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Mutations in a Plasmodium de-ubiquitinase UBP1 have been linked to antimalarial drug resistance. However, the UBP1-mediated drug-resistant mechanism remains unknown. Through drug selection, genetic mapping, allelic exchange, and functional characterization, here we show that simultaneous mutations of two amino acids (I1560N and P2874T) in the Plasmodium yoelii UBP1 can mediate high-level resistance to mefloquine, lumefantrine, and piperaquine. Mechanistically, the double mutations are shown to impair UBP1 cytoplasmic aggregation and de-ubiquitinating activity, leading to increased ubiquitination levels and altered protein localization, from the parasite digestive vacuole to the plasma membrane, of the P. yoelii multidrug resistance transporter 1 (MDR1). The MDR1 on the plasma membrane enhances the efflux of substrates/drugs out of the parasite cytoplasm to confer multidrug resistance, which can be reversed by inhibition of MDR1 transport. This study reveals a previously unknown drug-resistant mechanism mediated by UBP1 through altered MDR1 localization and substrate transport direction in a mouse model, providing a new malaria treatment strategy., (© 2024. The Author(s).)

Protein ubiquitination is an important posttranslational regulation mechanism that mediates Plasmodium development and modifies parasite responses to antimalarial drugs. Although mutations in several parasite ubiquitination enzymes have been linked to increased drug tolerance, the molecular mechanisms by which ubiquitination pathways mediate these parasite responses remain largely unknown. Here, we investigate the roles of a Plasmodium falciparum ring finger ubiquitin ligase (PfRFUL) in parasite development and in responses to antimalarial drugs. We engineered a transgenic parasite having the Pfrful gene tagged with an HA-2A-NeoR-glmS sequence to knockdown (KD) Pfrful expression using glucosamine (GlcN). A Western blot analysis of the proteins from GlcN-treated pSLI-HA-NeoR-glmS-tagged (PfRFULg) parasites, relative to their wild-type (Dd2) controls, showed changes in the ubiquitination of numerous proteins. PfRFUL KD rendered the parasites more sensitive to multiple antimalarial drugs, including mefloquine, piperaquine, amodiaquine, and dihydroartemisinin. PfRFUL KD also decreased the protein level of the P. falciparum multiple drug resistance 1 protein (PfMDR1) and altered the ratio of two bands of the P. falciparum chloroquine resistance transporter (PfCRT), suggesting contributions to the changed drug responses by the altered ubiquitination of these two molecules. The inhibition of proteasomal protein degradation by epoxomicin increased the PfRFUL level, suggesting the degradation of PfRFUL by the proteasome pathways, whereas the inhibition of E3 ubiquitin ligase activities by JNJ26854165 reduced the PfRFUL level. This study reveals the potential mechanisms of PfRFUL in modifying the expression of drug transporters and their roles in parasite drug responses. PfRFUL could be a potential target for antimalarial drug development.

Malaria is the most deadly parasitic disease, affecting hundreds of millions of people worldwide. Malaria parasites have been associated with their hosts for millions of years. During the long history of host-parasite co-evolution, both parasites and hosts have applied pressure on each other through complex host-parasite molecular interactions. Whereas the hosts activate various immune mechanisms to remove parasites during an infection, the parasites attempt to evade host immunity by diversifying their genome and switching expression of targets of the host immune system. Human intervention to control the disease such as antimalarial drugs and vaccination can greatly alter parasite population dynamics and evolution, particularly the massive applications of antimalarial drugs in recent human history. Vaccination is likely the best method to prevent the disease; however, a partially protective vaccine may have unwanted consequences that require further investigation. Studies of host-parasite interactions and co-evolution will provide important information for designing safe and effective vaccines and for preventing drug resistance. In this essay, we will discuss some interesting molecules involved in host-parasite interactions, including important parasite antigens. We also discuss subjects relevant to drug and vaccine development and some approaches for studying host-parasite interactions., (Copyright © 2020 Su, Zhang and Joy.)

Protozoan Plasmodium parasites are the causative agents of malaria, a deadly disease that continues to afflict hundreds of millions of people every year. Infections with malaria parasites can be asymptomatic, with mild or severe symptoms, or fatal, depending on many factors such as parasite virulence and host immune status. Malaria can be treated with various drugs, with artemisinin-based combination therapies (ACTs) being the first-line choice. Recent advances in genetics and genomics of malaria parasites have contributed greatly to our understanding of parasite population dynamics, transmission, drug responses, and pathogenesis. However, knowledge gaps in parasite biology and host-parasite interactions still remain. Parasites resistant to multiple antimalarial drugs have emerged, while advanced clinical trials have shown partial efficacy for one available vaccine. Here we discuss genetic and genomic studies of Plasmodium biology, host-parasite interactions, population structures, mosquito infectivity, antigenic variation, and targets for treatment and immunization. Knowledge from these studies will advance our understanding of malaria pathogenesis, epidemiology, and evolution and will support work to discover and develop new medicines and vaccines., (Copyright © 2019 American Society for Microbiology.)

Concerns about malaria parasite resistance to treatment with artemisinin drugs (ARTs) have grown with findings of prolonged parasite clearance t 1/2 s (>5 h) and their association with mutations in Plasmodium falciparum Kelch-propeller protein K13. Here, we describe a P. falciparum laboratory cross of K13 C580Y mutant with C580 wild-type parasites to investigate ART response phenotypes in vitro and in vivo. After genotyping >400 isolated progeny, we evaluated 20 recombinants in vitro: IC 50 measurements of dihydroartemisinin were at similar low nanomolar levels for C580Y- and C580-type progeny (mean ratio, 1.00; 95% CI, 0.62-1.61), whereas, in a ring-stage survival assay, the C580Y-type progeny had 19.6-fold (95% CI, 9.76-39.2) higher average counts. In splenectomized Aotus monkeys treated with three daily doses of i.v. artesunate, t 1/2 calculations by three different methods yielded mean differences of 0.01 h (95% CI, -3.66 to 3.67), 0.80 h (95% CI, -0.92 to 2.53), and 2.07 h (95% CI, 0.77-3.36) between C580Y and C580 infections. Incidences of recrudescence were 57% in C580Y (4 of 7) versus 70% in C580 (7 of 10) infections (-13% difference; 95% CI, -58% to 35%). Allelic substitution of C580 in a C580Y-containing progeny clone (76H10) yielded a transformant (76H10 C580Rev ) that, in an infected monkey, recrudesced regularly 13 times over 500 d. Frequent recrudescences of ART-treated P. falciparum infections occur with or without K13 mutations and emphasize the need for improved partner drugs to effectively eliminate the parasites that persist through the ART component of combination therapy., Competing Interests: Conflict of interest statement: R.M.F. and D.E.G. are coauthors of a paper published in 2017. N.F.G., D.A.F., and D.E.G. are coauthors of papers published in 2016 and 2018. K.M., C.A.L., and D.E.G. are coauthors of a paper published in 2014. R.S.R. and S.R.M. are coauthors of papers published in 2015 and 2017.

