A new high-throughput method for simultaneous detection of drug resistance associated mutations in Plasmodium vivax dhfr, dhps and mdr1 genes
- Author(s)
- Barnadas, C; Kent, D; Timinao, L; Iga, J; Gray, LR; Siba, P; Mueller, I; Thomas, PJ; Zimmerman, PA;
- Journal Title
- MALARIA JOURNAL
- Publication Type
- Journal Article
- Abstract
- Background: Reports of severe cases and increasing levels of drug resistance highlight the importance of improved Plasmodium vivax case management. Whereas monitoring P. vivax resistance to anti-malarial drug by in vivo and in vitro tests remain challenging, molecular markers of resistance represent a valuable tool for high-scale analysis and surveillance studies. A new high-throughput assay for detecting the most relevant markers related to P. vivax drug resistance was developed and assessed on Papua New Guinea (PNG) patient isolates. Methods: Pvdhfr, pvdhps and pvmdr1 fragments were amplified by multiplex nested PCR. Then, PCR products were processed through an LDR-FMA (ligase detection reaction -fluorescent microsphere assay). 23 SNPs, including pvdhfr 57-58-61 and 173, pvdhps 382-383, 553, 647 and pvmdr1 976, were simultaneously screened in 366 PNG P. vivax samples. Results: Genotyping was successful in 95.4% of the samples for at least one gene. The coexistence of multiple distinct haplotypes in the parasite population necessitated the introduction of a computer-assisted approach to data analysis. Whereas 73.1% of patients were infected with at least one wild-type genotype at codons 57, 58 and 61 of pvdhfr, a triple mutant genotype was detected in 65.6% of the patients, often associated with the 117T mutation. Only one patient carried the 173L mutation. The mutant 647P pvdhps genotype allele was approaching genetic fixation (99.3%), whereas 35.1% of patients were infected with parasites carrying the pvmdr1 976F mutant allele. Conclusions: The LDR-FMA described here allows a discriminant genotyping of resistance alleles in the pvdhfr, pvdhps, and pvmdr1 genes and can be used in large-scale surveillance studies.
- Publisher
- BIOMED CENTRAL LTD
- Keywords
- PAPUA-NEW-GUINEA; PLUS SULFADOXINE-PYRIMETHAMINE; DIHYDROPTEROATE SYNTHASE; THERAPEUTIC RESPONSE; MOLECULAR MARKERS; MALARIA; CHLOROQUINE; FALCIPARUM; INFECTION; POLYMORPHISMS
- Research Division(s)
- Infection And Immunity
- Publisher's Version
- https://doi.org/10.1186/1475-2875-10-282
- Open Access at Publisher's Site
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Creation Date: 2011-09-24 12:00:00