DIGE enables the detection of a putative serum biomarker of fungal origin in a mouse model of invasive aspergillosis.

Invasive aspergillosis (IA) is a major threat for immunocompromised patients. Diagnostic difficulties often delay specific treatment initiation, which increases mortality. Finding new biomarkers to improve and speed accurate diagnosis is thus vital. To investigate the ability of proteomic methods for discovering new biomarkers of IA, we used a DIGE approach to perform a proteomic analysis on both bronchoalveolar lavages (BAL) and sera at different time-points of infection in a mouse model of invasive pulmonary aspergillosis. Progression of the infection was monitored using a bioluminescent strain of Aspergillus fumigatus. Sera proteins were enriched using the ProteoMiner kit (Biorad). This method allowed us to identify a fungal protein, the A. fumigatus major allergen Asp f 2, in sera of mice one day after the infection. However, this fungal protein was not detected three days after the infection. Importantly, in BAL, this work provides evidence of an in vivo complement evasion mechanism through the cleavage of C3b into three fragments during aspergillosis. Finally, our results underlining the inflammatory host response to IA in both lung and blood compartments at different times of infection may provide new insights into the pathophysiology of this disease.

Genetic polymorphisms influence Plasmodium ovale PCR detection accuracy.

Detection of Plasmodium ovale by use of a nested PCR assay with a novel Plasmodium ovale primer set was superior to detection of Plasmodium ovale by real-time PCR assays. Nested PCR was also better at detecting P. malariae. The detection of P. ovale in many patients first admitted >2 months following their return to Italy indicated that P. ovale relapses are common.

Eimeria (Coccidia: Eimeridea) of hares in France: description of new taxa.

The oocysts of coccidian of the genus Eimeria were sought in the caecal contents of 46 Lepus granatensis and 18 L. europaeus captured in France. Parasites were found in 34 of the hares. Parasite load was mainly very low. However, species diversity was considerable. 21 species or subspecies were identified, of which 13 species and two subspecies were not previously described. Three of the taxa, E. robertsoni, E. semisculpta and E. townsendi, previously identified on numerous occasions in western Europe and, corresponding to forms or variants created before 1960 that have been subsequently elevated to a specific level, appear to be invalid. Indeed, the parasite descriptions from the material used to effect this modification do not correspond to the original descriptions. A stable equilibrium, as generally observed in the case of many congeneric species co-infection of the same host, was not observed in the hares. This has been attributed to the solitary habits of the host and of the probable polyphyletic nature of the genus Eimeria. Paleontological data for the Leporidae indicate that rabbit parasites are derived from those of the hare.

Development of a real-time PCR assay for detection of Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale for routine clinical diagnosis.

A TaqMan-based real-time PCR qualitative assay for the detection of three species of malaria parasites-Plasmodium falciparum, P. ovale, and P. vivax-was devised and evaluated using 122 whole-blood samples from patients who had traveled to areas where malaria is endemic and who presented with malaria-like symptoms and fever. The assay was compared to conventional microscopy and to an established nested-PCR assay. The specificity of the new assay was confirmed by sequencing the PCR products from all the positive samples and by the lack of cross-reactivity with Toxoplasma gondii and Leishmania infantum DNA. Real-time PCR assay showed a detection limit (analytical sensitivity) of 0.7, 4, and 1.5 parasites/ micro l for P. falciparum, P. vivax, and P. ovale, respectively. Real-time PCR, like nested PCR, brought to light errors in the species identification by microscopic examination and revealed the presence of mixed infections (P. falciparum plus P. ovale). Real-time PCR can yield results within 2 h, does not require post-PCR processing, reduces sample handling, and minimizes the risks of contamination. The assay can therefore be easily implemented in routine diagnostic malaria tests. Future studies are warranted to investigate the clinical value of this technique.

Taxonomic status and re-description of Plasmodium relictum (Grassi et Feletti, 1891), Plasmodium maior Raffaele, 1931, and description of P. bigueti n. sp. in sparrows.

The first accurate re-description of Plasmodium relictum (Grassi et Feletti, 1891) in its type host was provided by Raffaele in 1931, and the name relictum should thus refer to this work. In his article, Raffaele noted the presence of an associated but distinct species, P. maior. The work of Raffaele has since remained overlooked, and the taxon relictum has been applied rather loosely to parasites found in numerous birds of diverse geographic origin. Examination of Passer domesticus specimens collected in France has confirmed the presence of the two species above, and further revealed that two other species can also be found in these birds. P. bigueti n. sp. is described here, whereas the other Plasmodium sp. was not found in sufficient numbers to allow characterisation.

