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.

BackgroundNon-human primates (NHPs) as a source for Plasmodium infections in humans are a challenge for malaria elimination. In Brazil, two species of Plasmodium have been described infecting NHPs, Plasmodium brasilianum and Plasmodium simium. Both species are infective to man. Plasmodium brasilianum resembles morphologically, genetically and immunologically the human quartan Plasmodium malariae. Plasmodium brasilianum naturally infects species of non-human primates from all New World monkey families from a large geographic area. In the family Callitrichidae only the genus Saguinus has been described infected so far. The present study describes the natural infection of P. brasilianum in tamarins and marmosets of the genera Callithrix, Mico and Leontopithecus in the Atlantic forest.MethodsOne hundred and twenty-two NHPs of the family Callitrichidae housed in the Primate Centre of Rio de Janeiro (CPRJ) were sampled in June 2015, and January and July 2016. The CPRJ is located in the Atlantic forest in the Guapimirim municipality, in the Rio de Janeiro state, where human autochthonous cases of malaria have been reported. The samples were screened for the presence of Plasmodium using optical microscopy and nested PCR for detection of 18S small subunit rRNA gene. The amplicon was sequenced to confirm the molecular diagnosis.ResultsThe frequency of Plasmodium infections detected by nested PCR in New World monkeys of the family Callitrichidae was 6.6%. For the first time, Callitrichidae primates of genera Callithrix, Mico and Leontopithecus were found naturally infected with P. brasilianum. Infection was confirmed by sequencing a small fragment of 18S rRNA gene, although no parasites were detected in blood smears.ConclusionsThe reported P. brasilianum infection in NHP species maintained in captivity suggests that infection can be favoured by the presence of vectors and the proximity between known (and unknown) hosts of malaria. Thus, the list of potential malaria reservoirs needs to be further explored.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-017-1724-0) contains supplementary material, which is available to authorized users.

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