Merozoite Surface Protein Research Articles

A Molecular Analysis of Memory B Cell and Antibody Responses Against Plasmodium falciparum Merozoite Surface Protein 1 in Children and Adults From Uganda.

Memory B cells (MBCs) and plasma antibodies against Plasmodium falciparum (Pf) merozoite antigens are important components of the protective immune response against malaria. To gain understanding of how responses against Pf develop in these two arms of the humoral immune system, we evaluated MBC and antibody responses against the most abundant merozoite antigen, full-length Pf merozoite surface protein 1 (PfMSP1FL), in individuals from a region in Uganda with high Pf transmission. Our results showed that PfMSP1FL-specific B cells in adults with immunological protection against malaria were predominantly IgG+ classical MBCs, while children with incomplete protection mainly harbored IgM+ PfMSP1FL-specific classical MBCs. In contrast, anti-PfMSP1FL plasma IgM reactivity was minimal in both children and adults. Instead, both groups showed high plasma IgG reactivity against PfMSP1FL, with broadening of the response against non-3D7 strains in adults. The B cell receptors encoded by PfMSP1FL-specific IgG+ MBCs carried high levels of amino acid substitutions and recognized relatively conserved epitopes on the highly variable PfMSP1 protein. Proteomics analysis of PfMSP119-specific IgG in plasma of an adult revealed a limited repertoire of anti-MSP1 antibodies, most of which were IgG1 or IgG3. Similar to B cell receptors of PfMSP1FL-specific MBCs, anti-PfMSP119 IgGs had high levels of amino acid substitutions and their sequences were predominantly found in classical MBCs, not atypical MBCs. Collectively, these results showed evolution of the PfMSP1-specific humoral immune response with cumulative Pf exposure, with a shift from IgM+ to IgG+ B cell memory, diversification of B cells from germline, and stronger recognition of PfMSP1 variants by the plasma IgG repertoire.

Genetic Diversity of Plasmodium vivax Merozoite Surface Protein-3 Alpha and Beta from Diverse Geographic Areas of Thailand.

Malaria is parasitic disease cause by Plasmodium infection. In Thailand, coinfections of Plasmodium vivax and Plasmodium falciparum are commonly found. P. vivax infection has been increasing in the past decade. The objective of this study was to investigate the genetic diversity patterns of P. vivax merozoite surface protein 3 (PvMSP-3) genes in a total of 450 isolates collected from Thai-neighboring border areas during two different periods (2009-2014 and 2015-2016) using polymerase chain reaction (PCR)-restriction fragment length polymorphism method. Three major types of PvMSP-3α (A, B, and C) and PvMSP-3β (A, B, and C) were detected based on PCR product size. In total, 45 and 23 alleles of PvMSP-3α and 41 and 30 alleles of PvMSP-3β with different frequencies of samples were distinguished from the first and second period, respectively. Our results strongly indicate genetic diversity patterns of PvMSP-3 in isolates from the second period, especially in samples from the Thai-Myanmar border area. These two polymorphic genes could be used as molecular epidemiologic markers for genotyping P. vivax isolate in Thailand.

Prevalence of malaria among febrile patients and assessment of efficacy of artemether-lumefantrine and artesunate-amodiaquine for uncomplicated malaria in Dolisie, Republic of the Congo

