Potential of a soluble tetrazolium/formazan assay for the evaluation of filarial viability

Comley, J. C. W.; Turner, Claire H.

doi:10.1016/0020-7519(90)90107-x

Cited by 17 publications

(12 citation statements)

References 2 publications

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“…XTT-based assays have also been used in a variety of eukaryotic cell drug assays (3,17,21). We describe a drug susceptibility assay for yeasts based on XTT reduction and quantitation of the resulting colored formazan solution.…”

Section: Methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-mentioning

confidence: 99%

“…Recently a new tetrazolium salt, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT), which after reduction yields a water-soluble formazan, has been synthesized (14). XTT-based assays have also been used in a variety of eukaryotic cell drug assays (3,17,21). We describe a drug susceptibility assay for yeasts based on XTT reduction and quantitation of the resulting colored formazan solution.…”

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confidence: 99%

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Innovative endpoint determination system for antifungal susceptibility testing of yeasts

Tellier¹,

Krajden²,

Grigoriew³

et al. 1992

Antimicrob Agents Chemother

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Fungal infections and antifungal resistance are increasingly recognized. Antifungal susceptibility testing remains unstandardized, and a particularly important problem is endpoint determination. In this paper we propose the yeast metabolic reduction of the tetrazolium salt 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyll-2H-tetrazolium hydroxide (XTT) as a colorimetric endpoint which is quantitative and objective. Amphotericin B, fluorocytosine, and fluconazole dose-response curves were obtained, and a metabolic MIC could be determined by using precise criteria.Yeast infections are an increasingly important problem, particularly in immunocompromised patients. The recognition of antifungal resistance stresses the need for improved susceptibility testing. Unfortunately, antifungal drug susceptibility assays have been difficult to standardize (6,11,15,16,18). While considerable progress has been made, the determination of endpoints is still not satisfactory. In most assays endpoints are determined by visually grading turbidity, a subjective operation, particularly problematic when testing azole drugs for which trailing endpoints are common (4,7,9). In this paper we describe an innovative colorimetric, quantitative method of endpoint determination for susceptibility testing based on the reduction of a tetrazolium salt.Tetrazolium salts are widely used as indicators of reducing systems (2, 10). When reduced they give rise to a colored formazan crystal. Because these agents are cleaved by various dehydrogenase enzymes, active mitochondria will produce colored formazans and provide a colorimetric method to detect metabolically active cells. By using a spectrophotometer or an enzyme-linked immunosorbent assay (ELISA) plate reader, this methodology is amenable to quantitation. One tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (MTT), has been used for anticancer drug assays (1,13,20).Historically, formazans were water insoluble and could not easily be quantified without performing additional steps involving centrifugation of cells and removal of media, use of an organic solvent such as dimethyl sulfoxide, and occasionally sonication. Recently a new tetrazolium salt, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT), which after reduction yields a water-soluble formazan, has been synthesized (14). XTT-based assays have also been used in a variety of eukaryotic cell drug assays (3,17,21). We describe a drug susceptibility assay for yeasts based on XTT reduction and quantitation of the resulting colored formazan solution.MATERIALS AND METHODS Organisms. Most yeast strains were clinical isolates from the microbiology laboratory of the Toronto Hospital (numbers refer to the Toronto Hospital collection number). Ad-* Corresponding author. ditional strains of Candida lusitaniae (including C. lusitaniae FR-8900) were obtained from R. Summerbell (Provincial Health Laboratory, Toronto, Canada). Organisms were maintained on Sabouraud ...

