Airway Specimens Research Articles

Reliability of Quantitative Real-Time PCR for Bacterial Detection in Cystic Fibrosis Airway Specimens

The cystic fibrosis (CF) airway microbiome is complex; polymicrobial infections are common, and the presence of fastidious bacteria including anaerobes make culture-based diagnosis challenging. Quantitative real-time PCR (qPCR) offers a culture-independent method for bacterial quantification that may improve diagnosis of CF airway infections; however, the reliability of qPCR applied to CF airway specimens is unknown. We sought to determine the reliability of nine specific bacterial qPCR assays (total bacteria, three typical CF pathogens, and five anaerobes) applied to CF airway specimens. Airway and salivary specimens from clinically stable pediatric CF subjects were collected. Quantitative PCR assay repeatability was determined using triplicate reactions. Split-sample measurements were performed to measure variability introduced by DNA extraction. Results from qPCR were compared to standard microbial culture for Pseudomonas aeruginosa, Staphylococcus aureus, and Haemophilus influenzae, common pathogens in CF. We obtained 84 sputa, 47 oropharyngeal and 27 salivary specimens from 16 pediatric subjects with CF. Quantitative PCR detected bacterial DNA in over 97% of specimens. All qPCR assays were highly reproducible at quantities ≥102 rRNA gene copies/reaction with coefficient of variation less than 20% for over 99% of samples. There was also excellent agreement between samples processed in duplicate. Anaerobic bacteria were highly prevalent and were detected in mean quantities similar to that of typical CF pathogens. Compared to a composite gold standard, qPCR and culture had variable sensitivities for detection of P. aeruginosa, S. aureus and H. influenzae from CF airway samples. By reliably quantifying fastidious airway bacteria, qPCR may improve our understanding of polymicrobial CF lung infections, progression of lung disease and ultimately improve antimicrobial treatments.

Community-acquired Oseltamivir-Resistant Pandemic (H1N1) 2009 in Child, Israel

Community-acquired Oseltamivir-Resistant Pandemic (H1N1) 2009 in Child, Israel

Development of Oseltamivir Resistance During Oseltamivir Therapy in a Child With Severe 2009 H1N1 Influenza

Development of Oseltamivir Resistance During Oseltamivir Therapy in a Child With Severe 2009 H1N1 Influenza

Comparison of multiple typing methods for Aspergillus fumigatus

As part of studies on the spread of infections, risk factors and prevention, several typing methods were developed to investigate the epidemiology of Aspergillus fumigatus. In the present study, 52 clinical isolates of A. fumigatus from 12 airway specimens from patients with invasive aspergillosis (hospitalized in three different centres) were characterized by short tandem repeat (STR) typing and multilocus sequence typing (MLST). These isolates were previously typed by random amplified polymorphic DNA (RAPD), sequence-specific DNA polymorphism (SSDP), microsatellite polymorphism (MSP) and multilocus enzyme electrophoresis (MLEE). STR typing identified 30 genotypes and, for most patients, all isolates were grouped in one cluster of the unweighted pair group method with arithmetic mean dendrogram. Using MLST, 16 genotypes were identified among 50 isolates, while two isolates appeared untypeable. RAPD, MSP, SSDP and MLEE allowed identification of eight, 14, nine and eight genotypes, respectively. Combining the results of these methods led to the delineation of 25 genotypes and a similar clustering pattern as with STR typing. In general, STR typing led to similar results to the previous combination of RAPD, SSDP, MSP and MLEE, but had a higher resolution, whereas MLST was less discriminatory and resulted in a totally different clustering pattern. Therefore, this study suggests the use of STR typing for research concerning the local epidemiology of A. fumigatus, which requires a high discriminatory power.

