This was a retrospective study performed at the Affiliated Hospital of Shaoxing University between November 2017 and November 2019.
Study population
The medical records of all patients with suspected pulmonary TB who had the Xpert® MTB/RIF performed on bronchial washing or BALF at the Affiliate Hospital of Shaoxing University, between November 2017 and November 2019, were reviewed. These records investigated included clinical, radiological and laboratory data. Concurrently, Xpert® MTB/RIF assays performed on sputum samples during the same period were also reviewed.
Patient Selection Criteria
Inclusion criteria
Both previously treated TB and new patients with clinical suspicion of pulmonary TB(PTB), exhibiting any two of the following symptoms: dry or productive cough for more than 3 weeks together with loss of appetite, fatigue, intermittent fever > 3 weeks, drenching night sweat> 2weeks, weight loss>4kg,haemoptysis. Additionally, the patients must have any one of the following radiological features: cavitations, consolidation, ground-glass opacity and pleural effusion. Also, HIV status of patients should be known.
Exclusion criteria
Patients without full clinical history or all three laboratory investigations requested, or with a previous clinical history of lung cancer or fungal infections.
Bronchoscopy procedure
Under aseptic conditions, a flexible bronchoscope (Fujinon Fujifilm Light Source XL-4450) was used to inspect all segments of the bronchial tree. A biopsy was performed in areas that showed abnormality, as depicted by the computer tomography (CT) scan or as per the clinician’s discretion. Then, 50-100 mL of normal saline was instilled in that segment of the lung and was aspirated into 2 sterile specimen tubes. The BAL fluids collected were sent for fluorescent microscopy and culture, and an Xpert® MTB/RIF assay.
Fluorescent microscopy and liquid culture
The BALF was divided into two parts; one was sent for fluorescent microscopy and the other for culture. Smears were prepared from direct inoculation of BALF after centrifugation and were stained using auramine-rhodamine staining for 15 minutes. Next, the slides were rinsed with water and flooded with 0.5% acid alcohol for 2 minutes. Then, they were rinsed in water. The smears were counter-stained with potassium permanganate and left for 2 minutes. Slides were then rinsed in water, air-dried, and viewed using an LED fluorescent microscope Olympus BX41TT (Olympus Corporation, Tokyo, Japan). The same process was repeated for sputum samples.
For culture, BAL fluid sediments were first decontaminated by adding 4% NaOH at a ratio of 1 part BALF to 2 parts 4% NaOH. The mixture was then shaken vigorously for 15 minutes using a Vortex Mixer V5 (EssenScien, USA). Then, sterile normal saline was added up to 45 mL and the mixture was centrifuged at 4000xg for another 15 minutes at 4℃. The supernatant was discarded and 1 mL of sterile normal saline was added to the sediments. Then, 0.5 mL aliquots of the sample were inoculated into the BACTEC-MGIT 960 (M960) culture system (Becton-Dickinson, Sparks, Maryland, USA) and were cultured for 6 weeks. The same protocol was repeated using patient sputum samples [14].
Drug sensitivity test (DST)
BALF was inoculated with a BACTEC MGIT 960 Mycobacterium liquid culture system. The Mycobacterium was isolated and cultured, and the positive samples were identified. Sensitivity tests for 4 anti-tuberculosis drugs were carried out using a modified version of the Roche absolute concentration indirect method. The specific steps were follows: isoniazid (1, 10 ug/ ml), rifampin (50, 250 ug/m1), streptomycin (0, 100 ug/ml), ethambutol (5, 50 ug/ml)[14].
Xpert® MTB/RIF
According to the manufacturer’s instructions, one part (mL) reagent was added to one part (mL) BALF. The mixture was shaken vigorously using a Vortex Mixer V5 (EssenScien, USA) for 1 minute and was then left for 15 minutes at room temperature. Then, 2 mL of the mixture was inoculated into the cartridge containing washing buffer, reagents for lyophilized DNA extraction and PCR amplification, and fluorescent detection probes. The results of the MTB/RIF assay were ready after 2 hours, indicating whether the sample was MTB positive or negative and whether it was rifampin resistance. Tests were performed on sputum samples using the same procedure [15].
Diagnosis of TB
In our preliminary analysis, Mycobacterium tuberculosis positive culture was used as the gold standard for diagnosis of pulmonary TB. Subsequent analysis was based on the presence of histological or cytological evidence of caseous granuloma containing acid-fast bacilli.
Data analysis
The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for the diagnosis of active pulmonary TB were calculated using the website http://vassarstats.net/clin1.html. Further, 95% confidence intervals (CIs) were estimated according to an exact binomial distribution. Sensitivity and specificity values were compared using McNemar’s test. Statistical analyses were performed using GraphPad Prism 7. P<0 .05 indicated a statistically significant difference.