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What is the role of the EUCAST reference method for MIC testing of the Mycobacterium tuberculosis complex?

Published:August 05, 2020DOI:https://doi.org/10.1016/j.cmi.2020.07.037

      Keywords

      Drug-resistant tuberculosis (TB) is estimated to account for approximately 30% of annual deaths due to antimicrobial resistance (AMR) [
      Review on Antimicrobial Resistance
      Tackling drug-resistant infections globally: final report and recommendations.
      ]. This is mainly due to mortality of approximately 44% caused by multidrug-resistant (MDR) TB (defined by resistance to isoniazid and rifampicin) [
      • WHO
      Global tuberculosis report 2019.
      ]. Therefore, accurately diagnosing AMR for TB is crucial not only to select the most effective regimen with the least side effects but also to minimize costs (e.g. the median cost to treat MDR-TB is $6430 compared with $973 for drug-susceptible TB) [
      • WHO
      Global tuberculosis report 2019.
      ]. Owing to the slow growth rate of the Mycobacterium tuberculosis complex (MTBC), this is increasingly achieved using genotypic approaches [
      • Schön T.
      • Miotto P.
      • Köser C.U.
      • Viveiros M.
      • Böttger E.
      • Cambau E.
      Mycobacterium tuberculosis drug-resistance testing: challenges, recent developments and perspectives.
      ]. However, phenotypic antimicrobial susceptibility testing (pAST) is still needed to correlate the presence of mutations and their phenotypic expression, especially for new anti-TB agents for which resistance mutations are unknown.