Effect of Repeated Induced Airway Collapse During Total Liquid Ventilation

Negative pressure generated during the expiratory phase of total liquid ventilation (TLV) may induce airway collapse. Evaluation of the effect of repeated airway collapse is crucial to optimize this technique. A total of 24 New Zealand White rabbits were randomly divided into four groups. Ventilation was performed for 6 hours with different strategies: conventional gas ventilation, TLV without airway collapse, and TLV with collapse induced in either 75 or 150 sequential breaths. In the treated groups, airway collapse was induced by increasing the perfluorocarbon drainage velocity while maintaining the minute ventilation constant. Airway pressure, gas exchange, and blood pressure were monitored at 30-minute intervals. At the end of the experiment, airway and lung parenchyma specimens were processed for light microscopy. No evidence of fluorothorax was noticed in any of the four groups at autopsy examination. Minimal signs of inflammation were noticed in all airway and lung parenchyma specimens, but no evident structural alteration was visible. Adequate gas exchange and systemic blood pressure were maintained during all the studies. Repeated airway collapse is not associated with structural changes in the respiratory system and does not alter the gas exchange ability of the lungs.

94 High bacterial diversity of Staphylococcus aureus populations in airway specimens of cystic fibrosis patients

94 High bacterial diversity of Staphylococcus aureus populations in airway specimens of cystic fibrosis patients

Activation of Nuclear Factor-κB in Airway Epithelial Cells in Patients with Chronic Obstructive Pulmonary Disease

Background: Chronic obstructive pulmonary disease (COPD) is characterized by chronic airway inflammation. Nuclear factor-ĸB (NF-ĸB) is a transcription factor that mediates proinflammatory gene expression. NF-ĸB is activated when its inhibitor, IĸBα, is phosphorylated and degraded. Objectives: The aims of this study were to compare the number of phosphorylated IĸBα-immunopositive airway epithelial cells (AECs) in the peripheral airways of patients with COPD, asymptomatic smokers and asymptomatic nonsmokers. Methods: We examined lung tissues obtained from 10 smokers with COPD, 7 asymptomatic smokers and 10 asymptomatic nonsmokers. Paraffin-embedded sections and immunohistochemical techniques were used to assess the number of phosphorylated IĸBα-positive AECs as well as the numbers of tumor necrosis factor (TNF)α-positive, 8-hydroxy-2′-deoxyguanosine (8-OHdG)-positive and 4-hydroxy-2-nonenal (4-HNE)-positive AECs in the peripheral airway specimens. Results: The percentages of phosphorylated IĸBα-positive AECs and TNFα-positive AECs out of the total number of AECs were significantly higher in patients with COPD than in asymptomatic nonsmokers (p < 0.05). The percentage of phosphorylated IĸBα-positive AECs was positively correlated with the percentage of TNFα-positive AECs (r = 0.82, p < 0.01), but not with the percentage of 8-OHdG-positive AECs or the percentage of 4-HNE-positive AECs. Conclusions: The activation of NF-ĸB, which was evaluated by measuring the level of phosphorylated IĸBα, was enhanced in the peripheral airway epithelia of the COPD patients in this study. The activation of NF-ĸB in the peripheral airway epithelium is associated with an elevated level of TNFα and seems to occur through a mechanism independent of oxidative stress.

Obligatory Role for Interleukin-13 in Obstructive Lesion Development in Airway Allografts

The pathogenesis of bronchiolitis obliterans (BO), a common and devastating obliterative disorder of small airways following lung transplantation, remains poorly understood. Lesions are characterized in their early stages by lymphocyte influx that evolves into dense fibrotic infiltrates. Airway specimens taken from patients with histological BO revealed infiltrating myofibroblasts, which strongly expressed the signaling chain of the high affinity interleukin-13 (IL-13) receptor IL-13Ralpha1. Because IL-13 has proinflammatory and profibrotic actions, a contributory role for IL-13 in BO development was examined using murine models of orthotopic and heterotopic tracheal transplantation. Compared with airway isografts, allografts exhibited a significant increase in relative IL-13 mRNA and protein levels. Allogeneic tracheas transplanted into IL-13-deficient mice were protected from BO in both transplant models. Flow cytometric analysis of orthotopic transplant tissue digests revealed markedly fewer infiltrating mononuclear phagocytes and CD3(+) T lymphocytes in IL-13-deficient recipients. Furthermore, protection from luminal obliteration, collagen deposition, and myofibroblast infiltration was observed in heterotopic airways transplanted into the IL-13(-/-) recipients. Transforming growth factor-beta1 expression was significantly decreased in tracheal allografts into IL-13(-/-) recipients, compared to wild-type counterparts. These human and murine data implicate IL-13 as a critical effector cytokine driving cellular recruitment and subsequent fibrosis in clinical and ex-perimental BO.