Background: Mass drug administration (MDA), with or without low-dose primaquine (PMQLD), is being considered for malaria elimination programs. The potential of PMQLD to block malaria transmission by mosquitoes must be balanced against liabilities of its use., Methods: Artemisinin-piperaquine (AP), with or without PMQLD, was administered in 3 monthly rounds across Anjouan Island, Union of Comoros. Plasmodium falciparum malaria rates, mortality, parasitemias, adverse events, and PfK13 Kelch-propeller gene polymorphisms were evaluated., Results: Coverage of 85 to 93% of the Anjouan population was achieved with AP plus PMQLD (AP+PMQLD) in 2 districts (population 97164) and with AP alone in 5 districts (224471). Between the months of April-September in both 2012 and 2013, average monthly malaria hospital rates per 100000 people fell from 310.8 to 2.06 in the AP+PMQLD population (ratio 2.06/310.8 = 0.66%; 95% CI: 0.02%, 3.62%; P = .00007) and from 412.1 to 2.60 in the AP population (ratio 0.63%; 95% CI: 0.11%, 1.93%; P < .00001). Effectiveness of AP+PMQLD was 0.9908 (95% CI: 0.9053, 0.9991), while effectiveness of AP alone was 0.9913 (95% CI: 0.9657, 0.9978). Both regimens were well tolerated, without severe adverse events. Analysis of 52 malaria samples after MDA showed no evidence for selection of PfK13 Kelch-propeller mutations., Conclusions: Steep reductions of malaria cases were achieved by 3 monthly rounds of either AP+PMQLD or AP alone, suggesting potential for highly successful MDA without PMQLD in epidemiological settings such as those on Anjouan. A major challenge is to sustain and expand the public health benefits of malaria reductions by MDA.

Background: Malaria is still one of the serious public health problems in Grande Comore Island, although the number of annual cases has been greatly reduced in recent years. A better understanding of malaria parasite population diversity and transmission dynamics is critical for assessing the effectiveness of malaria control measures. The objective of this study is to investigate temporal changes in genetic diversity of Plasmodium falciparum populations and multiplicity of infection (MOI) in Grande Comore 10 years after introduction of ACT., Methods: A total of 232 P. falciparum clinical isolates were collected from the Grande Comore Island during two sampling periods (118 for 2006‒2007 group, and 114 for 2013‒2016 group). Parasite isolates were characterized for genetic diversity and complexity of infection by genotyping polymorphic regions in merozoite surface protein gene 1 (msp-1), msp-2, and msp-3 using nested PCR and DNA sequencing., Results: Three msp-1 alleles (K1, MAD20, and RO33), two msp-2 alleles (FC27 and 3D7), and two msp-3 alleles (K1 and 3D7) were detected in parasites of both sampling periods. The RO33 allele of msp-1 (84.8%), 3D7 allele of msp-2 (90.8%), and K1 allele of msp-3 (66.7%) were the predominant allelic types in isolates from 2006-2007 group. In contrast, the RO33 allele of msp-1 (63.4%), FC27 allele of msp-2 (91.1%), and 3D7 allele of msp-3 (53.5%) were the most prevalent among isolates from the 2013-2016 group. Compared with the 2006‒2007 group, polyclonal infection rates of msp-1 (from 76.7 to 29.1%, P < 0.01) and msp-2 (from 62.4 to 28.3%, P < 0.01) allelic types were significantly decreased in those from 2013‒2016 group. Similarly, the MOIs for both msp-1 and msp-2 were higher in P. falciparum isolates in the 2006-2007 group than those in 2013-2016 group (MOI = 3.11 vs 1.63 for msp-1; MOI = 2.75 vs 1.35 for msp-2). DNA sequencing analyses also revealed reduced numbers of distinct sequence variants in the three genes from 2006‒2007 to 2013‒2016: msp-1, from 32 to 23 (about 28% decline); msp-2 from 29 to 21 (about 28% decline), and msp-3 from 11 to 3 (about 72% decline)., Conclusions: The present data showed dramatic reduction in genetic diversity and MOI among Grande Comore P. falciparum populations over the course of the study, suggesting a trend of decreasing malaria transmission intensity and genetic diversity in Grande Comore Island. These data provide valuable information for surveillance of P. falciparum infection and for assessing the appropriateness of the current malarial control strategies in the endemic area.

