Baseline parameters and antimalarial trial outcome Title: early and late parasitological outcomes of antimalarial treatment in children ACCEPTED

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1 AAC Accepts, published online ahead of print on February 00 Antimicrob. Agents Chemother. doi:./aac.00-0 Copyright 00, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. 1 Borrmann et al. Baseline parameters and antimalarial trial outcome Title: The effects of Plasmodium falciparum parasite population size and patient age on early and late parasitological outcomes of antimalarial treatment in children Authors: Steffen Borrmann 1,*, Pierre-Blaise Matsiegui,*, Michel Anoumou Missinou,, Peter G. Kremsner, *These authors contributed equally to the work. Affiliations: 1. Institute of Hygiene, University of Heidelberg School of Medicine, Germany. Kenya Medical Research Institute, Centre for Geographical Medicine Research Coast, Kilifi, Kenya. Medical Research Unit, Albert Schweitzer Hospital, Lambaréné, Gabon. Department of Parasitology, Institute of Tropical Medicine, University of Tübingen, Tübingen, Germany Correspondence: Steffen Borrmann, Institute of Hygiene, University of Heidelberg School of Medicine, Germany, sborrmann@kilifi.kemri-wellcome.org Manuscript word count:,1 Abstract word count: 1 Keywords: malaria, Plasmodium falciparum, amodiaquine, chemotherapy, pharmacodynamics, in vivo test Downloaded from on August, 01 by guest 1

2 Borrmann et al. Abstract Background: Baseline parameters and antimalarial trial outcome The design and interpretation of trials assessing the chemotherapeutic Methods: Results: effects of antimalarials drugs depends on our understanding of how different selection criteria affect treatment outcomes. We analysed the effects of baseline parameters on the initial parasite elimination rate and the risk of subsequent recrudescence as a marker for incompletely eliminated asexual blood stage infections in paediatric patients with uncomplicated P. falciparum treated with amodiaquine in a high transmission area. We find that (1) parasite population size and patient age independently determine early and late parasitological treatment outcome measurements; () the rate of recrudescence is higher in patients 1-yrs old as compared to patients aged <1 or >yrs; () patients >yrs and with parasite densities between,000-,000/µl have a lower recrudescence rate (1%; %CI %-1%) than patients aged <yrs and with >,000 parasites/µl (0%; %CI 0%-0%) and () the sensitivity of detecting recrudescences outside this high risk group, i.e. in patients of >yrs or with parasite densities of <,000/µL, are as low as % and %, respectively. Conclusions: These findings highlight the need to use adequate selection criteria and to report parasitological outcome results adjusted for readily available determinants of chemotherapeutic failure, i.e. patient age and baseline parasitaemia. The thresholds may vary by transmission intensity and drug regimen. A better understanding of the limitations of an antimalarial regimen in high-risk subgroups of patients has important implications for setting policy recommendations. Downloaded from on August, 01 by guest

3 Introduction Antimalarial treatment trials provide the empirical evidence base for antimalarial treatment policies in endemic countries (). These trials use variable endpoints to address a range of public health questions. For instance, trials conducted to monitor the emergence or spread of in vivo resistance or to determine the chemotherapeutic efficacy of a new regimen are designed to measure the ability of treatment to completely eliminate asexual blood stage infections and thus, to prevent recrudescent infections. On the other hand, the delay of re- infections by slowly eliminated drugs through effects on the erythrocytic, and in some cases, hepatic stages of P. falciparum (1) can be an important additional clinical benefit of antimalarial treatment. This is investigated in studies that use a combined measurement of the chemotherapeutic and the post-treatment prophylactic effects ( recurrence rate ) or that simply determine the requirement for re-treatment (1). Regardless of the objectives in these trials, the characterisation of the limitations of an antimalarial regimen in important subgroups of patients at high risk of failure and who may also be disproportionately susceptible to adverse clinical outcomes will provide critical information for setting policy recommendations. In areas of high malaria transmission, the value of an antimalarial drug largely depends on its performance in non-immune populations at highest risk of P. falciparum malaria, i.e., young children and pregnant women in high endemicity areas. Differential effects of parasite population size and host age on the risk of recrudescence are known since long (,, 1, 1). These associations were subsequently, though not universally (), shown to be operating in diverse transmission settings (, 1,,, ). Fewer studies have addressed the interaction of these factors on the response to antimalarial drugs (, 1,,, ). The mechanisms of the underlying biological processes are poorly understood; thus far antibody responses to selected parasite antigens (ring-infected erythrocyte antigen [RESA], merozoite surface protein 1 [MSP-1]) have been implicated in age-dependent, anti-parasitic Downloaded from on August, 01 by guest