Restricted genetic and antigenic diversity of Plasmodium falciparum under mesoendemic transmission in the Venezuelan Amazon.

The study of genetic diversity in malaria populations is expected to provide new insights for the deployment of control measures. Plasmodium falciparum diversity in Africa and Asia is thought to reflect endemicity. In comprehensive epidemiological surveys reported here the genetic and antigenic structure of P. falciparum in the Venezuelan Amazon were studied over a 2-year period. DNA polymorphisms in glutamate-rich protein (GLURP), merozoite-surface protein 1 (MSP1) and MSP2 genes, in a multicopy element (PfRRM), all showed low diversity, 1 predominant genotype, and virtually no multi-clonal infections. Moreover, linkage disequilibrium was seen between GLURP, MSP1 and MSP2. Specific antibody responses against MSP1 and MSP2 recombinant antigens reflected the low genetic diversity observed in the parasite population. This is unexpected in a mesoendemic area, and suggests that the low diversity here may not only relate to endemicity but to other influences such as a bottleneck effect. Linkage disequilibrium and a predominant genotype may imply that P. falciparum frequently propagates with an epidemic or clonal population structure in the Venezuelan Amazon.

Stage-specific transcription of distinct repertoires of a multigene family during Plasmodium life cycle.

Members of a multigene family in the rodent malaria parasite Plasmodium yoelii yoelii code for 235-kilodalton proteins (Py235) that are located in the merozoite apical complex, are implicated in virulence, and may determine red blood cell specificity. We show that distinct subsets of py235 genes are expressed in sporozoites and hepatic and erythrocytic stages. Antibodies to Py235 inhibited sporozoite invasion of hepatocytes. The switch in expression profile occurred immediately after transition from one stage to another. The results suggest that this differential expression is driven by strong biological requirements and provide evidence that hepatic and erythrocytic merozoites differ.

Association of genetic mutations in Plasmodium vivax dhfr with resistance to sulfadoxine-pyrimethamine: geographical and clinical correlates.

Mutations in the Plasmodium falciparum gene (dhfr) encoding dihydrofolate reductase are associated with resistance to antifols. Plasmodium vivax, the more prevalent malaria parasite in Asia and the Americas, is considered antifol resistant. Functional polymorphisms in the dhfr gene of P. vivax (pvdhfr) were assessed by PCR-restriction fragment length polymorphism using blood samples taken from 125 patients with acute vivax malaria from three widely separated locations, Thailand (n = 100), India (n = 16), and Madagascar and the Comoros Islands (n = 9). Upon evaluation of the three important codons (encoding residues 57, 58, and 117) of P. vivax dhfr (pvdhfr), double- or triple-mutation genotypes were found in all but one case from Thailand (99%), in only three cases from India (19%) and in no cases from Madagascar or the Comoros Islands (P < 0.0001). The dhfr PCR products of P. vivax from 32 Thai patients treated with the antifolate sulfadoxine-pyrimethamine (S-P) were investigated. All samples showed either double (53%) or triple (47%) mutations. Following treatment, 34% of the patients had early treatment failures and only 10 (31%) of the patients cleared their parasitemias for 28 days. There were no significant differences in cure rates, but parasite reduction ratios at 48 h were significantly lower for patients whose samples showed triple mutations than for those whose samples showed double mutations (P = 0.01). The three mutations at the pvdhfr codons for residues 57, 58, and 117 are associated with high levels of S-P resistance in P. vivax. These mutations presumably arose from selection pressure.

Meiotic recombination, cross-reactivity, and persistence in Plasmodium falciparum.

We incorporate a representation of Plasmodium falciparum recombination within a discrete-event model of malaria transmission. We simulate the introduction of a new parasite genotype into a human population in which another genotype has reached equilibrium prevalence and compare the emergence and persistence of the novel recombinant forms under differing cross-reactivity relationships between the genotypes. Cross-reactivity between the parental (initial and introduced) genotypes reduces the frequency of appearance of recombinants within three years of introduction from 100% to 14%, and delays their appearance by more than a year, on average. Cross-reactivity between parental and recombinant genotypes reduces the frequency of appearance to 36% and increases the probability of recombinant extinction following appearance from 0% to 83%. When a recombinant is cross-reactive with its parental types, its probability of extinction is influenced by cross-reactivity between the parental types in the opposite manner; that is, its probability of extinction after appearance decreases. Frequencies of P. falciparum outcrossing are mediated by frequencies of mixed-genotype infections in the host population, which are in turn mediated by the structure of cross-reactivity between parasite genotypes. The three leading hypotheses about how meiosis relates to oocyst production lead to quantitative, but no qualitative, differences in these results.

