Plasmodium vivax populations in the western Greater Mekong Subregion evaluated using a genetic barcode.

An improved understanding of the Plasmodium vivax populations in the Great Mekong Subregion (GMS) is needed to monitor the progress of malaria elimination. This study aimed to use a P. vivax single nucleotide polymorphism (SNP) barcode to evaluate the population dynamics and explore the gene flow among P. vivax parasite populations in the western GMS (China, Myanmar and Thailand). A total of 315 P. vivax patient samples collected in 2011 and 2018 from four regions of the western GMS were genotyped for 42 SNPs using the high-throughput MassARRAY SNP genotyping technology. Population genetic analysis was conducted to estimate the genetic diversity, effective population size, and population structure among the P. vivax populations. Overall, 291 samples were successfully genotyped at 39 SNPs. A significant difference was observed in the proportion of polyclonal infections among the five P. vivax populations (P = 0.0012, Pearson Chi-square test, χ2 = 18.1), with western Myanmar having the highest proportion (96.2%, 50/52) in 2018. Likewise, the average complexity of infection was also highest in western Myanmar (1.31) and lowest in northeast Myanmar (1.01) in 2018. The older samples from western China in 2011 had the highest pairwise nucleotide diversity (π, 0.388 ± 0.046), expected heterozygosity (He, 0.363 ± 0.02), and the largest effective population size. In comparison, in the neighboring northeast Myanmar, the more recent samples in 2018 showed the lowest values (π, 0.224 ± 0.036; He, 0.220 ± 0.026). Furthermore, the 2018 northeast Myanmar parasites showed high and moderate genetic differentiation from other populations with FST values of 0.162-0.252, whereas genetic differentiation among other populations was relatively low (FST ≤ 0.059). Principal component analysis, phylogeny, and STRUCTURE analysis showed that the P. vivax population in northeast Myanmar in 2018 substantially diverged from other populations. Although the 42 SNP barcode is a valuable tool for tracking parasite origins of worldwide parasite populations, a more extended barcode with additional SNPs is needed to distinguish the more related parasite populations in the western GMS.

The DEAD-box RNA helicase PfDOZI imposes opposing actions on RNA metabolism in Plasmodium falciparum.

In malaria parasites, the regulation of mRNA translation, storage and degradation during development and life-stage transitions remains largely unknown. Here, we functionally characterized the DEAD-box RNA helicase PfDOZI in P. falciparum. Disruption of pfdozi enhanced asexual proliferation but reduced sexual commitment and impaired gametocyte development. By quantitative transcriptomics, we show that PfDOZI is involved in the regulation of invasion-related genes and sexual stage-specific genes during different developmental stages. PfDOZI predominantly participates in processing body-like mRNPs in schizonts but germ cell granule-like mRNPs in gametocytes to impose opposing actions of degradation and protection on different mRNA targets. We further show the formation of stress granule-like mRNPs during nutritional deprivation, highlighting an essential role of PfDOZI-associated mRNPs in stress response. We demonstrate that PfDOZI participates in distinct mRNPs to maintain mRNA homeostasis in response to life-stage transition and environmental changes by differentially executing post-transcriptional regulation on the target mRNAs.

Effectiveness of unsupervised primaquine regimen for preventing Plasmodium vivax malaria relapses in northeast Myanmar, a single-arm non-randomized observational study.

BACKGROUND: Plasmodium vivax presents a significant challenge for malaria elimination in the Greater Mekong Subregion (GMS). We evaluated the effectiveness of primaquine (PQ) for reducing relapses of vivax malaria. METHODS: Patients with uncomplicated P. vivax malaria from eastern Myanmar received chloroquine (CQ, 25 mg base/kg given in 3 days) plus unsupervised PQ (0.25 mg/kg/day for 14 days) without screening for glucose-6-phosphate dehydrogenase deficiency and were followed for a year. RESULTS: Totally 556 patients were enrolled to receive the CQ/PQ treatment from February 2012 to August 2013. During the follow-up, 38 recurrences were detected, presenting a cumulative rate of recurrence of 9.1% (95% confidence interval, 4.1-14.1%). Genotyping at the pvmsp1 and pvmsp3α loci by Amplicon deep sequencing and model prediction indicated that 13 of the 27 recurrences with genotyping data were likely due to relapses. Notably, all confirmed relapses occurred within the first six months. CONCLUSIONS: The unsupervised standard dose of PQ was highly effective as a radical cure for P. vivax malaria in eastern Myanmar. The high presumed effectiveness might have benefited from the health messages delivered during the enrollment and follow-up activities. Six-month follow-ups in the GMS are sufficient for detecting most relapses.

