Evidence for expression of a Ras-like and a stage specific GTP binding homologous protein by Plasmodium falciparum.

Plasmodium falciparum, the parasite responsible for the most severe form of malaria, undergoes an asexual multiplication in man and a sexual one in mosquito. The asexual cycle can be reproduced in vitro. The present work reports the isolation of a small guanosine triphosphate-binding protein in Plasmodium falciparum extracts. This protein, a 21,000 M(r) Ras-like molecule, was revealed by western blotting in each stage of the intraerythrocytic asexual life cycle. Conversely, a 46,000 M(r) G alpha subunit of a heterotrimeric GTP-binding protein was found to be expressed during a short period from mature schizonts to free merozoites. In order to provide additional evidence for the presence of these GTP-binding proteins in Plasmodium falciparum cultures and also to determine the kinetics, we tested two toxins that are involved in the cellular signalling transduction. We observed that pertussis toxin increases P. falciparum growth, whereas cholera toxin induces crisis forms, and subsequent parasite death within the following 24 h.

TNF-alpha enhances Toxoplasma gondii cyst formation in human fibroblasts through the sphingomyelinase pathway.

The cystogenesis event of Toxoplasma gondii is poorly understood. In order to throw light on it, the effect of tumor necrosis factor-alpha (TNF-alpha) was studied in the Prugniaud strain of the organism. This showed that TNF-alpha increased the number of cysts formed in vitro in human MRC5 fibroblasts. The sphingomyelinase pathway may be involved in mediating the TNF effect, since ceramide (natural form in permeabilized cells or cell-permeable analogue) could mimic the action of TNF. More precisely, our results strongly suggest the involvement of an acidic sphingomyelinase in mediating the effect of TNF; indeed, D609 inhibited both the TNF effect and cyst formation, while arachidonic acid had no effect. Moreover, protein kinase (PKC) seems also to play a role in the process, since phorbol-12-myristate-13-acetate (PMA) enhanced the cyst formation. However, chelerythrine chloride did not prevent the TNF effect, suggesting that several host-cell pathways can affect the cystogenesis event. Taken together, these results suggest the active participation of host-cell components in the cystogenesis of Toxoplasma gondii.

Chemokines in host-protozoan-parasite interactions.

Here, we review the interactions between parasites and chemokines and chemokine receptors in toxoplasmosis, trypanosomiasis, leishmaniasis, malaria and other diseases caused by protozoan parasites. The potential roles of chemokines after infection by these intracellular pathogens include host defence functions such as leukocyte recruitment, participation in cell-mediated immunity and antiprotozoal activity. However, these interactions can also help the parasite in, for example, the penetration of host cells.

Extracerebral toxoplasmosis in patients infected with HIV. A French National Survey.

A French nationwide survey of extracerebral toxoplasmosis (ECT) in HIV-infected patients was performed between January 1990 and September 1992. All French hospitals were surveyed, and all but a few responded. Data collected included epidemiologic, clinical, and biologic features; therapy; and outcome. During the 33-month survey, 199 cases were collected. The prevalence of ECT in patients with AIDS can be estimated at 1.5%-2%. Age, sex, and HIV risk factors were similar to those of the general AIDS population in France. Extracerebral toxoplasmosis appeared mainly in HIV-infected patients with advanced immunosuppression: the mean CD4+ lymphocyte count was 57/mm3(+/- 99). The localizations observed were: eyes (50% of patients); lung (26%); disseminated (at least 2 extracerebral visceral localizations) (11.5%); peripheral blood (acute febrile syndrome with isolated positive parasitemia) (3%); heart (3%); bone marrow (3%); bladder (1%); and isolated cases of rhinopharynx, skin, liver, lymph nodes, conus medullaris, and pericardium. In this survey, muscular and pancreatic localizations were always associated with other extracerebral localizations. A cerebral localization was diagnosed in 41% of cases. Serologic data provided little information. Ocular fundus examination, bronchoalveolar lavage, tissue biopsy, and search for parasitemia were the main diagnostic procedures. Treatment was the same as for cerebral toxoplasmosis. A clinical response was observed in 64% of cases; 19% relapsed. Death occurred in 106 (53%) cases and was related to ECT in 34% of cases.(ABSTRACT TRUNCATED AT 250 WORDS)

Monocyte tissue factor expression induced by Plasmodium falciparum-infected erythrocytes.

