| Malaria is one of the most serious infectious diseases in the world,and its pathogen is Plasmodium.Plasmodium falciparum is one of the five species of Plasmodium,it causes falciparum malaria with acute onset,severe symptoms,and even death.Africa is the most severely endemic area of malaria,and China currently has no local cases of malaria infection.However,in recent years,with the increasing international communication and trade exchanges,the number of cases of falciparum malaria imported from Africa to China remains high.Therefore,the suveillance of drug resistance to Plasmodium falciparum from Africa became more important for malaria control in China.Since 2006,the World Health Organization has recommended Artemisinin-based combination therapies(ACTs)as first-line antimalarial drugs to treat falciparum malaria.The use of ACTs has significantly reduced the incidence and mortality of malaria.However,artemisinin resistant strains appeared in Southeast Asia in 2008,and it has now developed into a serious public health problem in Southeast Asia.At present,although there is little evidence of resistance to artemisinin in Plasmodium falciparum in Africa,and artemisinin-based first-line compound drugs are still very effective in the treatment of uncomplicated falciparum malaria,but due to the high incidence and mortality of malaria,drug-resistant strains may become the biggest threat to malaria prevention and control in Africa.Therefor,surveillance of artemisinin resistance is very important.In 2014,a study found that artemisinin resistance is related to the K13-propeller(k13)gene mutations,which has become a molecular marker for artemisinin resistance for artemisinin resistance surveillance.In this study,1561 falciparum malaria cases samples from 36 countries in Africa were used.The artemisinin resistance molecular marker k13 gene was analyzed for polymorphism and drug selection pressure,and further selected by microsatellite markers analysis.And population genetic structure,and genetic background research on strains in areas where drug selection pressure may occur,to provide scientific basis for the clinical rational use of imported falciparum malaria,and to provide a reference for surveillance the spread of artemisinin-resistant Plasmodium falciparum.The content of this research is divided into three parts:Part 1.Mutation analysis of k13,artemisinin resistance marker of Plasmodium falciparum imported from Africa Objective To understand the mutation of the artemisinin resistance molecular marker k13 gene of Plasmodium falciparum imported from Africa,and to analyze drug selection pressure.Methods The blood samples of imported patients with falciparum malaria from 2011 to 2019 in Jiangsu Province were collected,and the kelch propeller domain of the k13 gene of Plasmodium falciparum was amplified by PCR and sequenced.Use DNAstar software to edit and proofread all bidirectional sequencing sequences based on peak diagrams,use Bio Edit software to compare sample sequences with reference sequences,use Dna SP software to analyze the polymorphisms of the k13 gene sequence and perform neutral testing,and use MEGA software to calculate non-uniformity.Synonymous base substitution rate(d N)and synonymous base substitution rate(d S).Use SPSS22.0 software to carry out statistical analysis of the data,and calculate the k13 mutation rate between each site and different regions and years.Results Totally 89 of the 1561 imported falciparum malaria samples from 36 African countries had mutations in 54 sites in the k13 gene,35 non-synonymous mutations,and 19 synonymous mutations.Mutations related to artemisinin resistance(M476I,A481 V,G533A,G533 S,N537I,I543 T,M579I,M579 V,C580Y,P667 S,F673I)were detected in 17 samples from central Africa,and 15 cases were from In Equatorial Guinea,1 case(C580Y)was from Gabon and 1 case(M579V)was from Cameroon.The non-synonymous mutation rate and polymorphism level of k13 gene in samples from central Africa,especially Equatorial Guinea,were significantly higher than other regions,with a d N/d S ratio of 3.187.There was no statistically significant difference in the mutation rate between the years.Conclusion The k13 gene of Plasmodium falciparum in most parts of Africa has a multi-site low-frequency mutation.The mutation frequency of the k13 gene of Plasmodium falciparum in Equatorial Guinea is significantly higher than that in other regions,and multiple mutations that may be related to artemisinin resistance have been found.And the d N/d S ratio is 3.187,which may be affected by drug selection pressure.Part 2.Polymorphism of flanking microsatellite of k13 gene of imported Plasmodium falciparum from AfricaObjective To compare the expected