4 immunity (, ). It appears plausible that humoral and/or cellular immune responses play a role both in the initial, rapid parasite elimination phase during treatment when drug concentrations are highest as well as in suppressing slowly replicating asexual parasites preventing patent recrudescence of infections. Similarly, the pre-treatment size of a given parasite population is likely linked to treatment outcome by affecting the probability of survival of a sub-population of asexual blood stage parasites. In case of multiclonal infections this sub-population will likely consist of drug resistant parasites () but the mechanisms for differential parasite survival in sensitive infections remain elusive (). Using data from previously published controlled trials of amodiaquine we studied in detail the relationships between (a) pre-treatment asexual parasite density, host age and other baseline parameters and (b) early and late response rates to antimalarial treatment. We used graphical analyses for visualising relationships and survival analysis to reflect specific biological processes ( time to event, i.e., time to recrudescence, is thought to be a function of the antiparasitic potency of a regimen ()). Based on these results we aimed to describe the effect of different sets of selection criteria on outcome estimates and how this affected the sensitivity to detect recrudescences in comparison to the subgroup at highest risk of parasitological failure. Methods Clinical study For the purpose of this analysis we pooled previously analysed datasets of patients treated with amodiaquine for uncomplicated P. falciparum malaria in two independent trials conducted from 1 to 000 at the Albert Schweitzer Hospital, Lambaréné, Gabon ( Study A () and Study B (1)). The study protocols were approved by the ethics committee of the International Foundation of the Albert Schweitzer Hospital. The study region is characterised by perennial malaria transmission (0). Chloroquine resistance is prevalent (). Patients Downloaded from on August, 01 by guest

5 aged 0.. years (0.. years in study A () and 1.-. years in study B (1)), attending the outpatient paediatric department of the hospital were eligible for enrolment if the following inclusion criteria were met: asexual P. falciparum parasitaemia of 1,000 00,000 parasites/µl, tympanic temperature >. C or a history of fever in the preceding hours, and written informed consent given by a parent or guardian of the patients. Exclusion criteria were signs of severe disease, adequate antimalarial treatment in the preceding hours and haemoglobin of < g/dl. Patients were actively followed up daily until day or until the second consecutive negative blood slide and thereafter on days, 1 (study B only), 1 (study B only) and. Parents were encouraged to return to the clinic in case of a change in the condition of their child (passive follow-up). Asexual parasite density was determined using the previously described Lambaréné method (). Amodiaquine was formulated as 1% suspension of amodiaquine base in study A (Flavoquine, Hoechst Marion Roussel, France) () or tablets of amodiaquine base in study B (00 mg, Camoquin, Parke-Davis, France) (1). Supervised treatment was administered in target doses of mg/kg once daily for three consecutive days. Doses of tablets were rounded to the nearest quarter tablet and sirup was administered in spoons of ml. Full doses of drugs were re-administered if patients vomited or spit out the first dose within one hour after administration. Patients who vomited the study drug more than once were withdrawn from the study and treated with rescue medication. The day per protocol cure rate was the primary endpoint of the two study protocols. Parasitological cure was defined as initial asexual parasite clearance before day and no subsequence recrudescence between day and day (). Early treatment failures were defined based on standard clinical and parasitological criteria (before day (1, )) and late treatment failures were defined parasitologically as microscopically detectable recrudescent infections occurring between day and day (1, ). PCR-based genotyping of pairs of parasite isolates from baseline and day of asexual parasite recurrence (>day 1) was used to classify recurrent infections as either recrudescence or new infection (). Statistical analyses Downloaded from on August, 01 by guest

