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The Lancet 2006; 368:1349-1356
DOI:10.1016/S0140-6736(06)69559-7
Efficacy, safety, and tolerability of amodiaquine plus sulphadoxine-pyrimethamine used alone or in combination for malaria treatment in pregnancy: a randomised trial
Dr Harry TagborDrPH
a 
, Jane BruceMSc c, Edmund BrownePhD b, Anna RandalMSc c, Prof Brian GreenwoodMD c and Daniel ChandramohanPhD c
See Comment
Summary
Introduction
Methods
Results
Discussion
References
Summary
BackgroundThe widespread increase in resistance of Plasmodium falciparum to chloroquine and sulphadoxine-pyrimethamine threatens the use of these drugs for malaria treatment in pregnancy. We aimed to assess the safety and efficacy of amodiaquine alone or in combination with sulphadoxine-pyrimethamine as alternative regimens.
MethodsPregnant women with a gestational age of 16 weeks or more who attended antenatal clinics at a district hospital in Ghana were screened for malaria with OptiMAL dipsticks. 900 pregnant women who had a positive test result and P falciparum asexual stage parasitaemia were enrolled and randomly assigned chloroquine, sulphadoxine-pyrimethamine, amodiaquine, or amodiaquine plus sulphadoxine-pyrimethamine. The primary outcome was parasitological failure by day 28 of treatment. Women were seen on days 3, 7, 14, and 28 after the start of treatment to assess the effect of treatment on peripheral parasitaemia, haemoglobin concentration, white-blood-cell count, and liver function. Additionally, reports of adverse effects were solicited and monitored during follow-up visits. Analysis was by intention to treat. This trial is registered with the US National Institute of Health clinical trials database number NCT00131703.
FindingsPCR-corrected parasitological failure by day 28 was 14%, 11%, 3%, and 0% in the women assigned chloroquine, sulphadoxine-pyrimethamine, amodiaquine, and amodiaquine plus sulphadoxine-pyrimethamine, respectively (p<0?0001). No serious liver toxic effects or white-blood-cell dyscrasias were noted. Minor side-effects were reported more often on day 3 by women receiving amodiaquine (86%) or amodiaquine plus sulphadoxine-pyrimethamine (90%) than those receiving sulphadoxine-pyrimethamine (48%) or no antimalarial drugs (34%; p<0?0001 for every comparison).
InterpretationAmodiaquine alone or in combination with sulphadoxine-pyrimethamine, although associated with minor side-effects, is effective when used to treat malaria in pregnancy.
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Introduction
Plasmodium falciparum infection in pregnancy is associated with maternal and perinatal morbidity.1?3 For malaria control in pregnancy, WHO recommends the use of insecticide-treated bednets, intermittent preventive treatment with sulphadoxine-pyrimethamine in the second and third trimesters, and prompt and effective treatment of malaria and anaemia.1,4 Chloroquine and sulphadoxine-pyrimethamine are the safest and most readily available and affordable drugs for the treatment or prevention of uncomplicated malaria in pregnancy in most African countries. However, these drugs are becoming ineffective against uncompleted malaria in many areas because of increasing parasite resistance. Therefore, alternative safe and efficacious antimalarial drugs are needed urgently for both treatment of malaria during pregnancy and intermittent preventive treatment. Most countries in Africa are adopting artesunate-based combination therapy (ACT) as the preferred drug for the treatment of uncomplicated malaria. However, very little information on the safety of ACT during pregnancy is available, and there are concerns about the possible teratogenicity of these drugs when given in the first trimester of pregnancy.5 In Ghana, chloroquine was the preferred treatment for uncomplicated malaria in children and pregnant women, until a decision was made to change to the combination of artesunate and amodiaquine.
Since amodiaquine is effective in some areas with chloroquine resistance,6,7 and because the extent of resistance to sulphadoxine-pyrimethamine in west Africa is not as high as that in east Africa,7?9 we postulated that amodiaquine, used either alone or in combination with sulphadoxine-pyrimethamine, might be a safe and efficacious drug option for malaria treatment in pregnancy and for intermittent preventive treatment in Ghana until the safety of ACTs in pregnancy has been established. However, little information exists about the safety or efficacy of amodiaquine in pregnancy. A review10 found only six reports on the use of amodiaquine in pregnancy, of which four described its use for chemoprophylaxis and two for treatment. Toxic effects were assessed specifically in only one of the chemoprophylaxis studies, and none reported the outcome of pregnancy. The two treatment studies, one in Burma11 and another in Kenya,12 were small (19 and 23 patients, respectively) and did not study toxic effects specifically. Because of the paucity of knowledge about the safety and efficacy of amodiaquine in pregnancy, we undertook a clinical trial in which amodiaquine, amodiaquine plus sulphadoxine-pyrimethamine, sulphadoxine-pyrimethamine, and chloroquine were compared in the treatment of pregnant women with proven malaria infection.
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Methods
Patients
The study was done at St Theresa's Hospital, Nkoranza, Ghana. Malaria transmission in this area is perennial but peaks in July and August during the rainy season. P falciparum is the main malarial parasite. The entomological inoculation rate in the neighbouring area of Kintampo is about 250 infectious bites per year.
The study population consisted of pregnant women of all parities with a gestational age of 16 weeks or more who attended the antenatal clinic between March, 2003, and September, 2004. The study protocol was approved by the ethics committees of the Health Research Unit of the Ministry of Health of Ghana and the London School of Hygiene and Tropical Medicine. After written informed consent had been obtained, all pregnant women were screened for malaria infection with OptiMAL dipsticks (DiaMed AG, Cressier, Switzerland). Women who had positive results were tested for malaria parasites by microscopy. Only women with peripheral blood parasitaemia were enrolled into the study. We excluded women who had a multiple pregnancy, had severe malaria, or were enrolled previously in the current study.