Parasitic protozoan diseases continue to rank among the world's greatest global health problems, which are also common among poor populations. Currently available drugs for treatment present drawbacks, urging the need for more effective, safer, and cheaper drugs. Artemisinin (ART) and its derivatives are some of the most important classes of antimalarial agents originally derived from Artemisia annua L. However, besides the outstanding antimalarial and antischistosomal activities, ART and its derivatives also possess activities against other parasitic protozoa. In this paper we review the activities of ART and its derivatives against protozoan parasites in vitro and in vivo, including Leishmania spp., Trypanosoma spp., Toxoplasma gondii, Neospora caninum, Eimeria tenella, Acanthamoeba castellanii, Naegleria fowleri, Cryptosporidium parvum, Giardia lamblia, and Babesia spp. We conclude that ART and its derivatives may be good alternatives for treating other non-malarial protozoan infections in developing countries, although more studies are necessary before they can be applied clinically., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Drug resistance has emerged as one of the greatest challenges facing malaria control. The recent emergence of resistance to artemisinin (ART) and its partner drugs in ART-based combination therapies (ACT) is threatening the efficacy of this front-line regimen for treating Plasmodium falciparum parasites. Thus, an understanding of the molecular mechanisms that underlie the resistance to ART and the partner drugs has become a high priority for resistance containment and malaria management. Using genome-wide association studies, we investigated the associations of genome-wide single nucleotide polymorphisms with in vitro sensitivities to 10 commonly used antimalarial drugs in 94 P. falciparum isolates from the China-Myanmar border area, a region with the longest history of ART usage. We identified several loci associated with various drugs, including those containing pfcrt and pfdhfr. Of particular interest is a locus on chromosome 10 containing the autophagy-related protein 18 (ATG18) associated with decreased sensitivities to dihydroartemisinin, artemether and piperaquine - an ACT partner drug in this area. ATG18 is a phosphatidylinositol-3-phosphate binding protein essential for autophagy and recently identified as a potential ART target. Further investigations on the ATG18 and genes at the chromosome 10 locus may provide an important lead for a connection between ART resistance and autophagy.

Background: In Comoros, the widespread of chloroquine (CQ)-resistant Plasmodium falciparum populations was a major obstacle to malaria control, which led to the official withdrawal of CQ in 2004. Continuous monitoring of CQ-resistant markers of the P. falciparum CQ resistant transporter (pfcrt) and the P. falciparum multiple drug resistance 1 (pfmdr-1) is necessary inder to obtain first-hand information on CQ susceptibility of parasite populations in the field. The objective of this study is to assess the prevalence and evolution of CQ-resistance in the P. falciparum populations on the Comoros' Grande Comore island after withdrawal of CQ., Methods: A total of 207 P. falciparum clinical isolates were collected from the island, including 118 samples from 2006 to 2007 and 89 samples from 2013 to 2014. Nucleotide substitutions in the pfcrt and pfmdr-1 genes linked to CQ response in parasite isolates were assessed using nested PCR and DNA sequencing., Results: From the pfcrt gene segment sequenced, we detected C72S, M74I, N75E, and K76T substitutions in the parasite isolates collected from both 2006-2007 to 2013-2014 periods. Significant decline of pfcrt resistant alleles at C72S (42.6 to 6.9 %), M74I (39.1 to 14.9 %), N75E (63.5 to 18.3 %), and K76T (72.2 to 19.5 %) from 2006-2007 to 2013-2014 were observed, and the frequency of pfcrt wild type allele was significantly increased from 19.1 % in 2006-2007 to 75.8 % in 2013-2014. Sequence analysis of pfmdr-1 also detected point mutations at codons N86Y, Y184F, and D1246Y, but not S1034C and N1042D, in the isolates collected from both examined periods. An increasing trend in the prevalence of the pfmdr-1 wild type allele (NYD, 4.3 % in 2006-2007; and 28.7 % in 2013-2014), and a decreasing trend for pfmdr-1 N86Y mutation (87.0 % in 2006-2007; and 40.2 % in 2013-2014) were observed in our samples., Conclusions: The present data indicate that the prevalence and patterns of mutant pfcrt and pfmdr-1 dramatically decreased in the Grande Comore isolates from 2006 to 2014, suggesting that the CQ-sensitive P. falciparum strains have returned after the withdrawal of CQ. The data also suggests that the parasites with wild type pfcrt/pfdmr-1 genes may have growth and/or transmission advantages over the mutant parasites. The information obtained from this study will be useful for developing and updating anti-malarial treatment policy in Grande Comore island.

Treatment of the symptomatic asexual stage of Plasmodium falciparum relies almost exclusively on artemisinin (ART) combination therapies (ACTs) in endemic regions. ACTs combine ART or its derivative with a long-acting partner drug to maximize efficacy during the typical three-day regimen. Both laboratory and clinical studies have previously demonstrated that the common drug resistance determinants P. falciparum chloroquine resistance transporter (PfCRT) and multidrug resistance transporter (PfMDR1) can modulate the susceptibility to many current antimalarial drugs and chemical compounds. Here we investigated the parasite responses to dihydroartemisinin (DHA) and various Ca(2+) and Na(+) channel blockers and showed positively correlated responses between DHA and several channel blockers, suggesting potential shared transport pathways or mode of action. Additionally, we demonstrated that PfCRT and PfMDR1 could also significantly modulate the pharmacodynamic interactions of the compounds and that the interactions were influenced by the parasite genetic backgrounds. These results provide important information for better understanding of drug resistance and for assessing the overall impact of drug resistance markers on parasite response to ACTs.

Background: Plasmodium falciparum malaria is a significant public health problem in Comoros, and artemisinin combination therapy (ACT) remains the first choice for treating acute uncomplicated P. falciparum. The emergence and spread of artemisinin-resistant P. falciparum in Southeast Asia, associated with mutations in K13-propeller gene, poses a potential threat to ACT efficacy. Detection of mutations in the P. falciparum K13-propeller gene may provide the first-hand information on changes in parasite susceptibility to artemisinin. The objective of this study is to determinate the prevalence of mutant K13-propeller gene among the P. falciparum isolates collected from Grande Comore Island, Union of Comoros, where ACT has been in use since 2004., Methods: A total of 207 P. falciparum clinical isolates were collected from the island during March 2006 and October 2007 (n = 118) and March 2013 and December 2014 (n = 89). All isolates were analysed for single nucleotide polymorphisms (SNPs) and haplotypes in the K13-propeller gene using nested PCR and DNA sequencing., Results: Only three 2006-2007 samples carried SNPs in the K13-propeller gene, one having a synonymous (G538G) and the other having two non-synonymous (S477Y and D584E) substitutions leading to two mutated haplotypes (2.2%, 2/95). Three synonymous mutations (R471R, Y500Y, and G538G) (5.9%, 5/85) and 7 non-synonymous substitutions (21.2%, 18/85) with nine mutated haplotypes (18.8%, 16/85) were found in isolates from 2013 to 2014. However, none of the polymorphisms associated with artemisinin-resistance in Southeast Asia was detected from any of the parasites examined., Conclusion: This study showed increased K13-propeller gene diversity among P. falciparum populations on the Island over the course of 8 years (2006-2014). Nevertheless, none of the polymorphisms known to be associated with artemisinin resistance in Asia was detected in the parasite populations examined. Our data suggest that P. falciparum populations in Grande Comore are still effectively susceptible to artemisinin. Our results provide insights into P. falciparum populations regarding mutations in the gene associated with artemisinin resistance and will be useful for developing and updating anti-malarial guidance in Comoros.

