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

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.


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/mm 3 with 79.3% neutrophils, 12.9% lymphocytes and 0.6% eosinophils. The platelet count was 159,000 cells/mm 3 . 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 atovaquoneproguanil (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 and publish all well-documented cases of treatment failures because it is the only way to evaluate the level of resistance to atovaquone.

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 [2-4, 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 atovaquoneproguanil 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].

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

Availability of data and materials
The datasets analysed in this study are available from the corresponding author on reasonable request.