Spatial cluster analysis of Plasmodium vivax and P. malariae exposure using serological data among Haitian school children sampled between 2014 and 2016

Background Estimation of malaria prevalence in very low transmission settings is difficult by even the most advanced diagnostic tests. Antibodies against malaria antigens provide an indicator of active or past exposure to these parasites. The prominent malaria species within Haiti is Plasmodium falciparum, but P. vivax and P. malariae infections are also known to be endemic. Methodology/Principal findings From 2014–2016, 28,681 Haitian children were enrolled in school-based serosurveys and were asked to provide a blood sample for detection of antibodies against multiple infectious diseases. IgG against the P. falciparum, P. vivax, and P. malariae merozoite surface protein 19kD subunit (MSP119) antigens was detected by a multiplex bead assay (MBA). A subset of samples was also tested for Plasmodium DNA by PCR assays, and for Plasmodium antigens by a multiplex antigen detection assay. Geospatial clustering of high seroprevalence areas for P. vivax and P. malariae antigens was assessed by both Ripley’s K-function and Kulldorff’s spatial scan statistic. Of 21,719 children enrolled in 680 schools in Haiti who provided samples to assay for IgG against PmMSP119, 278 (1.27%) were seropositive. Of 24,559 children enrolled in 788 schools providing samples for PvMSP119 serology, 113 (0.46%) were seropositive. Two significant clusters of seropositivity were identified throughout the country for P. malariae exposure, and two identified for P. vivax. No samples were found to be positive for Plasmodium DNA or antigens. Conclusions/Significance From school-based surveys conducted from 2014 to 2016, very few Haitian children had evidence of exposure to P. vivax or P. malariae, with no children testing positive for active infection. Spatial scan statistics identified non-overlapping areas of the country with higher seroprevalence for these two malarias. Serological data provides useful information of exposure to very low endemic malaria species in a population that is unlikely to present to clinics with symptomatic infections.


Introduction
Identification of Plasmodium reservoirs in low transmission settings relies on conventional diagnostics (microscopy and RDT) which underestimate the true incidence and prevalence [1,2]. When compared to microscopy and RDTs, antibody-based assays are a more powerful method for detection of active infection or past exposure to a Plasmodium spp. by expanding the time window for diagnosis. Although there is generally an overlap between active infection and exposure data [3,4], attaining exposure data through serology explores malaria transmission by approximating past malaria infections. Antibodies produced by activated B-cells have been identified for all four stages of the human malaria lifecycle (sporozoite, liver stage, blood stage, and sexual stage) [5][6][7], though the highest titers are elicited to blood-stage antigens [8]. IgG antibodies against the merozoite surface protein 1 (MSP1) antigens produced by different human malarias have been modeled to show half-lives in systemic circulation for years to decades, though this has been studied much more extensively for PfMSP1 compared to the other non-falciparum isoforms [9][10][11]. Recent advances in assay development have allowed serological data to be collected simultaneously in a multiplex format, making data collection for multiple Plasmodium spp. more feasible [5].
As a result of efforts in mass-microscopy to assist malaria elimination efforts in Haiti in the 1960s, three species of Plasmodia were identified in the country (P. falciparum, P. vivax, P. malariae), with P. falciparum single-species infections accounting for approximately 97% of all persons with parasitemia [12][13][14]. Though great success was seen in reducing parasite prevalence in the population through intensive vector control and mass-drug administration campaigns, the elimination program was suspended in the late 1960s with all three species still found throughout the country [13]. Since that time, only a handful of published reports and studies have documented the presence of P. vivax or P. malariae in Haiti [15][16][17]. Additionally, after the 2010 earthquake in Haiti, the large-scale deployment of histidine-rich protein 2 (HRP2)-based rapid diagnostic tests (RDTs) led to further under-diagnosis of P. vivax and P. malariae since these tests only identify P. falciparum infections, unless microscopy is also being utilized [18,19]. We present here malaria serological data from school-based surveys from 2014-2016 in Haiti. A multiplex bead assay (MBA) was used to collect IgG data from samples collected in these surveys to assess the seroprevalence of anti-Plasmodium MSP1 antibodies with specific focus on PvMSP1 and PmMSP1 responses and exploratory spatial data investigation of potential seroprevalence clustering in Haiti. This study aims to utilize IgG data to determine spatial transmission patterns for the very low endemic malaria species of P. malariae and P. vivax in Haiti.

