Document Type : Original Article


1 Zoology Department, Faculty of Science, Gombe State University, Gombe, Gombe State

2 Zoology Department, Faculty of Life Sciences, Modibbo Adama University, Yola, ‎Adamawa State

3 ‎Department of Biomedical and Pharmaceutical Technology, Federal Polytechnic, Mubi, ‎Adamawa Sate, Nigeria

4 ‎Biology Department, Faculty of Science, Federal University Kashere, Gombe State


Background: Malaria Chemoprevention depends on synthetic drugs, but the parasite is continuously developing resistance to the antimalarial armament, hence a consequential need for surveillance studies on the sensitivity of the drugs is felt. Therefore, the aim of this paper was to determine the presence of biomarkers associated with drug sensitivity in DHFR and DHPS gene of Plasmodium falciparum.
Methods: 200 blood samples were collected using vein puncture technique and they were analysed using Microscopy, RDT and PCR. DNA was extracted using Quick-DNA™ Miniprep extraction kit. Purity and concentration of the DNA were determined using Nanodrop Spectrophotometer. 57 samples were selected for molecular analysis. Nested PCR was used to amplify PFDHFR and PFDHPS genes; all PCR reactions were carried out in 25 µL reaction mixture (5 µL DNA template, 1 µL Primer, 6.5µldistilled water and 12.5 µL Master mix). The PCR products were subjected to electrophoresis using 2% agarose gel. The amplicons were purified, sequenced and subjected to BLAST software.
Results: Mutations were recorded from A16V 05(8.77%), N51I 18(31.58%), C59R 03(5.26%), I164L 12(21.05) variants of DHFR gene, while in DHPS gene, mutations were recorded from K540E 6(10.52%) variant.
Conclusion: Basic Biomarkers of resistance in DHFR and DHPS gene were recorded from Gombe. 

Graphical Abstract

Determining Molecular Markers Associated with Drug ‎Resistance in DHFR and DHPS genes of Plasmodium ‎Falciparum from Gombe L.G.A. Gombe State, Nigeria


