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Tropical Medicine and International Health volume 6 no 2 pp 92±98 february 2001

Parasitaemia and gametocytaemia after treatment with chloroquine, pyrimethamine/sulfadoxine, and pyrimethamine/ sulfadoxine combined with artesunate in young Gambians with uncomplicated malaria Lorenz von Seidlein1,2, Musa Jawara1, Rosalind Coleman1, Tom Doherty1,2, Gijs Walraven1 and Geoffrey Targett1,2 1 Medical Research Council Laboratories, Fajara, The Gambia 2 Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, UK

Summary

As part of a study to assess the infectivity of gametocytes after treatment with four antimalarial regimens, the ef®cacy of each treatment was also determined. From September to December 1998, 598 children with uncomplicated malaria were treated; 135 received chloroquine (CQ) alone, 276 received pyrimethamine/sulfadoxine (Fansidarã, PSD) alone, 113 received PSD with a single dose of artesunate (PSD + 1ART) and 74 received PSD combined with three doses of artesunate (PSD + 3ART). On day 28 19/63 (30.2%; 95% C.I. 19.2% to 43.1%) of children treated with CQ alone, 5/134 (3.7%; 95% C.I. 1.2% to 8.5%) treated with PSD alone, 1/71 (1.4%, 95% C.I. 0.0% to 7.9%) treated with PSD + 1ART and 0/45 (0.0%; 95% C.I. 0.0% to 7.9%) treated with PSD + 3ART were parasitaemic. The proportion of children with gametocytes on day 7 after treatment with CQ alone was 16/89 (18.0%; 95% C.I. 10.6% to 27.6%), 98/174 (56.3%; 95% C.I. 48.6% to 63.8%) after treatment with PSD alone, 8/70 (11.4%; 95% C.I. 5.1% to 21.3%) after treatment with PSD + 1ART and 4/46 (8.7%; 95% C.I., 2.4% to 20.8%) after treatment with PSD + 3ART. CQ thus has a lower ef®cacy than PSD or either of the PSD and artesunate combinations. Use of PSD alone as an alternative ®rst line treatment results in a very high post-treatment gametocyte prevalence that is likely to enhance transmission. There would be greater and more sustainable bene®ts from using PSD and artesunate combinations. keywords P. faliciparum, malaria, Africa, The Gambia, artemisinin, artesunate, sulfadoxinepyrimethamine, PSD, combination, gametocytes, chloroquine correspondence Lorenz von Seidlein, The International Vaccine Institute, P.O. Box 14 Kwanak, Seoul, Korea 151-600. Telephone: 00 82 2 872 2801; Fax: 00 82 2 872 2803; E-mail: [email protected]

Introduction Chloroquine (CQ) is probably the most widely used antimalarial drug in Africa. Introduced shortly after the Second World War, CQ has been widely used due to its tolerability, safety and low price. In many regions of subSaharan Africa CQ remains the only widely available antimalarial drug and indeed the only measure to control malaria; hence the progressive decline in CQ susceptibility of P. falciparum throughout sub-Saharan Africa is cause for concern (Burchard et al. 1984; Kyroenseppae et al. 1984; Sansonetti et al. 1985; Brandicourt et al. 1986;

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LeBras et al. 1986; Neequaye 1986; Menon et al. 1987; Basco et al. 1993; Ezedinachi 1996; Ekvall et al. 1998; Brasseur et al. 1999; Premji et al. 1999; Williams et al. 1999). In Senegal the increase in chloroquine resistance has been linked to an increase in malaria-related mortality (Trape et al. 1998). New strategies to treat and to control malaria are urgently needed. While most malaria endemic countries including The Gambia continue to recommend CQ as ®rst line treatment, several African countries, including Kenya, Tanzania and Malawi, recommend pyrimethamine/sulfadoxine (PSD, FansidarÒ). However compared to the

