Discussion
In this study, the largest pooled analysis of individual patient PK-PD data for any antimalarial to date, artemether-lumefantrine was generally highly effective with only 73 (3 %) P. falciparum recrudescences among the 2,528 patients included in the treatment outcome analysis. The most important determinants of therapeutic response were baseline parasite density and day 7 blood or plasma lumefantrine concentrations. Current artemether-lumefantrine dosing recommendations achieve day 7 lumefantrine concentrations ≥200 ng/ml and >98 % cure rates in most uncomplicated malaria patients. However, three groups were at increased risk of treatment failure: very young children, particularly those that are underweight-for-age; patients with high parasitemias; and patients in areas with very low transmission intensity and slow early parasitological responses (reflecting artemisinin resistance).
Young children had 17.5–52.8 % lower day 7 lumefantrine concentrations following supervised treatment despite their actual mg/kg dose being higher, as they have higher body weight normalized apparent clearance after oral administration [
35]. Optimal dosing of artemether-lumefantrine in young children requires urgent investigation. Children under 5 years of age are at particular risk as they account for 78 % of all malaria-related deaths [
4]. Although this large pharmacokinetic data set did not have sufficient recrudescences to confirm the trend towards a higher risk of recrudescence among underweight young children, this was confirmed in the larger WWARN artemether-lumefantrine dose impact analysis. Underweight African children between 1 and 3 years old had an increased risk of recrudescence when compared with those of the same age who were not underweight (adjusted HR 1.66; 95 % CI 1.05 to 2.63;
P = 0.028) and a 4-fold higher risk than patients aged ≥12 years (adjusted HR 4.05; 95 % CI 1.78 to 9.18;
P = 0.001) [
67]
.
Malaria and malnutrition are common co-morbidities, particularly in Sub-Saharan Africa, where 90 % of global malaria deaths occur [
4] and 30–33 % of children under 5 years of age are underweight [
68]. However, there have been few studies on the effect of malnutrition on malaria, and these have yielded conflicting results [
69‐
71]. The mechanisms underlying the effects of malnutrition on antimalarial treatment response are complex and poorly understood. Malnutrition has also been shown to compromise the efficacy of chloroquine, sulfadoxine-pyrimethamine, amodiaquine, dihydroartemisinin and piperaquine [
72‐
75]. Several physiological changes can occur with malnutrition that may decrease drug concentrations, including reduced drug absorption and/or an increased volume of distribution. Malnutrition may reduce protein binding and increase clearance, but concomitant hepatic dysfunction may reduce the metabolism of some drugs. The net effect is uncertain [
1,
76]. In addition, the innate and adaptive immune responses may be impaired by malnutrition and micronutrient deficiencies [
70,
77,
78], which could explain the increased risk of malaria recurrence observed in our underweight young children even after adjusting for their total day 7 lumefantrine concentrations (unfortunately unbound lumefantrine concentrations were not measured in any of the studies included). A limitation of this study is that we were unable to use the preferred anthropometric indices for determining nutritional status [
79]. As the studies pooled for this analysis were designed to assess antimalarial efficacy, most only recorded body weight on a single occasion and height was only recorded in <5 % of young children. Thus we were unable to differentiate acute under-nutrition (low weight-for-height or BMI-for-age) from chronic under-nutrition (low height-for-age), or distinguish tall, thin children from short, well-proportioned children.
At currently recommended doses, the absorption of lumefantrine appears close to saturation [
40], or constrained by limited solubility. This was confirmed by the small effect of body weight-adjusted (mg/kg) dose in our study. Thus a simple increase in the number of tablets given at each twice daily dose may not ensure adequate lumefantrine exposure. Administering the same recommended six doses of artemether-lumefantrine over 5 days, dosing at 0, 8, 24, 48, 72 and 96 hours, has been shown to increase the area under the lumefantrine concentration time-curve (AUC) in Asian adults [
6,
7,
30], but this may compromise adherence. Further studies of higher, more frequent, or prolonged dosage regimens are needed to determine which dosing adjustments would ensure that all young children, including those that are underweight, could safely achieve the day 7 concentrations required to achieve ≥95 % cure rates.
