Diverse origin of Plasmodium falciparum in northwest Ecuador

Background Ecuador plans to eliminate malaria by 2020, and the country has already seen a decrease in the number of cases from more than 100,000 in 2000 to only 618 in 2015. Around 30% of malaria infections in Ecuador are caused by Plasmodium falciparum. Most malaria population genetics studies performed in Latin America, especially in the Pacific Coast, indicate a high clonality and a clear structure of P. falciparum populations. It was shown that an outbreak of P. falciparum in northwest Ecuador was the result of a clonal expansion of parasites circulating at low levels in the country or re-invading Ecuador from neighbouring territories. However, general characteristics of P. falciparum circulating in the northwest coast of Ecuador have not been determined. The main goal of this study was to genetically characterize the population structure of P. falciparum in coastal Ecuadorian localities bordering with Colombia. Methods Molecular investigation of 41 samples collected from 2013 to 2016 in San Lorenzo County, northwest Ecuador was performed using seven neutral microsatellite markers. Results The genetic population structure of P. falciparum in northwest Ecuador is clearly defined as three different genetic groups previously reported in Ecuador, Peru and Colombia. Conclusions The limited number of P. falciparum clonal types that are circulating in northwest Ecuador, are related to ancestral parasite clonal lineages reported in the Pacific Coast. These parasites could be a product of migration from neighbouring regions or residual clonal types circulating in the country in low proportions. Studies of the genetic characterization of P. falciparum in eliminating areas help determine the possible origin of parasites in order to create strategies to prevent the entrance of new lineages and achieve local elimination of malaria.


Background
Malaria is present in 21 countries of Latin America, and about 126.8 million people were at risk of the disease in 2016 in the region. Plasmodium falciparum was responsible for approximately 30% of the reported malaria cases in the region [1][2][3][4][5]. Ecuador is one of the eight countries of the region with capacity to eliminate malaria by 2020 [1]; indeed, the country has decreased the number of cases from more than 100,000 in 2000 to 618 in 2015, and 1279 in 2017 [1,4,6]. In Ecuador, the presence of malaria is mostly restricted to the northwest coast and the Amazon region, where outbreaks of P. falciparum and Plasmodium vivax still occur [6,7].
Genetic characterization of circulating malaria parasites in a specific area, especially in areas targeted for elimination, provide insights about the genetic connectivity of currently circulating populations to ancestral lineages and determine if left over residual historical parasite lineages are contributing to local transmission. This will also help to determine if new parasite lineages that have migrated from other regions are contributing to current malaria transmission. In addition, it can provide data about drug resistant alleles that may be relevant for targeting appropriate drugs for treatment or for mass drug administration [8][9][10]. Moreover, the level of diversity and its distribution provide insights into trends in parasite transmission and population history [11,12].
Most malaria population genetics studies performed in Latin America indicate high clonality of P. falciparum populations [13][14][15][16]. Plasmodium falciparum from Ecuador, Colombia, Peru, Honduras, Brazil and Venezuela have undergone one or more bottleneck events in the recent past and current populations expanded from a limited number of P. falciparum ancestral lineages [7,[17][18][19][20]. Plasmodium falciparum populations in the region consist of a continuous mixture and reorganization of clonal lineages (genetically identical for a set of markers, but potentially variable for others [9]), mainly due to migration, even though the opportunities for outcrossing between the different lineages is limited because of low transmission [9,13,17,21]. In addition, P. falciparum from Latin America has had chloroquine (CQ) resistance since 1960 [20], as well as sulfadoxinepyrimethamine (SP) resistance [22,23]. Recently, artemisinin (ART) resistance-related mutations have been reported in Guyana [20,24,25].
A molecular investigation of Peruvian P. falciparum population determined the presence of five clonal lineages in the country in 1999-2000. The Peruvian P. falciparum population consisted of A, B, C, D and E lineages distributed across the country. In the Amazon interior region, the five clonal lineages were present and in the northern Pacific coast only one lineage was reported (clonal lineage E). Each clonet had a specific drug resistant allelic profile; while all clonets reported CQ resistance, the clonets D and E had dhfr and dhps alleles that confer SP sensitivity [21]. A P. falciparum outbreak in Tumbes (Pacific coast of Peru) during 2010-2012, had a genotype related to clonal lineage B (B v1 ) but was unrelated to clonal lineage E (previously present in the same area) [15] and suggested that this outbreak was caused by clonal lineages from the Amazon region of Peru. Similarly, in Colombia the P. falciparum population has undergone a bottleneck event, showing low genetic diversity and low polyclonal infections [26]. The P. falciparum population structure consisted of four major clusters along the Colombian Pacific coast between 1999 and 2009 [26]. A different study in Colombia showed several multilocus haplotypes persist in multiple years between 2003 and 2010 in most of the country in Amazonas, Cordoba, Nariño and Valle [27]. Clonal lineage B v1 (reported in Peru [14]) in the Amazon, two new clusters F in Nariño, Valle and Cauca and cluster E V1 in Antioquia were reported [14,16,[26][27][28]. Colombian parasites have reported CQ and SP resistance, in addition to an increase in the number of pfmdr-1 copies, carrying mefloquine (MQ) and quinine (QN) resistance. Neither Peru nor Colombia has reported ART resistance or mutations in the Kelch 13 propeller domain [20,[22][23][24][25]29].
The information about P. falciparum population genetic structure in Ecuador is limited. A molecular study of P. falciparum from Ecuador, during an outbreak in Esmeraldas city in the northwest of the country between November 2012 and November 2013, revealed that the parasites were the result of a clonal expansion of P. falciparum circulating at low levels or re-invading Ecuador from border countries [7]. The P. falciparum outbreak in northwest Ecuador had an identical microsatellite genotypic profile to P. falciparum E clonal lineages from the Peruvian Pacific coast. Interestingly, these parasites were related to a single historical isolate that was collected in the Ecuadorian coast in 1990 [7]. Esmeraldas outbreak samples carried CQ resistance (CVMNT haplotype) and dhfr and dhps alleles that were similar to E clonal type that were associated with SP sensitivity [7].
Molecular tools like neutral microsatellite markers (tandem repeats of motifs [18]) are a very important and powerful tool for the study of population structure because they can characterize and identify haplotypes and are extremely widespread in P. falciparum (2-3 kb throughout the genome) [12,18,30]. Neutral microsatellites are usually the markers of choice for P. falciparum population genetic analysis because these markers are not directly under selection and are able to show genetic signatures [13-16, 18, 30]. There is considerable amount of data using neutral microsatellite markers that have provided clues about genetic connectivity between parasite populations in Peru, Ecuador and Colombia.
The main goal of this study was to genetically characterize and geographically map the population structure of P. falciparum in northwest Ecuador (San Lorenzo county), between 2013 and 2016 using seven neutral microsatellites markers and compare them to previously characterized Ecuadorian parasites. In addition, Ecuadorian P. falciparum genotypes were compared to Peruvian and Colombian parasites.