ã 2001 Blackwell Science Ltd

Tropical Medicine and International Health

volume 6 no 2 pp 92±98 february 2001

L. von Seidlein et al. Comparison of CQ with PSD and PSD + artesunate combinations

emergence of CQ resistance, PSD-resistant strains of P. falciparum have spread much more rapidly (Ronn et al. 1996; Warsame et al. 1999). The loss of CQ as an effective malaria treatment and the likelihood of losing PSD in the near future may have a disastrous impact on childhood mortality in many regions of Africa where no obvious replacement for PSD is available. The combination of PSD with a short-acting artemisinin derivative such as artesunate may be a more robust ®rst line treatment, as one drug can protect the other against the emergence of resistance (White 1998). However, very few data are available which describe the safety of PSD combined with an artemisinin derivative (Li et al. 1984; Doherty et al. 1999). In 1998, we compared the ef®cacy of PSD combined with artesunate with PSD alone at different sites and in another group of children, none of whom participated in the study presented here (von Seidlein et al. 2000). No studies have compared CQ, PSD and PSD combined with an artemisinin derivative. As part of an open label study designed to investigate the infectivity of gametocytes after antimalarial therapy (Targett et al. 2000), we investigated the ef®cacy of CQ alone, PSD alone, PSD combined with a single dose of artesunate (PSD + 1ART), and PSD combined with three daily doses of artesunate(PSD + 3ART), in clearing asexual and sexual parasitaemias. Materials and methods Study patients The study was approved by the Medical Research Council (MRC) Laboratories Scienti®c Co-ordinating Committee and The Gambia Government/MRC Ethical Committee. It was conducted from September to December 1998 in Farafenni, a rural town on the border between Senegal and The Gambia, 170 km east of the Atlantic coast. Children over 5 kg body weight and under 18 years of age were included in the study if they had an asexual parasitaemia of more than 500 P. falciparum parasites/ll, a history of fever, lived within 5 km of the health centre and if a guardian gave informed consent. Children who required parenteral treatment, had been treated with PSD or CQ within 2 weeks, had a packed cell volume (PCV) below 15%, or had any evidence of chronic disease were excluded. Malaria in The Gambia is highly seasonal, most cases occurring between August and the end of December. During the ®rst two months, for logistic reasons, children were treated with either CQ alone or PSD alone. A computer generated random allocation system was used for the assignment of drugs. In November, the study was extended with children randomised to receive either CQ


alone or PSD alone or PSD combined with one dose artesunate or PSD combined with three doses of artesunate. The study was open label throughout. Children in the CQ alone group received 10 mg of CQ base per kg body weight orally at 0, 24, and 48 h. The children in the other three treatment groups received half a tablet of PSD (12.5 mg pyrimethamine/250 mg sulfadoxine) if their bodyweight was under 10 kg and an additional quarter tablet for every 5 kg increase in weight. The children in the combination treatment groups received 4 mg artesunate per kg bodyweight in the single-dose group and 4 mg/kg daily for three days in the three-dose group. The administration of all ®rst doses, was supervised, as was the administration of all artesunate doses. The administration of the ®rst dose of CQ, but not the administration of subsequent doses, was supervised. The children were discharged home after one hour of observation. Children who vomited the ®rst dose during the observation period were given a replacement. All children were asked to come to the study laboratory 4 days later. A ®eldworker visited the children on days 7, 14, and 28 when a blood ®lm was obtained. Symptomatic children who presented during the 28-day follow-up period with parasitaemia received rescue medication with PSD, if the treatment had been CQ or quinine if treatment had been PSD or parenteral treatment was indicated. Each individual received 10 mg/kg paracetamol under direct supervision and the parents were instructed to give paracetamol every 6 h until the child's fever had subsided. Open label CQ (Alkaloida Ltd, Hungary), PSD (Pharmamed Ltd, Malta) and paracetamol (Echo International Health Services, UK) were used in the trial. Artesunate was manufactured by Guilin Pharmaceutical Works, China and provided by Sano®, Gentilly, France. Laboratory methods Thick blood ®lms were stained with Giemsa. Parasitaemia was calculated on the basis of the number of parasites per 200 leucocytes on a thick ®lm. If gametocytes were present, the gametocyte count was extended to 1000 leucocytes. Analysis The proportions of children with trophozoites and gametocytes in each treatment group are reported with their 95% con®dence intervals. Only children without detectable gametocytes on enrolment were included in the analysis of gametocyte rates. Children with an axillary temperature of > 37.4°C were considered febrile. Normally distributed data were compared using the Student's t-test or A N O V A ; homogeneity of variance was 93