Methods to identify the bioactive diversity within natural product extracts (NPEs) continue to evolve. NPEs constitute complex mixtures of chemical substances varying in structure, composition, and abundance. NPEs can therefore be challenging to evaluate efficiently with high-throughput screening approaches designed to test pure substances. Here we facilitate the rapid identification and prioritization of antimalarial NPEs using a pharmacologically driven, quantitative high-throughput-screening (qHTS) paradigm. In qHTS each NPE is tested across a concentration range from which sigmoidal response, efficacy, and apparent EC50s can be used to rank order NPEs for subsequent organism reculture, extraction, and fractionation. Using an NPE library derived from diverse marine microorganisms we observed potent antimalarial activity from two Streptomyces sp. extracts identified from thousands tested using qHTS. Seven compounds were isolated from two phylogenetically related Streptomyces species: Streptomyces ballenaensis collected from Costa Rica and Streptomyces bangulaensis collected from Papua New Guinea. Among them we identified actinoramides A and B, belonging to the unusually elaborated nonproteinogenic amino-acid-containing tetrapeptide series of natural products. In addition, we characterized a series of new compounds, including an artifact, 25-epi-actinoramide A, and actinoramides D, E, and F, which are closely related biosynthetic congeners of the previously reported metabolites.

Drug resistance in Plasmodium parasites is a constant threat. Novel therapeutics, especially new drug combinations, must be identified at a faster rate. In response to the urgent need for new antimalarial drug combinations we screened a large collection of approved and investigational drugs, tested 13,910 drug pairs, and identified many promising antimalarial drug combinations. The activity of known antimalarial drug regimens was confirmed and a myriad of new classes of positively interacting drug pairings were discovered. Network and clustering analyses reinforced established mechanistic relationships for known drug combinations and identified several novel mechanistic hypotheses. From eleven screens comprising >4,600 combinations per parasite strain (including duplicates) we further investigated interactions between approved antimalarials, calcium homeostasis modulators, and inhibitors of phosphatidylinositide 3-kinases (PI3K) and the mammalian target of rapamycin (mTOR). These studies highlight important targets and pathways and provide promising leads for clinically actionable antimalarial therapy.

Background: Plasmodium vivax is the predominant species of human malaria parasites present in China. Although sulphadoxine-pyrimethamine (SP) and chloroquine (CQ) have been widely used for malaria treatment in China, the resistance profiles of these drugs are not available. Analysis of dihydrofolate reductase (dhfr), dihydropteroate synthase (dhps), and multidrug resistance (mdr-1) gene mutations in P. vivax isolates is a valuable molecular approach for mapping resistance to SP and CQ. This study investigates the prevalence of pvdhfr, pvdhps, and pvmdr-1 of P. vivax clinical isolates from China and provides baseline molecular epidemiologic data on SP- and CQ-associated resistance in P. vivax., Methods: Plasmodium vivax clinical isolates were collected from two malaria-endemic regions of China, subtropical (Xishuangbanna, Yunnan province) and temperate (Bozhou, Anhui province), from 2009 to 2012. All isolates were analysed for single nucleotide polymorphism haplotypes in pvdhfr, pvdhps, and pvmdr-1 using direct DNA sequencing., Results: In pvdhfr, 15% of Xishuangbanna isolates carried wild-type (WT) allele, whereas the majority of isolates carried mutant genes with substitutions at five codons. Eight mutant haplotypes of pvdhfr were detected, while limited polymorphism of pvdhfr was found in Bozhou isolates. A size polymorphism was present in pvdhfr, with the three-repeat type being the most predominate in both Xishuangbanna (79%) and Bozhou (97%) isolates. In pvdhps, mutations at four codons were detected in Xishuangbanna isolates leading to six haplotypes, including WT allele, single-mutation, double-mutation, and triple-mutation alleles. All Bozhou isolates carried WT pvhdps. In pvmdr-1, isolates from Xishuangbanna carried mutations at codons Y976F and F1076L, whereas all isolates from Bozhou had only a single mutation at codon F1076L., Conclusions: Plasmodium vivax isolates from subtropical and temperate zones of China are shown to have dramatically different frequencies and patterns of mutations in pvdhfr, pvdhps, and pvmdr-1. Whereas P. vivax populations in subtropical China are highly resistant to SP and CQ, those in the temperate zone may still be susceptible to SP and CQ. This information is useful for establishing treatment policy and provides a baseline for molecular surveillance of drug-resistant P. vivax in these areas.

Malaria parasites are transmitted by mosquitoes, and blocking parasite transmission is critical in reducing or eliminating malaria in endemic regions. Here, we report the pharmacological characterization of a new class of malaria transmission-blocking compounds that acts via the inhibition of Plasmodia CDPK4 enzyme. We demonstrate that these compounds achieved selectivity over mammalian kinases by capitalizing on a small serine gatekeeper residue in the active site of the Plasmodium CDPK4 enzyme. To directly confirm the mechanism of action of these compounds, we generated P. falciparum parasites that express a drug-resistant methionine gatekeeper (S147 M) CDPK4 mutant. Mutant parasites showed a shift in exflagellation EC50 relative to the wild-type strains in the presence of compound 1294, providing chemical-genetic evidence that CDPK4 is the target of the compound. Pharmacokinetic analyses suggest that coformulation of this transmission-blocking agent with asexual stage antimalarials such as artemisinin combination therapy (ACT) is a promising option for drug delivery that may reduce transmission of malaria including drug-resistant strains. Ongoing studies include refining the compounds to improve efficacy and toxicological properties for efficient blocking of malaria transmission.

