Merozoite Surface Protein Research Articles

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.

Genetic polymorphism of merozoite surface protein 1 and antifolate-resistant genes in Plasmodium falciparum from Mali and Niger.

Since 2015, countries in the Sahel region have implemented large-scale seasonal malaria chemoprevention (SMC). However, the mass use of sulfadoxine-pyrimethamine (SP) plus amodiaquine impacts the genetic diversity of malaria parasites and their sensitivity to antimalarials. This study aimed to describe and compare the genetic diversity and SP resistance of Plasmodium falciparum strains in Mali and Niger. We collected 400 blood samples in Mali and Niger from children aged 3-59 months suspected of malaria. Of them, 201 tested positive (Niger, 111, 55.2%; Mali, 90, 44.8%). Polymorphism of merozoite surface protein 1 (msp1) genetic marker showed 201 allotypes. The frequency of the RO33 allotype was significantly higher in Niger (63.6%) than in Mali (39.3%). There was no significant difference in the frequency of the K1 and MAD20 allotypes between the 2 countries. The multiplicity of infection was 2 allotypes per patient in Mali and one allotype per patient in Niger. The prevalence of strains with the triple mutants Pfdhfr51I/Pfdhfr59R/Pfdhps436A/F/H and Pfdhfr51I/Pfdhfr59R/Pfdhps437G was 18.1% and 30.2%, respectively, and 7.7% carried the quadruple mutant Pfdhfr51I/Pfdhfr59R/Pfdhps436A/F/H/Pfdhps437G. Despite the significant genetic diversity of parasite populations, the level of SP resistance was comparable between Mali and Niger. The frequency of mutations conferring resistance to SP still allows its effective use in intermittent preventive treatment in pregnant women and in SMC.

A new Plasmodium vivax reference genome for South American isolates

BackgroundPlasmodium vivax is the second most important cause of human malaria worldwide, and accounts for the majority of malaria cases in South America. A high-quality reference genome exists for Papua Indonesia (PvP01) and Thailand (PvW1), but is lacking for South America. A reference genome specifically for South America would be beneficial though, as P. vivax is a genetically diverse parasite with geographical clustering.ResultsThis study presents a new high-quality assembly of a South American P. vivax isolate, referred to as PvPAM (P. vivax Peruvian AMazon). The genome was obtained from a low input patient sample from the Peruvian Amazon and sequenced using PacBio technology, resulting in a highly complete assembly with 6497 functional genes. Telomeric ends were present in 17 out of 28 chromosomal ends, and additional (sub)telomeric regions are present in 12 unassigned contigs. A comparison of multigene families between PvPAM and the PvP01 genome revealed remarkable variation in vir genes, and the presence of merozoite surface proteins (MSP) 3.6 and 3.7. Three dhfr and dhps drug resistance associated mutations are present in PvPAM, similar to those found in other Peruvian isolates. Mapping of publicly available South American whole genome sequencing (WGS) data to PvPAM resulted in significantly fewer variants and truncated reads compared to the use of PvP01 or PvW1 as reference genomes. To minimize the number of core genome variants in non-South American samples, PvW1 is most suited for Southeast Asian isolates, both PvPAM and PvW1 are suited for South Asian isolates, and PvPAM is recommended for African isolates. Interestingly, non-South American samples still contained the least subtelomeric variants when mapped to PvPAM, indicating high quality of the PvPAM subtelomeric regions.ConclusionsOur findings show that the PvPAM reference genome more accurately represents South American P. vivax isolates in comparison to PvP01 and PvW1. In addition, PvPAM has a high level of completeness, and contains a similar number of annotated genes as PvP01 or PvW1. The PvPAM genome therefore will be a valuable resource to improve future genomic analyses on P. vivax isolates from the South American continent.

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.

Tracking the geographical origin of Plasmodium falciparum causing a rare severe case of malaria imported into Palestine, a zero-indigenous case area

