Conceived and designed the experiments: CP EZ MM SW DB PW. Performed the experiments: DB SN. Analyzed the data: DB MM. Contributed reagents/materials/analysis tools: CP SW MM. Wrote the paper: CP EZ MM SW DB PW.
Peter Winstanley and Steve Ward are unpaid members of the MMV-GSK product development team for chlorproguanil-dapsone-artesunate. None of the other authors declare any conflicts of interest.
In Malawi, there has been a return of
455 children aged 1–5 years were recruited into a double-blinded randomised trial comparing SP to the three combination therapies. Using intention to treat analysis with missing outcomes treated as successes, and without adjustment to distinguish recrudescence from new infections, the day 28 adequate clinical and parasitological response (ACPR) rate for SP was 25%, inferior to each of the three combination therapies (p<0.001). AQ+SP had an ACPR rate of 97%, higher than CQ+SP (81%) and ART+SP (70%), p<0.001. Nineteen children developed a neutropenia of ≤0.5×103 cells/µl by day 14, more commonly after AQ+SP (p = 0.03). The mutation
The combination AQ+SP was highly efficacious, despite the low efficacy of SP alone; however, we found evidence that AQ may exert selective pressure for resistance associated mutations many weeks after treatment. This study confirms the return of CQ sensitivity in Malawi and importantly, shows no evidence of the re-emergence of
Controlled-Trials.com
Malaria is responsible for around 1 million deaths annually in sub-Saharan Africa, especially in young children. Attempts to control malaria have been hampered because of resistance in
The combination amodiaquine (AQ) plus SP has been shown in some African countries to have efficacy similar to ACTs
Here we report the results of a randomised double-blind clinical trial comparing the efficacy and safety of 3 SP based combination therapies, CQ+SP, artesunate (ART)+SP and AQ+SP with that of SP alone for the treatment of uncomplicated malaria in young children in Malawi. At the time of the study, SP monotherapy was the treatment recommended for uncomplicated malaria by the Malawi National Malaria control committee. In addition, we compared the selection of resistance associated mutations in the parasite genes
The protocol for this trial and supporting CONSORT checklist are available as supporting information; see
The study was based at Chileka health centre near Blantyre, Malawi, where malaria transmission is perennial, peaking during December to April. Between September 2003 and December 2005, children presenting with an illness suggesting falciparum malaria were screened. Inclusion criteria were: i) age ≥12 and <60 months, ii) weight ≥6 kg, iii) axillary temperature ≥37.5°C, iv) no history of treatment with an antimalarial, cotrimoxazole or a tetracycline antibiotic in the previous week, v) no features suggesting severe malaria or a concomitant illness, vi) haemoglobin ≥5.0 g/dl using Hemocue®, and vii)
Children meeting all inclusion criteria on day 0 were recruited and randomised to one of four treatment groups. Randomisation was in blocks of 12 according to an off-site computer-generated code to assign patients equally to the four oral treatment groups: SP (25 mg/kg sulfadoxine and 1.25 mg/kg pyrimethamine as a single dose on day 0)+vitamin C 50 mg tablet (placebo) daily for 3 days; CQ (10 mg/kg on days 0 and 1, and 5 mg/kg on day 2)+SP; ART (4 mg/kg once daily for 3 days)+SP; or AQ (10 mg/kg daily for 3 days)+SP. In the case of children too young to swallow tablets, CQ syrup (50 mg per 5mls) and AQ syrup (50 mg per 5mls) were used (same doses as above). The other study drugs were not available as syrups and were crushed and given on a spoon with water if the child could not swallow a tablet. The different tablets were not identical in appearance or taste. A three-day supply of paracetamol (10 mg/kg) was given.
Each child was given a unique study number, assigned sequentially. A dedicated study ‘drug dispenser’ opened the corresponding randomisation envelope and directly observed all drug doses but was not involved in the assessment of children. All other members of the study team were blinded to the dispensing process and patients were uninformed of their treatment allocation for the duration of the study. Children were observed for 30 minutes after dosing. If the child vomited, a second dose was given. If vomiting occurred a second time, the child was withdrawn and treated with parenteral quinine.