6 We used the non-parametric rank sum test for univariate comparisons of continuous baseline variables with skewed distributions. The risk of recrudescence and the corresponding confidence interval (CI) were estimated with the non-parametric Kaplan Meier survivor function. Observations from patients who did not complete follow-up because of protocol violations, withdrawals and re-infections before day were right-censored in the survival analysis, i.e. the data from the last evaluable visit were used in the survival analysis. Whenever appropriate we also calculated incidence point estimates ( cure rate or failure rate ) and respective risk differences based on the per protocol population as well as corresponding confidence intervals. The per protocol population consisted of patients with a known efficacy outcome (patients with new infections between days 1 and were classified as cured ). Proportions were compared with the Fisher s exact test and confidence intervals were calculated using the accurate Wilson method for small sample sizes (). We constructed, via maximum-likelihood, Cox proportional hazard and logistic regression models for analysing the effect of several risk factors on survival and binary outcomes, respectively, using a forward selection approach to include baseline parameters with p values of <0.1. The proportional hazard assumption was tested using weighted Schoenfeld residuals (1) and reported if violated. The degree of correlation of independent variables included in the models were examined using the Spearman s rank coefficient. The reported unadjusted and adjusted hazard ratios indicate one-unit changes of the corresponding variables. To objectively define a combination of thresholds of baseline parameters with the best discriminatory performance in correctly identifying patients with recrudescent infections we compared the areas under the Receiver Operating Characteristic (ROC) curve for different combinations of incrementally changed thresholds (one-unit increments). We used fractional polynomial regression as a flexible parametric method for fitting smooth curves of unknown relationships between Y and X variables (). Log transformed parasite reduction ratios (PRR) were calculated as the fractional reduction of microscopically determined asexual parasite densities per asexual life cycle (average of hours; PRR ) (). The initial asexual parasite clearance time (PCT) was computed as the time from start of Downloaded from on August, 01 by guest

7 treatment until the first of two consecutive negative malaria blood slides taken on two subsequent days. In addition, we calculated stratified recrudescence rate ratios for the rate of negative/all slides (negativity) at and hours after start of treatment and tested their signficance using Mantel-Haenszel statistics. All statistical analyses, the construction of graphs and the fitting of curves were performed using Stata/MP version.0 for Mac OS X. Results Study cohort and baseline parameters A total of patients were studied (Table 1a); 1 patients (%) reached a defined efficacy endpoint. Fifty-three recrudescent infections were detected until day (corresponding to a failure rate of %; % CI %-0%). The Kaplan Meier survival estimate was % (% CI 0%-%) for the pooled dataset but there was a striking difference between study A and B with survival estimates of % (% CI 1%-%) and 0% (% CI 0%-%), respectively. Early treatment failures occurred in patients; of these in children < years. Patients aged < years had a 1% higher rate of recrudescent infections than older children (% CI %-%; p = 0.01) corresponding to a hazard ratio (HR) of. (% CI 1.-.; p<0.01). Patients with recrudescent infections were significantly younger (medians of. years versus. years) and harboured higher numbers of circulating ring stage parasites than patients who experienced recrudescent infections until day (medians of 0,000/µL versus 1,000/µL) (Table 1b). None of the other baseline parameters, including tympanic temperature and plasma creatinine concentration, were differentially distributed in unadjusted univariate comparisons among patients with or without recrudescent infections (Table 1b). We found no evidence for underdosing or differences in daily amodiaquine base doses among patients who did or did not experience recrudescence (Table 1b). There was also no difference in the combined rates of vomiting and readministration of drugs (Table 1b). Downloaded from on August, 01 by guest