heterozygosity of microsatellite locus between non-synonymous mutant strains and wild strains in various regions of Africa,and to further analyze whether the k13 gene of Plasmodium falciparum is affected by selective elimination.Methods The 10 microsatellites loci of the k13 gene flanking region were analysised.Gen Al Ex software was used to calculate the expected heterozygosity(He)of each population,and test whether the difference in He value between the populations,and judge whether there is selective sweeping.Arlequin software was used to analyze and calculate the value of genetic differentiation coefficient Fst,and the structure software was used to analyze the genetic structure of Plasmodium falciparum.Results The average number of alleles at the five microsatellites loci of Plasmodium falciparum was 16.7,and the average He was 0.831.A comparative analysis of He in different geographic regions(Central Africa,Southern Africa,Western Africa)and countries with more samples(Equatorial Guinea,Angola,Nigeria)showed that the difference was not found to be statistically significant(P>0.05).The samples are classified according to whether they are mutated(non-synonymous mutant strains and wild strains)and whether they have artemisinin k13 resistance-related mutation sites(artemisinin resistance-related mutant strains and wild strains),and compare the two He,the difference It is also not statistically significant(P>0.05).The He analysis of non-synonymous mutant strains and wild strains in Central Africa and Equatorial Guinea,Southern Africa and Angola,and Western Africa showed that the difference was also not statistically significant(P>0.05).The analysis of genetic structure showed that the Fst value of the coefficient of genetic differentiation in western and Southern Africa was the largest value of 0.028(P<0.05).The maximum Fst value between Angola and Nigeria between different countries is 0.048(P<0.05).The Fst of non-synonymous mutant strain and wild type strain was 0.006(P<0.05).However,when Fst is between 0 and 0.05,the genetic differentiation between populations is very small.Conclusion The analysis of the microsatellites polymorphisms of the k13 gene flanking region in P.falciparum showed that there was no selective sweep in Africa,and no significant differences in the genetic structure of P.falciparum were found between each regions.Part 3.Studies on other resistance molecular markers of Plasmodium falciparum imported from AfricaObjective To analyze the genetic background of k13 non-synonymous mutant strains of Plasmodium falciparum from Equatorial Guinea,and to analyze molecular markers related to other antimalarial drug resistance.Methods The Pfcrt,Pfmdr1,Pfdhfr,and Pfdhps genes of Plasmodium falciparum(33cases of non-synonymous mutations,30 cases of wild type)were amplified by PCR and sequenced,and the sample sequence was compared with the reference sequence using Bio Edit software to calculate frequency of mutations.Fluorescence quantitative PCR was used to detect the copy number of Pfpm2 and Pfmdr1 genes.Results Totally 62 cases of Pfcrt gene were successfully sequenced,33 cases of k13non-synonymous mutation samples and 29 k13 wild-type carrying Pfcrt CVIET mutations accounted for 21.2% and 13.8%,respectively,and the proportion of Pfcrt mutations in k13 non-synonymous mutants slightly higher,but the difference was not statistically significant(P>0.05).The Pfmdr1 gene was successfully sequenced in 59 cases,the proportions of N86Y/Y184 F mutations carrying Pfmdr1 in 31 k13non-synonymous mutation samples and 28 k13 wild-type samples were 48.4%/83.9%,50%/71.4%,respectively,with no statistical significance(P>0.05).Fifty samples of Pfdhfr gene were successfully sequenced,k13 non-synonymous mutation samples all carried N51 I,C59R,and S108 N mutations,was not statistically different(P>0.05)with the proportion of Pfdhfr mutations carried by wild type.Forty-eight Pfdhps genes were successfully sequenced,and there was no statistically significant difference(P>0.05)in the proportion of Pfdhps mutation in k13 non-synonymous mutations and wild-type samples.The Pfpm2 gene was successfully amplified in all63 samples,and 62 cases were successfully amplified with Pfmdr1.There was no significant difference in k13 non-synonymous mutations and wild-type in samples with a copy number greater than 1.5(P>0.05).Conclusion There is no statistically significant difference in the polymorphism of Pfcrt,Pfdhps,Pfdhfr,Pfmdr1,Pfpm2 genes between non-synonymous mutant strains and wild strains,and no association between the k13 mutation of Plasmodium falciparum and other drug-resistant molecules has been observed. |
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