Examination of the utility of cryobiopsy for tuberculous pleurisy in thoracoscopy: A report of seven cases

Abstract

Thoracoscopy under local anaesthesia is effective for the diagnosis of tuberculous pleurisy (TP), mesothelioma and pleural metastases of lung cancer, etc. It has recently been reported that cryobiopsy is useful for obtaining sufficient tissue, achieving a greater depth, and avoiding crush artefact than biopsy forceps. However, the utility of the tissue obtained by cryobiopsy for culture for diagnosing TP is unknown. We compared positivity rates of tissue culture obtained by biopsy forceps and cryobiopsy in seven TP patients. The median tissue size was 2 mm by biopsy forceps and 6 mm by cryobiopsy. The pathological diagnostic rate of pleural tissue was 85.7% with biopsy forceps and 100% with cryobiopsy. However, the positivity rate of tissue culture was 57.1% with biopsy forceps and 28.5% with cryobiopsy. Since rapid freezing with a cryoprobe makes it difficult for bacteria to grow, it is possible that cryobiopsy might not be useful for obtaining a tissue culture in TP cases. However, since the sample size of this study was small, analysis of more cases is required to confirm our results.

Highlights

• •Cryobiopsy is useful for obtaining sufficient tissue, achieving a greater depth, and avoiding crush artefact than biopsy forceps by thoracoscopy under local anaesthesia.
• •The pathological diagnostic rate of pleural tissue for tuberculous pleurisy was 85.7% with biopsy forceps and 100% with cryobiopsy.
• •The positivity rate of tissue culture for tuberculous pleurisy was 57.1% with biopsy forceps and 28.5% with cryobiopsy.

1. Introduction

Video-assisted thoracic surgery (VATS) provides a high diagnostic yield, although the procedure requires general anaesthesia and is highly invasive [1]. Thoracoscopy under local anaesthesia is less invasive, with a short examination time, and is more popular in Japan. With this procedure, biopsy of the parietal pleura can be performed under direct observation, which leads to a higher diagnostic yield as compared to cytological diagnosis and blind pleural biopsy [2]. Moreover, the complication rate is also lower, with major complications (pyothorax, pneumothorax, etc.) reportedly occurring in 1.8%, and minor complications (generation of heat, sharp pain, etc.) occurring in 7.3% of patients, indicating that it is a relatively safe procedure [3].

Thoracoscopy under local anaesthesia is effective for the pathological diagnosis of tuberculous pleurisy (TP), mesothelioma and pleural metastases of lung cancer, etc. It has recently been reported that cryobiopsy is also useful for obtaining sufficient tissue, achieving a greater depth, and avoiding crush artefact than biopsy forceps [[4], [5], [6]]. However, it has not been reported whether tissue culture of a cryobiopsy specimen is effective for diagnosing TP. We compared culture positivity rates for TP using tissue obtained by biopsy forceps and cryobiopsy in seven patients diagnosed by thoracoscopy under local anaesthesia at our hospital.

2. Methods

2.1. Patients

Of the 23 patients who underwent thoracoscopy under local anaesthesia at our institution from November 2019 to November 2021, this study included seven patients who were diagnosed with TP. We retrospectively examined the utility of tissue culture of samples obtained using biopsy forceps and cryobiopsy in these seven patients. This work was performed with the approval of the Ethics Committee of the National Center for Global Health and Medicine (NCGM-S-004431-00). Patients were provided information explaining the study and were excluded from analysis if they opted out.

2.2. Thoracoscopy

All thoracoscopic examinations under local anaesthesia were performed in the operating room. For the purpose of pain control, 15 mg of pentazocine was injected intramuscularly before the procedure. After establishment of a peripheral intravenous line, patients were placed in the lateral decubitus position, with the side of the pleural effusion being uppermost. In all patients, chest ultrasonography was performed before thoracoscopy to evaluate the pleural effusion. Midazolam 1 mg was then slowly administered intravenously for sedation. A 2-cm incision was made just above the level of the effusion under local anaesthesia with 15–20 ml of 1% lidocaine. A single thoracoscopic port was used for visualization of the thorax. Thoracoscopy under local anaesthesia was performed using the LTF-240 or LTF-H290 (Olympus, Tokyo, Japan) for pleural effusion (Fig. 1A). The biopsy forceps was used FB-231D (Olympus) (Fig. 1B). Cryobiopsy was performed after about seven tissues were collected with biopsy forceps. Cryobiopsy was used cryoprobe (1.9 mm probe, Erbe Elektromedizin, GmbH, Tuebingen, Germany). The cryobiopsy freeze time was 7 seconds and three tissue samples were collected from each patient (Fig. 1C). Two of the samples obtained by biopsy forceps and one obtained by cryobiopsy were cultured. The remaining samples underwent pathological evaluation. Biopsy forceps and cryobiopsy tissues were treated separately (Fig. 1D). At the end of the examination, a chest tube was placed to drain the effusion.