Preliminary characterization of Plasmodium vivax sporozoite antigens as pre-erythrocytic vaccine candidates.

Plasmodium vivax pre-erythrocytic (PE) vaccine research has lagged far behind efforts to develop Plasmodium falciparum vaccines. There is a critical gap in our knowledge of PE antigen targets that can induce functionally inhibitory neutralizing antibody responses. To overcome this gap and guide the selection of potential PE vaccine candidates, we considered key characteristics such as surface exposure, essentiality to infectivity and liver stage development, expression as recombinant proteins, and functional immunogenicity. Selected P. vivax sporozoite antigens were surface sporozoite protein 3 (SSP3), sporozoite microneme protein essential for cell traversal (SPECT1), sporozoite surface protein essential for liver-stage development (SPELD), and M2 domain of MAEBL. Sequence analysis revealed little variation occurred in putative B-cell and T-cell epitopes of the PE candidates. Each antigen was tested for expression as refolded recombinant proteins using an established bacterial expression platform and only SPELD failed. The successfully expressed antigens were immunogenic in vaccinated laboratory mice and were positively reactive with serum antibodies of P. vivax-exposed residents living in an endemic region in Thailand. Vaccine immune antisera were tested for reactivity to native sporozoite proteins and for their potential vaccine efficacy using an in vitro inhibition of liver stage development assay in primary human hepatocytes quantified on day 6 post-infection by high content imaging analysis. The anti-PE sera produced significant inhibition of P. vivax sporozoite invasion and liver stage development. This report provides an initial characterization of potential new PE candidates for a future P. vivax vaccine.

Multidisciplinary Investigations of Sustained Malaria Transmission in the Greater Mekong Subregion.

In the course of malaria elimination in the Greater Mekong Subregion (GMS), malaria epidemiology has experienced drastic spatiotemporal changes with residual transmission concentrated along international borders and the rising predominance of Plasmodium vivax. The emergence of Plasmodium falciparum parasites resistant to artemisinin and partner drugs renders artemisinin-based combination therapies less effective while the potential spread of multidrug-resistant parasites elicits concern. Vector behavioral changes and insecticide resistance have reduced the effectiveness of core vector control measures. In recognition of these problems, the Southeast Asian International Center of Excellence for Malaria Research (ICEMR) has been conducting multidisciplinary research to determine how human migration, antimalarial drug resistance, vector behavior, and insecticide resistance sustain malaria transmission at international borders. These efforts allow us to comprehensively understand the ecology of border malaria transmission and develop population genomics tools to identify and track parasite introduction. In addition to employing in vivo, in vitro, and molecular approaches to monitor the emergence and spread of drug-resistant parasites, we also use genomic and genetic methods to reveal novel mechanisms of antimalarial drug resistance of parasites. We also use omics and population genetics approaches to study insecticide resistance in malaria vectors and identify changes in mosquito community structure, vectorial potential, and seasonal dynamics. Collectively, the scientific findings from the ICEMR research activities offer a systematic view of the factors sustaining residual malaria transmission and identify potential solutions to these problems to accelerate malaria elimination in the GMS.

Population genomics in neglected malaria parasites.

