Confirmation of emergence of mutations associated with atovaquone-proguanil resistance in unexposed Plasmodium falciparum isolates from Africa

Christian T Happi, Grace O Gbotosho, Onikepe A Folarin, Danny Milner, Ousmane Sarr, Akintunde Sowunmi, Dennis E Kyle, Wilbur K Milhous, Dyann F Wirth, Ayoade M J Oduola, Christian T Happi, Grace O Gbotosho, Onikepe A Folarin, Danny Milner, Ousmane Sarr, Akintunde Sowunmi, Dennis E Kyle, Wilbur K Milhous, Dyann F Wirth, Ayoade M J Oduola

Abstract

Background: In vitro and in vivo resistance of Plasmodium falciparum to atovaquone or atovaquone-proguanil hydrochloride combination has been associated to two point mutations in the parasite cytochrome b (cytb) gene (Tyr268Ser and Tyr268Asn). However, little is known about the prevalence of codon-268 mutations in natural populations of P. falciparum without previous exposure to the drug in Africa.

Methods: The prevalence of codon-268 mutations in the cytb gene of African P. falciparum isolates from Nigeria, Malawi and Senegal, where atovaquone-proguanil has not been introduced for treatment of malaria was assessed. Genotyping of the cytb gene in isolates of P. falciparum was performed by PCR-restriction fragment length polymorphism and confirmed by sequencing.

Results: 295 samples from Nigeria (111), Malawi (91) and Senegal (93) were successfully analyzed for detection of either mutant Tyr268Ser or Tyr268Asn. No case of Ser268 or Asn268 was detected in cytb gene of parasites from Malawi or Senegal. However, Asn268 was detected in five out of 111 (4.5%) unexposed P. falciparum isolates from Nigeria. In addition, one out of these five mutant Asn268 isolates showed an additional cytb mutation leading to a Pro266Thr substitution inside the ubiquinone reduction site.

Conclusion: No Tyr268Ser mutation is found in cytb of P. falciparum isolates from Nigeria, Malawi or Senegal. This study reports for the first time cytb Tyr268Asn mutation in unexposed P. falciparum isolates from Nigeria. The emergence in Africa of P. falciparum isolates with cytb Tyr268Asn mutation is a matter of serious concern. Continuous monitoring of atovaquone-proguanil resistant P. falciparum in Africa is warranted for the rational use of this new antimalarial drug, especially in non-immune travelers.

Figures

Figure 1
Figure 1
Detection of Asn268 mutation in cytochrome b gene in P. falciparum by Restriction digest method. 174 bp amplification products with the secondary amplification primer pair cytb2/cytb7 (lanes 3, 5, 7, 9) digested with SspI (lanes 2, 4, 6, 8) and were run on 2% NuSieve® 3:1 agarose gel. DNA from NGATV01 containing the AAT (Asn) mutation remains uncut (lane 6), while DNA from K1 containing the TAT (Tyr) wild-type codon is digested (150 bp) by the enzyme (lane 8). DNA from patient ID011 showed a mixed infection consisting both the TAT (Tyr) wild-type digested (150 bp) and mutant undigested (174 bp) codons (lane 2). Patient ID024 had parasites harboring the mutant (Asn268) allele of cytb as their DNA remained uncut (lane 4) by the enzyme. Lanes 1 and 10 represent the low molecular weight DNA ladder (New England Biolabs, Beverly, MA) used as a marker for the electrophoresis
Figure 2
Figure 2
Multiple sequences alignment of cytochrome b gene (residues 137 to 279) of some Nigerian isolates of P. falciparum. Highlighted are residues 268 with amino acid changes from the wild-type tyrosine (Y) to the mutant asparagine (N) allele associated previously with atovaquone resistance in Plasmodium falciparum. In addition residue 266 in patient ID (Cytb012) where Pro (P) is changed to Thr (T) is also highlighted. Sequences of the atovaquone resistant (NGTV01) and sensitive (K1 and 3D7) control strains are also present.

