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) [5-7], 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 [9-11]. 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 [12-14]. 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 [15-17]. 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.

Methods

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 soil-transmitted 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.

Table 1

Summary of Plasmodium malariae serology samples collected in Haiti between 2014–2016.

		Total	Seropositive
Variable		N	n (%)
Schools a		680	189 (27.8)
Participants		21,719	278 (1.3)
Age
	6	9,015	124 (1.4)
	7	12,184	154 (1.3)
	8–10	4	0 (0.0)
Gender
	Female	10,531	143 (1.4)
	Male	10,674	135 (1.3)
Department
	Centre	1,213	46 (3.8)
	Grand’Anse	1,666	12 (0.7)
	L’Artibonite	3,189	15 (0.5)
	Nippes	44	0 (0.0)
	Nord	9,409	116 (1.2)
	Nord-Est	1,625	31 (1.9)
	Nord-Ouest	2,368	35 (1.5)
	Ouest	15	0 (0.0)
	Sud	2,190	23 (1.1)
	Sud-Est	-	-

a One or more students determined seropositive categorizes a school as seropositive

Table 2

Summary Plasmodium vivax serology samples collected in Haiti between 2014–2016.

		Total	Seropositive
Variable		N	n (%)
Participants		24,559	113 (0.5)
Schools a		788	93 (11.8)
Age
	6	10,152	46 (0.45)
	7	13,136	54 (0.41)
	8–10	741	10 (1.35)
Gender
	Female	12,045	54 (0.4)
	Male	11,986	56 (0.5)
Department
	Centre	1,213	5 (0.4)
	Grand’Anse	1,666	2 (0.1)
	L’Artibonite	3,189	0 (0.0)
	Nippes	1,366	14 (1.0)
	Nord	9,184	43 (0.5)
	Nord-Est	3,115	37 (1.2)
	Nord-Ouest	2,368	6 (0.3)
	Ouest	71	0 (0.0)
	Sud	2,234	1 (0.0)
	Sud-Est	153	5 (3.3)

a One or more students determined seropositive categorizes a school as seropositive

Laboratory assays

Elution of blood from DBS

For the antibody and antigen detection assays, whole blood was eluted from a single tab of the TropBio filter wheels to provide sample for assay. A single DBS tab (10 μL whole blood) was rehydrated in a blocking buffer (PBS pH 7.2, 0.5% Polyvinyl alcohol (SigmaAldrich, St. Louis, MO) 0.5% polyvinylpyrrolidine (SigmaAldrich), 0.1% casein (ThermoFisher, Waltham, MA), 0.5% bovine serum albumin (SigmaAldrich), 0.3% Tween-20, 0.05% sodium azide, and 0.01% E. coli extract to prevent non-specific binding) to a final dilution of 1:20. Eluted blood samples were stored at 4°C until assayed.

Bead-based IgG detection

Three separate bead regions (Bio-Plex non-magnetic beads, BioRad) were coupled with malaria antigens for IgG capture and subsequent detection. The antigens in the multiplex panel have all been reported before [5,23], and were the Plasmodium falciparum merozoite surface protein 1 19kD fragment (PfMSP119; coupled at pH 5 at 20 μg/mL), P. vivax merozoite surface protein 1 19kD fragment (PvMSP119; coupled at pH 5 at 20 μg/mL), and P. malariae merozoite surface protein 1 19kD fragment (PmMSP119; coupled at pH 5 at 20 μg/mL). Overall sequence consensus among these three recombinant 19kD antigens was low at 34.4%.