tested by the F-test. Discrete data were compared by Kruskal±Wallis test, Fisher's exact test or the Chi-squared test with Yate's correction, as appropriate. Results Baseline characteristics and follow-up Of 1419 individuals who presented with a history of febrile illness to the Farafenni health centre, 821 did not ful®l the inclusion criteria and 598 children were included in the study. One hundred and thirty-®ve children were treated with CQ alone, 276 children with PSD alone, 113 children with PSD + 1ART, and 74 children with PSD + 3ART. The baseline characteristics of the children in the four treatment groups are shown in Table 1. All four treatment groups had a similar average age (6.5 years) and weight (17.7 kg), but differed signi®cantly in terms of their temperature, parasite density and packed cell volume (PCV). Most children treated with CQ alone (109/135; 80.7%) and PSD alone (226/276; 81.9%) were enrolled during September and October. All children who received PSD + 1ART or PSD + 3ART were treated during November and December. Children treated in September and October were more frequently febrile (74.1% vs. 57.6%; P < 0.001) and more often had a

parasite density above 100,000/ll (25% vs. 3.8%; P < 0.001). By contrast, a higher proportion of children treated in November and December had gametocytaemia (9.0% vs. 4.5%; P 0.03), splenomegaly (33.3% vs. 18.9%; P < 0.001), and a PCV less than 33% (69.0% vs. 61.3%; P 0.06). Within 4 days of treatment 2/135 (1.5%) of children in the CQ alone group, 8/276 (2.9%) in the PSD alone group, 1/113 (0.9%) in the PSD + 1ART group and none of the 74 children treated with PSD + 3ART received parenteral rescue therapy and were admitted to hospital. All of these children had recovered completely by the time of discharge. One other child vomited the ®rst and the replacement dose PSD and received parenteral CQ. All children who had received parenteral medication were excluded from the study. One 5-year-old girl returned to hospital with her parents one day after the initial treatment with CQ, and received parenteral quinine, but died of severe malaria the following day. By day 4, 552 of the 598 children (92.3%) could be evaluated. By day 28 the ®eldworkers were able to follow 313 (52.3%) of the children. A comparison of each baseline parameter of children who could be followed to day 28 and children who did not complete follow-up to day 28 did not reveal any statistically signi®cant differences.

Table 1 Demographic and other baseline characteristics of children who could be evaluated

Number enrolled* Mean weight in kg (SD) Mean age in years (SD) Temperature (°C); mean (SD) Temperature > 37.5°C 95% C.I. P.falciparum density Geometric mean; 95% C.I. Baseline parasitaemia >106/microL; 95% C.I. Gametocytaemic 95% C.I. PCV mean; SD Anaemic (PCV < 33%) 95% C.I. Splenomegaly 95% C.I. Male: Female Tribe Fula: Wollof: Mandinka

CQ

PSD

PSD + ART1

PSD + ART3

135 18.1 (8.9) 6.4 (3.7) 38.4 (1.2) 100/135 74.1%; 65.8%-81.2% 21,116 14,803±30,123 27/135, 20.0% 13.6%-27.8% 3/135, 2.2% 0.4%-6.4% 30.6; 4.8 79/121 65.3%; 56.1%-73.7% 32/132 24.2%; 17.2%-32.5% 65 : 70 20 : 33 : 36

276 17.6 (8.2) 6.4 (3.6) 38.3 (1.3) 194/276 70.3%; 64.5%-75.6% 23,707 19,121±29,393 60/276, 21.7% 17.0%-27.1% 15/273, 5.5% 3.1%-8.9% 30.8; 5.3 153/251 61.0%; 54.6%-67.0% 57/271 21.0%; 16.3%-26.4% 168 : 108 19 : 36 : 39

113 17.6 (8.8) 6.6 (3.7) 38.0 (1.2) 68/113 60.2%; 50.5%-69.2% 9,080 6,488±12,707 3/113, 2.7% 0.6%-7.6% 11/112, 9.8% 5.0%-16.9% 29.0; 5.8 82/110 74.6%; 65.4%-82.4% 41/112 36.6%; 27.7%-46.2% 67 : 46 19 : 31 : 43

74 17.2 (8.2) 6.6 (3.4) 37.9 (1.3) 38/74 51.4%; 39.4%-63.1% 8,618 5,449±13,630 4/74, 5.4% 1.4%-13.3% 9/71, 12.7% 5.9%-22.7% 30.4; 5.5 46/73 63.0%; 50.9%-74.0% 19/74 25.7%; 16.2%-37.2% 32 : 42 15 : 33 : 47

*Includes children with gametocytaemia on enrolment.

