Emergence of atovaquone-proguanil resistance during treatment of Plasmodium falciparum malaria acquired by a non-immune north American traveller to west Africa

The importation of drug-resistant malaria is a growing public health problem in non-endemic countries. The combination of atovaquone and proguanil (Malarone) has become established as an agent of choice to prevent and treat chloroquine-resistant
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  EMERGENCE OF ATOVAQUONE-PROGUANIL RESISTANCE DURINGTREATMENT OF  PLASMODIUM FALCIPARUM   MALARIA ACQUIRED BYA NON-IMMUNE NORTH AMERICAN TRAVELLER TO WEST AFRICA SUSAN KUHN, M. JOHN GILL,  AND  KEVIN C. KAIN Department of Pediatrics, Medicine, Medical Microbiology & Infectious Diseases, University of Calgary, Calgary, Alberta, Canada;McLaughlin-Rotman Global Health Program, McLaughlin Center for Molecular Medicine, University of Toronto; Toronto, Ontario,Canada; Tropical Disease Unit, Division of Infectious Diseases, Department of Medicine, Toronto General Hospital,Toronto, Ontario, Canada  Abstract.  The importation of drug-resistant malaria is a growing public health problem in non-endemic countries.The combination of atovaquone and proguanil (Malarone™) has become established as an agent of choice to preventand treat chloroquine-resistant  Plasmodium falciparum  malaria in travelers. We describe the first reported case in NorthAmerica of genetically confirmed atovaquone/proguanil-resistant  P. falciparum  malaria. Polymerase chain reaction andsequence analysis of the primary and recrudescent isolates confirmed the acquisition of a point mutation (Tyr268Ser) inthe cytochrome b gene of the recrudescent isolate known to confer high-level resistance to atovaquone. Suboptimaltherapy may have played a contributory role in the emergence of resistance.INTRODUCTIONThe combination of atovaquone and proguanil (Ma-larone™; GlaxoSmithKline, Research Triangle Park, NC) isthe latest drug to be approved by the Health ProtectionBranch of Health Canada and by the U.S. Food and DrugAdministration for the treatment and prevention of chloro-quine-resistant  Plasmodium falciparum  malaria in travelers.As with all antimalarial drugs, it is critical to identify thedevelopment and spread of resistance to monitor their effi-cacy and extend the effective lifespan of a drug. We report acase of atovaquone/proguanil (ATQ/PRO)−resistant  P. falci- parum  malaria in a Canadian traveler to Africa.CASE REPORTA previously healthy 25-year-old non-pregnant Canadianwoman lived for two and a half years in Freetown, SierraLeone. She took daily pyrimethamine for malaria prophylaxisand had no past history of malaria. One day after returning toCanada, she developed fever, chills, sweats, and headache.Malaria was suspected and blood smears were performed twodays after the onset of symptoms. Smears revealed  P. falci- parum  malaria. Although quantification was not availablethrough the rural regional laboratory where she presented,she did not meet World Health Organization clinical criteriafor severe malaria. 1 Treatment was initiated with ATQ/PRO using two tablets(250 mg of ATQ/100 mg of PRO per tablet) twice a day forthree days as outlined in a standard resource. 2 Therapy wasnot directly observed. She developed thrombocytopenia andher attending physician advised her to discontinue treatmentwith two tablets remaining in the total course. She defer-vesced and malaria smears were negative by day six of treat-ment.Fever recurred 19 days after therapy, and she was treatedempirically with the same regimen of ATQ/PRO (two tabletstwice a day for three days). A malaria smear was not done atthat time. All doses were taken and symptoms resolved withina few days of completion. Fever recurred 15 days later, andblood smears performed at this time confirmed the presenceofasexualstagesof   P. falciparum .Treatmentconsistedoforalquinine (600 mg three times a day) and doxycycline (100 mgtwice a day) for seven days. Symptoms resolved and repeatmalaria smears on days 7 and 28 post-treatment remainednegative.Blood specimens from the first and third episodes of ma-laria were submitted to a reference laboratory for amplifica-tion by polymerase chain reaction (PCR), sequence, and ge-netic fingerprint analysis. Unfortunately, quantification of parasitemia was not performed by the referring laboratoryand additional specimens were not available. This study wasreviewed and approved by the Institutional Review Board of the Toronto General Hospital. The DNA was extracted fromthe malaria isolate at presentation and from the second re-currence using columns obtained from Qiagen (Chatsworth,CA). 