Drakeley, CJ

Fivelman, Q

Saeed, M

Sutherland, C

Targett, GAT

English

LSHTM Research Online

Fivelman, QL; Butcher, GA; Adagu, IS; Warhurst, DC; Pasvol, G(2002) Malarone treatment failure and in vitro conﬁrmation of resis-tance of Plasmodium falciparum isolate from Lagos, Nigeria. MalarJ, 1. p. 1. ISSN 1475-2875Downloaded from: http://researchonline.lshtm.ac.uk/16311/Usage GuidelinesPlease refer to usage guidelines at http://researchonline.lshtm.ac.uk/policies.html or alterna-tively contact researchonline@lshtm.ac.uk.Available under license: Creative Commons Attribution http://creativecommons.org/licenses/by/2.5/BioMed  CentralPage 1 of 4(page number not for citation purposes)Malaria JournalMalaria Journal  2002,  1 Case reportMalarone treatment failure and in vitro confirmation of resistance of Plasmodium falciparum isolate from Lagos, NigeriaQuinton L Fivelman*1, Geoffrey A Butcher2, Ipemida S Adagu1, David C Warhurst1 and Geoffrey Pasvol3Address: 1Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London, WC1E 7HT, UK, 2Department of Biology, Sir Alexander Fleming Building, Impeial College, Imperial College Road, London, SW7 2AZ, UK and 3Department of Infection and Tropical Medicine, Faculty of Medicine, Imperial College, Lister Unit, Northwick Park Hospital, Harrow, Middlesex, HA1 3UJ, UKE-mail: Quinton L Fivelman* - quinton.fivelman@lshtm.ac.uk; Geoffrey A Butcher - g.butcher@ic.ac.uk; Ipemida S Adagu - ipemida.adagu@lshtm.ac.uk; David C Warhurst - david.warhurst@lshtm.ac.uk; Geoffrey Pasvol - g.pasvol@ic.ac.uk*Corresponding authorAbstractWe report the first in vitro and genetic confirmation of Malarone® (GlaxoSmithKline; atovaquoneand proguanil hydrochloride) resistance in Plasmodium falciparum acquired in Africa. On presentingwith malaria two weeks after returning from a 4-week visit to Lagos, Nigeria without prophylaxis,a male patient was given a standard 3-day treatment course of Malarone®. Twenty-eight days laterthe parasitaemia recrudesced. Parasites were cultured from the blood and the isolate (NGATV01)was shown to be resistant to atovaquone and the antifolate pyrimethamine. The cytochrome b geneof isolate NGATV01 showed a single mutation, Tyr268Asn which has not been seen previously.IntroductionIncreasing reports of drug-resistant P. falciparum through-out the world have forced changes in both prevention andtreatment. Malarone® (GlaxoSmithKline; atovaquone andproguanil hydrochloride) is a recently introduced newdrug combination for the treatment [1,2] and prophylaxis[3,4] of falciparum malaria. We report the first in vitro andgenetic confirmation of Malarone® resistance in a case ofP. falciparum acquired in Africa.Case ReportA forty-five year old Nigerian male, resident in the UK,presented with a fever and 1.5% P. falciparum parasitae-mia two weeks after returning from a 4-week visit to La-gos, Nigeria without taking prophylaxis. The patient wasgiven a standard 3-day treatment course of Malarone®;four tablets daily (one tablet is equivalent to 250 mg ofatovaquone and 100 mg of proguanil hydrochloride)with food which he tolerated well without vomiting andwas later discharged. Twenty-eight days later, his malariasymptoms returned. After a further five days the patientwas readmitted to hospital with a parasitaemia of lessthan 1 %. A blood sample taken at this point was placedinto culture. The patient was successfully treated with qui-nine 600 mg three times per day for three days followedby doxycycline 100 mg per day for seven days.Drug sensitivity assays were performed at 1 % parasitae-mia and 1 % haematocrit using tritiated hypoxanthine up-take as a measure of parasite viability [5] and the isolate(NGATV01) was shown to be resistant to atovaquone (Ta-ble 1). The NGATV01 isolate was also resistant to the an-tifolate pyrimethamine. The standard laboratory strain K1was assayed as above and exhibited resistance to bothPublished: 8 February 2002Malaria Journal 2002, 1:1Received: 19 December 2001Accepted: 8 February 2002This article is available from: http://www.malariajournal.com/content/1/1/1© 2002 ; licensee BioMed Central Ltd. Verbatim copying and redistribution of this article are permitted in any medium for any purpose, provided this notice is preserved along with the article's original URL.Malaria Journal 2002, 1 http://www.malariajournal.com/content/1/1/1Page 2 of 4(page number not for citation purposes)Table 1: In vitro sensitivity of isolate NGATV01 and strain K1 to standard antimalarial drugs with standard deviations (nmol/L).Drug NGATV01 K1 Resistance Cut-off*   Mean IC50 ± SD Mean IC50 ± SDChloroquine    9.54 ± 1.18   133.29 ± 30.12 100Mefloquine  24.14 ± 5.20    8.55 ± 0.29 20Pyrimethamine  16012.80 ± 2643.55     8082.84 ± 1202.69 100Atovaquone   1888.15 ± 106.65     2.41 ± 1.01 20Proguanil   4205.50 ± 716.99 10239.94 ± 843.51 not determinedDihydroartemisinin    2.39 ± 0.07     1.26 ± 0.46 not determinedDrug assay was performed at 1 % parasitaemia and 1 % haematocrit. Experiment was repeated twice in duplicate.