This retrospective study was approved by the Institutional Ethics Review Board, China Medical University (2013070). Written informed consent was obtained from all the patients involved in this study. We enrolled a total of 403 patients with various pulmonary diseases hospitalized between October 2016 and January 2018, from 5 hospitals in China, including the First Affiliated Hospital of China Medical University, the Fourth Affiliated Hospital of China Medical University, the Fifth Affiliated Hospital of China Medical University, the Eleventh Affiliated Hospital of Shanxi Medical University, and the General Hospital of Shenyang Military Command. Clinical background information of patients was collected through the Case File System, available in the participating hospitals.
The patients with different symptoms and signs of pulmonary diseases, with non-HIV infections, without the clinical evidence or history of PJP symptoms, and without the definite diagnosis of PJP were included in the study [14, 19]. Besides considering the predisposing factors for the diseases, the pulmonary diseases were diagnosed not only on the basis of the clinical manifestations of the respiratory tract diseases (such as cough, fever, expectoration, wheezing, chest pain, chest tightness, hemoptysis, and dyspnea), but also chest imaging tests, bronchoscopic examination, and laboratory test, primarily including COPD [including the stable stage and acute exacerbations (AECOPD)], acute exacerbations of chronic bronchitis (AECB), interstitial lung diseases (ILDs), bronchiectasis, bronchial asthma, invasive pulmonary aspergillosis (IPA), and type I respiratory failure. Various diseases, included in the study, were diagnosed as follows: i) COPD was diagnosed mainly by confirming the presence of persistent airflow limitation according to a post-bronchodilator FEV1/FVC < 70%, history of risk factors such as exposure to cigarette smoke, and the Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Lung Disease 2017 Report: GOLD Executive Summary . In addition, the assessment of AECOPD met the GOLD guidelines, the diagnostic criteria of AECOPD given by American College of Chest Physicians (2011), and the European Respiratory Society guidelines ; ii) ILDs were primarily diagnosed by high-resolution computed tomography (HRCT) depicting the radiological manifestations of fibrosis in ILDs and sometimes, by bronchoalveolar lavage for the diagnosis of some particular types of ILDs [22, 23]; iii) AECB was diagnosed by considering the patients with mild to moderate syndromes of COPD (the diagnosis of COPD as mentioned above), patients who developed the exacerbated clinical presentations with increased dyspnea, increased sputum volume, and increased sputum purulence, and meanwhile, excluding pneumonia and other lung diseases by chest radiography ; iv) bronchiectasis was primarily diagnosed by HRCT of the chest , the gold standard for confirming the bronchiectasis, and considering the underlying causes such as post-infectious ; v) bronchial asthma was diagnosed through a detailed clinical history, physical examination, lung function, and detection of allergens ; vi) IPA was diagnosed by demonstrating Aspergillus in the respiratory samples and other contributions by 1,3-β-D-glucan assay, clinical manifestation of lower respiratory tract infection, and CT clinical characteristic with the air-crescent sign, halo sign, cavitation lesion, or nodules infarction; and vii) type I respiratory failure was diagnosed by laboratory studies such as evaluating the level of hypoxemia and hypercapnia via arterial blood gas and oxygen saturation, imaging examination, and pulmonary function test .
Respiratory tract specimens
Before patients received diagnosis, antibiotics, and other treatments, specimens including blood, bronchoalveolar lavage fluid (BALF), and sputum samples were collected. Venous blood was collected in vacutainer tubes with different anticoagulants or coagulants and used immediately for the following tests: CD4+ T-cell counts (BD FACS Canto, USA), erythrocyte sedimentation rate (ESR) (Vital Monitor-20, Italy), and serum biochemical parameters including lactate dehydrogenase (LDH) (ADVIA 2400, SIEMENS Healthineers, Germany), pro-calcitonin (PCT) (Cobas 8000, Roche Diagnostics GmbH, Germany), C-reactive protein (CRP) (BN ProSpec System, SIEMENS Healthineers, Germany), and 1,3-β-D-glucan (BDG) (Goldstream MB-80, Era Biology Technology Co. Ltd. Tianjin, China). Pulmonary function (Medisoft S.A. Hyp’ Air, Belgium) and high-resolution computed tomography (HRCT) (BN ProSpec System, SIEMENS Healthineers, Germany) were measured by the following standard protocols in clinical practice. Each patient underwent HIV-1 and 2 antibody testing using the anti-HIV-1 and 2 antibody enzyme-linked immunosorbent assay (ELISA) diagnostic kit (Intec Products, Inc. Xiamen, China) to determine HIV infection status.
Amongst 403 patients, 324 patients provided sputum samples and 79 patients provided BALF samples. As the nature of the study is retrospective and severity status of the pulmonary disease was not available in the Case File System, we can only assume that patients who provided BALF sample had more severe disease status, as compared to patients who provide sputum samples. Moreover, patients providing sputum samples were not eligible for the BAL procedure. To collect the sputum samples, patients gargled with saline solution approximately three times to prevent oral microbial contamination before coughing up their morning sputa from the deep respiratory tract. Sputa were then collected in sterile containers and sent to clinical laboratories for bacterial and/or fungal culture and identification, respectively using the VITEC-2 system (ATB system, BioMérieux, Marcy-l’Étoile, France) and the Sensititre™, Aris 2X system (Thermo Fisher Scientific, USA). The remaining of the sputum samples were used to exact DNA and make slide smear to identify P. jirovecii microscopically following GMS and Giemsa staining.
