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

Soumitesh Chakravorty, Jong Seok Lee, Eun Jin Cho, Sandy S. Roh, Laura E. Smith, Jiim Lee, Cheon Tae Kim, Laura E. Via, Sangnae Cho, Clifton E. Barry, David Alland

Research output: Contribution to journalArticle

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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.

Original languageEnglish
Pages (from-to)43-51
Number of pages9
JournalJournal of Clinical Microbiology
Volume53
Issue number1
DOIs
Publication statusPublished - 2015 Jan 1

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Kanamycin Resistance
Amikacin
Mycobacterium tuberculosis
Drug Resistance
Mutation
Kanamycin
5' Untranslated Regions
Sensitivity and Specificity
Genes
Republic of Korea
Growth
Gold
Pharmaceutical Preparations
DNA

All Science Journal Classification (ASJC) codes

  • Microbiology (medical)

Cite this

Chakravorty, Soumitesh ; Lee, Jong Seok ; Cho, Eun Jin ; Roh, Sandy S. ; Smith, Laura E. ; Lee, Jiim ; Kim, Cheon Tae ; Via, Laura E. ; Cho, Sangnae ; Barry, Clifton E. ; Alland, David. / 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. In: Journal of Clinical Microbiology. 2015 ; Vol. 53, No. 1. pp. 43-51.
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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.",
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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. / Chakravorty, Soumitesh; Lee, Jong Seok; Cho, Eun Jin; Roh, Sandy S.; Smith, Laura E.; Lee, Jiim; Kim, Cheon Tae; Via, Laura E.; Cho, Sangnae; Barry, Clifton E.; Alland, David.

In: Journal of Clinical Microbiology, Vol. 53, No. 1, 01.01.2015, p. 43-51.

Research output: Contribution to journalArticle

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T1 - 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

AU - Chakravorty, Soumitesh

AU - Lee, Jong Seok

AU - Cho, Eun Jin

AU - Roh, Sandy S.

AU - Smith, Laura E.

AU - Lee, Jiim

AU - Kim, Cheon Tae

AU - Via, Laura E.

AU - Cho, Sangnae

AU - Barry, Clifton E.

AU - Alland, David

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N2 - 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.

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