Danila V Zimenkov1, Elena V Kulagina2, Olga V Antonova2, Viacheslav Yu Zhuravlev3, Dmitry A Gryadunov2. 1. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russian Federation z@biochip.ru. 2. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russian Federation. 3. Research Institute of Phthisiopulmonology, St Petersburg, Russian Federation.
Abstract
BACKGROUND: Nucleic acid amplification tests are widely used in TB diagnostics. Priority tasks in their development consist of increasing the specificity and sensitivity of the detection of resistance to a wide spectrum of anti-TB drugs. METHODS: We developed a multiplexed assay allowing the detection of 116 drug resistance-determining mutations in the rpoB, katG, inhA, ahpC, gyrA, gyrB, rrs, eis and embB genes in the Mycobacterium tuberculosis complex genome and six SNPs to identify the main lineages circulating in Russia. The assay is based on the amplification of 17 fragments of the genome using the universal primer adapter technique and heat pulses at the elongation step, followed by hybridization on a microarray. RESULTS: The method was evaluated using 264 pairs of clinical samples and corresponding isolates. A significant proportion (25%) of smear-negative samples were correctly analysed by microarray analysis in addition to 96% of smear-positive samples. The sensitivity and specificity of the assay exceeded 90% for rifampicin, isoniazid, ofloxacin and second-line injection drugs. In agreement with previous studies, the specificity of ethambutol resistance was as low as 57%, while the sensitivity was 89.9%. Strong association of the Beijing lineage with a resistant phenotype was observed. Euro-American lineage strains, excluding Ural and LAM, were predominantly associated with the susceptible phenotype. CONCLUSIONS: The developed test has a high sensitivity and specificity and can be directly applied to clinical samples. The combination of mutation-based drug resistance profiling and basic genotyping could be useful for clinical microbiology studies and epidemiological surveillance of the M. tuberculosis complex.
BACKGROUND: Nucleic acid amplification tests are widely used in TB diagnostics. Priority tasks in their development consist of increasing the specificity and sensitivity of the detection of resistance to a wide spectrum of anti-TB drugs. METHODS: We developed a multiplexed assay allowing the detection of 116 drug resistance-determining mutations in the rpoB, katG, inhA, ahpC, gyrA, gyrB, rrs, eis and embB genes in the Mycobacterium tuberculosis complex genome and six SNPs to identify the main lineages circulating in Russia. The assay is based on the amplification of 17 fragments of the genome using the universal primer adapter technique and heat pulses at the elongation step, followed by hybridization on a microarray. RESULTS: The method was evaluated using 264 pairs of clinical samples and corresponding isolates. A significant proportion (25%) of smear-negative samples were correctly analysed by microarray analysis in addition to 96% of smear-positive samples. The sensitivity and specificity of the assay exceeded 90% for rifampicin, isoniazid, ofloxacin and second-line injection drugs. In agreement with previous studies, the specificity of ethambutol resistance was as low as 57%, while the sensitivity was 89.9%. Strong association of the Beijing lineage with a resistant phenotype was observed. Euro-American lineage strains, excluding Ural and LAM, were predominantly associated with the susceptible phenotype. CONCLUSIONS: The developed test has a high sensitivity and specificity and can be directly applied to clinical samples. The combination of mutation-based drug resistance profiling and basic genotyping could be useful for clinical microbiology studies and epidemiological surveillance of the M. tuberculosis complex.
Authors: Elena Y Nosova; Danila V Zimenkov; Anastasia A Khakhalina; Alexandra I Isakova; Ludmila Y Krylova; Marina V Makarova; Ksenia Y Galkina; Maria A Krasnova; Svetlana G Safonova; Vitaly I Litvinov; Dmitry A Gryadunov; Elena M Bogorodskaya Journal: PLoS One Date: 2016-11-30 Impact factor: 3.240
Authors: Ahsan Munir; Hassan Waseem; Maggie R Williams; Robert D Stedtfeld; Erdogan Gulari; James M Tiedje; Syed A Hashsham Journal: Microarrays (Basel) Date: 2017-05-29
Authors: Yvonne Linger; Christopher Knickerbocker; David Sipes; Julia Golova; Molly Franke; Roger Calderon; Leonid Lecca; Nitu Thakore; Rebecca Holmberg; Peter Qu; Alexander Kukhtin; Megan B Murray; Christopher G Cooney; Darrell P Chandler Journal: J Clin Microbiol Date: 2018-02-22 Impact factor: 5.948
Authors: Olga Smoldovskaya; Guzel Feyzkhanova; Sergei Voloshin; Alla Arefieva; Antonina Chubarova; Ludmila Pavlushkina; Tatiana Filatova; Eugenia Antonova; Elena Timofeeva; Veronika Butvilovskaya; Yuri Lysov; Alexander Zasedatelev; Alla Rubina Journal: World Allergy Organ J Date: 2018-12-03 Impact factor: 4.084