Study design and changes in the prevalence of microscopic P. falciparum infection following the IRS and SMC interventions in Bongo, Ghana.

(A) Four age-stratified cross-sectional surveys of ∼2,000 participants per survey were conducted in Bongo, Ghana at the end of the wet seasons in October 2012 (Survey 1, baseline pre-IRS, red), October 2014 (Survey 2, during IRS, orange), October 2015 (Survey 3, post-IRS, green), and October 2017 (Survey 4, SMC, purple) (Table supplement 1) (see Materials and Methods). The three rounds of IRS (grey areas) were implemented between 2013 and 2015 (Tiedje et al., 2022). SMC was distributed to all children < 5 years of age during the wet seasons in 2016 (two rounds between August – September 2016) and 2017 (four rounds between September – December 2017) (Gogue et al., 2020). Both IRS and SMC were implemented against a background of widespread LLIN usage (Tiedje et al., 2022). Prevalence of microscopic P. falciparum infections (%) in the (B) study population and (C) for all age groups (years) in each survey. Error bars represent the upper and lower limits of the 95% confidence interval (CI) calculated using the Wald Interval.

Sharing of upsA and non-upsA DBLα types among the DBLα isolate repertoires in 2012 (pre-IRS, red), 2014 (during IRS, orange), 2015 (post-IRS, green), and 2017 (SMC, purple).

The overlapping density and violin plots (upper right-hand corners) show the distribution of PTS scores (i.e., DBLα isolate repertoire similarity) between the (A) upsA and (B) non-upsA DBLα isolate repertoires for those isolates with DBLα sequencing data (Table supplement 2) in each survey. The PTS scales in the density plots have been zoomed-in to provide better visualization of the upsA and non-upsA DBLα types PTS distributions. The colored dashed lines in the density plots indicate the median PTS scores in each survey for the upsA (2012 (red) = 0.078, 2014 (orange) = 0.063, 2015 (green) = 0.054, and 2017 (purple) = 0.064) and non-upsA (2012 (red) = 0.020, 2014 (orange) = 0.013, 2015 (green) = 0.013, and 2017 (purple) = 0.016) DBLα types. Note: The non-upsA median PTS values in 2014 (orange) and 2015 (green) were both 0.013 and overlap in the figure. In the PTS violin plots the central box plots indicate the medians (centre line), interquartile ranges (IQR, upper and lower quartiles), whiskers (1.5x IQR), and outliers (points).

MOIvar distributions in 2012 (pre-IRS, red), 2014 (during IRS, orange), 2015 (post-IRS, green), and 2017 (SMC, purple) based on pooling the maximum a posteriori MOI estimates.

Estimated MOIvar distributions for the (A) study population and (B) for all age groups (years) in each survey for those isolates with DBLα sequencing data (Table supplements 2 and 7). The median MOIvar values are indicated with the black dashed lines and have been provided in the top right corner (median MOIvar value [interquartile range, upper and lower quartiles]) along with the percentage of P. falciparum infections that were multiclonal (MOIvar > 1) in each survey and age group (years).

Estimated number and relative change in the number of P. falciparum var repertoires in 2012 (pre-IRS, red), 2014 (during IRS, orange), 2015 (post-IRS, green), and 2017 (SMC, purple).

The estimated number of var repertoires (i.e., census population size) for those isolates with DBLα sequencing data (Table supplements 2 and 7) in the (A) study population and (B) for all age groups (years). The estimated number of var repertoires vs. P. falciparum prevalence for (C) study population and (D) for all age groups (years) (Table supplement 7). The percentage change in P. falciparum prevalence (black dotted line) and the estimated number of var repertoires (black solid line) in 2014, 2015, and 2017 compared to the 2012 baseline survey (red dashed horizontal line at 0% change) for the (E) study population and (F) for all age groups (years). Error bars in (A-D) represent the upper and lower limits of the 95% confidence intervals (95% CIs). The 95% CIs for the number of var repertoires (i.e., census population size) were calculated based on a bootstrap approach. We resampled 10,000 replicates from the original population-level distribution with replacement. Each resampled replicate has the same size as the original sample. We then derive the 95% CI based on the distribution of the resampled replicates. The 95% CIs for P. falciparum prevalence (%) were calculated using the Wald Interval.

UpsA and non-upsA DBLα type richness in 2012 (pre-IRS, red), 2014 (during IRS, orange), 2015 (post-IRS, green), and 2017 (SMC, purple).

Number of unique (A) upsA and (B) non-upsA DBLα types (i.e., richness) observed in each survey vs. P. falciparum prevalence based on those isolates with DBLα sequencing data (Table supplement 2). Error bars represent the upper and lower limits of the 95% confidence intervals (95% CIs) for the P. falciparum prevalence (%; x-axis) and ± 2 standard deviations (± 2SD) for the number of unique upsA and non-upsA DBLα types (y-axis). The 95% CIs for P. falciparum prevalence (%) were calculated using the Wald Interval. The ± 2SD for the number of unique upsA and non-upsA DBLα types were calculated based on a bootstrap approach. We resampled 10,000 replicates from the original population-level distribution with replacement. Each resampled replicate has the same size as the original sample. We then derive the standard deviation (SD) based on the distribution of the resampled replicates.

UpsA and non-upsA DBLα type frequencies and survival in 2012 (pre-IRS, red), 2014 (during IRS, orange), 2015 (post-IRS, green), and 2017 (SMC, purple).

Heatmaps showing the patterns of diversity for the (A) upsA and (B) non-upsA DBLα types. The columns represent all the upsA and non-upsA DBLα types observed in the four surveys, and the rows represent each of the 2,802 upsA DBLα types and the 50,436 non-upsA DBLα types (Table supplement 2). White rows are used to denote the absence of a specific DBLα type, while the presence of a DBLα type is indicated in colour and further categorised (colour gradations) based on the frequency or the number of times (i.e., number of isolates) a DBLα type was observed in each survey (Frequency categories: 1, 2-10, 11-20, > 20 isolates; Note the frequency category cut-offs were chosen based on the frequency distributions in Figure supplement 7). The proportions of (C) upsA and (D) non-upsA DBLα types in each survey based on the number of times (i.e., number of isolates) they were observed in each survey. Kaplan-Meier survival curves for the (E) upsA and (F) non-upsA DBLα types across time (2012 to 2017) categorised based on their frequency at baseline in 2012 (pre-IRS, red). The colored shaded areas represent the upper and lower limits of the 95% confidence intervals (95% CIs) with the number (N) of upsA and non-upsA DBLα types in each frequency category are provided in parenthesis. These survival curves include only those upsA (N = 2,218) and non-upsA (N = 33,159) DBLα types that were seen at baseline in 2012 (pre-IRS) as indicated in red (Table supplement 2). The x-axis indicates time where time “0” denotes 2012 (pre-IRS), “1” denotes 2014 (during IRS), “2” denotes 2015 (post-IRS), and finally “3” denotes 2017 (SMC). Note: In the survival curves the 11-20 and > 20 frequency categories for both the (E) upsA and (F) non-upsA DBLα types overlap in the figure.