The complex relationship of exposure to new Plasmodium infections and incidence of clinical malaria in Papua New Guinea

The molecular force of blood-stage infection (molFOB) is a quantitative surrogate metric for malaria transmission at population level and for exposure at individual level. Relationships between molFOB, parasite prevalence and clinical incidence were assessed in a treatment-to-reinfection cohort, where P.vivax (Pv) hypnozoites were eliminated in half the children by primaquine (PQ). Discounting relapses, children acquired equal numbers of new P. falciparum (Pf) and Pv blood-stage infections/year (Pf-molFOB = 0–18, Pv-molFOB = 0–23) resulting in comparable spatial and temporal patterns in incidence and prevalence of infections. Including relapses, Pv-molFOB increased >3 fold (relative to PQ-treated children) showing greater heterogeneity at individual (Pv-molFOB = 0–36) and village levels. Pf- and Pv-molFOB were strongly associated with clinical episode risk. Yearly Pf clinical incidence rate (IR = 0.28) was higher than for Pv (IR = 0.12) despite lower Pf-molFOB. These relationships between molFOB, clinical incidence and parasite prevalence reveal a comparable decline in Pf and Pv transmission that is normally hidden by the high burden of Pv relapses. Clinical trial registration: ClinicalTrials.gov NCT02143934


Introduction
Renewed emphasis on malaria control has resulted in substantial reductions in overall malaria prevalence and incidence in many endemic countries (World Health Organization, 2015). However, where transmission persists, it is highly heterogeneous even on small spatial scales (Bousema et al., 2012). Individual exposure is further influenced by factors such as use of bednets, attractiveness to mosquitoes, or behavioural differences. In Papua New Guinea (PNG), malaria prevalence has sharply declined in the last decade, largely as a result of two nationwide distributions of long-lasting insecticide treated bednets (LLIN) (Hetzel et al., 2015;Hetzel et al., 2014;Hetzel et al., 2012). P. vivax and P. falciparum PCR-prevalence in the general population was reduced from 32% and 39% in 2006 to 13% and 18% in 2010 (Koepfli et al., 2015). Already before this decline in malaria prevalence, studies in PNG had reported significant heterogeneity in malaria transmission attributed to local population structure and geographical diversity (Hetzel et al., 2015;Cattani et al., 1986;Genton et al., 1995;Müller et al., 2003;Mueller et al., 2009a).
Prior to the up-scaling of malaria control, P. vivax endemicity in PNG was among the highest worldwide (Hetzel et al., 2015). Clinical immunity to P. vivax was acquired very rapidly in PNG children, and the incidence of P. vivax clinical episodes peaked in children younger than two years with only very few P. vivax clinical episodes reported in children older than 5 years or adults (Genton et al., 2008;Michon et al., 2007;Lin et al., 2010;Betuela et al., 2012). In contrast, the risk for uncomplicated P. falciparum clinical episodes increased during early childhood (Lin et al., 2010) and significant reductions in incidence of clinical episodes or high-density infections were only observed in children aged 5 years and older (Michon et al., 2007). Compared to the incidence of clinical malaria, prevalence of P. falciparum and P. vivax peaked in older age groups, with asymptomatic infections remaining common until adulthood in PNG (Koepfli et al., 2015;Mueller et al., 2009a). Concordant with the species-specific pattern in the burden of clinical episodes, P. vivax prevalence peaked in younger age groups than P. falciparum prevalence (Koepfli et al., 2015;Mueller et al., 2009a).
As malaria transmission declines, it is important to understand the resulting changes in malaria prevalence and clinical incidence patterns, as well as the extent of heterogeneity in transmission within malaria endemic regions so that high-risk areas can be identified and targeted (Mosha et al., 2014). Most attempts to delineate high and low transmission areas made to-date, by both researchers and control programs, have used passive case surveillance or cross-sectional malaria indicator surveys. These surveillance strategies result in clinical incidence and prevalence estimates, both of which are surrogate markers for transmission. A more accurate understanding of the relationship between exposure to new infections and malaria prevalence or clinical incidence is needed to determine how accurately these surrogate markers represent heterogeneity in transmission at local scales. In addition, quantifying clinical incidence in relation to exposure to blood-stage eLife digest Malaria is caused by five different species of parasites that are transmitted to humans by bites from parasite-carrying mosquitos. Once in human blood, the parasites rapidly multiply. People who live in countries where malaria is common may become infected and never show any symptoms because their immune systems are able to keep parasite numbers low. Repeated infections, or infection with more than one species of malaria parasite also are common. Some species of malaria, including Plasmodium vivax, can hibernate in the liver for weeks or months after the infection and only become active later.
Asymptomatic infections, multi-parasite infections, and reactivating parasites make it hard to measure how often new malaria infections occur. One way scientists can determine if a new infection has occurred is by genotyping the parasites in a person's blood. Genotyping involves looking for small differences in the parasite DNA. For example, a study in Papua New Guinea, where P. vivax is very common, showed that reactivations of hibernating parasites were more common than new infections. Now, Hofmann et al. use the same study in Papua New Guinea to compare the frequency and consequences of new infections with P. vivax and another malaria parasite, Plasmodium falciparum. In the study, 466 children from 6 villages were followed for 8 months with tests every 2 to 4 weeks to genotype the parasites in their blood. Some of the children were treated with antimalarial drugs to help wipe out any existing parasites including hibernating ones. While P. vivax was about twice as common in blood samples-likely due to reactivation-genotyping showed that new infections with the two parasites occur at equal rates and often at the same times and locations.
