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Lee, Kim, Kim, Lee, and Chang: 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

Ann Clin Microbiol 2015;18(2):37-43.

Published online: 5 June 2015

DOI: 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

Correspondence: Chulhun L. Chang, Department of Laboratory Medicine, Pusan National University School of Medicine, 20, Geumo-ro, Mulgeum-eup, Yangsan 626-770, Korea. (Tel) 82-55-360-1870, (Fax) 82-55-360-1880, (E-mail) cchl@pusan.ac.kr

Received 26 November 201431 March 2015       Accepted 1 April 2015

Copyright © 2015 The Korean Society of Clinical Microbiology

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

초록

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-re-activity 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.

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

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Fig. 1.
Positive peptide nucleic acid probe-based fluorescence in situ hybridization for Mycobacterium tuberculosis (A) and M. kansasii (B) (×100).
acm-18-37f1.tif
Table 1.
Results of the PNA probe-based FISH assay for mycobacteria and other ordinary bacteria
Type and clinical strains (no. of strains)* Results obtained with the following probes
M. tuberculosis-specific NTM-specific
Mycobacteria
 M. tuberculosis ATCC 13950 P N
 M. kansasii ATCC 12479 N P
 M. fortuitum ATCC 6841 N N
 M. intracellulare (11) N P
 M. abscessus (6) N P
 M. avium (5) N P
 M. gordonae (1) N P
 M. chelonae (1) N P
Gram-positive bacteria
 Enterococcus faecalis ATCC 29212 N N
 Staphylococcus aureus ATCC 29213 N N
 Streptococcus pneumoniae ATCC 49619 N N
 Enterococcus faecium (1) N N
 Nocardia asteroides (1) N N
Gram-negative bacteria
 Escherichia coli ATCC 25922 N N
 Haemophilus influenzae ATCC 9007 N N
 Klebsiella pneumoniae ATCC 13883 N N
 Pseudomonas aeruginosa ATCC 27853 N N
 Proteus mirabilis (1) N N
 Legionella pneumophila (1) N N

∗ All clinical strains were isolated in Pusan National University Yangsan Hospital. Numbers in parentheses indicate number of clinical isolates.

Abbreviations: PNA, peptide nucleic acid; FISH, fluorescence in situ hybridization; NTM, nontuberculous mycobacteria; P, positive; N, negative.

Table 2.
PNA FISH results of sputum smears compared with culture results
PNA FISH results Culture results Total
Negative MTB NTM*
Negative 10 0 0 10
MTB+/NTM- 0 89 0 89
MTB-/NTM+ 0 0 29 29
Total 10 89 29 128

∗ Eleven M. intracellulare, seven M. kansasii, six M. abscessus and five M. avium isolates were included.

Abbreviations: PNA, peptide nucleic acid; FISH, fluorescence in situ hybridization; MTB, Mycobacterium tuberculosis; NTM, nontuberculous mycobacteria.

Table 3.
Smear grade of routine fluorescent AFB strain and PNA FISH test
Grade of PNA FISH test Grade of routine fluorescent AFB stain Total
Negative Trace 1+ 2+ 3+ 4+
Negative 0 10∗ 0 0 0 0 10
Trace 0 6 4† 0 0 0 10
1+ 0 0 15 0 0 0 15
2+ 0 0 2‡ 34 0 0 36
3+ 0 0 0 0 33 0 33
4+ 0 0 0 0 0 26 26
Total 0 16 21 34 33 26 128

∗ Under-treatment culture-negative specimens.

† Nontuberculous mycobacteria.

‡ Mycobacterium tuberculosis.

Abbreviations: PNA, peptide nucleic acid; FISH, fluorescence in situ hybridization; AFB, acid-fast bacillus.

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