Pyrosequencing for rapid detection of Mycobacterium tuberculosis second-line drugs and ethambutol resistance.

The aim of this work was to study the diagnostic accuracy of pyrosequencing to detect resistance to fluoroquinolones, kanamycin, amikacin, capreomycin, and ethambutol (EMB) in Mycobacterium tuberculosis clinical strains. One hundred four clinical isolates previously characterized by BACTEC 460TB/MGIT 960 were included. Specific mutations were targeted in gyrA, rrs, eis promoter, and embB. When there was a discordant result between BACTEC and pyrosequencing, Genotype MTBDRsl (Hain Lifescience, Nehren, Germany) was performed. Sensitivity and specificity of pyrosequencing were 70.6% and 100%, respectively, for fluoroquinolones; 93.3% and 81.7%, respectively, for kanamycin; 94.1% and 95.9%, respectively, for amikacin; 90.0% and 100%, respectively, for capreomycin; and 64.8% and 87.8%, respectively, for EMB. This study shows that pyrosequencing may be a useful tool for making early decisions regarding second-line drugs and EMB resistance. However, for a correct management of patients with suspected extensively drug-resistant tuberculosis, susceptibility results obtained by molecular methods should be confirmed by a phenotypic method.

[1]  L. Heifets.  Drug Susceptibility Testing , 1996 .

[2]  V. Ausina,et al.  GenoType MTBDRsl for Molecular Detection of Second-Line-Drug and Ethambutol Resistance in Mycobacterium tuberculosis Strains and Clinical Samples , 2011, Journal of Clinical Microbiology.

[3]  S. Gharbia,et al.  Single-nucleotide polymorphism-based differentiation and drug resistance detection in Mycobacterium tuberculosis from isolates or directly from sputum. , 2005, Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases.

[4]  P. Juréen,et al.  Comparison of clinical isolates and in vitro selected mutants reveals that tlyA is not a sensitive genetic marker for capreomycin resistance in Mycobacterium tuberculosis. , 2011, The Journal of antimicrobial chemotherapy.

[5]  S. Siddiqi,et al.  Multicenter Evaluation of Bactec MGIT 960 System for Second-Line Drug Susceptibility Testing of Mycobacterium tuberculosis Complex , 2009, Journal of Clinical Microbiology.

[6]  P. Juréen,et al.  Detection of First- and Second-Line Drug Resistance in Mycobacterium tuberculosis Clinical Isolates by Pyrosequencing , 2012, Journal of Clinical Microbiology.

[7]  Yulia D Isaeva,et al.  Analysis of mutations in the gyrA and gyrB genes and their association with the resistance of Mycobacterium tuberculosis to levofloxacin, moxifloxacin and gatifloxacin. , 2013, Journal of medical microbiology.

[8]  A. Moroz,et al.  The role of eis mutations in the development of kanamycin resistance in Mycobacterium tuberculosis isolates from the Moscow region. , 2012, The Journal of antimicrobial chemotherapy.

[9]  Feifei Wang,et al.  Underestimation of the resistance of Mycobacterium tuberculosis to second-line drugs by the new GenoType MTBDRsl test. , 2013, The Journal of molecular diagnostics : JMD.

[10]  S. Niemann,et al.  Significance of Mutations in embB Codon 306 for Prediction of Ethambutol Resistance in Clinical Mycobacterium tuberculosis Isolates , 2006, Antimicrobial Agents and Chemotherapy.

[11]  B. Metchock,et al.  Overexpression of the chromosomally encoded aminoglycoside acetyltransferase eis confers kanamycin resistance in Mycobacterium tuberculosis , 2009, Proceedings of the National Academy of Sciences.

[12]  V. Ausina,et al.  Pyrosequencing for Rapid Molecular Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Strains and Clinical Specimens , 2011, Journal of Clinical Microbiology.

[13]  Q. Jin,et al.  Characteristics of embB mutations in multidrug-resistant Mycobacterium tuberculosis isolates in Henan, China , 2011, The Journal of antimicrobial chemotherapy.

[14]  Q. Gao,et al.  Prediction of XDR/pre-XDR tuberculosis by genetic mutations among MDR cases from a hospital in Shandong, China. , 2014, Tuberculosis.

[15]  R. Sultana,et al.  Aminoglycoside Cross-Resistance in Mycobacterium tuberculosis Due to Mutations in the 5′ Untranslated Region of whiB7 , 2013, Antimicrobial Agents and Chemotherapy.

[16]  E. Fedorova,et al.  Characterization of multiple and extensively drug resistant Mycobacterium tuberculosis isolates with different ofloxacin-resistance levels. , 2013, Tuberculosis.

[17]  Pan‐Chyr Yang,et al.  Fluoroquinolone resistance in Mycobacterium tuberculosis isolates: associated genetic mutations and relationship to antimicrobial exposure. , 2007, The Journal of antimicrobial chemotherapy.

[18]  Faramarz Valafar,et al.  Predicting Extensively Drug-Resistant Mycobacterium tuberculosis Phenotypes with Genetic Mutations , 2013, Journal of Clinical Microbiology.

[19]  Magali Cavatore,et al.  Role of embB Codon 306 Mutations in Mycobacterium tuberculosis Revisited: a Novel Association with Broad Drug Resistance and IS6110 Clustering Rather than Ethambutol Resistance , 2005, Antimicrobial Agents and Chemotherapy.

