Lipidome of extracellular vesicles from Giardia lamblia.

Extracellular vesicles (EVs) (exossomes, microvesicles and apoptotic bodies) have been well acknowledged as mediators of intercellular communications in prokaryotes and eukaryotes. Lipids are essential molecular components of EVs but at the moment the knowledge about the lipid composition and the function of lipids in EVs is limited and as for now none lipidomic studies in Giardia EVs was described. Therefore, the focus of the current study was to conduct, for the first time, the characterization of the polar lipidome, namely phospholipid and sphingolipid profiles of G. lamblia trophozoites, microvesicles (MVs) and exosomes, using C18-Liquid Chromatography-Mass Spectrometry (C18-LC-MS) and Tandem Mass Spectrometry (MS/MS). A total of 162 lipid species were identified and semi-quantified, in the trophozoites, or in the MVs and exosomes belonging to 8 lipid classes, including the phospholipid classes phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), cardiolipins (CL), the sphingolipid classes sphingomyelin (SM) and ceramides (Cer), and cholesterol (ST), and 3 lipid subclasses that include lyso PC (LPC), lyso PE (LPE) and lyso PG (LPG), but showing different abundances. This work also identified, for the first time, in G. lamblia trophozoites, the lipid classes CL, Cer and ST and subclasses of LPC, LPE and LPG. Univariate and multivariate analysis showed clear discrimination of lipid profiles between trophozoite, exosomes and MVs. The principal component analysis (PCA) plot of the lipidomics dataset showed clear discrimination between the three groups. Future studies focused on the composition and functional properties of Giardia EVs may prove crucial to understand the role of lipids in host-parasite communication, and to identify new targets that could be exploited to develop novel classes of drugs to treat giardiasis.

Protozoa-Derived Extracellular Vesicles on Intercellular Communication with Special Emphasis on Giardia lamblia.

Parasitic diseases are an important worldwide problem threatening human health and affect millions of people. Acute diarrhea, intestinal bleeding, malabsorption of nutrients and nutritional deficiency are some of the issues related to intestinal parasitic infections. Parasites are experts in subvert the host immune system through different kinds of mechanisms. There are evidences that extracellular vesicles (EVs) have an important role in dissemination of the disease and in modulating the host immune system. Released by almost all types of cells, these nanovesicles are a natural secretory product containing multiple components of interest. The EVs are classified as apoptotic bodies, microvesicles, exosomes, ectosomes, and microparticles, according to their physical characteristics, biochemical composition and cell of origin. Interestingly, EVs play an important role in intercellular communication between parasites as well as with the host cells. Concerning Giardia lamblia, it is known that this parasite release EVs during it life cycle that modulate the parasite growth and adherence as well the immune system of the host. Here we review the recently updates on protozoa EVs, with particular emphasis on the role of EVs released by the flagellate protozoa G. lamblia in cellular communication and its potential for future applications as vaccine, therapeutic agent, drug delivery system and as diagnostic or prognostic biomarker.

Seroepidemiology study of Cytomegalovirus and Rubella in pregnant women in Luanda, Angola: geospatial distribution and its association with socio-demographic and clinical-obstetric determinants.

BACKGROUND: Both CMV and Rubella virus infections are associated with the risk of vertical transmission, fetal death or congenital malformations. In Angola, there are no reports of CMV and Rubella studies. Therefore, our objectives were to study the seroprevalence of anti-CMV and anti-Rubella antibodies in pregnant women of Luanda (Angola), identify the risk of primary infection during pregnancy and evaluate the socio-demographic risk factors associated with both infections. METHODS: A prospective cross-sectional study was conducted from August 2016 to May 2017. Specific anti-CMV and anti-Rubella antibodies were quantified by electrochemiluminescence and demographic and clinical data were collected using standardized questionnaire. Bivariate and multivariate logistic regression analysis were used to quantify the effect of clinical and obstetric risk factors on virus seroprevalence. RESULTS: We recruited 396 pregnant women aged from 15 to 47. Among them, 335 (84.6%) were immune to both CMV and Rubella virus infections, while 8 (2.0%) had active CMV infection and 4 (1.0%) active RV infection but none had an active dual infection. Five women (1.2%) were susceptible to only CMV infection, 43 (10.9%) to only RV infection, and 1 (0.3) to both infections. Multivariate analysis showed a significant association between Rubella virus infection and number of previous births and suffering spontaneous abortion. CONCLUSIONS: Overall, this study showed that there is a high prevalence of anti-CMV and anti-Rubella antibodies in pregnant women in Luanda. It also showed that a small but important proportion of pregnant women, about 11%, are at risk of primary infection with rubella during pregnancy. This emphasizes the need for vaccination.

