The efficacy of antimalarials has contributed to the reduction of morbidity and mortality rates of malaria⁵⁷. However, the spread and increase of antimalarial resistance contribute to the emergence of malaria outbreaks and hinder malaria elimination^24,42. This study focused on the molecular epidemiology of drug resistance genes (Pfcrt, Pfdhps, Pfdhfr, Pfmdr-1, Pfk13 and Pfaat1) in P. falciparum in northwestern Ecuador between 2019 and 2021. We show P. falciparum CQ and pyrimethamine resistance expansion and most importantly the appearance of a mutation associated with proguanil resistance for the first time in Ecuador.

The haplotypes found in Pfcrt (Fig. 2a) are clear evidence of the strong pressure that CQ has exerted over P. falciparum for more than 5 decades in Ecuador (Fig. 4)^49,58. It is evident that in Ecuador as in the rest of South America^20,21,26, CQ resistance has become fixed as all sampled parasites carried the K76T coding mutation in Pfcrt. Reversal of CQ resistance has not been seen in this region even though CQ is no longer used against P. falciparum^26,59. However, in French Guiana, reversal of CQ resistance has been acquired by the C350R mutation in Pfcrt⁶⁰. In particular, the indiscriminate use of CQ reported in several areas of the Pacific coast during the 1990s contributed to the selection of CQR parasites⁶¹. Besides, clonal expansions, may have caused the fast spread and increase of CQR parasites²¹, as well as the loss of CQ-sensitive parasites. Specifically, in Ecuador CQ is not used against P. falciparum since 2006 and CQ has not been used in neighboring countries since the beginning of the century.

Antimalarial treatments against P. falciparum in Ecuador. Timeline of antimalarial treatments since 1950 according to the Ministry of Public Health of Ecuador. Chloroquine (CQ), primaquine (PQ), sulfadoxine-pyrimethamine (SP), artemether-lumefantrine (AL), artesunate (AS), quinine (QN) and clindamycin (CM). Source: Aguilar et al.⁵⁸.

Other mutations such as H97Q, A220S, Q271E, N326D, C350S, I356T/L and R371I in PfCRT have been associated with CQR parasites^16,17. In this study, H97Q, A220S, N326D, S334N, I356L and R371T were found (Table 1). The presence of these mutant alleles may have contributed to CQ resistance in Ecuadorian P. falciparum because it has been suggested that at least three complementary mutations to K76T are necessary to confer high degrees of CQ resistance¹⁸.

Resistance mutations in Pfdhfr were found in 65% of Ecuadorian samples (Fig. 2c). In contrast, sulfadoxine resistance appears low as only one instance of a mutant PfDHPS haplotype, SGKAA, (Fig. 2b) was found among the parasites originating from Colombia, but not Ecuador. This difference may stem from the history of drug use in the region. In South America, pyrimethamine was used massively as a prophylactic against malaria in the 1950s³³. Later, in the 1970s, pyrimethamine was co-administrated with sulfadoxine to combat CQR parasites¹⁴. Consequently, SP resistance has been reported in South America since 1980²⁶. Specifically, in Ecuador, SP was implemented as a second-line treatment in 1950 and, in 2005, it was used as a first-line treatment until 2006 (Fig. 4)⁵⁸. Therefore, the emergence of resistant parasites was caused by the pressure exerted by SP for more than 5 decades. However, the intermittent use of SP temporarily reduced SP pressure, which may have caused SP-sensitive parasites to continue to circulate in the region.

Importantly, the genetic monitoring of Pfdhfr further suggests that resistance to proguanil has appeared in Ecuador. Plasmodium falciparum acquires resistance to cycloguanil, the active metabolite of proguanil via combined coding mutations for A16V and S108N/T in PfDHFR^14,28,62. In Colombia, A16V was detected for the first time in combination with CNCSI and CICNI in Nariño between 2012 and 2013³¹. In this study, A16V with CNCSI was identified in two samples from Tobar Donoso (Table 1) and was previously detected in isolates from San Lorenzo⁴⁹. While A16V was reported in Pacific coast parasites, the samples from San Lorenzo and Tobar Donoso are not the same clones. Instead, our IBD analysis shows that A16V was passaged onto a new genomic background through recombination⁴⁹. Atovaquone-proguanil has been commercialized in Nariño, Colombia for many years³¹, suggesting that the emergence of A16V in Colombia has been caused by the use of atovaquone-proguanil as prophylaxis against malaria³¹. So, the presence of A16V in Tobar Donoso could be explained by the recent recombination of parasites in the Pacific Coast of Colombia or Ecuador⁴⁹. Given the temporal order of sampling, the Tobar Donoso genomes are likely the progeny of a cross between clonal cluster G (Carrasquilla et al.⁴⁹), which carries the A16V allele, and another parasite, but we were unable to identify the second putative parent within our extended Pacific Coast data set. The possible spread of this mutation in Ecuador and Colombia raises an important point against the future use of proguanil in the region.

