Plasmodium falciparum genetic diversity and multiplicity of infection based on msp-1, msp-2, glurp and microsatellite genetic markers in sub-Saharan Africa: a systematic review and meta-analysis.

BACKGROUND: In sub-Saharan Africa (SSA), Plasmodium falciparum causes most of the malaria cases. Despite its crucial roles in disease severity and drug resistance, comprehensive data on Plasmodium falciparum genetic diversity and multiplicity of infection (MOI) are sparse in SSA. This study summarizes available information on genetic diversity and MOI, focusing on key markers (msp-1, msp-2, glurp, and microsatellites). The systematic review aimed to evaluate their influence on malaria transmission dynamics and offer insights for enhancing malaria control measures in SSA. METHODS: The review was conducted following the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) guidelines. Two reviewers conducted article screening, assessed the risk of bias (RoB), and performed data abstraction. Meta-analysis was performed using the random-effects model in STATA version 17. RESULTS: The review included 52 articles: 39 cross-sectional studies and 13 Randomized Controlled Trial (RCT)/cohort studies, involving 11,640 genotyped parasite isolates from 23 SSA countries. The overall pooled mean expected heterozygosity was 0.65 (95% CI: 0.51-0.78). Regionally, values varied: East (0.58), Central (0.84), Southern (0.74), and West Africa (0.69). Overall pooled allele frequencies of msp-1 alleles K1, MAD20, and RO33 were 61%, 44%, and 40%, respectively, while msp-2 I/C 3D7 and FC27 alleles were 61% and 55%. Central Africa reported higher frequencies (K1: 74%, MAD20: 51%, RO33: 48%) than East Africa (K1: 46%, MAD20: 42%, RO33: 31%). For msp-2, East Africa had 60% and 55% for I/C 3D7 and FC27 alleles, while West Africa had 62% and 50%, respectively. The pooled allele frequency for glurp was 66%. The overall pooled mean MOI was 2.09 (95% CI: 1.88-2.30), with regional variations: East (2.05), Central (2.37), Southern (2.16), and West Africa (1.96). The overall prevalence of polyclonal Plasmodium falciparum infections was 63% (95% CI: 56-70), with regional prevalences as follows: East (62%), West (61%), Central (65%), and South Africa (71%). CONCLUSION: The study shows substantial regional variation in Plasmodium falciparum parasite genetic diversity and MOI in SSA. These findings suggest a need for malaria control strategies and surveillance efforts considering regional-specific factors underlying Plasmodium falciparum infection.

A severe case of Plasmodium falciparum malaria in a traveler returning from Kazakhstan, a malaria-free country.

Following a 2-week trip to Kazakhstan, a 42-year-old woman presented at the emergency department in Germany with fever, headache, nausea, and neurological symptoms. An infection with Plasmodium falciparum was rapidly diagnosed. The patient was immediately treated with intravenous artesunate and transferred to an intensive care unit. The initial parasite density was as high as 30% infected erythrocytes with 845,880 parasites/µL. Since Kazakhstan was declared malaria-free in 2012, molecular testing for Plasmodium has been initiated to identify a possible origin. Genotyping of the msp-1 gene and microsatellite markers showed that the parasites are of African origin, with two different alleles indicating a polyclonal infection. After a hospitalization of 10 days, the patient was discharged in good health. Overall, our results emphasize that malaria must be on the list of differential diagnoses for patients with fever of unknown origin, even if they come from countries where malaria does not commonly occur.

Bone scan findings of Paget's disease of bone in patients with VCP Multisystem Proteinopathy 1.

Multisystem Proteinopathy 1 (MSP1) disease is a rare genetic disorder caused by mutations in the Valosin-Containing Protein (VCP) gene with clinical features of inclusion body myopathy (IBM), frontotemporal dementia (FTD), and Paget's disease of bone (PDB). We performed bone scan imaging in twelve patients (6 females, 6 males) with confirmed VCP gene mutation six (50%) of which has myopathy alone, four (33%) with both PDB and myopathy, and two (15%) were presymptomatic carriers. We aim to characterize the PDB in diagnosed individuals, and potentially identify PDB in the myopathy and presymptomatic groups. Interestingly, two patients with previously undiagnosed PDB had positive diagnostic findings on the bone scan and subsequent radiograph imaging. Among the individuals with PDB, increased radiotracer uptake of the affected bones were of typical distribution as seen in conventional PDB and those reported in other MSP1 cohorts which are the thoracic spine and ribs (75%), pelvis (75%), shoulder (75%) and calvarium (15%). Overall, we show that technetium-99m bone scans done at regular intervals are a sensitive screening tool in patients with MSP1 associated VCP variants at risk for PDB. However, diagnostic confirmation should be coupled with clinical history, biochemical analysis, and skeletal radiographs to facilitate early treatment and prevention complications, acknowledging its limited specificity.