All IgG assay reagents were diluted in buffer containing PBS, 0.05% Tween20, 0.5% bovine serum albumin (SigmaAldrich), and 0.02% NaN3. 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-hIgG4, 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 PmMSP119 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 PmMSP119 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 MSP119 antigens produced by those malarias [23-27]. These same studies have also shown high specificity among Plasmodium MSP119 isoforms, meaning infection with one malaria species will typically produce IgG with homologous binding to only that MSP119 isoform. With these two factors considered, samples with the highest IgG assay signals to the PmMSP119 or PvMSP119 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/12.5x106 beads, rabbit IgG anti-aldolase, Abcam, Cambridge, UK), histidine-rich protein 2 (HRP2; 20μg/12.5x106 beads, mouse anti-P. falciparum, Abcam), pan-Plasmodium lactate dehydrogenase (pLDH; 12.5ug/12.5x106 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% NaN3. 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 William & Mary Geolab.

Haiti is a country with total surface area of 27,750 km2. 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 K-function 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 [29-31]. 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]. K-functions 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 km2. The southern region included the departments, Grand’Anse, Sud, Nippes, and Sud-Est which provided a cumulative surface area of 7,868 km2. 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.

Results

Independence of the anti-Plasmodium MSP119 IgG assay signals

Histograms for the log-transformed IgG assay signal to PfMSP119, PvMSP119, and PmMSP119 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 MSP119 species’ isoforms, very few children provided a blood sample which had a substantial signal to more than one of the MSP19 antigens (Fig 1B).

Fig 1

Distribution and independence of assay signals for IgG against PfMSP119, PvMSP119, and PmMSP119.

A) Histograms of log-transformed assay signal for the three Plasmodium antigens. Vertical hashed lines indicate seropositivity threshold for each antigen assay signal. B) Scatterplots of non-transformed assay signal as compared among the three species’ MSP119 isoforms.

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 PmMSP119 and 113 (0.46%) seropositive for PvMSP119. 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 PmMSP119, and 93 (11.8%) schools enrolled one or more seropositive children for PvMSP119. 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.

Fig 2

Location of schools included in the TAS from 2014 to 2016 with malaria serology data collected.

Schools are represented by a dot where size and color change correspond to the count of children determined seropositive. White dot: no seropositive children; orange: 1–2; red: >3 seropositive children. Additionally, color intensity is provided for each Haitian department by total number of children with serology data enrolled within that department. Panel A shows seropositivity and enrollment for children with IgG data for PmMSP119, and panel B shows seropositivity and enrollment for children with IgG data for PvMSP119. Base map for administrative boundaries found at: https://www.geoboundaries.org/index.html#getdata.

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 I-value 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 (Fig 3B): Cluster C (RR = 17.5, p<0.001) containing a circular hot-spot with a radius of 2.7 km and was located in the Nord-Est department, and Cluster D (RR = 5.48, p = 0.037) contained a circular hot-spot with a radius of 14.8 km was found in the southern department of Nippes. When Kulldorff’s Poisson Spatial Scans were re-run in the northern and southern regions independently, clusters were verified (Figs 3 versus S2). The Kulldorff’s Poisson Scan suggested an additional P. vivax cluster closer to the southeast border with the Dominican Republic (S2 Fig).

Table 3

Summary of hotspot clustering for P. malariae and P. vivax through Kulldorff’s scan statistic among school children in Haiti.

Cluster	Species	No. Serospositive	Population	Schools	Major/Minor Radius (Km)	RR	P value
A	P. malariae	46	1182	49	9.1 / 13.7	3.45	0.001
B	P. malariae	18	255	8	2.0 / 10.0	5.83	0.005
C	P. vivax	12	170	5	2.7/ 2.7	17.05	0.001
D	P. vivax	12	521	13	14.8 / 14.8	5.48	0.037

Fig 3

Kulldorff’s spatial scan statistic of seropositive children per school.

Maps displayed for P. malariae (A) and P. vivax (B) with ellipses denoting hotspot cluster boundaries for significant spatial aggregation of seropositive counts. Base map for administrative boundaries found at: https://www.geoboundaries.org/index.html#getdata.