Serological prevalence of toxoplasmosis in pregnant women in Luanda (Angola): Geospatial distribution and its association with socio-demographic and clinical-obstetric determinants.

We report a study on toxoplasmosis in pregnant women in Luanda, Angola, determining the seroprevalence, geospatial distribution and its association with socio-economic features, dietary habits and hygiene and health conditions. Anti-Toxoplasma gondii IgG and IgM were quantified in serum samples of women attended at the Lucrecia Paim Maternity Hospital between May 2016 and August 2017. The IgG avidity test and qPCR assay were used for dating the primary infection. Data were collected by questionnaire after written consent, and spatial distribution was assessed through a Kernel Density Function. The potential risk factors associated with Toxoplasma infection were evaluated using bivariate and multivariate binomial logistic regression analysis. Anti-T. gondii antibodies were quantified in 878 pregnant women, and 346 (39.4%) samples were IgG positive, 2 (0.2%) positive for IgM and IgG, and 530 (60.4%) negative for both immunoglobulins. The longitudinal study showed that none of the seronegative women seroconverted during the survey. Regarding other infections, 226 (25.7%) were positive for hepatitis B, while 118 (13.4%) were HIV-positive. The seroprevalence of toxoplasmosis was similar in most municipalities: 43.8% in Cazenga (28 of 64); 42.5% in Viana (88 of 207); 42.3% in Cacuaco (22 of 52); and 41.1% in Luanda ((179 of 435). In contrast, the seroprevalence in municipality of Belas was lower (25.8%; 31 of 120) and bivariate and multivariate analysis has shown a lower risk for toxoplasmosis in this area (OR 0.479, CI: 0.305-0.737; OR 0.471, CI: 0.299-0.728). The multivariate analysis has shown a significant increased risk for toxoplasmosis in women in the last trimester of pregnancy (OR 1.457, CI: 1.011-2.102), suffering spontaneous abortion (OR 1.863, CI: 1.014-3.465) and having pets at home (OR 1.658, CI: 1.212-2.269). Also, women who tested positive for hepatitis B (OR 1.375, CI: 1.008-1.874) and HIV (OR 1.833, CI: 1.233-2.730) had a significant increased risk for T. gondii infection. In conclusion, our study showed that a large number of pregnant women are not immunized for toxoplasmosis and identified the risk factors for this infection in Luanda. It is crucial to establish the diagnosis of primary maternal infection as well as the diagnosis of congenital toxoplasmosis. Our results underlined the need for diagnostic and clinical follow-up of toxoplasmosis, HIV and hepatitis B during pregnancy.

Giardia lamblia Decreases NF-κB p65RelA Protein Levels and Modulates LPS-Induced Pro-Inflammatory Response in Macrophages.

The protozoan Giardia lamblia is the most common cause of parasitic gastrointestinal infection worldwide. The parasite developed sophisticated, yet not completely disclosed, mechanisms to escape immune system and growth in the intestine. To further understand the interaction of G. lamblia with host immune cells, we investigated the ability of parasites to modulate the canonical activation of mouse macrophages (Raw 264.7 cell line) and human monocyte-derived macrophages triggered by the TLR4 agonist, lipopolysaccharide (LPS). We observed that G. lamblia impairs LPS-evoked pro-inflammatory status in these macrophage-like cells through inhibition of cyclooxygenase-2 and inducible nitric oxide synthase expression and subsequent NO production. This effect was in part due to the activity of three G. lamblia proteases, a 135 kDa metalloprotease and two cysteine proteases with 75 and 63 kDa, that cleave the p65RelA subunit of the nuclear factor-kappa B (NF-κB). Moreover, Tnf and Ccl4 transcription was increased in the presence of the parasite. Overall, our data indicates that G. lamblia modulates macrophages inflammatory response through impairment of the NF-κB, thus silencing a crucial signaling pathway of the host innate immune response.

