Merozoite Surface Protein Research Articles

Acquired antibody responses against merozoite surface protein-119 antigen during Plasmodium falciparum and P.vivax infections in South Indian city of Mangaluru.

Merozoite surface protein-1 (MSP-1) of malaria parasites has been extensively studied as a malaria vaccine candidate and the antibody response to this protein is an important indicator of protective immunity to malaria. Mangaluru city and its surrounding areas in southwestern India are endemic to malaria with Plasmodium vivax being the most widespread and prevalent species although P. falciparum also frequently infects. However, no information is available on the level of protective immunity in this population. In this regard, a prospective hospital-based study was performed in malarial patients to assess antibody responses against the 19-kDa C-terminal portion of P. vivax and P. falciparum MSP-1 (MSP-119). Serum samples from 51 healthy endemic controls and 267 infected individuals were collected and anti-MSP-119 antibody levels were analyzed by ELISA. The possible association between the antibody responses and morbidity parameters such as malarial anemia and thrombocytopenia was investigated. Among the 267 infected cases, 144 had P. vivax and 123 had P. falciparum infections. Significant levels of anti-MSP-119 antibody were observed both in P. vivax (123/144; 85.4%) and P. falciparum (108/123; 87.9%) infected individuals. In both type of infections, the major antibody isotypes were IgG1 and IgG3. The IgG levels were found to be increased in patients with severe anemia and thrombocytopenia. The antibody levels were also higher in infected individuals who had several previous infections, although antibodies produced during previous infections were short lived. The predominance of cytophilic anti-MSP-119 IgG1 and IgG3 antibodies suggests the possibility of a dual role of Pv MSP-119 and Pf MSP-119 during malarial immunity and pathogenesis.

Exploration of Plasmodium vivax merozoite surface proteins 1 and 7 genetic diversity in Brazilian Amazon and Rio de Janeiro Atlantic Forest.

Plasmodium vivax merozoite surface proteins (PvMSP) 1 and 7 are considered vaccine targets. Genetic diversity knowledge is crucial to assess their potential as immunogens and to provide insights about population structure in different epidemiological contexts. Here, we investigate the variability of pvmsp-142, pvmsp-7E, and pvmsp-7F genes in 227 samples from the Brazilian Amazon (BA) and Rio de Janeiro Atlantic Forest (AF). pvmsp-142 has 63 polymorphisms - 57 nonsynonymous - generating a nucleotide diversity of π=0.009 in AF, and π=0.018 in BA. In pvmsp-7E, 134 polymorphisms - 103 nonsynonymous - generate the nucleotide diversity of π=0.027 in AF, and π=0.042 in BA. The pvmsp-7F has only two SNPs - A610G and A1054T -, with nucleotide diversity of π=0.0004 in AF, and π=0.0007 in BA. The haplotype diversity of pvmsp-142, pvmsp-7E, and pvmsp-7F genes is 0.997, 1.00, and 0.649, respectively. None of the pvmsp-142 or pvmsp-7E sequences are identical to the Salvador 1 strain's sequence. Conversely, most of pvmsp-7F sequences (94/48%) are identical to Sal-1. We evaluated eight B-cell epitopes in pvmsp-7E, four of them showed higher nucleotide diversity compared to pvmsp-7E's epitopes. Positive selection was detected in pvmsp-142, pvmsp-7E central region, and pvmsp-7F with Tajima's D. In pvmsp-7E, the significant nucleotide and haplotype diversities with low genetic differentiation, could be indicative of balancing selection. The genetic differentiation of pvmsp-142 (0.315) and pvmsp-7F (0.354) genes between AF and BA regions is significant, which is not the case for pvmsp-7E (0.193). We conclude that pvmsp-142 and pvmsp-7E have great genetic diversity even in AF region, an enclosure area with deficient transmission levels of P. vivax zoonotic malaria. In both Brazilian regions, pvmsp-119, pvmsp-7E, and pvmsp-7F are conserved, most likely due to their roles in parasite survival, and could be considered potential targets for a "blood-stage vaccine".

Whole genome sequencing of Plasmodium vivax isolates reveals frequent sequence and structural polymorphisms in erythrocyte binding genes.

