- Open Access
Clonal population expansion in an outbreak of Plasmodium falciparum on the northwest coast of Ecuador
© Sáenz et al. 2015
- Received: 16 June 2015
- Accepted: 26 November 2015
- Published: 10 December 2015
Determining the source of malaria outbreaks in Ecuador and identifying remaining transmission foci will help in malaria elimination efforts. In this study, the genetic signatures of Plasmodium falciparum isolates, obtained from an outbreak that occurred in northwest Ecuador from 2012 to 2013, were characterized.
Molecular investigation of the outbreak was performed using neutral microsatellites, drug resistance markers and pfhrp2 and pfhrp3 genotyping.
A majority of parasite isolates (31/32) from this outbreak were of a single clonal type that matched a clonal lineage previously described on the northern coast of Peru and a historical isolate from Ecuador. All but one isolate carried a chloroquine-resistant pfcrt genotype and sulfadoxine- and pyrimethamine-sensitive pfdhps and pfdhfr genotypes. Pfmdr1 mutations were identified in codons 184 and 1042. In addition, most samples (97 %) showed presence of pfhrp2 gene.
This study indicates that parasites from a single clonal lineage largely contributed to this outbreak and this lineage was found to be genetically related to a lineage previously reported in the Peruvian coast and historical Ecuadorian parasites.
- Plasmodium falciparum
- Microsatellite markers
Some countries in Latin America have achieved and surpassed the goals set by the World Health Assembly for reducing malaria cases (reduction of malaria burden by 75 % between 2000 and 2015) . In particular, Ecuador has decreased the number of malaria cases from more than 100,000 (approximately 13.6 cases per 1000 individuals) in 2000 to 378 cases in 2013 (approximately 0.05 cases per 1000 individuals), thus placing it in the pre-elimination phase [1–3]. Nevertheless, outbreaks of both falciparum and vivax malaria still occur in both the Amazon and Coastal regions of Ecuador.
Molecular tools are valuable in providing insight about the potential source of malaria parasites in outbreak investigations [4–8]. Neutral microsatellite markers, single nucleotide polymorphism (SNP) based molecular barcodes and genomics tools have been used to characterize Plasmodium falciparum parasites in South America [5, 9–17]. These studies have suggested that P. falciparum parasites in this region have undergone population bottlenecks and in many parts of Peru and Colombia there are clonal population of parasites that are highly prevalent in different geographical regions.
One molecular investigation of P. falciparum isolates from Peru, using seven neutral microsatellite markers, revealed that the parasite isolates collected between 1999 and 2000 (during peak malaria expansion after the malaria elimination era) comprised five distinct clonal lineages named A, B, C, D, and E . In the coastal region of Peru, all tested parasites belonged to the E clonal lineage while in the western region of the Peruvian Amazon, the C and D clonal lineages appeared more frequently; in the eastern and central Amazon A, B, C and D clonal lineages were found . It was proposed that C, D and E clonal lineages may have originated in Ecuador and Colombia while A and B clonal lineages may have originated in Brazil . The profile of drug resistance markers among these clonal lineages also varied and, by combining drug resistance marker profiles and microsatellite allele information, it was possible to further understand the genetic relationship among these parasite lineages [7, 8, 12, 13, 17]. Recent studies suggest that Peru has the highest known proportion of pfhrp2 deleted P. falciparum parasites in the region, followed by Colombia and Suriname [14–16, 18]. Thus, by combining genetic markers such as neutral microsatellite markers, drug resistance markers and pfhrp2 gene presence or absence one might identify parasite lineages originating in this region as demonstrated in recent outbreak investigations in Peru [7, 8].
In order to understand the source of this outbreak parasite population the following questions were answered: (a) are these parasite populations genetically similar to parasite populations in Ecuador, Peru (southern border) and Colombia (northern border)? (b) is there any evidence to suggest these parasites may have origins outside this region (Andean)? and (c) did these outbreak parasites belong to a single clonal lineage or to multiple lineages?