* Cut-off points for resistance as previously reported [16,17,6]Figure 1Sequence analysis of P. falciparum CYT b gene from isolate NGATV01 showing codons 70 to 309. Residue 268 highlightedshows the change from tyrosine (Y) to asparagine (N) compared to atovaquone-sensitive strain K1 and the change to serine (S)in the atovaquone-resistant strain TM93-C1088 [6].K1         SGWCFRYMHATGASLVFLLTYLHILRGLNYSYMYLPLSWISGLILFMIFIVTAFVGYVLP 109TM93-C1088 SGWCFRYMHATGASLVFLLTYLHILRGLNYSYMYLPLSWISGLILFMIFIVTAFVGYVLP 109NGATV01    SGWCFRYMHATGASLVFLLTYLHILRGLNYSYMYLPLSWISGLILFMIFIVTAFVGYVLP 109K1         WGQMSYWGATVITNLLSSIPVAVIWICGGYTVSDPTIKRFFVLHFILPFIGLCIVFIHIF 179TM93-C1088 WGQMSYWGATVITNLLSSIPVAVIWICGGYTVSDPTIKRFFVLHFILPFIGLCIVFIHIF 179NGATV01    WGQMSYWGATVITNLLSSIPVAVIWICGGYTVSDPTIKRFFVLHFILPFIGLCIVFIHIF 179K1         FLHLHGSTNPLGYDTALKIPFYPNLLSLDVKGFNNVIILFLIQSLFGIIPLSHPDNAIVV 249TM93-C1088 FLHLHGSTNPLGYDTALKIPFYPNLLSLDVKGFNNVIILFLIQSLFGIIPLSHPDNAIVV 249NGATV01    FLHLHGSTNPLGYDTALKIPFYPNLLSLDVKGFNNVIILFLIQSLFGIIPLSHPDNAIVV 249K1         NTYVTPSQIVPEWYFLPFYAMLKTVPSKPAGLVIVLLSLQLLFLLAEQRSLTTIIQFKMI 309TM93-C1088 NTYVTPSQIVPEWYFLPFSAMLKTVPSKPAGLVIVLLSLQLLFLLAEQRSLTTIIQFKMI 309NGATV01    NTYVTPSQIVPEWYFLPFNAMLKTVPSKPAGLVIVLLSLQLLFLLAEQRSLTTIIQFKMI 309K1         FGARD 314TM93-C1088 FGARD 314NGATV01    FGARD 314Malaria Journal 2002, 1 http://www.malariajournal.com/content/1/1/1Page 3 of 4(page number not for citation purposes)chloroquine and pyrimethamine. The DNA of NGATV01was extracted and the cytochrome b coding region of mi-tochondrial DNA (mtDNA) sequenced [6] in both direc-tions together with DNA samples from P. falciparumcontrol strains. The sequence showed a change from TATto AAT in codon 268 (Figure 1), specifying a change fromtyrosine (Tyr) to asparagine (Asn): Y268N. A different mu-tation in this codon leading to serine was reported earlierin a sample (TM93-C1088) from an atovaquone and py-rimethamine treatment failure in a Thai patient [6]DiscussionThe target of atovaquone, CYT b, plays an important rolein electron transport during mitochondrial respiration. Itis thought that the drug, an analogue of coenzyme Q (ubi-quinone), interrupts electron transport and leads to lossof the mitochondrial membrane potential [7,8]. Tyr268 isa conserved bulky hydrophobic contact of the drug in theQo II region of the ubiquinol oxidation site. Substitutionof the less bulky Asn268 should affect the fit and bindingof the drug (Figure 2).Resistance rapidly emerges when atovaquone is usedalone [9]. It has been hypothesised that the mode of ac-tion of the drug might contribute to the rapid appearanceof resistant parasites. During a stage in its interaction withthe site when the drug is partially oxidised, the semiqui-none formed would be capable of forming reactive oxy-gen species (ROS) capable of acting as local mutagensduring replication of the mtDNA. Proguanil is believed tospeed the loss of the membrane potential, and ensure thatreplication of DNA stops before mutagenesis can occur[10].ConclusionsThis is an unusual example of resistance detected during asingle course of Malarone® on only a moderate parasitae-mia. The atovaquone/proguanil combination has notbeen widely used yet in West Africa so it is unlikely thatthe patient was initially infected with an atovaquone-re-sistant strain. The presence of multidrug-resistant strainssuch as this example raises concern about the recent moveto consider using Malarone as first-line therapy in Africa[11]. The case questions the potential useful life of thiscombination, especially as atovaquone may persist alonein plasma for up to 6 weeks after treatment [12]. It appearsthat the synergistic interaction with proguanil is not seenin atovaquone-resistant mutants [13], and higher resist-ance levels are achievable.AcknowledgementsWe thank Dr. Watcharee Chokejindachai and Dr. Jill Curtis of the London School of Hygiene and Tropical Medicine for technical advice and encour-Figure 2Atovaquone (ATV) in P. falciparum cytochrome b active site. A: Atovaquone built and docked using HyperChem release 6, inthe active site of a model of P. falciparum cytochrome B. Homology model prepared using the structure of the chicken enzyme[14] with the aid of the SWISS-MODEL Protein Modelling Server and observed in the Swiss Model Viewer [15]. B: As A, withactive site tyrosine268 replaced by asparagine.Tyr268 Tyr268ATV ATVa a-16 -16A AAsn268 Asn268ATV ATVa a-16 -16B BMalaria Journal 2002, 1 http://www.malariajournal.com/content/1/1/1Page 4 of 4(page number not for citation purposes)agement. Quinton Fivelman was supported by the Association of Common-wealth Universities and David Warhurst thanks UK PHLS for financial support.References1. Llanos-Cuentas A, Campos P, Clendenes M, Canfield CJ, HutchinsonDB:  Atovaquone and proguanil hydrochloride comparedwith chloroquine or pyrimethamine/sulfadoxine for treat-ment of acute Plasmodium falciparum malaria in Peru. Braz JInfect Dis 2001, 5:67-722. Looareesuwan S, Chulay JD, Canfield CJ, Hutchinson DB: Malarone(atovaquone and proguanil hydrochloride): a review of itsclinical development for treatment of malaria. MalaroneClinical Trials Study Group. Am J Trop Med Hyg 1999, 60:533-5413. 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AB: Atovaquone, a broad spectrum antiparasitic drug, collapses mitochondrial membrane potential in a malarial parasite.

