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Late clinical failure associated with cytochrome b codon 268 mutation during treatment of falciparum malaria with atovaquone–proguanil in traveller returning from Congo

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Abstract

Background

The drug combination atovaquone–proguanil, is recommended for treatment of uncomplicated falciparum malaria in France. Despite high efficacy, atovaquone–proguanil treatment failures have been reported. Resistance to cycloguanil, the active metabolite of proguanil, is conferred by multiple mutations in the Plasmodium falciparum dihydrofolate reductase (pfdhfr) and resistance to atovaquone by single mutation on codon 268 of the cytochrome b gene (pfcytb).

Case presentation

A 47-year-old female, native from Congo and resident in France, was admitted in hospital for uncomplicated falciparum malaria with parasitaemia of 0.5%, after travelling in Congo (Brazzaville and Pointe Noire). She was treated with atovaquone–proguanil (250 mg/100 mg) 4 tablets daily for 3 consecutive days. On day 5 after admission she was released home. However, many weeks after this episode, without having left France, she again experienced fever and intense weakness. On day 39 after the beginning of treatment, she consulted for fever, arthralgia, myalgia, photophobia, and blurred vision. She was hospitalized for uncomplicated falciparum malaria with a parasitaemia of 0.375% and treated effectively by piperaquine–artenimol (320 mg/40 mg) 3 tablets daily for 3 consecutive days. Resistance to atovaquone–proguanil was suspected. The Y268C mutation was detected in all of the isolates tested (D39, D42, D47). The genotyping of the pfdhfr gene showed a triple mutation (N51I, C59R, S108N) involved in cycloguanil resistance.

Conclusion

This is the first observation of a late clinical failure of atovaquone–proguanil treatment of P. falciparum uncomplicated malaria associated with pfcytb 268 mutation in a traveller returning from Congo. These data confirm that the Y268C mutation is associated with delayed recrudescence 4 weeks or more after initial treatment. Although atovaquone–proguanil treatment failures remain rare, an increased surveillance is required. It is essential to declare and publish all well-documented cases of treatment failures because it is the only way to evaluate the level of resistance to atovaquone.

Background

The drug combination atovaquone–proguanil, trade name Malarone, is recommended as the second-line treatment of uncomplicated falciparum malaria in adults in France [1]. Despite high efficacy, atovaquone–proguanil treatment failures have been reported [2,3,4,5,6,7,8,9,10,11,12,13,14,15,16]. Resistance to cycloguanil, the active metabolite of proguanil, is conferred by multiple mutations in the Plasmodium falciparum dihydrofolate reductase (pfdhfr) (N51I, C59R and S108N) [17]. Resistance to atovaquone is conferred by single mutation Y268S on the cytochrome b gene (pfcytb) [2,3,4, 8,9,10,11, 13], and much more rarely Y268C [3, 5, 7, 16] and Y268N [14]. In addition, resistance to atovaquone was also reported in patients with parasites without codon 268 mutation [5, 6, 13, 15]. Parasites carrying pfcytb codon 268 mutations are associated with delayed recrudescence 4 weeks or more after initial treatment, whereas recrudescence of parasites without codon 268 mutation appears more precociously after initial treatment [5, 18]. However, well-documented atovaquone–proguanil treatment failure remains extremely rare. This is the first report of genetic confirmation of atovaquone–proguanil resistance in P. falciparum isolate acquired in Congo.

