Evaluation of Genetic Mutations Associated with <em>Mycobacterium tuberculosis</em> Resistance to Amikacin, Kanamycin and Capreomycin: A Systematic Review

<div><h3>Background</h3><p>Rapid molecular diagnostics for detecting multidrug-resistant and extensively drug-resistant tuberculosis (M/XDR-TB) primarily identify mutations in <em>Mycobacterium tuberculosis</em> (<em>Mtb</em>) genes associated with drug resistance. Their accuracy, however, is dependent largely on the strength of the association between a specific mutation and the phenotypic resistance of the isolate with that mutation, which is not always 100%. While this relationship is well established and reliable for first-line anti-TB drugs, rifampin and isoniazid, it is less well-studied and understood for second-line, injectable drugs, amikacin (AMK), kanamycin (KAN) and capreomycin (CAP).</p> <h3>Methodology/Principal Findings</h3><p>We conducted a systematic review of all published studies evaluating <em>Mtb</em> mutations associated with resistance to AMK, KAN, CAP in order to characterize the diversity and frequency of mutations as well as describe the strength of the association between specific mutations and phenotypic resistance in global populations. Our objective was to determine the potential utility and reliability of these mutations as diagnostic markers for detecting AMK, KAN and CAP resistance. Mutation data was reviewed for 1,585 unique clinical isolates from four continents and over 18 countries. Mutations in the <em>rrs</em>, <em>tlyA</em>, <em>eis</em> promoter and <em>gidB</em> genes were associated with AMK, KAN and/or CAP resistance.</p> <h3>Conclusions/Significance</h3><p>The <em>rrs</em> A1401G mutation was present in the majority of AMK, KAN and CAP resistant <em>Mtb</em> strains reviewed, but was also found in 7% of CAP susceptible strains. The 1401 mutation alone, however, was not found with sufficient frequency to detect more than 70–80% of global <em>Mtb</em> strains resistant to AMK and CAP, and 60% of strains resistant to KAN. Additional mutations in the <em>rrs</em>, <em>eis</em> promoter, <em>tlyA</em> and <em>gidB</em> genes appear to be associated with resistance and could improve sensitivity and specificity of future diagnostics.</p> </div

Details of Studies Included in Review and Source of <i>Mycobactrium tuberculosis</i> Isolates.

<p> <b>CDC = Center for Disease Control and Prevention, Atlanta.</b></p

Heatmap of Reviewed Studies that Evaluated <i>rrs</i> Gene Mutations in <i>Mycobacterium tuberculosis</i> isolates.

<p>Graphic shows the region of the <i>rrs</i> gene studied, the number of isolates tested in each study and the locations of the mutations found. The X-axis (nucleotide position) has a 25 base pair resolution. The numbers of isolates varies from 314 (black) to 10 (lightest grey). Red indicates that a mutation has been found in that 25 base pair region.</p

Study Selection Process and Reason for Exclusion of Studies.

<p>Study Selection Process and Reason for Exclusion of Studies.</p

Cumulative Frequencies of Selected Mutations within the <i>tlyA</i> Gene among <i>Mycobacterium tuberculosis</i> Isolates Resistant or Susceptible to Amikacin (AMK), Kanamycin (KAN) and/or Capreomycin (CAP).

*<p>Represents an amino acid change, as specific nucleotide changes were not provided for this mutation.</p><p>R = Resistant isolates.</p><p>S = Susceptible isolates.</p

Cumulative Frequencies of Multiple Mutations within the <i>rrs</i> Gene among <i>Mycobacterium tuberculosis</i> Isolates Resistant or Susceptible to Amikacin (AMK), Kanamycin (KAN) and/or Capreomycin (CAP).

<p>R = Resistant isolates.</p><p>S = Susceptible isolates.</p

Illustration of universal tail approach.

<p>In a multiplex PCR, all <i>M</i>. <i>tuberculosis</i> gene specific targets are amplified with universal tailed primers. A second PCR extends the amplicon with a sample specific index that allows for several samples to be pooled together for sequencing. GS: gene specific primer; UT: universal tail sequence; Index A: Primer that includes the UT1 sequence and Illumina index sequence; Index. B: primer that includes the UT2 sequence and the Illumina sequence to bind to the flowcell.</p

Comparison of standard sequencing and SMOR analysis.

<p>The percentage of base calls for the resistant allele compared to the erroneous alleles at six resistant SNP loci in five different genes, five replicates each, is shown. All 36 SNP loci were examined; however most SNP loci are consistent between the resistant and susceptible strains used in the mixtures. The seven different mixtures contain six known allelic differences in resistant conferring loci. Each circle represents the percent of calls for a particular allele for each replicate and the color represents the type of allele. The pure control was the pan susceptible isolate DNA 2–0112. A. Results from standard sequencing analysis, ignoring read pair information. B. Results from SMOR analysis. For the means and standard deviations see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126626#pone.0126626.s009" target="_blank">S6 Table</a>.</p

SMOR minor subpopulation examination of a single replicate of 7 mixtures at six known differing resistance SNP loci.

<p>Each circle represents the percent SMOR call, where color represents allele state, for a single sample at the six resistant SNP loci. All 36 SNP loci were examined; however the seven different mixtures contain six known allelic differences in resistant conferring SNP loci.</p

Minor subpopulation detection in two sputum samples from Moldova.

<p>Resistant and erroneous allele frequencies from three resistance SNP loci in the <i>inhA</i> promoter are shown. Patient 21–0067 with 0.05% resistant allele and patient 22–0129 with 11.39% resistant allele at <i>inhA</i> -15, compared to erroneous and resistant alleles below 0.01% at the other two SNP positions.</p