Background
According to World Health Organization (WHO), 132 million people were at risk of malaria infection in 2015 in the Americas. Between 2010 and 2015 there was an estimated 31% decrease in malaria incidence in this region, as well as a 37% decrease in malaria-related mortality. Nevertheless, approximately 450,000 cases were reported in 2015 in this region [
1], 30% from Venezuela, 11% from Colombia and 15% from Peru [
1]. Thirty per cent of all cases were caused by
Plasmodium falciparum. Even though, Ecuador accounted for less than 1% of the region malaria cases in 2015, malaria continues to be endemic in the coast and Amazon areas of Ecuador. From 2016 to 2018, Ecuador has seen increases in malaria cases (1191 in 2016, 1380 in 2017 and 1806 in 2018), where
P. falciparum was responsible for 10% of these cases [
2].
The current treatment for
P. falciparum infection in the Americas is based on artemisinin in combination with another anti-malarial (ACT) [
3]. Treatment for uncomplicated falciparum malaria in Ecuador relies on the combination artemether-lumefantrine + primaquine; for
Plasmodium vivax the treatment used is chloroquine + primaquine [
4].
Plasmodium falciparum has developed resistance to almost all available anti-malarials, necessitating the need for an adequate knowledge of anti-malarial drug effectiveness. This is especially true in low transmission areas, where malaria elimination is ongoing, as the inflow of resistant parasites can generate unwanted outbreaks.
Chloroquine (CQ) resistance in
P. falciparum was reported in 1957 on the Thailand-Cambodian border in Southeast Asia and almost at the same time in Colombia and Venezuela in South America, before spreading to the rest of the world [
5]. Mutations in the
P. falciparum chloroquine resistance transporter (PfCRT) are considered the main reason for CQ resistance [
6]. Currently, CQ resistance is found throughout South America [
5] and the PfCRT molecular marker K76
T, thought to be mainly responsible for CQ resistance, is considered fixed in this region [
7]. The PfCRT haplotype CVMN
T (positions 72–76) has been reported in Colombia and Peru, while CVM
ET and CV
EIT have been reported in Colombia and Venezuela and
SVMN
T has been reported in the Amazon region of Brazil and Peru [
8].
During the 1970s the combination sulfadoxine-pyrimethamine (SP) was introduced in South America as treatment against
P. falciparum. Shortly after the introduction, resistance to these drugs was reported [
8]. Colombian, Brazilian and Peruvian parasite isolates showed mutations in
Pfdhps mainly in positions 437, 540 and 581. The mutation A437
G is dominant in Colombia, while the mutations A437
G and K540
E are found in Peru [
7‐
10]. In addition, Venezuela and Bolivia have reported the mutation K540
E in 90% of the parasite samples tested [
7,
8].
Pfdhfr mutations C50
R, I165
L and S108
N/T are common throughout South America [
8,
9] and all mutations are associated with SP resistance [
11].
Plasmodium falciparum multidrug resistance 1 (
Pfmdr1) transporter gene encodes for a p-glycoprotein that is part of the adenosine triphosphate-binding cassette transporter family. Mutations in
Pfmdr1 are associated with multidrug resistance, and show reduced susceptibility to mefloquine (MQ), halofantrine (HF), quinine (QN), and possibly lumefantrine (LUMF) [
11,
12]. The PfMDR1 mutations N86Y and Y184F are common in Asia and Africa, while the mutations S1034C, N1042D and D1246Y are mostly found in South America [
13].
Several studies associate increases in
Pfmdr1 copy number to MQ resistance, and QN and CQ susceptibility [
13‐
16]. Recent research suggested that an increase in
Pfmdr1 copy number is related to artemisinin resistance [
13,
14]. In South America, there are reports of changes in
Pfmdr1 copy number, specifically in samples coming from the Pacific region, Atlantic region and southeastern Colombia, where an increase of 2 to 5 copies of
Pfmdr1 were found in 30% of the parasite samples [
15]. Peru reported single
Pfmdr1 copy numbers [
17].
Resistance to artemisinin (ART) in
P. falciparum has been reported in five Asian countries: China, Vietnam, Cambodia, Thailand, and Myanmar. The current management to control
P. falciparum infections is based on ART derivatives combined with a partner anti-malarial (e.g., MQ, LUMF, primaquine) [
18].