Procedures
Eligible women were randomly allocated to one of four groups. Group 1 received 600 mg chloroquine for 2 days and 300 mg on the third day; group 2 received 600 mg amodiaquine for 2 days and 300 mg on the third day; group 3 received 1500 mg sulphadoxine and 75 mg pyrimethamine as one dose; group 4 received 600 mg amodiaquine, 1500 mg sulphadoxine, and 75 mg pyrimethamine on day 1, 600 mg amodiaquine on day 2, and 300 mg amodiaquine on day 3. The first dose of treatment was given by a member of the study team, and the second and third doses were given to study participants to take at home. All drugs were provided by Kinapharma (Accra, Ghana). The solubility and content of test drugs were confirmed with high-pressure liquid chromatography at the London School of Hygiene and Tropical Medicine.
Women were visited at home on days 3, 7, 14, and 28 after the start of treatment to assess adverse events and to obtain blood samples for microscopy. Blood samples for haemoglobin, bilirubin, liver transaminases, and white-blood-cell counts were taken on days 14 and 28. We followed up women at delivery and at 6 weeks' postpartum to measure perinatal outcomes, including placental parasitaemia, cord-blood parasitaemia, stillbirth, birthweight, neonatal death, and presence of congenital abnormalities. Examination for congenital abnormalities was first done by a midwife. If any abnormalities were suspected, a full examination, including a neurological assessment, was undertaken by a physician. A control group of 220 women with negative results from the malaria antigen test were randomly selected and actively followed on days 3, 7, 14, and 28, to compare the occurrence of symptoms suggestive of adverse events associated with study drugs.
The primary outcome of the study was to determine parasitological failure by day 28 of amodiaquine, sulphadoxine-pyrimethamine, and amodiaquine plus sulphadoxine-pyrimethamine, compared with chloroquine. The secondary outcomes were to compare the effect of study drugs on maternal haemoglobin, occurrence of adverse events, liver and bone marrow function, and perinatal outcomes. Parasitological failure by day 28 was defined as a need for rescue medication between days 0 and 28, or the presence of parasitaemia at day 28 in women who did not receive rescue medication. Rescue medication was given if a woman had a parasite density higher than that at enrolment between day 3 and day 14, or if a woman had parasitaemia of any density on day 14. Parasitological failure values were corrected for re-infection by PCR genotyping. Any new symptom or sign noted during the 28-day post-treatment period was defined as an adverse event.
Blocks of 16 treatment courses (four per study group) were packed in a large sealed envelope. There were 57 of these large envelopes in total. Every treatment course, concealed in a smaller sealed envelope, had a unique numerical code assigned from a random list of numbers. The assignment of drug codes and packaging was done by an independent statistician who took no further part in the trial. Study participants were asked to pick a drug pack from the larger envelope. The next large envelope was opened only when the contents of the previous one were used completely. The 57th large envelope containing four drug packs was the last to be opened. The entire study team and study participants were masked to the drug codes. Study drugs were formulated in adult doses and had the same appearance in size and colour. A drug pack contained a silver strip of three white tablets (containing sulphadoxine-pyrimethamine or placebo) and a blister pack of two pink tablets and one yellow tablet (containing active chloroquine or amodiaquine or corresponding placebo). Two spare packs of every treatment course were kept at the pharmacy.
A laboratory technician, masked to treatment assignment, examined thin and thick blood films to quantify parasitaemia against 200 leucocytes. A thick blood film was declared negative only after examination of 100 high-power fields. A second microscopist from the Noguchi Memorial Institute of Medical Research (Accra, Ghana) read 10% of all blood slides for quality assurance and was masked to treatment assignment. The agreements between the two microscopists on days 0, 3, 7, 14, and 28 were 95%, 99%, 98%, 99%, and 92%, respectively. PCR amplification for parasite genotyping was done on paired samples of P falciparum DNA to distinguish between re-infection and recrudescence by use of merozoite surface protein 2 (MSP2) as a genetic marker.13
Haemoglobin concentration was measured with a Haemocue (HemoCue AB, Angelholm, Sweden). We counted white blood cells microscopically by using uncoagulated whole blood that was diluted with a white-blood-cell diluting fluid. Kits were used to measure concentrations of aspartate aminotransferase and alanine aminotransferase (RANDOX Laboratories, Antrim, UK), and bilirubin and γ-glutamyl transpeptidase (BIOLABO, Maizy, France).
Statistical analysis
We assumed that parasitological failure by day 28 would be 22% in the chloroquine group14 and that failure would be no more than 10% in any of the other three groups. We also estimated that a sample size of 225 pregnant women per treatment group (including a 15% loss to follow-up) would have 90% power to detect a difference in failure between the chloroquine group and any of the other groups (a 12% absolute difference) at the 5% significance level.
The main analysis of primary and secondary outcomes was by an intention-to-treat basis to include all randomised participants in their assigned treatment groups according to a statistical analysis plan approved by the data and safety monitoring board. The significance of differences between proportions was tested by use of the χ2 test. The significance of changes in haemoglobin concentration, liver enzymes, bilirubin, and white-blood-cell count was tested by the Kruskal-Wallis test. This trial is registered with the US National Institute of Health clinical trials database number NCT00131703.
Role of the funding source
The sponsor of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.
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Results
6370 OptiMAL tests were done on 4500 women, of whom 1338 were positive. We enrolled 900 women who met all inclusion criteria. Follow-up on day 28 after treatment ranged from 92% to 94% in individual study groups (figure). Table 1 shows the differences in demographic or clinical characteristics between treatment groups at enrolment. A woman who became parasitaemic before delivery after successfully completing the initial 28-day follow-up received another course of her assigned treatment but did not re-enter the study.
