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Keywords:

  • malaria;
  • efficacy trials;
  • artemether–lumefantrine;
  • artesunate + mefloquine;
  • treatment failure;
  • Cambodia
  • paludisme;
  • études cliniques;
  • artéméther–luméfantrine;
  • artésunate + méfloquine;
  • échec du traitement;
  • Cambodge
  • malaria;
  • ensayos de eficacia;
  • artemeter–lumefantrina;
  • artesunato + mefloquina;
  • fallo terapéutico;
  • Cambodia

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Objective  To determine the efficacy of artemether–lumefantrine malaria treatment, as an alternative to artesunate + mefloquine, which is becoming ineffective in some areas of the Thai–Cambodian border.

Methods  Two studies were conducted to monitor the efficacy of artemether–lumefantrine in Sampov Lun referral hospital, Battambang Province, in 2002 and 2003, and one study was conducted to assess the efficacy of mefloquine + artesunate in 2003 for comparison. The studies were performed according to the WHO standardized protocol with a follow-up of 28 days. The therapeutic efficacy tests were complemented with in vitro tests and in 2003, with the measurement of lumefantrine plasma concentration at day 7 for the patients treated with artemether–lumefantrine.

Results  A total of 190 patients were included: 55 were treated with artemether–lumefantrine in 2002 (AL2002), 80 with artemether–lumefantrine and food supplementation in 2003 (AL2003) and 55 with artesunate + mefloquine in 2003 (AM2003). With the per-protocol analysis, the cure rate was 71.1% in study AL2002, 86.5% in study AL2003 and 92.4% in study AM2003. All the data were PCR corrected. The artemether–lumefantrine cure rate was unexpectedly low in 2002, but it increased with food supplementation in 2003. There was a significant difference (P = 0.02) in lumefantrine plasma concentrations between adequate clinical and parasitological responses and treatment failure cases. In vitro susceptibility to lumefantrine was reduced for isolates sampled from patients presenting with treatment failure, but the difference was not statistically different from isolates sampled from patients who were successfully treated.

Conclusion  Treatment failure cases of artemether–lumefantrine are most probably because of low levels of lumefantrine blood concentration. Further investigations are necessary to determine whether resistance of Plasmodium falciparum isolates to lumefantrine is present in the region.

Objectif  Déterminer l'efficacité de l'artéméther–luméfantrine pour le traitement paludisme en tant qu'alternative à l'artésunate + méfloquine qui devient de plus en plus inefficace dans certaines zones dans la frontière Thailando–Cambodgienne.

Méthodes  Deux études ont été menées pour mesurer l'efficacité de l'artéméther–luméfantrine dans l'hôpital de référence de Sampov Lun dans la province de Battambang en 2002 et 2003 et, dans un but de comparaison, une étude a aussi été menée pour évaluer l'efficacité de l'artésunate + méfloquine en 2003. Les études ont été menées selon les protocoles standardisés de l'OMS avec un suivi de 28 jours. Les tests d'efficacité thérapeutique ont été complémentés par des tests in vitro et, en 2003 par la mesure de la concentration plasmatique de lumefantrine au jour 7 pour les patients traités à l'artéméther–luméfantrine.

Résultats  Au total 190 patients ont été inclus dans l’étude parmi lesquels 55 ont été traités à l'artéméther–luméfantrine en 2002 (AL2002), 80 à l'artéméther–luméfantrine en plus d'un complément de nourriture en 2003 (AL2003) et 55 à l'artésunate + méfloquine en 2003 (AM2003). L'analyse suivant un protocole standard a révélé un taux de guérison de 71,1% pour AL2002, 86,5% pour AL2003 et 92,4% pour AM2003. Toutes les données ont été ajustées avec les résultats de la PCR. Le taux de guérison pour l'artéméther–luméfantrine s'est avéré bas de façon inattendue en 2002 mais il a augmenté avec le complément de nourriture en 2003. Il y avait une différence significative (p = 0,02) pour les concentrations plasmatiques de lumefantrine entre d'une part les réponses cliniques et parasitologiques adéquates et d'autre part les cas d’échec thérapeutique. La susceptibilité in vitro pour la lumefantrine était réduite pour les souches isolées de patients présentant un échec thérapeutique mais les valeurs n’étaient pas statistiquement différentes de celles de souches isolées de patients traités avec succès.