Plasmodium vivax malaria is much less common in Africa than the rest of the world because the parasite relies primarily on the Duffy antigen/chemokine receptor (DARC) to invade human erythrocytes, and the majority of Africans are Duffy negative. Recently, there has been a dramatic increase in the reporting of P. vivax cases in Africa, with a high number of them being in Duffy negative individuals, potentially indicating P. vivax has evolved an alternative invasion mechanism that can overcome Duffy negativity. Here, we analyzed single nucleotide polymorphism (SNP) and copy number variation (CNV) in Whole Genome Sequence (WGS) data from 44 P. vivax samples isolated from symptomatic malaria patients in southwestern Ethiopia, where both Duffy positive and Duffy negative individuals are found. A total of 123,711 SNPs were detected, of which 22.7% were nonsynonymous and 77.3% were synonymous mutations. The largest number of SNPs were detected on chromosomes 9 (24,007 SNPs; 19.4% of total) and 10 (16,852 SNPs, 13.6% of total). There were particularly high levels of polymorphism in erythrocyte binding gene candidates including merozoite surface protein 1 (MSP1) and merozoite surface protein 3 (MSP3.5, MSP3.85 and MSP3.9). Two genes, MAEBL and MSP3.8 related to immunogenicity and erythrocyte binding function were detected with significant signals of positive selection. Variation in gene copy number was also concentrated in genes involved in host-parasite interactions, including the expansion of the Duffy binding protein gene (PvDBP) on chromosome 6 and MSP3.11 on chromosome 10. Based on the phylogeny constructed from the whole genome sequences, the expansion of these genes was an independent process among the P. vivax lineages in Ethiopia. We further inferred transmission patterns of P. vivax infections among study sites and showed various levels of gene flow at a small geographical scale. The genomic features of P. vivax provided baseline data for future comparison with those in Duffy-negative individuals and allowed us to develop a panel of informative Single Nucleotide Polymorphic markers diagnostic at a micro-geographical scale.

Analysis of Plasmodium vivax schizont transcriptomes from field isolates reveals heterogeneity of expression of genes involved in host-parasite interactions

Plasmodium vivax gene regulation remains difficult to study due to the lack of a robust in vitro culture method, low parasite densities in peripheral circulation and asynchronous parasite development. We adapted an RNA-seq protocol “DAFT-seq” to sequence the transcriptome of four P. vivax field isolates that were cultured for a short period ex vivo before using a density gradient for schizont enrichment. Transcription was detected from 78% of the PvP01 reference genome, despite being schizont-enriched samples. This extensive data was used to define thousands of 5′ and 3′ untranslated regions, some of which overlapped with neighbouring transcripts, and to improve the gene models of 352 genes, including identifying 20 novel gene transcripts. This dataset has also significantly increased the known amount of heterogeneity between P. vivax schizont transcriptomes from individual patients. The majority of genes found to be differentially expressed between the isolates lack Plasmodium falciparum homologs and are predicted to be involved in host-parasite interactions, with an enrichment in reticulocyte binding proteins, merozoite surface proteins and exported proteins with unknown function. An improved understanding of the diversity within P. vivax transcriptomes will be essential for the prioritisation of novel vaccine targets.

Genetic diversity of Merozoite surface protein 1-42 (MSP1-42) fragment of Plasmodium vivax from Indonesian isolates: Rationale implementation of candidate MSP1 vaccine.

Morbidity and mortality related to malaria in Indonesia are attributed to both Plasmodium falciparum and P. vivax parasites. In addition to vaccines for P. falciparum, vaccines against P. vivax are urgently needed for the prevention of the disease. An extensively studied antigen is the carboxyl-terminus of the 42kDa region of P. vivax merozoite surface protein-1 (PvMSP1-42). The design of a vaccine based on this antigen requires an understanding of the extent of polymorphism. However, there is no information on the genetic diversity of the antigen in Indonesia. This study aimed to profile the diversity of PvMSP1-42 and its two subdomains (PvMSP1-33 and PvMSP1-19) among Indonesian P. vivax isolates. A total of 52 P. vivax-infected blood samples were collected from patients in two different endemic areas in Indonesia: Banjarmasin (Kalimantan) and Sumba Timur (Nusa Tenggara Timur). The polymorphic characteristics and natural selection of PvMSP1-42 were analyzed using the DnaSP, MEGA, and Structure software. Thirty distinct haplotypes of PvMSP1-42 were identified. They displayed amino acid changes compared to the reference PVP01 sequence. Most of the mutations were concentrated in the 33kDa fragment. PvMSP1-42 of the Indonesian isolates appeared to be under positive selection. Recombination may also play a role in the resulting genetic diversity of PvMSP1. In conclusion, PvMSP1-42 of Indonesian isolates displayed allelic polymorphisms caused by mutation, recombination, and positive selection. These results will aid the understanding of the P. vivax population in Indonesia and to develop a PvMSP1 based vaccine against P. vivax.