Click to enlarge image
Figure. Trial profile showing the flow of study women through the initial 28-day follow-up periodSP=sulphadoxine-pyrimethamine. PS=parasitological success. PF=parasitological failure. *Some women were tested more than once before delivery if the interval between the first tests and the subsequent visit was 1 month or more. ?Eligible women had positive results who met selection criteria.
Table 1. Demographic and clinical characteristics of study women at enrolment
After PCR-correction for re-infection, failure rates were 14% for the chloroquine group, 11% for the sulphadoxine-pyrimethamine group, 3% for the amodiaquine group, and 0% for the amodiaquine plus sulphadoxine-pyrimethamine group (p<0?0001; table 2). Patients assigned amodiaquine or amodiaquine plus sulphadoxine-pyrimethamine had lower failure rates than did those assigned chloroquine (both p<0?0001); failure in the sulphadoxine-pyrimethamine group did not differ significantly from the chloroquine group (p=0?2). The pattern of failure across treatment groups was similar when women were grouped on the basis of whether their initial parasite count was more or less than 1000 per μL. High parasite densities at enrolment and low parity increased the risk of parasitological failure at day 28 (p<0?0001). Parasitological failure was also more common in anaemic women, but this association was not significant after adjustment for parity, baseline parasite density, and age (table 3). We recorded a general improvement in mean haemoglobin concentration after treatment in all groups (table 4), with significant increases by day 14 (p=0?04) and day 28 (p=0?01).
Table 2. Parasitological response to treatment by day 28
Table 3. Baseline factors associated with parasitological failure at day 28 after start of treatment
Table 4. Haematological and biochemical values in parasitaemic study women at enrolment and at days 14 and 28 after treatment
General weakness, vomiting, dizziness, and nausea were the most commonly reported adverse effects. They were recorded more frequently in the amodiaquine and amodiaquine plus sulphadoxine-pyrimethamine groups than in the sulphadoxine-pyrimethamine group (table 5). Compared with women assigned chloroquine, those assigned amodiaquine were twice as likely to report an adverse event (relative risk 1?9 [95% CI 1?7?3?2], p=0?01). Similarly, women assigned the amodiaquine plus sulphadoxine-pyrimethamine combination were three times more likely to report an adverse effect (3?0 [1?7?5?1], p<0?0001) than those assigned chloroquine. However, women receiving sulphadoxine-pyrimethamine reported an adverse event 70% less frequently than did those receiving chloroquinine (RR 0?3 [0?19?0?44], p<0?0001). A third of women who had not taken any antimalarial drug had symptoms or signs on days 3 and 7 after enrolment that would have been recorded as an adverse event in women receiving treatment. 13 women in the chloroquine group, six in the amodiaquine group, and 16 in the amodiaquine plus sulphadoxine-pyrimethamine group did not take the last dose of their treatment because of an adverse effect. The risk of reporting an adverse event overall was slightly higher in the amodiaquine plus sulphadoxine-pyrimethamine group by day 7, compared with the other treatment groups.
Table 5. Reporting of adverse effects
Table 4 shows median values and ranges of white blood cells, bilirubin, and liver enzymes at enrolment and at days 14 and 28 after treatment. Minor changes in total and differential white-blood-cell counts and biochemical measurements were seen after the start of treatment with no significant differences between treatment groups.
711 (79%) of 900 study women were followed to delivery. 174 (24%) deliveries occurred at home, 114 (16%) at a health centre and 423 (59%) in hospital. Follow-up at delivery (within 24 h of delivery) and at 6 weeks' postpartum were similar between the treatment groups (table 6). The proportion of women who had peripheral parasitaemia at delivery was significantly lower in the amodiaquine plus sulphadoxine-pyrimethamine group than in the chloroquine group (2% vs 10%; p=0?02; table 6), but the occurrence of placental parasitaemia did not differ significantly between treatment groups. We recorded no maternal deaths. Seven babies had extra digits (one chloroquine, five amodiaquine, and one amodiaquine plus sulphadoxine-pyrimethamine). One baby in the chloroquine group had a malformation of the external ear.
Table 6. Perinatal and postpartum outcomes
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Discussion
Our study has shown that, in Ghana, the parasitological failure by day 28 is high (PCR uncorrected rate 30%; PCR corrected rate 14%) in pregnant women treated with chloroquine. Thus, a change in treatment is needed because any parasitaemia in a pregnant woman, whether due to a new infection or recrudescence, is a threat to the mother and her fetus. Several studies in children have shown that amodiaquine alone or in combination with sulphadoxine-pyrimethamine or artesunate15?17 is more efficacious than chloroquine in clearing parasitaemia and our study confirms this effect in pregnant women. In pregnant Ghanaian women, amodiaquine is an efficacious alternative to sulphadoxine-pyrimethamine and the combination of amodiaquine with sulphadoxine-pyrimethamine is very effective. This combination could be considered as a replacement for chloroquine and sulphadoxine-pyrimethamine in areas where parasites remain sensitive to amodiaquine and sulphadoxine-pyrimethamine, which occurs in much of west Africa. High parasite density and low parity independently predicted parasitological failure, and these variables need to be considered when drug sensitivity is assessed in pregnant women. Anaemia also predicted treatment failure, although this was not significant after correction for other variables, as has been recorded in children.18
Amodiaquine and amodiaquine plus sulphadoxine-pyrimethamine resulted in the highest occurrence of minor side-effects, whereas sulphadoxine-pyrimethamine alone was the best tolerated regimen. However, only 4% of women did not finish the full course of treatment because of adverse effects. The study women could have complied with the treatment because they knew that they had malaria. Compliance with amodiaquine or amodiaquine plus sulphadoxine-pyrimethamine might not be as good if these drugs are used for intermittent preventive treatment in pregnancy because, in this situation, most women will not know whether or not they are infected.