Ethics statement
The parasite samples used in this study were obtained from the malaria surveillance program from the Ecuadorian Ministry of Health. The protocol was approved by the Ethical Review Committee of Pontificia Universidad Católica del Ecuador (approvals #: CBE-016-2013 and 20-11-14-01). Written informed consent was provided by study participants and/or their legal guardians.

Study sites
The study analysed samples from communities in San Lorenzo county, northwest Ecuador (Fig. 1). These samples were compared to previously reported samples from the same province [7]. All the samples used in this study were collected between 2013 and 2016 ( Fig. 1). Ecuadorian parasites were compared to previously reported Peruvian and Colombian parasites [21,26].

Samples and reference isolates
Samples used in this study were collected from patients between January 2013 and March 2016 by the personnel of the Ministry of Health of Ecuador. A total of 41 blood samples (4 samples in 2013; 15 in 2014; 14 in 2015 and 8 in 2016) were collected in San Lorenzo county from patients who initially reported to be microscopically positive for P. falciparum infection, and from whom informed consent was obtained. The blood samples were collected by finger prick or by drawing peripheral whole blood and spotted on 3MM Whatman filter paper. Thirty-two samples from an outbreak in Esmeraldas city, between November 2012 and November 2013 and reported in Saenz et al. [7] were also included in the study. Fifty-six percent of the samples (23/41) were collected by finger prick or by drawing peripheral whole blood and 44% of samples (18/41) were collected only by rapid diagnostic tests (RDT) with a positive result.

DNA extraction and confirmation of infection
DNA was isolated from all the samples (whole blood, filter paper or RDT) using a QIAamp DNA MINI KIT (QIAGEN Sample & Assay Technologies, Germantown, USA). Plasmodium falciparum was confirmed by two different molecular methods: nested PCR using primers for 18S ribosomal [31] and PET-PCR using photo-induced electron transfer fluorogenic primers [32].

