Maria do Céu Sousa

Monoterpenic aldehydes as potential anti-Leishmania agents: Activity of Cymbopogon citratus and citral on L. infantum, L. tropica and L. major

In order to contribute for the search of new drugs for leishmaniasis, we study the susceptibility of Leishmania infantum, Leishmania tropica and Leishmania major to Cymbopogon citratus essential oil and major compounds, mrycene and citral. C. citratus and citral were the most active inhibiting L. infantum, L. tropica and L. major growth at IC(50) concentrations ranging from 25 to 52 μg/ml and from 34 to 42 μg/ml, respectively. L. infantum promastigotes exposed to essential oil and citral underwent considerable ultrastructural alterations, namely mitochondrial and kinetoplast swelling, autophagosomal structures, disruption of nuclear membrane and nuclear chromatin condensation. C. citratus essential oil and citral promoted the leishmanicidal effect by triggering a programmed cell death. In fact, the leishmanicidal activity was mediated via apoptosis as evidenced by externalization of phosphatidylserine, loss of mitochondrial membrane potential, and cell-cycle arrest at the G(0)/G(1) phase. Taken together, ours findings lead us to propose that citral was responsible for anti-Leishmania activity of the C. citratus and both may represent a valuable source for therapeutic control of leishmaniasis.

Anti-Giardia activity of Syzygium aromaticum essential oil and eugenol: Effects on growth, viability, adherence and ultrastructure

The present work evaluates the anti-Giardia activity of Syzygium aromaticum and its major compound eugenol. The effects were evaluated on parasite growth, adherence, viability and ultrastructure. S. aromaticum essential oil (IC(50)=134 μg/ml) and eugenol (IC(50)=101 μg/ml) inhibited the growth of G. lamblia. The essential oil inhibited trophozoites adherence since the first hour of incubation and was able to kill almost 50% of the parasites population in a time dependent manner. The eugenol inhibited G. lamblia trophozoites adherence since the third hour and not induce cell lyses. The main morphological alterations were modifications on the cell shape, presence of precipitates in the cytoplasm, autophagic vesicles, internalization of flagella and ventral disc, membrane blebs, and intracellular and nuclear clearing. Taken together, our findings lead us to propose that eugenol was responsible for the anti-giardial activity of the S. aromaticum essential oil and both have potential for use as therapeutic agents against giardiasis.

Anti-Giardia activity of phenolic-rich essential oils: effects of Thymbra capitata, Origanum virens, Thymus zygis subsp. sylvestris, and Lippia graveolens on trophozoites growth, viability, adherence, and ultrastructure.

The present work evaluates the anti-Giardia activity of phenolic-rich essential oils obtained from Thymbra capitata, Origanum virens, Thymus zygis subsp. sylvestris chemotype thymol, and Lippia graveolens aromatic plants. The effects were evaluated on parasite growth, cell viability adherence, and morphology. The tested essential oils inhibited the growth of Giardia lamblia. T. capitata essential oil is the most active followed by O. virens, T. zygis subsp. sylvestris, and L. graveolens oils. The tested essential oils at IC50 (71–257) μg/ml inhibited parasite adherence (p < 0.001) since the first hour of incubation and were able to kill almost 50% of the parasites population in a time-dependent manner. The main ultrastructural alterations promoted by essential oils were deformations in typical trophozoite appearance, often roundly shape, irregular dorsal and ventral surface, presence of membrane blebs, electrodense precipitates in cytoplasm and nuclei, and internalization of flagella and ventral disc. Our data suggest that essential oils induced cell death probably by processes associated to the loss of osmoregulation caused by plasmatic membrane alterations. Experiments revealed that the essential oils did not present cytotoxic effects in mammalian cells. In conclusion, T. capitata, O. virens, T. zygis subsp. sylvestris chemotype thymol, and L. graveolens essential oils have antigiardial activity in vitro and seem to have potential for the treatment of the parasitic disease caused by the protozoan G. lamblia.

