Abstract. Artemisinin-resistant Plasmodium falciparum malaria has been documented in southeast Asia and may already be spreading in that region. Molecular markers are important tools for monitoring the spread of antimalarial drug resistance. Recently, single-nucleotide polymorphisms (SNPs) in the PF3D7_1343700 kelch propeller (K13-propeller) domain were shown to be associated with artemisinin resistance in vivo and in vitro. The prevalence and role of K13-propeller mutations are poorly known in sub-Saharan Africa. K13-propeller mutations were genotyped by direct sequencing of nested polymerase chain reaction (PCR) amplicons from dried blood spots of pre-treatment falciparum malaria infections collected before and after the use of artemisinin-based combination therapy (ACT) as first-line therapy in Mali. Although K13-propeller mutations previously associated with delayed parasite clearance in Cambodia were not identified, 26 K13-propeller mutations were identified in both recent samples and pre-ACT infections. Parasite clearance time was comparable between infections with non-synonymous K13-propeller mutations and infections with the reference allele. These findings suggest that K13-propeller mutations are present in artemisinin-sensitive parasites and that they preceded the wide use of ACTs in Mali.

Chemotherapy is a critical component of malaria control. However, the most deadly malaria pathogen, Plasmodium falciparum, has repeatedly mounted resistance against a series of antimalarial drugs used in the last decades. Southeast Asia is an epicenter of emerging antimalarial drug resistance, including recent resistance to the artemisinins, the core component of all recommended antimalarial combination therapies. Alterations in the parasitic membrane proteins Pgh-1, PfCRT and PfMRP1 are believed to be major contributors to resistance through decreasing intracellular drug accumulation. The pfcrt, pfmdr1 and pfmrp1 genes were sequenced from a set of P.falciparum field isolates from the Thai-Myanmar border. In vitro drug susceptibility to artemisinin, dihydroartemisinin, mefloquine and lumefantrine were assessed. Positive correlations were seen between the in vitro susceptibility responses to artemisinin and dihydroartemisinin and the responses to the arylamino-alcohol quinolines lumefantrine and mefloquine. The previously unstudied pfmdr1 F1226Y and pfmrp1 F1390I SNPs were associated significantly with artemisinin, mefloquine and lumefantrine in vitro susceptibility. A variation in pfmdr1 gene copy number was also associated with parasite drug susceptibility of artemisinin, mefloquine and lumefantrine. Our work unveils new candidate markers of P. falciparum multidrug resistance in vitro, while contributing to the understanding of subjacent genetic complexity, essential for future evidence-based drug policy decisions.

Abstract. Plasmodium falciparum resistance to artemisinins by delayed parasite clearance is present in Southeast Asia. Scant data on parasite clearance after artemisinins are available from Africa, where transmission is high, burden is greatest, and artemisinin use is being scaled up. Children 1-10 years of age with uncomplicated malaria were treated with 7 days of artesunate and followed for 28 days. Blood smears were done every 8 hours until negative by light microscopy. Only one participant still had parasites at 48 hours and no participant presented parasitemia at 72 hours. Artesunate was highly efficacious, with no evidence of delayed parasite clearance. We provide baseline surveillance data for the emergence or dissemination of P. falciparum resistance in sub-Saharan Africa.

Sulfadoxine–pyrimethamine (SP) treatment increases the rate of gametocyte carriage and selects SP resistance-conferring mutations in Plasmodium falciparum dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS), raising concerns of increased malaria transmission and spread of drug resistance. In a setting in Mali where SP was highly efficacious, we measured the prevalence of DHFR and DHPS mutations in P. falciparum infections with microscopy-detected gametocytes following SP treatment, and used direct feeding to assess infectivity to Anopheles gambiae sensu lato. Children and young adults presenting with uncomplicated malaria were treated with SP or chloroquine and followed for 28 days. Gametocyte carriage peaked at 67% 1 week after treatment with a single dose of SP. Those post-SP gametocytes carried significantly more DHFR and DHPS mutations than pre-treatment asexual parasites from the same population. Only 0.5% of 1728 mosquitoes fed on SP-treated gametocyte carriers developed oocysts, while 11% of 198 mosquitoes fed on chloroquine-treated gametocyte carriers were positive for oocysts. This study shows that in an area of high SP efficacy, although SP treatment sharply increased gametocyte carriage, the infectiousness of these gametocytes to the vector may be very low. Accurate and robust methods for measuring infectivity are needed to guide malaria control interventions that affect transmission.

Sulfadoxine-pyrimethamine (SP) is currently the drug of choice for intermittent preventive treatment of Plasmodium falciparum both in pregnancy and infancy. A prolonged parasite clearance time conferred by dhfr and dhps mutations is believed to be responsible for increased gametocyte prevalence in SP treated individuals. However, using a direct feeding assay in Mali, we showed that gametocytes present in peripheral venous blood post-SP treatment had reduced infectivity for Anopheles gambiae sensu stricto (ss) mosquitoes. We investigated the potential mechanisms involved in the dhfr and dhps quintuple mutant NF135 and the single dhps 437 mutant NF54. Concentrations of Sulfadoxine (S) and Pyrimethamine (P) equivalent to the serum levels of the respective drugs on day 3 (S = 61 ug/ml, P = 154.7 ng/ml) day 7 (S = 33.8 ug/ml, P = 66.6 ng/ml) and day 14 (S = 14.2 ug/ml, P = 15.7 ng/ml) post-SP treatment were used to study the effect on gametocytogenesis, gametocyte maturation and infectivity to Anopheles stephensi mosquitoes fed through an artificial membrane. The drugs readily induced gametocytogenesis in the mutant NF-135 strain but effectively killed the wild-type NF54. However, both drugs impaired gametocyte maturation yielding odd-shaped non-exflagelating mature gametocytes. The concomitant ingestion of both S and P together with gametocytemic blood-meal significantly reduced the prevalence of oocyst positivity as well as oocyst density when compared to controls (P < 0.001). In addition, day 3 concentrations of SP decreased mosquito survival by up to 65% (P < 0.001). This study demonstrates that SP is deleterious in vitro for gametocyte infectivity as well as mosquito survival.