Efficacy and safety of artesunate plus amodiaquine in routine use for the treatment of uncomplicated malaria in Casamance, southern Sénégal

This study was conducted at the outpatient clinics of four dispensaries (Mlomp, Oussouye, Kabrousse and Djembereng) all situated in the District of Oussouye, southern Casamance. The total population of the district is circa 70,000, mostly farmers. Malaria transmission is perennial and meso-endemic with an increase in cases during the rainy season (July to December). The entomological inoculation rate is 25 infected bites per person-year [5].

In this area, the rate of chloroquine-resistant strains has remained stable at around 66% between 1997 and 2004 [6]. Despite this high rate, trials over the past decade have shown that AQ alone or combined with AS are efficacious [7‐9].

All eligible patients were treated with AS + AQ. Initially (2000–2001), the use of AS+AQ was restricted to the rainy season, and later extended (2002–2005) to all year round. During 2000–2003, loose tablets were used in combination and patients were dosed by body weight. Subsequently a blister pack containing both drugs became available during 2004–2005 and patients were dosed by age (except 30 subjects in 2004 who were dosed by weight). The drugs used were:

▪ loose combination: Arsumax^® tablets (Sanofi-Aventis) containing 50 mg of AS and Camoquin^® tablets (Parke-Davis) containing 200 mg of AQ base tablets. The doses used were 4 mg/kg/day (AS) and 10 mg/kg/day (AQ) both for three days.

▪ the blister pack: Arsumax^® tablets (Sanofi-Aventis) containing 50 mg of AS and AQ tablets (Sanofi-Aventis) containing 153 mg of AQ base. There were three dosing blisters and both drugs were administered following the manufacturer's instructions: (i) children below one year of age = 1/2 tablet of each drug; (ii) children between one and six years of age = one tablet of each drug; (iii) children above six and below 13 years of age = two tablets of each drug, and (iv) above 13 years of age = four tablets of each drug.

All treatments were administered under supervision in the clinic, except in 2005 when only the first dose was supervised and patients were instructed to continue their treatment at home. Patients were seen on Days 0–3 inclusive during 2000–04 (Days 0, 2 and 3 in 2005), and then on Days 7, 14, 21, 28 or in between, as needed. Non attendees for scheduled clinic visits were actively sought by community health workers. Giemsa-stained thick films were read by trained microscopists and confirmed by one of the investigators (PB).

The end point for efficacy was the Day 28 crude cure rate calculated using Kaplan-Meier survival analysis [10] on the Intent-to-Treat (ITT) dataset (all patients who entered the study) for the whole period under study and by year; the log rank test was used to test for significance between years. Success was defined as parasite clearance that was sustained through Day 28. Failure to clear parasites and recurrent parasitaemia were considered as failures. Genotyping parasites was not done to distinguish between recrudescences and reinfections. All study withdrawals due to adverse events, irrespective of their relationship to study drug, were considered as failures. All patients lost to follow up were censored on the date they were last seen. Parasitological failures were rescued with quinine.

Safety was assessed by: (i) recording treatment emergent sign/symptom (TESS, i.e. events which were not present pre-treatment or worsened with treatment) and (ii) measuring liver (alanine, ALT and aspartic, AST transaminases and bilirubin), renal functions (creatinine) (KONELAB 60I analyser, Konelab, Finland) and haematology (haematocrit, WBC total counts), aiming for about 30% of the patients enrolled to have at least one baseline and one post-treatment sample. The common toxicity criteria for adverse events (CTCAE Version 3.0 08/09/2006) were used to evaluate and grade the severity of clinical events and laboratory measurements. Shift tables were done to show changes in severity of CTC grades between Day 0 and Day 28.

Data were recorded in a case record form comprising demography, parasite counts, signs and symptoms, laboratory data and adverse events at each scheduled visit. They were double keyed in Excel^® using an end-user formatted sheet with online edit-checks.

Descriptive statistics are presented as counts, percentages, means and standard deviations, as appropriate. One-way ANOVA was used to assess the comparability of the patients' baseline characteristics between years and sites. Continuous data were assessed for normality using the Kolmogorov-Smirnov test; if significant, data were log-transformed and analysed using the 't' test, if normally distributed. Otherwise the Mann-Witney U test was used for paired comparisons. Homogeneity of variance was assessed with the Bartlett test. The Welch adjusted ANOVA was carried out if variances were unequal. Between groups comparisons of continuous data were further investigated with the Tuckey-Kramer post hoc test using means (normally distributed data) or ranks (skewed data). Dichotomous variables were analysed using chi-squared or Fisher's exact tests (Freeman-Halton if more than two categories) test if the expected counts were lower than five in any cell.

