Drug discovery and resistanceTuberculosis ethambutol resistance: Concordance between phenotypic and genotypic test results
Introduction
The increasing numbers of drug resistant and multidrug resistant tuberculosis (MDR-TB, resistance at least to isoniazid [INH] and rifampicin [RMP]) strains observed in several parts of the world represent a serious challenge for global tuberculosis control.1, 2 In its recent report, the Global Project on Antituberculosis Drug Resistance documented a prevalence of MDR-TB among new cases of tuberculosis ranging from 0% to 22,3% in 81 countries surveyed from years 2002–2007.2 However, in some areas of the world such as Karakalpakstan (Uzbekistan) MDR-TB rates have reached much more significant levels of up to 14% among patients never treated and up to 40% among previously treated patients.2, 3
These data underline the importance of the rapid detection of drug resistance in clinical isolates in order to initiate effective therapy that avoids the creation of further resistance and also prevents the spread of drug resistant isolates. In addition to more rapid methods for conventional drug susceptibility testing such as the BACTEC MGIT 960 system,4 molecular assays (e.g. based on DNA sequencing, Real Time PCR, or stripe technologies) for the detection of mutations conferring resistance have been increasingly used and have the potential to shorten the time to detection of resistance to one working day.5, 6, 7
However, while molecular mechanisms involved in resistance to INH and RMP have been identified with clear cut associations between mutations in particular genes such as katG and rpoB with phenotypic resistance to INH and RMP, respectively,8 associations between mutations and phenotypic ethambutol (EMB) resistance are less clear. Ethambutol is a key component of the first-line antituberculosis treatment regimen, and is also an important drug to be included in second-line regimens for MDR-TB, where susceptibility can be demonstrated.
Mutations in the embCAB operon (mainly the embB gene) have been most commonly associated with EMB resistance with variations of codon embB306 as a hot spot for mutations. Furthermore, mutations have been described e.g. in embB406 and embB497. In a recent study, Safi et al. demonstrated that transferring embB306 point mutations into EMB susceptible clinical Mycobacterium tuberculosis strain 210 increased the EMB MIC.9 However, if clinical isolates are considered, the percentage of EMB resistant strains which show mutations in embB306 ranges from 30 to 68%.10, 11 Additionally, a number of recent investigations described the occurrence of embB306 mutations in EMB susceptible strains,12, 13, 14 and several studies reporting an association with broad drug resistance rather than with EMB resistance alone.15, 16 These contradictory results cast doubt on the potential use of embB306 mutations as molecular markers for predicting EMB resistance in clinical isolates.
To address this important question, we investigated the correlation between embB306 mutations and phenotypic EMB resistance in a population based study. Therefore, strains from a cross sectional survey performed in Karakalpakstan, Uzbekistan, a setting with high TB incidence and a high level of drug resistance,3 were investigated for sequence variations at embB codon 306 and phenotypic EMB resistance. Special attention was drawn to the analysis of strains with discrepant results that were re-tested and screened for mixed populations of mutants and wild type.
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Study setting
The Autonomous Republic of Karakalpakstan is located in the west of Uzbekistan, south of the Aral Sea. Karakalpakstan has a high TB burden with a total case notification rate of 482/100,000 in 2001.3 A cross-sectional drug resistance survey conducted in 2001/2002 found MDR-TB infection among 13% of new cases and 40% of previously treated patients.3 TB isolates from this survey were used in the current study, with recruitment as previously described.3
Strain cultivation and drug susceptibility testing
Primary isolation and cultivation of
Results and discussion
A total of 197 strains were investigated for mutations in embB codon 306 by direct sequencing of PCR fragments. Details of drug resistance rates and M. tuberculosis population structure have been presented in our previous publications.3, 20
Briefly, 124 of the 197 (62.9%) strains were resistant to at least one first-line antituberculosis drug tested (INH, RMP, EMB, PZA, and SM) and 50 (25%) were MDR-TB. Analysis of IS6110 DNA fingerprint and spoligotyping data showed that 107 strains (54.3%)
Acknowledgments
The authors like to thank B. Schlüter, I. Radzio, T. Ubben, L. Dost, and P. Vock for excellent technical assistance and both national and expatriate staff of Médecins Sans Frontières in the field. We thank Jonny Polonsky and Clair Mills for their helpful comments on earlier drafts of the manuscript.
References (22)
- et al.
Frequency of embB codon 306 mutations in ethambutol-susceptible and -resistant clinical Mycobacterium tuberculosis isolates in Kuwait
Tuberculosis (Edinb)
(2007) Characterisation of rpsL, rrs and embB mutations associated with streptomycin and ethambutol resistance in Mycobacterium tuberculosis
Res Microbiol
(2004)Anti-tuberculosis drug resistance in the world: third global report
(2004)Anti-tuberculosis drug resistance in the world. report number 4
(2008)Multidrug-resistant tuberculosis in central Asia
Emerg Infect Dis
(2004)- et al.
