Ethical statement
Approval for the study was obtained from Noguchi Memorial Institute for Medical Research (NMIMR) IRB. Permission was also sought from Ghana Education Service and written consent were obtained from parents or guardians prior to recruitment as stated and described in previous studies [5, 14].
Study site and sample collection
The longitudinal study enrolled school children aged between 6 and 12 years old, living in Obom and Abura in southern Ghana. Characteristic features of Obom, a town with high and perennial malaria transmission and Abura, a town with low and seasonal malaria transmission have been previously described [5]. This study used samples from 65 children from Obom and 72 children from Abura, who were present during the July 2015 (Peak season), October 2015 (end of the peak season) and January 2016 (off peak season) sample collection time points. At each time point, 2.5 ml of venous blood was collected into EDTA vacutainer tubes. A drop of blood (about 10 µl) was used to prepare thick and thin blood smears, after which the blood was separated via centrifugation into plasma and packed cells. The plasma was collected into a 1.5 ml Eppendorf tube and used for the IgG and relative avidity ELISAs. An aliquot (50 µl) of the packed cells were stored in 250 µl of Trizol reagent (Invitrogen, USA) and used for RNA extraction and subsequent cDNA preparation.
Microscopic examination of blood smears
Staining of the thick and thin blood smears were as previously described [6] and according to World Health Organization (WHO) guidelines [15]. Parasite density (PD) was estimated based on visual counting of the number of infected red blood cells counted per 200 white blood cells, as recommended by the WHO [16]. Two independent microscopists read each slide.
Molecular assays
Extraction of RNA and cDNA preparation
RNA extraction was done using the Quick RNA miniprep kit (Zymo Research, USA) that included an on-column DNase 1 treatment prior to elution according to manufacturer’s instructions as previously reported [17]. The purity and concentration of the extracted RNA was checked using a Nanodrop 2000c spectrophotometer (Thermo Fisher Scientific, USA).
Extracted RNA was converted into cDNA using the Protoscript II first strand cDNA synthesis kit (NEB, UK) according to manufacturer’s instructions. Briefly, a mix of 2.5 µM oligo dT (NEB, UK) and 3.0 µM Random primer mix (NEB, UK) were added to 6 µl of the extracted RNA and incubated at 65 °C for 5 min. Subsequently, 10 µl of 1× Protoscript II reaction mix and 2 µl of 1× Protoscript II enzyme mix were added to the mixture and incubated for 60 min at 42 °C and then for 5 min at 80 °C. The resulting cDNA was stored at − 20 °C until further use.
Pfg377 genotyping
Gametocyte diversity was assessed at the Pfg377 locus in genomic DNA (gDNA), cDNA and RNA samples using a previously published protocol [8]. The nested PCR of the Pfg377 gene was performed using the GOTaq hot start master mix (Promega Corporation, USA). The amplification for both primary and secondary PCR reactions contained 1× GoTaq master mix and 20 µM of the primers (Additional file 1). The volume of template used for gametocyte diversity was 1 µl of cDNA, whilst 3 µl of RNA was used to check for gDNA contamination of the RNA samples. Nuclease free water template was used as negative control and each sample corresponding gDNA as well as cDNA from laboratory cultured gametocytes from the 3D7 P. falciparum parasite isolate were used as positive controls for the reactions. The reactions cycling conditions for both RNA, DNA and cDNA comprised of an initial denaturation at 94 °C for 2 min, 30 cycles (94 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s), 72 °C for 5 min, and then 4 °C on hold.
The amplified PCR products were loaded onto a 2% agarose gel stained with ethidium bromide and electrophoresed for 30 min at 100 V. Resolved amplicons were visualized using a UV trans-illuminator (Vilber, Germany). The absence of amplified PCR products on the gel in the lanes containing RNA template serve as an indication that gDNA was not carried over during the RNA extraction and purification processes.
Immunological assays
Naturally induced antibody responses against P. falciparum gametocyte antigen Pfs230 in the plasma collected from children were determined using ELISA as previously described [5, 6]. Plasma samples that had previously been identified as containing high levels of Pfs230 [5, 6] were pooled and used as a positive control. Plasma from malaria naïve individuals from Denmark served as negative control samples. Recombinant IgG, (BP055, the Binding Site) was used as the standard calibrator for total IgG measurements. The Pfs230 antigen was diluted to 1 µg/ml in carbonate buffer, pH 9.0 and coated at 100 µl/well onto ELISA plates at 4 °C overnight. The measured OD values were converted into concentration using the ADAMSEL software (Ed Remarque).
Avidity ELISA
The relative avidity of IgG responses to Pfs230 was determined as previously reported [18]. The procedure was similar to the standard ELISA protocol detailed above, with the exception of an additional step where 100 µl of 2.4 M sodium thiocyanate (NaSCN) was add to the plate and incubated for 10 min. The plates were subsequently washed 4 times with wash buffer and then incubated with the secondary antibody. The proceeding processes were identical to that of the ELISA described above.
Data analysis
The number of infected red blood cells observed per 200 white blood cells (WBC) counted on the thick smear was recorded and converted into parasite density (PD) by multiplying the number by 40, based on the assumption of 8000 WBCs μl per blood.
ELISA data was analysed using the ADAMSEL software, which converts optical density (OD) values into weighed concentrations (wConcs), which represents the antibody (IgG) responses to the antigen. Graphs were plotted using GraphPad Prism version 5. Kruskal–Wallis test (GraphPad prism) was used to determine whether there were any statistically significant differences between the parasite density measured across the towns and between visits. This was followed by the Dunn’s multiple comparison test (a post hoc test), to find which specific means are significant from others (p < 0.05). Column statistics (GraphPad prism) was used to determine the medians and inter-quartile ranges of the wConcs (IgG levels) in order to determine the measure of dispersion. The mean wConcs (Antibody (IgG) responses) were compared between the sites using nonparametric Mann–Whitney U test (paired two tailed t-test). Independent samples T-test was used when comparing the ages of the children, PD and gametocyte prevalence. P-values less than 0.05 were considered significant.
The relative avidity index (RAI) for anti-Pfs230 IgG was calculated as the ratio of the OD of the sodium thiocyanate-treated sample (numerator) to the OD of the untreated sample (denominator) multiplied by 100. (RAI = [Sodium thiocyanate (NaSCN) treated IgG/untreated IgG] × 100).