The parasite samples used in this study were obtained from the malaria surveillance protocol approved by the Ethical Review Committee of Pontificia Universidad Católica del Ecuador. Written informed consent was provided by study participants and/or their legal guardians.
Esmeraldas city is the largest city in Esmeraldas Province in northwest Ecuador. All samples used in this study were collected in Esmeraldas city during a malaria outbreak that occurred between November 2012 and November 2013.
In this study, laboratory adapted P. falciparum isolates 3D7, Dd2 and HB3 were used as controls for genotyping. In addition, Ecu 1110, a P. falciparum culture-adapted strain that was originally isolated in Ecuador in 1990 (provided by Dr Tom Wellems, National Institutes of Health, Bethesda, USA) was included as a reference strain from Ecuador.
DNA extraction and confirmation of infection
DNA was isolated from filter paper or whole blood using the QIAamp DNA mini-spin kit (QIAGEN, Valencia, CA, USA) or Chelex-100 . Plasmodium falciparum infection status was further confirmed by two different molecular methods: nested PCR using primers for 18S ribosomal RNA , and photo-induced electron transfer (PET) real-time PCR using specific fluorogenic primers for Plasmodium (genus) and falciparum (species) .
Detection of the pfhrp2 and pfhrp3 genes by PCR
Nested PCR amplifications of pfhrp2 and pfhrp3 genes were performed using reaction conditions and primers reported previously [15, 22]. In vitro cultured isolate Dd2 was used as a negative control for pfhrp2 amplification experiments while HB3 served as a negative control for pfhrp3 amplification because the laboratory isolates lacked the respective genes. The resulting PCR amplicons were visualized on a 2 % agarose gel. If a visible band of the appropriate size was observed, the isolate was scored as positive for the presence of the respective gene and that result was recorded as final. If no amplification occurred, PCR amplification was repeated to confirm this observation. If no amplification occurred during the second amplification attempt, thus showing concordance with the original PCR result, no further testing was done and the outcome was reported as negative. However, if there was discordance between the first and second result, the PCR was repeated again and the two matching results out of three were scored as the final result.
Genomic DNA was used for microsatellite characterization. Samples were genotyped for seven neutral microsatellite loci spanning six chromosomes that had been used in previous studies (TA1, chromosome 6; Polyα, ch. 4; PfPK2, ch. 12; TA109, ch. 6; and 2490, ch. 10; C2M34, ch. 2; C3M69, ch. 3) [23–25]. Genomic DNA was amplified by PCR using previously described methods [12, 15, 23–25] and fluorescently labelled (HEX and FAM) PCR products were separated by capillary electrophoresis on an Applied Biosystems 3130xl genetic analyzer (Applied Biosystems Foster City, CA, USA). Alleles were then sized and scored using GeneMapper v3.7 (Applied Biosystems Foster City, CA, USA) and binned to the nearest two or three base pairs.
Drug resistance markers
Sanger sequencing was performed on all samples to identify drug resistance-associated mutations in pfcrt, pfdhfr, pfdhps, and pfmdr1 genes . Mutations in the codon positions 72–76 of pfcrt, 51, 59, 108, and 164 of pfdhfr, 436, 437, 540, 581, and 613 of pfdhps and 84, 134, 184, 1034, 1042, 1226 and 1246 of pfmdr1 were identified.
A median-joining network diagram was generated in Network 22.214.171.124  using the seven neutral microsatellites previously described to examine the genetic relationships among the 32 samples collected from the outbreak, in addition to Ecu 1110, the Peruvian historical isolates published previously  as well as five Colombian isolates published recently .
Analysis of molecular variance (AMOVA) was used to partition variation within the study parasite population and between the population of Ecuador and falciparum populations of western Peru. In addition, heterozygosity (He) and pair-wise fixation indices (Fst) were calculated. Arlequin 126.96.36.199 software (CMPG, Swiss Institute of Bioinformatics, Lausanne, Switzerland) was used for these analyses . Structure v2.3.4 software [29, 30] was used to assign outbreak samples to previously known populations and Structure Harvester  was used to define the number of expected populations.
