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Background  Extra-Amazonian autochthonous  Plasmodium vivax  infections have been reported in mountainous regions surrounded by the Atlantic Forest in Espírito Santo state, Brazil.    Methods  Sixty-five patients and 1,777 residents were surveyed between April 2001 and March 2004. Laboratory methods included thin and thick smears, multiplex-PCR, immunofluorescent assay (IFA) against  P. vivax  and  Plasmodium malariae  crude blood-stage antigens and enzyme-linked immunosorbent assay (ELISA) for antibodies against the  P. vivax -complex ( P. vivax  and variants) and  P. malariae / Plasmodium brasilianum  circumsporozoite-protein (CSP) antigens.    Results  Average patient age was 35.1 years. Most (78.5%) were males; 64.6% lived in rural areas; 35.4% were farmers; and 12.3% students. There was no relevant history of travel. Ninety-five per cent of the patients were experiencing their first episode of malaria. Laboratory data from 51 patients were consistent with  P. vivax  infection, which was determined by thin smear. Of these samples, 48 were assayed by multiplex-PCR. Forty-five were positive for  P. vivax , confirming the parasitological results, while  P. malariae  was detected in one sample and two gave negative results. Fifty percent of the 50 patients tested had IgG antibodies against the  P. vivax -complex or  P. malariae  CSP as determined by ELISA. The percentages of residents with IgM and IgG antibodies detected by IFA for  P. malariae ,  P. vivax  and  Plasmodium falciparum  who did not complain of malaria symptoms at the time blood was collected were 30.1% and 56.5%, 6.2% and 37.7%, and 13.5% and 13%, respectively. The same sera that reacted to  P. vivax  also reacted to  P. malariae . The following numbers of samples were positive in multiplex-PCR: 23 for  P. vivax ; 15 for  P. malariae ; 9 for  P. falciparum  and only one for  P. falciparum  and  P. malariae . All thin and thick smears were negative. ELISA against CSP antigens was positive in 25.4%, 6.3%, 10.7% and 15.1% of the samples tested for "classical"  P. vivax  (VK210), VK247,  P. vivax -like and  P. malariae , respectively. Anopheline captures in the transmission area revealed only zoophilic and exophilic species.    Conclusion  The low incidence of malaria cases, the finding of asymptomatic inhabitants and the geographic separation of patients allied to serological and molecular results raise the possibility of the existence of a simian reservoir in these areas.

Epidemiologic aspects of the malaria transmission cycle in an area of very low incidence in Brazil