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Free Content Fluoroquinolone resistance detection in Mycobacterium tuberculosis with locked nucleic acid probe real-time PCR

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SETTING: Pham Ngoc Thach Hospital for Tuberculosis and Lung Diseases, Ho Chi Minh City, Vietnam.

OBJECTIVE: Fluoroquinolones (FQs) are increasingly used in the treatment of tuberculosis (TB) and are the second-line drugs of choice for treatment of multidrug-resistant TB. We aimed to set up a polymerase chain reaction (PCR) based assay to detect the most common FQ-resistance-associated mutations in gyrase A (gyrA) of Mycobacterium tuberculosis.

DESIGN: A total of 42 FQ-resistant and 40 FQ-susceptible isolates were collected in 2005–2006 and sequenced in gyrA. Using sequencing results as gold standard, a real-time PCR using three locked nucleic acid probes (LNA-PCR) was designed to detect mutations at positions 90, 91 and 94 (97% of gyrA FQ-resistance-associated mutations) and evaluated.

RESULTS: Sequencing of 42 FQ-resistant isolates revealed no gyrA mutations in 10 isolates, 20 isolates had a single mutation and 12 isolates showed double peaks at resistance-associated alleles, suggesting a heterogeneous population. With LNA-PCR, all wild-type and 19/20 mutant isolates were correctly identified. Eleven of 12 heterogeneous isolates were correctly identified as resistant mutants. Overall, 71% ([19 + 11]/42) of phenotypically FQ-resistant isolates were detected. Specificity was 100% on 40 FQ-susceptible isolates.

CONCLUSION: This assay provides a simple and rapid means to reliably detect FQ-resistance-associated gyrA mutations in M. tuberculosis.

Keywords: fluoroquinolones; gyrase; mycobacteria; real-time PCR; tuberculosis

Document Type: Regular Paper

Affiliations: 1: Department of Medical Microbiology, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands; Oxford University Clinical Research Unit, Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam 2: Oxford University Clinical Research Unit, Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam 3: Pham Ngoc Thach Hospital for Tuberculosis and Lung Disease, Ho Chi Minh City, Vietnam 4: Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam

Publication date: July 1, 2008

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  • The International Journal of Tuberculosis and Lung Disease publishes articles on all aspects of lung health, including public health-related issues such as training programmes, cost-benefit analysis, legislation, epidemiology, intervention studies and health systems research. The IJTLD is dedicated to the continuing education of physicians and health personnel and the dissemination of information on tuberculosis and lung health world-wide.

    Certain IJTLD articles are selected for translation into French, Spanish, Chinese or Russian. They are available on the Union website

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