Direct Colorimetric Assay for Rapid Detection of Rifampin-Resistant Mycobacterium tuberculosis

The colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (MTT) assay was standardized for direct detection ofrifampin-resistant Mycobacterium tuberculosis in sputum samples.The sensitivity and specificity of the direct MTT assay matchedthose of the standard indirect susceptibility assay on 7H10medium, and interpretable results were obtained for 98.5% ofthe samples within 2 weeks. Traditional methods of in vitrodrug susceptibility testing are time consuming and laborious.Susceptibility tests on clinical isolates require 6 to 9 weeks,and tests conducted directly on smear-positive samples takeabout 3 weeks (International Union Against Tuberculosis andLung Disease, The public health service national tuberculosisreference laboratory and the national laboratory network. Minimumrequirements, role and operation in a low-income country, Paris,France, 1998, and P. T. Kent and G. P. Kubica, Public healthmycobacteriology. A guide for the level III laboratory, Centersfor Disease Control and Prevention, Atlanta, Ga., 1985). More-rapidmethods are available but are very expensive for routine useunder program conditions in countries with high levels of tuberculosisendemicity.

INTRODUCTION

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Introduction

A colorimetric assay based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was previously standardized and evaluatedusing a BACTEC radiometric method as a "gold standard" for indirectdetection of rifampin resistance (1, 8). MTT is a yellow tetrazoliumsalt that is converted into blue formazan by dehydrogenasesof live cells (7). The amount of blue or purple formazan formationis proportional to the number of live mycobacteria in a sample(8). The results of the MTT assay matched fully with the resultsobtained using the BACTEC method (1).

This study was conducted with the objectives of standardizingthe MTT assay for the detection of rifampin resistance directlyon smear-positive sputum samples and evaluating the assay againstthe traditional method as the gold standard.

Standardization and evaluation.

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Standardization and Evaluation.

Sputum samples from 16 new smear-positive cases of pulmonarytuberculosis were used to standardize the assay. Sputum samplesfrom 74 smear-positive retreatment cases were pelleted afterdigestion and decontamination with 4% NaOH (5). The pelletswere neutralized and resuspended in 3 ml of sterile 7H9 brotheach. An aliquot of 500 µl was added to tubes containing3 ml of 7H9 Middlebrook broth supplemented with 10% oleic-acid-albumin-dextrose-catalase,glycerol (0.05%), and PANTA (Becton Dickinson, Paramus, N.J.)(75 µl). Rifampin (Sigma) at a final concentration of2 µg/ml was added to some of the tubes. The experimentalapproach is summarized in Fig. 1. All tubes were incubated at37°C until the day of the MTT assay.

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FIG. 1. Flow chart of MTT assay. Abbreviations: BC, bacterial control without rifampin; R, tube with 2 µg of rifampin/ml; C, control without bacteria. LJ, Löwenstein-Jensen.

MTT assay.

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Mtt assay.

The MTT assay was done each week for 3 weeks. Contaminationwas checked by growing subcultures on nutrient agar medium.Each week, an MTT assay was done using one bacterial controltube with no rifampin and another tube with bacteria and rifampin.The assay was done as described previously (1). Relative opticaldensity (OD) unit (RODU) values were calculated for each sampleby dividing the OD value of the rifampin-containing tube bythe OD value of the drug-free control. Resistance was definedas RODU > 0.5, and susceptibility was defined as RODU <0.2. RODU values obtained each week for samples containing susceptibleisolates were compared (using the Mann-Whitney U test) withthose of samples containing resistant isolates. The lowest ODvalue which was considered indicative of growth was determinedby growing a subculture with an aliquot of vortexed broth mediumevery week before the MTT assay. The lowest OD value with apositive culture result was 0.10; therefore, the results wereconsidered interpretable when the OD value of the control was $>=$ 0.1.

Standard sensitivity testing.

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Standard sensitivity testing.

Standard biochemical tests were used to identify all isolatesas Mycobacterium tuberculosis (5). A proportion method (5) usingMiddlebrook 7H10 medium was used as a reference method for rifampinsusceptibility testing. Reference M. tuberculosis strains ATCC35836 (rifampin susceptible) and ATCC 35838 (rifampin resistant)were used as controls.