Achieving acceptable cure rates is particularly challenging for underweight young children with higher parasite densities (>100,000/uL), who require higher day 7 concentrations (up to 256 ng/ml). Hyperparasitemia is an important source of antimalarial drug resistance [
80] and occurs commonly in patients with otherwise uncomplicated malaria. In the large WWARN pooled analysis of 14,327 patients treated with artemether-lumefantrine, 9.5 % had parasite densities above 100,000/uL [
67]. This 9.5 % prevalence would be an underestimate of all uncomplicated hyperparasitemia, as the WHO recommends excluding hyperparasitemic patients from therapeutic efficacy studies [
59]. To exclude uncomplicated hyperparasitemia, microscopy should be used rather than rapid diagnostic tests when feasible, particularly in very young and underweight children. The administration of at least two doses of parenteral artesunate is the preferred treatment for hyperparasitemic patients [
1], and the threshold of >250,000/uL in the current WHO definition of uncomplicated malaria in areas of moderate to high transmission intensity [
59] appears too high for very young children, particularly if they are underweight.
The risk of artemether-lumefantrine failure was, as expected, highest in western Cambodia, the epicenter of antimalarial drug resistance [
81,
82], where day 7 lumefantrine concentrations >1,616 ng/ml appear necessary to achieve acceptable cure rates even for very low baseline parasite densities (<5,000/μL). It is of concern that patients in the very low transmission areas included in this study also required high lumefantrine concentrations (>1,000 ng/ml) to cure even low parasite densities. In these areas it seems unlikely that artemether-lumefantrine dosage regimens could be adjusted to achieve the predicted lumefantrine exposure needed to ensure acceptable cure rates for parasite densities of up to 100,000/μL (the WHO definition of uncomplicated malaria in areas of low intensity malaria transmission). The very low transmission intensity areas included in this analysis comprised only two small studies in Thailand, and data on the frequency of the
pfmdr1 86 N allele and copy number in our study were insufficient for determining the extent to which these findings simply reflect high levels of lumefantrine resistance, or whether the lack of immunity in these areas of very low transmission intensity further compromises therapeutic efficacy. The WWARN pooled analysis of the relationship between lumefantrine-resistant polymorphisms in
pfcrt and
pfmdr1 and artemether-lumefantrine treatment response showed that presence of the
pfmdr1 gene N86 (adjusted HR 4.74; 95 % CI 2.29 to 9.78) and increased
pfmdr1 copy number (adjusted HR 6.52; 95 % CI 2.36 to 17.97) were significant independent risk factors for recrudescence in patients treated with artemether-lumefantrine [
83].
Even after adjusting for covariates, including site effects (for Cambodia and the nearby very low intensity transmission areas included in our study) and artemether-lumefantrine (mg/kg) dose, slow early parasitological treatment response more than doubled the risk of recrudescence. Artemether pharmacokinetic data were not available for this pooled analysis, and previous publications have been inconsistent. While some reported that higher artemether or dihydroartemisinin exposure was found to decrease parasite clearance time, others have found no clinically meaningful correlation between exposure and parasite clearance times [
44,
84,
85]. Whether due to artemisinin resistance and/or inadequate artemether/dihydroartemisinin exposure, a higher residual parasite biomass remains that the partner lumefantrine is less able to clear. Thus ACT treatment failure rates increase, risking the development and spread of resistance to both the artemisinin and lumefantrine components. The slow parasite clearance rates that characterize artemisinin resistance were originally documented in western Cambodia [
81,
82]. Despite containment efforts, artemisinin resistance has now been confirmed in five countries across mainland Southeast Asia [
86‐
88], where a total of 331,551
P. falciparum malaria cases were notified in 2013 [
4], highlighting the urgent need for novel antimalarials.
The simplicity of collecting a single pharmacokinetic sample per patient as an accurate measure of lumefantrine exposure is very appealing, particularly in remote field study sites with minimal infrastructure. The feasibility of pharmacokinetic sampling is further enhanced by the use of capillary blood specimens dried on filter paper, although this method is more vulnerable to inter-operator variability and the effects of anemia. This pooled analysis shows that this matrix is less optimal, being 2- to 3-fold more variable, and 5-fold less sensitive. However, with the therapeutic threshold of 200 ng/ml, the filter paper limit of quantification of 25 ng/ml should be sufficient for the measurements of day 7, if not later, concentrations. Careful attention to dried blood spot sample collection methods may reduce inter-operator variability.