Oral treatment with T6-loaded yeast cell wall particles reduces the parasitemia in murine visceral leishmaniasis model.

Yeast cell wall particles isolated from Saccharomyces cerevisiae (scYCWPs) have a rich constitution of ß-glucan derived from the cell wall. After removing intracellular contents, ß-glucan molecules are readily recognized by dectin-1 receptors, present on the cytoplasmic membrane surface of the mononuclear phagocytic cells and internalized. Leishmania spp. are obligate intracellular parasites; macrophages are its primary host cells. An experimental murine model of visceral leishmaniasis caused by L. infantum was used to evaluate the antileishmanial activity of oral administration of these particles. A low-water soluble thiophene previously studied in vitro against L. infantum was entrapped into scYCWPs to direct it into the host cell, in order to circumvent the typical pharmacokinetic problems of water-insoluble compounds. We found that scYCWPs + T6 reduced the parasitic burden in the liver and spleen. There was an increase in IFN-γ levels related to nitric oxide production, explaining the reduction of the L. infantum burden in the tissue. Histological analysis did not show signals of tissue inflammation and biochemical analysis from plasma did not indicate signals of cytotoxicity after scYCWPs + T6 treatment. These findings suggested that scYCWPs + T6 administered through oral route reduced the parasitic burden without causing toxic effects, satisfying requirements for development of new strategies to treat leishmaniasis.

Activity and Cell-Death Pathway in Leishmania infantum Induced by Sugiol: Vectorization Using Yeast Cell Wall Particles Obtained From Saccharomyces cerevisiae.

Visceral leishmaniasis, caused by Leishmania infantum, is a neglected tropical disease, to which efforts in the innovation of effective and affordable treatments remain limited, despite the rising incidence in several regions of the world. In this work, the antileishmanial effects of sugiol were investigated in vitro. This compound was isolated from the bark of Cupressus lusitanica and showed promising activity against L. infantum. In spite of the positive results, it is known that the compound is a poorly water-soluble diterpene molecule, which hinders further investigation, especially in preclinical animal studies. Thus, in an alternative delivery method, sugiol was entrapped in glucan-rich particles obtained from Saccharomyces cerevisiae yeast cell walls (YCWPs). To evaluate the activity of sugiol, the experiments were divided into two parts: (i) the in vitro investigation of antileishmanial activity of free sugiol against L. infantum promastigotes after 24, 48, and 72 h of treatment and (ii) the evaluation of antileishmanial activity of sugiol entrapped in glucan-rich particles against intracellular L. infantum amastigotes. Free sugiol induced the cell-death process in promastigotes, which was triggered by enhancing cytosolic calcium level and promoting the autophagy up to the first 24 h. Over time, the presence of autophagic vacuoles became rarer, especially after treatment with lower concentrations of sugiol, but other cellular events intensified, like ROS production, cell shrinkage, and phosphatidylserine exposure. Hyperpolarization of mitochondrial membrane potential was found at 72 h, induced by the mitochondria calcium uptake, causing an increase in ROS production and lipid peroxidation as a consequence. These events resulted in the cell death of promastigotes by secondary necrosis. Sugiol entrapped in glucan-rich particles was specifically recognized by dectin-1 receptor on the plasma membrane of macrophages, the main host cell of Leishmania spp. Electron micrographs revealed particles containing sugiol within the infected macrophages and these particles were active against the intracellular L. infantum amastigotes without affecting the host cell. Therefore, the YCWPs act like a Trojan horse to successfully deliver sugiol into the macrophage, presenting an interesting strategy to deliver water-insoluble drugs to parasitized cells.

Clotrimazole-Loaded Mediterranean Essential Oils NLC: A Synergic Treatment of Candida Skin Infections.

