Campaign Reduced Malaria Risk on an Individual but Not Population Level in a

Background: Further reductions in malaria incidence as more countries approach malaria elimination require the identification and treatment of asymptomatic individuals who carry mosquito-infective Plasmodium gametocytes that are responsible for furthering malaria transmission. Assessing the relationship between total parasitemia and gametocytemia in field surveys can provide insight as to whether detection of low-density, asymptomatic Plasmodium falciparum infections using sensitive molecular methods can sufficiently detect the majority of infected individuals who are potentially capable of onward transmission. Methods: In a cross-sectional survey of 1,354 healthy children and adults in three communities in western Kenya across a gradient of malaria transmission (Ajigo, Webuye, and Kapsisywa-Kipsamoite), we screened for asymptomatic P. falciparum infections by rapid diagnostic tests, blood smear, and quantitative PCR of dried blood spots targeting the varATS gene in genomic DNA. A multiplex quantitative reverse-transcriptase PCR assay targeting female and male gametocyte genes ( pfs25 , pfs230p ), a gene with a transcriptional pattern restricted to asexual blood-stages ( piesp2 ), and human GAPDH was also developed to determine total parasite and gametocyte densities among parasitemic individuals. Results: The prevalence of varATS -detectable asymptomatic infections was greatest in Ajigo (42%), followed by Webuye (10%). Only two infections were detected in Kapsisywa. No infections were detected in Kipsamoite. Across all communities, children aged 11-15 years account for the greatest proportion total and sub-microscopic asymptomatic infections. In younger age groups, the majority of infections were detectable by microscopy, while 68% of asymptomatically infected adults (>21 years old) had sub-microscopic parasitemia. Piesp2 -derived parasite densities correlated poorly with microscopy-determined parasite densities in patent infections relative to varATS -based detection. In general, both male and female gametocytemia increased with increasing varATS - derived total parasitemia. A substantial proportion (41.7%) of individuals with potential for onward transmission had qPCR-estimated parasite densities below the limit of microscopic detection but above the detectable limit of


INTRODUCTION
Malaria remains a global public health burden with more than 200 million cases worldwide [1]. Transmission of malaria requires that sexual-stage Plasmodium parasites, gametocytes, present in the blood of infected humans be ingested by female Anopheles mosquitoes during feeding. Strategies that combine effective control of the mosquito vector through use of insecticide-treated nets (ITNs) and indoor residual spraying alongside rapid diagnosis and effective treatment of malaria with artemisinin-combination therapy (ACT) have reduced the prevalence of Plasmodium falciparum infection and the incidence of clinical malaria in endemic areas of Africa since 2000, albeit at a slower rate in recent years [1]. Further reductions in malaria incidence as more countries approach malaria elimination would require the identification and treatment of asymptomatic individuals who carry mosquito-infective gametocytes that are responsible for furthering malaria transmission [2].
Detection of asymptomatically infected individuals has been a major challenge given that individuals residing in areas of high-transmission intensity often carry parasitemia at densities below the detection limits of accessible field diagnostics, which currently includes microscopy and rapid-diagnostic tests (RDTs) [3]. Moreover, the proportion of low-density infections among all malaria infections in a community increases with decreasing malaria transmission [2], suggesting that more sensitive diagnostics are required for detecting parasitemia among individuals in low-transmission settings [4]. Several studies have examined whether lowdensity infections contribute to onward transmission using mosquito feeding assays [5][6][7][8][9][10][11]. A recent metaanalysis of eight such studies estimated that individuals with sub-patent parasitemia were approximately onethird as infectious to mosquitoes as individuals with blood-smear positive infections [4]. In general, gametocyte density directly correlates with mosquito infectivity and thus transmission, with infections with parasite densities below the limit of detection of conventional molecular diagnostics being unlikely to contribute significantly to transmission [12]. Assessing the relationship between total parasitemia and gametocytemia in field surveys can provide insight as to whether detection of low-density, asymptomatic P. falciparum infections using sensitive molecular methods can sufficiently identify the majority of infected individuals who are potentially capable of onward transmission.
In this study, we determined the prevalence and density of asymptomatic P. falciparum infections among children and adults in three communities of western Kenya that differed in transmission intensities using quantitative molecular assays. To better estimate the relationship between asexual parasite densities and gametocyte densities, we sought to develop a multiplex quantitative reverse-transcriptase PCR assay that could detect asexual-stage specific, female gametocyte-specific, and male gametocyte-specific genes in a single blood sample. Results were compared to microscopy and an established quantitative PCR-based diagnostic assay.

