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1.  A Class of Tricyclic Compounds Blocking Malaria Parasite Oocyst Development and Transmission 
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_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.
doi:10.1128/AAC.00920-12
PMCID: PMC3535893  PMID: 23129054
2.  Hundreds of microsatellites for genotyping Plasmodium yoelii parasites 
Genetic crosses have been employed to study various traits of rodent malaria parasites and to locate loci that contribute to drug resistance, immune protection, and disease virulence. Compared with human malaria parasites, genetic crossing of rodent malaria parasites is more easily performed; however, genotyping methods using microsatellites (MS) or large-scale single nucleotide polymorphisms (SNPs) that have been widely used in typing Plasmodium falciparum are not available for rodent malaria species. Here we report a genome-wide search of the Plasmodium yoelii yoelii (P. yoelii) genome for simple sequence repeats (SSRs) and the identification of nearly 600 polymorphic microsatellite (MS) markers for typing the genomes of P. yoelii and Plasmodium berghei. The MS markers are randomly distributed across the 14 physical chromosomes assembled from genome sequences of three rodent malaria species, although some variations in the numbers of MS expected according to chromosome size exist. The majority of the MS markers are AT-rich repeats, similar to those found in the P. falciparum genome. The MS markers provide an important resource for genotyping, lay a foundation for developing linkage maps, and will greatly facilitate genetic studies of P. yoelii.
doi:10.1016/j.molbiopara.2009.03.011
PMCID: PMC2787103  PMID: 19450732
Rodent malaria parasite; Simple sequence repeat (SSR); Genetic markers; Genotyping; MS
3.  Effects of Plasmodium falciparum Mixed Infections on in Vitro Antimalarial Drug Tests and Genotyping 
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 employed to detect mixed infections; infections with single genotype or having a minor allele smaller than a subjectively selected cut-off value are often considered single infection. We investigate 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 ≤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.
PMCID: PMC2680026  PMID: 18689621
4.  A Specific Inhibitor of PfCDPK4 Blocks Malaria Transmission: Chemical-genetic Validation 
The Journal of Infectious Diseases  2013;209(2):275-284.
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 (S147M) 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.
doi:10.1093/infdis/jit522
PMCID: PMC3873787  PMID: 24123773
Plasmodium falciparum; malaria transmission-blocking; calcium-dependent protein kinase 4; bumped kinase inhibitors
5.  Chemical genomics for studying parasite gene function and interaction 
Trends in parasitology  2013;29(12):10.1016/j.pt.2013.10.005.
With the development of new technologies in genome sequencing, gene expression profiling, genotyping, and high-throughput screening of chemical compound libraries, small molecules are playing increasingly important roles in studying gene expression regulation, gene-gene interaction, and gene function. Here we briefly review and discuss some recent advancements in drug target identification and phenotype characterization using combinations of high-throughput screening of small-molecule libraries and various genome-wide methods such as whole genome sequencing, genome-wide association studies, and genome-wide expressional analysis. These approaches can be used to search for new drugs against parasitic infections, to identify drug targets or drug-resistance genes, and to infer gene function.
doi:10.1016/j.pt.2013.10.005
PMCID: PMC3844153  PMID: 24215777
High-throughput screening; genetics; genomics; gene function
6.  Plasmodium genetic loci linked to host cytokine and chemokine responses 
Genes and immunity  2014;15(3):145-152.
Both host and parasite factors contribute to disease severity of malaria infection; however, the molecular mechanisms responsible for the disease and the host-parasite interactions involved remain largely unresolved. To investigate effects of parasite factors on host immune responses and pathogenesis, we measured levels of plasma cytokines/chemokines (CC) and growth rates in mice infected with two Plasmodium yoelii strains having different virulence phenotypes and in progeny from a genetic cross of the two parasites. Quantitative trait loci (QTL) analysis linked levels of many CCs, particularly IL-1β, IP-10, IFN-γ, MCP-1, and MIG, and early parasite growth rate to loci on multiple parasite chromosomes, including chromosomes 7, 9, 10, 12, and 13. Comparison of the genome sequences spanning the mapped loci revealed various candidate genes. The loci on chromosome 7 and 13 had significant (p < 0.005) additive effects on IL-1β, IL-5, and IP-10 responses, and the chromosome 9 and 12 loci had significant (p = 0.017) interaction. Infection of knockout mice showed critical roles of MCP-1 and IL-10 in parasitemia control and host mortality. These results provide important information for better understanding of malaria pathogenesis and can be used to examine the role of these factors in human malaria infection.