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Parasitaemia Four days after treatment with the combination PSD and artesunate all children except one had cleared the parasites (Fig. 1). On days 7, 14, 28 all children treated with the combination PSD and artesunate remained free of parasites, with the exception of one child who was found to be parasitaemic 28 days after treatment with PSD and 1 dose artesunate. After treatment with PSD alone 4/147 (2.7%; 95% C.I. 0.7% to 6.8%) children were parasitaemic on day 14 and 5/134 (3.7%; 95% C.I. 1.2% to 8.5%) children were parasitaemic on day 28. By contrast 8/90 (8.9%; 95% C.I. 3.9% to 16.8%) children remained parasitaemic 7 days after treatment with CQ alone, 15/74 (20.2%; 95% C.I. 11.8% to 31.2%) on day 14 and 19/63 (30.2%; 95% C.I. 19.2% to 43.1%) children on day 28. The proportion of children with asexual and sexual parasitaemia was similar among children treated with

PSD alone during the ®rst and second half of the malaria season. In September and October a signi®cantly higher proportion of children treated with CQ alone were parasitaemic on day 28 (18/47, 38.3%; 95% C.I. 24.5% to 53.6%) than in November and December (1/16, 6.3%; 95% C.I. 0.2% to 30.2%). No signi®cant difference was observed before day 28. Gametocytaemia By day 7, 16 of 89 children (18.0%; 95% C.I. 10.6% to 27.6%) treated with CQ alone, 8 of 70 children (11.4%; 95% C.I. 5.1% to 21.3%) treated with PSD and 1 dose artesunate and 4 of 46 children (8.7%; 95% C.I. 2.4% to 20.8%) treated with PSD and 3 doses of artesunate were gametocytaemic, whereas this was the case for 98 of 174 patients (56.3%; 95% C.I. 48.6% to 63.8%) treated with PSD alone (Fig. 2). By day 28 one of the children treated with PSD and artesunate had detectable gametocytes;

Figure 1 % of children with patent asexual P. falciparum parasitaemia (95% con®dence intervals are shown as vertical lines).

Figure 2 Percentage of children who were free of gametocytes on enrolment and developed gametocytaemia during follow-up (95% con®dence intervals are shown as vertical lines).


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8/61(13.1%; 95% C.I. 5.8% to 24.2%) of the children with CQ alone and 13/123 (10.6%; 95% C.I. 5.8% to 17.4%) of children treated with PSD alone were gametocytaemic. Discussion This study was planned to assess the infectivity of gametocytes 4 and 7 days after treatment with one of four treatment regimens. To be able to show a signi®cant difference in gametocyte infectivity, a sample of 600 children distributed over four groups was required. Due to the seasonal character of malaria in The Gambia, a 4-month period from September to December was available for recruitment. While PSD alone and CQ alone could be used throughout the malaria season, the PSD and artesunate combinations could only be allocated in November and December 1998 after a study had shown the safety of the combination (Doherty et al. 1999). The difference in timing of treatment allocation may help to explain the differences in baseline characteristics between the four treatment groups. This absence of randomization is a limitation of the study and may have introduced bias. It is reassuring to see that parasite and gametocyte clearance in the children treated with PSD alone, PSD combined with a single dose artesunate or PSD combined with three doses of artesunate was in the same range as during the randomised, double blind trial (von Seidlein et al. 2000). Fever and high parasite density were more frequently detected in children who entered the CQ alone and the PSD alone treatment groups, most of whom were enrolled during the ®rst half of the malaria season. Development of fever depends, among other factors, on parasite density (Rogier et al. 1996; Bouvier et al. 1997). Parasite density in turn is in¯uenced by the level of immunity of the individual (Collins & Jeffery 1999). Children undergo repeated P. falciparum infections during one malaria season, resulting in partial immunity and thus better control of parasite density towards the end of the malaria season. Anaemia and splenomegaly were more frequent in children treated with PSD combined with artesunate, all of whom received treatment in November and December. Seasonal changes in the anaemia status have been described previously in The Gambia (Topley 1968). The parasite prevalence after treatment in September and October was compared to the prevalence after treatment in the second half of the malaria season. The parasite prevalence was similar in children treated with PSD alone and in children treated with CQ alone up to day 28. Children treated with CQ alone during September and October were more frequently parasitaemic on day 28 96