METHODS
Ethics Statement. The study was reviewed and approved by the Kenya Medical Research Institute Ethics Review Committee and the Indiana University Institutional Review Board. Written informed consent was obtained from a parent or guardian of participants who were minors and from adult participants. Minors ≥ 15 years of age provided their own written informed assent, accompanied by written consent of a parent or guardian.

Study Sites and Study Participants.
The study was conducted from August to September 2016 at three sites in western Kenya that differed in malaria transmission intensity. Ajigo, located in the lowland area of Siaya County, where malaria transmission is typically intense [13]. Webuye Town, a town located in Bungoma County, exhibits moderate, perennial transmission with seasonal peaks from May to June, and the study area has been previously described [14][15][16]. Kipsamoite and Kapsisywa, two adjacent highland communities in Nandi County, have low and unstable malaria transmission that has recently been described in detail [17,18]. Healthy participants aged 1 to 85 years were sampled from a randomized community census of households for each site and enrolled over a four-week period. A brief questionnaire that included gender, age, recent travel history within the last month, recent use of ITNs, and relevant medical history was administered. Exclusion criteria at enrollment were axillary temperature ≥ 37.5°C, known acute systemic or chronic illness, use of antimalarial or immunosuppressive medications within 30 days prior, or pregnant at time of survey. Blood Collection. Drops of blood were collected by fingerprick for RDT (Paracheck Pf; Orchid Biomedical Systems), whole-blood RNA, thick and thin blood smears, and dried blood spots (DBS) on filter paper (903 Protein Saver; Whatman). Individuals who tested positive for asymptomatic P. falciparum infection by RDT were . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted April 6, 2021. ; treated at the point-of-care using the standard regimen recommended by the Ministry of Health in Kenya. For whole-blood RNA, 200 μl of peripheral fingerprick blood was collected using capillary blood collection tubes containing EDTA (Microvette CB300 K2E; Sarstedt) and transferred immediately in cryotubes pre-filled with 400 μl Tempus solution (Applied Biosystems). Filled sample tubes were agitated vigorously per the manufacturer's instructions and stored at -80°C within 24 hours of collection until use.
Microscopy. Giemsa-stained blood smears were examined for the presence of asexual parasites in 200 fields using the 100x oil immersion objective lens by two trained microscopists. Independent verification was performed by a third reader for samples that were discordant between the first two microscopists. For positive samples, the number of asexual parasites per 200 leukocytes was multiplied by 40 to convert to parasites per μl, assuming an average leukocyte count of 8,000 leukocytes per μl of blood.
Parasite Culture. To produce parasite gDNA for use in standard curves for parasite density determination, P. falciparum 3D7 parasites (Malaria Research and Reference Reagent Resource Center [MR4], BEI Resources) were cultured in vitro using standard techniques [19] with two rounds of synchronization by sorbitol treatment to achieve a high parasitemia. Ring and early trophozoite stage P. falciparum parasites were 10-fold serially diluted in whole blood of an uninfected North American donor to obtain a final density of 440,000 down to 0.44 parasites/µl and spotted on 903 Protein Saver cards. To produce parasite RNA for use in standard curves for gametocyte density estimates, P. falciparum NF54 (MR4) in vitro cultures were enriched for gametocytes by decreasing asexual parasitemia [16]. Total gametocytes (without differentiating for sex) were counted and 10fold serially diluted in whole blood to obtain a final density of 2580 down to 0.00258 gametocytes/μl immediately prior to RNA stabilization with Tempus solution at a 1:2 ratio. Asexual parasites (rings, trophozoites, and schizonts) were also counted in the same culture to allow parasite quantification using asexual-stage specific targets.
DNA and RNA isolation. Total DNA was extracted from three 0.32 cm diameter circles punched from each DBS using the QIAamp 96 Blood Kit (Qiagen, Valencia, CA) per the manufacturer's instructions and eluted in 50 µl Tris-EDTA buffer. RNA was extracted from whole-blood RNA in Tempus using Norgen RNA extraction kit (Norgen Biotek, Thorold, Ontario) and treated with RNase-Free DNase I Kit (cat 25710) to a final elution volume of 50 µl, per manufacturer's instructions. Extracted RNA samples were assessed for quality and quantity using automated parallel capillary electrophoresis (Fragment Analyzer System, Agilent).