Main Subjects

1. Wang S, Xu S, Geng J, Si Y, Zhao H, Li X, Yang Q, Zeng W, Xiang Z, Chen X. (2020). ‎Molecular Surveillance and in vitro Drug Sensitivity Study of Plasmodium falciparum ‎Isolates from the China–Myanmar Border. The American Journal of Tropical Medicine and Hygiene, 103(3): 1100. [Crossref], [Google Scholar], [Publisher]
‎2. Bazie V B, Ouattara A K, Sagna T, Compaore T R, Soubeiga S T, Sorgho P A, Yonli A T, ‎Simpore J. (2020). Resistance of Plasmodium falciparum to Sulfadoxine-Pyrimethamine ‎‎(Dhfr and Dhps) and Artemisinin and Its Derivatives (K13): A Major Challenge for ‎Malaria Elimination in West Africa. Journal of Biosciences and Medicines, 8(02): 82. [Crossref], [Google Scholar], [Publisher]
‎3. Garrido-Cardenas J A, González-Cerón L, Manzano-Agugliaro F, Mesa-Valle C. (2019). ‎Plasmodium genomics: an approach for learning about and ending human malaria. ‎Parasitology Research, 118(1): 1-27. [Crossref], [Google Scholar], [Publisher]
‎4. Mathieu L C, Cox H, Early A M, Mok S, Lazrek Y, Paquet J-C, Ade M-P, Lucchi N W, Grant ‎Q, Udhayakumar V. (2020). Local emergence in Amazonia of Plasmodium falciparum ‎k13 C580Y mutants associated with in vitro artemisinin resistance. Elife, 9: e51015. [Crossref], [Google Scholar], [Publisher]
‎5. Madkhali A M, Al-Mekhlafi H M, Atroosh W M, Ghzwani A H, Zain K A, Abdulhaq A A, ‎Ghailan K Y, Anwar A A, Eisa Z M. (2020). Increased prevalence of pfdhfr and pfdhps ‎mutations associated with sulfadoxine–pyrimethamine resistance in Plasmodium ‎falciparum isolates from Jazan Region, Southwestern Saudi Arabia: important ‎implications for malaria treatment policy. Malaria journal, 19(1): 1-11. [Crossref], [Google Scholar], [Publisher]
‎6. Tuedom A G B, Sarah-Matio E M, Moukoko C E E, Feufack-Donfack B L, Maffo C N, ‎Bayibeki A N, Awono-Ambene H P, Ayong L, Berry A, Abate L. (2021). Antimalarial drug ‎resistance in the Central and Adamawa regions of Cameroon: Prevalence of mutations ‎in P. falciparum crt, Pfmdr1, Pfdhfr and Pfdhps genes. PloS one, 16(8): e0256343. [Crossref], [Google Scholar], [Publisher]
‎7. Todd A, Akhter N, Cairns J-M, Kasim A, Walton N, Ellison A, Chazot P, Eldabe S, Bambra ‎C. (2018). The pain divide: a cross-sectional analysis of chronic pain prevalence, pain ‎intensity and opioid utilisation in England. BMJ open, 8(7): e023391. [Crossref], [Google Scholar], [Publisher]
‎8. Nsanzabana C, Ariey F, Beck H-P, Ding X C, Kamau E, Krishna S, Legrand E, Lucchi N, ‎Miotto O, Nag S. (2018). Molecular assays for antimalarial drug resistance surveillance: ‎a target product profile. PloS one, 13(9): e0204347. [Crossref], [Google Scholar], [Publisher]
‎9. Zhao D, Zhang H, Ji P, Li S, Yang C, Liu Y, Qian D, Deng Y, Wang H, Lu D. (2021). ‎Surveillance of Antimalarial Drug-Resistance Genes in Imported Plasmodium ‎falciparum Isolates From Nigeria in Henan, China, 2012–2019. Frontiers in cellular and infection microbiology, 11(Article 644576): 1–9. [Crossref], [Google Scholar], [Publisher]
‎10. Ebel E R, Reis F, Petrov D A, Beleza S. (2021). Historical trends and new surveillance of ‎Plasmodium falciparum drug resistance markers in Angola. Malaria journal, 20(1): 1-9. ‎‎[Crossref], [Google Scholar], [Publisher]
‎11. Rouhani M, Zakeri S, Pirahmadi S, Raeisi A, Djadid N D. (2015). High prevalence of ‎pfdhfr–pfdhps triple mutations associated with anti-malarial drugs resistance in ‎Plasmodium falciparum isolates seven years after the adoption of sulfadoxine–‎pyrimethamine in combination with artesunate as first-line treatment in Iran. Infection, Genetics and Evolution, 31: 183-189. [Crossref], [Google Scholar], [Publisher]
‎12. Ruh E, Bateko J P, Imir T, Taylan-Ozkan A. (2018). Molecular identification of ‎sulfadoxine-pyrimethamine resistance in malaria infected women who received ‎intermittent preventive treatment in the Democratic Republic of Congo. Malaria journal, ‎‎17(1): 1-7. [Crossref], [Google Scholar], [Publisher]
