Thesis: Community-based studies on the epidemiology, treatment and prevention of malaria in an area of intense malaria transmission in western Kenya

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1 Summary and discussion Thesis: Community-based studies on the epidemiology, treatment and prevention of malaria in an area of intense malaria transmission in western Kenya Anja Terlouw 16 October 2003 University of Amsterdam Promotor: Prof. dr. P.A. Kager, co-promotor Dr. F.O. ter Kuile Sickle cell haemoglobin In chapter 2 to 4 we presented several studies on the role of the sickle cell phenotype in the epidemiology and treatment of malaria and anaemia in young children in this area with intense malaria transmission. In chapter 2 we showed that sickle cell trait (HbAS) is associated with a strong protection against allcause mortality among children between the age of 2 to 16 months (protective efficacy = 55%). Although we did not have cause-specific mortality data, the finding that HbAS was associated with protection against severe acute malarial anaemia (defined as a combination of a high parasite density >10,000 parasites /µl and haemoglobin <6 g/dl), a major cause of mortality, and high-density parasitaemia, supported the hypothesis that the lower mortality risk associated with HbAS is likely due to its protection against malaria-related mortality. HbAS was also associated with protection against severe anaemia in the presence of lower parasitaemia levels in the same age period of 2 to16 months. This likely represents a similar protective effect against the more chronic state of anaemia, which has been associated with persistent low-grade parasitaemias and repeated episodes of mild malaria, and is thought to contribute substantially to mortality in this area. Our study also provided the opportunity to define the main time window of selection due to malaria in areas with intense perennial transmission and shows that the main burden of malaria occurs in the first 1.5 years of life. It is well recognized that infants in areas of stable malaria transmission have a reduced risk to the clinical consequences of P. falciparum infection during the first few months of life, regardless of their genetic make-up. Indeed, we found no evidence that HbAS children were at a relative advantage over children with the normal (HbAA) phenotype in the first two months of life. Various mechanisms such as the presence of foetal haemoglobin, passively acquired malaria-specific antibodies, dietary absence of p-amino benzoic acid and breast milk constituents such as lactoferrin have been proposed to account for this phenomenon. 1-3 Furthermore, the protection observed in sickle cell trait carriers was no longer apparent beyond 16 months, suggesting that children with the normal phenotype who survived up to this age, had acquired sufficient clinical immunity to be protected from severe malaria. This observation thus helps to

2 predict which age groups are most likely to benefit in terms of reduced mortality from antimalaria interventions such as insecticide treated bed nets. We also assessed the role of the sickle cell phenotype in the treatment of anaemia with iron supplementation in children 2 to 35 months of age (chapter 3). In contrast to previous observations in multigravid pregnant women in The Gambia, 4 we found no indication that the effect of iron supplementation on haematologic response was dependent on haemoglobin S phenotype. Mildly to moderately anaemic children with HbAS benefited as much from iron supplementation as did HbAA children, with haemoglobin levels in both groups increasing 0.8 g/dl and 0.9 g/dl, respectively, within 12 weeks of daily iron treatment. We found some indication that HbAS children receiving iron supplementation may experience higher parasite densities and a higher incidence of clinical malaria compared with HbAS children not receiving iron, but the number of clinical events was small, and these findings need further confirmation. Nevertheless, parasite densities and rates of clinical malaria in the HbAS children were generally low and never exceeded those in their HbAA peers. Thus, while the sickle cell trait phenotype may, or may not, modify the effect of iron on the risk of malaria, our data suggest that this should not be a constraint in prescribing iron, because the clear hematological benefits from iron supplementation in the treatment of anaemia are likely to outweigh any adverse effects caused by an increased risk of malaria in children with the sickle cell trait. Our genetic make-up may not only influence the effect of iron supplementation, but also may also affect the outcome of antimalarial treatment of uncomplicated infections. In chapter 4 we showed that the sickle cell phenotype is a determinant of sulfadoxine-pyrimethamine (SP) treatment outcome. In approximately 2700 treatments of uncomplicated falciparum malaria in children under the age of 5 years, SP was half as likely to fail for those with HbAS as for those with HbAA. Apart from age, sickle cell phenotype was the strongest determinant of treatment outcome in this study. The effect extended beyond the age interval of 