Among the 74 samples used to evaluate the MTT assay, 5 (6.8%)were excluded, 3 because the OD values of control tubes remainedbelow 0.1 and 2 because there was no growth on Löwenstein-Jensenmedium. Of the remaining 69 samples, 5 (7.2%) were contaminated;however, for each of the five samples there was at least onenoncontaminated interpretable result among the results of thethree sets of experiments prepared for the 3 weeks. The contaminationrates of 18 samples tested in the presence of PANTA (BectonDickinson) and in the presence of a much cheaper but similarantibiotic cocktail prepared in-house were the same. There wasno contamination in the presence of Löwenstein-Jensen medium.

Table 1 shows the number of interpretable results obtained ineach week. In the first week, 43 of 68 (63%) of the samplesgave interpretable results; the number of samples with interpretableresults grew in the second (98.5%) and third (100%) weeks. TheMTT assay identified 8 of 69 (11.6%) samples as containing rifampin-resistantM. tuberculosis and 61 of 69 (88.4%) as containing rifampin-susceptibleM. tuberculosis. The susceptibility (sensitivity and specificity)results obtained with MTT concurred fully with findings obtainedusing the standard assay on 7H10 agar medium. Figure 2 showsthat the RODU values of samples containing susceptible bacteriaremained below 0.2 in all weeks of experiments and that theRODU values of samples containing resistant bacteria were above0.5. The differences in the RODU values of samples containingsusceptible and resistant isolates were statistically significantfor each week (P < 0.01).

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TABLE 1. Contamination rate and time of interpretation of a direct MTT assay for detection of rifampin-resistant M. tuberculosis^a

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FIG. 2. Growth patterns of rifampin-susceptible (n = 37 samples in the first week, 56 in the second week, and 58 in the third week) and rifampin-resistant (n = 6 in the first week and 8 in the subsequent weeks) strains of M. tuberculosis as reflected by RODU values (means ± standard errors) [(RODU = OD₅₇₀ of rifampin-containing medium)/(OD₅₇₀ of drug-free medium)]. The difference in RODU values in each week was statistically significant (P < 0.01 [Mann-Whitney test]).

Our findings show that a direct assay based on a tetrazoliumsalt significantly reduces the time required to obtain reliablesusceptibility results. The standard direct methods of drugsensitivity testing on solid medium take 3 to 4 weeks (5, 6),and with these conventional methods, there is in addition aneed to prepare appropriate dilutions of a specimen as determinedon the basis of smear grading (5). Our application of a directMTT assay is not dependent on smear grading and shortens theturnaround time. Other direct rapid methods (such as the BACTEC460 system and the mycobacterial growth indicator tube system)have a turnaround time ranging from 9 to 12 days (3, 6). However,they are very expensive for routine use in most countries inwhich tuberculosis is endemic.

Rifampin resistance is a strong predictor of the presence ofmultidrug-resistant tuberculosis (2). Therefore, the resultsof our study focusing on the direct detection of rifampin-resistantM. tuberculosis indicate the potential of this simple and inexpensiveassay for control programs in countries with high levels oftuberculosis endemicity. The same assay could theoreticallybe used to rapidly screen for resistance to other antituberculosisdrugs. Our preliminary findings indicate that the same assaycould be used for reliable and rapid detection of isoniazidresistance. This new method should be evaluated under programconditions in a region with a high level of tuberculosis endemicityand optimized for program use.

ACKNOWLEDGMENTS

This project was supported by a grant from the UNDP/WORLD BANK/WHOSpecial Programme for Research and Training in Tropical Diseases(TDR).

FOOTNOTES

* Corresponding author. Mailing address: Division of Infectious Diseases & Immunology, Departments of Internal Medicine & Molecular Microbiology, Saint Louis University Health Sciences Center, 3635 Vista Ave., FDT-8N, St. Louis, MO 63110. Phone: (314) 577-8690. Fax: (314) 771-3816. E-mail: abateg{at}slu.edu.

REFERENCES

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