Mutations in Plasmodium knowlesi Kelch protein 13 and the dihydropteroate synthase gene in clinical samples

Abstract

Objective: To determine the genetic diversity, natural selection and mutations in Plasmodium ( P.) knowlesi drug resistant molecular markers Kelch 13 and dhps gene in clinical samples of Malaysia. Methods: P. knowlesi full-length gene sequences Kelch 13 gene (PkK13) from 40 samples and dhps gene from 30 samples originating from Malaysian Borneo were retrieved from public databases. Genetic diversity, natural selection, and phylogenetic analysis of gene sequences were analysed using DNAsp v5.10 and MEGA v5.2. Results: Seventy-two single nucleotide polymorphic sites (SNPs) across the full-length PkK13 gene (63 synonymous substitutions and 9 non-synonymous substitutions) with nucleotide diversity of π~0.005 was observed. Analysis of the full-length Pk dhps gene revealed 73 SNPs and π~0.006 (44 synonymous substitutions and 29 non-synonymous substitutions). A high number of haplotypes (PkK13; H=37 and Pk dhps; H=29) with haplotype diversity of Hd ~0.99 were found in both genes, indicating population expansion. Nine mutant alleles were identified in PkK13 amino acid alignment of which, 7 (Asp³Glu, Lys⁵⁰Gln, Lys⁵³Glu, Ser¹²³Thr, Ser¹²⁷Pro, Ser¹⁴⁹Thr and Ala¹⁶⁹Thr) were within the Plasmodium specific domain, 2 (Val³⁷²Ile and Lys⁴²⁴Asn) were in the BTB/POZ domain and no mutation was observed within the kelch propeller domain. The 29 non-synonymous mutations in the Pk dhps gene were novel and only presented in exon 1 and 2. Conclusions: Monitoring the mutations from clinical samples collected from all states of Malaysia along with clinical efficacy studies will be necessary to determine the drug resistance in P. knowlesi.

Keywords

Plasmodium knowlesi / Kelch 13 / dhps / Dihydropteroate synthase / Drug resistance / Mutation

References

[1] Ahmed MA, Cox-Singh J. Plasmodium knowlesi-an emerging pathogen. ISBT Sci Ser 2015; 10(Suppl 1):134-140.

[2] Barber BE, William T, Grigg MJ, Menon J, Auburn S, Marfurt J, et al. A prospective comparative study of knowlesi, falciparum, and vivax malaria in Sabah, Malaysia: High proportion with severe disease from Plasmodium knowlesi and Plasmodium vivax but no mortality with early referral and artesunate therapy. Clin Infect Dis 2013; 56(3):383-397.

[3] Dondero TJ Jr., Parsons RE, Ponnampalam JT. Studies on the resistance of malaria to chloroquine and to a combination of chloroquine and pyrimethamine in Peninsular Malaysia. Trans R Soc Trop Med Hyg 1976; 70(2):145-148.

[4] Grigg MJ, Barber BE, Marfurt J, Imwong M, William T, Bird E, et al. Dihydrofolate-reductase mutations in Plasmodium knowlesi appear unrelated to selective drug pressure from putative human-to-human transmission in Sabah, Malaysia. PLoS One 2016; 11(3):e0149519.

[5] Triglia T, Menting JG, Wilson C, Cowman AF : Mutations in dihydropteroate synthase are responsible for sulfone and sulfonamide resistance in Plasmodium falciparum. Proc Natl Acad Sci U S A 1997; 94(25):13944-13949.

[6] Brooks DR, Wang P, Read M, Watkins WM, Sims PF, Hyde JE. Sequence variation of the hydroxymethyldihydropterin pyrophosphokinase: Dihydropteroate synthase gene in lines of the human malaria parasite, Plasmodium falciparum, with differing resistance to sulfadoxine. Eur J Biochem 1994; 224(2):397-405.

