Metagenomic sequencing and evaluation of the host response in the pediatric aerodigestive population.

OBJECTIVES: To assess the diagnostic utility of metagenomic sequencing in pediatric aerodigestive clinic patients being evaluated for chronic aspiration. We hypothesize that using a metagenomics platform will aid in the identification of microbes not found on standard culture. STUDY DESIGN AND METHODS: Twenty-four children referred to an aerodigestive clinic were enrolled in a prospective, single-site, cross-sectional cohort study. At the time of clinical evaluation under anesthesia, two samples were obtained: an upper airway sample and a sample from bronchoalveolar lavage (BAL). Samples were sent for routine culture and analyzed using Explify® Respiratory, a CLIA Laboratory Developed Test which identifies respiratory commensals and pathogens through RNA and DNA sequencing. Since RNA was sequenced in the course of the metagenomic analysis to identify organisms (RNA viruses and bacteria), the sequencing approach also captured host derived messenger RNA during sample analysis. This incidentally obtained host transcriptomic data were analyzed to evaluate the host immune response. The results of these studies were correlated with the clinical presentation of the research subjects. RESULTS: In 10 patients, organisms primarily associated with oral flora were identified in the BAL. Standard culture was negative in three patients where clinical metagenomics led to a result with potential clinical significance. Transcriptomic data correlated with the presence or absence of dysphagia as identified on prior videofluoroscopic evaluation of swallowing. CONCLUSIONS: Clinical metagenomics allows for simultaneous analysis of the microbiota and the host immune response from BAL samples. As the technologies in this field continue to advance, such testing may improve the diagnostic evaluation of patients with suspected chronic aspiration.

Presence of Gastric Pepsinogen in the Trachea Is Associated with Altered Inflammation and Microbial Composition.

Gastroesophageal reflux is a common gastrointestinal issue that can lead to aspiration and contribute to respiratory problems. Little is known about how reflux can alter the respiratory microenvironment. We aimed to determine if the presence of gastric pepsinogen in the trachea was associated with changes in the microbial and inflammatory microenvironment. A pediatric cohort at high risk of reflux aspiration was prospectively recruited, and the tracheal microenvironment was examined. Pepsinogen A3 (PGA3) and cytokines were measured. The microbiome (bacterial and fungal) was profiled using 16S rRNA and internal transcribed spacer 2 (ITS2) amplicon sequencing. Increased bacterial richness and an altered composition driven by an enrichment of Prevotella correlated with high PGA3 levels. Fungal richness increased with PGA3, with higher Candida relative abundances observed in a subset of samples with high PGA3 levels. Source tracking of tracheal microbial taxa against taxa from matched oral and gastric samples revealed a significantly greater contribution of oral than of gastric taxa with higher PGA3 levels. Tracheal cytokines were differentially produced when stratified according to PGA3, with higher levels of interleukin-1 (IL-1)-related cytokines and IL-8 being associated with high PGA3 levels. Network analysis across cytokine and microbiome measures identified relationships between IL-1-related proteins and microbial taxa, with the presence of respiratory issues associated with higher levels of IL-1ß, IP-10, and Prevotella In conclusion, PGA3 levels in the trachea are correlated with increases in specific microbial taxa and inflammatory molecules, with an increase in oral microbes with increasing PGA3.

Invasive and non-invasive diagnostic approaches for microbiological diagnosis of hospital-acquired pneumonia.

