A novel peptide pair-based rapid fluorescent diagnostic system for malaria Plasmodium falciparum detection.

Advanced diagnostic materials, such as aptamers, are required due to the scarcity of efficient diagnostic antibodies and the low sensitivity of rapid diagnostic kits at detecting the malaria parasite, Plasmodium falciparum. METHODS: Two peptides M2.9 [(KPTAEQTESPELQSAPEN) and M2.17 (KILFNVYSPLGCTCECWV)] were designed using simple epitope prediction tools and modified against the merozoite surface antigen 2 of P. falciparum (Pf.MSP2) by 3-dimensional modeling based on binding affinity. Based on five prediction tools for hydropathy, M2.17 was selected as an appropriate capture peptide. A peptide-based fluorescence-linked immunosorbent assay (FLISA) and a peptide pair-based fluorescent immunochromatographic test strip (FICT) were developed to detect P. falciparum 3D7 (drug-sensitive) and P. falciparum K1 (multi drugs-resistant) strains. RESULTS: Bioinformatic analysis of two peptides demonstrated the potential binding affinity with the merozoite surface protein 2 of P. falciparum (Pf.MSP2) with a positive hydropathy value. The limit of detection (LOD) of FLISA was 10 parasites/µL and of a peptide pair-linked rapid FICT system was 5 and 200 parasites/µL for P. falciparum 3D7 and K1, respectively. Compared to commercial rapid detection systems (RDTs), a peptide pair-linked FICT system exhibited a 20-fold greater efficiency in detecting P. falciparum 3D7 and specifically discriminated another protozoan spp. CONCLUSION: A peptide pair-linked rapid diagnostic strip could be an alternative to conventional RDTs for monitoring wild-type and drug-resistant malaria parasites.

Geographical and temporal dynamics of genetic diversity of Plasmodium falciparum merozoite surface proteins 1/2 in India.

The high genetic diversity of Plasmodium falciparum (Pf) is a big obstacle to successful vaccine development programs. Here, the geographical and temporal dynamics of the genetic diversity of Indian Pf isolates from patients living in Ranchi, Raipur, Mewat, and Rourkela were analyzed. Typing and frequency of merozoite surface protein 1 and 2 genes (pfmsp1/2), their genotypes, clonality, heterozygosity, multiplicity of infection, and neutral evolution metrics were computed. A phylogenetic analysis was also performed for these two genes. The dominant allelic types were K1 (55%) and MAD20 (55%) for msp1, and FC27 (64.7%) for msp2. Infections were mainly monoclonal in Ranchi and Mewat while polyclonal in Raipur and Rourkela. Polyclonal infections dropped from 57.1 to 71.3% in 2013 to 33.3-33.4% in 2016 in Raipur. K1 and MAD20 sequences were highly diverse due to the organization of the amino acid units SGG, SVA, SVT, and SGN. The IC/3D7-related G,S,A-rich region showed a large variation of four to eight amino acid repeats, including mostly GAVASA (81.8%), GSGA (54.5%), and GASGSA (45.5%). The 32-amino acid sequence of the FC27 type was present in all isolates with several mutations. The msp1/2 sequences were not under neutral evolution, except the K1 family, which is under balancing selection. The msp1/2 sequences are phylogenetically closer to previous Indian sequences than those from Africa, Asia, the Americas, and Oceania. This study outlines a high genetic diversity of Pf infections with complex structure, and evolutionary signature changed with time. Supplementary Information: The online version contains supplementary material available at 10.1007/s12639-024-01698-8.

Spatio-temporal analysis of genetic diversity of merozoite surface protein-3 alpha in Myanmar Plasmodium vivax isolates.