2 to 16 months during which HbAS was found to offer protection against mortality. Children with sickle cell trait were older, less likely to have received SP in the previous month, had lower parasite densities and a better nutritional status. However, as only a small part of the observed difference between the two phenotypes could be attributed to these potential intermediate factors, we concluded that sickle cell trait enhances the efficacy of sulfadoxinepyrimethamine to clear P. falciparum parasites in young children with uncomplicated malaria. As this is also the group usually targeted for in vivo studies of antimalarial drug efficacy this means that in areas with a high population prevalence of sickle cell trait, true levels of drug resistance may be underestimated compared to those in areas where sickle cell trait is less common. Furthermore, the improved treatment efficacy conferred by HbAS may become more apparent with the emergence of higher levels of antimalarial drug resistance, which will result in more parasites escaping the static or killing effects of the drug, requiring that host factors provide a greater contribution to the radical

3 clearance of parasites. 5 An unequal distribution of HbAS by chance between treatment groups or over time in longitudinal monitoring studies might be an important source of bias, but may only be a problem in studies of small sample sizes. These results therefore suggest that the presence of haemoglobin S should be taken into account in studies that aim to determine factors that affect treatment outcome with sulfadoxine-pyrimethamine, and possibly also other antimalarials. Treatment with sulfadoxine-pyrimethamine In chapters 5 to 7 we further describe the longitudinal monitoring of SP efficacy over time. An increase in parasitological failure was observed within five years of its introduction as first-line treatment for uncomplicated falciparum malaria in young children in this study area (Chapter 6). The high failure risk is consistent with recent reports from a treatment trial in northeast Tanzania. 6 Although there was no evidence of increasing high-grade clinical resistance in our study, close monitoring in the Tanzanian study showed that two-thirds of children who failed to clear their parasites by Day-7 subsequently developed clinical malaria within 4 weeks. 6 Failure risk assessed by Day-7 is likely to underestimate the degree of resistance in the population, as with slowly eliminated drugs, such as SP, most recrudescences in areas with predominantly low-grade resistance occur more than 14 days after treatment. 7,8 This is the time period required to reach patent parasitaemia from low parasite numbers with multiplication rates being suppressed by host defenses and residual inhibitory concentrations of the drug. 9 In summary, these results raise concern about the longevity of SP as monotherapy in these settings. Could any of these failures have been prevented? In Chapter 5 we identified 2 potentially modifiable determinants of SP treatment failure, recent SP use, and low treatment dose, that combined explained approximately one-third of all observed treatment failures. Current SP treatment guidelines in Kenya recommend SP as the first-line drug for the treatment of infections without history of SP intake in the previous 14 days. 10 With slowly eliminating drugs most recrudescences in areas with predominantly low grade resistance occur more than 14 days after treatment as this is the time required to reach patent parasitaemia from low parasite numbers with multiplication rates being suppressed by host defenses and residual inhibitory concentrations of the drug. 7,8 Our results support this. Children who received SP within the previous month (15-35 days) had a markedly higher failure rate than those who had received SP more than 35 days ago, and this was particularly evident if SP had been used in the previous 14 to 21 days. Resistance to SP is related to specific point mutations to both dihydropteroate synthetase (DHPS) and dihydrofolate reductase (DHFR), which have been associated with varying degrees of SP resistance. 11 Selection of resistant mutant genotypes over sensitive genotypes may occur when parasites from the initially infecting population survive the antimalarial action of the drug, 7 particularly in young children with insufficient host-immunity to clear surviving

4 resistant parasites. 12,13 In the absence of molecular genotyping we could not distinguish between treatment failure and new infections. However, these results are consistent with the above mentioned study in Tanzanian children less than five years of age where treatment with SP within 14 to 28 days of the previous SP treatment resulted in a 1.4-fold higher failure risk due to selection of resistant genotypes over sensitive genotypes following treatment. 