Large Variations in Malaria Parasite Carriage by Afebrile School Children Living in Nearby Communities in the Central Region of Ghana.

Background Indicators of successful malaria control interventions include a reduction in the prevalence and densities of malaria parasites contained in both symptomatic and asymptomatic infections as well as a reduction in malaria transmission. Individuals harboring malaria parasites in asymptomatic infections serve as reservoirs for malaria transmission. This study determined the prevalence of asymptomatic malaria parasite carriage in afebrile children attending six different schools in two districts, the Cape Coast Metropolitan Assembly (CCMA) and the Komenda Edina Eguafo Abirem (KEEA) of the Central Region of Ghana. Methods This cross sectional study recruited afebrile children aged between 3 and 15 years old from six randomly selected schools in the Central Region of Ghana. Finger-pricked blood was collected and used to prepare thick and thin blood smears as well as spot a strip of filter paper (Whatman #3). Nested PCR was used to identify Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax in DNA extracted from the filter paper spots. The multiplicity of P. falciparum infection was determined using merozoite surface protein 2 genotyping. Results Out of the 528 children sampled, PCR identified 27.1% to harbor Plasmodium parasites in asymptomatic infections, whilst microscopy identified malaria parasites in 10.6% of the children. The overall PCR estimated prevalence of P. falciparum and P. malariae was 26.6% and 1.3%, respectively, with no P. ovale or P. vivax identified by PCR or microscopy. The RDT positivity rate ranged from 55.8% in Simiw to 4.5% in Kuful. Children from the Simiw Basic School accounted for 87.5% of all the asymptomatic infections. The multiplicity of P. falciparum infection was predominantly monoclonal and biclonal. Conclusions The low prevalence of asymptomatic malaria parasite carriage by the children living in the Cape Coast Metropolis suggests that the malaria control interventions in place in CCMA are highly effective and that additional malaria control interventions are required for the KEEA district to reduce the prevalence of asymptomatic malaria parasite carriers. No molecular evidence of P. ovale and P. vivax was identified in the afebrile children sampled from the selected schools.

Genetic diversity and complexity of Plasmodium falciparum infections in the microenvironment among siblings of the same household in North-Central Nigeria

BackgroundPlasmodium falciparum parasites are known to exhibit extensive genetic diversity in areas of high transmission intensity and infected individuals in such communities often harbour several complex mixtures of parasite clones with different genetic characteristics. However, in the micro-environment, the extent of genetic diversity of P. falciparum parasites remain largely unknown. In this study therefore, the complexity of P. falciparum infections in households was investigated among symptomatic siblings, living under the same roof in north-central Nigeria.MethodsChildren were enrolled into the study if they were at least two from a household and presented with symptoms of uncomplicated malaria. Clinical malaria was confirmed by light microscopy of Giemsa-stained thick and thin blood films. Genomic DNA was isolated from blood spots on filter paper. Molecular characterization of P. falciparum isolates was done by allele-specific nested PCR of the highly polymorphic merozoite surface protein-2 (msp-2) gene.ResultsNinety-three children from 43 households were enrolled into this study. A total of 26 different msp-2 alleles were identified from 215 fragments (range: 180–480 bp). Majority of the isolates [65.6% (n = 61)] were polyclonal infections consisting of 2–6 clones and were significantly more common with the FC27 allelic family (p = 0.036). The multiplicity of infection (MOI) per household ranged from 1.0 to 4.5 while the overall MOI in the study population was 2.31. The pattern of distribution of msp-2 allele types among the households fell into two categories: households where both msp-2 allele types (FC27 and 3D7) were present; households where only one msp-2 allele type (FC27 or 3D7) was present. Majority of the households [88.4% (n = 38)], had both msp-2 allele types but they were disproportionately distributed among the children while in a few households [11.6% (n = 5)], all the children were infected with only one type of msp-2 allele.ConclusionThese findings showed that P. falciparum isolates exhibit remarkable degree of genetic diversity in the micro-environment and are composed mainly of multiclonal infections, which is an indication of a high ongoing parasite transmission. This suggests that the micro-environment is an important area of focus for malaria control interventions and for evaluating intervention programmes.