Hofmann et al. also showed that some villages and some children had much higher rates of infection than others. This difference could not fully be explained by use of bednets or other preventive measures. Children were more likely to become ill from P. falciparum than P. vivax even though P. vivax was more common. But children with more frequent infections with P. falciparum seemed better able to manage the parasites and were less likely to develop symptoms that those with infrequent infections. The experiments show that genotyping may help scientists better track new malaria infections and develop better strategies to prevent or treat malaria.
infections can increase our insight into the development and maintenance of immunity to malaria in a setting of sustained malaria control Cameron et al., 2015).
The molecular force of blood-stage infection ( mol FOB) describes the number of new genotypes observed in consecutive blood samples from cohort participants over time Koepfli et al., 2013). Genotyping of highly polymorphic markers detects superinfecting parasite clones in asymptomatic (but parasitaemic) or symptomatic individuals. mol FOB thus provides a longitudinal, individual and quantitative measure for exposure to new blood-stage malaria infections Koepfli et al., 2013). For P. falciparum, mol FOB is closely linked to the number of infective mosquito bites and therefore is a direct proxy for the actual force of infection (FOI) and thus for transmission in endemic settings . For P. vivax, clones appearing in the blood-stream can either originate directly from an infective mosquito bite or from a relapsing liver hypnozoite (Koepfli et al., 2013). For P. vivax, mol FOB is thus a compound measure of exposure to newly acquired infections from mosquito bites and relapsing blood-stage infections.
The usefulness of mol FOB as a surrogate marker of individual exposure was validated originally in a cohort of young PNG children 1-4 years of age, in which species-specific mol FOB was the most important predictor of clinical incidence for both species Koepfli et al., 2013). Although some spatial heterogeneity of transmission was observed in that study for both species, due to the high level of transmission the species-specific difference in rate of immune acquisition was the predominant feature in that study. While P. vivax mol FOB (Pvmol FOB) did not change with age, the incidence of P. vivax clinical episodes decreased significantly with age, with a faster rate of decrease in children with high Pvmol FOB [12]. P. falciparum mol FOB (Pfmol FOB) in that cohort was lower compared to Pvmol FOB and the incidence of P. falciparum clinical episodes increased in parallel with an increasing Pfmol FOB in children 1-3 years, reaching a plateau thereafter (Lin et al., 2010;Mueller et al., 2012). These earlier results indicated that (i) immunity to P. vivax is acquired more rapidly in children with higher cumulative exposure, that (ii) this developing immunity led to proportionally fewer clinical P. vivax episodes in older children despite similar exposure to new P. vivax blood-stage infections, and that (iii) higher exposure to P. vivax blood-stage infections, compared to P. falciparum, resulted in a more advanced immunity to P. vivax in this age group compared to P. falciparum (Doolan et al., 2009;Longley et al., 2016). The major challenge to these cross-species comparisons lies within the intrinsic differences of Pf-and Pvmol FOB: whereas   Pfmol FOB is a direct marker of mosquito-borne transmission, Pvmol FOB is a composite measure reflecting both newly acquired infections and those caused by relapses of previously acquired infections.
In this study, we extend the analysis of mol FOB's relationship with incidence of clinical malaria episodes to older PNG children and a lower transmission scenario. In addition, given the unique study design that randomized blood-stage only or blood-plus liver-stage treatment at enrolment , we are now, for the first time, able to compare the incidence of newly acquired P. falciparum infections with both the incidence of newly acquired P. vivax infections and relapsing P. vivax infections. Advancing on previous studies that investigated each species individually Koepfli et al., 2013), we now provide a comparative analysis of P. falciparum and P. vivax, directly exploring the role of exposure to multiple Plasmodium species in the development of clinical immunity to P. falciparum and P. vivax malaria. We quantify in detail the extent of heterogeneity in mol FOB on a small geographical scale and relate this to heterogeneity in clinical episode incidence to investigate effects of small-scale variation in malaria transmission on local malaria epidemiology. By combining in-depth molecular parasitological data with demographic and clinical data, this study thus provides detailed insights into the changing epidemiology of malaria in PNG in response to intense malaria control efforts.

