In all patients, nested polymerase chain reaction for Mycobacterium tuberculosis is performed with formalin-fixed paraffin-embedded specimens obtained by endobronchial ultrasound-guided transbronchial needle aspiration.

In all patients, nested polymerase chain reaction for Mycobacterium tuberculosis is performed with specimens in sterile saline obtained by endobronchial ultrasound-guided transbronchial needle aspiration.

In all patients, real-time polymerase chain reaction for Mycobacterium tuberculosis is performed with specimens in sterile saline obtained by endobronchial ultrasound-guided transbronchial needle aspiration.

For nested PCR, formalin-fixed paraffin-embedded tissues were incubated in 1 mL of xylene at 60°C for 30 min and then centrifuged for 10 min. Paraffin and the supernatant were removed from the samples after centrifugation. The same procedures were repeated until deparaffinization was complete. After adding 1 mL of alcohol, the samples were centrifuged for 5 min, and the supernatant was removed. The sample was then air-dried as a pellet. DNA was extracted using QIAamp (Qiagen, Valencia, CA, USA). PCR amplifications were performed using Mycobacterium tuberculosis IS6110 primers. The first round using outer primers and the second round using inner primers amplified a 256- and 181-bp fragment, respectively. Finally, the PCR products were visualized in a 2% agarose gel.

In all participants with isolated intrathoracic lymphadenopathy, three modalities of TB-PCR (nested PCR for formalin-fixed paraffin-embedded tissues, nested PCR for fresh tissues and real-time PCR for fresh tissues) are performed using EBUS-TBNA samples. The numbers of participants who reported as positive for each TB-PCR modalities are collected. From these data, sensitivity, specificity and diagnostic accuracy of each TB-PCR modalities to diagnose tuberculous lymphadenitis will be calculated.