We evaluated markers of sulfadoxine-pyrimethamine (SP) resistance in Plasmodium falciparum among 254 returned migrant workers in China from Africa from 2013 to 2016. High prevalences of pfdhfr (97.2%) and pfdhps (96.5%) mutations were observed.
KEYWORDS: Africa, China, Plasmodium falciparum, SP resistance, genotype, malaria, mutation, pfdhfr, pfdhps
ABSTRACT
We evaluated markers of sulfadoxine-pyrimethamine (SP) resistance in Plasmodium falciparum among 254 returned migrant workers in China from Africa from 2013 to 2016. High prevalences of pfdhfr (97.2%) and pfdhps (96.5%) mutations were observed. The partially resistant genotype was homogeneously distributed in Africa with a modestly high prevalence (48%), whereas the super resistant genotype was only found in West Africa with a very low frequency (1.2%). The findings provided baseline data about the molecular markers of SP resistance.
INTRODUCTION
Emergence and spread of antimalarial resistance in Plasmodium falciparum parasites have been major obstacles to global malaria control and eradication in past decades (1). Because chloroquine (CQ) resistance has spread throughout Africa since the early 1990s, the antifolate combination of sulfadoxine-pyrimethamine (SP) was widely introduced for the treatment of uncomplicated P. falciparum malaria (2). Unfortunately, an increase of parasite resistance to SP occurred, leading to a situation similar to that involving CQ resistance. SP is no longer used for current clinical treatment of malaria in Africa due to drug resistance, but it still serves as prophylaxis and is implemented routinely as an intermittent preventive treatment (IPT) for malaria, particularly during pregnancy (IPTp) and in infants (IPTi) (3, 4). The emergence of SP resistance is caused by substitutions in two enzymes, dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS), in the folate synthesis pathway (5, 6). It has been associated with A16V, N51I, C59R, S108N, and I164L mutations in the pfdhfr gene, which confer pyrimethamine resistance, as well as I431V, S436A/F, A437G, K540E, A581G, and A613S/T mutations in the pfdhps gene, which confer sulfadoxine resistance (7). The high level of SP resistance was related to increasing numbers of combined mutations within the two genes.
Indigenous malaria cases have been absent in most regions of China since 2012; however, imported cases were increased dramatically due to the large number of migrant workers who returned from Africa and were infected with P. falciparum (8–10). However, little is known about the mutation profile of molecular markers associated with the SP resistance of these populations. This study aims to explore the prevalence of mutation and genotype distribution of pfdhfr and pfdhps in P. falciparum patients imported from Africa into China.
Blood samples were collected from migrant workers with uncomplicated P. falciparum infection who returned from Africa to the Shandong Province of China between 2013 and 2016 before antimalarial drug treatment. The final diagnosis was validated with microscopic examination of Giemsa-stained thick smears (11). Approximately 200 μl of finger-prick blood for each patient was spotted on Whatman 903 filter paper and then air dried. The dried blood samples were labeled with names, serial numbers, and dates and stored at −20°C until DNA extraction. The study was reviewed and approved by the Ethics Committee of the Shandong Institute of Parasitic Diseases (Jining, China). Informed consent was obtained from each patient before the research.
