Molecular Epidemiology Of Pyrimethamine Resistance-conferring Mutations In The Dihydrofolate Reductase Gene Of Plasmodium Vivax Isolates From China

Occurrence and development of resistance are the main stumbling blocks to tackling malaria in worldwide. The intensive use of drugs, such as chloroquine and SP (sulfadoxine and pyrimethamine), against malaria has increased the potential mutations related to drug resistance and prevalence among epidemic regions. The spread of drug resistance among isolates must be measured. The identification of geographic distribution of alleles that contribute to drug resistance and population genetic diversity forms the foundation for public policy on antimalarial drugs and development of new chemotherapy.To assess the spread and geographic distribution of pyrimethamine resistant Plasmodium vivax, we analyzed the single nucleotide polymorphism (SNP) and haplotypes based on sequencing the dhfr alleles that confer resistance to pyrimethamine. To infer patterns of selection and origin of resistant parasites in natural populations, we determined the microsatellite variation around the dhfr gene and Plasmodium vivax circumsporozoite protein (PvCSP) heterogeneity.Plasmodium vivax clinical isolates were collected in China from symptomatic patients in Yunnan, Hainan Island, Anhui, Hebei and Hubei. This study was approved by Ministry of Health of the Government of the People's Republic of China. We obtained 339 samples from Nu River upstream and downstream areas in Yunnan (June 2005 to January 2009). We collected 80 samples from higher incidence west side and southern mountainous from Hainan island (October 2003 to December 2007). We also obtained samples from Anhui, Henan and Hubei (July 2007 to August 2008) in 252, 260 and 48 respectively.DNA was extracted from filter paper blood spots or blood using QIAmp Mini kit. According to handbook, dried blood spot protocol and blood and body fluid spin protocol were applied to extraction. The P. vivax dhfr coding region was amplified by nested PCR using specific primers.We sequenced total 556 P.vivax isolates for analysis SNP at positions 13, 57, 58, 61, 117, 173 of the dhfr. Majority of the isolates carrying mutation 117N/T(69.6%). 58R, 57I/L,61M,99S mutations were detected among 39.2%, 29.7%, 28.2% and 18.9% in examined samples, respectively. Mutations at residue 13 and 173 were observed only in parasites from Yunnan and altered in 1 and 3 of 556 isolates respectively (1.8‰and 1.1%). Overall, the proportion of wildtype pvdhfr was identified in 15.1% of the samples (84/556). Besides, Sequence comparison of the full length dhfr domains from Chinese vivax isolates with X98123 (isolate ARI:Pakistan) showed there were insertions/deletions of the short repetitive repeat (GGDN). The 169 samples contained 2 repetitive motifs, 3 samples contained 4 repetitive motifs and 3 samples contained 5 repetitive motifs.There was a signicant difference of dhfr SNP and haplotype diversity in three regions. The greatest range of mutations was observed in the Yunnan Province, ranging from wild type to quadruple mutations. Majority of isolates showed presence of quadruple mutations at residues 57, 58, 61 and 117 (57.9%). Alleles with triple mutations and double mutations were identified in isolates from Yunnan as well (11.7% and 10.9%). The dhfr wild haplotype was represented in 14 samples (6.1%). The double mutant IFRTHNI haplotype was the exclusive mutated haplotype observed in Hainan Island. All samples displayed an identical nucleotide sequence even without any indels in the tandem repeat region.The 117N mutation was frequently observed in Central China (50.2%). Among the 131 isolates of Henan P. vivax, single mutated haplotype IFSTHNI were found in 63 isolates (48.1%) and double mutated haplotype IFSTSNI were found in 15 isolates (11.5%). The Pvdhfr molecular characters in Anhui samples closely paralleled that of Henan. The most prevalent haplotypes among Hubei examined samples were wild type (56.3%). The central China isolate displayed the relatively easy sequence variance as these wild-type parasites, with the exception of the indels in the tandem repeat region.The combination of policy with molecular characterization of dhfr and dhps helped us explain the resistant process clearer. For forty years, large mutations and relatively high resistance level in Yunnan samples and pyrimethamine drug pressure coincided. Hainan isolates showed mutation at codon 58R and 117N, which essentially formalized the twenty year period drug-use. In central China, which includes Henan, Hubei and Anhui, pyrimethamine was introduced for anti-recurrence malarial treatment and precaution in a large-scale in intermittent period of transmission and malaria transmission season. 