Study area and population
Equatorial Guinea is located on the west coast of Central Africa at approximately 3° N latitude and 9° E longitude. It is bordered by Cameroon to the north and Gabon to the west and south. This country consists of two parts: a mainland and an insular region (Figure 1). Bioko is the largest island with a population of 335,048 inhabitants, which is located in the gulf of Guinea, 160 km northwest of the mainland region. Bata is the largest district in the mainland region, with a population of 244,264 inhabitants [21]. Despite the efforts made by the Equatorial Guinea Malaria Control Initiative (EGMCI, 2006-2011), and the Bioko Island Malaria Control Project (BIMCP, 2004-2018) also has had a marked impact on malaria transmission[22], malaria is still the major public health problem in the country (www.mcdinternational.org).
Ethical approval
Verbal informed consent was obtained from all participating subjects or their parents, and this study and the consent process were approved by the Ethics Committee of Malabo Regional Hospital and Chaozhou People’s Hospital.
Study population and blood sample collection
The study was carried out in Malabo Regional Hospital, private diagnostic clinics (PDCs) in different regions of Bioko Island and Bata district, and in a clinic of the Chinese medical aid team to the Republic of Equatorial Guinea. Plasmodium falciparum clinical samples (from individuals 4 months to 80 years of age) were collected from 297 confirmed P. falciparum malaria cases identified by microscopic examination and an immuno-colloidal gold test kit (ICT Diagnostics) in 2018–2019. Blood samples were collected on filter paper (Whatman 3 mm, GE Healthcare, Pittsburg, USA) for further molecular analysis, air-dried and stored in plastic sealing bags at ambient temperature. Thick blood smears were air dried and stained with 10% fresh Giemsa following standard procedures. After coding and recording the patient medical records, the dried blood filters were stored in plastic sealing bags at -80 °C.
DNA extraction
Genomic DNA (gDNA) was extracted from the dried blood spots using 0.5% saponin (Sigma-Aldrich, Taufkirchen, Germany) to free parasites from red blood cells followed by Chelex®100 (Bio-Rad L-aboratories, CA, USA) method as previously described [23] and stored at -20 °C.
Allelic genotyping of the PfEBA-175
An improved nested PCR method for genotype determination of PfEBA-175 as described by Touré [24] was used. In the first-round PCR, 1 μL of gDNA was amplified with 10 μL 2×Taq Plus master Mix II (GenStar, Beijing, China), 1 μL of 10 μmol/L forward primer (EBA1: 5’-CAAGAAGCAGTTCCTGAGGAA-3’) and 1 μL of 10 μmol/L reverse primer (EBA2: 5’-TCTCAACATTCATATTAACAATTC-3’), and sterile ultrapure water was added to a final volume of 20 μL. Thermal cycling parameters for PCR were as follows: initial denaturation at 96 °C for 3 min; 30 cycles of 96 °C for 10 s, 57 °C for 10 s, 72 °C for 50 s; and final extension of 72 °C for 10 min. For the second-round PCR, 1 μL of the primary PCR product was amplified with 10 μL 2×Taq Plus master Mix Ⅱ (GenStar, Beijing, China), 1 μL of 10 μmol/L forward primer (EBA3: 5’-GAGGAAAACACTGAAATAGCACAC-3’) and 1 μL of 10 μmol/L reverse primer (EBA4: 5’-CAATTCCTCC-AGACTGTTGAACAT-3’), and sterile ultrapure water was added to a final volume of 20 μL. The nested PCR cycling parameters used in the second round were the same as those used for the primary reaction. Nested PCRs were carried out using a LifeECO® PCR System 9700 (Bioer Technology, China). An allele-specific positive control and DNA negative control were included in each set of reactions.
Detection of alleles of PfEBA-175
The PCR products were stained with StarGreen nucleic acid gel stain (GenStar, Beijing, China) and resolved by gel electrophoresis in 2% agarose gel. The fragment sizes were determined using a low molecular weight DNA ladder marker (250-5000 bp, Dongsheng Biotech, Guangzhou, China) and photographed with a Tanon 2500/2500R Gel Imaging System (Tanon Science & Technology Co., Ltd., Shanghai, China). Alleles of PfEBA-175 were categorized according to their molecular weights.