BACKGROUND: Data on the methods used for microbiological diagnosis of hospital-acquired pneumonia (HAP) are mainly extrapolated from ventilator-associated pneumonia. HAP poses additional challenges for respiratory sampling, and the utility of sputum or distal sampling in HAP has not been comprehensively evaluated, particularly in HAP admitted to the ICU. METHODS: We analyzed 200 patients with HAP from six ICUs in a teaching hospital in Barcelona, Spain. The respiratory sampling methods used were divided into non-invasive [sputum and endotracheal aspirate (EAT)] and invasive [fiberoptic-bronchoscopy aspirate (FBAS), and bronchoalveolar lavage (BAL)]. RESULTS: A median of three diagnostic methods were applied [range 2-4]. At least one respiratory sampling method was applied in 93% of patients, and two or more were applied in 40%. Microbiological diagnosis was achieved in 99 (50%) patients, 69 (70%) by only one method (42% FBAS, 23% EAT, 15% sputum, 9% BAL, 7% blood culture, and 4% urinary antigen). Seventy-eight (39%) patients underwent a fiberoptic-bronchoscopy when not receiving mechanical ventilation. Higher rates of microbiological diagnosis were observed in the invasive group (56 vs. 39%, p = 0.018). Patients with microbiological diagnosis more frequently presented changes in their empirical antibiotic scheme, mainly de-escalation. CONCLUSIONS: A comprehensive approach might be undertaken for microbiological diagnosis in critically ill nonventilated HAP. Sputum sampling determined one third of microbiological diagnosis in HAP patients who were not subsequently intubated. Invasive methods were associated with higher rates of microbiological diagnosis.

Effect of gastric fluid aspiration on the lung microbiota of laboratory rats.

AIM OF THE STUDY: The pulmonary microbiota is important for both normal homeostasis and the progression of disease, and may be affected by aspiration of gastric fluid. The aim of this study was to investigate changes in the lung microbiota induced by aspiration of gastric fluid in a laboratory rat model. MATERIAL AND METHODS: Using the intratracheal application method, male rats received aspiration with 0.9% normal saline (n = 11); gastric fluid (n = 24) or sterilized (gamma-irradiated) gastric fluid (n = 12) once-weekly for four weeks. On the fifth week, the animals were sacrificed, and the microbiota of the lung was assessed by 16S ribosomal RNA gene sequencing. RESULTS: Lungs without aspiration and lungs after aspiration with normal saline had similar microbial compositions, dominated by bacteria of the genera Serratia, Ralstonia and Brucella. Evaluation of the microbiota following aspiration of gastric fluid revealed a much different profile that was dominated by bacteria from the genera Romboutsia and Turicibacter and largely independent of sterilization of the gastric fluid. CONCLUSION: In a laboratory rat model, aspiration with gastric fluid caused a substantial shift of the lung microbiota that could be characterized as a shift from Proteobacteria towards Firmicutes, possibly of enteric origin. Bacteria contained in the gastric fluid are not apparently responsible for this change.

The bacterial species associated with aspirated foreign bodies in children.

OBJECTIVE: Inhaled foreign bodies in children are common and may be complicated by secondary airway tract infection. The inhaled foreign body may act as carrier of infectious material and the aim of this study was to explore the bacterial species associated with aspirated foreign bodies in a cohort of children. METHODS: Retrospective case series of 34 patients who underwent rigid laryngobronchoscopy because of foreign body aspiration. Each patient had a sample taken from tracheobronchial secretions during the procedure. RESULTS: The average patient age was 31.2 months and the average hospital stay was 2.5 days. Of the foreign bodies 24 (71%) were organic in nature and 10 (29%) were non-organic. Twenty eight (82.3%) patients had mixed oropharyngeal flora organisms growth. Fifteen (44%) samples were positive for organisms other than oropharyngeal flora with the most common cultured organisms being: Streptococcus pneumonia (4/12%), Haemophilus influenza (4/12%), Moraxella catarrhalis (4/12%). Four samples (12%) grew a fungus; Candida albicans was cultured in 3 patients and Aspergillus glaucus was identified in one sample. Of the non-oropharyngeal organisms 7(47%) demonstrated antibiotic resistance with four having resistance to amoxycillin, two resistant to penicillin and one resistant to cotrimoxazole. CONCLUSION: Some children who present with aspirated foreign body may be complicated with secondary airway infection. Antibacterial treatment might be considered in some of these cases. The regimen of antibiotics should aim to cover oropharyngeal flora, S. pneumonia, H. influenza and Moraxella catarrhalis.

The bacterial microbiota in inflammatory lung diseases.