Design of a point-of-care device for electrochemical detection of P.vivax infected-malaria using antibody functionalized rGO-gold nanocomposite

A point-of-care electrochemical “Tri-inlet” device integrated with an electrode that acts as a sensor is developed for early-stage diagnosis of Plasmodium vivax (Pv)-infected malaria in a human patient’s whole blood sample. The electrode is prepared in a step-wise manner involving a synthesis of rGO-based gold nanocomposite (Au-rGO) followed by conjugation with P.vivax specific antibody (merozoite surface protein-1, MSP1) and deposition of the antibody functionalized nanocomposite (Au-rGO-MSP1) over carbon strip. The crucial directional conjugation of the antibody to the nanocomposite is achieved through ethylenediamine (EDA) as a spacer for sensitive detection of the Pv-infected RBCs (Pv-iRBCs) in vivax malaria diagnosis. The Au-rGO-MSP1 based electrode (sensor) showed a decrease in peak-current density and an increase in charge-transfer resistance (Rct) as electrical signals in ferro/ferricyanide solution system due to the interaction between MSP1 antibody of the electrode and Pv-iRBCs. The Rct values increased proportionally with the number of iRBCs and varied linearly for cell concentration (R2 = 0.990). The performance analysis with the patient’s whole blood sample showed good sensitivity and selectivity with a detection limit of ∼40 vivax infected RBCs (Pv-iRBCs)/ 10 μL blood sample in 5 minutes which is comparable or even better than the most of the commercially available malaria kit. The present electrochemical approach demonstrated the proof-of-concept for designing a new disposable type sensor kit that could be easily used for early onsite detection of Plasmodium vivax-infected malaria.

Contribution of P. falciparum parasites with Pfhrp 2 gene deletions to false negative PfHRP 2 based malaria RDT results in Ghana: A nationwide study of symptomatic malaria patients

IntroductionFalse-negative malaria rapid diagnostic test (RDT) results amongst symptomatic malaria patients are detrimental as they could lead to ineffective malaria case management. This study determined the nationwide contribution of parasites with Pfhrp2 and Pfhrp 3 gene deletions to false negative malaria RDT results in Ghana.MethodsThis was a cross sectional study where whole blood (~2 ml) was collected from patients presenting with malaria symptoms at 100 health facilities in all the regions in Ghana from May to August 2018. An aliquot of the blood was used to prepare thin and thick blood smears, filter paper blood spots (DBS) and spot a PfHRP 2 RDT kit. The remaining blood was separated into plasma and blood cells and stored at -20°C. Plasmodium parasite density and species identity was estimated from the blood smears. Plasmodium falciparum specific 18S rRNA PCR, merozoite surface protein (msp 1) and glutamate rich protein (glurp) gene PCR were used to identify P. falciparum positive samples, which were subjected to Pfhrp 2/3 exon1-2 and exon2 genotyping.ResultsOf the 2,860 microscopically P. falciparum positive patients analyzed, 134 (4.69%) had false negative P. falciparum specific RDT results. Samples for PCR analysis was available for 127 of the false negative patients, and the analysis identified 116 (91.3%) as positive for P. falciparum. Only 58.1% (79/116) of the false negative RDT samples tested positive by msp 1 and glurp PCR. Genotyping of exon 1–2 and exon 2 of the Pfhrp 2 gene identified 12.9% (10/79) and 39.5% (31/79) of samples respectively to have deletions. Genotyping exon 1–2 and exon 2 of the Pfhrp 3 gene identified 15.2% (12/79) and 40.5% (32/79) of samples respectively to have deletions. Only 5% (4/79) of the false negative samples had deletions in both exon 1–2 and exon 2 of the Pfhrp 2 gene. Out of the 49 samples that tested positive for aldolase by luminex, 32.6% (16/49) and) had deletions in Pfhrp 2 exon 2 and 2% (1/49) had deletions in both exon 2 and exon 1–2 of the Pfhrp 2 gene.ConclusionsThe low prevalence of false negative RDT test results provides assurance that PfHRP 2 based malaria RDT kits remain effective in diagnosing symptomatic malaria patients across all the Regions of Ghana. Although there was a low prevalence of parasites with deletions in exon 2 and exon 1–2 of the Pfhrp 2 gene the prevalence of parasites with deletions in Pfhrp 2 exon 2 was about a third of the false negative RDT results. The need to ensure rapid, accurate and reliable malaria diagnosis requires continuous surveillance of parasites with Pfhrp 2 gene deletions.

