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IAI Accepts, published online ahead of print on 17 February 2009 Infect. Immun. doi:10.1128/IAI.01358-08 Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

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Immunity to febrile malaria in children: an analysis that distinguishes immunity from lack of exposure. *Philip Bejon1,2, George Warimwe1, Claire L Mackintosh1, Margaret J Mackinnon1, Sam M Kinyanjui1, Jennifer N Musyoki1, Pete Bull1, Kevin Marsh1,2

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Kenyan Medical Research Institute (KEMRI) Centre for Geographic Medicine Research (coast) PO Box 230 Kilifi 80108 Kenya

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Nuffield Dept Medicine Centre for Clinical Vaccinology and Tropical Medicine University of Oxford Churchill Hospital Oxford, UK

Short Title: Distinguishing immunity from exposure.

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The corresponding author is Philip Bejon: [email protected], Tel.

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+44 770 4344 007

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Abstract

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In studies of immunity to malaria, the absence of febrile malaria is commonly

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considered evidence of “protection”. However, apparent “protection” may be due to

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lack of exposure to infective mosquito bites or due to immunity. We studied a cohort

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that was given curative anti-malarials before monitoring began, and documented

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newly acquired asymptomatic parasitemia and febrile malaria episodes during 3

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months surveillance.

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With increasing age, there was a shift away from febrile malaria to acquiring

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asymptomatic parasitemia, without changing the overall incidence of infection.

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Antibodies to the infected red cell surface were associated with acquiring

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asymptomatic infection rather than febrile malaria or remaining uninfected. Bednet

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use was associated with remaining uninfected rather than acquiring asymptomatic

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infection or febrile malaria.

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These observations suggest that most uninfected children were unexposed, rather than

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“immune”. Had they been immune, we would expect the proportion of uninfected

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children to rise with age, and to be distinguished from children with febrile malaria by

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the protective antibody response. We show that removing these less exposed children

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from conventional analyses clarifies the effects of immunity, transmission intensity,

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bednets and age. Observational studies and vaccine trials will have increased power if

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they differentiate between unexposed and immune children.

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Introduction

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Malaria is a pressing global health problem (36). The correlates of immunity in

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observational field-based studies are often used to guide vaccine design (22), in

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which the chosen definition of immunity to malaria is usually the absence of febrile

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malaria. However, the findings with this approach are often inconsistent, with

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responses to a specific antigen associating with protection in some studies but not

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others (4, 6, 7, 9-12, 23, 29). This may be because of parasite polymorphisms (38),

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because of a confounding association between protective and non-protective

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responses, because the endpoint of mild febrile malaria is not specific (26), or because

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rapidly waning antibody responses are not a stable predictive measure for the period

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of follow up (15).

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In studies in Kilifi, associations between specific antibody responses and protection

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were stronger in those children who had asymptomatic parasitemia at the start of

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monitoring (5, 16, 20, 28, 30, 31). This might imply premunition, where a chronic

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low-level infection is required to provide immunity against further infection (35), and

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antibody responses are more long-lived in the presence of asymptomatic parasitemia

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(1). Alternatively, antibody responses measured in the presence of challenge with

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asymptomatic parasitemia may be more informative than antibody responses

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measured without current exposure: For instance, protection against Hepatitis B is

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predicted by the antibody titer shortly after vaccination, even where antibody titers

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subsequently become undetectable (32). However, it may simply be that parasitemia

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reflects greater exposure to malaria, and hence greater power to detect associations.

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In this study, we cleared asymptomatic parasitemia with highly effective anti-

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malarials, in order to identify newly acquired parasitemia during follow up. We

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compared children acquiring asymptomatic parasitemia with those who developed

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febrile malaria, by examining the associations with known markers of exposure and

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immunity. We then examined what impact excluding “unexposed” children made on

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conventional survival analyses, in order to determine whether such analyses should be

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more widely used to study outcomes in observational studies or clinical trials.

