Spatial cluster analysis of Plasmodium vivax and P. malariae exposure using serological data among Haitian school children sampled between 2014 and 2016

Background Estimation of malaria prevalence in very low transmission settings is difficult by even the most advanced diagnostic tests. Antibodies against malaria antigens provide an indicator of active or past exposure to these parasites. The prominent malaria species within Haiti is Plasmodium falciparum, but P. vivax and P. malariae infections are also known to be endemic. Methodology/Principal findings From 2014–2016, 28,681 Haitian children were enrolled in school-based serosurveys and were asked to provide a blood sample for detection of antibodies against multiple infectious diseases. IgG against the P. falciparum, P. vivax, and P. malariae merozoite surface protein 19kD subunit (MSP119) antigens was detected by a multiplex bead assay (MBA). A subset of samples was also tested for Plasmodium DNA by PCR assays, and for Plasmodium antigens by a multiplex antigen detection assay. Geospatial clustering of high seroprevalence areas for P. vivax and P. malariae antigens was assessed by both Ripley’s K-function and Kulldorff’s spatial scan statistic. Of 21,719 children enrolled in 680 schools in Haiti who provided samples to assay for IgG against PmMSP119, 278 (1.27%) were seropositive. Of 24,559 children enrolled in 788 schools providing samples for PvMSP119 serology, 113 (0.46%) were seropositive. Two significant clusters of seropositivity were identified throughout the country for P. malariae exposure, and two identified for P. vivax. No samples were found to be positive for Plasmodium DNA or antigens. Conclusions/Significance From school-based surveys conducted from 2014 to 2016, very few Haitian children had evidence of exposure to P. vivax or P. malariae, with no children testing positive for active infection. Spatial scan statistics identified non-overlapping areas of the country with higher seroprevalence for these two malarias. Serological data provides useful information of exposure to very low endemic malaria species in a population that is unlikely to present to clinics with symptomatic infections.

Protein interaction analysis of Plasmodium falciparum circumsporozoite protein variants with human immunoproteins explains RTS,S vaccine efficacy in Ghana

The world's first malaria vaccine RTS,S provides only partial protection against Plasmodium falciparum infections. The explanation for such low efficacy is unclear. This study examined the associations of parasite genetic variations with binding affinity to human immunological proteins including human leukocyte antigen (HLA) and T cell receptors (TCR) involved in RTS,S-induced immune responses. Multiplicity of infections was determined by amplicon deep sequencing of merozoite surface protein 1 (PfMSP1). Genetic variations in the C-terminal of circumsporozoite protein (PfMSP1) gene were examined across 88 samples of P. falciparum collected from high and low transmission settings of Ghana. Binding interactions of PfMSP1 variants and HLA/TCR were analyzed using NetChop} and HADDOCK predictions. Anti-CSP IgG levels were measured by ELISA in a subset of 10 samples. High polyclonality was detected among P. falciparum infections. A total 27 CSP haplotypes were detected among samples. A significant correlation was detected between the CSP and MSP multiplicity of infection (MOI). No clear clustering of haplotypes was observed by geographic regions. The number of genetic differences in PfCSP between 3D7 and non-3D7 variants does not influence binding interactions to HLA/T cells nor anti-CSP IgG levels. Nevertheless, PfCSP peptide length significantly affects its molecular weight and binding affinity to the HLA. The presence of multiple non-3D7 strains among P. falciparum infections in Ghana impact the effectiveness of RTS,S. Longer PfCSP peptides may elicit a stronger immune response and should be considered in future version RTS,S. The molecular mechanisms of RTS,S cell-mediated immune responses related to longer CSP peptides warrants further investigations.

