Artesunate plus sulfadoxine-pyrimethamine for uncomplicated malaria ...

TRANSACTIONSOF THE ROYALSOCIETYOF TROPICALMEDICINEAND HYGIENE(2003) 97, 585-591

Artesunate plus sulfadoxine-pyrimethamine for uncomplicated malaria in Kenyan children: a randomized, double-blind, placebo-controlled trial Charles O. O b o n y o 1, Francis O c h i e n g 2, Walter R. J. Taylor 3, S a m u e l A. O c h o l a 4, Kefas Mugitu 5, Piero O l l i a r o 3, Feiko ter Kuile t'6 and Aggrey J. O1001 1Centre for Vector Biology and Control Research, Kenya Medical Research Institute, Kisumu, Kenya; 2Siaya District Hospital, Siaya, Kenya; 3UNDP/World Bank~Special Programme for Research and Training in Tropical Diseases (TDR), World Health Organization, Geneva, Switzerland; 4National Malaria Control Programme, Kenyan Ministry of Health, Nairobi, Kenya; 5Ifakara Health Research and Development Centre (IHRDC), Ifakara, Tanzania; 6Unit of Infectious Diseases and Tropical Medicine, A M C , University of Amsterdam, Amsterdam, The Netherlands Abstract Plasmodium falciparum has developed resistance to almost all routinely used antimalarial drugs. Sulfadoxine-pyrimethamine (SP) has replaced chloroquine as first-line treatment of uncomplicated malaria infection in Kenya but resistance to SP is already reported. The addition of artemisinin derivatives to SP may delay the development of drug resistance, improve cure rates, and reduce transmission. The efficacy and safety of artesunate plus SP in the treatment of uncomplicated P. falciparum malaria was evaluated in a randomized trial of 600 children at Siaya District Hospital, western Kenya between October 1999 and March 2000. Children aged < 5 years were randomly assigned to receive SP alone (1.25 mg/kg based on pyrimethamine), or in combination with artesunate (4 mg/kg/d) for either 1 or 3 d. Parasitological failure by days 14 and 28 (polymerase chain reaction [PCR]-corrected for new infections) were the primary endpoints. Treatment failure rates by day 14 were 25.5% in the SP alone group, 16.2% (risk difference [zX]-9.3%, 95% CI -17.3 to -1.2%, P = 0.027) in the 1-dose artesunate group, and 9.4% (A-16.2%, 95% CI - 2 3 . 6 to - 8 . 7 % , P < 0.001) in the 3-dose artesunate group. Corresponding rates by day 28 were 46.0% in the SP alone group, 38.2% (A-7.8%, 95% CI - 1 7 . 7 to 2.1%, P = 0.16) in the 1-dose artesunate group, and 26.0% (A-20.0%, 95% CI - 2 9 . 4 to - 1 0 . 6 % , P < 0.001) in the 3-dose artesunate group. The artesunate and SP combination was well tolerated. There were no serious drug-related adverse events. Parasite clearance and gametocyte carriage were reduced significantly in both combination groups compared with SP alone. Three days of artesunate were required to reduce significantly the risk of treatment failure by day 28. However, the high background rate of parasitological failure with SP may make this combination unsuitable for widespread use in Kenya.

