Serum factors inhibitory for in vitro development of Plasmodium falciparum blood-stage parasites

Sera from 29 individuals residing in a malaria-endemic region of Colombia were evaluated by an inhibition assay for their capacity to retard the growth of Plasmodium falciparum in vitro. The inhibitory activity was found to be independent of antibody activity. Furthermore, the degree of inhibition of parasite development was variable, depending on the parasite isolate used for the assay and the season of malaria transmission. We selected sera with high inhibitory activity and carried out partial analytical characterization by anion exchange fast protein liquid chromatography (FPLC) to identify the chemical nature of the inhibitory factor(s). The results suggested that the in vitro inhibitory activity might result from the additive effect of different molecules. It appears that these molecules could be non-specifically induced by stimulation of the immune system, they seem to play a role in the immunity to malaria.

Classification of immature stage habitats of Culicidae (Diptera) collected in Córdoba, Argentina

In order to classify mosquito immature stage habitats, samples were taken in 42 localities of Córdoba Province, Argentina, representing the phytogeographic regions of Chaco, Espinal and Pampa. Immature stage habitats were described and classified according to the following criteria: natural or artificial; size; location related to light and neighboring houses; vegetation; water: permanence, movement, turbidity and pH. Four groups of species were associated based on the habitat similarity by means of cluster analysis: Aedes albifasciatus, Culex saltanensis, Cx. mollis, Cx. brethesi, Psorophora ciliata, Anopheles albitarsis, and Uranotaenia lowii (Group A); Cx. acharistus, Cx. quinquefasciatus, Cx. bidens, Cx. dolosus, Cx. maxi and Cx. apicinus (Group B); Cx. coronator, Cx. chidesteri, Mansonia titillans and Ps. ferox (Group C); Ae. fluviatilis and Ae. milleri (Group D). The principal component analysis (ordination method) pointed out that the different types of habitats, their nature (natural or artificial), plant species, water movement and depth are the main characters explaining the observed variation among the mosquito species. The distribution of mosquito species by phytogeographic region did not affect the species groups, since species belonging to different groups were collected in the same region.

Study on the Elimination of Angiostrongylus costaricensis First Stage Larvae in the Experimental Infection of Swiss Mice

Abdominal angiostrongylosis is a nematode infection of wild rodents. Human infection may result in severe abdominal disease and has been reported from several countries in the Americas. The domestic mouse, Mus musculus, has not been found with natural infection and, like other urban rodents, should not be considered a natural host for Angiostrongylus costaricensis. Quantification of parasitic forms released for transmission may better express the coevolutionary status in parasite-host relationship. With this objective, five groups of experimentally infected Swiss mice were followed for up to 155 days post-infection (PI) days and the quantification of first stage larvae (L1) output revealed: an irregular elimination of L1 and a huge variation in the patency period (1 to 114 days) and in the number of L1 eliminated daily by individual animals (1 to 6340 L1/g). Overall mortality was 72% (range: 28% to 100%) at seven weeks PI. In conclusion, abdominal angiostrongylosis in M. musculus presents high mortality and a very variable and irregular elimination of L1 in feces.

Acute intestinal anisakiasis in Spain: a fourth-stage Anisakis simplex larva

A case of acute intestinal anisakiasis has been reported; a nematode larva being found in the submucosa of the ileum of a woman in Jaén (Spain). The source of infection was the ingestion of raw Engraulis encrasicholus. On the basis of its morphology, the worm has been identified as a fourth-stage larva of Anisakis simplex. In Spain, this is the ninth report of human anisakiasis and also probably the first case of anisakiasis caused by a fourth-stage larva of A. simplex.

Bloodstream infections in late-stage acquired immunodeficiency syndrome patients evaluated by a lysis centrifugation system

Opportunistic infections, which affect acquired immunodeficiency syndrome (Aids) patients, are frequently disseminated and may cause bloodstream infections (BSI). The aim of this study was to evaluate the main causes of BSI in Aids patients with advanced stage of the disease, with special emphasis on the identification of fungemia. During a 21 months period, all patients with Aids (CD4 < 200) and febrile syndrome admitted to 3 university hospitals were systematically evaluated. For each patient presenting fever, a pair of blood cultures was collected and processed by using a commercial lysis-centrifugation system. One hundred and eleven patients (75 males) with a mean age of 36 years (median 33 years) and mean CD4 count of 64 cells/ml were included. Among the 111 patients evaluated we documented 54 episodes of BSI, including 46 patients with truly systemic infections and 8 episodes considered as contaminants. BSI were caused by gram-positive bacteria (43%), fungi (20%), gram-negative bacteria (15%), mycobacteria (15%), and mixed flora (7%). The crude mortality rate of our patients was 39%, being 50% for patients with BSI and 31% for the others. In conclusion, BSI are a common related to systemic infections on Aids patients with advanced stage of disease and is associated with a high rate of mortality.

