Validating a 14-drug microtitre plate containing bedaquiline and delamanid for large-scale research susceptibility testing ofMycobacterium tuberculosis

Abstract

Background: Universal access to drug susceptibility testing is key to ending TB. UKMYC5 is a 96-well microtitre plate designed by the Comprehensive ResistancePrediction for Tuberculosis: an International Consortium (CRyPTIC) which has potential to determine, at low cost, the MICs for 14 different anti-TB drugs, including several new and repurposed compounds. It is a dry-format plate and therefore easy to transport and store.Objectives: Determine how long to incubate the plates before reading, and the optimal reading method. Confirm the reproducibility of the UKMYC5 plate and compare it to established methods.* These authors contributed equally to this work § Senior Authors † To whom correspondence should be addressed: cirillo.daniela@hsr.it ‡ This is a corporate author; for the list of members, please see the section at the end of this manuscript.International license not peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/244731 doi: bioRxiv preprint first posted online Jan. 8, 2018; 2Methods: UKMYC5 plates were tested by seven laboratories on four continents using a panel of 19 external quality assessment (EQA) strains, including H37Rv.MICs were measured from each plate by two readers using three methods (mirroredbox, microscope and Vizion™ Digital viewing system) at four different timepoints. All EQA strains were whole-genome sequenced and phenotypically characterized by MGIT960, 7H10/7H11 agar and resazurin microtitre assay. Results:The optimum duration to incubate a plate is 14 days. The within-and between-laboratory reproducibilities for the best performing methods (mirrored-box and Vizion) were >95% and >92%, respectively. One site was identified as requiring re-training and one drug (para-aminosalicylic acid) produced inconsistent results. Conclusions:MICs measured using the UKMYC5 microtitre plate (i) are reproducible, (ii) compare well with the results of several established methods and (iii) correlate with the presence or absence of genetic mutations that confer resistance. This study provides the evidence that this assay can be deployed by TB reference laboratories world-wide as a diagnostic and research tool.

Standards for MIC testing that apply to the majority of bacterial pathogens should also be enforced for Mycobacterium tuberculosis complex

“…These problems also extend to the recently developed UKMYC5 microtitre plate, which can be used for MIC testing for traditional first-line and second-line drugs as well as newly approved agents, simultaneously [11]. Yet, the lower ends of the MIC distributions of H37Rv ATCC 27294 are again truncated for nine of the twelve drugs on the UKMYC5 plate [11].…”

“…These problems also extend to the recently developed UKMYC5 microtitre plate, which can be used for MIC testing for traditional first-line and second-line drugs as well as newly approved agents, simultaneously [11]. Yet, the lower ends of the MIC distributions of H37Rv ATCC 27294 are again truncated for nine of the twelve drugs on the UKMYC5 plate [11]. Importantly, this includes bedaquiline and clofazimine, which are key to the treatment of multidrugresistant tuberculosis using the long regimen, and amikacin, which is an important component of the modified short-course multidrug-resistant regimen and may be critical to complete an active regimen when additional resistances or drug intolerances exist.…”

BashTheBug: a crowd of volunteers reproducibly and accurately measure the minimum inhibitory concentrations of 13 antitubercular drugs from photographs of 96-well broth microdilution plates

“…Thirty one vials containing 19 external quality assessment (EQA) M. tuberculosis strains, including the reference strain H37Rv ATCC 27294 8 , were sent to seven participating laboratories as described previously 9 . Since some labs only received a subset of the 31 vials (Table S1), a total of 447 plates were inoculated and then incubated for 3 weeks (Fig.…”

Estimating MIC distributions and cutoffs through mixture models: an application to establish M. Tuberculosis resistance

“…Two researchers from each laboratory were asked to independently identify the MIC values for each plate at four moments after incubation (day 7, day 10, day 14 and day 21) with three reading methods: Vizion imaging system, a mirrored box and an inverted-light microscope. A full description of the experiment and of the results in terms of reproducibility of the plate are available in Rancoita et al (2018). Since the authors identified the highest level of reproducibility for readings at day 14 with the Vizion imaging system, we will focus only on data relative to this subset.…”

“…Since the authors identified the highest level of reproducibility for readings at day 14 with the Vizion imaging system, we will focus only on data relative to this subset. Moreover, Rancoita et al (2018) also shown that the compound PAS seems to not perform well on the plate, therefore it will be discarded in the following of the CRyPTIC study. For these reason, the outcome relative to PAS will be presented in this work, but considered more uncertain.…”