BackgroundIn the Republic of the Congo, malaria represents a major public health problem affecting all age groups. A regular surveillance of the current efficacy of first-line anti-malarial drugs is required in the face of possible emergence and spread of artemisinin-resistant Plasmodium falciparum strains in Africa. The purpose of this study was to determine the prevalence of malaria among febrile patients of all ages and assess the efficacy of artemether-lumefantrine (AL) and artesunate-amodiaquine (ASAQ) in Congolese children.MethodsFebrile patients of all ages were initially screened for malaria by both rapid diagnostic test (RDT) and microscopy. Patients less than 12 years of age, with parasitaemia ≥ 1000 asexual parasites of P. falciparum/µL of blood, without any signs of severity, were enrolled in a therapeutic efficacy study and treated after obtaining their parents' (or legal guardian’s) informed consent in two health centres in Dolisie. The patients were followed for 28 days in accordance with the 2009 World Health Organization standard protocol. If parasitaemia reappeared on or after day 7, the genetic profiles (genes expressing merozoite surface protein-1 [msp1], merozoite surface protein-2 [msp2], and glutamine-rich protein [glurp]) of pre-treatment and post-treatment isolates were compared by nested polymerase chain reaction (PCR) followed by capillary electrophoresis to make a distinction between recrudescence and re-infection. The clinical and parasitological outcome was analysed by the per-protocol method and Kaplan–Meier survival curves.ResultsA total of 994 febrile patients of all ages were screened by RDT and microscopy. Of 994 patients, 323 (32.5%) presented a positive RDT, and 266 (26.8%) were microscopy-positive. Based on microscopy as the reference diagnostic method, the sensitivity and the specificity of the RDT were 98.9 and 91.8%, respectively. The Cohen’s kappa coefficient was 0.86. A total of 121 children aged less than 12 years (61 in AL treatment group and 60 in ASAQ treatment group) were included in therapeutic efficacy study. Before PCR correction, the proportions of adequate clinical and parasitological response were 96.6% for AL and 86.0% for ASAQ in the per-protocol population (P < 0.05). The PCR-corrected efficacy rates were 98.2% and 94.2% for AL and ASAQ, respectively (P > 0.05). Both treatments were well tolerated.ConclusionsAL and ASAQ remain highly effective for the first-line treatment of uncomplicated P. falciparum malaria in Dolisie. Despite high efficacy of first- and second-line treatment, there is a continuing need to scale up effective malaria preventive interventions and vector control strategies in the country.Trial Registration Number: ACTRN12616001422415.

Studying the disease severity in clinical isolates of Plasmodiumvivax

Among the human malaria Plasmodium species, Plasmodium vivax is the most widespread species globally. In recent times, this historically benign species is now being recognized as also responsible for severe malaria infections in humans. Hence, a deeper insight of P.vivax immunopathogenesis in clinical patients is essential for malaria control and elimination strategies. Certain genes like vir genes, merozoite surface protein 3α genes (msp3α) and biomarkers like super oxide dismutase (SOD-1), tumor necrosis factor (TNF- α), interleukin (IL-10) are speculated to have some role in disease severity and thus can be useful as diagnostic markers. In the reported study, the clinical samples of P.vivax were genotyped for msp3α gene and cytokine analysis, expression profiling of vir genes were also carried out in these samples. A total of 84 P.vivax samples were collected (39 severe and 45 non-severe samples) and no correlation of parasitemia with severity of disease was seen in these samples (p-value = 0.38). On analysis four genotypes of msp3α were found, with type B (1.5 kb) as the predominant genotype. Cytokine analysis revealed SOD-1 and TNF-α levels to be significantly more in the severe group than in non-severe group, whereas for IL-10 no significant difference was observed between two clinical groups. The vir gene profiling revealed increased level of expression for vir-12, vir-14 related, and vir-17 like in severe group and vir-10 related gene expression was more in non-severe samples. There are multiple factors that bring phenotypic and genotypic changes in P.vivax malaria and thus, it is important to assess the potential diagnostic markers for detection of disease severity. In future, studies with more number of clinical samples should be undertaken for better insight of P.vivax disease severity.

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

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

Genetic Polymorphism of Merozoite Surface Protein-2 (MSP-2) in Plasmodium falciparum Isolates from Endemic Sub-Counties, A Case Study in Kisii County, Kenya

Background: Despite a lot of efforts being put in place globally to reduce malaria burden, malaria is still one of the major public health problem. P. falciparum analysis of the Msp2 gene gives paramount evidence in the evaluation of malaria epidemiology to assist in the ongoing malaria control and elimination programs. Therefore this study aimed at examining genetic polymorphism of msp-2 gene of P. falciparum and its multiplicity of infection (MOI) from endemic Sub-Counties in Kisii County, Kenya.&#x0D; Methodology: Dried blood spots were collected from 249 study participants who were positive for P. falciparum parasite in endemic Sub – Counties in Kisii County from January to April 2021. Parasites density/μl of blood was calculated by multiplying the number of parasites counted and 8000 white cells/μl then dividing by number of white cells counted. Parasite level was considered low if the parasite/μl of blood was &lt; 500, moderate 501 ≤ 5000 and high if it was &gt; 5000 Parasites DNA was extracted using chelex-100® method and nested PCR for amplification followed by fragment analysis using gel electrophoresis.&#x0D; Results: 246 samples were identified with msp2 alleles where 94% belonged to 3D7 and 83% belonged to FC27. 40 different polymorphic alleles were identified where 13 belonged to FC27 and 27 belonging to 3D7. 86% isolates had multiple msp2 alleles where the Multiplicity of infection (MOI) overall mean was 2.75.&#x0D; Conclusion: The study isolates showed high genetic polymorphism. Therefore, this suggests that there is high local malaria transmission, meaning there is failure to the scale up of malaria control interventions in the endemic areas of the County.