Stage-specific activity of potential antimalarial compounds measured in vitro by flow cytometry in comparison to optical microscopy and hypoxanthine uptake

The evaluation of new antimalarial agents using older methods of monitoring sensitivity to antimalarial drugs are laborious and poorly suited to discriminate stage-specific activity. We used flow cytometry to study the effect of established antimalarial compounds, cysteine protease inhibitors, and a quinolone against asexual stages of Plasmodium falciparum. Cultured P. falciparum parasites were treated for 48 h with different drug concentrations and the parasitemia was determined by flow cytometry methods after DNA staining with propidium iodide. P. falciparum erythrocytic life cycle stages were readily distinguished by flow cytometry. Activities of established and new antimalarial compounds measured by flow cytometry were equivalent to results obtained with microscopy and metabolite uptake assays. The antimalarial activity of all compounds was higher against P. falciparum trophozoite stages. Advantages of flow cytometry analysis over traditional assays included higher throughput for data collection, insight into the stage-specificity of antimalarial activity avoiding use of radioactive isotopes.

Ivermectin resistant and susceptible third-stage larvae of Haemonchus contortus: cholinesterase and phosphatase activities

Cholinesterase and acid phosphatase (AP), but not alkaline phosphatase activities, were detected in cytosolic and membrane-bound fractions of ivermectin resistant and susceptible Haemonchus contortus infective-stage larvae. Some differences in acetylcholinesterase activity of cytosolic fractions and in the AP activity of these fractions as well as in the response to AP inhibitors by membrane-bound fractions were detected. Data are discussed.

Beyond sepsis pathophysiology with cytokines: what is their value as biomarkers for disease severity?

Sepsis is a major challenge in medicine. It is a common and frequently fatal infectious condition. The incidence continues to increase, with unacceptably high mortality rates, despite the use of specific antibiotics, aggressive operative intervention, nutritional support, and anti-inflammatory therapies. Typically, septic patients exhibit a high degree of heterogeneity due to variables such as age, weight, gender, the presence of secondary disease, the state of the immune system, and the severity of the infection. We are at urgent need for biomarkers and reliable measurements that can be applied to risk stratification of septic patients and that would easily identify those patients at the highest risk of a poor outcome. Such markers would be of fundamental importance to decision making for early intervention therapy or for the design of septic clinical trials. In the present work, we will review current biomarkers for sepsis severity and especially the use of cytokines as biomarkers with important pathophysiological role.

Failure of neutrophil migration toward infectious focus in severe sepsis: a critical event for the outcome of this syndrome

Sepsis is a systemic inflammatory response commonly caused by bacterial infection. We demonstrated that the outcome of sepsis induced by cecal ligation and puncture (CLP) correlates with the severity of the neutrophil migration failure towards infectious focus. Failure appears to be due to a decrease in the rolling and adhesion of neutrophil to endothelium cells. It seems that neutrophil migration impairment is mediated by the circulating inflammatory cytokines, such as TNF-alpha and IL-8, which induce the nitric oxide (NO) production systemically. It is supported by the fact that intravenous administration of these cytokines reduces the neutrophil migration induced by different inflammatory stimuli, and in severe sepsis the circulating concentrations of the cytokines and chemokines are significantly increased. Moreover, the neutrophil migration failure and the reduction in the rolling/adhesion were not observed in iNOS-/- mice and, aminoguanidine prevented this event. We also demonstrated that the failure of neutrophil migration is a Toll-4 receptor (TLR4) dependent mechanism, since it was not observed in TLR4 deficient mice. Furthermore, it was also observed that circulating neutrophils obtained from septic patients present failure of neutrophil chemotaxis toward fMLP, IL-8, and LTB4 and an increased in sera concentrations of NO3 and cytokines. In conclusion, we demonstrated that, in sepsis, failure of neutrophil migration is critical for the outcome and that NO is involved in the process.