At PfMDR-1, a shift in haplotype frequencies across time was found (Fig. 3d). While both common haplotypes have been circulating in South America for many years, the most frequent haplotype in 2019–2021 (the triple mutant NEDFSDFY) has not been sampled previously in Ecuador. Mutations in Pfmdr-1 have an important role in response to different antimalarials such as CQ, QN, AQ, MQ and LF^6,14,15. However, South American P. falciparum isolates have not shown phenotypic changes in drug response even though Y184F, N1042D and D1246Y are commonly present^20,63. An in vitro assay tested parasites from Nariño with NEDFSDFD and NEDFSDFY haplotypes, which were shown to be sensitive to AQ, MQ and LF⁶³. In Esmeraldas, ESM-2013 (isolated from 2013) with Y184F and N1042D mutation showed sensitivity to QN, LF and AR²⁰. Mutations in other genes located in the DV such as Pfcrt and Pfaat1 may therefore influence resistance to these drugs^11,64.

The prevalence of MYRIC haplotype (wild-type) in Pfk13 (Fig. 2e) shows sensitivity to ARTs in 100% of Ecuadorian P. falciparum samples. Previous studies in Ecuador, Colombia, Peru, Venezuela, Brazil, Suriname and French Guiana have not found any mutation in Pfk13 associated with ART resistance^20,42,43. However, in Guyana, the C580Y mutation was detected in 2010^41,42. There is, therefore, concern that C580Y could spread to other regions of South America, and molecular studies are key to monitoring for any spread or new emergence. In addition, K189T mutation in PfK13, not associated with ART resistance, was identified in this study and recently reported in Ecuador, Colombia, Venezuela, Brazil, Guyana and French Guiana^42,49. In Ecuador, AS was used with SP as a first-line treatment in 2006; however, AL was already used as a second-line treatment in 2005⁵⁸. AL has been the official first-line treatment against P. falciparum since 2012 (Fig. 4)⁵⁸.

In the last decade, resistance to quinolines such as CQ, QN, AQ and MQ has been associated with mutations in Pfaat1^44,45,46. The S258L mutation identified in this study (Table 1) shows evidence of having been under selection in the Pacific coast of Ecuador and Colombia⁴⁹. Recently, Amambua-Ngwa et al.⁴⁸ found the same mutation segregating in Gambian parasites, and through CRISPR/Cas9 editing, validated its role in CQ resistance. Likewise, an in vitro study showed resistance to three compounds when P380S, K238N, V185L, F230L mutations and an insertion at codon 35 were present in Pfaat1⁴⁷. This pressure could have been caused mostly by CQ and QN, used historically in South America^26,65, and AQ which was introduced in several areas of South America, including the Pacific coast, since 1949^24,59,66.

The observed pattern of resistance does not match the current drug use as has been observed elsewhere in South America, Instead, the recent increase in proportions of mutant haplotypes in Ecuador such as CVMET (double mutant) in PfCRT, CICNI (double mutant) in PfDHFR and the appearance of NEDFSDFY (triple mutant) in PfMDR-1 (Fig. 3) could be explained by clonal expansions caused by parasites from migrant humans and not by the pressure resulting from CQ and SP treatments, because they have not been used against P. falciparum in the last 2 decades. A previous analysis of neutral microsatellites found that outbreaks that occurred in Tobar Donoso and Esmeraldas in 2019 and 2020, respectively, were mostly caused by a single parasite clone⁶⁷. Furthermore, CVMET in PfCRT, CICNI in PfDHFR and NEDFSDFY in PfMDR-1 were dominant in samples from migrant human hosts in this study (Fig. 2), and they have been found circulating in Nariño since 1999^24,26,31. While in northwest Ecuador, CVMET and CICNI were reported in low proportions in 2013–2015²⁰, and NEDFSDFY was absent, but they are now common in recent samples in this study. This suggests that recent outbreaks that occurred in Ecuador may have resulted from the spread of parasites by migrant humans from Nariño. In agreement, the findings of this study show that genetic diversity (both nucleotide and haplotype) in drug resistance genes (Pfcrt, Pfdhps, Pfdhfr and Pfmdr-1) is low. San Lorenzo and Nariño, Colombia presented relatively more genetic variability than other sampled locations in this study. The low diversity in Esmeraldas and Tobar Donoso was mostly due to the fact that samples came from clonal outbreaks in which single haplotypes were predominant. The drug resistance haplotype diversity mirrors diversity seen with neutral microsatellite markers.