THE USE OF ARCHIVED GIEMSA-STAINED BLOOD SMEARS AND RDT FOR PCR-BASED GENOTYPING OF Plasmodium vivax MEROZOITE SURFACE PROTEIN-1 IN CENTRAL KALIMANTAN PROVINCE, INDONESIA: PCR-Based Genotyping of Pvmsp-1 in Central Kalimantan Province, Indonesia

Background: Plasmodium vivax is transmitted most across the country of Indonesia. The country has set out a malaria elimination program by 2030. The information on genetic diversity of malarial parasites relates to malaria transmission in an endemic area may provide the information that can help the malaria control program to achieve the target. Therefore, the purpose of this study was to determine the genetic diversity of the Pvmsp-1 gene in Central Kalimantan Province. Materials and Methods: Samples were 140 of archived Giemsa-stained blood smear and rapid detection test. Samples were divided into the indigenous and migrant populations. After confirmation by single-step PCR, only P. vivax and mixed infection samples were amplified to nested PCR for genotyping of Pvmsp-1 allelic variation in segments F1, F2, and F3. Results: Genotyping of 23 PCR positive samples resulted in 13 genotypes. In segment F1, three allelic variants type A containing subtype A1 (1,050 bp), A2 (350 bp), A3 (150 bp), and type B (100 bp). In segment F2, mono genotypes were detected as variant type A (1,050 bp) and type B3 (150 bp), multiple genotypes were detected as type B containing subtype B1 (250 bp), B2 (200 bp), and B3 (150bp). In segment F3, three allelic variants generated from four mono genotypes were type A (350 bp), type B (300 bp), and two type C (250 bp). Conclusion: The low allelic variation of Pvmsp-1 gene may reflect the actual situation of the low malaria endemic status of the study sites.

An Evolution From a Predominant K1 Allelic Variant to MAD20 of msp1 Gene Between 2015 to 2019 in Metehara, Southeast Ethiopia

Background: The complexity and quantity of parasite populations circulating in a specific location are reflected in the genetic diversity of malaria parasites (s). Between 2015 and 2019, this study in Metehara, South east, Ethiopia. set out to investigate the temporal dynamics of genetic diversity and multiplicity as a result of evolutionary change in the genes that contribute to Plasmodium falciparum infection elimination. Method: Between 2015 and 2019, a cross-sectional study was carried out. from eighty-three dry blood spots from malaria patients who were screened for P. falciparum mono-infection by QPCR. From this seventy confirmed P. falciparum were genotyping to merozoite surface protein 1,2 and glutamate-rich protein using nested PCR.Result: Between 2015 and 2019, seventy (84.3%) of the isolates were successfully genotyped for all three target genes in both years. In 2015 and 2019, the allelic distributions of the three genes differed significantly (P= 0.001). Overall, the most common allelic families for msp1 and msp2 were K1 and FC27 respectively. For glurp, eight distinct genotypes were identified. In 2015, the genotyping of msp1, msp2 and glurp was 25 (86.2%), 25 (86.2%) and 24 (82.2%) respectively. K1, MAD20 and RO33 all have 19(65.5%), 3(10.3%) and 3(10.3%) msp1 allelic families respectively. In 2019 the genes were 30 (73.2%), 39 (95.1%) and 30 (73.2%). K1, MAD20, and RO33 were genotyped for 6 (14.6 percent), 18 (43.9 percent) and 6 (14.6 percent) genotyping respectively. Over all the multiplicity of infection was 1.67 (95 percent CI 1.54-1.74) and the heterozygosity index for msp1, msp2, and glurp was 0.48, 0.70, and 0.55 respectively.Conclusion: This study provides current information on the genetic diversity of P. falciparum populations in Metehara over five-year intervals, The progression of the dominant K1 variant from 2015 to MAD20 variant in 2019 was observed in this study.

Serology and Amplicon Deep Sequencing of MSP1-Block 2 Haplotypes as New Tools for Plasmodium Vivax Infections