Statistical analysis
Heterozygosity (He) was estimated using the formula He = n n−1 1 − k i=l p 2 1 , and pairwise fixation indices (Fst), estimated using the formula (δµ) 2 = (µ A − µ B ) 2 , where (δµ) 2 is the genetic distance and µ A and µ B are the average number of allelic size differences within population A and B, respectively. He and Fst were calculated using Arlequin 3.5.1.2 software (CMPG, Swiss Institute of Bio-informatics, Lausanne, Switzerland) [37,38]. To assess the parasite population structure, the genetic data was analysed with Structure 2.3.4 software, that assigned samples to different populations (K), and Structure Harvester was used to define the number of expected populations [39][40][41]. The data were evaluated using different K values (K = 1-15) and twenty independent repetitions were run for each K value with a burn-in period of 10,000 iterations followed by 1,000,000 iterations. In addition, software PHYLOViZ version 10 was used to construct a median-joining network diagram [42]. The samples were represented in a complete spanning tree using goe-BURST algorithm. This analysis shows the possible evolutionary relationships between strains (samples) [42]. San Lorenzo samples showed a higher heterozygosity (He = 0.4726) than Esmeraldas (He = 0.1018) [7] ( Table 1) and Linkage Disequilibrium (LD) in San Lorenzo (I A S = 0.0762, p < 1.00 × 10 −2 ) was much lower than in Esmeraldas (I A S = 0.3798, p < 1.00 × 10 −2 ) [7] (Table 1). Pairwise Fst between the two Ecuadorian counties (Esmeraldas and San Lorenzo) was high (0.39640) [43] ( Table 2).

Genetic characterization of Ecuadorian
Cluster analysis inferred a well differentiated population structure in this group of isolates. Three population clusters (K) were predicted in northwest Ecuador (San Lorenzo and Esmeraldas) (Fig. 2). Most samples from San Lorenzo county 68.3% (28/41) belonged to cluster one (Yellow); 24.4% (10/41) belonged to cluster two (Red) and 7.3% (3/41) belonged to cluster three (Pink) (Fig. 2). This information was confirmed by a Neighbour Joining network. Thirteen different haplotypes formed the yellow cluster, three haplotypes formed the red cluster, and two haplotypes were part of the pink cluster.

Genetic relatedness between Ecuadorian, Peruvian and Colombian isolates
The comparison between Ecuadorian (2013-2016), Peruvian (1999-2012) [21] and Colombian (2003-2008) samples [26] showed close relationship between samples of the three countries. Pairwise Fst was performed between samples from northwest Ecuador and samples from neighboring locations of Peru and Colombia. Ecuadorian samples from San Lorenzo had a close genetic relationship with Valle (0.06966) and Nariño (0.08481) but were distantly related from the Peruvian West Amazon and the Pacific Coast of Peru ( Table 2). The comparison of localities using Structure software predicted the presence of four clusters in San Lorenzo, northwest Ecuador and its neighbouring locations. All samples (41/41) from San Lorenzo corresponded to three clusters (yellow, red and pink). The yellow cluster was predominantly present in Nariño [26], Colombia (35%), the Red cluster was the most prevalent parasite in Esmeraldas (94%), the pacific Coast of Peru [21] (71%) and was present in Valle [26] (24%) and the West Amazon of Peru [21] (13%) (Fig. 3), while the pink cluster was predominant in Peru's West Amazon [21] (87%), it was present in Nariño [26], Colombia (24%), and in Esmeraldas [7] (3%).
In order to identify if the parasites from northwest Ecuador corresponded to any of the previously reported clonal lineages or genetic clusters, a median-joining network was completed using predominant haplotypes of clonets D, E [21] and F [27]. The network diagram shows that P. falciparum from San Lorenzo's Yellow cluster are closely related to genetic lineage F from Colombia (two haplotypes shared with previously reported haplotype F) [27]. In addition, parasites in the Red cluster are closely related to E clonal lineage parasites present in Esmeraldas and previously in the North coast of Peru [7,21]. Finally, parasites in the Pink cluster were related to clonal lineage D, first described in the Amazon of Peru and reported in Esmeraldas [7,21] (Fig. 4). The main haplotypes for each previously described lineage and the parasites in this study are shown in Table 3.

