Children are predisposed to a variety of prenatal, natal, or postnatal risk factors which may lead to the development of chronic lung diseases.1 The terminology of pediatric “chronic lung diseases” include abnormalities in airways, lung parenchyma, blood vessels, or pleura.2 These pathologies may result from congenital parenchymal lung defects (such as congenital lobar emphysema, congenital cystic lung, sequestrated lobes, etc), airway disease (such as bronchiectasis, primary ciliary dyskinesia, cystic fibrosis, etc), or acquired interstitial lung diseases.3
In contrast to the healthy population, distal airway bacterial colonization with potentially pathogenic microorganisms (PPMs) may occur in patients with chronic lung diseases, who often have altered pulmonary defenses.4,5
The primary objective of this study was to demonstrate frequency of microbial colonization and ongoing cell damage in distal airways of children with stable chronic lung diseases using bronchoalveolar lavage fluid (BALF) compared with sputum and blood samples. The second objective was to evaluate the safety and feasibility of bronchoscopic technique in this pediatric population.
MATERIALS AND METHODS
This cross-sectional descriptive study was performed over a 17-month period between July 2005 and November 2006.
The study included 40 children, 13 years old or younger (age ranged between 8 mo and 13 y, median 5.25 y, mean 6.36+5 y) who were divided into 2 groups:
(A) Thirty patients with chronic pulmonary diseases, 16 females while they were free of any exacerbation and off antibiotics for at least 2 weeks. A sample size of 30 patients was considered appropriate (confidence level 80% of study power more than 85%—calculated using Epi Info software for windows, version 3.3.2; Atlanta, CDCP; 2005) for the nature of this study.
Inclusion criteria for the study: 1—persistent chest symptoms for more than 6 weeks as: chronic cough, dyspnea, excess sputum, or wheezes. 2—Abnormal and persistent chest x-ray findings for more than 6 weeks as: persistent lobe collapse, miliary appearance, unresolving pneumonia, persistent consolidation, honeycombing, or ground glass appearance. Exclusion criteria were as follows: recent exacerbation of pulmonary symptoms, use of antibiotics in past 2 weeks, history of asthma, active tuberculosis, or any contraindications to perform flexible bronchoscopy with bronchoalveolar lavage (BAL) (severe bronchospasm, pulmonary hypertension, cardiopulmonary instability, and severe hypoxemia).6
(B) Control group: 10 clinically healthy children (with no history or manifestations of any chest illness) were randomly selected. They were being operated upon owing to elective nonpulmonary surgical causes (inguinoscrotal hernia, lymph node biopsy, etc) and BALF was collected while they underwent general anesthesia.
All studied patients were subjected to full medical history, thorough clinical examination, plain chest x-rays, and blood sample collection.
Informed consent was obtained from the parents. This study was approved by the Research Ethics Committee of Ain Shams University Hospital.
- Blood: 5 mL of sterile blood was collected from a peripheral vein in all subjects.
- Sputum: spontaneous sputum was collected from older and cooperative children. They were asked to expectorate immediately before the bronchoscopy. Induced sputum (with concentrated saline inhalation) was obtained from younger children who could not expectorate.7 Processing of sputum samples was carried out according to Angrill coworkers.8
- BAL: all BALs were performed by the same operating team using a Pantex flexible bronchoscope system (PENTAX FB-10X bronchoscope with: external diameter of 3.4 mm, working channel diameter=1.2 mm, working length=600 mm, and total length=900 mm).
Prebronchoscopy evaluation for cardiac condition, blood pressure, and arterial blood gases was carried out on all patients. All subjects studied were fasting for at least 6 hours before bronchoscopy. They underwent bronchoscopy under general anesthesia using deep Halothane inhalation, monitored by electrocardiogram, and pulse oximetry and noninvasive blood pressure monitor.3,6 Flexible bronchoscope was introduced via endotracheal tube, laryngeal mask, or through face mask using swivel connector to maintain ventilation under general anesthesia. The tip of the bronchoscope was wedged into the diseased segment as indicated by chest x-ray, or in the right middle lobe or lingula when the pathology was generalized. Aliquot (1 to 3 mL/kg)of prewarmed sterile normal saline 0.9% (maximum of 3 aliquots) was used for lavage and rapidly aspirated through the suction port of the bronchoscope.4
Samples were rapidly transferred to the laboratory. Macroscopic examination was performed for caseous, purulent or bloody, and necrotic material. Sputum and BALF samples were pooled and centrifuged at 300 rpm for 15 minutes and separated into supernatant and sediment. Blood samples were divided into 2 parts: the first part was used as whole blood for microbiologic assessment and the second part was centrifuged for serum collection used for biochemical markers.