6,14 In this group an alternative antimalarial was needed to cure the infection. These results have important policy implications and suggest that SP should not be recommended for patients with a history of SP intake in the previous month because of the increased probability that these are recrudescent infections with a selected parasite population of resistant genotypes. Such selection and persistence of resistant strains will not only have clinical consequences for individual patients. It will also increase the risk of transmission and spread of resistant mutants in the population. Patients whose infections respond slowly to treatment or recrudesce subsequently are more likely to carry gametocytes than those patients who respond rapidly. This will increase the biomass of resistant genotypes in a population Similar to the considerations regarding the treatment effect in children with sickle cell trait, these results also have implications for surveillance of SP efficacy. When including patients with a recent treatment history and thus potentially with selected clones of resistant genotypes, the true level of resistance to that antimalarial in the population may be over-estimated because of selection. Furthermore, with increasing resistance, a higher percentage of the study population is likely to have been exposed to the study drug within the previous month. Thus, the history of SP in the previous month should either be added as an exclusion criterion or should be adjusted for in subsequent analyses. The current studies also suggested that a high proportion of treatment failures could be attributed to the relatively low treatment dose in mg per kg body weight received by most children. We were surprised to find that, with the agebased dose regimen used before April 1998, three-quarters of the children received less than the dose of 25/1.25 mg/kg S/P recommended by the WHO. Doses below 27.5/1.375 mg/kg S/P, the most discriminative threshold concentration associated with treatment failure identified, explained a quarter of parasitological treatment failures by day 7. Under-dosing is one of the few determinants of the rate at which antimalarial drug resistance develops that is amendable. To investigate the magnitude of this problem, we reviewed recent and current international age-based dose recommendations for SP for children <5 years old from WHO, UNICEF, and key paediatric infectious diseases, and tropical medicine textbooks. This indicated that important variation exists in agebased dose recommendations. More importantly, we found that most age-based dose regimens deployed in sub-saharan Africa result in a large proportion of under dosing in children <5 years. The preliminary findings from this study led the Kenyan Ministry of Health to revise their SP dose recommendations in A similar change in dose regimen was implemented in our study site in April While the dose increase markedly reduced the proportion of under-dosing, this did not result in a

5 decrease in treatment failures and the rate of annual increase in failure risk remained unchanged (chapter 6). We hypothesize that the low treatment dose used in the first five years of the study contributed to the speed at which the initial SP resistance could develop in this population. As resistance, once established, is unlikely to be susceptible to increases in doses, it is critical to ensure that treatment guidelines result in adequate doses at the initial deployment of antimalarials. To address the concerns regarding under-dosing with some of the commonly used age-based dose regimens, WHO requested our assistance to update their dose recommendations for SP in A database of the body weights by age of >70,000 pre-school children and >15,000 older children and adults from malaria endemic countries in sub-saharan Africa was established to help devise an age-based dose regimen that aimed to minimize the risk of over and under dosing (chapter 7). 19 This analysis also helped to identify a few key determinants of age-dose cut-offs, namely the width of the therapeutic range (defined as the maximum tolerated dose minus the minimum effective dose), the relative importance given to over dosing versus under dosing, the minimal tablet fraction, and the number of different tablet formulations. Our results imply that, for antimalarials with a relatively narrow therapeutic range, such as SP, agebased dosage regimens consisting of 9 or more age-dose categories and allowing quarter tablet fractions in children <5 years of age are predicted to result in 0.5% and 3.0% of patients to be over dosed and under dosed, respectively. Similar results can be obtained with an age-based regimen consisting of 4 agedose categories for artesunate due to its wider therapeutic-range. It was also found that while most antimalarial drug formulations use a single tablet strength, use of a paediatric and an adult tablet with a standard ratio in tablet strength (mg/tablet) of 1:4 could reduce the risk of over and under-dosing while improving the ease-of-use of regimens. Thus, this method, which used weightfor-age-reference data from malaria endemic countries, provides a valuable tool in optimizing existing recommendations or designing dose recommendations for new antimalarials. The method can be applied to antimalarials used as monotherapy or in combination with other antimalarials in fixed dose combinations. In conclusion, these results have implications for SP as well as for other antimalarials. First of