Extracellular nicotinamide phosphoribosyltransferase (eNAMPT) neutralization counteracts T cell immune evasion in breast cancer.

BACKGROUND: Nicotinamide phosphoribosyltransferase (NAMPT) is a key intracellular enzyme that participates in nicotinamide adenine dinucleotide (NAD) homeostasis as well as a released cytokine (eNAMPT) that is elevated in inflammatory conditions and in cancer. In patients with breast cancer, circulating eNAMPT is elevated and its plasma levels correlate with prognosis and staging. In light of this, we investigated the contribution of eNAMPT in triple negative mammary carcinoma progression by investigating the effect of its neutralization via a specific neutralizing monoclonal antibody (C269). METHODS: We used female BALB/c mice injected with 4T1 clone 5 cells and female C57BL6 injected with EO771 cells, evaluating tumoral size, spleen weight and number of metastases. We injected two times a week the anti-eNAMPT neutralizing antibody and we sacrificed the mice after 28 days. Harvested tumors were analyzed by histopathology, flow cytometry, western blot, immunohistochemistry, immunofluorescence and RNA sequencing to define tumor characteristics (isolating tumor infiltrating lymphocytes and tumoral cells) and to investigate the molecular mechanisms behind the observed phenotype. Moreover, we dissected the functional relationship between T cells and tumoral cells using three-dimensional (3D) co-cultures. RESULTS: The neutralization of eNAMPT with C269 led to decreased tumor size and reduced number of lung metastases. RNA sequencing and functional assays showed that eNAMPT controlled T-cell response via the programmed death-ligand 1/programmed cell death protein 1 (PD-L1/PD-1) axis and its neutralization led to a restoration of antitumoral immune responses. In particular, eNAMPT neutralization was able to activate CD8+IFNγ+GrzB+ T cells, reducing the immunosuppressive phenotype of T regulatory cells. CONCLUSIONS: These studies indicate for the first time eNAMPT as a novel immunotherapeutic target for triple negative breast cancer.

Efficiency of a multi-barrier household system for surface water treatment combining a household slow sand filter to a Mesita Azul® ultraviolet disinfection device.

Low-cost household technologies for water treatment are crucial to improving drinking water quality and preventing health, social and economic impacts, mostly in middle- and low-income regions. This work assessed the removal efficiency of physical-chemical and bacteriological parameters from river water by a multi-barrier household water treatment system for 113 consecutive days. This system combines a pre-treatment step through a non-woven synthetic blanket, filtration by an intermittent household slow sand filter (HSSF) and a Mesita Azul® ultraviolet disinfection device. In general, the water quality was improved by the evaluated system. Turbidity was removed by an average of 73% (ranging from 33 to 94%), total coliforms (TC) of 3.88 log10 (ranging from 2.22 to 5.16 log10) and E. coli of 2.49 log10 (ranging from 1.81 to 3.30 log10). Filtration improvement was mostly correlated to HSSF biofilm development and influent water quality. Characterisation of HSSF schmutzdecke demonstrated a predominance of organic content, and a higher presence of carbohydrates than proteins on the sand and the blanket. Ultraviolet disinfection with Mesita Azul® inactivated most of the remaining bacteria after filtration and no regrowth was observed after 15 days of disinfection. In conclusion, the multi-barrier household water treatment system was efficient in treating river water, reducing risks of microbial contamination to achieve safe drinking water.

CFDA-AM staining to assess the metabolic activity of Giardia duodenalis cysts inactivated by chlorine, boiling and ultraviolet irradiation.

