Characterization of Pinus spp. needles by gas chromatography and mass spectrometry: Application to plant-insect interactions

Dissertação apresentada para obtenção do Grau de Doutor em Ciências do Ambiente pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecn

Evaluation of a rapid dipstick test, Malar-CheckTM, for the diagnosis of Plasmodium falciparum malaria in Brazil

The present study was carried out to evaluate the Malar-CheckTM Pf test, an immunochromatographic assay that detects Plasmodium falciparum Histidine Rich Protein II, does not require equipment, and is easy and rapid to perform. In dilution assays performed to test sensitivity against known parasite density, Malar-CheckTMwere compared with thick blood smear (TBS), the gold standard for diagnosis. Palo Alto isolate or P. falciparum blood from patients with different parasitemias was used. The average cut-off points for each technique in three independent experiments were 12 and 71 parasites/mm³ (TBS and Malar-CheckTM, respectively). In the field assays, samples were collected from patients with fever who visited endemic regions. Compared to TBS, Malar-CheckTMyielded true-positive results in 38 patients, false-positive results in 3, true-negative results in 23, and false-negative result in 1. Malar-CheckTMperformed with samples from falciparum-infected patients after treatment showed persistence of antigen up to 30 days. Malar-CheckTM should aid the diagnosis of P. falciparum in remote areas and improve routine diagnosis even when microscopy is available. Previous P. falciparum infection, which can determine a false-positive test in cured individuals, should be considered. The prompt results obtained with the Malar-CheckTM for early diagnosis could avoid disease evolution to severe cases.

PCR - based diagnosis to evaluate the performance of malaria reference centers

Although the Giemsa-stained thick blood smear (GTS) remains the gold standard for the diagnosis of malaria, molecular methods are more sensitive and specific to detect parasites and can be used at reference centers to evaluate the performance of microscopy. The description of the Plasmodium falciparum, P. vivax, P. malariae and P. ovale ssrRNA gene sequences allowed the development of a polymerase chain reaction (PCR) that had been used to differentiate the four species. The objective of this study was to determine Plasmodium species through PCR in 190 positive smears from patients in order to verify the quality of diagnosis at SUCEN's Malaria Laboratory. Considering only the 131 positive results in both techniques, GTS detected 4.6% of mixed and 3.1% of P. malariae infections whereas PCR identified 19.1% and 13.8%, respectively.

Cytotoxicity Induced by Extracts of Pisolithus tinctorius Spores on Human Cancer and Normal Cell Lines—Evaluation of the Anticancer Potential

Fungi have been considered a potential source of natural anticancer drugs. However, studies on these organisms have mainly focused on compounds present in the sporocarp and mycelium. The aim of this study was to assess the anticancer potential of fungal spores using a bioassay-guided fractionation with cancer and normal cell lines. Crude extracts from spores of the basidiomycetous fungus Pisolithus tinctorius were prepared using five solvents/solvent mixtures in order to select the most effective crude extraction procedure. A dichloromethane/methanol (DCM/MeOH) mixture was found to produce the highest extraction yield, and this extract was fractionated into 11 fractions. Crude extracts and fractions were assayed for cytotoxicity in the human osteocarcinoma cell line MG63, the human breast carcinoma cell line T47D, the human colon adenocarcinoma cell line RKO, and the normal human brain capillary endothelial cell line hCMEC/D3. Cytotoxicity was assessed by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction assay. The results showed a reduction in cancer cell viability of approximately 95% with 4 of 11 fractions without a significant reduction in viability of hCMEC/D3 cells. Data demonstrated that spores of P. tinctorius might serve as an interesting source of compounds with potential anticancer properties.

Analysis of the genetic polymorphism of Paracoccidioides brasiliensis and Paracoccidioides cerebriformis "Moore" by random amplified polymorphic DNA (RAPD) and 28S ribosomal DNA sequencing: Paracoccidioides cerebriformis revisited

Our purpose was to compare the genetic polymorphism of six samples of P. brasiliensis (113, 339, BAT, T1F1, T3B6, T5LN1), with four samples of P. cerebriformis (735, 741, 750, 361) from the Mycological Laboratory of the Instituto de Medicina Tropical de São Paulo, using Random Amplified Polymorphic DNA Analysis (RAPD). RAPD profiles clearly segregated P. brasiliensis and P. cerebriformis isolates. However, the variation on band patterns among P. cerebriformis isolates was high. Sequencing of the 28S rDNA gene showed nucleotide conservancy among P. cerebriformis isolates, providing basis for taxonomical grouping, and disclosing high divergence to P. brasiliensis supporting that they are in fact two distinct species. Moreover, DNA sequence suggests that P. cerebriformis belongs in fact to the Aspergillus genus.