References

    1. Milhous WK. Development of new drugs for chemoprophylaxis of malaria. Med Trop (Mars) 2001;61:48–50.
    1. Matsuu A, Koshida Y, Kawahara M, Inoue K, Ikadai H, Hikasa Y, Okano S, Higuchi S. Efficacy of atovaquone against Babesia gibsoni in vivo and in vitro. Vet Parasitol. 2004;124:9–18. doi: 10.1016/j.vetpar.2004.07.005.
    1. Araujo FG, Huskinson J, Remington JS. Remarkable in vitro and in vivo activities of the hydroxynaphthoquinone 566C80 against tachyzoites and tissue cysts of Toxoplasma gondii. Antimicrob Agents Chemother. 1991;35:293–299.
    1. Fry M, Pudney M. Site of action of the antimalarial hydroxynaphthoquinone, 2-[trans-4-(4'-chlorophenyl) cyclohexyl]-3-hydroxy-1,4-naphthoquinone (566C80) Biochem Pharmacol. 1992;43:1545–1553. doi: 10.1016/0006-2952(92)90213-3.
    1. Srivastava IK, Rottenberg H, Vaidya AB. Atovaquone, a broad spectrum antiparasitic drug, collapses mitochondrial membrane potential in a malarial parasite. J Biol Chem. 1997;272:3961–3966. doi: 10.1074/jbc.272.52.33360.
    1. Kessl JJ, Lange BB, Merbitz-Zahradnik T, Zwicker K, Hill P, Meunier B, Palsdottir H, Hunte C, Meshnick S, Trumpower BL. Molecular basis for atovaquone binding to the cytochrome bc1 complex. J Biol Chem. 2003;278:31312–31318. doi: 10.1074/jbc.M304042200.
    1. Kessl JJ, Ha KH, Merritt AK, Lange BB, Hill P, Meunier B, Meshnick SR, Trumpower BL. Cytochrome b mutations that modify the ubiquinol-binding pocket of the cytochrome bc1 complex and confer anti-malarial drug resistance in Saccharomyces cerevisiae. J Biol Chem. 2005;280:17142–17148. doi: 10.1074/jbc.M500388200.
    1. Chiodini PL, Conlon CP, Hutchinson DB, Farquhar JA, Hall AP, Peto TE, Birley H, Warrell DA. Evaluation of atovaquone in the treatment of patients with uncomplicated Plasmodium falciparum malaria. J Antimicrob Chemother. 1995;36:1073–1078.
    1. Looareesuwan S, Viravan C, Webster HK, Kyle DE, Hutchinson DB, Canfield CJ. Clinical studies of atovaquone, alone or in combination with other antimalarial drugs, for treatment of acute uncomplicated malaria in Thailand. Am J Trop Med Hyg. 1996;54:62–66.
    1. Looareesuwan S, Chulay JD, Canfield CJ, Hutchinson DB. Malarone (atovaquone and proguanil hydrochloride): a review of its clinical development for treatment of malaria. Malarone Clinical Trials Study Group. Am J Trop Med Hyg. 1999;60:533–541.
    1. Musset L, Pradines B, Parzy D, Durand R, Bigot P, Le Bras J. Apparent absence of atovaquone/proguanil resistance in 477 Plasmodium falciparum isolates from untreated French travellers. J Antimicrob Chemother. 2006;57:110–115. doi: 10.1093/jac/dki420.
    1. Musset L, Bouchaud O, Matheron S, Massias L, Le Bras J. Clinical atovaquone-proguanil resistance of Plasmodium falciparum associated with cytochrome b codon 268 mutations. Microbes and Infect. 2006. doi:10.1016/j.micinf.2006.07.011.
    1. Srivastava I, Vadiya A. A mechanism for the synergistic antimalarial action of atovaquone and proguanil. Antimicrob Agents Chemother. 1999;43:1334–1339.
    1. Anabwani G, Canfield CJ, Hutchinson DB. Combination atovaquone and proguanil hydrochloride vs. halofantrine for treatment of acute Plasmodium falciparum malaria in children. Pediatr Infect Dis J. 1999;18:456–461. doi: 10.1097/00006454-199905000-00011.
    1. Mulenga M, Sukwa TY, Canfield CJ, Hutchinson DB. Atovaquone and proguanil versus pyrimethamine/sulfadoxine for the treatment of acute falciparum malaria in Zambia. Clin Ther. 1999;21:841–852. doi: 10.1016/S0149-2918(99)80006-X.
    1. Llanos-Cuentas A, Campos P, Clendenes M, Canfield CJ, Hutchinson DB. Atovaquone and proguanil hydrochloride compared with chloroquine or pyrimethamine/sulfodaxine for treatment of acute Plasmodium falciparum malaria in Peru. Braz J Infect Dis. 2001;5:67–72. doi: 10.1590/S1413-86702001000200004.
    1. Camus D, Djossou F, Schilthuis HJ, Hogh B, Dutoit E, Malvy D, Roskell NS, Hedgley C, De Boever EH, Miller GB. Atovaquone-proguanil versus chloroquine-proguanil for malaria prophylaxis in nonimmune pediatric travelers: results of an international, randomized, open-label study. Clin Infect Dis. 2004;38:1716–1723. doi: 10.1086/421086.
    1. Petersen E. The safety of atovaquone/proguanil in long-term malaria prophylaxis of non-immune adults. J Travel Med. 2003;10:S13–15. discussion S21.
    1. Srivastava IK, Morrisey JM, Darrouzet E, Daldal F, Vaidya AB. Resistance mutations reveal the atovaquone-binding domain of cytochrome b in malaria parasites. Mol Microbiol. 1999;33:704–711. doi: 10.1046/j.1365-2958.1999.01515.x.
