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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

Rapid suppression of parasitemia following drug-treatment is a poor indicator of drug efficacy and parasitological cure.(A) Evolution of parasitemia after infection with 1×103 CL strain of T. cruzi on day 0 in untreated (▪), BZ-40 (▵), POS (○), NTLA-1 (▴), or BIS767 (□) treated mice. “BIS767, BZ-40, POS and NTLA-1” bars below x axis indicate period of treatments. (B) Parasitemias in untreated or treated mice at 120dpi, after administration of the immunosuppressant cyclophosphamide (cy) (days 105, 108, 111, 113 and 117).
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pntd-0000740-g007: Rapid suppression of parasitemia following drug-treatment is a poor indicator of drug efficacy and parasitological cure.(A) Evolution of parasitemia after infection with 1×103 CL strain of T. cruzi on day 0 in untreated (▪), BZ-40 (▵), POS (○), NTLA-1 (▴), or BIS767 (□) treated mice. “BIS767, BZ-40, POS and NTLA-1” bars below x axis indicate period of treatments. (B) Parasitemias in untreated or treated mice at 120dpi, after administration of the immunosuppressant cyclophosphamide (cy) (days 105, 108, 111, 113 and 117).

Mentions: In previous work, we showed that BZ treatment in the acute or chronic phase of the infection can provide cure of mice infected with T. cruzi [43]. We sought to explore whether the same compounds used in the in vivo short term assays were effective to cure mice infected with T. cruzi. To address this question, C57BL/6 mice were infected with the wild-type CL strain of T. cruzi and treated with either BZ, POS, NTLA-1 or BIS767 or left untreated. All mice exhibited detectable parasitemias by day 14 post-infection; in untreated mice this acute phase parasitemia peaked at 21 dpi and became undetectable by approximately 35 dpi (Figure 7A). All of the compounds evaluated in this study suppressed parasitemia, which became undetectable in all cases by 21 dpi (Figure 7A). However, only the mice treated for 40 days with BZ, the majority of POS-treated (90%) and a small fraction of NLTA-1-treated mice (20%) were able to clear the infection and cure, as by the failure to detect parasitemias after cyclophosphamide (cy) immunosuppression (Figure 7B). These results demonstrate that suppression of parasitemia soon after drug-treatment initiation Figure 7A) is a poor indicator of drug efficacy and the potential for a compound to achieve parasitological cure over a long-term course of treatment. Moreover, the results of the long-term-treatment assay and the short-term in vitro screens using measurement of parasite growth in the foot-pad following the injection of luminescent or fluorescent T. cruzi (Figures 5 and 6) are perfectly concordant, suggesting that the short-term in vivo assay is strong predictor of in vivo drug efficacy and clearly superior to measuring suppression of parasitemia.


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)

Rapid suppression of parasitemia following drug-treatment is a poor indicator of drug efficacy and parasitological cure.(A) Evolution of parasitemia after infection with 1×103 CL strain of T. cruzi on day 0 in untreated (▪), BZ-40 (▵), POS (○), NTLA-1 (▴), or BIS767 (□) treated mice. “BIS767, BZ-40, POS and NTLA-1” bars below x axis indicate period of treatments. (B) Parasitemias in untreated or treated mice at 120dpi, after administration of the immunosuppressant cyclophosphamide (cy) (days 105, 108, 111, 113 and 117).
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0000740-g007: Rapid suppression of parasitemia following drug-treatment is a poor indicator of drug efficacy and parasitological cure.(A) Evolution of parasitemia after infection with 1×103 CL strain of T. cruzi on day 0 in untreated (▪), BZ-40 (▵), POS (○), NTLA-1 (▴), or BIS767 (□) treated mice. “BIS767, BZ-40, POS and NTLA-1” bars below x axis indicate period of treatments. (B) Parasitemias in untreated or treated mice at 120dpi, after administration of the immunosuppressant cyclophosphamide (cy) (days 105, 108, 111, 113 and 117).
Mentions: In previous work, we showed that BZ treatment in the acute or chronic phase of the infection can provide cure of mice infected with T. cruzi [43]. We sought to explore whether the same compounds used in the in vivo short term assays were effective to cure mice infected with T. cruzi. To address this question, C57BL/6 mice were infected with the wild-type CL strain of T. cruzi and treated with either BZ, POS, NTLA-1 or BIS767 or left untreated. All mice exhibited detectable parasitemias by day 14 post-infection; in untreated mice this acute phase parasitemia peaked at 21 dpi and became undetectable by approximately 35 dpi (Figure 7A). All of the compounds evaluated in this study suppressed parasitemia, which became undetectable in all cases by 21 dpi (Figure 7A). However, only the mice treated for 40 days with BZ, the majority of POS-treated (90%) and a small fraction of NLTA-1-treated mice (20%) were able to clear the infection and cure, as by the failure to detect parasitemias after cyclophosphamide (cy) immunosuppression (Figure 7B). These results demonstrate that suppression of parasitemia soon after drug-treatment initiation Figure 7A) is a poor indicator of drug efficacy and the potential for a compound to achieve parasitological cure over a long-term course of treatment. Moreover, the results of the long-term-treatment assay and the short-term in vitro screens using measurement of parasite growth in the foot-pad following the injection of luminescent or fluorescent T. cruzi (Figures 5 and 6) are perfectly concordant, suggesting that the short-term in vivo assay is strong predictor of in vivo drug efficacy and clearly superior to measuring suppression of parasitemia.

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