Identification and determination of the viability of Giardia lamblia cysts and Cryptosporidium parvum and Cryptosporidium hominis oocysts in human fecal and water supply samples by fluorescent in situ hybridization (FISH) and monoclonal antibodies

In the present study, fluorescent in situ hybridization (FISH) and monoclonal antibodies (MAbs) were evaluated for species-specific detection and viability determination of Giardia lamblia, Cryptosporidium parvum, and Cryptosporidium hominis in human fecal and water supply samples. A total of 50 fecal human samples positive for G. lamblia cysts, 38 positive for C. parvum, and 23 positive for C. hominis were studied. Also, 18 water supply samples positive for Giardia spp. and Cryptosporidium spp. by the United States Environmental Protection Agency (USEPA) Method 1623 were studied by FISH and fluorescein isothiocyanate (FITC)-conjugated MAbs. Eighteen percent of the fecal samples parasitologically positive for G. lamblia presented viable and nonviable cysts, and 5% of those positive for Cryptosporidium spp. presented viable and nonviable oocysts. Of the 18 water supply samples analyzed, 6 (33%) presented Giardia spp. viable and nonviable cysts and 2 (11%) presented viable and nonviable Cryptosporidium spp. oocysts. G. lamblia identification was confirmed by polymerase chain reaction (PCR) and sequencing of the β-giardin gene in the fecal and water samples found positive by FISH and FITC-conjugated MAbs. C. parvum and Cryptosporidium muris were identified, by PCR and sequencing of the small subunit of ribosomal RNA gene, in seven and one water samples, respectively. Our results confirm that this technique enables simultaneous visualization, species-specific identification, and viability determination of the organisms present in human fecal and water supply samples.

Antileishmanial Activity of Semisynthetic Lupane Triterpenoids Betulin and Betulinic Acid Derivatives: Synergistic Effects with Miltefosine

Leishmaniasis is a neglected tropical disease (NTDs), endemic in 88 countries, affecting more than 12 million people. The treatment consists in pentavalent antimony compounds, amphotericin B, pentamidine and miltefosine, among others. However, these current drugs are limited due to their toxicity, development of biological resistance, length of treatment and high cost. Thus, it is important to continue the search for new effective and less toxic treatments. The anti-Leishmania activity of sixteen semisynthetic lupane triterpenoids derivatives of betulin (BT01 to BT09) and betulinic acid (AB10 to AB16) were evaluated. Drug interactions between the active compounds and one current antileishmanial drug, miltefosine, were assessed using the fixed ratio isobologram method. In addition, effects on the cell cycle, apoptosis/necrosis events, morphology and DNA integrity were studied. The derivatives BT06 (3β-Hydroxy-(20R)-lupan-29-oxo-28-yl-1H-imidazole-1-carboxylate) and AB13 (28-(1H-imidazole-1-yl)-3,28-dioxo-lup-1,20(29)-dien-2-yl-1H-imidazole-1-carboxylate) were found to be the most active, with IC50 values of 50.8 µM and 25.8 µM, respectively. Interactions between these two compounds and miltefosine were classified as synergistic, with the most effective association being between AB13 and miltefosine, where decreases of IC50 values to 6 µM were observed, similar to the miltefosine activity alone. AB13 induced significant morphological changes, while both derivatives produced anti-proliferative activity through cell cycle arrest at the G0/G1 phase. Neither of these derivatives induced significant apoptosis/necrosis, as indicated by phosphatidylserine externalization and DNA fragmentation assays. In addition, neither of the derivatives induced death in macrophage cell lines. Thus, they do not present any potential risk of toxicity for the host cells. This study has identified the betulin derivative BT06 and the betulinic acid derivative AB13 as promising molecules in the development of new alternative therapies for leishmaniasis, including those involving combined-therapy with miltefosine.

