Temperature-Mediated Heteroduplex Analysis for Detection of
 pncA
 Mutations Associated with Pyrazinamide Resistance and Differentiation between
 Mycobacterium tuberculosis
 and
 Mycobacterium bovis
 by Denaturing High- Performance Liquid Chromatography

Am, Mohamed; Bastola, Dhundy; Morlock, Glenn P.; Cooksey, Robert C.; Hinrichs, Steven H.

doi:10.1128/jcm.42.3.1016-1023.2004

Cited by 19 publications

(9 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Similar problems are expected for any molecular technique that probes for DNA alterations in pncA, probably including sequencing. For instance, Mohamed et al (25) reported problems detecting a 1-bp deletion within codon 24 using denaturing high-performance liquid chromatography. Branch migration inhibition (18) and single-stranded conformation polymorphism (6,34) have also been used to detect pncA mutations.…”

Section: Discussionmentioning

confidence: 99%

Detection by Denaturing Gradient Gel Electrophoresis of pncA Mutations Associated with Pyrazinamide Resistance in Mycobacterium tuberculosis Isolates from the United States-Mexico Border Region

McCammon¹,

Gillette²,

Thomas³

et al. 2005

Antimicrob Agents Chemother

View full text Add to dashboard Cite

Denaturing gradient gel electrophoresis (DGGE) was used to probe for mutations associated with pyrazinamide (PZA) resistance in the pncA gene of Mycobacterium tuberculosis. DGGE scans for mutations across large regions of DNA and rivals sequencing in its ability to detect DNA alterations. Specific mutations can often be recognized by their characteristic denaturation pattern, which serves as a molecular fingerprint. Five PCR target fragments were designed to scan for DNA alterations across 600 bp of pncA in 181 M. tuberculosis isolates from patients residing in the U.S-Mexico border states of Texas and Tamaulipas, respectively. A region of pncA was observed with a high GC content and a melting temperature approaching 90°C that was initially refractory to denaturation, and a DGGE target fragment was specifically designed to detect mutations in this region. DGGE detected pncA mutations in 82 of 83 PZA-resistant isolates. By contrast, only 1 of 98 PZA-susceptible isolates harbored a detectable DNA alteration. The pncA gene was sequenced from 41 isolates, and 32 DNA alterations in 32 PZA-resistant isolates were identified, including 11 new mutations. DGGE also detected nine isolates whose susceptibility to PZA appeared to be incorrect, and DNA sequencing confirmed these apparent errors in drug susceptibility testing. These results demonstrate the power and usefulness of DGGE in detecting mutations associated with PZA resistance in M. tuberculosis.Pyrazinamide (PZA) is a front-line drug used in the treatment of tuberculosis (TB). In a typical short-course (6-month) therapy, PZA is administered with rifampin (RIF), isoniazid (INH), and ethambutol (EMB) for the first 2 months of treatment, followed by 4 months of treatment with INH and RIF (1). During the initial acute phase, PZA and RIF are responsible for much of the killing of persisting Mycobacterium tuberculosis bacteria (23, 48). The mode of action of PZA is complex and not fully understood. It requires activation to pyrazinoic acid by a pyrazinamidase/nicotinamidase (PZase) encoded by the pncA gene (48). The activated acid form is excreted and then reabsorbed in a protonated form. It is thought that the acidification of the M. tuberculosis cells by the protonated form represents the primary mechanism by which PZA kills tubercle bacilli (48, 49). PZase inactivation is the primary mechanism for developing resistance to PZA (31,35,47,48). Any mutation in pncA that inactivates the encoded enzyme appears to be sufficient to confer resistance. Consequently, PZA resistance mutations are highly diverse and are found throughout pncA. Cumulative reports indicate that between 72 and 98% of PZAresistant isolates harbor pncA mutations (33,46,49). More recent reports, however, tend to support the upper end of this range (48). Earlier reports may have been subject to inaccurate susceptibility testing using culture methods. Susceptibility testing media must have a pH of 6 for PZA to be active against susceptible cells, and this pH is near the limit for growth of tubercle bacill...

show abstract

Section: Discussionmentioning

confidence: 99%

Detection by Denaturing Gradient Gel Electrophoresis of pncA Mutations Associated with Pyrazinamide Resistance in Mycobacterium tuberculosis Isolates from the United States-Mexico Border Region

