Genetic polymorphism of the N-terminal region in circumsporozoite surface protein of Plasmodium falciparum field isolates from Sudan

Abstract Background Malaria caused by Plasmodium falciparum parasite is still known to be one of the most significant public health problems in sub-Saharan Africa. Genetic diversity of the Sudanese P. falciparum based on the diversity in the circumsporozoite surface protein (PfCSP) has not been previously studied. Therefore, this study aimed to investigate the genetic diversity of the N-terminal region of the pfcsp gene. Methods A cross-sectional molecular study was conducted; 50 blood samples have been analysed from different regions in Sudan. Patients were recruited from the health facilities of Khartoum, New Halfa, Red Sea, White Nile, Al Qadarif, Gezira, River Nile, and Ad Damazin during malaria transmission seasons between June to October and December to February 2017–2018. Microscopic and nested PCR was performed for detection of P. falciparum. Merozoite surface protein-1 was performed to differentiate single and multiple clonal infections. The N-terminal of the pfcsp gene has been sequenced using PCR-Sanger dideoxy method and analysed to sequences polymorphism including the numbers of haplotypes (H), segregating sites (S), haplotypes diversity (Hd) and the average number of nucleotide differences between two sequences (Pi) were obtained using the software DnaSP v5.10. As well as neutrality testing, Tajima’s D test, Fu and Li’s D and F statistics. Results PCR amplification resulted in 1200 bp of the pfcsp gene. Only 21 PCR products were successfully sequenced while 29 were presenting multiple clonal P. falciparum parasite were not sequenced. The analysis of the N-terminal region of the PfCSP amino acids sequence compared to the reference strains showed five different haplotypes. H1 consisted of 3D7, NF54, HB3 and 13 isolates of the Sudanese pfcsp. H2 comprised of 7G8, Dd2, MAD20, RO33, Wellcome strain, and 5 isolates of the Sudanese pfcsp. H3, H4, and H5 were found in 3 distinct isolates. Hd was 0.594 ± 0.065, and S was 12. The most common polymorphic site was A98G; other sites were D82Y, N83H, N83M, K85L, L86F, R87L, R87F, and A98S. Fu and Li’s D* test value was − 2.70818, Fu and Li’s F* test value was − 2.83907, indicating a role of negative balancing selection in the pfcsp N-terminal region. Analysis with the global pfcsp N-terminal regions showed the presence of 13 haplotypes. Haplotypes frequencies were 79.4%, 17.0%, 1.6% and 1.0% for H1, H2, H3 and H4, respectively. Remaining haplotypes frequency was 0.1% for each. Hd was 0.340 ± 0.017 with a Pi of 0.00485, S was 18 sites, and Pi was 0.00030. Amino acid polymorphisms identified in the N-terminal region of global pfcsp were present at eight positions (D82Y, N83H/M, K85L/T/N, L86F, R87L/F, A98G/V/S, D99G, and G100D). Conclusions Sudanese pfcsp N-terminal region was well-conserved with only a few polymorphic sites. Geographical distribution of genetic diversity showed high similarity to the African isolates, and this will help and contribute in the deployment of RTS,S, a PfCSP-based vaccine, in Sudan.

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Genetic Diversity of Merozoite Surface Protein 2 (MSP2) Plasmodium falciparum Clinical Isolate in Wamena General Hospital

JURNAL BIOLOGI PAPUA ◽

10.31957/jbp.1319 ◽

2020 ◽

Vol 12 (2) ◽

pp. 123-132

Author(s):

Dirk Y.P. Runtuboi ◽

Rosye H.R. Tanjung ◽

Yulius Sarungu ◽

Meidy J. Imbiri ◽

Irma A. Resmol ◽

...

Keyword(s):