[7] Ndiaye D, Dieye B, Ndiaye YD, Van Tyne D, Daniels R, Bei AK, et al. Polymorphism in dhfr/dhps genes, parasite density and ex vivo response to pyrimethamine in Plasmodium falciparum malaria parasites in Thies, Senegal. Int J Parasitol Drugs Drug Resist 2013; 3:135-142.

[8] Flannery EL, Chatterjee AK, Winzeler EA. Antimalarial drug discovery approaches and progress towards new medicines. Nat Rev Microbiol 2017; 15(9):572.

[9] White NJ. Qinghaosu (artemisinin): The price of success. Science 2008; 320(5874):330-334.

[10] Arrow KJ, Panosian C, Gelband H. In, Saving lives, buying time: Economics of malaria drugs in an age of resistance. Washington DC: National Academies Press; 2004.

[11] Noedl H, Se Y, Schaecher K, Smith BL, Socheat D, Fukuda MM, et al. Evidence of artemisinin-resistant malaria in western Cambodia. N Engl J Med 2008; 359(24):2619-2620.

[12] Dondorp AM, Nosten F, Yi P, Das D, Phyo AP, Tarning J, et al. Artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med 2009; 361(5):455-467.

[13] Ashley EA, Dhorda M, Fairhurst RM, Amaratunga C, Lim P, Suon S, et al. Spread of artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med 2014; 371(5):411-423.

[14] Ariey F, Witkowski B, Amaratunga C, Beghain J, Langlois AC, Khim N, et al. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria. Nature 2014; 505(7481):50-55.

[15] WHO. Artemisinin resistance and artemisinin-based combination therapy efficacy. [Online]. Available from:https://www.who.int/docs/default-source/documents/publications/gmp/who-cds-gmp-2018-26-eng.pdf. [Accessed on 8 September 2022].

[16] Coppee R, Jeffares DC, Miteva MA, Sabbagh A, Clain J. Comparative structural and evolutionary analyses predict functional sites in the artemisinin resistance malaria protein K13. Sci Rep 2019; 9(1):10675.

[17] Wang X, Ruan W, Zhou S, Huang F, Lu Q, Feng X, et al. Molecular surveillance of Pfcrt and K13 propeller polymorphisms of imported Plasmodium falciparum cases to Zhejiang Province, China between 2016 and 2018. Malar J 2020; 19(1):59.

[18] Miotto O, Amato R, Ashley EA, MacInnis B, Almagro-Garcia J, Amaratunga C, et al. Genetic architecture of artemisinin-resistant Plasmodium falciparum. Nat Genet 2015; 47(3):226-234.

[19] Ocan M, Akena D, Nsobya S, Kamya MR, Senono R, Kinengyere AA, et al. K13-propeller gene polymorphisms in Plasmodium falciparum parasite population in malaria affected countries: A systematic review of prevalence and risk factors. Malar J 2019; 18(1):60.

[20] Nzoumbou-Boko R, Pante-Wockama CG, Ngoagoni C, Petiot N, Legrand E, Vickos U, et al. Molecular assessment of kelch13 non-synonymous mutations in Plasmodium falciparum isolates from Central African Republic (2017-2019). Malar J 2020; 19(1):191.

[21] Ooi CH, Phang WK, Liew JWK, Atroosh WM, Lau YL. Epidemiology of indigenous Plasmodium knowlesi infection in Sarawak, 2011-2019. Trop Med Int Health 2022; 27(8):705-718.

[22] Daneshvar C, Davis TM, Cox-Singh J, Rafa'ee MZ, Zakaria SK, Divis PC, et al. Clinical and laboratory features of human Plasmodium knowlesi infection. Clin Infect Dis 2009; 49(6):852-860.

[23] Barber BE, William T, Jikal M, Jilip J, Dhararaj P, Menon J, et al. Plasmodium knowlesi malaria in children. Emerg Infect Dis 2011; 17(5):814-820.

