3.1 General information
A total of 296 returnees from Africa and SEA who were diagnosed with P. falciparum and treated with ACTs were collected from 2011 to 2019 (Table S3). Including 122 cases in West Africa (WA), 78 cases in Central Africa (CA), 59 cases in South Africa (SA), 26 cases in East Africa (EA), 6 cases in North Africa (NA), and 5 cases in SEA. These cases were mainly from 29 countries in African and SEA, mainly concentrated in Congo (14.86%, 44/296), Nigeria (14.53%, 43/296), followed by Angola (11.15%, 33/296), Liberia (7.43%, 22/296), and Mozambique (4.72%, 14/296).
3.2 Mutation prevalence of pfubp1 gene
For pfubp1 investigation and evaluation, a 304-bp region was amplified and sequenced. A total of 296 samples were sequenced through quality assurance analysis, of which 23 were of low quality. Of the 273 samples successfully sequenced, these cases were mainly from Congo, Nigeria and Angola, accounting for 14.65% (40/273), 13.55% (37/273) and 12.09% (33/273), respectively (Table S3). The proportion of isolates with no mutations in the pfubp1 gene, that is wild type sequence as the 3D7 strain, was 60.07% (164/273). A total of 109 isolates (39.93%, 109/273) were found to harbour 10 different SNPs. There were 8 non-synonymous mutations including D1510N, E1518Y, E1519D, E1519K, E1519V, D1525E, E1528D and E1528G. And 2 synonymous mutations were N1515N and N1518N. Among, the unreported loci were D1510N and N1515N. Most isolates had more than one mutation in the pfubp1 gene (Fig. 1A).
This survey found that the main epidemic mutation sites were D1525E and E1528D. For the D1525E locus, the frequencies of wild type, mutant, and mixed types were 83.15% (227/273), 10.99% (30/273) and 5.86% (16/273), respectively. The prevalence of mutation in WA, CA, SA, EA, NA, and SEA were 16.07%, 22.86%, 12.28%, 8.33%, 40.00% and 20.00% respectively (Table 1). These mutations were also mainly focused in Congo (20%, 6/30), Nigeria (13.33%, 4/30), (10%, 3/30) each in Liberia and Equatorial Guinea. In addition, one case was found in SEA (Fig. 2A). For the D1525E, Among the WA, CA, and SA regions. After statistical analysis, but it was not statistically significant (P = 0.068). For the period from 2011 to 2019 (Table 2), the annual trend results showed that the mutation rate of D1525E was 28.57% in 2011 and 38.38% in 2012. By 2013 and 2014, it had decreased to 10.26% and 8.82% respectively. Then the mutation rate rose sharply to 24.24% in 2015 and 18.60% in 2016. By 2017 it had fallen to 0.00%. In 2018, it began to increase from 6.67–17.02% in 2019. Due to the extremely low number of samples in 2011 and 2017, they were excluded. After trend analysis was performed for the remaining samples, there was no statistical significance (F = 0.895, P = 0.388).
For the E1528D locus, the frequencies of wild type, mutant type, and mixed types were 79.12% (216/273), 13.19% (36/273) and 7.69% (21/273), respectively. The prevalence of mutation in WA, CA, SA, EA, NA, and SEA were 29.46%, 14.29%, 14.04%, 12.50%, 40.00%, and 20.00%, respectively (Table 1). For the E1528D, among the WA, CA, and SA regions. After statistical analysis, which was statistically significant (P = 0.016). And comparison between regions, WA and CA also showed differences (P = 0.042). These mutations were also mainly focused in Liberia (22.22%, 8/36), (13.89%, 5/36) each in Nigeria and Congo, and Sierra Leone (8.33%, 3/36). Similarly, one case has been found in SEA (Fig. 2B). From 2011 to 2019 (Table 2), the annual trend results showed that the mutation rate of E1528D was 28.57% in 2011 and 31.25% in 2012. By 2013 and 2014, it had decreased to 20.51% and 23.53% respectively. By 2015, the mutation rate was 27.27%. Between 2016 and 2017, it dropped again to 16.28% and 12.50%. It rose to 20.00% in 2018, and then fell to 12.77% in 2019. For trend analysis, which was statistically significant (F = 7.708, P = 0.039).
