The prevalence and density of asymptomatic Plasmodium falciparum infections among children and adults in three communities of western Kenya

The study was reviewed and approved by the Kenya Medical Research Institute Scientific and Ethics Review Unit 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 aged 13–17 years provided their own written informed assent, accompanied by written consent of a parent or guardian.

The study was conducted from August to September 2016 at three sites in western Kenya that differed in malaria transmission intensity. This time period was specifically chosen as it was one month after the primary malaria transmission peak but before the secondary peak for all three sites and thus increased the likelihood of recruiting individuals who had not experience symptomatic malaria within the last 30 days. Ajigo is located in the lowland area of Siaya County, where malaria transmission is intense and perennial, but with a seasonal peak from May to July [13, 14]. The Webuye township is in Bungoma County, which exhibits moderate, perennial transmission with a primary seasonal peak in May to June and a smaller, second peak in October [15‐17]. Kapsisywa and Kipsamoite are two adjacent highland communities in Nandi County with low and unstable malaria transmission. Transmission for these two sites is highly variable, with a typical primary peak in May to July and an occasional smaller peak in November to December that varies from year to year [18, 19]. Intended recruitment targets were 200 participants in each of the moderate-to-high-transmission sites (the Matulo sublocation of Webuye and Ajigo) and 450 participants in each of the low-transmission sites (Kapsisywa and Kipsamoite). Sample sizes of 200, 450, and 900 participants provided 95% confidence of detecting within 20% of the true proportion of asymptomatic infections if the unknown prevalence was estimated to be 43%, 25%, and 14%, respectively. Healthy participants aged 1 to 85 years were recruited from a randomized community census of households for each site in an age-stratified manner to ensure adequate sample sizes for each age group, with a maximum of two participants for any household. Recruitment and enrollment occurred over a -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 or history of fever, acute symptomatic illness, underlying chronic disease, malaria in the last 30 days, use of anti-malarial or immunosuppressive medications in the last 30 days, or pregnancy.

Drops of blood were collected by fingerprick for Paracheck Pf RDT (Orchid Biomedical Systems), which detects the presence of P. falciparum histidine-rich protein 2 in blood specimens; whole-blood ribonucleic acid (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 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 Tris-ethylenediaminetetraacetic acid (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 h of collection until use.

Giemsa-stained blood smears were examined for the presence of asexual parasites in 200 fields using the 100× oil immersion objective lens by two trained microscopists. Independent verification was performed by a third reader for samples that were qualitatively discordant for positivity 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 8000 leukocytes per μl of blood. The mean parasite density from the two concordant microscopists were used for analysis.

To produce parasite genomic deoxyribonucleic acid (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 [20] with two rounds of synchronization by sorbitol treatment to achieve a high parasitaemia. Ring and early trophozoite stage P. falciparum parasites were tenfold 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 parasitaemia [17]. Total gametocytes (without differentiating for sex) were counted and tenfold 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.

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 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 (Norgen Biotek) 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).

To detect the presence of P. falciparum genomic DNA isolated from the DBS, primers targeting P. falciparum varATS that were originally designed for use in a Taqman-based qPCR assay [21] were adapted for use with the PowerUp SYBR Green Master Mix System (Thermo Fisher Scientific, Waltham, Massachusetts) (Additional file 1: 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, PCR products were Sanger sequenced to verify that wells with a first calculated melt temperature (Tm1) > 71.14 °C contained varATS amplicons and wells with Tm1 < 71.14 °C contained primer dimers. Subsequently, the criteria for a P. falciparum positive sample were set 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 [22, 23] 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.

An initial aim of the study was 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 [24] and pfs230p were used as the female- and male-specific gametocyte 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) [25‐27]. Primers and probes for pfs25 were adapted from Wampfler et al. [28]. Primers and probes for pfs230p and piesp2 were developed de novo using Primer 3 software [29] following standard guidelines for qPCR primer design. All primer and probes are listed in Additional file 1: Table S1.

After generation of complementary DNA (cDNA) from 50 ng RNA for each sample replicate using LunaScript RT Supermix (New England Biolabs) under standard cycling conditions, 20× triplex master mix was prepared from appropriate final concentrations of primers and probes for the three parasite targets and combined with 20× 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 as positive for P. falciparum by varATS qPCR, no reverse transcriptase controls, amplification controls, and tenfold 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 (Additional file 1: 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 20 s (hold stage) followed by 40 cycles of 95.0 °C for 1 s and 60.0 °C for 20 s (PCR stage).

All statistical analyses were performed using R version 4.0.1 (https://​www.​r-project.​org). Sample size estimates were performed using the epiR package. Multiple logistic regression was performed with PCR-confirmed gametocytaemia as the dependent variable and gender, age (in years), recent bed net 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.