Evaluation of different methods for Plasmodia detection, in well defined population groups in an endemic area of Brazil

Mais de 500.000 casos novos de malaria foram notificados no Brasil, em 1992. P. falciparum and P. vivax sao as especies responsaveis por 99,3 por cento dos casos. O diagnostico precoce e indispensavel para inicio do tratamento adequado. Neste trabalho, apresentamos or resultados do metodo tradicional para a deteccao de plasmodios, gota espessa, e os resultados de metodos alternativos estudados: imunofluorescencia indireta com anticorpo policlonal anti-P. falciparum e QBC-metodo, em grupos populacionais bem definidos....

Characteristics of multidrug resistance in Plasmodium and Leishmania: detection of P-glycoprotein-like components.

Multidrug-resistance (MDR) in neoplastic cells is frequently characterized by the overexpression of P-glycoprotein (PGP), a 170 kDa transmembrane glycoprotein that binds multiple cytotoxic drugs as well as calcium channel antagonists. Chloroquine resistance in Plasmodium falciparum appears to be analogous to MDR in neoplastic cells, where the induction of resistance with one drug confers resistance to other structurally and functionally unrelated drugs. To test the hypothesis that chloroquine resistance in P. falciparum and antimony resistance in Leishmania is mediated by a similar mechanism of MDR in mammalian neoplastic cells, a PGP-specific monoclonal antibody (C219) was used to determine the presence of PGP genes in resistant and sensitive Plasmodium and Leishmania parasites by indirect immunofluorescence assays and Western blotting procedures. These PGP-like components were detected in both drug-sensitive and -resistant Plasmodium and Leishmania cells. A 40-42 kDa component was observed to be greater in a chloroquine-resistant P. berghei (C line) than in a chloroquine-susceptible P line. Differences observed between Pentostam-resistant and -sensitive Leishmania promastigote clones and isolates included the increased expression of 96-106 and 23-25 kDa peptides in drug-resistant L. enrietti, and increased amounts of two different peptides in two drug-resistant L. panamensis clones (i.e., 96-106 and 43-45 kDa in WR-746-CL4, and 53 and 23-25 kDa in kDa) in amastigotes as in MDR KB carcinoma cells (KB-V1). Comparative indirect immunofluorescent studies suggested that a correlation existed between the degree of antimony susceptibility and the concentration of the moiety recognized by C219 in two L. panamensis clones. Binding of the C219 monoclonal antibody to the PGP-like component of Leishmania was blocked by Pentostam, while the binding of C219 to multiple-drug resistant KB-V1 PGP was not inhibited by Pentostam, regardless of the PGP concentration. This suggests some degree of specificity in the binding of Pentostam to the Leishmania PGP-like components. In addition, these studies have demonstrated that drug-sensitive Leishmania accumulate two to five times more 125Sb-Pentostam than resistant clones.

Biochemical characterization of Plasmodium falciparum hemozoin.

Hemozoin, the pigment granule which develops within the blood stage food vacuole of the malaria parasite Plasmodium falciparum, was biochemically characterized. Hemozoin was found to be composed of 65% protein, 16% ferriprotoporphyrin-IX (hematin), 6% carbohydrate, and trace amounts of lipid and nucleic acids. The overwhelming majority of the protein component is a mixture of native and denatured human globin non-covalently associated with the metalloporphyrin. Immunoelectron microscopy, employing anti-human hemoglobin as a probe, identified in situ association of hemoglobin with hemozoin. Hemozoin produced within diabetic blood had a higher proportion of carbohydrate, suggesting that the carbohydrate component comes from non-enzymatic glycosylation of hemoglobin.

Immunodot assay of Plasmodium falciparum sporozoites in mosquitoes using a direct binding membrane system.

We describe a membrane based immunodot assay for the detection of Plasmodium falciparum sporozoites mixed with mosquitoes. A crude sodium dodecyl sulfate extract of mosquitoes and sporozoites is passed through a bi-layered membrane system, the top layer being a polyvinyldiene difluoride hydrophilic pre-filter which screens out debris but allows the passage of antigen. Sporozoite, as well as mosquito, proteins are bound to the hydrophobic membrane below. This membrane was probed with a monoclonal antibody to the repeat region of the P. falciparum circumsporozoite protein, a peroxidase labeled second antibody and a tetramethyl-benzidine substrate. The method detects as few as 10 sporozoites/mosquito or 100 sporozoites in a pool of 10.

Continuous in-vitro cultivation of Plasmodium falciparum in Ibadan: solutions to scientific and logistical problems.