Demographic and parasitological parameters at enrolment
This study was conducted in six villages in Maprik district, East Sepik Province, PNG between August 2009 and May 2010 . 524 children aged 5-10 years were enrolled and randomized to receive either chloroquine (CQ), artemeter-lumefantrine (AL) and primaquine (PQ); or CQ, AL and placebo. Demographic parameters of the 466 children that completed the full course of randomized treatment with PQ/CQ/AL (n = 233) or placebo/CQ/AL (n = 233), and were thereafter closely followed for 8 months, were comparable between the six villages ( Table 1). P. vivax was the most common infection at enrolment with 48% of children positive by quantitative PCR (qPCR), followed by P. falciparum (24%), P. malariae (15%) and P. ovale (3%; Table 2). 39% of children were not infected with any Plasmodium species at enrolment. The vast majority of P. malariae (75%) and almost all P. ovale infections (93%) occurred in children co-infected with either P. vivax and/or P. falciparum ( Table 2). Prevalence of each Plasmodium species varied between villages (P. falciparum, 9-71%; P. vivax, 38-67%; P. malariae, 8-40%; P. ovale, 0-11%; Table 2) and was highest in Bolumita for all species. Accordingly, mixed-species infections were also most prevalent in Bolumita ( Table 2). The multiplicity of infection (MOI), that is, the number of parasite genotypes per infection, also varied between villages for both species (mean P. falciparum MOI, 1.1-2.2 clones/ infection; mean P. vivax MOI, 1.6-2.9 clones/infection) and children from Bolumita carried more multi-clone infections with P. vivax and P. falciparum than children in other villages ( Table 2). Mean P. falciparum parasite density was almost two-to six-fold higher in Bolumita (331 18S rRNA gene copies/ml) than in other villages (56-192 18S rRNA gene copies/ml, Table 2). mol FOB and parasite prevalence after randomized radical cure treatment Children who had received PQ for clearance of P. vivax hypnozoites experienced similar numbers of new blood-stage infections with P. falciparum and P. vivax during follow-up (mean Pfmol FOB = 1.5 CI 95 [1.3-1.7] new blood-stage clones/year, Pvmol FOB = 1.6 [1.4-1.9] new blood-stage clones/year, Figure 1A, Figure 1-figure supplement 1). Pfmol FOB in the placebo arm was comparable to the PQ arm (mean Pfmol FOB = 1.4 [1.2-1.6] new blood-stage clones/year), whereas due to the hypnozoite reservoir Pvmol FOB was more than three times higher in the placebo arm compared to the PQ arm (mean Pvmol FOB = 5.4 [4.9-5.8] new blood-stage clones/year). Pvmol FOB in the placebo arm showed a pronounced peak at months 2-3 of follow-up, which likely represents a wave of fast-relapsing hypnozoites in children who did not receive PQ ( Figure 1A).
P. vivax prevalence in the PQ arm was comparable to P. falciparum prevalence throughout the study and increased steadily, irrespective of the diagnostic method used ( Figure 1B and C). P. vivax prevalence increased more rapidly in the placebo arm until month 3 of follow-up and dropped thereafter, similar to patterns in Pvmol FOB in the same arm. Prevalence as measured by qPCR did not reach pre-treatment levels until the end of the study for any of the four Plasmodium species ( At the end of follow-up, P. vivax prevalence by qPCR in the placebo arm was 25% [19-31%], and therefore more than two-fold higher than in the PQ arm (9% [6-14%]; Figure 1B). Also, throughout follow-up, P. vivax prevalence in the placebo arm was 2-3 fold higher compared to the PQ arm, suggesting that at least 50% of the overall P. vivax prevalence in this cohort can be attributed to the contribution of relapses.
Similarly, throughout and at the end of follow-up P. vivax prevalence in the placebo arm was 2-3 fold higher compared to P. falciparum (irrespective of treatment arm; P. falciparum prevalence at Table 4. Multivariable predictors of Pv-and Pfmol FOB per follow-up interval.
Model predictions from this model were used for mapping mol FOB in Figure 3A.  , which is in agreement with the prevalence pattern at enrolment. Assuming equal transmission from mosquitoes for both species, which was corroborated by a comparable Pfmol FOB and Pvmol FOB in the PQ arm, P. vivax relapses have contributed to a P. vivax prevalence twice as high as that of P. falciparum.

Risk of recurrent blood-stage infections and mol FOB during follow-up
In the present study design, recurrent blood-stage infection can either originate from a new transmission event (both arms and all species) or for P. vivax and P. ovale also from a relapse of any previous infection (placebo arm only). After adjusting for the effect of PQ treatment , village of residence and infection status by qPCR at enrolment were the main predictors for the risk of recurrent Plasmodium spp. during follow-up (Table 3). Interestingly, in addition to a protective effect against recurrent P. vivax and P. ovale, the risk of recurrent P. falciparum was also reduced by 27% [0-48%] after PQ treatment (p=0.064).
The risk of a recurrent infection (measured by qPCR) with P. falciparum, P. vivax and P. ovale varied more than 7-fold between villages, with a higher risk observed in Bolumita (78%, 77%, and 15% with recurrent P. vivax, P. falciparum and P. ovale, respectively) compared to the other villages (recurrent P. vivax, range 25-73%; recurrent P. falciparum, range 12-44%; recurrent P. ovale, range 0-7%). For P. falciparum and P. vivax, a mixed infection at enrolment as measured by qPCR was further associated with up to a two-fold increased risk of recurrent infection (P. falciparum: AHR = 2.08 [1.25-3.48], p=0.005; P. vivax: AHR = 1.74 [1.14-2.65], p=0.010; Table 3), supporting the idea that focal transmission within villages leads to the presence of high-risk and low-risk individuals. For P. malariae, the infection status at enrolment was a stronger predictor of risk of recurrent infection than village of residence. An infection with P. falciparum, P. malariae or a mixed infection at enrolment as measured by qPCR was associated with up to a 6-fold increase in risk of recurrent P. malariae (AHR  Table 3).