Keywords: malaria, artesunate, sulfadoxine-pyrimethamine, combination therapy, Kenya Introduction Plasmodium falciparum, resistant to most standard antimalarial drugs, poses a major problem for the treatment of malaria. Several countries in sub-Saharan Africa including Kenya, Malawi, Tanzania, and South Africa have replaced chloroquine with sulfadoxinepyrimethamine (SP) as the first-line drug for the treatment of uncomplicated P. falciparum malaria. Both the long unmatched half-lives of sulfadoxine and pyrimethamine (mean 180 h and 95 h, respectively) and the mechanism of resistance (single-point mutations) favour the selection of resistant parasites (Watkins & Mosobo, 1993; Nzila et al., 2000; Hastings et al., 2002). In other areas of the world where SP replaced chloroquine, such as South-East Asia, resistance to SP developed within a few years of its introduction. There is concern that in Africa the effective life of SP may be equally limited. SP has been used officially in East Africa for < 5 years and already resistance is present, resulting in a decrease in the effectiveness of this drug (Anabwani et al., 1996; R o n n e t al., 1996; van Dillen et al., 1999; Gorissen et al., 2000; Ogutu et al., 2000). In addition, alternatives to SP are few, costly, and may have more adverse effects. Protection of drugs against the development of resistance is a key factor in the fight against malaria. One strategy that may achieve this objective is the use of drug combinations with independent modes of action. The concept that resistance could be delayed or prevented by combining drugs with different targets was first developed in the treatment of tuberculosis (Grosett, 1980), and has been adopted widely for the treatment of HIV, leprosy, and cancer. Artemisinin-based Address for correspondence: Charles O. Obonyo, Centre for Vector Biology and Control, Kenya Medical Research Institute, P.O. Box 1578, Kisumn, Kenya; phone +254 35 22923/ 24, cellphone +254 733 837969, fax +254 35 22981, e-mail [email protected]

drug combinations have been proposed as an option for treatment of drug-resistant malaria (White & Olliaro, 1996). The artemisinins are currently the most potent of antimalarials, and to date no clinical resistance has been documented. The basis for adopting artemisininbased drug combinations is the ability of the artemisinin to reduce significantly the initial parasite biomass, leaving only a small residual number of parasites to be eliminated by high concentrations of the companion drug. This minimizes the risk of parasite exposure to suboptimal antimalarial drug levels thus reducing the risk of selection of resistant strains (White, 1999). Furthermore, because the artemisinins reduce gametocyte carriage their use may also lead to a reduction in malaria transmission (Price et al., 1996). In Thailand, an area of low malaria transmission, the introduction of the combination of artesunate plus mefloquine for the treatment of multidrug-resistant P. falciparum malaria has resulted in an improvement in cure rates, reduction in malaria transmission, and a sustained high efficacy of mefloquine (Price et al., 1996; Nosten et al., 2000). As of today, only one study has been published from Africa that has evaluated the effect of artesunate and SP in combination. In that study, in The Gambia, children with uncomplicated P. falciparum malaria treated with 3 d of artesunate plus SP had a faster resolution of fever, parasite clearance, and gametocyte carriage compared with those on SP alone (yon Seidlein et al., 2000). The efficacy of SP in vivo was high in the Gambian setting when the trial was undertaken. It is not known whether these findings would apply to areas of sub-Saharan Africa where parasitological resistance to SP is already substantial, or areas with higher malaria transmission. As a first step to the possible deployment of artemisinin-based combinations in sub-Saharan Africa, there is need to study the safety and efficacy of these combinations. We therefore evaluated in a randomized, doubleblind, placebo-controlled trial, the efficacy, safety and

C.O. OBONYOETAL.

586 tolerability of artesunate plus SP compared with SP alone in the treatment of uncomplicated P. falciparum malaria in western Kenya. This study was part of a multicentre trial sponsored by UNDP/World Bank/ W H O Special Programme for Research and Training in Tropical Diseases (TDR) to assess the role of artesunate-based combinations for treatment of uncomplicated P. falciparum malaria. Materials a n d M e t h o d s

Study site The study was conducted between October 1999 and March 2000 at Siaya District Hospital, a government health facility serving a rural population of approximately 600 000 people in Siaya District, western Kenya. The area has intense P. falciparum malaria transmission occurring throughout the year, with peak transmission associated with the 2 rainy seasons (March to June and September to October). The entomological inoculation rate is estimated at 100-300 per person-year (Beier et al., 1994). SP has been used in this hospital as second-line treatment for uncomplicated P. falciparum malaria since 1992. In 1999, there was a 27% parasitological failure rate in vivo in the surrounding community 7 d after treatment using SP in children aged < 5 years (F. ter Kuile, unpublished data).

