Molecular detection of drug resistance in Plasmodium vivax

Introduction: Surveillance of drug resistance is essential for early warning systems and to advocate for appropriate drug policies. Many countries experience a high prevalence of antimalarial drug resistance, which threatens malaria control and elimination programs. The molecular methods used to monitor chloroquine resistance in Plasmodium vivax, target a multidrug resistance 1 (Pvmdr1) gene, and a putative transporter protein (Pvcrt-o) gene. Molecular detection of resistance in sulphadoxine-pyrimethamine, which is often accidentally used to treat against P. vivax, has dihydrofolate reductase (Pvdhfr) gene, and dihydropteroate synthetase (Pvdhps) gene as targets. Objective: The aims of this study were (A) to observe the prevalence of antimalarial drug resistance in return travellers to monitor drug resistance of P. vivax in endemic regions, and (B) to focus on an area known to have drug resistant P. vivax and use the molecular markers to monitor the status of drug resistance. Southern Thailand was chosen as target area, because no previous investigation for drug resistance had been undertaken in this area. Methods: The first set of blood samples (n=122) were stored frozen samples from patients who submitted blood for malaria screening. These patients had returned from malaria endemic countries, such as India, Papua New Guinea, Pakistan, Indonesia, Thailand, Solomon Islands, South Korea, and Cambodia. The second set (n=157) was provided by the malaria clinics in Surat thani, Ranong, Chumphon, and Yala provinces in Southern Thailand, over a period of one year 2017-2018. Specific primers for the Pvmdr1 and Pvcrt-o genes and the Pvdhfr and Pvdhps genes were designed to detect P. vivax DNA. The obtained PCR products were subsequently sequenced and analysed for the presence of resistance-associated mutations by alignments against the following GenBank reference strains: Reference no. Pvdhfr; iv XM001615032 & X98123.1, Pvdhps; XM001617159, Pvcrt-o; AF314649, and Pvmdr; AY618622. Result: Chloroquine resistance biomarkers, Pvmdr1, showed a predominance of the double mutations (F976:L1076) and a single mutation (L1076). Isolates with K10 insertion in the Pvcrt-o were in low numbers from India, Cambodia and Ranong Provence in Southern Thailand. The SP resistance biomarkers showed a combination of quadruple mutations L/I7:R58:M61:T117 in Pvdhfr and a double mutation G383:G553 in Pvdhps in India, PNG, and Southern Thailand. Conclusions: Mutations in targeted genes show the presence of resistance genes and suggests that continued use of chloroquine will eventually lead to increase chloroquine resistance cases in these regions. Additionally, sulphadoxine-pyrimethamine should not be used as an alternative treatment for P. vivax as resistance biomarkers are now evident. The misguided use of this drug for P. vivax must be stopped. This study demonstrated the application of molecular tools for the surveillance of antimalarial resistance in P. vivax