During the P. falciparum malaria outbreak in Esmeraldas, 151 cases of P. falciparum were identified (Fig. 1). Eighty per cent of the cases were located in two parishes in the south part of Esmeraldas. Men accounted for 57 % of the cases. Fifty per cent of the total number of cases were reported in teenagers and young adults (11–25 years old), while 13 % were in children 10 years old or younger, 22 % were in adults 25–40 years old and 15 % were in adults over 40 years old. Most cases were reported between February and April 2013, which corresponds to the rainy season on the Ecuadorian coast (Fig. 1). Thirty-two of these samples were available for this molecular investigation and were confirmed positive by nested PCR and real time PET-PCR.
Genetic characterization using microsatellite markers
Microsatellite alleles amplified in isolates collected during the outbreak
Number of samples
2490 Ch. 10
C2M34 Ch. 2
C3M69 Ch 3.
A clonal lineage (PER)
B clonal lineage (PER)
C clonal lineage (PER)
D clonal lineage (PER)
E clonal lineage (PER)
Cluster 1 (COL)
Cluster 2 (COL)
Cluster 3 (COL)
Cluster 4 (COL)
Drug resistance markers
Drug resistance markers haplotypes and pfhrp2 and pfhrp3 presence
Number of samples
MS clonal type
Peru clonal lineage E 1999a
Ecu 1110 1990b
Pfhrp2 and pfhrp3 genotyping
The samples from this outbreak were genotyped for the presence of pfhrp2 and pfhrp3 genes. The results indicated that both genes were intact in all 31 E clonal lineage-related parasite isolates. In contrast, both pfhrp2 and pfhrp3 were deleted in the D clonal lineage isolate F31 (Table 3).
Overall, these findings suggest that the main source of this outbreak might be parasites belonging to the E clonal lineage. However, not all samples from this outbreak were available due to logistical challenges and the unwillingness of some participants to provide consent. Since not all outbreak samples were available for molecular analysis, it was not possible to identify the genetic profile of all the parasite isolates, which would have helped to unambiguously describe the origin of the entire outbreak parasite population. Nevertheless, this finding clearly illustrates the utility of molecular epidemiological investigations in determining the origin(s) of outbreak parasite populations in future investigations as Ecuador moves towards malaria elimination.
The high clonality of P. falciparum observed in this low-transmission area is consistent with reports from Peru [12, 13, 32], Colombia [10, 14, 21] and Panama . Using the same set of microsatellite markers used in this study, recent P. falciparum outbreaks in the north coastal region and Cusco area of Peru, were determined to have originated in a region of the Peruvian Amazon [7, 8]. A molecular epidemiological study conducted previously in the north coast of Peru determined that E clonal lineage parasites were circulating in the 1990s . Importantly, P. falciparum transmission was eliminated in the north coast of Peru from 2005 until the malaria outbreak of 2010–2012, during which a clonal population of parasites belonging to BV1 lineage from Peruvian Amazon, were introduced to the region . These findings demonstrate the utility of molecular tools for epidemiological investigations.
Since the genetic profile of P. falciparum parasites that were circulating in Ecuador is not known, it is difficult to clearly attribute whether the current major outbreak population expanded from a residual, vestigial population of parasites that may still be circulating in Ecuador. The finding that a single historical isolate from Ecuador collected about two decades ago genetically resembled the major outbreak population and that sporadic cases have been occurring in the area (Fig. 1) is consistent with the hypothesis that E clonal lineage-related P. falciparum strains were present in Ecuador throughout this time period. Based on this finding, one could argue for the possibility of a potential local expansion of residual reservoirs of parasites with E clonal genetic signatures in this outbreak due to some ecological changes. However, further genetic evidence using parasite isolates collected in recent times from this region is needed to support such a hypothesis.