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 PmMSP119 and PvMSP119 antigens were selected to undergo laboratory assays for the presence of Plasmodium antigens or DNA. As shown in S3 Fig, the majority of all children’s IgG signals to the PmMSP1 or PvMSP119 antigens were quite low (<10,000 MFI units on a scale of 0 to 32,000). In total, 27 samples were selected based on PmMSP119 antibody level, 24 samples selected based on PvMSP1 response, and 1 sample selected due to high responses for both PmMSP119 and PvMSP119. For all 52 selected samples, neither Plasmodium antigen detection nor PET-PCR assays produced positive test results. As no antigen/DNA positives were found among these samples with the highest IgG responses, no further samples were selected for antigen and DNA testing.

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 school-based 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 [34-36]. 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 MSP119 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 MSP119 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’ MSP119 antigens: P. malariae (1.3%) and P. vivax (0.5%). The IgG response to the two MSP119 antigens appeared to be largely independent from each other (as well as independent from PfMSP119), and the total number of children seropositive to both PmMSP119 and PvMSP119 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 PfMSP119, 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 [38-41]. 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 MSP119 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 PmMSP119 seroprevalence at 3.8% (Centre department) and the highest PvMSP119 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 over-estimated 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 PmMSP119 and PvMSP119 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.

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.

(TIF)

Click here for additional data file.

Kulldorff’s Spatial Scan for seropositive counts for P. malariae (top panel) and P. vivax (lower panel) as divided into the northern and southern sections of the country. Ellipses denote cluster borders, and shading of sub-communes indicate number of seropositive children enrolled during the TAS. Base map for administrative boundaries found at: https://www.geoboundaries.org/index.html#getdata.

(TIF)

Click here for additional data file.

Selection of samples with high IgG assay signal to PmMSP119 (A) and PvMSP119 (B) for further laboratory tests. Samples providing assay signals in the grey circles were selected.

(TIF)

Click here for additional data file.

Raw data used for analysis.

(XLSX)

Click here for additional data file.

11 Jul 2021

Dear Dr. Rogier,

Thank you very much for submitting your manuscript "Spatial Cluster Analysis of Plasmodium vivax and P. malariae Exposure Among Haitian School Children Sampled Between 2014 And 2016" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' and editors' comments.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

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[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Daniel M Parker

Associate Editor

PLOS Neglected Tropical Diseases

Amanda Ross

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: See all comments below

Reviewer #2: - Provide a clearer explanation on the differences between each of the three spatial/cluster analyses used. Each of the sections in the results talk about “clustering” but its hard to tell how the interpretations between the three methods fit together.

- Provide details in the statistical analysis section on the use of Bayesian logistic model, and it is not clear in the results as to why this was performed and why the OR estimates were inconsistent for NDVI and LST. This finding seems very strange for univariate multilevel logistic models – what were the priors, how influential? Also for the multilevel logistic model more details should be provided on the random effects – e.g. for school clustering.

Reviewer #3: The authors clearly stated their objective to establish seroprevalence of malaria species in Haiti. They used a young age group collected at schools to do this in order to look at recent infections and were able to establish appropriately large sample sizes (21,719 students from 680 schools). They used appropriate statistics to test for the degree of spatial aggregation of the schools, and autocorrelation of seropositivity counts, followed by Kulldorff’s Bernoulii scan to identify significant hotspots.

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: See all comments below

Reviewer #2: - Figure 1: need to have higher resolution on these figures.

- Table 1 – 2: It should be made clearer in Tables 1 and 2 that the OR presented for age, elevation, NDVI and LDT are for these as continuous covariates (also did the authors investigate if the association between these continuous covariates and the log odds of seropositivity was linear, as no details are provided in the statistical analysis section), and the units should be given in the variable column for age. Also the reference group for gender should be specified. ‘Variable’ is not spelt correctly in Table.

- Line 343 – 347: the 95% CI should be provided in brackets next to the OR estimate, and the exact p-value instead of a categorised p-value. (Should be “<” instead of “>” on line 343 – 344 if you decide to keep these p-values in text).

- Line 363: change “detail” to “correspond to”.