8 Relationships between host age, parasite population size and risk of recrudescence The rate of recrudescence was highest in children 1 to years old (%. /1; % CI %-%). Children <1 year old had a similar rate compared to children of > years (1%, /1; % CI %-% vs. 1%, 1/; % CI %-%) (Figure 1a). A one-sided comparison between the group of children <1 year to children 1- years was borderline significant (p =0.0). The two-way graph of log transformed parasite density data against the rate of recrudescent infections plotted separately by age group (< versus > years) suggested that the reduced rate of recrudescence in older children may be mainly mediated by their increased ability to deal with moderate to high parasite population sizes ranging from,000-0,000/µl (Figure 1b). Of note, parasite densities >0,000 were associated with a high rate of recrudescence regardless of the age of the patient but the numbers were small and confidence intervals proportionally large (1%, /1; % CI %-% vs. %, /; % CI 1%-%) (Figure 1b). Two third of patients with recrudescent infections had an asexual parasite density of >,000/µL at baseline. The Cox proportional hazard model on time to recrudescence corroborated the univariate associations of host and parasite density with the rate of recrudescence described in Table 1b. Age (in years) (adjusted hazard ratio [AHR] of 0., % CI ; p=0.0) and log transformed asexual parasite density (AHR of.1, % CI 1.-.; p <0.01), but not tympanic temperature (in ºC) (AHR of 1.1, % CI 0.-1.; p=0.), resulted in lower and higher hazards, respectively (likelihood ratio chi-square = 1; p value for whole model < 0.001; Nagelkerke s coefficient of determination = 0.). In our data-set host age and parasite density were not correlated (Spearman's rank correlation coefficient = -0.1; p=0.) (Figure ). However, baseline tympanic body temperature was significantly correlated with patient age and pre-treatment parasite density (Spearman's rank correlation coefficients of -0.; p<0.001 and 0.; p<0.01, respectively). The addition of study (A or B) as a categorical variable did not improve the model (p>0.1 for both variables) because study (A,B) and age were highly correlated (Spearman's rank correlation coefficient = 0.; p<0.001). A separate Downloaded from on August, 01 by guest

9 proportional hazard model where age was replaced by the study variable (A,B) gave comparable results to the first model: study and baseline parasitaemia, but not baseline body temperature, resulted in significantly lower and higher hazards, respectively (data not shown). This supports the conclusion that age is a key determinant of the hazard to experience a recrudescent infection. Since age and rate of recrudescence are non-linearly related (Figure 1a) we also evaluated a proportional hazard model by including patients age and baseline parasite density as dichotomous variables (age below or above years and parasitaemia below or above,000/µl). Age < years and baseline parasite density >,000/µL gave significantly higher hazards (AHRs of., % CI 1.-.; p=0.01 and 1., % CI 1.1-.; p=0.0). The Kaplan-Meier plots in Figure a and b illustrate the impact of age < years and baseline parasitaemia >,000/µL on survival estimates. A logistical regression analysis of the risk of recrudescence in patients classified according to age and density (Figure ) revealed that the adjusted odds for recrudescence were times higher (% CI -; p<0.001) in patients < years old and with >,000 parasites/µl compared to patients not falling in this category. Moreover, Receiver Operating Characteristic (ROC) curve analysis showed that the combination of age < years and parasitaemia > /µl resulted in the highest area under the ROC curve (0.). Relationship between host age, parasite population size and early treatment response rates. The time to asexual parasite clearance below the microscopic detection threshold (parasite clearance time; PCT) positively correlated with the baseline asexual parasite density (Spearman's rank correlation coefficient = 0.; p<0.01). Patient age was negatively correlated with the PCT (Spearman's rank correlation coefficient = -0.; p<0.01) (Figure ). In contrast to the differential recrudescence rates in infants compared to children 1- years, the PCTs in the <1 year and 1- years age groups were equivalent (geometric mean of hrs and hrs, respectively). Of note: out of parasitologically defined early treatment failures occurred in infants, all of whom were months old. A Cox proportional hazard model Downloaded from on August, 01 by guest