Discussion
In Ecuador Plasmodium infections are reported in the Amazon and Costal regions [1,7]. Specifically, the northwest coast of Ecuador has historically been endemic to P. falciparum where periodic transmission of this parasite at low levels has been reported [7]. This study was designed to understand the P. falciparum parasite population structure in parasite isolates collected in recent years and determine how this data can be used in support of malaria elimination efforts.
Plasmodium falciparum from northwest Ecuador have medium/low diversity (medium/low He and medium/ high linkage disequilibrium) similarly to what has been reported for other places of South America. This is partly because P. falciparum populations have undergone bottleneck events in the recent past due to elimination efforts by malaria programmes [14,21,26].
When comparing San Lorenzo (border locality) with Esmeraldas (150 km from border), it is clear that the border locality has more diversity and has different genetic composition from less endemic localities. This is due to two main factors: (1) regular migration from Colombia is common in the border areas and (2) most samples collected in Esmeraldas city were from a clonal P. falciparum outbreak [7]. Low LD in border localities matches higher number parasites entering from Colombia into Ecuador and a higher number of cases in the border county of San Lorenzo [45,46].
This study shows that northwest Ecuador has a simple, well defined structure. Indeed, between 2013 and 2016, three different genetic groups were present. These groups are related to previously reported groups in Colombia, Peru and Ecuador itself.
The majority of samples (68.3%) had genetic similarity to samples circulating in Colombia. This cluster was previously defined as genetic lineage F by Dorado et al. [27]. The parasites in San Lorenzo, Ecuador shared the majority of markers with the defined F haplotype ( Fig. 4 and Table 3) and only had some variations previously reported for F genetic lineage. This similarity is expected since the F clonet has been reported in the southern part of Colombia and human migration between Colombia and southern Ecuador is common. This human migration is related to several activities and some of the well-known include mining and palm oil agriculture. In addition, several Colombians and Ecuadorians cross the border on a Fig. 4 Median-joining network diagram of Ecuadorian P. falciparum and previously reported clonets. The network diagram shows the genetic relationship between samples from San Lorenzo county, Esmeraldas county [7], Colombian genetic group F [27] and Peruvian clonets D and E [21]. The circle size is proportional to the number of samples with the same haplotype. The colors represent clusters assigned by Structure software Table 3 Main microsatellite and drug resistance markers haplotypes [47] present in northwest Ecuador as compared to previously described haplotypes daily basis for other reasons. This genetic lineage presents the drug resistance haplotype CVMNT and wild type dhfr and dhps drug resistance markers. The mutations 184S and 1042D in Pfmdr1 are also related to genotypes reported previously [47].
One-fourth (28%) of the analysed samples from P. falciparum in San Lorenzo had genetic similarity or identity to parasites previously reported in an outbreak in Esmeraldas and the Pacific coast of Peru [7,21]. This genetic type has also been reported in Valle, Colombia and in Nariño, Colombia with some variations. In addition, the E clonet was present in early 1990 in Esmeraldas province [7] suggesting that this group has been present in the area for several years. The parasites from clonet E have a characteristic conserved drug resistance genotype that includes a mutation in the 76 position of Pfcrt (CVMNT), prevalence of wild type genotypes for Pfdhfr and Pfdhps and mutations 184S and 1042D in Pfmdr1 (Table 3) [7,47].
A small percentage of the samples (7.3%) have similarity with D clonal type previously reported in the West Amazon of Peru [21], Esmeraldas [7] and Colombia [27]. The D clonet was first reported in the West Amazon of Peru in 1999-2000 but found in the South Pacific Coast of Colombia in 2008 and in the Ecuadorian coast in 2013 [7,21,26,27]. The D clonet was found again in the north coast of Ecuador in this study in samples from 2013 and 2014 but not in more recent samples. As previously suggested by Griffing et al. [21], these clonal type parasites could have migrated to western Amazon of Peru from Ecuador which could have originated in Colombia and spread south to Ecuador [21]. The D clonet parasites in Ecuador have a characteristic pfcrt CVMET genotype and the synonymous mutation in the 540 position of pfdhps is common [7,47].
In summary, this study showed that all the parasites that were found in the reported study sites clearly belonged to one of the three mentioned clusters that have been known to be present in the Pacific Coast of Peru, Ecuador and Colombia. It was difficult to determine if there are further variations between these clonal types found in Colombia, Ecuador and Peru using these limited genetic markers. However, future efforts can focus on characterizing the genotypes of these parasite types using genomic analysis. This data suggests that some ancestral populations that have been known to have existed in this region are still continuing to cause transmission of malaria in this region. Previous studies have also found that most of these parasites are carrying markers associated with CQ resistance but sensitive to SP. No evidence for artemisinin resistant genotypes were found. Collectively, these data suggest using current anti-malarial drug policies implemented in Ecuador these parasites can be treated during elimination phase. Continuous characterization of parasite isolates from this region using genomic analysis may help to determine if human migration between border regions of Ecuador and Colombia is a primary cause of malaria importation to Ecuador.
This study increases the knowledge about P. falciparum populations circulating in Ecuador and in the region. It gives a better understanding of the parasites present for future surveillance and prevention of parasite re-introduction in an area that is in the process of eliminating malaria. New outbreaks can be studied based on the current situation and new haplotypes can be easily identified.

Conclusions
The P. falciparum diversity found in Ecuador could be a product of migration or the result of haplotypes circulating in the country in low proportions. The three genetic groups present in the north coast of Ecuador confirm the low transmission situation of the last endemic area of the coast of the country. Studies of the genetic characterization of P. falciparum in eliminating areas help determine the possible origin of parasites in order to create strategies to prevent the entrance of new lineages and achieve local elimination of malaria.