Histopathologic and Immunohistochemical Features of Fatal Influenza Virus Infection in Children during the 2003–2004 Season

The Centers for Disease Control and Prevention enhanced national surveillance for influenza-associated deaths among children because of early reports of pediatric deaths during the 2003-2004 influenza season. We studied lung and upper airway specimens from 47 case patients who died who had at least 1 positive result for influenza virus tests using hematoxylin and eosin, special stains for bacteria and fungi, and immunohistochemical (IHC) assays for influenza A and B viruses and other potential viral and bacterial respiratory pathogens. Nineteen (40%) of the 47 patients were <or=2 years old, and 26 (55%) were female. Influenza IHC testing identified type A antigens in 26 patients and type B antigens in 1 patient. Influenza antigens were observed focally in bronchoepithelial cells and mucous glands of trachea, bronchi, and larger bronchioli, showing submucosal mononuclear inflammatory infiltrates. IHC assays were the only confirmatory diagnostic test for 5 patients (11%). Significant life-threatening pathological conditions that could be considered the cause of death were present in 36 patients (77%) and included diffuse alveolar damage (12 cases), extensive secondary pneumonia (11 cases), extensive intraalveolar hemorrhage (10 cases), viral pneumonitis (10 cases), myocarditis (6 cases), and meningoencephalitis (1 case). For 9 patients with bronchopneumonia, a bacterial or fungal etiology was determined with IHC assay (3 Staphylococcus infections, 3 group A streptococci infections, 1 Streptococcus pneumoniae infection, 1 Bordetella pertussis infection, and 1 Aspergillus infection). IHC assays are useful for the diagnosis of influenza and bacterial pneumonia. This study underscores the importance of performing autopsies to identify the causes of death in patients with influenza infection.

Characterization of Wild-Type and ΔF508 Cystic Fibrosis Transmembrane Regulator in Human Respiratory Epithelia

Previous studies in native tissues have produced conflicting data on the localization and metabolic fate of WT and deltaF508 cystic fibrosis transmembrane regulator (CFTR) in the lung. Combining immunocytochemical and biochemical studies utilizing new high-affinity CFTR mAbs with ion transport assays, we examined both 1) the cell type and region specific expression of CFTR in normal airways and 2) the metabolic fate of deltaF508 CFTR and associated ERM proteins in the cystic fibrosis lung. Studies of lungs from a large number of normal subjects revealed that WT CFTR protein localized to the apical membrane of ciliated cells within the superficial epithelium and gland ducts. In contrast, other cell types in the superficial, gland acinar, and alveolar epithelia expressed little WT CFTR protein. No deltaF508 CFTR mature protein or function could be detected in airway specimens freshly excised from a large number of deltaF508 homozygous subjects, despite an intact ERM complex. In sum, our data demonstrate that WT CFTR is predominantly expressed in ciliated cells, and deltaF508 CFTR pathogenesis in native tissues, like heterologous cells, reflects loss of normal protein processing.

Practices in non-neutropenic ICU patients with Candida-positive airway specimens.