Genetic diversity of Plasmodium falciparum in Grande Comore Island

BackgroundDespite several control interventions resulting in a considerable decrease in malaria prevalence in the Union of the Comoros, the disease remains a public health problem with high transmission in Grande Comore compared to neighbouring islands. In this country, only a few studies investigating the genetic diversity of Plasmodium falciparum have been performed so far. For this reason, this study aims to examine the genetic diversity of P. falciparum by studying samples collected in Grande Comore in 2012 and 2013, using merozoite surface protein 1 (msp1), merozoite surface protein 2 (msp2) and single nucleotide polymorphism (SNP) genetic markers.MethodsA total of 162 positive rapid diagnostic test (RDT) samples from Grande Comore were used to extract parasite DNA. Allelic families K1, Mad20 and RO33 of the msp1 gene as well as allelic families IC3D7 and FC37 of the msp2 gene were determined by using nested PCR. Additionally, 50 out of 151 samples were genotyped to study 24 SNPs by using high resolution melting (HRM).ResultsTwo allelic families were predominant, the K1 family of msp1 gene (55%) and the FC27 family of msp2 gene (47.4%). Among 50 samples genotyped for 24 SNPs, 42 (84%) yielded interpretable results. Out of these isolates, 36 (85%) were genetically unique and 6 (15%) grouped into two clusters. The genetic diversity of P. falciparum calculated from msp1 and msp2 genes and SNPs was 0.82 and 0.61, respectively.ConclusionIn summary, a large genetic diversity of P. falciparum was observed in Grande Comore. This may favour persistence of malaria and might be one of the reasons for the high malaria transmission compared to neighbouring islands. Further surveillance of P. falciparum isolates, mainly through environmental management and vector control, is warranted until complete elimination is attained.

Evaluating the predictive performance of malaria antibodies and FCGR3B gene polymorphisms on Plasmodium falciparum infection outcome: a prospective cohort study

BackgroundMalaria antigen-specific antibodies and polymorphisms in host receptors involved in antibody functionality have been associated with different outcomes of Plasmodium falciparum infections. Thus, to identify key prospective malaria antigens for vaccine development, there is the need to evaluate the associations between malaria antibodies and antibody dependent host factors with more rigorous statistical methods. In this study, different statistical models were used to evaluate the predictive performance of malaria-specific antibodies and host gene polymorphisms on P. falciparum infection in a longitudinal cohort study involving Ghanaian children.MethodsModels with different functional forms were built using known predictors (age, sickle cell status, blood group status, parasite density, and mosquito bed net use) and malaria antigen-specific immunoglobulin (Ig) G and IgG subclasses and FCGR3B polymorphisms shown to mediate antibody-dependent cellular functions. Malaria antigens studied were Merozoite surface proteins (MSP-1 and MSP-3), Glutamate Rich Protein (GLURP)-R0, R2, and the Apical Membrane Antigen (AMA-1). The models were evaluated through visualization and assessment of differences between the Area Under the Receiver Operating Characteristic Curve and Brier Score estimated by suitable internal cross-validation designs.ResultsThis study found that the FCGR3B-c.233C>A genotype and IgG against AMA1 were relatively better compared to the other antibodies and FCGR3B genotypes studied in classifying or predicting malaria risk among children.ConclusionsThe data supports the P. falciparum, AMA1 as an important malaria vaccine antigen, while FCGR3B-c.233C>A under the additive and dominant models of inheritance could be an important modifier of the effect of malaria protective antibodies.

Virus‐like particles expressing Plasmodium berghei MSP‐8 induce protection against P. berghei infection

Merozoite surface protein 8 (MSP-8) of Plasmodium parasites plays an important role in erythrocyte invasion and is a potential malaria vaccine candidate. In this study, virus-like particles (VLPs) expressing MSP-8 of Plasmodium berghei on the surface of influenza virus matrix protein 1 (M1) core protein were generated for vaccine efficacy assessment. Mice were intramuscularly (IM) immunized with MSP-8 VLPs twice and challenge-infected with P. berghei. We found that VLP vaccination elicited higher levels of P. berghei-specific IgG antibody response in the sera, along with blood CD4+ and CD8+ T-cell response enhancement compared to the naïve control mice. CD4+ and CD8+ effector memory T-cell and memory B-cell responses in the spleen were found to be higher in VLP-immunized mice compared to control mice. VLP vaccination significantly reduced inflammatory cytokine (IFN-γ) response in the spleen and parasitemia levels in blood compared to naïve control mice. These results indicate that MSP-8 containing virus-like particles could be a vaccine candidate for blood-stage vaccine design.

Treatment of Plasmodium falciparum merozoites with the protease inhibitor E64 and mechanical filtration increases their susceptibility to complement activation.