‎13. Beigomi M, Biabangard A, Rohani R. (2021). Evaluation of antimicrobial effects of ‎Rosemary and Withania somnifera methanol extract prepared by ultrasound waveform ‎on Escherichia coli biofilm isolated from urinary tract infection. Micro Environer, 1(2): ‎‎17-25. [Crossref], [Google Scholar], [PDF]
‎14. Beigomi M, shakoory-moghadam V, Biabangard A, Behzadmehr R. (2021). Evaluation ‎of the antimicrobial activity of plant extracts on Escherichia coli and Candida albicans. ‎Micro Environer, 1(2): 86-92. [Crossref], [Google Scholar], [PDF]
‎15. Chagona P, Kwamboka N, Gaya H, Makonde H, Adem A, Osano K, Kawaka F. (2021). ‎Phytochemical Analysis and Antibacterial Activity of the Kenyan Wild Orchids. Micro Environer, 1(2): 93-100.. [Crossref], [Google Scholar], [Publisher]
‎16. Ghafari M, Beigomi Z, Javadian E. (2021). Evaluation of antibacterial activity of extract ‎plant against Staphylococcus aureus and Candida albicans isolated from women. Micro Environer, 1(2): 78-85.. [Crossref], [Publisher]
‎17. Olà D, Berenger A A, Brice B K, Noel D D, David C N g, Baba C, Joseph D A. (2020). ‎Assessing the polymorphism of DHFR gene from Plasmodium falciparum in the south of ‎Cte dIvoire. African Journal of Microbiology Research, 14(5): 158-165.. [Crossref], [Google Scholar], [Publisher]
‎18. Jahantigh M, ahmadi H. (2021). Analysis of the antimicrobial activity of Ashurak ‎extracts prepared with different solvents on Klebsiella pneumoniae and Shigella ‎dysentery isolated from poultry faeces. Micro Environer, 1(1): 54-62 ‎. [Crossref], [Google Scholar], [Publisher]‎ ‎
‎19. Karabulut F, Aydın S, Parray J A. (2021). Interactions of antioxidant defense ‎mechanisms developed by plants and microorganisms against pesticides, Micro Environer, 1(2): 63-77.. [Crossref], [Google Scholar], [PDF]
‎20. Karabulut F, Parray J A, Mir M Y. (2021). Emerging trends for Harnessing plant ‎metabolome and microbiome for sustainable food Production. Micro Environer, 1(1): ‎‎33-53. [Crossref], [Google Scholar], [PDF]
‎21. Sugaram R, Suwannasin K, Kunasol C, Mathema V B, Day N P, Sudathip P, Prempree P, ‎Dondorp A M, Imwong M. (2020). Molecular characterization of Plasmodium falciparum ‎antifolate resistance markers in Thailand between 2008 and 2016. Malaria journal, ‎‎19(1): 1-10.. [Crossref], [Google Scholar], [Publisher]
‎22. Jalei A A, Chaijaroenkul W, Na-Bangchang K. (2018). Plasmodium falciparum drug ‎resistance gene status in the Horn of Africa: a systematic review. African Journal of Pharmacy and Pharmacology, 12(25): 361-373.. [Crossref], [Google Scholar], [Publisher]
‎23. Mbaye A, Gaye A, Dieye B, Ndiaye Y D, Bei A K, Affara M, Deme A B, Yade M S, Diongue ‎K, Ndiaye I M. (2017). Ex vivo susceptibility and genotyping of Plasmodium falciparum ‎isolates from Pikine, Senegal. Malaria journal, 16(1): 1-7.. [Crossref], [Google Scholar], [Publisher]
‎24. Gikunju S W, Agola E L, Ondondo R O, Kinyua J, Kimani F, LaBeaud A D, Malhotra I, ‎King C, Thiong’o K, Mutuku F. (2020). Prevalence of pfdhfr and pfdhps mutations in ‎Plasmodium falciparum associated with drug resistance among pregnant women ‎receiving IPTp-SP at Msambweni County Referral Hospital, Kwale County, Kenya. ‎Malaria journal, 19(1): 1-7.. [Crossref], [Google Scholar], [Publisher]
‎25. Menard D, Dondorp A. (2017). Antimalarial drug resistance: a threat to malaria ‎elimination. Cold Spring Harbor Perspectives in Medicine, 7(7): a025619.. [Crossref], [Google Scholar], [Publisher]
‎26. Bansal D, Bharti P K, Acharya A, Abdelraheem M H, Patel P, Elmalik A, Abosalah S, Khan ‎F Y, ElKhalifa M, Kaur H. (2019). Molecular surveillance of putative drug resistance ‎markers of antifolate and artemisinin among imported Plasmodium falciparum in Qatar. ‎Pathogens and global health, 113(4): 158-166.. [Crossref], [Google Scholar], [Publisher]
‎27. Rahbar-Karbasdehi E, Rahbar-Karbasdehi F. (2021). Clinical challenges of stress ‎cardiomyopathy during coronavirus 2019 epidemic. Cellular, Molecular and Biomedical Reports, 1(2): 88-90.. [Crossref], [Google Scholar], [Publisher]
‎28. Abbas-Al-Khafaji Z K, Aubais-aljelehawy Q h. (2021). Evaluation of antibiotic resistance ‎and prevalence of multi-antibiotic resistant genes among Acinetobacter baumannii ‎strains isolated from patients admitted to al-yarmouk hospital. Cellular, Molecular and Biomedical Reports, 1(2): 60-68.. [Crossref], [Google Scholar], [Publisher]‎