Myanmar aims to eliminate malaria by 2030. However, recent increase of malaria incidence is a great challenge to archive that goal. Increasing prevalence of Plasmodium vivax also hinders this endeavor. Monitoring genetic structure of the parasite is necessary to understand genetic nature and evolutionary aspect of P. vivax population in Myanmar. Partial fragment flanking blocks I and II of merozoite surface protein-3 alpha of P. vivax (pvmsp-3α) was amplified from P. vivax isolates collected in Pyin Oo Lwin, Mandalay Region, Myanmar in 2013-2015. Sequence analysis of pvmsp-3α was performed to determine genetic diversity and natural selection of this gene. Spatio-temporal genetic changes of pvmsp-3α in Myanmar P. vivax population were also investigated via comparative analysis of gene sequences obtained in this study and previously reported Myanmar pvmsp-3α sequences. Genetic diversity of Myanmar pvmsp-3α was detected in P. vivax isolates analyzed. Size polymorphisms in block I and amino acid changes and recombination events in block II were main factors contributing to the genetic diversity of pvmsp-3α. Comparative spatio-temporal analysis with previously reported Myanmar pvmsp-3α populations revealed the presence of genetic differences by population with moderate genetic differentiation between populations. Similar pattern of natural selection was also detected in Myanmar pvmsp-3α populations. These suggested that enough size of the P. vivax population sufficient to generate or maintain the genetic diversity remains in the population. Thus, continuous molecular surveillance of genetic structure of Myanmar P. vivax is necessary.

Genetic Diversity and Natural Selection of Plasmodium vivax Merozoite Surface Protein 8 in Global Populations.

Plasmodium vivax Merozoite Surface Protein 8 (PvMSP8) is a promising candidate target for the development of multi-component vaccines. Therefore, determining the genetic variation pattern of Pvmsp8 is essential in providing a reference for the rational design of the P. vivax malaria vaccines. This study delves into the genetic characteristics of the Pvmsp8 gene, specifically focusing on samples from the China-Myanmar border (CMB) region, and contrasts these findings with broader global patterns. The study uncovers that Pvmsp8 exhibits a notable level of conservation across different populations, with limited polymorphisms and relatively low nucleotide diversity (0.00023-0.00120). This conservation contrasts starkly with the high polymorphisms found in other P. vivax antigens such as Pvmsp1. A total of 25 haplotypes and 14 amino acid mutation sites were identified in the global populations, and all mutation sites were confined to non-functional regions. The study also notes that most CMB Pvmsp8 haplotypes are shared among Burmese, Cambodian, Thai, and Vietnamese populations, indicating less geographical variance, but differ notably from those found in Pacific island regions or the Panama. The findings underscore the importance of considering regional genetic diversity in P. vivax when developing targeted malaria vaccines. Non departure from neutral evolution were found by Tajima's D test, however, statistically significant differences were observed between the kn/ks rates. The study's findings are crucial in understanding the evolution and population structure of the Pvmsp8 gene, particularly during regional malaria elimination efforts. The highly conserved nature of Pvmsp8, combined with the lack of mutations in its functional domain, presents it as a promising candidate for developing a broad and effective P. vivax vaccine. This research thus lays a foundation for the rational development of multivalent malaria vaccines targeting this genetically stable antigen.

Anti-Plasmodium vivax merozoite surface protein 3 ϒ (PvMSP3 ϒ) antibodies upon natural infection.

Merozoite surface protein 3 of Plasmodium vivax (PvMSP3) contains a repertoire of protein members with unique sequence organization. While the biological functions of these proteins await elucidation, PvMSP3 has been suggested to be potential vaccine targets. To date, studies on natural immune responses to this protein family have been confined to two members, PvMSP3α and PvMSP3ß. This study analyzed natural IgG antibody responses to PvMSP3γ recombinant proteins derived from two variants: one containing insert blocks (CT1230nF) and the other without insert domain (NR25nF). The former variant was also expressed as two subfragment proteins: one encompassing variable domain I and insert block A (CT1230N) and the other spanning from insert block B to conserved block III (CT1230C). Serum samples were obtained from 246 symptomatic vivax malaria patients in Tak (n = 50) and Ubon Ratchathani (n = 196) Provinces. In total, 176 (71.5%) patients could mount antibodies to at least one recombinant PvMSP3γ antigen. IgG antibodies directed against antigens CT1230nF, CT1230N, CT1230C and NR25nF occurred in 96.6%, 61.4%, 71.6% and 68.2% of samples, respectively, suggesting the widespread occurrence of B-cell epitopes across PvMSP3γ. The rates of seropositivity seemed to correlate with the number of previous malaria episodes. Isotype analysis of anti-PvMSP3γ antibodies has shown predominant cytophilic subclass responses, accounting for 75.4-81.7% for IgG1 and 63.6-77.5% for IgG3. Comparing with previous studies in the same cohort, the numbers of serum samples reactive to antigens derived from P. vivax merozoite surface protein 9 (PvMSP9) and thrombospondin-related anonymous protein (PvTRAP) were higher than those to PvMSP3γ, being 92.7% and 87.0% versus 71.5%, respectively. Three (1.22%) serum samples were nonresponsive to all these malarial proteins. Nevertheless, the relevance of naturally acquired antibodies to PvMSP3γ in host protection requires further studies.

