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J Clin Microbiol. 2015 Jan;53(1):43-51. doi: 10.1128/JCM.02059-14. Epub 2014 Oct 22.

Genotypic susceptibility testing of Mycobacterium tuberculosis isolates for amikacin and kanamycin resistance by use of a rapid sloppy molecular beacon-based assay identifies more cases of low-level drug resistance than phenotypic Lowenstein-Jensen testing.

Author information

1
Department of Medicine, New Jersey Medical School, Rutgers University, Newark, New Jersey, USA chakraso@njms.rutgers.edu.
2
Department of Microbiology, International Tuberculosis Research Center, Changwon, Gyeongsang, Republic of Korea.
3
Department of Medicine, New Jersey Medical School, Rutgers University, Newark, New Jersey, USA.
4
National Masan Tuberculosis Hospital, Changwon, Republic of Korea.
5
Tuberculosis Research Section, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland, USA.
6
Department of Microbiology, International Tuberculosis Research Center, Changwon, Gyeongsang, Republic of Korea Department of Microbiology and Institute of Immunology and Immunological Disease, Yonsei University, College of Medicine, Seoul, Republic of Korea.

Abstract

Resistance to amikacin (AMK) and kanamycin (KAN) in clinical Mycobacterium tuberculosis strains is largely determined by specific mutations in the rrs gene and eis gene promoter. We developed a rapid, multiplexed sloppy molecular beacon (SMB) assay to identify these mutations and then evaluated assay performance on 603 clinical M. tuberculosis DNA samples collected in South Korea. Assay performance was compared to gold-standard phenotypic drug susceptibility tests, including Lowenstein-Jensen (LJ) absolute concentration, mycobacterial growth indicator tubes (MGIT), and TREK Sensititre MycoTB MIC plate (MycoTB) methods. Target amplicons were also tested for mutations by Sanger sequencing. The SMB assay correctly detected 115/116 mutant and mixed sequences and 487/487 wild-type sequences (sensitivity and specificity of 99.1 and 100%, respectively). Using the LJ method as the reference, sensitivity and specificity for AMK resistance were 92.2% and 100%, respectively, and sensitivity and specificity for KAN resistance were 87.7% and 95.6%, respectively. Mutations in the rrs gene were unequivocally associated with high-level cross-resistance to AMK and KAN in all three conventional drug susceptibility testing methods. However, eis promoter mutations were associated with KAN resistance using the MGIT or MycoTB methods but not the LJ method. No testing method associated eis promoter mutations with AMK resistance. Among the discordant samples with AMK and/or KAN resistance but wild-type sequence at the target genes, we discovered four new mutations in the whiB7 5' untranslated region (UTR) in 6/22 samples. All six samples were resistant only to KAN, suggesting the possible role of these whiB7 5' UTR mutations in KAN resistance.

PMID:
25339395
PMCID:
PMC4290905
DOI:
10.1128/JCM.02059-14
[Indexed for MEDLINE]
Free PMC Article

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