BACKGROUNDDrug susceptibility assay is very important in tuberculosis therapy. Pyrazinamide is a first line antituberculosis drug and diagnosis of its resistance in Mycobacterium tuberculosis (M. tuberculosis) is difficult and time consuming by conventional methods. In this study, we aimed to evaluate the performance of the microscopic observation drug susceptibility (MODS) assay in the detection of pyrazinamide resistance in M. tuberculosis relative to the conventional Wayne assay and Lowenstein-Jensen (LJ) proportion method.

METHODSM. tuberculosis clinical isolates (n = 132) were tested by the MODS and the Wayne assay: the results were compared with those obtained by the LJ proportion method. Mutations in the gene were identified by direct sequencing of the pncA genes of all isolates in which pyrazinamide resistance was detected by any of the three methods.

RESULTSCompared to the LJ results, the sensitivity and specificity of the MODS assay were 97.8% and 96.5% respectively; the sensitivity and specificity of the Wayne assay were 87.0% and 97.7% respectively. Mutations in the pncA gene were found in 41 of 46 strains that were pyrazinamide resistant (3 tests), in 1 of the 4 strains (LJ only), in 42 of 48 strains (at least 1 test), but no mutations in 1 strain sensitive according to the MODS assay only. The MODS assay, Wayne assay and LJ proportion method provided results in a median time of 6, 7 and 26 days respectively.

CONCLUSIONSMODS assay offers a rapid, simple and reliable method for the detection of pyrazinamide resistance in M. tuberculosis and is an optimal alternative method in resource limited countries.

Antitubercular Agents ; pharmacology ; Microbial Sensitivity Tests ; Microscopy ; methods ; Mycobacterium tuberculosis ; drug effects ; Pyrazinamide ; pharmacology

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.
Amidohydrolases/*genetics ; Antitubercular Agents/*pharmacology ; Drug Resistance, Bacterial ; *Mutation ; Mycobacterium tuberculosis/*drug effects/genetics ; Pyrazinamide/*pharmacology

OBJECTIVETo understand the characteristics of pncA gene in multidrug-resistant Mycobacterium tuberculosis isolates and its correlation with drug resistance to pyrazinamide.

METHODSA total of 127 clinical isolates of multidrug-resistant mycobacterium tuberculosis were collected from Shenzhen from year 2007 to 2009. PZA susceptibility was determined by the BACTEC MGIT 960 PZA method. Pyrazinamidase (PZase) activity testing and pncA gene sequencing were performed in all the isolates. The type and frequency of mutations in pncA were determined. Correlation analysis among PZA susceptibility and PZase activity, pncA mutation was performed.

RESULTSAmong the 127 isolates, 62 isolates (48.8%) were found resistance to PZA. Among the 62 PZA resistant isolates, 45 isolates which had various pncA mutations were negative for PZase. Mutation rate was 77.4% (48/62) in total PZA resistance isolates. Different types of 48 resistant isolates were identified in the pncA gene, including base substitution (33 isolates), frame shift mutation (12 isolates) and codon mutation (3 isolates). No mutations except one isolate (N11D) existed in all PZA-susceptibility isolates which were positive for PZase. A total of 5 mutations which have not been described previously were found as follows: H57P, P62Q, G108R, D110Y and G162V. The correlation among the PZA susceptibility and the PZase activity (r = 0.895, P < 0.05), the pncA mutation (r = 0.779, P < 0.05) were significant in 127 multidrug-resistant isolates.

CONCLUSIONA high diversity of pncA gene mutation was found among PZA resistant strains of MTB. This study revealed five new mutations of the pncA gene that were not previously described, which scattered in the hot-spot regions located in the metal coordination site and active site of the enzyme. Mutations had a high correlation with the PZA resistance.

Antitubercular Agents ; pharmacology ; DNA, Bacterial ; genetics ; Humans ; Mutation ; Mycobacterium tuberculosis ; drug effects ; genetics ; isolation & purification ; Pyrazinamide ; pharmacology ; Tuberculosis, Multidrug-Resistant ; genetics ; microbiology