A phase IIa, randomized, double-blind, safety, immunogenicity and efficacy trial of Plasmodium falciparum vaccine antigens merozoite surface protein 1 and RTS,S formulated with AS02 adjuvant in healthy, malaria-naïve adults.

BACKGROUND: To improve the efficacy of Plasmodium falciparum malaria vaccine RTS,S/AS02, we conducted a study in 2001 in healthy, malaria-naïve adults administered RTS,S/AS02 in combination with FMP1, a recombinant merozoite surface-protein-1, C-terminal 42kD fragment. METHODS: A double-blind Phase I/IIa study randomized N = 60 subjects 1:1:1:1 to one of four groups, N = 15/group, to evaluate safety, immunogenicity, and efficacy of intra-deltoid half-doses of RTS,S/AS02 and FMP1/AS02 administered in the contralateral (RTS,S + FMP1-separate) or same (RTS,S + FMP1-same) sites, or FMP1/AS02 alone (FMP1-alone), or RTS,S/AS02 alone (RTS,S-alone) on a 0-, 1-, 3-month schedule. Subjects receiving three doses of vaccine and non-immunized controls (N = 11) were infected with homologous P. falciparum 3D7 sporozoites by Controlled Human Malaria Infection (CHMI). RESULTS: Subjects in all vaccination groups experienced mostly mild or moderate local and general adverse events that resolved within eight days. Anti-circumsporozoite antibody levels were lower when FMP1 and RTS,S were co-administered at the same site (35.0 µg/mL: 95 % CI 20.3-63), versus separate arms (57.4 µg/mL: 95 % CI 32.3-102) or RTS,S alone (62.0 µg/mL: 95 % CI: 37.8-101.8). RTS,S-specific lymphoproliferative responses and ex vivo ELISpot CSP-specific interferon-gamma (IFN-γ) responses were indistinguishable among groups receiving RTS,S/AS02. There was no difference in antibody to FMP1 among groups receiving FMP1/AS02. After CHMI, groups immunized with a RTS,S-containing regimen had âˆ¼ 30 % sterile protection against parasitemia, and equivalent delays in time-to-parasitemia. The FMP1/AS02 alone group showed no sterile immunity or delay in parasitemia. CONCLUSION: Co-administration of RTS,S and FMP1/AS02 reduced anti-RTS,S antibody, but did not affect tolerability, cellular immunity, or efficacy in a stringent CHMI model. Absence of efficacy or delay of patency in the sporozoite challenge model in the FMP1/AS02 group did not rule out efficacy of FMP1/AS02 in an endemic population. However, a Phase IIb trial of FMP1/AS02 in children in malaria-endemic Kenya did not demonstrate efficacy against natural infection. CLINICALTRIALS: gov identifier: NCT01556945.

Genetic diversity of merozoite surface protein-5 (MSP-5) of Plasmodium vivax isolates from Malaria patients in Iran.

