Molecular identification of bacteria in bronchoalveolar lavage fluid from children with cystic fibrosis.

Culture of bronchoalveolar lavage fluid (BALF) is the gold standard for detection of pathogens in the lower airways in cystic fibrosis (CF). However, current culture results do not explain all clinical observations in CF, including negative culture results during pulmonary exacerbation and inflammation in the absence of pathogens. We hypothesize that organisms not routinely identified by culture occur in the CF airway and may contribute to disease. To test this hypothesis we used a culture-independent molecular approach, based on use of rRNA sequence analysis, to assess the bacterial composition of BALF from children with CF and disease controls (DC). Specimens from 42 subjects (28 CF) were examined, and approximately 6,600 total clones were screened to identify 121 species of bacteria. In general, a single rRNA type dominated clone libraries from CF specimens, but not DC. Thirteen CF subjects contained bacteria that are not routinely assessed by culture. In four CF subjects, candidate pathogens were identified and include the anaerobe Prevotella denticola, a Lysobacter sp., and members of the Rickettsiales. The presumptive pathogens Tropheryma whipplei and Granulicatella elegans were identified in cases from the DC group. The presence of unexpected bacteria in CF may explain inflammation without documented pathogens and consequent failure to respond to standard treatment. These results show that molecular techniques provide a broader perspective on airway bacteria than do routine clinical cultures and thus can identify targets for further clinical evaluation.

Induced sputum matrix metalloproteinase-9 correlates with lung function and airway inflammation in children with cystic fibrosis.

Matrix metalloproteinases (MMPs) degrade extracellular matrix and are implicated in causing airway damage in chronic inflammatory lung diseases, including cystic fibrosis (CF). Our primary objective was to examine the relationship between matrix metalloproteinase-9 (MMP-9) and pulmonary function, as measured by forced expiratory volume in 1 sec (FEV1), in children with CF. We measured MMP-9 and its natural tissue inhibitor of metalloproteinase-1 (TIMP-1) in induced sputum from 18 clinically stable CF children with normal to mildly abnormal lung function and 7 healthy control children. Measures of airway inflammation from induced sputum included cell counts and differentials, interleukin-8 (IL-8), neutrophil elastase, MMP-9, and TIMP-1. Infection was assessed through quantitative bacterial counts. Induced sputum levels of MMP-9 and TIMP-1 were significantly increased in children with CF compared with healthy controls. Also, the MMP-9/TIMP-1 molar ratio was higher in the CF group. Among CF children, there was a significant inverse relationship between MMP-9 and FEV1. In addition, sputum MMP-9 and TIMP-1 concentrations significantly correlated with total white cells and neutrophils, IL-8, and neutrophil elastase. Neither MMP-9 nor TIMP-1 correlated with airway infection. We conclude that clinically stable CF children with normal to mildly abnormal lung function have an increased burden of MMP-9 in their airways. The observed relationships of MMP-9 with lung function and other measures of airway inflammation suggest that this enzyme may be a useful marker of airway injury and airflow obstruction in persons with CF.

Induced sputum inflammatory measures correlate with lung function in children with cystic fibrosis.

OBJECTIVE: To validate a sputum induction technique in cystic fibrosis (CF), we examined the relation between airway inflammation and pulmonary function in children with CF by correlating inflammatory indexes in induced sputum with FEV(1). STUDY DESIGN: We measured baseline spirometry and oxygen saturations and then performed sputum inductions with 3% hypertonic saline in 20 clinically stable children with CF (11 girls). We examined the relation of airway inflammation and lung function in the 19 individuals (95%) who expectorated an adequate sputum sample. Measures of airway inflammation in induced sputum included total cell counts, neutrophil (PMN) counts, interleukin-8 levels, and free neutrophil elastase activity. RESULTS: There were significant inverse relations between FEV(1) and total cell counts and PMN counts (r = -0.57, P <.01 for both), interleukin-8 (r = -0.72, P =.002), and elastase (r = -0.75, P =.001). Airway infection, as assessed by bacterial density in induced sputum, did not correlate with lung function or indexes of inflammation. CONCLUSIONS: We conclude that measures of inflammation in induced sputum correlate with FEV(1) in clinically stable children with CF with normal to mildly abnormal lung function and that they may be useful as surrogate outcome measures in clinical trials.