Characterization ofpncAMutations of Pyrazinamide-ResistantMycobacterium tuberculosisin Korea

Abstract

Pyrazinamide (PZA) is one of the most important drugs for the treatment of Mycobacterium tuberculosis infection. However, the increasing frequency of PZA-resistant strains limits its effectiveness. In Korea, most PZA-resistant strains also exhibit both isoniazid and rifampin resistance making it essential to identify these resistant strains accurately and rapidly for effective treatment of mycobacterial infection. In this study, the characteristics and frequency of mutations of the pncA gene encoding pyrazinamidase were investigated in PZA-resistant clinical isolates from Korea. Automated DNA sequencing was used to evaluate the usefulness of DNA-based detection of PZA resistance. Among 95 PZA-resistant clinical isolates, 92 (97%) exhibited mutations potentially affecting either the production or the activity of the enzyme. Mutations were found throughout the pncA gene including the upstream region. Single nucleotide replacement appeared to be the major mutational event (69/92), although multiple substitutions as well as insertion and deletion of nucleotides were also identified. The high frequency of pncA mutations observed in this study supports the usefulness of DNA-based detection of PZA-resistant M. tuberculosis. Having verified the scattered and diverse mutational characteristics of the pncA gene, automated DNA sequencing seems to be the best strategy for rapid detection of PZA-resistant M. tuberculosis.

Evaluation of Colorimetric Methods Using Nicotinamide for Rapid Detection of Pyrazinamide Resistance in Mycobacterium tuberculosis

“…18, 19, and 20), i.e., strains that were PZA sensitive by the Wayne assay but resistant with one of the other assays, showed no mutations in their pncA coding regions or in the 65 bases upstream of the first codon. The mutations found in our PZA-resistant strains were distributed throughout the pncA gene, confirming the high diversity and absence of mutation hot spots reported in other studies (10,15,18), but the L172P substitution was present in three strains and the V139A substitution in two. Perhaps these were multiple isolates of the same strain from different patients.…”

High Resolution Melting Curve Analysis for Rapid Detection of Pyrazinamide Resistance in <i>Mycobacterium tuberculosis</i> Clinical Isolates

“…However, mutations at codon 135 were found in five isolates, whereas mutations at codons 14 and 139 were shown by three isolates each. These observations of scattered mutations are supported by similar findings in previous studies [27] [36]. The high diversity of pncA mutations limits its inclusion in the current molecular techniques such as GenoType MTBDRplus (Hain Lifescience) [37].…”

Characterization of pncA Mutations in Pyrazinamide-Resistant Mycobacterium tuberculosis in Brazil

“…PCR products were purified with MicroSpin S-300 HR columns (Amersham Biosciences, Piscataway, N.J.) and sequenced using the Big Dye Terminator Cycle sequencing kit with AmpliTaq DNA polymerase (Applied Biosystems, Foster City, Calif.) in the ABI Prism 3100 DNA sequencer (Applied Biosystems). Nucleotide sequences were analyzed us- (24,27), but 12 of the mutations found have not been described previously (1,3,5,8,12,14,16,19,20,21,22,24,27,28,31,33) (Fig. 1).…”