Malaria has not yet been eradicated in Iran, and Plasmodium vivax (P. vivax) is the main cause of malaria in the country. This study aimed to investigate and analyze the amount of genetic diversity of Plasmodium vivax merozoite surface protein-5 (PvMSP-5) exon 1 gene in the southeast of Iran.Thirty-five patients with clinical symptoms of P. vivax malaria participated. The exon 1 of PvMSP-5 was amplified by PCR, and the PCR product of all isolates was sequenced, and genetic polymorphisms were determined using various genetic software.The analysis showed that studied isolates are different from one another in the DnaSP software version. Out of the 612 sites, 477 were monomorphic and 135 were segregated. The total number of mutations was 143. The singleton variable and the parsimony informative sites were 23 and 112, respectively. There were 17 specific haplotypes with haplotype diversity equal to 0.943. Nucleotide diversity was equal to 0.06766 in the isolates. The ratio of nonsynonymous (0.06446) to synonymous (0.07909) mutations was 0.815020. Tajima's D, which expressed coding, and non-coding regions, was 0.72403, which was not deemed significant (P > 0.10).The analysis of intrapopulation diversity revealed nucleotide and haplotype diversity in the msp-5 gene of Iranian P. vivax isolates. In addition to balancing or purifying selection, intragenic recombination also contributed to the variation observed in exon 1 of PvMSP-5, according to the findings.

A genomic platform for surveillance and antigen discovery in Plasmodium spp. using long-read amplicon sequencing.

Many vaccine candidate proteins in the malaria parasite Plasmodium falciparum are under strong immunological pressure and confer antigenic diversity. We present a sequencing and data analysis platform for the genomic surveillance of the insertion or deletion (indel)-rich antigens merozoite surface protein 1 (MSP1), MSP2, glutamate-rich protein (GLURP), and CSP from P. falciparum using long-read circular consensus sequencing (CCS) in multiclonal malaria isolates. Our platform uses 40 PCR primers per gene to asymmetrically barcode and identify multiclonal infections in pools of up to 384 samples. With msp2, we validated the method using 235 mock infections combining 10 synthetic variants at different concentrations and infection complexities. We applied this strategy to P. falciparum isolates from a longitudinal cohort in Tanzania. Finally, we constructed an analysis pipeline that streamlines the processing and interpretation of epidemiological and antigenic diversity data from demultiplexed FASTQ files. This platform can be easily adapted to other polymorphic antigens of interest in Plasmodium or any other human pathogen.

Genetic diversity of Plasmodium falciparum among asymptomatic pregnant women on intermittent preventive treatment with sulfadoxine-pyrimethamine in Nigeria.

This study investigated the genetic diversity of Plasmodium falciparum among asymptomatic pregnant women on intermittent preventive treatment with sulfadoxine-pyrimethamine (IPTp-Sp) in Osogbo, southwest Nigeria. Blood sample was obtained from consenting pregnant women attending antenatal clinics. Microscopy and Polymerase chain reaction (PCR) were employed to diagnose and analyse genetic diversity. Of the 301 samples, 53 (18%) and 83 (28%) were positive for P. falciparum by microscopy and PCR, respectively. Using the merozoite surface protein (msp)-1, msp-2, and glutamate-rich protein (glurp) genes of P. falciparum as polymorphic markers, the msp-1 gene showed nine alleles with R033 (66.7%) being predominant, followed by K1 (45.5%) and MAD20 (33.3%). The msp-2 gene had 16 alleles (eight each for FC27 and 3D7). The 3D7 alleles (82.1%) was significantly more than FC27 alleles (48.2%) (p = 0.0093). Nine alleles were detected with glurp gene, presenting with the highest monoclonal and the lowest polyclonal infection. The multiplicity of infection (MOI) of 1.5, 1.8, and 1.2 were obtained for msp-1, msp-2 and glurp genes. In light of the high P. falciparum genetic diversity among pregnant women on IPT-Sp in this study, additional strategies for preventing and controlling malaria in pregnancy might be required.

Genetic diversity and molecular evolution of Plasmodium vivax Duffy Binding Protein and Merozoite Surface Protein-1 in northwestern Thailand.

