The effectiveness of one dose of varicella vaccine
International Journal of Infectious Diseases
The commonly used drugs against T. cruzi are nifurtimox and benznidazole but they both have adverse effects for humans. Clinical symptoms of Chagas disease also depends on the strain present in each region. Drug screening methods are tedious and expensive. High-throughput systems are required for screening anti-T. cruzi agents. The use of transfected parasites expressing reporter genes likě -galactosidase has facilitated drug screening. However, luciferase (luc) has a higher sensibility. In our
... studies we found clinical and serological differences between the Peruvian and Bolivian strain. The Bolivian strain seems more virulent as it has been documented with a high number of acute and chronic cases. Here we developed transgenic parasites expressing luciferase gene for having a new drug screening method. Methods: Bolivian strain (DH29) was isolated from a child who acquired Chagas Disease by congenital infection based on microscopic examination and parasites were recovered by in vitro cultivation. The open reading frame of luc was inserted in two vectors: pBS:CL-Neo-01/BC-LacZ-10 (pLAC) and pTREX. Stable transfectans were isolated with G418 following integration of the circular transforming plasmids into the nucleus of epimastigotes by electroporation. Luciferase was detected by PCR, western blot and its activity with a luminometer. Results: Two plasmids were developed pBS-Neo-Luc and pR-Luc-Neo. Drug (G418)-resistant parasites were isolated after three weeks post electroporation. PCR (1.7Kb) and western blot (61 KD) detected luciferase gene. Transfected parasites incubated with luciferin showed luciferase activity. The clones that failed to express luciferase were found to have integrated the neomicin gene. The clones that expressed luciferase were stable for more than six months without selection. Two clones (L95 y L97) expressing luciferase were isolated from DH29 strain. There is a correlation between the enzyme activity and number of parasites. Luciferase assay was able to detect 102 parasites ml-1. Conclusion: The constructed plasmids pBS-Neo-Luc and el pR-Luc-Neo are useful for stable expression in T. cruzi. Two clones L95 and L97 from DH29 strain of T. cruzi expressing luciferase gene were developed.There is a linear correlation between luciferase activity and the number of parasites ml-1. Luciferase assay is able to detect at least 102 parásitos ml-1.