Case presentation

A 47-year-old female, native from Congo and resident in France, was admitted 15 June, 2019 to the Emergency Unit of a private hospital in Nice, France. She had presented fever, headache and abnormal weakness for 2 days prior to admission. She had no history of underlying diseases, but had recently travelled to Congo (Brazzaville and Pointe Noire) from 31 May to 13 June, 2019. During her stay she took halofantrine as anti-malarial prophylaxis medication. This anti-malarial drug is not recommended for malaria prophylaxis. Halofantrine presents a risk of cardiac toxicity and its absorption is unreliable. On admission, her physical examination revealed fever, headache, arthralgia, and myalgia. No neurological deficits were found and the patient was haemodynamically stable. Laboratory studies showed C reactive protein of 62 mg/L, a haemoglobin concentration of 10.2 g/dL, a haematocrit 34%, a mean corpuscular volume (MCV) of 76 fL and a white cell count 3190 cells/mm3 with 79.3% neutrophils, 12.9% lymphocytes and 0.6% eosinophils. The platelet count was 159,000 cells/mm3. The liver function showed a hepatic cytolysis (ASAT 151 U/L; ALAT 129 U/L). The peripheral blood smear revealed the presence of trophozoïtes of P. falciparum with 0.5% of parasitaemia. She weighed 90 kg. She was hospitalized and treated on 16 June, 2019 at 1h30 am by atovaquone–proguanil (250 mg/100 mg) (Malarone®) 4 tablets daily for 3 consecutive days. The drug intake with food was monitored by nurses. The patient experienced no vomiting or diarrhea after drug administration. The patient was apyretic the day after the first dose of atovaquone–malarone. Control of parasitaemia, performed 3 days after the beginning of treatment, was 0.1%. On day 5 after admission, she did not show complaints or signs of any disease and parasites and was released home. However, many weeks after this episode, without any subsequent travel, she again experienced fever and intense weakness. On day 39 (24 July) after the beginning of treatment and because of the aggravation of symptoms, she consulted for fever, arthralgia, myalgia, photophobia, and blurred vision at the Emergency Unit of the teaching hospital of Nice. A blood smear stained with Giemsa revealed the presence of trophozoïtes of P. falciparum with 0.375% of parasitaemia. Resistance to atovaquone–proguanil was suspected. She was hospitalized in the Internal Medicine unit where she received medication of piperaquine-artenimol (320 mg/40 mg) 3 tablets daily for 3 consecutive days. After initiation of treatment the patient’s clinical outcome improved: fever, headache and myalgia disappeared and only weakness remained and lasted for several weeks after her stay in the teaching hospital. Peripheral blood smear controls performed at day 42, day 49 and day 66 (day 3, 7 and 27 after the second cure of treatment, respectively) found no trophozoïte and the patient was considered cured from her malaria episode.

The blood sampled for malaria researches were sent to the French National Reference Centre for Imported Malaria Study Group of Marseille (France) where Sanger sequencing of pfctyb for atovaquone resistance, pfdhfr for cycloguanil resistance, P. falciparum chloroquine resistance gene (pfcrt) for chloroquine resistance, P. falciparum Kelch propeller gene (K13) for artemisinin resistance and P. falciparum multidrug resistance 1 gene (pfmdr1) for lumefantrine resistance was performed as previously described [19,20,21]. The Y268C mutation was detected in all of the isolates collected during clinical treatment failure and follow-up (D39, D42, D47). The pre-treatment (D0) was not available. The molecular data are presented in Table 1. The genotyping of the pfdhfr gene showed a triple mutation (N51I, C59R, S108N) involved in cycloguanil resistance. The haplotype CVIET, associated with chloroquine resistance, was found [22]. No mutation was found in K13 propeller domain. The parasites carried a wild haplotype NYSND (codons 86, 184, 1034, 1042, 1246 on pfmdr1).

Table 1 Single nucleotide polymorphisms identified in P. falciparum cytochrome b gene (pfcytb), P. falciparum dihydrofolate reductase gene (pfdhfr), P. falciparum chloroquine resistance transporter gene (pfcrt), P. falciparum multidrug resistance 1 gene (pfmdr1), P. falciparum kelch propeller gene (pfK13) of samples collected at day 39, 42 and 47

