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We describe the development and evaluation of a novel pair of real-time, fluorescence-based PCR assays using molecular beacon probes for rapid, sensitive, and specific detection and quantification of Plasmodium falciparum and other Plasmodium species organisms.
Invasion of erythrocytes by Plasmodium merozoites is an intricate process involving multiple receptor-ligand interactions. The glycophorins and an unknown trypsin sensitive factor are all erythrocyte receptors used during invasion by the major human pathogen Plasmodium falciparum. However, only one erythrocyte receptor, Glycophorin A, has a well-established cognate parasite ligand, the merozoite protein erythrocyte binding antigen-175 (EBA-175). The involvement of several other parasite proteins during invasion have been proposed, but no direct evidence links them with a specific invasion pathway. Here we report the identification and characterization of P. falciparum normocyte binding protein 1 (PfNBP1), an ortholog of Plasmodium vivax reticulocyte binding protein-1. PfNBP1 binds to a sialic acid dependent trypsin-resistant receptor on the erythrocyte surface that appears to be distinct from known invasion receptors. Antibodies against PfNBP1 can inhibit invasion of trypsinized erythrocytes and two P. falciparum strains that express truncated PfNBP1 are unable to invade trypsinized erythrocytes. One of these strain, 7G8, also does not invade Glycophorin B-negative erythrocytes. PfNBP1 therefore defines a novel trypsin-resistant invasion pathway and adds a level of complexity to current models for P. falciparum erythrocyte invasion.
BACKGROUND: Malaria is one of the most important parasitic infections in humans. A sensitive diagnostic test for malaria that could be applied at the community level could be useful in programs to control the disease. The aim of the present work was to develop a simple, inexpensive molecular test for Plasmodium falciparum. METHODS: Blood was collected from controls (n = 100) and from patients diagnosed with falciparum malaria infection (n = 102), who were recruited to the study. Heat-treated blood samples were tested by a loop-mediated isothermal amplification (LAMP) assay for P. falciparum. Results were interpreted by a turbidity meter in real time or visually at the end of the assay. To evaluate the assay, DNA from these samples was purified and tested by PCR. Results from the LAMP and PCR assays were compared. RESULTS: The LAMP assay detected P. falciparum directly from heat-treated blood. The quantitative data from the assay correlated to the parasite counts obtained by blood-film microscopic analyses. When we used the PCR assay as the comparison method, the sensitivity and specificity of the LAMP assay were 95% and 99%, respectively. CONCLUSIONS: Unlike PCR, the LAMP assay does not require purified DNA for efficient DNA amplification, thereby reducing the cost and turnaround time for P. falciparum diagnosis. The assay requires only basic instruments, and assay positivity can be verified by visual inspection.
Plasmodium falciparum malaria remains one of the most frequently lethal diseases affecting children in sub-Saharan Africa, yet the immune mediators that regulate pathogenesis are only partially defined. Since macrophage migration inhibitory factor (MIF) is important for regulating innate immunity in bacterial and parasitic infections, circulating MIF and peripheral blood mononuclear cell (PBMC) MIF transcripts were investigated in children with acute falciparum malaria. Peripheral blood levels of MIF-regulatory cytokines and effector molecules, including interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)-12, IL-10, transforming growth factor (TGF)-beta1, bicyclo-prostaglandin (PG) E2, and nitric oxide synthase activity were also determined. Circulating MIF and PBMC MIF mRNA were significantly lower in children with acute malaria relative to healthy, malaria-exposed children. Peripheral blood MIF levels showed no association with either parasitemia or hemoglobin concentrations. Circulating MIF was, however, significantly associated with IL-12 and TGF-beta1. Multiple regression analyses revealed that IFN-gamma was the most significant predictor of peripheral blood MIF concentrations. These findings suggest that reduced MIF production may promote enhanced disease severity in children with falciparum malaria.
Plasmodium falciparum malaria is responsible for 2 million deaths each year. Even in endemic regions, immunity to malaria builds slowly and is rarely complete. Strategies such as antigenic variation and antigenic diversity are critical to a parasite's ability to evade the host immune response and infect previously exposed individuals. In this short review, the phenomenon of antigenic variation is discussed in relation to immune evasion and its impact on parasite pathogenesis. Recent advances in the understanding of the underlying molecular mechanisms of antigenic variation are examined and questions posed for future research.
The Plasmodium falciparum genome sequence has boosted hopes for a new era of malaria research and for the application of comprehensive molecular knowledge to disease control, but formidable obstacles remain: approximately 60% of the predicted P. falciparum proteins have no known functions or homologues, and most life cycle stages of this haploid eukaryotic parasite are relatively intractable to cultivation and biochemical manipulation. Genetic mapping based on high-resolution maps saturated with single-nucleotide polymorphisms or microsatellites is now providing effective strategies for discovering candidate genes determining important parasite phenotypes. Here we review classical linkage studies using laboratory crosses and population associations that are now amenable to genome-wide approaches and are revealing multiple candidate genes involved in complex drug responses. Moreover, mapping by linkage disequilibrium is practicable in cases where chromosomal segments flanking drug-selected genes have been preserved in populations during relatively recent P. falciparum evolution. We discuss the advantages and limitations of these various genetic mapping strategies, results from which offer complementary insights to those emerging from gene knockout experiments and/or high-throughput genomic technologies.
GTP cyclohydrolase (EC 3.5.4.16), the first enzyme in the pteridine pathway leading to the de novo formation of folic acid, has been identified and isolated from the human malaria parasite, Plasmodium falciparum. The enzyme was purified 200-fold by high performance size-exclusion chromatography on a TSK-G-3000 SW protein column. The molecular weight was estimated at 300 000. Optimal enzyme activity was observed at pH 8.0 and 42 degrees C. The Km for GTP was 54.6 microM. Products of the enzyme reaction were identified as the carbon-8 of GTP and D-erythro-dihydroneopterin triphosphate. ATP was a competitive inhibitor (Ki = 600 microM) of the enzyme. Activity of the enzyme was Mg2+-independent, whereas Mn2+, Cu2+ and Hg2+ (5 mM) were inhibitory. GTP cyclohydrolase activity was also identified in a murine parasite, Plasmodium berghei, and a simian parasite, Plasmodium knowlesi. Activity of the enzyme in P. knowlesi, an intrinsically synchronous quotidian parasite, was found to be dependent on the stage of parasite development.
Malaria is caused by Plasmodium spp. and is one of the major infectious diseases leading to morbidity and mortality in tropical areas of the world. The model of protective immunity induced by immunization with radiation-attenuated Plasmodia sporozoites (SPZ) has become the framework for the elucidation of protective immune mechanisms and the prototype for a promising vaccine strategy. We have previously reported that although considered stage specific based on antibody and CD8+ cytolytic T lymphocyte responses directed against preerythrocytic stage antigens, in particular, the circumsporozoite protein and sporozoite surface protein 2, protective immunity induced in humans by attenuated Plasmodium falciparum SPZ may also involve CD4+ T cell responding to antigens present on parasitized red blood cells (pRBC). In this study we examined the functional role of pRBC responding CD4+ T cells by comparing in vitro pRBC-stimulated responses of CD4+ T cells from persons during preimmunity to irradiated SPZ, during induction of protection, and infection induced with SPZ. The results reported herein corroborate previously published observations that antigens associated with pRBC induce proliferative CD4+ lymphocytes responses in subjects exposed to malaria parasite-derived antigens and not malaria-naive persons; however, now we demonstrate that pRBC-proliferative CD4+ T cells did not coincide with protective immunity. Similarly, pRBC-induced IFN-gamma levels did not distinguish malaria protected from susceptible persons, although IFN-gamma was observed only in lymphocyte cultures from malaria parasite-exposed volunteers and not in lymphocyte cultures from malaria-naive persons. In contrast, we noted an increase in the IL-4-producing CD4+ T cells that also exhibited the memory phenotype, CD45RO, and an upregulated expression of CD25 in cultures from malaria protected persons as compared to malaria naive persons and subjects who became parasitemic. Hence, these observations suggest that the induction of memory CD4+ T cell subset distinguished by the expression of CD45RO and CD25 and production of IL-4 coincides with protective immune responses generated by immunization with attenuated SPZ.
The Plasmodium falciparum serine repeat antigen (SERA) and serine repeat protein homologue (SERPH) contain highly conserved domains that appear to encode cysteine proteases or related proteins. Humoral immune responses against the protease domains of SERA and SERPH were evaluated. Malaria-immune Africans, but not nonimmune controls, demonstrated potent humoral responses against the protease domains. As the SERA and SERPH protease domains are likely accessible to circulating antibody, these results suggest that humoral responses to the domains may contribute to antimalarial immunity.