Monocytes are active elements of the host response against Plasmodium falciparum. They are able to express tissue factor and trigger the extrinsic pathway of blood coagulation the activation of which remained unclear in malaria. Our aim was to assess the tissue factor expression of purified blood monocytes stimulated by cultured Plasmodium falciparum-infected erythrocytes. Malaria parasite induced an early generation of tissue factor with a peak between 8 and 12 h of stimulation. Maximum expression was observed for parasitemia ranging from 1 to 2%. Plasmodium falciparum culture supernatants had the same effect showing the existence of a soluble factor able to induce the tissue factor expression. These data, demonstrating an activation of the tissue factor pathway by the malaria parasite, emphasize thrombin generation. Therefore, thrombin could participate in malaria pathology either in the microcirculatory blockade via platelet and fibrinogen activation or as a mitotic.

Emerging parasite zoonoses: the role of host-parasite relationship.

Human-parasite relationships have played an essential role in the emergence or re-emergence of some parasitic diseases. These interactions are due to numerous causes. Some are linked to humans (immunodeficiencies due to AIDS among other causes, treatments, nosocomial contaminations, genetic predisposition), others concern the parasite (particular genotypes having modified their parasitic specificity). Several of these causes were predominant in the emergence of parasitoses such as cryptosporidiasis, microsporidioses or, to a certain point, pneumocystosis, the transmission of which has become zoonotic or even anthroponotic, inter-human. Re-emergent diseases (toxoplasmosis, leishmaniasis, giardiasis, strongyloidiasis, scabies) had already been described in human pathology, but their frequency or symptomatology have been drastically modified. In this case also, the unbalanced host-parasite relationship is largely responsible but it can not be dissociated from other causes, especially environmental and nutritional.

Relationships between clinical protection and antibodies to Plasmodium falciparum RESA (ring-infected erythrocyte surface antigen) peptides.

A longitudinal study involving 76 individuals living in Dafinso and Vallée du Kou (near Bobo-Dioulasso, Burkina Faso, West Africa) was performed in June 1987 (beginning of the transmission period), August-September 1987 (during) and January 1988 (after). The serological antibody (Ab) responses against synthetic peptides representing repeat amino acid sequences of the P. falciparum Ring-Infected Erythrocyte Surface Antigen (RESA): (EENV)5, (EENVEHDA)4, (DDEHVEEPTVA)2 were evaluated by ELISA. The clinical longitudinal study during the transmission period allowed us to define three different groups in terms of age and occurrence of clinical malarial attack (greater than 5000 parasites mm-3 of blood and axillary fever greater than 37.7 degrees C). Levels (A620) of Ab to (EENVEHDA)4 and (DDEHVEEPTVA)2 were correlated with age. The adult group (III) had the highest prevalences of Ab to RESA peptides. No significant difference was found between groups of children with or without malaria attack. Nevertheless, at the beginning of the transmission period, children who had at least one malaria attack during the study presented the lowest level of antibodies to RESA peptides.

Tumour necrosis factor alpha receptors: role in the physiopathology of protozoan parasite infections.

Tumour necrosis factor alpha (TNF alpha) is an important cytokine in immune regulation and resistance to various micro-organisms. It provides signals to the target cells through two different receptors: TNFR1 and TNFR2. The present report reviews the role of TNF receptors (TNFRs) in the immune response against protozoan parasite infections of medical interest (Toxoplasma gondii, Leishmania major, Trypanosoma cruzi, Plasmodium spp.). TNF alpha has been regarded as a modulator cytokine in host defence against protozoans infections and recent findings on experimental gene-deficient mice have showed that TNF alpha/TNFRs pathway may be beneficial for host protection during these infections.

Correlation between beta 2-microglobulin and soluble interleukin-2 receptor levels in plasma of individuals living in a malarial endemic region.

beta 2-Microglobulin (beta 2m) levels were related to the expected immunoprotection in 81 individuals living in a malarial mesoendemic area near Bobo-Dioulasso (Burkina Faso), who were longitudinally followed. Soluble interleukin-2 receptor (sIL-2R) levels were positively correlated to those of beta 2m (r = 0.44; n = 237; P less than 0.001). This suggests that most of the beta 2m could have originated from activated T and B cell membrane turnover. In our study, both beta 2m and sIL-2R were inversely related to IgG antibodies (Ab) against somatic antigen of Plasmodium falciparum (Som-Ag). Therefore, these molecules at high levels could have a down regulating activity, directly or indirectly, on B cells producing this kind of Ab.

Detection of Toxoplasma gondii in 94 placentae from infected women by polymerase chain reaction, in vivo, and in vitro cultures.

The biological diagnosis of congenital toxoplasmosis at birth is important to determine the infant's treatment. The aim of this study was to evaluate the placenta results in the congenital toxoplasmosis diagnosis and to compare them with those obtained with other samples collected at birth (cord blood and newborn blood). A total of 94 placentas, of which 33 came from fetuses suspected of or with proven congenital toxoplasmosis (CT+) and 61 from definitely or probably non-infected fetuses (CT-), was analysed by in vitro culture, mouse inoculation and polymerase chain reaction (PCR). The PCR sensitivity was higher (60.9 per cent) than that of cell culture (29.6 per cent) and mouse inoculation (51.5 per cent) but the number of PCR positive results in CT - patients was also higher (9.5 per cent). The presence of Toxoplasma gondii in the placenta tissues was the only argument at birth (IgM and neosynthesized Ig were negative) in three out of the 33 CT+ cases. The detection of IgM by ELISA and ISAGA and the detection of neosynthesized Ig by immunoblotting were more satisfactory to diagnose congenital toxoplasmosis but the placenta analysis was important to improve the sensitivity of the diagnosis at birth, especially when the prenatal diagnosis was negative or not performed.

Aspergillus and lung transplant recipients: a mycologic and molecular epidemiologic study.

BACKGROUND: After lung transplantation, filamentous fungi and more particularly Aspergillus fumigatus are commonly isolated, although the origin of contamination is unclear. METHODS: To investigate the fungal flora in bronchoscopic fluids, we retrospectively reviewed 20 cases of lung transplant recipients. Using sequence-specific DNA primers analysis, we typed the clinical strains of A. fumigatus isolated from 6 lung transplant recipients. For 4 of them, the strains of this species were isolated from their environment. RESULTS: At least once 90% of patients had filamentous fungi, and A. fumigatus was the most frequently isolated. Bronchial colonization was detected in 14 patients, invasive bronchial mycosis was diagnosed in 4 others, and no case of invasive pulmonary fungal infection was detected. Genome typing of the 47 clinical strains revealed that a given patient could be affected by several different strains. A very extensive polymorphism existed among the 38 environmental strains. Origin of contamination at home was possible in 1 case and in the hospital in 3 cases. CONCLUSIONS: Bronchial colonization is frequent after lung transplantation. Although the clinical strains show a polymorphism, it is less widespread than the polymorphism of environmental strains. The origin of acquisition may be in the patient's community.

Identification of differentially transcribed RNA and DNA helicase-related genes of Plasmodium falciparum.

Chloroquine antimalarial action was assessed by the analysis of changes in gene expression. With this aim, Plasmodium falciparum cultures were submitted to chloroquine and to other stresses to determine which transcripts were specifically induced. P. falciparum in vitro control culture was compared to cultures where chloroquine was added and to cultures where serum was omitted, or where higher partial oxygen pressure was used, and, finally, at a temperature of 40 degrees C instead of 37 degrees C. Poly (A)+RNAs were reverse-transcribed and detected by the differential display technique. Two specific cDNAs were obtained and cloned, and a part of the genes was sequenced. The deduced protein, referred to as Pfhel-1, was related to a RNA helicase and was thought to be involved in protein translation control. The second deduced protein, called Pfhel-2, possessed consensus sequences of ATP-dependent helicase domains. Pfhel-2 may be involved either in mitotic control or in DNA repair. The possible roles of both helicase-related genes in chloroquine therapeutic activity are discussed.

High human malarial infectivity to laboratory-bred Anopheles gambiae in a village in Burkina Faso.