Activity of Thymus capitellatus volatile extract, 1,8-cineole and borneol against Leishmania species.

In the search for new leishmanicidal agents, Thymus capitellatus Hoffmanns. & Link (family Lamiaceae) volatile extract and its major compounds, 1,8-cineole and borneol, were tested against Leishmania infantum, Leishmania tropica and Leishmania major. Plant volatile extract (essential oil) was analysed by GC and GC-MS and the activity of essential oil on Leishmania promastigotes viability was assessed using tetrazolium-dye colorimetric method (MTT). The MTT test was also used to assess the cytotoxicity of essential oil on macrophages and bovine aortic endothelial cells. Effects on parasites were also analyzed by flow cytometry in order to assess mitochondrial transmembrane electrochemical gradient (JC-1), analyze phosphatidylserine externalization (annexin V-FITC, propidium iodide) and evaluate cell cycle (DNase-free, RNase, PI). Morphological and ultrastructural studies were performed by light, scanning and transmission electron microscopy. T. capitellatus volatile extract exhibited anti-parasite activity on Leishmania species, with IC50 values ranging from 35 to 62 μg/ml. However, major compounds 1,8-cineole and borneol did not showed biological activity suggesting that these monoterpenes are not responsible for the antileishmanial activity of T. capitellatus essential oil. Appearance of aberrant-shaped cells, mitochondrial swelling and autophagosomal structures were some of the ultrastructural alterations exhibited among treated promastigote cells. T. capitellatus promoted leishmanicidal effect by triggering a programmed cell death as evidenced by externalization of phosphatidylserine, loss of mitochondrial membrane potential, and cell-cycle arrest at the G(0)/G(1) phase. The volatile extract did not induced cytotoxic effects on mammalian cells. Taken together, these results suggest that T. capitellatus may represent a valuable source for therapeutic control of leishmaniasis in humans and animals.

Geospatial distribution of intestinal parasitic infections in Rio de Janeiro (Brazil) and its association with social determinants

Background Intestinal parasitic infections remain among the most common infectious diseases worldwide. This study aimed to estimate their prevalence and provide a detailed analysis of geographical distribution of intestinal parasites in the metropolitan region of Rio de Janeiro, considering demographic, socio-economic, and epidemiological contextual factors. Methods/Principal findings The cross-section survey was conducted among individuals attending the Evandro Chagas National Institute of Infectious Diseases (FIOCRUZ, RJ) during the period from April 2012 to February 2015. Stool samples were collected and processed by sedimentation, flotation, Kato-Katz, Baermann-Moraes and Graham methods, iron haematoxylin staining and safranin staining. Of the 3245 individuals analysed, 569 (17.5%) were infected with at least one parasite. The most common protozoa were Endolimax nana (28.8%), Entamoeba coli (14.8%), Complex Entamoeba histolytica/Entamoeba dispar (13.5%), Blastocystis hominis (12.7%), and Giardia lamblia (8.1%). Strongyloides stercoralis (4.3%), Schistosoma mansoni (3.3%), Ascaris lumbricoides (1.6%), and hookworms (1.5%) were the most frequent helminths. There was a high frequency of contamination by protozoa (87%), and multiple infections were observed in 141 participants (24.8%). A positive association between age (young children) and gender (male) with intestinal parasites was observed. Geospatial distribution of the detected intestinal parasitic infections was not random or homogeneous, but was influenced by socioeconomic conditions (through the material deprivation index (MDI)). Participants classified in the highest levels of deprivation had higher risk of having intestinal parasites. Conclusions/Significance This study provides the first epidemiological information on the prevalence and distribution of intestinal parasitic infections in the Rio de Janeiro metropolitan area. Intestinal parasites, especially protozoa, are highly prevalent, indicating that parasitic infections are still a serious public health problem. MDI showed that intestinal parasites were strongly associated with the socioeconomic status of the population, thus making it possible to identify social vulnerable areas.