BACKGROUND: Plasmodium falciparum merozoite surface protein 3 is a malaria vaccine candidate that was identified, characterised, and developed based on a unique immuno-clinical approach. The vaccine construct was derived from regions fully conserved among various strains and containing B cell epitopes targeted by human antibodies (from malaria-immune adults) that are able to mediate a monocyte-dependent parasite killing effect. The corresponding long synthetic peptide was administered to 36 volunteers, with either alum or Montanide ISA720 as adjuvant. METHODS AND FINDINGS: Both formulations induced cellular and humoral immune responses. With alum, the responses lasted up to 12 mo. The vaccine-induced antibodies were predominantly of cytophilic classes, i.e., able to cooperate with effector cells. In vitro, the antibodies induced an inhibition of the P. falciparum erythrocytic growth in a monocyte-dependent manner, which was in most instances as high as or greater than that induced by natural antibodies from immune African adults. In vivo transfer of the volunteers' sera into P. falciparum-infected humanized SCID mice profoundly reduced or abrogated parasitaemia. These inhibitory effects were related to the antibody reactivity with the parasite native protein, which was seen in 60% of the volunteers, and remained in samples taken 12 mo postimmunisation. CONCLUSION: This is the first malaria vaccine clinical trial to clearly demonstrate antiparasitic activity by vaccine-induced antibodies by both in vitro and in vivo methods. The results, showing the induction of long-lasting antibodies directed to a fully conserved polypeptide, also challenge current concepts about malaria vaccines, such as unavoidable polymorphism, low antigenicity, and poor induction of immune memory.
Placental Plasmodium falciparum sequestration is associated with dysregulated immune function. Placental inflammatory responses via IFN-gamma and TNF-alpha are implicated in functional damage. However, they are needed during placental infection to control asexual stage parasites. To test the hypothesis that placental immunomodulation associated with malaria disturbs cytokine secretion differently in monocytes and lymphocytes, we have determined the proportion of monocytes and/or lymphocytes secreting IFN-gamma, TNF-alpha, IL-10 and IL-12. Intervillous and peripheral blood monocyte (CD14+) and lymphocyte (CD3/CD4+; CD3/CD8+) cytokine production was compared between 17 P. falciparum-infected and 12 non-infected Senegalese women. After culture with phorbolmyristate acetate/ionomycin (PMA/iono), lipopolysaccharide (LPS) or P. falciparum-infected erythrocytes (IE), the intracellular expression of cytokines in lymphocytes (IFN-gamma, TNF-alpha) and monocytes (IL-10, IL-12, TNF-alpha), was detected. In response to IE, CD4+ and CD8+ T-cells produced IFN-gamma and TNF-alpha at similar rates in both compartments. In response to PMA/iono, the frequencies of CD4+ and CD8+ T-cells producing IFN-gamma and TNF-alpha were similar in both compartments, but increased in P. falciparum-infected placentas. In response to LPS or IE, IL-12 secreting monocytes were increased in infected women, while the frequency of TNF-alpha secreting monocytes was decreased compared to that in non-infected placenta. The monocyte IL-12 response is not impaired in infected women. IL-12 is an important factor for inducing IFN-gamma in T-cells. Thus, IL-12 and IFN-alpha responses may synergistically allow a protective immune response in placental malaria. TNF-alpha production by CD4+ and CD8+ T-cells is up-regulated in P. falciparum-infected placentas, suggesting that T-cells actively participate to inflammatory responses.
Severe Plasmodium falciparum malaria is characterized by excessive sequestration of infected and uninfected erythrocytes in the microvasculature of the affected organ. Rosetting, the adhesion of P. falciparum-infected erythrocytes to uninfected erythrocytes is a virulent parasite phenotype associated with the occurrence of severe malaria. Here we report on the identification by single-cell reverse transcriptase PCR and cDNA cloning of the adhesive ligand P. falciparum erythrocyte membrane protein 1 (PfEMP1). Rosetting PfEMP1 contains clusters of glycosaminoglycan-binding motifs. A recombinant fusion protein (Duffy binding-like 1-glutathione S transferase; Duffy binding-like-1-GST) was found to adhere directly to normal erythrocytes, disrupt naturally formed rosettes, block rosette reformation, and bind to a heparin-Sepharose matrix. The adhesive interactions could be inhibited with heparan sulfate or enzymes that remove heparan sulfate from the cell surface whereas other enzymes or similar glycosaminoglycans of a like negative charge did not affect the binding. PfEMP1 is suggested to be the rosetting ligand and heparan sulfate, or a heparan sulfate-like molecule, the receptor both for PfEMP1 binding and naturally formed erythrocyte rosettes.