Genetic Diversity ◽

Plasmodium Falciparum ◽

General Hospital ◽

Surface Protein ◽

Merozoite Surface Protein ◽

Dominant Frequency ◽

Asexual Parasite ◽

Cross Sectional ◽

Single Allele ◽

Msp2 Gene

The genetic diversity of typical clinical isolated Plasmodium falciparum in the malaria population varies greatly, especially at the location where malaria disease were recorded at high incidence rate. MSP2 is known as glycoprotein expressed on the surface of merozoites, which is an antigenic protein and has a potential to act as vaccine candidate for malaria. The MSP2 gene has two main allelic groups called FC27 and 3D7/IC. Block 3 from MSP2 gene is the most polymorphic to describe the diversity of parasite populations. The P. falciparum parasite population is often characterized by wide genetic diversity in areas of high transmission intensity. Therefore, the study on P. falciparum diversity is useful to describe the level of malaria transmission. The study of genetic diversity focused on clinical isolated species at Wamena General Hospital was aimed to determine the presence of the MSP2 gene, variety of MSP2 gene allele and the dominant frequency of the MSP2 gene allele. This research has been carried out from March 2018 to February 2019 using a cross sectional approach. The research sample was taken and prepared from Wamena Regional Hospital and followed by the analyzing of DNA isolation, PCR, electrophoresis of the research samples was done at the genetic science laboratory in Jakarta, Indonesia. The samples studied were patients who met the inclusion criteria, namely a single P. falciparum infection with an asexual parasite density >1000 parasites/µl or >3+ (1-10 P/Lp), and were agreed to become respondents by signing an informed consent. A total of 26 clinical isolates of P. falciparum were isolated with the MSP2 gene distribution on the FC27 allele with the highest as many as 25 samples (96.2%), 22 samples (84.6%) of the 3D7 / IC allele while the mixture of the two alleles was 22 samples (84.6%). From a total of 26 samples, there were samples with the male gender category counted for 77.3% and female 41%. The results of the identification of clinical isolated P. falciparum at Wamena Hospital with a total of 26 samples were found in productive age, between 15-34 years with a single allele (95.8%), while 23 cases and mix (both alleles 87.5%) about 21 cases, meanwhile in cases of before-productive age, in which ages were 12 and 14 years of age with a single allele 100% (FC27) 2 cases and 50% (3D7/IC) found to be 1 case, The mixture of the two alleles is 50% was only 1 case and there was no sample at non-productive age observed. Key words: Malaria; MSP-2; P. falciparum; Wamena

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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

Frontiers in Genetics ◽

10.3389/fgene.2021.751552 ◽

2021 ◽

Vol 12 ◽

Author(s):

Shahid Niaz Khan ◽

Rehman Ali ◽

Sanaullah Khan ◽

Muhammad Rooman ◽

Sadia Norin ◽

...

Keyword(s):

Genetic Diversity ◽

Plasmodium Falciparum ◽

Pcr Amplification ◽

Surface Protein ◽

Polymorphic Marker ◽

Merozoite Surface Protein ◽

Drug Formulation ◽

Effective Control ◽

Endemic Region ◽

Khyber Pakhtunkhwa

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.

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Sequence Studies on the COOH-Terminal Region of the Merozoite Surface Protein-1 in Field Samples of Plasmodium falciparum from Diverse Geographic Areas

Annals of the New York Academy of Sciences ◽

10.1111/j.1749-6632.1996.tb52979.x ◽

1996 ◽

Vol 797 (1 Microbial Pat) ◽

pp. 282-284

Author(s):

YANG KANG ◽

CAROLE A. LONG

Keyword(s):

Plasmodium Falciparum ◽

Surface Protein ◽

Merozoite Surface Protein ◽

Terminal Region ◽

Merozoite Surface Protein 1 ◽

Field Samples

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Genetic Diversity in Merozoite Surface Protein.1 of Plasmodium falciparum in Highlands: Wamena Papua Indonesia

American Journal of Infectious Diseases ◽

10.3844/ajidsp.2018.106.109 ◽

2018 ◽

Vol 14 (4) ◽

pp. 106-109

Author(s):

Rosye Hefmi Rechnelty Tanjung ◽

Yulius Sarungu ◽

Meidy Johana Imbiri ◽

Ade Irma Resmol ◽

Dirk Yanes Persius Runtuboi ◽

...

Keyword(s):

Genetic Diversity ◽

Plasmodium Falciparum ◽

Surface Protein ◽

Merozoite Surface Protein ◽

Merozoite Surface Protein 1

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Comparative Immunogenicities of Full-Length Plasmodium falciparum Merozoite Surface Protein 3 and a 24-Kilodalton N-Terminal Fragment

Clinical and Vaccine Immunology ◽

10.1128/cvi.00064-11 ◽

2011 ◽

Vol 18 (8) ◽

pp. 1221-1228 ◽

Cited By ~ 6

Author(s):

Maryam Imam ◽

Yengkhom Sangeeta Devi ◽

Akhilesh K. Verma ◽

Virander Singh Chauhan

Keyword(s):

