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Optimization of a Fluorescence-Based Assay for Large-Scale Drug Screening against Babesia and Theileria Parasites.

ABSTRACT: A rapid and accurate assay for evaluating antibabesial drugs on a large scale is required for the discovery of novel chemotherapeutic agents against Babesia parasites. In the current study, we evaluated the usefulness of a fluorescence-based assay for determining the efficacies of antibabesial compounds against bovine and equine hemoparasites in in vitro cultures. Three different hematocrits (HCTs; 2.5%, 5%, and 10%) were used without daily replacement of the medium. The results of a high-throughput screening assay revealed that the best HCT was 2.5% for bovine Babesia parasites and 5% for equine Babesia and Theileria parasites. The IC50 values of diminazene aceturate obtained by fluorescence and microscopy did not differ significantly. Likewise, the IC50 values of luteolin, pyronaridine tetraphosphate, nimbolide, gedunin, and enoxacin did not differ between the two methods. In conclusion, our fluorescence-based assay uses low HCT and does not require daily replacement of culture medium, making it highly suitable for in vitro large-scale drug screening against Babesia and Theileria parasites that infect cattle and horses.

SUBMITTER: Rizk MA

PROVIDER: S-EPMC4411034 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Optimization of a Fluorescence-Based Assay for Large-Scale Drug Screening against Babesia and Theileria Parasites.

Rizk Mohamed Abdo MA El-Sayed Shimaa Abd El-Salam SA Terkawi Mohamed Alaa MA Youssef Mohamed Ahmed MA El Said El Said El Shirbini el Sel S Elsayed Gehad G El-Khodery Sabry S El-Ashker Maged M Elsify Ahmed A Omar Mosaab M Salama Akram A Yokoyama Naoaki N Igarashi Ikuo I

PloS one 20150427 4

A rapid and accurate assay for evaluating antibabesial drugs on a large scale is required for the discovery of novel chemotherapeutic agents against Babesia parasites. In the current study, we evaluated the usefulness of a fluorescence-based assay for determining the efficacies of antibabesial compounds against bovine and equine hemoparasites in in vitro cultures. Three different hematocrits (HCTs; 2.5%, 5%, and 10%) were used without daily replacement of the medium. The results of a high-throug ...[more]

PMID: 25915529

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Project description:BACKGROUND:The plenteous resistance to and undesirable consequences of the existing antipiroplasmic therapies have emphasized the urgent need for new chemotherapeutics and drug targets for both prophylaxis and chemotherapy. Hydroxyurea (HYD) is an antineoplastic agent with antitrypanosomal activity. Eflornithine (?-difluoro-methyl ornithine, DFMO) is the best choice therapy for the treatment of late-stage Gambian human African trypanosomiasis. METHODS:In this study, the inhibitory and combination efficacy of HYD and DFMO with existing babesicidal drugs (diminazene aceturate (DA), atovaquone (ATV), and clofazimine (CLF)) deoxyribonucleotide in vitro against the multiplication of Babesia and Theileria. As well as, their chemotherapeutic effects were assessed on B. microti strain that infects rodents. The Cell Counting Kits-8 (CCK-8) test was used to examine their cytotoxicity on human foreskin fibroblast (HFF), mouse embryonic fibroblast (NIH/3T3), and Madin-Darby bovine kidney (MDBK) cells. FINDINGS:HYD and DFMO suppressed the multiplication of all tested species (B. bigemina, B. bovis, B. caballi, B. divergens, and T. equi) in a dose-related manner. HFF, NIH/3T3, or MDBK cell viability was not influenced by DFMO at 1000 ?M, while HYD affected the MDBK cell viability at EC50 value of 887.5±14.4 ?M. The in vitro combination treatments of DFMO and HYD with CLF, DA, and ATV exhibited synergistic and additive efficacy toward all tested species. The in vivo experiment revealed that HYD and DFMO oral administration at 100 and 50 mg/kg inhibited B. microti multiplication in mice by 60.1% and 78.2%, respectively. HYD-DA and DFMO-DA combined treatments showed higher chemotherapeutic efficacy than their monotherapies. CONCLUSION:These results indicate the prospects of HYD and DFMO as drug candidates for piroplasmosis treatment, when combined mainly with DA, ATV, and CLF. Therefore, further studies are needed to combine HYD or DFMO with either ATV or CLF and examine their impact on B. microti infection in mice.

Project description:BackgroundThe emergence and spread of drug resistance in Trichomonas vaginalis parasites has become an important concern in trichomoniasis treatment. Fast and reliable growth assessment is critical for validating in vitro drug susceptibility and high-throughput screening of newly developed drugs.MethodsModified media without yeast extract were evaluated for their ability to support the growth of T. vaginalis parasites. The potential of the nucleic acid-binding dye SYBR Green I for detecting T. vaginalis drug resistance was characterized, and seeding parasite concentration and incubation time were optimized. The fluorescence assay based on SYBR Green I was further validated in four T. vaginalis isolates with different susceptibilities to the antibiotics metronidazole, tinidazole, ornidazole and secnidazole, and compared with the traditional method that detects minimum lethal concentrations (MLCs).ResultsA modified medium consisting of RPMI 1640 and Tryptone Plus as replacements for yeast extract and tryptone, respectively, in traditional trypticase-yeast extract-maltose (TYM) medium exhibited similar performance as TYM medium in maintaining T. vaginalis growth, while it showed much lower background fluorescent signals. The T. vaginalis SYBR Green I-based fluorescence (TSF) drug assay was found to have to satisfy one of two conditions to demonstrate the 50% inhibitory concentration of metronidazole for the sensitive isolate TV-334: (i) a seeding density of 3 × 104 parasites/ml and an incubation time of 48 h; or (ii) a seeding density of 1 × 104 parasites/ml and an incubation time of 72 h. Subsequent validation experiments revealed that the 48-h incubation/3 × 104 parasites/ml seeding density condition had a greater sensitivity to detect drug resistance than the 72-h condition. The TSF assay also exhibited high efficiency in identifying parasite drug resistance, as evidenced by its strong correlation with the standard MLC assay results (P = 0.003).ConclusionsThis study presents a robust TSF assay that has the potential to facilitate high-throughput, automated in vitro anti-trichomoniasis susceptibility testing for drug resistance monitoring and drug development. In comparison to the standard MLC method, this assay offers the advantages of reduced labor and elimination of subjective examination.

Dataset Information

Optimization of a Fluorescence-Based Assay for Large-Scale Drug Screening against Babesia and Theileria Parasites.

Publications

Optimization of a Fluorescence-Based Assay for Large-Scale Drug Screening against Babesia and Theileria Parasites.

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