Polymorphism Analysis Of Drug Resistance Molecular Markers And Population Genetic Study In Plasmodium Ovale

Plasmodium ovale is one of the major human malaria parasites.It is mainly prevalent in Africa,Middle East and Southeast Asia.Since Stevens Systematically described it in 1992,the difficulty of microscopic identification has led to an underestimation of Plasmodium ovale infection,and its mild clinical symptoms had received less attention.There are no reports of local infections of Plasmodium ovale in China by now.However,with the development of social economy,labor export and cross-border trade and tourism increasing,the number of imported P.ovale cases is increasing year by year.The main origin of infection is from Africa.Therefore,the study of African imported Plasmodium ovale is particularly important.With the widespread use of antimalarial drugs,Plasmodium falciparum and Plasmodium vivax drug resistant strains have been reported in many regions around the world.Plasmodium ovale shares the same vectors and human host,with other malaria parasites,and it is likely to be under the same drug selection pressure.At present,the monitoring methods for drug resistance mainly include in vivo efficacy test,in vitro drug sensitivity test and resistance molecular marker detection.Since the clinical cases of Plasmodium ovale are rare and the in vitro culture system has not been established,the resistance molecular markers has become the most suitable method for monitoring the drug resistance of Plasmodium ovale.In this study,168Plasmodium ovale cases imported from African were included in the study.The polymorphism analysis of the four genes and drug selection pressure analysis of Plasmodium ovale were carried out.For the genes that may under drug selection pressure were further validated by microsatellite markers.The genetic structure of Plasmodium ovale was analyzed to provide scientific basis for studying the surveillance and further research of Plasmodium ovale resistance.This study is divided into two parts:Part 1.Polymorphism analysis of drug resistance molecular markers in Plasmodium ovaleThe blood samples were collected from imported P.ovale malaria patients from2012 to 2016 in Jiangsu Province.The P.ovale K13,crt,cytb and dhfr genes was amplified by PCR.The acquired DNA sequences were aligned using BioEdit software.The mutations of the obtained DNA sequences were analyzed using DNAstar software.The polymorphism of genes was analyzed by DnaSP software.The phylogenetic tree was constructed using MEGA software.The drug selection pressure was analyzed by calculating d_N,d_S using MEGA software.The results showed that the four drug resistance-related genes have lower polymorphism levels expect for dhfr gene.the K13 gene contained two synonymous mutations.P.ovale curtisi crt gene contain E34G,L43V mutation in each one case,P.ovale walliker have E34G mutation in two cases,I102M mutation in one case and V111F mutation in other one case.The crt gene was under purifying selection.There were R66K,R75K and R95K mutations in the P.ovale cytb gene,but the mutation ratio was low.The cytb gene was not under positive selection.The mutation rates of S58R and S113B/T were 52.17%and 17.39%respectively in P.ovale curtisi isolates,d_N/d_S was 2.42,and the P.ovale curtisi isolates maybe under positive selection.The P.ovale wallikeri isolates contained S58R,T62R,S113T,S113N and I169T mutations,but the mutation ratio was low.Part2.Flanking Microsatellite analysis of drug resistance molecular marker dhfr gene in Plasmodium ovale curtisiPolymorphism analysis of K13,crt,cytb and dhfr genes of Plasmodium ovale revealed that the mutation rate of the dhfr gene of the Plasmodium ovale is relatively high and may be under positive selection.To further confirm the presence of drug selection pressure,we analyzed five microsatellite loci flanking the dhfr gene.The expected heterozygosity of each population was calculated by GenAlEx software,and the statistical difference of He value was tested for statistical significance to determine whether there was a selective sweep.Structure software was used to analyze the genetic structure of Plasmodium ovale,providing a basis for further exploring the origin and transmission of Plasmodium ovale resistant strains.The results showed that the average number of alleles in five microsatellite loci of Plasmodium ovale was 7.2.The mean expected heterozygous of five microsatellite sites from different sources was analyzed,with 0.619±0.041 in South Africa,0.678±0.036 in West Africa,and 0.636±0.054 in Central Africa,no statistical difference between the three regions(P=0.092>0.05).The mean of expected heterozygous in samples from three countries with more input sources was compared,with Angola being 0.612±0.038,Nigeria being 0.658.±0.033,Equatorial Guinea being 0.598±0.061,and no statistically significant difference between the three countries(P=0.131>0.05).The amino acid at position 58 and 113 of the dhfr gene were classified.The average heterozygosity of the 5 sites of the S58R mutant was0.575±0.069,and the Non-S58R mutant was 0.682±0.045.Statistical difference between the S58R and Non-S58R mutant was P=0.011(P<0.05).,Mean heterozygosity of 5 sites of S113B/T mutant was 0.511±0.074,Non-S113B/T mutant strain was0.661±0.060,the statistical difference between the two mutants was P=0.001(P<0.05),the non-mutant strain is expected to have higher heterozygosity than the mutant strain.The expected heterozygosity of non-mutant strains was higher than that of mutant strains,suggesting that there was selective sweep in dhfr gene,which might be under the pressure of drug selection.The genetic structure analysis of the P.ovale dhfr gene showed that there are 7 haplotypes,and 3 of them are dominant haplotypes.South Africa,West Africa and Central Africa share the same haplotype.The genetic differentiation coefficients of Plasmodium ovale in South Africa,West Africa and Central Africa were different.The Fst maximum between West Africa and Central Africa was 0.038,and the difference was not statistically significant(P>0.05).The genetic differentiation coefficient between Nigeria and Equatorial Guinea was the largest among different countries,and the Fst was 0.041.The difference was also not statistically significant(P>0.05).The S58R mutant and the wild-type strain were genetically differentiated to 0.103(P<0.05),and the S113B/T mutant and the wild-type strain were genetically differentiated to 0.087(P<0.05).The mutant strain and the wild strain showed a certain degree of genetic differentiation.Through the sequencing analysis of molecular marker genes associated with drug resistance and microsatellite marker analysis of Plasmodium ovale imported from Africa,the selective sweep of dhfr gene was observed.The dhfr gene may be under the pressure of drug selection.The study results provide a scientific basis for the monitoring of the drug resistance of Plasmodium ovale,it also laid the foundation for further research on the resistance of Plasmodium ovale.