Plasmodium falciparum malaria killed 451,000 people in 2017. Merozoites, the stage of the parasite that invades RBCs, are a logical target for vaccine development. Treatment with the protease inhibitor E64 followed by filtration through a 1.2 μm filter is being used to purify merozoites for immunologic assays. However, there have been no studies to determine the effect of these treatments on the susceptibility of merozoites to complement or antibodies. To address this gap, we purified merozoites with or without E64 followed by filtration through either a 1.2 or 2.7 μm filter, or no filtration. Merozoites were then incubated in either 10% fresh or heat-inactivated serum followed by surface staining and flow cytometry with monoclonal antibodies against the complement effector molecules C3b or C5b9. To determine the effect of anti-merozoite antibodies, we incubated merozoites with MAb5.2, a mouse monoclonal antibody that targets the merozoite surface protein 1. We used an amine-reactive fluorescent dye to measure membrane integrity. Treatment with E64 resulted in an insignificant increase in the proportion of merozoites that were C3b positive but in a significant increase in the proportion that were C5b9 positive. Filtration increased the proportion of merozoites that were either C3b or C5b9-positive. The combination of filtration and E64 treatment resulted in marked deposition of C3b and C5b9. MAb5.2 induced greater complement deposition than serum alone or an IgG2b isotype control. The combination of E64 treatment, filtration, and MAb5.2 resulted in very rapid and significant deposition of C5b9. Filtration through the 1.2 μm filter selected a population of merozoites with greater membrane integrity, but their integrity deteriorated rapidly upon exposure to serum. We conclude that E64 treatment and filtration increase the susceptibility of merozoites to complement and antibody. Filtered or E64-treated merozoites are not suitable for immunologic studies that address the efficacy of antibodies in vitro.

Determinants of Plasmodium falciparum multiplicity of infection and genetic diversity in Burkina Faso

BackgroundInvestigating malaria transmission dynamics is essential to inform policy decision making. Whether multiplicity of infection (MOI) dynamic from individual infections could be a reliable malaria metric in high transmission settings with marked variation in seasons of malaria transmission has been poorly assessed. This study aimed at investigating factors driving Plasmodium falciparum MOI and genetic diversity in a hyperendemic area of Burkina Faso.MethodsBlood samples collected from a pharmacovigilance trial were used for polymerase chain reaction genotyping of the merozoite surface proteins 1 and 2. MOI was defined as the number of distinct parasite genotypes co-existing within a particular infection. Monthly rainfall data were obtained from satellite data of the Global Precipitation Measurement Database while monthly malaria incidence aggregated data were extracted from District Health Information Software 2 medical data of the Center-West health regional direction.ResultsIn the study area, infected people harboured an average of 2.732 (± 0.056) different parasite genotypes. A significant correlation between the monthly MOI and the monthly malaria incidence was observed, suggesting that MOI could be a good predictor of transmission intensity. A strong effect of season on MOI was observed, with infected patients harbouring higher number of parasite genotypes during the rainy season as compared to the dry season. There was a negative relationship between MOI and host age. In addition, MOI decreased with increasing parasite densities, suggesting that there was a within-host competition among co-infecting genetically distinct P. falciparum variants. Each allelic family of the msp1 and msp2 genes was present all year round with no significant monthly fluctuation.ConclusionsIn high malaria endemic settings with marked variation in seasons of malaria transmission, MOI represents an appropriate malaria metric which provides useful information about the longitudinal changes in malaria transmission in a given area. Besides transmission season, patient age and parasite density are important factors to consider for better understanding of variations in MOI. All allelic families of msp1 and msp2 genes were found in both dry and rainy season. The approach offers the opportunity of translating genotyping data into relevant epidemiological information for malaria control.

Analysis of sulphadoxine-pyrimethamine resistance-associated mutations in Plasmodium falciparum isolates obtained from asymptomatic pregnant women in Ogun State, Southwest Nigeria.