In 2008, dihydroartemisinin (DHA)-piperaquine (PPQ) became the first-line treatment for uncomplicated Plasmodium falciparum malaria in western Cambodia. Recent reports of increased treatment failure rates after DHA-PPQ therapy in this region suggest that parasite resistance to DHA, PPQ, or both is now adversely affecting treatment. While artemisinin (ART) resistance is established in western Cambodia, there is no evidence of PPQ resistance. To monitor for resistance to PPQ and other antimalarials, we measured drug susceptibilities for parasites collected in 2011 and 2012 from Pursat, Preah Vihear, and Ratanakiri, in western, northern, and eastern Cambodia, respectively. Using a SYBR green I fluorescence assay, we calculated the ex vivo 50% inhibitory concentrations (IC50s) of 310 parasites to six antimalarials: chloroquine (CQ), mefloquine (MQ), quinine (QN), PPQ, artesunate (ATS), and DHA. Geometric mean IC50s (GMIC50s) for all drugs (except PPQ) were significantly higher in Pursat and Preah Vihear than in Ratanakiri (P ≤ 0.001). An increased copy number of P. falciparum mdr1 (pfmdr1), an MQ resistance marker, was more prevalent in Pursat and Preah Vihear than in Ratanakiri and was associated with higher GMIC50s for MQ, QN, ATS, and DHA. An increased copy number of a chromosome 5 region (X5r), a candidate PPQ resistance marker, was detected in Pursat but was not associated with reduced susceptibility to PPQ. The ex vivo IC50 and pfmdr1 copy number are important tools in the surveillance of multidrug-resistant (MDR) parasites in Cambodia. While MDR P. falciparum is prevalent in western and northern Cambodia, there is no evidence for PPQ resistance, suggesting that DHA-PPQ treatment failures result mainly from ART resistance.

We describe an analysis of genome variation in 825 P. falciparum samples from Asia and Africa that identifies an unusual pattern of parasite population structure at the epicenter of artemisinin resistance in western Cambodia. Within this relatively small geographic area, we have discovered several distinct but apparently sympatric parasite subpopulations with extremely high levels of genetic differentiation. Of particular interest are three subpopulations, all associated with clinical resistance to artemisinin, which have skewed allele frequency spectra and high levels of haplotype homozygosity, indicative of founder effects and recent population expansion. We provide a catalog of SNPs that show high levels of differentiation in the artemisinin-resistant subpopulations, including codon variants in transporter proteins and DNA mismatch repair proteins. These data provide a population-level genetic framework for investigating the biological origins of artemisinin resistance and for defining molecular markers to assist in its elimination.

Plasmodium falciparum malaria, an infectious disease caused by a parasitic protozoan, claims the lives of nearly a million children each year in Africa alone and is a top public health concern. Evidence is accumulating that resistance to artemisinin derivatives, the frontline therapy for the asexual blood stage of the infection, is developing in southeast Asia. Renewed initiatives to eliminate malaria will benefit from an expanded repertoire of antimalarials, including new drugs that kill circulating P. falciparum gametocytes, thereby preventing transmission. Our current understanding of the biology of asexual blood-stage parasites and gametocytes and the ability to culture them in vitro lends optimism that high-throughput screenings of large chemical libraries will produce a new generation of antimalarial drugs. There is also a need for new therapies to reduce the high mortality of severe malaria. An understanding of the pathophysiology of severe disease may identify rational targets for drugs that improve survival.

Malaria is a deadly infectious disease in many tropical and subtropical countries. Previous efforts to eradicate malaria have failed, largely due to the emergence of drug-resistant parasites, insecticide-resistant mosquitoes and, in particular, the lack of drugs or vaccines to block parasite transmission. ATP-binding cassette (ABC) transporters are known to play a role in drug transport, metabolism, and resistance in many organisms, including malaria parasites. To investigate whether a Plasmodium falciparum ABC transporter (Pf14&#95;0244 or PfABCG2) modulates parasite susceptibility to chemical compounds or plays a role in drug resistance, we disrupted the gene encoding PfABCG2, screened the recombinant and the wild-type 3D7 parasites against a library containing 2,816 drugs approved for human or animal use, and identified an antihistamine (ketotifen) that became less active against the PfABCG2-disrupted parasite in culture. In addition to some activity against asexual stages and gametocytes, ketotifen was highly potent in blocking oocyst development of P. falciparum and the rodent parasite Plasmodium yoelii in mosquitoes. Tests of structurally related tricyclic compounds identified additional compounds with similar activities in inhibiting transmission. Additionally, ketotifen appeared to have some activity against relapse of Plasmodium cynomolgi infection in rhesus monkeys. Further clinical evaluation of ketotifen and related compounds, including synthetic new derivatives, in blocking malaria transmission may provide new weapons for the current effort of malaria eradication.