Earlier studies19 of prophylaxis with amodiaquine plus sulphadoxine-pyrimethamine, and mefloquine plus sulphadoxine-pyrimethamine or chloroquine, have recorded increases in aspartate aminotransferase, alanine aminotransferase, and γ-glutamyl transferase activities above normal ranges that might not have been related to the drugs. We saw little change in liver enzyme concentrations in the women in our study. Earlier studies of patients receiving amodiaquine, mostly for prophylaxis, showed an association with leucopenia due to neutropenia20?24 or to lymphopenia,24 which we did not observe.
Rates of abortions, preterm deliveries, and stillbirths in the study women were similar to those for non-study women who delivered at St Theresa's Hospital (unpublished data) and did not differ between study groups. Some minor congenital abnormalities were seen, but with no significant differences between groups. However, the study was too small to detect an increase in rare congenital abnormalities.
Weaknesses of the study were the loss to follow-up for the maternal and fetal outcomes at delivery (21%) and for the 6 weeks' postpartum maternal outcomes (36%); lack of Dubowitz score to assess prematurity, since ultrasonography assessments obtained at enrolment might not be appropriate for estimating gestational age at delivery; and lack of placental histology. However, since the loss to follow-up was similar between treatment groups and the potential misclassification of prematurity was probably distributed equally between groups, we think that our conclusions are unbiased.
We conclude that the combination of amodiaquine plus sulphadoxine-pyrimethamine is an efficacious regimen for the treatment of pregnant women with parasitaemia. No serious side-effects were noted and the combination was reasonably well tolerated by pregnant women who understood that the benefit of treatment outweighed the minor discomfort caused by this regimen. Another study to assess the efficacy and tolerability of these study regimens when used for intermittent preventive treatment for malaria in pregnancy is currently in progress in Navrongo, Ghana. The enrolment phase of this study has been completed and the results will be available in early 2007 (Clerk C, Navrongo Health Research Centre, Navrongo, Ghana, personal communication).
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Conflict of interest statement
We declare that we have no conflict of interest.
Contributors
H Tagbor developed the study protocol, oversaw the implementation of the trial, and assisted with the data analysis and drafting of the manuscript. J Bruce contributed to the data analysis and to the manuscript. E Browne contributed to the development of the protocol and to the manuscript. A Randall undertook the PCR analysis. B Greenwood and D Chandramohan contributed to the development of the protocol, monitoring of the trial, data analysis, and drafting of the manuscript.
Acknowledgments
We thank the study participants and the staff of St Theresa's Hospital who assisted with running this project; Harparkash Kaur, Rosalind Ords, Francis Owusu Ansah, Frank Agyemang Bonsu, Elizabeth Dekyi, James Beard, Heather Naylor, Amit Bhasin, and Carol Aldous for their support for this project; and Seth Owusu-Agyei for allowing us to use the Kintampo Health Research Centre's laboratory for quality assurance. This study was funded by the Gates Malaria Partnership (GMP), which receives support from the Bill and Melinda Gates Foundation.
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<!--start tail=-->References
1. World Health Organization. A strategic framework for malaria prevention and control during pregnancy in the African region. Brazzaville, Democratic Republic of the Congo: WHO Regional Office for Africa, 2004:AFR/MAL/04/01.
2. Shulman CE, T Marshall EK, Dorman JN, et al. Malaria in pregnancy: adverse effects on haemoglobin levels and birthweight in primigravidae and multigravidae. Trop Med Int Health 2001; 6: 770-778. CrossRef
3. Steketee RW, Nahlen BL, Parise ME, Menendez C. The burden of malaria in pregnancy in malaria-endemic areas. Am J Trop Med Hyg 2001; 64: 28-35.
4. Garner P, Gulmezoglu AM. Prevention versus treatment for malaria in pregnant women. Cochrane Database Syst Rev 2000; 2:CD000169.
5. World Health Organization. Assessment of the safety of artemisinin compounds in pregnancy. Geneva: WHO, 2003:WHO/CDS/MAL 2003.1094, WHO/RBM/TDR/Artemisinin/03.1.
6. Mengesha T, Makonnen E. Comparative efficacy and safety of chloroquine and alternative antimalarial drugs: a meta-analysis from six African countries. East Afr Med J 1999; 76: 314-319.
7. Muller O, van Hensbroek MB, Jaffar S, et al. A randomized trial of chloroquine, amodiaquine and pyrimethamine-sulphadoxine in Gambian children with uncomplicated malaria. Trop Med Int Health 1996; 1: 124-132. MEDLINE
8. EANMAT. The efficacy of antimalarial monotherapies, sulphadoxine-pyrimethamine and amodiaquine in east Africa: implications for sub-regional policy. Trop Med Int Health 2003; 8: 860-867. CrossRef
9. Baird JK. Effectiveness of antimalarial drugs. N Engl J Med 2005; 352: 1565-1577. CrossRef
10. Thomas F, Erhart A, D'Alessandro U. Can amodiaquine be used safely during pregnancy?. Lancet Infect Dis 2004; 4: 235-239. Abstract | Full Text | PDF (112 KB) | CrossRef
11. Thet N, Hla W, Yin Yin N. Falciparum malaria and pregnancy: relationship and treatment response. S Asian J Trop Med Public Health 1988; 19: 253-258.
