Sensititre MYCOTB MIC plate for testing Mycobacterium tuberculosis susceptibility to first- and second-line drugs

Abstract

For Mycobacterium tuberculosis, phenotypic methods for drug susceptibility testing of second-line drugs are poorly standardized and technically challenging. The Sensititre MYCOTB MIC plate (MYCOTB) is a microtiter plate containing lyophilized antibiotics and configured for determination of MICs to first- and second-line antituberculosis drugs. To evaluate the performance of MYCOTB for M. tuberculosis drug susceptibility testing using the Middlebrook 7H10 agar proportion method (APM) as the comparator, we conducted a two-site study using archived M. tuberculosis isolates from Uganda and the Republic of Korea. Thawed isolates were subcultured, and dilutions were inoculated into MYCOTB wells and onto 7H10 agar. MYCOTB results were read at days 7, 10, 14, and 21; APM results were read at 21 days. A total of 222 isolates provided results on both platforms. By APM, 106/222 (47.7%) of isolates were resistant to at least isoniazid and rifampin. Agreement between MYCOTB and APM with respect to susceptibility or resistance was ≥92% for 7 of 12 drugs when a strict definition was used and ≥96% for 10 of 12 drugs when agreement was defined by allowing a ± one-well range of dilutions around the APM critical concentration. For ethambutol, agreement was 80% to 81%. For moxifloxacin, agreement was 83% to 85%; incorporating existing DNA sequencing information for discrepant analysis raised agreement to 91% to 96%. For MYCOTB, the median time to plate interpretation was 10 days and interreader agreement was ≥95% for all drugs. MYCOTB provided reliable results for M. tuberculosis susceptibility testing of first- and second-line drugs except ethambutol, and results were available sooner than those determined by APM.

FIG 1

Schematic of the Sensititre MYCOTB MIC plate showing antibiotics and concentrations in micrograms/milliliter. Abbreviations: OFL, ofloxacin; MXF, moxifloxacin; RIF, rifampin; AMI, amikacin; STR, streptomycin; RFB, rifabutin; PAS, para-aminosalicylic acid; ETH, ethionamide; CYC, cycloserine; INH, isoniazid; KAN, kanamycin; EMB, ethambutol; POS, antibiotic-free positive-control wells.

FIG 2

MICs as determined using the Sensititre MYCOTB MIC plate versus agar proportion method results for all tested isolates for moxifloxacin at 0.5 μg/ml (A), moxifloxacin at 2.0 μg/ml (B), and ofloxacin at 2.0 (C) μg/ml. Closed circles, DNA sequencing of gyrA and gyrB was not performed; open circles, DNA sequencing was performed but no mutation was identified; X, DNA sequencing was performed and a resistance-associated mutation was identified.

FIG 3

MICs as determined using the Sensititre MYCOTB MIC plate versus agar proportion method results for all tested isolates for (A) ethambutol at 5.0 μg/ml and (B) ethambutol at 10.0 μg/ml.