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‐
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‐
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‐
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‐
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 sulfadoxine–pyrimethamine (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‐
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‐
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.
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.
This study employed seven neutral microsatellites (TA1, Poly-α, PfPK2, TA109, 2490, C2M34 and C3M69 [
12,
18,
33]) to characterize
P. falciparum populations from Esmeraldas Province in northwest Ecuador. The same seven markers have been widely used in South America to characterize
P. falciparum populations in Peru [
15,
21], Colombia [
26,
27] and Brazil [
44].
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 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.
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