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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

DB: Atovaquone and proguanil hydrochloride compared with chloroquine or pyrimethamine/sulfadoxine for treatment of acute Plasmodium falciparum malaria in Peru.

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

et al: Atovaquone-proguanil versus chloroquine-proguanil for malaria prophylaxis in non-immune travellers: a randomised, double-blind study. Malarone International Study Team. Lancet

et al: Atovaquone-Proguanil versus Mefloquine for Malaria Prophylaxis in Nonimmune Travellers: Results from a Randomized, Double-Blind Study. Clin Infect Dis

et al: In vitro response of Plasmodium falciparum to atovaquone and correlation with other antimalarials: comparison between African and Asian strains. Am J Trop Med Hyg

et al: Pathways for proton release during ubihydroquinone oxidation by the bc(1) complex.

Inhibition of the mosquito transmission of Plasmodium berghei by Malarone (atovaquone-proguanil). Ann Trop Med Parasitol

MW: Atovaquone resistance in malaria parasites. Drug Resist Updat

Protein modelling for all. Trends Biochem Sci

Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob Agents Chemother

Site of action of the antimalarial hydroxynaphthoquinone, 2-[trans-4-(4'-chlorophenyl) cyclohexyl]-3-hydroxy-1,4-naphthoquinone (566C80). Biochem Pharmacol

Snow RW: Sustainability, affordability, and equity of corporate drug donations: the case of Malarone. Lancet

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Introducing GSA: surface antigens of P-falciparum gametocyte-infected erythrocytes

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