An alternative explanation for the source of this outbreak, based on epidemiological investigations, suggests that the migration of human cases between Colombia and Ecuador may be the cause of this outbreak (Ministry of Public Health of Ecuador, pers. comm.). However, there is no strong evidence to support this hypothesis as explained below. In a recent study, five parasite isolates closely related to the E clonal lineage were detected in sample collections from 2008 and 2009 obtained from Valle de Cauca Department, Colombia. However, three of these parasites differed from this outbreak samples in the chromosomal 10 locus 2490 and two differed in the chromosomal 6 locus TA109. While mostly CVMNK and CVMNT genotypes of pfcrt have been reported in Ecuador , Colombian parasites have been reported to be CVMET genotype . Since there was no availability of parasite specimens from the index case(s), it was not possible to link the genetic data obtained with travel history information.
The F31 sample donor, who reported travel outside of the Esmeraldas area and worked in the Amazon region of Ecuador (Ministry of Public Health, pers. comm.), had parasites belonging to the D clonal lineage. Interestingly, this parasite isolate carried pfcrt genotype of CVMET that is characteristic of Colombian parasites, as documented in previous studies [34, 35]. Based on the travel history and pfcrt genotype, one can hypothesize that this traveler may have acquired the D lineage parasites from Colombia or the Ecuador Amazon region; however, information about the genetic profile of P. falciparum parasites circulating in the Ecuadorian Amazon is lacking and so in order to validate the hypothesis, P. falciparum genotype data will need to be collected from the region.
Ecuador changed its P. falciparum anti-malarial treatment policy in 2006, switching from chloroquine (CQ) to artesunate-sulfadoxine-pyrimethamine (AS-SP) and recently to artemether-lumefantrine (AL) [33, 36]. It is important to note that even though the P. falciparum parasites from the outbreak have a CQ-resistant haplotype (CVMNT and CVMET), they carried wild type dhfr and dhps gentotypes associated with SP sensitivity. Mutations in pfmdr1 were restricted to only two codons as opposed to mutations in three or four codons of this gene in parasite isolates from the Amazon region of South America [13, 37]. Remarkably, the most common haplotype found in the Esmeraldas outbreak had the exact same drug resistance gene profile as Ecu 1110 [34, 35, 38], an isolate collected in Ecuador in 1990. These results suggest that if these outbreak parasites are from the local residual transmission foci then they have maintained the historical CQ-resistant haplotype CVMNT in the population. In addition, the profile of wild type pfdhr and pfdhps alleles including the limited mutation pattern in pfmdr1 in the E clonal outbreak parasites indicates that this is an ancestral lineage that may predate SP introduction.
The clonal population from the Esmeraldas outbreak does not have deletions of pfhrp2 or pfhrp3, which would imply that these parasites can be detected by PfHRP2-based rapid diagnostic tests (RDTs). However, the D clonal lineage-related F31 sample had deleted both pfhrp2 and pfhrp3. This finding highlights the need for vigilance when PfHRP2-based RDTs are used for control programmes in South America since pfhrp2 deletion has been documented in parts of South America [14–16, 18].
Taken together, this data suggest that the P. falciparum isolates from the Esmeraldas outbreak are the result of a clonal expansion of parasites either circulating at very low levels in Ecuador or re-invading Ecuador from border countries. Some of the limitations of this study include the lack of genetic and travel information from the index case(s), lack of population-level genotypic data for P. falciparum parasites in Ecuador and the availability of only a sub-set of samples from the outbreak for analysis. Nevertheless, this study highlights the importance of molecular studies to investigate the origin and relatedness of Plasmodium parasite populations in outbreak investigations. Further genetic characterization of historical malaria parasites from Ecuador will help in determining if low-level transmission of bottle-necked, vestigial populations of historical Ecuadorian parasites still contribute to local malaria transmission. The genetic characterizations will also help in future outbreak investigations to differentiate residual bottle-necked populations from newly introduced parasites from other regions as efforts are underway to eliminate malaria from this region.
FES and VU conceived and designed the study; FES, LCM, SAO, and VU wrote the manuscript; FES, LCM, SAO, GV, EVA, CVA, and NWL performed the experiments; LEC performed field work. All authors read and approved the final manuscript.