- Figure 2: consider giving one common legend for this figure rather than splitting between the two plots.

- Figure S2: explain the acronyms shown in the figures (L(d), Low Conf. Env., High Conf. Env.) in the figure legend.

- Line 389 - 414: give actual p-value rather than categorising as >0.05 and <0.01. Replace by 95% CI if this is available.

- Table 3: include information on method used in the legend, for example: “by Kulldorff’s scan statistics.” Please also provide confidence intervals for the RR estimates in the table and in text if available.

- Line 426: can remove “as described in Methods”.

- Line 426: remove the ’ at the end of children’s.

Reviewer #3: The results establish the geographical distribution of cases of various malaria species in Haiti as the authors intended. Drawing figure 3 as a superimposition over cases, as was done in S3, may help readers see the pattern more effectively.

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: See all comments below

Reviewer #2: - The discussion could be made stronger by having a summary of the key findings, which address the research questions detailed in the introduction, earlier in the discussion section (at least in the first paragraph).

- Line 462: consider removing the word “vanishingly”, small is enough.

- Line 512 – 513: “autocorrelation measures clearly suggest that high seropositive counts do not neighbor other high counts” – but in the results section autocorrelation results showed clustering of high P. malariae seropositivity in the north?

- Include in the discussion section recommendations on how the detection of hotspots using Kulldorff’s scan statistic (Table 3) could be used – do the authors recommend anything based on these hotspot results?

Reviewer #3: Conclusions are generally well supported by the data presented and limitations are mostly well addressed. One exception would be the exclusion of P. ovale within the study is not mentioned when discussing relative species prevalence.

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: See all comments below

Reviewer #2: - Line 1-3: consider including something about seroprevalence or serological data in the title.

- Line 28 – 29: include study aims at the end of the background section of the abstract.

- Line 33: can remove the word “later” for simplicity.

- Line 56 – 57: consider rewording this sentence to: “P. falciparum is the dominant malaria species worldwide and is often the primary, or only, focus of malaria surveys.”

- Line 58 and 62 – change “populace” to “population”.

- Line 70 – 71: consider rewording to “Identification of Plasmodium reservoirs in low transmission settings rely on conventional diagnostics (microscopy and RDT) which underestimate the true incidence and prevalence.”

- Line 72 – 73: antibody-based assays are a type of diagnostic. We would suggest rewording “When compared to diagnostic tests” to “When compared to microscopy and RDTs”.

- Line 73: suggest removing “more powerful indicator for detecting active infection…” in place of “more powerful method for detection of active infection…”.

- Line 74 – 75: consider simplifying the last part of this sentence to “expanding the time window for diagnosis.”

- Line 88: consider changing “reduction efforts” to “elimination efforts”.

- Line 100 – 104: an overview of what is done in the study is given but should also have clear aims stated.

- Line 100: remove the word “occurring”.

Reviewer #3: In figure S3, in the top panel, I suggest the P-value be written using scientific notation so that it is not P < 0.000

Line 132: extra parenthesis at the end of the sentence

Line 142: missing space

Line 379: S1F figure should likely be S2F

Line 399: Combine table & figure references to be more consistent with previous reference formats

Line 442-444: Sentence lacks appropriate subject

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: Oviedo and colleagues describe results from a large-scale sero-survey of >20,000 school children in Haiti, with a particular focus on P. malariae and P. vivax. They identified low-levels of sero-positivity against both P. malariae and P. vivax MSP1-19, with evidence of some (independent) spatial clusters. In a subset of samples tested, they found no evidence of active Plasmodium infections. The article is well written, logical and with sound data analysis. I have only minor comments that I as a reader would find helpful for the authors to address.

