Ethics statement and informed consent
The prospective study had been carried out in accordance with the Declaration of Helsinki on ethical principles for the use of human specimens for research. Each patient received written informed consent prior to participation. The study was approved by the Ethics Committee of Shanghai Pulmonary Hospital (approve number K18-170).
Study design and participants
This study was conducted in a national pulmonary diseases specialized hospital admitted patients mainly from East China. The ultrasound department of Shanghai Pulmonary Hospital performs more than 10,000 ultrasound-interventional puncture biopsies per year.
The inclusion criteria were as follows: Adults (≥ 18 years of age) admitted to the hospital with pleural lesions (with or without pleural effusion) based on chest CT and detectable by chest ultrasound. The pleural lesions may appeared as a pleural thickening or pleural nodularity [12]. The minimal cut-off for the thickness size of the pleural based lesions was ≥ 10 mm which was visible and suitable for obtained biopsy [13]. All patients had indication for CEUS guided biopsy, which were determined by clinical specialists and sonographer, with the prior sputum or tracheoscopic results were not sufficient to eatablish a definite diagnosis.
The exclusion criteria were: a definite diagnosis was obtained by means other than pleural effusion and pleural biopsy (e.g., sputum, tracheoscopy); presence of biopsy contraindications (low platelet count, other risks such as coagulation and bleeding, clinical instability or unsafe location of biopsy); elderly patients or patients with serious systemic diseases who could not tolerate the pleural biopsy; serum HIV positive; the patients refused a pleural biopsy; pleural lesions judged by the sonographer to be unsuitable for biopsy.
When clinically necessary and safe, we performed CEUS-guided pleural biopsy. Pleural tissue was evaluated by histopathology and microbiological tests.
Patients with pleural effusion received ultrasound-guided pleural catheter drainage, and pleural effusion was tested accordingly, including routine pleural effusion examination (biochemical and ADA), microbiological and cytological tests.
Diagnostic criteria and establishment of the final diagnosis
Diagnosis of pleural TB: We used a composite reference standard (CRS) as the diagnostic standard. CRS included both definite and possible cases. Definite cases: microbiological positive by either MTB culture or Xpert MTB/RIF(Xpert) positive on pleural effusion or pleural biopsy specimens; Possible cases: patients with TB imaging characteristics and clinical manifestations or positive TB immunological test, or histopathology confirmed as tuberculosis lesions on pleural biopsy, or biochemical examination of pleural effusion suggested TB and patients were responsive to anti-TB treatment [14].
Diagnosis of other pleural infectious diseases: Specific pathogens were identified in pleural effusion or pleural biopsy by microbiological, molecular biological or histopathological staining and targeted antibiotic treatment was effective.
Diagnosis of malignant pleural lesions: malignancy confirmed by cytological or histological examination on pleural effusion or pleural biopsy.
All patients were treated and followed up for at least 6 months. Thoracoscopy was performed on patients with undefinite diagnosis through all above examinations. A nonspecific pleurisy must be established in those with no clinical or radiological progression during follow-up [5].
Instruments and methods
Ultrasound instrument LOGIQ E9 ultrasonic diagnostic instrument from GE, USA, with convex array probe, frequency 1–6 MHz. The instrument has harmonic contrast imaging function with low mechanical index (MI).
For patients with pleural effusion, pleural fluid was collected by closed thoracic drainage guided by ultrasound. CEUS was then used to find the target biopsy site.
CEUS: Conventional ultrasound was used to observe and record the size, shape, boundary, internal echo and periphery of the lesions. All patients signed the informed consent before the angiography. The instrument MI was adjusted to 0.10 and the contrast gain was 20 DB. 1.5 mL SonoVue ultrasound contrast agent (Bracco, Italy) was injected through the elbow vein. The dual-contrast interface was adopted. At the same time of the injection of contrast agent, the ultrasonic instrument was started with a built-in chronograph for 3 min continuous observation, and the images were stored and analyzed.
CEUS-guided biopsy: ultrasound-guided biopsy was performed immediately after CEUS. Before the biopsy, two observers evaluated the enhancement pattern. They reached a consensus to identify the viable biopsy target areas. 2% lidocaine hydrochloride was subjected to local layer. When the needle tip reached the leading edge of the enhancement area of the lesion 2–3 mm, the max-core automatic biopsy needle (model 18 G × 10 cm or 16 G × 10 cm, BD Company, USA) was excited to complete a biopsy. Repeat the operation to obtain 3–4 more complete tissue strips (Figs. 1, 2).
Microbiological tests
The pleural tissue/pleural effusion was finely ground and suspended in 1 ml sterile saline, the pleural fluid was centrifuged (3000RPM, 15 min), the supernatant was discarded, and the precipitation solution was used for further microbiological tests (Bacteria/fungi culture), all procedures were performed according to the protocols.
BACTECTM MGIT 960 culture (MGIT 960, BD, USA) and Xpert were used for identification of mycobacterium, including mycobacterium tuberculosis (MTB) and non-Mycobacterium tuberculosis (NTM).
Pathological examination
The biopsy specimens were fixed with 10% neutral formalin. All formalin fixation and paraffin embedding (FFPE) sections stained with hematoxylin-eosin (HE) were observed by two experienced pathologists. Specific stain and immunohistochemistry (IHC) were performed if needed.
Complications
Complications were recorded and classified into four categories: no, self-limited pneumothorax or bleeding, pneumothorax or bleeding requiring a chest tube and hospitalization, and other major complications (death, prolonged hospitalization, etc.).
Statistical analysis
All datas were set up in Excel 2010 and analyzed statistically with SPSS 20.0. The measurement data was compared by Student's t test. Enumeration data was compared by chi-square test. McNemar χ2 test was used to compare the diagnostic yield of CEUS guided biopsy specimens and pleural effusion for pleural lesions combined pleural effusion. A p-value of < 0.05 was considered statistically significant.