Fluoroquinolone resistance detection in Mycobacterium tuberculosis with locked nucleic acid probe real-time PCR.
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 polymer...
| Main Authors: | , , , , , , , , , , , |
|---|---|
| Format: | Journal article |
| Language: | English |
| Published: |
2008
|
| _version_ | 1797078466056683520 |
|---|---|
| author | Van Doorn, H An, D Jong, d Lan, N Hoa, D Quy, H Chau, N Duy, P Tho, D Chinh, N Farrar, J Caws, M |
| author_facet | Van Doorn, H An, D Jong, d Lan, N Hoa, D Quy, H Chau, N Duy, P Tho, D Chinh, N Farrar, J Caws, M |
| author_sort | Van Doorn, H |
| collection | OXFORD |
| description | 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. |
| first_indexed | 2024-03-07T00:32:14Z |
| format | Journal article |
| id | oxford-uuid:8037753d-71e4-4eb2-8393-f62a31bd1f07 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T00:32:14Z |
| publishDate | 2008 |
| record_format | dspace |
| spelling | oxford-uuid:8037753d-71e4-4eb2-8393-f62a31bd1f072022-03-26T21:21:50ZFluoroquinolone resistance detection in Mycobacterium tuberculosis with locked nucleic acid probe real-time PCR.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:8037753d-71e4-4eb2-8393-f62a31bd1f07EnglishSymplectic Elements at Oxford2008Van Doorn, HAn, DJong, dLan, NHoa, DQuy, HChau, NDuy, PTho, DChinh, NFarrar, JCaws, MSETTING: 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. |
| spellingShingle | Van Doorn, H An, D Jong, d Lan, N Hoa, D Quy, H Chau, N Duy, P Tho, D Chinh, N Farrar, J Caws, M Fluoroquinolone resistance detection in Mycobacterium tuberculosis with locked nucleic acid probe real-time PCR. |
| title | Fluoroquinolone resistance detection in Mycobacterium tuberculosis with locked nucleic acid probe real-time PCR. |
| title_full | Fluoroquinolone resistance detection in Mycobacterium tuberculosis with locked nucleic acid probe real-time PCR. |
| title_fullStr | Fluoroquinolone resistance detection in Mycobacterium tuberculosis with locked nucleic acid probe real-time PCR. |
| title_full_unstemmed | Fluoroquinolone resistance detection in Mycobacterium tuberculosis with locked nucleic acid probe real-time PCR. |
| title_short | Fluoroquinolone resistance detection in Mycobacterium tuberculosis with locked nucleic acid probe real-time PCR. |
| title_sort | fluoroquinolone resistance detection in mycobacterium tuberculosis with locked nucleic acid probe real time pcr |
| work_keys_str_mv | AT vandoornh fluoroquinoloneresistancedetectioninmycobacteriumtuberculosiswithlockednucleicacidproberealtimepcr AT and fluoroquinoloneresistancedetectioninmycobacteriumtuberculosiswithlockednucleicacidproberealtimepcr AT jongd fluoroquinoloneresistancedetectioninmycobacteriumtuberculosiswithlockednucleicacidproberealtimepcr AT lann fluoroquinoloneresistancedetectioninmycobacteriumtuberculosiswithlockednucleicacidproberealtimepcr AT hoad fluoroquinoloneresistancedetectioninmycobacteriumtuberculosiswithlockednucleicacidproberealtimepcr AT quyh fluoroquinoloneresistancedetectioninmycobacteriumtuberculosiswithlockednucleicacidproberealtimepcr AT chaun fluoroquinoloneresistancedetectioninmycobacteriumtuberculosiswithlockednucleicacidproberealtimepcr AT duyp fluoroquinoloneresistancedetectioninmycobacteriumtuberculosiswithlockednucleicacidproberealtimepcr AT thod fluoroquinoloneresistancedetectioninmycobacteriumtuberculosiswithlockednucleicacidproberealtimepcr AT chinhn fluoroquinoloneresistancedetectioninmycobacteriumtuberculosiswithlockednucleicacidproberealtimepcr AT farrarj fluoroquinoloneresistancedetectioninmycobacteriumtuberculosiswithlockednucleicacidproberealtimepcr AT cawsm fluoroquinoloneresistancedetectioninmycobacteriumtuberculosiswithlockednucleicacidproberealtimepcr |