Dataset Information

Bacteria from bronchoalveolar lavage fluid from children with suspected chronic lower respiratory tract infection: results from a multi-center, cross-sectional study in Spain.

ABSTRACT: This cross-sectional study assessed the prevalence of bacteria isolated from Spanish children with suspected chronic lower respiratory tract infection (LRTI) for whom bronchoalveolar lavage (BAL) was indicated. BAL fluid (BALF) was collected from 191 children (aged ??6 months to ?104?colony-forming units/mL) in 10.5, 8.9, and 6.3% of children, respectively. Clinical characteristics were similar among participants, regardless of positivity status for any of the bacteria. Approximately 26% of pneumococcal isolates were PCV13 serotypes, and 96% of H. influenzae isolates were non-typeable (NTHi). Concordance between BALF and NPS isolates was 51.0% for S. pneumoniae, 52.1% for H. influenzae, and 22.0% for M. catarrhalis.S. pneumoniae, NTHi, and M. catarrhalis were the main bacteria detected in BALF and NPS. Children with suspected chronic LRTI may benefit from a vaccine protecting against NTHi. What is Known: • Chronic lower respiratory tract infection (LRTI) in children can cause high morbidity and is a major use of healthcare resources worldwide. Despite this, their etiology or potential preventive measures are poorly assessed. • Bronchoalveolar lavage can be used to determine bacterial etiology of chronic LRTI. What is New: • We used conventional and molecular techniques to show that Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis were present in the LRT of Spanish children with suspected chronic LRTI • Concordance between isolates from bronchoalveolar lavage fluid and nasopharyngeal swabs was low, suggesting that samples from the upper respiratory tract could not reliably predict the bacterial etiology of suspected chronic LRTI.

SUBMITTER: Escribano Montaner A

PROVIDER: S-EPMC5758651 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Bacteria from bronchoalveolar lavage fluid from children with suspected chronic lower respiratory tract infection: results from a multi-center, cross-sectional study in Spain.

Escribano Montaner Amparo A García de Lomas Juan J Villa Asensi José Ramón JR Asensio de la Cruz Oscar O de la Serna Blázquez Olga O Santiago Burruchaga Mikel M Mondéjar López Pedro P Torrent Vernetta Alba A Feng Yang Y Van Dyke Melissa K MK Reyes Janet J Garcia-Corbeira Pilar P Talarico Carla A CA

European journal of pediatrics 20171229 2

This cross-sectional study assessed the prevalence of bacteria isolated from Spanish children with suspected chronic lower respiratory tract infection (LRTI) for whom bronchoalveolar lavage (BAL) was indicated. BAL fluid (BALF) was collected from 191 children (aged ≥ 6 months to < 6 years, with persistent or recurrent respiratory symptoms, non-responders to usual treatment) and cultured. Nasopharyngeal swabs (NPSs) were also obtained and cultured to assess concordance of BALF and NPS findings in ...[more]

PMID: 29285648

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Project description:Three small antimicrobial anionic peptides (AP) were originally isolated from an ovine pulmonary surfactant. However, their presence in bronchoalveolar lavage (BAL) fluid and tissues of the respiratory tract is unknown. In this study, we made affinity-purified rabbit polyclonal and mouse monoclonal antibodies to synthetic H-DDDDDDD-OH. Antibody specificity was assessed by a competitive enzyme-linked immunosorbent assay (ELISA), and the exact epitope binding sites were determined with analog peptides synthesized on derivatized cellulose. These antibodies were used to detect AP in BAL fluid by ELISA and in respiratory tissues by Western blot analysis and immunocytochemistry. BAL fluid from 25 sheep contained 0.83 +/- 0.33 mM AP (mean +/- standard deviation; range, 0.10 to 1.59 mM) and was antimicrobial. The presence of AP in BAL fluid was confirmed by reverse-phase high-pressure liquid chromatography fractionation followed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry on those fractions which were positive by competitive ELISA and demonstrated antimicrobial activity. In Western blots, polyclonal antibody PAB96-1 and monoclonal antibody 1G9-1C2 (5.0 micrograms/ml) detected four bands in solubilized turbinate and tracheal epithelial cells (53.7, 31.2, 28.0, and 25.7 kDa) and five bands in lung homogenates (53.5, 37.1, 31.2, 28.0, and 25.7 kDa). Only a single band was seen in solubilized liver and small-intestine homogenates, and no bands were seen in blots containing BAL fluid, albumin, or kidney or spleen homogenates. In pulmonary-tissue sections, both antibodies PAB96-1 and 1G9-1C2 identified accumulated protein in the apical cytoplasm of the bronchial and bronchiolar epithelia, in the cytoplasm of pulmonary endothelial cells, and in an occasional alveolar macrophage. As a first step in identifying a candidate AP precursor gene(s), degenerate oligonucleotides representing all possible coding combinations for H-GADDDDD-OH and H-DDDDDDD-OH were synthesized and used to probe Southern blots of sheep genomic DNA. Following low-stringency washes and a 2-day exposure, strongly hybridizing bands could be identified. One degenerate oligonucleotide, SH87, was used as a hybridization probe to screen a sheep phage genomic library. Two independent phage contained the H-GADDDDD-OH coding sequence as part of a larger predicted protein. AP may originate as part of an intracellular precursor protein, with multistep processing leading to the release of the heptapeptide into mucosal secretions. There it may interact with other innate pulmonary defenses to prevent microbial infection.

Project description:BackgroundIt is now possible to comprehensively characterize the microbiota of the lungs using culture-independent, sequencing-based assays. Several sample types have been used to investigate the lung microbiota, each presenting specific challenges for preparation and analysis of microbial communities. Bronchoalveolar lavage fluid (BALF) enables the identification of microbiota specific to the lower lung but commonly has low bacterial density, increasing the risk of false-positive signal from contaminating DNA. The objectives of this study were to investigate the extent of contamination across a range of sample densities representative of BALF and identify features of contaminants that facilitate their removal from sequence data and aid in the interpretation of BALF sample 16S sequencing data.ResultsUsing three mock communities across a range of densities ranging from 8E+?02 to 8E+?09 16S copies/ml, we assessed taxonomic accuracy and precision by 16S rRNA gene sequencing and the proportion of reads arising from contaminants. Sequencing accuracy, precision, and the relative abundance of mock community members decreased with sample input density, with a significant drop-off below 8E+?05 16S copies/ml. Contaminant OTUs were commonly inversely correlated with sample input density or not reproduced between technical replicates. Removal of taxa with these features or physical concentration of samples prior to sequencing improved both sequencing accuracy and precision for samples between 8E+?04 and 8E+?06 16S copies/ml. For the lowest densities, below 8E+?03 16S copies/ml BALF, accuracy and precision could not be significantly improved using these approaches. Using clinical BALF samples across a large density range, we observed that OTUs with features of contaminants identified in mock communities were also evident in low-density BALF samples.ConclusionRelative abundance data and community composition generated by 16S sequencing of BALF samples across the range of density commonly observed in this sample type should be interpreted in the context of input sample density and may be improved by simple pre- and post-sequencing steps for densities above 8E+?04 16S copies/ml.

Dataset Information

Bacteria from bronchoalveolar lavage fluid from children with suspected chronic lower respiratory tract infection: results from a multi-center, cross-sectional study in Spain.

Publications

Bacteria from bronchoalveolar lavage fluid from children with suspected chronic lower respiratory tract infection: results from a multi-center, cross-sectional study in Spain.

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