10 on the time to asexual parasite clearance demonstrated that age (in years) (adjusted hazard ratio [AHR] of 0., % CI 0.-0.; p<0.01) and log transformed asexual parasite density (AHR of 1.1, % CI ; p=0.01), but not tympanic temperature (in ºC) (AHR of 0., % CI ; p=0.), resulted in lower and higher hazards, respectively. The -hour parasite reduction ratio (PRR ) was highly correlated with baseline parasite density (Spearman's rank correlation coefficient = 0.; p<0.001), though this is likely an artefact since supposedly high PRR s in patients with low baseline density will invariably be associated with sub-patent parasite densities by hours and hence, missing data ( out of 1 patients with an evaluable -hour slide result). Association between in vivo pharmacodynamic parameters and risk of recrudescence It has been suggested that the PCT and especially, the PRR are related to differential parasite drug sensitivities (). We therefore explored the relationship between these parameters and the risk of recrudescence. A Cox proportional hazard model on the time to recrudescence demonstrated that PCT (in hours) (adjusted hazard ratio [AHR] of 1.1, % CI ; p=0.001), but not the PRR (AHR of 1., % CI 0.-1.; p=0.), resulted in a significantly higher hazard (likelihood ratio chi-square = ; p value for whole model = 0.; Nagelkerke s coefficient of determination = 0.1). In an alternative approach, we used the slide negativity rates at and hours after start of treatment (Hour : %, /1 vs. 0%, 0/ and Hour : %, /1 vs. 0%, 1/ in patients without and with subsequent recrudescence) for calculating clinically more useful rate ratios of the risk of recrudescence but the rate ratios were low (<0.1) and non-significant (p=0.1 and p=0., respectively). Sensitivity to detect treatment failures in sub-populations of patients selected by host age and parasite population size Patients were categorised in groups below and above years of age (by neglecting the Downloaded from on August, 01 by guest

11 heterogeneous response rates to amodiaquine in children < years). Patients were further categorised by baseline parasitaemia using a threshold of,000 parasites/µl based on earlier results (Figure ). The sensitivity to detect failure in patients of different categories was calculated by defining the sub-population of patients < years and with >,000 parasites as the internal standard. Table a shows that the sensitivities for detecting failure in the comparison groups were %. The impact on selection criteria on failure estimates The use of selection criteria (< years and >,000 parasites/µl) that were more stringent than current WHO recommendations (< years and >,000 parasites/µl) produced a significantly higher estimate for the risk difference as compared to an unrestricted approach equivalent to the total patient population in this study (estimate of the risk difference = 1%; % CI %-%; p=0.01) (Table b). Downloaded from on August, 01 by guest

12 Discussion The interest in amodiaquine, especially in combinations with sulfadoxine-pyrimethamine or artesunate, as a replacement drug in areas of chloroquine resistance continues to grow (, 1, ) despite the important limitation of geographically heterogeneous response rates (1) and its potential for high degrees of cross-resistance with chloroquine (). An overall parasitological failure rate of % clearly does neither warrant the use of amodiaquine as monotherapy nor encourage its use in combinations. In this study we have attempted to systematically dissect the relationships between host age as the best surrogate marker for blood stage-specific immunity (1), the pre-treatment parasite population size and early as well as late outcome measurements of antimalarial treatment trials in children. Our results confirmed previously, mainly separately reported associations: (1) parasite population size and host age independently determine the risk of recrudescence and () patient age is nonlinearly related to the risk of recrudescence with the highest rates of recrudescence observed in patients 1 to years old compared to patients aged <1 or > years after treatment with amodiaquine in the high transmission study area. Moreover, we demonstrated that these associations equally applied to early and late outcome measurements. We could not confirm recently reported associations between body temperature and risk of recrudescence (, ), which may be due to the smaller sample size of our study and hence, its limited power, or alternatively, to the lack of a causal relationship. In contrast to rapidly acting artemisinin-containing combination therapies, the comparatively low per-cycle log parasite reduction ratio of amodiaquine (median PRR =.) unmasked a significant impact of host age on the time to parasite clearance. The observed age-dependent distribution of the risk of failure suggests an apparent rapid decline and subsequent slow acquisition of the ability of the host to assist the effect of the drug during the first and after the third year of life, respectively. This mirror image-like pattern is reminiscent of the phenomenon of age-dependent negative conversion rates () Downloaded from on August, 01 by guest 1

