Adding methylene blue to artemisinin-based combination therapy was safe in patients with malaria and resulted in faster parasite clearance than artemisinin-based combination therapy alone, according to results of a phase 2 trial.

The findings support previous research that show methylene blue (MB) has potent in vitro activity against Plasmodium falciparum, Teun Bousema, PhD, of the Radboud Institute for Health Sciences at Radboud University Medical Center, the Netherlands, and colleagues reported in The Lancet Infectious Diseases.

“There is already a body of evidence pointing toward an effect of methylene blue (MB) on malaria. We performed the first formal clinical trial to establish this effect in malaria patients,” Bousema told Infectious Disease News. “The novelty of our study is that MB is extremely potent against the transmission stages of malaria parasites that resist most common treatments.”

According to the researchers, sulfadoxine-pyrimethamine plus amodiaquine and diydroartemisinin-piperaquine are two regimens commonly used to treat malaria and prevent transmission. However, neither regimen targets the gametocyte stage of P. falciparum. During the gametocyte stage, parasites split in patients’ red blood cells to form male and female sex cells, according to a press release. If another mosquito bites the patient during this phase, it can ingest these cells, which then fertilize in the mosquito’s stomach. The offspring can travel to the mosquito’s salivary glands, where they are transmitted during the next bloodmeal.

Primaquine is a gametocytocidal drug that WHO currently recommends administering with standard therapy to help reduce P. falciparum transmission, according to the researchers. Although MB also has high gametocytocidal effects, its efficacy has not been confirmed in humans.

For the phase 2 trial, Bousema and colleagues investigated the impact of both primaquine and MB in patients with asymptomatic P. falciparum malaria in Mali, where malaria transmission is hyperendemic. The researchers randomly assigned 80 participants to receive treatment with sulfadoxine-pyrimethamine and amodiaquine (n = 20); sulfadoxine-pyrimethamine and amodiaquine plus 0.25 mg/kg of primaquine (n = 20); dihydroartemisinin-piperaquine (n = 20); or dihydroartemisinin-piperaquine plus 15 mg/kg of MB daily for 3 days (n = 20).

Anopheles gambiae mosquitoes fed on participants for 15 to 20 minutes before and after treatment. The mosquitoes were transported to an insectary, where they were dissected and assessed for infectivity after feeding. The primary endpoint was the change in mosquito infectivity 2 and 7 days after treatment.


After treatment, no patients in the dihydroartemisinin-piperaquine plus MB group were infectious on day 2 vs. 65% of patients who received only dihydroartemisinin-piperquine. Half of patients in the dihydroartemisinin-piperquine group were still infectious on day 7.

Among patients who received sulfadoxine-pyrimethamine and amodiaquine, 5% who also received primaquine were infectious at day 2 vs. 60% who did not receive primaquine. By day 7, infectivity was observed in 0 patients who received primaquine vs. 58% who did not.

Adverse events were similar across the treatment groups, suggesting primaquine and MB are safe, according to the researchers. However, blue urine was frequently reported among participants who received MB.

“This is something that we need to solve because it could stop people from using it,” Bousema said in the release.

Overall, 74% of participants reported a total of 162 adverse events, 90% of which were mild. Bousema said the research team’s next step is to identify the lowest optimal dose of MB in a formal dose-finding study.

“Once we know the lowest efficacious dose, we can work toward an easy dosing schedule and may expand our work to areas in Southeast Asia where antimalaria drugs are under considerable pressure and transmission-blocking drugs may be of particular use,” he said.

In a related editorial, Ric N. Price, MD, FRCP, FRCPath, FRACP, and Nicholas J. White, MD, FRCP, F Med Sci, DSc, FBPhS, of the department of clinical medicine at the University of Oxford, highlighted the need for additional gametocytocidal agents other than primaquine.

“Reliance on a single therapeutic intervention to reduce mosquito infectivity is risky,” they wrote. “Artemisinin-resistant P. falciparum has emerged in the Greater Mekong subregion and is spreading. In affected areas, patients treated with artemisinin-based combination therapy take longer to clear their peripheral parasitemia and are at risk of having patent gametocytemia and failing treatment, all of which fuel the spread of resistance to both artemisinin and its partner drugs. This study suggests that [MB] is a potential alternative gametocytocidal drug.” – by Stephanie Viguers

Disclosures: Bousema reports being supported by a grant from the Bill & Melinda Gates Foundation. Price and White report no relevant financial disclosures. Please see the study for all other authors’ relevant financial disclosures.