Microsatellites reveal high polymorphism and high potential for use in anti-malarial efficacy studies in areas with different transmission intensities in mainland Tanzania.

BACKGROUND: Tanzania is currently implementing therapeutic efficacy studies (TES) in areas of varying malaria transmission intensities as per the World Health Organization (WHO) recommendations. In TES, distinguishing reinfection from recrudescence is critical for the determination of anti-malarial efficacy. Recently, the WHO recommended genotyping polymorphic coding genes, merozoite surface proteins 1 and 2 (msp1 and msp2), and replacing the glutamate-rich protein (glurp) gene with one of the highly polymorphic microsatellites in Plasmodium falciparum to adjust the efficacy of antimalarials in TES. This study assessed the polymorphisms of six neutral microsatellite markers and their potential use in TES, which is routinely performed in Tanzania. METHODS: Plasmodium falciparum samples were obtained from four TES sentinel sites, Kibaha (Pwani), Mkuzi (Tanga), Mlimba (Morogoro) and Ujiji (Kigoma), between April and September 2016. Parasite genomic DNA was extracted from dried blood spots on filter papers using commercial kits. Genotyping was done using six microsatellites (Poly-α, PfPK2, TA1, C3M69, C2M34 and M2490) by capillary method, and the data were analysed to determine the extent of their polymorphisms and genetic diversity at the four sites. RESULTS: Overall, 83 (88.3%) of the 94 samples were successfully genotyped (with positive results for ≥ 50.0% of the markers), and > 50.0% of the samples (range = 47.6-59.1%) were polyclonal, with a mean multiplicity of infection (MOI) ranging from 1.68 to 1.88 among the four sites. There was high genetic diversity but limited variability among the four sites based on mean allelic richness (RS = 7.48, range = 7.27-8.03, for an adjusted minimum sample size of 18 per site) and mean expected heterozygosity (He = 0.83, range = 0.80-0.85). Cluster analysis of haplotypes using STRUCTURE, principal component analysis, and pairwise genetic differentiation (FST) did not reveal population structure or clustering of parasites according to geographic origin. Of the six markers, Poly-α was the most polymorphic, followed by C2M34, TA1 and C3M69, while M2490 was the least polymorphic. CONCLUSION: Microsatellite genotyping revealed high polyclonality and genetic diversity but no significant population structure. Poly-α, C2M34, TA1 and C3M69 were the most polymorphic markers, and Poly-α alone or with any of the other three markers could be adopted for use in TES in Tanzania.

msp1, msp2, and glurp genotyping to differentiate Plasmodium falciparum recrudescence from reinfections during prevention of reestablishment phase, Sri Lanka, 2014-2019.