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Section: Methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-mentioning

confidence: 99%

mentioning

confidence: 99%

Innovative endpoint determination system for antifungal susceptibility testing of yeasts

Tellier¹,

Krajden²,

Grigoriew³

et al. 1992

Antimicrob Agents Chemother

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“…Assays have been developed in recent years that score worm migration, feeding and development [3]–[19] as well as worm viability based on the MTT ((3-(4,5-dimethylthiazola-2-yl)-2, S-diphenyl tetrazolium bromide) assay [7], [8], [11]–[13], [20], [21], but these are not amenable to screening 1000's of compounds with a quick turnaround time on large worms such as filarid nematodes. Parasite movement is an important indication of the effectiveness of a drug and constitutes a crucial phenotype for HTS.…”

Section: Introductionmentioning

confidence: 99%

WormAssay: A Novel Computer Application for Whole-Plate Motion-based Screening of Macroscopic Parasites

et al. 2012

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Lymphatic filariasis is caused by filarial nematode parasites, including Brugia malayi. Adult worms live in the lymphatic system and cause a strong immune reaction that leads to the obstruction of lymph vessels and swelling of the extremities. Chronic disease leads to the painful and disfiguring condition known as elephantiasis. Current drug therapy is effective against the microfilariae (larval stage) of the parasite, but no drugs are effective against the adult worms. One of the major stumbling blocks toward developing effective macrofilaricides to kill the adult worms is the lack of a high throughput screening method for candidate drugs. Current methods utilize systems that measure one well at a time and are time consuming and often expensive. We have developed a low-cost and simple visual imaging system to automate and quantify screening entire plates based on parasite movement. This system can be applied to the study of many macroparasites as well as other macroscopic organisms.

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“…A new tetrazolium salt, sodium 3'{1-[(phenylamino)-carbonyl]-3,4-tetrazolium}-bis (4-methoxy-6-nitro)benzenesulfonic acid hydrate (XTT), with a corresponding watersoluble formazan has been synthesized recently (18). This tetrazolium salt appeared promising as an indicator of viability in assays with eucaryotic cells, including in vitro screening of chemotherapeutic drugs, estimation of filarial viability, and susceptibility of yeasts to antifungal drugs (4,24,28). These assays have required addition of an electroncoupling agent, such as phenazine methosulfate or menadione, to give satisfactory color accumulation.…”

mentioning

confidence: 99%

Application of a Tetrazolium Salt with a Water-Soluble Formazan as an Indicator of Viability in Respiring Bacteria

Roslev

King

1993

Appl Environ Microbiol

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The tetrazolium salt sodium 3'4{1-[(phenylamino)-carbonyll-3,4-tetrazolium}-bis (4-methoxy-6-nitro)benzenesulfonic acid hydrate (XTT) was examined for use as a colorimetric indicator of viability in respiring bacteria. XTT was reduced to an orange, water-soluble formazan product by Methylosinus trichosporium OB3b, Pseudomonas putida, Escherichia coli, and Bacilus subtilis. Formazan production was proportional to live cell biomass, and XTT was reduced by all cultures in the absence of added electron-coupling agents. XTT reduction by M. trichosporium OB3b was linear over several hours and was stimulated by the presence of an exogenous substrate (methanol). Addition of cyanide to cultures incubated under oxic conditions gave an initial 10-fold increase in XTT reduction. Viability of bacteria incubated in the absence of exogenous carbon substrates was measured as XTT reduction and compared with viability estimates from plate counts. Results obtained with the two methods were generally comparable, but the XTI7 assay was superior when cell recovery on plates was low. Incubation of E. coli for 7 days in the absence of exogenous carbon substrates decreased viability by 90%, whereas the corresponding decreases for cultures ofM. trichosporium OB3b, P. putida, and B. subtilis were less than 4%o.

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Potential of a soluble tetrazolium/formazan assay for the evaluation of filarial viability

Cited by 17 publications

References 2 publications

Innovative endpoint determination system for antifungal susceptibility testing of yeasts

Innovative endpoint determination system for antifungal susceptibility testing of yeasts

WormAssay: A Novel Computer Application for Whole-Plate Motion-based Screening of Macroscopic Parasites

Application of a Tetrazolium Salt with a Water-Soluble Formazan as an Indicator of Viability in Respiring Bacteria

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