Multicenter evaluation of fully automated BACTEC Mycobacteria Growth Indicator Tube 960 system for susceptibility testing of Mycobacterium tuberculosis
J Clin Microbiol
(2002) - et al.
Use of the genotype MTBDR assay for rapid detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis complex isolates
J Clin Microbiol
(2005) - et al.
Laboratory diagnostic aspects of drug resistant tuberculosis
Front Biosci
(2004) Evaluation of molecular-Beacon, TaqMan, and fluorescence resonance energy transfer probes for detection of antibiotic resistance-conferring single nucleotide polymorphisms in mixed Mycobacterium tuberculosis DNA extracts
J Clin Microbiol
(2006)- et al.
Transfer of embB codon 306 mutations into clinical Mycobacterium tuberculosis strains alters susceptibility to ethambutol, isoniazid, and rifampin
Antimicrob Agents Chemother
Cited by (30)
Genetics and roadblocks of drug resistant tuberculosis
2019, Infection, Genetics and EvolutionCitation Excerpt :On the other hand, M306V/L mutations appear to confer a higher level of EMB resistance than M306I (Campbell et al., 2011; Giri et al., 2018; Ramaswamy et al., 2000; Sreevatsan et al., 1997b; Sun et al., 2018). Yet, the molecular basis of EMB resistance is not straightforwardly determined as, over a decade ago, it has been reported that embB306 mutations occasionally fail to deliver a phenotypic resistance level that overcomes the critical concentration used in standardized drug susceptibility testing, with several authors reporting EMB susceptible isolates bearing embB306 mutations whereas others only detected embB306 mutations among EMB resistant isolates (Cambau et al., 2015; Campbell et al., 2011; Johnson et al., 2006; Mokrousov et al., 2002; Plinke et al., 2009; Plinke et al., 2006; Shen et al., 2007). As a result, a high degree of uncertainty fell upon embB306 mutations as a molecular determinant of EMB resistance and its eventual utility for rapid molecular testing.
Drug resistance in mycobacterium tuberculosis: Molecular mechanisms challenging fluoroquinolones and pyrazinamide effectiveness
2015, ChestCitation Excerpt :Nevertheless, the continuously evolving next-generation sequencing technology will provide novel tools for diagnostic purposes, as demonstrated by an increasing number of studies.76–78 The use of molecular assays led to an increasing number of studies highlighting discrepancies between conventional and genotypic DST for different drugs, including (but not limited to) first-line antibiotics.79–84 Three core elements are important to understand the individual mutation's contribution to the drug-resistance phenotype in MTB: (1) different mutations of the same genomic region can lead to different MIC values, (2) same mutations can cause variable levels of resistance to different members of the same drug class, and (3) same specific mutations can lead to different MIC levels in different strains, suggesting a role for the genetic background.85
Simple and rapid discrimination of embB codon 306 mutations in Mycobacterium tuberculosis clinical isolates by a real-time PCR assay using an LNA-TaqMan probe
2013, Journal of Microbiological MethodsCitation Excerpt :Among the mutations, the nucleotide substitutions at position 306 Met codon (ATG) of the embB gene most commonly occur in 50–70% of EMB-resistant isolates (Ahmad et al., 2004; Plinke et al., 2006; Sreevatsan et al., 1997). Different mutations have been found in this codon resulting in four amino acid shifts (Met to Ile, Leu, Val, or Thr) with seven different SNPs [ATG to AT(A/T/C), (C/T)TG, GTG, or ACG, respectively] (Lee et al., 2002; Plinke et al., 2009; Sreevatsan et al., 1997). Various molecular methods have been applied to detect M. tuberculosis embB gene mutations, including PCR-single-stranded conformation polymorphism assay, multiplex allele-specific PCR assay, PCR-restriction fragment length polymorphism analysis, PCR-based dot-blot hybridization, molecular beacon assay, and direct sequencing assay (Ahmad et al., 2004; Chan et al., 2007; Hazbón et al., 2005; Mokrousov et al., 2002a; Rinder et al., 2001; Ramaswamy et al., 2000; Victor et al., 1999).
Mutations and insights into the molecular mechanisms of resistance of Mycobacterium tuberculosis to first-line drugs
2023, Genetics and Molecular BiologyAntibiotic heteroresistance in Mycobacterium tuberculosis isolates: a systematic review and meta-analysis
2021, Annals of Clinical Microbiology and Antimicrobials
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These authors have contributed equally to this study.