BackgroundMalaria cases in non-endemic zero-indigenous case areas are most likely to have been imported whatever of the route of importation. In countries recently declared malaria-free and now without local transmission, imported cases remain a threat to re-introduction of the disease and a burden on the health system.Case presentationThree days after returning from a long trip to malaria- endemic countries; Abyei-Sudan, Chad and Uganda, a 41-year-old male resident from Jericho, Palestine, suffered paroxysms of fever, general fatigue, myalgia, arthralgia, headache, and a strong desire to vomit. Thin and thick Giemsa-stained blood smears were prepared and examined microscopically using oil immersion. Immature trophozoites (ring forms) were seen to parasitize approximately 10% of the erythrocytes revealing hyperparasitemia equivalent to > 100,000 parasites/ µl indicating severe malaria [1, 2]. The double chromatin configuration (headphones) and accolé (applique) position are both indicative of Plasmodium falciparum infection. The 18S rRNA- PCR targeting the rPLU6-rPLU5 region was used to confirm the diagnosis. The next-generation sequencing (NGS) method was carried out according to the manufacturer’s instructions (Illumina® DNA Prep, (M) Tagmentation kit (20060060), Illumina) to identify Plasmodium spp. Furthermore, NGS produced a whole-genome sequence of 22.8Mbp of the 14 chromosomes and 25Kbp of the apicoplast. A BLAST search of the apicoplast DNA and selected chromosomal DNA revealed that P. falciparum was the causative agent. The merozoite surface protein-1 (msp-1) was used to construct a phylogenetic tree of 26 P. falciparum, including the one isolated from the patient from Jericho, which clustered with the Sudanese isolate indicating genetic relatedness between the two.ConclusionThe travel history together with signs and symptoms of malaria, followed by prompt diagnosis using conventional microscopic inspection of Giemsa-stained films together with molecular DNA tracking tools like msp-1 were key means in tracking the place of origin of infection in the case of travel to multiple destination.

Severity of Schistosoma haematobium co-infection with malaria in school-children is potentially modulated by host CD14 gene variants

ObjectiveSchistosomiasis remains a chronic disease of global importance, especially in many rural areas of the world where co-infection with Plasmodium falciparum is common. It is critical to decipher the role of single or co-infected disease scenarios on immune system regulation in such individuals and how such co-infections can either ameliorate or complicate immune response and the consequent disease outcome. First, 10 ml of urine samples, collected between 10:00 am and 2:00 pm, was filtered for diagnosis of schistosomiasis, while egg count, indicative of disease severity, was determined by microscopy. Furthermore, genomic DNA samples extracted from dried blood spots collected on filter paper from one hundred and forty-four Schistosoma haematobium-infected school-children was tested for P. falciparum parasite positivity by an allele-specific nested-PCR analysis of merozoite surface protein (msp)-1 and -2 genes and a real-time PCR assay. In addition, among P. falciparum parasite-positive individuals, we carried out a Taqman SNP genotyping assay to extrapolate the effect of host CD14 (-159 C/T; rs2569190) genetic variants on schistosome egg count.ResultsOf the 144 individuals recruited, P. falciparum parasite positivity with msp-1 gene were 34%, 43% and 55% for MAD20, RO33 and K1 alleles respectively. Of the co-infected individuals, CD14 genetic variants ranged from 18.8% vs 21.5%, 33.3% vs 44.4%, 9.7% vs 11.8% for single versus schistosome co-infection for the wild type (CC), heterozygous (CT) and mutant (TT) variants respectively. Though the mean egg count for co-infected individuals with CD14 wild type (33.7 eggs per 10 ml of urine) and heterozygote variants (37.5 eggs per 10 ml of urine) were lower than that of schistosome infection alone (52.48 and 48.08 eggs/10 ml of urine respectively), it lacked statistical significance (p-value 0.12 and 0.29), possibly reflecting the benefit of the CD14 activation in schistosome plus malaria co-infection and not schistosome infection alone. In addition, the lower mean egg count in co-infected individuals reveal the benefit of downstream Th1 immune response mitigated by CD14 innate activation that is absent in schistosome infection alone.

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.

Recent increase in low complexity polygenomic infections and sialic acid-independent invasion pathways in Plasmodium falciparum from Western Gambia