The incidence of new P. falciparum and P. vivax blood-stage clones detected during follow-up, that is mol FOB, was highly variable between individual children and ranged from 0 to 18 new clones/ year for P. falciparum ( Figure 2A) and 0 to 36 or 23 new blood-stage clones/year for P. vivax in the placebo or PQ arm, respectively ( Figure 2B). Mean Pf-and Pvmol FOB varied significantly between villages and were higher in Bolumita (Pfmol FOB = 4.9 new blood-stage clones/year, Pvmol FOB PQ arm =4.4 new blood-stage clones/year, Pvmol FOB placebo arm =12.1 new blood-stage clones/year; Table 4; Figure 3) than in the other villages (Pfmol FOB, range 0.7-1.8 new blood-stage clones/year; Pvmol FOB PQ arm , range 0.03-2.2 new blood-stage clones/year; Pvmol FOB placebo arm , range 2.3-7.4 new blood-stage clones/year). In univariate analyses, new P. vivax infections were strongly associated with new P. falciparum infections per sampling interval and vice versa, suggesting concurrent exposure to the two species (Supplementary file 1 - Table 2). However, these effects were reduced when other variables of varying exposure such as village of residence or infection at enrolment were  Table 4). LLIN use, Table 5. Multivariable predictors for time to P. vivax and P. falciparum clinical episodes. Model predictions from this model were used for mapping the relative risk of clinical malaria episodes in Figure 3C and D.  P. vivax clinical episodes occurred mainly in the placebo arm shortly after directly observed treatment (DOT) , the time of peak Pvmol FOB due to relapsing hypnozoites ( Figure 1A). On an individual level, Pvmol FOB was positively associated with the risk of clinical episodes and each additional blood-stage P. vivax clone increased the risk of experiencing a P. vivax clinical episode slightly (AHR = 1.07 [1.04-1.09], p<0.001; Table 5). No significant differences in P. vivax clinical episode risk were observed between villages after adjusting for individual mol FOB. The risk for a P. vivax clinical episode decreased significantly with age (AHR = 0.62 [0.46-0.84], p=0.002; Table 5). This was paralleled by a decrease in P. vivax densities with age (by qPCR, exp(b)=0.90 [0.83-0.98], p=0.016; Supplementary file 3) indicative of more advanced immunity against P. vivax and thus better control of P. vivax densities in older children.
Patterns in the occurrence of P. falciparum clinical episodes during follow-up were more complex. Between-village variation in P. falciparum clinical episode risk remained significant even after adjusting for individual exposure. This effect was mainly apparent in Numangu, where children were at three-to six-fold higher risk for clinical episodes than children in other villages (Numangu AHR = 4.29 [2.06-8.97], other villages range AHR = 0.65 [0.20-2.08] to 1.39 [0.59-3.30]; Table 5). Overall, Pvmol FOB was positively associated with the risk of clinical episodes and each additional P. falciparum blood-stage clone slightly increased the risk for P. falciparum clinical episodes (AHR = 1.15 [1.11-1.21], p<0.001; Table 5); however, relative to the number of new blood-stage clones, P. falciparum clinical episodes were less frequent in highly exposed children compared to low-exposed children (Figure 4). One clinical episode per three new blood-stage clones was detected in the least exposed children (Pfmol FOB <4 new blood-stage clones/year), but only one clinical episode per 15 blood-stage clones in the highest exposed children (Pfmol FOB >9 new blood-stage clones/year, Fisher's exact test p<0.001). Age was not associated with the risk of P. falciparum clinical episodes. LLIN use at enrolment was associated with a 56% [13-78%] reduced risk of P. falciparum clinical episodes (p=0.018; Table 5).
A higher risk for P. falciparum clinical episodes in children that had received PQ treatment for clearance of P. vivax liver stages was observed (AHR = 1.79 [1.05-3.03], p=0.031; Table 5), suggesting a potential protective effect of P. vivax infections against P. falciparum clinical episodes. Analysis to further explore this revealed that a concurrent or recent infection (i.e., at the same or preceding follow-up visit) with P. vivax reduced the odds of a P. falciparum clinical episode by 65% [22-85%] (p=0.011; Table 6). Further indications for a potential interaction between the two species was also observed when analyzing P. falciparum parasite densities, which were reduced by 55% [19-75%] (p=0.008; Supplementary file 3) in mixed P. falciparum/P. vivax infections compared to P. falciparum single infections, indicative of suppression of one of the species in mixed infections.

Discussion
In the present study, we describe striking heterogeneity in malaria transmission not only between closely neighboring communities in Maprik district, PNG, but also substantial differences in exposure between individual children from the same village. On village level this heterogeneity is apparent both when using traditional markers such as prevalence of infection, as well as when using the novel 'reference standard' marker of individual exposure mol FOB. The increased resolution provided by mol-FOB further allows quantifying heterogeneity in exposure to new blood-stage infections between individual children. Extending an earlier study in a neighboring area in which younger children had been enrolled, and that had identified mol FOB as the most important predictor of malaria clinical episodes Koepfli et al., 2013), we confirmed that mol FOB remains significantly associated with the risk for clinical episodes, but other factors such as age (P. vivax), or a mixed Pf/ Pv infection and village factors not captured by any of the other parameters assessed (P. falciparum) have a stronger effect on the risk for clinical malaria ( Table 5 and 6).