      Rationale for the EUCAST MIC reference method

      Historically, several methods and culture media have been used for pAST of MTBC [
      • Schön T.
      • Miotto P.
      • Köser C.U.
      • Viveiros M.
      • Böttger E.
      • Cambau E.
      Mycobacterium tuberculosis drug-resistance testing: challenges, recent developments and perspectives.
      ,
      • Canetti G.
      • Froman S.
      • Grosset J.
      • Hauduroy P.
      • Langerova M.
      • Mahler H.T.
      • et al.
      Mycobacteria: laboratory methods for testing drug sensitivity and resistance.
      ]. The corresponding breakpoints, which were traditionally referred to as critical concentrations (CC), were first set in the 1960s by the World Health Organization (WHO) and subsequently revised by the Clinical and Laboratory Standards Institute (CLSI) and WHO (Table 1) [
      • WHO
      Technical report on critical concentrations for drug susceptibility testing of medicines used in the treatment of drug-resistant tuberculosis. WHO/CDS/TB/2018.5-Geneva.
      ,
      CLSI
      Susceptibility testing of mycobacteria, nocardiae, and other aerobic actinomycetes; approved standard.
      ]. Although CC values were determined based on microbiological and clinical evidence, this was done only for certain drugs and media, which complicates the use of these values today [
      • Schön T.
      • Miotto P.
      • Köser C.U.
      • Viveiros M.
      • Böttger E.
      • Cambau E.
      Mycobacterium tuberculosis drug-resistance testing: challenges, recent developments and perspectives.
      ]. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) sets clinical breakpoints (CBs) by evaluating epidemiological cut-off values (ECOFFs), based on MIC distributions, pharmacokinetic/pharmacodynamic (PK/PD) and clinical outcome data together [
      • Kahlmeter G.
      The 2014 Garrod Lecture: EUCAST – are we heading towards international agreement?.
      ]. This approach has now been used for most antibacterial and antifungal agents, enabling high-quality pAST globally. The time has come to bring the same rigorous approach to the TB field.
      Table 1List of the critical concentrations (mg/L) recommended by WHO for phenotypic antimycobacterial susceptibility testing of isolates of Mycobacterium tuberculosis complex using the indirect proportion method
      Antimicrobial agentLJ7H107H11MGIT
      Rifampicin40111
      Isoniazid0.20.20.20.1
      Ethambutol257.55
      Pyrazinamide100
      Levofloxacin211
      Moxifloxacin10.50.50.25
      Bedaquiline0.251
      Linezolid111
      Clofazimine1
      Cycloserine
      Delamanid0.0160.06
      Imipenem–cilastatin
      Meropenem
      Amikacin3021
      Streptomycin4221
      Ethionamide405105
      Prothionamide402.5
      P-aminosalicylic acid
      In 2018, WHO redefined the CCs to correspond to ECOFFs and performed an extensive systematic literature review of MIC distributions for WHO-endorsed media (i.e. egg-based Löwenstein–Jensen (LJ), Middlebrook 7H10 or 7H11 synthetic solid media and BACTEC 960 MGIT liquid medium, see Table 1) [
      • WHO
      Technical report on critical concentrations for drug susceptibility testing of medicines used in the treatment of drug-resistant tuberculosis. WHO/CDS/TB/2018.5-Geneva.
      ]. This revealed that some CCs had been too high, resulting in the misclassification of some resistant strains as susceptible. In general, the quality and quantity of MIC data available for the majority of agents were insufficient to define ECOFFs according to the criteria adopted by EUCAST [
      • Kahlmeter G.
      The 2014 Garrod Lecture: EUCAST – are we heading towards international agreement?.
      ,
      • WHO
      Technical manual for drug susceptibility testing of medicines used in the treatment of tuberculosis.
      ]. For example, MICs were often truncated because inappropriate concentration ranges were tested, which precluded a comprehensive assessment of the phenotypically wild-type MIC distributions [
      • Schön T.
      • Matuschek E.
      • Mohamed S.
      • Utukuri M.
      • Heysell S.
      • Alffenaar J.W.
      • et al.
      Standards for MIC testing that apply to the majority of bacterial pathogens should also be enforced for Mycobacterium tuberculosis complex.
      ,
      • Kaniga K.
      • Cirillo D.M.
      • Hoffner S.
      • Ismail N.A.
      • Kaur D.
      • Lounis N.
      • et al.
      A Multilaboratory, multicountry study to determine MIC quality control ranges for phenotypic drug susceptibility testing of selected first-line antituberculosis drugs, second-line injectables, fluoroquinolones, clofazimine, and linezolid.
      ]. In addition, systematic differences in the MIC distributions from different laboratories that supposedly used the same method became apparent, even for testing on 7H10 despite this method being standardized by CLSI [
      CLSI
      Susceptibility testing of mycobacteria, nocardiae, and other aerobic actinomycetes; approved standard.
      ]. This had gone largely unnoticed because of the lack of rigorously defined quality control (QC) ranges/targets [
      • Schön T.
      • Matuschek E.
      • Mohamed S.
      • Utukuri M.
      • Heysell S.
      • Alffenaar J.W.
      • et al.
      Standards for MIC testing that apply to the majority of bacterial pathogens should also be enforced for Mycobacterium tuberculosis complex.
      ].
      The need for greater standardisation in the TB field was underscored when the new anti-TB agents -delamanid and bedaquiline- were submitted to the European Medical Agency (EMA) in 2012 and 2013, respectively. Since EMA routinely expects EUCAST to set CBs, EUCAST established a subcommittee for antimycobacterial susceptibility testing (AMST) in 2016 and its first task was to develop a reference MIC method for MTBC. This was made possible by the strong involvement of the ESCMID study group of mycobacterial infections (ESGMYC). In 2019, AMST proposed a new protocol for broth microdilution (BMD) testing in Middlebrook 7H9 using a carefully standardised inoculum [
      • Schön T.
      • Werngren J.
      • Machado D.
      • Borroni E.
      • Wijkander M.
      • Lina G.
      • et al.
      Antimicrobial susceptibility testing of Mycobacterium tuberculosis complex isolates – The EUCAST broth microdilution reference method for MIC determination.
      ]. Following a public consultation, this protocol was validated in the four AMST laboratories. This new reference MIC method for MTBC was endorsed by EUCAST as the reference method to set QC ranges/targets, ECOFFs and CBs for the MTBC. As with other organisms, commercial methods, such as MGIT, could not be accepted as formal reference methods to avoid dependence on a single manufacturer that may go out of the business or decide to modify or discontinue a specific method. Instead, commercial methods have to be calibrated against the reference method. The choice of the reference method was based on a multicentre comparison of the intra- and interassay reproducibility of BMD with 7H10 as well as broader considerations, such as the labour requirements, costs, published MIC evidence and experiences with other bacteria (Table 2).
      Table 2Criteria for the selection of a reference method for MIC testing on Mycobacterium tuberculosis complex
      Criteria7H9 BMD EUCAST7H107H11MGITLJ
      Reproducibility+++++++++++++
      Media variability+
      Workload for preparation++++++++
      Ease of use++++++
      Material cost per MIC concentration (Euro)0.3–0.50.5–10.5–15–100.1–0.5
      Similar reference method for other species?YesNoNoNoNo
      Time to result (days)7–1421217–1428
      Availability of different media distributorsYesYesYesNoYes
      Availability of a commercial kitNoNoNoYesNo
      CLSI or WHO breakpoints availableNoYesYesYesYes
      Potential for future development+++++++++
      Biosafety risk++++++++
      BMD, broth microdilution; LJ, Löwenstein Jensen; MGIT, BACTEC 960 MGIT; —, low, +, acceptable, ++, high, +++, very high. CLSI, Clinical and Laboratory Standards Institute; WHO, World Health Organization.
      The reference method is described on the EUCAST website and is detailed in an accompanying article [
      • Schön T.
      • Werngren J.
      • Machado D.
      • Borroni E.
      • Wijkander M.
      • Lina G.
      • et al.
      Antimicrobial susceptibility testing of Mycobacterium tuberculosis complex isolates – The EUCAST broth microdilution reference method for MIC determination.
      ]. To ensure standardization, a QC target/range for H37Rv ATCC 27294 will be defined for each drug, as it is routinely done for other pathogens [
      • Schön T.
      • Matuschek E.
      • Mohamed S.
      • Utukuri M.
      • Heysell S.
      • Alffenaar J.W.
      • et al.
      Standards for MIC testing that apply to the majority of bacterial pathogens should also be enforced for Mycobacterium tuberculosis complex.
      ]. This will not only improve the reproducibility of MIC testing during routine clinical testing but also ensure comparability of MIC data from different clinical trials and scientific studies [
      • Schön T.
      • Matuschek E.
      • Mohamed S.
      • Utukuri M.
      • Heysell S.
      • Alffenaar J.W.
      • et al.
      Standards for MIC testing that apply to the majority of bacterial pathogens should also be enforced for Mycobacterium tuberculosis complex.
      ].

      Role of other pAST methods

      The EUCAST reference method is mostly suitable for specialized reference laboratories. Other non-commercial (e.g. LJ, 7H10 or 7H11) and commercial methods can be used instead for routine pAST or clinical trials as they may offer other distinct advantages (e.g. higher biosafety of MGIT, reduced labour requirements for Sensititre plates or lower cost of LJ, see Table 2) [
      • WHO
      Technical report on critical concentrations for drug susceptibility testing of medicines used in the treatment of drug-resistant tuberculosis. WHO/CDS/TB/2018.5-Geneva.
      ,
      • Kaniga K.
      • Cirillo D.M.
      • Hoffner S.
      • Ismail N.A.
      • Kaur D.
      • Lounis N.
      • et al.
      A Multilaboratory, multicountry study to determine MIC quality control ranges for phenotypic drug susceptibility testing of selected first-line antituberculosis drugs, second-line injectables, fluoroquinolones, clofazimine, and linezolid.
      ]. However, to interpret the results of surrogate methods using EUCAST CBs, these methods will need to be calibrated against the reference method. The calibration does not have to be carried out by each laboratory that would like to use a surrogate method. Rather, the calibration will be done once in a multicentre study according to the standard operating procedures provided by EUCAST. For other microbial pathogens, EUCAST considers a calibration successful if the surrogate method is equally reproducible (equal degree of random variation) and yields equivalent MICs (essential agreement; absence of systematic difference) compared with the reference method. For MTBC, however, EUCAST may accept methods that yield MICs that are systematically higher or lower, provided the difference is fully predictable, by introducing a conversion factor. This would allow for reference MICs to be predicted from the results of the surrogate method. This is needed for therapeutic drug monitoring using the PK/PD target that will be set based on the reference method.

      EUCAST strategy for setting CBs for MTBC

      From now on, it will be the responsibility of pharmaceutical companies to present MIC data generated by the reference method, along with PK/PD and clinical outcome data to allow EUCAST to set ECOFFs and CBs when new drugs are to be approved by EMA. To define ECOFFs, it is important to note that all known MTBC lineages and major genotypes should be included to ensure that the resulting MIC distribution is as representative as possible of the global MTBC diversity. The first task for EUCAST will be to set definitive CBs for bedaquiline and delamanid based on reference method generated MIC values (i.e. the current CBs are provisional since the reference method and associated guidelines did not exist when these agents were approved). The AMST network will facilitate the generation of necessary data to set CBs for traditional agents through transparent interactions with diagnostic companies and the pharmaceutical industry. For this extensive task, collaboration within the recently launched AMST general committee, to which representatives of all EU countries were invited, will be needed.
      In summary, the overarching goal of AMST is to create a scientifically rigorous framework to set CBs for MTBC. To achieve this, global collaboration between varieties of stakeholders is necessary, ranging from microbiologists, molecular biologists, pharmacologists and clinicians to pharmaceutical and diagnostic companies and regulatory agencies. This is foremost in the interest of TB patients but also provides crucial information to those involved in the measurement of antimicrobial resistance and those struggling to develop new agents.

      Transparency Declaration

      C.U.K. is a consultant for the World Health Organization (WHO) Global TB Programme, the WHO Regional Office for Europe, The Global Alliance for TB Drug Development, Becton Dickinson, and the Foundation for Innovative New Diagnostics, which involved work for Cepheid, Hain Lifescience and WHO. C.U.K is an unpaid advisor to GenoScreen. C.U.K. worked as a consultant for QuantuMDx. Hain Lifescience covered C.U.K.'s travel and accommodation to present at a meeting. Otsuka Novel Products GmbH has supplied C.U.K. with antibiotics for in vitro research. C.U.K. is collaborating with YD Diagnostics. The remaining authors declare that they have no conflicts of interest.

      Author contributions

      T.S. and C.U.K. designed the paper. J.W., M.V., S.G., G.K., C.G., F.M., G.L., J.T., K.v.I., M.J., D.G., D.M.C., M.S. and E.C. participated in the discussion. T.S., C.U.K., S.G. and E.C. wrote a draft of the manuscript and all authors participated in the final version and revisions.

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