To examine practices of French intensivists regarding the management of mechanically ventilated patients with Candida-positive airway specimens but no major risk factors for immunodepression. Design : Closed-item questionnaire with a clinical vignette. 564 French intensive care units (ICUs). 198 intensivists who have a special interest in infectious diseases and who answered the questionnaire (response rate, 35.1%). None. The respondents recommended bronchoalveolar lavage (62.6% of respondents), protected distal sampling and protected specimen brush (59.1%), transbronchial biopsy (38.9%), and tracheal aspiration (12.1%) for the diagnosis of candidal pneumonia. A positive airway specimen was felt by most respondents (83.3%) to indicate colonisation; 66.7% of respondents recommended tests for systemic candidiasis in this situation, and 56.5% serial sampling to compute the colonisation index. Azole derivatives were the preferred antifungal medications. The clinical vignette described a patient with chronic obstructive lung disease who required mechanical ventilation for an acute exacerbation and who had a tracheal aspirate positive for Candida. Responses varied widely, with 37.8% of respondents diagnosing clinically insignificant colonisation but 24.2% recommending antifungal treatment and 61.6% serial testing to assess the Candida colonisation index. Intensivists with greater experience with severely immunocompromised patients were more aggressive in their diagnostic management. Wide variations occur among practices of French intensivists regarding Candida-positive airway specimens in patients without major risk factors for immunodepression. Additional studies are needed to improve our understanding of the links between Candida colonisation and infection and to determine the indications for pre-emptive antifungal treatment in non-neutropenic critically ill patients.

In-depth analysis of molecular alterations within normal and tumour tissue from an entire bronchial tree.

Using laser capture microdissection (LCM), fluorescent microsatellite analysis and immunohistochemical analysis, we have constructed a detailed topographical molecular map of the entire bronchial tree surrounding a primary bronchial squamous carcinoma in order to establish the relationship between the molecular damage within the airway and that in the tumour itself. Allelic imbalance was analysed using markers on chromosomes 3, 9, 13 and 17. In addition, immunohistochemical analysis for p53 and cyclin D1 expression was performed. Analysis revealed allelic imbalance at several loci at the tumour site but also in 83% of the histologically normal airway specimens of the upper and lower lobes. The fractional allele loss (FAL) value was statistically higher (0.75+/-0.13) in the tumour site than in the distal site of the upper (0.42+/-0.09) and lower lobes (0.31+/-0.08). Immunohistochemical analysis revealed overexpression of p53 and cyclin D1 protein within histologically normal bronchial epithelium, thus confirming previous reports for their early involvement in lung tumour development. This is to date the largest in-depth study of allelic imbalance using LCM in a single individual. The patterns of allele-specific imbalance observed support a clonal or oligoclonal expansion model of outgrowths throughout the lung. The widespread incidence of genetic changes in the whole of lung most likely represents smoking-induced alterations and emphasize the complexity of the field cancerization concept. Our findings point to the need for in-depth studies of the whole bronchial tree tissue surrounding lung carcinomas, in order to identify the genetic changes that differentiate preneoplastic and neoplastic stages in lung carcinogenesis.

Nitric oxide treatment and acute pulmonary inflammatory response in very premature infants with intractable respiratory failure shortly after birth.

Premature infants with respiratory failure and early-onset pneumonia have low inducible nitric oxide synthase (NOS2) and no evidence of nitric oxide (NO) toxicity. However, inhalation of NO may not be indicated in sepsis because excessive NO generation has been reported. This prospective study was designed to test the hypothesis that inhaled NO is effective in a select group of small premature infants and that the responsiveness to NO is associated with low NOS2 enzyme. 246 very low birthweight infants (birthweight <1500 g, VLBW) were screened for severe, intractable respiratory failure (oxygenation index >40, arterial-alveolar ratio for oxygen tension <0.10) that does not respond to two doses of surfactant within 5 h from birth. Infants with severe cardiac failure or a bleeding disorder were excluded. Five of the nine eligible cases received inhaled NO. They all had prolonged rupture of foetal membranes, early-onset pneumonia and persistent pulmonary hypertension. All five responded strikingly, survived and appeared normal in follow-up. Airway specimens during the first day of life revealed very low NOS2, interleukin-1beta and surfactant protein A, compared with VLBW infants who had no acute infection despite histological chorioamnionitis. In early-onset pneumonia, NOS2 and other inflammatory mediators increased first during the recovery 1-2 d after birth. VLBW infants with progressive respiratory failure and infection at birth have deficient pulmonary NOS2 and cytokine response. After surfactant therapy, these infants responded strikingly to inhaled NO. An acute pulmonary inflammatory response may contribute to respiratory adaptation in early-onset pneumonia.

The Editors’ Choice

The Editors’ Choice

Diminished inducible nitric oxide synthase expression in fulminant early-onset neonatal pneumonia.

