Abstract
The emergence of chloroquine resistance in Plasmodium falciparum is a significant public health problem where malaria is endemic. We aimed to evaluate the efficacy of pyrosequencing to assess chloroquine resistance among P. falciparum isolates from the southwestern region of Saudi Arabia by analyzing the K76T and N86Y mutations in the P. falciparum chloroquine resistance transporter (PfCRT) and P. falciparum multidrug resistance 1 (PfMDR1) genes, respectively. Blood samples (n = 121) from microscopically positive P. falciparum cases were collected. DNA was extracted, and fragments from each of the genes were amplified by PCR using new sets of primers. The amplicons were sequenced using a pyrosequencer. All of the 121 samples were amplified for assessment of the PfCRT K76T and PfMDR1 N86Y mutations. All of the samples amplified for the PfCRT 76T mutation harbored the ACA codon (121/121; 100%), indicating the presence of the 76T mutation. For the PfMDR1 N86Y mutation, 72/121 samples (59.5%) had the sequence AAT at that position, indicating the presence of the wild-type allele (86N). However, 49/121 samples (40.5%) had a TAT codon, indicating the mutant allele (Y) at position 86. This study shows that pyrosequencing could be useful as a high throughput, rapid, and sensitive assay for the detection of specific single nucleotide polymorphisms in drug-resistant P. falciparum strains. This will help health authorities in malaria-endemic regions to adopt new malaria control strategies that will be applicable for diagnostic and drug resistance assays for malaria and other life-threatening pathogens that are endemic in their respective countries.
Similar content being viewed by others
References
Adagu IS, Warhurst DC (2001) Plasmodium falciparum: linkage disequilibrium between loci in chromosomes 7 and 5 and chloroquine selective pressure in Northern Nigeria. Parasitology 123:219–224
Ahmadian A, Ehn M, Hober S (2006) Pyrosequencing: history, biochemistry and future. Clin Chim Acta 363:83–94
Al-Tawfiq JA (2006) Epidemiology of travel-related malaria in a non-malarious area in Saudi Arabia. Saudi Med J 27:86–89
Arnold C, Westland L, Mowat G, Underwood A, Magee J, Gharbia S (2005) Single-nucleotide polymorphism-based differentiation and drug resistance detection in Mycobacterium tuberculosis from isolates or directly from sputum. Clin Microbiol Infect 11:122–130
Babiker HA, Pringle SJ, Abdel-Muhsin A, Mackinnon M, Hunt P, Walliker D (2001) High-level chloroquine resistance in Sudanese isolates of Plasmodium falciparum is associated with mutations in the chloroquine resistance transporter gene pfcrt and the multidrug resistance gene pfmdr1. J Infect Dis 183:1535–1538
Basco LK, Le Bras J, Rhoades Z, Wilson CM (1995) Analysis of pfmdr1 and drug susceptibility in fresh isolates of Plasmodium falciparum from subsaharan Africa. Mol Biochem Parasitol 74:157–166
Bashwari LA, Mandil AM, Bahnassy AA, Al-Shamsi MA, Bukhari HA (2001) Epidemiological profile of malaria in a university hospital in the eastern region of Saudi Arabia. Saudi Med J 22:133–138
Bin Dajem SM, Al-Qahtani A (2010) Analysis of gene mutations involved in chloroquine resistance in Plasmodium falciparum parasites isolated from patients in the southwest of Saudi Arabia. Ann Saudi Med 30:187–192
Bray PG et al (2006) PfCRT and the trans-vacuolar proton electrochemical gradient: regulating the access of chloroquine to ferriprotoporphyrin IX. Mol Microbiol 62:238–251
Chima RI, Goodman CA, Mills A (2003) The economic impact of malaria in Africa: a critical review of the evidence. Health Policy 63:17–36
Djimde A et al (2001) A molecular marker for chloroquine-resistant falciparum malaria. N Engl J Med 344:257–263
Dorsey G, Kamya MR, Singh A, Rosenthal PJ (2001) Polymorphisms in the Plasmodium falciparum pfcrt and pfmdr-1 genes and clinical response to chloroquine in Kampala, Uganda. J Infect Dis 183:1417–1420
Duraisingh MT, Cowman AF (2005) Contribution of the pfmdr1 gene to antimalarial drug-resistance. Acta Trop 94(3):181–190
Foley M, Tilley L (1998) Quinoline antimalarials: mechanisms of action and resistance and prospects for new agents. Pharmacol Ther 79:55–87
Hisaeda H et al (2008) Malaria parasites require TLR9 signaling for immune evasion by activating regulatory T cells. J Immunol 180:2496–2503
Krogstad DJ, Gluzman IY, Kyle DE, Oduola AM, Martin SK, Milhous WK, Schlesinger PH (1987) Efflux of chloroquine from Plasmodium falciparum: mechanism of chloroquine resistance. Science 238:1283–1285
Kublin JG et al (2002) Molecular markers for failure of sulfadoxine-pyrimethamine and chlorproguanil-dapsone treatment of Plasmodium falciparum malaria. J Infect Dis 185:380–388