Conclusion  Les cas d’échec thérapeutique à l'artéméther–luméfantrine sont plus probablement dus à des concentrations sanguines basses de luméfantrine. Des investigations supplémentaires sont nécessaires pour déterminer s'il existe une résistance de P. falciparumà la luméfantrine dans la région.

Objetivo  Determinar la eficacia del tratamiento de malaria con artemeter-lumefantrina como alternativa al artesunato + mefloquina, el cual está dejando de ser efectivo en algunas áreas de la frontera entre Tailanda y Cambodia.

Métodos  Se llevaron a cabo dos estudios para monitorizar la eficacia de artemeter-lumefantrina en el hospital de referencia de Sampov Lun, provincia de Battambang, entre el 2002 y 2003, así como un estudio durante el 2003, y a modo de comparación, para evaluar la eficacia de la mefloquina + artesunato. Estos estudios se realizaron siguiendo el protocolo estándar de la OMS con un seguimiento de 28 días. Las pruebas de eficacia terapéutica se complementaron con pruebas in vitro y, durante el 2003, con la medición de la concentración de lumefantrina en plasma en el día 7 para los pacientes tratados con artemeter-lumefantrina.

Resultados  Se incluyeron 190 pacientes: 55 fueron tratados con artemeter-lumefantrina en el 2002 (AL2002), 80 con artemeter-lumefantrina y suplementación alimenticia en el 2003 (AL2003), y 55 con artesunato + mefloquina en el 2003. Con el análisis por protocolo, la tasa de curación fue de 71.1% en el estudio AL2002, 86.5% en el estudio AL2003, y 92.4% en el estudio AM2003. Todos los datos fueron corregidos mediante PCR. La tasa de curación para el tratamiento con artemeter-lumefantrina fue sorprendentemente baja en el 2002, pero aumentó con la suplementación alimenticia en el 2003. Se encontró una diferencia significativa (p = 0.02) en las concentraciones de lumefantrina en plasma entre casos con una respuesta clínica y parasitológica adecuadas y aquellos con fallo terapéutico. La susceptibilidad in vitro frente a la lumefantrina se redujo para cepas aisladas de pacientes que presentaban fallo en el tratamiento, pero la diferencia no era estadísticamente significativa de aquella para cepas aisladas de pacientes que hubiesen respondido adecuadamente al tratamiento.

Conclusións  Los casos de fallo terapéutico con artemeter-lumefantrina probablemente son debidos a bajos niveles de concentración de lumefantrina en la sangre. Se necesitan más estudios para determinar si existe resistencia a la lumefantrina entre las cepas de P. falciparum de la región.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Malaria is still a major public health problem in Southeast Asia. Malaria treatment and control are hampered by the spread of resistance to common antimalarial drugs, especially along the Thai–Cambodian border where multidrug-resistant malaria is highly prevalent. Chloroquine-resistant parasites were first detected in Cambodia at the beginning of the 1960s (Eyles et al. 1963). Cambodia had used chloroquine as the first-line treatment for uncomplicated falciparum malaria until 1991. Quinine plus tetracycline combination was proposed as the first-line treatment along the Thai–Cambodian border and as the second-line treatment in other areas where resistance is less marked (Meek et al. 1986; Giboda & Denis 1988). However, this treatment regimen has never become popular in Cambodia. Between 1993 and 2000, mefloquine monotherapy was rather used in the northwest part of the country. As a result of the high recrudescence rates of mefloquine monotherapy between 1993 and 2000, artemisinin-based combination therapy (ACT) was deployed and used as first-line treatment all over the country in 2000 in the form of co-blister packs designed for three different age groups. In the same year, studies of the clinical efficacy of the combination of artesunate + mefloquine were conducted and confirmed 100% efficacy with 14-day follow-up in the northwest and central parts of the country. In 2001, monitoring studies (following the standard WHO 28-day protocol) in two provinces in the northwest part of the country at the Thai–Cambodian and the Vietnamese border confirmed the high efficacy of the combination: 96% and 100% of adequate clinical and parasitological response (ACPR), respectively. Failure rates of the artesunate + mefloquine combination began to rise in 2002. The efficacy of the combination treatment ranged from 0% in the northeast part to 14.3% in the northwest part of the country along the Thai–Cambodian border.