Fulminant early-onset neonatal pneumonia is associated with ascending intrauterine infection (IUI), prematurity, persistent pulmonary hypertension (PPHN), and septicemia. Nitric oxide (NO) as an inflammatory mediator is included in antimicrobial defense and has a role in pathogenesis of septic shock. The aim was to study the role of inflammatory NO in neonatal pneumonia. Lungs from 36 autopsies were studied: 12 had fulminant early-onset neonatal pneumonia, 5 pneumonia of later onset, and 19 controls had similar gestational and postnatal age. In addition, airway specimens from 21 intubated newborns were analyzed: 7 with fulminant early-onset pneumonia, 7 apparently noninfected infants born prematurely attributable to IUI, and 7 premature infants of similar gestation. Specimens were analyzed for inducible NO synthase (NOS2) and nitrotyrosine, an indicator of NO toxicity. The degree of staining was analyzed. In fulminant pneumonia, alveolar macrophages (AM) showed significantly less NOS2 immunoactivity than the controls. In the airway specimens, the infants with fulminant pneumonia 0 to 2 days after birth had significantly lower intracellular NOS2 and nitrotyrosine and significantly lower interleukin-1beta and surfactant protein-A than apparently noninfected IUI infants. NOS2 and the other indices increased significantly during the recovery. For the first time, we report NOS2 expression by macrophages from human neonates. In fulminant early-onset neonatal pneumonia, delayed production rather than excess of pulmonary inflammatory NO is associated with severe symptoms.

Granulocyte-macrophage colony-stimulating factor increases surfactant phospholipid in premature rabbits.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic cytokine that is low in airway specimens from immature lungs at birth. In adult mice, an absence of GM-CSF causes excessive accumulation of alveolar surfactant due to a lack of catabolism. Our aim was to investigate whether recombinant human GM-CSF (rhGM-CSF) affects the pool sizes or the turnover of disaturated phosphatidylcholine (DPC) in preterm (gestation 29 d) rabbits at birth and in term rabbits, age 3 d. 3H-labeled dipalmitoyl phosphatidylcholine, 14C-acetate, and either rhGM-CSF (125 or 25 microg/kg body weight) or placebo were given intratracheally. Thereafter, the intra- and extracellular surfactant fractions were isolated and quantified for DPC and radioactivity. In preterm animals, GM-CSF increased dose-dependently within 24 h both the pool sizes of surfactant DPC and the 3H,14C-labeling of surfactant DPC (p < 0.05). The expression of surfactant protein B mRNA was unaffected, whereas surfactant protein B in bronchoalveolar lavage increased. The number of cells in the whole lung, the type II alveolar epithelial cells, and the lavageable alveolar macrophages were unaffected. At term, rhGM-CSF increased the turnover but did not affect the pool sizes of surfactant DPC. Intraperitoneal rhGM-CSF increased blood eosinophils but had no effect on surfactant DPC. Depending on the degree of lung maturity, GM-CSF in the alveolar space may either up-regulate the pool size or increase the turnover of surfactant phospholipid after birth.

Expression and localization of COX-2 in human airways and cultured airway epithelial cells.

Cyclo-oxygenase is the rate-limiting enzyme in the prostanoid pathway. Although expression of the inducible isoform of cyclo-oxygenase (COX-2) is associated with cytokine-mediated inflammation, recent evidence suggests a homeostatic role for epithelial COX-2 in the gastrointestinal tract. The aim of this study was to examine the expression and localization of COX-2 in human airway epithelium both in vivo and in vitro. Human airway specimens from patients undergoing lung resection surgery for primary lung tumours (n=10) or nasal mucosal resection for non-inflammatory nasal obstruction (n=5) were examined for COX-2 expression by in situ hybridization and immunohistochemistry. COX-2 expression was also studied in two human airway epithelial cell lines (BEAS-2B and A549) using reverse transcription polymerase chain reaction and Northern and Western blot analysis. COX-2 messenger ribonucleic acid (mRNA) and protein were localized to individual columnar epithelial cells and to airway resident inflammatory cells in 9/10 lower and 5/5 upper airway specimens. Expression of COX-2 did not correlate with evidence of airway inflammation. Focal expression of COX-2 mRNA and protein was observed in bronchus-associated lymphoid tissue. Both COX-2 mRNA and protein were detected in BEAS-2B and A549 cells cultured under standard conditions. In conclusion, expression of COX-2 in human airway epithelium occurs in the upper and lower airways, is widespread in airway epithelial and airway resident inflammatory cells in the absence of overt airway inflammation, and is detectable in cultured human airway epithelial cells in the absence of inflammatory cytokine stimulation. These data suggest a potentially important homeostatic role for COX-2 in the regulation of human airway contractility, inflammation and immune responses.

