This study is a single-center, parallel-group, assessor-blinded randomized controlled trial (RCT) to be conducted at the Seoul National University Hospital in South Korea. Consenting and eligible participants who are scheduled to undergo EBUS-TBNA will be randomly assigned to either the intervention group or the control group at a 1:1 ratio. The flow diagram of this trial is shown in Figure 2. The protocol of this trial is described in accordance with the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) guidelines [13] (Figure 3).

Hospitalized patients aged 19 years and older who are scheduled to undergo EBUS-TBNA using a convex probe will be included in this trial. Patients will be excluded if they meet any of the following criteria: (1) antiseptic mouthrinse within 7 days before inclusion; (2) active infection or antibiotic treatment within 7 days before inclusion; (3) being immunocompromised status; or (4) tracheostomy status. We will also exclude patients who have already undergone gastroscopy on the same day when EBUS-TBNA is scheduled to be administered.

Our dedicated bronchoscopy suite is composed of a preprocedural room, two procedural rooms, and a disinfection and reprocessing room. The postprocedural area is the same as the preprocedural room. In the preprocedural room, all patients will gargle with 20 mL of 1% lidocaine for topical oropharyngeal anesthesia (Figure 4).

Following topical oropharyngeal anesthesia, participants who are assigned to the intervention group will gargle for 1 minute with 100 mL of 0.12% chlorhexidine gluconate in the preprocedural room. Participants in the control group will not be provided with mouthrinse. The chlorhexidine mouthrinse intervention will be performed approximately 10 minutes before insertion of the bronchoscope (Figure 4).

After participants move to a procedural room, conscious sedation will be initiated with intravenous midazolam and adjuvant fentanyl administered by an experienced nurse and titrated by the attending bronchoscopist. As the flexible bronchoscope is advanced orally, 1% lidocaine solution will be used for “spray-as-you-go” administration to the trachea. Prior to EBUS-TBNA, an endobronchial examination of the bronchial trees will be routinely performed using a flexible bronchoscope (BF-1T260, BF-Q290, BF-260, BF-P260F, Olympus, Tokyo, Japan) unless it has been performed within the previous several days. During the procedure, basic diagnostic sampling methods (bronchial wash, bronchoalveolar lavage, bronchial brush, transbronchial lung biopsy, and endobronchial biopsy) can be performed at the bronchoscopist’s discretion. For EBUS-TBNA, an EBUS bronchoscope with a convex probe (BF-UC260FW, Olympus) will be inserted orally into the airway. Following identification of mediastinal or hilar lymph nodes by ultrasound, a 22-gauge aspiration needle (NA-201SX-4022 or NA-U401SX-4022, Olympus) will be advanced through the working channel of the bronchoscope to puncture the airway wall at the predetermined position. Paratracheal and peribronchial lung masses can also be sampled. The bronchoscopist can replace the aspiration needle with a new one during the procedure. Transesophageal bronchoscopic ultrasound-guided fine needle aspiration (EUS-B-FNA) will be allowed. For EUS-B-FNA, the same EBUS bronchoscope will be re-inserted into the esophagus after the completion of EBUS-TBNA. A 22-gauge aspiration needle (NA-201SX-4022 or NA-U401SX-4022, Olympus) will be used for sampling through the esophageal wall (either the same needle used in EBUS-TBNA or a new one). The NA-201SX-4022 is usually used during the procedure; however, the U401SX-4022 can also be used, especially when the supply of NA-201SX-4022 from the manufacturer is insufficient. All bronchoscopic procedures including EBUS-TBNA will be performed by an experienced pulmonologist or a fellow in pulmonary and critical care medicine under the close supervision of the pulmonologist. As part of standard care, patients will be monitored by an experienced nurse under the direction of the bronchoscopist. Prescription of prophylactic antibiotics after EBUS-TBNA is allowed, and its use, type, dosage, and duration will be determined by the bronchoscopist or care provider.

Immediately after completion of EBUS-TBNA on all targeted lesions with an aspiration needle and ejection of all aspirated materials from the needle for pathologic evaluation, a needle wash sample will be taken by instilling 5 mL of sterile physiological saline solution into the used needle, and collecting it in a sterile 10 mL test tube. This collection method has been reported previously [10,14]. If the bronchoscopist uses two or more needles during the procedure, each needle wash sample will be collected in each sterile tube immediately after completing the procedure. The samples will be immediately refrigerated at 5°C and processed on the same day.

