High Frequency Mutations in pfdhfr and pfdhps of Plasmodium falciparum in Response to Sulfadoxine-Pyrimethamine: A Cross-Sectional Survey in Returning Chinese Migrants From Africa

Background Sulfadoxine-pyrimethamine (SP) is recommended for intermittent preventive treatment in Africa against Plasmodium falciparum infection. However, increasing SP resistance (SPR) of P. falciparum affects the therapeutic efficacy of SP, and pfdhfr (encoding dihydrofolate reductase) and pfdhps (encoding dihydropteroate synthase) genes are widely used as molecular markers for SPR surveillance. In the present study, we analyzed single nucleotide polymorphisms (SNPs) of pfdhfr and pfdhps in P. falciparum isolated from infected Chinese migrant workers returning from Africa. Methods In total, 159 blood samples from P. falciparum-infected workers who had returned from Africa to Anhui, Shangdong, and Guangxi provinces were successfully detected and analyzed from 2017 to 2019. The SNPs in pfdhfr and pfdhps were analyzed using nested PCR. The genotypes and linkage disequilibrium (LD) were analyzed using Haploview. Results High frequencies of the Asn51Ile (N51I), Cys59Arg(C59R), and Ser108Asn(S108N) mutant alleles were observed, with mutation frequencies of 97.60, 87.43, and 97.01% in pfdhfr, respectively. A triple mutation (IRN) in pfdhfr was the most prevalent haplotype (86.83%). Six point mutations were detected in pfdhps DNA fragment, Ile431Val (I431V), Ser436Ala (S436A), Ala437Gly (A437G), Lys540Glu(K540E), Ala581Gly(A581G), Ala613Ser(A613S). The pfdhps K540E (27.67%) was the most predominant allele, followed by S436A (27.04%), and a single mutant haplotype (SGKAA; 62.66%) was predominant in pfdhps. In total, 5 haplotypes of the pfdhfr gene and 13 haplotypes of the pfdhps gene were identified. A total of 130 isolates with 12 unique haplotypes were found in the pfdhfr-pfdhps combined haplotypes, most of them (n = 85, 65.38%) carried quadruple allele combinations (CIRNI-SGKAA). Conclusion A high prevalence of point mutations in the pfdhfr and pfdhps genes of P. falciparum isolates was detected among Chinese migrant workers returning from Africa. Therefore, continuous in vitro molecular monitoring of Sulfadoxine-Pyrimethemine combined in vivo therapeutic monitoring of artemisinin combination therapy (ACT) efficacy and additional control efforts among migrant workers are urgently needed.

Background: Sulfadoxine-pyrimethamine (SP) is recommended for intermittent preventive treatment in Africa against Plasmodium falciparum infection. However, increasing SP resistance (SPR) of P. falciparum affects the therapeutic efficacy of SP, and pfdhfr (encoding dihydrofolate reductase) and pfdhps (encoding dihydropteroate synthase) genes are widely used as molecular markers for SPR surveillance. In the present study, we analyzed single nucleotide polymorphisms (SNPs) of pfdhfr and pfdhps in P. falciparum isolated from infected Chinese migrant workers returning from Africa.
Methods: In total, 159 blood samples from P. falciparum-infected workers who had returned from Africa to Anhui, Shangdong, and Guangxi provinces were successfully detected and analyzed from 2017 to 2019. The SNPs in pfdhfr and pfdhps were analyzed using nested PCR. The genotypes and linkage disequilibrium (LD) were analyzed using Haploview.

INTRODUCTION
Imported Plasmodium falciparum infections are of great concern in China during malaria elimination and post-elimination stages, because China achieved zero indigenous malaria cases in 2017 (Feng et al., 2018). Appropriate treatment for P. falciparum according to the national plan is important; however, some studies have demonstrated mutations in molecular markers related to artemisinin combination therapy (ACT) resistance among isolates from Africa, suggesting that careful surveillance of African parasite populations is still warranted (Feng et al., 2015a;Feng et al., 2019, Yan et al., 2020. Sulfadoxine-pyrimethamine (SP) is a second-line antimalarial for uncomplicated P. falciparum malaria treatment, as recommended by the World Health Organization (WHO), which is used as an intermittent preventive treatment in pregnancy (IPTp) and as an intermittent preventive treatment in infants (IPTi) in malariaendemic regions (Gosling et al., 2010;Konate et al., 2011). Resistance to SP is caused mainly by point mutations in pfdhfr (encoding dihydrofolate reductase) and pfdhps (encoding dihydropteroate synthase). Mutations in pfdhfr and pfdhps have been associated with decreased parasite sensitivity to the SP, because the products of these genes could incrementally increase the parasite's tolerance to the drugs in vitro (Chulay et al. 1984). Studies have identified point mutations in codons N51I, C59R, and S108N, I164L of pfdhfr located on chromosome 4 and codons I431V, S436A, A437G, K540E, A581G, and A613S of pfdhps located on chromosome 8, all of which were associated with P. falciparum SP treatment failure (Triglia et al., 1997;Berglez et al., 2004;Pearce et al., 2009). Monitoring drug resistance and the pattern of mutations is essential for early detection and subsequent prevention of the spread of drug resistance. The present study identified the polymorphisms in pfdhfr and pfdhps in P. falciparum among returned migrant workers from Africa in 2017-2019 reported in eastern China. The results provide a deeper understanding of the disease as well as baseline information on antimalarial drug resistance among imported P. falciparum in China.