Real-time quantitative PCR using genomic DNA
To detect the presence of P. falciparum genomic DNA isolated from the DBS, we adapted primers targeting P. falciparum varATS that were originally designed for use in a Taqman-based qPCR assay [20] for use with the PowerUp SYBR Green Master Mix System (Thermo Fisher Scientific, Waltham, Massachusetts) ( Table S1). Samples were assayed in triplicate in 384-well plates on a QuantStudio 6 Flex Real Time PCR System (Thermo Fisher Scientific, Waltham, Massachusetts) using standard cycling conditions and a melt curve analysis. During assay development, we verified that wells with a first calculated melt temperature (Tm1) >71.14°C contained varATS amplicons by Sanger sequencing, whereas wells with Tm1 <71.14°C contained primer dimers. Subsequently, we set the criteria for a P. falciparum positive sample as having ≥2 of 3 replicate wells with a Ct <39 AND a Tm1 >71.14°C. Using these criteria, genomic DNA samples isolated from the blood of 20 of 20 (100%) healthy North American controls with no malaria exposure history were confirmed to be P. falciparum negative, and 86 of 87 (98.9%) samples positive by conventional P. falciparum 18s rRNA PCR [21,22] were confirmed as positive with the modified varATS-based assay. The one discordant sample had only 1 of 3 replicate wells meeting the Ct and Tm1 criteria. A standard curve of gDNA extracted from serially diluted P. falciparum-spiked DBS samples (described above) and no-template negative controls were run on every plate, which allowed for estimation of parasite densities using Ct values.

Multiplex real-time quantitative reverse transcription PCR
We initially sought to develop a four-plex real-time quantitative reverse transcription PCR (RT-qPCR) that would detect female gametocytes, male gametocytes, and asexual parasites, as well as a human housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which served as a control for RNA extraction and relative quantification. The genes pfs25 [23] and pfs230p were used as the female-and male-specific gametocyte . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 6, 2021. targets, respectively. The gene encoding for parasite-infected erythrocyte surface protein (piesp2, also called PFE60 and PF3D7_0501200) was chosen based on a transcriptional pattern restricted to asexual blood-stages, particularly trophozoites, in three P. falciparum gene expression datasets available on PlasmoDB (http://plasmodb.org) [24][25][26]. Primers and probes for pfs25 were adapted from Wampfler et al [27]. Primers and probes for pfs230p and piesp2 were developed de novo using Primer 3 software [28] following standard guidelines for qPCR primer design. All primer and probes are listed in Table S1.
After generation of cDNA from 50 ng RNA for each sample replicate using LunaScript RT Supermix (New England Biolabs) under standard cycling conditions, 20X triplex master mix was prepared from appropriate final concentrations of primers and probes for the three parasite targets and combined with 20X human GAPDH master mix (Applied Biosystems), Taqman multiplex master mix (Applied Biosystems), and cDNA to a final reaction volume of 10 μl. Field samples identified a positive for P. falciparum by varATS qPCR, no reverse transcriptase controls, amplification controls, and 10-fold parasite RNA dilution standards were assayed in triplicate in 384-well MicroAMP Optical PCR plates (Applied Biosystems). The targets pfs25, pfs230p, piesp2, and human GAPDH were run in a QuantStudio6 Flex qPCR system (Applied Biosystems) with NFQ-MGB Quencher and VIC, FAM, ABY, and JUN reporter dyes, respectively (Table S1). Mustang Purple was selected as the reference dye. Multiplex assay was run under standard cycling conditions: initial denaturation at 95.0°C for 20s (hold stage) followed by 40 cycles of 95.0° C for 1s and 60.0°C for 20s (PCR stage).
Statistical analysis. All statistical analysis was performed using R version 4.0.1(https://www.r-project.org). Multiple logistic regression was performed with PCR-confirmed gametocytemia as the dependent variable and gender, age (in years), recent bednet use, recent travel, log10 transformed parasite density, and community as independent variables. Plots were rendered using the ggplot2 package. Statistical tests used to determine significance are indicated in tables and figure legends, and p values <0.05 were considered significant.