13 and resembles the dynamics of drug-resistant parasite clearance (). The lower risk of recrudescence in infants corresponds to a short period of post-natal protection (1, ); possibly mediated by maternal antibodies (, ) and fetal haemoglobin (0) among other factors. The lower risk for recrudescent breakthrough infections in infants may also provide a credible alternative explanation for recently reported, age-dependent protective effects of intermittent preventive treatment regimens (1). Intriguingly, the time to parasite clearance was prolonged in infants and three out of four parasitologically defined early treatment failures occurred in this age group. If this finding is not due to chance it may indicate that the immune mechanisms related to the initial parasite clearance under drug treatment (e.g., related to splenic clearance ()) differ from those that govern the control of slowly replicating sub-patent infections and cause differential survival rates of asexual parasites (0, ). In favour of this hypothesis, the body temperature - a crude measure of unspecific immune responsiveness - was also elevated in infants. Clearly, more data from infants are needed to elucidate the dynamic nature of these relationships. The proportional hazard model on risk of recrudescence used in this study could only account for 0% of the data variation. This may indicate a lack of consideration of other important determinants - our model did not include other important independent variables, e.g. ex vivo chemosensitivity of isolates and pharmacokinetic parameters () - or simply a large stochastic element in the distribution of the risk. We attempted to use calculated parasite reduction ratios to estimate in vivo pharmacodynamic effects but failed to detect any meaningful relationship between these ratios and treatment outcome. Recrudescences clustered in a sub-population of patients characterised by age < years and baseline parasite density >,000/µL. The sensitivity to detect treatment failures in older children and those with <,000 asexual parasites/µl was as low as 1%. Against the backdrop of this figure, we clearly endorse earlier recommendations () for using adequate protocols and to report study results stratified or adjusted by age and ideally, pre-treatment 1 Downloaded from on August, 01 by guest

14 parasite density. This is especially important for large meta-analyses of antimalarial trials, which have until now, mainly reported averaged treatment outcomes (). For proof-ofprinciple studies, e.g. phase II trials, trials in semi-immune populations in highly endemic areas (older children, adults) may be desirable due to safety and ethical considerations - with the important limitation that the results may o nly tell about % of the truth of the performance of a drug in particularly susceptible high risk populations. Obviously, this figure may represent a worst-case scenario since our findings cannot easily be extrapolated to other transmission settings and different drugs (). In summary, a better understanding of the limitations of an antimalarial regimen in high-risk subgroups of patients can provide important information for setting policy recommendations. Conflict of interest disclosure The authors have no potential conflict of interest to declare. The funding sources had no influence on the design, analysis and reporting of this study. Funding This work was supported by a German Research Foundation (DFG) Junior Group grant to SB (A, SFB ). Acknowledgment We would like to thank the patients and their parents for participating in the clinical studies. We are grateful to our colleagues who participated in the collection of the clinical data. We would also like to acknowledge the valuable comments by one of the reviewers of an earlier version of this manuscript. Downloaded from on August, 01 by guest 1

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18 Mayxay, M., K. Chotivanich, S. Pukrittayakamee, P. Newton, S. Looareesuwan, and N. J. White Contribution of humoral immunity to the therapeutic response in falciparum malaria. Am J Trop Med Hyg :1-.. Molineaux, L., and G. Gramiccia.. The Garki Project. World Health Organisation, Geneva.. Myint, L., G. A. Targett, and M. J. Doenhoff. 1. Reduced efficacy of chemotherapy of Plasmodium chabaudi in T cell-deprived mice. Trans R Soc Trop Med Hyg 1:-0.. Newton, P. N., K. Chotivanich, W. Chierakul, R. Ruangveerayuth, P. Teerapong, K. Silamut, S. Looareesuwan, and N. J. White A comparison of the in vivo kinetics of Plasmodium falciparum ring-infected erythrocyte surface antigen-positive and -negative erythrocytes. Blood :0-.. Nzila, A. M., E. Nduati, E. K. Mberu, C. Hopkins Sibley, S. A. Monks, P. A. Winstanley, and W. M. Watkins Molecular evidence of greater selective pressure for drug resistance exerted by the long-acting antifolate Pyrimethamine/Sulfadoxine compared with the shorter-acting chlorproguanil/dapsone on Kenyan Plasmodium falciparum. J Infect Dis :0-.. Olliaro, P., and P. Mussano. 00. Amodiaquine for treating malaria. Cochrane Database Syst Rev:CD Pasvol, G., D. J. Weatherall, R. J. Wilson, D. H. Smith, and H. M. Gilles. 1. Fetal haemoglobin and malaria. Lancet 1: Peters, W. 1. Chemotherapy and drug resistance in malaria. Academic Press, London.. Price, R. N., F. Nosten, C. Luxemburger, M. van Vugt, L. Phaipun, T. Chongsuphajaisiddhi, and N. J. White. 1. Artesunate/mefloquine treatment of multi-drug resistant falciparum malaria. Trans R Soc Trop Med Hyg 1:-.. Price, R. N., A. C. Uhlemann, M. van Vugt, A. Brockman, R. Hutagalung, S. Nair, D. Nash, P. Singhasivanon, T. J. Anderson, S. Krishna, N. J. White, and F. Nosten. 00. Molecular and pharmacological determinants of the therapeutic Downloaded from on August, 01 by guest 1