Patient screening and recruitment Children aged < 5 years with a history of fever attending the hospital's outpatient department were referred to the study team for evaluation. They were eligible for inclusion in the study if they had a consenting guardian, weighed at least 5.0 kg, had a history of fever during the preceding 48 h, and had a smearconfirmed monoinfection with P. falciparum of at least 4000 asexual parasites/mm 3. Children were excluded if they had mixed plasmodial infections, signs of severe malaria (WHO, 1990), danger signs (e.g. inability to drink, repeated vomiting, convulsions, lethargy, or abnormal sleepiness), a history of allergy to any of the study drugs, evidence of chronic disease (heart, liver, renal, malnutrition), or a clear history of an adequate malaria treatment in the preceding 72 h. Specifically, children were excluded if they had a recent history of treatment using a drug with antimalarial activity, e.g. co-trimoxazole, erythromycin, tetracycline, or doxycycline. Children recently treated with chloroquine were included. On admission to the study, a standardized medical history was taken and a clinical examination performed. The children were weighed, axillary temperatures taken (using a digital thermometer), and capillary blood samples obtained for malaria films, haemoglobin (Hb) determination, and filter-paper blots for parasite genotyping. From the first 70 children, a 2.0 mL blood sample was taken by venepuncture for a full blood cell count and biochemistry (creatinine, total bilirubin, and alanine aminotransferase). These were repeated on days 7 and 28 after enrolment.

Study design, randomization and treatment We used a randomized, placebo-controlled, doubleblind trial design. Parasitological failure rate by day 28 after starting treatment was the primary endpoint used for computing the sample size. On the assumption of a 15% failure rate with SP and 5% with the artesunate and SP combination, we required 200 patients (after 10% adjustment for attrition) in each treatment group to detect this 10% difference in cure rate with 80% power using a 2-sided a of 0.05. The randomization code was computer-generated in blocks of 12. Patients were randomized to receive SP (Fansidar®, 500 mg/25 mg, Hoffman-La Roche, Basel, Switzerland) plus placebo (SP alone group); SP plus 1 dose of artesunate and 2 doses of placebo (AS 1 group);

or SP plus 1 dose of artesunate daily (AS3 group) for 3 d. Artesunate (Arsumax 50 mg; Sanofi-Synth61abo, Gentilly Cedex, France) and placebo tablets, of exact size, colour, and shape were pre-packed in aluminium sachets and serially labelled with the randomization number. There were 4 drug sachets, one for each of days 0, 1, 2, and an extra for study drug replacement. Both artesunate (4 mg/kg bodyweight) and SP (single dose, 25 mg/kg sulfadoxine and 1.25mg/kg pyrimethamine) were administered orally by a nurse in the clinic. Children were observed for 30 min following drug administration for vomiting. Those who vomited within 30 min received a replacement dose of the study drugs, and were observed for another 30 min. Further vomiting resulted in study withdrawal and parenteral treatment. If the axillary temperature was >~ 38.0 °C paracetamol was dispensed to be administered every 8 h for 2 d. Parents, children, clinicians, and investigators remained masked to the treatment allocation throughout the study.

Patient follow-up Children were followed-up for 28 d. They returned daily to the hospital for evaluations until parasites cleared. Thereafter, they were seen weekly, i.e. on days 7, 14, 21, and 28. Guardians were encouraged to return at other times if their children were unwell or developed a fever. At each follow-up visit, children were clinically assessed, an adverse events questionnaire was completed, temperatures were taken, and capillary blood samples were obtained by finger-prick. If children did not return for scheduled follow-up visits, they were visited at home. Children who failed treatment received rescue treatment and were withdrawn from the study. A treatment failure was defined as the development of any of the following: (i) danger sign or signs of severe malaria, or a clinical requirement for parenteral treatment; (ii) rising or unchanged parasitaemia at 48 h; (iii) day 3 parasitaemia/> 25% of enrolment parasitaemia and patient unwell; (iv) day 4 parasitaemia i> 25% of enrolment parasitaemia; (v) any parasitaemia on day 7; (vi) recurrent parasitaemia at any time; and (vii) any adverse event requiring treatment withdrawal. Rescue treatment consisted of oral amodiaquine (25 mg/kg as 10 mg/kg on days 0 and 1, and 5 mg/kg on day 2) for uncomplicated malaria and parenteral quinine for severe malaria.

Ethical considerations This study was approved by the Ethical Review Committee of the Kenya Medical Research Institute, Nairobi, Kenya and also by the WHO Steering Committee for Research Involving H u m a n Subjects. Written informed consent was obtained from all parents or guardians of children participating in the study.