Kaplan-Meier survival analysis was performed to evaluate cure rates: (i) for all the years combined, (ii) between years, and (iii) between the loose and the co-blistered products [10]. The log rank test was used to test for homogeneity between survival curves of each year and the loose and the co-blistered products. A Cox proportional hazard model of the probability of failure was done to test for the contribution of the year of treatment and products. A descending stepwise manual modelling strategy based on the likelihood ratio test between subsequent models was carried out, beginning with a saturated model containing all factors.

A p value of < 0.05 was considered statistically significant. All tests were two-tailed. Statistical analyses were conducted with the statistical package SAS version 9.3.1 (SAS Institute, Cary, NC, USA)

Because the dosing of AS and AQ changed during the study, the doses of AS and AQ taken by patients with the loose and the blister combinations are presented in relation to two dosing schedules: (i) the recommended weight based dosing of 4 mg/kg/d × 3 d for AS and 10 mg/kg/d × 3 d for AQ base, and (ii) the new age based dosing regimen with newly defined therapeutic windows of 2–10 and 7.5–15 mg/kg/d, respectively [11].

During 2000–2005, 966 patients were enrolled, of whom 723 in Mlomp (75%), 110 in Diembereng, 27 in Kabrousse and 106 in Oussouye. The loose combination was given to 731 patients during 2000–03 and the co-blistered product to 235 patients during 2004–05. The dose was calculated on body weight for 761 patients (731 treated with the loose and 30 with the co-blistered products) and on age for 205 (all co-blister). Treatment was given supervised to 810 patients and unsupervised to 156 (co-blistered product in 2005). Table 1 shows the patients' baseline characteristics. Overall, for all the years combined, there were 30% more male than female patients, the mean age, weight and temperature values were 13.8 years, 33.3 kg and 38.4°C. The geometric mean baseline parasitaemia was 31,850/μL. The mean (± SD) daily doses of AQ and AS for all the study years were 359 (± 179) mg and 131 (± 66) mg, respectively.

There were significant statistical differences for certain baseline parameters between most of the years. In particular, there was a difference in (i) body weight between 2000–2004, 2001–2003, 2001–2004, (ii) AQ dose between 2000–2004, 2000–2005, 2001–2003, 2001–2004, 2001–2005, 2002–2004, 2002–2005, and (iii) AS dose between 2001–2004 and 2004–2005.

The age structure of the population treated is presented in Figure 1. Overall, 16% of patients were ≤ 5 years of age, 30% were 6–10 years of age, 27% were 11–15 years of age, 11% were 16–20, 8% were 21–30 and 7% ≥ 30 years of age. Using these categories there was no difference between years and sites (p > 0.05). Just over half, 57% (n = 556), of the patients were aged between 6–15 years; 46% (n = 449) were under 11 years of age.

The doses of AS and AQ taken by patients compared with the weight- and age-based dosing regimens are shown in Figure 2. With both products used, doses were well within the newly defined, therapeutic windows for both drugs. For AS, doses with the loose product and the co-blistered product were similar and very close to the target dose of 4 mg/day; the co-blister mean doses were slightly lower with wider 95% CIs. For AQ, doses were higher with both products than the target dose of 10 mg/day with a tighter 95% CIs for the loose combination except for 11–15 years old.

The Kaplan-Meier estimates of the crude cure efficacy rate was 94.6% (95% CI 93.0; 95.9) for all years combined. By individual years cure rates were 96.7% [93.2; 98.4] in 2000, 94.0% [90.6; 96.2] in 2001, 95.7% [90.0; 98.2] in 2002, 94.9% [88.2; 97.8] in 2003, 88.5% [79.0; 93.8] in 2004 and 95.9% [91.1; 98.1] in 2005. There were no differences (p = 0.12) in cure rates between years by the log rank test for homogeneity over time (Figure 3) All patients cleared their parasites by Day 3 (no early treatment failure, ETF); 36 patients returned with parasites during follow-up (late treatment failures, LTF) and nine were withdrawn due to an adverse event (considered as failures in our analysis – see below); 32 were lost to follow-up (censored) (Table 2). All treatment failures were retreated successfully with injectable quinine. All 36 LTFs occurred in patients under 16 years of age (20 in the age range 6–10). Efficacy was 95% in 0–10 years and 97% in 11 and above (log rank test, p = 0.03). In 2005 the losses to follow up amounted to 15%, while they were ≤3% in the other years.

