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.

Molecular investigation of ready-to-eat salads for Giardia duodenalis and Cryptosporidium spp. in Portugal.

Foodborne outbreaks are often associated with the consumption of salads. However, published studies on the detection of foodborne pathogens in ready-to-eat salads are scarce. The aim of this study was to detect Giardia duodenalis and Cryptosporidium DNA in ready-to-eat salads, by applying techniques of molecular biology to study the frequency of contamination in salads. A total of 100 packages of ready-to-eat salads containing assorted leafy green vegetables were randomly purchased from hypermarkets located in central regions of Portugal (Coimbra and Viseu). Nested-PCR and qPCR methods were used to detect G. duodenalis and Cryptosporidium DNA. Species and assemblages of the parasites were identified by sequence analysis and PCR. Eighteen of the 100 samples (18%) were positive for G. duodenalis and twelve were sequenced and identified as assemblage A. Cryptosporidium spp. were not detected in any salads. Overall, pre-harvest and post-harvest preventive measures may be need for G. duodenalis control throughout the food production industry, from the field to consumers.

Evaluation of the drinking water quality surveillance system in the metropolitan region of Rio de Janeiro.

The present work evaluated the surveillance of the drinking water quality information system database and correlated the findings of the microbiological analysis with the distribution of intestinal protozoa from the metropolitan region of Rio de Janeiro. From the database, we obtained 1,654 georeferenced monitoring stations that were used in the analysis. The results indicate that the minimum number of samples collected per parameter (free residual chlorine, turbidity, counts of total and fecal coliforms (Escherichia coli)) was not fulfilled, the collection of samples throughout the year was irregular and the representability of sampling points considered strategic was low (48% of municipalities). Besides, municipalities with a high prevalence for intestinal parasite protozoa were also the ones that had the highest counts for coliforms and the reverse can also be observed, indicating a transmission through contaminated drinking water. Despite the increased participation of municipalities in water surveillance actions during the studied period, it is necessary to implement managerial measures to improve the system, aiming to correct flaws and inconsistencies in the application of the water quality monitoring protocol.

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.

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.

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.

Merozoite surface protein 2 allelic variation influences the specific antibody response during acute malaria in individuals from a Brazilian endemic area.

The antibody response to Plasmodium falciparum parasites of naturally infected population is critical to elucidate the role of polymorphic alleles in malaria. Thus, we evaluated the impact of antigenic diversity of repetitive and family dimorphic domains of the merozoite surface protein 2 (MSP-2) on immune response of 96 individuals living in Peixoto de Azevedo (MT-Brazil), by ELISA using recombinant MSP-2 proteins. The majority of these individuals were carrying FC27-type infections. IgG antibody responses were predominantly directed to FC27 parasites and were correlated to the extension of polymorphism presented by each MSP-2 region. This finding demonstrated the impact of the genetic polymorphism on antibody response and therefore, its importance on malaria vaccine efficacy.

Merozoite surface protein 2 allelic variation influences the specific antibody response during acute malaria in individuals from a Brazilian endemic area

The antibody response to Plasmodium falciparum parasites of naturally infected population is critical to elucidate the role of polymorphic alleles in malaria. Thus, we evaluated the impact of antigenic diversity of repetitive and family dimorphic domains of the merozoite surface protein 2 (MSP-2) on immune response of 96 individuals living in Peixoto de Azevedo (MT-Brazil), by ELISA using recombinant MSP-2 proteins. The majority of these individuals were carrying FC27-type infections. IgG antibody responses were predominantly directed to FC27 parasites and were correlated to the extension of polymorphism presented by each MSP-2 region. This finding demonstrated the impact of the genetic polymorphism on antibody response and therefore, its importance on malaria vaccine efficacy.