Evaluation of the accuracy of the microplate alamar blue assay and the proportion method for the prompt detection of Mycobacterium tuberculosis and susceptibility of multidrug-resistant Mycobacterium tuberculosis clinical isolates

Background: Tuberculosis is appraised to cause the deaths of more than a billion people in the last decades. Objectives: The current study compares the performance of microplate Alamar blue assay for clinical isolates of Mycobacterium tuberculosis and multidrug-resistant tuberculosis. Microplate Alamar blue assay was performed in a central tuberculosis laboratory at Golestan University of Medical Sciences in Gorgan, Iran. Methods: In the first step, the microplate Alamar blue assay was used for the detection of 78 clinical isolates in the Golestan Regional Tuberculosis Reference Laboratory, and the results were compared with those of the proportion assay. In the second step, the microplate Alamar blue assay and the proportion assay were used for the drug susceptibility of 35 isolates. Results: In the microplate Alamar blue assay, the sensitivity was 100 (90.97 - 100), with a specificity of 74.36 (57.87 - 86.96), positive predictive value of 79.59 (65.66 - 89.76), and negative predictive value of 100 (88.06 - 100). For the microplate Alamar blue assay with rifampin, the sensitivity was 100 (89.11 - 100), specificity was 100 (29.24 - 100), positive predictive value was 100 (89.11 - 100), and negative predictive value was 100 (29.24 - 100). For the microplate Alamar blue assay with isoniazid, the sensitivity was 84.38 (67.21 - 94.72), specificity was 66.67 (9.43 - 99.16), positive predictive value was 96.43 (81.65 - 99.91), and negative predictive value was 28.57 (3.67 - 70.96). Conclusions: We found high accuracy between the microplate Alamar blue assay with rifampin and the proportion assay. The rapid and low-cost microplate Alamar blue assay is an inexpensive and appropriate assay for the detection of rifampin-resistant tuberculosis in low-income countries.

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Rapid, Low-Technology MIC Determination with Clinical Mycobacterium tuberculosis Isolates by Using the Microplate Alamar Blue Assay

Journal of Clinical Microbiology ◽

10.1128/jcm.36.2.362-366.1998 ◽

1998 ◽

Vol 36 (2) ◽

pp. 362-366 ◽

Cited By ~ 552

Author(s):

Scott G. Franzblau ◽

Richard S. Witzig ◽

James C. McLaughlin ◽

Patricia Torres ◽

Guillermo Madico ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Low Cost ◽

Multidrug Resistant ◽

Appropriate Technology ◽

Alamar Blue Assay ◽

Alamar Blue ◽

Solid Media ◽

Resistant Strains ◽

Stable Reagent ◽

Initial Results

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 PeruvianMycobacterium 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.

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Molecular Diagnosis of Fluoroquinolone Resistance in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04058-14 ◽

2014 ◽

Vol 59 (3) ◽

pp. 1519-1524 ◽

Cited By ~ 22

Author(s):

Christine Bernard ◽

Nicolas Veziris ◽

Florence Brossier ◽

Wladimir Sougakoff ◽

Vincent Jarlier ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

False Negative ◽

Real Life ◽

Multidrug Resistant ◽

Clinical Isolates ◽

Fluoroquinolone Resistance ◽

Single Mutation ◽

Content Type ◽

Negative Results ◽

Proportion Method

ABSTRACTAs a consequence of the use of fluoroquinolones (FQ), resistance to FQ has emerged, leading to cases of nearly untreatable and extensively drug-resistant tuberculosis. Mutations in DNA gyrase represent the main mechanism of FQ resistance. A full understanding of the pattern of mutations found in FQ-resistant (FQr) clinical isolates, and of their proportions, is crucial for improving molecular methods for the detection of FQ resistance inMycobacterium tuberculosis. In this study, we reviewed the detection of FQ resistance in isolates addressed to the French National Reference Center for Mycobacteria from 2007 to 2012, with the aim of evaluating the performance of PCR sequencing in a real-life context.gyrAandgyrBsequencing, performed prospectively onM. tuberculosisclinical isolates, was compared for FQ susceptibility to 2 mg/liter ofloxacin by the reference proportion method. A total of 605 isolates, of which 50% were multidrug resistant, were analyzed. The increase in FQrstrains among multidrug-resistant (MDR) strains during the time of the study was alarming (8% to 30%). The majority (78%) of the isolates withgyrAmutations were FQr, whereas only 36% of those withgyrBmutations were FQr. Only 12% of the FQrisolates had a single mutation ingyrB. CombinedgyrAandgyrBsequencing led to >93% sensitivity for detecting resistance. The analysis of the four false-positive and the five false-negative results ofgyrAandgyrBsequencing illustrated the actual limitations of the reference proportion method. Our data emphasize the need for combinedgyrAandgyrBsequencing in the investigation of FQ susceptibility inM. tuberculosisand challenge the validity of the current phenotype-based approach as the diagnostic gold standard for determining FQ resistance.

