Study Of Chloroquine Resistance Genes Polymorphisms And Msp3 Gene Typing Of Plasmodium Faciparum In Hainan, China

Malaria remains uncontrolled to-date due to various reasons: emergence of the drug resistant parasite, pesticide resistant mosquito vector and nonavailability of suitable and effective malaria vaccine. The disease burden is increasing in almost all the tropical countries since malaria creates socioeconomic problems and also causes large number of deaths. The situation is becoming more difficult because the most widely used antimalarial drug chloroquine is losing its effectiveness. Though, exact molecular mechanism of chloroquine resistance remains elusive, three food vacuole membrane proteins , namely, Pgh1, a ~330 kDa protein and PfCRT, have been identified to play a significant role and their respective genes pfmdr1, cg2 and pfcrt have been cloned and sequenced. Otherwise Plasmodium falciparum isolates with different gene-types probably have different pathogenicity, antigenicity and drug-sensitivity. This study included two main parts: one was the polymorphism analysis of Chloroquine resistant genes of Plasmodium falciparum. The other was the gene typing of merozoite surface protein 3.[Objective]To identify genetic polymorphisms in the pfcrt gene and pfmdr1 gene in 42 Plasmodium falciparum isolates collected from Hainan Province and to determine their relationship with chloroquine resistance in vitro.To identify allelic type of pfmsp3 gene in 42 Plasmodium falciparum isolates collected from Hainan Province.[Methods]For the polymorphism analysis of these genes as above, nested PCR was applied to amplify fragments of pfcrt gene coding the 76 and 356 amino acids and fragments of pfmdr1 gene coding the 86, 1042 and 1246 amino acides. Then PCR products were digested with special restriction endonucleases to detect whether point mutations existed in those genes and their relations with Chloroquine resistance.Based on the different allele type sequences of pfmsp3, 3 pairs primers were designed to amplify the two different allele gene of pfmsp3 by nested PCR respectively.[Results]22 of the 42 samples collected were chloroquine resistant, the others were chloroquine sensitive, resistant ratio is 52.38%.The N86Y mutation of pfmdr1 was not detected in all 42 samples tested. D1246Y mutation of pfmdr1 was found in 8 of 42 isolates, except one mixinfection, the others were all chloroquine resistant. N1042D mutation of pfmdr1 was detected in 10 of 37 isolates, nine of which were chloroquine resistant. The 356 mutation of pfcrt was not detected in all 42 samples, while K76T mutation of pfcrt was found in 30(with 2 of mix-infection) of 42 isolates, 18 of which were chloroquine resistant.The pfmsp3 gene fragments of in 36 out of 42 blood samples were amplified, of which the majority was the K1-type allele (69.44%) and the minority was the 3D7-type allele (63.89%). The mixed infection rate of two different allelic types was 33.33%.[Conclusion]The K76T mutation was shown certain association with chloroquine resistance while no relationship of the 356 mutation with chloroquine resistance was found. The N86Y mutation in pfmdr1 seemed to have no association with chloroquine resistance as the N1042D and D1246Y did.K1-type allele of the msp3 gene was found more popular than 3D7-type allele, but was not dominant. The mixed infection rate was not high.