Unexpected high prevalence of resistance-associated Rv0678 variants in MDR-TB patients without documented prior use of clofazimine or bedaquiline.

YesObjectives: Resistance-associated variants (RAVs) in Rv0678, a regulator of the MmpS5-MmpL5 efflux pump, have been shown to lead to increased MICs of bedaquiline (2- to 8- fold) and clofazimine (2- to 4-fold). The prevalence of these Rv0678 RAVs in clinical isolates and their impact on treatment outcomes are important factors to take into account in bedaquiline treatment guidelines. Methods: Baseline isolates from two bedaquiline MDR-TB clinical trials were sequenced for Rv0678 RAVs and corresponding bedaquiline MICs were determined on 7H11 agar. Rv0678 RAVs were also investigated in non-MDRTB sequences of a population-based cohort. Results: Rv0678 RAVs were identified in 23/347 (6.3%) of MDR-TB baseline isolates. Surprisingly, bedaquiline MICs for these isolates were high (>0.24 mg/L, n¼8), normal (0.03 0.24 mg/L, n¼11) or low(<0.03 mg/L, n¼4). A variant at position 11 in the intergenic region mmpS5–Rv0678 was identified in 39 isolates (11.3%) and appeared to increase the susceptibility to bedaquiline. In non-MDR-TB isolates, the frequency of Rv0678 RAVs was lower (6/ 852 or 0.7%). Competition experiments suggested that rifampicin was not the drug selecting for Rv0678 RAVs. Conclusions: RAVs in Rv0678 occur more frequently in MDR-TB patients than previously anticipated, are not associated with prior use of bedaquiline or clofazimine, and in the majority of cases do not lead to bedaquiline MICs above the provisional breakpoint (0.24mg/L). Their origin remains unknown. Given the variety of RAVs in Rv0678 and their variable effects on the MIC, only phenotypic drug-susceptibility methods can currently be used to assess bedaquiline susceptibility.This work was supported by Janssen Pharmaceutica. N. C. was supported by a Baekeland PhD scholarship from the Flemish Institute for Scientific Technology (IWT 130308, Belgium). C. J. M., L. R. and B. d. J. were supported by a European Research Council Starting Grant INTERRUPTB (311725)

Mycobacterium tuberculosis whole genome sequencing and protein structure modelling provides insights into anti-tuberculosis drug resistance.

BACKGROUND: Combating the spread of drug resistant tuberculosis is a global health priority. Whole genome association studies are being applied to identify genetic determinants of resistance to anti-tuberculosis drugs. Protein structure and interaction modelling are used to understand the functional effects of putative mutations and provide insight into the molecular mechanisms leading to resistance. METHODS: To investigate the potential utility of these approaches, we analysed the genomes of 144 Mycobacterium tuberculosis clinical isolates from The Special Programme for Research and Training in Tropical Diseases (TDR) collection sourced from 20 countries in four continents. A genome-wide approach was applied to 127 isolates to identify polymorphisms associated with minimum inhibitory concentrations for first-line anti-tuberculosis drugs. In addition, the effect of identified candidate mutations on protein stability and interactions was assessed quantitatively with well-established computational methods. RESULTS: The analysis revealed that mutations in the genes rpoB (rifampicin), katG (isoniazid), inhA-promoter (isoniazid), rpsL (streptomycin) and embB (ethambutol) were responsible for the majority of resistance observed. A subset of the mutations identified in rpoB and katG were predicted to affect protein stability. Further, a strong direct correlation was observed between the minimum inhibitory concentration values and the distance of the mutated residues in the three-dimensional structures of rpoB and katG to their respective drugs binding sites. CONCLUSIONS: Using the TDR resource, we demonstrate the usefulness of whole genome association and convergent evolution approaches to detect known and potentially novel mutations associated with drug resistance. Further, protein structural modelling could provide a means of predicting the impact of polymorphisms on drug efficacy in the absence of phenotypic data. These approaches could ultimately lead to novel resistance mutations to improve the design of tuberculosis control measures, such as diagnostics, and inform patient management

Cross-cultural Psychometric Evaluation of the Dutch McGill-QoL Questionnaire for Breast Cancer Patients

AIM OF THE STUDY: Assessing the cross-cultural psychometric properties of the Dutch-MQoL for breast cancer patients. METHODS: 26 patients were recruited at the Antwerp University Hospital. Eligible patients filled in the MQoL on different moments in time in order to evaluate clinimetric properties. To determine the validity; MQoL was correlated to the EORTC QLQ-C30. Internal consistency was analysed using Cronbach's and test-retest reliability was determined by ICC. For statistical responsiveness, S.E.M and MDC were calculated. RESULTS: A strong correlation was found between the 'QoL score' of the MQoL and the domain 'existential well- being' of the EORTC QLQ-C30 (r = 0.72). An excellent test-retest reliability (ICC (1,1)) was demonstrated with intraclass coefficients ranging from 0.82 to 0.92. A MDC in total score of only 1.22 (12%) was seen, needed to detect a factual change within a patients' QoL. Psychometric properties of the Dutch MQoL were found comparable to the properties of the original questionnaire. CONCLUSION: The Dutch version of the MQoL is a valid and reliable questionnaire for breast cancer patients and shows statistical responsiveness. Due to the strong to excellent reliability, this version of the MQoL is useful in clinical as well as scientific setting.status: publishe

Correlation of different phenotypic drug susceptibility testing methods for four fluoroquinolones in Mycobacterium tuberculosis

BACKGROUND: Molecular resistance testing fails to explain all fluoroquinolone resistance, with a continued need for a suitable rapid phenotypic drug susceptibility testing method. OBJECTIVE: To evaluate the optimal method for phenotypic fluoroquinolone susceptibility testing. METHODS: Using Löwenstein–Jensen medium, Middlebrook 7H11 agar, BACTEC-MGIT 960 and the resazurin microtitre plate assay, we determined susceptibility to fluoroquinolones in Mycobacterium tuberculosis and investigated cross-resistance between ofloxacin, levofloxacin, moxifloxacin and gatifloxacin. We compared MICs of all four fluoroquinolones for 91 strains on Löwenstein–Jensen (as the gold standard) with their MICs in resazurin plates, and with ofloxacin susceptibility at a single concentration in MGIT and on 7H11 agar, in addition to sequencing of the gyrAB genes. RESULTS AND CONCLUSIONS: Applying a cut-off of 2 mg/L ofloxacin, 1 mg/L levofloxacin and 0.5 mg/L moxifloxacin and gatifloxacin in all methods, some discordance between solid medium and MGIT methods was observed, yet this tended to be explained by MICs around the cut-off. The high discordance between Löwenstein–Jensen (LJ) and resazurin plates suggests that the currently applied cut-offs for all fluoroquinolones in the resazurin method should decrease and minor changes in colour (from blue to purple) be considered as meaningful. High-level resistance in all assays to all drugs correlated well with the presence of gyrA mutations, in support of recent findings that fluoroquinolone resistance should be tested at different concentrations, as patients with lower levels of resistance may continue to benefit from high-dose fluoroquinolone-based therapy

Acquired Resistance of <i>Mycobacterium tuberculosis</i> to Bedaquiline

<div><p>Bedaquiline (BDQ), an ATP synthase inhibitor, is the first drug to be approved for treatment of multi-drug resistant tuberculosis in decades. <i>In vitro</i> resistance to BDQ was previously shown to be due to target-based mutations. Here we report that non-target based resistance to BDQ, and cross-resistance to clofazimine (CFZ), is due to mutations in Rv0678, a transcriptional repressor of the genes encoding the MmpS5-MmpL5 efflux pump. Efflux-based resistance was identified in paired isolates from patients treated with BDQ, as well as in mice, in which it was confirmed to decrease bactericidal efficacy. The efflux inhibitors verapamil and reserpine decreased the minimum inhibitory concentrations of BDQ and CFZ <i>in vitro</i>, but verapamil failed to increase the bactericidal effect of BDQ in mice and was unable to reverse efflux-based resistance <i>in vivo</i>. Cross-resistance between BDQ and CFZ may have important clinical implications.</p></div

Regulation of efflux pumps proteins in EH3.6 and EH3.2.

<p>Efflux pump proteins were selected from the complete dataset of the proteome analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102135#pone.0102135.s003" target="_blank">Table S1</a>). The descriptions of the genes were retrieved from TubercuList (<a href="http://tuberculist.epfl.ch/" target="_blank">http://tuberculist.epfl.ch/</a>).</p

Mechanism of BDQ and CFZ resistance in <i>Rv0678</i> mutants.

<p><b>A.</b> Regulation of <i>mmpS5</i> and <i>mmpL5</i> transcription by wild type Rv0678 repressor. Rv0678 protein binds to the intergenic region located between <i>Rv0678</i> and <i>mmpS5</i>, which contains the-10 consensus boxes of promoters for both <i>Rv0678</i> and <i>mmpS5</i>. This prevents the RNA polymerase to start transcription, resulting in the decrease of expression of MmpS5, MmpL5 and Rv0678 proteins. In response to an unknown stimulus, the regulator detaches from DNA and transcription can be resumed. <b>B.</b> Lack of regulation in <i>Rv0678</i> mutants. The strains carrying frame shifting mutations in <i>Rv0678</i> will not produce a functional repressor, thus the transcription of <i>mmpS5, mmpL5</i> and <i>Rv0678</i> will be increased. If the mutation results in an amino acid polymorphism, the protein may still be functional, but with reduced DNA-binding ability depending on the location of the mutation. In either case, the final consequence will be an increase in the expression levels of the proteins MmpS5, MmpL5 and Rv0678.</p