BackgroundThe malaria parasite Plasmodium falciparum utilizes multiple alternative receptor-ligand interactions for the invasion of human erythrocytes. While some P. falciparum clones make use of sialic acid (SA) residues on the surface of the human glycophorin receptors to invade the erythrocyte, others use alternative receptors independent of sialic acid residues. We hypothesized that over the years, intensified malaria control interventions and declining prevalence in The Gambia have resulted in a selection of parasites with a dominant invasion pathways and ligand expression profiles.MethodsBlood samples were collected from 65 malaria-infected participants with uncomplicated malaria across 3 years (2015, 2016, and 2021). Genetic diversity was determined by genotyping the merozoite surface protein 2 (msp2) polymorphic gene of P. falciparum. Erythrocyte invasion phenotypes were determined using neuraminidase, trypsin, and chymotrypsin enzymes, known to cleave different receptors from the surface of the erythrocyte. Schizont-stage transcript levels were obtained for a panel of 6 P. falciparum invasion ligand genes (eba175, eba181, Rh2b, Rh4, Rh5, and clag2) using 48 successfully cultured isolates.ResultsThough the allelic heterozygosity of msp2 repeat region decreased as expected with reduced transmission, there was an increase in infections with more than a single msp2 allelotype from 2015 to 2021. The invasion phenotypes of these isolates were mostly SA independent with a continuous increase from 2015 to 2021. Isolates from 2021 were highly inhibited by chymotrypsin treatment compared to isolates from 2015 and 2016. Higher invasion inhibition for 2021 isolates was further obtained following erythrocyte treatment with a combination of chymotrypsin and trypsin. The transcript levels of invasion ligand genes varied across years. However, levels of clag2, a rhoptry-associated protein, were higher in 2015 and 2016 isolates than in 2021 isolates, while Rh5 levels were higher in 2021 compared to other years.ConclusionsOverall, these findings suggest increasing mixed infections with an increase in the use of sialic-acid independent invasion pathways by P. falciparum clinical isolates in the Western part of Gambia.Graphical

Plasmodium falciparum msp-1 and msp-2 genetic diversity and multiplicity of infection in isolates from Congolese patients in the Republic of Congo.

With limited up to date data from the Republic of Congo, the aim of this study was to investigate allelic polymorphism of merozoite surface protein-1 (msp-1) and merozoite surface protein-2 (msp-2). This will help assess the genetic diversity and multiplicity of Plasmodium falciparum infection (MOI), from uncomplicated malaria individuals living in Brazzaville. Between March and October 2021, a cross-sectional study was carried out at a health center in Madibou District located in the south of Brazzaville. Plasmodium infection was diagnosed in human blood by microscopy and the block 2 of P. falciparum msp-1 and block 3 of msp-2 genes were genotyped by nested PCR. Overall, 57 genotypes with fragment sizes ranging from 110 to 410 bp were recorded for msp-1, among which 25, 21, and 11 genotypes identified for K1, MAD20, and RO33 allelic families respectively. RO33 (34.3%) and MAD20 (34.3%) allelic families were more frequent compared to K1 (31.4%) although the difference was not statistically significant. Also, 47 msp-2 genotypes were identified, including 26 FC27 genotypes type, and 21 genotypes belonging to the 3D7 allelic family. FC27 was more frequent (52.3%) compared to 3D7 (47.7%). The prevalence of the polyclonal infection was 90.0% while the MOI was 2.90 ± 1.0. The MOI and polyclonal infection were not significantly associated with the parasitaemia and anaemia. This study reveals a high genetic diversity and the trend of increasing MOI of P. falciparum isolates from the south of Brazzaville, compared to the reports from the same setting before the COVID-19 pandemic.

Artemisinin resistance in P. falciparum: probing the interacting partners of Kelch13 protein in parasite

Artemisinin (ART) resistance in Plasmodium is threatening the artemisinin combination therapies-the first line of defence against malaria. ART resistance has been established to be mediated by the Plasmodium Kelch13 (PfK13) protein. For the crucial role of PfK13 in multiple pathways of the Plasmodium life cycle and ART resistance, it is imperative that we investigate its interacting partners. We recombinantly expressed PfK13-p (Bric a brac/Poxvirus and zinc finger and propeller domains), generating anti-PfK13-p antibodies to perform co-immunoprecipitation assays and probed PfK13 interacting partners. Surface plasmon resonance and pull-down assays were performed to establish physical interactions of representative proteins with PfK13-p. The co-immunoprecipitation assays identified 17 proteins with distinct functions in the parasite life cycle- protein folding, cellular metabolism, and protein binding and invasion. In addition to the overlap with previously identified proteins, our study identified 10 unique proteins. Fructose-biphosphate aldolase and heat shock protein 70 demonstrated strong biophysical interaction with PfK13-p, with KD values of 6.6 µM and 7.6 µM, respectively. Additionally, Plasmodium merozoite surface protein 1 formed a complex with PfK13-p, which is evident from the pull-down assay. This study adds to our knowledge of the PfK13 protein in mediating ART resistance by identifying new PfK13 interacting partners. Three representative proteins-fructose-biphosphate aldolase, heat shock protein 70, and merozoite surface protein 1-demonstrated clear evidence of biophysical interactions with PfK13-p. However, elucidation of the functional relevance of these physical interactions are crucial in context of PfK13 role in ART resistance.