Laboratory procedures The Hb concentration was measured using a Hemocue machine ® (Mission Viejo, CA, USA). The total blood cell count and Hb for the first 70 children was done using Coulter counter. Thick and thin blood films were stained with 3% Giemsa's stain for 30 min and read by microscopists who were blinded to treatment allocation and clinical outcomes. Parasite density was calculated as the number of parasites counted per 200 white blood cells (WBC) on a thick film assuming a mean WBC count of 8000/pL. IfP. falciparum gametocytes were detected, a gametocyte count was done per 1000 leucocytes. Serum alanine aminotransferase, creatinine, and total bilirubin were measured using commercial kits (Boehringer Ingelheim, Germany). If a child had recurrent parasitaemia, blood samples (blotted on IsoCode StixTM, Schleicher and Schuell, Dassel, Germany) from the enrolment and recurrent episodes were genotyped by the polymerase chain reac-

ARTESUNATEAND SULFADOXINE-PYRIMETHAMINEFOR UNCOMPLICATEDMALARIA

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tion (PCR) for merozoite surface protein (MSP1 and MSP2), and glutamate rich protein (GLURP) to distinguish recrudescence of the original parasite strain from reinfection with a new parasite strain (Snounou & Beck, 1998). In addition, point mutations at codons 51, 59, and 164 of the dihydrofolate reductase (DHFR) gene and at codons 437, 540, and 581 of the dihydropteroate synthetase (DHPS) gene were detected by allele-specific PCR and restriction fragment length polymorphism (RFLP) as previously described (Duraisingh et al., 1998). In our study we did not routinely include the detection of point mutation at codon 108 of dhfr, because we assumed that subsequent point mutations at codons 51, 59, or 164 almost never occur before this mutation. Parasite genotyping for dhfr and dhps was restricted to children who had recurrent parasitaemia between 14 and 28 days after enrolment.

Data were analysed using SPSS 11.0 (SPSS Inc., Chicago, IL, USA). The analysis excluded children who were wrongly randomized or lost to follow-up. Proportions were compared between treatment groups using the X2 test. For all pairwise comparisons of failure rates, the SP alone group formed the reference category and results are presented as risk differences (A), together with their 95% CI. Normally distributed continuous variables were compared using Student's t test and analysis of variance (ANOVA). Data not conforming to a normal distribution were compared using the Kruskal-Wallis one-way ANOVA. For some analyses we pooled data for the 2 artesunate groups, since they received the same treatment on day 1. All reported Pvalues are two-tailed.

Outcomes

A total of 2492 children presenting to the trial site during the study period with a history of fever or measured fever were screened and 600 eligible children were enrolled. The baseline characteristics of enrolled children are shown in Table 1. The treatment groups were similar in distribution of children by age, gender, clinical features, and parasite density.

The 2 primary endpoints were defined as parasitological failure rate by days 14 and 28. The day 28 failure rate was adjusted using the results of PCR genotyping to differentiate recurrent parasitaemia after day 14 from a reinfection. A recurrent parasitaemia was classified as either recrudescent (treatment failure) or a new infection (cure). Failure after day 14 was defined as a recurrent parasitaemia with the same genotype as the initial parasitaemia occurring between days 14 and 28, inclusive. Missing P C R results (due to either missing samples, a failed PCR analysis, or unequivocal PCR results), were analysed in 2 ways: as treatment failures, or excluded from the analysis. The secondary endpoints were fever (defined as an axillary temperature ~> 37.5 °C) resolution rates, parasite clearance rates, change in Hb from day 0 to 28, and presence of gametocytes on days 7, 14, 21, and 28. Drug safety and tolerability were evaluated clinically and by laboratory tests. An adverse event was defined as a sign, symptom, intercurrent illness, or abnormal laboratory value not present on day 0 that occurred during follow-up. The relationship to the study drug was determined by the clinicians in the field and designated as definite, probable, possible, unlikely, not related or unknown. A serious adverse event was defined as lethal, life-threatening, and/or requiring hospital admission (Edwards & Aronson, 2001).