doi:10.1038/gene.2013.74
PMCID: PMC3999244  PMID: 24452266
genetic mapping; cytokine; chemokine; inflammation; pathogenesis; virulence
7.  Molecular surveillance of pvdhfr, pvdhps, and pvmdr-1 mutations in Plasmodium vivax isolates from Yunnan and Anhui provinces of China 
Malaria Journal  2014;13(1):346.
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.
Electronic supplementary material
The online version of this article (doi:10.1186/1475-2875-13-346) contains supplementary material, which is available to authorized users.
doi:10.1186/1475-2875-13-346
PMCID: PMC4161776  PMID: 25179752
Plasmodium vivax; Polymorphism; Drug resistance markers; China
8.  Identification and characterisation of functional expressed sequence tags-derived simple sequence repeat (eSSR) markers for genetic linkage mapping of Schistosoma mansoni juvenile resistance and susceptibility loci in Biomphalaria glabrata 
Biomphalaria glabrata susceptibility to Schistosoma mansoni has a strong genetic component, offering the possibility for investigating host–parasite interactions at the molecular level, perhaps leading to novel control approaches. The identification, mapping and molecular characterisation of genes that influence the outcome of parasitic infection in the intermediate snail host is, therefore, seen as fundamental to the control of schistosomiasis. To better understand the evolutionary processes driving disease resistance/susceptibility phenotypes, we previously identified polymorphic random amplification of polymorphic DNA and genomic simple sequence repeats from B. glabrata. In the present study we identified and characterised polymorphic expressed simple sequence repeats markers (Bg-eSSR) from existing B. glabrata expressed sequence tags. Using these markers, and with previously identified genomic simple sequence repeats, genetic linkage mapping for parasite refractory and susceptibility phenotypes, the first known for B. glabrata, was initiated. Data mining of 54,309 expressed sequence tag, produced 660 expressed simple sequence repeats of which dinucleotide motifs (TA)n were the most common (37.88%), followed by trinucleotide (29.55%), mononucleotide (18.64%) and tetranucleotide (10.15%). Penta- and hexanucleotide motifs represented <3% of the Bg-eSSRs identified. While the majority (71%) of Bg-eSSRs were monomorphic between resistant and susceptible snails, several were, however, useful for the construction of a genetic linkage map based on their inheritance in segregating F2 progeny snails derived from crossing juvenile BS-90 and NMRI snails. Polymorphic Bg-eSSRs assorted into six linkage groups at a logarithm of odds score of 3. Interestingly, the heritability of four markers (Prim1_910, Prim1_771, Prim6_1024 and Prim7_823) with juvenile snail resistance were, by t-test, significant (P < 0.05) while an allelic marker, Prim24_524, showed linkage with the juvenile snail susceptibility phenotype. On the basis of our results it is possible that the gene(s) controlling juvenile resistance and susceptibility to S. mansoni infection in B. glabrata are not only on the same linkage group but lie within a short distance (42 cM) of each other.
doi:10.1016/j.ijpara.2013.03.007
PMCID: PMC4038333  PMID: 23643514
Expressed sequence tags; Genetic linkage mapping; Resistance; Susceptibility; Biomphalaria glabrata; Schistosoma mansoni
9.  Efficient Editing of Malaria Parasite Genome Using the CRISPR/Cas9 System 
mBio  2014;5(4):e01414-14.
