Annals of Clinical Microbiology, The official Journal of the Korean Society of Clinical Microbiology

6

Weeks in Review

2

Weeks to Publication
Indexed in KCI, KoreaMed, Synapse, DOAJ
Open Access, Peer Reviewed
pISSN 2288-0585 eISSN 2288-6850

Evaluation of Peptide Nucleic Acid Probe-Based Fluorescence In Situ Hybridization for the Detection of Mycobacterium tuberculosis Complex and Nontuberculous Mycobacteria in Clinical Respiratory Specimens

Original article

Annals of Clinical Microbiology (Ann Clin Microbiol) 2015 June, Volume 18, Issue 2, pages 37-43.

https://doi.org/10.5145/ACM.2015.18.2.37

Evaluation of Peptide Nucleic Acid Probe-Based Fluorescence In Situ Hybridization for the Detection of Mycobacterium tuberculosis Complex and Nontuberculous Mycobacteria in Clinical Respiratory Specimens

Seung Hee Lee, Shine Young Kim, Hyung Hoi Kim, Eun Yup Lee, Chulhun L. Chang

Department of Laboratory Medicine, Pusan National University School of Medicine, Yangsan, Korea

Abstract

Background: Tuberculosis is globally the most important cause of death from single pathogen. Rapid and accurate identification of mycobacteria is essential for the control of tuberculosis. We evaluated a fluorescence in situ hybridization (FISH) method using peptide nucleic acid (PNA) probes for the differentiation of Mycobacterium tuberculosis complex (MTB) and nontuberculous mycobacteria (NTM) in direct smears of sputum specimens.

Methods: The cross-reactivity of MTB- and NTM-specific PNA probes was examined with reference strains of M. tuberculosis ATCC 13950, Mycobacterium kansasii ATCC 12479, Mycobacterium fortuitum ATCC 6841, several clinical isolates of mycobacteria (Mycobacterium abscessus, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium gordonae and Mycobacterium chelonae), and 11 frequently isolated respiratory bacterial species other than mycobacteria. A series of 128 sputa (89 MTB culture positive, 29 NTM culture positive, and 10 under treatment culture negative) with grades of trace to 4+ were used to evaluate the performance of the method.

Results: The MTB- and NTM-specific PNA probes showed specific reactions with the reference strains of MTB and M. kansasii and clinical isolates of mycobacteria except M. fortuitum ATCC 6841, and no cross-reactivity with other tested bacteria. The PNA probe-based FISH assay for detection of MTB had a sensitivity and specificity of 100%, respectively. The sensitivity and specificity of the NTM-specific PNA probe was 100%. The smear grades of the PNA FISH test were same as with those of the fluorescence AFB stain in 2+ or higher grade.

Conclusion: Detection and differentiation based on PNA FISH is sensitive and accurate for detecting mycobacteria and for differentiating MTB from NTM in clinical sputum smears. (Ann Clin Microbiol 2015;18:37-43)

Keywords

Fluorescence, In situ hybridization, Mycobacterium tuberculosis, Nontuberculous mycobacteria, Peptide nucleic acids