‎29. Alavi M, Rai M, Martinez F, Kahrizi D, Khan H, Rose Alencar de Menezes I, Douglas ‎Melo Coutinho H, Costa J G M. (2022). The efficiency of metal, metal oxide, and ‎metalloid nanoparticles against cancer cells and bacterial pathogens: different ‎mechanisms of action. Cellular, Molecular and Biomedical Reports, 2(1): 10-21. [Crossref], [Google Scholar], [Publisher]
‎30. Cowell A N, Winzeler E A. (2019). The genomic architecture of antimalarial drug ‎resistance. Briefings in Functional Genomics, 18(5): 314-328. [Crossref], [Google Scholar], [Publisher]
‎31. Abaza S, El-Tonsy M. (2017). Gene mutations in parasitic diseases Part II: Parasite gene ‎mutations. Parasitologists United Journal, 10(1-2): 4-22. [Crossref], [Google Scholar], [Publisher]
‎32. Upadhyay R K. (2016). Emergence of drug resistance in Plasmodiun falciparum: ‎reasons of its dispersal and transmission in different climatic regions of the world: a ‎review. Clin Microbiol Infect Dis, 1(2): 45-55.. [Crossref], [Google Scholar], [PDF]
‎33. Zakeri S, Farahani M S, Afsharpad M, Salehi M, Raeisi A, Djadid N D. (2010). High ‎prevalence of the 437G mutation associated with sulfadoxine resistance among ‎Plasmodium falciparum clinical isolates from Iran, three years after the introduction of ‎sulfadoxine–pyrimethamine. International Journal of Infectious Diseases, 14: e123-e128.. [Crossref], [Google Scholar], [Publisher]
‎34. Peek R, Van Gool T, Panchoe D, Greve S, Bus E, Resida L. (2005). Drug resistance and ‎genetic diversity of Plasmodium falciparum parasites from Suriname. The American Journal of Tropical Medicine and Hygiene, 73(5): 833-838.. [Crossref], [Google Scholar], [Publisher]
‎35. Quan H, Igbasi U, Oyibo W, Omilabu S, Chen S-B, Shen H-M, Okolie C, Chen J-H, Zhou X-‎N. (2020). High multiple mutations of Plasmodium falciparum-resistant genotypes to ‎sulphadoxine-pyrimethamine in Lagos, Nigeria. Infectious Diseases of Poverty, 9(1): 1-‎‎11.. [Crossref], [Google Scholar], [Publisher]
‎36. Ould Ahmedou Salem M S, Mint Lekweiry K, Bouchiba H, Pascual A, Pradines B, Ould ‎Mohamed Salem Boukhary A, Briolant S, Basco L K, Bogreau H. (2017). ‎Characterization of Plasmodium falciparum genes associated with drug resistance in ‎Hodh Elgharbi, a malaria hotspot near Malian–Mauritanian border. Malaria journal, ‎‎16(1): 1-9. [Crossref], [Google Scholar], [Publisher]
‎37. Biswas S, Escalante A, Chaiyaroj S, Angkasekwinai P, Lal A. (2000). Prevalence of point ‎mutations in the dihydrofolate reductase and dihydropteroate synthetase genes of ‎Plasmodium falciparum isolates from India and Thailand: a molecular epidemiologic ‎study. Tropical Medicine & International Health, 5(10): 737-743. [Crossref], [Google Scholar], [Publisher]
‎38. Balogun S T, Sandabe U K, Sodipo O A, Okon K O, Akanmu A O. (2021). Single ‎Nucleotide Polymorphisms of Pfdhfr and Pfdhps Genes: Implications for Malaria ‎Prophylactic Strategies in Maiduguri, Northeast Nigeria. Journal of tropical medicine, ‎‎2021: Article ID: 8840089.. [Crossref], [Google Scholar], [Publisher]
‎39. Zhao Y, Liu Z, Soe M T, Wang L, Soe T N, Wei H, Than A, Aung P L, Li Y, Zhang X. ‎‎(2019). Genetic variations associated with drug resistance markers in asymptomatic ‎Plasmodium falciparum infections in Myanmar. Genes, 10(9): 692.. [Crossref], [Google Scholar], [Publisher]
‎40. Hussien M, Abdel Hamid M M, Elamin E A, Hassan A O, Elaagip A H, Salama A H A, ‎Abdelraheem M H, Mohamed A O. (2020). Antimalarial drug resistance molecular ‎makers of Plasmodium falciparum isolates from Sudan during 2015–2017. PloS one, ‎‎15(8): e0235401. [Crossref], [Google Scholar], [Publisher]
‎41. Voumbo-Matoumona D F, Kouna L C, Madamet M, Maghendji-Nzondo S, Pradines B, ‎Lekana-Douki J B. (2018). Prevalence of Plasmodium falciparum antimalarial drug ‎resistance genes in Southeastern Gabon from 2011 to 2014. Infection and drug resistance, 11: 1329-1338. [Crossref], [Google Scholar], [Publisher]