The technique of continuous in-vitro cultivation of Plasmodium falciparum has not been widely applied in malaria-endemic areas, due to scientific as well as purely logistical problems. Methods for solving or coping with these problems are described. They have already proved effective for over 4 years. The parasites harvested have been satisfactory, as judged by reproducible logarithmic growth curves and normal morphology in Leishman-stained smears as well as phase-contrast microscopy of wet-preparations. They have also been successfully used as antigen sources in the malarial fluorescent-antibody test and to investigate enhanced platelet aggregation in experimental malaria.

Genetic diversity of Plasmodium falciparum shows geographical variation.

Sixty Plasmodium falciparum isolates, 20 each from Thailand, Zimbabwe, and Brazil, were characterized for 20 variant genetic markers, including the enzymes glucose phosphate isomerase, adenosine deaminase and peptidase, 11 other proteins detected by 2-dimensional electrophoresis (2D-PAGE), 2 merozoite surface antigens (MSA-1 and MSA-2), one exported antigen (Exp-1), and sensitivity to the drugs chloroquine, pyrimethamine, and mefloquine. The study examines the extent of diversity between individual isolates and the differences in the frequency of certain variants of the markers between the 3 countries. The principal conclusions to be drawn from the study are that there is extensive polymorphism in many of the genetically determined characters of this parasite, multiple infections with greater than 1 genetically distinct parasite are common, and there are geographical variations in the frequencies with which variant forms of certain markers occur.

A circumsporozoite-like protein is present in micronemes of mature blood stages of malaria parasites.

We demonstrate for the first time the presence of a circumsporozoite (CS)-like protein in invasive blood stages of malaria parasites. Immunogold electron microscopy using antisporozoite monoclonal antibodies localized these antigens in the micronemes of merozoites. Western immunoblot and two-dimensional gel electrophoresis of mature blood stage extracts of Plasmodium falciparum, P. berghei, P. cynomolgi, and P. brasilianum identified polypeptides having the same apparent molecular mass and isoelectric points as the corresponding sporozoite (CS) proteins. The CS-like protein of merozoites is present in relatively minor amounts, compared to the CS protein of sporozoites. Mice with long-term P. berghei blood-induced infections develop antibodies which react with sporozoites.

A 60-kDa Plasmodium falciparum protein at the moving junction formed between merozoite and erythrocyte during invasion.

Invasion of erythrocytes by malaria merozoites requires the formation of a junction of attachment between erythrocyte and merozoite membranes. The attachment junction initially forms at the apical region of the merozoite. It then moves around to the posterior of the merozoite as invasion proceeds. A monoclonal antibody against a 60-kDa merozoite protein (termed MCP-1 for merozoite capping protein 1) of Plasmodium falciparum reacts in an immunofluorescence pattern resembling the moving junction. By two-color immunofluorescence, MCP-1 was located at the attachment site formed between the merozoite apical region and erythrocyte. During invasion, MCP-1 separated and migrated around merozoites at the orifice of the parasitophorous vacuole. In newly-invaded erythrocytes, MCP-1 persisted at the pole of the young parasite nearest the erythrocyte membrane, suggesting its anterior-to-posterior movement. MCP-1 exhibited no variability in molecular mass among the FCR-3, Camp and 7G8 strains of P. falciparum, and the epitope was invariant in the P. falciparum strains studied. We conclude that MCP-1 may participate in merozoite invasion of erythrocytes by facilitating attachment or movement of the junction along the parasite cytoskeletal network.

Purification and partial characterization of an unusual protein of Plasmodium falciparum: histidine-rich protein II.

The human malarial parasite Plasmodium falciparum secretes a histidine-rich protein (HRP-II) from infected erythrocytes. HRP-II has a very high content of histidine (H) (34%), alanine (A) (37%) and aspartic acid (D) (10%) and many contiguous repeats of the sequences AHH and AHHAAD. The histidine content of the protein suggested the potential to bind metal ions. We have demonstrated by metal chelate chromatography an extraordinary capacity of HRP-II to bind zinc ions (Zn2+) and employed this characteristic to isolate the extracellular protein. The HRP-II was further purified by antibody affinity chromatography. The identity of the purified protein was verified by relative molecular weight on denaturing polyacrylamide gels, by reactivity with monoclonal antibodies and monospecific rabbit antiserum, and by comparison of the amino-acid analysis with that derived from the cloned gene sequence. Analysis of the sequence for periodicities using the hydrophobic moment method indicated that HRP-II may potentially form a 3/10 helix. Immunoprecipitation of HRP-II from culture supernatants of parasites metabolically labeled with tritiated sugars showed that the extracellular form of HRP-II is a glycoprotein containing galactose.

