Korean J Lab Med.  2006 Jun;26(3):161-167. 10.3343/kjlm.2006.26.3.161.

Isolation of Nontuberculous Mycobacteria Using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism

• Park CM
• Heo SR
• Park KU
• Song J
• Lee JH
• Lee CT
• Kim EC

• KMID: 2238881
• DOI: http://doi.org/10.3343/kjlm.2006.26.3.161

Abstract

BACKGROUND: The isolation rate of nontuberculous mycobacteria (NTM) in clinical laboratories and the incidence of NTM infections are on the increase recently in Korea, but there have been only a few studies that reveal the general aspect of NTM isolation or species distribution. Therefore, this study was performed to examine the isolation rate of NTM, species identification, and the clinical significance of mycobacterial cultures.
METHODS
From August 2004 to May 2005, we examined mycobacterial isolates by AccuProbe test to differentiate NTM from Mycobacterium tuberculosis complex. NTM was then identified by polymerase chain reaction-restriction fragment length analysis (PCR-RFLP).
RESULTS
A total of 6,742 specimens from 2,784 patients were requested for mycobacterial culture. Mycobacteria were isolated from 776 specimens (11.5%). The isolation rates of NTMs among the total culture positive specimens and culture positive sputum specimens were 24.4% (189/776) and 25.3% (169/667), respectively. Fourteen species of NTM identified in 172 of the 175 specimens tested included M. avium (39.0%), M. intracellulare (22.7%), and M. abscessus (19.8%).
CONCLUSIONS
Using AccuProbe tests and PCR-RFLP method for mycobacterial cultures processed in a clinical laboratory, we were able to identify NTMs to the species level. The isolation rate of NTM in this study was similar to that reported in past studies in Korea. In addition, we found that some of the NTMs isolated in this study could cause pulmonary diseases.

Keyword

Nontuberculous mycobacteria; Polymerase chain reaction; Restriction fragment length polymorphism; Pulmonary disease

MeSH Terms

Humans
Incidence
Korea
Lung Diseases
Mycobacterium tuberculosis
Nontuberculous Mycobacteria*
Polymerase Chain Reaction
Polymorphism, Restriction Fragment Length
Sputum

Reference

References

1. Falkinham JO 3rd. Nontuberculous mycobacteria in the environment. Clin Chest Med. 2002; 23:529–51.

2. Prince DS, Peterson DD, Steiner RM, Gottlieb JE, Scott R, Israel HL, et al. Infection with Mycobacterium avium complex in patients without predisposing conditions. N Engl J Med. 1989; 321:863–8.

3. British Thoracic Society. Management of opportunistic mycobacterial infections: Joint Tuberculosis Committee Guidelines 1999. Thorax. 2000; 55:210–8.

4. American Thoracic Society. Diagnosis and treatment of disease caused by nontuberculous mycobacteria. Am J Respir Crit Care Med. 1997; 156(s):):1–25.

5. Sakatani M. The non-tuberculous mycobacteriosis. Kekkaku. 2005; 80:25–30.

6. Koh WJ, Kwon OJ, Yu CM, Jeon K, Suh GY, Chung MP, et al. Recovery rate of nontuberculous mycobacteria from acid-fast-bacilli smear-positive sputum specimens. Tuberc Respir Dis. 2003; 54:22–32.

7. Lee JY, Choi HJ, Lee H, Joung EY, Huh JW, Oh Y, et al. Recovery rate and characteristics of nontuberculous mycobacterial isolates in a university hospital in Korea. Tuberc Respir Dis. 2005; 58:385–91.

8. Centers for Disease Control and Prevention. Update: Nucleic acid amplification tests for tuberculosis. MMWR Morb Mortal Wkly Rep. 2000; 49:593–4.

9. Kim MN, Lee SH, Yang SE, Pai CH. Mycobacterial testing in hospital laboratories in Korea: results of a survey of 40 university or tertiary-care hospitals. Korean J Clin Pathol. 1999; 19:86–91.

10. Korean Academy of Tuberculosis and Respiratory Disease. National survey of mycobacterial diseases other than tuberculosis in Korea. Tuberc Respir Dis. 1995; 42:277–94.

11. American Thoracic Society. Diagnostic standards and classification of tuberculosis in adults and children. Am J Respir Crit Care Med. 2000; 161:1376–95.

12. Chung J, Lee SH, Jeong US, Chang CH, Kim SR. Identification of mycobacteria using high performance liquid chromatography in clinical specimens. Korean J Clin Microbiol. 2004; 7:148–55.

13. Lee HW, Kim M, Shim TS, Bai GH, Pai CH. Nontuberculous mycobacterial pulmonary infection in immunocompetent patients. Tuberc Respir Dis. 2002; 53:173–82.

14. Lee JY, Kim M, Chung H, Jun KR, Choi HJ, Lee H, et al. Clinical significance of low-colony count scotochromogen nontuberculous mycobacteria. Tuberc Respir Dis. 2005; 59:39–46.

15. Park HS, Chin DI, Chong Y, Kwon OH. Effect of N-acetyl-L-cysteine decontamination on the contamination rate of medium and culture positive rate of Mycobacterium. Clin Pathol & Qual Control. 1991; 13:223–7.

16. Nah J, Huh JW, Lee SH, Kim BC, Koh YS, Pai CH. Identification of Mycobacterium tuberculosis complex using a Gene Probe method. Korean J Clin Pathol. 1997; 17:71–7.

17. Yi JY, Kim JP, Shin JH, Suh SP, Ryang DW. Detection of Mycobacterium tuberculosis using BACTEC Mycobacteria Growth Indicator Tube (MGIT) 960 system – comparison with BACTEC 460 TB system and Ogawa media. Korean J Clin Pathol. 2000; 20:384–91.

18. Butler WR, Guthertz LS. Mycolic acid analysis by high-performance liquid chromatography for identification of Mycobacterium species. Clin Microbiol Rev. 2001; 14:704–26.

19. Kim HK, Kim YR, Park JP, Kim NH, Ok CH, Jung MH, et al. Isolation of nontuberculous mycobacteria by DNA probe and clinical characteristics of patients with NTM pulmonary disease. Tuberc Respir Dis. 2005; 58:248–56.

20. Cheunoy W, Prammananan T, Chaiprasert A, Foongladda S. Comparative evaluation of polymerase chain reaction and restriction enzyme analysis: Two amplified targets, hsp65 and rpoB, for identification of cultured mycobacteria. Diag Microbiol Infect Dis. 2005; 51:165–71.

21. Devallois A, Goh KS, Rastogi N. Rapid identification of mycobacteria to species level by PCR-restriction fragment length polymorphism analysis of the hsp65 gene and proposition of an algorithm to differentiate 34 mycobacterial species. J Clin Microbiol. 1997; 35:2969–73.

22. Lee H, Park HJ, Cho SN, Bai GH, Kim SJ. Species identification of mycobacteria by PCR-restriction fragment length polymorphism of the rpoB gene. J Clin Microbiol. 2000; 38:2966–71.

23. Koh WJ, Kwon OJ. Diagnosis and treatment of pulmonary tuberculosis. Tuberc Respir Dis. 2005; 58:438–51.