Giardia duodenalis is responsible for several waterborne gastrointestinal outbreaks worldwide. In addition to limitations presented by the main disinfection methods, assessing the inactivation efficiency of cysts after the treatment also poses challenges. Thus, this study aimed to use the 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM) staining protocol to evaluate the viability of G. duodenalis cysts inactivated by different UV and chlorination doses and boiling times. Under epifluorescent microscopy, metabolically active cysts that presented green fluorescence were considered viable. In contrast, when no green fluorescence could be observed, organisms were considered non-viable. Although statistical analysis revealed that increasing the UV dose did not significantly decrease the percentage of viable cysts, the fluorescence signal intensity decreased considerably when the cysts were irradiated with a dose equal to or greater than 80 mJ cm-2. Regarding chlorination and boiling treatments, this study demonstrated that no cyst showed fluorescence at the lowest NaClO concentration (0.5 mg/L) and in the shortest boiling time (2 min). Despite some limitations regarding the use of metabolic activity as a viability marker, this methodology is rapid, easy to run and cost-effective. Thus, we conclude that the CFDA-AM staining protocol has the potential to be used to assess Giardia cyst inactivation, although further research is required.

Label-free detection and enumeration of Giardia cysts in agitated suspensions using in situ microscopy.

Laboratory procedures performed in water treatment studies frequently require the characterization of (oo)cyst suspensions. Standard methods commonly used are laborious, expensive and time-consuming, besides requiring well-trained personnel to prepare samples with fluorescent staining and perform analysis under fluorescence microscopy. In this study, an easy cost-effective in situ microscope was assessed to acquire images of Giardia cysts directly from agitated suspensions without using any chemical labels or sample preparation steps. An image analysis algorithm analyzes the acquired images, and automatically enumerates and provides morphological information of cysts within 10 min. The proposed system was evaluated at different cyst concentrations, achieving a limit of detection of ~30 cysts/mL. The proposed system overcomes cost, time and labor demands by standard methods and has the potential to be an alternative technique for the characterization of Giardia cyst suspensions in resource-limited facilities, since it is independent of experts and free of consumables.

Household slow sand filters in continuous and intermittent flows and their efficiency in microorganism's removal from river water.

This study aimed to evaluate the efficiency of four household slow sand filter (HSSF) models for the removal of microorganisms from river water throughout the development of their biological layers (schmutzdecke). Two models were designed to be operated continuously (HSSF-CC and HSSF-CT) and two intermittently (HSSF-ID and HSSF-IF). Filters were fed daily with 48 L pre-treated river water (24 h sedimentation followed by filtration through a non-woven synthetic blanket). Water samples were quantified by coliform group bacteria and analysed by bright field microscopy to visualize the microorganisms. Total coliform reduction was between 1.42 ± 0.59 log and 2.96 ± 0.58 log, with continuous models showing a better performance (p-values < 0.004). Escherichia coli reduction varied from 1.49 ± 0.58 log to 2.09 ± 0.66 log and HSSF-IF, HSSF-CC and HSSF-CT presented a similar performance (p-values > 0.06), slightly better than the one presented by HSSF-ID (p-value=0.04). Microorganisms, such as algae, protozoa and helminths were detected by microscopy in raw water and pre-treated water. Algae were the most significant group in these samples, although they were not visualized by bright field microscopy in the filtered water. Results showed the potential of HSSF in microbiological risk reduction from river water, which increases the range of point-of-use water treatments in rural communities. However, additional studies of the HSSF biological layer must be performed.

Evaluation of a multi-barrier household system as an alternative to surface water treatment with microbiological risks.

Household Water Treatment and Safe Storage (HWTS) are recommended to supply the demand for drinking water in communities without conventional water supply systems. However, there is a lack of long-term laboratory studies regarding such technologies. We evaluated the contributions of each step of a multi-barrier system with pretreatment (sedimentation and fabric filtration), filtration in Household Slow Sand Filters (HSSFs) and disinfection (sodium hypochlorite) treating surface water for more than 14 consecutive months. Removal of turbidity, colour, organic matter, coliform group bacteria and protozoa were evaluated. Two HSSF models were compared, one with a diffuser vessel (HSSF-d) and one with a gravity float equipped vessel (HSSF-f). Correlations between efficiency and operational parameters were assessed. Overall, the multi-barrier system removed more than 90% of turbidity and more than 3.5 log of Escherichia coli. HSSF removed up to 3.0 log of Giardia spp. and 2.4 log of Cryptosporidium spp. HSSF-f presented significantly higher removal rates for turbidity, apparent colour and E. coli. Disinfection resulted in water with E. coli concentration lower than 1 CFU 100mL-1, however it was not able to inactivate protozoa. The evaluated system was able to reduce microbiological risks from water and could indeed be an alternative to communities that depend on surface water as their main source of supply. Nevertheless, further studies are recommended to include a low-cost disinfectant for protozoa inactivation.

