We examined the mitogenomes of a large global collection of human malaria parasites to explore how and when Plasmodium falciparum and P. vivax entered the Americas. We found evidence of a significant contribution of African and South Asian lineages to present-day New World malaria parasites with additional P. vivax lineages appearing to originate from Melanesia that were putatively carried by the Australasian peoples who contributed genes to Native Americans. Importantly, mitochondrial lineages of the P. vivax-like species P. simium are shared by platyrrhine monkeys and humans in the Atlantic Forest ecosystem, but not across the Amazon, which most likely resulted from one or a few recent human-to-monkey transfers. While enslaved Africans were likely the main carriers of P. falciparum mitochondrial lineages into the Americas after the conquest, additional parasites carried by Australasian peoples in pre-Columbian times may have contributed to the extensive diversity of extant local populations of P. vivax.

Blood infection by the simian parasite, Plasmodium simium, was identified in captive (n = 45, 4.4%) and in wild Alouatta clamitans monkeys (n = 20, 35%) from the Atlantic Forest of southern Brazil. A single malaria infection was symptomatic and the monkey presented clinical and haematological alterations. A high frequency of Plasmodium vivax-specific antibodies was detected among these monkeys, with 87% of the monkeys testing positive against P. vivax antigens. These findings highlight the possibility of malaria as a zoonosis in the remaining Atlantic Forest and its impact on the epidemiology of the disease.

Zoonotic malaria poses a unique problem for malaria control. Autochthonous cases of human malaria in the Atlantic Forest have recently been attributed to Plasmodium simium, a parasite that commonly infects non-human primates in this Brazilian biome. However, due to its close similarity at both the morphological and molecular level to Plasmodium vivax, the diagnosis of P. simium in this region remains problematic. Therefore, a diagnostic assay able to accurately identify P. simium is important for malaria surveillance. Based on mitochondrial genome sequences, primers were designed to amplify a region containing a SNP specific to P. simium. This region can then be digested with the restriction enzyme HpyCH4III, which results in digestion of P. simium sequences, but not of any other malaria parasite. Fifty-two human and monkey blood samples from different regions and infected with different Plasmodium species were used to validate this protocol. This easy and inexpensive tool can be used for the diagnosis of P. simium in non-human primates and human infections from the Atlantic Forest region to monitor zoonotic malaria transmission in Brazil.

Background The Americas were the last continent to be settled by modern humans, but how and when human malaria parasites arrived in the New World is uncertain. Here, we apply phylogenetic analysis and coalescent-based gene flow modeling to a global collection of Plasmodium falciparum and P. vivax mitogenomes to infer the demographic history and geographic origins of malaria parasites circulating in the Americas. Importantly, we examine P.vivax mitogenomes from previously unsampled forest-covered sites along the Atlantic Coast of Brazil, including the vivax-like species P. simium that locally infects platyrrhini monkeys. ResultsThe best-supported gene flow models are consistent with migration of both malaria parasites from Africa and South Asia to the New World, with no genetic signature of a population bottleneck upon parasite's arrival in the Americas. We found evidence of additional gene flow from Melanesia in P. vivax (but not P. falciparum) mitogenomes from the Americas and speculate that some P. vivax lineages might have arrived with the Australasian peoples who contributed genes to Native Americans in pre-Columbian times. Mitochondrial haplotypes characterized in P. simium from monkeys from the Atlantic Forest are shared by local humans.These vivax-like lineages have not spread to the Amazon Basin, are much less diverse than P. vivax circulating elsewhere in Brazil, and show no close genetic relatedness with P. vivax populations from other continents. ConclusionsEnslaved peoples brought from a wide variety of African locations were major carriers of P. falciparum mitochondrial lineages into the Americas, but additional human migration waves are likely to have contributed to the extensive genetic diversity of present-day New World populations of P. vivax. The reduced genetic diversity of vivax-like monkey parasites, compared with human P. vivax from across this country, argues for a recent humanto-monkey transfer of these lineages in the Atlantic Forest of Brazil. Word count: 299 Author summaryMalaria is currently endemic to the Americas, with over 400,000 laboratory-confirmed infections reported annually, but how and when human malaria parasites entered this continent remains largely unknown. To determine the geographic origins of malaria parasites currently circulating in the Americas, we examined a global collection of Plasmodium falciparum and P. vivax mitochondrial genomes, including those from understudied isolates of P. vivax and P. simium, a vivax-like species that infect platyrrhini monkeys, from the Atlantic Forest of Brazil.We found evidence of significant historical migration to the New World of malaria parasites from Africa and, to a lesser extent, South Asia, with further genetic contribution of Melanesian lineages to South American P. vivax populations. Importantly, mitochondrial haplotypes of P. simium are shared by monkeys and humans from the Atlantic Forest, most likely as a result of a recent human-to-monkey transfer. Interestingly, these potentially zoonotic lineages are not found in the ...

Oxford, Headington, Oxford, OX3 9DU, UK 41 42 §) Contributed equally, arranged alphabetically 43 †) Corresponding authors 44 45 46 Summary 47 48 Plasmodium simium, a malaria parasite of non-human primates in the Atlantic forest region of 49Brazil was recently shown to cause zoonotic infection in humans in the region. Phylogenetic 50 analyses based on the whole genome sequences of six P. simium isolates infecting humans 51 and two isolates from brown howler monkeys revealed that P. simium is monophyletic within 52 the broader diversity of South American Plasmodium vivax, consistent with the hypothesis 53 that P. simium first infected non-human primates as a result of a host-switch from humans 54 carrying P. vivax. We provide molecular evidence that the current zoonotic infections of 55 people have likely resulted from multiple independent host switches, each seeded from a 56 different monkey infection. Very low levels of genetic diversity within P. simium genomes 57 and the absence of P. simium-P. vivax hybrids suggest that the P. simium population emerged 58 recently and has subsequently experienced a period of independent evolution in Platyrrhini 59 monkeys. We further find that Plasmodium Interspersed Repeat (PIR) genes, Plasmodium 60Helical Interspersed Subtelomeric (PHIST) genes and Tryptophan-Rich Antigens (TRAg) 61 genes in P. siumium are genetically divergent from P. vivax and are enriched for non-62 synonymous single nucleotide polymorphisms, consistent with the rapid evolution of these 63 genes. Analysis of genes involved in erythrocyte invasion revealed several notable differences 64 between P. vivax and P. simium, including large deletions within the coding region of the 65 Duffy Binding Protein 1 (DBP1) and Reticulocyte Binding Protein 2a (RBP2a) genes in P. 66 simium. Genotyping of P. simium isolates from non-human primates (NHPs) and zoonotic 67 human infections showed that a precise deletion of 38 amino acids in DBP1 is exclusively 67 present in all human infecting isolates, whereas non-human primate infecting isolates were 68 polymorphic for the deletion. We speculate that these deletions in the parasite-encoded key 69 erythrocyte invasion ligands and the additional rapid genetic changes have facilitated zoonotic 70 transfer to humans. Non-human primate malaria parasites can be considered a reservoir of 71 potential infectious human parasites that must be considered in any attempt of malaria 72 elimination. The genome of P. simium will thus form an important basis for future functional 73 characterizations on the mechanisms underlying malaria zoonosis. 74 75 76 Introduction 77 78 There are currently eight species of malaria parasites known to cause disease in humans; 79 Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale 80 curtisi, Plasmodium ovale wallikeri, Plasmodium knowlesi, Plasmodium cynomolgi and 81 Plasmodium simium. The latter three species are more commonly parasitic on non-human 82 primates and have only relatively recently been shown to infect humans 1-3 . ...