Comparative recognition by human IgG antibodies of recombinant proteins representing three asexual erythrocytic stage vaccine candidates of Plasmodium vivax

In previous immuno-epidemiological studies of the naturally acquired antibody responses to merozoite surface protein-1 (MSP-1) of Plasmodium vivax, we had evidence that the responses to distinct erythrocytic stage antigens could be differentially regulated. The present study was designed to compare the antibody response to three asexual erythrocytic stage antigens vaccine candidates of P. vivax. Recombinant proteins representing the 19 kDa C-terminal region of MSP-1(PvMSP19), apical membrane antigen n-1 ectodomain (PvAMA-1), and the region II of duffy binding protein (PvDBP-RII) were compared in their ability to bind to IgG antibodies of serum samples collected from 220 individuals from the state of Pará, in the North of Brazil. During patent infection with P. vivax, the frequency of individuals with IgG antibodies to PvMSP1(19), PvAMA-1, and PvDBP-RII were 95, 72.7, and 44.5% respectively. Although the frequency of responders to PvDBP-RII was lower, this frequency increased in individuals following multiple malarial infections. Individually, the specific antibody levels did not decline significantly nine months after treatment, except to PvMSP1(19). Our results further confirm a complex regulation of the immune response to distinct blood stage antigens. The reason for that is presently unknown but it may contribute to the high risk of re-infection in individuals living in the endemic areas.

Trypanosoma cruzi: a stage-specific calpain-like protein is induced after various kinds of stress

Calpains are calcium-dependent cysteine proteinases found in all living organisms and are involved in diverse cellular processes. Calpain-like proteins have been reported after in silico analysis of the Tritryps genome and are believed to play important roles in cell functions of trypanosomatids. We describe the characterization of a member of this family, which is differentially expressed during the life-cycle of Trypanosoma cruzi.

Spontaneous stage differentiation of mouse-virulent Toxoplasma gondii RH parasites in skeletal muscle cells: an ultrastructural evaluation

Although the predilection for Toxoplasma gondii to form cysts in the nervous system and skeletal and heart muscles has been described for more than fifty years, skeletal muscle cells (SkMCs) have not been explored as a host cell type to study the Toxoplasma-host cell interaction and investigate the intracellular development of the parasite. Morphological aspects of the initial events in the Toxoplasma-SkMC interaction were analysed and suggest that there are different processes of protozoan adhesion and invasion and of the subsequent fate of the parasite inside the parasitophorous vacuole (PV). Using scanning electron microscopy,Toxoplasma tachyzoites from the mouse-virulent RH strain were found to be attached to SkMCs by the anterior or posterior region of the body, with or without expansion of the SkMC membrane. This suggests that different types of parasite internalization occurred. Asynchronous multiplication and differentiation of T. gondii were observed. Importantly, intracellular parasites were seen to display high amounts of amylopectin granules in their cytoplasm, indicating that tachyzoites of the RH strain were able to differentiate spontaneously into bradyzoites in SkMCs. This stage conversion occurred in approximately 3% of the PVs. This is particularly intriguing as tachyzoites of virulent Toxoplasma strains are not thought to be prone to cyst formation. We discuss whether biological differences in host cells are crucial to Toxoplasma stage conversion and suggest that important questions concerning the host cell type and its relevance in Toxoplasma differentiation are still unanswered.

Development of physiological resistance and its stage specificity in Culex quinquefasciatus after selection with deltamethrin in Assam, India

The study investigated the development and stage specificity of physiological resistance to insecticides in a colony of Culex quinquefasciatus Say (Diptera: Culicidae) mosquitoes, which are vectors of bancroftian filariasis in India, after selection with deltamethrin. Resistance was selected by exposing the larvae to the concentration of deltamethrin that caused 50% mortality in the tested population (i.e., LC50). Under continuous selection pressure, the LC50 increased steadily in subsequent generations. The estimated LC50 for the F0 generation was 0.409 μg/L; the LC50 first displayed a substantial increase in the F5 generation (5.616 μg/L) and reached 121.902 μg/L in the F10 generation. The objective of this study was to establish a deltamethrin-resistant colony to develop a research programme that will study the evolution of physiological resistance patterns and stage-specific resistance responses in Cx. quinquefasciatus larvae and adults under laboratory conditions. An approximately 298-fold increase in resistance was recorded after 10 generations, as evidenced by the resistance ratio (RR50). The progress and effect of the selection pressure in the adult stage was monitored with the World Health Organisation (WHO) diagnostic test. The mortality, as observed using the WHO diagnostic test, declined significantly from the F5 generation (85%) onwards and the highest rate of survival (65%) was observed in the F10 generation.