Background: Relapses of Plasmodium vivax (P. vivax) infections are major causes of malaria morbidity, and tools for distinguishing relapses from reinfections are needed in malaria endemic areas. Methods: Herein, a panel of plasmas of 72 P. vivax-infected pregnant women, of whom 31 had had at least a recurrence of P. vivax infection, was used in a serology for IgM and IgG against 6 P. vivax-merozoite surface protein-1 (P. vivax-MSP1-Block 2) haplotype-specific peptides, in order to identify re-expositions to same haplotypes in the recurrences during the pregnancy. In parallel, we used the amplicon deep sequencing (ADS) with P. vivax-MSP1-Block 2 amplicons of the in eight blood samples of non-pregnant P. vivax-infected patients to identify multi or monoclonal infections based on MSP1-Block-2 haplotypes, and to quantify the reads of different haplotypes between those with multiclonal infections. We synthetized a new panel of overlapping peptides mapping each one of the six P. vivax-MSP1-Block 2 haplotypes and we validated with new IgM and IgG serology. Results: Most pregnant women presented IgM that recognized more than one peptide, thus indicating multiple infections by P. vivax-MSP1-Block 2 haplotypes. The same IgM anti-peptides remained in several women in the recurrent episodes most likely indicates re-exposure to the same haplotype of MSP1 Block 2. The IgG reactivity the IgM to IgG switch were low. The ADS using next-generation sequencing (NGS) identified multi- and monoinfection by P. vivax-MSP1-Block 2 haplotypes. Of eight patients, two of them had had the first P. vivax-infection. Monoinfections with P. vivax-MSP1-Block 2 haplotypes were observed in two prime-infected patients and three of patients with previous malaria. In all P. vivax-MSP1-Block 2 haplotype-monoinfected patients, the reactivity of IgM was observed only against overlapped peptides of the same haplotype detected in ADS, while for IgG, no reactivity was observed for any of the peptides of the same haplotype or the others.We were able to identify multiclonal infections through three haplotypes of P. vivax MSP1 Block 2 in three remaining patients, among which, there was always one majority haplotype that predominated with more of 95% of high-quality reads. The levels of haplotype-specific IgM in the serology correlated with the read ratios of each haplotype, but IgG levels not, including in one of the multiclonal infections, a minority haplotype was recognized with higher levels of IgG than that of the majority one. Conclusion: Our findings suggest that the combination of ADS and serology for P. vivax-MSP1-Block 2 haplotypes may be used as a new tool for distinguishing reinfections from relapses in malaria.

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

Background Thailand 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. Methods Under 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. Results The 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. Conclusions This 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.

Genetic diversity and immunogenicity of the merozoite surface protein 1 C-terminal 19-kDa fragment of Plasmodium ovale imported from Africa into China

Background Merozoite surface protein 1 (MSP1) plays an essential role in erythrocyte invasion by malaria parasites. The C-terminal 19-kDa region of MSP1 has long been considered one of the major candidate antigens for a malaria blood-stage vaccine against Plasmodium falciparum. However, there is limited information on the C-terminal 19-kDa region of Plasmodium ovale MSP1 (PoMSP119). This study aims to analyze the genetic diversity and immunogenicity of PoMSP119. Methods A total of 37 clinical Plasmodium ovale isolates including Plasmodium ovale curtisi and Plasmodium ovale wallikeri imported from Africa into China and collected during the period 2012–2016 were used. Genomic DNA was used to amplify P. ovale curtisi (poc) msp119 (pocmsp119) and P. ovale wallikeri (pow) msp119 (powmsp119) genes by polymerase chain reaction. The genetic diversity of pomsp119 was analyzed using the GeneDoc version 6 programs. Recombinant PoMSP119 (rPoMSP119)-glutathione S-transferase (GST) proteins were expressed in an Escherichia coli expression system and analyzed by western blot. Immune responses in BALB/c mice immunized with rPoMSP119-GST were determined using enzyme-linked immunosorbent assay. In addition, antigen-specific T cell responses were assessed by lymphocyte proliferation assays. A total of 49 serum samples from healthy individuals and individuals infected with P. ovale were used for the evaluation of natural immune responses by using protein microarrays. Results Sequences of pomsp119 were found to be thoroughly conserved in all the clinical isolates. rPoMSP119 proteins were efficiently expressed and purified as ~ 37-kDa proteins. High antibody responses in mice immunized with rPoMSP119-GST were observed. rPoMSP119-GST induced high avidity indexes, with an average of 92.57% and 85.32% for rPocMSP119 and rPowMSP119, respectively. Cross-reactivity between rPocMSP119 and rPowMSP119 was observed. Cellular immune responses to rPocMSP119 (69.51%) and rPowMSP119 (52.17%) induced in rPocMSP119- and rPowMSP119-immunized mice were found in the splenocyte proliferation assays. The sensitivity and specificity of rPoMSP119-GST proteins for the detection of natural immune responses in patients infected with P. ovale were 89.96% and 75%, respectively. Conclusions This study revealed highly conserved gene sequences of pomsp119. In addition, naturally acquired humoral immune responses against rPoMSP1 were observed in P. ovale infections, and high immunogenicity of rPoMSP119 in mice was also identified. These instructive findings should encourage further testing of PoMSP119 for rational vaccine design. Graphical abstract

Genetic Diversity of Polymorphic Marker Merozoite Surface Protein 1 (Msp-1) and 2 (Msp-2) Genes of Plasmodium falciparum Isolates From Malaria Endemic Region of Pakistan

Background: Understanding the genetic diversity of Plasmodium species through polymorphic studies can assist in designing more effective control strategies of malaria like new drug formulation and development of a vaccine. Pakistan is moderate endemic for Plasmodium falciparum, but little is known about the genetic diversity of this parasite. This study aimed to investigate the molecular diversity of P. falciparum based on msp-1 and msp-2 genes in the malaria-endemic regions of Khyber Pakhtunkhwa, Pakistan.Methods: A total of 199/723 blood samples, tested positive by microscopy for falciparum malaria, were collected from four districts (Dera Ismail Khan, Karak, Mardan, and Peshawar) of Khyber Pakhtunkhwa. Nested PCR amplification technique was employed to target block 2 of msp-1 and the central domain of msp-2 genes, including their respective allelic families K1, MAD20, RO33, FC27, and 3D7/IC, and to detect the extent of genetic diversity of P. falciparum clinical isolates.Results: Among the 199 microscopy-positive P. falciparum samples, a total of 192 were confirmed using PCR. Ninety-seven amplicons were observed for msp-1 and 95 for msp-2. A total of 33 genotypes, 17 for msp-1 (eight K1, six MAD20, and three RO33) and 16 for msp-2 (nine FC27 and seven 3D7/IC), were identified. The specific allelic frequency of the K1 family was higher (44.3%) than that of MAD20 (33.0%) and RO33 (23.0%) for msp-1, while the FC27 allelic family was dominant (60.0%) compared with 3D7/IC (40.0%) for msp-2. No polyclonal infection was observed in msp-1 and msp-2. The expected heterozygosity was 0.98 and 0.97 for msp-1 and msp-2, respectively.Conclusion: It was concluded that the P. falciparum populations are highly polymorphic, and diverse allelic variants of msp-1 and msp-2 are present in Khyber Pakhtunkhwa, Pakistan.

A comparative 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 merozoite antigens are important components of the protective immune response against malaria. To gain understanding of how responses against P. falciparum develop in these two arms of the humoral immune system, we evaluated MBC and antibody responses against the most abundant merozoite antigen, merozoite surface protein 1 (MSP1), in individuals from a region in Uganda with high P. falciparum transmission. Our results showed that MSP1-specific B cells in adults with immunological protection against malaria were predominantly IgG+ classical MBCs, while children with incomplete protection mainly harbored IgM+ MSP1-specific classical MBCs. In contrast, anti-MSP1 plasma IgM reactivity was minimal in both children and adults. Instead, both groups showed high plasma IgG reactivity against MSP1 and whole merozoites, with broadening of the response against non-3D7 strains in adults. The antibodies encoded by MSP1-specific IgG+ MBCs carried high levels of amino acid substitutions and recognized relatively conserved epitopes on the highly variable MSP1 protein. Proteomics analysis of MSP119-specific IgG in plasma of an adult revealed a limited repertoire of anti-MSP1 antibodies, most of which were IgG1 or IgG3. Similar to MSP1-specific MBCs, anti-MSP1 IgGs had relatively 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 MSP1-specific humoral immune response with cumulative P. falciparum exposure, with a shift from IgM+ to IgG+ B cell memory, diversification of B cells from germline, and stronger recognition of MSP1 variants by the plasma IgG repertoire.