High Genetic Diversity of Plasmodium falciparum in the Low-Transmission Setting of the Kingdom of Eswatini

Abstract Background To better understand transmission dynamics, we characterized Plasmodium falciparum genetic diversity in Eswatini, where transmission is low and sustained by importation. Methods Twenty-six P. falciparum microsatellites were genotyped in 66% of confirmed cases (2014–2016; N = 582). Population and within-host diversity were used to characterize differences between imported and locally acquired infections. Logistic regression was used to assess the added value of diversity metrics to classify imported and local infections beyond epidemiology data alone. Results Parasite population in Eswatini was highly diverse (expected heterozygosity [HE] = 0.75) and complex: 67% polyclonal infections, mean multiplicity of infection (MOI) 2.2, and mean within-host infection fixation index (FWS) 0.84. Imported cases had comparable diversity to local cases but exhibited higher MOI (2.4 vs 2.0; P = .004) and lower mean FWS (0.82 vs 0.85; P = .03). Addition of MOI and FWS to multivariate analyses did not increase discrimination between imported and local infections. Conclusions In contrast to the common perception that P. falciparum diversity declines with decreasing transmission intensity, Eswatini isolates exhibited high parasite diversity consistent with high rates of malaria importation and limited local transmission. Estimates of malaria transmission intensity from genetic data need to consider the effect of importation, especially as countries near elimination.


Maxima Window
Final filter to remove close peaks. For each peak identified, take only largest peaks that fall within window size of each other 10 Relative Maximum Window Number of points on each side of prospective peak to identify relative maximum 6 Smoothing Window Window size passed to the Savitzky-Golay filter 11 Smoothing Order Order of the polynomial used in the Savitzky-Golay filter to smooth the data 7 Tophat Factor Size factor to apply the white-tophat filter. Tophat filter footprint size is (Tophat Factor)*(length(signal)) 0.005 Filtering Parameters Min Peak Height Peaks with peak height lower than Min Peak Height are filtered out 300 Max Peak Height Peaks with peak height greater than Max Peak Height are filtered out 40000 Min Peak Height Ratio Secondary peaks with a height ratio compared to the primary peak less than Min Peak Height Ratio are filtered out 0

Max Bleedthrough Ratio
Let signal strength be the sum of the signal from -1 to +1 indices of a peak. Bleedthrough ratio is defined as the peak signal strength divided by the maximum signal strength of other channels in the same well at the same peak index. Peaks with a bleedthrough ratio greater than Max Bleedthrough Ratio are filtered out 10

Max Crosstalk Ratio
Crosstalk ratio is defined as the peak signal strength divided by the maximum signal strength of other channels in the same color in surrounding wells (surrounding wells defined as surrounding capillaries as they feed into the machine, not surrounding wells within the plate.) 10

Min Peak Distance
Peaks that are less than Min Peak Distance away (in nucleotides) are filtered out, leaving only the tallest peaks 2.5 Genotyping Parameters

Min Relative Peak Height
Peaks with a height relative to the tallest peak that do not exceed Min Relative Peak Height are classified as artifact 0.03 Min Absolute Peak Height Minimum peak height for a peak to not be classified as artifact 500 (1000) Bleedthrough Limit Maximum bleedthrough ratio before a peak is classified as bleedthrough 2 Crosstalk Limit Maximum crosstalk ratio before a peak is classified as crosstalk 2 Failure Threshold At least one peak's height must exceed the failure threshold, otherwise the run is deemed a failure and rejected 800 Soft Artifact SD Limit Peaks with a height that falls within the Soft Artifact SD Limit times the artifact error plus the estimated artifact contribution have a peak probability calculated when probabilistic annotation is enabled 6 Hard Artifact SD Limit Peaks with a height minus (artifact contribution + artifact error * Hard Artifact SD Limit) < Min Absolute Peak Height are classified as artifact 0 Genotyping Probability Threshold Peaks with a probability less than the Genotyping Probability Threshold are classified as artifact 0.95

Iterative Probability Threshold
Peaks with a probability less than the Bootstrap Probability Threshold are culled during the iterative peak frequency and sample MOI estimation steps during probabilistic peak annotation 0.99 Off-scale Threshold Runs with a peak that exceeds the Off-scale Threshold are flagged as off-scale and are avoided if possible 32000 * The default minimum absolute peak height was 500 florescence units, except for PfPK2, TA81, PolyA, and PFG377, where setting was raised to 1000.