Discussion and conclusion

This is the first described observation of a late clinical failure of atovaquone–proguanil treatment of P. falciparum uncomplicated malaria associated with pfcytb 268 mutation in a traveller returning from Congo. The Y268C mutation was identified in recrudescence on day 39 after initial treatment by atovaquone–proguanil. These data confirm analyses that showed that pfcytb codon 268 mutations are associated with delayed recrudescence 4 weeks or more after initial treatment [5, 18]. The cases of early treatment failures are not associated with codon 268 mutation [5, 6, 13, 15]. Although atovaquone–proguanil treatment failures remain rare, increased surveillance is required. It is essential to declare and publish all well-documented cases of treatment failures because it is the only way to evaluate the level of resistance to atovaquone. It is difficult to monitor atovaquone resistance by using in vitro testing or pfcytb mutation detection in general surveys in local and global parasites or in pre-treatment isolates. The codon 268 mutation or in vitro decreased susceptibility are rarely found in initial P. falciparum parasites before atovaquone–proguanil treatment and clinical failure [2, 4, 5, 8,9,10,11,12,13,14,15,16, 23] and in general surveys on unexposed P. falciparum parasites to atovaquone–proguanil due to low selective pressure in endemic areas [4, 19, 24,25,26,27,28,29]. As no D0 sample was available, it is difficult to conclude on whether this resistance was acquired during atovaquone–proguanil treatment or transmitted. However, this resistance is in almost all the cases described associated with acquisition and selection of cytochrome b mutation by parasites already resistant to cycloguanil during atovaquone–proguanil treatment [2,3,4, 10, 13, 16, 30]. Another hypothesis which argues for acquired resistance during atovaquone–proguanil treatment is that parasites carrying the 268C mutation in P. berghei ANKA cyb gene are unable to produce sporozoïte stages in the mosquito salivary glands or to infect mouse [31]. These parasites successfully generated oocysts but these oocysts had developmental defects. This lack of transmission also explains the low prevalence of pfcytb 268 mutations in endemic areas and the spread of atovaquone–proguanil resistance.

Plasma drug concentration is a factor of treatment failure. In absence of atovaquone plasma concentration measurement, underdosing seems anyway to be ruled out. Atovaquone–proguanil was correctly administered with food and the intake was monitored by nurses. The patient experienced no vomiting or diarrhea after drug administration. She weighed 90 kg. Patients with a body weight > 100 kg have a marked increased chance of treatment failure by underdosing compared with < 100 kg [32].

The investigation of atovaquone–proguanil treatment failure should continue and be reinforced in order to identify emergence and to monitor the spread of atovaquone resistance, and even more, if atovaquone–proguanil would be associated with artesunate or not as an alternative to artemisinin combination therapy in areas where P. falciparum parasites are multi-drug resistant, such as in Cambodia [29, 33].

Availability of data and materials

The datasets analysed in this study are available from the corresponding author on reasonable request.

Abbreviations

ALAT:

alanine aminotransferase

ASAT:

aspartate aminotransferase

MCV:

mean corpuscular volume

pfcrt :

P. falciparum chloroquine resistance gene

pfcytb :

P. falciparum cytochrome b gene

pfdhfr :

P. falciparum dihydrofolate reductase

K13 :

P. falciparum Kelch propeller gene

pfmdr1 :

P. falciparum multidrug resistance 1 gene

References

  1. 1.

    Epelboin L, Rapp C, Faucher JF, Méchaï F, Bottieau E, Matheron S, et al. Management and treatment of uncomplicated imported malaria in adults. Update of the French malaria clinical guidelines. Med Mal Infect. 2019. https://doi.org/10.1016/j.medmal.2019.07.011.

  2. 2.

    Savini H, Bogreau H, Bertaux L, Bouchiba H, Kraemer P, Parzy D, et al. First case of emergence of atovaquone–proguanil resistance in Plasmodium falciparum during treatment in a traveler in Comoros. Antimicrob Agents Chemother. 2008;52:2283–4.

  3. 3.

    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 Infect. 2006;8:2599–604.

  4. 4.

    Musset L, Pradines B, Parzy D, Durand D, Bigot P, Le Bras J. Apparent absence of atovaquone/proguanil resistance in 477 Plasmodium falciparum isolates from untreated French travellers. J Antimicrob Chemother. 2006;57:110–5.

  5. 5.

    Sutherland CJ, Laundy M, Price N, Burke M, Fivelman QL, Pasvol G, et al. Mutations in the Plasmodium falciparum cytochrome b gene are associated with delayed parasite recrudescence in malaria patients treated with atovaquone–proguanil. Malar J. 2008;7:240.

  6. 6.

    Wurtz N, Pascual A, Marin-Jauffre A, Bouchiba H, Benoit N, Desbordes M, et al. Early treatment failure during treatment of Plasmodium falciparum malaria with atovaquone–proguanil in the Republic of Ivory Coast. Malar J. 2012;11:146.

  7. 7.

    Perry TL, Pandey P, Grant JM, Kain KC. Severe atovaquone-resistant Plasmodium falciparum malaria in a Canadian traveller returned from the Indian subcontinent. Open Med. 2009;3:e10–6.

  8. 8.

    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;72:407–9.

  9. 9.