Kelch 13 (
k13) propeller mutations have been associated with ART resistance and can be used as molecular markers to monitor the possible emergence of ART resistance [
3,
18]. ACT treatment continues to be effective in South America. New studies in Brazilian, Peruvian and Colombian isolates show no
k13 mutations associated with ART resistance [
5,
19,
20].
In addition to genetic variability studies, drug resistant phenotypes can be characterized using in vitro assays. In particular, Colombia reported low in vitro susceptibility to CQ and amodiaquine (AQ) in almost 90% of the isolates analysed, showing IC
50 values for both anti-malarials greater than 100 nM [
14,
21]. Furthermore, all samples showed high susceptibility to dihydroartemisinin (DHA), LUMF and artemether (ATM) [
14,
21,
22]. Brazilian samples from the Amazon region also showed resistance to CQ and AQ, with an elevated IC
50 [
23]. In vitro assays with field parasites in South America have been limited, since culture adaptation of field parasites to laboratory conditions require a long time and are usually challenging [
24].
In 2002, studies from Ecuador reported mutations in isolates collected in Esmeraldas. The parasites presented the CVMN
T Pfcrt genotype and one, (position 108
N) two (positions 108
N, 164
L) or three (51
I, 108
N, 164
L) mutations in
Pfdhfr [
25]. In 2013, parasite isolates showed wild type genotypes for
Pfdhfr and
Pfdhps, the CVMN
T and CVM
ET Pfcrt genotypes, and the mutations Y184
F and N1042
D in
Pfmdr1 in an outbreak that occurred in Esmeraldas [
26]. These genotypes indicated that Ecuadorian strains were CQ resistant and mostly sensitive to sulfadoxine and pyrimethamine [
26]. This genotype was shared with Ecu 1110, a 1990 isolate from the same area. Ecu1110 has an in vitro CQ resistance phenotype (IC
50 > 90 nM) [
27].
In this study, in vitro assays were used to determine drug susceptibility phenotypes. In addition, drug resistance genotypes were analysed in Pfcrt, Pfdhfr, Pfdhps, pfmdr1, and k13 of Ecuadorian P. falciparum isolates. The aim of this study was to understand current anti-malarial resistance in Ecuador, in order to support malaria elimination efforts in the country.
Discussion
Ecuador has been very successful in reducing the number of malaria cases in the country. It is estimated that more than 99% prevalence reduction took place from 2000 to 2015 [
34]. In this context, understanding drug resistance genotypes and phenotypes of Ecuadorian
Plasmodium isolates constitutes crucial information for supporting the National Malaria Programme to achieve elimination of the disease in the country.
Ninety per cent of the samples analysed in this study were collected in Esmeraldas province (Esmeraldas and San Lorenzo counties), where most
P. falciparum cases are known to occur. In 2016, this province reported 125
P. falciparum cases, roughly 45% of the total of
P. falciparum cases in the country [
2].
This study determined the mutations associated with five different genes involved in CQ, SP, MQ, QN, and ART resistance present in Ecuador. The results showed that Ecuadorian parasites presented the CQ resistance haplotypes CVMN
T, CVM
ET and
SVMN
T in
Pfcrt (72–76). The genotype CVMN
T was found in Esmeraldas, San Lorenzo, Carchi and Sucumbíos while the CVM
ET genotype was found in San Lorenzo and Sucumbíos, the
SVMN
T genotype was found in one sample from Orellana. CVMN
T and CVM
ET genotypes have previously been reported in Colombia [
8]. CVMN
T was reported in the Pacific coast of Peru and the CVM
ET genotype was reported in the Amazon region of Peru [
10] The genotype
SVMN
T has been found in the Peruvian [
10] and Brazilian Amazon [
9].
The CVMN
T haplotype had been previously reported in a parasite isolate obtained in Esmeraldas in the 1990 [
27]. Additionally, this genotype was reported in Ecuador during an outbreak occurred in 2013 [
26]. This suggests that this haplotype has been circulating in Ecuador for decades and is still maintained in the country. Griffing and collaborators suggest that parasites carrying this haplotype, were circulating in Colombia and then crossed into Ecuador and later entered Peru [
10].
The resistance to CQ is considered fixed in South America, since CVMN
T, CVM
ET and
SVMN
T are common genotypes in the region [
7,
9]. However, in Ecuador, the national treatment regime was changed from CQ in 2004 to artesunate + SP [
35] and, more recently, to ATM-LUMF [
26].
