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

Primary Purpose

Lymphadenopathy, Tuberculosis, Sarcoidosis

Status
Unknown status
Phase
Not Applicable
Locations
Korea, Republic of
Study Type
Interventional
Intervention
Nested PCR for formalin-fixed tissues
Nested PCR for fresh tissues
Real-time PCR for fresh tissues
Sponsored by
Pusan National University Hospital
About
Eligibility
Locations
Arms
Outcomes
Full info

About this trial

This is an interventional diagnostic trial for Lymphadenopathy focused on measuring Bronchoscopy, Ultrasonography, Tuberculous lymphadenitis, Polymerase chain reaction, Sensitivity and specificity

Eligibility Criteria

18 Years - undefined (Adult, Older Adult)All SexesDoes not accept healthy volunteers

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

Sites / Locations

  • Pusan National University HospitalRecruiting

Arms of the Study

Arm 1

Arm 2

Arm 3

Arm Type

Experimental

Experimental

Experimental

Arm Label

Nested PCR for formalin-fixed tissues

Nested PCR for fresh tissues

Real-time PCR for fresh tissues

Arm Description

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.

Outcomes

Primary Outcome Measures

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.

Secondary Outcome Measures

Full Information

First Posted
July 8, 2015
Last Updated
July 16, 2015
Sponsor
Pusan National University Hospital
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1. Study Identification

Unique Protocol Identification Number
NCT02497079
Brief Title
Diagnostic Accuracy of Polymerase Chain Reaction for Mycobacterium Tuberculosis Using EBUS-TBNA Samples
Official Title
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
Study Type
Interventional

2. Study Status

Record Verification Date
July 2015
Overall Recruitment Status
Unknown status
Study Start Date
July 2015 (undefined)
Primary Completion Date
December 2018 (Anticipated)
Study Completion Date
December 2018 (Anticipated)

3. Sponsor/Collaborators

Responsible Party, by Official Title
Principal Investigator
Name of the Sponsor
Pusan National University Hospital

4. Oversight

Data Monitoring Committee
No

5. Study Description

Brief Summary
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
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).

6. Conditions and Keywords

Primary Disease or Condition Being Studied in the Trial, or the Focus of the Study
Lymphadenopathy, Tuberculosis, Sarcoidosis
Keywords
Bronchoscopy, Ultrasonography, Tuberculous lymphadenitis, Polymerase chain reaction, Sensitivity and specificity

7. Study Design

Primary Purpose
Diagnostic
Study Phase
Not Applicable
Interventional Study Model
Single Group Assignment
Masking
None (Open Label)
Allocation
Non-Randomized
Enrollment
100 (Anticipated)

8. Arms, Groups, and Interventions

Arm Title
Nested PCR for formalin-fixed tissues
Arm Type
Experimental
Arm Description
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.
Arm Title
Nested PCR for fresh tissues
Arm Type
Experimental
Arm Description
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.
Arm Title
Real-time PCR for fresh tissues
Arm Type
Experimental
Arm Description
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.
Intervention Type
Device
Intervention Name(s)
Nested PCR for formalin-fixed tissues
Other Intervention Name(s)
MTB-PCR kit; Biosewoom, Seoul, South Korea
Intervention Description
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.
Intervention Type
Device
Intervention Name(s)
Nested PCR for fresh tissues
Other Intervention Name(s)
MTB-PCR kit; Biosewoom, Seoul, South Korea
Intervention Description
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.
Intervention Type
Device
Intervention Name(s)
Real-time PCR for fresh tissues
Other Intervention Name(s)
AdvanSure TB/NTM real-time PCR kit; LG, Seoul, South Korea
Intervention Description
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.
Primary Outcome Measure Information:
Title
Number of participants who detected each polymerase chain reactions for Mycobacterium tuberculosis
Description
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.
Time Frame
6 months

10. Eligibility

Sex
All
Minimum Age & Unit of Time
18 Years
Accepts Healthy Volunteers
No
Eligibility Criteria
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
Central Contact Person:
First Name & Middle Initial & Last Name or Official Title & Degree
Jung Seop Eom Eom, Master
Phone
82-01-2081-0859
Email
ejspulm@gmail.com
Overall Study Officials:
First Name & Middle Initial & Last Name & Degree
Jeong Ha Mok, Master
Organizational Affiliation
Pusan National University Hospital
Official's Role
Principal Investigator
First Name & Middle Initial & Last Name & Degree
Jung Seop Eom, Master
Organizational Affiliation
Pusan National University Hospital
Official's Role
Principal Investigator
Facility Information:
Facility Name
Pusan National University Hospital
City
Busan
ZIP/Postal Code
602-739
Country
Korea, Republic of
Individual Site Status
Recruiting
Facility Contact:
First Name & Middle Initial & Last Name & Degree
Jung Seop Eom, Master
Phone
+821020810859
Email
ejspulm@gmail.com
First Name & Middle Initial & Last Name & Degree
Jeong Ha Mok, Master
Phone
+820185196183
Email
mokgamokga@gmail.com

12. IPD Sharing Statement

Citations:
PubMed Identifier
22652031
Citation
Navani N, Lawrence DR, Kolvekar S, Hayward M, McAsey D, Kocjan G, Falzon M, Capitanio A, Shaw P, Morris S, Omar RZ, Janes SM; REMEDY Trial Investigators. Endobronchial ultrasound-guided transbronchial needle aspiration prevents mediastinoscopies in the diagnosis of isolated mediastinal lymphadenopathy: a prospective trial. Am J Respir Crit Care Med. 2012 Aug 1;186(3):255-60. doi: 10.1164/rccm.201203-0393OC. Epub 2012 May 31.
Results Reference
background
PubMed Identifier
21813622
Citation
Navani N, Molyneaux PL, Breen RA, Connell DW, Jepson A, Nankivell M, Brown JM, Morris-Jones S, Ng B, Wickremasinghe M, Lalvani A, Rintoul RC, Santis G, Kon OM, Janes SM. Utility of endobronchial ultrasound-guided transbronchial needle aspiration in patients with tuberculous intrathoracic lymphadenopathy: a multicentre study. Thorax. 2011 Oct;66(10):889-93. doi: 10.1136/thoraxjnl-2011-200063. Epub 2011 Aug 3.
Results Reference
background
PubMed Identifier
24035300
Citation
Sun J, Teng J, Yang H, Li Z, Zhang J, Zhao H, Garfield DH, Han B. Endobronchial ultrasound-guided transbronchial needle aspiration in diagnosing intrathoracic tuberculosis. Ann Thorac Surg. 2013 Dec;96(6):2021-7. doi: 10.1016/j.athoracsur.2013.07.005. Epub 2013 Sep 12.
Results Reference
background
PubMed Identifier
10430755
Citation
Statement on sarcoidosis. Joint Statement of the American Thoracic Society (ATS), the European Respiratory Society (ERS) and the World Association of Sarcoidosis and Other Granulomatous Disorders (WASOG) adopted by the ATS Board of Directors and by the ERS Executive Committee, February 1999. Am J Respir Crit Care Med. 1999 Aug;160(2):736-55. doi: 10.1164/ajrccm.160.2.ats4-99. No abstract available.
Results Reference
background
PubMed Identifier
12421444
Citation
Bezabih M, Mariam DW, Selassie SG. Fine needle aspiration cytology of suspected tuberculous lymphadenitis. Cytopathology. 2002 Oct;13(5):284-90. doi: 10.1046/j.1365-2303.2002.00418.x.
Results Reference
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Diagnostic Accuracy of Polymerase Chain Reaction for Mycobacterium Tuberculosis Using EBUS-TBNA Samples

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