Polymorphism in the Plasmodium vivax msp 3: gene in field samples from Tierralta, Colombia

We evaluated the Plasmodium vivax polymorphism by studying the Pvmsp-3 gene's polymorphic region by PCR-RFLP in 55 samples from patients living in Tierralta, Colombia. Three different sizes of the Pvmsp-3 gene were found, type A (1,900 bp), type B (1,500 bp) and type C (1,100 bp); most of the samples were type A (96.4 %). The Pvmsp-3 gene exhibited high polymorphism. Seven restriction patterns were found when using Alu I, and nine were found with Hha I; 12 different alleles were obtained when these patterns were combined. The findings suggest that this gene could be used in Colombia as a molecular epidemiologic marker for genotyping P. vivax.

Improving the production of the denatured recombinant N-terminal domain of rhoptry-associated protein 2 from a Plasmodium falciparum target in the pathology of anemia in falciparum malaria

Rhoptry-associated protein 2 (RAP2) is known to be discharged from rhoptry onto the membrane surface of infected and uninfected erythrocytes (UEs) ex vivo and in vitro and this information provides new insights into the understanding of the pathology of severe anemia in falciparum malaria. In this study, a hexahistidine-tagged recombinant protein corresponding to residues 5-190 of the N-terminal of Plasmodium falciparum RAP2 (rN-RAP2) was produced using a new method of solubilization and purification. Expression was induced with D-lactose, a less expensive alternative inducer to the more common isopropyl-²-D-thio-galactopyranosidase. The recombinant protein was purified using two types of commercially-available affinity columns, iminodiacetic and nitrilotriacetic. rN-RAP2 had immunogenic potential, since it induced high titers of anti-RAP2 antibodies in mice. These antibodies recognized full-length RAP2 prepared from Triton X-100 extracts from two strains of P. falciparum. In fact, the antibody recognized a 29-kDa product of RAP2 cleavage as well as 82 and 70-kDa products of RAP1 cleavage. These results indicate that the two antigens share sequence epitopes. Our expressed protein fragment was shown to contain a functional epitope that is also present in rhoptry-derived ring surface protein 2 which attaches to the surface of both infected and UEs and erythroid precursor cells in the bone marrow of malaria patients. Serum from malaria patients who developed anemia during infection recognized rN-RAP2, suggesting that this protein fragment may be important for epidemiological studies investigating whether immune responses to RAP2 exacerbate hemolysis in falciparum malaria patients.

Study of the relationship between Aedes (Stegomyia) aegypti egg and adult densities, dengue fever and climate in Mirassol, state of São Paulo, Brazil

The purpose of this study was to examine the relationship between Aedes aegypti egg and adult density indices, dengue fever and climate in Mirassol, state of São Paulo, Brazil, between November 2004-November 2005. Weekly collections of adults and eggs were made using, respectively, manual aspirators and oviposition traps that produced four entomological indices (positivity and average of females and eggs). Weekly incidence coefficients were calculated based on dengue cases. Each week, the data obtained from entomological indices were related to each other, dengue, and climate variables. The first index to show an association with dengue transmission was the female average, followed by female positivity and egg average. Egg positivity did not show a relationship with risk for dengue, but was sensitive to identifying the presence of the vector, principally in dry seasons. The relationship between climatic factors, the vector and the disease found in this study can be widely employed in planning and undertaking dengue surveillance and control activities, but it is a tool that has not been considered by the authorities responsible for controlling the disease. In fact, this relationship permits the use of information about climate for early detection of epidemics and for establishing more effective prevention strategies than currently exist.

Asymptomatic Plasmodium spp. infection in Tierralta, Colombia

With the aim of determining the prevalence of asymptomatic Plasmodium spp. infection by thick smear and PCR and its association with demographic and epidemiological characteristics in the village of Nuevo Tay, Tierralta, Córdoba, Colombia, a cross-sectional population study was carried out, using random probabilistic sampling. Venous blood samples were taken from 212 people on day 0 for thick smear and PCR. Clinical follow-up and thick smears were carried out on days 14 and 28. The prevalence of Plasmodium spp. infection was 17.9% (38/212; 95% CI: 12.5-23.3%) and the prevalence of asymptomatic Plasmodiumspp. infection was 14.6% (31/212; 95% CI: 9.6-19.6%). Plasmodium vivax was found more frequently (20/31; 64.5%) than Plasmodium falciparum (9/31; 29%) and mixed infections (2/31; 6.5%). A significantly higher prevalence of asymptomatic infection was found in men (19.30%) than in women (9.18%) (prevalence ratio: 2.10; 95% CI: 1.01-4.34%; p = 0.02). People who developed symptoms had a significantly higher parasitemia on day 0 than those who remained asymptomatic, of 1,881.5 ± 3,759 versus 79 ± 106.9 (p = 0.008). PCR detected 50% more infections than the thick smears. The presence of asymptomatic Plasmodium spp. infection highlights the importance of carrying out active searches amongst asymptomatic populations residing in endemic areas.