Virulence profile of ten Paracoccidioides brasiliensis isolates: association with morphologic and genetic patterns

Ten isolates of Paracoccidioides brasiliensis were examined for differences in virulence in outbred mice intravenously inoculated with the fungus, associated with mycelial morphology, and genetic patterns measured by random amplified polymorphic DNA (RAPD). Virulence was evaluated by viable yeast cell recovery from lungs and demonstration of histopathologic lesions in different organs. The results showed that the isolates presented four virulence degrees: high virulence, intermediate, low and non-virulence. RAPD clustered the isolates studied in two main groups with 56% of genetic similarity. Strains with low virulence, Pb265 or the non-virulent, Pb192, showed glabrous/cerebriform morphology and high genetic similarity (98.7%) when compared to the other isolates studied. The same was observed with Bt79 and Bt83 that shared 96% genetic similarity, cottony colonies and high virulence. The RAPD technique could only discriminate P. brasiliensis isolates according to glabrous/cerebriform or cottony colonies, and also high from low virulence strains. Isolates with intermediate virulence such as Pb18, Pb18B6, Bt32 and Bt56 showed variability in their similarity coefficient suggesting that RAPD was able to detect genetic variability in this fungal specie. Virulence profile of P. brasiliensis demonstrated that both mycelial morphologic extreme phenotypes may be associated with fungal virulence and their in vitro subculture time. Thus, RAPD technique analysis employed in association with virulence, morphologic and immunologic aspects might prove adequate to detect differences between P. brasiliensis isolates.

Inhibitory effect of deferoxamine on Paracoccidioides brasiliensis survival in human monocytes: reversal by holotransferrin not by apotransferrin

The mechanisms used by Paracoccidioides brasiliensis to survive into phagocytic cells are not clear. Cellular iron metabolism is of critical importance to the growth of several intracellular pathogens whose capacity to multiply in mononuclear phagocytes is dependent on the availability of intracellular iron. Thus, the objective of this paper was to investigate the role of intracellular iron in regulating the capacity of P. brasiliensis yeast cells to survive within human monocytes. Treatment of monocytes with deferoxamine, an iron chelator, suppressed the survival of yeasts in a concentration-dependent manner. The effect of deferoxamine was reversed by iron-saturated transferrin (holotransferrin) but not by nonsaturated transferrin (apotransferrin). These results strongly suggest that P. brasiliensis survival in human monocytes is iron dependent.

Epidemiological and ecological aspects related to malaria in the area of influence of the lake at Porto Primavera dam, in western São Paulo State, Brazil

A study was carried out in the area of influence of the Porto Primavera Hydroelectric Power Station, in western São Paulo State, to investigate ecological and epidemiological aspects of malaria in the area and monitor the profile of the anopheline populations following the environmental changes brought about by the construction of the lake. Mosquitoes captured were analyzed by standardized indicator species analysis (ISA) before and during different flooding phases (253 m and 257 m elevations). The local human population was studied by means of parasitological (thin/thick blood smears), molecular (PCR) and serological tests. Serological tests consisted of Enzyme Linked Immunosorbent Assay (ELISA) with synthetic peptides of the circumsporozoite protein (CSP) from classic Plasmodium vivax, P. vivax variants (VK247 and "vivax-like"), P. malariae and P. falciparum and Indirect Immunofluorescence Assay (IFA) with asexual forms of P. vivax, P. malariae and P. falciparum. The results of the entomological survey indicated that, although the Anopheles darlingi population increased after the flooding, the population density remained very low. No malaria, parasite infection or DNA was detected in the inhabitants of the study area. However, there was a low frequency of antibodies against asexual forms and a significant prevalence of antibodies against P. vivax, P. vivax variants, P. falciparum and P. malariae; the presence of these antibodies may result from recent or less recent contact with human or simian Plasmodium (a parallel study in the same area revealed the existence of a sylvatic cycle). Nevertheless, these results suggest that, as in other places where malaria is present and potential vectors circulate, the local epidemiological conditions observed could potentially support the transmission of malaria in Porto Primavera Lake if infected individuals are introduced in sufficient numbers. Further studies are required to elucidate the phenomena described in this paper.