BACKGROUND: Sri Lanka after eliminating malaria in 2012, is in the prevention of re-establishment (POR) phase. Being a tropical country with high malariogenic potential, maintaining vigilance is important. All malaria cases are investigated epidemiologically and followed up by integrated drug efficacy surveillance (iDES). Occasionally, that alone is not adequate to differentiate Plasmodium falciparum reinfections from recrudescences. This study evaluated the World Health Organization and Medicines for Malaria Venture (MMV) recommended genotyping protocol for the merozoite surface proteins (msp1, msp2) and the glutamate-rich protein (glurp) to discriminate P. falciparum recrudescence from reinfection in POR phase. METHODS: All P. falciparum patients detected from April 2014 to December 2019 were included in this study. Patients were treated and followed up by iDES up to 28 days and were advised to get tested if they develop fever at any time over the following year. Basic socio-demographic information including history of travel was obtained. Details of the malariogenic potential and reactive entomological and parasitological surveillance carried out by the Anti Malaria Campaign to exclude the possibility of local transmission were also collected. The msp1, msp2, and glurp genotyping was performed for initial and any recurrent infections. Classification of recurrent infections as recrudescence or reinfection was done based on epidemiological findings and was compared with the genotyping outcome. RESULTS: Among 106 P. falciparum patients, six had recurrent infections. All the initial infections were imported, with a history of travel to malaria endemic countries. In all instances, the reactive entomological and parasitological surveillance had no evidence for local transmission. Five recurrences occurred within 28 days of follow-up and were classified as recrudescence. They have not travelled to malaria endemic countries between the initial and recurrent infections. The other had a recurrent infection after 105 days. It was assumed a reinfection, as he had travelled to the same malaria endemic country in between the two malaria attacks. Genotyping confirmed the recrudescence and the reinfection. CONCLUSIONS: The msp1, msp2 and glurp genotyping method accurately differentiated reinfections from recrudescence. Since reinfection without a history of travel to a malaria endemic country would mean local transmission, combining genotyping outcome with epidemiological findings will assist classifying malaria cases without any ambiguity.

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.

Merozoite surface protein 1 paralog is involved in the human erythrocyte invasion of a zoonotic malaria, Plasmodium knowlesi.

The zoonotic malaria parasite Plasmodium knowlesi is an important public health concern in Southeast Asia. Invasion of host erythrocytes is essential for parasite growth, and thus, understanding the repertoire of parasite proteins that enable this process is vital for identifying vaccine candidates and how some species are able to cause zoonotic infection. Merozoite surface protein 1 (MSP1) is found in all malaria parasite species and is perhaps the most well-studied as a potential vaccine candidate. While MSP1 is encoded by a single gene in P. falciparum, all other human infective species (P. vivax, P. knowlesi, P. ovale, and P. malariae) additionally encode a divergent paralogue known as MSP1P, and little is known about its role or potential functional redundancy with MSP1. We, therefore, studied the function of P. knowlesi merozoite surface protein 1 paralog (PkMSP1P), using both recombinant protein and CRISPR-Cas9 genome editing. The recombinant 19-kDa C-terminus of PkMSP1P (PkMSP1P-19) was shown to bind specifically to human reticulocytes. However, immunoblotting data suggested that PkMSP1P-19-induced antibodies can recognize PkMSP1-19 and vice versa, confounding our ability to separate the properties of these two proteins. Targeted disruption of the pkmsp1p gene profoundly impacts parasite growth, demonstrating for the first time that PkMSP1P is important in in vitro growth of P. knowlesi and likely plays a distinct role from PkMSP1. Importantly, the MSP1P KO also enabled functional characterization of the PkMSP1P-19 antibodies, revealing clear immune cross-reactivity between the two paralogues, highlighting the vital importance of genetic studies in contextualizing recombinant protein studies.

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 Diversity of Merozoite Surface Antigens in Global Babesia bovis Populations.

Cattle can be severely infected with the tick-borne protozoa Babesia bovis, giving rise to serious economic losses. Invasion of the host's RBCs by the parasite merozoite/sporozoites depends largely on the MSA (merozoite surface antigens) gene family, which comprises various fragments, e.g., MSA-1, MSA-2a1, MSA-2a2, MSA-2b and MSA-2c, highlighting the importance of these antigens as vaccine candidates. However, experimental trials documented the failure of some developed MSA-based vaccines to fully protect animals from B. bovis infection. One reason for this failure may be related to the genetic structure of the parasite. In the present study, all MSA-sequenced B. bovis isolates on the GenBank were collected and subjected to various analyses to evaluate their genetic diversity and population structure. The analyses were conducted on 199 MSA-1, 24 MSA-2a1, 193 MSA-2b and 148 MSA-2c isolates from geographically diverse regions. All these fragments displayed high nucleotide and haplotype diversities, but the MSA-1 was the most hypervariable and had the lowest inter- and intra-population gene flow values. This fragment also displayed a strong positive selection when testing its isolates for the natural selection, which suggests the potential occurrence of more genetic variations. On the contrary, the MSA-2c was the most conserved in comparison to the other fragments, and displayed the highest inter- and intra-population gene flow values, which was evidenced by a significantly negative selection and negative neutrality indices (Fu's Fs and Tajima's D). The majority of the MSA-2c tested isolates had two conserved amino acid repeats, and earlier reports have found these repeats to be highly immunogenic, which underlines the importance of this fragment in developing vaccines against B. bovis. Results of the MSA-2a1 analyses were also promising, but many more MSA-2a1 sequenced isolates are required to validating this assumption. The genetic analyses conducted for the MSA-2b fragment displayed borderline values when compared to the other fragments.