    1. Korsinczky M, Chen N, Kotecka B, Saul A, Rieckmann K, Cheng Q. Mutations in Plasmodium falciparum cytochrome b that are associated with atovaquone resistance are located at a putative drug-binding site. Antimicrob Agents Chemother. 2000;44:2100–2108. doi: 10.1128/AAC.44.8.2100-2108.2000.
    1. Suswam E, Kyle D, Lang-Unnasch N. Plasmodium falciparum: the effects of atovaquone resistance on respiration. Exp Parasitol. 2001;98:180–187. doi: 10.1006/expr.2001.4639.
    1. Fivelman QL, Butcher GA, Adagu IS, Warhurst DC, Pasvol G. Malarone treatment failure and in vitro confirmationof resistance of Plasmodium falciparum isolate from Lagos, Nigeria. Malar J. 2002;1:1. doi: 10.1186/1475-2875-1-1.
    1. Fivelman QL, Adagu IS, Warhurst DC. Modified fixed-ratio isobologram method for studying in vitro interactions between atovaquone and proguanil or dihydroartemisinin against drug-resistant strains of Plasmodium falciparum. Antimicrob Agents Chemother. 2004;48:4097–4102. doi: 10.1128/AAC.48.11.4097-4102.2004.
    1. Farnert A, Lindberg J, Gil P, Swedberg G, Berqvist Y, Thapar MM, Lindegardh N, Berezcky S, Bjorkman A. Evidence of Plasmodium falciparum malaria resistant to atovaquone and proguanil hydrochloride: case reports. Bmj. 2003;326:628–629. doi: 10.1136/bmj.326.7390.628.
    1. Schwobel B, Alifrangis M, Salanti A, Jelinek T. Different mutation patterns of atovaquone resistance to Plasmodium falciparum in vitro and in vivo: rapid detection of codon 268 polymorphisms in the cytochrome b as potential in vivo resistance marker. Malar J. 2003;2:5. doi: 10.1186/1475-2875-2-5.
    1. David KP, Alifrangis M, Salanti A, Vestergaard LS, Ronn A, Bygbjerg IB. Atovaquone/proguanil resistance in Africa: a case report. Scand J Infect Dis. 2003;35:897–898.
    1. Kuhn S, Gill MJ, Kain KC. Emergence of atovaquone-proguanil resistance during treatment of Plasmodium falciparum malaria acquired by a non-immune north American traveller to west Africa. Am J Trop Med Hyg. 2005;72:407–409.
    1. Wichmann O, Muehlberger N, Jelinek T, Alifrangis M, Peyerl-Hoffmann G, Muhlen M, Grobusch MP, Gascon J, Matteelli A, Laferl H, Bisoffi Z, Ehrhardt S, Cuadros J, Hatz C, Gjorup I, McWhinney P, Beran J, da Cunha S, Schulze M, Kollaritsch H, Kern P, Fry G, Richter J, European Network on Surveillance of Imported Infectious Diseases Screening for mutations related to atovaquone/proguanil resistance in treatment failures and other imported isolates of Plasmodium falciparum in Europe. J Infect Dis. 2004;190:1541–1546. doi: 10.1086/424469.
    1. Gil JP, Nogueira F, Stromberg-Norklit J, Lindberg J, Carrolo M, Casimiro C, Lopes D, Arez AP, Cravo PV, Rosario VE. Detection of atovaquone and Malarone® resistance conferring mutations in Plasmodium falciparum cytochrome b gene (cytb) Mol Cell Probes. 2003;17:85–89. doi: 10.1016/S0890-8508(03)00006-9.
    1. Pimentel S, Nogueira F, Benchimol C, Quinhentos V, Bom J, Varandas L, do Rosario V, Bernardino L. Detection of atovaquone-proguanil resistance conferring mutations in Plasmodium falciparum cytochrome b gene in Luanda, Angola. Malar J. 2006;5:30. doi: 10.1186/1475-2875-5-30.
    1. Happi CT, Gbotosho GO, Folarin OA, Akinboye DO, Yusuf BO, Ebong OO, Sowunmi A, Kyle DE, Milhous W, Wirth DF, Oduola AMJ. Polymorphisms in Plasmodium falciparum dhfr and dhps genes and age related in vivo sulfadoxine-pyrimethamine resistance in malaria-infected patients from Nigeria. Acta Trop. 2005;95:183–193. doi: 10.1016/j.actatropica.2005.06.015.
    1. Plowe CV, Djimde A, Bouare M, Doumbo O, Wellems TE. Pyrimethamine and proguanil resistance-conferring mutations in Plasmodium falciparum dihydrofolate reductase: polymerase chain reaction methods for surveillance in Africa. Am J Trop Med Hyg. 1995;52:565–568.
    1. Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004;32:1792–1797. doi: 10.1093/nar/gkh340.
    1. Fisk DT, Meshnick S, Kazanjian PH. Pneumocystis carinii pneumonia in patients in the developing world who have acquired immunodeficiency syndrome. Clin Infect Dis. 2003;36:70–78. doi: 10.1086/344951.
    1. Basselin M, Hunt SM, Abdala-Valencia H, Kaneshiro ES. Ubiquinone synthesis in mitochondrial and microsomal subcellular fractions of Pneumocystis spp.: differential sensitivities to atovaquone. Eukaryot Cell. 2005;4:1483–1492. doi: 10.1128/EC.4.8.1483-1492.2005.

Source: PubMed

Sponsors en medewerkers

Medische omstandigheden

Geneesmiddelinterventies

3
Abonneren