The local diversity and population structure of malaria parasites vary across different regions of the world, reflecting variations in transmission intensity, host immunity, and vector species. This study aimed to use amplicon sequencing to investigate the genotypic patterns and population structure of P. vivax isolates from a highly endemic province of Thailand in recent years. Amplicon deep sequencing was performed on 70 samples for the 42-kDa region of pvmsp1 and domain II of pvdbp. Unique haplotypes were identified and a network constructed to illustrate genetic relatedness in northwestern Thailand. Based on this dataset of 70 samples collected between 2015 and 2021, 16 and 40 unique haplotypes were identified in pvdbpII and pvmsp142kDa, respectively. Nucleotide diversity was higher in pvmsp142kDa than in pvdbpII (π = 0.027 and 0.012), as was haplotype diversity (Hd = 0.962 and 0.849). pvmsp142kDa also showed a higher recombination rate and higher levels of genetic differentiation (Fst) in northwestern Thailand versus other regions (0.2761-0.4881). These data together suggested that the genetic diversity of P. vivax in northwestern Thailand at these two studied loci evolved under a balancing selection, most likely host immunity. The lower genetic diversity of pvdbpII may reflect its stronger functional constrain. In addition, despite the balancing selection, a decrease in genetic diversity was observed. Hd of pvdbpII decreased from 0.874 in 2015-2016 to 0.778 in 2018-2021; π of pvmsp142kDa decreased from 0.030 to 0.022 over the same period. Thus, the control activities must have had a strong impact on the parasite population size. The findings from this study provide an understanding of P. vivax population structure and the evolutionary force on vaccine candidates. They also established a new baseline for tracking future changes in P. vivax diversity in the most malarious area of Thailand.

Inter-Population Genetic Diversity and Clustering of Merozoite Surface Protein-1 (pkmsp-1) of Plasmodium knowlesi Isolates from Malaysia and Thailand.

The genetic diversity of pkmsp-1 of Malaysian Plasmodium knowlesi isolates was studied recently. However, the study only included three relatively older strains from Peninsular Malaysia and focused mainly on the conserved blocks of this gene. In this study, the full-length pkmsp-1 sequence of recent P. knowlesi isolates from Peninsular Malaysia was characterized, along with Malaysian Borneo and Thailand pkmsp-1 sequences that were retrieved from GenBank. Genomic DNA of P. knowlesi was extracted from human blood specimens and the pkmsp-1 gene was PCR-amplified, cloned, and sequenced. The sequences were analysed for genetic diversity, departure from neutrality, and geographical clustering. The pkmsp-1 gene was found to be under purifying/negative selection and grouped into three clusters via a neighbour-joining tree and neighbour net inferences. Of the four polymorphic blocks in pkmsp-1, block IV, was most polymorphic, with the highest insertion-deletion (indel) sites. Two allelic families were identified in block IV, thereby highlighting the importance of this block as a promising genotyping marker for the multiplicity of infection study of P. knowlesi malaria. A single locus marker may provide an alternate, simpler method to type P. knowlesi in a population.

Genetic diversity of Plasmodium vivax populations from the China-Myanmar border identified by genotyping merozoite surface protein markers.

BACKGROUND: Parasite diversity and population structure influence malaria control measures. Malaria transmission at international borders affects indigenous residents and migrants, defying management efforts and resulting in malaria re-introduction. Here we aimed to determine the extent and distribution of genetic variations in Plasmodium vivax populations and the complexity of infections along the China-Myanmar border. METHODS: We collected clinical P. vivax samples from local and migrant malaria patients from Laiza and Myitsone, Kachin State, Myanmar, respectively. We characterized the polymorphisms in two P. vivax merozoite surface protein markers, Pvmsp-3α and Pvmsp-3ß, by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis. We sought to determine whether these genetic markers could differentiate these two neighboring parasite populations. RESULTS: PCR revealed three major size variants for Pvmsp-3α and four for Pvmsp-3ß among the 370 and 378 samples, respectively. PCR-RFLP resolved 26 fragment-size alleles by digesting Pvmsp-3α with Alu I and Hha I and 28 alleles by digesting Pvmsp-3ß with Pst I. PCR-RFLP analysis of Pvmsp-3α found that infections in migrant laborers from Myitsone bore more alleles than did infections in residents of Laiza, while such difference was not evident from genotyping Pvmsp-3ß. Infections originating from these two places contained distinct but overlapping subpopulations of P. vivax. Infections from Myitsone had a higher multiplicity of infection as judged by the size of the Pvmsp-3α amplicons and alleles after Alu I/Hha I digestion. CONCLUSIONS: Migrant laborers from Myitsone and indigenous residents from Laiza harbored overlapping but genetically distinct P. vivax parasite populations. The results suggested a more diverse P. vivax population in Myitsone than in the border town of Laiza. PCR-RFLP of Pvmsp-3α offers a convenient method to determine the complexity of P. vivax infections and differentiate parasite populations.