3 The gene encoding merozoite surface protein 1 (MSP-1) was amplified by a PCR and subjected to genetic finger-printing with single-strand conformational polymorphism(SSCP) analysis as described. 4 The MSP-1 gene fragmentfrom the recurrent isolate had an identical SSCP fingerprintto the day 0 isolate, confirming true treatment failure. 4 Para-site DNA was extracted from the malaria isolate at presen-tation and from the day of recurrence as above, and the cy-tochrome b and the dihydrofolate reductase (DHFR) geneswere amplified by PCR and sequenced to detect mutationsassociated with resistance to atovaquone and proguanil. 3,5 Sequence analysis of the clinical isolate at presentation hada wild-type sequence of cytochrome b. However, the recru-descent isolate had a mutation at position 268 specifying achange from tyrosine to serine (Figure 1). Both isolates hadmutations in the DHFR gene (S108N, C59R) associated withresistance to cycloguanil, the active metabolite of proguanil. 3 DISCUSSIONAtovaquone/proguanilisausefulagentforthetreatmentof imported uncomplicated  P. falciparum  malaria due to its con-venient route of administration (oral), short treatment course(three days), and attractive adverse-effect profile. Atova-quone causes parasite mitochondrial membrane collapse andinhibition of mitochondrial electron transport via the cyto-chrome b-c complex. 6 Resistance can result from a singlepoint mutation in plasmodial cytochrome b, resulting in re-duced binding affinity of atovaquone. 5  Am. J. Trop. Med. Hyg.,  72(4), 2005, pp. 407–409Copyright © 2005 by The American Society of Tropical Medicine and Hygiene 407  The pro-drug proguanil, rather than the major metabolitecycloguanil, appears to act synergistically with ATQ. 1,6 Re-sistance to ATQ/PRO  in vivo  has rarely been reported. Asof April 2004, a total of five cases of genetically confirmedATQ/PRO resistance had been reported, with all of theseassociated with single amino acid mutation at codon 268 of cytochrome b resulting in a change from tyrosine to serine (infour cases and the present case) 7 – 9 or tyrosine to alanine (onecase). 10 Several factors may contribute to emergence of ATQ orPRO resistance, including high parasite burdens, rapid me-tabolism of proguanil, exposure to suboptimal drug concen-trations, or prior exposure to related drugs. 11 In this case, ahigh parasitemia was not documented. Caucasians are knownto metabolize proguanil to cycloguanil relatively fast com-pared with other ethnic groups, 12 theoretically leaving longertime periods in which the parasites were exposed to ATQalone. However, there was no clear evidence to implicate thismechanism as a factor in the emergence of ATQ/PRO resis-tance in this case.Suboptimal drug levels may result from dosing errors, pa-tient non-adherence, or malabsorption. Although there wasno evidence of diarrhea, vomiting, or voluntary non-compli-anceinthiscase,thefirsttwoclinicalepisodesofmalariaweretreated with a divided dose of ATQ/PRO, which may haveresulted in a reduced peak drug level and a reduced timeabove the minimum parasiticidal concentration. Incompletetherapy with the first episode may also have been contributory.Most clinical trials of ATQ/PRO treatment have usedsingle daily dosing. One commonly used source recommendsa divided dose regimen to reduce gastrointestinal adverse ef-fects. 2 A single open label study has been published showingclinical success using this approach in 12 patients, 13 althoughunpublished pharmacokinetic data from the manufacturer(GlaxoSmithKline) also support efficacy when the drug isused in this manner.This patient had been taking pyrimethamine for long-termprophylaxis while living in west Africa and had mutationsobserved in the DHFR gene of the  P. falciparum  isolate.Pyrimethamine is not considered an effective drug for pre-vention, and like cycloguanil, it is a DHFR inhibitor. How-ever it does not have the same synergistic effect in combina-tion with atovaquone. 14 It is unlikely that there is cross-overresistance to proguanil when used in combination with atova-quone. However once cytochrome b mutations at position 268occur, then anti-malarial activity would be dependent on theanti-folate activity of cycloguanil and this would be expectedto be compromised by the observed mutations in DHFR. 3 F IGURE  1. DNA sequence analysis of the  Plasmodium falciparum  cytochrome b gene from the isolate at presentation ( left panel ) and at thetime of recrudescence ( right panel ).  