In order to determine the efficacy of an alternative ACT, the national malaria control programme conducted a therapeutic efficacy study with artemether + lumefantrine in 2002 in Sampov Lun district, Battambang Province, located in the northwest part of the country at the Thai–Cambodian border. The result showed an unexpected high level of treatment failures contrary to other clinical trials conducted in the region (Vugt et al. 1999; van Vugt et al. 2000; Stohrer et al. 2004). One possible explanation was that the high failure rate was a consequence of poor bioavailability of lumefantrine when taken without fatty food (Ezzet et al. 1998; White et al. 1999). Therefore, the national malaria control programme decided to conduct in 2003 a similar trial with artemether + lumefantrine but to give each dose with food supplementation. Day 7 plasma concentration of lumefantrine was measured, as it has been shown to be a main determinant of therapeutic success for the combination (Ezzet et al. 1998; White et al. 1999). A therapeutic efficacy study with artesunate + mefloquine was simultaneously conducted in 2003 in the same site for comparison.

Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Patients and clinical procedures

The three therapeutic efficacy studies were conducted in Sampov Lun referral hospital, Battambang Province: artemether–lumefantrine between June and August 2002 (AL2002), artemether–lumefantrine between October 2003 and March 2004 (AL2003) and artesunate + mefloquine between October 2003 and March 2004 (AM2003). The studies followed the standard WHO protocol for the assessment of the efficacy of antimalarial drugs (WHO 2003). Briefly, all patients over 6 years of age and weight > 16 kg presenting with fever defined as axillary temperature ≥ 37.5 °C or history of fever and a positive Plasmodium falciparum mono-infection with parasite density of 1000–150 000 parasites/μl were included in the trial. Informed consent was given by the patient or by a parent/guardian for children. Enrolled patients had to stay in hospital until parasite clearance and had to be committed to complete the 28-day follow-up. The presence of one of the following conditions was an exclusion criteria for the study: one or more of the general danger signs or any sign of severe and complicated malaria, pregnancy, febrile diseases other than malaria and severe malnutrition, and known hypersensitivity or contraindication to the study drugs. The therapeutic response was classified as early treatment failure (ETF), late clinical failure (LCF), late parasitological failure (LPF) and ACPR.

Treatment regimen

Patients were treated orally under supervision. Coartem® (Novartis, Basel, Switzerland) tablets containing 20 mg artemether and 120 mg lumefantrine were given the first day at 0 and 8 h and twice daily in the following 2 days in the morning and late afternoon, i.e. 8–12 h apart (total course of 24 tablets for an adult as recommended by the manufacturer). The total course comprised 12 tablets and 18 tablets for children weighing between 16–24 and 25–34 kg, respectively. Artesunate tablets (50 mg) were imported from Mediplantex (Hanoi, Vietnam) and mefloquine tablets (250 mg mefloquine hydrochloride equivalent to 225 mg base) were imported from Pharmadanica (Lynge, Denmark). A total dose of 12 mg/kg of artesunate and 25 mg/kg of mefloquine were given over 3 days. On day 0, the 4 mg/kg of artesunate was divided into two equal doses, one in the morning and one in the evening, and on days 1 and 2, the 4 mg/kg was given as single doses. The 25 mg/kg of mefloquine was also divided into two equal doses in the morning and evening on day 0. The maximum dose of mefloquine was limited to 1500 mg for adults, considering the potential side effects. Health staff was provided with a dosage table based on body weight for practical use.

Patients received food for lunch and dinner from the hospital, usually rice and soup with fish or chicken. In the morning, patients normally ate rice porridge prepared by themselves or their family. In the studies AL2003 and AM2003, patients were provided with 250 ml milk and five pieces of coconut biscuits with each dose of artemether–lumefantrine. For children who could not swallow tablets, the appropriate dose (mg/kg body weight) was crushed and mixed with water and given through a syringe. If vomiting occurred within 1 h of taking the drugs, the medication was re-administered.