Human subjects
Samples were collected from 2014 to 2016 as part of lymphatic filiariasis (LF) transmission assessment surveys (TASs) in Haiti, with integration of malaria RDTs and microscopy for soiltransmitted helminths in stool specimens [20]. Fingerprick blood was collected on filter papers (TropBio filter wheels, Cellabs, Sydney, Australia), dried (creating a dried blood spot, DBS), and packaged individually with desiccant for later laboratory analysis at the Centers for Disease Control and Prevention in Atlanta, GA. This activity was considered a program evaluation activity by CDC Human Subjects Office (#2014-256). Persons consented to future laboratory testing of DBS, and CDC laboratory staff did not have access to any personal identifiers.

Survey design
Surveys were conducted in evaluation units (EUs) that had met World Health Organization (WHO) criteria to conduct LF TAS, with the current WHO recommendation to conduct a school-based TAS in areas where the net primary-school enrollment rate is �75%. Haitian school enrollment data for 2014 was utilized along with population census data to determine sampling approach employed in each EU, which are program defined and dependent on baseline LF prevalence found during initial mapping surveys [21]. Using the tables provided by WHO [22], the target sample size and the critical cut-off threshold was determined, and if the number of positive children identified fell below the critical cut-off threshold, the recommendation was made to stop mass-drug administration (MDA) for LF. Upon completion of MDA, TAS surveys were conducted to determine whether populations have reached the critical threshold of infection prevalence (<2% antigenemia), below which LF transmission is likely no longer sustainable [20]. At each school, all children in second grade (overwhelmingly aged 6 or 7 years, Tables 1 and 2) were asked to participate.
All IgG assay reagents were diluted in buffer containing PBS, 0.05% Tween20, 0.5% bovine serum albumin (SigmaAldrich), and 0.02% NaN 3 . Hyperimmune positive and negative controls were included on each IgG detection plate to ensure appropriate assay performance. A bead mix was prepared so approximately 1,000 beads/region would be in each assay well. Samples (50 μL of 1:200 dilution whole blood) were incubated with beads for 90 min at room temperature (25˚C) under gentle shaking protected from light in MultiScreen-BV filter plates (SigmaAldrich). After three washes (wash buffer: PBS, 0.05% Tween 20), beads were incubated with 50 μL biotinylated detection antibody (a mixture of 1:500 anti-hIgG and 1:625 anti-hIgG 4 , both produced by Southern Biotech, Birmingham, AL) for 45 min with same incubation conditions as above. After three washes, 50 μL streptavidin-phycoerythrin (Invitrogen, Waltham, MA) was added to all wells (1:250x of 1 mg/mL) for a 30 min incubation. After three washes, samples beads were incubated with 50 μL reagent buffer for 30 min, washed once, and resuspended in 100 μL PBS. Assay plates were briefly shaken and read on a Bio-Plex 200 machine (BioRad) by generating the median fluorescence intensity (MFI) for 50 beads. The final measure, denoted as MFI minus background (MFI-bg), was reported by subtracting MFI values of beads on each plate only exposed to sample diluent during the sample incubation step. The MFI-bg threshold for true positive IgG assay signal against Plasmodium antigens was ascertained if the sample MFI-bg was higher than the mean + 3SD of the MFI-bg signal of a panel of 92 known anti-malaria IgG negative DBS samples. The testing of TAS samples was already underway before the PmMSP1 19 antigen was available for use. For this reason, the first two EUs consisting of the Sud-Est and Nippes departments in southern Haiti are lacking PmMSP1 19 IgG data.