Leoratti FM, Farias L, Alves FP, Suarez-Mutis MC, Coura JR, Kalil J, Camargo EP, Moraes SL, Ramasawmy R (2008) Variants in the toll-like receptor signaling pathway and clinical outcomes of malaria. J Infect Dis 198:772–780
Malik GM, Seidi O, El-Taher A, Mohammed AS (1998) Clinical aspects of malaria in the Asir Region, Saudi Arabia. Ann Saudi Med 18:15–17
Marfurt J et al (2008) Molecular markers of in vivo Plasmodium vivax resistance to amodiaquine plus sulfadoxine-pyrimethamine: mutations in pvdhfr and pvmdr1. J Infect Dis 198:409–417
Nagesha HS, Din S, Casey GJ, Susanti AI, Fryauff DJ, Reeder JC, Cowman AF (2001) Mutations in the pfmdr1, dhfr and dhps genes of Plasmodium falciparum are associated with in-vivo drug resistance in West Papua, Indonesia. Trans R Soc Trop Med Hyg 95:43–49
Nahlen BL, Korenromp EL, Miller JM, Shibuya K (2005) Malaria risk: estimating clinical episodes of malaria. Nature 437:E3, discussion E4-5
Nair S, Brockman A, Paiphun L, Nosten F, Anderson TJ (2002) Rapid genotyping of loci involved in antifolate drug resistance in Plasmodium falciparum by primer extension. Int J Parasitol 32:852–858
Nguyen-Dinh P, Payne D (1980) Pyrimethamine sensitivity in Plasmodium falciparum: determination in vitro by a modified 48-hour test. Bull World Health Organ 58:909–912
Omar MS, Malik GM, Al-Amari OM, Abdalla SE, Moosa RA (1999) The rapid manual ParaSight-F test for diagnosing Plasmodium falciparum malaria in Saudi Arabia. Ann Saudi Med 19:159–162
Plowe CV (2003) Monitoring antimalarial drug resistance: making the most of the tools at hand. J Exp Biol 206:3745–3752
Plowe CV (2009) The evolution of drug-resistant malaria. Trans R Soc Trop Med Hyg 103(Suppl 1):S11–S14
Rayavara K, Rajapandi T, Wollenberg K, Kabat J, Fischer ER, Desai SA (2009) A complex of three related membrane proteins is conserved on malarial merozoites. Mol Biochem Parasitol 167:135–143
Ronaghi M (2001) Pyrosequencing sheds light on DNA sequencing. Genome Res 11:3–11
Schonfeld M, Barreto Miranda I, Schunk M, Maduhu I, Maboko L, Hoelscher M, Berens-Riha N, Kitua A, Loscher T (2007) Molecular surveillance of drug-resistance associated mutations of Plasmodium falciparum in south-west Tanzania. Malar J 6:2
Schwab AE, Boakye DA, Kyelem D, Prichard RK (2005) Detection of benzimidazole resistance-associated mutations in the filarial nematode Wuchereria bancrofti and evidence for selection by albendazole and ivermectin combination treatment. Am J Trop Med Hyg 73:234–238
Shaio MF, Wang P, Lee CS, Sims PF, Hyde JE (1998) Development and comparison of quantitative assays for the dihydropteroate synthetase codon 540 mutation associated with sulfadoxine resistance in Plasmodium falciparum. Parasitology 116(Pt 3):203–210
Sreekumar C, Hill DE, Miska KB, Vianna MC, Yan L, Myers RL, Dubey JP (2005) Genotyping and detection of multiple infections of Toxoplasma gondii using pyrosequencing. Int J Parasitol 35:991–999
Takala SL, Smith DL, Stine OC, Coulibaly D, Thera MA, Doumbo OK, Plowe CV (2006) A high-throughput method for quantifying alleles and haplotypes of the malaria vaccine candidate Plasmodium falciparum merozoite surface protein-1 19kDa. Malar J 5:31
von Seidlein L, Duraisingh MT, Drakeley CJ, Bailey R, Greenwood BM, Pinder M (1997) Polymorphism of the Pfmdr1 gene and chloroquine resistance in Plasmodium falciparum in The Gambia. Trans R Soc Trop Med Hyg 91:450–453
Wahab T, Hjalmarsson S, Wollin R, Engstrand L (2005) Pyrosequencing Bacillus anthracis. Emerg Infect Dis 11:1527–1531
Wellems TE, Plowe CV (2001) Chloroquine-resistant malaria. J Infect Dis 184:770–776
Wernsdorfer WH, Noedl H (2003) Molecular markers for drug resistance in malaria: use in treatment, diagnosis and epidemiology. Curr Opin Infect Dis 16:553–558
WHO (2006) Guidelines for the treatment of malaria. WHO/HTM/MAL/2006.1108
WHO-EMRO. 2008. World malaria report 2008. http://www.who.int/malaria/publications/atoz/9789241563697/en/index.html
Wilson PE, Alker AP, Meshnick SR (2005) Real-time PCR methods for monitoring antimalarial drug resistance. Trends Parasitol 21:278–283
Zhou Z, Poe AC, Limor J, Grady KK, Goldman I, McCollum AM, Escalante AA, Barnwell JW, Udhayakumar V (2006) Pyrosequencing, a high-throughput method for detecting single nucleotide polymorphisms in the dihydrofolate reductase and dihydropteroate synthetase genes of Plasmodium falciparum. J Clin Microbiol 44:3900–3910
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Bin Dajem, S.M., Al-Sheikh, A.A.H., Bohol, M.F. et al. Detecting mutations in PfCRT and PfMDR1 genes among Plasmodium falciparum isolates from Saudi Arabia by pyrosequencing. Parasitol Res 109, 291–296 (2011). https://doi.org/10.1007/s00436-011-2251-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00436-011-2251-5