Patients were assessed on days 0, 1, 2, 3, 7, 14, 28 and 42 and any other day if unwell. Blood was collected for parasite microscopy, storage on Whatman 3M filter paper and, at specified visits, for determination of the full blood count and biochemical parameters. Clinical outcome was assessed using the 2003 WHO therapeutic efficacy protocol for areas of intense malaria transmission
The planned sample size of 100 evaluable patients per treatment arm was calculated to have 90% power to detect the difference between an “adequate clinical and parasitological response” (ACPR) rate of 80% with SP alone and 95% with combination therapies using the 5% significance level for each comparison with SP alone. The primary endpoint was the day 28 ACPR rate and the major analysis strategy for the primary endpoint was intention to treat (ITT). Patients with missing outcomes were all classified as successes in one analysis and then as failures in a separate analysis. Per protocol (PP) analysis was also done using polymerase chain reaction (PCR) corrected data to distinguish recrudescences from reinfections. When PCR analysis indicated that a post-treatment parasitaemia was a reinfection, the outcome was classified as a treatment success on that day, but excluded from subsequent analyses. If PCR was inconclusive, the case was excluded from the analysis.
Secondary endpoints included day 14 and 42 ACPR rates, time to fever resolution (axillary temperature≤37.5°C), time to parasite clearance, change in haemoglobin from day 0 to day 14 and the appearance of gametocytes by day 28 after treatment. We also compared adverse events (AEs) between the treatment groups, including self-reported AEs and laboratory AEs; rises in alanine transferase (ALT), total bilirubin, and creatinine between days 0 and 14.
Data were double entered and validated prior to the analyses. Data analysis was performed using Stata 8. Binomial regression was used to obtain risk differences between treatments and 95% confidence intervals. Fisher's exact p-values were reported. Tests of significance were performed using the 5% level to infer significance for the planned analyses. Pair wise comparisons between combination therapies were not planned and in these comparisons we adjusted the significance level to 1.7% (i.e. p<0.017) using Bonferroni's approach.
To look for evidence of selection of resistance mutations in the genes
Blood films were stained with Fields stain and parasite densities estimated from thick films by counting the number of parasites per 200 white blood cells (WBC) assuming a total count of 8000/µl. These parasite counts and haemoglobin (Hb) estimates using Hemocue® were used for screening purposes. In addition, on days 0 and 14, the full blood count was measured using a Beckman Coulter HMX and plasma ALT, total bilirubin and creatinine using a Vitros DTII dry biochemistry analyser. The actual WBC count from the coulter was subsequently used to calculate an accurate parasite count for the analyses. The presence or absence of gametocytes was noted on each blood film.
Parasite DNA was extracted from dried blood on filter paper. A nested PCR was used to distinguish recrudescent infections from new infections in all patients with parasitaemias appearing from day 12 onwards. The
We screened 1625 children and 455 met all inclusion criteria and were enrolled. Baseline characteristics are shown in
SP | CQ+SP | ART+SP | AQ+SP | |
Number of patients | 114 | 113 | 114 | 114 |
Median age (months), (IQR) | 22.6 (17.6) | 22.2 (14.0) | 21.6 (8.2) | 21.0 (14.2) |
Number (%) female | 62 (54.4%) | 54 (47.8%) | 51 (44.7%) | 56 (49.1%) |
Number of days of fever | 3.1 (1.8) | 2.9 (1.8) | 3.1 (1.9) | 3.1 (1.7) |
Weight (kg) | 10.9 (2.2) | 10.8 (2.3) | 10.8 (2.1) | 10.8 (2.4) |
Initial temperature (°C) | 38.8 (0.9) | 38.7 (0.9) | 38.8 (0.9) | 38.8 (0.9) |
Geometric mean (range) parasite count | 66,171 (973–301,294) | 59,874 (2146–280,720) | 36,315 (1077–306,722) | 44,356 (1892–288,353) |
Parasite count>200,000/µl (%) | 10 (8.8%) | 16 (14.2%) | 9 (7.9%) | 13 (11.4%) |
Gametocytes seen on Day 0 | 18 (15.8%) | 15 (13.3%) | 18 (15.8%) | 19 (16.7%) |