1. Cross-reactivity between Pf, Pm, Pv MSP1-19

The authors seem confident that there would be minimal cross-reactive antibody responses against the various MSP1-19 proteins tested from the three species, based on past literature and their results in Figure 1.

a. I suggest in the methods that the authors do detail the sequence identity between the Pf-Pv Pf-Pm and Pv-Pm MSP1-19 protein sequences (I believe it is moderate at least for Pv-Pm around 56%).

b. In the results description for Figure 1, it would be helpful to quantify how many children had a positive signal to more than one MSP1-19 antigen out of the total children positive with any positive signal. This is detailed around lines 318/319 for Pm and Pv but doesn’t include Pf, and clearly from Figure 1 there are quite a few positive antibody signals for Pf.

2. Results for Pf MSP1-19

I appreciate that the authors have focused their results on Pv and Pm given the prevalence/transmission of these species in recent years in Haiti remains unknown. However they did measure antibody against PfMSP1-19 so I think it is worth at least documenting/quantifying the proportion of the children seropositive to Pf-MSP1-19 even if the subsequent detailed analysis is only on Pm and Pv. This could be added to the text description for Figure 1, and added into Line 459 in the discussion.

3. Remaining minor points

a. Line 75 seems to be an aberrant comma

b. Line 81 define MSP1 for first use

c. Line 142 missing space between C and until

d. Line 164 text says City, STATE – needs adding in

e. Lines 155, 173, 204 please provide more details. What is the positive control? How many negative controls were run to generate the seropositivity cut-off?

f. Would Figure 1B be clearer on a log scale like Figure 1A?

g. Line could be helpful to give more context – latest % prevalence estimate? Or # cases per year?

h. Line 456 would antibody longevity be expected to be similar between adults and children?

i. In the discussion, the authors could comment on their use of binary seropositivty data, would added value be gained from using the actual antibody magnitude?

j. For the discussion, can the authors comment on whether there are any geographic or human features that associate with the clusters? Like village boundaries, or rivers/mountains/roads etc? And perhaps why Cluster D is so large (is this an effect of less schools being tested in that region)?

k. Do the authors think it is worthwhile doing their PCR assays/antigen assays on the whole sample set in the future to search for active Pm and Pv infections or would that be waste of time/resources?

I selected "no" for is all the underlying data available as it was not included as a Supplement so I personally could not access it. The corresponding author does say it is available upon request so the Editor can determine whether this is appropriate or not.

Reviewer #2: The manuscript “Spatial cluster analysis of Plasmodium vivax and Plasmodium malariae exposure among Haitian school children sampled between 2014 and 2016” presents an interesting analysis of a very important issue. As malaria declines it is essential that we understand the changing dynamics of transmission. This study will provide useful information to those working in malaria elimination in Haiti and those who are interested in how serological data can supplement existing diagnostic methods.

The comments provided here recommend further improvements to the manuscript.

Reviewer #3: The study by Oviedo et al provides a serosurvey of P. malariae, P. vivax and P. falciparum in Haiti between 2014 and 2016. The study focuses on young children who have just a few years of potential exposure so the data presented is valuable in its ability to inform scientists on the degree to which these species have been endemic in Haiti over the last few years. The use of spatial clustering methods established areas where each species is likely to be most endemic and may be useful in elimination efforts. The methodology seems appropriate for the conclusions. While there are some limitations that result from sampling bias, the authors address this multiple times in their results and discussion. I recommend this manuscript be accepted, though I have some minor recommendations:

Sources 5 and 23 both appear to reveal plausibility for detection of P. ovale, and the authors mention that the last surveys in this area were in the 60s. Further, they assert that results may indicate P. malariae is the second most common on lines 470-472 without having data for P. ovale available. Authors should discuss why they chose to not include P. ovale and to consider this in their assertions.

Authors should comment on why they chose not to adjust for collection or population numbers.