A—Microscopic examination: the sediment was spread on slides and stained by Giemsa stain to be examined microscopically to evaluate cellular pattern. The differential cell count was reported as percentages of the total cell count.
B—Quantitative bacteriologic cultures were performed on suitable media including blood agar, MacConckey agar, and chocolate agar medium. All cultures were incubated at 37°C under aerobic and anaerobic conditions and in a CO2-enriched atmosphere. Cultures were evaluated for growth after 24 and 48 hours and discarded, if negative, after 5 days. Bacterial growth was identified by: appearance of growth, rate of growth, colony pigmentation, type of media, and microscopic examination.
Bacterial agents were classified into PPMs or non-PPMs. PPMs were those microorganisms recognized as agents causing respiratory infections, whether or not belonging to the gastrointestinal or oropharyngeal flora; Gram-negative rods, such as Pseudomonas aeruginosa, Enterobacteriaceae, and Haemophilus spp.; Gram-positive cocci, such as Staphylococcus aureus and Streptococcus pneumoniae. Non-PPMs were those microorganisms belonging to the oropharyngeal or gastrointestinal flora that are not usually involved in respiratory infections in nonimmunocompromised patients (Streptococcus viridans group, Neisseria spp., Corynebacterium spp., Candida spp.,and others).
Colonization of lower airways cultures was considered present if BAL cultures yielded ≥103 colony-forming units (cfu)/mL, and was considered absent if they were sterile or yielded <103 cfu/mL. Bacterial sensitivity and antimicrobial susceptibility of the isolated organism were routinely performed.
(2) Cell Damage and Inflammatory Markers
Biochemical assessment of serum and the supernatant lactate dehydrogenase enzyme and alkaline phosphatase (ALP) enzyme levels in IU/L using enzyme kinetic assay on automated Hitachi 917 was performed.9 All samples were analyzed by the same microbiologist and the same hematologist, who were blinded as to the origin of the samples.
Descriptive data are expressed as means±SD. Continuous data were compared by the Student paired t test or Wilcoxon signed-rank test. Categorical data were compared by χ2 test analysis. Receiver operating curve (ROC) was obtained to calculate cut off values. κ test was performed for diagnostic agreement. Significant results were expressed as P value, odds ratio (OR) and 95% confidence interval (CI). Statistical significance was established at P<0.05. The statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) for Windows (version 11.5; SPSS Inc, Chicago, IL; 2006).
Demographic data: 30 patients were included in the study. Demographic data for the 30 patients are presented in Table 1.
Distal Airways Bacterial Colonization
The overall colonization rate among patient group was 73.3% (22/30). BALF culture resulted in growth of 32 PPMs from 22 patients. Gram-negative organisms were the predominating organisms detected in the airways of clinically stable patients with chronic lung diseases. Sputum culture resulted in growth of only 14 different PPMs from 10 patients (OR=5.5; 95% CI: 1.6-19.7, P=0.004).
Colonizing organisms detected by BALF culture: total Gram negative (n=22) (68.8%) and total Gram positive (n=10) (31.2%). Isolated Gram-negative organisms: E. coli (n=6), P. aeruginosa (n=5), Citrobacter freundii (n=4), Serratia odorifera (n=2), Haemophilus influenzae (n=1), Klebsiella spp. (n=1), Enterobacter cloaca (n=1), and Enterobacter agglomerans (n=2). Isolated Gram-positive organisms: S. aureus (n=6), methicillin-resistant S. aureus (n=2), and Pneumococcus pneumoniae (n=2).
Types of colonizing organisms detected by sputum culture: total Gram negative (n=5) (35.7%) and total Gram positive (n=9) (64.3%). Isolated Gram-negative organisms: E. coli (n=1), P. aeruginosa (n=2), and S. odorifera (n=2). Isolated Gram-negative organisms: S. aureus (n=6) and P. pneumoniae (n=3). Blood cultures showed poor results regarding airways colonization compared with BALF (OR=38.5; 95% CI: 6.4-302, P=0.0001).
