Diagnostic Accuracy of Polymerase Chain Reaction for Mycobacterium Tuberculosis Using EBUS-TBNA Samples

Study Overview
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Brief Summary
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The purpose of this study is to compare the diagnostic efficacy of nested and realtime polymerase chain reaction for Mycobacterium tuberculosis using EBUS-TBNA samples in patients with isolated intrathoracic lymphadenopathy.

Detailed Description
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Although endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) has been widely used to perform mediastinal lymph node sampling, little information is available on polymerase chain reaction for Mycobacterium tuberculosis (TB-PCR) using EBUS-TBNA samples in patients with isolated intrathoracic lymphadenopathy. In addition, two methods of TB-PCR (nested PCR and realtime PCR) are available for the detection of Mycobacterium tuberculosis and which method is superior for detection of Mycobacterium tuberculosis has not been established in patients with isolated intrathoracic lymphadenopathy. Thus, this study was design to compare the diagnostic efficacy of nested and realtime TB-PCR using EBUS-TBNA samples in patients with isolated intrathoracic lymphadenopathy in South Korea, where the incidence of tuberculosis is intermediate (97/100,000 per year).

Official Title
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Comparison of the Diagnostic Accuracy of Nested and Real-time Polymerase Chain Reaction for Mycobacterium Tuberculosis Using EBUS-TBNA Samples in Patients With Isolated Intrathoracic Lymphadenopathy

Conditions
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Lymphadenopathy, Tuberculosis, Sarcoidosis

Intervention / Treatment
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* Device: Nested PCR for formalin-fixed tissues
* Device: Nested PCR for fresh tissues
* Device: Real-time PCR for fresh tissues


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Patients with isolated intrathoracic lymphadenopathy Exclusion Criteria: Suspicion of primary lung cancer on CT or PET scan Suspicion of pulmonary tuberculosis on CT or PET scan Suspicion of sarcoidosis of lung parenchyma on CT or PET scan

Ages Eligible for Study
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Minimum Age: 18 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Diagnostic
Allocation: Non-Randomized
Intervention Model: Single Group Assignment
Masking: None (Open Label)


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: Nested PCR for formalin-fixed tissues<br>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. | Device: Nested PCR for formalin-fixed tissues<br>* 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.<br>* Other names: MTB-PCR kit; Biosewoom, Seoul, South Korea;|
| Experimental: Nested PCR for fresh tissues<br>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. | Device: Nested PCR for fresh tissues<br>* For nested PCR with specimens in sterile saline, 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.<br>* Other names: MTB-PCR kit; Biosewoom, Seoul, South Korea;|
| Experimental: Real-time PCR for fresh tissues<br>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. | Device: Real-time PCR for fresh tissues<br>* For real-time PCR, specimens in sterile saline were filtered and 1 mL of phosphate based saline was added. After centrifugation for 3 min, the supernatant was removed. Next, 50 μL of extraction buffer was added to the sample, and the sample was heated at 100°C for 20 min. After centrifugation for 3 min, the supernatant was used in PCR. Real-time PCR was performed using the AdvanSure TB/NTM real-time PCR kit. Three channels were used in the real-time PCR reaction (Mycobacterium tuberculosis complex, mycobacteria, and internal control). Signals for FAM, HEX, and Cy5 were measured in each channel. Mycobacterium tuberculosis was considered present if the cycle threshold of rpoB was less than 35 on each signal and greater than or equivalent to that of IS6110.<br>* Other names: AdvanSure TB/NTM real-time PCR kit; LG, Seoul, South Korea;|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Number of participants who detected each polymerase chain reactions for Mycobacterium tuberculosis | 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. | 6 months | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Bronchoscopy, Ultrasonography, Tuberculous lymphadenitis, Polymerase chain reaction, Sensitivity and specificity