Background: Artemisinin-resistant Plasmodium falciparum has been reported in Pailin, western Cambodia, detected as a slow parasite clearance rate in vivo. Emergence of this phenotype in western Thailand and possibly elsewhere threatens to compromise the effectiveness of all artemisinin-based combination therapies. Parasite genetics is associated with parasite clearance rate but does not account for all variation. We investigated contributions of both parasite genetics and host factors to the artemisinin-resistance phenotype in Pursat, western Cambodia., Methods: Between June 19 and Nov 28, 2009, and June 26 and Dec 6, 2010, we enrolled patients aged 10 years or older with uncomplicated falciparum malaria, a density of asexual parasites of at least 10,000 per μL of whole blood, no symptoms or signs of severe malaria, no other cause of febrile illness, and no chronic illness. We gave participants 4 mg/kg artesunate at 0, 24, and 48 h, 15 mg/kg mefloquine at 72 h, and 10 mg/kg mefloquine at 96 h. We assessed parasite density on thick blood films every 6 h until undetectable. The parasite clearance half-life was calculated from the parasite clearance curve. We genotyped parasites with 18 microsatellite markers and patients for haemoglobin E, α-thalassaemia, and a mutation of G6PD, which encodes glucose-6-phosphate dehydrogenase. To account for the possible effects of acquired immunity on half-life, we used three surrogates for increased likelihood of exposure to P falciparum: age, sex, and place of residence. This study is registered with ClinicalTrials.gov, number NCT00341003., Findings: We assessed 3504 individuals from all six districts of Pursat province seeking treatment for malaria symptoms. We enrolled 168 patients with falciparum malaria who met inclusion criteria. The geometric mean half-life was 5·85 h (95% CI 5·54-6·18) in Pursat, similar to that reported in Pailin (p=0·109). We identified two genetically different parasite clone groups: parasite group 1 (PG1) and parasite group 2 (PG2). Non-significant increases in parasite clearance half-life were seen in patients with haemoglobin E (0·55 h; p=0·078), those of male sex (0·96 h; p=0·064), and in 2010 (0·68 h; p=0·068); PG1 was associated with a significant increase (0·79 h; p=0·033). The mean parasite heritability of half-life was 0·40 (SD 0·17)., Interpretation: Heritable artemisinin resistance is established in a second Cambodian province. To accurately identify parasites that are intrinsically susceptible or resistant to artemisinins, future studies should explore the effect of erythrocyte polymorphisms and specific immune responses on half-life variation., Funding: Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The recent reports of artemisinin (ART) resistance in the Thai-Cambodian border area raise a serious concern on the long-term efficacy of ARTs. To elucidate the resistance mechanisms, we performed in vitro selection with dihydroartemisinin (DHA) and obtained two parasite clones from Dd2 with more than 25-fold decrease in susceptibility to DHA. The DHA-resistant clones were more tolerant of stressful growth conditions and more resistant to several commonly used antimalarial drugs than Dd2. The result is worrisome as many of the drugs are currently used as ART partners in malaria control. This study showed that the DHA resistance is not limited to ring stage, but also occurred in trophozoites and schizonts. Microarray and biochemical analyses revealed pfmdr1 amplification, elevation of the antioxidant defence network, and increased expression of many chaperones in the DHA-resistant parasites. Without drug pressure, the DHA-resistant parasites reverted to sensitivity in approximately 8 weeks, accompanied by de-amplification of pfmdr1 and reduced antioxidant activities. The parallel decrease and increase in pfmdr1 copy number and antioxidant activity and the up and down of DHA sensitivity strongly suggest that pfmdr1 and antioxidant defence play a role in in vitro resistance to DHA, providing potential molecular markers for ART resistance., (© 2012 Blackwell Publishing Ltd.)

The recent emergence of artemisinin (ART) resistance in Plasmodium falciparum in western Cambodia, manifested as delayed parasite clearance, is a big threat to the long-term efficacy of this family of antimalarial drugs. Among the multiple candidate genes associated with ART resistance in P. falciparum, the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase PfATP6 has been postulated as a specific target of ARTs. The PfATP6 gene harbors multiple single-nucleotide polymorphisms in field parasite populations, and S769N has been associated with decreased sensitivity to artemether in parasite populations from French Guiana. In this study, we used an allelic exchange strategy to engineer parasite lines carrying the S769N mutations in P. falciparum strain 3D7 and evaluated whether introduction of this mutation modulated parasite sensitivity to ART derivatives. Using three transgenic lines carrying the 769N mutation and two transgenic lines carrying the wild-type 769S as controls, we found that S769N did not affect PfATP6 gene expression. We compared the sensitivities of these parasite lines to three ART derivatives, artemether, artesunate, and dihydroartemisinin, in 18 biological experiments and detected no significant effect of the S769N mutation on parasite response to these ART derivatives. This study provides further evidence for the lack of association of PfATP6 with ART resistance.

Malaria remains a devastating disease largely because of widespread drug resistance. New drugs and a better understanding of the mechanisms of drug action and resistance are essential for fulfilling the promise of eradicating malaria. Using high-throughput chemical screening and genome-wide association analysis, we identified 32 highly active compounds and genetic loci associated with differential chemical phenotypes (DCPs), defined as greater than or equal to fivefold differences in half-maximum inhibitor concentration (IC(50)) between parasite lines. Chromosomal loci associated with 49 DCPs were confirmed by linkage analysis and tests of genetically modified parasites, including three genes that were linked to 96% of the DCPs. Drugs whose responses mapped to wild-type or mutant pfcrt alleles were tested in combination in vitro and in vivo, which yielded promising new leads for antimalarial treatments.

In an effort to expand the stereochemical and structural complexity of chemical libraries used in drug discovery, the Center for Chemical Methodology and Library Development at Boston University has established an infrastructure to translate methodologies accessing diverse chemotypes into arrayed libraries for biological evaluation. In a collaborative effort, the NIH Chemical Genomics Center determined IC(50)'s for Plasmodium falciparum viability for each of 2,070 members of the CMLD-BU compound collection using quantitative high-throughput screening across five parasite lines of distinct geographic origin. Three compound classes displaying either differential or comprehensive antimalarial activity across the lines were identified, and the nascent structure activity relationships (SAR) from this experiment used to initiate optimization of these chemotypes for further development.

Artemisinin (ART)-based combination therapies (ACTs) are the first-line drugs-and often the last treatments-that can effectively cure Plasmodium falciparum infections. Unfortunately, the decreased clinical efficacy of artesunate, one of the major ART derivatives, was recently reported along the Thailand-Cambodia border. Through long-term artemisinin pressure in vitro, we have obtained an ART-tolerant strain that can survive extremely high doses of ART. We showed that drug pressure could induce a subpopulation of ring stages into developmental arrest, which can explain the ART tolerance in P. falciparum. We also observed interesting transcriptomic modifications possibly associated with the acquisition of ART tolerance. These modifications include the overexpression of heat shock and erythrocyte surface proteins and the downexpression of a cell cycle regulator and a DNA biosynthesis protein. This study highlights a new phenomenon in the Plasmodium response to ART that may explain the delayed clearance of parasites after artesunate treatment observed on the Thailand-Cambodia border and that provides important information for achieving a better understanding of the mechanisms of antimalarial resistance.

Antimalarial drugs impose strong selective pressure on Plasmodium falciparum parasites and leave signatures of selection in the parasite genome; screening for genes under selection may suggest potential drug or immune targets. Genome-wide association studies (GWAS) of parasite traits have been hampered by the lack of high-throughput genotyping methods, inadequate knowledge of parasite population history and time-consuming adaptations of parasites to in vitro culture. Here we report the first Plasmodium GWAS, which included 189 culture-adapted P. falciparum parasites genotyped using a custom-built Affymetrix molecular inversion probe 3K malaria panel array with a coverage of approximately 1 SNP per 7 kb. Population structure, variation in recombination rate and loci under recent positive selection were detected. Parasite half-maximum inhibitory concentrations for seven antimalarial drugs were obtained and used in GWAS to identify genes associated with drug responses. This study provides valuable tools and insight into the P. falciparum genome.

