Clin Exp Vaccine Res.  2018 Jul;7(2):111-118. 10.7774/cevr.2018.7.2.111.

Evaluation of EZplex MTBC/NTM Real-Time PCR kit: diagnostic accuracy and efficacy in vaccination

Affiliations
  • 1Department of Biotechnology, The Catholic University of Korea, Bucheon, Korea. jhnam@catholic.ac.kr
  • 2Department of Research and Development, Genetree Research, Seoul, Korea.

Abstract

PURPOSE
Tuberculosis (TB) is mainly caused by Mycobacterium tuberculosis, which is a pathogenic mycobacterial species grouped under Mycobacterium tuberculosis complex (MTBC) with four other pathogenic mycobacterial species. The mycobacteria not included in MTBC are known as nontuberculous mycobacteria (NTM), and cause several pulmonary diseases including pneumonia. Currently, NTM occurrences in TB-suspected respiratory specimens have increased, due to which, precise detection of MTBC and NTM is considered critical for the diagnosis and vaccination of TB. Among the various methods available, real-time PCR is frequently adopted for MTBC/NTM detection due to its rapidness, accuracy, and ease of handling. In this study, we evaluated a new real-time PCR kit for analytical and clinical performance on sputum, bronchial washing, and culture specimens.
MATERIALS AND METHODS
For assessing its analytical performance, limit of detection (LOD), reactivity, and repeatability test were performed using DNA samples. To evaluate clinical performance, 612 samples were collected and clinically tested at a tertiary hospital.
RESULTS
LOD was confirmed as 0.584 copies/µL for MTBC and 47.836 copies/µL for NTM by probit analysis (95% positive). For the reactivity test, all intended strains were detected and, in the repeatability test, stable and steady results were confirmed with coefficient of variation ranging from 0.36 to 1.59. For the clinical test, sensitivity and specificity were 98.6%-100% and 98.8%-100% for MTBC and NTM, respectively.
CONCLUSION
The results proved the usefulness of the kit in TB diagnosis. Furthermore, it could be adopted for the assessment of vaccine efficacy.

Keyword

Tuberculosis; Mycobacterium tuberculosis complex; Nontuberculous mycobacteria; Real-time polymerase chain reaction; BCG vaccine

MeSH Terms

BCG Vaccine
Diagnosis
DNA
Limit of Detection
Lung Diseases
Mycobacterium tuberculosis
Nontuberculous Mycobacteria
Pneumonia
Real-Time Polymerase Chain Reaction*
Sensitivity and Specificity
Sputum
Tertiary Care Centers
Tuberculosis
Vaccination*
BCG Vaccine
DNA