Malaria elimination includes neglected human malaria parasites Plasmodium vivax, Plasmodium ovale spp., and Plasmodium malariae. Biological features such as association with low-density infection and the formation of hypnozoites responsible for relapse make their elimination challenging. Studies on these parasites rely primarily on clinical samples due to the lack of long-term culture techniques. With improved methods to enrich parasite DNA from clinical samples, whole-genome sequencing of the neglected malaria parasites has gained increasing popularity. Population genomics of more than 2200 P. vivax global isolates has improved our knowledge of parasite biology and host-parasite interactions, identified vaccine targets and potential drug resistance markers, and provided a new way to track parasite migration and introduction and monitor the evolutionary response of local populations to elimination efforts. Here, we review advances in population genomics for neglected malaria parasites, discuss how the rich genomic information is being used to understand parasite biology and epidemiology, and explore opportunities for the applications of malaria genomic data in malaria elimination practice.

Distinct Histone Post-translational Modifications during Plasmodium falciparum Gametocyte Development.

Histones are the building units of nucleosomes, which constitute chromatin. Histone post-translational modifications (PTMs) play an essential role in epigenetic gene regulation. The Plasmodium falciparum genome encodes canonical and variant histones and a collection of conserved enzymes for histone PTMs and chromatin remodeling. Herein, we profiled the P. falciparum histone PTMs during the development of gametocytes, the obligatory stage for parasite transmission. Mass spectrometric analysis of histones extracted from the early, middle, and late stages of gametocytes identified 457 unique histone peptides with 90 PTMs, of which 50% were novel. The gametocyte histone PTMs display distinct patterns from asexual stages, with many new methylation sites in histones H3 and H3.3 (e.g., K14, K18, and K37). Quantitative analyses revealed a high abundance of acetylation in H3 and H4, mono-methylation of H3/H3.3 K37, and ubiquitination of H3BK112, suggesting that these PTMs play critical roles in gametocytes. Gametocyte histones also showed extensive and unique combinations of PTMs. These data indicate that the parasite harbors distinct transcription regulation mechanisms during gametocyte development and lay the foundation for further characterization of epigenetic regulation in the life cycle of the malaria parasite.

New Plasmodium vivax Genomes From the China-Myanmar Border.

Plasmodium vivax is increasingly the dominant species of malaria in the Greater Mekong Subregion (GMS), which is pursuing regional malaria elimination. P. vivax lineages in the GMS are poorly characterized. Currently, P. vivax reference genomes are scarce due to difficulties in culturing the parasite and lack of high-quality samples. In addition, P. vivax is incredibly diverse, necessitating the procurement of reference genomes from different geographical regions. Here we present four new P. vivax draft genomes assembled de novo from clinical samples collected in the China-Myanmar border area. We demonstrate comparable length and content to existing genomes, with the majority of structural variation occurring around subtelomeric regions and exported proteins, which we corroborated with detection of copy number variations in these regions. We predicted peptides from all PIR gene subfamilies, except for PIR D. We confirmed that proteins classically labeled as PIR D family members are not identifiable by PIR motifs, and actually bear stronger resemblance to DUF (domain of unknown function) family DUF3671, potentially pointing to a new, closely related gene family. Further, phylogenetic analyses of MSP7 genes showed high variability within the MSP7-B family compared to MSP7-A and -C families, and the result was comparable to that from whole genome analyses. The new genome assemblies serve as a resource for studying P. vivax within the GMS.

Population genomics identifies a distinct Plasmodium vivax population on the China-Myanmar border of Southeast Asia.

Plasmodium vivax has become the predominant malaria parasite and a major challenge for malaria elimination in the Greater Mekong Subregion (GMS). Yet, our knowledge about the evolution of P. vivax populations in the GMS is fragmental. We performed whole genome sequencing on 23 P. vivax samples from the China-Myanmar border (CMB) and used 21 high-coverage samples to compare to over 200 samples from the rest of the GMS. Using genome-wide single nucleotide polymorphisms (SNPs), we analyzed population differentiation, genetic structure, migration and potential selection using an array of methods. The CMB parasites displayed a higher proportion of monoclonal infections, and 52% shared over 90% of their genomes in identity-by-descent segments with at least one other sample from the CMB, suggesting preferential expansion of certain parasite strains in this region, likely resulting from the P. vivax outbreaks occurring during this study period. Principal component, admixture, fixation index and phylogenetic analyses all identified that parasites from the CMB were genetically distinct from parasites from eastern parts of the GMS (Cambodia, Laos, Vietnam, and Thailand), whereas the eastern GMS parasite populations were largely undifferentiated. Such a genetic differentiation pattern of the P. vivax populations from the GMS parasite was largely explainable through geographic distance. Using the genome-wide SNPs, we narrowed down to a set of 36 SNPs for differentiating parasites from different areas of the GMS. Genome-wide scans to determine selection in the genome with two statistical methods identified genes potentially under drug selection, including genes associated with antifolate resistance and genes linked to chloroquine resistance in Plasmodium falciparum.