McCammon¹,

Gillette²,

Thomas³

et al. 2005

Antimicrob Agents Chemother

View full text Add to dashboard Cite

show abstract

“…The "bubble" type is formed between DNA fragments with single base differences and the bulge type is formed when there are deletions or insertions present within the two fragments. Recently, temperature mediated HA has been applied to the detection of mutations associated with mutations in rpoB, katG, rpsL, embB and pncA genes (Mohamed et al, 2004;Cooksey et al, 2002). Neither HA nor the SSCP analysis are 100% sensitive although Rosetti et al found that HA detected more mutants (Nataraj et al, 1999).…”

Section: Heteroduplex Analysis (Ha)mentioning

confidence: 99%

Drug Resistance inMycobacterium tuberculosis

Johnson

Streicher

Louw³

et al. 2006

Current Issues in Molecular Biology

View full text Add to dashboard Cite

show abstract

“…Double point mutation of gyrA is relatively rare (2,5,12) and is generally thought to be uncommon in clinical isolates. The Ala 74 Ser mutation has been reported only for other bacteria (8,12). This indicates that fluoroquinolone resistance is already emerging in China.…”

mentioning

confidence: 99%

“…DHPLC is a relatively new technique utilizing heteroduplex formation between wild-type and mutated DNA strands to identify mutations and was predicted to be a potentially useful genotypic screening method for gene mutations conferring drug resistance in M. tuberculosis (3,8,10). In this study, by introducing M. tuberculosis H37Rv and M. tuberculosis Erdman as two reference strains, the interference from the codon 95 AGC3ACC natural polymorphism was successfully avoided.…”

mentioning

confidence: 99%

Emergence of Ofloxacin Resistance in Mycobacterium tuberculosis Clinical Isolates from China as Determined by gyrA Mutation Analysis Using Denaturing High-Pressure Liquid Chromatography and DNA Sequencing

Shi

Zhang

et al. 2006

J Clin Microbiol

View full text Add to dashboard Cite

A high rate of double point mutations in gyrA (56% of 87 ofloxacin-resistant Mycobacterium tuberculosis clinical isolates) indicates the emergence of fluoroquinolone resistance. This is the first report to describe denaturing high-pressure liquid chromatography analysis of mutations in gyrA of M. tuberculosis in a large number of clinical isolates.Up to the present, fluoroquinolones have been studied as a first-line treatment for tuberculosis (9). However, fluoroquinolone resistance among Mycobacterium tuberculosis strains is emerging, with important implications for treatment (4, 6). Fluoroquinolones have been widely used for tuberculosis treatment in China for more than 10 years and have been given routinely as monotherapy for the empirical treatment of numerous outpatient infections. Thus, China may be one of the countries with the highest rate of fluoroquinolone abuse and resistance in the world. The goal of this work was to identify quinolone resistance-determining regions (QRDRs) of gyrA in ofloxacin-resistant isolates from China by denaturing highpressure liquid chromatography (DHPLC) and DNA sequencing methods.( All the isolates contain a naturally occurring polymorphism, codon 95 AGC3ACC. Seventy-three (84%) of the 87 ofloxacin-resistant clinical isolates were found to carry a codon 94 mutation, and 49 (56%) were found to harbor a double point mutation. 4566on May 9, 2018 by guest

show abstract

Temperature-Mediated Heteroduplex Analysis for Detection of pncA Mutations Associated with Pyrazinamide Resistance and Differentiation between Mycobacterium tuberculosis and Mycobacterium bovis by Denaturing High- Performance Liquid Chromatography

Cited by 19 publications

References 30 publications

Detection by Denaturing Gradient Gel Electrophoresis of pncA Mutations Associated with Pyrazinamide Resistance in Mycobacterium tuberculosis Isolates from the United States-Mexico Border Region

Detection by Denaturing Gradient Gel Electrophoresis of pncA Mutations Associated with Pyrazinamide Resistance in Mycobacterium tuberculosis Isolates from the United States-Mexico Border Region

Drug Resistance inMycobacterium tuberculosis

Emergence of Ofloxacin Resistance in Mycobacterium tuberculosis Clinical Isolates from China as Determined by gyrA Mutation Analysis Using Denaturing High-Pressure Liquid Chromatography and DNA Sequencing

Contact Info

Product

Resources

About