Results Baseline characteristics

Withdrawals Four children randomized in error were excluded from the efficacy analysis: 2 [AS1 (n = 1), AS3 (n = 1)] had severe anaemia (Hb < 5.0 g/dL), 1 (SP alone) had persistent vomiting, and 1 (AS1) had both severe anaemia and persistent vomiting. A total of 22 [SP alone (n = 7), AS1 (n = 6), AS3 (n = 9)] children were lost to follow-up: 13 on day 7, 5 on day 14, and 4 on day 28. Three [AS3 (n = 2), AS1 (n = 1)] children were absent on day 7, but were seen on day 14. Three [AS3 (n = 2), SP alone (n = 1)] other children were absent on day 14 and were seen on day 28. Twelve children [AS3 (n = 4), AS1 (n = 3), SP alone (n = 5)] did not receive the full course of treatment because of either loss to follow-up (n = 7) or early withdrawal (n = 5). Of these, 7 missed treatment on day 1, and a further 5 on day 2. Treatment outcome was known for 96% (578/600) by day 7, 97% (581/600) by day 14, and 95% (572/600) by day 28.

Early treatment response Statistical analysis Patients' crude data were recorded on the case record form, double-entered and validated using Epi-Info 6.04d (CDC, Atlanta, GA, USA).

During the week following treatment, there were 31 treatment failures: 21/191 (11.0%) in the SP alone group, 6/194 (3.1%) in the AS1 group, and 4/193 (2.1%) in the AS3 group. The risk of failure was

Table 1. B a s e l i n e characteristics o f study children with u n c o m p l i c a t e d m a l a r i a attending a district hospital, w e s t e r n K e n y a , O c t o b e r 1999 to M a r c h 2000 Treatment group

Male/female Age (years) Body weight (kg) Temperature (°C) Parasite count/pL (range) Gametocyte prevalence (%) Haemoglobin (g/dL) Leucocyte count (X 109/L) Alanine aminotransferase (IU/L) Creatinine (pmol/L) Total bilirubin (pmol/L)

SP alone (n = 200)

SP plus 1-d artesunate (n = 200)

SP plus 3-d artesunate (n = 200)

97/103 1.4 ± 1.1 9.4 ± 3.0 38.0 -4- 1.2 19172 (3360-77880) 14.5% 8.4 -4- 1.8 10.8 ± 4.1 18.8 ± 6.7 0.5 -4- 0 1.6 ± 0.4

105/95 1.5 ± 1.2 9.6 zk 3.2 37.8 ± 1.2 19858 (4040-100720) 16.5% 8.4 -4- 2.0 10.0 ± 3.9 13.4 + 2.8 0.5 ± 0 1.0 ± 0.4

104/96 1.3 4- 1.0 9.2 ± 2.8 37.8 ± 1.2 20429 (4040-86040) 12.0% 8.4 ± 1.8 11.0 ± 3.5 11.4 + 3.6 0.5 ± 0 1.2 ± 0.6

SP, sulfadoxine-pyrimethamine. Data are presented as mean 2_SD except for parasite count [geometric mean (range)] and gametocyte prevalence (%).

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significantly reduced among those receiving an artesunate and SP combination: AS3 vs. SP alone (risk ratio [RR] = 0.19, 95% CI 0.07 to 0.54, P < 0.0001); AS1 vs. SP alone ( R R = 0 . 2 8 , 95% CI 0.12 to 0.68, P < 0.001). Five [SP alone (n = 2), AS1 (n = 1), AS3 (n = 2)] of these developed severe malaria or danger signs, 5 (SP alone) had day 2 parasitaemia /> 25% of day 0, 5 [SP alone (n = 4), AS1 (n = 1)], had day 3 parasitaemia ~> 25% of day 0, 13 [AS1 ( n = 4 ) , SP alone (n = 9)] had persistent parasitaemia on day 7, 2 (AS3) used other drugs with antimalarial activity, and 1 (SP alone) developed an adverse event (vomiting).

OBONYO

ETAL.

100! 90-

AS3

80....