The alternative treatment in the case of failure was quinine + tetracycline for 7 days. Those who developed severe disease were treated with intramuscular artemether for 5 days. Potential drug side effects were recorded. Ethical clearance for the study was granted by the National Ethics Committee for Health Research of the Ministry of Health, Cambodia.

Laboratory investigations and outcome measures

Clinical examination and parasite counts were performed every day until parasites were completely cleared for two consecutive days and then weekly from day 7. Axillary temperatures were recorded every 8 h during hospitalization. Patients were followed up until day 28. Primary end point of the study was the parasitological and clinical response to treatment. Parasite clearance time (defined as the first thick film negative for two consecutive days) and fever clearance time (axillary temperature below 37.5 °C for two consecutive days) were also analysed. For quality assurance, blood slides were cross-checked by the National Center for Parasitology, Entomology and Malaria Control, Phnom Penh. All slides on day 0, all positive slides after day 7 (regardless of the parasite species) and 10% randomly selected negative slides were cross-checked by a senior laboratory technician with more than 10 years experience in malaria diagnosis.

Sample size and statistical analysis

Sample sizes were determined according to WHO protocol based on the estimation of the prevalence with a confidence interval of 95% and a precision of 10%. Therefore, the sample size was 50 cases in study AL2002 with the assumption of less than 5% treatment failures, 80 cases for study AL2003 based on the result of the study AL2002, and 50 cases for study AM2003 with the assumption of less than 15% treatment failures. Although the studies in 2003 were not designed to compare the efficacy of artemether–lumefantrine and artesunate + mefloquine, the first 110 patients were assigned randomly to each treatment group; the last 25 patients were treated with artemether–lumefantrine. Data were entered into the WHO Excel spreadsheet for drug efficacy data, based on Excel software (Microsoft Corporation, Redmond, WA, USA) and further analysed with spss software (SPSS Inc., Chicago, IL, USA). Analysis of the treatment outcome was performed by per-protocol method and by survival curve method. All re-infections and Plasmodium vivax infection were excluded from the analysis according to the WHO protocol. anova was used for normally distributed data, and Mann–Whitney U-test was used to compare data that were not normally distributed. Chi-square test with Yate's correction was used to compare proportion.

PCR for parasite genotyping

As re-infection within the 28-day follow-up was possible, blood samples were collected onto filter paper at day 0 and at the day of the detection of the reappearance of asexual parasitaemia. Re-infection and recrudescence were distinguished for all the failures by PCR genotyping of three P. falciparum polymorphic genes msp1, msp2 and glurp at the Institut Pasteur du Cambodge (Contamin et al. 1995).

In vitro assay

Venous blood was collected on EDTA in Venoject® tubes (Termo Europen.V.3001, Leuven, Belgium) before patient treatment and transported to Phnom Penh at 4 °C, generally within 24–48 h of collection, and in any case in a delay never exceeding 72 h. For the in vitro testing, quinine hydrochloride was purchased from Sigma (Munich, Germany). Mefloquine, chloroquine diphosphate, artemether and artesunate were obtained from WHO/TDR Drug Discovery Research, and lumefantrine from Novartis Pharma (Ho Chi Minh City, Vietnam). Stock solutions of chloroquine diphosphate were prepared in water, while stock solutions of quinine hydrochloride, mefloquine, lumefantrine, artemether and artesunate were prepared in ethanol. The classical isotopic 48-h test was performed to assess the in vitro sensitivity of the P. falciparum isolates at the Institut Pasteur du Cambodge (Desjardins et al. 1979). The results of in vitro assay were expressed as the 50% inhibitory concentration (IC50), defined as the drug concentration at which 50% of the incorporation of hypoxanthine (Meek et al. 1986) is inhibited, when compared with the drug-free control wells.

