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Howler monkeys are the reservoir of malaria parasites causing zoonotic infections in the Atlantic forest of Rio de Janeiro

Background Although malaria transmission was eradicated from southeast Brazil, a significant increase in the number of Plasmodium vivax-like autochthonous human cases has been reported in remote areas of the Atlantic Forest in the last decades in Rio de Janeiro (RJ) state, including an outbreak in 2015-2016. The singular clinical and epidemiological aspects of several human cases combined with molecular and genetic data revealed that they were due to the non-human primate (NHP) parasite P. simium. The full understanding of the epidemiology of the autochthonous malaria in southeastern Brazil depends, however, upon the knowledge on the circulation of NHP Plasmodium in the foci and the determination of its reservoirs. Methodology A large sampling effort was carried out in the Atlantic forest of RJ and its bordering states (Minas Gerais, São Paulo, Espírito Santo) for capture and examination of free-living NHPs. Blood and/or viscera were analyzed for Plasmodia infections through molecular and microscopic techniques. Principal findings In total, 146 NHPs of six species, from 30 counties in four states were tested. Howler monkeys (A. guariba clamitans) were the only NHP species found infected. In RJ, 26% of howlers were positive, among them 17% were found to be infected with P. simium. Importantly, specific single nucleotide polymorphisms were detected in all P. simium infected howlers regardless geographical origin of malaria foci. Interestingly, 71% of P. simium infected NHP were from the coastal slope of a mountain chain (Serra do Mar), where most human cases have been occurring. P. brasilianum/malariae was detected for the first time in 14% free-living howlers in RJ as well as in 25% of those from the Espírito Santo state. Moreover, malarial pigment was detected in spleen fragments of 50% of a subsample composed of howler monkeys found dead in both RJ and ES. All NHPs were negative for P. falciparum. Conclusions/Significance Our data indicate the howler monkeys as the main reservoir of the Atlantic forest human malaria in RJ and other sites in Southeast Brazil and reinforce its zoonotic nature. Author summary The present work consists of an unprecedented capture effort and large-scale field survey of plasmodial species in Non-human primates (NHPs) in RJ, a state recording a three-decade history of autochthonous human cases of benign tertian malaria pending epidemiological clarification of their origin. For the first time, we describe infection rates by Plasmodium sp.in free-living NHP, match the spatial distribution of P. simium in NHP with that of local human cases of benign tertian malaria due to this parasite, disclose howler monkeys as the only confirmed reservoir of this zoonotic malaria in the state and showed that specific single nucleotide polymorphisms were present in all P. simium infected howlers, regardless of the geographical origin of malaria foci. It is also the first time that P. brasilianum/malariae is recorded in free-living NHPs from Rio de Janeiro and the widespread distribution of this quartan-malaria parasite of zoonotic potential in the state is illustrated. Together, these findings increase the understanding about the simian malaria parasites in Atlantic Forests, as well as on the zoonotic character of autochthonous human malaria in Rio de Janeiro, providing subsidies for shaping surveillance and control.