Sample Collection and DNA Extraction
The study was conducted in Anhui, Shandong, and Guangxi Provinces in Eastern and Southern China, where imported P. falciparum cases were predominantly. Anhui Province covers 105 counties with 70.6 million people and experienced a malaria resurgence in 2005-2008 that was mainly caused by the accumulation of residual foci of P. vivax (Feng et al., 2015b). Shandong Province, located in eastern China, has a long coastline measuring 3,024.4 kilometers. It contains 137 counties and has a population of 97.9 million. Economic trade overseas is frequent. Another province, Guangxi, is known for its gold miners who returned from Ghana in 2013, which was mainly reported in Shanglin County (Feng et al., 2015a). The number of imported P. falciparum cases, especially those from Africa, has increased significantly in these three provinces, and 360 P. falciparum cases were reported in 2019, accounting for 18.5% of all P. falciparum cases nationwide (Zhang L et al., 2020).
A total of 206 P. falciparum-infected blood samples were collected from the travelers returning from Africa from 2017 to 2019. The samples distribution were shown as Figure 1. Approximately 100 ml of finger-prick blood was spotted onto a piece of 3MM Whatman filter paper (GE Healthcare, Boston, MA, USA) and air dried.
The Plasmodium falciparum genomic DNA from the approximately 100 ml of collected blood sample was extracted using a QIAamp DNA blood kit (QIAGEN, Valencia, CA, USA) as described previously (Yan et al., 2020). Each of the samples was labeled with a study number and stored at −4°C until extraction. Individual epidemiological information was also collected using a web-based reporting system (China Information System for Diseases Control and Prevention) and analyzed.

Detection of pfdhfr and pfdhps Polymorphisms
Point mutations at codons 16, 51, 59, 108, and 164 of the pfdhfr gene and codons 431, 436, 437, 540, 581, and 613 of the pfdhps gene were evaluated using nested PCR amplification. The sequences of the primers used for pfdhfr and pfdhps genotyping were as described previously (Zhang et al., 2014). The primary amplification was performed using the following parameters: 1 cycle of 95°C for 3 min; 35 cycles of 95°C for 30 s, 55°C for 30 s, and 65°C for 6 for 30 s, and 1 cycle of 65°C for 60 s; and 65°C for 5 min. The second amplifcation was performed using the following parameters: 1 cycle of 95°C for 3 min; 35 cycles of 95°C for 30 s, 52°C for 30 s, and 65°C for 60 s; and 1 cycle of 65°C for 5 min. 750bp product of pfdhps were sent for Sanger sequencing (Shanghai Bunan Biological Co., Ltd., Shanghai, China).

Data Analysis
Sequences were analyzed using the Blast program (http://blast. ncbi.nlm.nih.gov/). Multiple nucleotide sequence alignments and analysis were carried out using the MAFFT web-based tool with the Cluster Omega Sequence Alignment Editor (https://www.ebi. ac.uk/Tools/msa/clustalo/). Sequences with poor quality after three sequencing attempts or those with more than one peak at one locus were not included in the analysis. The map showing the imported of countries with number of the isolates was created by ArcGIS 10.1 (Environmental Systems Research Institute, Inc., Redlands, CA, USA). SPSS18.0 (IBM Corp., Armonk, NY, USA) was used to conduct the statistical analyses, and the Chi-squared test was employed to test the different constituent ratios of pfdhfr and pfdhps gene polymorphisms. The Fisher's precision probability test was used as the sample size is less 50; For the sample size less than five would be discarded when analyzing the geographical distribution difference. The inter and intragenic SNP Linkage disequilibrium (LD) associations of pfdhfr and pfdhps were analyzed using Haploview 15 (Patel et al., 2017).

Ethical Considerations
This study was reviewed and approved by the ethical committee of the National Institute of Parasitic Diseases, Chinese Centre for Disease Control and Prevention (NIPD, China CDC, No. 2019008).

Epidemiological Information
Among the 206 P. falciparum isolates collected in this study, 202 and 177 isolates were successfully amplified and sequenced for the pfdhfr and pfdhps genes, respectively; however, only single infections and both of pfdhfr and pfdhps successful amplicons were involved in the final analysis. Therefore, a total of 159 P. falciparum isolates were successfully detected and analyzed in this study (Figure 1). Their distribution was identified as 2 from North Africa, 23 from East Africa, 54 from West Africa, and 80 from Central Africa. Among them, the Democratic Republic of the Congo (n = 28), Nigeria (n = 27), and Angola (n = 16) were considered as the top three imported source countries.