Plasma lumefantrine assays

In study AL2003, heparinized venous blood samples for lumefantrine plasma level determination were taken on day 7 from each patient who received the artemether–lumefantrine treatment. Whole blood was centrifuged and plasma was transferred to a cryotube and frozen in liquid nitrogen. Lumefantrine concentrations were determined at the Wellcome Unit, Faculty of Tropical Medicine, Mahidol University in Bangkok using solid phase extraction and liquid chromatography, as described elsewhere (Lindegardh et al. 2005).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Patient enrolment and baseline characteristics

In study AL2002, 55 patients with acute uncomplicated falciparum malaria were enrolled. In 2003, 135 were eligible and willing to participate in the study. Among the 135 cases, 80 cases were included in study AL2003 and 55 in study AM2003. The baseline characteristics of the study populations are summarized in Table 1. The admission demographic and clinical features among the three study groups were similar, except in the proportion of the patients’ gender: more males were included in study AL2003 compared with the two other studies. In all the studies, more than 80% of the patients were aged between 15 and 50.

Table 1.   Patients’ characteristics
 20022003 to 2004
AL2002AL2003AM2003
Number of patients enrolled558055
Gender (M/F)38/1765/1539/16
Mean age (years) (SD)26.1 (12.2)24 (8.9)26.3 (12.9)
Mean weight (kg) (SD)47.7 (12.1)47.7 (11)45.0 (9.4)
Mean temperature at admission (°C) (SD)38.6 (0.9)38.0 (1.1)38.2 (1.1)
Geometric mean parasitaemia at admission (per μl) (range)21 020 (1020-149 750)16 000 (1020–100 000)22 210 (2640–95 240)
Mean packed cell volume (SD)0.39 (0.06)0.37 (0.05)0.34 (0.06)

Outcome measures are summarized in Table 2. In study AL2002, six patients were lost to follow-up (all at day 8), two cases showed re-infection (day 26 and day 28), two cases showed P. vivax infection (both day 28). In study AL2003, three patients were lost to follow-up (day 8, day 8 and day 22), two cases showed re-infection (day 21 and day 26), one case showed P. vivax infection (day 28). In study AM2003, three patients were lost to follow-up (day 8, day 15 and day 22). No ETF was observed, all failures were late treatment failures (LTF = LCF + LPF). With the per-protocol analysis, treatment success rate (95% confidence interval) was the highest for artesunate + mefloquine, 92.3% (81.5–97.9). The success rate did not differ significantly for artemether–lumefantrine between 2002 and 2003: 71.1% (55.7–83.6) and 86.5% (76.5–93.3), respectively. There was no statistically significant difference of the fever and the parasite clearance times among the three studies. With the survival curve analysis, the results are similar (Figure 1). The cumulative success rate was highest for artesunate + mefloquine, 92.4% (85.3–99.6) compared with artemether–lumefantrine in 2002 and 2003, 72.8% (60.8–85.5) and 86.7% (79.1–94.4), respectively. In all three studies, all patients with treatment failures were successfully treated with a combination of quinine and tetracycline. Patients with vivax malaria were successfully treated with chloroquine. Artemether–lumefantrine was well tolerated and minor symptoms of nausea and headache occurred in studies AL2002 and AL2003, whereas dizziness, insomnia, headache, vomiting and nausea were reported commonly in the AM2003 group.

Table 2.   Outcome measures
Study20022003 to 2004
AL2002AL2003AM2003
Number of patients analysed457452
28 day cure rate, no. of patients (%)32/45 (71.1%)64/74 (86.5%)48/52 (92.3%)
Mean fever clearance time (hours) (SD)49.5 (12.5)29.5 (13.6)28.8 (15.1)
Mean parasite clearance time (days) (SD)3.2 (0.8)2.6 (0.8)2.5 (0.8)
Late clinical failure1141
Late parasitological failure263
Late treatment failure13 (28.9%)10 (13.5%)4 (7.7%)
image

Figure 1.  Cumulative success rate of patients treated with artemether–lumefantrine in 2002 (AL2002), 2003 (AL2003) or with mefloquine + artesunate (AM2003).

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Because of the low number of failures, the in vitro data of the studies AL2002 and AL2003 were merged (Table 3). Geometric mean of the IC50 (GMIC50) of artemether and lumefantrine were lower in the ACPR group compared with the LTF group, but this difference is not statistically significant. For the other drugs (mefloquine, quinine, chloroquine and artesunate), the GMIC50 were identical.

Table 3. In vitro responses of Plasmodium falciparum isolates during studies AL2002 and AL2003
 ArtemetherLumefantrineMefloquineArtesunateChloroquineQuinine
  1. ACPR, adequate clinical and parasitological response; LTF, late treatment failure; n, number of samples; GMIC50, geometric mean of 50% inhibitory concentration expressed in nmol/l.