12. Steketee RW, Brandling-Bennett AD, Kaseje DCO, Schwartz IK, Churchill FC. In vivo response of Plasmodium falciparum to chloroquine in pregnant and non-pregnant women in Siaya District, Kenya. Bull World Health Organ 1987; 65: 885-890. MEDLINE
13. Snounou G, Zhu N, Siripoon W, et al. Biased distribution of msp1 and msp2 allelic variants in Plasmodium falciparum populations in Thailand. Trans R Soc Trop Med Hyg 1999; 93: 369-374. CrossRef
14. Marfo C. Ghana malaria baseline situational analysis survey report. Accra, Ghana: MOH/GHANA, WHO-RBM/AFRO, 2001:.
15. Mockenhaupt FP, Ehrhardt S, Dzisi SY, et al. A randomized, placebo-controlled, double-blind trial on sulfadoxine-pyrimethamine alone or combined with artesunate or amodiaquine in uncomplicated malaria. Trop Med Int Health 2005; 10: 512-520. CrossRef
16. Oduro AR, Anyorigiya T, Hodgson A, et al. A randomized comparative study of chloroquine, amodiaquine and sulphadoxine-pyrimethamine for the treatment of uncomplicated malaria in Ghana. Trop Med Int Health 2005; 10: 279-284. CrossRef
17. Olliaro P, Nevill C, LeBras J, et al. Systematic review of amodiaquine treatment in uncomplicated malaria. Lancet 1996; 348: 1196-1201. Abstract | Full Text | PDF (230 KB) | MEDLINE | CrossRef
18. Meerman L, Ord R, Bousema JT, et al. Carriage of chloroquine-resistant parasites and delay of effective treatment increase the risk of severe malaria in Gambian children. J Infect Dis 2005; 192: 1651-1657. MEDLINE | CrossRef
19. Kollaritsch H, Stemberger H, Mailer H, et al. Tolerability of long-term malaria prophylaxis with the combination mefloquine+sulfadoxine+pyrimethamine (Fansimef): results of a double blind field trial versus chloroquine in Nigeria. Trans R Soc Trop Med Hyg 1988; 82: 524-529. MEDLINE | CrossRef
20. Phillips-Howard PA, West LJ. Serious adverse drug reactions to pyrimethamine-sulphadoxine, pyrimethamine-dapsone and to amodiaquine in Britain. J R Soc Med 1990; 83: 82-85. MEDLINE
21. Hatton CSR, Peto TEA, Bunch C, et al. Frequency of severe neutropenia associated with amodiaquine prophylaxis against malaria. Lancet 1986; 327: 411-414. CrossRef
22. Neftel KA, Woodtly W, Schmid M, Frick PG, Fehr J. Amodiaquine induced agranulocytosis and liver damage. BMJ 1986; 292: 721-723.
23. Rhodes EGH, Ball J, Franklin M. Amodiaquine induced agranulocytosis: inhibition of colony growth in bone marrow by antimalarial agents. BMJ 1986; 292: 717-718.
24. Sturchler D, Schar M, Gyr N. Leucopenia and abnormal liver function in travellers on malaria chemoprophylaxis. J Trop Med Hyg 1987; 90: 239-243. MEDLINE
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DOI:10.1016/S0140-6736(06)69559-7
Efficacy, safety, and tolerability of amodiaquine plus sulphadoxine-pyrimethamine used alone or in combination for malaria treatment in pregnancy: a randomised trial
Dr Harry TagborDrPH
a , Jane BruceMSc c, Edmund BrownePhD b, Anna RandalMSc c, Prof Brian GreenwoodMD c and Daniel ChandramohanPhD cSee Comment
Summary
Introduction
Methods
Results
Discussion
References
Summary
BackgroundThe widespread increase in resistance of Plasmodium falciparum to chloroquine and sulphadoxine-pyrimethamine threatens the use of these drugs for malaria treatment in pregnancy. We aimed to assess the safety and efficacy of amodiaquine alone or in combination with sulphadoxine-pyrimethamine as alternative regimens.
MethodsPregnant women with a gestational age of 16 weeks or more who attended antenatal clinics at a district hospital in Ghana were screened for malaria with OptiMAL dipsticks. 900 pregnant women who had a positive test result and P falciparum asexual stage parasitaemia were enrolled and randomly assigned chloroquine, sulphadoxine-pyrimethamine, amodiaquine, or amodiaquine plus sulphadoxine-pyrimethamine. The primary outcome was parasitological failure by day 28 of treatment. Women were seen on days 3, 7, 14, and 28 after the start of treatment to assess the effect of treatment on peripheral parasitaemia, haemoglobin concentration, white-blood-cell count, and liver function. Additionally, reports of adverse effects were solicited and monitored during follow-up visits. Analysis was by intention to treat. This trial is registered with the US National Institute of Health clinical trials database number NCT00131703.
FindingsPCR-corrected parasitological failure by day 28 was 14%, 11%, 3%, and 0% in the women assigned chloroquine, sulphadoxine-pyrimethamine, amodiaquine, and amodiaquine plus sulphadoxine-pyrimethamine, respectively (p<0?0001). No serious liver toxic effects or white-blood-cell dyscrasias were noted. Minor side-effects were reported more often on day 3 by women receiving amodiaquine (86%) or amodiaquine plus sulphadoxine-pyrimethamine (90%) than those receiving sulphadoxine-pyrimethamine (48%) or no antimalarial drugs (34%; p<0?0001 for every comparison).
InterpretationAmodiaquine alone or in combination with sulphadoxine-pyrimethamine, although associated with minor side-effects, is effective when used to treat malaria in pregnancy.