Immunogenicity, reactogenicity and consistency of production of a Brazilian combined vaccine against diphtheria, tetanus, pertussis and Haemophilus influenzae type b

A randomized, double-blinded study evaluating the immunogenicity, safety and consistency of production of a combined diphtheria-tetanus-pertussis-Haemophilus influenzae type b vaccine entirely produced in Brazil by Bio-Manguinhos and Instituto Butantan (DTP/Hib-BM) was undertaken. The reference vaccine had the same DTP vaccine but the Hib component was produced using purified materials supplied by GlaxoSmithKline (DTP/Hib-GSK), which is registered and has supplied the Brazilian National Immunization Program for over more than five years. One thousand infants were recruited for the study and received vaccinations at two, four and six months of age. With respect to immunogenicity, the vaccination protocol was followed in 95.6% and 98.4% of infants in the DTP/Hib-BM and DTP/Hib-GSK groups, respectively. For the Hib component of the study, there was 100% seroprotection (>0.15 µg/mL) with all three lots of DTP/Hib-BM and DTP/Hib-GSK. The geometric mean titer (GMT) was 9.3 µg/mL, 10.3 µg/mL and 10.3 µg/mL for lots 1, 2 and 3 of DTP/Hib-BM, respectively, and the GMT was 11.3 g/mL for DTP/Hib-GSK. For diphtheria, tetanus and pertussis, seroprotection was 99.7%, 100% and 99.9%, respectively, for DTP/Hib-BM, three lots altogether and 99.2%, 100% and 100% for DTP/Hib-GSK. GMTs were similar across all lots and vaccines. Adverse events rates were comparable among the vaccine groups. The Brazilian DTP/Hib vaccine demonstrated an immunogenicity and reactogenicity profile similar to that of the reference vaccine.

Heterologous expression and biochemical characterization of an α1,2-mannosidase encoded by the Candida albicans MNS1 gene

Protein glycosylation pathways, commonly found in fungal pathogens, offer an attractive new area of study for the discovery of antifungal targets. In particular, these post-translational modifications are required for virulence and proper cell wall assembly in Candida albicans, an opportunistic human pathogen. The C. albicans MNS1 gene is predicted to encode a member of the glycosyl hydrolase family 47, with 1,2-mannosidase activity. In order to characterise its activity, we first cloned the C. albicans MNS1 gene into Escherichia coli, then expressed and purified the enzyme. The recombinant Mns1 was capable of converting a Man9GlcNAc2 N-glycan core into Man8GlcNAc2 isomer B, but failed to process a Man5GlcNAc2-Asn N-oligosaccharide. These properties are similar to those displayed by Mns1 purified from C. albicansmembranes and strongly suggest that the enzyme is an ±1,2-mannosidase that is localised to the endoplasmic reticulum and involved in the processing of N-linked mannans. Polyclonal antibodies specifically raised against recombinant Mns1 also immunoreacted with the soluble ±1,2-mannosidases E-I and E-II, indicating that Mns1 could share structural similarities with both soluble enzymes. Due to the high degree of similarity between the members of family 47, it is conceivable that these antibodies may recognise ±1,2-mannosidases in other biological systems as well.

The interaction between Histoplasma capsulatum cell wall carbohydrates and host components: relevance in the immunomodulatory role of histoplasmosis

Histoplasma capsulatum is an intracellular fungal pathogen that causes respiratory and systemic disease by proliferating within phagocytic cells. The binding of H. capsulatum to phagocytes may be mediated by the pathogen's cell wall carbohydrates, glucans, which consist of glucose homo and hetero-polymers and whose glycosydic linkage types differ between the yeast and mycelial phases. The ±-1,3-glucan is considered relevant for H. capsulatum virulence, whereas the ²-1,3-glucan is antigenic and participates in the modulation of the host immune response. H. capsulatum cell wall components with lectin-like activity seem to interact with the host cell surface, while host membrane lectin-like receptors can recognize a particular fungal carbohydrate ligand. This review emphasizes the relevance of the main H. capsulatum and host carbohydrate-driven interactions that allow for binding and internalization of the fungal cell into phagocytes and its subsequent avoidance of intracellular elimination.