3.3 Mutation prevalence of pfap2mu gene
In order to investigate and evaluate pfap2mu, which divided into three regions for amplification and sequencing. A total of 296 samples were amplified and sequence. The sizes are 841 bp, 578 bp, and 753 bp, respectively. The quality assurance analysis determined that the three fragments of the pfap2mu gene failed to be sequenced in 29 cases, 51 cases and 16 cases respectively. A total of 32 different SNPs were found to harbour in the samples successfully sequenced. There were 20 non-synonymous mutations including V127L, R146K, Q149H, S160N, N162I, N184H, V196I, K199T, N200Y, A236T, N239K, G315S, A337S, L363H, Y407H, S476A, I478F, L579F, S594F and *595Q (*, the stop codon). And 12 synonymous mutations including Y6Y, I100I, E163E, K186K, R188R, K199K, T287T, L442L, T443T, S476S, N533N and D548D. Among the unreported loci, non-synonymous mutations including V127L, R146K, N184H, V196I, A236T, N239K, G315S, L363H, Y407H, L579F, S594F and *595Q. And synonymous mutations including Y6Y, I100I, K186K, T287T, L442L, T443T, N533N and D548D. Most isolates had more than one mutation in the pfap2mu gene (Fig. 1B).
Among them, Y6Y to I100I were detected in the first segment. According to sequencing quality assurance determined, 17.23% (51/296) were sequencing failures. The successful sequencing rate was 82.77% (245/296). Most cases were concentrated in Congo 13.47% (33/245), Nigeria 13.06% (32/245) and Angola 12.24% (30/245). The prevalence of I100I mutation was 4.49% (11/245) and 1.22% (3/245) of mixed type. Mutation cases were mainly focused on Congo 36.36 (4/11) and Mozambique 18.18% (2/11). Only one mutant of Y6V was detected in Ivory Coast. And one case mixed type of V127L occurred in South Sudan (Table S3).
The second amplified fragment detected sites from R146K to V196I. According to sequencing quality assurance determined, 9.79% (29/296) were sequencing failures. The successful sequencing rate was 90.20% (267/296) (Table S3). Most cases were concentrated in Congo 14.98% (40/267), Nigeria 14.61% (39/267) and Angola 11.99% (32/267). The most prevalent mutation was S160N, E163E, R188R, and K199T in these isolates. The prevalence of S160N mutation was 11.24% (30/267) and 1.87% (5/267) of mixed type. Mutations were mainly concentrated in Angola and Congo with 5 cases each, and Cameroon and Equatorial Guinea with 4 cases each (Fig. 3). The mutation prevalence of E163E, R188R, and K199T were 6.37% (17/267), 2.25% (6/267) and 5.24% (14/267), respectively. The R188R mutations were mainly concentrated in Angola and Congo, and the K199T mutations were mainly concentrated in Angola, Congo and Zambia (Table S3).
The third fragment examined sites from Y407H to *595Q. A total of 296 samples were sequenced and 16 were of low quality as determined by quality assurance analysis. Among the 280 samples successfully sequenced, these cases were mainly from Congo, Nigeria and Angola, accounting for 15.36% (43/280), 13.93% (39/280) and 11.79% (33/280), respectively (Table S3).
For known delayed clearance locus S160N. The prevalence of S160N mutation was 11.24% (30/267). The prevalence of mutation in WA, CA, SA, EA, NA, and SEA were 9.26 %, 19.72%, 25.00%, 10.53%, 13.64% and 20.00%, respectively (Table 1). For the S160N, among the WA, CA, and SA regions. After statistical analysis, but it was not statistically significant (P = 0.102). Mutations were mainly concentrated in Angola and Congo with 5 cases each, and Cameroon and Equatorial Guinea with 4 cases each (Fig. 3). From 2011 to 2019, according to the annual trend results of S160N (Table 2). During 2011, the mutation rate was 50%. In 2012, 2013 and 2014, the values dropped sharply to 19.35%, 8.33% and 6.06% respectively. Then it rose to 20.00% and 18.00% during 2015 and 2016, and from 2017 to 2019 it began to decline gradually again, to 14.90%, 9.38% and 7.84% respectively. For trend analysis, which was no statistically significant (F = 0.327, P = 0.592).
3.4 Prevalence of the pfubp1 and pfap2mu haplotypes
The results showed a number of SNPs that were being inherited together on the gene as haplotypes in some of the isolates. However, the prevalence of the haplotypes was low (Table 3). Eighteen different haplotypes were observed: two for pfubp1 and sixteen for pfap2mu. The most prevalent haplotype for the pfbup1 gene was D1525E-E1528D, which was observed in 0.73% (2/273) of the isolates with mutations. They were distributed in Congo and Liberia. The other haploids were all present singly.