

Nutritional status and bacterial binding in the lower respiratory tract in patients with chronic traceostromy

Ann Intern Med

Adherence of Pseudomonas aeruginosa to tracheal cells injured by influenza infection or by endotracheal intubation

Infect Immun

Bacterial adherence to epithelial cells in bacillary colonization of the respiratory tract

Am Rev Respir Dis

Augmented bacterial adherence to tracheal epithelial cells is associated with Gram-negative pneumonia in an intensive care unit population

Am Rev Respir Dis

Role of salivary protease activity in adherence of Gram-negative bacilli to mammalian buccal epithelial cells in vivo

J Clin Invest

Increased salivary elastase precedes Gram-negative bacillary colonization in postoperative patients

Am Rev Respir Dis

Influence of sputum IgA and elastase on tracheal cell bacterial adherence

Am Rev Respir Dis

Clinical Investigations in Critical Care
Tracheal Aspirates in Long-term Mechanically Ventilated Patients: A Human Model of Gram-Negative Infection and Airway Inflammation