Induction of apoptosis in A549 pulmonary cells by two Paracoccidioides brasiliensis samples

Paracoccidioidomycosis presents a variety of clinical manifestations and Paracoccidioides brasiliensis can reach many tissues, most importantly the lungs. The ability of the pathogen to interact with host surface structures is essential to its virulence. The interaction between P. brasiliensis and epithelial cells has been studied, with particular emphasis on the induction of apoptosis. To investigate the expression of different apoptosis-inducing pathways in human A549 cells, we infected these cells with P. brasiliensis Pb18SP (subcultured) and 18R (recently isolated from cell culture and showing a high adhesion pattern) samples in vitro. The expressions of Bcl-2, Bak and caspase 3 were analysed by flow cytometry and DNA fragmentation using the TUNEL technique. Apoptosis of human A549 cells was induced by P. brasiliensis in a sample and time-dependent manner. Using an in vitro model, our data demonstrates that caspase 3, Bak, Bcl-2 and DNA fragmentation mediate P. brasiliensis-induced apoptosis in A549 cells. The overall mechanism is a complex process, which may involve several signal transduction pathways. These findings could partially explain the efficient behaviour of this fungus in promoting tissue infection and/or blood dissemination.

Potential of laticifer fluids for inhibiting Aedes aegypti larval development: evidence for the involvement of proteolytic activity

It has been shown previously that the laticifer fluid of Calotropis procera (Ait.) R.Br. is highly toxic to the egg hatching and larval development of Aedes aegypti L. In the present study, the larvicidal potential of other laticifer fluids obtained from Cryptostegia grandiflora R.Br., Plumeria rubra L. and Euphorbia tirucalli L. was evaluated. We attempted to correlate larvicidal activity with the presence of endogenous proteolytic activity in the protein fraction of the fluids. After collection, the fluids were processed by centrifugation and dialysis to obtain the soluble laticifer protein (LP) fractions and eliminate water insoluble and low molecular mass molecules. LP did not visibly affect egg hatching at the doses assayed. LP from Cr. grandiflora exhibited the highest larval toxicity, while P. rubra was almost inactive. E. tirucalli was slightly active, but its activity could not be correlated to proteins since no protein was detected in the fluid. The larvicidal effects of LP from C. procera and Cr. grandiflora showed a significant relationship with the proteolytic activity of cysteine proteinases, which are present in both materials. A purified cysteine proteinase (papain) from the latex of Carica papaya (obtained from Sigma) was similarly effective, whereas trypsin and chymotrypsin (both serine proteinases) were ineffective. The results provide evidence for the involvement of cysteine proteinase activity in the larvicidal action of some laticifer fluids. C. procera is an invasive species found in areas infested with Ae. aegypti and thus could prove useful for combating mosquito proliferation. This is the first report to present evidence for the use of proteolytic enzymes as chemical agents to destroy Ae. aegypti larvae.

Chemical composition and antiprotozoal activities of Colombian Lippia spp essential oils and their major components

The chemical composition and biological activities of 19 essential oils and seven of their major components were tested against free and intracellular forms of Leishmania chagasi and Trypanosoma cruzi parasites as well as Vero and THP-1 mammalian cell lines. The essential oils were obtained from different species of Lippia, a widely distributed genus of Colombian plants. They were extracted by microwave radiation-assisted hydro-distillation and characterised by GC-FID and GC-MS. The major components were geranial, neral, limonene, nerol, carvacrol, p-cymene, γ-terpinene, carvone and thymol. The essential oil of Lippia alba exhibited the highest activity against T. cruzi epimastigotes and intracellular amastigotes with an IC50 of 5.5 μg/mL and 12.2 μg/mL, respectively. The essential oil of Lippia origanoides had an IC50 of 4.4 μg/mL in L. chagasi promastigotes and exhibited no toxicity in mammalian cells. Thymol (IC50 3.2 ± 0.4 μg/mL) and S-carvone (IC50 6.1 ± 2.2 μg/mL), two of the major components of the active essential oils, were active on intracellular amastigotes of T. cruziinfected Vero cells, with a selective index greater than 10. None of the essential oils or major components tested in this study was active on amastigotes of L. chagasi infected THP-1 cells.

Interobserver variability of ultrasound parameters in portal hypertension

The aim of this study was to assess interobserver agreement of ultrasound parameters for portal hypertension in hepatosplenic mansonic schistosomiasis. Spleen size, diameter of the portal, splenic and superior mesenteric veins and presence of thrombosis and cavernous transformation were determined by three radiologists in blinded and independent fashion in 30 patients. Interobserver agreement was measured by the kappa index and intraclass correlation coefficient. Interobserver agreement was considered substantial (κ = 0.714-0.795) for portal vein thrombosis and perfect (κ = 1) for cavernous transformation. Interobserver agreement measured by the intraclass correlation coefficient was excellent for longitudinal diameter of the spleen (r = 0.828-0.869) and splenic index (r = 0.816-0.905) and varied from fair to almost perfect for diameter of the portal (r = 0.622-0.675), splenic (r = 0.573-0.913) and superior mesenteric (r = 0.525-0.607) veins. According to the results, ultrasound is a highly reproducible method for the main morphological parameters of portal hypertension in schistosomiasis patients.