Bronchoscopic examination was performed in 79 patients. BALF was collected using strict aseptic technique and filtered through a 2-layered nylon gauze to remove the mucus. The filtered BALF was divided for DNA extraction (described below) and slide smear staining by GMS and Giemsa staining.
BALF samples were centrifuged at 1,500 rpm for 15 minutes and cell pellets were collected. The sputa were pretreated with 4% NaOH (w/v %), washed with saline solution approximately three times, and then centrifuged at 12,000 rpm for 10 minutes. After centrifugation, the supernatant was discarded. All cell pellets were washed three times with PBS and then subjected to DNA extraction following the traditional protocol involving proteinase K digestion, phenol-chloroform extraction, and ethanol precipitation. The DNA extracts were quantitated using the NanoDrop UV-Vis spectrophotometer (Thermo Fisher Scientific, USA) and stored at -80°C before use.
Real-time fluorescence LAMP assay
All primers used in this study, including the external primer set (F3-B3), internal primer set (FIP-BIP), and loop primer set (LF-LB) have been described previously . The LAMP reaction mixture contained 1.6 µM FIP-BIP, 0.2 µM F3-B3, 0.4 µM Loop F-B, and 2.5 μL 10× Isothermal Amplification Buffer including 0.1% Triton X-100 or 100 μg/ml BSA. The preferred buffer was 50 mM KCl, 10 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 1 mM DTT, 0.1% Triton X-100, and 50% glycerol (New England Biolabs, USA), 3.5 µL 10 mM dNTPs (Takara Biotechnology Co., LTD., Dalian, China), 1 μl 8 U/μl Bst DNA Polymerase, Large Fragment (New England Biolabs, USA), 1 µL100 mM MgSO4 (New England Biolabs, USA), 0.5 µL 10μM SYTO 9.0 (Thermo Fisher Scientific, USA), 2 µL 5.0 M Betaine (Sigma-Aldrich, USA), 2 µL DNA template solution, and 6.5 µL RNase-free water (Takara Biotechnology Co., LTD., Dalian, China) up to a final volume of 25 µL. Each experiment included a no template control (with distilled water). The LAMP reaction was performed in the ABI 7500 system (ThermoFisher Scientific, USA) at 63℃ for 60 min, and terminated at 80℃ for 10 min.
The entire LAMP amplification process was monitored in real time and the fluorescent signals recorded automatically by the ABI 7500 system. Compared with the conventional LAMP, the real-time LAMP was analyzed by the amplification curves. At the end of the reaction, white precipitates formed at the bottom of the test tube. In the analysis of conventional LAMP, visual detection was observed by adding 2 µL 1000× SYBR Green I (Thermo Fisher Scientific, USA) and the detection of LAMP amplicons were evaluated by 2% agarose gel electrophoresis and stained with GoodView (SBS Genetech Co., Ltd.) for visualization under UV radiation.
PCR was performed using the external primers targeting the 18S rRNA gene of P. jirovecii described elsewhere . PCR mixture contained 12.5 µL of DNA polymerase Premix Taq™ (Takara Biotechnology Co., LTD., Dalian, China), 1-3 µL of DNA samples, 1 µL of 10 μM F3-B3 primer pair, and RNase-free water up to a final volume of 25 µL. Ultrapure distilled water was used as a negative control. PCR was performed under the following conditions: 94℃ for 5 min; 30 cycles of 94℃ for 30 s, 55 ℃ for 30 s, 72℃ for 30 s, and 72℃ for 8 min. PCR products were separated by electrophoresis on 1.5% agarose gels, stained with GoodView (SBS Genetech Co., LTD. Beijing, China), and visualized by UV radiation.
Sensitivity and specificity of conventional PCR and the real-time fluorescence LAMP assay
To prepare a plasmid DNA standard, the PCR product, amplified using the external primers of the P. jirovecii 18S rRNA gene described above, was excised from the gel and purified using the agarose gel DNA extraction kit (Tiangen Biotech Co., LTD., Beijing, China). The recovered fragment was cloned into the TA cloning vector pMD18-T (Takara, Biotechnology Co., LTD., Dalian, China) with a standard TA cloning technique. DNA was extracted from one recombinant plasmid clone containing the correct sequence. DNA concentration was measured using the NanoDrop UV-Vis spectrophotometer (Thermo Fisher Scientific, USA). The sensitivities of the LAMP and PCR methods were compared using 10-fold serial dilutions of recombinant plasmid DNA (from 1×100 copies/µL to 1×108 copies/µL) in triplicate.
The specificities of the real-time fluorescence LAMP assay and conventional PCR were determined using genomic DNA from 10 other common respiratory microorganisms, including Candida albicans, C. tropicalis, C. krusei, C. glabrata, C. parapsilosis, Aspergillus niger, Streptococcus pneumoniae, Escherichia coli, Acinetobacter baumannii, and Methicillin resistant Staphylococcus aureus (MRSA). The genomic DNA of each microorganism was extracted using the Plant Genomic DNA Kit (Tiangen Biotech Co., LTD., Beijing, China) or the TIANamp Bacteria DNA Kit (Tiangen Biotech Co., LTD., Beijing, China). An aliquot of 10-100 ng of genomic DNA from each microorganism was used in each reaction.
All data were analyzed using the statistical analysis software SPSS (version 20.0, IBM Corporation, Chicago, IL, USA). Counting variables are represented as frequencies or percentages. Continuous variables are represented as the mean ± SD. Data related to clinical findings, imaging examinations, serologic parameters, and other indicators were compared using the c2 test. Fisher’s exact test was used for the clinical data analysis when the number of samples was less than 40 or the expected value was less than 1. P values less than 0.05 were considered as statistically significant.