The increasing development of resistance of Candida species to traditional drugs represents a great challenge to the medical field for the treatment of skin infections. Essential oils were recently proposed to increase drug effectiveness. Herein, we developed and optimized (23 full factorial design) Mediterranean essential oil (Rosmarinus officinalis, Lavandula x intermedia "Sumian", Origanum vulgare subsp. hirtum) lipid nanoparticles for clotrimazole delivery, exploring the potential synergistic effects against Candida spp. Small sized nanoparticles (<100 nm) with a very broad size distribution (PDI < 0.15) and long-term stability were successfully prepared. Results of the in vitro biosafety on HaCaT (normal cell line) and A431 (tumoral cell line), allowed us to select Lavandula and Rosmarinus as anti-proliferative agents with the potential to be used as co-adjuvants in the treatment of non-tumoral proliferative dermal diseases. Results of calorimetric studies on biomembrane models, confirmed the potential antimicrobial activity of the selected oils due to their interaction with membrane permeabilization. Nanoparticles provided a prolonged in vitro release of clotrimazole. In vitro studies against Candida albicans, Candida krusei and Candida parapsilosis, showed an increase of the antifungal activity of clotrimazole-loaded nanoparticles prepared with Lavandula or Rosmarinus, thus confirming nanostructured lipid carriers (NLC) containing Mediterranean essential oils represent a promising strategy to improve drug effectiveness against topical candidiasis.

In vitro activities of glycoalkaloids from the Solanum lycocarpum against Leishmania infantum

ABSTRACT Leishmania infantum is an etiologic agent of visceral leishmaniasis. This disease is a neglected disease that can be fatal if not treated and additionally, the few therapeutic option present several drawbacks, including difficult route of administration and toxicity, which turn the search for new therapeutic alternatives necessary. Herein, we evaluated the leishmanicidal in vitro activity of the solanum extract from Solanum lycocarpum A. St.-Hil., Solanaceae, and the isolated alkaloids solasodine, solamargine and solasonine against promastigotes and intracellular amastigotes of L. infantum. Solasodine (IC50-pro = 4.7 µg/ml; IC50-ama = 10.8 µg/ml) and solamargine (IC50-pro = 8.1 µg/ml; IC50-ama = 3.0 µg/ml) exhibited interesting leishmanicidal ativity. Solasonine was approximately four-times (Selective Index 3.7) more selective to the parasite than to the host cells. This data suggest that solasonine might be considered as a potential drug candidate for leishmaniasis treatment.

In vitro anti-Leishmania activity of T6 synthetic compound encapsulated in yeast-derived ß-(1,3)-d-glucan particles.

The objective of this study was to encapsulate a synthetic compound, the 4-[(2E)-N'-(2,2'-bithienyl-5-methylene)hydra-zinecarbonyl]-6,7-dihydro-1-phenyl-1H-pyrazolo[3,4-d]pyridazin-7-one (T6) in glucan-rich particles mainly composed by the cell wall of Saccharomyces cerevisiae (GPs) and to study their individual and combined activity on Leishmania infantum. The possible mechanism of action of T6 was also investigated. Our results showed the activity of T6 compound in both promastigote (IC50 = 2.5 µg/mL) and intracellular amastigote (IC50 = 1.23 µg/mL) forms. We also found activity against intracellular amastigote forms (IC50 = 8.20 µg/mL) when the T6 compound was encapsulated in GPs. Another interesting finding was the fact that T6 encapsulated in GPs showed a significant decrease in J774A1 macrophage toxicity (CC50 ≥ 18.53 µg/mL) compared to the T6 compound alone (IC50 = 2.27 µg/mL). Through electron microscopy and biochemical methodologies, we verified that the activity of T6 in promastigote forms of L. infantum was characterized by events of cell death by apoptosis like increased ROS production, cell shrinkage, phosphatidylserine exposure and DNA fragmentation. We conclude that T6 can be considered a promising anti-Leishmania compound, and that the use of GPs for drug encapsulation is an interesting approach to the development of new effective and less toxic formulations.

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 ß-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.

Associations of Giardia lamblia assemblages with HIV infections and symptomatology: HIV virus and assemblage B were they born to each other?

