High genetic complexity but low relatedness in Plasmodium falciparum infections from Western Savannah Highlands and coastal equatorial Lowlands of Cameroon

Figures & data

Figure 1. Sampled populations from Limbe Health District (LHD) of the South West Region (SWR) and Ndop Health District (NHD) of the North West Region of Western Cameroon. (a) A zoomed-out map of collection sites from the NHD (Bambalang, Bamukumbit and Bamunka) and LHD (Mile 1, Bota and Bonadikombo). The insert shows the infection density (transmission intensity) from WHO, 2018 malaria report. Each red dot depicts a local hospital used for patient sampling. (b) Distribution of complexity of infection measured by MOI. (c) Distribution of diversity of infection measured by Nei expected heterozygosity index (He) between individual collection sites.

Table 1. Clinical and demographic characteristics of clinical isolates.

Characteristic	North West Region (NWR) NHD				South West Region (SWR) LHD				NHD + LHD	P value
	BMK	BBL	BKB	ALL	ML1	BOT	BDK	ALL
DBS extracted DNA	77	33	25	135	72	43	47	154	289	–
Var ATS Pos	73	30	23	126	62	38	42	142	266	–
Successfully genotyped	56	28	22	106	59	34	33	126	232	–
Male sex	27	11	11	49	29	18	14	61	110	0.096
Female sex	29	17	11	57	30	16	19	65	122	0.231
Age, yrs (median ± SD)	8.1	7.5	7.4	7.7 ± 0.18	6.4	6.2	8.0	6.5 ± 0.02	7.1 ± 0.22	0.083
Parasitemia (%) (median ± SD)	6.3	5.5	5.9	5.9 ± 0.11	6.4	5.9	6.8	6.3 ± 0.23	5.4 ± 0.04	0.041

P value by Kruskall-Wallis test for comparison between NHD and LHD. VarATS (variable gene acidic terminal sequence) - P. falciparum diagnostic PCR (50copies/genome). Collection sites included BMK-bamunka, BBL-bambalang and BKB-bamukumbit from the Ndop health district (NHD) of the northwest region (NWR) and ML1-mile one, BOT-bota, and BDK-bonadikombo of the Limbe health district (LHD) of the southwest region (SWR) of Cameroon.

Table 2. Multi-locus linkage disequilibrium of P. falciparum infections from western highlands in the North West region and coastal lowland areas in the South West region of Cameroon.

Population (n)	Standardized Index of Association, I^SA (p value)
Bamunka (56)	0.3637 (0.001)
Bambalang (28)	0.2487 (0.005)
Bamukumbit (22)	0.5154 (0.001)
Mile 1 (59)	0.0746 (0.021)
Bota (34)	0.1720 (0.013)
Bonadikombo (33)	0.0315 (0.703)
Total (232)	0.1628 (0.001)

n = number of isolates; I^S_A  = standardized index of association. The Monte Carlo method was used to test the significance of LD.

Table 3. Pairwise Fst (bottom- half) and Jost’s D (top- half) between P. falciparum infections from western highlands in the North West region and coastal lowlands in the South West region of Cameroon.

Study Site ap < 0.01	BMK	BBL	BKB	ML1	BOT	BDK
BMK	–	0.0564	0.0873	0.287	0.219	0.251
BBL	0.004	–	0.0501	0.246	0.233	0.196
BKB	0.001	0.012	–	0.107	0.442	0.274
ML1	0.021	0.012	0.022	–	0.063	0.107
BOT	0.028	0.0115	0.197	0.005	–	0.133
BDK	0.025	0.012	0.020	0.001	0.002	–

^ap value between all comparisons by Monte Carlo method. BMK-bamunka, BBL-bambalang and BKB-bamukumbit from the Ndop health district (NHD) of the northwest region (NWR) and ML1-mile one, BOT-bota, and BDK-bonadikombo of the Limbe health district (LHD) of the southwest region (SWR).

Figure 2. Population structures and Ancestral origin of P. falciparum infections from two contrasting geo-ecological sites of Cameroon. (a) Scatter plot of discriminant analysis of principal components (DAPC) showing genetic clusters of P. falciparum infections (color-coded by collection site within the two regions) from collection sites. (b) Population clustering based on ancestral origin showing ancestry coefficients and optimum k means (k = 2 and k = 3). k = 2 produced two major population clusters (dark blue and red) while k = 3 produced the two major population clusters of microsatellite haplotypes and a common cluster (light blue). Black borders separate individual populations.

Figure 3. Genetic Relatedness of P. falciparum Infections from western highland areas and coastal lowland areas in Cameroon. (a) Average pairwise population differentiation with genetic and spatial distance between populations. (b) Distribution of p-values from simulated pairwise genetic distance versus physical distance relationships, showing significant correlation. (c) Pairwise Identity by state (IBS) between isolates within and between sites with threshold for relatedness, IBS > 0.5 demarcated by the gray line shown.