Molecular and epidemiological characterization of imported malaria cases in Chile

Malaria is a mosquito-borne infectious disease caused by parasites of the genus Plasmodium. The four species of Plasmodium that commonly infect humans are Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae. The fifth human malaria species is Plasmodium knowlesi, known to cause simian malaria, which has been reported in nearly all countries from Southeast Asia and in travellers visiting these countries [1, 2].

In 2017, there were 219 million estimated cases of malaria worldwide, resulting in 435,000 deaths [3]. In South America, the most prevalent species are P. vivax and P. falciparum, with 773,000 malaria cases estimated in 2017; 97% of these cases were found in Brazil, Colombia, Peru, and Venezuela [1, 4]. Recently, there has been a significant increase in the number of cases in Venezuela due to the political unrest leading to lack of available treatment [3, 5]. Colombia and Brazil also reported more confirmed cases compared to previous years, which shows an increase of the transmission in the region for this period [3, 6, 7].

Chile has been certified as a malaria-free country by the World Health Organization (WHO), with no indigenously-acquired cases reported since 1945 [8]. This was possible due to the implementation of an anti-malaria campaign in the early 1940s directed towards vector (Anopheles pseudopunctipennis) control and the prompt treatment of malaria patients [8]. Between 1945 and 2001, there were 90 imported cases of malaria and five malaria-related deaths reported by the Chilean Ministry of Health [8]. Malaria is a disease of immediate notification according to decree DTO N° 158/04 as part of the Emerging Diseases Surveillance of the Ministry of Health. Samples from all suspected cases are sent to the Public Health Institute of Chile (ISP) for diagnostic confirmation.

Currently, An. pseudopunctipennis (known malaria vector) breeding sites have been found in close proximity to residential areas in Valle de Azapa, Valle de Lluta, Quebrada de Camarones and Chaca in the Arica y Parinacota Region and in the Tarapacá Region. Additionally, González and Sallum, reported the presence of a new species of Anopheles with unknown vector potential in Atacama Region, northern Chile [9].

For many years, the recommended malaria treatment was chloroquine (CQ) followed by sulfadoxine–pyrimethamine (SP) until resistant parasites spread worldwide. Currently, artemisinin-based combination therapy (ACT) is the first-line treatment recommended by the WHO for P. falciparum infected patients in countries where CQ and SP resistance has been reported [10]. However, the treatment for P. vivax infections is chloroquine plus primaquine (CQ + PQ), with a variation of 7 or 14 days of therapy with PQ depending on the country [4].

Publications reporting delayed parasite clearance following ACT in Southeast Asia [11‐13] placed a worldwide alert on the importance of monitoring malaria drug resistance. In South America, the first P. falciparum isolates harbouring the C580Y mutation in P. falciparum kelch 13 gene were reported in samples collected in 2010 in Guyana [14]. This mutation is highly prevalent in Southeast Asia and is a confirmed marker of artemisinin resistance [12, 15, 16]. Although ACT is still highly effective in many parts of the world, there is a serious concern about the emergence of artemisinin resistance in other parts of the world.

While malaria transmission is not known to occur in Chile, importation of malaria cases from other South American countries and other parts of the world requires proper monitoring in order to inform treatment policy. In this study, a molecular characterization of parasites from imported malaria cases was undertaken to confirm infecting Plasmodium species, mixed infections, and to assess the drug resistance profile of parasites. Additionally, microsatellite (MS) markers were analysed in P. falciparum samples to explore the presence of clusters of cases and/or polyclonal infections.

The increase in the number of suspected and confirmed malaria cases in Chile might be associated with the high movement of Chileans traveling for work and/or pleasure to endemic countries, such as Brazil, Colombia, and Peru. Additionally, according to the National Institute of Statistics of Chile (INE), 746,465 immigrants (particularly from Haiti, Venezuela, Colombia, and Peru) live in Chile and it is estimated that this number has increased more than 300% compared to the previous decade (2010–2017: 471,285 and 2016–2017: 220,090) [23].

Although malaria has been eliminated in Chile, globalization and cultural exchange has contributed to raising awareness of the disease and for that reason early detection of malaria cases have improved in recent years. It is necessary that health care providers in Chile obtain the travel history of patients in order to diagnose malaria promptly, notify health authorities and send samples for confirmation to the ISP. Moreover, malaria should be included in the differential diagnosis for every patient with fever who has travelled to an area where malaria is endemic [24].

In this study, the most frequent malaria cases were due to P. vivax infections. Unfortunately, complete travel history, prophylaxis, and anti-malarial treatment could not be obtained for all cases. Nevertheless, as expected, the travel history matched the infecting species expected, with P. vivax infection identified in people who visited P. vivax endemic countries in South America and Asia and P. falciparum mainly from Africa. While no clusters were detected in our analysis, a cluster of P. vivax cases was previously reported among Chilean travellers returning from Peru [25]. This highlights the need to promote the use of chemoprophylaxis in travellers, especially, in those visiting specific locations in endemic countries with high malaria transmission.