[24] Fatih FA, Staines HM, Siner A, Ahmed MA, Woon LC, Pasini EM, et al. Susceptibility of human Plasmodium knowlesi infections to anti-malarials. Malar J 2013; 12:425.

[25] Grigg MJ, William T, Barber BE, Rajahram GS, Menon J, Schimann E, et al. Artemether-lumefantrine versus chloroquine for the treatment of uncomplicated Plasmodium knowlesi malaria: An open-label randomized controlled trial CAN KNOW. Clin Infect Dis 2018; 66(2):229-236.

[26] Assefa S, Lim C, Preston MD, Duffy CW, Nair MB, Adroub SA, et al. Population genomic structure and adaptation in the zoonotic malaria parasite Plasmodium knowlesi. Proc Natl Acad Sci U S A 2015; 112(42):13027-13032.

[27] Librado P, Rozas J. DnaSP v5: A software for comprehensive analysis of DNA polymorphism data. Bioinformatics 2009; 25(11):1451-1452.

[28] Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 2011; 28(10):2731-2739.

[29] Daneshvar C, Davis TM, Cox-Singh J, Rafa'ee MZ, Zakaria SK, Divis PC, et al. Clinical and parasitological response to oral chloroquine and primaquine in uncomplicated human Plasmodium knowlesi infections. Malar J 2010; 9:238.

[30] Grigg MJ, William T, Menon J, Dhanaraj P, Barber BE, Wilkes CS, et al. Artesunate-mefloquine versus chloroquine for treatment of uncomplicated Plasmodium knowlesi malaria in Malaysia (ACT KNOW): An open-label, randomised controlled trial. Lancet Infect Dis 2016; 16(2):180-188.

[31] Pinheiro MM, Ahmed MA, Millar SB, Sanderson T, Otto TD, Lu WC, et al. Plasmodium knowlesi genome sequences from clinical isolates reveal extensive genomic dimorphism. PLoS One 2015; 10(4):e0121303.

[32] Ahmed MA, Chu KB, Quan FS. The Plasmodium knowlesi Pk41 surface protein diversity, natural selection, sub population and geographical clustering: A 6-cysteine protein family member. Peer J 2018; 6:e6141.

[33] Ahmed MA, Lau YL, Quan FS. Diversity and natural selection on the thrombospondin-related adhesive protein (TRAP) gene of Plasmodium knowlesi in Malaysia. Malar J 2018; 17(1):274.

[34] Yusof R, Ahmed MA, Jelip J, Ngian HU, Mustakim S, Hussin HM, et al. Phylogeographic evidence for 2 genetically distinct zoonotic Plasmodium knowlesi parasites, Malaysia. Emerg Infect Dis 2016; 22(8):1371-1380.

[35] Ahmed MA, Fong MY, Lau YL, Yusof R. Clustering and genetic differentiation of the normocyte binding protein (nbpxa) of Plasmodium knowlesi clinical isolates from Peninsular Malaysia and Malaysia Borneo. Malar J 2016; 15:241.

[36] Ahmed MA, Deshmukh GY, Zaidi RH, Saif A, Alshahrani MA, Wazid SW, et al. Identification, mapping, and genetic diversity of novel conserved cross-species epitopes of RhopH2 in Plasmodium knowlesi with Plasmodium vivax. Front Cellu Infect Microbiol 2022. doi:10.3389/fcimb.2021.810398.

[37] Ahmed MA, Zaidi RH, Deshmukh GY, Saif A, Alshahrani MA, Salam SS, et al. Genetic diversity and population genetic structure analysis of Plasmodium knowlesi thrombospondin-related apical merozoite protein (TRAMP) in clinical samples. Genes 2022; 13(11):1944.

[38] Muhammad AB, Nor Azman E, Eddie NA, Azmi NI, Yee VCT, Idris ZM. The rise of Plasmodium knowlesi cases: Implication to Malaysia’s malaria-free status. Asian Pac J Trop Med 2022; 15(8):337-338.