Amplification and sequencing of domains II and III of PfEBA-175
For sequencing domains II and III of PfEBA-175 gene, the samples were amplified by nested PCR. In the first-round PCR, 1 μL of gDNA was amplified with 10 μL PrimeSTAR Max Premix (Takara Bio, Dalian, China), 1 μL of 10 μmol/L forward primer (EBA175F1: 5’-ATTAACGCTGTACGTGTGTCTAG-3’) and 1 μL of 10 μmol/L reverse primer (EBA2: 5’-TCTCAACATTCATATTAACAATTC-3’), 1 μL of DMSO and sterile ultrapure water was added to a final volume of 20 μL. The thermal cycling parameters for PCR were as follows: initial denaturation at 96 °C for 3 min; 30 cycles of 96 °C for 15 s, 57.7 °C for 10 s, 72 °C for 2 min; a final extension of 72 °C for 10 min. For the second-round PCR, 1 μL of the primary PCR product was amplified with 10 μL PrimeSTAR Max Premix (Takara Bioko, Beijing, China), 1 μL of 10 μmol/L forward primer (EBA175F2: 5’-AAGAAATACTTCATCTAATAACG-3’) and 1 μL of 10 μmol/L reverse primer (EBA4: 5’-CAATTCCTCCAGACTGTTGAACAT-3’), and sterile ultrapure water was added to a final volume of 20 μL. The nested PCR cycling parameters of the second round were the same as those of the primary reaction. Nested PCRs were carried out using a LifeECO® PCR System 9700 (Bioer Technology, China). Nested PCRs were carried out using a LifeECO® PCR System 9700 (Bioer Technology, China). All PCR products were analyzed using 1% agarose gel electrophoresis. Then, a random selection of 70 samples with suitable F type or C type PfEBA-175 amplicons (only single infection) were purified and then sequenced by using an ABI 3730×L automated sequencer (BGI, Shenzhen, China). The sequencing primers were the same as the primers for the second-round PCR. All sequences were analyzed and integrated by BioEdit and MEGA 7.0 software.
Sequence analysis of domain II of the PfEBA-175
The MEGA 7.0 was used for the analysis of the nucleotide sequence polymorphism, and the amino acid sequences of domain II of PfEBA-175 from Equatorial Guinea were also analyzed by this program [25]. The numbers of segregating sites (S), haplotypes (H), haplotype diversity (Hd), nucleotide diversity (π), and average number of pairwise nucleotide differences within a population (K) were estimated by using DnaSP 6.0 [26]. The value of π was calculated to estimate stepwise the diversity of domain II of PfEBA-175 based on a sliding window of 100 bp with a step size of 5 bp. Values of nonsynonymous (dN) and synonymous (dS) substitutions were estimated and compared using the Z test (P < 0.05 was considered significant) in the MEGA 7.0 program[25] based on the method of Nei and Gojobori [27] with Jukes and Cantor correction. Tajima’s D value [28] and Fu and Li’s D and F values [29] were analyzed using DnaSP 6.0 to evaluate the neutral theory of evolution [26]. Recombination parameters (R), which included the effective population size and probability of recombination between adjacent nucleotides per generation, and the minimum number of recombination events (Rm) were analyzed by DnaSP 6.0 [26]. Linkage disequilibrium (LD) between different polymorphic sites was computed based on the R2 index using DnaSP 6.0 [26].
Global sequences acquisition and global diversity analysis
Genetic diversity of PfEBA-175 domain II in other global P. falciparum isolates was analyzed. Parasite populations from Kenya (n = 39, DQ092087 - DQ092125), Thailand (Year = 2006, n = 48, DQ092039 - DQ092085), Thailand (Year = 2015, n = 33, LC008232 - LC008263), Benin (n = 8, KJ419497 - KJ419504), Camopi (n = 23, KJ419512 - KJ419608), Colombia (n = 20, KJ419512 - KJ419531), Madagascar (n = 7, KJ419506 - KJ419546), Maripasoula (n = 15, KJ419532 - KJ419546), Peru (n = 31, KJ419547 - KJ419576), Venezuela (n = 9, KJ419577 - KJ419585), and Nigeria (n = 30, AJ438799 - AJ438828) were included in this analysis. All publicly available sequences covered the domain II of PfEBA-175. Nucleotide sequence polymorphism analysis and neutrality test were performed for each population using programs DnaSP 6.0[26] and MEGA 7.0 [25] as described above. Genetic differentiation among parasite populations was calculated based on the fixation index (Fst) to estimate pairwise DNA sequence diversity between and within populations using Arlequin 3.5 [30]. To investigate relationships among PfEBA-175 haplotypes, the haplotype network for a total of 312 PfEBA-175 sequences, including 49 Bioko Island and Bata district sequences and the 263 publicly available sequences from Kenya, Thailand, Benin, Colombia, Madagascar, Maripasoula, Peru, Venezuela, and Nigeria, was constructed using the Median Joining algorithm of the PopART program [31]. Nucleotide diversity and natural selection of each region were analyzed using DnasP 6.0 as described above [26].
Prediction of the impact of amino acid change upon protein structure
The potential impact of amino acid substitutions on the structure or function was predicted by the PolyPhen-2 [32] online server and PROVEAN [33]. The FOLDX plugin [34] in YASARA [35] was used to predict the changes in free energy before and after the mutations: ΔΔG(change) = ΔG(mutation)–ΔG(wild-type). Generally, we considered ΔΔG (change) > 0 to indicate a destabilizing mutation and ΔΔG (change) < 0 to indicate a stabilizing mutation.
Statistical analysis
All statistical analyses were performed using the software Statistical Package for Social Sciences version 17.0 (SPSS, Inc., Chicago, IL, USA). The chi-square test was used in the univariate analysis to compare proportions. Statistical significance was set at alpha = 0.05 for all tests.