Dynamics of IgG antibody response against Plasmodium antigens among Nigerian infants and young children.

Plasmodium falciparum malaria is a leading cause of child mortality in Nigeria. Neonates are born with maternal antibodies from placental transfer which may protect against malaria infection in the first months of life. The IgG dynamics of the transition from passively transferred antimalarial antibodies to actively acquired IgG from natural exposure have not been well elucidated. Blood samples collected during a 2018 Nigeria nationwide HIV/AIDS household survey were available for 9,443 children under 5 years of age, with a subset of infants under 2 months of age having maternal samples available (n=41). Samples were assayed for the P. falciparum HRP2 antigen and anti-malarial IgG antibodies. LOESS regression examined the dynamics in IgG response in the first 5 years of life. Correlation with maternal IgG levels was assessed for mother/child pairs. Consistent decreases were observed in median IgG levels against all Plasmodium spp. antigen targets for the first months of life. At a population level, P. falciparum apical membrane antigen-1 (AMA1) and merozoite surface protein-1 19kD (PfMSP1) IgG decreased during the first 12 months of life before reaching a nadir, whereas IgGs to other targets only declined for the first 4 months of life. Seropositivity showed a similar decline with the lowest seropositivity against AMA1 and PfMSP1 at 10-12 months, though remaining above 50% during the first 2 years of life in higher transmission areas. No protective association was observed between IgG positivity and P. falciparum infection in infants. Maternal antibody levels showed a strong positive correlation with infant antibody levels for all P. falciparum antigens from birth to 2 months of age, but this correlation was lost by 6 months of age. Maternally transferred anti-malarial IgG antibodies rapidly decline during the first 6 months of life, with variations among specific antigens and malaria transmission intensity. From 3-23 months of age, there was a wide range in IgG levels for the blood-stage antigens indicating high individual variation in antibody production as children are infected with malaria. Non-falciparum species-specific antigens showed similar patterns in waning immunity and correlation with paired mother's IgG levels compared to P. falciparum antigens.

Distinct Allelic Diversity of Plasmodium vivax Merozoite Surface Protein 3-Alpha (PvMSP-3α) Gene in Thailand Using PCR-RFLP.

Considering the importance of merozoite surface proteins (MSPs) as vaccine candidates, this study was conducted to investigate the polymorphism and genetic diversity of Plasmodium vivax merozoite surface protein 3-alpha (PvMSP-3α) in Thailand. To analyze genetic diversity, 118 blood samples containing P. vivax were collected from four malaria-endemic areas in western and southern Thailand. The DNA was extracted and amplified for the PvMSP-3α gene using nested PCR. The PCR products were genotyped by PCR-RFLP with Hha I and Alu I restriction enzymes. The combination patterns of Hha I and Alu I RFLP were used to identify allelic variants. Genetic evaluation and phylogenic analysis were performed on 13 sequences, including 10 sequences from our study and 3 sequences from GenBank. The results revealed three major types of PvMSP-3α, 91.5% allelic type A (∼1.8 kb), 5.1% allelic type B (∼1.5 kb), and 3.4% allelic type C (∼1.2 kb), were detected based on PCR product size with different frequencies. Among all PvMSP-3α, 19 allelic subtypes with Hha I RFLP patterns were distinguished and 6 allelic subtypes with Alu I RFLP patterns were identified. Of these samples, 73 (61%) and 42 (35.6%) samples were defined as monoallelic subtype infection by Hha I and Alu I PCR-RFLP, respectively, whereas 77 (65.3%) samples were determined to be mixed-allelic subtype infection by the combination patterns of Hha I and Alu I RFLP. These results strongly indicate that PvMSP-3α gene is highly polymorphic, particularly in blood samples collected from the Thai-Myanmar border area (the western part of Thailand). The combination patterns of Hha I and Alu I RFLP of the PvMSP-3α gene could be considered for use as molecular epidemiologic markers for genotyping P. vivax isolates in Thailand.