ABSTRACT
Malaria parasites are unicellular organisms residing inside the red blood cells, and current methods for editing the parasite genes have been inefficient. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and Cas9 endonuclease-mediated genome editing) system is a new powerful technique for genome editing and has been widely employed to study gene function in various organisms. However, whether this technique can be applied to modify the genomes of malaria parasites has not been determined. In this paper, we demonstrated that Cas9 is able to introduce site-specific DNA double-strand breaks in the Plasmodium yoelii genome that can be repaired through homologous recombination. By supplying engineered homologous repair templates, we generated targeted deletion, reporter knock-in, and nucleotide replacement in multiple parasite genes, achieving up to 100% efficiency in gene deletion and 22 to 45% efficiencies in knock-in and allelic replacement. Our results establish methodologies for introducing desired modifications in the P. yoelii genome with high efficiency and accuracy, which will greatly improve our ability to study gene function of malaria parasites.
IMPORTANCE
Malaria, caused by infection of Plasmodium parasites, remains a world-wide public health burden. Although the genomes of many malaria parasites have been sequenced, we still do not know the functions of approximately half of the genes in the genomes. Studying gene function has become the focus of many studies; however, editing genes in malaria parasite genomes is still inefficient. Here we designed several efficient approaches, based on the CRISPR/Cas9 system, to introduce site-specific DNA double-strand breaks in the Plasmodium yoelii genome that can be repaired through homologous recombination. Using this system, we achieved high efficiencies in gene deletion, reporter tagging, and allelic replacement in multiple parasite genes. This technique for editing the malaria parasite genome will greatly facilitate our ability to elucidate gene function.
doi:10.1128/mBio.01414-14
PMCID: PMC4161241  PMID: 24987097
10.  Multi-Strain Infections and ‘Relapse’ of Leucocytozoon sabrazesi Gametocytes in Domestic Chickens in Southern China 
PLoS ONE  2014;9(4):e94877.
Leucocytozoon parasites infect many species of avian hosts, including domestic chicken, and can inflict heavy economic loss to the poultry industry. Although the prevalence and distribution of two Leucocytozoon species (L. sabrazesi and L. caulleryi) have been reported in China previously, there are many questions related to the parasite infection that remain unanswered, including population diversity and transmission dynamics in domestic chickens. Here we surveyed chicken blood samples from seven sites in four provinces of China to identify Leucocytozoon infection, characterized parasite diversity within individual infected hosts and between sampling sites, and investigated the dynamics of gametocytemia in chickens over time. We found high infection rates in three of the seven sites. Clustering parasite sequences of the mitochondrial cytochrome oxidase III (coxIII) and cytochrome b (cytb) genes showed lack of grouping according to geographic origins and individual hosts carrying large numbers of L. sabrazesi strains. Monitoring gametocytemia in blood samples from infected chickens over time showed ‘relapse’ or persistence of low-level gametocytemia for 4–5 months, which could be explored as an in vivo model for testing drugs against liver stages of Apicomplexan parasites. This study provides important information on population diversity and transmission dynamics of L. sabrazesi and for disease control.
doi:10.1371/journal.pone.0094877
PMCID: PMC3984278  PMID: 24728499
11.  Genome-wide profiling of chromosome interactions in Plasmodium falciparum characterizes nuclear architecture and reconfigurations associated with antigenic variation 
Molecular microbiology  2013;90(3):519-537.
Summary
Spatial relationships within the eukaryotic nucleus are essential for proper nuclear function. In Plasmodium falciparum, the repositioning of chromosomes has been implicated in the regulation of the expression of genes responsible for antigenic variation, and the formation of a single, peri-nuclear nucleolus results in the clustering of rDNA. Nevertheless, the precise spatial relationships between chromosomes remain poorly understood, because, until recently, techniques with sufficient resolution have been lacking. Here we have used chromosome conformation capture and second-generation sequencing to study changes in chromosome folding and spatial positioning that occur during switches in var gene expression. We have generated maps of chromosomal spatial affinities within the P. falciparum nucleus at 25 Kb resolution, revealing a structured nucleolus, an absence of chromosome territories, and confirming previously identified clustering of heterochromatin foci. We show that switches in var gene expression do not appear to involve interaction with a distant enhancer, but do result in local changes at the active locus. These maps reveal the folding properties of malaria chromosomes, validate known physical associations, and characterize the global landscape of spatial interactions. Collectively, our data provide critical information for a better understanding of gene expression regulation and antigenic variation in malaria parasites.