I also noted the following minor writing errors:

Line 132: extra parenthesis at the end of the sentence

Line 142: missing space

Line 379: S1F figure should likely be S2F

Line 399: Combine table & figure references to be more consistent with previous reference formats

Line 442-444: Sentence lacks appropriate subject

Figure S3, in the top panel, I suggest the P-value be written using scientific notation so that it is not P < 0.000

Deputy editor:

The statistical analysis appears a little weak. The methods chosen are not well justified, and there is a heavy reliance on scan statistics which while simple and easy to run can only say whether there is a significantly higher prevalence in an area but not how much higher (which would be useful for control activities). In figure 3, the hotspots for P vivax are very large, but the prevalence is unlikely to be uniformly high across this area, there are relatively few cases and it does not correspond well visually to Fig 2. (A prevalence map may be an option, although would require more sophisticated statistical methods). I suggest reducing the emphasis on the cluster analysis, and removing the section on spatial randomness since malaria is well known to be heterogenous on many scales. The size of the clusters does not seem to be justified - ideally it would bear relevance to control activities or whatever the information is used for. In Fig S3, the elipses cover areas with no data and so the shape seems rather inflexible. Whether you choose scan statistics or other analysis, the methods should be justified and limitations mentioned in the discussion.

For the univariate logistic regression models, clustering within schools does not look as if it has been taken into account, and the choice of univariate is neither obvious nor justified. A statistician would be able to advise on this.

--------------------

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Figure Files:

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To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

29 Sep 2021

Submitted filename: Oviedo_etal_Response_to_Reviewers.docx

Click here for additional data file.

19 Oct 2021

Dear Dr. Rogier,

Thank you very much for submitting your manuscript "Spatial Cluster Analysis of Plasmodium vivax and P. malariae Exposure Using Serological Data Among Haitian School Children Sampled Between 2014 and 2016" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Daniel M Parker

Associate Editor

PLOS Neglected Tropical Diseases

Amanda Ross

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: The authors addressed my comments re some additional clarifications in the serological methods appropriately.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: The authors considered my suggestions and ultimately disagreed, which they appropriately justified.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: The authors have better acknowledged the limitations of their study following a suggestion by the editor.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: In all Tables, needs to have the Plasmodium species in italics.

In Table 1 and Table 2, the "a" after schools could be in superscript then it would look less like a typographical error.

For clarity, I would suggest noting in the results or discussion that analyses and presentation of the Pf data will be in an upcoming publication.

Reviewer #2: Line 59: change “populace” to “population” – make sure “population” used throughout text for consistency

Line 66: change “test for antibodies” to “testing for antibodies” or “tests for antibodies”

Line 67: change “burden to” to “burden of”

Line 72: change “rely” to “relies”

Line 88: change “of years” to “for years”

Line 105: change “will only identify” to “only identify”

Line 119: change “helminth” to “helminths”

Line 295 – 297: would be helpful to have an estimate of the area covered by the north and south regions, and the region as a whole (in km2 for example) to give context into how large each of these sub-areas are compared to the whole region.

Line 336 – 342: when you specify the departments with more than 100 children sampled you should also state species to make it clear which you are talking about. For example: “For departments with more than one-hundred children sampled, P. vivax seroprevalence was highest in Sud-Est (3.3%)…”

Line 344: would be helpful to have a range alongside the average age of positive children.

Line 348: use a symbol like a cross or star instead of a, or make “a” a superscript to make it clear it is a comment not a misplaced letter

Line 388: remove “the” from “in the northern”

Line 395: make sure “k” in K-function is capitalized

Line 395: could be good to remind the reader here how the K-functions are weighted

Line 431: make sure malaria spp is italicized throughout text (also unitalicized on line 528)

Line 460: remove “which”

Line 521: remove “not”

Reviewer #3: In figure 3, I maintain that adjusting for sampling would be useful. the number of seropositive students per school isn't very informative if sampling isn't entirely uniform between schools and therefore I think the color coding of schools by seropositive students per school could be misleading. I believe this to be a rather minor concern that doesn't threaten overall conclusions.

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: (No Response)

Reviewer #2: The authors have responded well to the first round of comments and suggestions which have improved the manuscript. The comments provided here recommend further improvements to the manuscript. Line numbers provided refer to the location in the tracked changes document.

