Bronchoalveolar Fluid Specimens Research Articles

1224. Investigation of Infectious Etiologies in the Lower Respiratory Tract from Pediatric Patients with Unexpected Cardiopulmonary Deterioration using Next-Generation Sequencing

BackgroundIn pediatric patients, unexpected cardiopulmonary deterioration with or without following cardiopulmonary arrest (CPA) are rare events, but can be caused by any of several etiologies, including infectious diseases. The most common cause of out-of-hospital CPA in children ≤12 years old was sudden infant death syndrome (SIDS), whereas infectious diseases were responsible for approximately 10% of the CPA cases. However, the role of infection may have been underestimated as triggers of SIDS or CPA. This study aimed to investigate the infectious etiologies in pediatric patients with unexpected cardiopulmonary deterioration using next-generation sequencing (NGS).MethodsA total of 16 pediatric patients who were admitted to the pediatric intensive care unit with unexpected cardiopulmonary deterioration with or without following CPA were enrolled. Ten bronchoalveolar fluid (BALF) and six transtracheal aspirates (TTA) samples obtained in the acute phase were used to prepare NGS libraries. The libraries were sequenced on HiSeq and analyzed using metagenome analysis tools.ResultsIn ten of 16 patients, one or more bacterial/viral pathogens were detected in the BALF or TTA specimens using NGS. Compared to the conventional culture and viral antigen test results, an additional 6 bacterial (e.g., Chlamydia trachomatis) and 4 viral pathogens (e.g., coxsackievirus A6 and human coronavirus NL63) were identified by NGS in four of ten patients in whom no causative pathogen had been identified by conventional culture and viral antigen tests. A summary of the detected pathogens is listed in Table 1. Notably, sequencing results allowed us to define genotypes for all of the detected viruses in a single NGS assay per patient. Furthermore, based on phylogenetic analysis of the VP1 region, the coxsackievirus A6 strain detected in this study belongs to lineage E2 and harbors an amino acid change (T283A), a substitution that has potential to cause severe illness.Table 1 ConclusionOur results suggest that viral and bacterial infection are common triggers in unexpected cardiopulmonary deterioration in pediatric patients. NGS has the potential to contribute to the clarification of the etiology of pediatric critical illness.Disclosures All Authors: No reported disclosures

An Outbreak of Pneumocystis jiroveci Pneumonia with 1 Predominant Genotype among Renal Transplant Recipients: Interhuman Transmission or a Common Environmental Source?

An outbreak of Pneumocystis jiroveci pneumonia (PCP) occurred among renal transplant recipients attending the outpatient department at the Leiden University Medical Centre (Leiden, The Netherlands) from 1 March 2005 through 1 February 2006. Clinical, epidemiological, and molecular data were analyzed to trace the outbreak's origin. Renal transplant recipients with a clinical suspected diagnosis of PCP were included in the study. The diagnosis had to be confirmed by direct microscopy or real-time polymerase chain reaction of the dihydropteroate synthase gene in a bronchoalveolar fluid specimen. To detect contacts between patients, a transmission map was constructed. A case-control analysis was performed to asses whether infection was associated with certain wardrooms. Genotyping of Pneumocystis isolates was performed by sequence analysis of the internal transcribed spacer (ITS) number 1 and 2 gene regions. Twenty-two confirmed PCP cases were identified; approximately 0-1 would have been expected over the same time period. No risk factor was predominantly present, and standard immunosuppressive regimens had not changed. Liver transplant recipients who used the same outpatient facilities had not acquired PCP. The transmission map findings were compatible with interhuman transmission on multiple occasions. The case-control study did not point to wardrooms as a common source. Genotyping by sequencing of the ITS1 and ITS2 gene regions revealed type Ne in 12 of 16 successfully typed samples. Genotype Ne was found in only 2 of 12 reference samples. The clinical data and genotyping results are compatible with either interhuman transmission or an environmental source of infection. More complex models may account for PCP clusters.

Promoter hypermethylation of resected bronchial margins: a field defect of changes?

Histologically positive bronchial margins after resection for non-small cell lung cancer are associated with shortened patient survival due to local recurrence. We hypothesized that DNA promoter hypermethylation changes at bronchial margins could be detected in patients with no histological evidence of malignancy and that they would reflect epigenetic events in the primary tumor. Bronchial margins, primary tumor, bronchoalveolar fluid, and paired nonmalignant lung were obtained from 20 non-small cell lung cancer patients who underwent a lobectomy or greater resection. Disease-specific recurrence was the primary end point. The methylation status of p16, MGMT, DAPK, SOCS1, RASSF1A, COX2, and RARbeta was examined using methylation-specific polymerase chain reaction. All malignancies had methylation in at least one locus. Concordance of one gene with an identical epigenetic change in the tumor or bronchial margin was observed in 85% of patients. Only one patient had methylation at the bronchial margin for a gene that was not methylated in the corresponding tumor. Median time to recurrence was 37 months (range, 5-71 months). There were two local recurrences and five metastases. There were no significant correlations between DNA methylation in tumor, margins, or bronchoalveolar fluid specimens and either regional recurrence or distant metastases. Histologically negative bronchial margins of resected non-small cell lung cancer exhibit frequent hypermethylation changes in multiple genes. These hyper-methylation abnormalities are also present in the primary tumor and thus may represent a field defect of preneoplastic changes that occurs early in carcinogenesis.

Rapid detection of Mycoplasma pneumoniae by an assay based on PCR and probe hybridization in a nonradioactive microwell plate format.

A new molecular assay, based on a rapid DNA extraction protocol, PCR, and hybridization to a specific probe in a nonradioactive microwell plate format was used to detect Mycoplasma pneumoniae in bronchoalveolar fluid specimens. The sensitivity of the assay was determined to be 10 to 100 organisms with M. pneumoniae reference strains. Specificity testing with different bacteria capable of producing pneumonia showed no cross-reactivity. In a prospective study, bronchoalveolar lavage fluids obtained from patients with pneumonia were investigated with the PCR assay and compared to culture. Twelve positive samples were detected with the PCR assay. Seven of them were subsequently confirmed by culture. All patients with positive PCR results seroconverted. Application of the PCR assay described may lead to safe and early diagnosis of M. pneumoniae in patients with pneumonia.

Rapid detection of Legionella species in bronchoalveolar lavage fluids with the EnviroAmp Legionella PCR amplification and detection kit.

A molecular assay based on a rapid DNA extraction protocol and the EnviroAmp Legionella Kits was used to detect Legionella species in bronchoalveolar fluid specimens. All Legionella strains isolated from tap water in hospitals could be detected distinctly. Both sensitivity and specificity were tested. In a prospective study, bronchoalveolar lavage fluids obtained from patients with atypical pneumonia were investigated. Three positive samples were detected with the molecular techniques and were subsequently confirmed by culture. Application of the system described may lead to safe and early diagnosis of Legionnaires' disease in patients with atypical pneumonia.