The malarial infectivity of an African village population was tested by selecting a demographically representative sample of individuals for study, regardless of parasitemia or gametocytemia. The infectivity of this population people to laboratory-bred mosquitoes was investigated using membrane feeding techniques. Tests on 322 subjects (greater than four years of age) indicated that approximately 48.4% were capable of infecting mosquitoes. There were similar proportions of infectious individuals among gametocyte carriers (52.5%) and nongametocyte carriers (46.6%). All age groups appeared to contribute equally to this infective reservoir. Most of the infections resulted in low oocyst loads (1.8 oocysts) on the midgut of the positive mosquitoes and only a few mosquitoes per batch were infected (11.5%). A previous entomologic survey estimated 90 infected bites/person/year and a low parity index in Anopheles gambiae (< 60%) as well as in An. funestus (< 40%), the two main malaria vectors in this region. This low parity index could indicate a low life expectancy for infected mosquitoes and could therefore explain an inoculation rare lower than expected considering the high degree of infectivity of the human population studied.

Diagnosis of malaria in non-endemic countries by the ParaSight-F test.

QBC, examination of thin blood smears, and Parasight-F were performed for every case of malaria suspected between May 1997 and December 1998. Data from 310 patients were reported. Fifty had malaria infection diagnosed by QBC and thin blood film, among whom 39 had Plasmodium falciparum infection. Three of these 39 were negative with the Parasight-F test. Eleven patients had a positive ParaSight-F test but microscopic diagnosis methods were negative. Interpretation of these 11 positive ParaSight-F results is proposed to depend on clinical criteria.

Determination of anti-Toxoplasma gondii immunoglobulin G avidity: adaptation to the Vidas system (bioMérieux).

The determination of specific anti-Toxoplasma gondii IgG avidity has been proposed to improve the determination of the date of toxoplasmic seroconversion in pregnant women. In this study, we adapted this serological technique to the Vidas system (bioMérieux) using 6 M urea as the dissociating agent. We studied 356 sera, including 42 sequential sera from sero-conversions in pregnant women. Our results show that the test is easy to use, and that an avidity index higher than 0.300 allows the exclusion of a recent infection acquired less than 4 months before serum sampling.

Specific anti-toxoplasmic IgG antibody immunoblot profiles in patients with AIDS-associated Toxoplasma encephalitis.

Among 186 suspected cases of Toxoplasma encephalitis (TE) in HIV-infected patients, 113 were classified as TE and 73 as non-TE. Serum Toxoplasma gondii (T.g.) antibodies were detected by ELISA in 97% of TE vs 71% of non-TE cases (p < 0.001). In the 164 patients positive for T. g. antibodies, the IgG 27 and 32 bands were more frequently present in TE than in non-TE (p = 0.003, p = 0.002, respectively). Among patients with positive T.g. serology, multivariate analysis showed that the presence of an IgG 32 (OR 3.1) or IgG 27 band (OR 2.7) on Western blot was highly indicative of TE independently of each other. Positive T.g. serology, but not anti-T.g. IgG antibody titres, was predictive. Thus, the positivity of IgG 27 and 32 bands on Western blot analysis provides useful data for improving the diagnosis of presumptive TE in HIV-infected patients with suspected TE and positive Toxoplasma serology.

A new set of primers for the detection of Toxoplasma gondii in amniotic fluid using polymerase chain reaction.

A new PCR system including a pair of primers, a probe and an internal control were designed from the B1 gene of Toxoplasma gondii. The system described allowed the detection of less than 10 tachyzoites of the RH strain of T. gondii. Among 21 amniotic fluid samples, this system diagnosed the cases of congenital toxoplasmosis which were simultaneously diagnosed using mice inoculation, in vitro culture, and serology from both amniotic fluid and fetal blood. These results show that these new primers allow for a highly sensitive detection of T. gondii DNA.

Virulence of Aspergillus fumigatus strains investigated by random amplified polymorphic DNA analysis.

A possible relationship between the ability of Aspergillus fumigatus strains to invade tissues and genetic polymorphism was studied by random amplified polymorphic DNA (RAPD) analysis. One hundred randomly designed oligonucleotide decamers were examined with DNA of three reference strains, eight environmental isolates and 21 isolates from two distinct clinical situations: non-invasive aspergillosis (predominantly aspergilloma) and invasive aspergillosis. One primer (OPQ 6) was found to generate a reproducible amplification product that enabled distinction between the two groups according to the presence or absence of a 0.95-kb fragment that correlated with the nature of the infection (non-invasive or invasive) and immune status of the patient. The results indicated that the pathogenicity of A. fumigatus was related not only to the host's immune status but also to the virulence of the strain of A. fumigatus.