We would like to thank the personnel of SNEM, Ecuadorian Ministry of Public Health taking part in the sample collection and all the patients participating in this study. SAO was also supported in part by the Atlanta Research and Education Foundation (AREF). We thank Dr Alexandre Macedo de Oliveira, Dr Roberto Montoya and the Pan American Health Organization for encouragement in the study and support in training of Esmeraldas microscopists. We acknowledge César Yumiseva for the help in the preparation of the Figures.
This work was partly supported by Amazon Malaria Initiative-RAVREDA and PUCE grants J130158 and K13058. The Amazon Malaria Initiative is financially supported by the United States Agency for International Development. This work was also partially supported by the Atlanta Research and Education Foundation.
The authors declare that they have no competing interests.
The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- WHO. World malaria report. World Health Organization. 2013.Google Scholar
- SIVEMAE 2 S, Ministerio de Salud Pública Reporte de casos de malaria en Ecuador. Guayaquil, Ecuador 2013.Google Scholar
- PAHO: Report on the Situation of Malaria in the Americas 2012. 2013.Google Scholar
- Patel JC, Taylor SM, Juliao PC, Parobek CM, Janko M, Gonzalez LD, et al. Genetic evidence of importation of drug-resistant Plasmodium falciparum to Guatemala from the Democratic Republic of the Congo. Emerg Infect Dis. 2014;20:932–40.PubMed CentralView ArticlePubMedGoogle Scholar
- Obaldia N 3rd, Baro NK, Calzada JE, Santamaria AM, Daniels R, Wong W, et al. Clonal outbreak of Plasmodium falciparum infection in Eastern Panama. J Infect Dis. 2015;211:1087–96.View ArticlePubMedGoogle Scholar
- Arez AP, Snounou G, Pinto J, Sousa CA, Modiano D, Ribeiro H, et al. A clonal Plasmodium falciparum population in an isolated outbreak of malaria in the Republic of Cabo Verde. Parasitology. 1999;118:347–55.View ArticlePubMedGoogle Scholar
- Okoth SA, Chenet SM, Arrospide N, Gutierrez S, Cabezas C, Matta JA, et al. Molecular investigation into a malaria outbreak in Cusco, Peru: Plasmodium falciparum BV1 lineage is linked to a second outbreak in recent times. Am J Trop Med Hyg. 2015 [Epub ahead of print].Google Scholar
- Baldeviano GC, Okoth SA, Arrospide N, Gonzalez RV, Sanchez JF, Macedo S, et al. Molecular Epidemiology of Plasmodium falciparum Malaria Outbreak, Tumbes, Peru, 2010–2012. Emerg Infect Dis. 2015;21:797–803.PubMed CentralView ArticlePubMedGoogle Scholar
- Hernandez DC, Guerra AP, Cucunuba ZM, Nicholls RS, Barrera SM. Haplotypes associated with resistance to sulfadoxine-pyrimethamine in Plasmodium falciparum in two malaria endemic locations in Colombia. Infect Genet Evol. 2013;18:183–90.View ArticlePubMedGoogle Scholar
- Echeverry DF, Nair S, Osorio L, Menon S, Murillo C, Anderson TJ. Long term persistence of clonal malaria parasite Plasmodium falciparum lineages in the Colombian Pacific region. BMC Genet. 2013;14:2.PubMed CentralView ArticlePubMedGoogle Scholar
- Arango EM, Samuel R, Agudelo OM, Carmona-Fonseca J, Maestre A, Yanow SK. Genotype comparison of Plasmodium vivax and Plasmodium falciparum clones from pregnant and non-pregnant populations in North-west Colombia. Malar J. 2012;11:392.PubMed CentralView ArticlePubMedGoogle Scholar