DISCUSSION
The emergence and spread of Sulfadoxine-Pyrimethamine (SP) resistance has narrowed its usage to IPTp and IPTi in Africa (Rieckmann and Cheng, 2002). In 2019, the WHO covered 33 African countries for IPTp, in which at least nearly 62% of pregnant women received a first dose of IPTp (of four doses of ITPp)) (World Health Organization, 2020). The presents study aimed to determine the prevalence of SP resistance-associated pfdhfr and pfdhps genes in P. falciparum isolated from returned migrant workers from Africa reported in China. The study showed the pfdhfr triple haplotype mutation (IRN, including N51I, C59R, S108N) was highly prevalent, which was similar to other publications concerning African isolates (Jiang et al., 2019;Gikunju et al., 2020;Quan et al., 2020). This high prevalence was also found in the migrant workers returning to Guangxi from Ghana, suggesting that SP resistant genotype was widespread in Central and West Africa (Zhao et al., 2020). It was noted pfdhfr I164L, which was also associated with high resistance to cycloguanil, was detected in Ghanaian isolates, but was not found in our study (Zhao et al., 2020). It is worth noting that I164L is common in East Africa and Asia (Basuki et al., 2018,;Lynch et al., 2017), but rarely seen in Central and West Africa. One of the explanations was that this mutation site carries a high fitness cost to the parasite and therefore it is unable to survive the immune response of hosts in West Africa (Nzila et al., 2002). Further studies are needed to assess the effect of this mutation on the phenotype of parasites carrying this haplotype. Mutations in pfdhps haplotypes at S436A and K540E, which are associated with decreased parasite sensitivity to Sulfadoxine drugs (Berglez et al., 2004), carried a higher allele frequency, which was also found in Uganda and Tanzania (Alifrangis et al., 2009;Mbonye et al., 2015). The WHO recommended that IPTp should not implemented in the regions when K540E exceed 50%; in our study, this mutation was present at 27.67%, which might favor the continued efficacy of IPTp treatment in these countries. Another mutation, A581G, is considered to have an important modulatory role in SP resistance. IPTp and SP could not protect pregnant women from delivering low birth weight infants when the frequency of this mutation is above 10% (Chico et al., 2015). In our study, the frequency of this mutation was 8.81% and half of these mutated isolates were found in Nigeria. For the mutation A437G, which was associated with resistance to Sulfadoxine in endemic regions because of the drug pressure selection, showed a frequency of only 9.43% in our study, which was lower than that reported in Equatorial Guinea, Pakistan, and Iran (Rouhani et al., 2015;Yaqoob et al., 2018;Jiang et al., 2019). The I431V mutation was detected in Nigeria, Cameroon, Equatorial Guinea, and Chad. The most frequent haplotype of I431V was VAAKGA, similar to that found in isolates from Cameroon and Nigeria (Chauvin et al., 2015;Oguike et al., 2016). These haplotypes occurring in Central and West Africa suggested that SP conferred a selective advantage, and ongoing drug pressure is relative strong because SP was used as IPTp in these regions. It is noted A437G was widely spread in central and western African countries, which indicating in vivo SP drug resistance in these regions (Pearce et al., 2009). Some other studies combined with the clinical study indicated the pfdhfr triple mutant genotype was associated with SP treatment resistance (Basco et al, 2000;Naidoo et al., 2013). However, in this study, the A437G was not widely spread in central and western African region, even less than S436A, which was also different with our previous study in China-Myanmar border (Zhang et al., 2014), which may partially ascribe as the sample size limitation, the loss of drug pressure, and time passage may also the potential reason. In our study, the frequency of the quadruple mutation IRN/ ISAEAA was significantly higher in East Africa (100% in Uganda, 85.7% in Mozambique) than western and central African countries (P < 0.01), suggesting that the clinical implications of such haplotypes require further combined genotype and phenotype analysis. SP was limited for malaria control among the general population in many countries in Africa because of the high frequency of drug resistance developed by P. falciparum; therefore, we expected to obtain SP sensitive strains. Indeed, in our study, 39.62% of the isolates harbored the non-mutated pfdhps gene. This may be similar to the process for chloroquine, which tends to recover its effectiveness against the parasite after a long period during which its use in malaria control activities is halted (Lu et al., 2017).

CONCLUSION
This study showed a high frequency of SP-resistance associated SNPs in the pfdhfr and pfdhps genes of P. falciparum isolated since 2017 in returned migrant workers from Africa. The high resistance may be linked to the unsuccessful withdrawal of the SP treatment, and thus might affect the efficacy of IPTp for pregnant women and IPTi for infants. Mutations such as K540E, and the pfdhfr-pfdhps haplotype IRN/ISAEAA, which occurred at moderate frequencies in East Africa, such as in Uganda and Mozambique, the other regions showed as high frequency triple mutations of pfdhfr, but relatively scatter site mutations in pfdhps gene. The present data could provide the evidence for molecular surveillance in the post-elimination stage in China, focusing on the risk population among returning migrant workers from Africa, and could be used to determine the treatment policy for imported malaria in China.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the ethical committee of the National Institute of Parasitic Diseases (NIPD), Chinese Centre for Disease Control and Prevention, Chinese Center for Tropical Diseases Research. The patients/participants provided their written informed consent to participate in this study.