Mosquitoes as a feasible sentinel group for anti-malarial resistance surveillance by Next Generation Sequencing of Plasmodium falciparum

Table 3 Comparison of wild type and mutant frequencies of P. falciparum in mosquito samples using Sanger sequencing and NGS

Gene	Sanger sequencing of mosquito samples			NGS of mosquito samples^a		Sequenced samples by both methods	p-value
Gene	Wild type %	Mutant %	Mixture %	Wild type %	Mutant %	Sequenced samples by both methods	p-value
K13
Y493H R539T I543T C580Y	100	0	0	100	0	66	1
pfcrt
CVMNK (72–76)	100	0	0	100	0	93	1
pfmdr1 F1
N86Y/F	98.8	0	1.2	100	0	81	3.44 × 10⁻⁴
Y184F	46.9	21.0	32.1	50.3	49.3	81	0.59
pfmdr1 F2
S1034C N1042D D1246Y	100	0	0	100	0	70	1
pfdhps F1
S436F/A	100	0	0	100	0	86	1
A437G	2.3	95.3	2.3	0	100	86	1.32 × 10⁻⁷
pfdhps F2
K540E	2.3	96.6	1.1	6.2	93.7	87	0.17
A581G A613T/S	100	0	0	100	0	87	1
pfdhfr
C50R/S	99	1.0	0	100	0	95	1.64 × 10⁻⁴
N51I	0	100	0	0.9	98.4		1
C59R	3.2	32.6	64.2	3.6	96.3		1
S108N	0	100	0	0.3	96.3		1
164L	100	0	0	100	0		1

Fisher’s exact test was utilized for all comparisons except for pfmdr1 position Y184F, for which χ-squared test was performed. Sanger sequencing mixtures were assumed as mutant for statistical calculations. F1: fragment 1. F2: fragment 2. Wild-type haplotypes are indicated on the left and mutant amino acids on the right of the position number (see Additional file 1: Tables S5 and S7)
^aNGS allele frequency estimates were calculated by dividing individual unfiltered allele depth by total filtered depth, hence total values do not always add up to 100 (see “Methods” section) [61]

ISSN: 1475-2875