- Griffing SM, Mixson-Hayden T, Sridaran S, Alam MT, McCollum AM, Cabezas C, et al. South American Plasmodium falciparum after the malaria eradication era: clonal population expansion and survival of the fittest hybrids. PLoS One. 2011;6:e23486.PubMed CentralView ArticlePubMedGoogle Scholar
- Bacon DJ, McCollum AM, Griffing SM, Salas C, Soberon V, Santolalla M, et al. Dynamics of malaria drug resistance patterns in the Amazon basin region following changes in Peruvian national treatment policy for uncomplicated malaria. Antimicrob Agents Chemother. 2009;53:2042–51.PubMed CentralView ArticlePubMedGoogle Scholar
- Murillo Solano C, Akinyi Okoth S, Abdallah JF, Pava Z, Dorado E, Incardona S, et al. Deletion of Plasmodium falciparum histidine-rich protein 2 (pfhrp2) and histidine-rich protein 3 (pfhrp3) genes in Colombian parasites. PLoS One. 2015;10:e0131576.PubMed CentralView ArticlePubMedGoogle Scholar
- Akinyi S, Hayden T, Gamboa D, Torres K, Bendezu J, Abdallah JF, et al. Multiple genetic origins of histidine-rich protein 2 gene deletion in Plasmodium falciparum parasites from Peru. Sci Rep. 2013;3:2797.PubMed CentralView ArticlePubMedGoogle Scholar
- Akinyi Okoth S, Abdallah JF, Ceron N, Adhin MR, Chandrabose J, Krishnalall K, et al. Variation in Plasmodium falciparum histidine-rich protein 2 (Pfhrp2) and Plasmodium falciparum histidine-rich protein 3 (Pfhrp3) gene deletions in Guyana and Suriname. PLoS One. 2015;10:e0126805.PubMed CentralView ArticlePubMedGoogle Scholar
- Griffing SM, Viana GM, Mixson-Hayden T, Sridaran S, Alam MT, de Oliveira AM, et al. Historical shifts in Brazilian P. falciparum population structure and drug resistance alleles. PLoS One. 2013;8:e58984.PubMed CentralView ArticlePubMedGoogle Scholar
- Gamboa D, Ho MF, Bendezu J, Torres K, Chiodini PL, Barnwell JW, et al. A large proportion of P. falciparum isolates in the Amazon region of Peru lack pfhrp2 and pfhrp3: implications for malaria rapid diagnostic tests. PLoS One. 2010;5:e8091.PubMed CentralView ArticlePubMedGoogle Scholar
- Kain KC, Lanar DE. Determination of genetic variation within Plasmodium falciparum by using enzymatically amplified DNA from filter paper disks impregnated with whole blood. J Clin Microbiol. 1991;29:1171–4.PubMed CentralPubMedGoogle Scholar
- Snounou G. Detection and identification of the four malaria parasite species infecting humans by PCR amplification. Methods Mol Biol. 1996;50:263–91.PubMedGoogle Scholar
- Lucchi NW, Narayanan J, Karell MA, Xayavong M, Kariuki S, DaSilva AJ, et al. Molecular diagnosis of malaria by photo-induced electron transfer fluorogenic primers: PET-PCR. PLoS One. 2013;8:e56677.PubMed CentralView ArticlePubMedGoogle Scholar
- Abdallah JF, Okoth S, Fontecha GA, Torres R, Banegas EI, Matute M, et al. Prevalence of pfhrp2 and pfhrp3 gene deletions in Puerto Lempira, Honduras. Malar J. 2015;14:19.PubMed CentralView ArticlePubMedGoogle Scholar
- Anderson TJ, Haubold B, Williams JT, Estrada-Franco JG, Richardson L, Mollinedo R, et al. Microsatellite markers reveal a spectrum of population structures in the malaria parasite Plasmodium falciparum. Mol Biol Evol. 2000;17:1467–82.View ArticlePubMedGoogle Scholar
- Anderson TJ, Su XZ, Bockarie M, Lagog M, Day KP. Twelve microsatellite markers for characterization of Plasmodium falciparum from finger-prick blood samples. Parasitology. 1999;119:113–25.View ArticlePubMedGoogle Scholar
