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.
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These authors have contributed equally to this study.