Behavior of Plasmodium falciparum against first-line regimens for malaria: evaluation of in vitro sensitivity and artemisinin combination therapyinduced parasite dormancy

Author(s)

Rosa Del Carmen Miluska Vargas Rodriguez

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina (FM/SBD)
• Defense date: 2016-12-06

Examining board members

Sílvia Maria Fátima di Santi; André Gustavo Tempone Cardoso; Maria Irma Seixas Duarte; José Angelo Lauletta Lindoso

• Advisor: Sílvia Maria Fátima di Santi

Abstract

The phenotypic characterization of Plasmodium falciparum is useful for the knowledge of parasite sensitivity against antimalarial used in endemic countries. In this study we evaluated the sensitivity of clinical isolates of P. falciparum from the African continent and the Caribbean. The sensitivity to dihydroartemisinin (DHA: 4 - 1,000nM), artesunate (AS: 0.1 - 100 nM), lumefantrine (LMF: 3.1 to 200 nM), and mefloquine (MFQ: 0.2 to 1,000 nM) was investigated through four techniques: (a) ex vivo and in vitro microtests, (b) dormancy assay, (c) flow cytometry assay and (d) survival assay using young trophozoites (Ring Stage Survival Assay - RSA). In the ex vivo and in vitro experiments, the IC50 was calculated and was 0.4 - 66.6 nM for DHA; 3.8 - 48.8 nM for LMF; 0.3 - 25.9 nM for AS and 2 - 439 nM for MFQ. According to dormancy assays, schizonts were observed in the P. falciparum reference isolate NF54 and in the clinical isolate S-01/15 after pressure with 62.5 nM, 250 nM and 1,000 nM DHA. The recovery period ranged from 4 to 40 days. For LMF was observed the growth to the schizont stage in NF54, in the days 7 e 12 after exposure to 66.6 nM and 200 nM of the drug, respectively. Schizonts were seen in the P. falciparum clinical isolate FS-08/15 after pressure with 100 nM of AS, right after incubation period, with no dormancy of trophozoites. In flow cytometry assays, viable young trophozoites of P. falciparum labeled with DAPI and Rhodamine 123 were observed at the maximum concentrations of DHA (1,000 nM) and LMF (200 nM). Finally in RSA, the growth rate (GR) and percentage of survival (PS) of the reference isolate was 2.92 and 4.19%, respectively, after pressure with 700 nM of DHA. The same isolate pressed with 3,500 nM of LMF presented GR of 3.6% and PS of 2.25%. In conclusion, microscopic evaluation of ex vivo and in vitro sensitivity tests underestimates the P. falciparum response to artemisinin-based combination therapy (ACT). Our results suggest that the dormancy, main mechanism of tolerance to artemisinin (ART), is not presented in all clinical isolates of P. falciparum. Flow cytometry was able to confirm the parasite viability accurately. In this study, for the first time the dormancy of P. falciparum after pressure with LMF was reported (AU)

• FAPESP's process: 13/19588-8 - Behavior of Plasmodium falciparum against first-line regimens for malaria: evaluation of in vitro sensitivity and dormancy mechanism of artemisinin combination therapies
• Grantee: Rosa Del Carmen Miluska Vargas Rodriguez
• Support Opportunities: Scholarships in Brazil - Doctorate