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

Cline Barnadas David Kent 0 Lincoln Timinao Jonah Iga Laurie R Gray 1 Peter Siba Ivo Mueller Peter J Thomas 0 Peter A Zimmerman 1 0 Department of Mathematics, Case Western Reserve University , Cleveland , USA 1 Center for Global Health and Diseases, Case Western Reserve University , Cleveland , USA 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. - Background Plasmodium vivax is the most widespread of the malaria parasites infecting human hosts and may be responsible for up to 400 million infections each year [1,2]. The widespread belief that this parasite causes only benign malaria has been challenged recently by reports of severe pathology, including cerebral malaria, acute respiratory distress, acute renal failure, and severe anaemia in patients with P. vivax infections [3-6]. In addition, there are increasing worldwide reports of treatment failure after administration of the standard drug regimens, mainly chloroquine (CQ) [7-15]. New drugs are now available, but they are significantly more costly than CQ plus primaquine, the standard treatment for vivax malaria for the last 50 years [16]. In order to change their treatment policies based on local evidence, countries need efficient methods to assess potential for resistance to current standard treatments. Monitoring in vivo drug resistance remains challenging due to the ability of P. vivax to relapse from longlasting liver stages. At the same time, the recurrence of parasitaemia due to de novo infection can confound evaluation of treatment efficacy. Genotyping of recurrent infections [17] makes it possible to distinguish infection by parasites with different genotypes (new infections) [18,19] from infection by parasites with identical genotypes (whether due to relapse or to recrudescence from blood-stage parasites that survived drug treatment) [20]. Of course, even molecular diagnosis cannot distinguish persistent infection from de novo infection by a genetically identical parasite. In vitro assays, which should provide drug susceptibility data free from the effects of confounding factors, such as host immunity, are still difficult to conduct because of the lack of stable, continuous P. vivax in vitro culture [5,21]. Molecular markers of resistance, therefore, represent a useful tool to monitor the introduction and spread of anti-malarial resistance. For P. vivax, these markers include mutations in genes encoding the dihydrofolate reductase (PvDHFR) and the dihydropteroate synthase (PvDHPS) that are involved in drug resistance to antifolates (pyrimethamine) [22], and sulphonamides (sulphadoxine) [23], respectively. The 976F mutation in the gene encoding the multidrug resistance 1 protein (PvMDR1), which has been associated with 4-aminoquinolines (amodiaquine, CQ) resistance in some [21,24] but not all studies [21,25]. Further investigations are needed to assess the predictive value of molecular markers; their use in monitoring of P. vivax drug resistance needs to be addressed, geographically, at both a local and a large-scale level [26]. For these reasons, a new assay was developed to detect single nucleotide polymorphisms (SNPs) potentially associated with P. vivax drug resistance, in pvdhfr, pvdhps and pvmdr1 genes (total of 23 allelic variants). This post-PCR multiplex assay uses ligase detection reaction and fluorescent-microsphere technologies. A similar approach was previously developed to screen for Plasmodium falciparum drug resistance-associated SNPs [27]. The assay was validated using sequenced isolates and a subset of samples from Papua New Guinea (PNG), where CQ resistant P. vivax was first described in 1989 [12]. Resistance to sulphadoxine-pyrimethamine (SP) was observed to be low until 2000 [28], when PNG introduced CQ or amodiaquine plus SP as the national first line treatment for both P. falciparum and P. vivax. Since then, levels of resistance to this combination have increased [24,29]. Here the development and application of this multiplex assay is described; the frequency of P. vivax genetic markers associated with multiple drug resistance in PNG patient isolates is presented. Methods Study population and blood sample collection Samples were collected between 2006 and 2007 through active detection of malaria infections in a cohort of children 1-4 yrs of age in Ilaita, East Sepik Province, Papua New Guinea [30]. The study was approved by the PNG Medical Research Advisory Council. Plasmodium vivax infected monkey blood (SalI and Thai-3 strains) was kindly provided by W. E. Collins (Centers for Disease Control and Prevention, Atlanta, GA). Plasmodium falciparum positive samples were obtained from a culture of the 3D7 strain. DNA template preparation DNA was extracted from cell pellets (250L) using the QIAamp 96 DNA blood kit (Qiagen, Valencia, CA). Genomic DNA was extracted from P. vivax-infected monkey blood or from P. falciparum culture using the QIAamp DNA blood minikit (Qiagen, Valencia, CA). Plasmodium species diagnosis Plasmodium species diagnosis was performed using a post-PCR ligase detection reaction - fluorescent microsphere assay (LDR-FMA) as described previously [30,31]. Positive samples for P. vivax infections were identified and then assessed for mutations in pvdhfr, pvdhps and pvmdr1 genes. Multiplex PCR a (...truncated)