Arrows  indicate the development of a single nucleotide substitution in the recrudescent isolate encodinga tyrosine (TAT) to serine (TCT) amino acid change at position 268. KUHN AND OTHERS 408  Atovaquone/proguanil remains an effective treatment op-tion for chloroquine-resistant  P. falciparum  malaria. It is notentirely clear what contributed to the emergence of resistancein this case, although incomplete therapy may have played arole. Given that there may not be a wide margin for dosingerrors, particularly in the face of a large parasite load, clini-cians should ensure close adherence to the complete treat-ment course of ATQ/PRO to increase the chance of success-ful treatment of   P. falciparum  malaria in a non-immune host.In addition, an effective alternative antimalarial therapy suchas quinine plus doxycycline, or artemether/lumefantrine (if available) should be used to treat recrudescent malaria afterclinical ATQ/PRO failure. 7,8 Received May 7, 2004. Accepted for publication September 17, 2004.Financial support: This work was supported by the Canadian Insti-tutes of Health Research (CIHR, MT-13721). Kevin C. Kain is sup-ported by a Career Scientist Award from the Ontario Ministry of Health and a Canada Research Chair (CIHR).Disclosure: Susan Kuhn is on the speaker ’ s bureau for GlaxoSmith-Kine, which produces Malarone ™ . This statement is made in theinterest of full disclosure, and not because the author considers this aconflict of interest.Authors ’  addresses: Susan Kuhn and M. John Gill, Department of Pediatrics, Medicine, Medical Microbiology & Infectious Diseases,University of Calgary, Calgary T2N 1N4, Alberta, Canada. Kevin C.Kain, Toronto General Hospital, 200 Elizabeth Street, ES 9-412,Toronto M5G 2C4, Ontario, Canada, Telephone: 416-340-3535, Fax:416-595-5826, E-mail: REFERENCES 1. Srivastava IK, Vaidya AB, 2003. A mechanism for the synergisticantimalarial action of atovaquone and proguanil.  Antimicrob Agents Chemother 43:  1334 – 1339.2. 2002. Drugs for parasitic infections.  Med Lett Drugs Ther 44:  32.3. Labbe AC, Patel S, Crandall I, Kain KC, 2003. Molecular sur-veillance system for global patterns of drug resistance in im-ported malaria.  Emerg Infect Dis 9:  33 – 36.4. Ohrt C, Mirabelli-Primdahl L, Looareesuwan S, Walsh D, KainKC, 1999. Determination of   Plasmodium falciparum  treatmentfailure by fingerprinting with PCR-SSCP.  Clin Infect Dis 28: 847 – 852.5. Korsinczky M, Chen N, Kotecka B, Saul A, Rieckmann K, ChengQ, 2000. Mutations in  Plasmodium falciparum  cytochrome bthat are associated with atovaquone resistance are located at aputative drug-binding site.  Antimicrob Agents Chemother 44: 2100 – 2108.6. McKeage K, Scott LJ, 2003. Atovaquone/proguanil.  Drugs 63: 597 – 623.7. Farnert A, Linkberg J, Gil P, Swedberg G, Berqvist Y, ThaparMM, Lindegardh N, Berezcky S, Bjorkman A, 2003. Evidenceof   Plasmodium falciparum  malaria resistance to atovaquoneand proguanil hydrochloride: case reports.  BMJ 326:  628 – 629.8. Schwartz E, Bujanover S, Kain KC, 2003. Genetic confirmationof atovaquone-proguanil-resistant  Plasmodium falciparum malaria acquired by a nonimmune traveler to east Africa.  Clin Infect Dis 37:  450 – 451.9. Schwobel B, Alifrangis M, Salanti A, Jelinek T, 2003. Differentmutation patterns of atovaquone resistance to  Plasmodium fal-ciparum in vitro  and  in vivo. Malar J 2:  5.10. Fivelman QL, Butcher GA, Adagu IS, Warhust DC, Pasvol G,2002. Malarone treatment failure and  in vitro  confirmation of resistance of   Plasmodium falciparum  isolate from Lagos, Ni-geria.  Malar J 1:  1.11. White NJ, 1998. Why is it that antimalarial drug treatments donot always work?  Ann Trop Med Parasitol 92:  449 – 458.12. Hussein Z, Eaves CJ, Hutchinson DB, Canfield CK, 1996. Popu-lation pharmacokinetics of proguanil in patients with acute P. falciparum  malaria after combined therapy with atova-quone.  Br J Clin Pharmacol 42:  589 – 597.13. Edstein MD, Looareesuwan S, Viravan C, Kyle DE, 1996. Phar-macokinetics of proguanil in malaria patients treated withproguanil plus atovaquone.  Southeast Asian J Trop Med PublicHealth 27:  216 – 220.14. Looareesuwan S, Viravan C, Webster HK, Kyle DE, HutchinsonDB, Canfield CK, 1996. Clinical studies of atovaquone, aloneor in combination with other antimalarial drugs, for treatmentof acute uncomplicated malaria in Thailand.  Am J Trop MedHyg 54:  62 – 66. ATOVAQUONE/PROGUANIL-RESISTANT  P. FALCIPARUM   MALARIA  409
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