all, they show that there is an urgent need to reevaluate the dosing guidelines for SP, because most existing age-based dosage recommendations result in considerable under dosing. This is particularly timely, as it is expected that several more African countries will replace chloroquine with SP, or combination therapy including SP, within the near future. Unsuccessful treatments increase the biomass of resistant genotypes in a population. As such, they increase transmission and potentially contribute to the rate at which SP resistance develops. Thus, the rate at which antimalarial drug resistance develops may be reduced by re-treating patients with a history of recent SP intake with an alternative antimalarial. Further, it is critical to ensure that treatment guidelines result in adequate doses at the initial deployment of

6 antimalarials when countries alter first-line treatment for non-severe falciparum malaria. Insecticide-treated bed nets In chapters 8, 9 and 10 we investigated the impact of a large reduction of malaria exposure on malaria, non-malarial morbidity and nutritional parameters in the Asembo area. These studies were all part of the large-scale western Kenya Insecticide-treated Bednet Trial, 20 which aimed to determine whether ITNs are efficacious in areas with intense perennial malaria transmission. Although ITNs have the potential to confer considerable health benefits, any intervention that substantially reduces malaria exposure may also have undesirable consequences by delaying the acquisition of clinical immunity against malaria. This has been a particular concern in areas of intense malaria transmission, such as our study area. To address the risks and benefits of ITNs in these circumstances, we studied their impact during pregnancy and in the first few years of life in both longitudinal and cross-sectional studies. In chapter 8 we reported the efficacy of ITNs when used throughout pregnancy. In the first four pregnancies, ITNs were associated with clinically significant reductions in parasitaemia during pregnancy as well as at the time of delivery. This resulted in improved maternal haemoglobin concentrations and a 28% reduction in the risk of low birth weight, mainly as a consequence of improved fetal growth. In gravidae 5 or greater, no beneficial impact on birth outcome was observed. As birth weight is associated with neonatal, post-neonatal, infant and child mortality and morbidity, these findings suggest that ITNs may be a valuable public health tool for the control of malaria in pregnancy. Care should be taken when predicting the impact of ITNs if distributed to individuals in the second or third trimester as part of a pre-natal care package. Our study was a group-randomized controlled trial with a very high population based coverage of ITNs, which resulted in mass killing of malaria transmitting mosquitoes and areawide reductions in malaria transmission ( community or mass effect). Furthermore, women were protected prior to, as well as throughout pregnancy. Our results, however, are consistent with that of two other recently published studies of ITNs in pregnancy, both conducted in similar areas with intense malaria transmission. One of these was a non-randomized study of socially marketed ITNs in southern Tanzania with a lower ITN coverage than ours. 25 The other study was a randomized controlled trial of ITN-use distributed as part of antenatal care in the second and third trimester. 26 These studies also found that a beneficial impact of ITNs extends beyond primigravidae and includes women of higher gravidity. These findings, together with the increasing evidence that in areas with a high prevalence of HIV all pregnancies are adversely affected by malaria, 27 add further weight to the argument that pregnant women, regardless of their parity, should be targeted for protection against malaria in highly endemic areas.

7 One of the attractive features of ITN distribution to pregnant women is that with continued use after delivery, the newborn will benefit from reduced malaria exposure in the first few months of life as shown in chapter 9. In this chapter we present the results of the impact of ITNs in a birth cohort of children born to the pregnant women referred to in chapter 8. ITNs were found to reduce malariaassociated morbidity in infants by 60%, regardless of birth order. The ITNs were equally as effective in the second year of life as during infancy. Thus, there was no evidence that, with continued use of properly re-treated bed nets, a 74% reduction in the force of infection in infancy was associated with an increased risk of malaria morbidity in one-year old children. ITNs were also associated with nutritional benefits as indicated by the improvements in mid-upper-arm circumference, weight, and length. While the weight gain benefit was only apparent in infancy, the beneficial effect on linear growth was maintained in the second year of life. Thus, apart from the reduction of malaria-associated morbidity, ITN use in infancy, the period of maximum annual growth velocity in life, could potentially result in sustained benefits, since the opportunity for catchup growth later in life is limited for children who remain in the same unfavorable environment. 