- McCollum AM, Mueller K, Villegas L, Udhayakumar V, Escalante AA. Common origin and fixation of Plasmodium falciparum dhfr and dhps mutations associated with sulfadoxine-pyrimethamine resistance in a low-transmission area in South America. Antimicrob Agents Chemother. 2007;51:2085–91.PubMed CentralView ArticlePubMedGoogle Scholar
- Alam MT, de Souza DK, Vinayak S, Griffing SM, Poe AC, Duah NO, et al. Selective sweeps and genetic lineages of Plasmodium falciparum drug -resistant alleles in Ghana. J Infect Dis. 2011;203:220–7.PubMed CentralView ArticlePubMedGoogle Scholar
- Bandelt HJ, Forster P, Rohl A. Median-joining networks for inferring intraspecific phylogenies. Mol Biol Evol. 1999;16:37–48.View ArticlePubMedGoogle Scholar
- Excoffier L, Laval G, Schneider S. Arlequin (version 3.0): an integrated software package for population genetics data analysis. Evol Bioinform Online. 2005;1:47–50.PubMed CentralGoogle Scholar
- Pritchard JK, Stephens M, Donnelly P. Inference of population structure using multilocus genotype data. Genetics. 2000;155:945–59.PubMed CentralPubMedGoogle Scholar
- Hubisz MJ, Falush D, Stephens M, Pritchard JK. Inferring weak population structure with the assistance of sample group information. Mol Ecol Resour. 2009;9:1322–32.PubMed CentralView ArticlePubMedGoogle Scholar
- Earl DA, von Holt BM. STRUCTURE HARVESTER: a website and program for visualizing STRUCTURE output and implementing the Evanno method. Conserv Genet Resour. 2012;4:359–61.View ArticleGoogle Scholar
- Branch OH, Sutton PL, Barnes C, Castro JC, Hussin J, Awadalla P, et al. Plasmodium falciparum genetic diversity maintained and amplified over 5 years of a low transmission endemic in the Peruvian Amazon. Mol Biol Evol. 2011;28:1973–86.PubMed CentralView ArticlePubMedGoogle Scholar
- Arrospide N, Hijar-Guerra G, de Mora D, Diaz-Cortéz CE, Veloz-Perez R, Gutierrez S, et al. Alelos mutantes asociados a la resistencia a cloroquina y sulfadoxina-pirimetamina en Plasmodium falciparum de las fronteras Ecuador-Perú y Ecuador-Colombia. Rev Peru Med Exp Salud Publica. 2014;31:282–7.PubMedGoogle Scholar
- Sa JM, Twu O, Hayton K, Reyes S, Fay MP, Ringwald P, et al. Geographic patterns of Plasmodium falciparum drug resistance distinguished by differential responses to amodiaquine and chloroquine. Proc Natl Acad Sci USA. 2009;106:18883–9.PubMed CentralView ArticlePubMedGoogle Scholar
- Cortese JF, Caraballo A, Contreras CE, Plowe CV. Origin and dissemination of Plasmodium falciparum drug-resistance mutations in South America. J Infect Dis. 2002;186:999–1006.View ArticlePubMedGoogle Scholar
- Jiménez M, Valdez C, Marmion J, Barillas E. Situación de la gestión del suministro de medicamentos para el tratamiento de la malaria en los países que comparten la cuenca Amazónica y Centroamérica. Strengthening Pharmaceutical Systems, Management Sciences for Health. 2011. pp. 21.Google Scholar
- Griffing S, Syphard L, Sridaran S, McCollum AM, Mixson-Hayden T, Vinayak S, et al. pfmdr1 amplification and fixation of pfcrt chloroquine resistance alleles in Plasmodium falciparum in Venezuela. Antimicrob Agents Chemother. 2010;54:1572–9.PubMed CentralView ArticlePubMedGoogle Scholar
- Fidock DA, Nomura T, Talley AK, Cooper RA, Dzekunov SM, Ferdig MT, et al. Mutations in the P. falciparum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance. Mol Cell. 2000;6:861–71.PubMed CentralView ArticlePubMedGoogle Scholar