Despite great international efforts, malaria still inflicts an enormous toll on human lives, especially in Africa. Throughout history, antimalarial medicines have been one of the most powerful tools in malaria control. However, the acquisition and spread of parasite strains that are resistant to multiple antimalarial drugs have become one of the greatest challenges to malaria treatment, and are associated with the increase in morbidity and mortality in many malaria-endemic countries. To deal with this grave situation, artemisinin-based combinatory therapies (ACTs) have been introduced and widely deployed in malarious regions. Artemisinin is a new class of antimalarial compounds discovered by Chinese scientists from the sweet wormwood Artemisia annua. The potential development of resistance to artemisinins by Plasmodium falciparum threatens the usable lifespan of ACTs, and therefore is a subject of close surveillance and extensive research. Studies at the Thai-Cambodian border, a historical epicenter of multidrug resistance, have detected reduced susceptibility to artemisinins as manifested by prolonged parasite-clearance times, raising considerable concerns on resistance development. Despite this significance, there is still controversy on the mode of action of artemisinins. Although a number of potential cellular targets of artemisinins have been proposed, they remain to be verified experimentally. Here, we review the history of artemisinin discovery, discuss the mode of action and potential drug targets, and present strategies to elucidate resistance mechanisms.

Studies of gene function and molecular mechanisms in Plasmodium falciparum are hampered by difficulties in characterizing and measuring phenotypic differences between individual parasites. We screened seven parasite lines for differences in responses to 1,279 bioactive chemicals. Hundreds of compounds were active in inhibiting parasite growth; 607 differential chemical phenotypes, defined as pairwise IC(50) differences of fivefold or more between parasite lines, were cataloged. We mapped major determinants for three differential chemical phenotypes between the parents of a genetic cross, and we identified target genes by fine mapping and testing the responses of parasites in which candidate genes were genetically replaced with mutant alleles. Differential sensitivity to dihydroergotamine methanesulfonate (1), a serotonin receptor antagonist, was mapped to a gene encoding the homolog of human P-glycoprotein (PfPgh-1). This study identifies new leads for antimalarial drugs and demonstrates the utility of a high-throughput chemical genomic strategy for studying malaria traits.

ATP-binding cassette transporters play an important role in drug resistance and nutrient transport. In the human malaria parasite Plasmodium falciparum, a homolog of the human p-glycoprotein (PfPgh-1) was shown to be involved in resistance to several drugs. More recently, many transporters were associated with higher IC(50) levels in responses to chloroquine (CQ) and quinine (QN) in field isolates. Subsequent studies, however, could not confirm the associations, although inaccuracy in drug tests in the later studies could contribute to the lack of associations. Here we disrupted a gene encoding a putative multidrug resistance-associated protein (PfMRP) that was previously shown to be associated with P. falciparum responses to CQ and QN. Parasites with disrupted PfMRP (W2/MRPDelta) could not grow to a parasitemia higher than 5% under normal culture conditions, possibly because of lower efficiency in removing toxic metabolites. The W2/MRPDelta parasite also accumulated more radioactive glutathione, CQ, and QN and became more sensitive to multiple antimalarial drugs, including CQ, QN, artemisinin, piperaquine, and primaquine. PfMRP was localized on the parasite surface membrane, within membrane-bound vesicles, and along the straight side of the D-shaped stage II gametocytes. The results suggest that PfMRP plays a role in the efflux of glutathione, CQ, and QN and contributes to parasite responses to multiple antimalarial drugs, possibly by pumping drugs outside the parasite.

Drug resistance in malaria parasites is a serious public health burden, and resistance to most of the antimalarial drugs currently in use has been reported. A better understanding of the molecular mechanisms of drug resistance is urgently needed to slow or circumvent the spread of resistance, to allow local treatments to be deployed more effectively to prolong the life span of the current drugs, and to develop new drugs. Although mutations in genes determining resistance to drugs such as chloroquine and the antifolates have been identified, we still do not have a full understanding of the resistance mechanisms, and genes that contribute to resistance to many other drugs remain to be discovered. Genetic mapping is a powerful tool for the identification of mutations conferring drug resistance in malaria parasites because most drug-resistant phenotypes were selected within the past 60 years. High-throughput methods for genotyping large numbers of single nucleotide polymorphisms (SNPs) and microsatellites (MSs) are now available or are being developed, and genome-wide association studies for malaria traits will soon become a reality. Here we discuss strategies and issues related to mapping genes contributing to drug resistance in the human malaria parasite Plasmodium falciparum.

Studying drug resistance in Plasmodium falciparum requires accurate measurement of parasite response to a drug. Factors such as mixed infection of drug-resistant and -sensitive parasites can influence drug test outcome. Polymorphic DNA sequences are frequently used to detect mixed infections; infections with a single genotype or having a minor allele smaller than a subjectively selected cut-off value are often considered single infection. We studied the effects of mixed parasite populations containing various ratios of parasites resistant and sensitive to chloroquine on outcomes of drug tests and how ratios of parasite mixtures correlated with genotypes using polymerase chain reaction-based methods. Our results show that a mixture with a resistant population as low as 10% could greatly impact a drug test outcome. None of the genotyping methods could reliably detect minor DNA alleles at < or = 10%. Mixed infection presents a serious problem for drug tests, and genotyping using microsatellite or other methods may not reliably reflect true ratios of alleles.