doi:10.1111/mmi.12381
PMCID: PMC3894959  PMID: 23980881
Plasmodium falciparum; antigenic variation; genome conformation capture; 3C; HiC
12.  Genetic diversity of Plasmodium vivax population in Anhui province of China 
Malaria Journal  2014;13:13.
Background
Although the numbers of malaria cases in China have been declining in recent years, outbreaks of Plasmodium vivax malaria were still being reported in rural areas south of the Yellow River. To better understand the transmission dynamics of P. vivax parasites in China, the extent of genetic diversity of P. vivax populations circulating in Bozhou of Anhui province of China were investigated using three polymorphic genetic markers: merozoite surface proteins 1 and 3α (pvmsp-1 and pvmsp-3α) and circumsporozoite protein (pvcsp).
Methods
Forty-five P. vivax clinical isolates from Bouzhou of Anhui province were collected from 2009 to 2010 and were analysed using PCR/RFLP or DNA sequencing.
Results
Seven and six distinct allelic variants were identified using PCR/RFLP analysis of pvmsp-3α with HhaI and AluI, respectively. DNA sequence analysis of pvmsp-1 (variable block 5) revealed that there were Sal-I and recombinant types but not Belem type, and seven distinct allelic variants in pvmsp-1 were detected, with recombinant subtype 2 (R2) being predominant (66.7%). All the isolates carried pvcsp with VK210 type but not VK247 or P. vivax-like types in the samples. Sequence analysis of pvcsp gene revealed 12 distinct allelic variants, with VK210-1 being predominant (41.5%).
Conclusions
The present data indicate that there is some degree of genetic diversity among P. vivax populations in Anhui province of China. The genetic data obtained may assist in the surveillance of P. vivax infection in endemic areas or in tracking potential future disease outbreak.
doi:10.1186/1475-2875-13-13
PMCID: PMC3893497  PMID: 24401153
Plasmodium vivax; pvmsp-1; pvmsp-3α; pvcsp; Anhui; China
13.  Optimized protocols for improving the likelihood of cloning recombinant progeny from Plasmodium yoelii genetic crosses 
Experimental parasitology  2012;133(1):44-50.
Genetic cross is a powerful tool for studying malaria genes contributing to drug resistance, parasite development, and pathogenesis. Cloning and identification of recombinant progeny (RP) is laborious and expensive, especially when a large proportion of progeny derived from self-fertilization are present in the uncloned progeny of a genetic cross. Since the frequency of cross-fertilization affects the number of recombinant progeny in a genetic cross, it is important to optimize the procedure of a genetic cross to maximize the cross-fertilization. Here we investigated the factors that might influence the chances of obtaining RP from a genetic cross and showed that different Plasmodium yoelii strains/subspecies/clones had unique abilities in producing oocysts in a mosquito midgut. When a genetic cross is performed between two parents producing different numbers of functional gametocytes, the ratio of parental parasites must be adjusted to improve the chance of obtaining RP. An optimized parental ratio could be established based on oocyst counts from single infection of each parent before crossing experiments, which may reflect the efficiency of gametocyte production and/or fertilization. The timing of progeny cloning is also important; cloning of genetic cross progeny from mice directly infected with sporozoites (vs. frozen blood after needle passage) at a time when parasitemia is low (usually <1%) could improve the chance of obtaining RP. This study provides an optimized protocol for efficiently cloning RPs from a genetic cross of malaria parasites.
doi:10.1016/j.exppara.2012.10.011
PMCID: PMC3530016  PMID: 23116600
Malaria; genetic cross; rodent; parasite cloning; microsatellite
14.  Peripheral Blood Stem Cell Transplant Related Plasmodium falciparum Infection in a Patient with Sickle Cell Disease 
Transfusion  2012;52(12):2677-2682.