19 response to artemether-lumefantrine in multidrug-resistant Plasmodium falciparum malaria. Clin Infect Dis :-.. Royston, P., and D. G. Altman. 1. Approximating statistical functions by using fractional polynomial regression. The Statistician :-.. Sapak, P., P. Garner, M. Baea, A. Narara, P. Heywood, and M. Alpers.. Ineffectiveness of amodiaquine against Plasmodium falciparum malaria in symptomatic young children living in an endemic malarious area of Papua New Guinea. J Trop Pediatr :1-0.. Snow, R. W., J. A. Omumbo, B. Lowe, C. S. Molyneux, J. O. Obiero, A. Palmer, M. W. Weber, M. Pinder, B. Nahlen, C. Obonyo, C. Newbold, S. Gupta, and K. Marsh. 1. Relation between severe malaria morbidity in children and level of Plasmodium falciparum transmission in Africa. Lancet :-.. Sowunmi, A., B. A. Fateye, A. A. Adedeji, G. O. Gbotosho, T. C. Happi, A. E. Bamgboye, O. M. Bolaji, and A. M. Oduola. 00. Predictors of the failure of treatment with pyrimethamine-sulfadoxine in children with uncomplicated falciparum malaria. Acta Trop :-1.. Staedke, S. G., M. R. Kamya, G. Dorsey, A. Gasasira, G. Ndeezi, E. D. Charlebois, and P. J. Rosenthal Amodiaquine, sulfadoxine/pyrimethamine, and combination therapy for treatment of uncomplicated falciparum malaria in Kampala, Uganda: a randomised trial. Lancet :-.. Staedke, S. G., H. Sendagire, S. Lamola, M. R. Kamya, G. Dorsey, and P. J. Rosenthal. 00. Relationship between age, molecular markers, and response to sulphadoxine-pyrimethamine treatment in Kampala, Uganda. Trop Med Int Health :-. 0. Sylla, E. H., B. Lell, J. F. Kun, and P. G. Kremsner Plasmodium falciparum transmission intensity and infection rates in children in Gabon. Parasitol Res : Targett, G. A. 1. Malaria: drug use and the immune response. Parasitology Suppl:S Downloaded from on August, 01 by guest

20 . ter Kuile, F. O., C. Luxemburger, F. Nosten, K. L. Thwai, T. Chongsuphajaisiddhi, and N. J. White. 1. Predictors of mefloquine treatment failure: a prospective study of patients with uncomplicated falciparum malaria. Trans R Soc Trop Med Hyg :0-.. White, N. J. 1. Assessment of the pharmacodynamic properties of antimalarial drugs in vivo. Antimicrob Agents Chemother 1:-.. WHO. 00. Assessment and monitoring of antimalarial drug efficacy for the treatment of uncomplicated falciparum malaria. WHO. Downloaded from on August, 01 by guest 0

21 Figure legends Figure 1a. Two-way graph of the proportion of recrudescent infections over age. Error Figure 1b. Figure a. Figure b. Figure. bars indicate upper and lower limits of binomial exact % confidence intervals. Two-way graph of the proportion of recrudescent infections over pre- treatment asexual parasite density/µl stratified by age of patients. Error bars indicate upper and lower limits of % confidence intervals. Kaplan-Meier plot of the survivor function of recrudescence in patients with pre-treatment asexual parasite densities of <,000/µL or,000/µl. Log- rank test for equality of survivor functions; p<0.01. Kaplan-Meier plot of the survivor function of recrudescence in patients aged years or 1 to years. Log-rank test for equality of survivor functions; p<0.01. Inclusion of patients <1 year in the < year age group did neither change the shape of curves nor the significance level. Scatter plot of pre-treatment asexual density over age. Failures cluster within category 1 (age < years and >,000 parasites/µl). Figure. Two-way graph of geometric mean times to asexual parasite clearance over age. Parasite clearance signifies the time point when asexual parasite densities become undetectable via light microscopy, i.e. subpatent. Error bars indicate upper and lower limits of % confidence intervals. Downloaded from on August, 01 by guest 1