Plasmodium falciparum population structure inferred by msp1 amplicon sequencing of parasites collected from febrile patients in Kenya.

BACKGROUND: Multiplicity of infection (MOI) is an important measure of Plasmodium falciparum diversity, usually derived from the highly polymorphic genes, such as msp1, msp2 and glurp as well as microsatellites. Conventional methods of deriving MOI lack fine resolution needed to discriminate minor clones. This study used amplicon sequencing (AmpliSeq) of P. falciparum msp1 ï»¿(Pfmsp1) to measure spatial and temporal genetic diversity of P. falciparum. METHODS: 264 P. falciparum positive blood samples collected from areas of differing malaria endemicities between 2010 and 2019 were used. Pfmsp1 gene was amplified and amplicon libraries sequenced on Illumina MiSeq. Sequences were aligned against a reference sequence (NC_004330.2) and clustered to detect fragment length polymorphism and amino acid variations. RESULTS: Children < 5 years had higher parasitaemia (median = 23.5 ± 5 SD, p = 0.03) than the > 5-14 (= 25.3 ± 5 SD), and those > 15 (= 25.1 ± 6 SD). Of the alleles detected, 553 (54.5%) were K1, 250 (24.7%) MAD20 and 211 (20.8%) RO33 that grouped into 19 K1 allelic families (108-270 bp), 14 MAD20 (108-216 bp) and one RO33 (153 bp). AmpliSeq revealed nucleotide polymorphisms in alleles that had similar sizes, thus increasing the K1 to 104, 58 for MAD20 and 14 for RO33. By AmpliSeq, the mean MOI was 4.8 (± 0.78, 95% CI) for the malaria endemic Lake Victoria region, 4.4 (± 1.03, 95% CI) for the epidemic prone Kisii Highland and 3.4 (± 0.62, 95% CI) for the seasonal malaria Semi-Arid region. MOI decreased with age: 4.5 (± 0.76, 95% CI) for children < 5 years, compared to 3.9 (± 0.70, 95% CI) for ages 5 to 14 and 2.7 (± 0.90, 95% CI) for those > 15. Females' MOI (4.2 ± 0.66, 95% CI) was not different from males 4.0 (± 0.61, 95% CI). In all regions, the number of alleles were high in the 2014-2015 period, more so in the Lake Victoria and the seasonal transmission arid regions. CONCLUSION: These findings highlight the added advantages of AmpliSeq in haplotype discrimination and the associated improvement in unravelling complexity of P. falciparum population structure.

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.

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.

Analysis of post-translational modification dynamics unveiled novel insights into Rice responses to MSP1.

Magnaporthe oryzae snodprot1 homologous protein (MSP1) is known to function as a pathogen-associated molecular pattern (PAMP) and trigger PAMP-triggered immunity (PTI) in rice including induction of programmed cell death and expression of defense-related genes. The involvement of several post-translational modifications (PTMs) in the regulation of plant immune response, especially PTI, is well established, however, the information on the regulatory roles of these PTMs in response to MSP1-induced signaling is currently elusive. Here, we report the phosphoproteome, ubiquitinome, and acetylproteome to investigate the MSP1-induced PTMs alterations in MSP1 overexpressed and wild-type rice. Our analysis identified a total of 4666 PTMs-modified sites in rice leaves including 4292 phosphosites, 189 ubiquitin sites, and 185 acetylation sites. Among these, the PTM status of 437 phosphorylated, 53 ubiquitinated, and 68 acetylated peptides was significantly changed by MSP1. Functional annotation of MSP1 modulated peptides by MapMan analysis revealed that these were majorly associated with cellular immune responses including signaling, transcription factors, DNA and RNA regulation, and protein metabolism, among others. Taken together, our study provides novel insights into post-translational mediated regulation of rice proteins in response to M. oryzae secreted PAMP which help in understanding the molecular mechanism of MSP1-induced signaling in rice in greater detail. SIGNIFICANCE: The research investigates the effect of overexpression of MSP1 protein in rice leaves on the phosphoproteome, acetylome, and ubiquitinome. The study found that MSP1 is involved in rice protein phosphorylation, particularly in signaling pathways, and identified a key component, PTAC16, in MSP1-induced signaling. The analysis also revealed MSP1's role in protein degradation and modification by inducing ubiquitination of the target rice proteins. The research identified potential kinases involved in the phosphorylation of rice proteins, including casein kinase II, 14-3-3 domain binding motif, ß-adrenergic receptor kinase, ERK1,2 kinase substrate motif, and casein kinase I motifs. Overall, the findings provide insights into the molecular mechanisms underlying of MSP1 induced signaling in rice which may have implications for improving crop yield and quality.

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.

Polymorphisms in Toll-Like receptors genes and their associations with immunological parameters in Plasmodium vivax malaria in the Brazil-French Guiana Border.

BACKGROUND: The innate immune response plays an important role during malaria. Toll-like receptors (TLR) are capable of recognizing pathogen molecules. We aimed to evaluate five polymorphisms in TLR-4, TLR-6, and TLR-9 genes and their association with cytokine levels and clinical parameters in malaria from the Brazil-French Guiana border. METHODS: A case-control study was conducted in Amapá, Brazil. P. vivax patients and individuals not infected were evaluated. Genotyping of five SNPs was carried out by qPCR. Circulating cytokines were measured by CBA. The MSP-119 IgG antibodies were performed by ELISA. RESULTS: An association between TLR4 A299G with parasitemia was observed. There was an increase for IFN-ɤ, TNF-ɑ, IL-6, and IL-10 in the TLR-4 A299G and T3911, TLR-6 S249P, and TLR-9 1486C/T, SNPs for the studied malarial groups. There were significant findings for the TLR-4 variants A299G and T3911, TLR-9 1237C/T, and 1486C/T. For the reactivity of MSP-119 antibodies levels, no significant results were found in malaria, and control groups. CONCLUSIONS: The profile of the immune response observed by polymorphisms in TLRs genes does not seem to be standard for all types of malaria infection around the world. This can depend on the human population and the species of Plasmodium.

The diversity of Plasmodium falciparum isolates from asymptomatic and symptomatic school-age children in Kinshasa Province, Democratic Republic of Congo.

BACKGROUND: Understanding Plasmodium falciparum population diversity and transmission dynamics provides information on the intensity of malaria transmission, which is needed for assessing malaria control interventions. This study aimed to determine P. falciparum allelic diversity and multiplicity of infection (MOI) among asymptomatic and symptomatic school-age children in Kinshasa Province, Democratic Republic of Congo (DRC). METHODS: A total of 438 DNA samples (248 asymptomatic and 190 symptomatic) were characterized by nested PCR and genotyping the polymorphic regions of pfmsp1 block 2 and pfmsp2 block 3. RESULTS: Nine allele types were observed in pfmsp1 block2. The K1-type allele was predominant with 78% (229/293) prevalence, followed by the MAD20-type allele (52%, 152/293) and RO33-type allele (44%, 129/293). Twelve alleles were detected in pfmsp2, and the 3D7-type allele was the most frequent with 84% (256/304) prevalence, followed by the FC27-type allele (66%, 201/304). Polyclonal infections were detected in 63% (95% CI 56, 69) of the samples, and the MOI (SD) was 1.99 (0.97) in P. falciparum single-species infections. MOIs significantly increased in P. falciparum isolates from symptomatic parasite carriers compared with asymptomatic carriers (2.24 versus 1.69, adjusted b: 0.36, (95% CI 0.01, 0.72), p = 0.046) and parasitaemia > 10,000 parasites/µL compared to parasitaemia < 5000 parasites/µL (2.68 versus 1.63, adjusted b: 0.89, (95% CI 0.46, 1.25), p < 0.001). CONCLUSION: This survey showed low allelic diversity and MOI of P. falciparum, which reflects a moderate intensity of malaria transmission in the study areas. MOIs were more likely to be common in symptomatic infections and increased with the parasitaemia level. Further studies in different transmission zones are needed to understand the epidemiology and parasite complexity in the DRC.

High prevalence of persistent residual parasitemia on days 3 and 14 after artemether-lumefantrine or pyronaridine-artesunate treatment of uncomplicated Plasmodium falciparum malaria in Nigeria.

BACKGROUND: Microscopic evaluation of parasite clearance is the gold standard in antimalarial drug efficacy trials. However, the presence of sub-microscopic residual parasitemia after artemisinin-based combination therapy (ACT) needs to be investigated. METHODS: One hundred and twenty (AL: n = 60, PA: n = 60) days 3 and 14 dried blood spots, negative by microscopy were analysed for residual parasitemia using nested PCR. Isolates with residual parasitemia on days 3 and 14 were further genotyped with their corresponding day-0 isolates using merozoite surface proteins msp-1, msp-2, and glurp genes for allelic similarity. RESULTS: Persistent PCR-determined sub-microscopic residual parasitemia at day 3 post ACT treatment was 83.3 (AL) and 88.3% (PA), respectively (ρ = 0.600), while 63.6 and 36.4% (ρ = 0.066) isolates were parasitemic at day 14 for AL and PA, respectively. Microscopy-confirmed gametocytemia persisted from days 0 to 7 and from days 0 to 21 for AL and PA. When the alleles of day 3 versus day 0 were compared according to base pair sizes, 59% of parasites shared identical alleles for glurp, 36% each for 3D7 and FC27, while K1 was 77%, RO33 64%, and MAD20 23%, respectively. Similarly, day 14 versus day 0 was 36% (glurp), 64% (3D7), and 32% (FC27), while 73% (K1), 77% (RO33), and 41% (MAD20), respectively. CONCLUSION: The occurrence of residual parasitemia on days 3 and 14 following AL or PA treatment may be attributable to the presence of either viable asexual, gametocytes, or dead parasite DNAs, which requires further investigation.

Low genetic diversity of Plasmodium falciparum merozoite surface protein 1 and 2 and multiplicity of infections in western Ethiopia following effective malaria interventions.

BACKGROUND: Genetic diversity of malaria parasites can inform the intensity of transmission and poses a major threat to malaria control and elimination interventions. Characterization of the genetic diversity would provide essential information about the ongoing control efforts. This study aimed to explore allelic polymorphism of merozoite surface protein 1 (msp1) and merozoite surface protein 2 (msp2) to determine the genetic diversity and multiplicity of Plasmodium falciparum infections circulating in high and low transmission sites in western Ethiopia. METHODS: Parasite genomic DNA was extracted from a total of 225 dried blood spots collected from confirmed uncomplicated P. falciparum malaria-infected patients in western Ethiopia. Of these, 72.4% (163/225) and 27.6% (62/225) of the samples were collected in high and low transmission areas, respectively. Polymorphic msp1 and msp2 genes were used to explore the genetic diversity and multiplicity of falciparum malaria infections. Genotyping of msp1 was successful in 86.5% (141/163) and 88.7% (55/62) samples collected from high and low transmission areas, respectively. Genotyping of msp2 was carried out among 85.3% (139/163) and 96.8% (60/62) of the samples collected in high and low transmission sites, respectively. Plasmodium falciparum msp1 and msp2 genes were amplified by nested PCR and the PCR products were analysed by QIAxcel ScreenGel Software. A P-value of less or equal to 0.05 was considered significant. RESULTS: High prevalence of falciparum malaria was identified in children less than 15 years as compared with those ≥ 15 years old (AOR = 2.438, P = 0.005). The three allelic families of msp1 (K1, MAD20, and RO33) and the two allelic families of msp2 (FC27 and 3D7), were observed in samples collected in high and low transmission areas. However, MAD 20 and FC 27 alleles were the predominant allelic families in both settings. Plasmodium falciparum isolates circulating in western Ethiopia had low genetic diversity and mean MOI. No difference in mean MOI between high transmission sites (mean MOI 1.104) compared with low transmission area (mean MOI 1.08) (p > 0.05). The expected heterozygosity of msp1 was slightly higher in isolates collected from high transmission sites (He = 0.17) than in those isolates from low transmission (He = 0.12). However, the heterozygosity of msp2 was not different in both settings (Pfmsp2: 0.04 in high transmission; pfmsp2: 0.03 in low transmission). CONCLUSION: Plasmodium falciparum from clinical malaria cases in western Ethiopia has low genetic diversity and multiplicity of infection irrespective of the intensity of transmission at the site of sampling. These may be signaling the effectiveness of malaria control strategies in Ethiopia; although further studies are required to determine how specific intervention strategies and other parameters that drive the pattern.

