Document Type : Original Article


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

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

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


Chloroquine was one of the most cheapest and effective chemotherapeutic drugs for Plasmodium falciparum-malaria, but for a long, the drug has been officially withdrawn in almost all malaria-endemic countries including Nigeria, due to the development of resistance by the parasite. Withdrawal of the drug may make the drug regains its efficacy. Therefore, this study aimed to determine the presence of Biomarkers associated with chloroquine resistance from Gombe Local Government Area, Gombe State, Nigeria after its withdrawal in 2005. Twenty hundred blood samples were collected from consented study subjects and analysed using Microscopy, RDT and PCR. DNA was extracted using Quick-DNA™ Miniprep (No. D4069), Purity and Concentration of the DNA were determined using Nanodrop Spectrophotometer. 57 true positive samples were selected for molecular analysis. Nested PCR was used to amplify the required codon (C72S, M74I, K76T and N75E) position of PCRT the gene of P. falciparum. Both Primary and Secondary PCR was carried out. The PCR products were subjected to electrophoresis in 2% agarose and stained with ethidium bromide. The amplicons were purified and sequenced, after which the sequenced products were subjected to BLAST software. Single Nucleotide Polymorphism was recorded from C72S and K76T with a prevalence of 05(8.80%) and 46(80.70%) respectively. Confirmed biomarkers of Chloroquine resistance are still present in P. falciparum isolate from Gombe L.G.A. 

Graphical Abstract

Molecular analysis of bio-makers of Chloroquine resistance in Plasmodium falciparum isolate from gombe local government area, Gombe State, Nigeria


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ِDr. Ismail Muhammad
Gombe State University

