Studies On Plasmodium Falciparum Chloroquine Resistance Gene Pfcrt

Malaria is one of most harmful parasitic diseases which remains one of the leading causes of morbidity and mortality in tropical and subtropical regions. Plasmodium falciparum causes the most severe disease In four human malaria parasites and it is still endemic in some provinces of China such as Hainan and Yunnan provinces. Chloroquine resistance was first reported in Southeast Asia and South America and has now spread to the vast majority of malaria-endemic countries. Chloroquine resistance necessitates the use of drugs which are more expensive and may have dangerous side effects. Study of chloroquine resistance focused on the in vivo and in vitro drug sensitivity assay during a long period. But pfcrt gene was rencently identified as a candidate gene for chloroquine resistance after the analysis of a genetic cross between a chloroquine-resistant clone (Dd2) and a chloroquine sensitive clone (HB3). Objective: To develop a method for detecting the mutation of P.falciparum chloroquine resistant transport gene 76 codon. To compare the haplotypes of pfcrt polymorphism of Hainan province with that of other areas of the world. Methods: (1) According to the sequence characteristic of pfcrt gene, nested polymerase chain reaction was used to amplify the polymorphic region including codon 76 of pfcrt gene.The PCR products were digested by ApoI restriction endonuclease and the digested fragments were observed by 2% agarose gel electrophpresis. The allelic types were determined according to the banding pattern. (2) Another nested PCR was used to amplify the polymorphic region including codon 72 to 76 and 97. The PCR products were digested by ApoI restriction endonuclease to determine the allelic types. According to the allelic types, each 2 products with mutant type and wild type were sequenced on single pass sequencing to analyze the haplotypes of pfcrt polymorphism. (3) 19 blood samples collected on filter paper of P. falciparum patients from Ledong county Hainan province in 2004 were detected. Results: (1)Bands in size of 145 bp appeared in all 31 samples. After ApoI digestion, two fragments (97 bp and 48 bp)were obtained from 17 samples, and 14 of 31 samples were defined to be the mutant type in codon 76 of pfcrt gene with a mutant rate of 45.16%. One of 14 samples collected from malaria patients were defined to be chloroquine sensitive in vivo. (2) Bands in size of 195 bp appeared in all 31 samples. After ApoI digestion, consistent with the result of part 1, fragments of 100 bp were obtained from 17 samples which were defined to be wild type in codon 76 of pfcrt gene. According to DNA sequencing, 2 CQRP.falciparum samples carry pfcrt alleles encoding an amino acid haplotype of CVIET(residues 72~76) and the haplotype of CVMNK were found in other 2 samples with wild type pfcrt gene. We observed a previously unreported mutation H97Y in codon 97. (3) 8 out of 19 samples collected from patients were defined to be mutant type pfcrt gene with a mutant rate of 42.10%. All these 8 samples were defined to be chloroquine resistance in vitro. We found the CVIET haplotype of Hainan province was the same as that of Southeast Asia and Africa and no mutation was found in codon 97. Conclusion: (1) The nested PCR-RELP is rapid and effective to detect the point mutation of pfcrt gene 76 codon. And it also can be applied in the surveillance of the change of CQR. (2) Haplotypes of pfcrt polymorphism from Hainan province were the same as those of Southeast Asia and Africa.