Sensititre MYCOTB MIC Plate for Testing Mycobacterium tuberculosis Susceptibility to First- and Second-Line Drugs

Abstract

f 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.T he emergence and spread of drug-resistant strains of Mycobacterium tuberculosis comprise a serious threat to tuberculosis (TB) control (1, 2). Knowledge of M. tuberculosis drug susceptibility is important in optimizing individual patient management and TB control in populations. Genotypic methods have the potential for a very short time to results, but to date, the knowledge of the full spectrum of genetic loci and mutations associated with resistance to many antituberculosis drugs is incomplete (3,4,5,6,7). Phenotypic methods therefore remain important. The reference phenotypic method-the indirect agar proportion method (APM) using Middlebrook solid media-is qualitative and based on drug critical concentrations. Limitations of the APM and related methods include lack of standardization and in some cases the need for in-laboratory preparation of drug stocks and agar plates, which can be a source of variability over time and between laboratories. Critical concentrations are based on historical epidemiological data and for some drugs are not well-aligned with achievable drug serum concentrations or accurate in predicting clinical failure (8,9,10). Studies on molecular drug resistance mechanisms in M. tuberculosis have shown that, at least for some antibiotics, different mutations are associated with different MICs, furthe...

The epidemiology, pathogenesis, transmission, diagnosis, and management of multidrug-resistant, extensively drug-resistant, and incurable tuberculosis

“…231 The results are typically reported as susceptible or resistant and are less precise than those generated by semiquantitative methods, whereby bacteria are grown at a range of drug concentrations so the MIC can be established. 232 Semiquantitative data have therapeutic implications because it differentiates between susceptible and resistant strains, and it also detects moderate or low-level resistance where increased dosage might be beneficial to the patient. 233,234 The first DST methods were developed and validated for solid culture using Löwenstein-Jensen slopes or agar plates.…”

“…MICs of test strains are compared with the upper MIC limit or the epidemiological cutoff value of wildtype strains. 232 WHO defines the MIC for M tuberculosis as the lowest concentration of drug that inhibits 95% of a wild-type strain (that has never been exposed to drug) while allowing clinically resistant strains to grow.…”

Genotypic Susceptibility Testing of Mycobacterium tuberculosis Isolates for Amikacin and Kanamycin Resistance by Use of a Rapid Sloppy Molecular Beacon-Based Assay Identifies More Cases of Low-Level Drug Resistance than Phenotypic Lowenstein-Jensen Testing

“…Phenotypic drug susceptibility testing was performed on all 603 isolates by the absolute concentration method on LJ medium to determine the susceptibility to AMK and KAN using a critical concentration of 40 g/ml (the standard concentration used during 2012 when the isolates were tested) for both the antibiotics (27) at the International Tuberculosis Research Center (ITRC), South Korea. MICs of AMK and KAN for 173/603 samples were also evaluated using TREK Sensititre MycoTB MIC plates (MycoTB; TREK Diagnostic Systems, Cleveland, OH, USA) as described previously (28). For 560/603 samples, resistance to KAN was also evaluated using a mycobacterial growth indicator tube (MGIT) system (Becton Dickinson, Franklin Lakes, NJ, USA) at a critical concentration of 2.5 g/ml.…”

Investigation of Susceptibility of Mycobacterium tuberculosis Complex Strains Isolated from Clinical Samples Against the First and Second-Line Anti-tuberculosis Drugs by the Sensititre MycoTB Plate Method

“…Lee ve arkadaşları duyarlı ve dirençli izolatlardan oluşan toplam 222 MTC izolatının duyarlılığı-nı MycoTB ve 7H10 Agar proporsiyon yöntemiy-le karşılaştırmışlar ve MycoTB'nin birinci ve ikinci basamak ilaçların (etambutol hariç) duyarlılığında güvenilir sonuçlar sağladığı ve Agar proporsiyon yönteminden daha erken sonuç verdiği kanısına varmışlardır [8] .…”