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

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)

Identification of nontuberculous mycobacteria using multilocous sequence analysis of 16S rRNA, hsp65, and rpoB

“…However, these tests are time‐consuming and cumbersome and sometimes produce ambiguous results. Many molecular methods have been developed for NTM identification to overcome the limitations of conventional methods . Gene sequencing is considered the gold standard for identification of Mycobacterium species.…”

Peptide nucleic acids: Advanced tools for biomedical applications

“…PNA-FISH was found to be useful for the detection of pathogenic oomycete Aphanomyces invadans which is responsible for ulcerative mycosis, a skin disease caused by a fungus-like agent of wild and cultured fish (Vandersea et al, 2006). In another study, PNA probe-FISH assay was used for detecting mycobacteria with great specificity and sensitivity which helps in the fast and effective treatment of tuberculosis (Lee et al, 2015).…”