Quantification and viability analyses of Pseudokirchneriella subcapitata algal cells using image-based cytometry

This work aims to evaluate the feasibility of using image-based cytometry (IBC) in the analysis of algal cell quantification and viability, using Pseudokirchneriella subcapitata as a cell model. Cell concentration was determined by IBC to be in a linear range between 1 × 105 and 8 × 106 cells mL−1. Algal viability was defined on the basis that the intact membrane of viable cells excludes the SYTOX Green (SG) probe. The disruption of membrane integrity represents irreversible damage and consequently results in cell death. Using IBC, we were able to successfully discriminate between live (SG-negative cells) and dead algal cells (heat-treated at 65 °C for 60 min; SG-positive cells). The observed viability of algal populations containing different proportions of killed cells was well correlated (R 2 = 0.994) with the theoretical viability. The validation of the use of this technology was carried out by exposing algal cells of P. subcapitata to a copper stress test for 96 h. IBC allowed us to follow the evolution of cell concentration and the viability of copper-exposed algal populations. This technology overcomes several main drawbacks usually associated with microscopy counting, such as labour-intensive experiments, tedious work and lack of the representativeness of the cell counting. In conclusion, IBC allowed a fast and automated determination of the total number of algal cells and allowed us to analyse viability. This technology can provide a useful tool for a wide variety of fields that utilise microalgae, such as the aquatic toxicology and biotechnology fields.

Use of a fluorescence-based approach to assess short-term responses of the alga Pseudokirchneriella subcapitata to metal stress

This work explores the use of fluorescent probes to evaluate the responses of the green alga Pseudokirchneriella subcapitata to the action of three nominal concentrations of Cd(II), Cr(VI), Cu(II) and Zn(II) for a short time (6 h). The toxic effect of the metals on algal cells was monitored using the fluorochromes SYTOX Green (SG, membrane integrity), fluorescein diacetate (FDA, esterase activity) and rhodamine 123 (Rh123, mitochondrial membrane potential). The impact of metals on chlorophyll a (Chl a) autofluorescence was also evaluated. Esterase activity was the most sensitive parameter. At the concentrations studied, all metals induced the loss of esterase activity. SG could be used to effectively detect the loss of membrane integrity in algal cells exposed to 0.32 or 1.3 μmol L−1 Cu(II). Rh123 revealed a decrease in the mitochondrial membrane potential of algal cells exposed to 0.32 and 1.3 μmol L−1 Cu(II), indicating that mitochondrial activity was compromised. Chl a autofluorescence was also affected by the presence of Cr(VI) and Cu(II), suggesting perturbation of photosynthesis. In conclusion, the fluorescence-based approach was useful for detecting the disturbance of specific cellular characteristics. Fluorescent probes are a useful diagnostic tool for the assessment of the impact of toxicants on specific targets of P. subcapitata algal cells.

Responses of the alga Pseudokirchneriella subcapitata to long-term exposure to metal stress

The green alga Pseudokirchneriella subcapitata has been widely used in ecological risk assessment, usually based on the impact of the toxicants in the alga growth. However, the physiological causes that lead algal growth inhibition are not completely understood. This work aimed to evaluate the biochemical and structural modifications in P. subcapitata after exposure, for 72 h, to three nominal concentrations of Cd(II), Cr(VI), Cu(II) and Zn(II), corresponding approximately to 72 h-EC10 and 72 h-EC50 values and a high concentration (above 72 h-EC90 values). The incubation of algal cells with the highest concentration of Cd(II), Cr(VI) or Cu(II) resulted in a loss of membrane integrity of ~16, 38 and 55%, respectively. For all metals tested, an inhibition of esterase activity, in a dose-dependent manner, was observed. Reduction of chlorophyll a content, decrease of maximum quantum yield of photosystem II and modification of mitochondrial membrane potential was also verified. In conclusion, the exposure of P. subcapitata to metals resulted in a perturbation of the cell physiological status. Principal component analysis revealed that the impairment of esterase activity combined with the reduction of chlorophyll a content were related with the inhibition of growth caused by a prolonged exposure to the heavy metals.

Geographical distribution and intra-domiciliary capture of sylvatic triatomines in La Convención province, Cusco, Peru

The geographical distribution and intra-domiciliary capture of sylvatic triatomines in three districts of the province of La Convención, Cusco, Peru are presented. In the district of Vilcabamba, eight adults of Rhodnius pictipes and five adults of Panstrongylus geniculatus were found. In the district of Ocobamba, 19 adults, 14 nymphs, and eggs of P. rufotuberculatus were found. In the district of Echarate, six adults and 10 nymphs of Eratyrus mucronatus, an adult of R. pictipes and P. geniculatus, and a nymph of P. rufotuberculatus were also found. The geographical distribution of E. mucronatus has extended to Cusco. This is the first report in Peru of household colonization by this triatomine.