Parasite DNA was extracted through the QIAamp DNA minikit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. For each sample, a 594-bp fragment (covering loci 16, 51, 59, 108, and 164 on the pfdhfr gene) and a 711-bp fragment (covering loci 431, 436, 437, 540, 581, and 613 on the pfdhps gene) were amplified using nested PCR, as previously described (12). The products were analyzed through agarose (1.5%) gel stained with ethidium bromide. The fragments were sequenced by the BGI Corporation (Beijing, China). Mutations on nucleotide sequences were examined using BioEdit 7.0. The reference sequences of the pfdhfr gene (PF3D7_0417200) and the pfdhps gene (PF3D7_0810800) were acquired from PlasmoDB (http://plasmodb.org/plasmo/). Samples with mixed alleles were considered to be mutated for the purpose of mutation frequency estimation. Data were entered in Microsoft Excel 2013 and analyzed using SPSS 19.0. The χ2 test or Fisher’s exact test was used to evaluate differences of mutation frequency among the groups. A P value of <0.05 was considered to be statistically significance. The map was created by MapInfo v15.0 (Pitney Bowes, Troy, NY). The parasite genotypes that presented a combination of multiple mutations in both the pfdhfr and pfdhps genes were classified as follows: a quadruple mutant (pfdhfr 51I + 59R + 108N and pfdhps 437G [IRNG]) was considered to be “partially resistant”; a quintuple mutant (pfdhfr 51I + 59R + 108N and pfdhps 437G + 540E [IRNGE]) was considered to be “fully resistant”; and a sextuple mutant (pfdhfr 51I + 59R + 108N and pfdhps 437G + 540E + 581G or 613S/T [IRNGEG or IRNGES/T]) was considered to be “super resistant,” as described previously (13).
A total of 254 samples collected from P. falciparum cases were successfully sequenced and enrolled in this study. The areas from which patients returned were as follows: Nigeria, Ghana, and Guinea of West Africa; Equatorial Guinea, Congo, and Cameroon of Central Africa; and Angola and Mozambique of Southern Africa (Table 1). There were 252 male patients and 2 female patients (126:1). The age range was 21 to 60, and the mean age was 39 (±8.7) years. All patients accepted the artemisinin-based combination therapies (ACTs) treatment according to antimalarial drug policy in China (2009) (14). Clinical features showed that patients recovered well from malaria after the treatment.
TABLE 1.
Prevalences of mutation and genotype in pfdhfr and pfdhps genes of P. falciparum isolates imported from Africa
| Gene | Mutation/genotype | Prevalences in: |
Total no. (%) (n = 254) |
|||||||
|---|---|---|---|---|---|---|---|---|---|---|
| West Africa (na = 70) |
Central Africa (n = 98) |
Southern Africa (n = 86) |
||||||||
| No. (%) from Nigeria (n = 36) |
No. (%) from Ghana (n = 21) |
No. (%) from Guinea (n = 13) |
No. (%) from Equatorial Guinea (n = 80) |
No. (%) from Congo (n = 12) |
No. (%) from Cameroon (n = 6) |
No. (%) from Angola (n = 69) |
No. (%) from Mozambique (n = 17) |
|||
| pfdhfr | N51I | 31 (86.1) | 19 (90.5) | 12 (92.3) | 76 (95) | 11 (91.7) | 6 (100) | 65 (94.2) | 17 (100) | 237 (93.3) |
| C59R | 29 (80.6) | 20 (95.2) | 12 (92.3) | 74 (92.5) | 9 (75) | 6 (100) | 51 (73.9) | 17 (100) | 218 (85.8) | |
| S108N | 32 (88.9) | 20 (95.2) | 13 (100) | 79 (98.8) | 12 (100) | 6 (100) | 68 (98.6) | 17 (100) | 247 (97.2) | |
| 51I + 59R + 108N (IRN) | 29 (80.6) | 19 (90.5) | 12 (92.3) | 71 (88.8) | 9 (75) | 6 (100) | 48 (69.6) | 17 (100) | 211 (83.1) | |
| pfdhps | I431V | 3 (8.3) | 0 | 0 | 5 (6.3) | 0 | 1 (16.7) | 0 | 0 | 9 (3.5) |
| S436A/F | 17 (47.2) | 6 (28.6) | 1 (7.7) | 23 (28.8) | 1 (8.3) | 2 (33.3) | 3 (4.3) | 0 | 53 (20.9) | |
| A437G | 33 (91.7) | 21 (100) | 13 (100) | 73 (91.3) | 12 (100) | 5 (83.3) | 63 (91.3) | 17 (100) | 237 (93.3) | |
| K540E | 4 (11.1) | 3 (14.3) | 2 (15.4) | 10 (12.5) | 2 (16.7) | 1 (16.7) | 12 (17.4) | 14 (82.4) | 48 (18.9) | |
| A581G | 5 (13.9) | 0 | 0 | 0 | 0 | 1 (16.7) | 0 | 0 | 6 (2.4) | |
| A613S | 5 (13.9) | 4 (19) | 1 (7.7) | 4 (5) | 0 | 1 (16.7) | 0 | 0 | 15 (5.9) | |
| pfdhf + pfdhps | 51I + 59R + 108N + 437G (IRNG) | 12 (33.3) | 12 (57.1) | 8 (61.5) | 43 (53.8) | 6 (50) | 3 (50) | 35 (50.7) | 3 (17.6) | 122 (48) |
| 51I + 59R + 108N + 437G + 540E (IRNGE) | 1 (2.8) | 1 (4.8) | 2 (15.4) | 8 (10) | 2 (16.7) | 1 (16.7) | 8 (11.6) | 14 (82.4) | 37 (14.6) | |
| 51I + 59R + 108N + 437G + 540E + 581G or 613S (IRNGEG/S) | 2 (5.6) | 1 (4.8) | 0 | 0 | 0 | 0 | 0 | 0 | 3 (1.2) | |
n, number of samples.
