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In vitro and in vivo high-throughput assays for the testing of anti-Trypanosoma cruzi compounds.

Canavaci AM, Bustamante JM, Padilla AM, Perez Brandan CM, Simpson LJ, Xu D, Boehlke CL, Tarleton RL - PLoS Negl Trop Dis (2010)

Bottom Line: The two available drugs for treatment of T. cruzi infection, nifurtimox and benznidazole (BZ), have potential toxic side effects and variable efficacy, contributing to their low rate of use.In this work, we describe the development and validation of improved methods to test anti-T. cruzi compounds in vitro and in vivo using parasite lines expressing the firefly luciferase (luc) or the tandem tomato fluorescent protein (tdTomato).This method was highly reproducible and had the added advantage of requiring relatively low numbers of parasites and no additional indicator reagents, enzymatic post-processes or laborious visual counting.

View Article: PubMed Central - PubMed

Affiliation: Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, Georgia, United States of America.

ABSTRACT

Background: The two available drugs for treatment of T. cruzi infection, nifurtimox and benznidazole (BZ), have potential toxic side effects and variable efficacy, contributing to their low rate of use. With scant economic resources available for antiparasitic drug discovery and development, inexpensive, high-throughput and in vivo assays to screen potential new drugs and existing compound libraries are essential.

Methods: In this work, we describe the development and validation of improved methods to test anti-T. cruzi compounds in vitro and in vivo using parasite lines expressing the firefly luciferase (luc) or the tandem tomato fluorescent protein (tdTomato). For in vitro assays, the change in fluorescence intensity of tdTomato-expressing lines was measured as an indicator of parasite replication daily for 4 days and this method was used to identify compounds with IC(50) lower than that of BZ.

Findings: This method was highly reproducible and had the added advantage of requiring relatively low numbers of parasites and no additional indicator reagents, enzymatic post-processes or laborious visual counting. In vivo, mice were infected in the footpads with fluorescent or bioluminescent parasites and the signal intensity was measured as a surrogate of parasite load at the site of infection before and after initiation of drug treatment. Importantly, the efficacy of various drugs as determined in this short-term (<2 weeks) assay mirrored that of a 40 day treatment course.

Conclusion: These methods should make feasible broader and higher-throughput screening programs needed to identify potential new drugs for the treatment of T. cruzi infection and for their rapid validation in vivo.

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Related in: MedlinePlus

Fluorescent T. cruzi-tdTomato expressing parasites imaged post-treatment.Mice (10 per group) were infected in the hind foot pads with 2.5×105 T. cruzi tdTomato trypomastigotes and the images were taken every two days from day 1 to 11 post infection. (A) Images from days 5, 7 and 9 post infection. (B) Quantification of the fluorescent signal from mice in panel A at all imaging points.
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pntd-0000740-g005: Fluorescent T. cruzi-tdTomato expressing parasites imaged post-treatment.Mice (10 per group) were infected in the hind foot pads with 2.5×105 T. cruzi tdTomato trypomastigotes and the images were taken every two days from day 1 to 11 post infection. (A) Images from days 5, 7 and 9 post infection. (B) Quantification of the fluorescent signal from mice in panel A at all imaging points.

Mentions: Both the tdTomato and the luc-based assays revealed a rapid in vivo parasite clearance activity for BZ and POS, with dramatic control of parasite load within 1 day with BZ and slightly longer in the case of POS (Figures 5 and 6). In contrast, none of the other test compounds exhibited significant effects on in vivo parasite growth, despite their previously demonstrated in vitro anti-T. cruzi activity.


In vitro and in vivo high-throughput assays for the testing of anti-Trypanosoma cruzi compounds.

Canavaci AM, Bustamante JM, Padilla AM, Perez Brandan CM, Simpson LJ, Xu D, Boehlke CL, Tarleton RL - PLoS Negl Trop Dis (2010)

Fluorescent T. cruzi-tdTomato expressing parasites imaged post-treatment.Mice (10 per group) were infected in the hind foot pads with 2.5×105 T. cruzi tdTomato trypomastigotes and the images were taken every two days from day 1 to 11 post infection. (A) Images from days 5, 7 and 9 post infection. (B) Quantification of the fluorescent signal from mice in panel A at all imaging points.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2903469&req=5

pntd-0000740-g005: Fluorescent T. cruzi-tdTomato expressing parasites imaged post-treatment.Mice (10 per group) were infected in the hind foot pads with 2.5×105 T. cruzi tdTomato trypomastigotes and the images were taken every two days from day 1 to 11 post infection. (A) Images from days 5, 7 and 9 post infection. (B) Quantification of the fluorescent signal from mice in panel A at all imaging points.
Mentions: Both the tdTomato and the luc-based assays revealed a rapid in vivo parasite clearance activity for BZ and POS, with dramatic control of parasite load within 1 day with BZ and slightly longer in the case of POS (Figures 5 and 6). In contrast, none of the other test compounds exhibited significant effects on in vivo parasite growth, despite their previously demonstrated in vitro anti-T. cruzi activity.

Bottom Line: The two available drugs for treatment of T. cruzi infection, nifurtimox and benznidazole (BZ), have potential toxic side effects and variable efficacy, contributing to their low rate of use.In this work, we describe the development and validation of improved methods to test anti-T. cruzi compounds in vitro and in vivo using parasite lines expressing the firefly luciferase (luc) or the tandem tomato fluorescent protein (tdTomato).This method was highly reproducible and had the added advantage of requiring relatively low numbers of parasites and no additional indicator reagents, enzymatic post-processes or laborious visual counting.

View Article: PubMed Central - PubMed

Affiliation: Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, Georgia, United States of America.

ABSTRACT

Background: The two available drugs for treatment of T. cruzi infection, nifurtimox and benznidazole (BZ), have potential toxic side effects and variable efficacy, contributing to their low rate of use. With scant economic resources available for antiparasitic drug discovery and development, inexpensive, high-throughput and in vivo assays to screen potential new drugs and existing compound libraries are essential.

Methods: In this work, we describe the development and validation of improved methods to test anti-T. cruzi compounds in vitro and in vivo using parasite lines expressing the firefly luciferase (luc) or the tandem tomato fluorescent protein (tdTomato). For in vitro assays, the change in fluorescence intensity of tdTomato-expressing lines was measured as an indicator of parasite replication daily for 4 days and this method was used to identify compounds with IC(50) lower than that of BZ.

Findings: This method was highly reproducible and had the added advantage of requiring relatively low numbers of parasites and no additional indicator reagents, enzymatic post-processes or laborious visual counting. In vivo, mice were infected in the footpads with fluorescent or bioluminescent parasites and the signal intensity was measured as a surrogate of parasite load at the site of infection before and after initiation of drug treatment. Importantly, the efficacy of various drugs as determined in this short-term (<2 weeks) assay mirrored that of a 40 day treatment course.

Conclusion: These methods should make feasible broader and higher-throughput screening programs needed to identify potential new drugs for the treatment of T. cruzi infection and for their rapid validation in vivo.

Show MeSH
Related in: MedlinePlus