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Methods

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Study Design

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The data presented here were generated during a randomised controlled trial of a

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candidate malaria vaccine. The details of study design are described elsewhere (3).

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Participants were aged 1-6 years old (inclusive), healthy, and resident in Junju

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sublocation, Kilifi District, Kenya. Vaccination had no effect on either the incidence

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of febrile episodes, prevalence of asymptomatic parasitemia, parasite density (3) or

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anti-VSA antibodies (p=0.57), and is not considered further here. Ethical approval

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was obtained from the Kenyan Medical Research Institute National Ethics

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Committee, the Central Oxford Research Ethics Committee, and the London School

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of Hygiene and Tropical Medicine Ethics Committee. Parents of all children were

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approached for informed consent before research began. Blood was taken for plasma

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and cross-sectional assessments of malaria parasitemia before all children were

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treated with antimalarials at the start of follow up, and again after 3 months.

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Drug treatment

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Immediately following the first cross-sectional bleed, curative anti-malarial treatment

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was given with 7 days of directly observed dihydroartemisinin monotherapy (2mg per

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kg on the first day, followed by 1mg per kg for 6 days). This regimen is highly

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effective when directly observed (18, 39), and parasite clearance was confirmed by

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slides taken 7 days after completing treatment.

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Follow up

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Children were visited every week by fieldworkers. When the temperature was above

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37.5 degrees, a blood film was made and a rapid near-patient test for malaria

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conducted.

When the mother reported fever, but the temperature was below 37.5

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degrees, blood films and rapid testing was not performed. Instead, the field worker

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returned to the child a further three times in the next 24 hours. Rapid testing and

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blood films were performed if the temperature was elevated on any of these visits.

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Parents could bring their children for assessment in between regular weekly visits if

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they thought the child had developed fever. Field workers were resident in the villages

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in which the study was conducted, and readily accessible to the parents. Treatment for

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episodes of malaria was with the Government of Kenya recommended first line

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treatment, artemether-lumefantrine. Cross-sectional blood films were performed on

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all children before clearing asymptomatic parasitemia, and once after 3 months of

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follow up.

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Bednet use was assessed by fieldworkers visiting the subject’s home at the beginning

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of the study. They obseerved whether the bednet was hung over the child’s sleeping

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space, asked about recent treatment, and examined the number of holes that could

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admit a fingertip. Children were considered net users if an untreated bednet had less

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than 3 holes that could admit a fingertip (25). Children without nets or with untreated

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nets with 3 or more holes were considered to be non-users.

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Laboratory studies

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The A4 parasite clone of Plasmodium falciparum was cultured in vitro to the mature

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trophozoite stage in blood group O erythrocytes. A4 was previously derived from the

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endothelial binding line ITO4, for CD36 and ICAM-1 binding (33). A4 was chosen

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because of the results of previous studies, showing that the responses measured

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correlated well with immunity to malaria (20). The major target of immunity to

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parasite derived antigen on the red cell surface antigen appears to be to VSA (6, 27),

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and flow cytometry based assays to detect this response are widely used (2, 14).

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Briefly, the infected erythrocyte suspension was adjusted to a 4% haematocrit in

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phosphate buffered saline (PBS) containing 0.5% bovine serum albumin (BSA) and

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Ethidium bromide (10µg/ml). 11.5µl of the suspension was incubated with 1µl of

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each individual’s plasma in 96-well U-bottomed plates (Falcon, Becton Dickinson,

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USA) for 30 minutes at room temperature. The cells were then washed three times

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with 0.5% BSA/PBS by centrifuging at 1000rpm for 3 minutes each time. 50ul of

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FITC-conjugated sheep anti-human IgG (Binding Site, UK) at a 1:50 dilution was

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then added to the cells and incubated for 30 minutes at room temperature. Following a

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further series of washes, 1000 infected erythrocytes were acquired on an FC500 flow

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cytometer (Beckman Coulter) and the proportion binding IgG determined using CXP

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analysis software (Beckman Coulter). Non-specific IgG binding was controlled for by

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subtracting the proportion of infected erythrocytes binding IgG in plasma from 8 non-

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exposed UK donors.