Occurrence and Molecular Identification of Hemoparasites in Wild Mammals Kept in Rehabilitation Centers in Brazil.

PURPOSE: Hepatozoonosis and piroplasmosis are diseases caused by apicomplexan protozoa that affect different types of animals, including mammals. The present study aimed to evaluate the occurrence of Hepatozoon spp. and piroplasms in wild mammals kept in captivity in rehabilitation centers in the states of Minas Gerais and Goiás, Brazil. METHODS: For this, blood samples from 152 animals were collected and analyzed by conventional optical microscopy and polymerase chain reaction (PCR). In addition, positive PCR samples were submitted to sequencing for molecular characterization of the specimens found. RESULTS: Microscopic analysis revealed 53 of the 152 animals (28.3%) parasitized by piroplasms. No Hepatozoon sp. was observed. On the other hand, using the primers HepF300/HepR900 and Piro1F/Piro5R, both amplifying fragments of the 18S rDNA gene, eight animals (5.2%) were positive for Hepatozoon spp. and 40 (26.3%) for piroplasms. From the sequencing of the positive samples Hepatozoon canis, Hepatozoon felis, Theileria cervi, Theileria equi and Cytauxzoon felis were identified. In addition to the aforementioned hemoparasites, some animals were found parasitized by microfilaria. Such data ratify the presence of hemoparasites in captive wild animals, and are unprecedented in the two geographical regions covered by the present study. 19.7% of mammals harbored ectoparasites of the genera Amblyomma and Rhipicephalus. CONCLUSION: Wild mammals are infected by several pathogens that can also infect domestic animals, some of them potentially zoonotic which can directly contribute to mortality and species reduction. Therefore, a deep understanding of the parasites, the hosts and the diseases is extremely necessary so that prevention, control and treatment measures are effectively applied.

Does each bead count? A reduced-cost approach for recovering waterborne protozoa from challenge water using immunomagnetic separation.

Giardia duodenalis and Cryptosporidium spp. are two of the most prominent aetiological agents of waterborne diseases. Therefore, efficient and affordable methodologies for identifying and quantifying these parasites in water are increasingly necessary. USEPA Method 1623.1 is a widely used and validated protocol for detecting these parasites in water samples. It consists of a concentration step, followed by parasite purification and visualization by immunofluorescence microscopy. Although efficient, this method has a high cost particularly due to the immunomagnetic separation (IMS) step, which is most needed with complex and highly contaminated samples. Based on this, the present study aimed to determine whether it is possible to maintain the efficiency of Method 1623.1 while reducing the amount of beads per reaction, using as a matrix the challenge water recommended by the World Health Organization. As for Giardia cysts, a satisfactory recovery efficiency (RE) was obtained using 50% less IMS beads. This was evaluated both with a commercial cyst suspension (56.1% recovery) and an analytical quality assessment (47.5% recovery). Although RE rates obtained for Cryptosporidium parvum did not meet Method 1623.1 criteria in any of the experimental conditions tested, results presented in this paper indicated the relevance of the described adaptations, even in challenge water.

Microbiome and oral squamous cell carcinoma: a possible interplay on iron metabolism and its impact on tumor microenvironment.

