Abstract
Background: Plasmodium vivax isolate resistance to Chloroquine (CQ) has been reported from many endemic regions in the world. P. vivax is responsible for 95% of malaria cases in Afghanistan and CQ is the first-line treatment given for treatment of vivax malaria. The pvmdr-1 and pvcrt-o (K10 insertion) genes are the possible markers for CQ-resistance P. vivax isolates. There have been no studies done on the presence or absence of molecular markers for CQ-resistance P. vivax in Afghanistan. In the present work, we aimed to evaluate the frequency of mutations in the pvmdr-1 and K10 insertion in the pvcrt-o genes of P. vivax.Methods: P. vivax isolates were collected from Laghman, Baghlan and Khost provinces. For investigation of polymorphisms of desired regions in pvmdr-1 and pvcrt-o genes, sequencing was applied on the PCR products. We developed a new asymmetric qPCR and melting analysis assay based on unlabeled probe for scanning of K10 insertion in pvcrt-o gene. Results: The analysis of sequencing data of the pvmdr-1 gene showed wild type Y976 and K997 and mutant M958 and L1076 in 33 isolates from three provinces and submitted in GenBank (accession number MK419882-MK419914)Of 36 samples that evaluated for K10 insertion in pvcrt-o, 2/18(11%), 0/10(0%) and 0/8(0%) isolates from Laghman, Baghlan and Khost province possessed K10 insertion, respectively, that confirmed by either sequencing and unlabeled probes and submitted in GenBank (accession number MK292011-MK292046).Conclusion: Two samples with K10 insertion and 33 samples with pvmdr1 polymorphism, indicating on the possibility of CQ resistance P. vivax populations in Afghanistan. Furthermore, unlabeled probes are simple and inexpensive alternative tools for screening of P. vivax mutations.