Regulatory elements involved in the post-transcriptional control of stage-specific gene expression in Trypanosoma cruzi: a review

Trypanosoma cruzi, a protozoan parasite that causes Chagas disease, exhibits unique mechanisms for gene expression such as constitutive polycistronic transcription of protein-coding genes, RNA editing and trans-splicing. In the absence of mechanism controlling transcription initiation, organized subsets of T. cruzi genes must be post-transcriptionally co-regulated in response to extracellular signals. The mechanisms that regulate stage-specific gene expression in this parasite have become much clearer through sequencing its whole genome as well as performing various proteomic and microarray analyses, which have demonstrated that at least half of the T. cruzi genes are differentially regulated during its life cycle. In this review, we attempt to highlight the recent advances in characterising cis and trans-acting elements in the T. cruzi genome that are involved in its post-transcriptional regulatory machinery.

Recent advances in the study of avian malaria: an overview with an emphasis on the distribution of Plasmodium spp in Brazil

Avian malaria parasites (Plasmodium) have a worldwide distribution except for Antarctica. They are transmitted exclusively by mosquito vectors (Diptera: Culicidae) and are of particular interest to health care research due to their phylogenetic relationship with human plasmodia and their ability to cause avian malaria, which is frequently lethal in non-adapted avian hosts. However, different features of avian Plasmodium spp, including their taxonomy and aspects of their life-history traits, need to be examined in more detail. Over the last 10 years, ecologists, evolutionary biologists and wildlife researchers have recognized the importance of studying avian malaria parasites and other related haemosporidians, which are the largest group of the order Haemosporida by number of species. These studies have included understanding the ecological, behavioral and evolutionary aspects that arise in this wildlife host-parasite system. Molecular tools have provided new and exiting opportunities for such research. This review discusses several emerging topics related to the current research of avian Plasmodium spp and some related avian haemosporidians. We also summarize some important discoveries in this field and emphasize the value of using both polymerase chain reaction-based and microscopy-based methods in parallel for wildlife studies. We will focus on the genus Plasmodium, with an emphasis on the distribution and pathogenicity of these parasites in wild birds in Brazil.

Depletion of CD25+ cells during acute toxoplasmosis does not significantly increase mortality in Swiss OF1 mice

The interleukin (IL)-2R alpha chain (CD25) is expressed on regulatory T cells (Treg), which constitute more than 85% of the CD25+ T cell population in a naïve mouse. CD25 is also expressed on effector T cells in mice suffering from an acute infection by the obligate intracellular protozoan parasite, Toxoplasma gondii. Lethal toxoplasmosis is accompanied by a significant loss of Treg in mice naturally susceptible to toxoplasmosis. The present study was done to explore the role of Treg cells using an anti-CD25 antibody-mediated depletion in mice naturally resistant to toxoplasmosis. Although a significant decrease in the percentage of Treg cells was observed following anti-CD25 monoclonal antibody injections, the depletion of CD25+ cells during acute toxoplasmosis did not significantly increase the mortality of Swiss OF1 mice and no significant difference was observed in the brain parasitic load between the mice in the depleted-infected and isotype-infected groups. We found no significant difference between the titres of total IgG in the sera of the mice from the two groups in the chronic phase. However, CD25+ cells depletion was followed by significantly higher levels of IL-12 in the serum of depleted mice than in that of mice injected with the isotype control antibody.

The zinc finger protein TcZFP2 binds target mRNAs enriched during Trypanosoma cruzi metacyclogenesis

Trypanosomes are parasitic protozoa in which gene expression is primarily controlled through the regulation of mRNA stability and translation. This post-transcriptional control is mediated by various families of RNA-binding proteins, including those with zinc finger CCCH motifs. CCCH zinc finger proteins have been shown to be essential to differentiation events in trypanosomatid parasites. Here, we functionally characterise TcZFP2 as a predicted post-transcriptional regulator of differentiation in Trypanosoma cruzi. This protein was detected in cell culture-derived amastigotes and trypomastigotes, but it was present in smaller amounts in metacyclic trypomastigote forms of T. cruzi. We use an optimised recombinant RNA immunopreciptation followed by microarray analysis assay to identify TcZFP2 target mRNAs. We further demonstrate that TcZFP2 binds an A-rich sequence in which the adenosine residue repeats are essential for high-affinity recognition. An analysis of the expression profiles of the genes encoding the TcZFP2-associated mRNAs throughout the parasite life cycle by microarray hybridisation showed that most of the associated mRNAs were upregulated in the metacyclic trypomastigote forms, also suggesting a role for TcZFP2 in metacyclic trypomastigote differentiation. Knockdown of the orthologous Trypanosoma brucei protein levels showed ZFP2 to be a positive regulator of specific target mRNA abundance.