Assessment of a 96-Well Plate Assay of Quantitative Drug Susceptibility Testing for Mycobacterium Tuberculosis Complex in China

Abstract

ObjectiveTo evaluate the performance of the Sensitire MYCOTB MIC Plate (MYCOTB) which could measure the twelve anti-tuberculosis drugs susceptibility on one 96-wells plate.MethodsA total of 140 MDR-TB strains and 60 non-MDR strains were sub-cultured and 193 strains were finally tested for drug resistance using MYCOTB and agar proportion method (APM) and another 7 strains failed of subculture. The drugs included ofloxacin (Ofx), moxifloxacin (Mfx), rifampin (RFP), amikacin (Am), rifabutin (Rfb), para-aminosalicylic acid (PAS), ethionamide (Eth), isoniazid (INH), kanamycin (Km), ethambutol (EMB), streptomycin (Sm), and cycloserine(Cs). The categorical agreement, conditional agreement, sensitivity and specificity of MYCOTB were assessed in comparison with APM. For strains with inconsistent results between MYCOTB and APM, the drug resistance related gene fragments were amplified and sequenced: gyrA for Ofx and Mfx; rpoB for RFP and Rfb; embB for EMB; rpsl for Sm; katG and the promoter region of inhA for INH, ethA and the promoter region of inhA for Eth. The sequence results were compared with results of MYCOTB and APM to analyze the consistency between sequence results and MYCOTB or APM.ResultsThe categorical agreement between two methods for each drug ranged from 88.6% to 100%. It was the lowest for INH (88.6%). The sensitivity and specificity of MYCOTB ranged from 71.4% to 100% and 84.3% to 100%, respectively. The sensitivity was lowest for Cs(71.4%), EMB at 10μg/ml (80.0%) and INH at 10.0μg/ml (84.6%). The specificity was lowest for Rfb (84.3%). Overall discordance between the two phenotypic methods was observed for 96 strains, of which 63 (65.6%) were found susceptible with APM and resistant with MYCOTB and the remaining 33(34.4%) strains were resistant by APM and susceptible with MYCOTB. 34/52 (65.4%) sequenced APM susceptible and MYCOTB resistant(APM-S/MYCOTB-R) strains had mutations or insertions in the amplified regions. 20/30 (66.7%) sequenced APM resistant and MYCOTB susceptible strains had mutations in the sequenced genes. MICs of twenty-nine of these thirty isolates were equal to or within 1 doubling dilution of the critical concentration.ConclusionMYCOTB had good performance for most of tested drugs and could be used as an alternative to the more labor demanding and longer turnaround time solid culture based DST method for detection of drug susceptibility in China.

Drug resistance mechanisms and drug susceptibility testing for tuberculosis

“…[123][124][125] A dryformat, 96-well microtitre plate assay (Sensititre MYCOTB plate; Thermo Fisher Scientific Inc., Waltham, MA, USA) has been commercially available since 2010 and early validation studies have returned promising results. [126][127][128][129][130] This assay performs MIC for 12 anti-TB drugs on the same plates. New layouts, custommade, microtitre plates have been recently designed and evaluated in multicentric studies.…”

“…Broth microdilution methods, including using several colorimetric indicators, have been developed and used as research tools or to establish the ECOFF for new drugs . A dry‐format, 96‐well microtitre plate assay (Sensititre MYCOTB plate; Thermo Fisher Scientific Inc., Waltham, MA, USA) has been commercially available since 2010 and early validation studies have returned promising results . This assay performs MIC for 12 anti‐TB drugs on the same plates.…”

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

“…Since no critical concentrations exist for UKMYC5 plate, all 19 EQA strains (including H37Rv) were then categorized as resistant or susceptible using the same CC as the comparator method (as it was done in 13,14): (i) the 7H10/711 agar proportion method (APM) and (ii) the mycobacterial growth indicator tube (MGIT , Table S22). Where possible, critical concentrations for APM were taken from recent guidance (20).…”

“…The categorical and conditional agreements between UKMYC5 MIC mode and those obtained from MGIT and the APM were thereby computed (Table S13). Since no critical concentrations exist for the UKMYC5 plate, we assumed shared breakpoints with each comparator method (APM or MGIT) to infer whether each strain was 'susceptible' or 'resistant' (13,14).…”

“…Broth microdilution methods, including several using colorimetric indicators, have previously been developed that assess the MICs for a panel of compounds using a single microtitre plate (5,10,11). A dry-format, 96-well microtitre plate assay (the Sensititre™ MYCOTB plate; Thermo Fisher Scientific Inc., USA) containing 12 drugs has been commercially available since 2010 and early validation studies have returned promising results (12)(13)(14)(15)(16)(17). At present however, neither clinical breakpoints nor epidemiological cutoffs have been defined for any microtitre plate.…”