Malaria diagnosis: standardization of a polymerase chain reaction for the detection of Plasmodium falciparum parasites in individuals with low-grade parasitemia

Abstract

This work was supported by the Science and Technology for Development Program of the Commission of the European Communities and by the Brazilian National Research Council (CNPq). Maria de Fátima Ferreira-da-Cruz is the recipient of a CNPq fellowship (Pesq 302325/84–0 NV).Universidade Federal do Rio de Janeiro. Department of Biophysics. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Department of Immunology. World Health Organization Collaborating Center for Research and Training in the Immunology of Parasitic Diseases, Rio de Janeiro, RJ, Brazil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Department of Immunology. World Health Organization Collaborating Center for Research and Training in the Immunology of Parasitic Diseases, Rio de Janeiro, RJ, Brazil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Department of Immunology. World Health Organization Collaborating Center for Research and Training in the Immunology of Parasitic Diseases, Rio de Janeiro, RJ, Brazil.Instituto de Medicina Tropical. Manaus, AM, Brazil.Ministério da Saúde. Fundação Nacional de Saúde. Instituto Evandro Chagas. Belém, PA, Brasil.Institut Pasteur de Paris. FranceFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Department of Immunology. World Health Organization Collaborating Center for Research and Training in the Immunology of Parasitic Diseases, Rio de Janeiro, RJ, Brazil.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 Palo Alto strain (culture samples) was serially diluted in blood from an uninfected donor to a final level of parasitemia corresponding to 10–8% and was processed for PCR amplification. In each of these dilutions a parasitological examination was performed to compare the sensitivity with that of PCR amplification. Blood samples (field samples) were obtained from 51 malarious patients with positive thick blood smears (TBS) who were living in endemic regions of the Brazilian Amazon. They corresponded to 42 P. falciparum and 9 P. vivax cases, with parasitemia levels ranging from only 16 to 20,200 parasites/µl for P. falciparum disease and from 114 to 11,000 parasites/µl for P. vivax malaria. We demonstrate that the use of nested PCR allows the detection of 0.005 parasites/µl without the use of radioactive material. The use of a 1-ml sample volume and the organic DNA extraction method should be suitable in blood banks and for the evaluation of patients during and after drug treatment