There is increasing evidence showing positive association between changes in oral microbiome and the occurrence of oral squamous cell carcinoma (OSCC). Alcohol- and nicotine-related products can induce microbial changes but are still unknown if these changes are related to cancerous lesion sites. In an attempt to understand how these changes can influence the OSCC development and maintenance, the aim of this study was to investigate the oral microbiome linked with OSCC as well as to identify functional signatures and associate them with healthy or precancerous and cancerous sites. Our group used data of oral microbiomes available in public repositories. The analysis included data of oral microbiomes from electronic cigarette users, alcohol consumers, and precancerous and OSCC samples. An R-based pipeline was used for taxonomic and functional prediction analysis. The Streptococcus spp. genus was the main class identified in the healthy group. Haemophilus spp. predominated in precancerous lesions. OSCC samples revealed a higher relative abundance compared with the other groups, represented by an increased proportion of Fusobacterium spp., Prevotella spp., Haemophilus spp., and Campylobacter spp. Venn diagram analysis showed 52 genera exclusive of OSCC samples. Both precancerous and OSCC samples seemed to present a specific associated functional pattern. They were menaquinone-dependent protoporphyrinogen oxidase pattern enhanced in the former and both 3',5'-cyclic-nucleotide phosphodiesterase (purine metabolism) and iron(III) transport system ATP-binding protein enhanced in the latter. We conclude that although precancerous and OSCC samples present some differences on microbial profile, both microbiomes act as "iron chelators-like" potentially contributing to tumor growth.

Filter media depth and its effect on the efficiency of Household Slow Sand Filter in continuous flow.

This study evaluated the impact of a 50% reduction of filter media depth in Household Slow Sand Filters (HSSFs) on continuous flow to remove physicochemical and microbiological parameters from river water. Furthermore, simple pre-treatment and disinfection processes were evaluated as additional treatments. Two filter models with different filtration layer depths were evaluated: a traditional one with 50 cm media depth (T-HSSF) and a compact one (C-HSSF) with 25 cm. HSSFs were fed with pre-treated river water (24-h water sedimentation followed by synthetic fabric filtration) for 436 days at a constant filtration rate of 0.90 m3 m-2 day-1 with a daily production of 48 L day-1. Sodium hypochlorite (2.0 mg L-1 of NaOCl 2.5% for 30 min) was used to disinfect the filtered water. Water samples were analysed weekly for parameters such as turbidity, organic matter, colour and E. coli, among others. Removal of protozoan cysts and oocysts by the HSSFs were also evaluated. After pretreatment, turbidity from the HSSF river water was reduced to 13.2 ± 14.6 NTU, allowing the filters to operate. Statistical analysis indicated no significant difference (p > 0.05) between T-HSSF and C-HSSF efficiencies in all evaluated parameters throughout operation time. Hence, media depth reduction did not hinder continuous HSSF performance for almost all the evaluated parameters. However, it may have affected Giardia cysts retaining, which passed through the thinner media on one evaluation day. Disinfection was effective in reducing remaining bacteria from filtered water; however, it was ineffective to inactivate protozoa. The reduction in the filtration layer did not affect the overall filtered water quality or quantity showing that a compact HSSF model may be a viable option for decentralized water treatment.

Phylogenetic relationships among Toxocara spp. and Toxascaris sp. from different regions of the world.

Toxocara and Toxascaris are parasitic nematodes that infect canids and felids although species of the genus Toxocara also infect humans. This work aimed to establish the phylogenetic and phylogeographic relationship between specimens of T. canis, T. cati, T. malaysiensis and Toxascaris leonina and to evaluate the degree of host specificity. In total, 437 samples (adults and pools of eggs) were collected from canids and felids from eight countries. Parasites were identified by morphology, PCR linked Restriction Fragment Length Polymorphism (PCR-RFLP) and partial sequencing of the mitochondrial gene cox1. Phylogenetic trees were constructed and genetic distance among isolates was estimated. Based on the molecular characterization all worms were identified in agreement with their respective hosts with the exception of three samples; two from cats and one from dogs identified as T. canis and T. cati, respectively. There was no clear geographical clustering of the samples despite this study including parasites from three continents. This is the first study, to our knowledge, to use molecular methods to identify T. canis in cats and T. cati in dogs with host specificity being the most common finding. Our developed PCR-RFLP method was found to be a facile and reliable method for identifying Toxocara species.