Previously, an important human migratory flow that contributes to the spread of parasites on the Pacific coast of Colombia has already been reported⁵¹. In the present study, the main routes of cross-border and intra-border human migration contributing to the spread of malaria parasites in the North coast of Ecuador and the South coast of Colombia are proposed (Fig. 5). Specifically, the spread of resistant parasites to Ecuador could have occurred in two ways: (1) by Ecuadorians who traveled to Colombia and became infected with parasites from there, and (2) directly by infected Colombians who arrived in Ecuador. The main migration route between Nariño and San Lorenzo for several years has been by their seaports. Therefore, Colombian parasites could have reached the coast of Ecuador by the constant migratory flow along the Pacific coast due to the shellfish trade, legal and illegal mining, agriculture and illegal activities such as drug trafficking through the seaports of Nariño, San Lorenzo and Esmeraldas^68,69,70. The opening of a new road between Colombia and Ecuador, and the bridge over Mataje River that connects Nariño and San Lorenzo⁷¹, could represent an important passage of malaria infected people between Ecuador and Colombia. In addition, the E15 road that connects San Lorenzo and Esmeraldas is an important corridor in the spread of malaria parasites (Fig. 5). The presence of A16V in PfDHFR (Table 1) in Tobar Donoso also suggests a process of human migration because this mutation has already found in San Lorenzo⁴⁹ and is common in Nariño³¹.

Cross-border and intra-border routes of entry of P. falciparum in northwestern Ecuador. Possible routes of the human migration used for the passage of P. falciparum in the south coast of Colombia and the north coast of Ecuador. Elaborated according to the information collected from oral reports. Map created using QGIS software (QGIS 3.34 Prizren) (https://www.qgis.org) and edited with Adobe Illustrator version 27 (https://www.adobe.org).

Even though, cross border migration maintains transmission in eliminating areas, only a small number of studies have focused on cross border migration that maintains malaria transmission and spreads drug resistance mutations throughout the region. The study by Carrasquilla et al.⁴⁹ performed whole-genome sequencing of parasites from Ecuador and Colombia and found high relatedness and shared circulating clones. Previously, a study by Griffing et al.³⁰ has shown how P. falciparum lineages with unique drug resistance mutations repopulated Peru after near elimination in the 1990s. Similarly, a study by Ventocilla et al.⁷² suggests population flow between P. vivax parasites in Ecuador and Peru. This current study complements these previous findings by suggesting that migratory routes carry new drug resistance mutations to areas where they were not previously reported.

A possible limitation of this study is the low number of samples in some of the localities such as San Lorenzo. The number of samples mirrors the small number of reported cases in those localities and shows that even a small number of parasites circulating can cause important outbreaks that bring large shifts in genotype frequencies and continue spatio-temporal allele fluctuations. Consequently, in a region in pre-elimination with these epidemiological characteristics, genotype predictions will be difficult and continued monitoring is needed.

Factors such as drug pressure and host human migration influence the emergence and spread of resistant parasites which contribute to the appearance of malaria outbreaks and complicate malaria control and elimination in Ecuador. The findings of this work indicate that CQ and pyrimethamine-resistant parasites have increased in recent years, while artemisinin-sensitive parasites prevail in Ecuador. Therefore, this study shows that the current treatment (artemether + lumefantrine) likely remains effective against P. falciparum and supports the continuation of its use as a main treatment, but warns against the use of CQ, SP or proguanil. We note, however, that we employ a candidate gene approach and cannot rule out the presence of novel resistance-linked mutations elsewhere in the genome.

In addition, the genotypes of P. falciparum show rapid fluctuations within the region, likely due to the boom-bust dynamics of clonal lineages coupled with frequent migration of human hosts across the Ecuador-Colombia border which may make the spread of drug resistant parasites in the region less predictable, increasing the need for continued monitoring of genetic drug resistance profiles in low transmission areas.

Finally, this study demonstrates the importance of the epidemiological characterization of resistance in P. falciparum in a region in the process of eliminating malaria: genetic surveillance of drug resistance genes is key to responding in a timely manner in case of therapeutic failure because it informs treatment success or failure before clinical observations to prevent the spread of malaria.