The colonization rate was highest between patients with bronchiectasis (92.3%), high in patients with persistent or recurrent pneumonia and/or collapse (63.3%), and lowest in patients with interstitial lung disease (50%).
After acquisition of the microbiologic results patient group was divided into 2 subgroups: group 1 with bacterial colonization and group 2 without bacterial colonization.
All statistical comparison between groups 1 and 2 regarding clinical symptoms was nonsignificant.
Bronchoscopic visualization of the airways manifested different degrees of inflamed mucosa, which directed the BAL samples. Adverse events during the procedure were benign, transient, and did not require any treatment: lowest oxygen saturation noted was 80% and lasted for less than 2 minutes; bradycardia and a drop in blood pressure occurred in 2 patients but resolved spontaneously within seconds. These complications were observed in 50% of all subjects.
Forty samples of BAL were analyzed. All specimens were considered satisfactory (<1% squamous epithelial cells). The amount of fluid injected was 18.1±5.4 mL, the amount of aspirated fluid was 7.2±2.5 mL, and the ratio of fluid aspirated to fluid injected was appropriate (40%±12%).
In the present study, levels of inflammatory markers (LDH and ALP) in BAL were found to be significantly higher in patients than in the control group (P<0.05). Mean BALF (ALP) level in group 1 (68.95±7 IU/L) was statistically higher than group 2 mean level (52±12 IU/L)with P<0.05. Similarly, mean BALF (LDH) level in group 1 (639.4±188 IU/L) was statistically higher than group 2 mean level (226.6±132 IU/L) with P<0.05.
On comparing the levels of total cell count in BALF in the 3 groups, it was found that BALF of groups 1 and 2 contained significantly higher levels than that of control group (P<0.001) and BALF of group 1 contained significantly higher levels than that of group 2 (P<0.05).
On drawing the ROC for total leukocytic counts in BAL, area under the curve was 0.798, with best cut off level=1250×103 cell/mL BALF, giving sensitivity (to detect colonization)=95.5%, specificity=75%, positive predictive value=77.8%, and negative predictive value=66.7%.
In the sputum of our patients, there was no significant difference between groups 1 and 2 regarding the mean values of total and differential cells (P>0.05). However, sputum total leukocytic cell count showed significantly higher values in patients compared with healthy controls. On drawing ROC for total leukocytic counts in sputum, area under the curve=0.679, with best cut off level=1350×103 cell/mL sputum, giving sensitivity (to detect airway inflammation)=86.4%, specificity=37.5%, positive predictive value=79.2%, and negative predictive value=50% (Fig. 1).
Nevertheless, levels of BALF both neutrophil % and lymphocyte % in patients group were significantly higher than that of control group, their counts in BALF of group 1 was almost the same as group 2, with no significant difference.
In contrast, BALF macrophage % was found to be significantly higher in healthy control compared with patients group (and without any statistical difference between groups 1 and 2).
Agreement between sputum and BAL results of pathogens was poor for detection of bacterial colonization in children with chronic lung diseases (κ value=0.07, P>0.05).
BAL Diagnostic Specimen
Bronchoscopic BAL specimens seem ideal for the pediatric population, regarding reproducibility and safety. Even though bronchoscopic BAL is considered fairly safe, complications of BAL were frequent (in 50% of children) in this study, but most were transitory and benign. Meticulous monitoring of the patients' hemodynamics while performing the bronchoscopic lavage together with shortening the length of the procedure and the use of the appropriately sized scope are important precautions for performing a safe BAL in pediatric patients. Complications could probably be avoided if the procedure is performed by an experienced bronchoscopist and the children are monitored carefully during and after the procedure.6,8
Distal Airways Bacterial Colonization
The lower respiratory tract of a healthy child is sterile. In contrast, patients with bronchiectasis and chronic pulmonary diseases are often colonized with PPMs.10
Scientific evidence suggests that colonization of distal airways by PPMs may be specifically harmful to this group of patients. These microorganisms represent a potential risk for lung infections and may secrete several inflammatory mediators that cause progressive tissue damage and airway obstruction.11
The phenomenon of chronic pulmonary colonization, secondary inflammatory reaction, and progressive lung injury is a “vicious cycle” and is the reason why appropriate evaluation of distal airway colonization is needed. To break this vicious cycle, it is necessary to identify the colonizing bacteria and to know which antibiotic to be administered.1,12 However, in this study no relevant infection was noticed in all the children up to 4 weeks.