Background
Although transmission of Plasmodium falciparum (Pf) infection during red blood cell transfusion from an infected donor has been well documented, malaria parasites are not known to infect hematopoietic stem cells. We report a case of Pf infection in a patient 11 days after peripheral blood stem cell transplant for sickle cell disease.
Study Design and Methods
Malaria parasites were detected in thick blood smears by Giemsa staining. Pf HRP2 antigen was measured by ELISA on whole blood and plasma. Pf DNA was detected in whole blood and stem cell retention samples by real-time PCR using Pf species–specific primers and probes. Genotyping of 8 Pf microsatellites was performed on genomic DNA extracted from whole blood.
Results
Pf was not detected by molecular, serologic or parasitologic means in samples from the recipient until day 11 post-transplant, coincident with the onset of symptoms. In contrast, Pf antigen was retrospectively detected in stored plasma collected 3 months prior to transplant from the asymptomatic donor. Pf DNA was detected in whole blood from both the donor and recipient post-transplant, and genotyping confirmed shared markers between donor and recipient Pf strains. Look back analysis of red blood cell donors was negative for Pf infection.
Conclusions
These findings are consistent with transmission by the stem cell product and have profound implications with respect to the screening of potential stem cell donors and recipients from malaria-endemic regions.
doi:10.1111/j.1537-2995.2012.03673.x
PMCID: PMC3408807  PMID: 22536941
Plasmodium falciparum; Sickle Cell Disease; Peripheral Blood Stem Cell Transplant; Real-Time PCR; PfHRP2 Antigen ELISA
15.  Anti-Pfs25 Human Plasma Reduces Transmission of Plasmodium falciparum Isolates That Have Diverse Genetic Backgrounds 
Infection and Immunity  2013;81(6):1984-1989.
Pfs25 is a leading candidate for a malaria transmission-blocking vaccine whose potential has been demonstrated in a phase 1 trial with recombinant Pfs25 formulated with Montanide ISA51. Because of limited sequence polymorphism, the anti-Pfs25 antibodies induced by this vaccine are likely to have transmission-blocking or -reducing activity against most, if not all, field isolates. To test this hypothesis, we evaluated transmission-blocking activities by membrane feeding assay of anti-Pfs25 plasma from the Pfs25/ISA51 phase 1 trial against Plasmodium falciparum parasites from patients in two different geographical regions of the world, Thailand and Burkina Faso. In parallel, parasite isolates from these patients were sequenced for the Pfs25 gene and genotyped for seven microsatellites. The results indicate that despite different genetic backgrounds among parasite isolates, the Pfs25 sequences are highly conserved, with a single nonsynonymous nucleotide polymorphism detected in 1 of 41 patients in Thailand and Burkina Faso. The anti-Pfs25 immune plasma had significantly higher transmission-reducing activity against parasite isolates from the two geographical regions than the nonimmune controls (P < 0.0001).
doi:10.1128/IAI.00016-13
PMCID: PMC3676035  PMID: 23509152
16.  Artemisinin-resistant Plasmodium falciparum in Pursat province, western Cambodia: a parasite clearance rate study 
The Lancet infectious diseases  2012;12(11):851-858.
Summary
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.
doi:10.1016/S1473-3099(12)70181-0
PMCID: PMC3786328  PMID: 22940027
17.  Ex Vivo Susceptibility of Plasmodium falciparum to Antimalarial Drugs in Western, Northern, and Eastern Cambodia, 2011-2012: Association with Molecular Markers 
Antimicrobial Agents and Chemotherapy  2013;57(11):5277-5283.
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.
doi:10.1128/AAC.00687-13
PMCID: PMC3811250  PMID: 23939897
18.  Multiple populations of artemisinin-resistant Plasmodium falciparum in Cambodia 
Nature genetics  2013;45(6):10.1038/ng.2624.