The N-terminal amino acid sequences of the Plasmodium falciparum (FCB1) merozoite surface antigens of 42 and 36 kilodalton, both derived from the 185-195-kilodalton precursor.

The N-terminal amino acid sequences of the 42- and 36-kDa Plasmodium falciparum (strain FCB1) merozoite surface polypeptides, both processing fragments from the 185-195-kDa polymorphic glycoprotein, have been obtained. The N-terminus of the 42-kDa fragment is located in the amino acid sequence of the precursor molecule at amino acid residue 1255 (numbering according to Mackay, M., Goman, M., Bone, N., Hyde, J.E., Scaife, J., Certa, U., Stunnenberg, H. and Bujard, H. (1985) EMBO J. 4, 3823-3829). The peptide bond cleaved during processing is Glu-Ala. This fragment is derived from the C-terminal end of the precursor molecule. The N-terminus of the 36-kDa fragment is located in the precursor molecule at amino acid residue 902 (numbering as above), and the bond cleaved is Asn-Asp.

The composition of haemozoin from Plasmodium falciparum.

Haemozoin (malaria pigment) was isolated from 2 strains of Plasmodium falciparum cultivated in vitro. The purest preparations contained 41 to 45% ferriprotoporphyrin IX and a glycine-rich polypeptide ('apohaemozoin') of approximately 14 kDa molecular weight which is synthesized by the parasite. In the two strains studied, NF54 and K1, it was calculated that about 15 and 18 iron porphyrin molecules, respectively, were associated with each molecule of apohaemozoin, which contained more hydrophobic amino acid residues in strain K1. One molecule of iron porphyrin was associated with every 9-10 amino acid residues in the haemozoin of both strains. Our observations support the idea that the intraerythrocytic malaria parasite, incapable of cleaving the haem ring, detoxifies the iron porphyrin residuum from haemoglobin digestion in a crystalline complex with a specially synthesized protein.

46-53 kilodalton glycoprotein from the surface of Plasmodium falciparum merozoites.

A 46-53 kDa glycoprotein antigen of Plasmodium falciparum merozoites has been identified using a murine monoclonal antibody that inhibits infection of human erythrocytes in vitro. Immunofluorescence screening with the antibody of greater than 250 isolates of the parasite finds the inhibitory epitope expressed by only 18% of strains. The glycoprotein is metabolically labelled with methionine, cysteine, histidine and glucosamine but incorporates little lysine or leucine. It is synthesized early in schizogony and remains, without any apparent processing, on the surface of released merozoites where it is demonstrated by immuno-electronmicroscopy and also by vectorial radio-iodination.

Patterns of pigment accumulation in Plasmodium falciparum trophozoites in peripheral blood samples.

Ninety-five samples of peripheral blood from patients with Plasmodium falciparum malaria in southwest Saudi Arabia were examined by Giemsa staining and darkfield microscopy under flow condition. Eighty-four samples contained trophozoites (ring forms) only and 11 samples contained gametocytes and trophozoites. Two patterns of pigmentation were observed in the trophozoite-containing samples: 48 (57%) contained trophozoites in which no pigment could be detected, 32 (38%) contained trophozoites with clearly detectable pigment, and 4 (5%) contained both pigmented and nonpigmented forms. Trophozoite pigmentation did not correlate with percent parasitemia or age or sex of the patients. These results indicate that microscopically observable pigment accumulation in trophozoites of P. falciparum is not required during the asexual multiplication cycle. Pigment accumulation may be triggered later in infection, perhaps as a feature of the differentiation process leading to the formation of gametocytes.

Characterization of a Plasmodium falciparum polypeptide associated with membrane vesicles in the infected erythrocytes.