Mutations and/or overexpression of various transporters are known to confer drug resistance in a variety of organisms. In the malaria parasite Plasmodium falciparum, a homologue of P-glycoprotein, PfMDR1, has been implicated in responses to chloroquine (CQ), quinine (QN) and other drugs, and a putative transporter, PfCRT, was recently demonstrated to be the key molecule in CQ resistance. However, other unknown molecules are probably involved, as different parasite clones carrying the same pfcrt and pfmdr1 alleles show a wide range of quantitative responses to CQ and QN. Such molecules may contribute to increasing incidences of QN treatment failure, the molecular basis of which is not understood. To identify additional genes involved in parasite CQ and QN responses, we assayed the in vitro susceptibilities of 97 culture-adapted cloned isolates to CQ and QN and searched for single nucleotide polymorphisms (SNPs) in DNA encoding 49 putative transporters (total 113 kb) and in 39 housekeeping genes that acted as negative controls. SNPs in 11 of the putative transporter genes, including pfcrt and pfmdr1, showed significant associations with decreased sensitivity to CQ and/or QN in P. falciparum. Significant linkage disequilibria within and between these genes were also detected, suggesting interactions among the transporter genes. This study provides specific leads for better understanding of complex drug resistances in malaria parasites.

Widespread use of antimalarial agents can profoundly influence the evolution of the human malaria parasite Plasmodium falciparum. Recent selective sweeps for drug-resistant genotypes may have restricted the genetic diversity of this parasite, resembling effects attributed in current debates to a historic population bottleneck. Chloroquine-resistant (CQR) parasites were initially reported about 45 years ago from two foci in southeast Asia and South America, but the number of CQR founder mutations and the impact of chlorquine on parasite genomes worldwide have been difficult to evaluate. Using 342 highly polymorphic microsatellite markers from a genetic map, here we show that the level of genetic diversity varies substantially among different regions of the parasite genome, revealing extensive linkage disequilibrium surrounding the key CQR gene pfcrt and at least four CQR founder events. This disequilibrium and its decay rate in the pfcrt-flanking region are consistent with strong directional selective sweeps occurring over only approximately 20-80 sexual generations, especially a single resistant pfcrt haplotype spreading to very high frequencies throughout most of Asia and Africa. The presence of linkage disequilibrium provides a basis for mapping genes under drug selection in P. falciparum.

Despite significant improvement in the malaria situation of the Greater Mekong Subregion (GMS), malaria control for the region continues to face a multitude of challenges. The extremely patchy malaria distribution, especially along international borders, makes disease surveillance and targeted control difficult. The vector systems are also diverse with dramatic differences in habitat ecology, biting behavior, and vectorial capacity, and there is a lack of effective transmission surveillance and control tools. Finally, in an era of heavy deployment of artemisinin-based combination therapies, the region acts as an epicenter of drug resistance, with the emergence of artemisinin resistant Plasmodium falciparum posing a threat to both regional and global malaria elimination campaigns. This problem is further exacerbated by the circulation of counterfeit and substandard artemisinin drugs. Accordingly, this Southeast Asian Malaria Research Center, consisting of a consortium of US and regional research institutions, has proposed four interlinked projects to address these most urgent problems in malaria control. The aims of these projects will help to substantially improve our understanding of malaria epidemiology, vector systems and their roles in malaria transmission, as well as the mechanisms of drug resistance in parasites. Through the training of next-generation scientists in malaria research, this program will help build up and strengthen regional research infrastructure and capacities, which are essential for sustained malaria control in this region.

The Greater Mekong Subregion (GMS), comprised of six countries including Cambodia, China's Yunnan Province, Lao PDR, Myanmar (Burma), Thailand and Vietnam, is one of the most threatening foci of malaria. Since the initiation of the WHO's Mekong Malaria Program a decade ago, malaria situation in the GMS has greatly improved, reflected in the continuous decline in annual malaria incidence and deaths. However, as many nations are moving towards malaria elimination, the GMS nations still face great challenges. Malaria epidemiology in this region exhibits enormous geographical heterogeneity with Myanmar and Cambodia remaining high-burden countries. Within each country, malaria distribution is also patchy, exemplified by 'border malaria' and 'forest malaria' with high transmission occurring along international borders and in forests or forest fringes, respectively. 'Border malaria' is extremely difficult to monitor, and frequent malaria introductions by migratory human populations constitute a major threat to neighboring, malaria-eliminating countries. Therefore, coordination between neighboring countries is essential for malaria elimination from the entire region. In addition to these operational difficulties, malaria control in the GMS also encounters several technological challenges. Contemporary malaria control measures rely heavily on effective chemotherapy and insecticide control of vector mosquitoes. However, the spread of multidrug resistance and potential emergence of artemisinin resistance in Plasmodium falciparum make resistance management a high priority in the GMS. This situation is further worsened by the circulation of counterfeit and substandard artemisinin-related drugs. In most endemic areas of the GMS, P. falciparum and Plasmodium vivax coexist, and in recent malaria control history, P. vivax has demonstrated remarkable resilience to control measures. Deployment of the only registered drug (primaquine) for the radical cure of vivax malaria is severely undermined due to high prevalence of glucose-6-phosphate dehydrogenase deficiency in target human populations. In the GMS, the dramatically different ecologies, diverse vector systems, and insecticide resistance render traditional mosquito control less efficient. Here we attempt to review the changing malaria epidemiology in the GMS, analyze the vector systems and patterns of malaria transmission, and identify the major challenges the malaria control community faces on its way to malaria elimination.

Summary Type I interferon (IFN) is critical for controlling pathogen infection; however, its regulatory mechanisms in plasmacytoid cells (pDCs) still remain unclear. Here, we have shown that nucleic acid sensors cGAS-, STING-, MDA5-, MAVS-, or transcription factor IRF3-deficient mice produced high amounts of type I IFN-α and IFN-β (IFN-α/β) in the serum and were resistant to lethal plasmodium yoelii YM infection. Robust IFN-α/β production was abolished when gene encoding nucleic acid sensor TLR7, signaling adaptor MyD88, or transcription factor IRF7 was ablated or pDCs were depleted. Further, we identified SOCS1 as a key negative regulator to inhibit MyD88-dependent type I IFN signaling in pDCs. Finally, we have demonstrated that pDCs, cDCs, and macrophages were required for generating IFN-α/β-induced subsequent protective immunity. Thus, our findings have identified a critical regulatory mechanism of type I IFN signaling in pDCs and stage-specific function of immune cells in generating potent immunity against lethal YM infection. [ABSTRACT FROM AUTHOR]