Ethics Statement and Informed Consent
The study was performed according to the Declaration of Helsinki with respect to ethical principles for research involving application of human specimens. Written informed consent for a retrospective-review protocol approved by The Ethics Committee of Shanghai Pulmonary Hospital, Tongji University School of Medicine in China had been provided by all of the patients.
Clinical specimen collection
All patients with clinically suspected NTM-PD attending Shanghai Pulmonary Hospital between January 3, 2018, and November 28, 2019, were eligible for screening if they met the following inclusion criteria: 1) negative for HIV test; 2) providing sputum specimens for examinations, or bronchial alveolar lavage fluids (BALF) from sputum-scarce patients. Two sputum samples or BALF were obtained for MGIT 960 and PCR-REBA at screening. Respiratory samples should be processed within 24 hours of collection (or refrigerated at 4°C if delays are anticipated).
Fiberoptic bronchoscopy
Bronchoscopy procedures were performed according to our institute’s infection regulation and manufacturer’s instruction guidelines (BF-1 T260, Olympus, Tokyo, Japan). Inspectors wore N95 masks, goggles and gowns during the procedure. The bronchoscopy room was equipped with negative pressure isolation and an air disinfection system.
Routine Identification Methods
MGIT960 culture: Each specimen of approximately 5 ml was decontaminated using the Nacetyl-L-cysteine (NALC)–NaOH method (25). The final concentration of NaOH is 4%. The samples exposed to NaOH for 15–20 min. The processed sediment was washed once using a sterile 0.9% NaCl solution and resuspended in 1.5 ml sterile 0.9% NaCl solution; two separate 500-μl aliquots were prepared in 1.5-ml tubes for PCR-REBA and MGIT 960 culture. Specimens were cultured by MGIT 960 (Becton Dickinson Diagnostic Systems, Sparks, MD) for 6 weeks following the standard procedure of the manufacturer (26). Then samples from positive signaled MGIT 960 tubes were Ziehl-Neelsen (ZN) -stained and Gram-stained to confirm the presence of mycobacteria and exclude contamination.
TBc identification test (TBc ID): TBc ID (Becton Dickinson, Sparks, MD) is an immunochromatographic assay for the detection of MPT64 Ag, which is a mycobacterial protein secreted only by MTBC and has been shown to differentiate MTBC from NTM. 100-μl of liquid media from the positive MGIT tubes was added to the TBc ID card. Then the cards were incubated for 15 min at room temperature and the results were visually assessed. A positive test result indicated MTBC while a negative test result indicated NTM.
Para-nitrobenzoic acid (PNB) test: The PNB media was prepared locally, stored between 2 °C – 8 °C and quality controlled according to our standard operating procedures. The test required two Lowenstein Jensen slants one with and another without PNB reagent, the latter serving as negative control. Both slants were inoculated with 500 µL of liquid media from the negative MPT64 Ag tubes, incubated at 37 °C and read weekly until colonial growth was observed. Identification of NTM was made based on presence of growth in the tube with PNB, with no growth in the control tube (without PNB).
PCR-reverse blot hybridization assay (PCR-REBA): a) DNA Isolation: 500 μl bacterial precipitate were treated with DNA Lysis Buffer (10 mmol/L NaCl, 1 mg/ml SDS, 0.15 g/ml Chelex-100 glass beads, 1% Tween 20) at 50°C for 1 hour, then at 100°C for 10 minutes, and centrifuged at 10000r/min for 2min. The supernatant containing genomic DNA was transferred into new tube and stored at -20°C for PCR amplification. b) Amplification: PCR was carried out in a 50μl reaction mixture containing 5 μl DNA template, 10 mM Tris/HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 0.2 mM dNTP, 0.4 mM each primer, and 2 U AmpliTaq Gold polymerase. The mixture was first incubated at 94°C to activate the Taq polymerase, followed by 40 cycles of amplification (94°C for 1 minute, annealing and extension for 30 seconds at 65°C, 72°C for 1 minute), and finally 72°C for 10 minutes. A 5μl aliquot of the PCR product was electrophoresed on 6% polyacrylamide gel with silver staining. c) Hybridization: The amplified PCR products were then subjected to REBA using Mycobacterium species identification detecting Kit (Yaneng BioSciences, Shenzhen) according to the manufacturer’s instructions. In brief, biotinylated PCR products were denatured at 25°C for 5min and added to the REBA membrane strip in the provided blotting tray. Denatured single-stranded PCR products were hybridized with the probes on the strip at 55°C for 30min. The strips were then washed twice with gentle shaking in 1.0ml of washing solution for 10min at 55°C, incubated at 25°C for 30min, and washed twice with 1.0ml of CDS at room temperature for 1min. Finally, the colorimetric hybridization signals were visualized and the band pattern was read.
High Resolution Melting (HRM) analysis: To confirm mycobacterium species inconsistently identified by the two different assays, fluorescence PCR-HRM Assay was performed using Mycobacterium Identification Kit (Zeesan Biotech, Xiamen). The amplification of rpoB gene was performed on the following conditions: a pre-incubation step at 95°C for 10 min, followed by 45 cycles of denaturation at 95°C for 10 s, annealing at 65°C for 30 s, and extension at 72°C for 10 s followed by the Tm analysis with increasing temperatures from 60 to 95°C in a 0.2°C s-1 slope increment for 10 s. The HRM analysis was performed using Gene Scanning Software Version 1.5.0 (Roche Instrument Centre, Switzerland). The clustering of the melting curves was based on the regions of the melting curve corresponding to the pre-melting, melting, and post-melting regions. Distilled water was used instead of the DNA template as the non-template control (NTC).
Statistical Analysis
SPSS for Windows (Version 19.0, SPSS Inc., Chicago) was used for data analysis. The patients’ characteristics and detection results were integrated together. All patients were followed up for at least 6 months with assessment of symptoms, radiological change and mycobacterial culture results. We calculated sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of PCR-REBA assay and MGIT 960- TBc ID-PNB test. The categorical variables were analyzed using Fisher exact or Pearson X2 tests where appropriate and 2-tailed tests were used. The concordance of agreement between MGIT 960- TBc ID-PNB test and PCR-REBA assay was evaluated using Cohen’s kappa test (k > 0.75, excellent agreement; 0.4 < k < 0.75, moderate agreement; and k < 0.4, poor agreement). Receiver operating characteristic (ROC) curve analysis was performed to determine the power of MGIT 960- TBc ID-PNB test and PCR-REBA assay to distinguish PTB and NTM patients from non-mycobacterium patients. The area under curve(AUC)with a 95% confidence interval (CI) was further calculated. P <0.05 was considered statistically significant.