Lineage-Specific Expansion of Plasmodium falciparum Parasites With pfhrp2 Deletion in the Greater Mekong Subregion.

Deletion of the pfhrp2 gene in Plasmodium falciparum can lead to false-negative rapid diagnostic test (RDT) results, constituting a major challenge for evidence-based malaria treatment. Here we analyzed the whole genome sequences of 138 P. falciparum clinical samples collected from the China-Myanmar boarder for pfhrp2 and pfhrp3 gene deletions. We found pfhrp2 and pfhrp3 deletions in 9.4% and 3.6% of samples, respectively, with no samples harboring deletions of both genes. The pfhrp2 deletions showed 2 distinct breakpoints, representing 2 different chromosomal deletion events. A phylogenetic analysis performed using genome-wide single-nucleotide polymorphisms revealed that the 2 pfhrp2 breakpoint groups as well as all the pfhrp3-negative parasites formed separate clades, suggesting they might have resulted from clonal expansion of pfhrp2- and pfhrp3-negative parasites. These findings highlight the need for urgent surveys to determine the prevalence of pfhrp2-negative parasites causing false-negative RDT results and a plan for switching of RDTs pending the survey results.

A glance of the blood stage transcriptome of a Southeast Asian Plasmodium ovale isolate.

Plasmodium ovale accounts for a disproportionate number of travel-related malaria cases. This parasite is understudied since there is a reliance on clinical samples. We collected a P. ovale curtisi parasite isolate from a clinical case in western Thailand and performed RNA-seq analysis on the blood stage transcriptomes. Using both de novo assembly and alignment-based methods, we detected the transcripts for 6628 out of 7280 annotated genes. For those lacking evidence of expression, the vast majority belonged to the PIR and STP1 gene families. We identified new splicing patterns for over 2500 genes, and mapped at least one untranslated region for over half of all annotated genes. Our analysis also detected a notable presence of anti-sense transcripts for over 10% of P. ovale curtisi genes. This transcriptomic analysis provides new insights into the blood-stage biology of this neglected parasite.

Plasmodium falciparum Falcipain-2a Polymorphisms in Southeast Asia and Their Association With Artemisinin Resistance.

Background: Falcipain-2a ([FP2a] PF3D7_1115700) is a Plasmodium falciparum cysteine protease and hemoglobinase. Functional FP2a is required for potent activity of artemisinin, and in vitro selection for artemisinin resistance selected for an FP2a nonsense mutation. Methods: To investigate associations between FP2a polymorphisms and artemisinin resistance and to characterize the diversity of the enzyme in parasites from the China-Myanmar border, we sequenced the full-length FP2a gene in 140 P falciparum isolates collected during 2004-2011. Results: The isolates were grouped into 8 different haplotype groups. Haplotype group I appeared in samples obtained after 2008, coinciding with implementation of artemisinin-based combination therapy in this region. In functional studies, compared with wild-type parasites, the FP2a haplotypes demonstrated increased ring survival, and all haplotype groups exhibited significantly reduced FP2a activity, with group I showing the slowest protease kinetics and reduced parasite fitness. Conclusions: These results suggest that altered hemoglobin digestion due to FP2a mutations may contribute to artemisinin resistance.

Further evaluation of the NWF filter for the purification of Plasmodium vivax-infected erythrocytes.

