Donor and patients characteristics.
Donor lungs inclusion criteria: a. Age <60 years old, smoking history <20 packs/year. b. No chest injury. c. Continuous mechanical ventilation < 1 week. d. PaO2＞300 mmHg (FiO2 = 100%, PEEP = 5 cmH2O). e. X-ray or CT shows that the lung field is relatively clear. f. There is no abscess secretion in the lung bronchus at all levels though bronchoscopy.
Donor lungs exclusion criteria: a. Age >60 years old, smoking history > 20 packs/year. b. Chest trauma and lung contusion. c. Continuous mechanical ventilation > 1 week. d. PaO2<300 mmHg (FiO2 = 100%, PEEP = 5 cmH2O). e. X-ray or CT shows that the lung field is infected. f. There are purulent secretions at bronchoscopy in the donor lower airways. g. The percentage of white blood cells, neutrophils, C-reactive protein, and procalcitonin increases gradually comparing with the situation at onset of disease. h. The donor's body temperature is higher than normal. i. Blood culture is positive.
From March 1st, 2018 to June 30th 2018, we completed 48 lung transplants totally, 17 of which were included in the study. The inclusion criteria of the patients included: Patients had no pulmonary infection, no mechanical ventilation or tracheotomy; patients met the indications for lung transplantation and there were no contraindications for lung transplantation.
Sample collection and preservation.
According to the National Lung Transplantation Data Center for donor lung selection criteria, this study included 12 donor lungs. 17 lung transplantations were performed, including 12 single lung transplantation and 5 lung transplantation from March 2018 to June 2018 at Wuxi People's Hospital affiliated to Nanjing Medical University (Wuxi, China). This study included 12 donor lung tissue samples which were biopsied with a cutting suturing device from basal segment of the lower lobe, the size of tissues was about 0.5 cm × 0.5 cm. The lung tissue samples were frozen in solid carbon dioxide immediately. NGS is used for bacterial detection of the donor lungs (BGI PathoGenesis Pharmaceutical Technology). Swabs were used to collect the secretions from the bronchi of the donor lungs during the operation of donor lung procurement for bacterial culture. After lung transplantation, sputum was collected from the recipients for bacterial culture. The specimens of lung tissue and bronchial secretions of the donor lung were collected one hour before transplantation. In order to avoid the influence of secondary infections, the postoperative sputum was collected within one week, and the sputum was collected multiple times within one week after the operation. This study was approved by Ethics Committee of Wuxi People's Hospital affiliated to Nanjing Medical University, and each patient has signed an informed consent form.
The nucleic acid sequence of the pathogenic microorganism in the sample was analyzed, and the microorganism was identified by comparing with the nucleic acid sequence of the existing microorganism in the database. The detection process included: nucleic acid extraction, library construction, sequencing, information analysis and report interpretation.
Tissue sample: Sample Processing and DNA Extraction
Tissue sample from patient was collected and cut into small pieces according to standard procedures. A 1.5mL microcentrifuge tube with 0.7mL lysis buffer, together with pieces of tissue sample and 1g 0.5mm glass bead were attached to a horizontal platform on a vortex mixer and agitated vigorously at 2800-3200RPM for 30 min. 0.3mL sample was separated into a new 1.5mL microcentrifuge tube and DNA was extracted using the TIANamp Micro DNA Kit (DP316, TIANGEN BIOTECH) according to the manufacturer’s recommendation.
Construction of DNA libraries
DNA libraries were constructed through DNA-fragmentation，end-repair, adapter-ligation and PCR amplification. Agilent 2100 was used for quality control of the DNA libraries. Quality qualified libraries were sequenced by BGISEQ-50 platform .
Sequencing and bioinformatic analysis
High-quality sequencing data were generated by removing low-quality, and short (length < 35bp) reads, followed by computational substraction of human host sequences mapped to the human reference genome (hg19) using Burrows-Wheeler Alignment.The remaining data by removal of low-complexity reads were classified by simultaneously aligning to four Microbial Genome Databases, consisting of viruses, bacteria, fungi, and parasites.
The classification reference databases were downloaded from NCBI (ftp://ftp.ncbi.nlm.nih.gov/genomes/). RefSeq contains 4,061 whole genome sequence of viral taxa, 2,473 bacteral genomes or scaffolds, 199 fungi related to human infection, and 135 parasites associated with human diseases.
Descriptive statistics were computed for the overall sample and stratified by presence of positive bacteria detected by NGS or bacterial culture positive on donor lung samples. Mean ± standard deviation (SD) or median (interquartile range, IQR) was used for describe the continuous variables. We used t test/ANOVA or non-parametric Wilcoxon-Mann-Whitney (for continuous variables) and chi-squared or Fisher’s Exact test for categorical variables to compare the difference between two groups. The significance level was set at 0.05. SPSS 19.0 for Windows (SPSS Inc, Chicago, IL, USA) was used for statistical analysis.