RESULTS
A total of 1,354 participants were enrolled across all communities for this study ( Table 1). The RDTs used for point-of-care diagnosis of asymptomatic infections demonstrated a 4.7% false positive rate using varATS qPCR as the reference standard. In contrast, microscopy showed no false positives. Given this, RDT data was not used for subsequent analyses. The prevalence of asymptomatic infections was greatest in Ajigo, followed by Webuye, regardless of diagnostic modality ( Table 1). Only two asymptomatic infections were detected by PCR in Kapsisywa, and no infections were detected in Kipsamoite. Parasite densities were not statistically different across the four communities ( Table 1) and did appear to vary with infection prevalence (Figure S1). Given the similarities in prevalence of asymptomatic infections in the two highlands communities Kapsisywa and Kipsamoite, they were treated as a single community "Kap-Kip" for all subsequent analyses.
Across all communities, children aged 11-15 years account for the greatest proportion total and sub-microscopic asymptomatic infections ( Figure 1A). In contrast to the younger age groups, in which the majority of infections are detectable by microscopy, 68% of asymptomatically infected adults >21 years of age have sub-microscopic parasitemia (Figure 1A), which suggests acquisition of blood-stage immunity [29,30]. Similar findings were observed in Ajigo and Webuye when asymptomatic infection prevalence was separated by community with the notable observation that in individuals aged 6-20 years the majority of asymptomatic infections in Ajigo were detectable by microscopy, whereas in Webuye, the majority of infections in this age range were sub-microscopic ( Figure 1B).
For individuals identified as parasitemic by varATS qPCR, we sought to further quantify both asexual parasite densities and sexual parasite densities within the same sample by four-plex RT-qPCR (see Methods). We quantified female and male gametocytes using qRT-PCR targeting pfs25 and pfs230p targets, respectively, and identified 122 of 1354 (9.0%) as having gametocytemia based on the quantifiable expression of either gene. Among individuals with varATS-detectable parasitemia (n=122), there were no significant differences in gender distribution, age, use of ITNs, history of recent travel, or site distribution between those with and without gametocytes by univariate analysis ( Table 2). To determine the relationship between gametocytemia and total parasitemia, we plotted female and male gametocyte densities estimated by RT-qPCR against estimated asexual parasite density. We initially planned to use parasite densities estimated from piesp2 Ct values obtained from the same multiplex RT-qPCR assay, which would maintain internal consistency for each sample. However, . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 6, 2021. ; piesp2-derived parasite densities demonstrated poorer correlation with microscopy-determined parasite densities in patent infections and less sensitivity than gDNA-based detection using varATS ( Figure S2). Thus, we used total parasite densities derived from the varATS-based assay to approximate asexual parasite densities for the remainder of the study. We included varATS-estimated total parasite densities in a multiple logistic regression model that revealed a decreased risk of gametocytemia in the lower transmission communities relative to the high-transmission community of Ajigo and among individuals who reported recent travel ( Table 3). As expected, increased total parasite densities greatly increased the likelihood of gametocytemia independent of site ( Table 3).
A substantial proportion (41.7%) of individuals with potential for onward transmission, defined in our study as having at least 1.25 female and four male gametocytes per 2.5 µl of blood (thresholds adapted from a prior study [12] to account for sex-specific gametocytemia overestimation), had qPCR-estimated parasite densities above the detectable limit of conventional, 18s ribosomal RNA-based nested PCR (1 parasite per µl) [21] and below the limit of detection of microscopy (40 parasites per µl), which corresponded well to the actual proportion potential transmitters with submicroscopic infections (36.9%; Figure 3A-B). These data provide evidence of a substantial sub-patent infectious reservoir among asymptomatic carriers in these communities but also demonstrates that the vast majority of infections capable of onward transmission can be detectable with conventional molecular diagnostics [12].

