Plasmodium vivax infections often recur due to relapse of hypnozoites from the liver. In malaria-endemic areas, tools to distinguish relapse from reinfection are needed. We applied amplicon deep sequencing to P. vivax isolates from 78 Cambodian volunteers, nearly one-third of whom suffered recurrence at a median of 68 days. Deep sequencing at a highly variable region of the P. vivax merozoite surface protein 1 gene revealed impressive diversity-generating 67 unique haplotypes and detecting on average 3.6 cocirculating parasite clones within individuals, compared to 2.1 clones detected by a combination of 3 microsatellite markers. This diversity enabled a scheme to classify over half of recurrences as probable relapses based on the low probability of reinfection by multiple recurring variants. In areas of high P. vivax diversity, targeted deep sequencing can help detect genetic signatures of relapse, key to evaluating antivivax interventions and achieving a better understanding of relapse-reinfection epidemiology.

Our approach for detecting drug resistance identifies rare resistant parasites in polyclonal infections through their phenotypic signature. By translating genetic data to clearance phenotypes of parasite subpopulations in malaria infections, we found rare slow clearing parasites in Tanzania where resistance to front-line drugs is not thought to be a problem.

Abstract. Plasmodium vivax accounts for an increasing fraction of malaria infections in Thailand and Cambodia. We compared P. vivax genetic complexity and antimalarial resistance patterns in the two countries. Use of a heteroduplex tracking assay targeting the merozoite surface protein 1 gene revealed that vivax infections in both countries are frequently polyclonal (84%), with parasites that are highly diverse (H E = 0.86) but closely related (G ST = 0.18). Following a history of different drug policies in Thailand and Cambodia, distinct patterns of antimalarial resistance have emerged: most Cambodian isolates harbor the P. vivax multidrug resistance gene 1 ( pvmdr1) 976F mutation associated with chloroquine resistance (89% versus 8%, P 0.001), whereas Thai isolates more often display increased pvmdr1 copy number (39% versus 4%, P 0.001). Finally, genotyping of paired isolates from individuals suspected of suffering relapse supports a complex scheme of relapse whereby recurrence of multiple identical variants is sometimes accompanied by the appearance of novel variants.

BackgroundHumans living in regions with high falciparum malaria transmission intensity harbour multi-strain infections comprised of several genetically distinct malaria haplotypes. The number of distinct malaria parasite haplotypes identified from an infected human host at a given time is referred to as the complexity of infection (COI). In this study, an amplicon-based deep sequencing method targeting the Plasmodium falciparum apical membrane antigen 1 (pfama1) was utilized to (1) investigate the relationship between P. falciparum prevalence and COI, (2) to explore the population genetic structure of P. falciparum parasites from malaria asymptomatic individuals participating in the 2007 Demographic and Health Survey (DHS) in the Democratic Republic of Congo (DRC), and (3) to explore selection pressures on geospatially divergent parasite populations by comparing AMA1 amino acid frequencies in the DRC and Mali.ResultsA total of 900 P. falciparum infections across 11 DRC provinces were examined. Deep sequencing of both individuals, for COI analysis, and pools of individuals, to examine population structure, identified 77 unique pfama1 haplotypes. The majority of individual infections (64.5%) contained polyclonal (COI > 1) malaria infections based on the presence of genetically distinct pfama1 haplotypes. A minimal correlation between COI and malaria prevalence as determined by sensitive real-time PCR was identified. Population genetic analyses revealed extensive haplotype diversity, the vast majority of which was shared across the sites. AMA1 amino acid frequencies were similar between parasite populations in the DRC and Mali.ConclusionsAmplicon-based deep sequencing is a useful tool for the detection of multi-strain infections that can aid in the understanding of antigen heterogeneity of potential malaria vaccine candidates, population genetics of malaria parasites, and factors that influence complex, polyclonal malaria infections. While AMA1 and other diverse markers under balancing selection may perform well for understanding COI, they may offer little geographic or temporal discrimination between parasite populations.Electronic supplementary materialThe online version of this article (10.1186/s12936-017-2137-9) contains supplementary material, which is available to authorized users.

f Using a newly developed Plasmodium vivax merozoite surface protein 1 gene (Pvmsp1) heteroduplex tracking assay, we genotyped 107 P. vivax infections in individuals from Cambodia, 45 of whom developed recurrent parasitemia within 42 days. The majority of isolates were polyclonal, but recurrent parasitemias displayed fewer variants compared to initial parasitemias. Two Pvmsp1 gene variants occurred more frequently in the initial genotypes of those who developed recurrent parasitemia, representing the first time P. vivax variants associated with a higher risk of relapse have been described. P lasmodium vivax is the most prevalent malaria species outside Africa and causes significant morbidity in the developing world. It uniquely maintains its transmission in a wide range of latitudes by causing periodic relapses through reactivation of liver stage parasites called hypnozoites. Being able to identify those at risk for relapse has the potential to guide clinical management and drug policy (4, 9). However, studying P. vivax relapse in areas of endemicity has been difficult because genotyping methods have not been able to precisely differentiate relapse from new infection or recrudescence due to treatment failure.It is known that P. vivax infections are frequently polyclonal, even in relatively low-transmission settings (3, 7). We sought to exploit this complexity of infection to better describe genotypic patterns of relapsing parasites and search for genotypic variants associated with relapse. To do this, we developed a P. vivax heteroduplex tracking assay (HTA) to evaluate pretreatment and recurrent parasitemias from 107 patients treated with chloroquine monotherapy in Chumkiri, Cambodia, 45 of whom developed recurrent P. vivax infection within 42 days of therapy.HTAs have previously been shown to be more sensitive than nested PCR for determining the multiplicity of infection (MOI) in malaria infections due to their ability to detect both sequence and size polymorphisms (11). We developed an HTA targeting the P. vivax merozoite surface protein 1 gene (Pvmsp1), a highly polymorphic gene that is commonly used for genotyping P. vivax malaria. The probe was constructed as previously described, using primers based on a previously published assay and genomic DNA obtained from MR4 (Nicaragua strain, catalog no. MRA-340; MR4, Manassas, VA) (10, 11) (GenBank accession number JN674534). Pvmsp1 PCR products were amplified using the same primers from patient DNA that was extracted from filter paper blood spots. HTAs were performed as previously described. All gels were run with probe alone and nontemplate control lanes.Patient samples were acquired from a clinical trial that enrolled persons Ն1 year old presenting with uncomplicated P. vivax malaria to the Chumkiri health center in Kampot Province, Cambodia, between August 2006 and December 2007 (14). In this study, subjects were given a total dose of 25 mg/kg of body weight of chloroquine base over 3 days with directly observed therapy, followed with weekly blood smears until...