Multiplicity of infection of Plasmodium knowlesi in Malaysia: an application of Pkmsp-1 block IV

@#In Malaysia presently, the main cause of human malaria is by the zoonotic monkey parasite Plasmodium knowlesi. A previous study has suggested that the P. knowlesi merozoite surface protein 1 (Pkmsp-1) block IV to be a suitable multiplicity of infection (MOI) genotyping marker for knowlesimalaria. This study therefore aimed to investigate the usefulness of Pkmsp-1 block IV in assessing the MOI of P. knowlesi in clinical isolates from Malaysia. Two allele-specific PCR primer pairs targeting the two allelic families of block IV (T1 and T2) were designed, and used to genotype P. knowlesi in 200 blood samples (100 from Peninsular Malaysia and 100 from Malaysian Borneo). Results showed that the mean MOI in Malaysian Borneo was slightly higher as compared to Peninsular Malaysia (1.58 and 1.40, respectively). Almost half of the total blood samples from Malaysian Borneo (52%) had polyclonal infections (i.e., more than one allele of any family type) as compared to Peninsular Malaysia (33%) samples. The T1 allelic family was more prevalent in Peninsular Malaysia (n=75) than in Malaysian Borneo (n=60). The T2 allelic family, however, was more prevalent in the Malaysian Borneo (n=87 vs n=53 respectively). This study shows that the single locus Pkmsp-1 block IV can serve as a simple alternative genetic marker for estimating knowlesi malaria MOI in a population. Future MOI studies should focus on macaque populations as macaques are the natural host of P. knowlesi.

Genetic polymorphism of Plasmodium falciparum msp-1, msp-2 and glurp vaccine candidate genes in pre-artemisinin era clinical isolates from Lakhimpur district in Assam, Northeast India.

Background: Northeast India shares its international border with Southeast Asia and has a number of malaria endemic zones. Monitoring genetic diversity of malaria parasites is important in this area as drug resistance and increasing genetic diversity form a vicious cycle in which one favours the development of the other. This retrospective study was done to evaluate the genetic diversity patterns in Plasmodium falciparum strains circulating in North Lakhimpur area of Assam in the pre-artemisinin era and compare the findings with current diversity patterns. Methods: Genomic DNA extraction was done from archived blood spot samples collected in 2006 from malaria-positive cases in Lakhimpur district of Assam, Northeast India. Three antigenic markers of genetic diversity were studied - msp-1 (block-2), msp-2 (block-3) and the glurp RII region of P. falciparum using nested PCR. Results: Allelic diversity was examined in 71 isolates and high polymorphism was observed. In msp-1, eight genotypes were detected; K1 (single allele), MAD20 (six different alleles) and RO33 (single allele) allelic families were noted. Among msp-2 genotypes, 22 distinct alleles were observed out of which FC27 had six alleles and IC/3D7 had 16 alleles. In RII region of glurp, nine genotypes were obtained. Expected heterozygosity (H E) values of the three antigenic markers were 0.72, 0.81 and 0.88, respectively. Multiplicity of infection (MOI) values noted were 1.28, 1.84 and 1.04 for msp-1, msp-2 and glurp, respectively. Conclusion: Results suggest a high level of genetic diversity in P. falciparum msp (block-2 of msp-1 and block-3 of msp-2) and the glurp RII region in Northeast India in the pre-artemisinin era when chloroqunine was the primary drug used for uncomplicated falciparum malaria. Comparison with current studies have revealed that the genetic diversity in these genes is still high in this region, complicating malaria vaccine research.

Genotyping of Plasmodium falciparum to Assess Clone Composition in Parasite Cultures.

Defining clone composition in Plasmodium falciparum cultures is key to verify that in vitro experiments are performed on the parasite line of interest. Genotyping of the highly polymorphic merozoite surface protein 2 gene (msp2) is a widely established method to define P. falciparum clones. Specific size variants from the two msp2 families (IC and FC27) can be used as "fingerprints" to identify individual clones in parasite mixtures. Size variant genotyping of msp2 using fluorescent nested PCR followed by fragment analysis by capillary electrophoresis (CE) provides accurate information about the presence of one or multiple parasite clones. Here, we describe an adaptation of this approach to assess the integrity and purity of P. falciparum lines kept in in vitro culture. In addition, we describe the use of synthetic mock parasite mixtures with the msp2 sequences from the parasite lines kept in culture that can provide a good estimate of the assay sensitivity, specificity, and reproducibility. We suggest that genotyping of P. falciparum lines should be performed on a regular basis as part of the standard procedures of in vitro parasite culture, as a way to secure that the parasite lines of interest are cultivated, and to monitor any cross-contamination and/or recombination events.

Why Plasmodium vivax and Plasmodium falciparum are so different? A tale of two clades and their species diversities.

The global malaria burden sometimes obscures that the genus Plasmodium comprises diverse clades with lineages that independently gave origin to the extant human parasites. Indeed, the differences between the human malaria parasites were highlighted in the classical taxonomy by dividing them into two subgenera, the subgenus Plasmodium, which included all the human parasites but Plasmodium falciparum that was placed in its separate subgenus, Laverania. Here, the evolution of Plasmodium in primates will be discussed in terms of their species diversity and some of their distinct phenotypes, putative molecular adaptations, and host-parasite biocenosis. Thus, in addition to a current phylogeny using genome-level data, some specific molecular features will be discussed as examples of how these parasites have diverged. The two subgenera of malaria parasites found in primates, Plasmodium and Laverania, reflect extant monophyletic groups that originated in Africa. However, the subgenus Plasmodium involves species in Southeast Asia that were likely the result of adaptive radiation. Such events led to the Plasmodium vivax lineage. Although the Laverania species, including P. falciparum, has been considered to share "avian characteristics," molecular traits that were likely in the common ancestor of primate and avian parasites are sometimes kept in the Plasmodium subgenus while being lost in Laverania. Assessing how molecular traits in the primate malaria clades originated is a fundamental science problem that will likely provide new targets for interventions. However, given that the genus Plasmodium is paraphyletic (some descendant groups are in other genera), understanding the evolution of malaria parasites will benefit from studying "non-Plasmodium" Haemosporida.

P. falciparum msp1 and msp2 genetic diversity in P. falciparum single and mixed infection with P. malariae among the asymptomatic population in Southern Benin.

Plasmodium falciparum and Plasmodium malariae infections are prevalent in malaria-endemic countries. However, very little is known about their interactions especially the effect of P. malariae on P. falciparum genetic diversity. This study aimed to assess P. falciparum genetic diversity in P. falciparum and mixed infection P. falciparum/P. malariae isolates among the asymptomatic populations in Southern Benin. Two hundred and fifty blood samples (125 of P. falciparum and 125 P. falciparum/P. malariae isolates) were analysed by a nested PCR amplification of msp1 and msp2 genes. The R033 allelic family was the most represented for the msp1 gene in mono and mixed infection isolates (99.2% vs 86.4%), while the K1 family had the lowest frequency (38.3% vs 20.4%). However, with the msp2 gene, the two allelic families displayed similar frequencies in P. falciparum isolates while the 3D7 allelic family was more represented in P. falciparum/P. malariae isolates (88.7%). Polyclonal infections were also lower (62.9%) in P. falciparum/P. malariae isolates (p < 0.05). Overall, 96 individual alleles were identified (47 for msp1 and 49 for msp2) in P. falciparum isolates while a total of 50 individual alleles were identified (23 for msp1 and 27 for msp2) in P. falciparum/P. malariae isolates. The Multiplicity of Infection (MOI) was lower in P. falciparum/P. malariae isolates (p < 0.05). This study revealed a lower genetic diversity of P. falciparum in P. falciparum/P. malariae isolates using msp1 and msp2 genes among the asymptomatic population in Southern Benin.

Antibody Responses Against Plasmodium falciparum MSP3 Protein During Natural Malaria Infection in Individuals Living in Malaria-Endemic Regions of India.