    Schwartz E, Bujanover S, Kain KC. Genetic confirmation of atovaquone–proguanil-resistant Plasmodium falciparum malaria acquired by a nonimmune traveler to East Africa. Clin Infect Dis. 2003;37:450–1.

  10. 10.

    Rose GW, Suh KN, Kain KC, Le Saux N, McCarthy AE. Atovaquone–proguanil resistance in imported falciparum malaria in a young child. Pediatr Infect Dis J. 2008;27:567–9.

  11. 11.

    Färnert A, Lindberg J, Gil P, Swedberg G, Berqvist Y, Thapar MM, et al. Evidence of Plasmodium falciparum malaria resistant to atovaquone and proguanil hydrochloride: case reports. BMJ. 2003;326:628–9.

  12. 12.

    Musset L, Le Bras J, Clain J. Parallel evolution of adaptative mutations in Plasmodium falciparum mitochondrial DNA during atovaquone–proguanil treatment. Mol Biol Evol. 2007;24:1582–5.

  13. 13.

    Plucinski MM, Huber CS, Akinyi S, Dalton W, Eschete M, Grady K, et al. Novel mutation in cytochrome B of Plasmodium falciparum in one of two atovaquone–proguanil treatment failures in travelers returning from same site in Nigeria. Open Forum Infect Dis. 2014;1:ofu059.

  14. 14.

    Fivelman QL, Butcher GA, Adagu IS, Warhurst DC, Pasvol G. Malarone treatment failure and in vitro confirmation of resistance of Plasmodium falciparum isolate from Lagos, Nigeria. Malar J. 2002;1:1.

  15. 15.

    Wichmann O, Muehlen M, Gruss H, Mockenhaupt FP, Suttorp N, Jelinek T. Malarone treatment failure not associated with previously described mutations in the cytochrome b gene. Malar J. 2004;3:14.

  16. 16.

    Aurégan C, Argy N, Hubert V, Aprahamian A, Clain J, Cheron G. Résistance acquise à l’atovaquone–proguanil dans un cas importé d’accès palustre simple à Plasmodium falciparum chez un jeune enfant. Presse Med. 2017;46:344–6.

  17. 17.

    Parzy D, Doerig C, Pradines B, Rico A, Fusai T, Doury C. Proguanil resistance in Plasmodium falciparum African isolates: assessment by mutation-specific polymerase chain reaction and in vitro susceptibility testing. Am J Trop Med Hyg. 1997;57:646–50.

  18. 18.

    Staines HM, Burrows R, Teo BH, Chis Ster I, Kremsner PG, Krishna S. Clinical implications of Plasmodium resistance to atovaquone/proguanil: a systematic review and meta-analysis. J Antimicrob Chemother. 2018;73:581–95.

  19. 19.

    Parola P, Pradines B, Simon F, Carlotti MP, Minodier P, Ranjeva MP, et al. Antimalarial drug susceptibility and point mutations associated with drug resistance in 248 Plasmodium falciparum isolates imported from Comoros to Marseille, France in 2004 2006. Am J Top Med Hyg. 2007;77:431–7.

  20. 20.

    Madamet M, Kounta MB, Wade KA, Lo G, Diawara S, Fall M, et al. Absence of association between polyporphisms in the K13 gene and the presence of Plasmodium falciparum parasites at day 3 after treatment with artemisinin derivatives in Senegal. Int J Antimicrob Agents. 2017;49:754–6.

  21. 21.

    Boussaroque A, Fall B, Madamet M, Wade KA, Fall M, Nakoulima A, et al. Prevalence of anti-malarial resistance genes in Dakar, Senegal from 2013 to 2014. Malar J. 2016;15:347.

  22. 22.

    Fidock DA, Nomura T, Talley AK, Cooper RA, Djekunov SM, Ferdig MT, et al. Mutations in the P. falciparum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance. Mol Cell. 2000;6:861–71.

  23. 23.

    Wichmann O, Muehlberger N, Jelinek T, Alifrangis M, Peyerl-Hoffmann G, Muhlen M, et al. Screening for mutations related to atovaquone/proguanil resistance in treatment failures and other imported isolates of Plasmodium falciparum in Europe. J Infect Dis. 2004;190:1541–6.

  24. 24.

    Pradines B, Hovette P, Fusai T, Atanda HL, Baret E, Cheval P, et al. Prevalence of in vitro resistance to eleven standard or new antimalarial drugs among Plasmodium falciparum isolates from Pointe-Noire, Republic of Congo. J Clin Microbiol. 2006;44:2404–8.