DISCUSSION
The current descriptive study provides a cross-sectional assessment of asymptomatic P. falciparum infections of three communities in western Kenya with differing malaria transmission intensities from August to September 2016. [12]. We confirmed that transmission intensity remained low in Kap-Kip [17,31], where asymptomatic P. falciparum parasitemia was rarely detected by varATS qPCR (0.22% prevalence), and established that high malaria transmission occurs in Ajigo, where 42% of individuals had asymptomatic parasitemia. Webuye demonstrated moderate transmission with 10% prevalence of asymptomatic parasitemia, which is lower than what has been previously described at this site [32,33], possibly reflecting micro-heterogeneity or seasonal differences, as our study was performed during months when rainfall is historically lower in western Kenya.
Our data showing the substantial reservoir among 6-15 year old children in Ajigo and Webuye is consistent with a prior study in the Kakamega district of western Kenya that showed PCR-confirmed asymptomatic P. falciparum infections were more prevalent in younger children age 5-14 years (~34%) relative to older children >14 years (~9%) [34] but contrasts with a study conducted in a high transmission area (Suba district) that demonstrated the greatest prevalence of blood-smear positive asymptomatic infections in young children <5 years (74% vs. 30-50% in older children) [35]. The differences in relative contribution to the asymptomatic infectious reservoir by age groups may be attributable to intense malaria transmission in Suba, where clinical immunity may be acquired more rapidly, and differences in assay sensitivity.
To determine whether sensitive molecular assays can sufficiently detect the majority of individuals carrying lowdensity P. falciparum infections who are also potentially capable of onward transmission, we assessed the relationship between asexual parasitemia and gametocytemia. We initially intended to correlate gametocyte densities with asexual parasite densities using a multiplex RT-qPCR that would contain targets specific to female gametocytes, male gametocytes, and asexual blood-stage parasites in a single assay, which we had hoped would facilitate comparisons as this strategy eliminates both within subject differences in template preparation and assay variability. However, parasite densities determined using the chosen asexual-specific target piesp2, which encodes for parasite-infected erythrocyte surface protein and previously shown to be maximally transcribed in the trophozoite stage in laboratory isolates [24][25][26]36], showed weaker correlation with microscopy-determined parasite density than densities derived from varATS qPCR using gDNA (Figure S2). The weaker correlation for piesp2 could be due to lower piesp2 expression in ring stages, which had previously been thought to be the predominant asexual form of P. falciparum found in peripheral circulation. However, a recent study in Mali revealed that more developed trophozoite stages were commonly found in asymptomatic P. falciparum infections [37]. Expression of piesp2 could also vary among the field isolates, perhaps due to differential transcriptional regulation related to precise stage at the time of collection or host immune pressure. Although speculative, these potential explanations are intriguing given that antibodies against PIESP2 associate with protection from malaria [38], and PIESP2 has recently been observed to bind to brain microvascular endothelial cells in vitro to induce an inflammatory response [39]. Our data, combined with these prior findings, suggest that piesp2 is a poor target for quantifying asexual parasite densities.
Our observation that a sizable proportion of low-density infections (<40 parasites/μl) had estimated gametocyte densities that would favor onward transmission is consistent with prior studies that demonstrated a considerable sub-microscopic infectious reservoir [4,10,40,41]. Although we only determined the presence of gametocytemia among individuals who were parasitemic by varATS qPCR, which had a limit of detection of ~0.4 parasites/μl using dried blood spots, our data also shows that the proportion of individuals with potential for onward transmission drops off below 10 parasite/μl. This finding is in line with recent studies suggesting that mosquito infectivity occurs primarily when parasitemia is >1 parasite/μl [12,42], which is the limit of detection of standard molecular diagnostics. Taking together, the main implication is that ultra-sensitive molecular diagnostics capable of detecting infections <1 parasite/μl may not be necessary to achieve significant reductions in malaria transmission using a screen-and-treat strategy.
There are several limitations to our study. The cross-sectional study design provides only a snapshot of infection prevalence in these communities during the relatively dry season and our observations may not be generalizable to the rainy season when malaria transmission is more intense. We estimated gametocyte densities using molecular quantification of male and female gametocyte-specific gene expression as a surrogate of potential for onward transmission and did not directly measure mosquito infectivity using direct or indirect feeding assays. Such a surrogate based solely on gametocyte density along ignores the relative contribution of anti-gametocyte immunity in reducing malaria transmission [43]. Although we use male and female gametocyte targets for gametocyte quantification, we did not differentiate gametocyte sex when determining gametocyte densities by microscopy for our standard curves, which would lead to overestimates of sex-specific gametocytemia. This is especially true for male-specific gametocytemia given that natural infections are biased towards females, with 3-5 times more females [44]. However, we made no assessments using sex ratio in this study. Furthermore, gametocytemia overestimates would affect all samples consistently and thus would not affect our ranked correlation analyses. Importantly, in our determination of number of individuals capable of onward transmission, we adjusted for sex-specific overestimates by using higher thresholds for minimum male and female gametocyte densities.
In summary, our cross-sectional survey of the prevalence and densities of P. falciparum infections among asymptomatic individuals in western Kenya provides an assessment of the relationship between parasitemia and gametocytemia in three communities with different transmission intensities. Experimental studies are needed to definitively determine whether the low-density infections in communities such as Ajigo and Webuye contribute significantly to malaria transmission.
. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.   potential and unlikely transmitters by parasite density and blood smear positivity. Individuals with potential for onward transmission are defined in as having at least 1.25 female and four male gametocytes per 2.5 µl of blood, which are increased thresholds adapted from a prior study [12] to account for sex-specific gametocytemia overestimation.
. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted April 6, 2021. ; . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

TABLES
The copyright holder for this preprint this version posted April 6, 2021. ; Table 3. Multiple logistic regression to assess the risk of gametocytemia