Genetic diversity and allelic frequency of antigenic markers in Plasmodium falciparum isolates from Nnewi district in Nigeria.

The genetic diversity of Plasmodium falciparum poses a threat to the development and implementation of malaria control strategies. Thus, there is a need for continuous surveillance of its genetic diversity, especially amongst the parasite's reservoir's asymptomatic population. Three cohorts comprising children under ten years old, pregnant women and other adults were recruited into this study. Blood sample was collected from all consenting individuals and screened by the polymerase chain reaction (PCR) method. The genetic diversity of P. falciparum was determined by genotyping the merozoite surface protein-1 (msp-1), merozoite surface protein-2 (msp-2) and glutamate-rich protein (glurp). The size of alleles was visualized on the agarose gel. The multiplicity of infection (MOI) and expected heterozygosity (He) were determined. The majority of the patients showed polyclonal infections, while the multiplicity of infection with msp-2 and glurp of isolates from pregnant women were 2.5 and 1.8, respectively. Children and adults were 2.3 and 1.1; 2.4 and 1.3, respectively. The estimated number of genotypes was 10 msp-1 (4 KI; 4 MAD; 2 RO33), 27 msp-2 (14 FC27; 13 IC/3D7) and 8 glurp. K1 (36/100) was more frequent than the MAD20 (22.33/100) allele, which was, in turn, more frequent than the RO33 (13.59/100). The samples with the 3D7 allele (53.40/100) of msp-2 occurred more frequently than the FC27 type (45.63/100). Polymorphism in the glurp gene occurred most frequently (72.82/100). The study samples exhibited a high degree of genetic polymorphism in msp-2 allele typing with multiple clones, reflecting the complexity of parasite populations.

Prefoldin subunit 6 of Plasmodium falciparum binds merozoite surface protein-1.

Malaria is a human disease caused by eukaryotic protozoan parasites of the Plasmodium genus. Plasmodium falciparum (Pf) causes the most lethal form of human malaria and is responsible for widespread mortality worldwide. Prefoldin is a heterohexameric molecular complex that binds and delivers unfolded proteins to chaperonin for correct folding. The prefoldin PFD6 is predicted to interact with merozoite surface protein-1 (MSP-1), a protein well known to play a pivotal role in erythrocyte binding and invasion by Plasmodium merozoites. We previously found that the P. falciparum (Pf) genome contains six prefoldin genes and a prefoldin-like gene whose molecular functions are unidentified. Here, we analyzed the expression of PfPFD-6 during the asexual blood stages of the parasite and investigated its interacting partners. PfPFD-6 was found to be significantly expressed at the trophozoite and schizont stages. Pull-down assays suggest PfPFD-6 interacts with MSP-1. In silico analysis suggested critical residues involved in the PfPFD-6-MSP-1 interaction. Our data suggest PfPFD-6 may play a role in stabilizing or trafficking MSP-1.

Investigation of liposomal self-adjuvanting peptide epitopes derived from conserved blood-stage Plasmodium antigens.

Malaria is a vector born parasitic disease causing millions of deaths every year. Despite the high mortality rate, an effective vaccine against this mosquito-borne infectious disease is yet to be developed. Up to date, RTS,S/AS01 is the only vaccine available for malaria prevention; however, its efficacy is low. Among a variety of malaria antigens, merozoite surface protein-1(MSP-1) and ring-infected erythrocyte surface antigen (RESA) have been proposed as promising candidates for malaria vaccine development. We developed peptide-based Plasmodium falciparum vaccine candidates that incorporated three previously reported conserved epitopes from MSP-1 and RESA into highly effective liposomal polyleucine delivery system. Indeed, MSP-1 and RESA-derived epitopes conjugated to polyleucine and formulated into liposomes induced higher epitope specific antibody titres. However, immunized mice failed to demonstrate protection in a rodent malaria challenge study with Plasmodium yoelii. In addition, we found that the three reported P. falciparum epitopes did not to share conformational properties and high sequence similarity with P. yoelii MSP-1 and RESA proteins, despite the epitopes were reported to protect mice against P. yoelii challenge.

Quantification of the dynamics of antibody response to malaria to inform sero-surveillance in pregnant women

BackgroundMalaria remains a major public health threat and tools sensitive to detect infections in low malaria transmission areas are needed to progress elimination efforts. Pregnant women are particularly vulnerable to malaria infections. Throughout pregnancy they access routine antenatal care, presenting a unique sentinel population to apply novel sero-surveillance tools to measure malaria transmission. The aim of this study was to quantify the dynamic antibody responses to multiple antigens during pregnancy so as to identify a single or multiple antibody response of exposure to malaria in pregnancy.MethodsThis study involved a secondary analysis of antibody responses to six parasite antigens [five commonly studied merozoite antigens and the variant surface antigen 2-chondroitin sulphate A (VAR2CSA), a pregnancy-specific erythrocytic antigen] measured by enzyme-linked immunosorbent assay (ELISA) over the gestation period until delivery (median of 7 measurements/woman) in 250 pregnant women who attended antenatal clinics located at the Thai-Myanmar border. A multivariate mixture linear mixed model was used to cluster the pregnant women into groups that have similar longitudinal antibody responses to all six antigens over the gestational period using a Bayesian approach. The variable-specific entropy was calculated to identify the antibody responses that have the highest influence on the classification of the women into clusters, and subsequent agreement with grouping of women based on exposure to malaria during pregnancy.ResultsOf the 250 pregnant women, 135 had a Plasmodium infection detected by light microscopy during pregnancy (39% Plasmodium falciparum only, 33% Plasmodium vivax only and 28% mixed/other species), defined as cases. The antibody responses to all six antigens accurately identified the women who did not have a malaria infection detected during pregnancy (93%, 107/115 controls). Antibody responses to P. falciparum merozoite surface protein 3 (PfMSP3) and P. vivax apical membrane antigen 1 (PvAMA1) were the least dynamic. Antibody responses to the antigens P. falciparum apical membrane antigen 1 (PfAMA1) and PfVAR2CSA were able to identify the majority of the cases more accurately (63%, 85/135).ConclusionThese findings suggest that the combination of antibodies, PfAMA1 and PfVAR2CSA, may be useful for sero-surveillance of malaria infections in pregnant women, particularly in low malaria transmission settings. Further investigation of other antibody markers is warranted considering these antibodies combined only detected 63% of the malaria infections during pregnancy.

Surveillance of Plasmodium vivax transmission using serological models in the border areas of China\u2013Myanmar

BackgroundTo understand the Plasmodium vivax malaria transmission intensity and to assess the effectiveness of prevention and control measures taken along the China–Myanmar border, a catalytic model was used to calculate the seroconversion rate, an important indicator of malaria transmission intensity with high sensitivity, which is particularly useful in areas of low transmission.MethodsFive counties in Yunnan Province bordering Myanmar were selected as survey sites, and subjects were obtained in each county by stratified random sampling in 2013–2014. Fingerstick blood was collected from each subject and tested for antibodies to P. vivax Merozoite Surface Protein 1-19 (PvMSP1-19) using indirect ELISA. A catalytic conversion model was used to assess the transmission intensity of P. vivax malaria based on the maximum likelihood of generating a community seroconversion rate.ResultsA total of 3064 valid blood samples were collected. Antibody levels were positively correlated with age. The seroconversion rate (SCR) values for each village were Luoping (0.0054), Jingqiao (0.0061), Longpen (0.0087), Eluo (0.0079), Banwang (0.0042) and Banbie (0.0046), respectively.ConclusionOverall, the intensity of P. vivax malaria transmission in the border areas of Yunnan Province is low and not entirely consistent across counties. Consecutive serological surveys are needed to provide a sensitive evaluation of transmission dynamics and can help to confirm areas where infection is no longer present.

Multi-Dose Priming Regimens of PfSPZ Vaccine: Safety and Efficacy against Controlled Human Malaria Infection in Equatoguinean Adults.

ABSTRACT.Plasmodium falciparum sporozoite (PfSPZ) Vaccine is composed of radiation-attenuated, aseptic, purified cryopreserved PfSPZ. Multiple clinical trials empirically assessing two to six doses have shown multi-dose priming (two to four doses the first week) to be optimal for protection in both 4- and 16-week regimens. In this randomized, double-blind, normal saline (NS) placebo-controlled trial, four groups (G) of 18- to 32-year-old Equatoguineans received multi-dose priming regimens with or without a delayed final dose at 4 or 16 weeks. The regimens were G1: days 1, 3, 5, 7, and 113; G2: days 1, 3, 5, and 7; G3: days 1, 3, 5, 7, and 29; and G4: days 1, 8, and 29. All doses were 9 × 105 PfSPZ. Tolerability, safety, immunogenicity, and vaccine efficacy (VE) against homologous controlled human malaria infection (CHMI) 6–7 weeks after vaccination were assessed to down-select the best regimen. All four regimens were safe and well tolerated, with no significant differences in adverse events (AEs) between vaccinees (N = 84) and NS controls (N = 20) or between regimens. Out of 19 controls, 13 developed Pf parasitemia by quantitative polymerase chain reaction (qPCR) after CHMI. Only the vaccine regimen administered on study days 1, 8, and 29 gave significant protection (7/21 vaccinees versus 13/19 controls infected, VE 51.3%, P = 0.03, Barnard’s test, two-tailed). There were no significant differences in antibodies against Pf circumsporozoite protein (PfCSP), a major SPZ antigen, between protected and nonprotected vaccinees or controls pre-CHMI. The six controls not developing Pf parasitemia had significantly higher antibodies to blood stage antigens Pf exported protein 1 (PfEXP1) and Pf merozoite surface protein 1 (PfMSP1) than the controls who developed parasitemia, suggesting naturally acquired immunity against Pf limited infections in controls. This study identified a safe, protective, 4-week, multi-dose prime vaccination regimen for assessment in future trials of PfSPZ Vaccine.

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

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

Genetic Diversity and Molecular Surveillance of Antimalarial Drug Resistance of Plasmodium falciparum among Hospitals Patients in Benue State Nigeria

Introduction: Malaria is a febrile illness caused by parasites of the genus Plasmodium and transmitted by female Anopheles mosquitoes. The genetic diversity and antimalarial drug resistance of Plasmodium falciparum are some of the major challenges of malaria control programme in Nigeria.&#x0D; Aim: This study was aimed at determining the genetic diversity, and molecular surveillance of antimalarial drug resistance among patients attending Government hospitals in Benue State, Nigeria.&#x0D; Methodology: Plasmodium falciparum deoxyribonucleic acid was extracted from dried blood spots of 60 positive malariacases among the patients. The diversity of Plasmodium falciparum was done by genotyping 3D7 and FC27 families of merozoite surface protein- 2 alleles. The Plasmodium falciparum multidrug resistance 1 and Plasmodium falciparum kelch13 genes of Plasmodium falciparum were also amplified and assessed by restriction fragment length polymorphism (RFLP) to survey molecular resistance to antimalarial drugs.&#x0D; Results: The results showed that the frequency of 3D7 allele 37(61.7%) was higher than FC27 allele 18(30.0%). The frequency of merozoite surface protein- 2 infections with both allelic types was 5(8.3%). There was a significant difference in the distribution of the merozoite surface protein two alleles (χ2=25.9,df=2 P&lt;.0.001). Both the Plasmodium falciparum multidrug resistance 1 Asparagine 86Tyrosine (N86Y) and Aspartic acid 1246Tyrosine (D1246Y), had 100 % mutant while the 100% while the Plasmodium falciparum kelch13 G449A had 100% wild type allele.&#x0D; Conclusion: The current study underscores the need for frequent monitoring of indicators of antimalaria drug resistance in Nigeria.

Plasmodium falciparum merozoite surface protein 3 as a vaccine candidate: a brief review

ABSTRACTDespite the many efforts of researchers around the world, there is currently no effective vaccine for malaria. Numerous studies have been developed to find vaccine antigens that are immunogenic and safe. Among antigen candidates, Plasmodium falciparum merozoite surface protein 3 (MSP3) has stood out in a number of these studies for its ability to induce a consistent and protective immune response, also being safe for use in humans. This review presents the main studies that explored MSP3 as a vaccine candidate over the last few decades. MSP3 formulations were tested in animals and humans and the most advanced candidate formulations are MSP3-LSP, a combination of MSP3 and LSP1, and GMZ2 (a vaccine based on the recombinant protein fusion GLURP and MSP3) which is currently being tested in phase II clinical studies. This brief review highlights the history and the main formulations of MSP3-based vaccines approaches against P. falciparum .

Plasmodium falciparum Merozoite Surface Protein 2 Genetic Diversity and Multiplicity of Infection in Asymptomatic Malaria Cases in Lagos, Nigeria

Background: The burden of malaria due to Plasmodium falciparum is high in Nigeria. Continuous surveillance of the genetic diversity of malaria parasites can provide important information on transmission intensity in an area. Objective: This study investigated the genetic diversity in the highly polymorphic merozoite surface protein 2 (msp2) gene of P. falciparum in in asymptomatc malaria cases. Methods: A total of 59 P. falciparum-positive DNA samples collected from asymptomatic cases in Igbogbo-Bayekun, Lagos, were analyzed targeting block 3 of msp2 gene. Primers were specific for the two allelic families of msp2 block 3 (FC27 and 3D7). Results: Overall, the frequencies of 3D7 and FC27 alleles were 71.2% (42/59) and 67.8% (40/59) respectively. Twenty-three (39%) of the samples had polyinfections (3D7+FC27), while monoinfections with 3D7 and FC27 were observed in 19 (32%) and 17 (29%) samples respectively. Similar frequencies of 3D7 19/25(76%) and FC27 18/25(72%) were observed in samples with submicroscopic parasitaemia. Twenty-seven distinct msp2 genotypes were identified: 14 3D7 (range 175–480 bp) and 13 FC27 (range 125–500bp). Overall, there were more monoclonal infections 35(59.3%) than polyclonal infections 24 (40.7%). The overall mean multiplicity of infection (MOI) was 1.44 while the expected heterozygosity (He) was 0.51. The means of MOI were similar when compared on the basis of malaria status (microscopic or submicroscopic parasitemia) (P=0.166), gender (P=0.057) or age (P=0.866). Also, there was no association between polyclonal infection and submicroscopic parasitaemia (P=0.129). Conclusion: The genetic diversity of falciparum infections in the study area is high while moderate multiplicity of infection was observed. The genetic diversity and MOI of msp2 gene were similar in submicroscopic and microscopic malaria parasitaemia.

Plasmodium falciparum msp1 and msp2 genetic diversity in parasites isolated from symptomatic and asymptomatic malaria subjects in the South of Benin.

Symptomatic and asymptomatic malaria patients are considered as the reservoirs of human Plasmodium. In the present study, we have evaluated the Plasmodium falciparum merozoite surface protein-1 (Pfmsp1) and protein-2 (Pfmsp2) genetic diversity among the symptomatic and asymptomatic malaria infection from health facilities in Cotonou, Benin Republic. A cross-sectional study recruited 158 individuals, including 77 from the asymptomatic and 81 from the symptomatic groups. The parasites were genotyped using Nested Polymerase Chain Reaction. Samples identified as Plasmodium falciparum were genotyped for their genetic diversity. No significant difference was observed in the overall multiplicity of infection (MOI) between the asymptomatic and symptomatic groups. In the symptomatic group, the overall frequency of K1, MAD20, and RO33 allelic family was more predominant (98.5%) followed by 3D7 (87.3%) and FC27 (83.1%). However, in asymptomatic group, the K1 alleles were the most prevalent (100%) followed by FC27 (89.9%), 3D7 (76.8%), MAD20 (60.5%), and RO33 (35.5%). The frequency of multiple allelic types (K1+MAD20+RO33) at the Pfmsp1 loci in the symptomatic infections was significantly higher when compared to that of the asymptomatic ones (97% vs. 34%, p < 0.05), whereas no difference was observed in the frequency of multiple allelic types (3D7 and FC27) at the Pfmsp2 loci between the two groups. The high presence of msp1 multiple infections in the symptomatic group compared to asymptomatic ones suggests an association between the genetic diversity and the onset of malaria symptoms. These data can provide valuable information in the development of a vaccine that could reduce the symptomatic disease.