Intermittent preventive treatment in pregnancy with sulphadoxine-pyrimethamine (IPTp-SP) is one of the main strategies for protecting pregnant women, fetus, and their new-born against adverse effects of P. falciparum infection. The development of the drug resistance linked to mutations in P. falciparum dihydrofolate reductase gene (pfdhfr) and P. falciparum dihydropteroate synthase gene (pfdhps), is currently threatening the IPTp-SP approach. This study determined the prevalence of pfdhfr and pfdhps mutations in isolates obtained from pregnant women with asymptomatic P. falciparum infection in Nigerian. Additionally, P. falciparum genetic diversity and multiplicity of infection (MOI) was assessed by genotyping the P. falciparum merozoite surface Protein 1 and 2 (pfmsp-1 and pfmsp-2) genes. The pfdhfr and pfdhps were genotyped by direct sequencing, and the pfmsp-1 and pfmsp-2 fragment analysis by polymerase chain reaction was used to determine P. falciparum genetic diversity. Of the 406 pregnant women recruited, 123 had P. falciparum infection by PCR, and of these, 52 were successfully genotyped for pfdhfr and 42 for pfdhps genes. The pfdhfr triple-mutant parasites (N51I, C59R, and S108N) or the IRN haplotype were predominant (98%), whereas pfdhfr mutations C50R and I164L did not occur. For pfdhps gene, the prevalence of A437G, A581G, A436A, and A613S mutations were 98, 71, 55, and 36%, respectively. Nineteen (44%) isolates with quintuple mutations (CIRNI- SGKGA) had the highest combined pfdhfr-pfdhps haplotype. Isolates with sextuple mutants; CIRNI- AGKAS and CIRNI- AGKGA had a prevalence of 29 and 14%, respectively. High genetic diversity (7 pfmsp-1 alleles and 10 pfmsp-2 alleles) and monoclonal infection rate (76%) was observed. This study demonstrated a continuous high prevalence of pfdhfr mutation and an increase in pfdhps mutations associated with SP-resistance in southwest Nigeria. Continuous surveillance of IPTp-SP effectiveness and consideration of alternative IPTp strategies is recommended.

Prevalence of Asymptomatic Malaria Parasitemia in Odisha, India: A Challenge to Malaria Elimination.

The prevalence of malaria in India is decreasing, but it remains a major concern for public health administration. The role of submicroscopic malaria and asymptomatic malaria parasitemia and their persistence is being explored. A cross-sectional survey was conducted in the Kandhamal district of Odisha (India) during May-June 2017. Blood samples were collected from 1897 individuals for screening of asymptomatic parasitemia. Samples were screened using rapid diagnostic tests (RDTs) and examined microscopically for Plasmodium species. Approximately 30% of randomly selected samples (n = 586) were analyzed using real-time PCR (qPCR), and the genetic diversity of Plasmodium falciparum was analyzed. The prevalence of Plasmodium species among asymptomatic individuals detected using qPCR was 18%, which was significantly higher than that detected by microscopy examination (5.5%) or RDT (7.3%). Of these, 37% had submicroscopic malaria. The species-specific prevalence among asymptomatic malaria-positive cases for P. falciparum, Plasmodium vivax, and mixed infection (P. falciparum and P. vivax) by qPCR was 57%, 29%, and 14%, respectively. The multiplicity of infection was 1.6 and 1.2 for the merozoite surface protein-1 gene (msp1) and (msp2), respectively. Expected heterozygosity was 0.64 and 0.47 for msp1 and msp2, respectively. A significant proportion of the study population, 105/586 (18%), was found to be a reservoir for malaria infection, and identification of this group will help in the development of elimination strategies.

Protective Immunity Induced by Virus-Like Particle Containing Merozoite Surface Protein 9 of Plasmodium berghei

Merozoite surface protein 9 (MSP-9) from Plasmodium has shown promise as a vaccine candidate due to its location and possible role in erythrocyte invasion. In this study, we generated virus-like particles (VLPs) targeting P. berghei MSP-9, and investigated the protection against lethal doses of P. berghei in a mouse model. We found that VLP vaccination induced a P. berghei-specific IgG antibody response in the sera and CD4+ and CD8+ T cell populations in blood compared to a naïve control group. Upon challenge infection with P. berghei, vaccinated mice showed a significant increase in CD4+ and CD8+ effector memory T cell and memory B cell populations. Importantly, MSP-9 VLP immunization inhibited levels of the pro-inflammatory cytokines IFN-γ and IL-6 in the spleen and parasite replication in blood, resulting in significantly prolonged survival time. These results suggest that the MSP-9 VLP vaccine may constitute an effective malaria vaccine.

Structural diversity, natural selection and intragenic recombination in the Plasmodium vivax merozoite surface protein 9 locus in Thailand