Additionally, the drug resistance profiles of the parasites are important to establish the guidelines for an appropriate treatment policy. Mutations in the pfcrt gene reported in this study (CVMNT, CVIET, CVMET, S_tctVMNT) are known chloroquine resistance markers found worldwide. Their frequency in endemic countries and the possible origin of each genotype is associated with different geographical locations [26, 27]. For instance, the CVIET genotype is typically found in Africa, in countries such as Uganda, Guinea and Benin [26, 28‐34]. The literature also reports the presence of this genotype in countries such as India, Thailand, Vietnam, Cambodia and Papua New Guinea [27]. In South America, it has been reported in Peru, Brazil and Guyana. The S_tctVMNT genotype, found in samples from Venezuela, has two possible independent origins, one in South America and one in Papua New Guinea [20, 27]. The CVMET genotype found in a sample from a patient with travel history to Colombia and Nicaragua is mainly reported in the northern part of South America. The CVMNT genotype identified in two samples from Colombia is the closest to the ancestral sensitive genotype, which has been previously reported in Brazil, Colombia, Ecuador and Peru. The wild-type pfcrt genotype is commonly found in Central America [27, 28, 35].

There were several pfmdr1 alleles identified in the samples analyzed, showing single, triple, and quadruple mutants. Mutations at pfmdr1 N86Y and D1246Y, which are common in Africa, have been linked to decreased sensitivity to chloroquine and amodiaquine, but increased sensitivity to lumefantrine, mefloquine, and artemisinins [36]. Other polymorphisms, primarily seen outside Africa (including 1034C and 1042D), are associated with altered sensitivity to lumefantrine, mefloquine, and artemisinins [36]. Samples from patients who traveled to Uganda, Nigeria, and the Dominican Republic showed a single mutation in codon 184 (Y184F), which has been previously reported in these countries [37‐39]. MS analysis of P. falciparum confirmed that the samples analyzed were all imported, clusters or clonal expansions were not found. Furthermore, the haplotypes identified in this study, were all different (Table 3). The MS alleles found in the sample from Uganda, were consistent with previous findings of these alleles in this country. The analysis of alleles showed that four of the seven loci belong to haplotypes previously described in Uganda: TA1 (183), Polyα (165), Pfpk2 (162/171/180) and TA109 (163) [40].

In addition, the 2490 allele “81” found in the sample from Uganda has also been found in other African countries such as Kenya [41]. Furthermore, the TA1 (141/165) and Polyα (156) alleles found in the patient returning from Kenya, are commonly found in several localities of this country [41, 42]. Also, the patient’s sample with travel history to Guinea revealed a consistent MS pattern with a previous report from this country [43]. The TA1 allele (171) found in isolates from Guinea and Colombia, has been previously reported in other countries from South and Central America but also in low frequency in Guinea and Mozambique [35, 44‐46]. The sample from Nigeria also revealed a consistent pattern with previous reports on loci PfPK2 and TA109 [47]. The haplotypes found in samples from Colombia and Venezuela in this study, also presented alleles previously detected in both countries and in others from South America [44, 45]. The samples from Colombia showed a haplotype profile akin to one previously reported in Colombia, from the town of Nariño [44]. One of the samples from Colombia corresponded to a patient with travel history to Nicaragua. However, the haplotype profile suggests the infection was acquired in Colombia. Finally, the MS profile identified in the sample from the Dominican Republic was similar to the one reported by Carter et al., in the Island of Hispaniola in Central America [48].

An important aspect within an Integrated Surveillance System, such as the one Chile is currently implementing, is to join efforts to obtain complete epidemiological information on all cases reported, including using adequate diagnostic tools in primary health centers, following up all malaria cases, recommending appropriate prophylaxis to Chilean travellers, and ensuring the availability of appropriate drugs for malaria treatment. Currently, atovaquone–proguanil (malarone) or mefloquine could be used as the first-line treatment for patients with uncomplicated malaria in Chile [49]. Among the patients included in this study, four reported post-treatment relapses with incomplete epidemiological information, such as prophylaxis, treatment and/or patient compliance with the treatment. It is worth noting that patients in Table 1 reported recurrent symptoms after more than 1 month. This may be attributable to a parasite relapse from latent hypnozoites, since all cases were P. vivax and patients did not report additional travels during or after treatment. Also, it is possible that a lack of anti-hypnozoite therapy has increased the probability of these relapses (which could be higher than 20%, according to previous reports) [50].

It is essential to strengthen the malaria surveillance system in Chile, which should include complete epidemiological data, travel history, previous diagnostic results and prophylaxis or treatment given. This information is valuable to better assist patients, provide recommendations to travellers and further analyse the current malaria situation in the country. Moreover, the introduction of malaria cases in Chile has brought the presence of resistant strains from all over the world, which requires proper control and surveillance.