117N mutation was selected during the constant drug pressure resulting from pyrimethamine intensive precaution.We tested the linkage disequilibrium between the codons involved in anti-folates resistance of dhfr and dhps among Hainan and Yunnnan isolates. We observed significant LD between codons 58 and 117 in dhfr and codons 382 and 383, 383 and 553 in dhps, as well as codons 117 in dhfr (chromosome 5) and codons 382 in dhps (chromosome 14).We genotyped 6 microsatellite markers around dhfr to make certain the selection sweeps of P.vivax pyrimethamine resistance alleles and the origins and spread of dhfr mutations in different populations, which remain fully be elucidated. The limit was set at a minimum repeat unit length two, minimum unit length of the array 8 and maximum repeat unit length 100 bp. Then pure microsatellites, compound microsatellites and interrupted microsatellites were included. Lastly, microsatellites should be placed in corresponding regions in the upstream and downstream respectively. 6 microsatellites markers which were located in non-coding region have been selected are u93, u38, u2.6, d4.9, d37, d94.We grouped the samples according to resistant haplotypes. IFSTH/SSI, IFSTHNI, IFRTHNI, II/LRMHTI represent as wild type, low, middle, high level of resistance respectively. Then we genotyped the haplotypes according to the different sample sources.Every microsatellites were amplified by nested or semi-nested PCR with primers designed by Oligo, then the microsatellite polymorphisms were detected by QIAxcel system and data acquisited by Bio Calculator. The data was processed and analyzed with the following statistic methods: GENALEx, MS tools and Network.6 microsatellites were polymorphic and 57 alleles were identified. The allele frequency and heterozygosity (He) varies according to the groups of resistance. Isolates bearing wild type dhfr alleles were highly variable and the mean value for He was 0.76472, which exhibited variation similar to that seen for neutral markers. And four remote microsatellites were found to be highly polymorphic in the population where number of mutant alleles varied from 5 to 12. In comparison, He at markers situated around 2.6kb in IFSTHNI group (He=0.32) and 4.9kb in II/LRMHTI group was low (He=0.29) and different from He at farther or farthest microsatellites. We also see a reduction in He surrounding mutant dhfr alleles compared to that for the sensitive alleles for the pivotal loci. The lowest curves are seen for the IFSTHNI group and II/LRMHTI group. This lack of variation is characteristic of a possible selective sweep. The main microsatellites size at u2.6 locus were 265,260,290/295 in IFSTHNI group,IFRTHNI group,II/LRMHTI group respectively. The main microsatellites size at d4.9 locus were 112/114,122,112 in order same as above.The most widespread antifolates resistance lineage in Yunnan province was the quadruple haplotype IIRMHTI and ILRMHTI, which appears to have originated in this location independently. The existence of two origins was unlikely to substantially contribute to the counterbalance of the two main microsatellites polymorphisms in u2.6 locus. Microsatellite instability or other peculiarity could have a more basis. HM3 and HM4 from II/LRMHTI group showed similar characters at locus u2.6 with samples from Central China rather than Yunnan quadruple haplotypes. We believed the development of triple or quadruple dhfr allele was on the basis of local single mutant dfhr allele (117N). The double mutant (58R/117N) dhfr allele has evolved as 2 origins, because Yunnan and Hainan showed disparity in allele frequency distribution of key locus. However, we could not preclude the possibility of trans-regional malaria transmission. Moreover, we do see the wide separation between 3 level of resistance groups from Network diagram, which supported that the quadruple (II/LRMHTI), double (IFRTHNI) and single (IFSTHNI) mutant alleles had at least three independent origins in Yunnan, Hainan, and Central China.This research also selected 90 samples having haplotype and microsatellites data for amplifying and examining the central motif of PvCSP. The CSP, binds specifically to salivary glands, is one of the major structural proteins in the mosquito stage. Local adaptations that optimize transmission success can reflect by central repeat motif of CSP. The different composition of nonapeptide repeats between Central China and Yunnan /Hainan samples have been identified, which could be attributed to the type of vector. In this way, even if the Yunnan or Hainan resistant parasite infected the Central China residents, the parasite will unable to adapt to local arthropods host as to be unspreading nextly or unsurvivble. But we found the consistent sequence of nonapeptide repeats in Yunnan and Hainan samples. As set forth, we could not preclude the possibility of trans-regional malaria transmission, but according to the distinction of dhfr haplotypes and microsatellite polymorphisms in two regions, we consider that the indigenous resistant lineages still occupied absoluteness predominance.Summarily, Plasmodium vivax from Yunnan, Hainan Island and Central China fields commonly exists point mutations in dhfr genes that confer resistance to pyrimethamine, but there was a signicant difference of dhfr SNP and haplotype diversity in three regions. Meanwhile, from the allele frequency of the nearest microsatellite markers we can identified mutations in P.vivax dhfr have arisen three times in Yunnan, Hainan and Central China independently. The molecular data from this project will provide important information for making decisions on population based drug use in China.