Molecular Characterization of Giardia lamblia: First Report of Assemblage B in Human Isolates from Rio de Janeiro (Brazil).

Background Despite the high prevalence of giardiasis, the genetic characterization of Giardia lamblia has been poorly documented in Brazil and molecular epidemiology research has only been conducted in the last few years. The aim of this study was to determine the prevalence of different G. lamblia assemblages and detect mixed infections among patients with giardiasis. Methods and Principal Findings The cross-section survey was conducted among patients attending the FIOCRUZ in Rio de Janeiro. In order to discriminate the genetic assemblages/sub-assemblages, G. lamblia isolates were characterized by PCR-RFLP and qPCR using four loci genes (bg, gdh, tpi and orfC4). Of the 65 positive samples, 41 (63.1%) were successfully amplified by nested-PCR of bg and gdh genes. Among them, 16 were typed as sub-assemblage AII, 7 as BIII, 4 as BIV and 8 as a mixture of BIII and BIV. After the analysis by qPCR assay, a total of 55 (84.6%) samples were amplified using at least one locus: bg gene was amplified in 38 (58.5%) samples, gdh in 41 (63.1%), tpi in 39 (60%), and orfC4 in 39 (60%). Multilocus genotyping results showed that 29 (52.7%) samples belonged to Assemblage A and 26 (47.3%) samples belonged to Assemblage B. In 2011 and 2012, 20 (74.1%) samples belonged to Assemblage A and 7 (25.9%) belonged to Assemblage B. In subsequent years (2013–2015) there was a predominance of Assemblage B, 19 (67.9%) versus 9 (32.1%) Assemblage A. Conclusions This is the first time that Assemblage B of G. lamblia was reported in human clinical samples from Rio de Janeiro (Brazil) and is the first report about genetic characterization using four genes. The qPCR assemblage-specific showed no mixed infections by Assemblages A and B. A switch in genetic profile over the years was observed, firstly predominance of Assemblage A and lastly of Assemblage B.

Draft Genome Sequence of Stenotrophomonas maltophilia Strain EPM1, Found in Association with a Culture of the Human Parasite Giardia duodenalis

ABSTRACT We report the draft genome sequence of the Stenotrophomonas maltophilia strain EPM1, found in association with a culture of Giardia duodenalis. The draft genome sequence of S. maltophilia strain EPM1, obtained with Roche 454 GS-FLX Titanium technology, is composed of 19 contigs totaling 4,785,869 bp, with a G+C content of 66.37%.

New multilocus genotypes of Giardia lamblia human isolates

Giardia lamblia is considered a species complex, whose members show little differences in their morphology, but have remarkable genetic variability. The aim of this study was to identify inter- and intra-assemblage genetic variation in G. lamblia among patients in Rio de Janeiro. The parasitological study was performed on faeces, and DNA was extracted from the samples which tested positive for G. lamblia. The genetic assemblages and subtypes were determined via multilocus sequence typing (MLST) using β-giardin, triose phosphate isomerase and glutamate dehydrogenase gene loci. Fourteen assemblage A samples were successfully genotyped at the three MLST loci (bg/tpi/gdh). Two previously identified multilocus genotypes were found (AII-1 and AII-4), and two novel multilocus genotypes are proposed (AII-8, profile A2/A2/A4; AII-9, profile A3/A2/A2). Sequence analysis showed that assemblage B isolates have a higher nucleotide variation than those from assemblage A. Novel assemblage B sequences are described and most (66.7%) have heterogeneous nucleotides, which prevent the definition of multilocus genotypes. This is the first time that MLST has been used to characterise G. lamblia isolates in human clinical samples from Rio de Janeiro. In addition, MLST has enabled the detection of novel subtypes in both assemblages and the description of two novel multilocus genotypes in assemblage A. This study provides new insights into the genetic diversity of assemblage A and shows that MLST should be used to characterise G. lamblia both in Brazil and globally.