MALARIA DIAGNOSIS - STANDARDIZATION OF A POLYMERASE CHAIN-REACTION FOR THE DETECTION OF PLASMODIUM-FALCIPARUM PARASITES IN INDIVIDUALS WITHLOW-GRADE PARASITEMIA

In Brazil, no study has been done concerning the detection of malaria parasites by polymerase chain reaction (PCR) related to the diagnosis of Plasmodium falciparum malaria. In the present report we describe a highly sensitive methodology for malaria diagnosis using a nested PCR method based on amplification of the p126 P. falciparum gene detected by simple ethidium bromide staining. The P. falciparum Pale Alto strai n (culture samples) was serially diluted in blood from an uninfected d onor to a final level of parasitemia corresponding to 10(-8)% and was processed for PCR amplification. In each of these dilutions a parasito logical examination was performed to compare the sensitivity with that of PCR amplification. Blood samples (field samples) were obtained fro m 51 malarious patients with positive thick blood smears (TBS) who wer e living in endemic regions of the Brazilian Amazon. They corresponded to 42 P. falciparum and 9 P. vivax cases, with parasitemia levels ran ging from only 16 to 20,200 parasites/mu l for P. falciparum disease a nd from 114 to 11,000 parasites/mu l for P. vivax malaria. We demonstr ate that the use of nested PCR allows the detection of 0.005 parasites /mu l without the use of radioactive material. The use of a 1-ml sampl e volume and the organic DNA extraction method should be suitable in b lood banks and for the evaluation of patients during and after drug tr eatment.