Malaria transmission is often estimated by investigating the more accessible human host rather than the mosquito vector (Tusting et al., 2014). Because P. falciparum blood-stage infections are a direct outcome of mosquito-to-human transmission, infection parameters assessed in the human blood closely reflect P. falciparum transmission. In contrast, relapses arising from dormant hypnozoites contribute substantially to P. vivax blood-stage infections , thus complicating the assessment of mosquito-to-human P. vivax transmission via infection parameters measured in the human blood. The unique design of this study, that combined clearance of hypnozoites in half of the study participants with subsequent measurement of Pvmol FOB, allowed us to identify the burden of P. vivax infections due to mosquito-to-human transmission (in hypnozoitecleared individuals) and compare it to the total burden of P. vivax infections. We found a highly similar incidence and comparable temporal and spatial heterogeneity of P. falciparum and P. vivax infection acquired through renewed exposure to infected mosquito bites. In children that experienced the full burden of relapses we found two-fold higher P. vivax infection prevalence and 4-times higher incidence ( mol FOB) compared to P. falciparum. This first quantitative comparative assessment of P. falciparum and P. vivax transmission using non-entomological molecular parameters thus indicates that the observed differences in epidemiology between the two species are largely due to the high burden of relapsing P. vivax blood-stage infections . Our molecular results thus support recent entomological data from Dreikikir district, 50 km from Maprik in East Sepik Province (Reimer et al., 2016) as well as earlier studies in East Sepik (Hii et al., 2001) that found similar sporozoite rates for P. falciparum and P. vivax.
Our previous analysis of this cohort had investigated the contribution of the hypnozoite reservoir to P. vivax infection and disease in order to inform strategies for achieving a sustained reduction of the P. vivax burden in PNG . Here, we now describe the post-treatment reinfection dynamics in higher temporal detail. Through a detailed comparison of these patterns for P. vivax and P. falciparum in PQ and placebo-treated children we further elucidate the contribution of relapses to P. vivax prevalence and clinical incidence, which are the most commonly used parameters for planning and monitoring of malaria control strategies. P. vivax relapses accounted for more than half of the observed P. vivax prevalence in this cohort, which is lower than what was previously estimated as the contribution of relapses towards Pvmol FOB by comparison of treatment arms (77%, Robinson et al., 2015). This difference can be accounted for by the higher number of P. vivax multiple clone infections that will accumulate more rapidly in the placebo-arm, where additional parasite clones from relapses and/or new infections may overlap with without a corresponding change in overall prevalence.
While we previously described a sustained effect of PQ treatment with significant reductions in Pvmol FOB observed up to eight months post treatment , here, we describe temporal variation in relapse rate with a rapid and wave-like recurrence of P. vivax in children from the placebo arm, who had retained their hypnozoites ( Figure 1A). The concurrent, modest peaks in Pfmol FOB and Pvmol FOB in the PQ arm represent seasonal variation in transmission, which is highest in December and January in the study area ; corresponding to weeks 8-14 of follow-up). A much higher peak and subsequent drop in appearance of new clones within three months after blood-stage only treatment, which was mirrored by a corresponding peak and drop in P. vivax prevalence ( Figure 1B andC), suggests that the incidence of relapse infections in the blood was not constant during follow-up. P. vivax infections are often observed following treatment of P. falciparum malaria (Douglas et al., 2011), and it can thus been hypothesized that the frequency of relapses may be temporarily increased after blood-stage antimalarial treatment (White and Imwong, 2012). It is thus conceivable that the blood-stage antimalarial at baseline either triggered P. vivax relapses directly, or indirectly by allowing more hypnozoites to establish blood-stage infections in parasite-free hosts. Alternatively, blood-stage P. vivax infections from hypnozoites relapsing shortly after baseline treatment (during a period when antimalarial drugs were present at sub-curative levels) may be suppressed to sub-detectable densities until complete waning of drug levels, resulting in simultaneous proliferation and detection of many new blood-stage clones within the first weeks after treatment (Douglas et al., 2011;Tarning et al., 2014). More detailed modeling of the dynamics of individual P. vivax blood-stage infections and their association with potential triggers such as treatment or febrile illness will be required to determine the existence and importance of proposed relapse-triggers.
Malaria transmission showed high micro-spatial heterogeneity with more than 10-fold differences in Pf-and Pvmol FOB (in the PQ arm) between villages despite an overall high LLIN use by the study participants (during follow-up; village average use,>90%; individual use,>50%). Individual LLIN use at enrolment was nevertheless associated with a reduced risk of recurrent P. falciparum and P. vivax in univariate analyses. However, after adjustment for other related variables (i.e., village of residence or infection status) this association became non-significant. Children living in Bolumita, where both P. falciparum and P. vivax mol FOB and prevalence were highest, had a modestly lower LLIN use (mean during follow-up, 92%; at enrolment, 77%) compared to children from other villages (mean during follow-up, 97-100%; at enrolment, 91-100%). It is conceivable that LLIN use in the Bolumita community may be less effective in reducing malaria transmission (Killeen et al., 2007;Smith et al., 2009). Potential differences between villages in mosquito density, behavior, sporozoite rate, proximity of house or play areas to mosquito breeding sites, or human behavioral factors (related to LLIN use or other risk factors) are however likely to be more important determinants for exposure to infective bites. Small-scale variations in vector species and distribution between and within villages in PNG have been described previously (Cattani et al., 1986;Reimer et al., 2016;Charlwood et al., 1986;Hii et al., 1997;Burkot et al., 1988) and likely account in a large part for the micro-geographic heterogeneity in malariological parameters observed in this and other studies.