Melanoides tuberculata as intermediate host of Philophthalmus gralli in Brazil

Melanoides tuberculata that naturally harbored trematode larvae were collected at the Pampulha dam, Belo Horizonte (Minas Gerais, Brazil), during malacological surveys conducted from 2006 to 2010. From 7,164 specimens of M. tuberculata collected, 25 (0.35%) were infected by cercariae, which have been morphologically characterized as belonging to the Megalurous group, genus Philophthalmus. Excysted metacercariae were used for successful experimental infection of Gallus gallus domesticus, and adult parasites recovered from the nictitating membranes of chickens were identified as Philophthalmus gralli. This is the first report of P. gralli in M. tuberculata in Brazil.

The use of long acting sulfonamides, alone or with pyrimethamine, in malaria (with special reference to sulformetoxine)

Review of the early literature as well as more recent results show that sulfonamides possess a distinct antimalarial activity. However, when give alone, their action is less marked and slower than that of the antimalarials commonly used in the treatment of the acute attack. Combinations with pyrimethamine provide better results, even in cases of pyrimethamine and chloroquine resistance. This warrants further investigations in an attempt to develop a therapeutic agent suitable for the treatment of such resistant cases. It may also be possible with an appropriate combination of pyrimethamine with a sulfonamide to achieve a satisfactory method for suppressive treatment both in areas with and without pyrimethamine resistance. Three main points must still be carefully studied: 1) the risk of developing malaria resistance against one or both of the components of the combination. 2) The risk of developing bacterial resistance to sulfonamides if these substances are used on a large scale in too low doses. It seems indeed that antimalarial effect with the combination of sufonamides + pyrimethamine can be obtained with doses of sulfonamides which are below those usually employed in bacterial diseases. Since the range of the ratios providing potentiation is rather large, only ratios of the combination sulfonamides: pyrimethamine should be chosen in which an antfbacterial sulfonamidemia is guaranteed. 3) It goes without sayinq that, although both pyrimethamine and modem sulfonamides, when given by themselves, have proved tc possess a large margin of safety, long term administration of their combination should be careful studied from the point of view of possible side effects. Substantial evidence has already been produced to show that the long acting sulfonamide Fanasil (Ro 4-4393) given once or once weekly possesses marked schizonticidal activity against P. falciparum. Although its action is slower than that of 4-aminoquinolines, it may be useful as a second choice drug in semi-immune subjects for the therapy of falciparum malaria. Preliminary results show that, when combined with pyrimethamine, Fanasil is highly effective in suppressing fever and asexual parasitemia due to P. falciparum. Single doses of 1 g Fanasil together with 50 mg pyrimethamine seem to be adequate for the treatment of acute falciparum malaria in semi-immune patients. The onset of action of the combination is much more rapid than that of the single components. Weekly doses of 500 mg Fanasil and 25 mg pyrimeihamine appear to provide satisfactory suppressive effects against P. falciparum at least in East Africa. This combination is active on strains which do not respond satisfactorily to the standard doses of pyrimethamine and/or chloroquine and seems to have a satisfactory sporontocidal effect. Preliminary results indicate that Fanasil alone cannot be recommended for use against the other human malaria parasites. The combination with pyrimethamine appears to be much more effective. East African strains of P. malariae seem to respond better to the combination than do Malayan strains of P. vivax but further trials are required before definite assessment can be made. Fanasil by itself has no gametocytoddal or sporontocidal action but seems to potentiate the effect of pyrimethamine at least on sporogony of P. falciparum.

Laboratory infection of the wild rodent, Sciurus alphonsei with Pasteurella pestis

The author studied the susceptibility of the wild rodent, Sciurus alphonsei (Sciuridae) from Brazil to Pasteurella pestis. Two strains of P. pestis isolated from wild rodents were used: one, P. EXU 19, was isolated from O. subflavus in the county of Em, Pernambuco (Brazil), and other, RANGEL, isolated from S. hispidus of Camp. Rangel (Venezuela) . Six animais were tested by inoculation through different routes (percutaneous, subcutaneous and peritoneal). All the animals died as a result of the infection.