Amodiaquine

In a previous analysis of the samples collected 24 hours apart, we found that not all clones present in a host were detected within a single sample. Twenty-one percent of all msp1F3 alleles and 28% of all MS16 clones were missed on a single day [30] (link). Thus, we used the combined genotyping data from both day 1 and day 2, except for samples from enrolment and final visits, where only 1 venous blood sample was taken.
The force of new P. vivax blood-stage infections (_molFOB) was generated by counting the number of genotypes in each interval that had not been present in the preceding interval. An 8 to 9 weeks interval started on the first day after a regular cross-sectional visit and included all samples collected during passive case detection over two months plus the samples collected at the end of the interval. _molFOB was also determined for both markers combined, msp1F3 and MS16. In case of discrepancy between the markers for an 8-weeks interval the higher estimate from either marker was used. This approach corrected for imperfect resolution and detectability of a single marker.
Genotyping cannot directly identify relapses; _molFOB measures the combination of primary blood-stage infections and those caused by relapsing hypnozoites. Thus, homologous relapses occurring in two subsequent 2-month intervals would be misclassified as persisting clones. New Guinean P. vivax strains are known to relapse rapidly [31] (link), however in regions of high transmission, relapsing clones are usually of a different genotype than the initial blood stage infection [24] (link). As a consequence the number of homologous relapses that were not detected is expected to be relatively small.
In line with the pharmacokinetic properties of the drugs [32] (link)–[34] (link), children were not considered at risk for two weeks after treatment with artemether-lumefantrine and four weeks after treatment with amodiaquine (AQ) plus sulphadoxine-pyramethamine (SP). The force of blood-stage infection for each child and interval was subsequently converted into the number of new clones acquired per year-at-risk.
Similar to previous analyses of P. falciparum_molFOI [23] (link), generalized linear mixed models (GLMMs) were used for analyses of force of blood-stage infection as well as for incidence of P. vivax episodes. These models were chosen because they allowed the fixed effects to be specified separately from the random effects (i.e. repeated measurements from the same child over time and unmeasured village factors). Furthermore, the random-effects model allowed for decomposition of the error into between-village and within-village variation.
We fit a Poisson GLMM model with a log link to relate the fixed and (Gaussian) random effects to the number of clinical episodes experienced during a two month interval (defined as febrile illness plus P. vivax >500 parasites/µl). Covariates were selected based on earlier analyses of the same data [27] (link). Seasonality was characterized by two readily interpretable parameters: the amplitude, which was half the range between the peak and trough, and the phase, which was the location of the first zero crossing in a cycle relative to the origin in time (in this case, the first week of the year). For computational convenience, they were replaced by sine and cosine terms with fixed phases. For all outcomes except prevalence, an offset was fit to adjust for years at risk. Estimation of these models was done using the LME4 package in R version 2.12 [35] . All point estimates provided throughout the text (except those for seasonal effects) were obtained from cubic splines fit using generalized additive models (Figure 1) using the MGCV package in R version 2.12 [36] . For a more detailed description of the statistical approaches see [23] (link). Point estimates for seasonal peaks and troughs were obtained from the GLMMs by setting all other values of the covariates at their means. For the analyses of the effect of exposure on the relationship between age and incidence of P. vivax malaria, children were stratified into terciles according to the average _molFOB during the entire follow-up.