A Plasmodium falciparum polypeptide (46 kDa) associated with the infected erythrocytes of all asexual stages as well as immature gametocytes was identified by the monoclonal antibody (Mab) 30B8.3. The expression of this protein was not dependent upon the knobby phenotype and was detected in parasites grown either in human or Aotus erythrocytes. The antigen was heatstable, did not label with [14C]glucosamine, and was not sensitive to periodate oxidation. Immunofluorescent staining patterns of Mab 30B8.3 on in vitro cultured parasites varied from punctate (rings and trophozoites) to patchy (trophozoites and schizonts) fluorescence. The Mab 30B8.3 antigen was not detected on the infected erythrocyte surface by conventional wet-mount IFA procedure. However, when parasites were cultured in the presence of Mab 30B8.3, the epitope was detected by the monoclonal antibodies present in the culture medium. Differential extraction of the polypeptide from infected erythrocytes and immune electron microscopy of cryosectioned parasites localized the 30B8.3 epitope primarily on membranes of Maurer's clefts within the infected erythrocyte's cytosol. This 46 kDa polypeptide is unique because it seemed to be an integral membrane protein of the Maurer's clefts/vesicles and it was not secreted into the culture medium nor deposited on the infected erythrocyte membrane. Previous studies indicate that several parasite proteins, excreted extracellularly or deposited on infected erythrocyte membrane, are found to be associated with Maurer's cleft membranes and vesicles. The 46 kDa polypeptide described in this study may play an important role in the transport of the parasite antigens.

The 140/130/105 kilodalton protein complex in the rhoptries of Plasmodium falciparum consists of discrete polypeptides.

Four monoclonal antibodies (MAbs) recognise an antigen localised in the rhoptries of Plasmodium falciparum merozoites using both indirect immunofluorescence assay and immunoelectron microscopy with immunogold labeling. All MAbs immunoprecipitated bands at 140, 130 and 105 kDa from [35S]methionine-labeled parasites; however, one MAb immunoblotted only the 130 kDa protein and another MAb immunoblotted the 105 kDa protein. The affinity purified antigen complex consisted of proteins of 140, 130, 105 and 98 kDa. The individual proteins were subjected to peptide mapping with Staphylococcus aureus V8 protease; the 98 kDa protein was a degradation product of the 105 kDa protein and the 140, 130, and 105 kDa proteins were found to be unrelated. The antigen complex was synthesised at the mid trophozoite stage and was considered to be soluble as judged by release from mature schizonts by freeze/thaw lysis. One of the MAbs inhibited parasite growth and/or merozoite invasion of erythrocytes, in vitro, to a small but significant extent.

Formation of the circumsporozoite protein of Plasmodium falciparum in Anopheles stephensi.

The place and time of synthesis of the circumsporozoite protein of P. falciparum was analysed with a monoclonal antibody directed against the (NANP)3 repetitive epitope of the CS protein. By using an indirect fluorescent antibody test the epitope could be detected on the oocyst 7 days after infection and 3 days before the appearance of mature sporozoites. Using the Western blot technique, 3 polypeptides from midgut preparations were recognized by the (NANP)3-specific monoclonal antibody from the day 9 of infection onwards. The circumsporozoite precipitation reaction could be induced in sporozoites from either the midgut or the salivary glands 11 days after a blood meal. A similar reactivity of midgut-sporozoites and salivary gland-sporozoites was observed with antisporozoite antisera. Not all sporozoites recovered from the midgut showed a precipitation reaction.

Stage-dependent processing and localization of a Plasmodium falciparum protein of 130,000 molecular weight.

A Plasmodium falciparum protein of 130,000 molecular weight (m.w.) has been identified, cloned in Escherichia coli, and completely sequenced (Kochan et al. 1986). The protein appeared to bind to soluble glycophorin, a host erythrocyte surface protein. In the present study, extracts of parasites from different intraerythrocytic stages were immunoblotted with antibodies, raised against a 30,000 m.w. fusion protein corresponding to the 3' end of the 130,000 m.w. protein. It was demonstrated that the protein is synthesized at the trophozoite stage, accumulates at the schizont stage, and is processed at the merozoite stage to a triplet of three polypeptides. The processed proteins are present in the culture supernatant at the time of merozoite burst from the red cell. Immunofluorescent staining of the parasite at different intracellular stages indicates that the protein is localized on the parasite at the trophozoite stage. At late trophozoite stage, it appears to be transported to the erythrocyte cytoplasm, where it is present in small vesicles or inclusions. In mature schizonts the protein accumulates around the plasma membrane of the erythrocyte. At the segmenter stage, just prior to merozoite release, it appears also to surround the intracellular merozoite, as well as the erythrocyte plasma membrane. The soluble 130,000 m.w. protein binds to erythrocytes but binds significantly greater to erythrocyte membranes, suggesting it binds to an internal domain of glycophorin rather than the domain exposed on the surface. The 130,000 m.w. protein is present in 11 different geographic isolates of P. falciparum from diverse geographic origins. Its molecular weight is similar in all isolates.