Study Design and Study Subjects
This was a single-center retrospective cohort study from the Third Xiangya Hospital, Central South University, China. All renal transplant recipients with a diagnosis of pneumonia who admitted to our center from March 1, 2019 to October 31, 2020 were enrolled, divided into two groups: PJP or non-PJP. Clinical data of renal transplant recipients diagnosed with PJP or non-PJP mainly by mNGS and confirmed by PCR were analyzed. All patients included were at least 18 years old at renal transplantation. Trimethoprim-sulfamethoxazole (at doses of 80 mg and 400 mg, respectively, once daily, for at least four months) was given as a prophylaxis to all recipients enrolled after renal transplantation. No organs from executed prisoners were used. This retrospective non-interventional study involved 14 RTRs with PJP and 10 non-PJP RTRs, which was approved by the Research Ethics Committee at the Third Xiangya Hospital of Central South University, consistent with principles in the Declaration of Helsinki (protocol no. IRB-2020-S636). The ethics committee waived the need for informed consent.
Routine Immunosuppression
Induction immunosuppression consisted of basiliximab and/or antithymocyte globulin and methylprednisolone (0.5g given during the operative procedure, 200-250mg on postoperative day 1-3). Maintenance immunosuppression included mycophenolate mofetil, prednisone and a calcineurin inhibitor, tacrolimus or cyclosporin A.
Sample Collection and Processing
Routine cultures for bacteria and fungi were performed for all samples. A total volume of 8 ml blood samples was collected in BD vacutainer plasma preparation tubes for viral and microbial analysis using mNGS, or 3 ml BALF/sputum samples were provided for mNGS detection. Blood samples were stored at room temperature and BALF/sputum samples were store with ice bags at 2-8 degrees Celsius.
DNA Extraction, Library Construction and Sequencing
Viscous BALF or sputum samples were homogenized with normal saline. Blood samples were centrifuged at 1600 rpm and BALF/sputum samples were centrifuged at 12000 rpm for 10 minutes at 4°C to eliminate debris. Pyrolysis liquid GHL was added to release total DNA for BALF/sputum samples. Cell-free DNA from plasma and total DNA from BALF/sputum samples was extracted and purified using a Nucleic Acid Extraction (DNA) or Purification Kit (Sansure Biotech, Magnetic beads method, S1005). Extracted DNA was measured by Qubit dsDNA HS Assay Kits (Thermo Fisher Scientific, Art. No. Q32854), and the total mass of DNA needs to be more than 1 ng as a quality control. The total DNA was further fragmented using dsDNA fragmentase. DNA libraries for sequencing were then constructed. The 5' end of DNA fragment was phosphorylated and a Da tail was added at the 3' end, following adapters were attached to the nucleic acid templates for subsequent sequencing and sample identification. Sequencing libraries were pooled with environmental control samples, which were used to monitor sample-to-sample contamination and were processed alongside the test samples in every batch. Pooled sample libraries up to 18 libraries per batch were multiplexed and sequenced on Illumina NextSeq500 sequencers using a 75-cycle single-end, single index sequencing kit.
Bioinformatic Analysis
High-quality sequencing data were generated by removing low quality and adapter sequences using Trimmomatic. To remove the human original sequences, the cleaning reads were mapped to the human genome (containing GRCh38 and Homo sapien clone satellite sequences) using Burrows-Wheeler Alignment tool. Subsequently, the remaining data were aligned to the bacterial, viral, fungal and parasite databases using Burrows-Wheeler Alignment tool. The reference databases were downloaded from NCBI RefSeq (ftp://ftp.ncbi.nlm.nih.gov/genomes/refseq) and Genbank (ftp://ftp.ncbi.nlm.nih.gov/genomes/genbank). The bacterial database contained 6600 different species, the fungal database contained 980 different species, the viral database contained 5000 different species, parasite database contained 220 different species. The mapping standard criteria included: 1) continuous match numbers were more than 50bp. 2) mismatch numbers were less than 3bp.
The mapped reads were aligned to the NCBI-nt database using Blastn for verification and filtration. We further calculated the coverage and abundance of species using Samtools. Some non-specific reads would be filtered if they were mapped to different genus and different species within the standard mapping criteria. Only the reads mapped to unique species could be classified to the species level. If one read was mapped to a unique genus and different species, it would be classified to the genus level. According to the classification results, we obtained the information of genus and species for the tested samples.
Criteria for Reporting a Positive mNGS Result
The database curated from books was used for pathogenic comments, which was after the positives were selected [13,14]. The background database (containing species with detection rate greater than 50% in all whole blood samples) and the environmental control were used for filtering out the environmental contaminations. The criteria for reporting positive pathogens would be as follows:
- For fungi and viruses, pathogens with the mapping read number greater than or equal to 3 and the confidence level more than 99% will be selected;
- For bacteria (Mycobacterium tuberculosis excluded) and parasites, the pathogens will be reported if the mapping read number was in the top 5 of bacteria or parasitic list and not in the background library [15];
- Mycobacterium tuberculosis would be considered positive if at least 1 read was mapped to either the species or genus level, due to the difficulty of DNA extraction and low possibility of contamination [16];
PCR Validation
Sterile centrifuge tubes were used to collect blood, sputum or alveolar lavage fluid from patients. The sample volume after liquefaction should not be less than 1mL. The sample to be tested could be placed at 2℃～8℃ for less than 72 hours. Long-term storage should be placed under the condition of ≤ -20℃ to avoid repeated freezing and thawing. The specimens were transported in a curling pot with ice or a foam box with ice sealed for transportation.
200μL of the sample to be tested were put into a 1.5mL centrifuge tube, to perform nucleic acid extraction using the nucleic acid extraction or purification reagent (S1006, Sansure Biotech ) according to its instruction.
The PCR validation was carried out using the pneumocystis nucleic acid detection kit (PCR-fluorescent probe method，Sansure Biotech). The fluorescence detection used the ROX channel to detect human pneumocystis, and the CY5 channel to detect the internal standard.
If the ROX channel detected a typical S-type amplification curve, and Ct ≤ 40, the human pneumocystis was positive. If Ct > 40 or no Ct, it was negative. If the internal standard did not detect Ct or Ct > 40 in the CY5 channel, which meant that the concentration of the test sample was too low or there were interfering substances that inhibited the reaction, the experiment was repeated again.
Statistical Analysis
All analyses were performed using the statistical package SPSS for Windows, version 22.0 (IBM Corporation, Armonk, NY, USA). Results were listed as mean (±SD) for continuous variables. Univariate analysis was applied to inspect the distinction of characteristics between PJP and non-PJP groups. Continuous variables were compared using Student’s unpaired t-test. Categorical variables were compared with Pearson’s χ2 test or Fisher’s exact test. Statistical tests were two-tailed and statistical significance was defined as P<0.05.