Fig. 1.

Chest CT shows a right-sided pleural effusion (A). Thoracoscopy shows reddening and swelling of the entire pleura with scattered nodules. Conventional forceps biopsied the nodule (B). The tip pf the cryoprobe was attached to the nodules and cooled using carbon dioxide (C). A tissue by conventional biopsy forceps ( blue arrow) and a tissue at 7 seconds (red arrow) by cryobiopsy (D). Histological analysis of the specimen showed inflammation with lymphocytes and epithelioid cell granulomas with caseous necrosis (E).

2.3. Pathological diagnosis

Biopsy samples were submitted for pathology evaluation, using additional staining as appropriate for the disease. For a diagnosis of TP, tissue samples or tissue cultures had to be positive for M. tuberculosis by auramine staining and Ziehl-Neelsen staining or pathological assessment had to demonstrate the presence of caseating granulomas (Fig. 1E). Tissues submitted for tissue culture were crushed prior to submission for Mycobacteria growth indicator tube culture methods. Mycobacterium tuberculosis contains a large amount of fat in its cell wall. The presence of fat impedes passage of the dye during staining. For that reason, the collected tissue was cut with a scalpel, placed in physiological saline, crushed with a pean clamp, and submitted for culture.

Thoracoscopy shows reddening and swelling of the entire pleura with scattered nodules. Conventional forceps biopsied the nodule (B). The tip pf the cryoprobe was attached to the nodules and cooled using carbon dioxide (C). A tissue by conventional biopsy forceps (blue arrow) and a tissue at 7 seconds (red arrow) by cryobiopsy (D).

Histological analysis of the specimen showed inflammation with lymphocytes and epithelioid cell granulomas with caseous necrosis (E).

3. Results

The median age of the seven patients was 44 years (20–86), with 5 men (71.4%) and 2 women (28.6%). QuantiFERON-TB Gold Plus (Qiagen, Venlo Netherlands) was positive in four (57.1%) and negative in three (42.9%) patients. Pleural effusion occurred only on the left side in four (57.1%) and only on the right side in three (42.9%). The proportions of small, medium and large size of pleural effusions were seen in one (14.3%), four (57.1%) and two (28.6%) patients. The median lymphocyte ratio of the pleural effusion was 67% (50–100), and median adenosine deaminase (ADA) level was 82 IU/l (49.2–101.7). The median tissue size was 2 mm [[2], [3], [4]] by biopsy forceps and 6 mm [[5], [6], [7], [8], [9], [10]] by cryobiopsy. The pathological diagnostic rate was 85.7% for tissue obtained with biopsy forceps and 100% for cryobiopsy tissue. In one case, biopsy forceps showed only lymphocyte infiltration and cryobiopsy showed epithelioid cell granulomas with caseous necrosis. The tissue culture positivity rate was 57.1% with biopsy forceps samples and 28.5% with cryobiopsy samples (Table 1).

Table 1.

Patient characteristics.

Characteristics of subjects (n = 7)
Age median (range) years	44 (20–86)
Sex n (%)
Men	5 (71.4)
Women	2 (28.6)
QuantiFERON-TB Gold Plus
Positive	4 (57.1)
Negative	3 (42.9)
Side of pleural effusion n (%)
Left side	4 (57.1)
Right side	3 (42.9)
Side of effusion n (%)
Small	1 (14.3)
Medium	4 (57.1)
Large	2 (28.6)
Lymphocyte ratio of pleural effusion median (range) %	67 (50–100)
Adenosine deaminase of pleural effusion median (range) IU/l	82 (49.2–101.7)

	Pleural tissue with conventional biopsy forceps	Pleural tissue with cryobiopsy
Tissue size median (range) mm	2 (2–4)	6 (5–10)
Pathological diagnostic rate (%)	85.7	100
Positive rate of tissue culture (%)	57.1	28.5

Minor complications included minor bleeding in all cases, although the bleeding stopped spontaneously. No major complications were observed.