22 Figure 1a Proportion of recrudescent infections Figure 1b Proportion of recrudescent infections 0 Age in years Proportion of recrudescent infections Fitted curve Downloaded from on August, 01 by guest... Log pre-treatment asexual parasite density Proportion of recrudescent infections in children aged < years Proportion of recrudescent infections in children aged >= years Fitted curve for children aged < years Fitted curve for children aged >= years

23 Figure a. Kaplan-Meier survival estimates Cumulative risk of recrudescence Figure b. Cumulative risk of recrudescence Follow-up time in days Log asexual parasite density < /µl Log asexual parasite density >= /µl Kaplan-Meier survival estimates Downloaded from on August, 01 by guest Follow-up time in days Patients aged >= years Patients aged 1 to < years

24 Figure. Log pre-treatment parasite density... 0 Age in years Patients remaining without recrudescence Patients with recrudescent infection Fitted curve for patients without recrudescence Fitted curve for patients with recrudescent infections Downloaded from on August, 01 by guest

25 Figure. Geometric mean PCT in hours Age in years Geometric mean time to asexual parasite clearance Fitted curve Downloaded from on August, 01 by guest

26 Table 1a. Outcomes of paediated patients treated with amodiaquine for uncomplicated P. falciparum malaria Age range in years Outcome classification 0. to < to < Total Cured (%) (1) (%) (1) Early treatment failure (%) (1) 1 (%) (1) Late treatment failure (%) (1) (1%) (1) Per protocol population 1 Lost to follow-up () 1 (%) () (%) () Protocol violations, SAE () 1 (%) () (%) () Total 1 (1) Denominator corresponds to per protocol population () Patients were included in survival analysis on the risk of recrudescence when drop-out occurred after day () Denominator corresponds to total patient population () Serious Adverse Event Downloaded from on August, 01 by guest Table 1b. Univariate analysis of the relationships between baseline parameters and risk of subsequent recrudescence (median or percentage and % confidence interval) No recrudescence Recrudescence p value Demographics

27 Age in years. (.-.). (1.-.1) <0.01 Tympanic temperature in ºC.0 (.-.).0 (.-.) 0.0 Parasitology Asexual parasite density /µl 1,000 (1,000-,000) 0,000 (,000-,000) <0.001 Haematology & Chemistry Haemoglobin conc. in g/dl. (.-.). (.1-.1) 0. Creatinine conc. in mmol/l (-) (0-) 0.1 Alanine aminotransferase in U/L (-) (1-) 0. Pharmacology Amodiaquine dose/day. (.1-.).1 (.-.) 0. Frequency of vomiting on days 0- % (1/, %- 1%) % (/0, %-1%) 0. Frequency of diarrhea on days % (/, 1%- % (/0, 1% %) %) 1 Downloaded from on August, 01 by guest

28 Table a. Sensitivity and specificity to detect recrudescence in subgroups of patients classified by using threshold criteria for host age and parasite population size Age in years Classification Sensitivity Specificity Parasite density 1 <,000 1% % Any <,000 % % Any % % <,000 0% % Table b. Impact of selection criteria on the estimated risk of recrudescence by day. Selection criteria # correspond to WHO protocol recommendations for high transmission settings [1]. Criteria Recrudescence rate by day (% CI) Difference of risk estimates (% CI) Age in years Parasite density p value 1 < >,000-00,000 % (0%-%) - - < >,000-00,000 0% (%-%) % (1) (-%-1%) 0. <,000-00,000 0% (0%-0%) 1% (1) (%-%) 0.01 % () (-%-%) 0.1 (1) comparison to #1, () comparison to # Downloaded from on August, 01 by guest