Numerous lines of evidence, ranging from recent studies back to those in the 1920s, have demonstrated that the lungs are NOT bacteria-free during health. We have recently proposed that the entire respiratory tract should be considered a single ecosystem extending from the nasal and oral cavities to the alveoli, which includes gradients and niches that modulate microbiome dispersion, retention, survival and proliferation. Bacterial exposure and colonization of the lungs during health is most likely constant and transient, respectively. Host microanatomy, cell biology and innate defenses are altered during chronic lung disease, which in turn, alters the dynamics of bacterial turnover in the lungs and can lead to longer term bacterial colonization, as well as blooms of well-recognized respiratory bacterial pathogens. A few new respiratory colonizers have been identified by culture-independent methods, such as Pseudomonas fluorescens; however, the role of these bacteria in respiratory disease remains to be determined.

Potential role of periodontal infection in respiratory diseases - a review.

Respiratory diseases are responsible for a significant number of deaths and considerable suffering in humans. Accumulating evidence suggests that oral disorders, particularly periodontal disease, may influence the course of respiratory infections like bacterial pneumonia and chronic obstructive pulmonary disease (COPD). Oral periodontopathic bacteria can be aspirated into the lung causing aspiration pneumonia. The teeth may also serve as a reservoir for respiratory pathogen colonization and subsequent nosocomial pneumonia. The overreaction of the inflammatory process that leads to the destruction of the connective tissue is present in both periodontal disease and emphysema. This overreaction may explain the association between periodontal disease and chronic obstructive pulmonary disease. The mechanisms of infection could be the aspiration into the lung of oral pathogens capable of causing pneumonia, colonization of dental plaque by respiratory pathogens followed by aspiration, or facilitation of colonization of the upper airway by pulmonary pathogens by periodontal pathogens. The present article briefly reviews the epidemiologic evidence & role of periodontopathogens in causing respiratory infections.

PCR improves diagnostic yield from lung aspiration in Malawian children with radiologically confirmed pneumonia.

BACKGROUND: Accurate data on childhood pneumonia aetiology are essential especially from regions where mortality is high, in order to inform case-management guidelines and the potential of prevention strategies such as bacterial conjugate vaccines. Yield from blood culture is low, but lung aspirate culture provides a higher diagnostic yield. We aimed to determine if diagnostic yield could be increased further by polymerase chain reaction (PCR) detection of bacteria (Streptococcus pneumoniae and Haemophilus influenzae b) and viruses in lung aspirate fluid. METHODS: A total of 95 children with radiological focal, lobar or segmental consolidation had lung aspirate performed and sent for bacterial culture and for PCR for detection of bacteria, viruses and Pneumocystis jirovecii. In children with a pneumococcal aetiology, pneumococcal bacterial loads were calculated in blood and lung aspirate fluid. RESULTS: Blood culture identified a bacterial pathogen in only 8 patients (8%). With the addition of PCR on lung aspirate samples, causative pathogens (bacterial, viral, pneumocystis) were identified singly or as co-infections in 59 children (62%). The commonest bacterial organism was S.pneumoniae (41%), followed by H. influenzae b (6%), and the commonest virus identified was adenovirus (16%), followed by human bocavirus (HBoV) (4%), either as single or co-infection. CONCLUSIONS: In a select group of African children, lung aspirate PCR significantly improves diagnostic yield. Our study confirms a major role of S.pneumoniae and viruses in the aetiology of childhood pneumonia in Africa.

[Early fibrobronchoscopy and the microbial flora composition of bronchoalveolar lavage fluid in patients with concomitant injury during treatment in intensive care units].

Resuscitative bronchofibroscopy in victims with severe injury and aspiration has been shown to be both a method for diagnosing damages to the bronchus and lung and that for recovering airway patency in them. The most common involvement (47% of cases) in chest injury has been noted to be the lower lung with the development of pyoinflammatory processes caused by etiologically significant microorganisms, such as Staphylococcus aureus and types of Enterobacteriacea, in most cases (more than 50%).