Reviewer #3: (No Response)

--------------------

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Deputy editor:

The authors have improved the paper.

I remained concerned about the implausibly large hotspot – it does not tally with the data in Figure 2. I think this is likely to be just a matter of changing the maximum size of the scan window to something justifiable. (It does not need to be justifiable on the grounds of interventions if they are not to be used, but should be justifiable for some reason other than that it was the default software setting).

I agree with Reviewer 2 (L404-430) who requested actual p-values rather than p<0.05 or p<0.01. Statistics has moved away from these cut-offs now and textbooks recommend using the exact p-value since they contain more information and also give a more accurate impression of the test that was carried out. Please use the exact p-values.

The sentence added at the end of the Introduction about the rationale for the analysis does not seem accurate. An ‘analysis’ does not aim to provide a rationale for something, it only answers a specific question. Do you mean the aim of the ‘study’ is to provide a rationale for something else? It would make more sense if the aim of the study would be to summarize the spatial clusters of Pf and Pm in Haiti, and to demonstrate the use of IgG. Otherwise it does rather sound as if the aim of research is to justify research.

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

18 Nov 2021

Submitted filename: Oviedo_etal_Resubmission2_Response to Reviewers.docx

Click here for additional data file.

30 Nov 2021

Dear Dr. Rogier,

Thank you very much for submitting your manuscript "Spatial Cluster Analysis of Plasmodium vivax and P. malariae Exposure Using Serological Data Among Haitian School Children Sampled Between 2014 and 2016" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

The authors have adequately addressed all reviewer critiques, with one exception. Please address the following issue:

Line 269 "The north region included the departments Nord, Nord-Est, Nord-Ouest, Artibonite, and Centre. The southern region included the departments, Grand'Anse, Sud, Nippes, and Sud-Est."

Please give total land areas for these subregions. As you state in the previous rebuttal, you do already provide the total landmass of Haiti. However, it is useful for the reader to understand the two subareas that are analyzed, especially since one part of the nation is excluded (Oueste). This should be easy to calculate/estimate with the GIS software used. Without this it would be easy for a reader to think that the entire nation was included in this analysis. Also consider a very brief statement about why Oueste is excluded.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Daniel M Parker

Associate Editor

PLOS Neglected Tropical Diseases

Amanda Ross

Deputy Editor

PLOS Neglected Tropical Diseases

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The authors have adequately addressed all reviewer critiques, with one exception. Please address the following issue:

Line 269 "The north region included the departments Nord, Nord-Est, Nord-Ouest, Artibonite, and Centre. The southern region included the departments, Grand'Anse, Sud, Nippes, and Sud-Est."

Please give total land areas for these subregions. As you state in the previous rebuttal, you do already provide the total landmass of Haiti. However, it is useful for the reader to understand the two subareas that are analyzed, especially since one part of the nation is excluded (Oueste). This should be easy to calculate/estimate with the GIS software used. Without this it would be easy for a reader to think that the entire nation was included in this analysis. Also consider a very brief statement about why Oueste is excluded.

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

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References

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

30 Nov 2021

Submitted filename: Oviedo_etal_Resubmission3_Response to Editor.docx

Click here for additional data file.

3 Dec 2021

Dear Dr. Rogier,

We are pleased to inform you that your manuscript 'Spatial Cluster Analysis of Plasmodium vivax and P. malariae Exposure Using Serological Data Among Haitian School Children Sampled Between 2014 and 2016' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Daniel M Parker

Associate Editor

PLOS Neglected Tropical Diseases

Amanda Ross

Deputy Editor

PLOS Neglected Tropical Diseases

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24 Dec 2021

Dear Dr. Rogier,

We are delighted to inform you that your manuscript, "Spatial Cluster Analysis of Plasmodium vivax and P. malariae Exposure Using Serological Data Among Haitian School Children Sampled Between 2014 and 2016," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Editorial, Viewpoint, Symposium, Review, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript will be published online unless you opted out of this process. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases