The use of chloroquine dates back to 1955 being one of the oldest synthetic drugs used in the treatment of malaria [18]. However, the first chloroquine resistant (CQR) strains of Plasmodium falciparum began to be reported in Southeast Asia as early as 1957. CQR rapidly spread to sub-Saharan Africa and new resistant variants appeared in South America and Asia during the following decades [5]. Surprisingly, northern Central America and the island of Hispaniola in the Caribbean are the only region in the world where P. falciparum strains are still susceptible to chloroquine (CQS) [10, 19]. Therefore, there are six countries in the Americas that still use chloroquine and primaquine as first line of treatment against uncomplicated P. falciparum malaria.: Guatemala, Honduras, Nicaragua, Costa Rica, Haiti, and the Dominican Republic [1]. Given the imminent threat of the spontaneous appearance of mutant parasite strains due to drug exerted pressure, as well as increasing human migration to North America through the Central American isthmus, it is important to periodically monitor parasite populations for evidence of CQR [20, 21]. This study is an effort to contribute to the routine surveillance of CQR in P. falciparum that has been carried out in Central America since 2010.
We sequenced the pfcrt gene region spanning codons 72 to 76 in 205 P. falciparum isolates collected between 2018 and 2021 from Honduras and Nicaragua. All samples showed a wild-type amino acid sequence CVMNK associated with susceptibility to CQ. This result is consistent with previously published studies. The first study carried out with 30 samples collected in five departments of Honduras between 2004 and 2009 showed 100% of isolates with wild genotype, except for two individuals whose infection had been contracted in Asia and Africa [12]. A study of the therapeutic efficacy of CQ included 68 samples collected in Puerto Lempira, in the Honduran Mosquitia, revealing only wild genotypes in the pfcrt gene [11]. Likewise, an efficacy trial conducted in the North Atlantic Region (RAAN) of Nicaragua during 2005 and with samples from a surveillance study from 2011 showed that 96 of 98 samples had the CVMNK phenotype, while two samples had the CVIET CQR phenotype. Unfortunately, the authors were unable to confirm whether these cases were imported or not [22]. In a 2014 publication, the authors evaluated 160 samples collected from several municipalities in Honduras between 2010 and 2013 and showing 100% wild genotypes of pfcrt [10]. Unpublished data revealed that 16 samples collected in 2015 from Guatemala also showed the wild genotype. In a more recent study including 16 specimens that underwent next generation sequencing, 3 specimens were found with a CQR haplotype SVMNT. The authors indicate that two of these samples were imported cases from Africa and that the third was a local case collected in 2013 in the Choluteca region [9]. However, no epidemiological information is offered on the case that can ensure that it was indeed a local case or a foreign migrant passing through the country.
In the case of Hispaniola, where circulating parasites still show a wild CQS genotype [19, 23, 24], the presence of imported cases from countries with CQR strains in Central America and Hispaniola [9, 22, 24–29] puts at risk the efforts made in recent decades to achieve malaria elimination [2]. The continued finding of CQ susceptibility in recent years, indicates that the notable increase in malaria cases in Nicaragua from 2014 to the present (Fig. 1) is not caused by the appearance of CQR strains but to other phenomena that exceed the purposes of this study and must be promptly analyzed in an integrated way.
The second gene analyzed in this study was pfmdr1, a transporter on the membrane of the digestive vacuole that mediates the transfer of antimalarial drugs from the cytosol to the vacuole [30]. At least 5 SNPs have been described in pfmdr1 presumably associated with resistance to different antimalarial drugs [31–33]. Unlike pfcrt, the influence of the pfmdr1 gene on P. falciparum CQR is still not entirely clear, and the response to antimalarials is likely to be a multigenic phenomenon that is affected by the sum of mutations in different transporter genes [34]. To shed some light on this topic, we sequenced three fragments of the pfmdr1 gene encompassing codons 86, 184, 1034, 1042, and 1246 in 51 samples that had shown a wild type pfcrt genotype. All samples showed an NFCDD genotype, with wild type alleles at positions 86 and 1246, and mutant alleles at 184, 1034, and 1042. There are two published studies on this gene in samples from Honduras that coincide with our results. A first study in 2011 showed that all 30 samples tested had the wild N86 genotype [12]. Similarly, a second study revealed the genotype N86, 184F, D1246 in all 16 samples [9]. To our knowledge, this would be the first study to analyze the five codons of interest in the pfmdr1 gene in samples from Honduras and Nicaragua.
A study carried out in Haiti found the haplotype N86/184F in 108 samples analyzed after the 2010 earthquake [27]. A second study carried out in Haiti amplified 54 samples and analyzed all five codons, finding mutations only in codon 184 (NFSND) [23]. Likewise, six patients infected by P. falciparum in Punta Cana in the Dominican Republic were described as carriers of the 184F mutation [24]. On the other hand, there are several studies that reported different haplotypes of pfmdr1 in South America. As in the present study, all reported exclusively the wild-type N86 [35–40] that seems to be a common characteristic in the strains of the continent. Some authors propose that CQR mediated by pfcrt mutations is modulated somehow by mutations in pfmdr1, and that the mutant alleles 86Y and 184F are the most relevant [41, 42]. According to the literature all the P. falciparum isolates show the 184F mutant allele in Central America [9], Haiti [23, 27] and South America [36–40, 43–45]. Mutation 184F is believed to have a limited effect in the absence of a mutation at codon 86 [46, 47]. Consequently, a CVMNK genotype in pfcrt together with a wild type N86 allele in most parasite strains in Central America and Haiti (despite the presence of 184F mutants), would allow predicting that it is unlikely that short-term CQR will appear in the region due to an accumulation of mutations in both genes.
Codon 1034 is more heterogeneous on the American continent. Both the wild genotype S1034, and the mutant 1034C, have been described in Colombia, Venezuela, Peru, and Suriname. The wild genotypes 1042D and 1246Y are the most frequent in the region [36, 37, 39, 40, 45, 48–51]. The “NFCDD” signature found in this study in all samples analyzed has also been described in Peru (94.5–100%) [37, 40], French Guiana (0.2%) [51], Ghana (43.5%) [52], and Yemen (57%) [53]. It is complex to interpret the role that mutations in pfmdr1 play on the modulation of resistance to the different antimalarial drugs available. However, in the absence of mutations in pfcrt in parasites from Honduras and Nicaragua, where CQ remains the first line of treatment for uncomplicated malaria, mutant haplotypes in pfmdr1 do not appear to be an important variable to consider at present.