New strategies for the diagnosis and screening of malaria.

Thin and thick blood film microscopy are the "gold standard" for malaria diagnosis. In recent years, there have been important developments in malaria diagnostic tests including fluorescence microscopy of malaria parasites stained with acridine orange, dipstick immunoassays that detect species-specific parasite antigens, and more recently, detection of parasite nucleic acids after amplification by PCR. With some of these methods, sensitivities and specificities approaching and even exceeding those of the thin and thick film can be attained. In particular, PCR-based tests for plasmodium DNA or RNA are more sensitive and specific than other tests for malarial parasites. A specific application for PCR diagnosis of malaria could be blood donor screening. Clinical trials of blood donor sreening for malarial parasites by PCR are being conducted, in which pooled donor samples are screened to increase efficiency and reduce costs. Some of the new diagnostic methods may have specific applications in particular settings, depending on the purpose and location of testing, and other factors such as cost, desired sensitivity and specificity, speed and ease of use.

Immune-mediated parasite clearance in mice infected with Plasmodium berghei following treatment with manzamine A.

Manzamine A, a sponge-derived alkaloid, was recently shown to possess in vivo antimalarial activity against the blood stages of the rodent malaria parasite Plasmodium berghei. A single intraperitoneal dose of 100 micromol/kg of manzamine A suppressed parasite growth but was followed by parasite recrudescence. Forty percent of mice with recrudescing parasites were able to recover and clear the fulminating parasitaemia. Examination of sera from these mice revealed that infected mice treated with manzamine A had a suppressed IFN-gamma production but an increase in their IL-10 and IgG production. The prolonged survival of infected mice treated with manzamine A and the eventual clearance of recrudescing parasites in some of these mice involve a down-regulation of Thl responses and a switch to antibody dependent-Th2 responses.

New manzamine alkaloids with potent activity against infectious diseases.

The isolation of the new enantiomers of 8-hydroxymanzamine A (1), manzamine F (2), along with the unprecedented manzamine dimer, neo-kauluamine from an undescribed genus of Indo-Pacific sponge (family Petrosiidae, order Haplosclerida) is reported. The relative stereochemistry of neo-kauluamine was established through detailed analysis of NOE-correlations combined with molecular modeling. The significance of the manzamines as in vivo antimalarial agents with superior activity to the clinically used drugs artemisinin and chloroquine is discussed along with the activity in vitro against the AIDS-opportunistic infectious diseases tuberculosis and toxoplasmosis. Reexamination of the sponges identified as Prianos, and Pachypellina, in earlier publications has confirmed that these are members of the same genus as the sponge described here, but differ at the species level.

In vivo antimalarial activity of the beta-carboline alkaloid manzamine A.

Manzamine A, a beta-carboline alkaloid present in several marine sponge species, inhibits the growth of the rodent malaria parasite Plasmodium berghei in vivo. More than 90% of the asexual erythrocytic stages of P. berghei were inhibited after a single intraperitoneal injection of manzamine A into infected mice. A remarkable aspect of manzamine A treatment is its ability to prolong the survival of highly parasitemic mice, with 40% recovery 60 days after a single injection. Oral administration of an oil suspension of manzamine A also produced significant reductions in parasitemia. The plasma manzamine A concentration peaked 4 h after injection and remained high even at 48 h. Morphological changes of P. berghei were observed 1 h after treatment of infected mice. (-)-8-Hydroxymanzamine A also displayed antimalarial activity, whereas manzamine F, a ketone analog of manzamine A, did not. Our results suggest that manzamine A and (-)-8-hydroxymanzamine A are promising new antimalarial agents.

Detection and species determination of malaria parasites by PCR: comparison with microscopy and with ParaSight-F and ICT malaria Pf tests in a clinical environment.

A rapid procedure for the diagnosis of malaria infections directly from dried blood spots by PCR amplification was evaluated with samples from 52 patients. Plasmodium infections were identified with a genus-specific primer set, and species differentiation between Plasmodium falciparum and Plasmodium vivax was analyzed by multiplex PCR. The PCR test with any of the three primer sets was able to detect as few as four parasites per microliter by gel electrophoresis or by nonisotopic paper hybridization chromatography. The diagnoses obtained by PCR correlated closely with those obtained by Giemsa staining except for two samples observed to have mixed P. falciparum-P. vivax infections. These were initially missed by microscopic analysis. In comparison with antigen-capture assays for P. falciparum, the PCR assays were able to detect three infections that were missed by the ParaSight-F test. The PCR test was negative for nine ParaSight-F-positive samples and one ICT Malaria Pf-positive sample, and these were confirmed to be false-positive results. The PCR thus gave no false-negative or false-positive results. Patients undergoing antimalarial therapy were also monitored by the PCR assay. Four of seven patients who were PCR positive for P. vivax at the time of discharge were later readmitted to the hospital with a recurrence of P. vivax infection. We would like to propose that PCR is a sensitive and easy method that can serve as a useful addition to microscopy for the diagnosis and the clinical monitoring of treatment of malaria.