Multifunctional IgG/IgM antibodies and cellular cytotoxicity are elicited by the full-length MSP1 SumayaVac-1 malaria vaccine

Radical control of malaria likely requires a vaccine that targets both the asymptomatic liver stages and the disease-causing blood stages of the human malaria parasite Plasmodium falciparum. While substantial progress has been made towards liver stage vaccines, the development of a blood stage vaccine is lagging behind. We have recently conducted a first-in-human clinical trial to evaluate the safety and immunogenicity of the recombinant, full-length merozoite surface protein 1 (MSP1FL) formulated with GLA-SE as adjuvant. Here, we show that the vaccine, termed SumayaVac-1, elicited both a humoral and cellular immune response as well as a recall T cell memory. The induced IgG and IgM antibodies were able to stimulate various Fc-mediated effector mechanisms associated with protection against malaria, including phagocytosis, release of reactive oxygen species, production of IFN-γ as well as complement activation and fixation. The multifunctional activity of the humoral immune response remained for at least 6 months after vaccination and was comparable to that of naturally acquired anti-MSP1 antibodies from semi-immune adults from Kenya. We further present evidence of SumayaVac-1 eliciting a recallable cellular cytotoxicity by IFN-γ producing CD8+ T cells. Our study revitalizes MSP1FL as a relevant blood stage vaccine candidate and warrants further evaluation of SumayaVac-1 in a phase II efficacy trial.

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.

Hemoglobinopathies, merozoite surface protein-2 gene polymorphisms, and acquisition of Epstein Barr virus among infants in Western Kenya

BackgroundEpstein Barr virus (EBV)-associated endemic Burkitt’s Lymphoma pediatric cancer is associated with morbidity and mortality among children resident in holoendemic Plasmodium falciparum regions in western Kenya. P. falciparum exerts strong selection pressure on sickle cell trait (SCT), alpha thalassemia (-α3.7/αα), glucose-6-phosphate dehydrogenase (G6PD), and merozoite surface protein 2 (MSP-2) variants (FC27, 3D7) that confer reduced malarial disease severity. The current study tested the hypothesis that SCT, (-α3.7/αα), G6PD mutation and (MSP-2) variants (FC27, 3D7) are associated with an early age of EBV acquisition.MethodsData on infant EBV infection status (< 6 and ≥ 6–12 months of age) was abstracted from a previous longitudinal study. Archived infant DNA (n = 81) and mothers DNA (n = 70) samples were used for genotyping hemoglobinopathies and MSP-2. The presence of MSP-2 genotypes in maternal DNA samples was used to indicate infant in-utero malarial exposure. Genetic variants were determined by TaqMan assays or standard PCR. Group differences were determined by Chi-square or Fisher’s analysis. Bivariate regression modeling was used to determine the relationship between the carriage of genetic variants and EBV acquisition.ResultsEBV acquisition for infants < 6 months was not associated with -α3.7/αα (OR = 1.824, P = 0.354), SCT (OR = 0.897, P = 0.881), or G6PD [Viangchan (871G > A)/Chinese (1024 C > T) (OR = 2.614, P = 0.212)] and [Union (1360 C > T)/Kaiping (1388G > A) (OR = 0.321, P = 0.295)]. There was no relationship between EBV acquisition and in-utero exposure to either FC27 (OR = 0.922, P = 0.914) or 3D7 (OR = 0.933, P = 0.921). In addition, EBV acquisition in infants ≥ 6–12 months also showed no association with -α3.7/αα (OR = 0.681, P = 0.442), SCT (OR = 0.513, P = 0.305), G6PD [(Viangchan (871G > A)/Chinese (1024 C > T) (OR = 0.640, P = 0.677)], [Mahidol (487G > A)/Coimbra (592 C > T) (OR = 0.948, P = 0.940)], [(Union (1360 C > T)/Kaiping (1388G > A) (OR = 1.221, P = 0.768)], African A (OR = 0.278, P = 0.257)], or in utero exposure to either FC27 (OR = 0.780, P = 0.662) or 3D7 (OR = 0.549, P = 0.241).ConclusionAlthough hemoglobinopathies (-α3.7/αα, SCT, and G6PD mutations) and in-utero exposure to MSP-2 were not associated with EBV acquisition in infants 0–12 months, novel G6PD variants were discovered in the population from western Kenya. To establish that the known and novel hemoglobinopathies, and in utero MSP-2 exposure do not confer susceptibility to EBV, future studies with larger sample sizes from multiple sites adopting genome-wide analysis are required.

Comparative study of Plasmodium falciparum msp-1 and msp-2 Genetic Diversity in Isolates from Rural and Urban Areas in the South of Brazzaville, Republic of Congo.