The Kaplan-Meier estimates of the crude cure efficacy rates were similar (p = 0.14) between the loose (n = 731) and the co-blistered (n = 235) products: 95.1% (93.3;96.5) vs. 93.1% (88.8;95.8), respectively. This held true whether dosing was weight (n = 761) or age (n = 205) based: 94.8% (92.9;96.2) vs. 94.2% (89.7;96.7), respectively (p = 0.51) and whether treatment was supervised (n = 810) or unsupervised (n = 156): 94.5% (92.7;95.9) vs. 95.9% (91.1;98.1), respectively (p = 0.75).

Year of study and product used were non significant contributors to failure in a Cox proportional hazard model. Using a saturated model containing year, product, site, age, sex, weight, dose AS and dose AQ, the contribution to hazard of failure was border-line for year (p = 0.06) but significant for the total daily dose of AS (p = 0.004) for a hazard of failure of 0.993 [0.988; 0.998]. Only year 2004 was statistically different from the reference year 2000 (p = 0.003): the hazard of failure was four times greater in 2004 than in 2000 (hazard ratio = 4.496 95% CI = [1.661; 12.168]).

Complete safety records are available for 752 patients enrolled during 2001–2005. At presentation (Day 0), all patients reported fever or had a measured fever in the clinic. Other malaria associated symptoms/signs on presentation were weakness [n = 296 (29%)], headache [n = 291 (29%)], vomiting [n = 133 (13%)] and nausea [n = 113 (11%)]. There were no significant differences in the frequency of symptoms/signs between the different age groups or sexes.

After treatment, 69 patients (7.1%) experienced at least one treatment emergent sign/symptom (TESS) which was either not present pre-treatment or worsened post-treatment: 54 patients suffered one TESS, 14 had two and 1 had three for a total of 85 TESSs: 36 were vomiting, 19 vertigo, 11 asthenia, 8 pruritus without a rash, 5 abdominal pain, 3 diarrhoea, 2 headaches and 1 nausea (Table 3). TESSs were more likely to be reported by the co-blister recipients: 54 (5.6%) vs. 15 (1.5%) (p < 0.0001) and were independent of age. For the co-blister 39 patients suffered one TESS during the study, 14 suffered 2 TESSs and one suffered 3 TESSs; of patients treated with the loose combination, 15 suffered one TESSs (p < 0.0001).

Nine patients, all treated with the co-blistered product (two weight-based and seven age-based), were withdrawn from the study because of a TESS for an overall, crude withdrawal rate of 0.9%: 3.8% [(co-blister) vs. 0% (loose), p < 0.0001(Table 4)]. All events were considered probably related to AS+AQ except one case of vomiting (possibly related). In five such cases, the daily dose of AQ exceeded the target dose by 20% or more. Two patients with Grade 2 vomiting were admitted to hospital for intravenous quinine and symptomatic treatment; they recovered well. These hospitalizations define these AEs as serious adverse events (SAEs).

For the laboratory investigations, pretreatment results were available in 33%, 22% and 17% of patients for haematocrit (Hct), total white blood cells (WBC) and biochemistry, respectively (Table 5). No CTC grade 4 values were present at baseline and at Day 28. One patient had a grade 4 creatinine value on Day 7 [16.5 > 6 × ULN, ULN = 1,20 mg/dL] and returned to grade 0 at Day 28.

There were no shifts between Day 0 and Day 28 from grades 0 – 2 to grades 3 or 4 (Table 6). Out of 12 changes in the total WBCs, three patients changed from grade 0 to grade 1 and one patient from grade 0 to grade 2, while the remaining eight patients changed to lower grades. There were 28 changes in CTC grade for AST; four had increased grades, from grade 0 to 1 (n = 3) and from grade 0 to grade 2 (n = 1). Out of 29, only two increased shifts were observed with ALT; one from grade 0 to grade 1 and one from grade 1 to grade 2. One shift from grade 0 to grade 1 was observed for the serum creatinine. For total bilirubin there were one change in CTC grades from 0 to 1 and two from 0 to 2. There were also nine decreases from grade 1 to grade 0 and four decreases from grade 2 to 0.

There were no significant changes in mean values between Day 0 and Day 28 for the haematocrit (mean diff. 0.31 ± 6.10%, p = 0.51) and total WBC (mean diff. -395 ± 2940, p = 0.21) but there were significant decrease from Day 0 to Day 7 (2.52 ± 5.56%, p < 0.0001) and increase from Day 7 to Day 28 (-1.91 ± 4.70%, p < 0.0001) for haematocrit. The mean AST, ALT and bilirubin decreased significantly while there was a small (0.16 mg/dL) but statistically significant increase in the mean creatinine values between Day 0 and Day 28 (Figure 4).