compared to children treated during November and December. A likely explanation for this observation is the decreased transmission pressure towards the end of the malaria season. Children receiving PSD were protected by its long half life which is suf®cient to inhibit parasite growth. By contrast, CQ levels drop rapidly below an inhibitory concentration. Which proportion of the parasitaemias on day 28 was due to a reinfection is not known, as a genotypic analysis to distinguish reinfections from recrudescences was not available. A study conducted in The Gambia in the early part of the 1994 malaria season found a similar proportion: 36/100 (36.0%, 95% C.I. 26.6% to 46.2%) of children parasitaemic 28 days after treatment with CQ alone (Muller et al. 1996). CQ resistance was ®rst described in The Gambia in 1987 (Menon et al. 1987). In 1998 91.1% of children treated with CQ cleared their parasitaemia within 7 days. The present level of CQ resistance in The Gambia does not warrant a change in ®rst line treatment of malaria according to WHO guidelines (World Health Organization 1 1997). However, ineffective ®rst line treatment is likely to cause an increasing proportion of patients to develop severe malaria (White 1999). There should be no doubt that CQ resistance will result in an increase in malariarelated mortality, which has already been observed in Mlomp, Senegal (Trape et al. 1998). There were very few parasitological failures following treatment with PSD alone in this study. Parasite rates 28 days after treatment with PSD alone in The Gambia have been 8.0% (8/100; 95% C.I. 3.5% to 15.2%) in 1994, 10.0% (15/150; 95% C.I. 5.7% to 16.0%) in 1995 and 6.4% (12/187; 3.4% to 10.9%) in 1998 (Muller et al. 1996; Bojang et al. 1998). PSD is well tolerated, requires only a single dose and is in the same low price class as chloroquine. However, in this study more than half of the children treated with PSD alone became gametocytaemic, about three times more than children treated with PSD combined with artesunate. Gametocytes, the sexual form of P. falciparum, are responsible for transmission of malaria, and high gametocyte rates may accelerate its spread. PSD resistance has also spread rapidly after the introduction of PSD monotherapy as ®rst line treatment for uncomplicated malaria in East Africa (Ronn et al. 1996; Warsame et al. 1999). Loss of PSD as an effective oral antimalarial is a real danger in West Africa if PSD is introduced as monotherapy. Children treated with PSD combined with artesunate had a lower prevalence of asexual parasitaemia as well as gametocytaemia than children treated with either CQ alone or PSD alone. In addition to the clinical bene®t of being free of parasites, there are theoretical reasons why the introduction of the combination therapy may be ã 2001 Blackwell Science Ltd




advantageous. Artemisinin derivatives have a half life measured in hours and are the most potent and rapidly acting antimalarial drugs (White et al. 1999). The P. falciparum biomass in the individual is therefore drastically reduced and the probability of harbouring a resistant strain of P. falciparum is smaller compared to single drug regimens. However, to achieve a 28-day cure with artemisinin drugs alone, treatment courses of 5±7 days are required (Meshnick et al. 1996). Treatment with a single dose of artesunate combined with PSD resulted in excellent parasite clearance and the treatment can easily be given under direct observation. Emergence of artemisinin resistance is unlikely, since PSD ± which has a relatively long half life measured in weeks ± is likely to protect artemisinin derivatives, and the probability of double mutants emerging is exceedingly small (White 1998). Concerns that resource-poor countries cannot afford combination therapy are probably not justi®ed. A single dose of PSD and artesunate could be available for less than $1.00, a fraction of the price of alternative highly effective drug regimens (me¯oquine, atovaquone/proguanil) which may be needed once resistance has rendered PSD ineffective. The introduction of combination therapy should also reduce the cost for return visits to health facilities. Further studies are required to assess the bene®t and the cost effectiveness of combination therapy. But time is running out for a strategic implementation of PSD combined with artesunate as ®rst line treatment of uncomplicated malaria in sub-Saharan Africa. Acknowledgements We thank Margaret Pinder for her help, support and advice, Shabbar Jaffar for his advice on the analysis of the study, Brian Greenword for advice on the study design, Tumani Corrah for his diligent help as safety monitor, Sister Pam Njai for pharmaceutical support and Kunle Okunoye for data management. We are grateful to Dr Jean-Pascal Ducret and Marie Helen Corel (Sano®, France) for generously providing artesunate and placebo. We wish to acknowledge the ®nancial support of Novartis Pharma AG and the Sir Halley Stewart Trust. References Basco L, Ringwald P, Simon F, Doury J & LeBras J (1993) Evolution of chloroquine resistance in Central and West Africa. Tropical Medicine and Parasitology 44, 111±112. Bojang KA, Schneider G, Forck S et al. (1998) A trial of Fansidar plus chloroquine or Fansidar alone for the treatment of uncomplicated malaria in Gambian children. Transactions of the Royal Society of Tropical Medicine and Hygiene 92, 73±76.


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