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Introduction
Plasmodium falciparum infection in pregnancy is associated with maternal and perinatal morbidity.1?3 For malaria control in pregnancy, WHO recommends the use of insecticide-treated bednets, intermittent preventive treatment with sulphadoxine-pyrimethamine in the second and third trimesters, and prompt and effective treatment of malaria and anaemia.1,4 Chloroquine and sulphadoxine-pyrimethamine are the safest and most readily available and affordable drugs for the treatment or prevention of uncomplicated malaria in pregnancy in most African countries. However, these drugs are becoming ineffective against uncompleted malaria in many areas because of increasing parasite resistance. Therefore, alternative safe and efficacious antimalarial drugs are needed urgently for both treatment of malaria during pregnancy and intermittent preventive treatment. Most countries in Africa are adopting artesunate-based combination therapy (ACT) as the preferred drug for the treatment of uncomplicated malaria. However, very little information on the safety of ACT during pregnancy is available, and there are concerns about the possible teratogenicity of these drugs when given in the first trimester of pregnancy.5 In Ghana, chloroquine was the preferred treatment for uncomplicated malaria in children and pregnant women, until a decision was made to change to the combination of artesunate and amodiaquine.
Since amodiaquine is effective in some areas with chloroquine resistance,6,7 and because the extent of resistance to sulphadoxine-pyrimethamine in west Africa is not as high as that in east Africa,7?9 we postulated that amodiaquine, used either alone or in combination with sulphadoxine-pyrimethamine, might be a safe and efficacious drug option for malaria treatment in pregnancy and for intermittent preventive treatment in Ghana until the safety of ACTs in pregnancy has been established. However, little information exists about the safety or efficacy of amodiaquine in pregnancy. A review10 found only six reports on the use of amodiaquine in pregnancy, of which four described its use for chemoprophylaxis and two for treatment. Toxic effects were assessed specifically in only one of the chemoprophylaxis studies, and none reported the outcome of pregnancy. The two treatment studies, one in Burma11 and another in Kenya,12 were small (19 and 23 patients, respectively) and did not study toxic effects specifically. Because of the paucity of knowledge about the safety and efficacy of amodiaquine in pregnancy, we undertook a clinical trial in which amodiaquine, amodiaquine plus sulphadoxine-pyrimethamine, sulphadoxine-pyrimethamine, and chloroquine were compared in the treatment of pregnant women with proven malaria infection.
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Methods
Patients
The study was done at St Theresa's Hospital, Nkoranza, Ghana. Malaria transmission in this area is perennial but peaks in July and August during the rainy season. P falciparum is the main malarial parasite. The entomological inoculation rate in the neighbouring area of Kintampo is about 250 infectious bites per year.
The study population consisted of pregnant women of all parities with a gestational age of 16 weeks or more who attended the antenatal clinic between March, 2003, and September, 2004. The study protocol was approved by the ethics committees of the Health Research Unit of the Ministry of Health of Ghana and the London School of Hygiene and Tropical Medicine. After written informed consent had been obtained, all pregnant women were screened for malaria infection with OptiMAL dipsticks (DiaMed AG, Cressier, Switzerland). Women who had positive results were tested for malaria parasites by microscopy. Only women with peripheral blood parasitaemia were enrolled into the study. We excluded women who had a multiple pregnancy, had severe malaria, or were enrolled previously in the current study.
Procedures
Eligible women were randomly allocated to one of four groups. Group 1 received 600 mg chloroquine for 2 days and 300 mg on the third day; group 2 received 600 mg amodiaquine for 2 days and 300 mg on the third day; group 3 received 1500 mg sulphadoxine and 75 mg pyrimethamine as one dose; group 4 received 600 mg amodiaquine, 1500 mg sulphadoxine, and 75 mg pyrimethamine on day 1, 600 mg amodiaquine on day 2, and 300 mg amodiaquine on day 3. The first dose of treatment was given by a member of the study team, and the second and third doses were given to study participants to take at home. All drugs were provided by Kinapharma (Accra, Ghana). The solubility and content of test drugs were confirmed with high-pressure liquid chromatography at the London School of Hygiene and Tropical Medicine.
Women were visited at home on days 3, 7, 14, and 28 after the start of treatment to assess adverse events and to obtain blood samples for microscopy. Blood samples for haemoglobin, bilirubin, liver transaminases, and white-blood-cell counts were taken on days 14 and 28. We followed up women at delivery and at 6 weeks' postpartum to measure perinatal outcomes, including placental parasitaemia, cord-blood parasitaemia, stillbirth, birthweight, neonatal death, and presence of congenital abnormalities. Examination for congenital abnormalities was first done by a midwife. If any abnormalities were suspected, a full examination, including a neurological assessment, was undertaken by a physician. A control group of 220 women with negative results from the malaria antigen test were randomly selected and actively followed on days 3, 7, 14, and 28, to compare the occurrence of symptoms suggestive of adverse events associated with study drugs.
The primary outcome of the study was to determine parasitological failure by day 28 of amodiaquine, sulphadoxine-pyrimethamine, and amodiaquine plus sulphadoxine-pyrimethamine, compared with chloroquine. The secondary outcomes were to compare the effect of study drugs on maternal haemoglobin, occurrence of adverse events, liver and bone marrow function, and perinatal outcomes. Parasitological failure by day 28 was defined as a need for rescue medication between days 0 and 28, or the presence of parasitaemia at day 28 in women who did not receive rescue medication. Rescue medication was given if a woman had a parasite density higher than that at enrolment between day 3 and day 14, or if a woman had parasitaemia of any density on day 14. Parasitological failure values were corrected for re-infection by PCR genotyping. Any new symptom or sign noted during the 28-day post-treatment period was defined as an adverse event.
Blocks of 16 treatment courses (four per study group) were packed in a large sealed envelope. There were 57 of these large envelopes in total. Every treatment course, concealed in a smaller sealed envelope, had a unique numerical code assigned from a random list of numbers. The assignment of drug codes and packaging was done by an independent statistician who took no further part in the trial. Study participants were asked to pick a drug pack from the larger envelope. The next large envelope was opened only when the contents of the previous one were used completely. The 57th large envelope containing four drug packs was the last to be opened. The entire study team and study participants were masked to the drug codes. Study drugs were formulated in adult doses and had the same appearance in size and colour. A drug pack contained a silver strip of three white tablets (containing sulphadoxine-pyrimethamine or placebo) and a blister pack of two pink tablets and one yellow tablet (containing active chloroquine or amodiaquine or corresponding placebo). Two spare packs of every treatment course were kept at the pharmacy.
