Patients
The Ethics Committee of the Shanghai Pulmonary Hospital approved this prospective study and written informed consent was obtained from each participant before enrollment.
All adult patients admitted to Shanghai Pulmonary Hospital were screened between April 2014 and January 2015. Inclusion criteria were: suspected active PTB; age ≥ 17 years; no previous history of anti-TB treatment; inability to provide sputum for examinations; negative HIV status. Exclusion criteria were: inability to tolerate bronchofibroscopy; no finalized diagnosis after examination and treatment (obscure diagnosis). When FOB was performed, BALF specimens from the affected lung were acquired for acid-fast bacilli (AFB) smear, mycobacterial culture and SAT-TB assays.
A standard questionnaire was completed by each patient before enrollment, including basic demographic data, history of TB contacts, previous TB, current TB symptoms, anti-TB treatments, as well as underlying diseases and concurrent therapies.
All patients to be evaluated for active TB were tested using the SAT-TB assay, AFB smear test, as well as culture tests at enrollment, in addition to physical, pathological and radiographic examinations.
Active PTB was confirmed when radiographic or chest computed tomography (CT) manifestations were in accordance with the PTB pattern and accompanied by one of the following criteria at enrollment or later during the study period: bacteriological diagnosis based on positive MTBC in cultures and pathological diagnosis of PTB based on analyses of AFB in resected lung tissues.
In patients who did not meet one of the above two criteria, clinical diagnosis was further confirmed by meeting all of the following categories: signs or symptoms of PTB and typical manifestation of TB on chest CT scans; received anti-TB medication for 2 months with a favorable response, based on improved signs or symptoms and chest CT results. For these cases, chest CT scans were reviewed every 2 months until the patients had been treated for 6 months.
At the end of the study, each patient was classified into one of three predefined clinical categories namely active PTB, non-TB pulmonary disease or without a final diagnosis (with obscure diagnosis or the patients lost to follow-up) by clinical experts who were blinded to the results of the SAT-TB assays.
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.
Microbiological identification
Acid-fast bacilli smears and culture assays
BALF specimens were routinely tested by AFB smears and cultures with Lowenstein–Jensen medium, as well as with the Bactec MGIT 960 System (Becton Dickinson Diagnostic Systems, Sparks, MD) by following the standard procedure of the manufacturer [
20]. Each specimen of approximately 5 ml was decontaminated using the N-acetyl-L-cysteine (NALC)–NaOH method [
21]. 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; three separate 500-μl aliquots were prepared in 1.5-ml tubes for the SAT-TB and Bactec MGIT 960 culture tests. The detection threshold of a positive smear was smear positive grade 1. All tests were performed at the tuberculosis reference laboratory in Shanghai Pulmonary Hospital, and quality controls were routinely performed.
M. tuberculosis H37Rv (ATCC 27294) and 20 reference strains were gifts of the National Tuberculosis Reference Laboratory (Beijing, China).
Simultaneous amplification and testing method for detection of Mycobacterium tuberculosis (MTBC)
To process the clinical samples for the SAT-TB assay, the aliquoted processed sediments were centrifuged, the supernatants were discarded, and 50 μl TB dilution solution (10 mM sodium citrate, pH 8.0) was added to each tube and vortexed. Each sample was sonicated for 15 min at room temperature in a water bath sonicator (Shanghai Sheng-Yan Ultrasound Machines Co. Ltd., Shanghai, China) at 300 W and then centrifuged, and the supernatant was used in the SAT-TB assay. The live M. tuberculosis attenuated strain H37Ra (ATCC 25177) was used as a positive control, and the negative control was double-distilled water. For the assay, 2 μl of processed supernatant and 30 μl of a reaction solution containing 40mMTris-HCl, pH 8.1, 8mMMgCl2, 25mMNaCl, 2 mM spermidine hydrochloride, 5 mM dithiothreitol, 80 μg/ml bovine serum albumin, 200 μM dATP, 200 μM dTTP, 200 μM dGTP, 200 μM dCTP, 1 mM ATP, 1 mM GTP, 1 mM CTP, 1 mM UTP, and 0.5 mM (each) primers and probe were prepared in a 200-_l PCR tube. The mixture was preincubated at 60 °C for 10 min, followed by 42 °C for 5 min, and then a 10-μl aliquot containing 2000 units M-MLV reverse transcriptase and 2000 units T7 RNA polymerase (RD Bioscience, Inc., San Diego, CA) was added and gently mixed. The reaction mix was then placed immediately into a model 7500 real-time PCR system (Applied Biosystems Inc., Foster City, CA). RNA isothermal amplification was conducted at 42 °C for 1 min, with a total of 40 cycles, and FAM fluorescence data were collected after each cycle of amplification. Specimens with cycle thresholds (CT) of≦40 were classified as TB positive. The above processes were performed in a biological safety cabinet (class II B2) in a biosafety level PII laboratory. RNase-free tubes and tips were required for the SAT-TB assay.
Statistical analysis
The data were entered into a computer and analyzed using SPSS for Windows (Version 18.0:, SPSS Inc., Chicago). Sensitivity, specificity, positive and negative predictive values, as well as accuracy rates of the SAT-TB assay, BALF smear and cultures were calculated. 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 SAT-TB assays and smear and culture data was assessed using Cohen’s kappa test (k > 0.75, excellent agreement; 0.4 < k < 0.75, moderate agreement; and k < 0.4, poor agreement). P values <0.05 were considered to be statistically significant. Receiver operating characteristic (ROC) curve analysis was performed to determine the diagnostic power of SAT-TB assays.