Babesiosis caused by Babesia vogeli in dogs from Uberlândia State of Minas Gerais, Brazil.

Babesia is tick-transmitted protozoan parasites that infect mammalian hosts and have a major impact on farm and pet health-associated costs worldwide. This study aimed to test the prevalence of Babesia spp. infection in a small cohort of dogs at a veterinary hospital and to perform molecular characterization of the Babesia species causing the infection. For the PCR assay, 5 mL of blood was collected by venipuncture of the cephalic or radial veins in 300 dogs of different ages, sex, and breeds, which were presented to the veterinary hospital of the Federal University of Uberlândia between March 2015 and April 2016. In addition, a drop of blood was collected from the marginal blood vessels of the ear of dogs included in this study. Ninety-two (30.67%) were positive for Babesia spp., as determined by microscopic observation of the blood smear, revealing the presence of intra-erythrocyte merozoites. For molecular characterization by PCR, 17 samples were chosen from dogs who were tested positive for Babesia spp. by blood smears. Among them, B. vogeli was found to infect all 17 dogs, as determined by 99-100% sequence identity (closest GenBank match KT246307) using primers PIRO A/PIRO B. Our results indicate that the species observed in these dogs was B. vogeli.

TRICHOMONAS VAGINALIS in female prison inmates from Uberlândia, Minas Gerais, Brazil: prevalence, diagnosis and epidemiological aspects

Trichomonas vaginalis is the etiological agent of trichomoniasis one of the most prevalent sexually transmitted infections worldwide. This paper aimed to determine the prevalence and the risk factors associated to the dissemination of the parasite in the prison environment, as well as comparing the diagnostic methods used for its detection. The present study included 56 female inmates at Professor Jacy de Assis Penitentiary, in Uberlandia, Minas Gerais, Brazil, regardless of ethnicity, socioeconomic status, age and sexual orientation. To diagnose T. vaginalis, wet mount and culture in TYM medium were utilized. The results were compared to the Papanicolaou test, the routine diagnostic method used in prisons. To outline the socioepidemiological profile of the participants an investigative survey was applied during an interview preceding the medical consultation. Of the 56 women included in the present study, six were diagnosed positive for T. vaginalis, by the three methods resulting in a prevalence of 10.7%. Culture and wet mount presented 100% specificity and sensitivity. On the other hand, sensitivity and specificity of the Papanicolaou test were 75% and 96%, respectively. None of the variables analysed, herein, could be associated with the infection. Despite the presence of the parasite, it was not possible to set an epidemiological pattern for positive patients, highlighting the particularities of this population. Regarding the diagnostic methods, wet mount and culture were equally efficient and superior to Papanicolaou in detecting T. vaginalis.

Distribution and risk factors of Ascarididae and other geohelminths in the soil of Uberlandia, Minas Gerais, Brazil.

Among the geohelminths, parasites from the Ascarididae family have a significant role in the human and animal health. This research aims to determine the prevalence of Ascarididae species in the soil in different areas located in a city of Minas Gerais State, Brazil (South America). The study was developed in squares, parks, sports clubs, orchards and municipal children's schools. Samples of sand/soil/grass were collected from January to August 2014. The optical microscope screening was performed through formal-ether and spontaneous sedimentation techniques. Out of the 183 collected samples, eight (4.4%) belonged to parks, 16 (8.7%) to sports clubs, 76 (41.5%) to squares, 23 (12.6%) to orchards, and 60 (32.8%) to schools. Out of the total, 28 (15.3%) contained Ascarididae eggs. Higher levels of positivity were demonstrated in the raining season (25.0%), in samples collected in the southern region of the town (25.1%), on ground soils (27.3%). Twenty-three (12.6%) were detected by the formal-ether sedimentation technique and 10 (5.5%) by spontaneous sedimentation technique. Therefore, it was concluded that the soils in the city of Uberlandia are contaminated with eggs and larvae of geohelminths, enabling dissemination of illnesses among animals and human beings.