The study was carried out in Dielmo, a village situated in a Sudan-savannah region of central Senegal, on the marshy bank of a small permanent stream, where anopheline mosquitoes breed all year round [22] (link). Malaria transmission is intense and perennial, with a mean 258 and 132 infected bites per person per year during 1990–2006 and 2007–2010, respectively [21] (link). From 1990 to 2010, we did a longitudinal study involving most of the population of the village (all 247 inhabitants of the village in June 1990 at the beginning of the project, 468 of 509 inhabitants in December 2010).
The study area, the procedures of medical, parasitological, entomological and epidemiological surveillance and the main characteristics of malaria in this village have been described previously [21] (link), [22] (link). Briefly, during 20 years, we visited all households daily, and collected nominative information on the presence or absence in the village of each individual we had enrolled, their location when absent, and the presence of fever or other symptoms. We systematically recorded body temperature at home three times a week (every second day except Sunday) in children younger than 5 years, and in older children and adults in case of suspected fever or fever-related symptoms. In cases of fever or other symptoms, blood testing was done by finger prick at our dispensary located in the village, and we provided detailed medical examination and specific treatment. The dispensary created for our project was open 24 h a day, 7 days a week, to allow both active and passive case detection. To investigate asymptomatic malaria carriage, we performed cross-sectional surveys at least quarterly in all individuals enrolled in the project. Blood was taken by finger prick and we examined 200 oil-immersion fields. We measured the parasite: leukocyte ratio for each plasmodial species and we enumerated separately the gametocytes of P. falciparum.
Between June 1990 and December 2010, four first-line drugs regimens were successively used for antimalarial treatment: oral quinine (Quinimax®) (October 1990–December 1994), chloroquine (January 1995–October 2003), sulfadoxine/pyrimethamine+amodiaquine (SP+AQ) (November 2003–May 2006) and artesunate+amodiaquine (AS+AQ) (June 2006–December 2010). Antimalarials were systematically given to young children in case of fever associated with a parasite: leukocyte ratio ≥2 [22] (link). When parasitemia was lower, the requirement for antimalarial treatment was decided taking into account all the patient's clinical, biological and epidemiological data [22] (link). Among older children (≥10 years) and adults permanently living in the village, it was rapidly observed that clinical malaria attacks lasted only a few hours even in cases where specific malaria treatment was delayed or not taken [23] (link), and thus in most cases (except pregnant women) only symptomatic treatment was given under close clinical surveillance (three daily visit at home until recovery) in order to reduce the selection of drug resistant malaria parasites. Urine tests carried out to detect the presence of antimalarials indicated that almost all positive results (>99%) were explained by treatments given in our clinic [22] (link). There were no chemoprohylaxis, intermittent preventive treatment nor presumptive malaria treatment in children or adults during the time period 1990–2010. At the beginning of the project, 48.6% (children: 51.1%, adults: 47.1%) of the villagers used traditional mosquito nets, which were untreated, and this proportion remained almost unchanged until July 2008 when LLINs were distributed to all villagers.

This was a longitudinal, single-arm, prospective study to evaluate P. falciparum tolerance to ART and its derivatives in children with uncomplicated malaria aged 6 months to 14 years in 3 health facilities in the Greater Accra region of Ghana. The study focused mainly on day 3 post artemether-lumefantrine (AL) treatment parasitaemia, 72-h ex vivo RSA after dihydroartemisinin (DHA) exposure, 72-h parasite clearance in vitro against a panel of 6 drugs (ART, AS, artemether [AM], DHA, amodiaquine [AQ], lumefantrine [LUM], and the following molecular markers of drug tolerance / resistance: Single Nucleotide Polymorphisms (SNPs), Multiple Nucleotide Polymorphisms (MNPs), Insertions & Deletions (INDEL) in Pfk13, Pfcoronin, P. falciparum multidrug resistance protein 1 (Pfmdr1), multidrug resistance protein 2 (Pfmdr2), dihydrofolate reductase (Pfdhfr), dihydropteroate synthetase (Pfdhps), signal peptide peptidase (Pfspp), and multidrug resistance-associated protein 2 (Pfmrp2) genes. It sought to set up correlates of ART tolerance.

Using HLM as the enzyme source, the CYP inhibition potential was employed to assess the inhibitory effect of the TbB ligands on the CYP1A2, 2B6, 2C8, 2C9, 2C19, and 2D6 3A4 isoforms. Phenacetin (CYP1A2, 10 mM), tolbutamide (CYP2C9, 20 mM), bupropion (CYP2B6, 10 mM), mephenytoin (CYP2C19, 100 mM), midazolam (CYP3A4, 5 mM), amodiaquine (CYP2C8, 0.02 mM), and dextromethorphan (CYP2D6, 5 mM) were used as probe substrates. The test substance included TbB ligand (final concentrations of 10 M for each) and HLMs (final concentration of 0.1 mg protein per mL), with or without 1.0 mM NADPH. The reaction was started by adding pre-incubated 25 $\mathsf{\mu }$ L of NADPH into all wells and incubate at 37 °C for 10 min for 3A4 and 20 min for remaining all isoforms. The reaction was stopped by adding 100 $\mathsf{\mu }$ L of stop solution to all wells. The samples were centrifuged at 4000 rpm for 10 min at 4 °C. Supernatant (100 $\mathsf{\mu }$ L) was withdrawn, mixed with 200 $\mathsf{\mu }$ L of water followed by LC-MS/MS analysis.