The Plasmodium falciparum major merozoite surface protein gp195 is a candidate antigen for a vaccine against human malaria. The significance of allelism and polymorphism in vaccine-induced immunity to gp195 was investigated in this study. Rabbits were immunized with each of two allelic forms of gp195 that were affinity purified from the FUP and FVO parasite isolates. gp195-specific antibodies raised against one allelic form of gp195 cross-reacted extensively with the gp195 of the heterologous allele in enzyme-linked immunosorbent assays (ELISAs) and immunofluorescence assays. Competitive binding ELISAs with homologous and heterologous gp195s confirmed that a majority of the anti-gp195 antibodies produced against either allelic protein were cross-reactive. Moreover, the biological activities of the gp195 antibody responses were also highly cross-reactive, since anti-gp195 sera inhibited the in vitro growth of the homologous and heterologous parasites with equal efficiency. The degree of cross-reactivity with strain-specific and allele-specific determinants of gp195 in the ELISA was low. These results suggest that the immunological cross-reactivity between the two gp195 proteins is due to recognition of conserved determinants. They also suggest that a gp195-based vaccine may be effective against blood-stage infection with a diverse array of parasite isolates.
Plasmodium falciparum FCC1/HN-infected human erythrocytes were embedded with LR White resin at low temperature. The 185 kDa and 82/41 kDa proteins in erythrocytic stages of P. falciparum were then immunolabeled by using the protective monoclonal antibodies (McAb)F6-D3 and F6-C2 with protein A-colloidal gold probe. The electron-microscopical observation showed that the 185kDa protein recognized by McAb F6-D3 was located on the surface of free and intracellular merozoites as well as the cytoplasm, plasma membrane, and parasitophorous vacuole membrane of immature schizonts. The 82/41 kDa proteins identified by McAb F6-C2 was located within the rhoptries of immature schizonts and mature merozoites. These results demonstrated ultrastructurally that the 185 kDa and 82/41 kDa protective antigens were merozoite surface antigen and merozoite rhoptry antigens of P. falciparum FCC1/HN, respectively.
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A Plasmodium falciparum schizont lysate has been previously described as being a powerful inducer of proliferation for human peripheral T lymphocytes. In this report we study the phenotype of cycling T cells from unexposed donors and examine how the P. falciparum lysate compares with the conventional T cell mitogen phytohemagglutinin (PHA), a known superantigen staphylococcal enterotoxin B (SEB), and a classical antigen pure protein derivative (PPD). We show that for this lymphoproliferative activity interaction with the MHC class II molecule is required and that in the presence of P. falciparum the great majority of the cycling cells at day 6 are gamma delta T cells, all of them bearing V gamma 9 V delta 2. Our results suggest that P. falciparum induces a T cell proliferative response that resembles a response of human peripheral blood gamma delta T cells to superantigen. This observation is in agreement with the elevated level of peripheral gamma delta lymphocytes observed during and after malaria acute infection.
Plasmodium falciparum strains from different malaria-endemic regions were grown in continuous culture without human serum. The medium was complemented with Nutridoma-SR instead of serum. All parasite strains developed well in serum-free medium. Two strains were thawed without serum and showed good multiplication in subsequent continuous culture.
Plasmodium falciparum var genes encode a diverse family of proteins, located on the surfaces of infected erythrocytes, which are implicated in the pathology of human malaria through antigenic variation and adhesion of infected erythrocytes to the microvasculature. We have constructed a complete representative telomere-to-telomere yeast artificial chromosome (YAC) contig map of the P. falciparum chromosome 8 for studies on the chromosomal organization, distribution, and expression of var genes. Three var gene loci were identified on chromosome 8, two of which map close to the telomeres at either end of the chromosome. Analysis of the previously described chromosome 2 contig map and random P. falciparum telomeric YAC clones revealed that most, if not all, 14 P. falciparum chromosomes contain var genes in a subtelomeric location. Mapping the chromosomal location of var genes expressed in a long-term culture of the P. falciparum isolate Dd2 revealed that four of the five different expressed var genes identified map within subtelomeric locations. Expression of var genes from a chromosomal domain known for frequent rearrangements has important implications for the mechanism of var gene switching and the generation of novel antigenic and adhesive phenotypes.