Expression of the erythrocyte-binding antigen 175 in sporozoites and in liver stages of Plasmodium falciparum.

Screening of a Plasmodium falciparum genomic expression library for antigens expressed at the pre-erythrocytic stages resulted in the isolation of a recombinant phage (DG249) whose insert corresponded to regions II and III of a 175-kDa erythrocyte-binding antigen (EBA-175). EBA-175 is a parasite ligand implicated in red blood cell invasion. Reverse-transcriptase polymerase chain reaction, indirect immunofluorescent antibody test, and Western blot analysis confirmed that EBA-175 is expressed not only in blood-stage parasites but also in infected hepatocytes and on the sporozoite surface. The presence of EBA-175 on pre-erythrocytic parasites enhances the vaccine potential of this antigen by adding another target to the immune responses elicited by immunization.

Human antibodies against Plasmodium falciparum liver-stage antigen 3 cross-react with Plasmodium yoelii preerythrocytic-stage epitopes and inhibit sporozoite invasion in vitro and in vivo.

The Plasmodium falciparum liver-stage antigen 3 (LSA3), a recently identified preerythrocytic antigen, induces protection against malaria in chimpanzees. Using antibodies from individuals with hyperimmunity to malaria affinity purified on recombinant or synthetic polypeptides of LSA3, we identified four non-cross-reactive B-cell epitopes in Plasmodium yoelii preerythrocytic stages. On sporozoites the P. yoelii protein detected has a molecular mass similar to that of LSA3. T-cell epitopes cross-reacting with P. yoelii were also demonstrated using peripheral blood lymphocytes from LSA3-immunized chimpanzees. In contrast, no cross-reactive epitopes were found in Plasmodium berghei. LSA3-specific human antibodies exerted up to 100% inhibition of in vitro invasion of P. yoelii sporozoites into mouse hepatocytes. This strong in vitro activity was reproduced in vivo by passive transfer of LSA3 antibodies. These results indicate that the homologous epitopes may be biologically functional and suggest that P. yoelii could be used as a model to assess the antisporozoite activity of anti-LSA3 antibodies.

Genotyping of Plasmodium falciparum infections by PCR: a comparative multicentre study.

Genetic diversity of malaria parasites represents a major issue in understanding several aspects of malaria infection and disease. Genotyping of Plasmodium falciparum infections with polymerase chain reaction (PCR)-based methods has therefore been introduced in epidemiological studies. Polymorphic regions of the msp1, msp2 and glurp genes are the most frequently used markers for genotyping, but methods may differ. A multicentre study was therefore conducted to evaluate the comparability of results from different laboratories when the same samples were analysed. Analyses of laboratory-cloned lines revealed high specificity but varying sensitivity. Detection of low-density clones was hampered in multiclonal infections. Analyses of isolates from Tanzania and Papua New Guinea revealed similar positivity rates with the same allelic types identified. The number of alleles detected per isolate, however, varied systematically between the laboratories especially at high parasite densities. When the analyses were repeated within the laboratories, high agreement was found in getting positive or negative results but with a random variation in the number of alleles detected. The msp2 locus appeared to be the most informative single marker for analyses of multiplicity of infection. Genotyping by PCR is a powerful tool for studies on genetic diversity of P. falciparum but this study has revealed limitations in comparing results on multiplicity of infection derived from different laboratories and emphasizes the need for highly standardized laboratory protocols.

The Plasmodium falciparum knob-associated PfEMP3 antigen is also expressed at pre-erythrocytic stages and induces antibodies which inhibit sporozoite invasion.

The expression of the pfemp3 gene and the corresponding PfEMP3 knob-associated protein in the pre-erythrocytic stages of Plasmodium falciparum was demonstrated by RT-PCR, Western blots, IFAT and IEM. The antigen was found on the surface of the sporozoite and in the cytoplasm of mature hepatic stage parasites. Immunological cross-reactivity was observed with sporozoites from the rodent malaria parasites Plasmodium yoelii yoelii and Plasmodium berghei and was exploited to assess a potential role of this protein at the pre-erythrocytic stages. Specific antibodies from immune individuals were found to inhibit P. yoelii yoelii and P. berghei sporozoite invasion of primary hepatocyte cultures. PfEMP3 should now be added to the small list of proteins expressed at the pre-erythrocytic stages of P. falciparum, and its vaccine potential now deserves to be investigated.

Malaria multigene families: the price of chronicity.

