• Differences in selective pressure on dhps and dhfr drug resistant mutations in western Kenya

      McCollum, AM; Schneider, KA; Griffing, SM; Zhou, Z; Kariuki, S; Ter-Kuile, F; Shi, YP; Slutsker, L; Lal, AA; Udhayakumar, V; Escalante, AA (2012-03-23)
      Background: Understanding the origin and spread of mutations associated with drug resistance, especially in the context of combination therapy, will help guide strategies to halt and prevent the emergence of resistance. Unfortunately, studies have assessed these complex processes when resistance is already highly prevalent. Even further, information on the evolutionary dynamics leading to multidrug-resistant parasites is scattered and limited to areas with low or seasonal malaria transmission. This study describes the dynamics of strong selection for mutations conferring resistance against sulphadoxine-pyrimethamine (SP), a combination therapy, in western Kenya between 1992 and 1999, just before SP became first-line therapy (1999). Importantly, the study is based on longitudinal data, which allows for a comprehensive analysis that contrasts with previous cross-sectional studies carried out in other endemic regions. Methods. This study used 236 blood samples collected between 1992 and 1999 in the Asembo Bay area of Kenya. Pyrosequencing was used to determine the alleles of dihydrofolate reductase (dhfr) and dihydropterote synthase (dhps) genes. Microsatellite alleles spanning 138 kb around dhfr and dhps, as well as, neutral markers spanning approximately 100 kb on chromosomes 2 and 3 were characterized. Results: By 1992, the South-Asian dhfr triple mutant was already spreading, albeit in low frequency, in this holoendemic Kenyan population, prior to the use of SP as a first-line therapy. Additionally, dhfr triple mutant alleles that originated independently from the predominant Southeast Asian lineage were present in the sample set. Likewise, dhps double mutants were already present as early as 1992. There is evidence for soft selective sweeps of two dhfr mutant alleles and the possible emergence of a selective sweep of double mutant dhps alleles between 1992 and 1997. The longitudinal structure of the dataset allowed estimation of selection pressures on various dhfr and dhps mutants relative to each other based on a theoretical model tailored to P. falciparum. The data indicate that drug selection acted differently on the resistant alleles of dhfr and dhps, as evidenced by fitness differences. Thus a combination drug therapy such as SP, by itself, does not appear to select for "multidrug"-resistant parasites in areas with high recombination rate. Conclusions: The complexity of these observations emphasizes the importance of population-based studies to evaluate the effects of strong drug selection on Plasmodium falciparum populations. © 2012 McCollum et al; licensee BioMed Central Ltd.
    • Genetic variation and recurrent parasitaemia in Peruvian Plasmodium vivax populations

      McCollum, AM; Soberon, V; Salas, CJ; Santolalla, ML; Udhayakumar, V; Escalante, AA; Graf, PC; Durand, S; Cabezas, C; Bacon, DJ (2014-02-24)
      Background: Plasmodium vivax is a predominant species of malaria in parts of South America and there is increasing resistance to drugs to treat infections by P. vivax. The existence of latent hypnozoites further complicates the ability to classify recurrent infections as treatment failures due to relapse, recrudescence of hyponozoites or re-infections. Antigen loci are putatively under natural selection and may not be an optimal molecular marker to define parasite haplotypes in paired samples. Putatively neutral microsatellite loci, however, offer an assessment of neutral haplotypes. The objective here was to assess the utility of neutral microsatellite loci to reconcile cases of recurrent parasitaemia in Amazonian P. vivax populations in Peru. Methods. Patient blood samples were collected from three locations in or around Iquitos in the Peruvian Amazon. Five putatively neutral microsatellite loci were characterized from 445 samples to ascertain the within and amongst population variation. A total of 30 day 0 and day of recurrent parasitaemia samples were characterized at microsatellite loci and five polymorphic antigen loci for haplotype classification. Results: The genetic diversity at microsatellite loci was consistent with neutral levels of variation measured in other South American P. vivax populations. Results between antigen and microsatellite loci for the 30 day 0 and day of recurrent parasitaemia samples were the same for 80% of the pairs. The majority of non-concordant results were the result of differing alleles at microsatellite loci. This analysis estimates that 90% of the paired samples with the same microsatellite haplotype are unlikely to be due to a new infection. Conclusions: A population-level approach was used to yield a better estimate of the probability of a new infection versus relapse or recrudescence of homologous hypnozoites; hypnozoite activation was common for this cohort. Population studies are critical with the evaluation of genetic markers to assess P. vivax biology and epidemiology. The additional demonstration of microsatellite loci as neutral markers capable of distinguishing the origin of the recurrent parasites (new infection or originating from the patient) lends support to their use in assessment of treatment outcomes. © 2014 McCollum et al.; licensee BioMed Central Ltd.
    • Historical Shifts in Brazilian P. falciparum Population Structure and Drug Resistance Alleles

      Griffing, SM; Viana, GMR; Mixson-Hayden, T; Sridaran, S; Alam, MT; de Oliveira, AM; Barnwell, JW; Escalante, AA; Povoa, MM; Udhayakumar, V (2013-03-15)
      Previous work suggests that Brazilian Plasmodium falciparum has limited genetic diversity and a history of bottlenecks, multiple reintroductions due to human migration, and clonal expansions. We hypothesized that Brazilian P. falciparum would exhibit clonal structure. We examined isolates collected across two decades from Amapá, Rondônia, and Pará state (n = 190). By examining more microsatellites markers on more chromosomes than previous studies, we hoped to define the extent of low diversity, linkage disequilibrium, bottlenecks, population structure, and parasite migration within Brazil. We used retrospective genotyping of samples from the 1980s and 1990s to explore the population genetics of SP resistant dhfr and dhps alleles. We tested an existing hypothesis that the triple mutant dhfr mutations 50R/51I/108N and 51I/108N/164L developed in southern Amazon from a single origin of common or similar parasites. We found that Brazilian P. falciparum had limited genetic diversity and isolation by distance was rejected, which suggests it underwent bottlenecks followed by migration between sites. Unlike Peru, there appeared to be gene flow across the Brazilian Amazon basin. We were unable to divide parasite populations by clonal lineages and pairwise FST were common. Most parasite diversity was found within sites in the Brazilian Amazon, according to AMOVA. Our results challenge the hypothesis that triple mutant alleles arose from a single lineage in the Southern Amazon. SP resistance, at both the double and triple mutant stages, developed twice and potentially in different regions of the Brazilian Amazon. We would have required samples from before the 1980s to describe how SP resistance spread across the basin or describe the complex internal migration of Brazilian parasites after the colonization efforts of past decades. The Brazilian Amazon basin may have sufficient internal migration for drug resistance reported in any particular region to rapidly spread to other parts of basin under similar drug pressure.
    • Origin and evolution of sulfadoxine resistant Plasmodium falciparum

      Vinayak, S; Alam, T; Mixson-Hayden, T; McCollum, AM; Sem, R; Shah, NK; Lim, P; Muth, S; Rogers, WO; Fandeur, T; Barnwell, JW; Escalante, AA; Wongsrichanalai, C; Ariey, F; Meshnick, SR; Udhayakumar, V (2010-03-01)
      The Thailand-Cambodia border is the epicenter for drug-resistant falciparum malaria. Previous studies have shown that chloroquine (CQ) and pyrimethamine resistance originated in this region and eventually spread to other Asian countries and Africa. However, there is a dearth in understanding the origin and evolution of dhps alleles associated with sulfadoxine resistance. The present study was designed to reveal the origin(s) of sulfadoxine resistance in Cambodia and its evolutionary relationship to African and South American dhps alleles. We sequenced 234 Cambodian Plasmodium falciparum isolates for the dhps codons S436A/F, A437G, K540E, A581G and A613S/T implicated in sulfadoxine resistance. We also genotyped 10 microsatellite loci around dhps to determine the genetic backgrounds of various alleles and compared them with the backgrounds of alleles prevalent in Africa and South America. In addition to previously known highly-resistant triple mutant dhps alleles SGEGA and AGEAA (codons 436, 437, 540, 581, 613 are sequentially indicated), a large proportion of the isolates (19.3%) contained a 540N mutation in association with 437G/581G yielding a previously unreported triple mutant allele, SGNGA. Microsatellite data strongly suggest the strength of selection was greater on triple mutant dhps alleles followed by the double and single mutants. We provide evidence for at least three independent origins for the double mutants, one each for the SGKGA, AGKAA and SGEAA alleles. Our data suggest that the triple mutant allele SGEGA and the novel allele SGNGA have common origin on the SGKGA background, whereas the AGEAA triple mutant was derived from AGKAA on multiple, albeit limited, genetic backgrounds. The SGEAA did not share haplotypes with any of the triple mutants. Comparative analysis of the microsatellite haplotypes flanking dhps alleles from Cambodia, Kenya, Cameroon and Venezuela revealed an independent origin of sulfadoxine resistant alleles in each of these regions.
    • Real-Time Loop-Mediated Isothermal Amplification (RealAmp) for the Species-Specific Identification of Plasmodium vivax

      Patel, JC; Oberstaller, J; Xayavong, M; Narayanan, J; DeBarry, JD; Srinivasamoorthy, G; Villegas, L; Escalante, AA; DaSilva, A; Peterson, DS; Barnwell, JW; Kissinger, JC; Udhayakumar, V; Lucchi, NW (2013-01-29)
      Plasmodium vivax infections remain a major source of malaria-related morbidity and mortality. Early and accurate diagnosis is an integral component of effective malaria control programs. Conventional molecular diagnostic methods provide accurate results but are often resource-intensive, expensive, have a long turnaround time and are beyond the capacity of most malaria-endemic countries. Our laboratory has recently developed a new platform called RealAmp, which combines loop-mediated isothermal amplification (LAMP) with a portable tube scanner real-time isothermal instrument for the rapid detection of malaria parasites. Here we describe new primers for the detection of P. vivax using the RealAmp method. Three pairs of amplification primers required for this method were derived from a conserved DNA sequence unique to the P. vivax genome. The amplification was carried out at 64°C using SYBR Green or SYTO-9 intercalating dyes for 90 minutes with the tube scanner set to collect fluorescence signals at 1-minute intervals. Clinical samples of P. vivax and other human-infecting malaria parasite species were used to determine the sensitivity and specificity of the primers by comparing with an 18S ribosomal RNA-based nested PCR as the gold standard. The new set of primers consistently detected laboratory-maintained isolates of P. vivax from different parts of the world. The primers detected P. vivax in the clinical samples with 94.59% sensitivity (95% CI: 87.48-98.26%) and 100% specificity (95% CI: 90.40-100%) compared to the gold standard nested-PCR method. The new primers also proved to be more sensitive than the published species-specific primers specifically developed for the LAMP method in detecting P. vivax.
    • South American plasmodium falciparum after the malaria eradication era: Clonal population expansion and survival of the fittest hybrids

      Griffing, SM; Mixson-Hayden, T; Sridaran, S; Alam, MT; McCollum, AM; Cabezas, C; Quezada, WM; Barnwell, JW; de Oliveira, AM; Lucas, C; Arrospide, N; Escalante, AA; Bacon, DJ; Udhayakumar, V (2011-09-16)
      Malaria has reemerged in many regions where once it was nearly eliminated. Yet the source of these parasites, the process of repopulation, their population structure, and dynamics are ill defined. Peru was one of malaria eradication's successes, where Plasmodium falciparum was nearly eliminated for two decades. It reemerged in the 1990s. In the new era of malaria elimination, Peruvian P. falciparum is a model of malaria reinvasion. We investigated its population structure and drug resistance profiles. We hypothesized that only populations adapted to local ecological niches could expand and repopulate and originated as vestigial populations or recent introductions. We investigated the genetic structure (using microsatellites) and drug resistant genotypes of 220 parasites collected from patients immediately after peak epidemic expansion (1999-2000) from seven sites across the country. The majority of parasites could be grouped into five clonal lineages by networks and AMOVA. The distribution of clonal lineages and their drug sensitivity profiles suggested geographic structure. In 2001, artesunate combination therapy was introduced in Peru. We tested 62 parasites collected in 2006-2007 for changes in genetic structure. Clonal lineages had recombined under selection for the fittest parasites. Our findings illustrate that local adaptations in the post-eradication era have contributed to clonal lineage expansion. Within the shifting confluence of drug policy and malaria incidence, populations continue to evolve through genetic outcrossing influenced by antimalarial selection pressure. Understanding the population substructure of P. falciparum has implications for vaccine, drug, and epidemiologic studies, including monitoring malaria during and after the elimination phase.