In the pfdhfr gene, mutations were detected in 97.2% (247/254) of P. falciparum isolates. None of the isolates carried a mutation at codons 16 and 164. The highest frequency of mutation was S108N (97.2%), and its frequency was a significant difference in isolates among West Africa, Central Africa, and Southern Africa (χ2 = 6.94; P = 0.030000). Nevertheless, there was no significant difference in the prevalence of N51I (χ2 = 3.49; P = 0.180000) in isolates among the three areas as well as in that of C59R (χ2 = 5.33; P = 0.070000). Regarding the pfdhps gene, point mutations were observed in 96.5% (245/254) of the samples. Of these, A437G was predominant (93.3%) and also occurred at a prevalence of more than 90% in West Africa, Central Africa, and Southern Africa, with no significant differences among these areas (χ2 = 1.00; P = 0.610000). The frequency of the K540E mutation that was detected in Southern Africa (30.2%) was higher than the frequencies in West Africa and Central Africa (χ2 = 10.9; P = 0.004290). The I431V mutation was found in isolates from West Africa and Central Africa, with 4.3% and 6.1% prevalence, respectively. No mutation was observed at codons 431, 581, and 613 in isolates from Southern Africa, and the frequency of the S436A/F mutation in this area was obviously less than the frequencies in West Africa and Central Africa (χ2 = 25.27; P = 0.000003). Detailed information about the pfdhfr and pfdhps mutations is shown in Table 1.
The triple mutant 51I + 59R + 108N (IRN) of pfdhfr was detected in 83.1% of isolates, and there was no significant difference in mutation frequency among isolates from West Africa, Central Africa, and Southern Africa (χ2 = 5.31; P = 0.070000). The partially resistant 51I + 59R + 108N + 437G (IRNG) mutant was observed in 48% of the samples, and the genotype did not differ significantly among the three areas (χ2 = 1.65; P = 0.440000). The overall prevalence of the fully resistant 51I + 59R + 108N + 437G + 540E (IRNGE) mutant was 14.6%, of which the genotype frequency in Southern Africa was greater than in the other two areas (χ2 = 13.67; P = 0.001070). Moreover, 1.2% of the samples from West Africa were detected to be the super resistant 51I + 59R + 108N + 437G + 540E + 581G/613S (IRNGEG/S) genotype. The genotype distributions of pfdhfr and pfdhps are shown in Table 1 and Fig. 1.
FIG 1.
Distribution of pfdhfr and pfdhps genotypes of P. falciparum isolates imported from Africa. The source countries of isolates are shown in different colors on the map. The histogram presents the frequencies of different genotypes detected in the isolates from West Africa, Central Africa, and Southern Africa.