Partial nucleotide sequence and organisation of extrachromosomal plastid-like DNA in Plasmodium berghei.

The murine malaria parasite Plasmodium berghei contains a plastid-like extrachromosomal genome. This genome is 30.7 kb in size and is transcriptionally active as shown by RT-PCR. DNA sequence analysis of the genome reveals 69.9-95.5% homology to sequences of the 35-kb extrachromosomal circle found in the human malaria species Plasmodium falciparum. Homologous sequences include regions of genes for the ssu-rRNA, lsu-rRNA, rpo B and clusters of t-RNAs. Sequence variation between the two Plasmodium species exists in the non-coding interspacing regions. A physical map has been constructed for the P. berghei circle, indicating the EcoRI and HindIII restriction sites as well as the arrangement of the rRNA, rpo B and tRNA genes. Arrangement of these genes is similar to that found on the P. falciparum 35-kb circle. The P. berghei circular element is distinct from the mitochondrial 6-kb DNA of both the murine and the human Plasmodium species. Preliminary results indicate that the circle may be a useful target for drug therapy.

Direct PCR amplification and sequence analysis of extrachromosomal Plasmodium DNA from dried blood spots.

The Plasmodium parasite possesses two extrachromosomal genomes; the mitochondrial genetic element and the extrachromosomal plastid-like DNA. The latter has only been fully described for one culture strain of P. falciparum. In this study, a rapid procedure for amplifying plastid DNA from dried blood spots of blood infected with different malaria species was developed. PCR amplification of a 595 bp fragment within the plastid-like large subunit ribosomal-RNA (LSU-rRNA) gene was achieved using primers derived from the P. falciparum sequence. The PCR product was observed in all Plasmodium species examined. Sequence analysis of amplified products homologous to an LSU-rRNA fragment of the plastid-like extrachromosomal circle revealed extensive conservation between Plasmodium species including P. falciparum, P. vivax, P. malariae and P. berghei.

Identification of a Plasmodium berghei antigen sharing common features with P. falciparum and P. chabaudi parasitophorous-vacuole membrane antigens.

On the basis of immunological cross-reactivity, we identified a 43-kDa Plasmodium berghei antigen with homology to the exp-1 antigen from P. falciparium. The P. berghei antigen was recognized by an antibody directed against an epitope on the C-terminus of the P. falciparum exp-1 protein. This antigen is localized on the surface of the parasite and shares peptide sequence homology with the P. chabudi antigen Ag3008. To investigate further the role of the P. berghei antigen, we designed antisense phosphorothioate oligodeoxynucleotides (PS oligos) complementary to sequences of the exp-1 mRNA from P. falciparum. The PS oligos were capable of inhibiting the development of P. falciparum in vitro by 47%. In vivo, experiments in mice showed that the same PS oligos had the potential to extend the life span of mice infected with P. berghei by a factor of 2-4. The immunological cross-reactivity and the antisense inhibition of P. berghei parasite development in vivo indicate that this antigen may be a homologue of exp-1 from P. falciparum that has functional importance for parasite survival.

Sorting of malarial antigens into vesicular compartments within the host cell cytoplasm as demonstrated by immunoelectron microscopy.

A double and triple immunogold labeling technique has been applied to demonstrate that several malarial antigens of the erythrocytic stages of Plasmodium falciparum are exported from the parasite into distinct compartments within the host cell cytoplasm. Multiple species of vesicles, each with specifically packaged contents, are consistent with a sorting function of vesicular structures in the Plasmodium infected erythrocyte. During schizogony, two parasite antigens, an S-antigen and a parasitophorous vacuole membrane antigen, QF 116, become packaged into such vesicles and are transported into the erythrocyte cytoplasm. At this stage of parasite development, host cell material is taken in through the parasitophorous vacuole membrane into the vacuolar space surrounding the parasite.

Inhibitory monoclonal antibody against a (myristylated) small-molecular-weight antigen from Plasmodium falciparum associated with the parasitophorous vacuole membrane.

A small-molecular-weight antigen that occurs in asexual blood stages in synchronized cultures of Plasmodium falciparum was detected by a monoclonal antibody which inhibits parasite growth in vitro. This antigen, QF116, showed a molecular weight of 15,000 in parasite strain FCR-3K+ from The Gambia and 19,000 in strain FCQ-27 from Papua New Guinea. The protein did not show significant glycosylation by galactose or glucosamine labeling but was found to be acylated by myristic acid. By using immunogold labeling and electron microscopy, the location of the antigen could be attributed to the parasitophorous vacuole membrane and to inclusions and vesicles residing within the cytoplasm of the erythrocyte host cell.

The parasitophorous vacuole membrane of Plasmodium falciparum: demonstration of vesicle formation using an immunoprobe.

We have applied several immunolabeling techniques using a monoclonal antibody to a Plasmodium falciparum antigen to differentiate morphologically dissimilar membranous structures present in infected erythrocytes. Evidence is presented that cytoplasmic clefts, multimembranous structures and vesicles within the infected cell originate from the parasitophorous vacuole membrane by a process described as budding off. The parasitophorous vacuole membrane and related structures in infected, parasitized erythrocytes reacted with the cyanine dye Merocyanine 540, demonstrating that they are accessible to molecules from the extracellular environment. Immunogold labeling of freeze-fractured preparations and of thin sections of parasitized cells using pre- and post-embedding techniques revealed that each of the membranous structures carried a common parasite antigen, QF 116, which was identified by monoclonal antibody 8E7/55.

Localisation of internal antigens of Plasmodium falciparum using monoclonal antibodies and colloidal gold.

By the examination of several defined malarial antigens, we have demonstrated the necessity for etching pretreatments to be used in conjunction with post-embedding immunolabelling of LR White-embedded parasite material. In general, etching procedures markedly enhanced immunolabelling of the various antigens, while in some cases etching was essential for obtaining positive immunolabelling. Of the etching pretreatments evaluated, a combination of an alcoholic solution of sodium hydroxide followed by sodium metaperiodate gave optimal labelling with minimal background. A number of fixation regimes were also compared for their applicability to immunolabelling of malaria-infected erythrocytes. Generally, fixation with low concentrations of glutaraldehyde was found to be appropriate. We have also successfully used paraformaldehyde fixation coupled with etching to localise a rhoptry-associated antigen, which is presumably sensitive to glutaraldehyde fixation. Due to the high specificity of monoclonal antibodies, however, different fixation regimes may need to be considered for various combinations of antigen and antibody.

Glycosyltransferase activities in Golgi complex and endoplasmic reticulum fractions isolated from African trypanosomes.

Highly enriched Golgi complex and endoplasmic reticulum fractions were isolated from total microsomes obtained from Trypanosoma brucei, Trypanosoma congolense, and Trypanosoma vivax, and tested for glycosyltransferase activity. Purity of the fractions was assessed by electron microscopy as well as by biochemical analysis. The relative distribution of all the glycosyltransferases was remarkably similar for the three species of African trypanosomes studied. The Golgi complex fraction contained most of the galactosyltransferase activity followed by the smooth and rough endoplasmic reticulum fractions. The dolichol-dependent mannosyltransferase activities were highest for the rough endoplasmic reticulum, lower for the smooth endoplasmic reticulum, and lowest for the Golgi complex. Although the dolichol-independent form of N-acetylglucosaminyltransferase was essentially similar in all the fractions, the dolichol-dependent form of this enzyme was much higher in the endoplasmic reticulum fractions than in the Golgi complex fraction. Inhibition of this latter activity in the smooth endoplasmic reticulum fraction by tunicamycin A1 suggests that core glycosylation of the variable surface glycoprotein may occur in this organelle and not in the rough endoplasmic reticulum as previously assumed.

Membranes of protein bodies. I. Isolation from cotyledons of germinating cucumber seeds.

Protein bodies were prepared from cotyledons of germinating seeds of cucumber (Cucumis sativus) in different ways: the organelles either obtained from protoplasts by lysis or from cotyledons by mechanical disintegration were separated on sucrose-density gradients. In addition, a non-aqueous procedure was employed to isolate protein bodies. Marker proteins indicative of membranes of other organelles were carefully assayed. By this means contaminations in the purified protein-body fractions could be ruled out. Isolated protein bodies were separated into crystalloids, matrix, and membranes. The membranes were purified and characterized according to their equilibrium density (rho = 1.20 kg/l) on sucrose gradients by flotation or sedimentation. Protein-body membranes labelled in the phospholipid moiety were prepared and analyzed after application of [methyl-14C] choline or [32P] phosphate in vivo.

Membranes of protein bodies. II. Detection and partial characterization of membrane glycoproteins.

The synthesis of organelle proteins was studied in cotyledons of Cucumis sativus. Protein constituents of protein-body membranes were shown to be synthesized and assembled, at a stage characterized by mobilization of the storage globulin. Besides L-[35S]methionine various labelled hexoses were incorporated into protein bodies of cucumber cotyledons. While D-[U-14C] glucose functioned as precursor of a broad spectrum of glycoproteins, D-[6-3H]glucosamine was selectively incorporated into four glycoproteins of the protein-body membrane. Labelled galactose and mannose, respectively, were preferentially transferred into another set of membrane glycoproteins. The four glycoproteins revealed by labelling with glucosamine were solubilized and purified by chromatography on concanavalin-A-Sepharose.