Giardia lamblia is an intestinal parasite that has an extensive genetic variation among isolates. This species is divided into eight different assemblages (A-H), but only assemblages A and B have been associated with human infections. Studies on the associations of G. lamblia assemblages and symptoms have been done but were inconclusive. The aim of this study was to correlate G. lamblia assemblages with symptoms in patients with and without HIV/AIDS and its association with the CD4T cell count. The cross-sectional survey was conducted among patients attending the Evandro Chagas National Institute of Infectious Diseases (INI/FIOCRUZ) in Rio de Janeiro from January 2011 to February 2015. Thirty-eight of 65 microscopically positive stool samples for G. lamblia were from HIV positive patients and 27 were from HIV negative patients. Of the HIV infected patients, 19 (55.9%) were genotyped as assemblage B of which 9 (47.4%) had a CD4Tcell count below 200cells/mm3. In addition, we found a greater number of samples belonging to assemblage B in symptomatic cases (11 of 19; 57.9%). Our data suggest that assemblage B is very likely to be found in HIV infected patients and probably the lower CD4T count gives advantages for assemblage B replication. Furthermore, assemblage B seems to be associated with symptomatology, particularly abdominal pain, asthenia, diarrhea, fever, headache and myalgia. This study provides information on G. lamblia assemblages and symptoms in patients with and without HIV/AIDS virus and their association with CD4Tcell counts.

Composition and leishmanicidal activity of the essential oil of Vernonia polyanthes Less (Asteraceae).

The composition of the essential oil (EO) from leaves of Vernonia polyanthes and the evaluation of its leishmanicidal potential are reported here for the first time. The oil obtained by hydrodistillation was analysed by combination of GC and GC/MS. Thirty-five compounds were identified, representing 91.8% of the oil composition. The oil consists primarily of monoterpenes (37.1%), sesquiterpenes (26.3%) and oxygenated sesquiterpenes (23.9%), myrcene (34.3%), zerumbone (15.8%), bicyclogermacrene (8.9%), α-humulene (4.8%) and germacrene D (4.3%) being the major constituents. Activity against Leishmania infantum was determined using the tetrazolium dye (MTT) colorimetric method. The oil, as well as zerumbone, one of its major constituents, showed significant leishmanicidal activity, with IC50 values of 19.4 and 9.0 µg/ml, respectively. Cytotoxicity in macrophages cells was evaluated using the MTT colorimetric assay. The EO showed the CC50 < 10 µg/ml to macrophages cells.

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.

(R)-(-)-Aloesaponol III 8-Methyl Ether from Eremurus persicus: A Novel Compound against Leishmaniosis.

Leishmaniosis is a neglected tropical disease which affects several millions of people worldwide. The current drug therapies are expensive and often lack efficacy, mainly due to the development of parasite resistance. Hence, there is an urgent need for new drugs effective against Leishmania infections. As a part of our ongoing study on the phytochemical characterization and biological investigation of plants used in the traditional medicine of western and central Asia, in the present study, we focused on Eremurus persicus root extract in order to evaluate its potential in the treatment of leishmaniosis. As a result of our study, aloesaponol III 8-methyl ether (ASME) was isolated for the first time from Eremurus persicus root extract, its chemical structure elucidated by means of IR and NMR experiments and the (R) configuration assigned by optical activity measurements: chiroptical aspects were investigated with vibrational circular dichroism (VCD) and electronic circular dichroism (ECD) spectroscopies and DFT (density functional theory) quantum mechanical calculations. Concerning biological investigations, our results clearly proved that (R)-ASME inhibits Leishmania infantum promastigotes viability (IC50 73 µg/mL), inducing morphological alterations and mitochondrial potential deregulation. Moreover, it is not toxic on macrophages at the concentration tested, thus representing a promising molecule against Leishmania infections.

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.

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) microg/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.

Effects of essential oils on the growth of Giardia lamblia trophozoites.

Giardia lamblia is one of the most important worldwide causes of intestinal infections produced by protozoa. Current therapy for giardiasis is unsatisfactory due to high incidence of undesirable side effects and significant failure in clearing parasites from the gastrointestinal tract. In the search for new therapeutic agents, we report on the effect of several essential oils on G. lamblia growth. Among eighteen tested essential oils, those with phenolic compositions were the most active, particularly if containing high contents of carvacrol, such as Thymbra capitata and Origanum virens (IC50 values of 71 and 85 microg x mL(-1), respectively). The oils from Syzygium aromaticum and Thymus zygis subsp. sylvestris (IC50 values from 100 to 200 microg x mL(-1)), as well as, those from Mentha x piperita and Lippia graveolens (IC50 values over 200 microg x mL(-1)) were less active. Results support the concept that several essential oils or some of their constituents may be useful in the clinical management of Giardia infections.