Bacterial culture of the needle wash samples will be performed according to a routine clinical protocol. Needle wash samples will be plated on blood agar and chocolate agar plates, and incubated for 2 days under aerobic conditions. The samples will also be plated on Brucella agar plates, and incubated for 2-4 days under anaerobic conditions. If two or more needles are used per participant, each needle wash sample will be plated and incubated under both aerobic and anaerobic conditions. The bacteria will be identified by matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry using a microflex LT (Bruker Daltonics, Bremen, Germany). A colony will be placed on the target plate by the direct transfer method and analyzed with MALDI Biotyper RTC software 3.1 and the BDAL database (6903 MSP). A colony forming unit (CFU) will be measured according to the colony morphology and the result of identification.

The primary outcome is CFU counts in aerobic culture. If two or more needle wash samples are obtained in a participant, the primary outcome is the average of CFU counts in the aerobic plates. Secondary outcomes include CFU counts in anaerobic culture, fever within 24 hours after EBUS-TBNA, and infectious complications within 4 weeks after EBUS-TBNA. If two or more needle wash samples are obtained in a participant, the average of CFU counts in the anaerobic cultures will be used as a secondary outcome measure. Fever is defined as a temperature ≥37.8°C. Infectious complications include pneumonia, lung abscess, empyema, mediastinal adenitis/abscess, mediastinitis, pericarditis, and sepsis.

Sample size calculations are based on an available RCT of 100 patients who underwent gastroscopy with or without chlorhexidine mouthrinse due to a lack of similar previous studies on EBUS-TBNA [15]. To test whether chlorhexidine mouthrinse reduces the CFU counts of needle wash samples by 50% with a power of 90% and an alpha of 0.05, the required sample size per group is 50. Considering a potential dropout rate of 10%, a sample size of 56 per group (112 in total) is required.

A web-based randomization system has been implemented by the Medical Research Collaborating Center of the Seoul National University Hospital (https://mrcc.snuh.org/). A clinical research coordinator will perform randomization through the website. Each patient will be assigned an alphanumeric code. Investigators and other research personnel are unable to access the website. Only the clinical research coordinator is able to view intervention allocation on the website.

The following personnel will be blinded to the intervention assignment: investigators including bronchoscopists performing EBUS-TBNA, technicians assisting with the procedure and instilling saline into aspiration needles to collect needle wash samples, outcome assessors including laboratory personnel, and care providers in general wards. Mouthrinsing with chlorhexidine will be done in the preprocedural room, which is separate from the procedural room, and bronchoscopists and assisting technicians in the procedural room can be blinded. However, participants are not blinded to the intervention assignment. Participants in the control group receive usual care (no mouthrinse). A placebo control such as pure water with or without a flavored additive will not be used in this trial because participants will be able to distinguish between chlorhexidine and placebos due to the odor and taste of chlorhexidine. Unblinding to an attending bronchoscopist performing EBUS-TBNA is possible if serious adverse events such as shock or seizure occur and if a relationship between the events and the chlorhexidine mouthrinse is suspected by the attending bronchoscopist. However, this unblinding has no consequence on the primary outcome measure.

The below data will be collected.

In all analyses, patients will be analyzed according to their randomization groups, with the exception of those who withdraw informed consent or do not undergo scheduled EBUS-TBNA (e.g., cancellation of scheduled EBUS-TBNA after successful diagnostic procedures such as endobronchial biopsy). CFU counts in the aerobic and anaerobic cultures of needle wash samples will be expressed as the mean±standard deviation and analyzed by the independent samples t test if distributed normally. Otherwise, they will be described as the median (interquartile range) and analyzed using the Mann-Whitney U test. Fever within 24 hours after EBUS-TBNA and infectious complications within 4 weeks after the procedure will be presented as numbers and percentages and analyzed by either the chi-square test or Fisher exact test. Subgroup analysis according to the use of EUS-B-FNA and prophylactic antibiotics will be conducted. For the subgroup analysis according to prophylactic antibiotic use, only fever and infectious complications will be analyzed as outcomes. All comparisons will be two-sided and p-values of less than 0.05 will be considered statistically significant.

This study was approved by the Institutional Review Board of Seoul National University Hospital (H-2011-096-1173). This trial will be conducted in accordance with the principles of the Declaration of Helsinki and Good Clinical Practice. Written informed consent will be obtained from all participants before randomization.

All data will be anonymized and identified by a coded ID number only to maintain participant confidentiality. All records containing personal identifiers such as informed consent forms will be stored separately from records identified by the coded ID number. All data will be stored in either a locked filing cabinet or in an electronic password-protected computer in a locked office. The access to data is restricted to authorized personnel only.