Papers

Evidence of Plasmodium falciparum malaria resistant to atovaquone and proguanil hydrochloride: case reports

Anna Färnert, medical doctor ^a, Johan Lindberg, associate professor ^b, Pedro Gil, post doctoral researcher ^d, Göte Swedberg, associate professor ^c, Yngve Berqvist, associate professor ^e, Mita M Thapar, PhD student ^a, Niklas Lindegårdh, PhD student ^e, Sándor Berezcky, PhD student ^a, A Björkman, professor ^a. 

^a Department of Medicine, Division of Infectious Diseases, Karolinska Institute, Karolinska Hospital, S-171 76 Stockholm, Sweden, ^b Department of Infectious Diseases, Sahlgrenska University Hospital, Östra, Gothenburg, Sweden, ^c Department of Pharmaceutical Biosciences, Division of Microbiology, Uppsala University, Uppsala, Sweden, ^d Centro de Malária e outras Doencas Tropicais, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisboa, Portugal, ^e Department of Clinical Chemistry, Falun Central Hospital, Falun and Dalarna University College, Borlänge, Sweden

Correspondence to: A Färnert anna.farnert{at}medks.ki.se

The increased spread of drug resistant malaria highlights the need for alternatives for treatment and chemoprophylaxis. The combination of atovaquone and proguanil hydrochloride (Malarone, GlaxoSmithKline, NC) has shown high efficacy against Plasmodium falciparum with only mild side effects and has been registered for use in several countries, including Denmark, Germany, Sweden, the United Kingdom, and the United States.¹ Treatment failures have been attributed to suboptimal dosage, reinfections, or to a point mutation in the cytochrome b gene. ^{1 2} Bioavailability of atovaquone depends on the concomitant intake of a fatty diet, yet drug concentrations were not analysed in these reports. We provide evidence of resistance in two patients treated with atovaquone and proguanil hydrochloride for P falciparum infection.

	Methods and results

Top Methods and results Comment References

In September 2000, two boys and their mother were diagnosed as having P falciparum malaria at the university hospital in Gothenburg, after returning from an eight week visit to the Ivory Coast. They had taken chloroquine weekly and proguanil daily for chemoprophylaxis against malaria. Case 1, the youngest boy (18 months), had fever, convulsions, and 1% infected erythrocytes. He was treated with atovaquone and proguanil hydrochloride (table). His fever continued, and he had a few convulsions but no impaired consciousness or other signs of severe malaria. On day 2 the parasitaemia was 4%. Treatment was changed to mefloquine, which was successful. His 4 year old brother, case 2, was febrile with 0.5% infected erythrocytes. His symptoms resolved with atovaquone and proguanil hydrochloride, and the parasites were cleared after three days. On day 28 he was again febrile with P falciparum parasites and was successfully treated with mefloquine. The mother (case 3), although asymptomatic, had a few P falciparum rings. She was treated with atovaquone and proguanil hydrochloride, which cleared the parasites without recrudescence.

Drug concentrations were measured by high pressure liquid chromatography in repeated serum samples. The concentrations of atovaquone (7.6-13.9 µM), proguanil (300-1200 nM), and cycloguanil (125-400 nM) were all above the levels considered therapeutic in children and adults.³ Treatment failure could therefore not be attributed to poor bioavailability of the drug.

Cases 1 and 3 were infected with single clones whereas case 2 had five genetically diverse parasite populations, detected by analysis of merozoite surface proteins 1 and 2. Mutation A803G (changing tyrosine to serine in 268) in cytochrome b, related to resistance to atovaquone, was detected in cases 2 and 3 by polymerase chain reaction and restriction fragment length polymorphism of loci 133, 268, 272, 275, 280, and 284, and confirmed by sequencing (table).⁴ However, only wild types were found in case 1. Analyses of loci 51, 59, 108, and 164 in the dihydrofolate reductase gene, related to resistance to proguanil and cycloguanil, revealed wild types in all samples except those from case 2, in which triple mutation were found at recrudescence.⁵

	Comment

Top Methods and results Comment References

Treatment of three patients with atovaquone and proguanil hydrochloride for P falciparum malaria was unsuccessful in two non-immune children but successful in an adult with probable partial protective immunity. The patients had adequate blood concentrations of the drugs, indicating resistance by P falciparum. Mutation in cytochrome b may have contributed to treatment failure but cannot be the only mechanism for resistance to the drug combination because it was also detected in the patient who responded well and was not detected in the patient with early treatment failure. Atovaquone and proguanil hydrochloride represents one of the main new developments in malaria chemotherapy, but because of the resistance shown at this early stage there is a need for careful surveillance of drug efficacy.

	Acknowledgments

Contributors: JL treated the patients. AF coordinated the analyses and wrote the paper with the assistance of the other authors, specifically AB. NL, YB, and MT performed the pharmacological analyses. PG, GS, and SB performed the genetic analyses. AF will act as guarantor for the paper.

	Footnotes

Funding: None.

Competing interests: JL has been reimbursed by GlaxoSmith Kline for speaking at a meeting.

	References

Top Methods and results Comment References

1.	Looareesuwan S, Chulay JD, Canfield CJ, Hutchinson DB. Malarone (atovaquone and proguanil hydrochloride): a review of its clinical development for treatment of malaria. Am J Trop Med Hyg 1999; 60: 533-541[Abstract].
2.	Fivelman QL, Butcher GA, Adagu IS, Warhurst DC, Pasvol G. Malarone treatment failure and in vitro confirmation of resistance of Plasmodium falciparum isolates from Lagos, Nigeria. Malaria J 2002; 1: 1.
3.	Sabchareon A, Attanath P, Phanuaksook P, Chanthavanich P, Poonpanich Y, Mookmanee D, et al. Efficacy and pharmacokinetics of atovaquone and proguanil in children with multidrug-resistant Plasmodium falciparum malaria. Trans R Soc Trop Med Hyg 1998; 92: 201-206[CrossRef][Web of Science][Medline].
4.	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[Abstract/Free Full Text].
5.	Sirawaraporn W, Sathitkul T, Sirawaraporn R, Yuthavong Y, Santi DV. Antifolate-resistant mutants of Plasmodium falciparum dihydrofolate reductase. Proc Natl Acad Sci USA 1997; 94: 1124-1129[Abstract/Free Full Text].

(Accepted 9 January 2003)