It was proved by using BALF culture that, among 30 patients with different chronic pulmonary disorders, 22 patients (73.3%) were colonized by different types of PPM. On using sputum as a sample for culture, it yielded colonization in only 10 children with a rate 33.3% (Table 2).
This result was in concordance with the study carried out by Angrill and coworkers,8 2001 who studied bronchial colonization by PPMs in 60 patients with bronchiectasis in a stable clinical situation using BALF culture and found that colonization rate was 60%.
In this study, BALF culture resulted in growth of 32 PPMs from 22 patients. Gram-negative organisms were the predominating organisms detected in the airways of clinically stable patients with chronic lung diseases. Sputum culture resulted in growth of only 14 different PPMs from 10 patients. Also, S. aureus was the most common organism in BALF cultures followed by E. coli, P. aeruginosa, C. freundii, other Enterobacteriacae, P. pneumoniae, H. influenzae, and finally Klebsiella spp. Two BALF samples resulted in heavy growth of methicillin-resistant S. aureus.
Indicators of Bacterial Colonization
In the present study, colonization rate was highest between patients with bronchiectasis (92.3%), high in patients with persistent or recurrent pneumonia and/or collapse (63.3%), and lowest in patients with interstitial pulmonary fibrosis (50%).
These data came in concordance with the study carried out by Cabello and coworkers3 in 1997. When they studied BALF samples from the airways of 100 patients with different chronic pulmonary problems, they found the highest colonization rate in patients with bronchiectasis.3 So category of chronic lung disease in children may point to higher or lower risk of distal airway bacterial colonization.
Parameters most often used to detect ongoing pulmonary inflammation in BALF are quantitative measures of the degree of the inflammatory response. Cellular changes observed in BALF during inflammation include an activation of alveolar macrophages (AMs) and an influx of polymorphonuclear neutrophils (PMNs).
Nevertheless, biochemical changes in BALF are suggested to be useful to detect pulmonary injury. The inflammatory process in the bronchial tree is complex. It involves the release of many mediators including chemo-attractants and cytokines. In the presence of neutrophils mainly, measuring myeloperoxidase activity and active neutrophil elastase may be helpful. However, it is not the case when AMs are predominant.13
In contrast, an increase of the activity of lactate dehydrogenase and ALP enzymes (which are normally intracellular) in the recovered BALF, reflects lung parenchyma cell damage or cell death of the predominant cell type.9
The ALP activity in BALF has been associated with type II cell damage or as an injury marker of cellular membranes from bronchial epithelium. These former cells are normally not present in BALF. That is why it was associated with bronchopulmonary pathology from different etiologies.14
Several pulmonary disorders have been associated with elevated LDH activity in serum and in BALF. Lung parenchymal cells and/or local inflammatory cells, including both AMs and PMNs, may be potential sources of LDH in BALF.13
In the present study, levels of inflammatory markers (LDH and ALP) in BAL were found to be significantly higher in patients than in the control group; and in group 1 (colonized) they were significantly higher than in group 2 (noncolonized). Nevertheless, the lack of statistically significant correlation between the levels of inflammatory markers in the BAL samples and those in the serum suggests that these markers are locally produced and not simply derived from the blood by simple diffusion.
However, these poor correlations were close to the adult BALF study performed by Hoffman and Rogers,15 other adult study has shown good correlation and mirror reflection of these parameters in both BAL and serum.16 At the same time no previous pediatric study has documented such comparison.
The relatively low levels of serum ALP and LDH compared with their levels in BALF and the poor correlations between them suggest that the inflammatory process in the airways is mostly compartmentalized and that serum enzymes cannot be used as a reflection for the ongoing airway inflammatory damage.
In 2002, Angrill and coworkers5 studied the levels of some inflammatory markers in BALF samples from patients with bronchiectasis and compared them to the serum levels. They found that patients showed a slight systemic inflammatory response. The levels of the different markers in BALF were significantly higher than in serum.5 They concluded that some increase in LDH is caused by transudation of serum, but to a minor component. The LDH in BALF seemed to originate from lung cells, probably AMs or PMNs.
Similarly, in the study by Cobben and coworkers9 in 1999, results of ALP and LDH levels in BALF of children with chronic lung diseases were very similar to that found by the present study.
These significantly high levels of chemical markers in children with chronic lung diseases may reflect the aggressive ongoing tissue damage and cell death—even in absence of exacerbation—that may eventually lead to progressive nonstop impairment and deterioration of pulmonary functions.17 So analysis of LDH level in BALF is a potentially useful tool for evaluating local lung tissue damage.18
Cytologic assessment showed that total leukocyte counts in BALF might have a specific diagnostic value for colonization. In this study, cut off level=1250×103&!thinsp;cell/mL BALF, giving sensitivity (to detect colonization) =95.5%, specificity=75% for detection of distal airways bacterial colonization. It is a reliable test to prove colonization and acceptable enough to exclude it based on predictive values.
Levels of BALF neutrophil percent and lymphocyte percent in all patients were significantly higher than that of the control group. This observation, again, reflects the tissue inflammation that is going on even without clinical exacerbation or colonization.
The BALF of group 1 contained more neutrophil and lymphocytes counts than group 2, however, this difference was not statistically significant.
Interestingly, our findings suggest that airway inflammation may occur even in the absence of colonization as demonstrated by the significant increase in levels of the different inflammatory mediators and cells among patients with negative BAL cultures, and may be intensified by the presence of colonizing microorganisms.
Angrill and coworkers8 in 2001 studied the bronchial inflammatory reaction in patients with bronchiectasis. Patients with bronchiectasis and negative cultures for PPM in the BALF had a more intense inflammatory reaction than did control subjects, with a higher percentage of neutrophils and higher concentrations of inflammatory markers. The group of patients with PPM in the airways had a higher BAL neutrophil count and higher BALF concentrations of inflammatory markers. So they concluded that inflammatory reaction might be intensified by the presence of PPM colonizing the lower respiratory tract.8
Further studies are needed to prove the correlation of the intensity of the cellular infiltrate to the intensity of the bacterial load.
In this study, BALF macrophage % was found to be significantly higher in controls than in patients (Table 3). This suggests that the differential leukocytic count in BALF of healthy children is relatively made by macrophages. This could be explained by the immunologic theory of self-protection during active inflammatory processes (like chronic lung diseases). As activation of proinflammatory cytokines (including macrophage inhibitory factor) are potent down regulators for macrophage production and activity.3
These findings were in concordance with the results of the European Respiratory Society task force 2000 on BAL in children.4
Ratjen and coworkers19 in 1994 studied the differential cytology of BAL fluid in 48 children aged 3 to 16 years (mean age±SD 7.9±3.5 y) undergoing elective surgery for nonpulmonary illnesses in a trial to put standard levels for pediatric population. Macrophages were the relative predominant cell type, with a mean percentage of 81.2%±12.7%, and this goes well with the results of our control group (Table 4).
Although different studies3,5,10 had used sputum bacteriology as a diagnostic tool to evaluate the presence of bronchial infection during exacerbations in children with bronchiectasis, very few have evaluated the pattern of bronchial colonization in clinically stable children (while not in exacerbation) and fewer studies dealt with BALF samples from children.
In our study, BAL cultures and sputum cultures gave different results. Sputum cultures did not reliably reflect conditions in the lower airways and have limited value. The results of BAL and sputum analysis agreed in 8 patients (26.7%), in whom both techniques resulted in negative cultures. Also they agreed in detection of 9 Gram-positive strains and 5 Gram-negative strains.
Sputum culture had missed 18 organisms in 12 patients. The defect was so evident in detection of the Gram-negative PPMs as it detected only 5 PPMs out of totally detected 22 PPMs. This poor agreement with the results of BALF culture may be explained by the fact that children cannot produce adequate amount of sputum and sputum arises from upper respiratory tract and oropharyngeal flora contaminate it, so it does not reflect the condition in the peripheral airways.
In conclusion, E. coli, P. aeruginosa, and S. aureus were the most common organisms in these patients and BALF was evident to be superior in detection of airways colonization. Knowledge of the type of colonizing agents may be important for future antibiotic prophylactic strategies and for the empirical antibiotic regimens when exacerbations occur in these patients.
The authors thank Ain Shams Pediatric Bronchology Laboratory staff for their help and support as well as, the participant children and their guardians for their cooperation and understanding.