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 manufacturer’s recommendations (Qiagen, France). Confirmation of
P. falciparum mono-infection was performed by real-time LightCycler
® 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,
20‐
22]. 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 patient’s 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 μm) maintained at 40°C. The mobile phase consisted of solvent A (0.5% acetic acid in purified water) and solvent B (acetonitrile). Two gradient programmes were used, one for the quantification of proguanil, cycloguanil and doxycycline and the second for the quantification of atovaquone. The flow rate was 0.8 mL/min. The injection volume and total run time were, respectively, 5 μL and 3 min. A purification step was performed before analysis using i) an OASIS
® HLB SPE cartridge (Waters, Milford, MA, USA) for proguanil, cycloguanil and doxycycline and ii) protein precipitation by the addition of two volumes of an ACN/H
2O/acetic acid solution (85:14:1, v/v/v).