Plasmodium falciparum parasites circulating in the region continue to have the K76
T genotype of
Pfcrt. This could be related to continuous drug pressure in the parasite population, since CQ remains the main treatment to control
P. vivax infections.
The wild type genotype CNCSI for
Pfdhfr was the main genotype found in all locations sampled in this study (Ecuador, Peru, Colombia, and in the Ecu 1110 isolate) [
27]. The genotype with a simple mutation in position 108 (CNC
NI) was only found in San Lorenzo county. These genotypes have previously been reported in Ecuador [
25], Colombia [
8] and Peru [
10]. The C
IC
NI resistance genotype was found in San Lorenzo county and Orellana; Peru, Colombia and Brazil have also reported this genotype [
8,
9].
The
Pfdhfr polymorphisms were more diverse in San Lorenzo than in the other Ecuadorian locations. San Lorenzo is located close to the Colombian border suggesting that the migration of parasites from Colombia to Ecuador could be related to the distribution of these mutations. In fact, double and triple mutations in
Pfdhfr and resistance to SP have frequently been reported in Colombia [
8]. In addition, it has recently been reported using neutral microsatellites that
P. falciparum populations from San Lorenzo are shared with the south of Colombia [
36].
The wild type genotype SAKAA for
Pfdhps presented the highest frequency in all studied locations, as well as for Ecu 1110 [
27]. Only the sample F50 from Orellana presented mutation in this position (SA
EAA). This mutation is common in Brazil [
9], Venezuela, Bolivia, and Peru [
8,
10]. The wild type genotype was previously found in samples from Esmeraldas in 2002 and 2013. No mutations in
Pfdhps have ever been previously reported in Ecuador [
25,
26].
Despite of the presence of mutations in
Pfdhfr and
Pfdhps in samples from Ecuador, the high prevalence of wild type genotypes suggests ongoing sensitivity to SP in the country. In 2002, 90% of samples collected in Esmeraldas presented at least one mutation in
Pfdhfr (position 108 N). In addition, double (positions 108
N, 164
L) and triple (51
I, 108
N, 164
L)
Pfdhfr mutations were reported [
25]. The decrease in the frequency of parasites carrying
Pfdhfr mutations in Esmeraldas province could be related to the change in treatment from artesunate + SP to ATM-LUMF, which reduced the parasites from drug pressure.
Pfmdr1 codes for a transmembrane P-glycoprotein in the DV of the parasites involved in transport of substrates from the cytosol to the DV. This protein belongs to the adenosine triphosphate-binding cassette transporter family [
12]. Two factors have been associated with alteration of function in PfMDR1: mutations present in
Pfmdr1 and copy number increase [
32]. The mutations N86
Y and Y184
F are more common in Asia and Africa. In contrast, in South America the mutations S1034
C, N1042
D and D1246
Y are found to be more common [
13]. These mutations are associated with multidrug resistance [
37].
In Ecuador, the mutations 184
F and 1042
D were found frequently and were present in the majority of samples from an outbreak that occurred in Esmeraldas in 2013 [
26]. These double mutants were found in this study in samples from Esmeraldas, San Lorenzo, Carchi and Sucumbíos. Furthermore, this genotype was found in Orellana and previously reported for the Ecu 1110 parasite [
27]. The
Pfmdr1 polymorphisms (86, 1034, 1042, 1246) have been associated with resistance to QN, MQ, DHA, and HF [
12]. The 184
F mutation has not been associated with any specific drug resistance and the mutation 1042
D has been linked to MQ and QN resistance [
32]. Ecuadorian parasites do not present clinical or in vitro resistance to MQ or QN, suggesting that these drugs can be considered as an alternative to current treatment in the future.
The increase in
Pfmdr1 copy number has been associated with
P. falciparum resistance to MQ, QN and ART [
14‐
16]. All Ecuadorian
P. falciparum parasites in this study showed one copy of this gene, suggesting that these parasites are sensitive to MQ. Efficacy in vivo studies of artesunate and MQ combination showed that these drugs were an effective treatment in Ecuador in 2000 [
38]. In South America, there are reports of modifications in copy number in samples from the Pacific region, Atlantic region and southeast of Colombia (2009–2012), where 32% of the isolates had
Pfmdr1 copy numbers increase to two to five copies [
15]. Peru reported single copy number for
Pfmdr1 in 2009 in the Amazon region [
17].
The mutation 1042
D and the increase in copy number have been associated with MQ resistance [
32]. MQ forms hydrogen bonds with the residue 1042 of PfMDR1 and the change of N (asparagine) to D (acid aspartic) in this position may result in the inhibition of MQ passage through the DV membrane [
39]. In order to test this hypothesis, live cell imaging using Fluo 4 AM was performed.