Reference

1. Global tuberculosis report 2017 [Internet]. Geneva: World Health Organization;2017. cited 2018 Jul 23. Available from: http://www.who.int/tb/publications/global_report/en/.
2. Korean Guidelines for Tuberculosis Third Edition 2017 [Internet]. Seoul: Joint Committee for the Revision of Korean Guidleines for Tuberculosis;2017. Available from: http://www.lungkorea.org/bbs/skin/guide/download.php?code=guide&number=9939.
3. Gordon SV, Parish T. Microbe profile: Mycobacterium tuberculosis: humanity's deadly microbial foe. Microbiology. 2018; 164:437–439. PMID: 29465344.
Article
4. van Soolingen D, Hoogenboezem T, de Haas PE, et al. A novel pathogenic taxon of the Mycobacterium tuberculosis complex, Canetti: characterization of an exceptional isolate from Africa. Int J Syst Bacteriol. 1997; 47:1236–1245. PMID: 9336935.
Article
5. Niemann S, Rusch-Gerdes S, Joloba ML, et al. Mycobacterium africanum subtype II is associated with two distinct genotypes and is a major cause of human tuberculosis in Kampala, Uganda. J Clin Microbiol. 2002; 40:3398–3405. PMID: 12202584.
6. Niobe-Eyangoh SN, Kuaban C, Sorlin P, et al. Genetic biodiversity of Mycobacterium tuberculosis complex strains from patients with pulmonary tuberculosis in Cameroon. J Clin Microbiol. 2003; 41:2547–2553. PMID: 12791879.
7. Pfyffer GE, Auckenthaler R, van Embden JD, van Soolingen D. Mycobacterium canettii, the smooth variant of M. tuberculosis, isolated from a Swiss patient exposed in Africa. Emerg Infect Dis. 1998; 4:631–634. PMID: 9866740.
Article
8. Panteix G, Gutierrez MC, Boschiroli ML, et al. Pulmonary tuberculosis due to Mycobacterium microti: a study of six recent cases in France. J Med Microbiol. 2010; 59:984–989. PMID: 20488936.
Article
9. Diagnosis and treatment of disease caused by nontuberculous mycobacteria. This official statement of the American Thoracic Society was approved by the Board of Directors, March 1997. Medical Section of the American Lung Association. Am J Respir Crit Care Med. 1997; 156(2 Pt 2):S1–S25. PMID: 9279284.
10. Kwon YS, Koh WJ. Diagnosis and treatment of nontuberculous Mycobacterial lung disease. J Korean Med Sci. 2016; 31:649–659. PMID: 27134484.
Article
11. Koh WJ, Kwon OJ, Lee KS. Diagnosis and treatment of nontuberculous mycobacterial pulmonary diseases: a Korean perspective. J Korean Med Sci. 2005; 20:913–925. PMID: 16361797.
Article
12. Koh WJ, Kwon OJ, Jeon K, et al. Clinical significance of nontuberculous mycobacteria isolated from respiratory specimens in Korea. Chest. 2006; 129:341–348. PMID: 16478850.
Article
13. Park YS, Lee CH, Lee SM, et al. Rapid increase of non-tuberculous mycobacterial lung diseases at a tertiary referral hospital in South Korea. Int J Tuberc Lung Dis. 2010; 14:1069–1071. PMID: 20626955.
14. Escalante P. In the clinic: tuberculosis. Ann Intern Med. 2009; 150:ITC61–ITC614. PMID: 19487708.
15. Metcalfe JZ, Everett CK, Steingart KR, et al. Interferon-gamma release assays for active pulmonary tuberculosis diagnosis in adults in low- and middle-income countries: systematic review and meta-analysis. J Infect Dis. 2011; 204(Suppl 4):S1120–S1129. PMID: 21996694.
16. Diagnostics for tuberculosis: global demand and market potential/TDR, FIND SA [Internet]. Geneva: World Health Organization;2006. cited 2018 Jul 23. Available from: http://apps.who.int/iris/handle/10665/43543.
17. Sester M, Sotgiu G, Lange C, et al. Interferon-gamma release assays for the diagnosis of active tuberculosis: a systematic review and meta-analysis. Eur Respir J. 2011; 37:100–111. PMID: 20847080.
18. Darban-Sarokhalil D, Imani Fooladi AA, Maleknejad P, et al. Comparison of smear microscopy, culture, and real-time PCR for quantitative detection of Mycobacterium tuberculosis in clinical respiratory specimens. Scand J Infect Dis. 2013; 45:250–255. PMID: 23113553.
Article
19. Huh HJ, Kwon HJ, Ki CS, Lee NY. Comparison of the genedia MTB detection kit and the cobas TaqMan MTB assay for detection of Mycobacterium tuberculosis in respiratory specimens. J Clin Microbiol. 2015; 53:1012–1014. PMID: 25568443.
Article
20. Perng CL, Chen HY, Chiueh TS, Wang WY, Huang CT, Sun JR. Identification of non-tuberculous mycobacteria by real-time PCR coupled with a high-resolution melting system. J Med Microbiol. 2012; 61:944–951. PMID: 22493281.