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  1. Owoloye A, Olufemi M, Idowu ET, Oyebola KM (2021) Prevalence of potential mediators of artemisinin resistance in African isolates of Plasmodium falciparum. Malaria journal 20(1):1-12. doi:
  2. Shafik SH, Cobbold SA, Barkat K, Richards SN, Lancaster NS, Llinás M, Hogg SJ, Summers RL, McConville MJ, Martin RE (2020) The natural function of the malaria parasite’s chloroquine resistance transporter. Nature communications 11(1):1-16. doi:
  3. Ahouidi A, Oliveira R, Lobo L, Diedhiou C, Mboup S, Nogueira F (2021) Prevalence of pfk13 and pfmdr1 polymorphisms in Bounkiling, Southern Senegal. PloS one 16(3):e0249357. doi:
  4. De-Dios T, van Dorp L, Gelabert P, Carøe C, Sandoval-Velasco M, Fregel R, Escosa R, Aranda C, Huijben S, Balloux F (2019) Genetic affinities of an eradicated European Plasmodium falciparum strain. Microbial genomics 5(9):PMC6807384. doi:
  5. Tola M, Ajibola O, Idowu ET, Omidiji O, Awolola ST, Amambua-Ngwa A (2020) Molecular detection of drug resistant polymorphisms in Plasmodium falciparum isolates from Southwest, Nigeria. BMC Research Notes 13(1):1-7. doi:
  6. Voumbo-Matoumona DF, Kouna LC, Madamet M, Maghendji-Nzondo S, Pradines B, Lekana-Douki JB (2018) Prevalence of Plasmodium falciparum antimalarial drug resistance genes in Southeastern Gabon from 2011 to 2014. Infection and drug resistance 11:1329-1338. doi:
  7. Adam R, Mukhtar MM, Abubakar UF, Damudi HA, Muhammad A, Ibrahim SS (2021) Polymorphism Analysis of pfmdr1 and pfcrt from Plasmodium falciparum Isolates in Northwestern Nigeria Revealed the Major Markers Associated with Antimalarial Resistance. Diseases 9(1):1-12
  8. Beargie SM, Higgins CR, Evans DR, Laing SK, Erim D, Ozawa S (2019) The economic impact of substandard and falsified antimalarial medications in Nigeria. PloS one 14(8):e0217910. doi:
  9. Muhammad I, Sale PM, Midala AL (2022) Absence of Biomakers of Resistance in K13 Propeller Gene of Plasmodium falciparum from Gombe LGA of Gombe State, Nigeria. Advances 3(1):25-33
  10. Stokes BH, Dhingra SK, Rubiano K, Mok S, Straimer J, Gnädig NF, Deni I, Schindler KA, Bath JR, Ward KE (2021) Plasmodium falciparum K13 mutations in Africa and Asia impact artemisinin resistance and parasite fitness. Elife 10:e66277. doi:
  11. Parray JA, Ali U, Mir MY, Shameem N (2021) A high throughputs and consistent method for the sampling and isolation of Endophytic bacteria allied to high altitude the medicinal plant Arnebia benthamii (Wall ex. G. Don). Micro Environer 1(1):1-6. doi:
  12. Karabulut F, Parray JA, Mir MY (2021) Emerging trends for Harnessing plant metabolome and microbiome for sustainable food Production. Micro Environer 1(1):33-53 doi:
  13. 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. doi:
  14. 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. doi:
  15. Karabulut F, Aydın S, Parray JA (2021) Interactions of antioxidant defense mechanisms developed by plants and microorganisms against pesticides Micro Environer 1(2):63-77. doi:
  16. Kayiba NK, Yobi DM, Tshibangu-Kabamba E, Tuan VP, Yamaoka Y, Devleesschauwer B, Mvumbi DM, Wemakoy EO, De Mol P, Mvumbi GL (2021) Spatial and molecular mapping of Pfkelch13 gene polymorphism in Africa in the era of emerging Plasmodium falciparum resistance to artemisinin: a systematic review. The Lancet Infectious Diseases 21(4):e82-e92. doi:
  17. 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. doi:
  18. 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. doi:
  19. 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. doi:
  20. Simon-Oke IA, Ade-Alao AO, Ologundudu F (2020) The impact of HIV-associated immunosuppression on the Plasmodium falciparum chloroquine resistance transporter gene (PfCRT) of HIV patients in Akure, Nigeria. Bulletin of the National Research Centre 44(1):1-7. doi:
  21. Xu C, Wei Q, Yin K, Sun H, Li J, Xiao T, Kong X, Wang Y, Zhao G, Zhu S (2018) Surveillance of antimalarial resistance Pfcrt, Pfmdr1, and Pfkelch13 polymorphisms in African Plasmodium falciparum imported to Shandong Province, China. Scientific reports 8(1):1-9. doi:
  22. Solomon VR, Lee H (2009) Chloroquine and its analogs: a new promise of an old drug for effective and safe cancer therapies. European journal of pharmacology 625(1-3):220-233. doi:
  23. Balikagala B, Sakurai-Yatsushiro M, Tachibana S-I, Ikeda M, Yamauchi M, Katuro OT, Ntege EH, Sekihara M, Fukuda N, Takahashi N (2020) Recovery and stable persistence of chloroquine sensitivity in Plasmodium falciparum parasites after its discontinued use in Northern Uganda. Malaria journal 19(1):1-12. doi:
  24. 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. doi:
  25. Agomo CO, Oyibo WA, Sutherland C, Hallet R, Oguike M (2016) Assessment of markers of antimalarial drug resistance in Plasmodium falciparum isolates from pregnant women in Lagos, Nigeria. PloS one 11(1):e0146908. doi:
  26. Mwanza S, Joshi S, Nambozi M, Chileshe J, Malunga P, Kabuya J-BB, Hachizovu S, Manyando C, Mulenga M, Laufer M (2016) The return of chloroquine-susceptible Plasmodium falciparum malaria in Zambia. Malaria journal 15(1):1-6. doi:
  27. Oladipo OO, Wellington OA, Sutherland CJ (2015) Persistence of chloroquine-resistant haplotypes of Plasmodium falciparum in children with uncomplicated Malaria in Lagos, Nigeria, four years after change of chloroquine as first-line antimalarial medicine. Diagnostic pathology 10(1):1-8. doi:
  28. Pathak A, Mårtensson A, Gawariker S, Sharma A, Diwan V, Purohit M, Ursing J (2020) Stable high frequencies of sulfadoxine–pyrimethamine resistance associated mutations and absence of K13 mutations in Plasmodium falciparum 3 and 4 years after the introduction of artesunate plus sulfadoxine–pyrimethamine in Ujjain, Madhya Pradesh, India. Malaria journal 19(1):1-7. doi:
  29. Menard D, Dondorp A (2017) Antimalarial drug resistance: a threat to malaria elimination. Cold Spring Harbor Perspectives in Medicine 7(7):a025619. doi:
  30. Mahende C, Ngasala B, Lusingu J, Yong T-S, Lushino P, Lemnge M, Mmbando B, Premji Z (2016) Performance of rapid diagnostic test, blood-film microscopy and PCR for the diagnosis of malaria infection among febrile children from Korogwe District, Tanzania. Malaria journal 15(1):1-7. doi:
  31. Ippolito MM, Moser KA, Kabuya J-BB, Cunningham C, Juliano JJ (2021) Antimalarial drug resistance and implications for the WHO global technical strategy. Current Epidemiology Reports 8(2):46-62. doi:
  32. Khan HM, Kirmani S, Khan PA, İslam U (2016) Prevalence of chloroquine resistant Plasmodium falciparum malaria in pregnant females attending North Indian hospital. Eastern Journal Of Medicine 21(2):64-68
  33. Nneji C, Adedapo A, Okorie P, Ademowo P (2015) Chloroquine resistance and host genetic factors among nigerian children with uncomplicated P. falciparum Infection. Arch Med 7(4):1-9
  34. Bello SO, Chika A, Bello AY (2010) Is Chloroquine better than Artemisinin combination therapy as first line treatment in adult Nigerians with uncomplicated malaria? a cost effective analysis. African Journal of Infectious Diseases 4(2):29-42. doi:
  35. Adyanthaya S, Jose M (2013) Quality and safety aspects in histopathology laboratory. Journal of oral and maxillofacial pathology: JOMFP 17(3):402. doi:
  36. García-Alegría AM, Anduro-Corona I, Pérez-Martínez CJ, Guadalupe Corella-Madueño MA, Rascón-Durán ML, Astiazaran-Garcia H (2020) Quantification of DNA through the nanodrop spectrophotometer: methodological validation using standard reference material and Sprague Dawley Rat and human DNA. International journal of analytical chemistry 2020:Article ID: 8896738. doi:
  37. Mogeni P, Williams TN, Omedo I, Kimani D, Ngoi JM, Mwacharo J, Morter R, Nyundo C, Wambua J, Nyangweso G (2017) Detecting malaria hotspots: a comparison of rapid diagnostic test, microscopy, and polymerase chain reaction. The Journal of infectious diseases 216(9):1091-1098. doi:
  38. Londono BL, Eisele TP, Keating J, Bennett A, Chattopadhyay C, Heyliger G, Mack B, Rawson I, Vely J-F, Désinor O (2009) Chloroquine-resistant haplotype Plasmodium falciparum parasites, Haiti. Emerging infectious diseases 15(5):735. doi:
  39. Atroosh WM, Al-Mekhlafi HM, Mahdy MA, Surin J (2012) The detection of pfcrt and pfmdr1 point mutations as molecular markers of chloroquine drug resistance, Pahang, Malaysia. Malaria journal 11(1):1-7. doi:
  40. Olukosi YA, Oyebola MK, Ajibaye O, Orok BA, Aina OO, Agomo CO, Iwalokun BA, Akindele SK, Enya VN, Okoh HI (2014) Persistence of markers of chloroquine resistance among P. falciparum isolates recovered from two Nigerian communities. Mal World J 5(3):3-6
  41. Aguiar AC, Murce E, Cortopassi WA, Pimentel AS, Almeida MM, Barros DC, Guedes JS, Meneghetti MR, Krettli AU (2018) Chloroquine analogs as antimalarial candidates with potent in vitro and in vivo activity. International Journal for Parasitology: Drugs and Drug Resistance 8(3):459-464. doi:
  42. Zhao Y, Liu Z, Soe MT, Wang L, Soe TN, Wei H, Than A, Aung PL, Li Y, Zhang X (2019) Genetic variations associated with drug resistance markers in asymptomatic Plasmodium falciparum infections in Myanmar. Genes 10(9):1-17. doi:
  43. Mittra P, Vinayak S, Chandawat H, Das MK, Singh N, Biswas S, Dev V, Kumar A, Ansari MA, Sharma YD (2006) Progressive increase in point mutations associated with chloroquine resistance in Plasmodium falciparum isolates from India. The Journal of infectious diseases 193(9):1304-1312. doi:
  44. Patel P, Bharti PK, Bansal D, Ali NA, Raman RK, Mohapatra PK, Sehgal R, Mahanta J, Sultan AA, Singh N (2017) Prevalence of mutations linked to antimalarial resistance in Plasmodium falciparum from Chhattisgarh, Central India: a malaria elimination point of view. Scientific reports 7(1):1-8. doi:
  45. Huang F, Yan H, Xue J-B, Cui Y-W, Zhou S-S, Xia Z-G, Abeyasinghe R, Ringwald P, Zhou X-N (2021) Molecular surveillance of pfcrt, pfmdr1 and pfk13-propeller mutations in Plasmodium falciparum isolates imported from Africa to China. Malaria journal 20(1):1-11. doi:
  46. Antony HA, Das S, Parija SC, Padhi S (2016) Sequence analysis of pfcrt and pfmdr1 genes and its association with chloroquine resistance in Southeast Indian Plasmodium falciparum isolates. Genomics data 8:85-90. doi:
  47. Folarin O, Gbotosho G, Sowunmi A, Olorunsogo O, Oduola A, Happi T (2008) Chloroquine resistant Plasmodium falciparum in Nigeria: relationship between pfcrt and pfmdr1 polymorphisms, in-vitro resistance and treatment outcome. The open tropical medicine journal 1:74-82. doi:
  48. Idowu AO, Oyibo WA, Bhattacharyya S, Khubbar M, Mendie UE, Bumah VV, Black C, Igietseme J, Azenabor AA (2019) Rare mutations in Pfmdr1 gene of Plasmodium falciparum detected in clinical isolates from patients treated with anti-malarial drug in Nigeria. Malaria journal 18(1):1-9. doi:
  49. TAS M, Timothy S, Emenike I (2016) Assessment of chloroquine tablets used in the treatment of malaria in northern nigeria. International Journal 7(1):2671-2677
  50. Yang Z, Zhang Z, Sun X, Wan W, Cui L, Zhang X, Zhong D, Yan G, Cui L (2007) Molecular analysis of chloroquine resistance in Plasmodium falciparum in Yunnan Province, China. Tropical Medicine & International Health 12(9):1051-1060. doi:
  51. Xiao X, Shen J, Wang S, Xiao H, Tong G (2010) The variation of the southwest monsoon from the high resolution pollen record in Heqing Basin, Yunnan Province, China for the last 2.78 Ma. Palaeogeography, Palaeoclimatology, Palaeoecology 287(1-4):45-57. doi:
  52. Ocan M, Akena D, Nsobya S, Kamya MR, Senono R, Kinengyere AA, Obuku EA (2019) Persistence of chloroquine resistance alleles in malaria endemic countries: a systematic review of burden and risk factors. Malaria journal 18(1):1-15. doi:
  53. Plummer WB, Pereira LMP, Carrington CV (2004) Pfcrt and pfmdr1 alleles associated with chloroquine resistance in Plasmodium falciparum from Guyana, South America. Memorias do Instituto Oswaldo Cruz 99(4):389-392
  54. Farcas GA, Soeller R, Zhong K, Zahirieh A, Kain KC (2006) Real-time polymerase chain reaction assay for the rapid detection and characterization of chloroquine-resistant Plasmodium falciparum malaria in returned travelers. Clinical infectious diseases 42(5):622-627. doi:
  55. Cheruiyot J, Ingasia LA, Omondi AA, Juma DW, Opot BH, Ndegwa JM, Mativo J, Cheruiyot AC, Yeda R, Okudo C (2014) Polymorphisms in Pf mdr1, Pf crt, and Pf nhe1 genes are associated with reduced in vitro activities of quinine in Plasmodium falciparum isolates from Western Kenya. Antimicrobial agents and chemotherapy 58(7):3737-3743. doi:
  56. Singh G, Singh R, Urhehar AD (2016) Simple molecular methods for early detection of chloroquine drug resistance in Plasmodium vivax and Plasmodium falciparum. Journal of clinical and diagnostic research: JCDR 10(7):DC19. doi:
  57. Djimdé A, Doumbo OK, Cortese JF, Kayentao K, Doumbo S, Diourté Y, Coulibaly D, Dicko A, Su X-z, Nomura T (2001) A molecular marker for chloroquine-resistant falciparum malaria. New England Journal of Medicine 344(4):257-263. doi:
  58. Shrivastava SK, Gupta RK, Mahanta J, Dubey ML (2014) Correlation of molecular markers, Pfmdr1-N86Y and Pfcrt-K76T, with in vitro chloroquine resistant Plasmodium falciparum, isolated in the malaria endemic states of Assam and Arunachal Pradesh, Northeast India. PloS one 9(8):e103848. doi: