Differential diagnosis of Plasmodium simium infection by nested/PCR followed by a digestion with Hpy CH4III restriction enzyme of 9 infected human samples from Atlantic Forest in Rio de Janeiro/RJ (H2 – H9) and one from Amazon endemic region (H1) according to Additional file 1. 3% agarose gel stained with ethidium bromide. MM:1 kb Plus Ladder (ThermoFischer, Calrsbad, CA, USA). Reactions were performed simultaneously in the same thermocycler and splited in different gels.D: Digested; ND: Non Digested. PC Pv: Positive Control for P. vivax , PC Ps: positive control for P. simium . NC: Negative Control (without DNA).