Evidence for increased oxidative stress in preterm infants eventually developing chronic lung disease

According to present evidence, prematurity and acute neonatal lung disease are principal risk factors of chronic lung disease originating from the perinatal-neonatal period (CLD). Likewise, oxidant stress is associated with degree of prematurity and with acute lung disease. High free radical activity — quantitated as expired ethane and pentane, other lipid peroxidation products in airway specimens or as protein oxidation products — is associated with high risk of CLD. Oxidant stress increases after birth to a maximum by 2–6 days of age. Thus, there is an interim period for potential antioxidant therapies. Inflammatory cells, intravenously administered lipids and supplemental oxygen are among the enhancers of oxidant stress in infants developing CLD. Low antioxidant enzymes, vitamins, excess free iron and deficient iron binding/metabolizing capacity are additional features favouring oxidant stress in the premature infant. Evidence of highly reactive free radical activity — hydroxyl radicals and metabolism products of NO-peroxynitrite — known to cause many pathological features characteristic of CLD, have been detected in infants at risk. The importance of free radicals in CLD and in other chronic organ injuries originating from the perintal-neonatal period remain to be confirmed in therapeutic trials.

Expression of epidermal growth factor and epidermal growth factor receptor immunoreactivity in the asthmatic human airway.

Chronic airway inflammation, one of the pathophysiologic features of bronchial asthma, is suspected to be responsible for irreversible pathological changes of airways, called airway remodeling. To examine the mechanisms of airway remodeling in asthma, we investigated the expression of epidermal growth factor (EGF) and its receptor immunohistochemically in asthmatic human airways. Airway specimens from seven patients with asthma were obtained from autopsied and surgically resected lungs. Control specimens were obtained from lungs of eight subjects without asthma and other pulmonary complications at autopsy. We stained those specimens by the avidin-biotin-peroxidase complex (ABC) method with anti-human polyclonal EGF antibody and monoclonal EGF receptor antibodies. Three different portions of airways-large bronchi (about 1 cm in diameter), small bronchi (about 3 mm in diameter), and peripheral airways (less than 2 mm in diameter)-were examined. The thickness of the bronchial smooth muscle and basement membrane was significantly greater in the asthmatic airways than in controls. Clear immunoreactivities of EGF were widely observed on bronchial epithelium, glands, and smooth muscle in asthmatic airways. In the controls, the bronchial epithelium and the bronchial glands partially expressed faint EGF immunoreactivity. For the EGF receptor, clear immunoreactivities were also observed on bronchial epithelium, glands, smooth muscle, and basement membrane in asthmatic airways. In control airways, only part of the bronchial epithelium and smooth muscle weakly expressed EGF receptor immunoreactivity. These results suggest a possible contribution of EGF to the pathophysiology of bronchial asthma, including airway remodeling.

Pulmonary toxicity associated with nitric oxide in term infants with severe respiratory failure

We prospectively analyzed airway specimens from 24 newborn infants. Inhaled nitric oxide (≤20 ppm for 1 to 4 days to 12 infants) did not affect the concentrations of the lipid peroxidation product, the surface activity, or the cytokines (interleukin-1, granulocyte-macrophage colony-stimulating factor, interleukin-1 receptor antagonist). Nitrotyrosine was detected after 10 days of life in the two infants requiring prolonged ventilation, suggesting toxicity of endogenous nitric oxide. (J Pediatr 1998;132:827-9)