ACPR group
 n425656625657
 GMIC501.118.812.91176.2112.4
 95% CI0.7–1.815.1–23.610–16.60.9–1.2150.1–206.895.2–132.8
 Range0.1–42.21.3–84.82.4–72.10.14–5.654.1–739.720.5–538
LTF group
 n131716171517
 GMIC501.928.912.81.1167.293.5
 95% CI0.9–4.120.7–40.27.2–22.80.7–1.5105.4–265.370.2–124.4
 Range0.2–12.68.9–1172.3–131.20.3–4.611.4–356.826.1–230.2

Of 80 patients in study AL2003, 76 plasma samples from day 7 were available for lumefantrine plasma concentration measurement. The mean lumefantrine plasma concentration on day 7 of the ACPR group was 0.86 μg/ml in 64 samples and the mean concentration of the LTF group was 0.51 μg/ml in 10 samples. Two patients were lost to follow-up and not included in the analysis. Their lumefantrine concentrations were 0.64 and 1.05 μg/ml. This difference between the mean lumefantrine values of the ACPR group and the LTF group was statistically significant (P = 0.02). Of 64 analysed cases of ACPR, 43 cases (67.2%) showed lumefantrine concentrations > 0.5 μg/ml compared with 3/10 (30%) cases with treatment failure cases (P < 0.01). Of 46 patients with a concentration of lumefantrine > 0.5 μg/ml, 43 patients (93.5%) were successfully treated.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In analogy to the treatment of tuberculosis or HIV infection, uncomplicated malaria should be treated with a combination of antimalarial drugs if possible based on an artemisinin derivative (ACT). At present, WHO recommends to countries five combinations (artesunate + amodiaquine, artesunate + sulphadoxine–pyrimethamine, artesunate + mefloquine, artemether–lumefantrine and amodiaquine + sulphadoxine–pyrimethamine as an interim option) (WHO 2001). Among these five options, only artemether–lumefantrine is available as a co-formulated product. Monotherapy of artemisinin derivatives is not recommended. Artemisinin and its derivatives act extremely rapidly by reducing the parasite biomass and clinical symptoms. They have broad stage specificity and reduce gametocyte carriage. Nevertheless, short courses lead to a high failure rate. A better option is to combine artemisinin derivatives with another antimalarial drug with longer half-life, which destroys residual parasites. During the clinical trials conducted in Africa by WHO/TDR, chloroquine was not a good partner drug for artesunate, because of the widespread high level of chloroquine resistance over the continent (Taylor et al. 2003). On the contrary, the combination of artesunate + mefloquine was introduced in Thailand after the increasing spread of mefloquine resistance. The combination proved to be highly effective at the Thai–Myanmar border, but most importantly, in vitro studies showed a significant reduction in mefloquine IC50 values (Nosten et al. 2000). Resistance to artemisinin has not been formally confirmed. In vitro monitoring over time has not shown evidence of resistance to artemisinin and its derivatives in many parts of the world including Cambodia, but only reduced sensitivity in China and Vietnam (Huong et al. 2001; Yang et al. 2003). Therefore, failures of an ACT are likely to be explained by the failure of the partner drug. Two main reasons could explain the unexpected high failure rate of artemether–lumefantrine: pharmacokinetic factors (i.e. inadequate plasma concentrations) and resistance to lumefantrine.

The combination artemether–lumefantrine has been marketed since a few years. The outcome of the first clinical trials in Asia, in which four doses were used, showed high rates of recrudescence (Looareesuwan et al. 1999; Kshirsagar et al. 2000). The regimen was therefore increased to six doses divided over 3 days. The pharmacokinetics of lumefantrine varies considerably among individuals, and its absorption is enhanced by concomitant intake of fatty foods. Studies of the pharmacokinetics and pharmacodynamics of lumefantrine indicate that the main determinant of the efficacy of the artemether–lumefantrine combination is the area under the curve for the plasma concentration of lumefantrine or its surrogate, the plasma concentration of lumefantrine on day 7. A lumefantrine plasma concentration >0.5 μg/ml on day 7 is associated with a treatment success rate >90% (Ezzet et al. 1998; White et al. 1999). As a six-dose treatment increases the plasma concentration of lumefantrine, this should be made a common policy. In our studies, the treatment success rate of artemether–lumefantrine increased by 15% when the treatment was supplemented with fatty food. Patients with treatment success presented with significantly higher plasma concentration of lumefantrine than patients with treatment failure. Over 90% of patients with a concentration of lumefantrine >0.5 μg/ml were cured. Several studies in which therapeutic efficacy tests were combined with sampling of plasma or whole blood for drug concentration measurements at various times during follow-up have shown that cured patients have higher drug concentrations than those in whom treatment failed. There are two possible explanations for the latter finding. First, failures are associated with inadequate drug concentrations rather than resistance – this could be the case in our studies AL2002 and AL2003; secondly, when drug resistance emerges, there is a higher likelihood that a resistant strain will emerge if the drug is present at a suboptimal concentration.

Multidrug resistance is well documented in Cambodia and Thailand, and failure of artesunate–mefloquine has been reported on both sides of the Thai–Cambodian border (WHO 2005). Artesunate + mefloquine remains effective in 2003 in Sampov Loum, the failure rate did not exceed 10%; however, the follow-up was only 28 days. Higher failures rates, up to 14.3%, have already been reported in Cambodia. Results of in vitro studies in Cambodia confirmed the in vivo results. Chloroquine-resistant phenotypes are highly prevalent in Cambodia. Similarly, a high proportion of isolates displayed elevated IC50 of mefloquine. But isolates with decreased susceptibility to chloroquine and mefloquine were more common along the border with Thailand. In contrast, most of the isolates from eastern Cambodia were susceptible to these two compounds. A small proportion of isolates presented with a decreased susceptibility to quinine and artesunate, and isolates collected in the western and eastern parts of the country did not respond differently to artesunate (Lim et al. 2005). In our study, the geometric mean of lumefantrine IC50 was increased in the LTF group compared with the ACPR group with a ratio of 1.5 only. The difference was not statistically significant but the sample size was rather small. Despite the few isolates with high lumefantrine IC50, the lumefantrine in vitro results do not differ from studies carried out in different parts of the world and are not in favour of lumefantrine resistance. Moreover, artemether–lumefantrine has not been widely used in the study area. However, cross-resistance between mefloquine and lumefantrine has been well described (Basco et al. 1998; Pilz et al. 2004). Mefloquine is still available as single treatment in many parts of the region and could induce selection pressure because of its long half-life on isolates with reduced sensitivity to amino alcohols. Studies conducted in Thailand showed that an increased copy number of the pfmdr1 gene is responsible not only for resistance to mefloquine but also for reduced sensitivity to artemisinin (Price et al. 2004). Moreover, in Thailand, the number of pfmdr1 copies was also associated with reduced in vitro sensitivity to lumefantrine and with a higher risk of treatment failure for a four-dose regimen of artemether–lumefantrine (S. Krishna, pers. comm.). Molecular studies to detect the presence of amplified pfmdr1 gene in the samples collected during studies AL2002 and AL2003 are ongoing and will hopefully bring further information on the lumefantrine resistance pattern in the region.

To date, ACTs are the most effective drug combinations we have for uncomplicated malaria. These medicines must be correctly used and it is vital that the countries closely monitor their efficacy. The situation at the Thai–Cambodian border is worrying. It is crucial that several other ACTs using partner drugs with a different mode of action and unavailable in monotherapy are developed rapidly.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank all the patients involved in this study. We are very grateful to Thay Khieng Heng, Kong Nareth, Va Soch, Muth Sinuon and Siv Sovannaroth, National Center for Parasitology, Entomology and Malaria Control for blood slide examination, data entry and quality assurance and to Pheaktra Chim, Sandra Incardona, Sem Rithy and Nhem Sina, Institut Pasteur du Cambodge for their collaboration. We also thank the health staff in Sampov Lun Referral Hospital for their cooperation. This study was supported by a grant from the US Agency for International Development through WHO. The Wellcome Trust of Great Britain funded the pharmacological work of Niklas Lindegardh and Anna Annerberg.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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