[20]  J. Musser,et al.  Molecular Genetic Analysis of Nucleotide Polymorphisms Associated with Ethambutol Resistance in Human Isolates ofMycobacterium tuberculosis , 2000, Antimicrobial Agents and Chemotherapy.

[21]  S. Niemann,et al.  A Pyrosequencing assay for rapid recognition of SNPs in Mycobacterium tuberculosis embB306 region. , 2005, Journal of microbiological methods.

[22]  Thomas M. Shinnick,et al.  Molecular Analysis of Cross-Resistance to Capreomycin, Kanamycin, Amikacin, and Viomycin in Mycobacterium tuberculosis , 2005, Antimicrobial Agents and Chemotherapy.

[23]  P. de Rijk,et al.  High Level of Cross-Resistance between Kanamycin, Amikacin, and Capreomycin among Mycobacterium tuberculosis Isolates from Georgia and a Close Relation with Mutations in the rrs Gene , 2009, Antimicrobial Agents and Chemotherapy.

[24]  R. Garfein,et al.  Evaluation of Genetic Mutations Associated with Mycobacterium tuberculosis Resistance to Amikacin, Kanamycin and Capreomycin: A Systematic Review , 2012, PloS one.

[25]  I. Mokrousov,et al.  Detection of embB306 Mutations in Ethambutol-Susceptible Clinical Isolates of Mycobacterium tuberculosis from Northwestern Russia: Implications for Genotypic Resistance Testing , 2002, Journal of Clinical Microbiology.

[26]  Ying Zhang,et al.  Mechanisms of drug resistance in Mycobacterium tuberculosis. , 2009, Frontiers in bioscience : a journal and virtual library.

[27]  V. Ausina,et al.  Diagnostic accuracy study of multiplex PCR for detecting tuberculosis drug resistance. , 2015, The Journal of infection.

[28]  V. Jarlier,et al.  Novel Gyrase Mutations in Quinolone-Resistant and -Hypersusceptible Clinical Isolates of Mycobacterium tuberculosis: Functional Analysis of Mutant Enzymes , 2006, Antimicrobial Agents and Chemotherapy.

[29]  A. Telenti,et al.  Cloning and nucleotide sequence of Mycobacterium tuberculosis gyrA and gyrB genes and detection of quinolone resistance mutations , 1994, Antimicrobial Agents and Chemotherapy.

[30]  T. Sterling,et al.  A systematic review of gyrase mutations associated with fluoroquinolone-resistant Mycobacterium tuberculosis and a proposed gyrase numbering system. , 2012, The Journal of antimicrobial chemotherapy.

[31]  V. Ausina,et al.  AID TB resistance line probe assay for rapid detection of resistant Mycobacterium tuberculosis in clinical samples. , 2015, The Journal of infection.

[32]  M. Hui,et al.  Multiplex PCR Amplimer Conformation Analysis for Rapid Detection of gyrA Mutations in Fluoroquinolone-Resistant Mycobacterium tuberculosis Clinical Isolates , 2004, Antimicrobial Agents and Chemotherapy.

[33]  R. Grant,et al.  Pharmacological considerations for tenofovir and emtricitabine to prevent HIV infection. , 2011, The Journal of antimicrobial chemotherapy.

[34]  S. Shenai,et al.  Drug susceptibility testing of Mycobacterium tuberculosis against second-line drugs using the Bactec MGIT 960 System. , 2008, The international journal of tuberculosis and lung disease : the official journal of the International Union against Tuberculosis and Lung Disease.

[35]  A. Lacoma,et al.  Recent Advances in Tuberculosis Diagnosis: IGRAs and Molecular Biology , 2014, Current Treatment Options in Infectious Diseases.

[36]  S. Rüsch-Gerdes,et al.  Drug-susceptibility testing in TB: current status and future prospects , 2009, Expert review of respiratory medicine.

[37]  Tracy L. Dalton,et al.  Molecular Detection of Mutations Associated with First- and Second-Line Drug Resistance Compared with Conventional Drug Susceptibility Testing of Mycobacterium tuberculosis , 2011, Antimicrobial Agents and Chemotherapy.

[38]  P. Juréen,et al.  Detection of Heteroresistant Mycobacterium tuberculosis by Pyrosequencing , 2013, Journal of Clinical Microbiology.

[39]  T. Whittam,et al.  Restricted structural gene polymorphism in the Mycobacterium tuberculosis complex indicates evolutionarily recent global dissemination. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[40]  P. V. van Helden,et al.  gyrA mutations and phenotypic susceptibility levels to ofloxacin and moxifloxacin in clinical isolates of Mycobacterium tuberculosis. , 2012, The Journal of antimicrobial chemotherapy.

[41]  A. Lacoma,et al.  GenoType MTBDRplus Assay for Molecular Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Strains and Clinical Samples , 2008, Journal of Clinical Microbiology.

[42]  J. Kornblum,et al.  Multicenter Laboratory Validation of Susceptibility Testing of Mycobacterium tuberculosis against Classical Second-Line and Newer Antimicrobial Drugs by Using the Radiometric BACTEC 460 Technique and the Proportion Method with Solid Media , 1999, Journal of Clinical Microbiology.

[43]  T. Victor,et al.  Pyrosequencing for Rapid Detection of Extensively Drug-Resistant Mycobacterium tuberculosis in Clinical Isolates and Clinical Specimens , 2013, Journal of Clinical Microbiology.

[44]  L. Heifets,et al.  Qualitative and quantitative drug-susceptibility tests in mycobacteriology. , 1988, The American review of respiratory disease.