A laboratory technician, masked to treatment assignment, examined thin and thick blood films to quantify parasitaemia against 200 leucocytes. A thick blood film was declared negative only after examination of 100 high-power fields. A second microscopist from the Noguchi Memorial Institute of Medical Research (Accra, Ghana) read 10% of all blood slides for quality assurance and was masked to treatment assignment. The agreements between the two microscopists on days 0, 3, 7, 14, and 28 were 95%, 99%, 98%, 99%, and 92%, respectively. PCR amplification for parasite genotyping was done on paired samples of P falciparum DNA to distinguish between re-infection and recrudescence by use of merozoite surface protein 2 (MSP2) as a genetic marker.13
Haemoglobin concentration was measured with a Haemocue (HemoCue AB, Angelholm, Sweden). We counted white blood cells microscopically by using uncoagulated whole blood that was diluted with a white-blood-cell diluting fluid. Kits were used to measure concentrations of aspartate aminotransferase and alanine aminotransferase (RANDOX Laboratories, Antrim, UK), and bilirubin and γ-glutamyl transpeptidase (BIOLABO, Maizy, France).
Statistical analysis
We assumed that parasitological failure by day 28 would be 22% in the chloroquine group14 and that failure would be no more than 10% in any of the other three groups. We also estimated that a sample size of 225 pregnant women per treatment group (including a 15% loss to follow-up) would have 90% power to detect a difference in failure between the chloroquine group and any of the other groups (a 12% absolute difference) at the 5% significance level.
The main analysis of primary and secondary outcomes was by an intention-to-treat basis to include all randomised participants in their assigned treatment groups according to a statistical analysis plan approved by the data and safety monitoring board. The significance of differences between proportions was tested by use of the χ2 test. The significance of changes in haemoglobin concentration, liver enzymes, bilirubin, and white-blood-cell count was tested by the Kruskal-Wallis test. This trial is registered with the US National Institute of Health clinical trials database number NCT00131703.
Role of the funding source
The sponsor of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.
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Results
6370 OptiMAL tests were done on 4500 women, of whom 1338 were positive. We enrolled 900 women who met all inclusion criteria. Follow-up on day 28 after treatment ranged from 92% to 94% in individual study groups (figure). Table 1 shows the differences in demographic or clinical characteristics between treatment groups at enrolment. A woman who became parasitaemic before delivery after successfully completing the initial 28-day follow-up received another course of her assigned treatment but did not re-enter the study.
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Figure. Trial profile showing the flow of study women through the initial 28-day follow-up periodSP=sulphadoxine-pyrimethamine. PS=parasitological success. PF=parasitological failure. *Some women were tested more than once before delivery if the interval between the first tests and the subsequent visit was 1 month or more. ?Eligible women had positive results who met selection criteria.
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Table 1. Demographic and clinical characteristics of study women at enrolment
After PCR-correction for re-infection, failure rates were 14% for the chloroquine group, 11% for the sulphadoxine-pyrimethamine group, 3% for the amodiaquine group, and 0% for the amodiaquine plus sulphadoxine-pyrimethamine group (p<0?0001; table 2). Patients assigned amodiaquine or amodiaquine plus sulphadoxine-pyrimethamine had lower failure rates than did those assigned chloroquine (both p<0?0001); failure in the sulphadoxine-pyrimethamine group did not differ significantly from the chloroquine group (p=0?2). The pattern of failure across treatment groups was similar when women were grouped on the basis of whether their initial parasite count was more or less than 1000 per μL. High parasite densities at enrolment and low parity increased the risk of parasitological failure at day 28 (p<0?0001). Parasitological failure was also more common in anaemic women, but this association was not significant after adjustment for parity, baseline parasite density, and age (table 3). We recorded a general improvement in mean haemoglobin concentration after treatment in all groups (table 4), with significant increases by day 14 (p=0?04) and day 28 (p=0?01).
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Table 2. Parasitological response to treatment by day 28
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Table 3. Baseline factors associated with parasitological failure at day 28 after start of treatment
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Table 4. Haematological and biochemical values in parasitaemic study women at enrolment and at days 14 and 28 after treatment
General weakness, vomiting, dizziness, and nausea were the most commonly reported adverse effects. They were recorded more frequently in the amodiaquine and amodiaquine plus sulphadoxine-pyrimethamine groups than in the sulphadoxine-pyrimethamine group (table 5). Compared with women assigned chloroquine, those assigned amodiaquine were twice as likely to report an adverse event (relative risk 1?9 [95% CI 1?7?3?2], p=0?01). Similarly, women assigned the amodiaquine plus sulphadoxine-pyrimethamine combination were three times more likely to report an adverse effect (3?0 [1?7?5?1], p<0?0001) than those assigned chloroquine. However, women receiving sulphadoxine-pyrimethamine reported an adverse event 70% less frequently than did those receiving chloroquinine (RR 0?3 [0?19?0?44], p<0?0001). A third of women who had not taken any antimalarial drug had symptoms or signs on days 3 and 7 after enrolment that would have been recorded as an adverse event in women receiving treatment. 13 women in the chloroquine group, six in the amodiaquine group, and 16 in the amodiaquine plus sulphadoxine-pyrimethamine group did not take the last dose of their treatment because of an adverse effect. The risk of reporting an adverse event overall was slightly higher in the amodiaquine plus sulphadoxine-pyrimethamine group by day 7, compared with the other treatment groups.