In this article, Georges Snounou, William Jarra and Peter Preiser discuss the survival strategy of malaria parasites in the light of a novel mechanism of clonal phenotypic variation recently described for a multigene family of Plasmodium yoelii yoelii. The 235 kDa rhoptry proteins (Py235) encoded by these genes may be involved in the selection of red blood cells for invasion by merozoites. The new mechanism may explain the ability of individual parasites to adapt to natural variations in red blood cell subsets, while ensuring that sufficient merozoites escape immune attack, thus maintaining a chronic infection for extended periods. This counterpoints the antigenic variation exemplified by PfEMP1 proteins (a large family of proteins derived from P. falciparum), which operates at the population level. The possibility of manipulating the expression of functionally similar genes in other Plasmodium species could lead to therapies aimed at reducing clinical severity without compromising the acquisition and maintenance of immunity.

Polymorphism at the merozoite surface protein-3alpha locus of Plasmodium vivax: global and local diversity.

Allelic diversity at the Plasmodium vivax merozoite surface protein-3alpha (PvMsp-3alpha) locus was investigated using a combined polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) protocol. Symptomatic patient isolates from global geographic origins showed a high level of polymorphism at the nucleotide level. These samples were used to validate the sensitivity, specificity, and reproducibility of the PCR/RFLP method. It was then used to investigate PvMsp3alpha diversity in field samples from children living in a single village in a malaria-endemic region of Papua New Guinea, with the aim of assessing the usefulness of this locus as an epidemiologic marker of P. vivax infections. Eleven PvMsp-3alpha alleles were distinguishable in 16 samples with single infections, revealing extensive parasite polymorphism within this restricted area. Multiple infections were easily detected and accounted for 5 (23%) of 22 positive samples. Pairs of samples from individual children provided preliminary evidence for high turnover of P. vivax populations.

Sampling and storage of blood and the detection of malaria parasites by polymerase chain reaction.

Polymerase chain reaction (PCR) is now widely used in malaria research for analysis of field samples. However, little has been reported regarding loss of sensitivity due to field methodology. Therefore, studies were carried out in relation to blood sampling (anticoagulants, culture medium, filter paper), storage (temperature, time and immediate lysis) and handling (repeated thawing and freezing). The PCR was unaffected by citrate and EDTA but partly inhibited by heparin (inhibition was reversed by heparinase at optimal concentrations). Samples collected on filter paper showed a significant 100-fold lower sensitivity (compared to control samples frozen immediately after collection) when stored at 30 degrees C and 60% humidity; and the paper quality appeared to be critical. Storage of unprocessed whole blood at 4 degrees C, 20 degrees C or 30 degrees C rarely resulted in any loss of sensitivity. Repeated thawing generally resulted in 10-fold loss of sensitivity compared to blood kept frozen until DNA extraction. The presence of antimalarial drug did not apparently affect sensitivity. We conclude that the mode of collection and storage of blood samples may influence the sensitivity of detection of malaria parasites by PCR. This may be critical in studies including individuals with low parasitaemia, mixed infections and comparison of data from different settings.

Reduction in the mean number of Plasmodium falciparum genotypes in Gambian children immunized with the malaria vaccine SPf66.

SPf66, a synthetic peptide Plasmodium falciparum vaccine, did not protect young Gambian children against clinical attacks of malaria. Nevertheless, Gambian children who had been vaccinated with SPf66 and who were parasitaemic at the end of the first malaria transmission season after vaccination had significantly fewer detectable P. falciparum genotypes than control children, as determined by polymerase chain reaction analysis of 3 polymorphic loci--the msp1 block 2 repeat region, the msp2 repeat region, and the R11 region of the glutamate-rich protein gene (glurp). Geometric mean numbers of genotypes were 1.66 vs. 1.87, 1.95 vs. 2.43, and 1.21 vs. 1.50 for msp1, msp2 and glurp, respectively (P = 0.31, P = 0.04 and P < 0.01). Differences between groups became a little more marked for msp1 and msp2 when children with symptomatic malaria were excluded. No significant difference was found between parasites obtained from SPf66-vaccinated or control children in the prevalences of amino acid alleles at positions 44 and 47 in the 11 amino acid sequence of the merozoite surface protein 1 molecule, which is present in SPf66. The reduction in the number of genotypes observed could not be explained by a difference in parasite densities between SPf66-vaccinated and control children, as geometric mean parasite densities were almost identical in the 2 groups. These observations suggest that SPf66 vaccine may have induced an immune response which reduced the incidence of new infections in immunized children or accelerated the rate of clearance of parasites of individual genotypes. However, no reduction in the prevalence or density of parasitaemia was recorded in SPf66-vaccinated children, suggesting the existence of some kind of density-dependent mechanism for controlling low levels of malaria parasitaemia.