Methodology

A new high-throughput method for simultaneous detection of drug resistance associated mutations in Plasmodium vivax dhfr, dhps and mdr1 genes

Céline Barnadas^1,2†, David Kent^3†, Lincoln Timinao¹, Jonah Iga¹, Laurie R Gray⁴, Peter Siba¹, Ivo Mueller^1,2,5, Peter J Thomas^3* and Peter A Zimmerman^4*

• * Corresponding authors: Peter J Thomas pjthomas@case.edu - Peter A Zimmerman paz@case.edu

• † Equal contributors

Author Affiliations

¹ Vector Borne Diseases Unit, Papua New Guinea Institute of Medical Research (PNGIMR), Goroka, Papua New Guinea

² Infection and Immunity Unit, Walter & Eliza Hall Institute of Medical Research, Parkville, Australia

³ Department of Mathematics, Case Western Reserve University, Cleveland, USA

⁴ Center for Global Health and Diseases, Case Western Reserve University, Cleveland, USA

⁵ Centre de Recerca en Salut Internacional de Barcelona (CRESIB), Barcelona, Spain

For all author emails, please log on.

Malaria Journal 2011, 10:282 doi:10.1186/1475-2875-10-282

Published: 24 September 2011

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.