Sample selection for detection of active Plasmodium infections. Persons (and animals) with active P. malariae or P. vivax infections typically show high IgG titers to the MSP1 19 antigens produced by those malarias [23][24][25][26][27]. These same studies have also shown high specificity among Plasmodium MSP1 19 isoforms, meaning infection with one malaria species will typically produce IgG with homologous binding to only that MSP1 19 isoform. With these two factors considered, samples with the highest IgG assay signals to the PmMSP1 19 or PvMSP1 19 antigens were selected for malaria antigen and PCR assays in an attempt to identify active infections to these malarias. For these additional lab assays, initial target sample selection was approximately 25 of the highest IgG signals for each antigen, and if active infections were found, the next 25 highest responders would also be selected for antigen detection and PCRs.
Bead-based malaria antigen detection. For detection of Plasmodium antigens, three separate bead regions (Bio-Plex non-magnetic beads, BioRad) were coupled by the EDC/Sulfo-NHS intermediate reaction with antibodies against antigen targets: pan-Plasmodium aldolase (12.5μg/12.5x10 6 beads, rabbit IgG anti-aldolase, Abcam, Cambridge, UK), histidine-rich protein 2 (HRP2; 20μg/12.5x10 6 beads, mouse anti-P. falciparum, Abcam), pan-Plasmodium lactate dehydrogenase (pLDH; 12.5ug/12.5x10 6 beads, mouse IgG anti-LDH, BBI Solutions, Cardiff, UK). All detection antibodies were previously biotinylated by Thermo Scientific EZ-Link Micro Sulfo-NHS-Biotinylation Kit (ThermoFisher Scientific) according to the manufacturer's protocol. All antigen detection reagents were diluted in buffer containing PBS, 0.05% Tween20, 0.5% bovine serum albumin (SigmaAldrich), and 0.02% NaN 3 . Recombinant antigen positive and negative blood controls were included on each antigen detection plate to ensure appropriate assay performance. A bead mix was prepared so approximately 800 beads/region would be in each assay well. Bead mix was pipetted into MultiScreen-BV plates, washed twice with wash buffer, and incubated for 90min with 50μL sample (at 1:20 dilution). After three washes, beads were incubated for 45min with a 50uL mix of detection antibodies (1:1000 anti-aldolase [Abcam], 1:500x of 2:1:1 mixture [BBI Solutions BM355-P4A2:BioRad Pv-pLDH HCA156: BioRad Pf-pLDH HCA158]), and 1:500 anti-HRP2 [Abcam]. Following three washes, 50 μL streptavidin-phycoerythrin (Invitrogen,) was added to all wells (1:250x of 1 mg/mL) for a 30 min incubation. After three washes, sample beads were incubated with 50 μL reagent buffer for 30 min, washed once, and resuspended in 100 μL PBS. Assay plates were shaken briefly and read on a Bio-Plex 200 machine (Bio-Rad) by generating the median fluorescence intensity (MFI) for 50 beads. The final measure, denoted as MFI-bg, was reported by subtracting MFI values from beads on each plate only exposed to sample diluent during the sample incubation step. The MFI-bg threshold for a true positive assay signal was the mean + 3SD of the MFI-bg signal of a panel of known malaria antigen negative DBS samples.
DNA extraction and Photoelectron-induced Electron Transfer (PET) PCR. DNA was extracted from DBS by using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) as recommended by the manufacturer. Briefly, a DBS tab (equivalent to 10 μL whole blood) was placed into a 1.5 mL tube and processed according to instructions. The DNA was eluted in 150 μL of elution buffer and stored at -20˚C until use. Real-time PCR reactions for Plasmodium DNA were carried out as described previously using the multiplex PET-PCR assay as previously described [28], with positive and negative controls included on each PCR plate.

Exploratory spatial analysis
All data cleaning were performed using SAS (Version 9.4). Geocoordinates were available for all schools enrolled in the TAS. Administrative boundaries for Haitian departments were obtained from geoBoundries (https://www.geoboundaries.org/), a service produced by the Haiti is a country with total surface area of 27,750 km 2 . School latitude and longitude coordinates were geocoded and converted from World Geodetic System 1984 into Universal Transverse Mercator Zone 18 projections using ArcMap (Version 10.5.1). Point pattern spatial randomness measures were calculated using Ripley's K-function through ArcMap. Ripley's Kfunction quantifies how often events are found within a certain distance of one another and the level of aggregation, randomness, or dispersion within the boundaries of a set of observations [29][30][31]. If observations are not spatially random, it would suggest evidence of biological, environmental, or sampling association with the clustering of cases. K-function permutations allow for confidence interval calculation around an expected random distribution [31]. Kfunctions in this study was calculated using a maximum radius of 50 km with 2.5 km intervals. Weighted K-function was calculated using seropositive counts per school with the same parameters. Confidence intervals were calculated using 999 permutations for weighted and unweighted K-functions. Autocorrelation was calculated using Moran's I in ArcMap. Autocorrelation measures such as Moran's I allow for the quantification of the degree to which values spatially correlate to similar values [31]. If there is no spatial autocorrelation, Moran's I is assumed to equal zero, whereas values above zero indicate autocorrelation of data values among the locations. Moran's I was calculated using inverse distance weights, 999 permutations, and a threshold distance of 20 km. Cluster analysis was performed by Kulldorff's spatial scan statistic using Satscan (Version 9.6) to identify clusters with an increased relative risk of malaria exposure. Kulldorff's scan statistic considers moving "windows" with variable radii ranging from the smallest observed distance to a pre-determined upper bound [31], and was calculated using elliptical windows with a maximum of 50% of the population, using discrete purely spatial Poisson modeling, 999 Monte Carlo simulations, non-overlapping windows, and maximum likelihood estimations with an alpha level of 0.05. In addition, spatial analyses were run after the study area was also divided into northern and southern regions. This was performed in order to compare global results with more localized results and explore the possibility of a biased clustering effect due to the large area of unsampled departments previously mentioned for each species. The north region included the departments Nord, Nord-Est, Nord-Ouest, Artibonite, and Centre which provided a cumulative surface area of 14,315 km 2 . The southern region included the departments, Grand'Anse, Sud, Nippes, and Sud-Est which provided a cumulative surface area of 7,868 km 2 . The department of Ouest was excluded for clustering analyses as so few children were enrolled for collection of both P. malariae and P. vivax serology data.

Independence of the anti-Plasmodium MSP1 19 IgG assay signals
Histograms for the log-transformed IgG assay signal to PfMSP1 19 , PvMSP1 19 , and PmMSP1 19 are shown in Fig 1A. In this low malaria transmission setting, a strong unimodal distribution can be observed which would be the "background" signal of the assay for seronegative individuals' blood samples. The seropositivity thresholds are indicated for each antigen, and seropositive sample signals indicated on the right side of each plot. When comparing the assay signal among the three Plasmodium MSP1 19 species' isoforms, very few children provided a blood sample which had a substantial signal to more than one of the MSP 19 antigens (Fig 1B).

Characteristics of children seropositive for P. malariae and/or P. vivax antigens
A total of 21,719 children were tested for P. malariae MSP1 exposure in 680 schools (Table 1), and 24,559 children were tested for P. vivax MSP1 exposure in 788 schools ( Table 2). Of those children tested, 278 (1.27%) were seropositive for PmMSP1 19 and 113 (0.46%) seropositive for PvMSP1 19 . Of the 21,415 students with assay results for both species, 12 (0.06%) were seropositive for both the P. malariae and P. vivax antigens. Of the schools surveyed, 189 (27.8%) enrolled one or more seropositive children for PmMSP1 19 , and 93 (11.8%) schools enrolled one or more seropositive children for PvMSP1 19 . By department (administrative district), P. malariae seroprevalence remained relatively similar to national seroprevalence in the Nord (1.2%), Nord-Ouest (1.5%), and Sud (1.1%) departments, but was more variable in other regions (Table 1). For departments with more than one-hundred children sampled, P. malariae seroprevalence was highest in Centre (3.8%) and lowest in Artibonite (0.5%). By department, P. vivax seroprevalence remained similar to national seroprevalence in the Centre (0.4%), Nord (0.5%), and Nord-Ouest (0.3%) but was more variable in other regions (Table 2).
For departments with more than one-hundred children sampled, P. vivax seroprevalence was highest in Sud-Est (3.3%) and lowest in Artibonite (0.0%). The 12 participants seropositive for both species were widely dispersed across the departments of Sud, Centre, Nord, Nord-Ouest, and Nord-Est. Average age among seropositive children was 6.7 years for P. vivax and 6.6 years for P. malariae. In addition, among P. malariae and P. vivax seropositive children, 51.4% and 49.1% were female respectively.

Spatial distribution of enrolled schools and seropositive children
Spatial distribution of schools enrolled in the 2014-2016 TASs and had Plasmodium IgG data collected is shown Fig 2. Indicators on the maps are provided for count of seropositive children per school for P. malariae and P. vivax antigens.

Assessment of spatial randomness
Ripley's K function for spatial randomness of schools determined a significant spatial clustering at all intervals tested up to 50 km for both species in northern and southern Haiti (S1A, S1C, S1E and S1G Fig). Weighted K-function of schools tested for P. malariae in northern Haiti using seropositive counts as weights suggests a significant spatial aggregation of cases per school at all intervals between 2.5 km and 15 km with a peak at 12.5 km (S1B Fig). The   weighted K-function of schools tested for P. vivax in northern Haiti also suggest a significant spatial aggregation of cases at all intervals between 2.5 km and 17.5 km with a peak at 12.5 km and again between 30 km and 50 km with a peak at 45 km (S1F Fig). Weighted K-functions for both species suggest spatial randomness at all distances in southern Haiti (S1D and S1H Fig). Although seropositive counts for P. vivax have no statistically significant aggregation at any interval, a very definitive arch can be noted between 5 and 50 km with a peak difference at 17.5 km (S1H Fig). This could suggest a borderline significant aggregation that has been underestimated by the spatial configuration of schools in the south (S1G Fig). Cluster analyses for P. malariae and P. vivax seropositivity At a national level, spatial autocorrelation analysis using Moran's I resulted in values above zero for both P. malariae (I = 0.16, p<0.001) and P. vivax (I = 0.04, p = 0.13), suggesting statistically significant spatial autocorrelation on a national level for P. malariae but not for P. vivax. Moran's I for P. malariae suggests a significant autocorrelation of high seropositive counts in the north with an I-value of 0.153 (p<0.001) and no autocorrelation in the south with an Ivalue of -0.001 (p = 0.99)(S2 Fig). The Moran's I for P. vivax in the north does not suggest autocorrelation with an I-value of -0.016 (p = 0.80). P. vivax Moran's I in the south however does suggest autocorrelation but not to a significant level with an I-value of 0.062 (p = 0.08). Cluster location analysis of the entire country using Kulldorff's Bernoulii scan produced four hot-spots with a significant likelihood for seroprevalence: two for P. malariae and two for P. vivax (Table 3 and Fig 3). Two significant clusters were observed for P. malariae (Fig 3A): Cluster A (RR = 3.45, p<0.001) containing an ellipse with a minor radius of 9.1 km and major radius of 13.7 km, and Cluster B (RR = 5.38, p = 0.005) containing an ellipse with a minor radius of 2 km and major radius of 10 km. Two significant clusters were observed for P. vivax

Testing for active P. malariae and P. vivax infections through laboratory assays
In attempt to identify children with active P. malariae or P. vivax infections, blood samples with the highest IgG responses to the PmMSP1 19 and PvMSP1 19 antigens were selected to undergo laboratory assays for the presence of Plasmodium antigens or DNA. As shown in S3  Fig, the

Discussion
Despite large-scale elimination efforts in the 1960s, malaria remains endemic in Haiti. The WHO-lead elimination program clearly identified three species of malaria in the population: Plasmodium falciparum, P. malariae, and P. vivax [13,14,32], but more recent malaria studies have largely overlooked the presence of non-P. falciparum species. The lymphatic filariasis (LF) TAS sampling methodology was developed to assess LF transmission [20,33], but schoolbased sampling designs are readily transferable to malaria exposure surveillance. The use of school children as a sentinel population with a biological peak determinacy of intensity and prevalence, is equally beneficial for malaria survey purposes [34][35][36]. With an extensive sample size of 21,719 children tested for antibodies against P. malariae and 24,559 children tested for P. vivax, this large seroepidemiological study also benefits from the very narrow age range of enrolled participants since high numbers of children of the same age (i.e. same number of years for possible exposure) were collected throughout the country. As IgG antibodies to Plasmodium antigens could have only been induced in this population from natural exposure, presence of IgG to these antigens in 6-or 7-year-old children would clearly indicate P. malariae or P. vivax exposure in these areas in the past 6 or 7 years. Recent studies have estimated that IgG against Plasmodium MSP1 19 antigens would have a duration of years to decades following natural exposure [9,11]. As the persons enrolled in this study were young, it is a reasonable assumption that presence of this long-lived IgG against the MSP1 19 antigens is a reliable indicator of lifetime P. malariae and/or P. vivax exposure. For all Haitian children sampled, the IgG seroprevalence was very low for both species' MSP1 19 antigens: P. malariae (1.3%) and P. vivax (0.5%). The IgG response to the two MSP1 19 antigens appeared to be largely independent from each other (as well as independent from PfMSP1 19 ), and the total number of children seropositive to both PmMSP1 19 and PvMSP1 19 was small (n = 12, 0.06%). To the authors' knowledge, the most recent surveys to investigate the presence of P. falciparum, P. malariae, and P. vivax in Haiti was through the WHO elimination program with data from 1964 to 1967 [13]. During these four years, P. falciparum remained the dominant malaria species, accounting for approximately 98% of all microscopically-confirmed cases, whereas P. malariae accounted for~2.5% and P. vivax~0.5% of all case counts. It is not surprising that, even today, P. falciparum remains the dominant malaria in Haiti [12,37]. Though we present here data showing IgG concordance with antibodies against PfMSP1 19 , a complete report of P. falciparum seroprevalence and spatial estimates will be forthcoming. From the serological data in our current study, it is interesting to note that P. malariae seroprevalence is over 2-fold higher than P. vivax-providing evidence that P. malariae may still be the second most prevalent malaria species in Haiti followed by P. vivax. Recently, more evidence has been generated that P. vivax can sustain transmission in largely-Duffy negative populations through alternate blood cell invasion routes [38][39][40][41]. This appears to be the situation in Haiti as well, with a largely Duffy negative population [42] showing a very low transmission rate for P. vivax [15,17].
When looking at seroprevalence by Haitian department, substantial differences were noted between IgG carriage against the P. malariae and P. vivax MSP1 19 antigens. However, even with significant differences among the department seroprevalence estimates, the absolute magnitude of IgG positivity for these antigens was still very low with the highest PmMSP1 19 seroprevalence at 3.8% (Centre department) and the highest PvMSP1 19 seroprevalence at 3.3% (Sud-Est department).