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Antituberculosis Activity of Brotowali (Tinospora crispa) Extract and Fractions against Mycobacterium tuberculosis using Microplate Alamar Blue Assay Method

Majalah Obat Tradisional ◽

10.22146/tradmedj.27925 ◽

2017 ◽

Vol 22 (2) ◽

pp. 124

Author(s):

Retno Wahyuningrum ◽

Ritmaleni Ritmaleni ◽

Tatang Irianti ◽

Subagus Wahyuono ◽

Takushi Kaneko ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Pathogenic Bacteria ◽

Ethanolic Extract ◽

Multidrug Resistant ◽

Alamar Blue Assay ◽

Assay Method ◽

Drug Candidate ◽

Antituberculosis Activity ◽

Alamar Blue ◽

Resistant Tuberculosis

Tuberculosis (TB), in which caused by pathogenic bacteria, Mycobacterium tuberculosis, has become the major causes of death among all of infectious diseases. The increasing incidence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) has created a need to discover a new antituberculosis drug candidate. The aim of this study was to screen extract and fractions of Tinospora crispa for activity against Mycobacterium tuberculosis H37Rv using the Microplate Alamar Blue Assay (MABA) method. T. crispa extract was prepared by maceration in ethanol (96%) and antituberculosis activity was carried out using MABA method. The result of this study showed that ethanolic extract of T. crispa exhibit antituberculosis activity with minimum inhibition concentration of 12.5 mg/ml.

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Resazurin Microtiter Assay Plate: Simple and Inexpensive Method for Detection of Drug Resistance in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.8.2720-2722.2002 ◽

2002 ◽

Vol 46 (8) ◽

pp. 2720-2722 ◽

Cited By ~ 630

Author(s):

Juan-Carlos Palomino ◽

Anandi Martin ◽

Mirtha Camacho ◽

Humberto Guerra ◽

Jean Swings ◽

...

Keyword(s):

Drug Resistance ◽

Mycobacterium Tuberculosis ◽

Multidrug Resistant ◽

Clinical Isolates ◽

Inexpensive Method ◽

Antituberculosis Drugs ◽

Specificity And Sensitivity ◽

Proportion Method ◽

Microtiter Assay

ABSTRACT A method for detecting multidrug-resistant Mycobacterium tuberculosis by using a reduction of resazurin is described. Eighty clinical isolates were evaluated against isoniazid and rifampin; results at 7 days were compared with those of the proportion method. Specificity and sensitivity were excellent. The method is simple, inexpensive, and rapid and might be used with other antituberculosis drugs.

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Rapid drug susceptibility testing for Mycobacterium tuberculosis in Thin layer agar media

Bangladesh Journal of Medical Microbiology ◽

10.3329/bjmm.v7i1.19313 ◽

2013 ◽

Vol 7 (1) ◽

pp. 02-06 ◽

Cited By ~ 1

Author(s):

Habiba Binte Alam ◽

Md. Ruhul Amin Miah ◽

S. M. Mostafa Kamal ◽

Chandan Kumar Roy ◽

Ahmed Abu Saleh

Keyword(s):

Mycobacterium Tuberculosis ◽

Thin Layer ◽

Rapid Detection ◽

Drug Susceptibility ◽

Multidrug Resistant ◽

Drug Susceptibility Testing ◽

Clinical Isolates ◽

Resistant Tuberculosis ◽

Proportion Method ◽

Petri Plate

There is a great need to determine the susceptibility of individual Mycobacterium tuberculosis strains as rapidly as possible because emergence of multidrug-resistant and extensively drug-resistant tuberculosis in developing countries. The study was conducted to evaluate the thin layer agar (TLA) media for rapid detection of resistance of M.tuberculosis to rifampicin (RMP) and isoniazid (INH) in clinical isolates and to determine the sensitivity and time to positivity compared to the proportion method. One hundred clinical isolates of M.tuberculosis were studied. For the TLA method, three compartment Petri plate containing 7H11 agar and 7H11 agar with RMP and INH. Results were compared to the proportion method for RMP and INH. The sensitivity for INH and RMP+INH was 85.7 % and 100%. The use of a TLA plate enables the rapid detection of resistance to the two prime anti-tuberculosis drugs RMP and INH in a median time of 9.60 days. TLA was a rapid method for the detection of resistance of M.tuberculosis in the two drugs studied. This faster method is simple to perform, providing an alternative method when more sophisticated techniques are not available in low-resource settings.DOI: http://dx.doi.org/10.3329/bjmm.v7i1.19313 Bangladesh J Med Microbiol 2013; 07(01): 2-6

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