Early treatment failure during treatment of Plasmodium falciparum malaria with atovaquone-proguanil in the Republic of Ivory Coast

Malaria Journal Early treatment failure during treatment of Plasmodium falciparum malaria with atovaquone-proguanil in the Republic of Ivory Coast Nathalie Wurtz 0 1 3 4 Aurlie Pascual 0 1 3 4 Adeline Marin-Jauffre 1 4 Housem Bouchiba 1 4 Nicolas Benoit 0 2 3 Marc Desbordes 0 2 3 Maryse Martelloni 0 2 3 Vincent Pommier de Santi 6 Georges Richa 5 Nicolas Taudon 0 2 3 Bruno Pradines 0 1 3 4 Sbastien Briolant 1 4 0 Centre National de Reference du Paludisme , Marseille , France 1 Unite de Parasitologie - Unite de Recherche pour les Maladies Infectieuses et Tropicales Emergentes - UMR 6236, Institut de Recherche Biomedicale des Armees , Marseille , France 2 UMR MD3 Infections Parasitaires: Transmission, Physiopathologie et Therapeutique, Aix-Marseille Universite, Institut de Recherche Biomedicale des Armees , Marseille , France 3 Centre National de Reference du Paludisme , Marseille , France 4 Unite de Parasitologie - Unite de Recherche pour les Maladies Infectieuses et Tropicales Emergentes - UMR 6236, Institut de Recherche Biomedicale des Armees , Marseille , France 5 Centre medical des armees de Nimes Orange Laudun, antenne colonel De Chabrieres , Nimes , France 6 Centre d'Epidemiologie et de Sante Publique des Armees , Marseille , France The increased spread of drug-resistant malaria highlights the need for alternative drugs for treatment and chemoprophylaxis. The combination of atovaquone-proguanil (MalaroneW) has shown high efficacy against Plasmodium falciparum with only mild side-effects. Treatment failures have been attributed to suboptimal dosages or to parasite resistance resulting from a point mutation in the cytochrome b gene. In this paper, a case of early treatment failure was reported in a patient treated with atovaquone-proguanil; this failure was not associated with a mutation in the parasite cytochrome b gene, with impaired drug bioavailability, or with re-infection. Malaria; Plasmodium falciparum; MalaroneW; Atovaquone-proguanil; Cytochrome b; Resistance; Clinical failure; in vitro; Anti-malarial drug - Background Increasing reports of drug-resistant Plasmodium falciparum throughout the world have forced changes in both prevention and treatment. Atovaquone-proguanil (A-P, MalaroneW, GlaxoSmithKline Inc) is one of the common first-line agent for the prophylaxis [1-3] and treatment [2,4,5] of falciparum malaria in France and causes only mild side-effects. Since the introduction of the A-P combination, several cases of treatment failure have been observed in travellers returning from Africa [6-14]. Treatment failures have been attributed to suboptimal dosage or impaired bioavailability, re-infection or to a point mutation in the cytochrome b gene (pfcytb) [4,9,15]. In this paper, a case of A-P treatment failure in a military employee stationed in the Republic of Ivory Coast was reported; this treatment failure was not due to low plasma levels of the drug, mutations in the pfcytb gene, or re-infection. Case presentation The patient was a 45-year-old military employee deployed to Abidjan in the Republic of Ivory Coast (Port Bouet camp). He had been stationed in Abidjan for six months and had repeatedly forgotten to take the prescribed anti-malarial chemoprophylaxis drug, doxycycline. He presented general malaise and headaches on 27 September, 2011, and was treated by self-medication with paracetamol. Two days later (29 September, 2011 (day 0)), the patient consulted the medical centre of the camp, where he presented headache, myalgias, chills and fever with a temperature of 39.7C. A blood smear examination revealed P. falciparum at a parasitaemia of 0.74%, and a rapid diagnosis test confirmed Plasmodium infection. The patient was hospitalized and immediately treated with the standard treatment of MalaroneW, four 250 mg tablets daily on day 0, day 1 and day 2, associated with paracetamol and the continuation of chemoprophylaxis with doxycycline. The tablets were taken with fatty food and were well tolerated. No vomiting or diarrhoea occurred during hospitalization. At day 1, the patient was asymptomatic and afebrile. At day 2, after the last intake of MalaroneW, the patient experienced a febrile peak at 38.5C and blood smear examination revealed a parasitaemia of 1.3%. Because of the recrudescent parasitaemia and according to WHO guidelines for the assessment and monitoring of anti-malarial drug efficacy for the treatment of uncomplicated falciparum malaria [16], the possibility of early A-P treatment failure was considered, and the patient was treated with 625 mg of quinine base (QuinimaxW) orally three times daily for seven days, doxycycline (100 mg twice daily for seven days) and paracetamol. On 4 October (day 5), the parasitaemia decreased to 0.02%, and on 7 October (day 8), a QBC test was negative. Methods Whole blood specimens from the first (day 0) and second (day 2) episodes of malaria were submitted to the reference laboratory for gene amplification by polymerase chain reaction (PCR), sequencing, genetic analysis and quantification of the plasma concentrations of drugs. No in vitro assays of the P. falciparum isolates could be performed. The DNA of both samples was extracted from blood samples using the QIAamp DNA Mini Kit according to the manufacturers recommendations (Qiagen, France). Confirmation of P. falciparum mono-infection was performed by realtime LightCyclerW PCR (Roche, Meylan, France), as described elsewhere [17]. The pfcytb and dihydrofolate reductase (pfdhfr) genes were amplified by PCR and sequenced for both isolates to detect mutations associated with resistance to A-P, respectively, as described [15,18,19]. Molecular markers of resistance [19], such as pfcrt (chloroquine resistance transporter), pfmdr1 (multidrug resistance 1 protein), pfnhe1 (Na+/H+ exporter 1), pfdhps (dihydropteroate synthetase), pftetQ (tetQ family GTPase) and pfmdt (metabolite/drug transporter) were also assessed. The single-nucleotide polymorphism and copy number assays for these different genes were performed as previously described [15,2022]. The parasite diversity between day 0 and day 2 was determined by genotyping the TRAP, 7A11, C4M79, Pf2802, and Pf2689 microsatellite loci; the highly polymorphic loci of the merozoite surface protein 1 and 2 antigen genes (msp1-msp2); and the highly polymorphic loci of the glutamate-rich protein gene (glurp) using fluorescent end-labelled nested PCR and restriction fragment length polymorphism analysis. The primer sequences, PCR conditions, and genotyping methods have been described elsewhere [23-25]. The drug absorption and compliance were estimated by quantification of the drug levels in the patients plasma; these assays were performed using a Waters Acquity UPLC instrument (Milford, MA, USA). Separation was carried out on an Acquity BEH C8 column (50 mm 2.1 mm, 1.7 m) maintained at 40C. The mobile phase consisted of solvent A (0.5% acetic acid in purified water) and solvent B (acetonitrile). Two gradient prog (...truncated)