Methylene blue inhibits the asexual development of vivax malaria parasites from a region of increasing chloroquine resistance

Abstract

Objectives: Methylene blue, once discarded due to its unsettling yet mild side effects, has now found a renewed place in the pharmacopoeia of modern medicine. The continued spread of drug-resistant Plasmodium vivax and Plasmodium falciparum has also led to a recent re-examination of methylene blue's potent antimalarial properties. Here we examine the ex vivo susceptibility profile of Plasmodium spp. isolates to methylene blue; the isolates were from a region on the Thai-Myanmar border where there are increasing rates of failure when treating vivax malaria with chloroquine.

Methods: To do this we used a newly developed ex vivo susceptibility assay utilizing flow cytometry and a portable flow cytometer with a near-UV laser.

Results: P. vivax (median methylene blue IC50 3.1 nM, IQR 1.7-4.3 nM) and P. falciparum (median methylene blue IC50 1.8 nM, IQR 1.6-2.3 nM) are susceptible to methylene blue treatment at physiologically relevant levels. Unfortunately, the addition of chloroquine to combination treatments with methylene blue significantly reduces the ex vivo effectiveness of this molecule.

Conclusions: Our data support further efforts to employ methylene blue as a safe, low-cost antimalarial to treat drug-resistant malaria.

Keywords: Plasmodium falciparum; Plasmodium vivax; drug sensitivity assays; drug susceptibility assays.

Figure 1.

(a) Flow cytometry gating strategy for determination of schizont parasitaemia in a 42 h ex vivo culture of P. vivax in the drug-free (DF) control. (b) A series of scatter plots from the Accuri C6 (with near-UV and blue lasers) comparing the effect of three antimalarials [methylene blue (MB), artesunate (AS) and chloroquine (CQ)] at various concentrations (the first panel being the DF control) on the maturation of P. vivax schizonts (shown in the gate). The y-axis represents the Hoechst signal and the x-axis represents the ethidium signal. Below the MB panels are a series of Giemsa-stained thick films from the same wells measured by flow cytometry. The thick films clearly show complete schizont inhibition at the highest concentration and a sequential increase in the maturity of stages as the concentration of MB decreases (the lowest concentration predominated by late schizont forms).

Figure 2.

Comparison of the antimalarial effect of methylene blue (MB), artesunate (AS) and chloroquine (CQ) on (a) P. vivax (n = 25) and (b) P. falciparum (n = 16) isolates from the Thai–Myanmar border. The lines represent the median IC₅₀ values (nM) (also indicated by the numerical values on the graph). The repeated measure medians were compared using Friedman's and Dunn's post hoc tests.

Figure 3.

Repeated-measures comparison of methylene blue (MB) and chloroquine (CQ) treatments, alone and in combination, against the ex vivo maturation of P. vivax schizonts (n = 17). (a) The effect on IC₅₀ values (nM) if P. vivax is exposed to methylene blue for 20 h from the early ring stage (1–6 h post-invasion) and (b) shows the effect of methylene blue on late trophozoites (∼21–26 h post-invasion). Each line connects the same P. vivax isolate. Isolates are listed in decreasing order of susceptibility to chloroquine. *, <0.05; **, <0.01; ***, <0.001.