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

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

Effects of SPAG6 silencing and decitabine treatment on apoptosis and phosphatase and tensin homolog methylation in SKM-1 cells

目的探讨SPAG6基因沉默和地西他滨在体内外对SKM-1细胞凋亡和PTEN启动子甲基化的作用。方法慢病毒载体转染SKM-1细胞沉默SPAG6基因的表达，地西他滨处理细胞后CCK-8检测细胞存活率，流式细胞术检测细胞凋亡，Western blot和甲基化特异性PCR检测PTEN的蛋白表达和甲基化情况，并构建非肥胖糖尿病/重度联合免疫缺陷（NOD/SCID）小鼠异体移植瘤模型，TUNEL法观察肿瘤组织凋亡，免疫组化检测PTEN在组织中表达情况。结果慢病毒转染后SPAG6干扰组SPAG6基因被成功沉默；CCK-8检测结果提示地西他滨处理能够降低SKM-1细胞的存活率，同时流式细胞术检测显示地西他滨处理组的细胞凋亡率［（17.35±3.37）％］高于未处理组［（5.09±2.06）％］，且SPAG6基因沉默联合地西他滨处理组的凋亡率最高［（36.34±4.00）％］；地西他滨处理后DNMT1的表达降低而PTEN表达升高，同时PTEN启动子甲基化程度降低，而经地西他滨处理后的SPAG6干扰组的PTEN去甲基化引起的蛋白表达升高效果最明显；NOD/SCID小鼠异体瘤移植模型中地西他滨组的肿瘤体积明显小于生理盐水组，而地西他滨处理SPAG6干扰组的小鼠肿瘤体积最小，且经TUNEL检测发现其凋亡程度最高（阳性比值为3.57±0.48）。结论SPAG6基因沉默在SKM-1细胞中能增强地西他滨诱导的凋亡和PTEN去甲基化作用，并且在NOD/SCID异体瘤移植模型小鼠中能够增强地西他滨的抗肿瘤特性。

Genetic population of Plasmodium knowlesi during pre-malaria elimination in Thailand

BackgroundThailand is committed to eliminating malaria by 2024. From 2013 to 2020, the total number of malaria cases have decreased, from 37,741 to 4474 (an 88.1% reduction). However, infections with Plasmodium knowlesi, a monkey malarial pathogen that can also infect humans, have been increasingly observed. This study focused on the molecular analysis of P. knowlesi parasites causing malaria in Thailand.MethodsUnder Thailand’s integrated Drug Efficacy Surveillance (iDES), which includes drug-resistance monitoring as part of routine case-based surveillance and responses, specimens were collected from malaria patients (n = 966) between 2018 and 2020. Thirty-one mono P. knowlesi infections (3.1%), most of which were from eastern and southern Thailand, were observed and confirmed by nested PCR assay and DNA sequencing. To evaluate whether these pathogens were from different lineages, cluster analysis based on seven microsatellite genotyping markers and the merozoite surface protein 1 (pkmsp1) gene was carried out. The P. knowlesi pyrimethamine resistance gene dihydrofolate reductase (pkdhfr) was sequenced and homology modelling was constructed.ResultsThe results of analysing the seven microsatellite markers and pkmsp1 sequence demonstrated that P. knowlesi parasites from eastern Thailand were of the same lineage as those isolated in Cambodia, while the parasites causing malaria in southern Thailand were the same lineage as those isolated from Malaysia. The sequencing results for the pkdhfr genes indicated the presence of two mutations, Arg34Leu and a deletion at position 105. On analysis with homology modelling, the two mutations were not associated with anti-malarial drug resistance.ConclusionsThis report compared the genetic populations of P. knowlesi parasites in Thailand from 2018 to 2020 and have shown similar lineages as those isolated in Cambodia and Malaysia of P. knowlesi infection in Thailand and demonstrated that the P. knowlesi parasites were of the same lineages as those isolated in Cambodia and Malaysia. The parasites were also shown to be sensitive to pyrimethamine.