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Assessing bacterial populations in the lung by replicate analysis of samples from the upper and lower respiratory tracts.

ABSTRACT: Microbes of the human respiratory tract are important in health and disease, but accurate sampling of the lung presents challenges. Lung microbes are commonly sampled by bronchoscopy, but to acquire samples the bronchoscope must pass through the upper respiratory tract, which is rich in microbes. Here we present methods to identify authentic lung microbiota in bronchoalveolar lavage (BAL) fluid that contains substantial oropharyngeal admixture. We studied clinical BAL samples from six selected subjects with potential heavy lung colonization. A single sample of BAL fluid was obtained from each subject along with contemporaneous oral wash (OW) to sample the oropharynx, and then DNA was extracted from three separate aliquots of each. Bacterial 16S rDNA sequences were amplified and products analyzed by 454 pyrosequencing. By comparing replicates, we were able to specify the depth of sequencing needed to reach a 95% chance of identifying a bacterial lineage of a given proportion--for example, at a depth of 5,000 tags, OTUs of proportion 0.3% or greater would be called with 95% confidence. We next constructed a single-sided outlier test that allowed lung-enriched organisms to be quantified against a background of oropharyngeal admixture, and assessed improvements available with replicate sequence analysis. This allowed identification of lineages enriched in lung in some BAL specimens. Finally, using samples from healthy volunteers collected at multiple sites in the upper respiratory tract, we show that OW provides a reasonable but not perfect surrogate for bacteria carried into to the lung by a bronchoscope. These methods allow identification of microbes that can replicate in the lung despite the background due to oropharyngeal microbes derived from aspiration and bronchoscopic carry-over.

SUBMITTER: Charlson ES

PROVIDER: S-EPMC3435383 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Assessing bacterial populations in the lung by replicate analysis of samples from the upper and lower respiratory tracts.

Charlson Emily S ES Bittinger Kyle K Chen Jun J Diamond Joshua M JM Li Hongzhe H Collman Ronald G RG Bushman Frederic D FD

PloS one 20120906 9

Microbes of the human respiratory tract are important in health and disease, but accurate sampling of the lung presents challenges. Lung microbes are commonly sampled by bronchoscopy, but to acquire samples the bronchoscope must pass through the upper respiratory tract, which is rich in microbes. Here we present methods to identify authentic lung microbiota in bronchoalveolar lavage (BAL) fluid that contains substantial oropharyngeal admixture. We studied clinical BAL samples from six selected s ...[more]

PMID: 22970118

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Project description:Background Respiratory tract infection (RTI) is a common disease among children of all ages that causes high hospitalization and mortality rates. Infection with more than one pathogen has been reported in RTI; however, the association of the pathogen spectrum in upper and lower respiratory tract infections remains unclear. Methods A prospective study was conducted during February to October 2016. Fifty-five nasopharyngeal swabs (NPS) and 30 bronchoalveolar lavage fluid (BALF) samples from 55 hospitalized children aged less than 14 years (mean age 40 months) and diagnosed with an RTI were collected. All samples were detected for 18 respiratory pathogens using the Filmarray assay, real-time PCR, or nested PCR methods. Detection results and clinical characteristics of all cases were analyzed using chi-square and t tests. Results Forty-one of 55 (74.5%) NPS obtained from children were positive for at least one pathogen by the Filmarray assay. Of these cases, 53.7% (22/41) were co-infected. The most commonly detected pathogen was rhinovirus (RV), followed by Mycoplasma pneumoniae (MP) and respiratory syncytial virus (RSV). Infection by both RV and MP was the most frequently observed pattern of co-infection. Similar results were observed using real-time PCR. The pathogens in the NPS from 76.6% of cases detected by Filmarray and 80.0% of cases by real-time PCR included all the pathogens detected in the BALF sample from the same individual. The Filmarray assay showed an 80% concordance rate with real-time PCR and had a turnaround time of less than 1.2 h. No significant differences were observed between the association of single-infection and co-infection with clinical characteristics, neither by Filmarray nor real-time PCR. Conclusion The spectrum of pathogens is mostly concordant in the upper and lower respiratory tract. Collecting NPS for detection can be a non-invasive and more convenient option compared with BALF. Although co-infection is common in children with an RTI, the clinical significance of co-infection remains unclear and warrants further analysis.

Project description:UNLABELLED:In March 2013, three fatal human cases of infection with influenza A virus (H7N9) were reported in China. Since then, human cases have been accumulating. Given the public health importance of this virus, we performed a pathogenicity study of the H7N9 virus in the cynomolgus macaque model, focusing on clinical aspects of disease, radiographic, histological, and gene expression profile changes in the upper and lower respiratory tracts, and changes in systemic cytokine and chemokine profiles during infection. Cynomolgus macaques developed transient, mild to severe disease with radiographic evidence of pulmonary infiltration. Virus replicated in the upper as well as lower respiratory tract, with sustained replication in the upper respiratory tract until the end of the experiment at 6 days after inoculation. Virus shedding occurred mainly via the throat. Histopathological changes in the lungs were similar to those observed in humans, albeit less severe, with diffuse alveolar damage, infiltration of polymorphonuclear cells, formation of hyaline membranes, pneumocyte hyperplasia, and fibroproliferative changes. Analysis of gene expression profiles in lung lesions identified pathways involved in tissue damage during H7N9 infection as well as leads for development of therapeutics targeting host responses rather than virus replication. Overall, H7N9 infection was not as severe in cynomolgus macaques as in humans, supporting the possible role of underlying medical complications in disease severity as discussed for human H7N9 infection (H. N. Gao et al., N. Engl. J. Med. 368:2277-2285, 2013, doi:10.1056/NEJMoa1305584). IMPORTANCE:Influenza A virus H7N9 emerged early in 2013, and human cases have continued to emerge since then. Although H7N9 virus-induced disease in humans is often very severe and even lethal, the majority of reported H7N9 cases occurred in older people and people with underlying medical conditions. To better understand the pathogenicity of this virus, healthy cynomolgus macaques were inoculated with influenza A virus H7N9. Cynomolgus macaques were used as a model because the receptor distribution for H7N9 virus in macaques was recently shown to be more similar to that in humans than that of other frequently used animal models. From comparison with previous studies, we conclude that the emerging H7N9 influenza virus was more pathogenic in cynomolgus macaques than seasonal influenza A viruses and most isolates of the pandemic H1N1 virus but less pathogenic than the 1918 Spanish influenza virus or highly pathogenic avian influenza (HPAI) H5N1 virus.

Dataset Information

Assessing bacterial populations in the lung by replicate analysis of samples from the upper and lower respiratory tracts.

Publications

Assessing bacterial populations in the lung by replicate analysis of samples from the upper and lower respiratory tracts.

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