Polymorphisms in the genes encoding the merozoite surface proteins msp-1 and msp-2 are widely used markers for characterizing the genetic diversity of Plasmodium falciparum. This study aimed to compare the genetic diversity of circulating parasite strains in rural and urban settings in the Republic of Congo after the introduction of artemisinin-based combination therapy (ACT) in 2006. A cross-sectional survey was conducted from March to September 2021 in rural and urban areas close to Brazzaville, during which Plasmodium infection was detected using microscopy (and nested-PCR for submicroscopic infection). The genes coding for merozoite proteins-1 and -2 were genotyped by allele-specific nested PCR. Totals of 397 (72.4%) and 151 (27.6%) P. falciparum isolates were collected in rural and urban areas, respectively. The K1/msp-1 and FC27/msp-2 allelic families were predominant both in rural (39% and 64%, respectively) and urban (45.4% and 54.5% respectively) areas. The multiplicity of infection (MOI) was higher (p = 0.0006) in rural areas (2.9) compared to urban settings (2.4). The rainy season and the positive microscopic infection were associated with an increase in MOI. These findings reveal a higher P. falciparum genetic diversity and MOI in the rural setting of the Republic of Congo, which is influenced by the season and the participant clinical status.

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 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 of Plasmodium vivax Field Isolates from the Thai–Myanmar Border during the Period of 2006–2016

High levels of genetic variants of Plasmodium vivax have previously been reported in Thailand. Circumsporozoite surface protein (CSP), merozoite surface protein (MSP), and microsatellite markers were used to determine the genetic polymorphisms of P. vivax. This study aimed to investigate the molecular epidemiology of P. vivax populations at the Thai-Myanmar border by genotyping the PvCSP, PvMSP-3α, and PvMSP-3β genes. Four hundred and forty P. vivax clinical isolates were collected from the Mae Sot and Sai Yok districts from 2006-2007 and 2014-2016. Polymerase chain reaction with restriction fragment length polymorphism (RFLP) was used to investigate the genetic polymorphisms of the target genes. Based on PCR band size variations, 14 different PvCSP alleles were identified: eight for VK210 and six for VK247. The VK210 genotype was the dominant variant during both sample collection periods. Based on PCR genotyping, three distinct types (A, B, and C) for both PvMSP-3α and PvMSP-3β were observed. Following RFLP, 28 and 14 allelic variants of PvMSP-3α and 36 and 20 allelic variants of PvMSP-3β with varying frequencies were identified during the first and second periods, respectively. High genetic variants of PvMSP-3 and PvCSP were found in the study area. PvMSP-3β exhibited a higher level of genetic diversity and multiple-genotype infection versus PvMSP-3α.

B-Cell Epitope Mapping of the Plasmodium falciparum Malaria Vaccine Candidate GMZ2.6c in a Naturally Exposed Population of the Brazilian Amazon.

The GMZ2.6c malaria vaccine candidate is a multi-stage P. falciparum chimeric protein that contains a fragment of the sexual-stage Pfs48/45-6C protein genetically fused to GMZ2, an asexual-stage vaccine construction consisting of the N-terminal region of the glutamate-rich protein (GLURP) and the C-terminal region of the merozoite surface protein-3 (MSP-3). Previous studies showed that GMZ2.6c is widely recognized by antibodies from Brazilian exposed individuals and that its components are immunogenic in natural infection by P. falciparum. In addition, anti-GMZ2.6c antibodies increase with exposure to infection and may contribute to parasite immunity. Therefore, identifying epitopes of proteins recognized by antibodies may be an important tool for understanding protective immunity. Herein, we identify and validate the B-cell epitopes of GMZ2.6c as immunogenic and immunodominant in individuals exposed to malaria living in endemic areas of the Brazilian Amazon. Specific IgG antibodies and subclasses against MSP-3, GLURP, and Pfs48/45 epitopes were detected by ELISA using synthetic peptides corresponding to B-cell epitopes previously described for MSP-3 and GLURP or identified by BepiPred for Pfs48/45. The results showed that the immunodominant epitopes were P11 from GLURP and MSP-3c and DG210 from MSP-3. The IgG1 and IgG3 subclasses were preferentially induced against these epitopes, supporting previous studies that these proteins are targets for cytophilic antibodies, important for the acquisition of protective immunity. Most individuals presented detectable IgG antibodies against Pfs48/45a and/or Pfs48/45b, validating the prediction of linear B-cell epitopes. The higher frequency and antibody levels against different epitopes from GLURP, MSP-3, and Pfs48/45 provide additional information that may suggest the relevance of GMZ2.6c as a multi-stage malaria vaccine candidate.