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Background   Plasmodium vivax  resistance to chloroquine (CQ) has been reported from many endemic regions in the world.  Plasmodium vivax  is responsible for 95% of malaria cases in Afghanistan and CQ is the first-line treatment given for vivax malaria. The  pvmdr - 1  and  pvcrt - o  (K10 insertion) genes are possible markers for CQ-resistance in  P. vivax  isolates. There have been no studies done on the presence or absence of molecular markers for CQ-resistance  P. vivax  in Afghanistan. The present work aimed to evaluate the frequency of mutations in the  pvmdr - 1  and K10 insertion in the  pvcrt - o  genes of  P. vivax.Methods   Plasmodium 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. A new asymmetric qPCR and melting analysis assay based on unlabelled probe developed 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. Of the 36 samples evaluated for K10 insertion in  pvcrt - o , 2/18(11%), 0/10(0%) and 0/8(0%) isolates from Laghman, Baghlan and Khost province, respectively, possessed K10 insertion, confirmed by either sequencing and unlabelled probes.    Conclusion  Two samples with K10 insertion and 33 samples with  pvmdr1  polymorphism, indicating on the possibility of CQ resistance in  P. vivax  populations in Afghanistan. Furthermore, unlabelled probes are simple and inexpensive alternative tools for screening of  P. vivax  mutations.

malaria journal

An unlabelled probe-based real time PCR and modified semi-nested PCR as molecular tools for analysis of chloroquine resistant Plasmodium vivax isolates from Afghanistan