Assessing the incidence of new infections from consecutive blood samples using molecular methods (as is necessary to determine mol FOB), is complicated by fluctuating densities of clonal parasitemia that may temporarily fall below the limit of detection of the genotyping PCR, leading to imperfect detectability of clones (Bretscher et al., 2010;Felger et al., 2012;Koepfli et al., 2011). For P. falciparum, periodical sequestration of clones and absence from the peripheral blood at time of sampling may further contribute to imperfect detectability. For P. vivax, generally low parasite densities aggravate the problem of imperfect detectability, and dis-and re-appearance of clones may be a result of imperfect detectability or relapsing hypnozoites. The overall estimates of mol FOB presented here may thus be biased. Accurately assessing the effects of this imperfect detectability on parameters estimated from longitudinal genotyping data, such as mol FOB, requires complex mathematical modeling (Bretscher et al., 2010;Felger et al., 2012;Sama et al., 2005;Sama et al., 2006). However, although clonal detectability has been shown to decrease with age Sama et al., 2006) and MOI (Koepfli et al., 2011), it is unlikely to vary substantially within our cohort's age range and transmission setting. Hence the observed differences in mol FOB are likely to accurately reflect the relative differences in individual exposure as well as in population transmission levels within the study area.
Evaluating the impact of malaria control efforts requires monitoring changes in malariological metrics over extended periods of time. Drawing comparisons between studies performed at different times in different age groups is particularly challenging because of the interplay of past and current exposure to infective bites and the resulting anti-malarial immunity in the study population of a certain age. In our cohort, fewer P. vivax clinical episodes than P. falciparum clinical episodes were detected despite a higher incidence of P. vivax blood-stage infections, which is consistent with earlier studies in children of similar age (Michon et al., 2007). The very low incidence of clinical P. vivax episodes in our cohort, at 0.16 clinical episodes/year (placebo arm ), contrasts drastically with that of 2.46 P. vivax clinical episodes/year observed in an earlier observational cohort of younger children aged 1-4 years from the same area (Lin et al., 2010). The 3-fold difference in Pvmol FOB between the two cohorts seems modest when compared to the 15-fold difference in the incidence of clinical Pv episodes (Koepfli et al., 2013). This suggests that the much lower incidence of P. vivax clinical illness in 5-10 years old children of this study is more likely explained by an advanced state of immunity to P. vivax compared to the younger children of the earlier cohort than the drop in P. vivax transmission. Consistently, age emerged as the strongest factor associated with protection against P. vivax clinical episodes, Pvmol FOB and P. vivax parasite density. Like in the previous cohort of younger children from neighboring villages (Lin et al., 2010) the incidence clinical P. vivax clinical episodes dropped significantly with age. Unlike in the previous cohort of younger children (Koepfli et al., 2013), in this cohort we additionally observed a drop in Pvmol FOB as well as P. vivax densities with age (Table 4 and Supplementary file 3). This is a further indication of the substantial clinical immunity to P. vivax acquired during years of past exposure in the children of this study, which is still ongoing after the age of five.
In sharp contrast to the age-dependent decline of P. vivax clinical episode incidence, no agedependent decrease in the incidence of clinical episodes was observed for P. falciparum. In a previous cohort study conducted in 2004 in 5-14 year old children in an area from PNG with substantially higher transmission levels (mean incidence risk 5.0 versus 0.8 infections/year, Michon et al., 2007;Robinson et al., 2015), the risk of moderate-to high-density P. falciparum infections decreased significantly with age (Michon et al., 2007). Clinical immunity to P. falciparum in children of this earlier cohort was not only significantly further advanced compared to children of this cohort, but in addition, transmission was more homogeneous in the area of that study. As a consequence age was a much better marker of life-time exposure and thus immune status compared to the present cohort.
In this cohort, exposure to P. falciparum infections was highly heterogeneous between study participants. Mathematical modeling suggests that at heterogeneous transmission, changes of parasite prevalence and clinical episode incidence with age are less pronounced compared to settings with homogeneous transmission (Ross and Smith, 2010). Although no age trends were observed for P. falciparum in this cohort, when children were stratified into groups ranging from low to high exposure we found that the proportion of P. falciparum clinical episodes relative to new infections decreased with increasing exposure. This could either reflect the development of clinical immunity in highly exposed children, or premunition, a proposed mechanism by which established infections help to control superinfections by immunological cross-protection (Sergent and Parrot, 1935;Smith et al., 1999). In settings of decreasing and heterogeneous transmission, age alone may therefore not be a suitable marker of immunity to P. falciparum. Instead, combining age and mol FOB to estimate cumulative life-time exposure may provide a more accurate surrogate measure of the extent of acquired clinical immunity. With P. falciparum transmission declining in PNG due to successful malaria control strategies (Koepfli et al., 2015), it is conceivable that immunity against P. falciparum will develop more slowly, shifting the burden of disease towards older age groups or towards more complex, non-linear age patterns. This delay in immune acquisition is however more than compensated by the overall much lower incidence of clinical malaria clinical episodes: in cohort studies in children younger than 4 years from Maprik district, clinical P. falciparum incidence had dropped from 2.56 clinical episodes/year before (observational cohort, [Lin et al., 2010;Mueller et al., 2012]) to 0.67 clinical episodes/year immediately after the free LLIN distribution campaign (placebo arm, [Betuela et al., 2012]).