As the determinants of therapeutic response are multi-factorial, studies of the pharmacokinetics of antimalarial drugs often have inadequate power to define optimal dosage recommendations. Pooled individual patient PK-PD data analysis makes the best use of available data for distinguishing treatment failures resulting from inadequate drug exposure from those caused by drug-resistant parasites. The main limitation of pooling individual patient pharmacokinetic data is differences in assay methods between studies. Only two of the studies included in this pooled analysis [
42,
44] used mass spectrometry to determine lumefantrine concentrations; early attempts failed due to matrix effects [
40]. More recently, several tandem mass spectrometry methods reported having addressed this issue [
42,
51,
89]. The risks of one study compromising the overall results of a pooled analysis decrease as the number of studies included increase; in our sensitivity analysis excluding each study one at a time, no individual study was shown to be influential and the main results were shown to be highly robust. Heterogeneity can be reduced by method standardization following the WHO/WWARN consensus document,
Methods and techniques for assessing exposure to antimalarial drugs in clinical field studies [
90]. The WWARN reference material program and, for more stable antimalarial medicines, external proficiency testing have further contributed to reducing inconsistency between antimalarial assays [
91].
Dose optimization is best informed when the pharmacokinetic parameters that drive artemether-lumefantrine exposure, particularly bioavailability (including doses above which absorption becomes saturated), volume of distribution and clearance, are characterized adequately in patients with uncomplicated malaria, including high-risk populations. Thus all available drug concentration-over-time data, and not just day 7 concentrations, need to be analyzed using a population PK-PD model. In collaboration with researchers worldwide, WWARN has obtained data from 4,122 malaria patients with a total of 9,258 lumefantrine concentrations (Fig.
1). This WWARN study group will continue to explore this expanded data set to answer key questions more fully, such as: ‘can Day 7 lumefantrine concentrations serve as a convenient surrogate for AUCs in all key target populations?’; ‘what proportion of treatment failures are explained by inadequate drug exposure?’; and ‘which modified dosage regimens should be investigated for important target populations, such as pregnant women, underweight young children or patients with co-morbid conditions (such as HIV/AIDS), or those who are taking drugs that reduce antimalarial exposure (such as efavirenz or rifampicin)?’.
Acknowledgments
We would like to thank all the patients and staff that participated in the 21 clinical trials included in this pooled analysis. We appreciate helpful comments from Michael Hendricks (Department of Paediatrics, University of Cape Town, Cape Town, South Africa) on the assessment of malnutrition in young children, and the assistance of Jennifer Flegg, Pete Gething and Simon Hay in obtaining Malaria Atlas Project estimates for study sites. The WorldWide Antimalarial Resistance Network (WWARN) is funded by a grant from the Bill & Melinda Gates Foundation. No funding bodies had any role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The members of the WorldWide Antimalarial Resistance Network (WWARN) Lumefantrine PK/PD Study Group are the authors of this paper: Elizabeth A Ashley, Shoklo Malaria Research Unit, Mae Sot, Thailand and Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Francesca Aweeka, Department of Clinical Pharmacology, University of California, San Francisco, CA, USA; Karen I Barnes, WorldWide Antimalarial Resistance Network (WWARN), Oxford, UK and Division of Clinical Pharmacology, Department of Medicine, University of Cape Town, Cape Town, South Africa; Quique Bassat, ISGlobal, Barcelona Centre for International Health Research (CRESIB), Hospital Clínic - Universitat de Barcelona, Barcelona, Spain and Centro de Investigação em Saúde de Manhiça (CISM), Maputo, Mozambique; Steffen Borrmann, Kenya Medical Research Institute/Wellcome Trust Research Programme, Kilifi, Kenya and Institute für Tropical Medicine, University of Tübingen, Germany; Prabin Dahal, WorldWide Antimalarial Resistance Network (WWARN), Oxford, UK and Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK; Timothy ME Davis, School of Medicine and Pharmacology, University of Western Australia, Crawley, Australia; Philippe Deloron, Institut de Recherché pour le Development, Mother and Child Faced with Tropical Infections Research Unit, Paris, France and PRES Paris Sorbonne Cité, Université Paris Descartes, Paris, France; Mey Bouth Denis, National Center for Parasitology, Entomology and Malaria Control, Phnom Penh, Cambodia and World Health Organization (WHO), Phnom Penh, Cambodia; Abdoulaye A Djimde, Malaria Research and Training Center, Department of Epidemiology of Parasitic Diseases, Faculty of Medicine, Pharmacy and Odonto-Stomatology, University of Bamako, Bamako, Mali; Jean‐François Faucher, Department of Infectious Diseases, Besançon University Medical Center, Hôpital Minjoz, Besançon, France and Institut de Recherche pour le Développement (IRD), Mother and Child Faced with Tropical Infections Research Unit, Paris, France; Blaise Genton, Department of Epidemiology and Public Health, Swiss Tropical and Public Health Institute, Basel, Switzerland and Division of Infectious Diseases and Department of Ambulatory Care and Community Medicine, University Hospital, Lausanne, Switzerland; Philippe J Guérin, World Wide Antimalarial Resistance Network (WWARN), Oxford, UK and Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK; Kamal Hamed, Novartis Pharmaceuticals Corporation, East Hanover, NJ, USA; Eva Maria Hodel, Parasitology Department, Liverpool School of Tropical Medicine, Liverpool, UK and Swiss Tropical Institute and Public Health Institute, Basel, Switzerland; Liusheng Huang, Department of Clinical Pharmacology, University of California, San Francisco, CA, USA; Vincent Jullien, INSERM U663, Universite Paris Descartes, Paris, France; Harin A Karunajeewa, Walter and Eliza Hall Institute, Parkville, Australia and Medicine Unit, School of Medicine and Pharmacology, University of Western Australia, Fremantle, Australia; Jean-René Kiechel, Drugs for Neglected Diseases Initiative, Geneva, Switzerland; Poul-Erik Kofoed, Projecto de Saúde de Bandim, Bissau, Guinea-Bissau and Health Services Research Unit, Lillebaelt Hospital/IRS University of Southern Denmark, Vejle, Denmark and Department of Paediatrics, Kolding Hospital, Kolding, Denmark; Gilbert Lefèvre, Novartis Pharma AG, Basel, Switzerland; Niklas Lindegardh (deceased), WorldWide Antimalarial Resistance Network (WWARN), Oxford, UK and Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Kevin Marsh, Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, Oxford, UK and Kenya Medical Research Institute/Wellcome Trust Research Programme, Kilifi, Kenya; Andreas Mårtensson, Malaria Research, Department of Medicine Solna, and Department of Public Health Sciences, Division of Global Health (IHCAR), Karolinska Institutet, Stockholm, Sweden; Mayfong Mayxay, Wellcome Trust – Mahosot Hospital – Oxford Tropical Medicine Research Collaboration, Mahosot Hospital, Vientiane, Lao PDR and Faculty of Medical Science, National University of Laos, Vientiane, Lao PDR; Rose McGready, Shoklo Malaria Research Unit, Mae Sot, Thailand and Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand and Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK; Clarissa Moreira, World Wide Antimalarial Resistance Network (WWARN), Oxford, UK and Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK; Paul N Newton, Wellcome Trust-Mahosot Hospital-Oxford University Tropical Medicine Research Collaboration, Mahosot Hospital, Vientiane, Lao PDR and Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK; Billy E Ngasala, Department of Parasitology, Muhimbili University of Health