Identification of Babesia bovis MSA-1 functionally constraint regions capable of binding to bovine erythrocytes.

Merozoite surface antigen-1 is a glycoprotein expressed by Babesia bovis and is considered a vaccine candidate given that antibodies against it are able to partially block in vitro invasion of bovine erythrocytes. Despite this, no study to date has confirmed the target cell binding properties of the full MSA-1 or its fragments. This research has thus been focused on identifying protein regions playing a role in erythrocyte attachment, based on genetic diversity and natural selection analysis. Two regions under functional constraint (nucleotides 134-428 and 464-629) having a preponderance of negatively-selected signals were identified in silico. Three non-overlapping peptides derived from functionally constraint regions (42422 (39PEGSFYDDMSKFYGAVGSFD58), 42424 (91NALIKNNPMIRPDLFNATIV110) and 42426 (150TDIVEEDREKAVEYFKKHVY169)) were able to specifically bind to a sialoglycoprotein located on the bovine erythrocyte surface as confirmed by sensitive and specific peptide-cell interaction competition assays using both enzymatically treated and untreated red blood cells. Interestingly, it was predicted that peptides 42422 and 42426 have a helical structure and conserved motifs in all strain/isolates. These findings provide evidence, for the first time, related to B. bovis MSA-1 short regions used by the parasite in erythrocyte binding which could be predicted using natural selection analysis. Future work focused on evaluating these peptides' antigenic ability during natural infection, and their ability to induce protection in immunisation assays are needed to confirm their usefulness as synthetic vaccine candidates.

Temporal dynamics of Plasmodium falciparum population in Metehara, east-central Ethiopia.

BACKGROUND: Plasmodium falciparum is the most serious, genetically most complex and fastest-evolving malaria parasite. Information on genetic diversity of this parasite would guide policy decision and malaria elimination endeavors. This study explored the temporal dynamics of P. falciparum population in two time points in Metehara, east-central Ethiopia. METHODS: The participants were quantitative real-time polymerase chain reaction-confirmed patients who were recruited for uncomplicated falciparum malaria therapeutic efficacy test in 2015 and 2019. Dry blood spot samples were analysed by the nested PCR to genotype P. falciparum merozoite surface protein (msp1, msp2) and glutamate-rich protein (glurp) genes. RESULTS: While msp1, msp2 and glurp genotypes were successfully detected in 26(89.7%), 24(82.8%) and 14(48.3%) of 2015 samples (n = 29); the respective figures for 2019 (n = 41) were 31(68.3%), 39(95.1%), 25(61.0%). In 2015, the frequencies of K1, MAD20 and RO33 allelic families of msp1, and FC27 and IC/3D7 of msp2 were 19(73.1%), 8(30.6%), 14(53.8%), 21(87.5%), 12(50.5%); and in 2019 it was 15(48.4%), 19(61.3%), 15(48.4%), 30(76.9%), 27(69.2%) respectively. MAD20 has shown dominance over both K1 and RO33 in 2019 compared to the proportion in 2015. Similarly, although FC27 remained dominant, there was shifting trend in the frequency of IC/3D7 from 50.5% in 2015 to 69.2% in 2019. The multiplicity of infection (MOI) and expected heterozygosity index (He) in 2015 and 2019 were respectively [1.43 ± 0.84] and [1.15 ± 0.91], 0.3 and 0.03 for msp1. However, there was no significant association between MOI and age or parasitaemia in both time points. CONCLUSION: The lower genetic diversity in P. falciparum population in the two time points and overall declining trend as demonstrated by the lower MOI and He may suggest better progress in malaria control in Metehara. But, the driving force and selective advantage of switching to MAD20 dominance over the other two msp1 allelic families, and the dynamics within msp2 alleles needs further investigation.