The pfdhfr I164L mutation was mainly found in East Africa and also appeared in Uganda and Equatorial Guinea in previous studies (15, 16). However, in our study, I164L was absent in isolates from Equatorial Guinea, and the reason might be attributed to sample differences. The core pfdhfr mutation, S108N, was detected with very high frequency in the samples and reached 100% in Guinea, Congo, Cameroon, and Mozambique, indicating that the mutation was fixed in the parasite population. The mutation I431V, a newly discovered mutant allele of pfdhps, was widespread throughout Nigeria and has emerged in neighboring Cameroon recently (17, 18). In this study, we detected I431V in isolates from Nigeria and Cameroon, with 8.3% and 16.7% prevalences, respectively. Particularly, we also detected I431V in 6.3% of the isolates from Equatorial Guinea. To our knowledge, I431V was detected for the first time in Equatorial Guinea, suggesting the emergence of I431V in more regions of Central Africa. It is known that A437G is very common throughout Africa. In our samples, the prevalence of A437G was significantly higher than those of other pfdhps mutations with no obvious geographical differences (P > 0.05), which was consistent with the above conclusion. The prevalence of K540E in Southern Africa (30.2%) was higher than the prevalences of K540E in the two other areas (P < 0.05). Perhaps this was mainly attributed to the fact that 82.4% of isolates that carried the mutation were derived from Mozambique, which suggested that the geographical distribution of K540E was heterogeneous in Africa.
Regarding the genotypes of parasites, the prevalences of IRN ranged from 69.6% to 100% among the 8 countries in our study, which was consistent with the published data describing a prevalence of IRN that was more than 50% in the vast majority of Africa (13). This was probably because SP was used as a first-line treatment for a long time, which resulted in a strong selection pressure on these mutations. The prevalence of IRNG exceeded 50% in 6 out of 8 African countries, and there were no obvious differences among West Africa, Central Africa, and Southern Africa (P > 0.05), suggesting that it was homogeneously distributed in Africa with a modestly high prevalence. A previous study indicated that IRNGE was associated with clinical SP treatment failure (19). In our study, the prevalence of IRNGE in isolates from Southern Africa was higher than in the two other regions (P < 0.05), of which a major contributor was an 82.4% prevalence of the genotype in Mozambique. The early clinical evidence demonstrated that the molecular markers of the super resistant genotype might be important predictors for the disappearing protective efficacy of SP-IPTp (13). In this study, very few isolates (1.2%) carried IRNGEG/S, which was only found in isolates from Nigeria and Ghana in West Africa, indicating that SP-IPT policy remains effective in most areas of Africa.
Our study had some limitations. First, the total number of study samples was small. Second, there was a limited geographic range, and as a result, the situation of parasites from other African countries could not be understood. Third, there were no pregnant or child patient samples; the information about these populations was unknown. However, the findings still provided available molecular surveillance data associated with SP resistance in P. falciparum isolates imported from Africa to China.
Taken together, high prevalences of pfdhfr and pfdhps mutations were presented in African P. falciparum isolates among returned migrant workers in China. In particular, the pfdhps I431V mutation was detected to emerge in Equatorial Guinea. The partially resistant IRNG was homogeneously distributed in Africa with a modestly high frequency, whereas the super resistant IRNGEG/S was observed only in West Africa with a very low frequency. The increasing number of returned migrants from Africa to China could lead to the risk of SP (partial, full, and super) resistance transmission between imported isolates and local isolates through resistant gene flow, which might present a potential impact to malaria elimination efforts by 2020 in China. Given the important role of SP in current prophylaxis and IPT strategy against malaria, further molecular surveillance needs to be enhanced, and it might be beneficial to prevent the spread of SP resistance in Africa as an indicator in China.
ACKNOWLEDGMENTS
This work was supported by the Projects of Medical and Health Technology Development Program in Shandong Province (2016WS0394, 2017WS103); the Natural Science Foundation of Shandong Province (ZR2014YL036, ZR2017YL003, ZR2016YL019, ZR2018LH016); the Project of Shandong Academy of Medical Sciences (2017-42); and the Innovation Project of Shandong Academy of Medical Sciences.
We declare no competing interests.
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