Rapid, low-technology MIC determination with clinical Mycobacterium tuberculosis isolates by using the microplate Alamar Blue assay

Abstract

A colorimetric, microplate-based Alamar Blue assay (MABA) method was used to determine the MICs of isoniazid (INH), rifampin, streptomycin (SM), and ethambutol (EMB) for 34 Peruvian Mycobacterium tuberculosis isolates (including both pansensitive and multidrug-resistant strains) and the H37Rv strain by using bacterial suspensions prepared directly from solid media. Results for all isolates were available within 8 days. Discordant results were observed on initial tests for 3 of 16 INH-susceptible isolates, 5 of 31 EMB-susceptible isolates, and 2 of 4 SM-resistant isolates (by the BACTEC 460 system). The overall agreements between the MICs obtained by MABA and the results obtained with the BACTEC 460 system were 87.9% for initial results and 93.6% after retesting 12 of 17 samples with discrepant results. Interpretation of MABA endpoints improved with technical experience. The MABA is a simple, rapid, low-cost, appropriate technology which does not require expensive instrumentation and which makes use of a nontoxic, temperature-stable reagent.

FIG. 1

Correlation between MICs by MABA and BACTEC 460 system classification for INH. •, isolates for which results were concordant (and isolates for which discordant results were confirmed upon repeat testing by MABA); ■, isolates for which results were discordant and for which MICs by MABA were not redetermined; ▾, isolates for which results were discordant but for which results were concordant upon repeat MABA testing by MABA. The vertical lines in the figures separate the standard breakpoints for the BACTEC 460 system. PR, partially resistant. The horizontal lines separate the interpretive breakpoints for colorimetric MICs, which were selected on the basis of the best fit of the MABA results with the BACTEC 460 system results.

FIG. 2

Correlation between MICs by MABA and BACTEC 460 system classification for RMP. The vertical lines separate the standard breakpoints for the BACTEC 460 system. The horizontal lines separate the interpretive breakpoints for colorimetric MICs, which were selected on the basis of the best fit of the MABA results with the BACTEC 460 system results.

FIG. 3

Correlation between MABA MICs and BACTEC 460 system classification for EMB. •, isolates for which results were concordant (and isolates for which discordant results were confirmed upon repeat testing by MABA); ■, isolates for which results were discordant and for which MICs by MABA were not redetermined; ▾, isolates for which results were discordant but for which results were concordant upon repeat testing by MABA. The vertical lines in the figures separate the standard breakpoints for the BACTEC 460 system. PR, partially resistant. The horizontal lines separate the interpretive breakpoints for colorimetric MICs, which were selected on the basis of the best fit of the MABA results with the BACTEC 460 system results.

FIG. 4

Correlation between MABA MICs and BACTEC 460 system classification for SM. •, isolates for which results were concordant (and isolates for which discordant results were confirmed upon repeat testing by MABA);  isolates for which results were discordant and for which MICs by MABA were not redetermined; ▾, isolates for which results were discordant but for which results were concordant upon repeat testing by MABA. The vertical lines in the figures separate the standard breakpoints for the BACTEC 460 system. PR, partially resistant. The horizontal lines separate the interpretive breakpoints for colorimetric MICs, which were selected on the basis of the best fit of the MABA results with the BACTEC 460 system results.