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

NCT02497079 | Unknown

• 1 other identifier: PNUH-P-1
• Study Type: interventional
• Target: 100
• Locations: 1 country
• Sites: 1

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.

Conditions

Lymphadenopathy; Tuberculosis; Sarcoidosis

Keywords

Bronchoscopy; Ultrasonography; Tuberculous lymphadenitis; Polymerase chain reaction; Sensitivity and specificity

Outcome Measures

Primary Outcomes (1)

• 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

Study Arms (3)

Nested PCR for formalin-fixed tissues

EXPERIMENTAL

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

Nested PCR for fresh tissues

EXPERIMENTAL

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

Real-time PCR for fresh tissues

EXPERIMENTAL

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

Interventions

Nested PCR for formalin-fixed tissues DEVICE

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.

Also known as: MTB-PCR kit; Biosewoom, Seoul, South Korea

Nested PCR for formalin-fixed tissues

Nested PCR for fresh tissues DEVICE

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.

Also known as: MTB-PCR kit; Biosewoom, Seoul, South Korea

Nested PCR for fresh tissues

Real-time PCR for fresh tissues DEVICE

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.

Also known as: AdvanSure TB/NTM real-time PCR kit; LG, Seoul, South Korea

Real-time PCR for fresh tissues

Eligibility Criteria

• Age: 18 Years+
• Sex: all
• Healthy Volunteers: No
• Age Groups: Adult (18-64), Older Adult (65+)

You may qualify if:

• Patients with isolated intrathoracic lymphadenopathy

You may not qualify if:

• 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

Sponsors & Collaborators

• Pusan National University Hospital: lead

Study Sites (1)

Pusan National University Hospital

Busan, Busan, 602-739, South Korea

RECRUITING

Related Publications (5)

• 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.

  PMID: 22652031 BACKGROUND

• 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.

  PMID: 21813622 BACKGROUND

• 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.

  PMID: 24035300 BACKGROUND

• 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.

  PMID: 10430755 BACKGROUND

• 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.

  PMID: 12421444 BACKGROUND

MeSH Terms

Conditions

Lymphadenopathy; Tuberculosis; Sarcoidosis; Tuberculosis, Lymph Node; Hypersensitivity

Interventions

Polymerase Chain Reaction; Real-Time Polymerase Chain Reaction

Condition Hierarchy (Ancestors)

Lymphatic Diseases; Hemic and Lymphatic Diseases; Mycobacterium Infections; Actinomycetales Infections; Gram-Positive Bacterial Infections; Bacterial Infections; Bacterial Infections and Mycoses; Infections; Lymphoproliferative Disorders; Hypersensitivity, Delayed; Immune System Diseases; Tuberculosis, Extrapulmonary

Intervention Hierarchy (Ancestors)

Nucleic Acid Amplification Techniques; Genetic Techniques; Investigative Techniques

Study Officials

• Jeong Ha Mok, Master

  Pusan National University Hospital

  PRINCIPAL INVESTIGATOR

• Jung Seop Eom, Master

  Pusan National University Hospital

  PRINCIPAL INVESTIGATOR

Central Study Contacts

Jung Seop Eom Eom, Master

CONTACT

82-01-2081-0859; ejspulm@gmail.com

Study Design

• Study Type: interventional
• Phase: not applicable
• Allocation: NON RANDOMIZED
• Masking: NONE
• Purpose: DIAGNOSTIC
• Intervention Model: SINGLE GROUP
• Sponsor Type: OTHER
• Responsible Party: PRINCIPAL INVESTIGATOR
• PI Title: Clinical Assistant Professor

Study Record Dates

• First Submitted: July 8, 2015
• First Posted: July 14, 2015
• Study Start: July 1, 2015
• Primary Completion: December 1, 2018
• Study Completion: December 1, 2018
• Last Updated: July 20, 2015

Record last verified: 2015-07

Locations

KR (1)