The merozoite surface protein 9 (MSP9) of malarial parasite forms co-ligand complex with the 19 kDa fragment of merozoite surface protein 1 (MSP1) prior to erythrocyte invasion. Interruption of this process could hamper subsequent asexual erythrocytic development of malaria parasites; therefore, these proteins are considered potential vaccine candidates. In Plasmodium vivax, MSP9 (PvMSP9) contains both conserved and polymorphic repetitive domains that were immunogenic upon natural malaria exposure and conferred protection in vaccination studies in animal models. To investigate the extent of sequence diversity at this locus, 104 P. vivax isolates from 4 major malaria endemic areas of Thailand were analyzed. Results revealed that pvmsp9 contained 3 repeat domains (R1–R3) flanked by conserved domains. Repeat domains exhibit extensive sequence and length variation, in which 14, 39 and 16 haplotypes for domains R1–R3, respectively, circulated in this country. Sequence diversity in pvmsp9 among P. vivax isolates from each endemic area displayed population structure. The extent of sequence diversity in pvmsp9 isolates from the provinces of Tak, Chanthaburi, Ubon Ratchathani and Prachuap Khiri Khan in northwestern, eastern, northeastern and southwestern areas, respectively, was almost comparable and was remarkably higher than that from Yala/Narathiwat population in southern Thailand. Evidence for intragenic recombination in this locus was observed within each P. vivax population except among isolates from Yala and Narathiwat. Synonymous nucleotide diversity significantly exceeded nonsynonymous nucleotide diversity in domains R2 and R3, indicating purifying selection. However, micro-scale signatures of positive and negative selections occurred in both conserved and repeat domains, implying two opposing forces, probably from functional or structural constraint and host immune pressure, could have influenced diversity at this locus. The immunodominant T and B cell epitopes so far identified were invariant or highly conserved across isolates. Further analysis of global isolates is warranted for vaccine design based on this protein.

Genetic Diversity of Plasmodium falciparum Isolated from Symptomatic and Asymptomatic Individuals in Parts of Kaduna Metropolis, Kaduna State, Nigeria

Malaria due to Plasmodium falciparum is still a major public health problem and cause of high morbidity and mortality in Nigeria despite many efforts and interventions programmes put in place to control malaria, with transmission occurring throughout the year. Characterization of P. falciparum isolates from different geographical locations of Nigeria could provide the much needed information on the genetic composition of P. falciparum natural populations with regard to three polymorphic genes: Merozoite Surface Protein 1 (MSP 1), Merozoite Surface Protein (MSP 2) and Glutamate-Rich Protein (GLURP) genes, the findings of this study will assist in adopting more strategies and intervention programmes aim at controlling P. falciparum infection in Nigeria. This study was conducted to determine the genetic diversity and elucidate possible genomic variation between isolates of P. falciparum from symptomatic and asymptomatic malaria cases in Kaduna metropolis. Plasmodium falciparum genomic DNA was extracted from 42 positive blood samples collected onto whatmani¯s filter paper using the phenol-chloroform DNA extraction method. Genotyping of P. falciparum was based on the amplification of MSP 1, MSP 2 and glurp genes. Analysis of PCR products of P. falciparum isolates shows the presence of 33 alleles spread across MSP 1, MSP 2 and glurp genes at a frequency of 5, 24 and 4 respectively. Detection of the three genes; (MSP1); MAD20 and KI, (MSP2); FC27 and 3D7/ICI and (glurp) gene and the occurrence of single and multiple genotypic infection recorded showed the diverse genetic composition of P. falciparum population, an indication of the endemicity of malaria. This information is essential for effective malaria control programme.

Insights into the molecular diversity of Plasmodium vivax merozoite surface protein-3\u03b3 (pvmsp3\u03b3), a polymorphic member in the msp3 multi-gene family

Plasmodium vivax merozoite surface protein 3 (PvMSP3) is encoded by a multi-gene family. Of these, PvMSP3α, PvMSP3β and PvMSP3γ, are considered to be vaccine targets. Despite comprehensive analyses of PvMSP3α and PvMSP3β, little is known about structural and sequence diversity in PvMSP3γ. Analysis of 118 complete pvmsp3γ sequences from diverse endemic areas of Thailand and 9 reported sequences has shown 86 distinct haplotypes. Based on variation in insert domains, pvmsp3γ can be classified into 3 types, i.e. Belem, Salvador I and NR520. Imperfect nucleotide repeats were found in six regions of the gene; none encoded tandem amino acid repeats. Predicted coiled-coil heptad repeats were abundant in the protein and displayed variation in length and location. Interspersed phase shifts occurred in the heptad arrays that may have an impact on protein structure. Polymorphism in pvmsp3γ seems to be generated by intragenic recombination and driven by natural selection. Most P. vivax isolates in Thailand exhibit population structure, suggesting limited gene flow across endemic areas. Phylogenetic analysis has suggested that insert domains could have been subsequently acquired during the evolution of pvmsp3γ. Sequence and structural diversity of PvMSP3γ may complicate vaccine design due to alteration in predicted immunogenic epitopes among variants.