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

• Lee SH
• Kim SY
• Kim HH
• Lee EY
• Chang CL

• KMID: 1971110
• DOI: http://doi.org/10.5145/ACM.2015.18.2.37

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.

Keyword

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

MeSH Terms

Bacteria
Cause of Death
Fluorescence*
In Situ Hybridization*
Mycobacterium
Mycobacterium avium
Mycobacterium avium Complex
Mycobacterium fortuitum
Mycobacterium kansasii
Mycobacterium tuberculosis*
Nontuberculous Mycobacteria*
Peptide Nucleic Acids
Sputum
Tuberculosis
Peptide Nucleic Acids

Figure

• 

  Fig. 1. Positive peptide nucleic acid probe-based fluorescence in situ hybridization for Mycobacterium tuberculosis (A) and M. kansasii (B) (×100).

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