Baseline and multinormal distribution of ex vivo susceptibilities of Plasmodium falciparum to methylene blue in Africa, 2013-18

Plasmodium falciparum resistance to most antimalarial compounds has emerged in Southeast Asia and spread to Africa. In this context, the development of new antimalarial drugs is urgent. To determine the baseline in vitro activity of methylene blue (Proveblue®) on African isolates and to determine whether parasites have different phenotypes of susceptibility to methylene blue. Ex vivo susceptibility to methylene blue was measured for 609 P. falciparum isolates of patients hospitalized in France for malaria imported from Africa. A Bayesian statistical analysis was designed to describe the distribution of median effective concentration (EC50) estimates. The EC50 ranged from 0.16 to 87.2 nM with a geometric mean of 7.17 nM (95% CI = 6.21-8.13). The 609 EC50 values were categorized into four components: A (mean = 2.5 nM; 95% CI = 2.28-2.72), B (mean = 7.44 nM; 95% CI = 7.07-7.81), C (mean = 16.29 nM; 95% CI = 15.40-17.18) and D (mean = 38.49 nM; 95% CI = 34.14-42.84). The threshold value for in vitro reduced susceptibility to methylene blue was estimated at 35 nM using the geometric mean of EC50 plus 2 SDs of the 609 isolates. This cut-off also corresponds to the lower limit of the 95% CI of the methylene blue EC50 of component D. Thirty-five isolates (5.7%) displayed EC50 values above this threshold. Methylene blue exerts a promising efficacy against P. falciparum and is a potential partner for triple combinations.