28,29 In chapter 10 we describe the results of a series of cross-sectional surveys to measure the impact of ITNs on the prevalence of all-cause and malaria-specific morbidity, in children less than three years of age. Similar to our findings in infants, these cross-sectional study results indicate that ITNs substantially reduced malaria and malaria-associated morbidity. ITNs were also associated with a greater increase in mid-upper-arm circumference and body weight by age. The impact on nutritional status was not just restricted to children at risk, but also included children not classified as malnourished (i.e., with Z-scores -2), and add further evidence to the importance of malaria as a preventable cause of malnutrition. Overall, these results present a consistent picture. A properly implemented though imperfect intervention such as ITNs, which reduced exposure by approximately 74%, had considerable beneficial effects in pregnant women and their newborns. The study also showed that ITNs prevented approximately one in four infant deaths, as described elsewhere. 30 While these results helped define the target groups for ITN programs in areas of intense perennial malaria transmission as infants and pregnant women, other studies of this bed net trial suggested that this does not necessarily imply that ITNs should be distributed solely to these target groups. ITNs provide personal protection to individuals using them. For individuals to enjoy the optimal effect of ITNs, it was found that individuals should not only properly deploy their nets each night, and retreat them every 6 six months, but coverage in the surrounding households should also be high. 31 This dependency on coverage supports a growing body of evidence for the existence of a community-wide effect due to marked reduction of the infective mosquito population This implies that when all members of a population use ITNs, this would further improve the benefit for target groups. 23

8 Despite the clear beneficial effects of ITNs, it is also important to note the limitations of the studies. These findings are based on a limited follow-up period of 2 years. Although our results indicate no reduction in the ability of the immune system to mount antibody responses to certain antigens, 32 and show no evidence that a marked exposure reduction in infants, with continued ITN use, results in increased morbidity in one-year old children, 33 it is by no means clear what this implies for long-term malaria transmission reduction. Further investigations of the effect of four years of continuous use of properly implemented ITNs are currently under way to answer some of these questions. Optimal malaria control, is likely to consist of a combination of strategies. ITNs, although they have a substantial impact on malaria transmission, morbidity and mortality, will not constitute a stand-alone solution. Communities using ITNs will still have children infected with and falling sick from malaria. Prompt, effective and safe treatment for uncomplicated malaria will thus remain of vital importance to prevent the progression of an infection to severe illness, and effectively clear all parasites. 34 Currently, little is known about the interaction between malaria control tools. Future research should aim to clarify the relationship between different malaria interventions. For example, the development of antimalarial resistance may be slower in the presence of widespread use of other interventions such as ITNs by reducing the frequencies of sick children seeking treatment and therewith drug pressure in the population. It is also unclear whether a high coverage with ITNs alters the need for, or frequency of, intermittent preventative treatment of malaria in pregnant women and children, or whether these interventions will act synergistically. In conclusion, the studies presented in this thesis were part of large-scale community-based malaria research projects conducted by CDC/KEMRI that aimed to contribute to effective malaria control using currently available tools in this area of intense transmission. The results support the notion that we do have malaria interventions that are efficacious, feasible, and relatively affordable. 35 They also indicate that considerable public health gain can be achieved with their improved use and widespread application. ITNs have proved to be a valuable control tool over a wide diversity of transmission levels, and efforts are underway for their widespread introduction in sub-saharan Africa. Further, the current switch from chloroquine to more effective antimalarials, including the introduction of malaria combination therapy in sub-saharan Africa is a promising development that could, when properly implemented, have a major impact on the control of malaria in this continent. References 1. Sehgal VM, Siddjiqui WA, Alpers MP. A seroepidemiological study to evaluate the role of passive maternal immunity to malaria in infants. Trans R Soc Trop Med Hyg 1989; 83: Suppl: Edozian JC, Gilles HM, Udeozo IO. Adult and cord blood gammaglobulin and immunity to malaria in Nigerians. Lancet 1962; ii:951-5.