We describe an analysis of genome variation in 825 Plasmodium falciparum samples from Asia and Africa that reveals an unusual pattern of parasite population structure at the epicentre of artemisinin resistance in western Cambodia. Within this relatively small geographical 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 remarkably high levels of haplotype homozygosity, indicative of founder effects and recent population expansion. We provide a catalogue of SNPs that show high levels of differentiation in the artemisinin-resistant subpopulations, including codon variants in various transporter proteins and DNA mismatch repair proteins. These data provide a population genetic framework for investigating the biological origins of artemisinin resistance and for defining molecular markers to assist its elimination.
doi:10.1038/ng.2624
PMCID: PMC3807790  PMID: 23624527
19.  Mechanisms of in vitro resistance to dihydroartemisinin in Plasmodium falciparum 
Molecular microbiology  2012;86(1):111-128.
Summary
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 since 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 defense network, and increased expression of many chaperones in the DHA-resistant parasites. Without drug pressure, the DHA resistant parasites reverted to sensitive in approximately eight 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 defense play a role in in vitro resistance to DHA, providing potential molecular markers for ART resistance.
doi:10.1111/j.1365-2958.2012.08180.x
PMCID: PMC3455110  PMID: 22812578
artemisinin resistance; drug selection; mechanism; pfmdr1; antioxidant defense
20.  Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing 
Nature  2012;487(7407):375-379.
Malaria elimination strategies require surveillance of the parasite population for genetic changes that demand a public health response, such as new forms of drug resistance. 1,2 Here we describe methods for large-scale analysis of genetic variation in Plasmodium falciparum by deep sequencing of parasite DNA obtained from the blood of patients with malaria, either directly or after short term culture. Analysis of 86,158 exonic SNPs that passed genotyping quality control in 227 samples from Africa, Asia and Oceania provides genome-wide estimates of allele frequency distribution, population structure and linkage disequilibrium. By comparing the genetic diversity of individual infections with that of the local parasite population, we derive a metric of within-host diversity that is related to the level of inbreeding in the population. An open-access web application has been established for exploration of regional differences in allele frequency and of highly differentiated loci in the P. falciparum genome.
doi:10.1038/nature11174
PMCID: PMC3738909  PMID: 22722859
21.  Macrophage migration inhibitory factor homolog from Plasmodium yoelii modulates monocyte recruitment and activation in spleen during infection 
Parasitology research  2011;110(5):1755-1763.
Macrophage migration inhibitory factor (MIF) has been shown to be involved in the pathogenesis of severe malaria. Malaria parasites express an MIF homolog that may play a role in regulating host immune responses and a recent study showed that overexpression of MIF reduced parasitemia in a mouse malaria model. Another recent study showed migration of monocytes to the spleen contributed to the control of blood stage infection. However, there are few papers describing the effect of MIF on monocyte recruitment/activation during the infection. We generated recombinant P. yoelii MIF (rPyMIF) and investigated its function on purified mouse CD11b+ cells in vitro and monocyte responses in vivo. The result shows that rPyMIF protein bound to mouse CD11b+ cells and inhibited their random migration in vitro. On the other hand, rPyMIF did not induce cytokine release from the cells directly or modulate LPS-induced cytokine release. Mice immunized with rPyMIF showed transient, but significantly lower parasitemia than the control mice at day 3 after lethal Py17XL challenge. The total number of CD11b+ cells in the spleens was significantly higher in rPyMIF-immunized group. Further investigation revealed that there were significantly higher numbers of recruited and activated monocytes in the spleens of rPyMIF immunization group on day 3. These results indicate that PyMIF potentially modulates monocyte recruitment and activation during infection of P. yoelii erythrocytic stages.
doi:10.1007/s00436-011-2696-6
PMCID: PMC3427932  PMID: 22015474
malaria; macrophage migration inhibitory factor; mouse; monocyte; antibody
22.  A new malaria antigen produces partial protection against Plasmodium yoelii challenge 
Parasitology research  2011;110(4):1337-1345.