The humoral immune responses to blood-stage malaria proteins are requisite for the inhibition of parasite invasion. Plasmodium falciparum merozoite surface protein 3 (MSP3) is a secretory, expressed abundantly, merozoite surface protein that is important for the parasite invasion process. It has been shown to induce antibody responses during natural infections and is, therefore, considered to be the potential vaccine candidates against Plasmodium. Elucidating the immunogenicity and prevalence of anti-parasite antibodies is important in identifying potential targets as candidates for malarial diagnosis and anti-malarial vaccine. The present study concerns the presence of antibodies against the MSP3 proteins of human malaria parasite- P. falciparum in infected individuals from endemic regions of India. Seventy-one anonymized P. falciparum infected serum samples were procured from the malaria fever clinic of ICMR-National Institute of Malaria Research (NIMR), New Delhi to detect the presence of antibodies against MSP3 protein by ELISA. The IgM antibody response against recombinant MSP3 was detected at significantly higher levels during acute malaria. The protein was found to be immunogenic and did not demonstrate any cross-reactivity with the serum of uninfected individuals or individuals infected with other Plasmodium species. The protein has hydrophilic regions in its N- and C-terminus which may contain immunogenic linear and conformational B-cell epitopes. The results from this study suggest that the MSP3 is immunogenic and likely a potential candidate for antibody-based diagnosis or vaccine development against the blood-stage of P. falciparum.

Protection and Alleviated Inflammation Induced by Virus-like Particle Vaccines Containing Plasmodium berghei MSP-8, MSP-9 and RAP1.

Virus-like particles (VLP) are a highly efficient vaccine platform used to present multiple antigenic proteins. Merozoite surface protein 8 (MSP-8), 9 (MSP-9) and rhoptry-associated protein 1 (RAP1) of Plasmodium berghei are the important proteins in erythrocyte invasion and the replication of parasites. In this study, we generated three VLPs expressing MSP-8, MSP-9 or RAP1 together with influenza virus matrix protein M1 as a core protein, and the protection and alleviated inflammation induced by VLP immunization were investigated. Mice were immunized with a mixture of three VLPs, MSP-8, MSP-9 and RAP1, and challenge-infected with P. berghei. As a result, VLPs immunization elicited higher levels of P. berghei or VLPs-specific IgG antibody responses in the sera upon boost compared to that upon prime and naive. Upon challenge infection with P. berghei, higher levels of CD4+ T cell and memory B cell responses in the spleen were also found in VLPs-immunized mice compared to non-immunized control. Importantly, VLP immunization significantly alleviated inflammatory cytokine responses (TNF-α, IFN-γ) both in the sera and spleen. VLP vaccine immunization also assisted in diminishing the parasitic burden in the peripheral blood and prolonged the survival of immunized mice. These results indicated that a VLPs vaccine containing MSP-8, MSP-9 and RAP1 could be a vaccine candidate for P. berghei infection.

Spatiotemporal Changes in Plasmodium vivax msp142 Haplotypes in Southern Mexico: From the Control to the Pre-Elimination Phase.

For 20 years, Plasmodium vivax has been the only prevalent malaria species in Mexico, and cases have declined significantly and continuously. Spatiotemporal genetic studies can be helpful for understanding parasite dynamics and developing strategies to weaken malaria transmission, thus facilitating the elimination of the parasite. The aim of the current contribution was to analyze P. vivax-infected blood samples from patients in southern Mexico during the control (1993-2007) and pre-elimination phases (2008-2011). Nucleotide and haplotype changes in the pvmsp142 fragment were evaluated over time. The majority of multiple genotype infections occurred in the 1990s, when the 198 single nucleotide sequences exhibited 57 segregating sites, 64 mutations, and 17 haplotypes. Nucleotide and genetic diversity parameters showed subtle fluctuations from across time, in contrast to the reduced haplotype diversity and the increase in the R2 index and Tajima's D value from 2008 to 2011. The haplotype network consisted of four haplogroups, the geographical distribution of which varied slightly over time. Haplogroup-specific B-cell epitopes were predicted. Since only high-frequency and divergent haplotypes persisted, there was a contraction of the parasite population. Given that 84% of haplotypes were exclusive to Mesoamerica, P. vivax flow is likely circumscribed to this region, representing important information for parasite surveillance.