Fluo 4 AM is a fluorochrome that has been used to determine PfMDR1 transport of substrates from the cytosol to the DV of the parasite [
32]. The parasites that present N1042 (wild type genotype) show an increased Fluo-4 fluorescence in the DV, showing that the fluorochrome is readily transported into this compartment [
30,
32,
33]. In contrast, the parasites with 1042
D (mutated) PfMDR1 show no increase in Fluo-4 fluorescence in the DV (there is rather an increase in fluorescence in the cytosol of the parasites), suggesting that the fluorochrome is not transported into the DV [
30,
32,
33].
The mutation N1042D was found in most Ecuadorian P. falciparum isolates tested in this study, including ESM-2013. The Fluo 4 AM assay was performed in the isolate ESM-2013 from Esmeraldas to confirm that this mutation inhibited the transport of this marker. The fluorescence intensity of Fluo-4 in the DV of Dd2 (N1042) was higher than in ESM-2013. The Ecuadorian P. falciparum presented the mutation 1042D and inhibited the transport of Fluo 4 into the DV and did not present in vitro resistance to MQ. This suggests that resistance to MQ can be related to the synergy between polymorphisms and increase in copy number of Pfmdr1. The results showed that PfMDR1 of ESM-2013 is not completely functional, since there was inhibition of Fluo 4 transport. These results are not directly related with the current treatment for P. falciparum in Ecuador but should be considered in case a treatment change is planned.
Plasmodium falciparum resistance to ART has been reported in Southeast Asian countries, particularly in the Grand Mekong area (China, Vietnam, Cambodia, Thailand, Myanmar). ART resistance has been associated with mutations in
k13 [
18,
19,
21,
40]. All Ecuadorian samples presented the wild type CRYGI (positions 476, 493, 539, 543, 580) genotype for this gene. Similarly, recent studies in Brazilian, Peruvian and Colombian isolates have shown a lack of
k13 mutations and the ACT treatment appears to be effective in these South American regions. The mutation C580
Y (associated with ART susceptibility in Southeast Asia) was found in 5% of
P. falciparum isolates from Guyana, even though ART showed 100% efficacy [
20]. Although no ATM resistance mutations were found in the studied samples, the spread of other mutations related to ART resistance cannot be ruled out.
Drug resistance phenotypes can also be characterized by in vitro assays. In this study, in vitro assays were used to associate drug susceptibility phenotypes to drug-resistant genotypes. In vitro studies are used to monitor the drug susceptibility of P. falciparum and help guide the drug policy in each country. These studies can give a better idea of how the interaction between the parasite and the drug occur and can help establish parasite sensitivity.
Ecuadorian parasites were cultured and exposed to common anti-malarial drugs to establish their drug susceptibility. The ESM-2013 parasite showed an IC
50 of 93.7 nM, confirming a CQ-resistant phenotype in Ecuadorian
P. falciparum isolates having the mutation K76
T. The in vitro resistance to CQ in Ecuadorian parasites has been previously reported in Ecu1110. It presented an IC
50 > 90.9 nM [
27] in comparison with 3D7 that showed an IC
50 < 10 nM. Other in vitro studies have shown resistance to CQ in Colombia, where 90% of parasites analysed presented IC
50 > 100 nM [
22]. CQ resistance has been present in Ecuador since the 1980s, suggesting that the resistance to CQ is fixed in Ecuadorian
P. falciparum parasites. ESM-2013 showed in vitro sensitivity to QN, MQ, DHA, LUMF, and ATM; similarly, Colombian samples had in vitro sensitivity to DHA, LUMF and ATM [
15].
The current treatment for
P. falciparum in Ecuador is ATM-LUMF. The results of this study suggest that this treatment continues to be effective in the country, as well as in the rest of Latin America where there is no reported resistance to ACT treatment [
5]. It is important to note that, even though Ecuadorian parasites have CQ-resistant genotype and phenotype and present mutations in
Pfdhfr and
Pfmdr1, they have the same resistance profile as Ecu 1110, an isolate collected in 1990 [
26]. These results suggest that the mutations in drug resistance genes have been maintained for almost 30 years in spite of a lack of selective pressure. This could be explained by a fixation of drug resistant mutations and the presence of parasites as asymptomatic reservoirs [
41].