Haemoglobin | 9.1 (1.7) | 9.0 (1.6) | 8.9 (1.4) | 9.3 (1.6) |
White cell count (x109/L) | 10.2 (4.3) | 10.8 (4.6) | 9.9 (4.4) | 10.3 (4.7) |
Neutrophil count (x109/L) | 5.1 (3.6) | 5.2 (3.6) | 4.1 (2.9) | 5.3 (4.1) |
Platelet count (x109/L) | 149 (91) | 165 (93) | 163 (100) | 162 (103) |
Alanine Transferase (IU/L) | 19 (13) | 18 (17) | 18 (16) | 22 (19) |
Total Bilirubin (mg/dl) | 0.97 (0.5) | 0.95 (0.7) | 0.88 (0.7) | 0.85 (0.6) |
Creatinine (mg/dl) | 0.4 (0.1) | 0.4 (0.1) | 0.4 (0.1) | 0.4 (0.1) |
For all, data shows mean (SD) unless otherwise indicated
Parasite count calculated using coulter HB and WCC
HB from coulter counter. If not available, the Hemocue HB was used (n = 5)
Using the ITT approach with missing outcomes treated as successes, the day 28 ACPR rate was lowest with SP alone at 25% and inferior to each of the three SP combination therapies (p<0.001),
Comparison | ITT analysis (Withdrawals counted as successes) | ITT analysis (Withdrawals counted as failures) | ||||
Success rate | Difference (95% confidence interval) | P-value | Success rate | Difference (95% confidence interval) | P-value | |
SP | 25% | 19% | ||||
CQ+SP | 81% | 56% (45%, 67%) | <0.001 | 67% | 48% (37%, 59%) | <0.001 |
ART+SP | 70% | 45% (33%, 56%) | <0.001 | 60% | 40% (29%, 52%) | <0.001 |
AQ+SP | 97% | 72% (63%, 80%) | <0.001 | 84% | 64% (54%, 74%) | <0.001 |
CQ+SP vs. ART+SP | −11% (−22%, −0.2%) | 0.063 | −8% (−20%, 5%) | 0.271 | ||
CQ+SP vs. AQ+SP | 16% (8%, 24%) | <0.001 | 16% (5%, 27%) | 0.006 | ||
ART+SP vs. AQ+SP | 27% (18%, 36%) | <0.001 | 24% (12%, 35%) | <0.001 |
156 recurrent parasitaemias occurred 12 or more days after treatment. PCR analysis showed that 97 (62%) were recrudescences, 56 (36%) were reinfections and for 3 (2%), the analysis failed.
Ninety-five percent of children had cleared their parasite by day 2 in the ART+SP group compared to 35% for SP, 47% for CQ+SP, and 55% for AQ+SP (p<0.001 for each comparison with AQ+SP). By days 3 and 7, there were no differences between the three combination therapies and they were all superior to SP alone, p = 0.005. In the SP group, there was no association between the day 0 parasitaemia and time to parasite clearance or between day 0 parasitaemia and clinical outcome. Fever resolution was slower with SP alone; the percentage of children who still had fever on day 1 were 18% for SP, 5% for CQ+SP, 6% for ART+SP and 5% for AQ+SP (p<0.008 for each comparison with SP).
Mean haemoglobin concentration rose in all treatment groups. Compared to SP alone, the adjusted mean on day 14 was greater after CQ+SP (p = 0.03) and AQ+SP (p = 0.002) but not after ART+SP (p = 0.81). Gametocytes were present on day 0 in 73 (16%) children,
284 clinical AEs were reported in 185 children. Cough was commonest, making up 45% of all AEs. Compared to SP alone, cough was more commonly reported after ART+SP, p = 0.04. No other statistically significant differences were found. There were 8 serious adverse events (SAEs) in the study with no more than 4 in any treatment group. There were no deaths. The SAEs included 2 cases of pneumonia requiring intravenous antibiotics, 1 child with gastroenteritis requiring intravenous fluids and 5 treatment failures requiring hospitalisation for intravenous quinine. For two of these children, this was due to the occurrence of seizures shortly after receiving their medication on day 0 (1 AQ+SP, 1 CQ+SP), and 1 child had persisted vomiting on day 1 and was unable to continue oral treatment (AQ+SP). The other two children had early treatment failures (1 SP, 1 AQ+SP), and were too unwell to continue oral therapy.
Neutrophil counts fell after treatment in all treatment groups but the proportion of children with neutrophil counts of ≤0.5×103/µl on day 14 (having been ≥1.0×103/µl on recruitment) was greatest in the AQ+SP group (11.5%, n = 7), higher than the SP alone group (1.5%, n = 1), p = 0.03. For 17 of the 19 children in which this low neutrophil count was observed, a repeat sample between day 28 and 42 showed neutrophils ≥1.0×103/µl. For the remaining 2 children no further sample was obtained. We noted no evidence of any ill effects due to these transient low neutrophil counts.