Finally, given that four Plasmodium species co-exist in PNG, there has long been considerable interest in potential mechanisms of cross-species immunity and mixed species interactions (Mueller et al., 2009a;Bruce et al., 2000;Smith et al., 2001;Mehlotra et al., 2000). However, there is so far no consistent evidence for the presence or absence of cross-protection among Plasmodium species. P. vivax and P. falciparum infections in our study were concentrated in the same children and villages (Figure 3), likely due to overlapping focal transmission for P. falciparum and P. vivax and thus potentially high co-infection rates in mosquitoes. Contrary to an earlier cohort in younger PNG children that found a decreased risk of P. falciparum clinical episodes after PQ radical cure (Betuela et al., 2012), we found indications for an increased risk of P. falciparum illness after clearance of P. vivax hypnozoites using PQ. Our data suggests that in individuals with substantial clinical immunity against P. vivax, a concurrent P. vivax infection may provide protection against P. falciparum clinical episodes by limiting P. falciparum densities ( Table 6, Supplementary file 3). However, the comparably small number of clinical episodes in this and the earlier contrasting study does not allow an in-depth analysis of causal relationships and therefore does not allow firm conclusions on the potential effects and mechanisms of cross-species interactions in mixed infections.
In conclusion, this study provides detailed insight into the changing epidemiology of malaria in PNG children under sustained malaria control, by using mol FOB as a powerful measure to quantitatively investigate patterns of new mosquito-derived P. falciparum and P. vivax infections versus those for P. vivax relapsing infections, as well as spatial and age trends in exposure to these infections. Striking heterogeneity in malaria transmission between villages as well as in individual exposure to new P. falciparum and P. vivax infections persisted in our study area despite very high use of LLINs. This presents a significant challenge for on-going malaria control efforts. The comparable patterns of new mosquito-derived P. falciparum and P. vivax infections indicate that sustained use of LLINs does result in a comparable reduction in transmission of both species. The higher incidence and prevalence of P. vivax infections observed in our data is thus directly linked to its ability to cause relapsing infections, highlighting the crucial role of hypnozoites for P. vivax epidemiology and the need to effectively intervene against these hidden stages. Together, these insights provide a crucial link to evaluate the level of P. vivax mosquito-based transmission against that of P. falciparum and serve to calibrate other standard malaria indicators such as parasite prevalence or incidence of clinical episodes and to ultimately inform new approaches to surveillance and response systems.

Study design and participants
This study was conducted in six villages in the Albinama and Balif areas, Maprik district, East Sepik Province, PNG between August 2009 and May 2010. The area is serviced by the Albinama health sub-center, Balif aid post and a network of health workers in all study villages. The study design has been described in detail elsewhere . Briefly, 524 children aged 5-10 years whose parents provided written informed consent for their participation were enrolled and randomized to receive either chloroquine (CQ, days 1-3, total dose 25 mg/kg), artemeter-lumefantrine (Coartem, AL, days 11-13, 2 mg/kg A, 12 mg/kg L) and primaquine (PQ, days 1-20, 0.5 mg/kg/day); or CQ (days 1-3), AL (days 11-13), and placebo (days 1-20) over 20 days of directly observed treatment (DOT 1-20 ) in a double-blinded manner. Children were actively visited and examined for signs and symptoms of malaria fortnightly at their schools for 8 months. In addition, passive surveillance was provided by the local health centre, aid post and village health workers throughout the study period. Finger-prick blood samples (250 ml) were collected at fortnightly active-follow-up visits in the first 12 weeks and monthly thereafter, as well as from symptomatic children detected during active or passive morbidity surveillance. Symptomatic children were tested for malaria infection with rapid diagnostic test (RDT, CareStartMalaria pLDH/HRP2 Combo, AccessBio, USA), and only RDT and or LM-confirmed Plasmodium infections of any density were treated with a 3 day course of AL.
Household, village and health facility location data was collected using a handheld GPS receiver (Garmin GPSmap62sc) and maps were prepared using ArcGIS 10.2 (Esri, USA).
The study received ethical clearance from the PNG IMR Institutional Review Board (0908), the PNG Medical Advisory Committee (09.11), the Ethics Committee of Basel 237/11 and was conducted in full concordance with the Declaration of Helsinki. The study was registered on Clinical-Trials.gov (NCT02143934).

Laboratory methods
All blood samples were examined by LM and qPCR for detection and speciation of Plasmodium infections as described earlier . Each blood slide was read independently by two skilled microscopists and re-read by an expert microscopist in case of discrepancies in positivity, speciation or density (2 x log 10 difference). Thick blood films were examined by LM for 200 fields (1000x magnification) before being declared parasite-negative. Parasite density was converted from the number of parasites per 200-500 white blood cells (WBC) to parasites/ml assuming 8000 WBC/ml (WHO malaria microscopy training guide) and calculated as the geometric mean of all positive reads.