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Thick and thin blood smear was stained with 10% Giemsa and examined at x1000

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magnification for asexual forms of Plasmodium falciparum and results expressed per

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µl using an assumed white or red cell count. Films were read in duplicate, and by a

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third reader if one film was positive and the other negative or if the calculated

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densities differed by more than tenfold. The final result was a geometric mean of the

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only two or two closest readings.

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Categorisation of outcome

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Children were assigned one of three categories; febrile malaria (i.e. one or more

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episode of an axillary temperature greater than 37.5 degrees centigrade, with a

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Plasmodium falciparum parasitemia greater than 2,500 parasites per µl (26) during the

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three months follow up), acquiring asymptomatic infection (i.e. no detected episode of

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febrile malaria but any asymptomatic parasitemia at the second cross-sectional bleed

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after 3 months), or uninfected (no episode of febrile malaria was identified and the 3

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month cross-sectional sample showed no parasites). Children with episodes of

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parasitemia 2,500/µl) during the three months of follow up. 85 (22%) acquired

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asymptomatic parasitemia during the three months (without a febrile episode) and 208

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children (55%) remained uninfected. 22 (6%) had a febrile episode with less than

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2,500 parasites/µl and were excluded from further analysis. There were no significant

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differences among this excluded group for age (p=0.43), anti-VSA levels (p=0.61),

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village (p=0.61) or parasitemia at baseline (p=0.83).

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We studied this categorization of outcome by examining the effect of factors of

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known effect against malaria. We used age (known to be associated with immunity to

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malaria), a specific protective antibody response associated with protection (20),

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bednets (17), village and parasitemia on the first cross-sectional bleed (before

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treatment with anti-malarials). These data are displayed graphically in Figures 1, 2

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and 3. Age was associated with a shift away from febrile malaria to acquiring

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asymptomatic parasitemia, without changing the overall incidence of infection

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(Figure 1). Antibodies to the infected red cell surface were associated with acquiring

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asymptomatic infection rather than febrile malaria or remaining uninfected (Figure

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2). Bednet use was associated with remaining uninfected rather than acquiring

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asymptomatic infection or febrile malaria (Figure 2).

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We studied the strength of these associations and their significance by logistic

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regression models (Table 1). Increasing age was associated with a significant trend in

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odds ratios in favour of developing asymptomatic parasitemia rather than febrile

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malaria. However, age had no clear effect on the risk of febrile malaria compared to

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uninfected status. The anti-VSA antibodies acted similarly, with increasing antibody

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levels significantly favouring the development of asymptomatic parasitemia rather

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than febrile malaria, but not effecting febrile malaria compared with uninfected status.

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Bednet use was not associated with an altered risk of asymptomatic parasitemia

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compared with febrile malaria, but was associated with increased odds of remaining

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uninfected compared with either developing febrile disease (p=0.017) or with

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acquiring asymptomatic parasitemia (p=0.13).

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Hence, anti variant surface antibodies and age were negatively associated with

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developing febrile disease versus developing asymptomatic parasitemia, but not

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associated with developing febrile disease rather than remaining uninfected. This

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suggests that many uninfected children were simply unexposed. This is consistent

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with bednet use being associated with uninfected status, but not with asymptomatic

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parasitemia compared with febrile malaria. We therefore examined the impact that the

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less exposed group had on standard survival analyses of time to febrile malaria

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(Figure 4)

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Survival analyses

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Time to febrile disease increased with age (Hazard Ratio=0.49, 95%CI 0.3-0.8,

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p=0.007), but this difference was increased once the less exposed children were

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removed from the analysis (HR=0.36, 95%CI 0.2-0.6, p