Analysis describing the distribution of the schools through un-weighted K-functions determined whether any clustering was simply a result of the spatial location of the sample sites. Ripley's K-function for spatial randomness determined that schools are spatially clustered at all intervals tested for north and south Haiti for both species. Therefore, any clustering of seropositive children could potentially be dependent on school location and therefore be overestimated due to the aggregation of school sample locations [29,30]. The suggested unweighted spatial distribution of schools calculated through this software was significantly clustered at all intervals for both species. Although the weighted K-function for P. malariae and P. vivax in north Haiti suggests borderline aggregation of cases across various intervals and Moran's I for P. malariae in the north suggest autocorrelation, all other autocorrelation measures clearly suggest that high seropositive counts do not neighbor other high counts-i.e. these areas are isolated from each other. If dividing the country into northern or southern sections or maintaining the area of the country as a whole, the overall concordance in defining areas of statistically significant seropositive clustering (Figs S2 versus S3) provides higher confidence in the finding of true geographical areas with higher P. malariae or P. vivax exposure. It is interesting to note that: 1) among the 4 clusters identified, none were overlapping between P. malariae and P. vivax, and 2) very few places in the country had a complete absence of children seropositive for either species. Practically, detection of hotspots of seropositivity for non-dominant malaria species could provide rationale for follow-up studies in these specific areas in attempt to obtain more granular spatial estimates for tangible programmatic purposes such as targeted mass-drug administration (MDA) in smaller populations. Additionally, health facilities within hotspot areas could be put on notice and given appropriate diagnostic tools to take into account non-dominant species when diagnosing a patient with 'malaria-like symptoms'.
A limitation to this current study is that children were enrolled at their respective schools, and not at primary residence where they would likely be spending the majority of their time, especially nights when the Anopheline mosquitoes are more active [43]. In this same manner, previous travel history for these children remains unknown and therefore malaria exposure (and IgG acquisition) could have occurred in a different area than at the area of the country where they were sampled. In addition, it is important to note that the spatial tests used serve as a descriptive measure of case distribution and can only determine general locations of high prevalence but not the magnitude of difference. The methods also do not suggest a high measure of granularity of malaria exposure.
As active infection with P. vivax or P. malariae is known to induce high levels of IgG, we selected the highest IgG responders to PmMSP1 19 and PvMSP1 19 in an attempt to identify current infections at time of sampling. Through qPCR and antigen detection assays, we were unable to identify any active malaria infections, so a limitation to this study is that no assertions could be made about P. malariae or P. vivax prevalence during the time of sampling between 2014-2016. Though providing an overall large sample size, many areas of Haiti did not have schools enrolled in the 2014-2016 TAS, so no assumptions could be made regarding malaria exposure in those areas not sampled. The sampling distribution of schools and low parasite prevalence of non-falciparum species in Haiti may also bias geospatial models as a large amount of empty space may exist between aggregate clusters of schools that were sampled [3,29,44,45].
In Haiti and other areas of the world where non-dominant malaria species are neglected in the country's surveillance and diagnostic efforts, serological data provides an objective measure to estimate population-level exposure to all endemic malaria species. In addition, as the design of diagnostic tests is intended to identify symptomatic and high parasite density infections, the use of IgG data can assist to approximate all forms of malaria exposure and infection, not just those in the population seeking treatment.
Supporting information S1 Fig. K-functions and weighted K-functions detailing spatial randomness of schools surveyed for IgG antibodies against P. malariae (A-D) and P. vivax (E-H) in Haiti. Spatial scan area was divided into northern and southern regions in order to avoid empty space bias caused by lack of sampling in the Ouest department. For unweighted fields, confidence envelopes are generated by distributing points randomly in the study area and calculating k-values for 999 permutations. For each distance, the highest and lowest deviation from the expected K-value construct the envelope. The same methods are used for weighted fields with the exception that only the weighted values are randomly distributed to generate confidence envelopes; point locations remain fixed.