Validation of Bedaquiline Phenotypic Drug Susceptibility Testing Methods and Breakpoints: a Multilaboratory, Multicountry Study

Kaniga, Koné; Ando, Akio; Borroni, Emanuele; Cirillo, Daniela María; Desmaretz, Christel; Hasan, Rumina; Joseph, Lavania; Mitarai, Satoshi; Shakoor, Sadia; Torrea, Gabriela; Ismail, Nazir; Omar, Shaheed V.

doi:10.1128/jcm.01677-19

Cited by 35 publications

(39 citation statements)

References 25 publications

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“…The ECOFFs determined in this study for both the frozen and dry BMDs matched those in the Ismail et al study based on ECOFFs of 95, 97.5, and 99% (17). Although the ECOFF 99% is preferred, the Kaniga et al study showed that using a well-characterized EQA panel, the ECOFF of 0.12 g/ml based upon 97.5% was the most robust concentration to clearly delineate wild-type from non-wild-type populations (22). In the present study, applying this cutoff yielded good overall performance for correctly classifying susceptibility.…”

Section: Discussionsupporting

confidence: 68%

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A Multimethod, Multicountry Evaluation of Breakpoints for Bedaquiline Resistance Determination

Ismail

Ando

Borroni

et al. 2020

Antimicrob Agents Chemother

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Criteria defining bedaquiline resistance for tuberculosis have been proposed addressing an emerging concern. We evaluated bedaquiline phenotypic drug susceptibility testing (pDST) criteria using drug-resistant tuberculosis clinical isolates tested at five reference laboratories. Isolates were tested at the proposed bedaquiline MGIT960 and 7H11 Agar Proportion (AP) critical concentrations, and dilutions higher. The epidemiological cut-off value for the broth microdilution (BMD) plates (frozen and dry) was investigated. Any isolate testing resistant had Sanger sequencing performed (atpE and Rv0678 genes). The composite reference standard (CRS) defined susceptibility or resistance as is, if all pDST methods agreed. If pDST was discordant sequencing results was used for final classification. Geographically-diverse and bedaquiline unexposed isolates were tested (N=495). The epidemiological cutoff value for BMD was confirmed to be 0.12 μg/mL. The majority of isolates were susceptible by all methods (467/495; 94.3%) and 28 were resistant by at least one method; of which 4 were resistant by all methods. 12/28 harboured Rv0678 mutations exclusively. Isolates with insertions/deletions were more likely to be resistant by more than one method (5/7) compared with isolates having a single nucleotide polymorphism (1/5). Applying the CRS to 24 discordant pDST, BMD dry correctly detected most (15/24; 63%), followed by MGIT960 and BMD frozen (13/24; 61%) and lastly AP (12/24; 50%). Applying the CRS, the prevalence of bedaquiline resistance was 2.2% and ranged from 1.4% to 3.4%, dependent on the method used. All methods performed well for bedaquiline susceptibility determination, however resistance detected should be investigated by a second alternative method.

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Section: Discussionsupporting

confidence: 68%

“…The WHO-proposed CC was based on multiple studies that lacked standardization and did not include BMD. An external quality assessment (EQA) study by Kaniga et al confirmed the appropriateness of the susceptible interpretive criterion by Ismail et al and WHO interim CCs using a set of well-characterized isolates (22).…”

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confidence: 77%

A Multimethod, Multicountry Evaluation of Breakpoints for Bedaquiline Resistance Determination

Ismail

Ando

Borroni

et al. 2020

Antimicrob Agents Chemother

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“…The minimum inhibitory concentration (MIC) of bedaquiline (Biochempartner, China) for all MTB isolates was determined by microplate alamarBlue assay using two-fold dilutions ranging from 0.008 mg/mL to 4 mg/mL, as described previously (Kaniga et al, 2020;Pang et al, 2017). Briefly, bacterial clones were harvested from the surface of LJ slants.…”

Section: Minimum Inhibitory Concentrationmentioning

confidence: 99%

Molecular characteristics and in vitro susceptibility to bedaquiline of Mycobacterium tuberculosis isolates circulating in Shaanxi, China

Yang

Zhang²,

Xian³

et al. 2020

International Journal of Infectious Diseases

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The aim of this study was to investigate the molecular characteristics and in vitro susceptibility to bedaquiline of Mycobacterium tuberculosis (MTB) isolates from Shaanxi, China. Methods: The minimum inhibitory concentration (MIC) of bedaquiline was determined using the microplate alamarBlue assay for 518 MTB isolates from Shaanxi. Isolates with MIC values of bedaquiline !0.12 mg/mL were sequenced for the atpE, Rv0678, and pepQ genes. Drug susceptibility testing and spoligotyping were also conducted for all strains. Results: Ten (1.93%) bedaquiline-resistant strains were isolated from 518 tuberculosis patients. The resistance rate of bedaquiline was not correlated to sex, age, treatment history, region, or genotype. Five bedaquiline-resistant isolates and one bedaquiline-susceptible isolate were found to carry Rv0678 mutations; six mutation types were identified, including G5T, A263G, C185T, G19deletion, C265T, and T323C. No mutations within the atpE and pepQ genes were observed. Conclusions: Bedaquiline showed strong in vitro antibacterial activity against MTB isolates, and the Rv0678 gene serves as the major mechanism contributing to bedaquiline resistance among MTB isolates from Shaanxi, China. Two novel mutation types (C265 T and T323 C) of the Rv0678 gene were associated with resistance to bedaquiline. Furthermore, in addition to the current three resistance-associated genes (atpE, Rv0678, and pepQ), other mechanisms of resistance to bedaquiline may exist that need further study.

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“…The detection of resistance to BDQ and DLM is critical to ensuring effective treatment and care for DR-TB patients and preventing ongoing transmission. Although evidence for the validation and standardization of efficient methods for MICs and the setting of breakpoints for BDQ and DLM continues to expand ( 22, 34, 50, 51 ), there is still a notable lack of suitable data on resistance-related genomic variants ( 52 ). Moreover, phenotypic methods are too slow to provide early indication of susceptibility status at the time of treatment initiation.…”

Section: Discussionmentioning

confidence: 99%

Characterization of genomic variants associated with resistance to bedaquiline and delamanid in naïve Mycobacterium tuberculosis clinical strains

Battaglia¹,

Spitaleri

Cabibbe³

et al. 2020

Preprint

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The role of genetic mutations in genes associated to phenotypic resistance to bedaquiline (BDQ) and delamanid (DLM) in Mycobacterium tuberculosis complex (MTBc) strains is poorly understood. However, a clear understanding of the role of each genetic variant is crucial to guide the development of molecular-based drug susceptibility testing (DST). In this work, we analysed all mutations in candidate genomic regions associated with BDQ- and DLM-resistant phenotypes using a whole genome sequencing (WGS) dataset from a collection of 4795 MTBc clinical isolates from six high-burden countries of tuberculosis (TB). From WGS analysis, we identified 61 and 158 unique mutations in genomic regions potentially involved in BDQ- and DLM-resistant phenotypes, respectively. Importantly, all strains were isolated from patients who likely have never been exposed to the medicines. In order to characterize the role of mutations, we performed an energetic in silico analysis to evaluate their effect in the available protein structures Ddn (DLM), Fgd1 (DLM) and Rv0678 (BDQ), and minimum inhibitory concentration (MIC) assays on a subset of MTBc strains carrying mutations to assess their phenotypic effect. The combination of structural protein information and phenotypic data allowed for cataloging the mutations clearly associated with resistance to BDQ (n= 4) and DLM (n= 35), as well as about a hundred genetic variants without any correlation with resistance. Importantly, these results show that both BDQ and DLM resistance-related mutations are diverse and distributed across the entire region of each gene target, which is of critical importance to the development of comprehensive molecular diagnostic tools.ImportancePhenotypic drug susceptibility tests (DST) are too slow to provide an early indication of drug susceptibility status at the time of treatment initiation and very demanding in terms of specimens handling and biosafety. The development of molecular assays to detect resistance to bedaquiline (BDQ) and delamanid (DLM) requires accurate categorization of genetic variants according to their association with phenotypic resistance. We have evaluated a large multi-country set of clinical isolates to identify mutations associated with increased minimum inhibitory concentrations (MICs) and used an in silico protein structure analysis to further unravel the potential role of these mutations in drug resistance mechanisms. The results of this study are an important source of information for the development of molecular diagnostic tests to improve the provision of appropriate treatment and care to TB patients.

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Validation of Bedaquiline Phenotypic Drug Susceptibility Testing Methods and Breakpoints: a Multilaboratory, Multicountry Study

Cited by 35 publications

References 25 publications

A Multimethod, Multicountry Evaluation of Breakpoints for Bedaquiline Resistance Determination

A Multimethod, Multicountry Evaluation of Breakpoints for Bedaquiline Resistance Determination

Molecular characteristics and in vitro susceptibility to bedaquiline of Mycobacterium tuberculosis isolates circulating in Shaanxi, China

Characterization of genomic variants associated with resistance to bedaquiline and delamanid in naïve Mycobacterium tuberculosis clinical strains

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