Plasmodium Falciparum ◽

Surface Protein ◽

Merozoite Surface Protein ◽

Terminal Region ◽

Content Type ◽

Conserved Domain ◽

Terminal Fragment ◽

Falciparum Merozoite ◽

Form Part ◽

Parasite Lysate

ABSTRACTRecombinantPlasmodium falciparummerozoite surface protein 3 (PfMSP3F) and a 24-kDa fragment from its N terminus (MSP3N) that includes the essential conserved domain, which elicits the maximum antibody (Ab)-dependent cellular inhibition (ADCI), were expressed as soluble proteins inEscherichia coli. Both proteins were found to be stable in both soluble and lyophilized forms. Immunization with MSP3F and MSP3N formulated separately with two human-compatible adjuvants, aluminum hydroxide (Alhydrogel) and Montanide ISA 720, produced significant antibody responses in mice and rabbits. Polyclonal Abs against both antigens recognized native MSP3 in the parasite lysate. These two Abs also recognized two synthetic peptides, previously characterized to possess B cell epitopes from the N-terminal region. Antibody depletion assay showed that most of the IgG response is directed toward the N-terminal region of the full protein. Anti-MSP3F and anti-MSP3N rabbit antibodies did not inhibit merozoite invasion or intraerythrocytic development but significantly reduced parasitemia in the presence of human monocytes. The ADCI demonstrated by anti-MSP3N antibodies was comparable to that exhibited by anti-MSP3F antibodies (both generated in rabbit). These results suggest that the N-terminal fragment of MSP3 can be considered a vaccine candidate that can form part of a multigenic vaccine against malaria.

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Molecular epidemiology of Plasmodium falciparum by multiplexed amplicon deep sequencing in Senegal

Malaria Journal ◽

10.1186/s12936-020-03471-7 ◽

2020 ◽

Vol 19 (1) ◽

Author(s):

Tolla Ndiaye ◽

Mouhamad Sy ◽

Amy Gaye ◽

Katherine J. Siddle ◽

Daniel J. Park ◽

...

Keyword(s):

Genetic Diversity ◽

Plasmodium Falciparum ◽

Molecular Epidemiology ◽

Deep Sequencing ◽

Haplotype Diversity ◽

Surface Protein ◽

Merozoite Surface Protein ◽

The South ◽

The North ◽

Significant Difference

Abstract Background Molecular epidemiology can provide important information regarding the genetic diversity and transmission of Plasmodium falciparum, which can assist in designing and monitoring elimination efforts. However, malaria molecular epidemiology including understanding the genetic diversity of the parasite and performing molecular surveillance of transmission has been poorly documented in Senegal. Next Generation Sequencing (NGS) offers a practical, fast and high-throughput approach to understand malaria population genetics. This study aims to unravel the population structure of P. falciparum and to estimate the allelic diversity, multiplicity of infection (MOI), and evolutionary patterns of the malaria parasite using the NGS platform. Methods Multiplex amplicon deep sequencing of merozoite surface protein 1 (PfMSP1) and merozoite surface protein 2 (PfMSP2) in fifty-three P. falciparum isolates from two epidemiologically different areas in the South and North of Senegal, was carried out. Results A total of 76 Pfmsp1 and 116 Pfmsp2 clones were identified and 135 different alleles were found, 56 and 79 belonged to the pfmsp1 and pfmsp2 genes, respectively. K1 and IC3D7 allelic families were most predominant in both sites. The local haplotype diversity (Hd) and nucleotide diversity (π) were higher in the South than in the North for both genes. For pfmsp1, a high positive Tajima’s D (TD) value was observed in the South (D = 2.0453) while negative TD value was recorded in the North (D = − 1.46045) and F-Statistic (Fst) was 0.19505. For pfmsp2, non-directional selection was found with a highly positive TD test in both areas and Fst was 0.02111. The mean MOI for both genes was 3.07 and 1.76 for the South and the North, respectively, with a statistically significant difference between areas (p = 0.001). Conclusion This study revealed a high genetic diversity of pfmsp1 and pfmsp2 genes and low genetic differentiation in P. falciparum population in Senegal. The MOI means were significantly different between the Southern and Northern areas. Findings also showed that multiplexed amplicon deep sequencing is a useful technique to investigate genetic diversity and molecular epidemiology of P. falciparum infections.

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