BACKGROUND: Isolation of Plasmodium-infected red blood cells (iRBCs) from clinical blood samples is often required for experiments, such as ex vivo drug assays, in vitro invasion assays and genome sequencing. Current methods for removing white blood cells (WBCs) from malaria-infected blood are time-consuming or costly. A prototype non-woven fabric (NWF) filter was developed for the purification of iRBCs, which showed great efficiency for removing WBCs in a pilot study. Previous work was performed with prototype filters optimized for processing 5-10 mL of blood. With the commercialization of the filters, this study aims to evaluate the efficiency and suitability of the commercial NWF filter for the purification of Plasmodium vivax-infected RBCs in smaller volumes of blood and to compare its performance with that of Plasmodipur® filters. METHODS: Forty-three clinical P. vivax blood samples taken from symptomatic patients attending malaria clinics at the China-Myanmar border were processed using the NWF filters in a nearby field laboratory. The numbers of WBCs and iRBCs and morphology of P. vivax parasites in the blood samples before and after NWF filtration were compared. The viability of P. vivax parasites after filtration from 27 blood samples was examined by in vitro short-term culture. In addition, the effectiveness of the NWF filter for removing WBCs was compared with that of the Plasmodipur® filter in six P. vivax blood samples. RESULTS: Filtration of 1-2 mL of P. vivax-infected blood with the NWF filter removed 99.68% WBCs. The densities of total iRBCs, ring and trophozoite stages before and after filtration were not significantly different (P > 0.05). However, the recovery rates of schizont- and gametocyte-infected RBCs, which were minor parasite stages in the clinical samples, were relatively low. After filtration, the P. vivax parasites did not show apparent morphological changes. Culture of 27 P. vivax-infected blood samples after filtration showed that parasites successfully matured into the schizont stage. The WBC removal rates and iRBC recovery rates were not significantly different between the NWF and Plasmodipur® filters (P > 0.05). CONCLUSIONS: When tested with 1-2 mL of P. vivax-infected blood, the NWF filter could effectively remove WBCs and the recovery rates for ring- and trophozoite-iRBCs were high. P. vivax parasites after filtration could be successfully cultured in vitro to reach maturity. The performance of the NWF and Plasmodipur® filters for removing WBCs and recovering iRBCs was comparable.

Genome-wide association analysis identifies genetic loci associated with resistance to multiple antimalarials in Plasmodium falciparum from China-Myanmar border.

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.

Artemisinin resistance at the China-Myanmar border and association with mutations in the K13 propeller gene.

Artemisinin resistance in Plasmodium falciparum parasites in Southeast Asia is a major concern for malaria control. Its emergence at the China-Myanmar border, where there have been more than 3 decades of artemisinin use, has yet to be investigated. Here, we comprehensively evaluated the potential emergence of artemisinin resistance and antimalarial drug resistance status in P. falciparum using data and parasites from three previous efficacy studies in this region. These efficacy studies of dihydroartemisinin-piperaquine combination and artesunate monotherapy of uncomplicated falciparum malaria in 248 P. falciparum patients showed an overall 28-day adequate clinical and parasitological response of >95% and day 3 parasite-positive rates of 6.3 to 23.1%. Comparison of the 57 K13 sequences (24 and 33 from day 3 parasite-positive and -negative cases, respectively) identified nine point mutations in 38 (66.7%) samples, of which F446I (49.1%) and an N-terminal NN insertion (86.0%) were predominant. K13 propeller mutations collectively, the F446I mutation alone, and the NN insertion all were significantly associated with day 3 parasite positivity. Increased ring-stage survival determined using the ring-stage survival assay (RSA) was highly associated with the K13 mutant genotype. Day 3 parasite-positive isolates had ∼10 times higher ring survival rates than day 3 parasite-negative isolates. Divergent K13 mutations suggested independent evolution of artemisinin resistance. Taken together, this study confirmed multidrug resistance and emergence of artemisinin resistance in P. falciparum at the China-Myanmar border. RSA and K13 mutations are useful phenotypic and molecular markers for monitoring artemisinin resistance.