Evaluation of Dual-Color Fluorescence In Situ Hybridization With Peptide Nucleic Acid Probes for the Detection of Mycobacterium tuberculosis and Non-Tuberculous Mycobacteria in Clinical Specimens

Abstract

BackgroundPeptide nucleic acid (PNA) probes are artificial DNA analogues with a hydrophobic nature that can penetrate the mycobacterial cell wall. We evaluated a FISH method for simultaneous detection and identification of Mycobacterium tuberculosis (MTB) and non-tuberculous mycobacteria (NTM) in clinical respiratory specimens using differentially labeled PNA probes.MethodsPNA probes targeting the mycobacterial 16S ribosomal RNA were synthesized. The cross-reactivity of MTB- and NTM-specific probes was examined with reference strains and 10 other frequently isolated bacterial species. A total of 140 sputum specimens were analyzed, comprising 100 MTB-positive specimens, 21 NTM-positive specimens, and 19 MTB/NTM-negative specimens; all of them were previously confirmed by PCR and culture. The PNA FISH test results were graded by using the United States Centers for Disease Control and Prevention-recommended scale and compared with the results from the fluorochrome acid-fast bacterial stain.ResultsThe MTB- and NTM-specific PNA probes showed no cross-reactivity with other tested bacterial species. The test results demonstrated 82.9% agreement with the culture results with diagnostic sensitivity of 80.2% and diagnostic specificity of 100.0% (kappa=0.52, 95% confidence interval: 0.370-0.676).ConclusionsDual-color PNA FISH showed high specificity for detecting and identifying mycobacteria in clinical specimens. However, because of its relatively low sensitivity, this method could be more applicable to culture confirmation. In application to direct specimens, the possibility of false-negative results needs to be considered.

Non-Tuberculous Mycobacteria: Molecular and Physiological Bases of Virulence and Adaptation to Ecological Niches

“…In the same work, while testing the MTBC-MAC FISH commercial kit, only the six MTC members among the 44 reference species reacted to the MTC-specific probe, and the 14 reference MAC members reacted to MAC-specific probe [ 331 ]. PNA probes have been developed for MTC members as well as for NTM, in which a sensitivity of 71.4% and specificity of 100% was obtained in clinical specimens [ 332 ]. In another study, a specific PNA probe targeting the 16S rRNA of MAA hybridized with MAP besides its initial target, being successful in MAA identification in both biofilm and potable-water samples, however, the struggle around background fluorescence was also reported [ 333 ].…”

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.…”

Rapid method for detecting and differentiating Mycobacterium tuberculosis complex and non-tuberculous mycobacteria in sputum by fluorescence in situ hybridization with DNA probes

“…A DNA probe-based FISH assay has been used to identify MTB in a large number of sputum samples, with a reported sensitivity of 82% and specificity of 98% compared with traditional culture-based identification, but this FISH assay does not differentially identify MTB and NTM (Yuan et al, 2015). A PNA-based FISH assay has been shown to differentiate MTB from several NTM species, but to have relatively low sensitivity with sputum samples (Kim et al, 2015). The present authors have described two dual fluorescence FISH assaysthe Mycobacterium/Nocardia Genus (MN Genus)-MTBC assay and the MAC-MTBC assaywhich identified MTBC and MAC with specificity and sensitivity of 100% in a large number of cultures and also differentiated them from other NTM and Nocardia species (Shah et al, 2017a).…”

“…Several fluorescence in situ hybridization (FISH) assays that target multiple copies of rRNA present in mycobacterial cells, and therefore do not require NAA, have been described (Yuan et al, 2015;Shah et al, 2017a;Amand St et al, 2005a,b;Stender et al, 1999;Lehtola et al, 2006;Kim et al, 2015;Lefmann et al, 2006). Such FISH assays are based on DNA (Yuan et al, 2015;Shah et al, 2017a;Amand St et al, 2005a,b) or peptide nucleic acid (PNA) (Stender et al, 1999;Lehtola et al, 2006;Kim et al, 2015;Lefmann et al, 2006) probes but are not yet in widespread clinical use. As NAA is not necessary, FISH assays are not affected by inhibitors of NAA and do not require the expensive equipment and infrastructure needed for NAA.…”

“…They are therefore more suitable for resource-limited tuberculosis-endemic countries. With two exceptions (Yuan et al, 2015;Kim et al, 2015), the FISH assays reported for MTB and other mycobacteria have largely been applied to cultures and biopsied tissues. The application of FISH assays to sputum samples is more complex than for cultures because of a lower MTB concentration, the presence of other bacteria in sputum, and the need to liquefy sputum before preparing smears for the assays.…”