DNA was extracted from the red blood cell pellet using the FavorPrep 96-well genomic DNA extraction kit (Favorgen). Samples carrying any Plasmodium spp. infection were identified using a generic qPCR (Wampfler et al., 2013) and positives were subsequently tested in species-specific qPCRs (Rosanas-Urgell et al., 2010;Wampfler et al., 2013). All qPCRs targeted the small subunit (18S) ribosomal RNA gene and were performed as simplex (P. vivax and P. falciparum) or duplex qPCR (P. malariae, P. ovale). The concentration of target copies per ml of DNA was determined relative to a dilution row of standard plasmid as previously described (Rosanas-Urgell et al., 2010). The qPCR limit of detection (LOD) was determined using a standard plasmid dilution row and defined as the last point with more than 50% of replicates positive. The LOD was 2 target copies/ml DNA, equaling 4 target copies/reaction, for all qPCRs. All samples that crossed the fluorescence threshold were scored as positive for species-specific qPCRs. In all samples positive in P. falciparum and/or P. vivax qPCRs, individual parasite clones were distinguished by genotyping the length-polymorphic Pf-msp2 or Pv-msp1F3 marker genes using capillary electrophoresis for highly precise fragment sizing (Koepfli et al., 2013;Koepfli et al., 2011;Falk et al., 2006;Schoepflin et al., 2009). MOI was determined by counting the number of detected Pf-msp2 or Pv-msp1F3 alleles per sample. mol FOB was calculated from the number of new parasite clones detected per child or per sampling interval in the peripheral blood, divided by the individual time at risk or length of the interval. A new infection was defined as a Pf-msp2 or Pv-msp1F3 allele not present in the two preceding genotypingpositive samples collected during active or passive surveillance (Figure 1-figure supplement 1). Imperfect diagnostic detectability was not further adjusted for.

Statistical analysis
Children were considered at risk for clinical malaria clinical episodes until the end of the study or until they were censored (on the last visit before two consecutively missed scheduled follow-up visits ). For clinical endpoints, time-at-risk (TAR) was not further adjusted for interim missed follow-up visits because the intense active and passive case detection presumably led to detection of all malaria clinical episodes. In contrast, TAR for analysis of molecular data (e.g., mol-FOB) was reduced by the duration of the missed interval if a child was not seen by the study team for six weeks or more (42 days). Children with a TAR of less than 3 months (<84 days) were excluded. This resulted in an analyzed population of 466 children (characterized in Table 1) of which 430 (92.3%) completed the whole follow-up period, with a median of 15 (IQR: 13-17) study contacts and mean TAR of 186 days (IQR 168-223 days).
Time to first Plasmodium infection by qPCR and LM and its association with covariates were modeled using Cox regression, and the proportional hazards assumption was checked using the test based on the Schoenfeld residuals. Multiple failure Cox regression was used to model the time to P. vivax and P. falciparum clinical episodes.
For statistical analysis, a malaria clinical episode was defined as fever (>37.5˚C axillary) plus the presence of LM-detectable parasites, irrespective of RDT result or antimalarial treatment during the field visit. Negative binomial generalized estimating equations (GEE) with log link function using an exchangeable correlation structure were used to model incidence of new infections with P. falciparum and P. vivax per sampling interval. For these analyses, the time at risk was restricted to the intervals where mol FOB could be estimated (i.e., starting in the third follow-up interval). A binomial GEE with logit link function using an exchangeable correlation structure was used to model the odds of a P. falciparum clinical episode per interval. Gaussian GEEs with log link function using an exchangeable correlation matrix were used to model log-transformed qPCR parasite densities in qPCR-positive samples, measured as 18S rRNA copy numbers/ml blood. In the GEE and Cox models where mol FOB was a covariate, it was included as a time-varying covariate. When modelling mol FOB ( Table 4) and the odds of clinical episodes (Table 6) using GEEs, mol FOB was calculated for each follow-up interval and used as predictor. In Cox models investigating the risk of clinical episodes (Table 5), mol FOB was calculated based on the new infections up to the time of failure and used as predictor. In exploratory preliminary analyses we tested for a wide variety of interactions between covariates including interactions between all combinations of mol FOB, enrolment infection status, age, village and bednet-usage. All analyses were done using STATA v14 and R.
Maps were drawn using Arcgis 10.1 (Esri Inc.). Ordinary kriging was used to generate the contour maps. Semivariograms were used as the mathematical forms used to express autocorrelation. Input variables for the spatial models were (i) mol FOB (prediction of independent variable, mol FOB) using the model presented in Table 4 (negative binomial GEE) for Pf and Supplementary file 4 for Pv (same as that shown in Table 4 but with primaquine and placebo arms combined), resulting in Figure 3 Panels A and B; (ii) relative risk of clinical episodes as predicted by the model shown in Table 5, resulting in Panels C and D of Figure 3. Relative standard error maps were generated by dividing the absolute standard error map by the model prediction map.
#1052760. IM is supported by an NHMRC Senior Research Fellowship (#1043345). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
was conducted in full concordance with the Declaration of Helsinki. Written informed consent was obtained from the parents/guardians of all children enrolled in the study. . Supplementary file 4. Multivariable predictors of Pvmol FOB (combining primaquine and placebo arms) per follow-up interval. This model is similar to that presented in Table 4 in the main text but combines the treatment arms for P. vivax. Model predictions from this model were used for mapping molFOI in Figure 3B.

Major datasets
The following dataset was generated: