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Background: Plasmodium falciparum erythrocyte binding antigen-175 (PfEBA-175) is a candidate antigen for a blood-stage malaria vaccine, while various polymorphisms in the PfEBA-175 gene among global P. falciparum populations have prevented the development of effective vaccines based on this gene. At the same time, the dimorphism of the F- and C-fragments associated with high endemic of severe malaria has been described. This study aimed to investigate the dimorphism of PfEBA-175 on both the Bioko island and continent of Equatorial Guinea, as well as the genetic polymorphism and natural selection of global PfEBA-175.Methods: A total of 218 blood samples were collected from patients with P. falciparum malaria on Bioko Island and Bata district in 2018 and 2019. The allelic dimorphism of PfEBA-175 region II was investigated by nested polymerase chain reaction and sequencing. Polymorphic characteristics and the effect of natural selection were analyzed using MEGA 7.0, DnaSP 6.0 and PopART programs. Genetic diversity in 312 global PfEBA-175 region II sequences was also analyzed. Protein function prediction of new amino acid mutation sites was performed using PolyPhen-2 and Foldx program.Results: Allelic dimorphism of PfEBA-175 was identified in the study area, and the frequency of the F-fragment was higher than that of the C-fragment in both Bioko Island and Bata district populations. Additionally, single infections (87.80%) were more frequent than mixed infections (12.20%). A total of 49 monoclonal PfEBA-175 region II sequences of Bioko Island and Bata district were sequenced successfully. PfEBA-175 of Bioko Island and Bata district isolates showed a high degree of genetic variability and heterogeneity, with π values of 0.00407 & 0.00411 and Hd values of 0.958 & 0.976 for nucleotide diversity, respectively. The values of Tajima's D of PfEBA-175 on Bata district and Bioko Island were 0.56395 and -0.27018, respectively. Globally, PfEBA-175 isolates from Asia were more diverse than those from Africa and South America, and genetic differentiation quantified by the fixation index between Asian and South American countries populations was significant (Fst>0.15, P1), indicating destabilization of the protein structure.Conclusions: This study proved the dimorphism of PfEBA-175, and also demonstrated that the F-fragment was remarkably predominant in the study area. The distribution patterns and genetic diversity of PfEBA-175 in Equatorial Guinea isolates were similar to those of isolates worldwide. High levels of recombination events were observed in PfEBA-175 isolates globally, suggesting that natural selection and intragenic recombination might be the main drivers of genetic diversity in global PfEBA-175. These results have important reference value for the development of blood-stage malaria vaccine based on this antigen.

Background Thrombospondin-related adhesive protein (TRAP) is a transmembrane protein that plays a crucial role during the invasion of Plasmodium falciparum into liver cells. As a potential malaria vaccine candidate, the genetic diversity and natural selection of PfTRAP was assessed and the global PfTRAP polymorphism pattern was described. Methods 153 blood spot samples from Bioko malaria patients were collected during 2016–2018 and the target TRAP gene was amplified. Together with the sequences from database, nucleotide diversity and natural selection analysis, and the structural prediction were preformed using bioinformatical tools. Results A total of 119 Bioko PfTRAP sequences were amplified successfully. On Bioko Island, PfTRAP shows its high degree of genetic diversity and heterogeneity, with π value for 0.01046 and Hd for 0.99. The value of dN–dS (6.2231, p PfTRAP on Bioko Island. Globally, the African PfTRAPs showed more diverse than the Asian ones, and significant genetic differentiation was discovered by the fixation index between African and Asian countries (Fst > 0.15, p 1) indicated a destabilization of protein structure. Conclusions Evidences in the present investigation supported that PfTRAP gene from Bioko Island and other malaria endemic countries is highly polymorphic (especially at T cell epitopes), which provided the genetic information background for developing an PfTRAP-based universal effective vaccine. Moreover, some mutations have been shown to be detrimental to the protein structure or function and deserve further study and continuous monitoring.

Background Plasmodium falciparum circumsporozoite protein (PfCSP) is a potential malaria vaccine candidate, but various polymorphisms of the pfcsp gene among global P. falciparum population become the major barrier to the effectiveness of vaccines. This study aimed to investigate the genetic polymorphisms and natural selection of pfcsp in Bioko and the comparison among global P. falciparum population. Methods From January 2011 to December 2018, 148 blood samples were collected from P. falciparum infected Bioko patients and 96 monoclonal sequences of them were successfully acquired and analysed with 2200 global pfcsp sequences mined from MalariaGEN Pf3k Database and NCBI. Results In Bioko, the N-terminus of pfcsp showed limited genetic variations and the numbers of repetitive sequences (NANP/NVDP) were mainly found as 40 (35%) and 41 (34%) in central region. Most polymorphic characters were found in Th2R/Th3R region, where natural selection (p > 0.05) and recombination occurred. The overall pattern of Bioko pfcsp gene had no obvious deviation from African mainland pfcsp (Fst = 0.00878, p pfcsp displayed the various mutation patterns and obvious geographic differentiation among populations from four continents (p pfcsp C-terminal sequences were clustered into 138 different haplotypes (H_1 to H_138). Only 3.35% of sequences matched 3D7 strain haplotype (H_1). Conclusions The genetic polymorphism phenomena of pfcsp were found universal in Bioko and global isolates and the majority mutations located at T cell epitopes. Global genetic polymorphism and geographical characteristics were recommended to be considered for future improvement of malaria vaccine design.

Background Glucose‐6‐phosphate dehydrogenase (G6PD) is an essential enzyme that protects red blood cells from oxidative damage. Although G6PD‐deficient alleles appear to confer a protective effect of malaria, the link with clinical protection against Plasmodium infection is conflicting. Methods A case–control study was conducted on Bioko Island, Equatorial Guinea and further genotyping analysis used to detect natural selection of the G6PD A‐ allele. Results Our results showed G6PD A‐ allele could significantly reduce the risk of Plasmodium falciparum infection in male individuals (adjusted odds ratio [AOR], 0.43; 95% confidence interval [CI], 0.20–0.93; p, In this work, we firstly analysed a large case–control study of 342 malaria cases and 1,287 health controls on Bioko Island, Equatorial Guinea. Our findings demonstrate G6PD A‐ allele could reduce the risk of Plasmodium falciparum infection in African population and indicate malaria has recent positive selection on G6PD A‐ allele.

BackgroundPlasmodium falciparumapical membrane antigen-1 (PfAMA-1) is a promising candidate antigen for a blood-stage malaria vaccine. However, antigenic variation and diversity ofPfAMA-1 are still major problems to design a universal malaria vaccine based on this antigen, especially against domain I (DI). Detail understanding of thePfAMA-1 gene polymorphism can provide useful information on this potential vaccine component. Here, general characteristics of genetic structure and the effect of natural selection of DIs among BiokoP. falciparumisolates were analysed.Methods214 blood samples were collected from Bioko Island patients withP. falciparummalaria between 2011 and 2017. A fragment spanning DI ofPfAMA-1 was amplified by nested polymerase chain reaction and sequenced. Polymorphic characteristics and the effect of natural selection were analysed using MEGA 5.0, DnaSP 6.0 and Popart programs. Genetic diversity in 576 globalPfAMA-1 DIs were also analysed. Protein function prediction of new amino acid mutation sites was performed using PolyPhen-2 program.Results131 different haplotypes ofPfAMA-1 were identified in 214 Bioko IslandP. falciparumisolates. Most amino acid changes identified on Bioko Island were found in C1L. 32 amino acid changes identified inPfAMA-1 sequences from Bioko Island were found in predicted RBC-binding sites, B cell epitopes or IUR regions. Overall patterns of amino acid changes of BiokoPfAMA-1 DIs were similar to those in globalPfAMA-1 isolates. Differential amino acid substitution frequencies were observed for samples from different geographical regions. Eight new amino acid changes of Bioko island isolates were also identified and their three-dimensional protein structural consequences were predicted. Evidence for natural selection and recombination event were observed in global isolates.ConclusionsPatterns of nucleotide diversity and amino acid polymorphisms of Bioko Island isolates were similar to those of globalPfAMA-1 DIs. Balancing natural selection across DIs might play a major role in generating genetic diversity in global isolates. Most amino acid changes in DIs occurred in predicted B-cell epitopes. Novel sites mapped on a three dimensional structure ofPfAMA-1 showed that these regions were located at the corner. These results may provide significant value in the design of a malaria vaccine based on this antigen.

Background Malaria is still a serious public health problem on Bioko Island (Equatorial Guinea), although the number of annual cases has been greatly reduced since 2004 through the Bioko Island Malaria Control Project (BIMCP). A better understanding of malaria parasite population diversity and transmission dynamics is critical for assessing the effectiveness of malaria control measures. The objective of this study is to investigate the genetic diversity of Plasmodium falciparum populations and multiplicity of infection (MOI) on Bioko Island 7 years after BIMCP. Methods A total of 181 patients with uncomplicated P. falciparum malaria diagnosed with microscopy were collected from Bioko Island from January 2011 to December 2014. Parasite DNA was extracted using chelex-100 and species were identified using a real-time PCR followed by high-resolution melting. Plasmodium falciparum msp1 and msp2 allelic families were determined using nested PCR. Results Three msp1 alleles (K1, MAD20, and RO33) and two msp2 alleles (FC27 and 3D7) were analysed in all samples. In msp1, the MAD20 allelic family was predominant with 96.69% (175/178) followed respectively by the K1 allelic family with 96.07% (171/178) and R033 allelic family with 70.78% (126/178). In msp2, the FC27 allelic family was the most frequently detected with 97.69% (169/173) compared to 3D7 with 72.25% (125/173). Twenty-six different alleles were observed in msp1 with 9 alleles for K1, 9 alleles for MAD20 and 8 alleles for R033. In msp2, 25 individual alleles were detected with 5 alleles for FC27 and 20 alleles for 3D7. The overall MOI was 5.51 with respectively 3.5 and 2.01 for msp1 and msp2. A significant increase in overall MOI was correlated with the age group of the patients (P = 0.026) or parasite densities (P = 0.04). Conclusions The present data showed high genetic diversity and MOI values among the P. falciparum population in the study, reflecting both the high endemic level and malaria transmission on Bioko Island. These data provide valuable information for surveillance of P. falciparum infection and for assessing the appropriateness of the current malarial control strategies in the endemic area.

Apolipoprotein E (APOE) gene polymorphism can affect APOE gene transcription, serum lipid levels and repair of tissue damage, which could place individuals at serious risk of cardiovascular disease or certain infectious diseases. Recently, high-resolution melting (HRM) analysis was reported to be a simple, inexpensive, accurate and sensitive method for the genotyping or/and scanning of rare mutations. For this reason, an HRM analysis was used in the present study for APOE genotyping in the Southern Chinese Han and African Fang populations. A total of 100 healthy Southern Chinese Han and 175 healthy African Fang individuals attended the study. Polymerase chain reaction-DNA sequencing was used as a reference method for the genotyping of these samples. The six APOE genotypes could all be rapidly and efficiently identified by HRM analysis, and 100% concordance was found between the HRM analysis and the reference method. The allele frequencies of APOE in the Southern Chinese Han population were 7.0, 87.5 and 5.5% for ɛ2, ɛ3 and ɛ4, respectively. In the African Fang population, the allele frequencies of APOE were 24.3, 65.7 and 10.0% for ɛ2, ɛ3 and ɛ4, respectively. A statistically significant difference was found between the allele frequencies between the populations (P

Background Antimalarial drug resistance is a primary public health problem. Haplotypes of pfcrt and pfmdr1 gene have been implicated to be molecular markers of chloroquine (CQ) resistance. This study aims to explore mutation distribution of Pfcrt and Pfmdr1 in Bioko Island, Equatorial Guinea (EG). Methods Blood samples were collected from different districts of Bioko. The single nucleotide polymorphisms in Pfcrt (codons 72 to 76) and Pfmdr1 (codons 86, 130, 184, 1034, 1042, 1109 and 1246) were assessed by nested PCR with DNA sequencing and haplotype prevalences were also determined. Results Analysis of Pfcrt and Pfmdr1 mutations was successful in 151 and 157 samples respectively out of the 172 samples taken for this study. The mutations of Pfcrt and Pfmdr1 were found in 98.67% and 89.81% isolates, respectively. The Pfcrt 74–76, Pfmdr1 86, and Pfmdr1 184 were 92.05%, 50.32%, and 87.26% found mostly of mutation type, respectively. Three haplotypes coding 72–76 of Pfcrt were found including CVMNK, CV IET , and CV M/I N/E K/T , which accounted for 1.33%, 92.05%, and 6.62%, respectively. No mutation in Pfmdr1-N1 codon at 130 and Pfmdr1-N2 (S1034C, N1042D, V1109I, and D1246Y) was detected. The types coding 86 and 184 in Pfmdr1 were found including NY, Y Y, N F , YF , N Y/F and YY/F , which accounted for 10.19%, 2.55%, 33.76%, 45.22%, 5.73% and 2.55%, respectively. Conclusion High prevalence of Pfcrt CV IET and Pfmdr1 86 Y , 184 F double mutations confirm high-level CQ resistance (CQR) and might suggest reduced susceptibility of Plasmodium falciparum isolates to AQ in Bioko, EG. It establishes fundamental data for detection of P. falciparum CQR with molecular markers and will promotes the surveillance level of drug resistance in Bioko, EG.

Objective With emergence and geographically expanding of antimalarial resistance worldwide, molecular markers are essential tool for surveillance of resistant Plasmodium parasites. Recently, single-nucleotide polymorphisms (SNPs) in the PF3D7_1343700 kelch propeller (K13-propeller) domain are shown to be associated with artemisinin (ART) resistance in vivo and in vitro. This study aims to investigate the ART resistance-associated polymorphisms of K13-propeller and PfATPase6 genes in Plasmodium falciparum isolates from Bioko Island, Equatorial Guinea (EG). Methods A total of 172 samples were collected from falciparum malaria patients on Bioko Island between 2013 and 2014. The polymorphisms of K13-propeller and PfATPase6 genes were analyzed by Nest-PCR and sequencing. Results Sequences of K13-propeller and PfATPase6 were obtained from 90.74% (98/108) and 91.45% (139/152) samples, respectively. The 2.04% (2/98) cases had non-synonymous K13-propeller A578S mutation but no found the mutations associated with ART resistance in Southeast Asia. For PfATPase6, the mutations were found at positions N569K and A630S with the mutation prevalence of 7.91% (11/139) and 1.44% (2/139), respectively. In addition, a sample with the mixed type at position I723V was discovered (0.72%, 1/139). Conclusions This study initially offers an insight of K13-propeller and PfATPase6 polymorphisms on Bioko Island, EG. It suggests no widespread ART resistance or tolerance in the region, and might be helpful for developing and updating guidance for the use of ART-based combination therapies (ACTs)., Graphical abstract, Highlights • The PfATPase6 mutations were found at positions N569K (7.91%), A630S (1.44%) and I723V (0.72%). • Only one non-synonymous K13-propeller mutation A578S (2.04%) was observed. • Limited SNPs of PfATPase6 and K13-propeller indicated no widespread ART on Bioko Island, EG.