and Allied Sciences, Dar es Salaam, Tanzania and Malaria Research, Infectious Disease Unit, Department of Medicine Solna, Karolinska Institutet, Stockholm, Sweden; Francois Nosten, Shoklo Malaria Research Unit, Mae Sot, Thailand and Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand and Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK; Christian Nsanzabana, World Wide Antimalarial Resistance Network (WWARN), Oxford, UK and Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK; Sunil Parikh, Yale Schools of Public Health and Medicine, New Haven, CT, USA and Department of Clinical Pharmacology, University of California, San Francisco, CA, USA; Patrice Piola, WorldWide Antimalarial Resistance Network (WWARN), Oxford, UK and Institut Pasteur de Madagascar, Antananarivo, Madagascar; Ric N Price, World Wide Antimalarial Resistance Network (WWARN), Oxford, UK and Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK and Menzies School of Health Research, Charles Darwin University, Darwin, Australia; Pascal Ringwald, Global Malaria Programme, World Health Organization (WHO), Geneva, Switzerland; Lars Rombo, Malaria Research, Infectious Disease Unit, Department of Medicine Solna, Karolinska Institutet, Stockholm, Sweden and Centre for Clinical Research Sormland County Council, Uppsala University Uppsala, Sweden; Birgit Schramm, Epicentre, MSF, Paris, France; Carol Hopkins Sibley, World Wide Antimalarial Resistance Network (WWARN), Oxford, UK and Department of Genome Sciences, University of Washington, Seattle, WA, USA; Kasia Stepniewska, World Wide Antimalarial Resistance Network (WWARN), Oxford, UK and Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK; Joel Tarning, Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand and WorldWide Antimalarial Resistance Network (WWARN), Oxford, UK and Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK; Johan Ursing, Projecto de Saúde de Bandim, Indepth Network, Bissau, Guinea-Bissau and Malaria Research, Infectious Disease Unit, Department of Medicine Solna, Karolinska Institutet, Stockholm, Sweden; Michele Van Vugt, Shoklo Malaria Research Unit, Mae Sot, Thailand and Division of Infectious Diseases, Center for Tropical Medicine and Travel Medicine, University of Amsterdam, the Netherlands; Nicholas J White, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand and Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK; Lesley J Workman, WorldWide Antimalarial Resistance Network (WWARN), Oxford, UK and Division of Clinical Pharmacology, Department of Medicine, University of Cape Town, Cape Town, South Africa.
Authors’ contributions
Conceived and designed the experiments: KIB, KS, JT, NJW, RNP, SP, TMED, PP, CHS and PJG. Performed the original experiments: EAA, FA, QB, SB, TMED, PDeleron, MBD, AAD, JFF, BG, EMH, KH, LH, VJ, HAK, JRK, PEK, GL, NL, AM, KM, MM, RM, PNN, BEN, FN, SP, PP, RNP, LR, PR, BS, JT, JU, MVV and NJW. Analyzed the pooled individual patient data: KS, LJW, PDahal, CN and CM. Wrote the first draft of the manuscript: KIB, KS and LJW. Contributed to the writing of the manuscript: EAA, FA, KIB, QB, SB, PDahal, TMED, PDeleron, MBD, AAD, JFF, BG, PJG, KH, EMH, VJ, HAK, JRK, PEK, GL, NL, LH, KM, AM, MM, RM, CM, PNN, BEN, FN, CN, SP, PP, RNP, PR, LR, BS, CHS, KS, JT, JU, MVV, NJW and LJW. International Committee of Medical Journal Editors (ICMJE) criteria for authorship read and met: EAA, FA, KIB, QB, SB, PDahal, TMED, PDeleron, MBD, AAD, JFF, BG, PJG, KH, EMH, VJ, HAK, JRK, PEK, GL, NL, LH, KM, AM, MM, RM, CM, PNN, BEN, FN, CN, SP, PP, RNP, PR, LR, BS, CHS, KS, JT, JU, MVV, NJW and LJW. Agree with manuscript results and conclusions: EAA, FA, KIB, QB, SB, PDahal, TMED, PDeleron, MBD, AAD, JFF, BG, PJG, KH, EMH, VJ, HAK, JRK, PEK, GL, NL, LH, KM, AM, MM, RM, CM, PNN, BEN, FN, CN, SP, PP, RNP, PR, LR, BS, CHS, KS, JT, JU, MVV, NJW and LJW. Enrolled patients: EAA, FA, QB, SB, TMED, PDeleron, MBD, AAD, JFF, BG, EMH, HAK, PEK, LH, AM, MM, RM, PNN, BEN, FN, SP, PP, RNP, LR, BS, CHS, JU and MVV. All authors read and approved the final manuscript.