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Table 5. Reporting of adverse effects
Table 4 shows median values and ranges of white blood cells, bilirubin, and liver enzymes at enrolment and at days 14 and 28 after treatment. Minor changes in total and differential white-blood-cell counts and biochemical measurements were seen after the start of treatment with no significant differences between treatment groups.
711 (79%) of 900 study women were followed to delivery. 174 (24%) deliveries occurred at home, 114 (16%) at a health centre and 423 (59%) in hospital. Follow-up at delivery (within 24 h of delivery) and at 6 weeks' postpartum were similar between the treatment groups (table 6). The proportion of women who had peripheral parasitaemia at delivery was significantly lower in the amodiaquine plus sulphadoxine-pyrimethamine group than in the chloroquine group (2% vs 10%; p=0?02; table 6), but the occurrence of placental parasitaemia did not differ significantly between treatment groups. We recorded no maternal deaths. Seven babies had extra digits (one chloroquine, five amodiaquine, and one amodiaquine plus sulphadoxine-pyrimethamine). One baby in the chloroquine group had a malformation of the external ear.
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Table 6. Perinatal and postpartum outcomes
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Discussion
Our study has shown that, in Ghana, the parasitological failure by day 28 is high (PCR uncorrected rate 30%; PCR corrected rate 14%) in pregnant women treated with chloroquine. Thus, a change in treatment is needed because any parasitaemia in a pregnant woman, whether due to a new infection or recrudescence, is a threat to the mother and her fetus. Several studies in children have shown that amodiaquine alone or in combination with sulphadoxine-pyrimethamine or artesunate15?17 is more efficacious than chloroquine in clearing parasitaemia and our study confirms this effect in pregnant women. In pregnant Ghanaian women, amodiaquine is an efficacious alternative to sulphadoxine-pyrimethamine and the combination of amodiaquine with sulphadoxine-pyrimethamine is very effective. This combination could be considered as a replacement for chloroquine and sulphadoxine-pyrimethamine in areas where parasites remain sensitive to amodiaquine and sulphadoxine-pyrimethamine, which occurs in much of west Africa. High parasite density and low parity independently predicted parasitological failure, and these variables need to be considered when drug sensitivity is assessed in pregnant women. Anaemia also predicted treatment failure, although this was not significant after correction for other variables, as has been recorded in children.18
Amodiaquine and amodiaquine plus sulphadoxine-pyrimethamine resulted in the highest occurrence of minor side-effects, whereas sulphadoxine-pyrimethamine alone was the best tolerated regimen. However, only 4% of women did not finish the full course of treatment because of adverse effects. The study women could have complied with the treatment because they knew that they had malaria. Compliance with amodiaquine or amodiaquine plus sulphadoxine-pyrimethamine might not be as good if these drugs are used for intermittent preventive treatment in pregnancy because, in this situation, most women will not know whether or not they are infected.
Earlier studies19 of prophylaxis with amodiaquine plus sulphadoxine-pyrimethamine, and mefloquine plus sulphadoxine-pyrimethamine or chloroquine, have recorded increases in aspartate aminotransferase, alanine aminotransferase, and γ-glutamyl transferase activities above normal ranges that might not have been related to the drugs. We saw little change in liver enzyme concentrations in the women in our study. Earlier studies of patients receiving amodiaquine, mostly for prophylaxis, showed an association with leucopenia due to neutropenia20?24 or to lymphopenia,24 which we did not observe.
Rates of abortions, preterm deliveries, and stillbirths in the study women were similar to those for non-study women who delivered at St Theresa's Hospital (unpublished data) and did not differ between study groups. Some minor congenital abnormalities were seen, but with no significant differences between groups. However, the study was too small to detect an increase in rare congenital abnormalities.
Weaknesses of the study were the loss to follow-up for the maternal and fetal outcomes at delivery (21%) and for the 6 weeks' postpartum maternal outcomes (36%); lack of Dubowitz score to assess prematurity, since ultrasonography assessments obtained at enrolment might not be appropriate for estimating gestational age at delivery; and lack of placental histology. However, since the loss to follow-up was similar between treatment groups and the potential misclassification of prematurity was probably distributed equally between groups, we think that our conclusions are unbiased.
We conclude that the combination of amodiaquine plus sulphadoxine-pyrimethamine is an efficacious regimen for the treatment of pregnant women with parasitaemia. No serious side-effects were noted and the combination was reasonably well tolerated by pregnant women who understood that the benefit of treatment outweighed the minor discomfort caused by this regimen. Another study to assess the efficacy and tolerability of these study regimens when used for intermittent preventive treatment for malaria in pregnancy is currently in progress in Navrongo, Ghana. The enrolment phase of this study has been completed and the results will be available in early 2007 (Clerk C, Navrongo Health Research Centre, Navrongo, Ghana, personal communication).
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Conflict of interest statement
We declare that we have no conflict of interest.
Contributors
H Tagbor developed the study protocol, oversaw the implementation of the trial, and assisted with the data analysis and drafting of the manuscript. J Bruce contributed to the data analysis and to the manuscript. E Browne contributed to the development of the protocol and to the manuscript. A Randall undertook the PCR analysis. B Greenwood and D Chandramohan contributed to the development of the protocol, monitoring of the trial, data analysis, and drafting of the manuscript.
Acknowledgments
We thank the study participants and the staff of St Theresa's Hospital who assisted with running this project; Harparkash Kaur, Rosalind Ords, Francis Owusu Ansah, Frank Agyemang Bonsu, Elizabeth Dekyi, James Beard, Heather Naylor, Amit Bhasin, and Carol Aldous for their support for this project; and Seth Owusu-Agyei for allowing us to use the Kintampo Health Research Centre's laboratory for quality assurance. This study was funded by the Gates Malaria Partnership (GMP), which receives support from the Bill and Melinda Gates Foundation.
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