Evaluation ofPlasmodium vivaxGenotyping Markers for Molecular Monitoring in Clinical Trials

Abstract

Even for the most diverse markers, the highest allelic frequencies reached 6% (MS16) or 13% (Pv3.27). To reduce the theoretical probability of superinfection with parasites that have the same haplotype as that detected at baseline, we propose to combine at least 2 markers for genotyping individual P. vivax infections.

High Genetic Diversity of Plasmodium vivax on the North Coast of Papua New Guinea

“…MS16 and msp1F3 were selected to genotype P. vivax isolates in the present study, as it has been shown previously that diversity of the two markers is sufficiently high to enable high-resolution differentiation between genetically distinct strains. 13,14 The MS16 marker is highly diverse and lacking in dominant alleles. Less diverse than MS16, the coding sequence of the msp1F3 marker is more complex than a microsatellite; therefore, artifacts introduced by slippage of the polymerase chain reaction (PCR) polymerase are less likely to occur.…”

“…14 Genotyping was performed as previously described, 14 with the following alterations: a multiplex first-round PCR was performed using the HotMaster Taq DNA Polymerase Kit (5 Prime, Murarrie, Queensland, Australia) and the published MS16 and msp1F3 primer sequences of Koepfli and others. 13 The primary PCR mastermix, in a final volume of 20 μL, consisted of 1 μL WGA template DNA, 2 μL 10 + HotMaster Taq buffer with Mg 2+ , 0.25 μM each primer (Geneworks, Hindmarsh, South Australia, Australia), 0.2 mM deoxynucleotide triphosphates (dNTPs) (Roche, Castle Hill, New South Wales, Australia), and 1 U HotMaster Taq DNA polymerase (5 Prime). Thermocycling was performed under the following conditions: denaturation at 94 C for 2 minutes; 30 cycles of denaturation at 94 C for 30 seconds, primer annealing at 56 C for 30 seconds, and primer extension at 65 C for 1 minute; final extension at 65 C for 5 minutes.…”

“…A panel of nine markers for genotyping P. vivax isolates was previously evaluated using samples collected in Papua New Guinea (PNG). 13 Three markers-MS16, pv3.27, and msp1F3-were identified as the most genetically diverse in the PNG parasite population investigated and therefore, best suited to identify individual clones. In a subsequent study by Koepfli and others, 14 it was reported that using the unlinked MS16 and msp1F3 markers combined was sufficient to identify genetically distinct clones from the highly endemic Maprik region in East Sepik Province of PNG, and also that the genetic diversity of P. vivax within this region is very high.…”

“…The diversity of a given marker and the number of markers required to accurately genotype P. vivax infections will, however, differ between geographical locations due to variations in malaria epidemiology. [11][12][13] As such, the suitability of genotyping markers for a specific geographic location or region should be assessed before use. A panel of nine markers for genotyping P. vivax isolates was previously evaluated using samples collected in Papua New Guinea (PNG).…”

Diagnosis and Treatment of Plasmodium vivax Malaria

“…A variety of methodologies have been developed for P. vivax with as few as three polymorphic markers proving to be sufficient to discriminate homologous from heterologous infections. [73][74][75][76] However, recurrence of P. vivax genetically identical to the pretreatment isolate can occur from either a true recrudescence of the initial infection or a relapse from hypnozoites generated from the prior blood-stage infection 74,77 ; unsurprisingly molecular methods are unable to distinguish between these alternatives. Relapses are also commonly genetically heterologous.…”