AS1

70 i

60-

•

SPalone

50-

o 403020-

Day 14 treatment failure By day 14 the parasitological failure rates for AS3 vs. SP alone and AS1 vs. SP alone were 18/192 (9.4%) vs. 49/192 (25.5%) (A-16.2%, 95% CI - 2 3 . 6 to - 8 . 7 % , P < 0.001), and 32/197 (16.2%) vs. 49/192 (25.5%) (A-9.3%, 95% CI - 1 7 . 3 to - 1 . 2 % , P = 0.027), respectively (Table 2).

Day 28 treatment failure The corresponding day 28 crude failure rates were 90/192 (46.9%) vs. 123/189 (65.1%) (A-18.2%, 95% CI - 2 8 . 0 to - 8 . 4 % , P < 0.001), and 114/191 (59.7%) vs. 123/189 (65.1%) (A - 5 . 4 % , 95% CI - 1 5 . 1 to 4.3%, P = 0.30), respectively (Table 2). A total of 291 children developed recurrent parasitaemia during follow-up. Sixty-five occurred by day 14 and 226 after day 14. Of those occurring after day 14, 210 (93%) were genotyped: 57 [AS3 (n = 13), AS1 ( n = 2 1 ) , SP alone ( n = 23)] were recrudescent, 117 [AS3 (n = 40), AS1 (n = 41), SP alone (n = 36)] were reinfections and the rest had no results [AS3 (n = 13), AS1 (n = 14), SP alone (n = 10)]. The day 28 failure rates adjusted for PCR were significantly higher in the SP alone group compared with the AS3 group. They were also higher in the SP alone group than in the AS 1 group, although not significantly (Table 2).

10...................... 0~ Study day

Fig. 1. Rate of parasite clearance: the proportion (%) of children with malaria parasitaemia during the first week after treatment with sulfadoxine-pyrimethamine (SP) alone or in combination with either one or three days of artesunate (AS 1 or AS3). Vertical bars indicate 95% CI.

about 1.2 times for those on AS1 (RR = 1.19, 95% CI 0.91 to 1.56, P = 0.19) compared with those on SP alone. For instance, on day 1, 190/198 (96%) treated with SP alone compared with 252/396 (63.6%) treated with an artesunate and SP combination were still parasitaemic (X2 = 72.0, P < 0.001) (Fig. 1). Fever clearance was similarly faster among children who received an artesunate and SP combination compared with those who received SP alone. On day 1, 36/197(18.3%) of children treated with SP alone were still febrile compared with 17/396 (4.3%) treated with an artesunate and SP combination (X2 = 3 1 . 6 , P < 0.001). Corresponding proportions on day 2 were 32/196 (16.3%) treated with SP alone, 6/199 (3.0%) treated with AS1, and 5/196 (2.6%) treated with AS3.

Parasite and fever clearance Children receiving an artesunate and SP combination cleared their parasitaemia faster than those who received SP alone (Fig. 1). The 2 groups receiving artesunate had similar clearance rates. The rate of parasite clearance was about twice for those on AS3 ( R R = 1 . 9 8 , 95% CI 1.5 to 2.60, P < 0 . 0 0 0 1 ) and

Gametocyte carriage On day 0, gametocytes were detected in only 86/600 (14.3%) children. The proportion of children with gametocytes during follow-up increased significantly among those treated with SP alone on day 7, peaked on day 14, and then decreased (Fig. 2). Those treated

T a b l e 2. P a r a s i t o l o g i c a l failure rates on days 14 a n d 28 for s t u d y c h i l d r e n w i t h u n c o m p l i c a t e d m a l a r i a t r e a t e d with s u l f a d o x i n e - p y r i m e t h a m i n e (SP) a l o n e or in c o m b i n a t i o n with e i t h e r one or three days of a r t e s u n a t e (AS1 or AS3)

Failure rate (%) Efficacy on AS3-SP AS1-SP SP alone Efficacy on AS3-SP AS1-SP SP alone Efficacy on AS3-SP AS1-SP SP alone Efficacy on AS3-SP AS1-SP SP alone

Risk differencea

(95% CI)

P

day 14 18/192 (9.4) 32/197 (16.2) 49/192 (25.5)

-16.2 -9.3 -

(-23.6; - 8 . 7 ) (-17.3; - 1 . 2 ) -