RESUMO
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/microliter for P. falciparum disease and from 114 to 11,000 parasites/microliter for P. vivax malaria. We demonstrate that the use of nested PCR allows the detection of 0.005 parasites/microliter 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.
Assuntos
Malária Falciparum/diagnóstico , Parasitemia/diagnóstico , Reação em Cadeia da Polimerase/métodos , Proteínas de Protozoários/genética , Brasil , Genes de Protozoários , Humanos , Sensibilidade e EspecificidadeAssuntos
Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Vacinas Antimaláricas/imunologia , Plasmodium falciparum/imunologia , Adulto , Animais , Brasil/epidemiologia , Humanos , Malária Falciparum/epidemiologia , Malária Falciparum/prevenção & controle , Fatores de TempoRESUMO
The need for an alternative methodology to assess disease activity in the case of malaria led us to evaluate the usefulness of studying the humoral immune response to establish the diagnosis of past or recent malaria. For this purpose, we analyzed sera from 439 individuals living in endemic areas of the Amazon region (Ariquemes, Rondonia). Individuals were classified according to the number and the date of past crises. The enzyme-linked immunosorbent assay (ELISA) was performed to evaluate the IgG/IgM ratio so as to discriminate acute or recent malaria from past infections against crude and defined (SPF70) Plasmodium falciparum asexual blood-stage antigens. We also analyzed the humoral immune response against components presented in crude P. falciparum antigen by the immunoblot technique. Use of the IgG/IgM ratio values did not allow us to differentiate acute from past infections. However, when we analyzed the humoral immune response to parasite components, we were capable of identifying a polypeptide with a molecular weight ranging up to 40 kDa, which was recognized by all parasitized polyinfected individuals studied but not by individuals with negative thick blood smears. In view of these data, we conclude that the 40-kDa polypeptide may represent a powerful tool in the diagnosis of acute malaria, mainly for screening blood donors in endemic areas.
Assuntos
Anticorpos Antiprotozoários/biossíntese , Antígenos de Protozoários/imunologia , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Malária Falciparum/diagnóstico , Plasmodium falciparum/imunologia , Adulto , Animais , Reações Antígeno-Anticorpo , Brasil/epidemiologia , Feminino , Humanos , Malária Falciparum/epidemiologia , Malária Falciparum/parasitologia , Masculino , Plasmodium falciparum/isolamento & purificaçãoAssuntos
Malária/epidemiologia , Adulto , Brasil/epidemiologia , Feminino , Humanos , Malária/prevenção & controle , Masculino , Estudos de AmostragemRESUMO
The distribution of the surface proteins of Toxoplasma gondii radiodinated were studied using the phase separation technique and ability of binding in the phenyl-Sepharose column. Eight polypeptides with Mr 22 to 180 distributed exclusively in the detergent rich-phase, while six polypeptides with mol. wt. 15,000 to 76,000 distributed exclusively in the detergent poor-phase. Two polypeptides with 15,000 and 70,000 distributed in both phase. All the polypeptides present in the detergent rich-phase binding in the phenyl-Sepharose column, and can be isolated in two peak according with their relative hydrophobicities. Two polypeptides hydrophobic with Mr 60 and 66 recognized by human serum were isolated by the association of the two technique. Our result showed that the surface proteins of T. gondii present different degrees of hydrophobicity and that the use of hydrophobic interaction chromatography after Triton X-114 extraction may be an important isolation method of membrane proteins.