Article
21. Centers for Disease Control and Prevention (CDC). Availability of an assay for detecting Mycobacterium tuberculosis, including rifampin-resistant strains, and considerations for its use, United States, 2013. MMWR Morb Mortal Wkly Rep. 2013; 62:821–827. PMID: 24141407.
22. Yang HY, Lee HJ, Park SY, Lee KK, Suh JT. Comparison of In-house polymerase chain reaction assay with conventional techniques for the detection of Mycobacterium tuberculosis. Korean J Lab Med. 2006; 26:174–178. PMID: 18156721.
Article
23. Park CM, Heo SR, Park KU, et al. Isolation of nontuberculous mycobacteria using polymerase chain reaction-restriction fragment length polymorphism. Korean J Lab Med. 2006; 26:161–167. PMID: 18156719.
Article
24. Nagy A, Vitaskova E, Cernikova L, et al. Evaluation of TaqMan qPCR system integrating two identically labelled hydrolysis probes in single assay. Sci Rep. 2017; 7:41392. PMID: 28120891.
Article
25. Armstrong PM, Prince N, Andreadis TG. Development of a multi-target TaqMan assay to detect eastern equine encephalitis virus variants in mosquitoes. Vector Borne Zoonotic Dis. 2012; 12:872–876. PMID: 22835151.
Article
26. Wang HY, Jin H, Bang H, et al. Evaluation of MolecuTech Real MTB-ID for MTB/NTM detection using direct specimens. Korean J Clin Microbiol. 2011; 14:103–109.
Article
27. Baek YK. The evaluation of tuberculosis examination kit using a real-time PCR [thesis]. Cheongju: Chungbuk National University Graduate School of Biology;2014.
28. Park KS, Kim JY, Lee JW, et al. Comparison of the Xpert MTB/RIF and Cobas TaqMan MTB assays for detection of Mycobacterium tuberculosis in respiratory specimens. J Clin Microbiol. 2013; 51:3225–3227. PMID: 23863563.
Article
29. Yang YC, Lu PL, Huang SC, Jenh YS, Jou R, Chang TC. Evaluation of the Cobas TaqMan MTB test for direct detection of Mycobacterium tuberculosis complex in respiratory specimens. J Clin Microbiol. 2011; 49:797–801. PMID: 21177901.
Article
30. Lim J, Kim J, Kim JW, et al. Multicenter evaluation of Seegene Anyplex TB PCR for the detection of Mycobacterium tuberculosis in respiratory specimens. J Microbiol Biotechnol. 2014; 24:1004–1007. PMID: 24786527.
Article
31. Lee MR, Chung KP, Wang HC, et al. Evaluation of the Cobas TaqMan MTB real-time PCR assay for direct detection of Mycobacterium tuberculosis in respiratory specimens. J Med Microbiol. 2013; 62:1160–1164. PMID: 23657531.
Article
32. Choe W, Kim E, Park SY, Chae JD. Performance evaluation of Anyplex plus MTB/NTM and AdvanSure TB/NTM for the detection of Mycobacterium tuberculosis and nontuberculous mycobacteria. Ann Clin Microbiol. 2015; 18:44–51.
33. Lee JH, Kim BH, Lee MK. Performance evaluation of Anyplex Plus MTB/NTM and MDR-TB detection kit for detection of mycobacteria and for anti-tuberculosis drug susceptibility test. Ann Clin Microbiol. 2014; 17:115–122.
Article
34. Ahn YO. Concepts and necessity of preventive medical services for the 21st century. J Korean Med Assoc. 2011; 54:246–249.
Article
35. Global Vaccine Action Plan 2011–2020 [Internet]. Geneva: World Health Organization;2012. cited 2018 Jul 23. Available from: http://www.who.int/immunization/global_vaccine_action_plan/GVAP_doc_2011_2020/.
36. Nguipdop-Djomo P, Heldal E, Rodrigues LC, Abubakar I, Mangtani P. Duration of BCG protection against tuberculosis and change in effectiveness with time since vaccination in Norway: a retrospective population-based cohort study. Lancet Infect Dis. 2016; 16:219–226. PMID: 26603173.
Article
37. Orme IM. Tuberculosis vaccine types and timings. Clin Vaccine Immunol. 2015; 22:249–257. PMID: 25540272.
Article
38. Kaufmann SH, Fortune S, Pepponi I, Ruhwald M, Schrager LK, Ottenhoff TH. TB biomarkers, TB correlates and human challenge models: New tools for improving assessment of new TB vaccines. Tuberculosis (Edinb). 2016; 99(Suppl 1):S8–S11. PMID: 27402312.
Article
39. Bedwell J, Kairo SK, Behr MA, Bygraves JA. Identification of substrains of BCG vaccine using multiplex PCR. Vaccine. 2001; 19:2146–2151. PMID: 11228387.
Article
40. Minassian AM, Satti I, Poulton ID, Meyer J, Hill AV, McShane H. A human challenge model for Mycobacterium tuberculosis using Mycobacterium bovis bacille Calmette-Guerin. J Infect Dis. 2012; 205:1035–1042. PMID: 22396610.
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