Evaluation of the chemosensitivity of asexual and sexual stages of P. falciparum field isolates by pLDH assay

Introduction: The number of malaria cases reported in Italy is low (500-600 per year). The L.Sacco Hospital is the reference infectious disease hospital of the metropolitan area of Milano. Cultivation and in vitro adaptation of fresh clinical isolates of Plasmodium falciparum is challenging and even more demanding if the growth of both sexual and asexual stages is attempted. This is crucial for in vitro screening of novel antimalarials and transmission blocking agents, as requested by the ongoing global elimination/eradication efforts. The aim of the present work was to attempt the long term cultivation and gametocytes differentiation of the P. falciparum isolated from patients in the L. Sacco Hospital independently from their geographic origin. Materials and methods: During the study period, 20 blood samples from malaria patients were received. The parasites were immediately put in culture using RPMI 1640 medium, sodium bicarbonate, hypoxanthine, HEPES and glutamine, in the presence of 1% AlbuMAX II (lipid-rich bovine serum albumin). Growing and stabilized cultures were then cultivated in the presence of 10% naturally clotted heat-inactivated human serum to allow gametocyte production. Gametocytogenesis was triggered by diluting the cultures to 0.5% parasitemia, and changing medium daily without the addition of erythrocytes. When a parasitemia of 5% was obtained and the parasites were stressed by nutrient deprivation, the cultures were treated with N-acetylglucosamine to clear residual asexual parasites and gametocyte differentiation was followed for 9-10 days. Chemosensitivity tests were done on both asexual and sexual stages of parasites using a panel of known antimalarial drugs in a modified version of the pLDH method. Results: The results indicated that 65% of fresh isoltates of P.falciparum adapted successfully to asexual culture. Moreover, 69% of the adapted isolates were able to produce mature gametocytes. Both asexual parasites and gametocytes were obtained in almost all cases in adequate quantity to perform the chemosensitivity assays. Within the field isolates, in the asexual stage, the distribution of results indicates a 100% susceptibility to DHA, atovaquone and mefloquine, whereas the response to CQ and quinine was differently distributed among the samples. The response of gametocytes was within the expected range seen using laboratory strains. Discussion and conclusions: We demonstrated that the pLDH method can be easily adapted to evaluate the chemosensitivity of field isolates to known or novel antimalarials, including transmission-blocking compounds. This confirms that the pLDH method has several advantages over other methods, it is reproducible, low cost, useful for both sexual and asexual stages and for field isolates.