Antituberculous drugs were administered for 6 months to a case diagnosed as TP.

There was no re-exacerbation of pleural effusion, and the final diagnosis remained unchanged in all cases.

4. Discussion

Thoracoscopy under local anaesthesia permits parietal pleural biopsies to be performed under direct vision. In recent years, cryobiopsy has often been used for thoracoscopic biopsy. Pleural cryobiopsy is safe and consistently yields larger tissue specimens than conventional biopsy forceps [5]. Chen et al. reported that the possible rate of immunostaining for malignant diseases was 87.0% with forceps biopsy and 98.9% for cryobiopsy in 92 patients who underwent thoracoscopy under local anaesthesia [7]. Shafiq et al. reported their review of 311 cryobiopsy procedures, and major bleeding was not reported in even one case [8].

Dhooria et al. performed cryobiopsy and conventional biopsy in 50 cases. In their study, a 2.4 mm cryoprobe was used. The freezing time was 3 seconds, which was increased to 6 seconds when the biopsy was unsuccessful. Four cryobiopsies were performed in each case. The results showed that conventional biopsy forceps led to a diagnosis of tuberculous pleurisy in thirteen patients. Cryobiopsy was diagnostic in twelve patients and non-diagnostic in one patient.

Cryobiopsy was difficult in cases with thickened pleura. In such cases, by repeatedly closing and opening the jaws of the forceps at the same sites on the pleural surface, a small flap of the tissue can be raised that can be subsequently pulled off [9].

Many reports have shown that cryobiopsy is useful for diagnosing undiagnosed pleural effusion. To the best of our knowledge, few studies have reported that cryobiopsy is not useful of tissue culture for TP.

In our study, the pathological diagnostic rate of pleural tissue with biopsy forceps was 85.7% and cryobiosy was 100%. Cryobiopsy was superior to biopsy forceps in pathological diagnostic rate of TP in our study.

In cases of TP, it is not only necessary to diagnosis but also to determine the presence of resistant tuberculosis. Treatment differs depending on whether resistance is found. Normally, treatment comprises of four medications, isoniazid (INH), rifampicin (RFP), ethambutol (EB), and pyrazinamide (PZA), but if INH resistance is present, then RFP, PZA, EB, and streptomycin are mainly used, with the addition of levofloxacin and ethionamide in cases with a high bacterial load [10]. However, positive rate of tissue culture of conventional biopsy forceps were 57.1% and cryobiopsy were 28.5%. Cryobiopsy can collect larger tissue than biopsy forceps, but the positivity rates of tissue culture is low.

Shu et al. reported slow freezing is better than quick for the preservation of M. tuberculosis [11]. In our study, using the cryoprobe, the tissue was frozen with carbon dioxide for 7 seconds to approximately −80 °C. Rapid freezing with a cryoprobe makes it difficult for bacteria to grow. Hence, it is possible that cryobiopsy might not be for obtaining tissue specimens for culture in TP cases.

This study has several limitations. First, the sample size was small. Future studies including a larger number of cases are required to further evaluate our results. Second, cryobiopsy was performed with freezing of the tissue for 7 seconds, which is rather long. This might have affected the survival of the bacteria.

In our patients, total of twenty-four times cryobiopsies were performed in seven patients, and sufficient tissue was not obtained in three times. This could have been because the probe adhered strongly to the pleura due to the long biopsy seconds.

Third, in Japan, the prevalence of tuberculosis is one of the lowest in the world, and thoracoscopy under local anaesthesia is performed for the differential diagnosis of various diseases and for detailed examination of resistant tuberculosis.

However, countries with a high prevalence of tuberculosis do not consider it necessary to perform thoracoscopy under local anaesthesia in all patients with high ADA levels in pleural effusion unless there is a strong possibility of another diagnosis or drug resistance.

Fourth, in this study polymerase chain reaction tests were only performed on sputum samples and not for the pleural effusion or pleural tissue.

In conclusion, cryobiopsy was superior to biopsy forceps in pathological diagnostic rate because it provides a sufficient amount of tissue with less crush. However, cryobiopsy might not be useful for tissue culture in TP cases. Our results need to be corroborated in future large-scale study.

Funding

The authors have no conflict of interest.

Ethics approval

This work was performed with the approval of the Ethics Committee of the National Center for Global Health and Medicine (NCGM-S-004431-00).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.