  25. 25.

    Muehlen M, Schreiber J, Ehrhardt S, Otchwemah R, Jelinek T, Bienzle U, et al. Short communication: prevalence of mutations associated with resistance to atovaquone and to the antifolate effect of proguanil in Plasmodium falciparum isolates from northern Ghana. Trop Med Int Health. 2004;9:361–3.

  26. 26.

    Ekala MT, Khim N, Legrand E, Randrianarivelojosia M, Jambou R, Fandeur T, et al. Sequence analysis of Plasmodium falciparum cytochrome b in multiple geographic sites. Malar J. 2007;6:164.

  27. 27.

    Pimentel S, Nogueira F, Benchimol C, Quinhentos V, Bom J, Varandas L, et al. Detection of atovaquone–proguanil resistance conferring mutations in Pasmodium falciparum cytochrome b gene in Luanda, Angola. Malar J. 2006;5:30.

  28. 28.

    Saunders DL, Chaorattanakawee S, Gosi P, Lanteri C, Somethy S, Kuntawunginn W, et al. Atovaquone–proguanil remains a potential stopgap therapy for multidrug-resistant Plasmodium falciparum in areas along the Thai-Cambodian border. Antimicrob Agents Chemother. 2015;60:1896–8.

  29. 29.

    Happi CT, Gbotosho GO, Folarin OA, Milner D, Sarr O, Sowunmi A, et al. Confirmation of emergence of mutations associated with atovaquone–proguanil resistance in unexposed Plasmodium falciparum isolates from Africa. Malar J. 2006;5:82.

  30. 30.

    Cottrell G, Musset L, Hubert V, Le Bras J, Clain J. Emergence of resistance to atovaquone–proguanil in malaria parasites: insights from computational modeling and clinical case reports. Antimicrob Agents Chemother. 2014;58:4504–14.

  31. 31.

    Goodman CD, Siregar JE, Mollard V, Vega-Rodriguez J, Syafruddin D, Matsuoka H, et al. Parasites resistant to the antimalarial atovaquone fail to transmit by mosquitoes. Science. 2016;352:349–53.

  32. 32.

    Nixon GL, Moss DM, Shane AE, Lalloo DG, Fisher N, O’Neill PM, et al. Antimalarial pharmacology and therapeutics of atovaquone. J Antimicrob Chemother. 2013;68:977–85.

  33. 33.

    Wojnarski M, Lon C, Vanachayangkul P, Gosi P, Sok S, Rachmat A, et al. Atovaquone–proguanil in combination with artesunate to treat multidrug-resistant P. falciparum malaria in Cambodia: an open label randomized trial. Open Forum Infect Dis. 2019;6:ofz314.

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Funding

This research was supported by the French Institute for Public Health Surveillance (Santé Publique France, grant CNR paludisme).

Author information

LM, CP, AC, VM, PYJ, PD and PM carried out diagnostic, monitoring of the patient, collection of clinical and epidemiological data. MM, NB, IF, RA, JM carried out the molecular studies. LM, MM and BP analysed the data and drafted the manuscript. All authors read and approved the final manuscript.

Correspondence to Bruno Pradines.

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Bio-banking of human clinical samples used for malaria diagnostics and secondary uses for scientific purposes is possible as long as the corresponding patients are informed and have not indicated any objections. This requirement was fulfilled here by giving verbal information to the patients, and no immediate or delayed patient opposition was reported to the hospital clinicians. Informed consent was not required for this study because the sampling procedures and testing are part of the French national recommendations for the care and surveillance of malaria. This work was performed under the statutory auspices of the French national reference centre for imported malaria, and isolates were anonymized by re-coding.

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Not applicable.

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The authors declare that they have no competing interests.

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Massamba, L., Madamet, M., Benoit, N. et al. Late clinical failure associated with cytochrome b codon 268 mutation during treatment of falciparum malaria with atovaquone–proguanil in traveller returning from Congo. Malar J 19, 37 (2020). https://doi.org/10.1186/s12936-020-3126-y

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Keywords

  • Malaria
  • Plasmodium falciparum
  • Anti-malarial drug
  • Resistance
  • In vitro
  • Atovaquone
  • Proguanil
  • Cytochrome b

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