9 3. Maegraith BG, Deegan T, Sherwood JE. Suppression of malaria, Plasmodium berghei, by milk. BMJ 1952; ii: Menendez C, Todd J, Alonso PL, et al. The response to iron supplementation of pregnant women with the haemoglobin genotype AA or AS. Trans R Soc Trop Med Hyg 1995; 89: Cravo P, Culleton R, Hunt P, Walliker D, Mackinnon MJ. Antimalarial drugs clear resistant parasites from partially immune hosts. Antimicrob Agents Chemother 2001; 45: Mutabingwa T, Nzila A, Mberu E, et al. Chlorproguanil-dapsone for treatment of drug-resistant falciparum malaria in Tanzania. Lancet 2001; 358: White NJ. Why is it that antimalarial drug treatments do not always work? Ann Trop Med Parasitol 1998; 92: Nakazawa S, Kanbara H, Aikawa M. Plasmodium falciparum: recrudescence of parasites in culture. Exp Parasitol 1995; 81: White NJ. The assessment of antimalarial drug efficacy. Trends Parasitol 2002; 18: Kenya Ministry of Health. A simplified national guideline on malaria control for community resource persons. Nairobi: Ministry of Health, Nzila AM, Mberu EK, Sulo J, et al. Towards an understanding of the mechanism of pyrimethamine-sulfadoxine resistance in Plasmodium falciparum: genotyping of dihydrofolate reductase and dihydropteroate synthase of Kenyan parasites. Antimicrob Agents Chemother 2000; 44: Simpson JA, Watkins ER, Price RN, Aarons L, Kyle DE, White NJ. Mefloquine pharmacokinetic-pharmacodynamic models: implications for dosing and resistance. Antimicrob Agents Chemother 2000; 44: Kun JF, Lehman LG, Lell B, Schmidt-Ott R, Kremsner PG. Low-dose treatment with sulfadoxine-pyrimethamine combinations selects for drugresistant Plasmodium falciparum strains. Antimicrob Agents Chemother 1999; 43: Nzila AM, Nduati E, Mberu EK, et al. 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 2000; 181: Price RN, Nosten F, Luxemburger C, et al. Effects of artemisinin derivatives on malaria transmissibility. Lancet 1996; 347: Price R, Nosten F, Simpson JA, et al. Risk factors for gametocyte carriage in uncomplicated falciparum malaria. Am J Trop Med Hyg 1999; 60: Robert V, Molez JF, Trape JF. Short report: gametocytes, chloroquine pressure, and the relative parasite survival advantage of resistant strains of falciparum malaria in west Africa. Am J Trop Med Hyg 1996; 55: Kenya Ministry of Health. National Guidelines for Diagnosis, treatment & prevention of malaria for health workers. Nairobi, WHO. Drug resistance in malaria. Geneva: World Health Organization, Phillips-Howard PA, ter Kuile FO, Nahlen BL, et al. The efficacy of permethrin-treated bed nets on child mortality and morbidity in western Kenya

10 II. Study design and methods. Am J Trop Med Hyg 2003; 68: WHO. Physical Status: The Use of and Interpretation of Anthropometry. Geneva: World Health Organization, 1995: McCormick MC. The contribution of low birth weight to infant mortality and childhood morbidity. N Engl J Med 1985; 312: Medicine/National Io. Sciences Ao. Nutrition during pregnancy. Washington, DC: National Academy Press, Teberg AJ, Walther FJ, Pena IC. Mortality, morbidity, and outcome of the small-for-gestational-age infant. Seminars in perinatology 1988; 12: Marchant T, Schellenberg JA, Edgar T, et al. Socially marketed insecticidetreated nets improve malaria and anaemia in pregnancy in southern Tanzania. Trop Med Int Health 2002; 7: Njagi JK. The effects of sulfadoxine-pyrimethamine intermittent treatment and pyrethroid impregnated bed nets on malaria morbidity in pregnancy and birth weight in Bondo District, Kenya. PhD: University of Nairobi, van Eijk AM AJ, ter Kuile FO, Misore AO, Otieno JA, Rosen DH, Kager PA, Steketee RW, Nahlen BL. HIV increases the risk of malaria in women of all gravidities in Kisumu, Kenya. 2003; 17: Martorell R, Khan LK, Schroeder DG. Reversibility of stunting: epidemiological findings in children from developing countries. Eur J Clin Nutr 1994; 48 Suppl 1:S Golden MH. Is complete catch-up possible for stunted malnourished children? Eur J Clin Nutr 1994; 48 Suppl 1:S58-70; discussion S Phillips-Howard PA, Nahlen BL, Kolczak MS, et al. Efficacy of permethrintreated bed nets in the prevention of mortality in young children in an area of high perennial malaria transmission in western Kenya. Am J Trop Med Hyg 2003; 68: Hawley WA, Phillips-Howard PA, ter Kuile FO, et al. Community-wide effects of permethrin-treated bed nets on child mortality and malaria morbidity in western Kenya. Am J Trop Med Hyg 2003; 68: Kariuki SK, Lal AA, Terlouw DJ, et al. Effects of permethrin-treated bed nets on immunity to malaria in western Kenya II. Antibody responses in young children in an area of intense malaria transmission. Am J Trop Med Hyg 2003; 68: ter Kuile FO, Terlouw DJ, Kariuki SK, et al. Impact of permethrin-treated bed nets on malaria, anemia, and growth in infants in an area of intense perennial malaria transmission in western Kenya. Am J Trop Med Hyg 2003; 68: Hawley WA, ter Kuile FO, Nahlen BL, et al. Implications of the western Kenya permethrin-treated bed net study for policy, program implementation, and future research. Am J Trop Med Hyg 2003; 68: WHO. Africa malaria report Geneva: WHO, 2003.