Of all the parasitic diseases, malaria is the number one killer. Despite tremendous efforts in disease control and research, nearly a million people, primarily children, still die from the disease each year, partly due to drug resistance and the lack of an effective vaccine. Many parasite antigens have been identified and evaluated for vaccine development; however, none has been approved for human use. Antigenic variation, complex life cycle, and inadequate understanding of the mechanisms of parasite-host interaction and of host immune response all contribute to the lack of an effective vaccine for malaria control. In a recent search of genome-wide polymorphism in Plasmodium falciparum, several molecules were found to be recognized by sera from patients infected with the P. falciparum parasite. Here, we have expressed a 350-amino acid N-terminus from one of the homologous candidate antigen genes from the rodent malaria parasite Plasmodium yoelii (Py01157, a putative dentin phosphorin) in bacteria and evaluated the immune response and protection generated after immunization with the recombinant protein. We showed that the recombinant protein was recognized by sera from both mice and humans infected with malaria parasites. Partial protection was observed after challenge with non-lethal P. yoelii 17XNL but not with the lethal P. yoelii 17XL parasite. Further tests using a full-length protein or the conserved C-terminus may provide additional information on whether this protein has the potential for being a malaria vaccine.
doi:10.1007/s00436-011-2630-y
PMCID: PMC3437252  PMID: 21915626
Malaria; Vaccine candidate; Recombinant protein; Immune protection; Mouse
23.  Genetic Diversity and Lack of Artemisinin Selection Signature on the Plasmodium falciparum ATP6 in the Greater Mekong Subregion 
PLoS ONE  2013;8(3):e59192.
The recent detection of clinical Artemisinin (ART) resistance manifested as delayed parasite clearance in the Cambodia-Thailand border area raises a serious concern. The mechanism of ART resistance is not clear; but the P. falciparum sarco/endoplasmic reticulum Ca2+-ATPase (PfSERCA or PfATP6) has been speculated to be the target of ARTs and thus a potential marker for ART resistance. Here we amplified and sequenced pfatp6 gene (∼3.6 Kb) in 213 samples collected after 2005 from the Greater Mekong Subregion, where ART drugs have been used extensively in the past. A total of 24 single nucleotide polymorphisms (SNPs), including 8 newly found in this study and 13 nonsynonymous, were identified. However, these mutations were either uncommon or also present in other geographical regions with limited ART use. None of the mutations were suggestive of directional selection by ARTs. We further analyzed pfatp6 from a worldwide collection of 862 P. falciparum isolates in 19 populations from Asia, Africa, South America and Oceania, which include samples from regions prior to and after deployments ART drugs. A total of 71 SNPs were identified, resulting in 106 nucleotide haplotypes. Similarly, many of the mutations were continent-specific and present at frequencies below 5%. The most predominant and perhaps the ancestral haplotype occurred in 441 samples and was present in 16 populations from Asia, Africa, and Oceania. The 3D7 haplotype found in 54 samples was the second most common haplotype and present in nine populations from all four continents. Assessment of the selection strength on pfatp6 in the 19 parasite populations found that pfatp6 in most of these populations was under purifying selection with an average dN/dS ratio of 0.333. Molecular evolution analyses did not detect significant departures from neutrality in pfatp6 for most populations, challenging the suitability of this gene as a marker for monitoring ART resistance.
doi:10.1371/journal.pone.0059192
PMCID: PMC3608609  PMID: 23555629
24.  Malaria in the Greater Mekong Subregion: Heterogeneity and Complexity 
Acta Tropica  2011;121(3):227-239.
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 P. 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.
doi:10.1016/j.actatropica.2011.02.016
PMCID: PMC3132579  PMID: 21382335
malaria; the Greater Mekong Subregion; epidemiology; Anopheles vectors; drug resistance; border malaria; elimination
25.  Challenges and prospects for malaria elimination in the Greater Mekong Subregion 
Acta Tropica  2011;121(3):240-245.
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 P. 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.
doi:10.1016/j.actatropica.2011.04.006
PMCID: PMC3155744  PMID: 21515238
malaria; the Greater Mekong Subregion; epidemiology; vector systems; drug resistance; counterfeit drugs

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