Genetic Mutations Associated with Isoniazid Resistance in <i>Mycobacterium tuberculosis</i>: A Systematic Review

<div><p>Background</p><p>Tuberculosis (TB) incidence and mortality are declining worldwide; however, poor detection of drug-resistant disease threatens to reverse current progress toward global TB control. Multiple, rapid molecular diagnostic tests have recently been developed to detect genetic mutations in <i>Mycobacterium tuberculosis (Mtb)</i> genes known to confer first-line drug resistance. Their utility, though, depends on the frequency and distribution of the resistance associated mutations in the pathogen population. Mutations associated with rifampicin resistance, one of the two first-line drugs, are well understood and appear to occur in a single gene region in >95% of phenotypically resistant isolates. Mutations associated with isoniazid, the other first-line drug, are more complex and occur in multiple <i>Mtb</i> genes.</p><p>Objectives/Methodology</p><p>A systematic review of all published studies from January 2000 through August 2013 was conducted to quantify the frequency of the most common mutations associated with isoniazid resistance, to describe the frequency at which these mutations co-occur, and to identify the regional differences in the distribution of these mutations. Mutation data from 118 publications were extracted and analyzed for 11,411 <i>Mtb</i> isolates from 49 countries.</p><p>Principal Findings/Conclusions</p><p>Globally, 64% of all observed phenotypic isoniazid resistance was associated with the <i>kat</i>G315 mutation. The second most frequently observed mutation, <i>inhA</i>-15, was reported among 19% of phenotypically resistant isolates. These two mutations, <i>katG</i>315 and <i>inhA</i>-15, combined with ten of the most commonly occurring mutations in the <i>inhA</i> promoter and the <i>ahpC-oxyR</i> intergenic region explain 84% of global phenotypic isoniazid resistance. Regional variation in the frequency of individual mutations may limit the sensitivity of molecular diagnostic tests. Well-designed systematic surveys and whole genome sequencing are needed to identify mutation frequencies in geographic regions where rapid molecular tests are currently being deployed, providing a context for interpretation of test results and the opportunity for improving the next generation of diagnostics.</p></div

Heat map of Reviewed Studies that Evaluated <i>gyrA</i> Gene Mutations in <i>Mtb</i>.

<p>Heat map of individual papers indicating the number of isolates and the region of the <i>gyrA</i> gene studied. The number of isolates testes ranges from 8 (light grey) to 227 (black). Red indicates that a mutation has been found.</p

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

Frequency and Geographic Distribution of <i>gyrA</i> and <i>gyrB</i> Mutations Associated with Fluoroquinolone Resistance in Clinical <i>Mycobacterium Tuberculosis</i> Isolates: A Systematic Review

<div><p>Background</p><p>The detection of mutations in the <i>gyrA</i> and <i>gyrB</i> genes in the <i>Mycobacterium tuberculosis</i> genome that have been demonstrated to confer phenotypic resistance to fluoroquinolones is the most promising technology for rapid diagnosis of fluoroquinolone resistance.</p><p>Methods</p><p>In order to characterize the diversity and frequency of gyrA and gyrB mutations and to describe the global distribution of these mutations, we conducted a systematic review, from May 1996 to April 2013, of all published studies evaluating Mycobacterium tuberculosis mutations associated with resistance to fluoroquinolones. The overall goal of the study was to determine the potential utility and reliability of these mutations as diagnostic markers to detect phenotypic fluoroquinolone resistance in Mycobacterium tuberculosis and to describe their geographic distribution.</p><p>Results</p><p>Forty-six studies, covering four continents and 18 countries, provided mutation data for 3,846 unique clinical isolates with phenotypic resistance profiles to fluoroquinolones. The gyrA mutations occurring most frequently in fluoroquinolone-resistant isolates, ranged from 21–32% for D94G and 13–20% for A90V, by drug. Eighty seven percent of all strains that were phenotypically resistant to moxifloxacin and 83% of ofloxacin resistant isolates contained mutations in gyrA. Additionally we found that 83% and 80% of moxifloxacin and ofloxacin resistant strains respectively, were observed to have mutations in the gyrA codons interrogated by the existing MTBDR<i>sl</i> line probe assay. In China and Russia, 83% and 84% of fluoroquinolone resistant strains respectively, were observed to have gyrA mutations in the gene regions covered by the MTBDR<i>sl</i> assay.</p><p>Conclusions</p><p>Molecular diagnostics, specifically the Genotype MTBDR<i>sl</i> assay, focusing on codons 88–94 should have moderate to high sensitivity in most countries. While we did observe geographic differences in the frequencies of single gyrA mutations across countries, molecular diagnostics based on detection of all gyrA mutations demonstrated to confer resistance should have broad and global utility.</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

Additional file 1: Table S1. of Shedding light on the performance of a pyrosequencing assay for drug-resistant tuberculosis diagnosis

Pyrosequencing (PSQ) Success of Each Gene Target by Smear- and Culture-Status. Table S2. Proportion of Successful Pyrosequencing (PSQ) Reactions by Smear- and Culture-Status. (DOCX 18 kb