We have analyzed the in vitro chemosensitivity profiles of 115 Kenyan isolates for chloroquine (CQ), piperaquine, lumefantrine (LM), and dihydroartemisinin in association with polymorphisms in pfcrt at codon 76 and pfmdr1 at codon 86, as well as with variations of the copy number of pfmdr1. The median drug concentrations that inhibit 50% of parasite growth (IC 50 s) were 41 nM (interquartile range [IQR], 18 to 73 nM), 50 nM (IQR, 29 to 96 nM), 32 nM (IQR, 17 to 46 nM), and 2 nM (IQR, 1 to 3 nM) for CQ, LM, piperaquine, and dihydroartemisinin, respectively. The activity of CQ correlated inversely with that of LM (r 2 ‫؍‬ ؊0.26; P ‫؍‬ 0.02). Interestingly, parasites for which LM IC 50 s were higher were wild type for pfcrt-76 and pfmdr1-86. All isolates had one pfmdr1 copy. Thus, the decrease in LM activity is associated with the selection of wild-type pfcrt-76 and pfmdr1-86 parasites, a feature that accounts for the inverse relationship between CQ and LM. Therefore, the use of LM-artemether is likely to lead to the selection of more CQ-susceptible parasites.Chemotherapy is still the main approach for the control of malaria, and current strategies for malaria treatment rely on the use of combinations of drugs that include artemisinin compounds. Although this strategy is designed to reduce the chance of resistance emerging, there is considerable concern that this will inevitably occur.For instance, the combination of lumefantrine (LM) and artemether (ATM), known as Coartem, has become the firstline treatment for malaria in many African countries, including Kenya (19). ATM is converted in vivo to dihydroartemisinin (DHA). Emerging reports indicate that the use of LM (in Coartem) selects for parasites that show increased tolerance to Coartem, and these parasites select for a wild-type pfmdr1 genotype or show increased copy numbers of pfmdr1, a gene associated with chloroquine (CQ) and mefloquine (MFQ) resistance (7,13,15,20,36,38). Thus, there is concern that resistance to LM could emerge rapidly. On the other hand, recent reports from Southeast Asia indicate that resistance to artemisinin derivatives is increasing, threatening the concept of artemisinin-based combinations (8).Another combination, piperaquine (PQ) and DHA, known as Artekin, is undergoing clinical evaluation (17,39,42). This drug is efficacious, safe, and affordable and thus is likely to become an alternative to Coartem. PQ is a bisquinoline derivative consisting of two linked CQ molecules. Although reports indicate that PQ retains potency against CQ-resistant parasites (3), there is concern that PQ could become less susceptible against a backdrop of high CQ resistance (17,22).In this paper, we sought to analyze the in vitro activities of the antimalarials LM, DHA, and PQ in relation to polymorphisms in pfcrt at codon 76 (pfcrt-76) and in pfmdr1 at codon 86 (pfmdr1-86) and in relation to pfmdr1 copy number variations in Kenyan isolates. We used CQ as a reference drug. MATERIALS AND METHODSCQ was purchased from Sigma Chemical Co. (Poole, Dorset, United...

BackgroundThe emergence of artemisinin-resistant P. falciparum malaria in South-East Asia highlights the need for continued global surveillance of the efficacy of artemisinin-based combination therapies.MethodsOn the Kenyan coast we studied the treatment responses in 474 children 6–59 months old with uncomplicated P. falciparum malaria in a randomized controlled trial of dihydroartemisinin-piperaquine vs. artemether-lumefantrine from 2005 to 2008. (ISRCTN88705995)ResultsThe proportion of patients with residual parasitemia on day 1 rose from 55% in 2005–2006 to 87% in 2007–2008 (odds ratio, 5.4, 95%CI, 2.7–11.1; P<0.001) and from 81% to 95% (OR, 4.1, 95%CI, 1.7–9.9; P = 0.002) in the DHA-PPQ and AM-LM groups, respectively. In parallel, Kaplan-Meier estimated risks of apparent recrudescent infection by day 84 increased from 7% to 14% (P = 0.1) and from 6% to 15% (P = 0.05) with DHA-PPQ and AM-LM, respectively. Coinciding with decreasing transmission in the study area, clinical tolerance to parasitemia (defined as absence of fever) declined between 2005–2006 and 2007–2008 (OR body temperature >37.5°C, 2.8, 1.9–4.1; P<0.001). Neither in vitro sensitivity of parasites to DHA nor levels of antibodies against parasite extract accounted for parasite clearance rates or changes thereof.ConclusionsThe significant, albeit small, decline through time of parasitological response rates to treatment with ACTs may be due to the emergence of parasites with reduced drug sensitivity, to the coincident reduction in population-level clinical immunity, or both. Maintaining the efficacy of artemisinin-based therapy in Africa would benefit from a better understanding of the mechanisms underlying reduced parasite clearance rates.Trial RegistrationControlled-Trials.com ISRCTN88705995

Early identification of causal genetic variants underlying antimalarial drug resistance could provide robust epidemiological tools for timely public health interventions. Using a novel natural genetics strategy for mapping novel candidate genes we analyzed >75,000 high quality single nucleotide polymorphisms selected from high-resolution whole-genome sequencing data in 27 isolates of Plasmodium falciparum. We identified genetic variants associated with susceptibility to dihydroartemisinin that implicate one region on chromosome 13, a candidate gene on chromosome 1 (PFA0220w, a UBP1 ortholog) and others (PFB0560w, PFB0630c, PFF0445w) with putative roles in protein homeostasis and stress response. There was a strong signal for positive selection on PFA0220w, but not the other candidate loci. Our results demonstrate the power of full-genome sequencing-based association studies for uncovering candidate genes that determine parasite sensitivity to artemisinins. Our study provides a unique reference for the interpretation of results from resistant infections.

Intestinal epithelial cells (IECs) constitute the primary barrier that separates us from the outside environment. These cells, lining the surface of the intestinal tract, represent a major challenge that enteric pathogens have to face. How IECs respond to viral infection and whether enteric viruses have developed strategies to subvert IECs innate immune response remains poorly characterized. Using mammalian reovirus (MRV) as a model enteric virus, we found that the intermediate subviral particles (ISVPs), which are formed in the gut during the natural course of infection by proteolytic digestion of the reovirus virion, trigger reduced innate antiviral immune response in IECs. On the contrary, infection of IECs by virions induces a strong antiviral immune response that leads to cellular death. Additionally, we determined that virions can be sensed by both TLR and RLR pathways while ISVPs are sensed by RLR pathways only. Interestingly, we found that ISVP infected cells secrete TGF-β acting as a pro-survival factor that protects IECs against virion induced cellular death. We propose that ISVPs represent a reovirus strategy to initiate primary infection of the gut by subverting IECs innate immune system and by counteracting cellular-death pathways.