Two children in the AQ+SP treatment group developed plasma ALT levels on day 14 greater than three times the upper limit of normal having been in the normal range (15–45 U/L) on recruitment. The possible association of this adverse effect with AQ+SP was not significant, p = 0.24. One of these children had a day 14 ALT of 1540 U/L with a total bilirubin of 1.1 mg/dl (0.1–1.4 mg/dl). By day 42 the ALT had returned to normal. The other child had a day 14 ALT of 473 U/L with a total bilirubin of 2.2 mg/dl. No further samples were collected for analysis. Both children were well and completed 42 days of follow up and both had day 14 neutrophil counts ≥1.0×103/µl.
The resistance mutations
The prevalence of
Ten years after the introduction of SP as first line treatment for uncomplicated malaria in Malawi the day 28 ACPR rate lies below 30%. Over 90% of pre-treatment parasites were “quintuple” mutants; this genotype has been shown to be strongly predictive of SP failure in young children in Malawi
Low cost alternatives to artemether-lumefantrine might have been attractive to the Malawi authorities and AQ+SP was one of the combinations considered. In most countries in Africa, antimalarials are obtained without prescription and often taken unnecessarily
This study confirms the return of CQ sensitivity in Malawi and importantly, shows no evidence of the re-emergence of
AQ+SP was significantly more efficacious than CQ+SP, ART+SP and SP alone. AQ+SP also appears to have a longer period of post-treatment prophylaxis than the other treatments; reinfection rates on days 14, 28 and 42 were similar after treatment with SP, CQ+SP or ART+SP but with AQ+SP, no reinfections were seen until after day 28 (
The combination of ART+SP for 3 days was the least effective of the three combination therapies. This is not surprising given the poor efficacy of SP. As a monotherapy, ART is usually taken for 7 days; a 3-day ACT course is only efficacious when the second drug retains adequate efficacy. The combination of ART+SP has proven ineffective in other sites in Africa with significant background SP resistance
AQ+SP has shown excellent efficacy in several African studies and a recent meta-analysis concluded that the efficacy of AQ+SP in Africa was similar to that of AQ+ART but inferior to artemether-lumefantrine
We saw evidence of the selection of the
We were unable to detect
AQ was withdrawn as a prophylaxis against malaria in 1986, because of agranulocytosis (1 in 2,100 subjects) and hepatitis (1 in 15,650)
SP has failed in Malawi after a decade of useful service, and its role in intermittent presumptive therapy in pregnancy (IPT) should also now be re-evaluated. There has been a return of CQ sensitivity in Malawi, and CQ could be considered as a possible replacement for SP in IPT programmes. The potential for CQ to be used as part of combination therapy should be considered with caution unless and until CQ-sensitive parasites predominate throughout the region. Given the extensive cross-border movement of people, it seems likely that CQ-resistant parasites from neighbouring countries would be selected rapidly on redeployment of this drug.
The combination AQ+SP was highly efficacious, despite the low efficacy of SP alone; however, we found evidence that in these circumstances AQ may exert selective pressure for resistance mutations many weeks after treatment. In parts of West Africa where SP and AQ both remain efficacious, the combination AQ+SP could be considered for first line treatment for uncomplicated malaria. The drugs have similar pharmacokinetic profiles and the combination may offer a cheaper, longer lasting and readily available alternative to ACTs, with the benefit of longer post-treatment prophylaxis. AQ+ART is being increasingly used and it will be interesting to see whether this combination, with its mismatched kinetics, can prevent the development of AQ resistance. In addition, it will be important to monitor for potential toxicities of AQ after repeated treatment doses.
CONSORT Checklist
(0.06 MB DOC)
Original study protocol as submitted to the Malawi Research Ethics Committee for approval
(0.07 MB DOC)
The authors would like to thank the children and their parents for taking part in this study and the study team, Alice Mbekyani, Rose Paligolo, Mary Kandapo, Albert Malenga, Joseph Bwanali, Andrew Khoriyo and David Misomali for their hard work. We would also like to thank the District Health Officer for permission to site the study at Chileka Health Centre and all the other staff at the health centre. Thank you to Dr Sarah White for her statistical advice and supervision of MM and to John White for generation of the study randomisation code. We are indebted to Drs Shaf Ahmed, Frank Bell and Grace Malenga as the study Safety Management Committee and to Dr Neena Bodasing for acting as the study monitor.