Haemogregarina podocnemis sp. nov.: description of a new species of Haemogregarina Danilewsky 1885 (Adeleina: Haemogregarinaidae) in free-living and captive yellow-spotted river turtles Podocnemis unifilis (Testudines: Podocnemididae) from Brazil.

Based on morphological, morphometric, and molecular data, we describe a new hemoparasite of the genus Haemogregarina Danilewsky 1885, isolated from the Brazilian aquatic turtle Podocnemis unifilis (Testudines: Podocnemididae). The new species, Haemogregarina podocnemis sp. nov. (Apicomplexa: Haemogregarinidae), is characterized by small trophozoites with a single cytoplasmic vacuole on one side; pre-meronts with nuclear chromatin dispersed in the cytoplasm, with or without cytoplasmic vacuoles; meronts that are usually broad and slightly curved (kidney-shaped), with an average of eight small rectangular nuclei; immature gamonts (bean-shaped) with two morphological types: one with nuclear chromatin dispersed in the cytoplasm and the other with nuclei in the middle of the cell; mature gamonts of two morphological types: one with a length equal to or greater than that of the erythrocyte and the width of the nuclei similar to that of the hemoparasite and the other smaller than the erythrocyte with the width of the nuclei less than that of the hemoparasite. This is the first hemogregarine species described that infects the Brazilian turtle Po. unifilis. These findings highlight the need for further studies of Haemogregarina spp. to better determine the biodiversity of this understudied parasite group.

Multilocus genotyping of Giardia duodenalis in Brazilian children.

BACKGROUND: Giardia duodenalis is a parasite of several mammalian species, including humans, distributed worldwide. This research aimed to identify the molecular assemblages/sub-assemblages of G. duodenalis and to determine the intra-assemblage genetic variation of the different genes of assemblages A and B in pre-school children in the cities of Araguari and Uberlândia, Minas Gerais, Brazil. METHODS: The molecular characterization followed ß-giardin (bg), glutamate dehydrogenase (gdh) and triose phosphate isomerase (tpi) protocols. RESULTS: Of 226 stool samples, G. duodenalis cysts were found in 45 (19.9%). The tpi gene was amplified in 34 samples: 16 assemblage A, 14 B and four mixed samples A/B. The gdh gene was amplified in 32 samples, including 14 A, 16 B and two A/B. For the bg gene, 19 samples were sequenced: nine assemblage A, five B, three E, and two mixed, A/E and B/E. Animal-specific assemblage E were identified by bg, but were not confirmed for other genes. Twelve samples were characterized by full agreement of the three genes. Two new multilocus genotyping (MLGs) for assemblage A and two new MLGs for assemblage B were also described. CONCLUSIONS: These findings substantiate the importance of using more than one gene protocol since the sensitivity and genetic variability changes with the locus used.Access numbers: The GenBank access numbers for the nucleotide sequences reported in this article are: JQ794877-JQ794890, JX033113-JX033118.

Molecular typing of canine Giardia duodenalis isolates from Minas Gerais, Brazil.

Giardia duodenalis (syn. Giardia intestinalis and Giardia lamblia) is a widespread intestinal parasite in mammals, including humans and pets worldwide. It should be considered a species complex and comprises eight assemblage (A-H). This works aimed to determine the genotypic variability among G. duodenalis isolates from dogs from Minas Gerais state, Brazil. Fecal samples of 97 dogs, from 1-to-10 months old from 15 commercial kennels, were collected and analyzed by the zinc sulfate centrifugal flotation technique, to determine their positivity for G. duodenalis cysts. Cysts pellets were stored and submitted to PCR and nested-PCR reactions with gdh and tpi primers, and then sequencing. Among positive samples (n = 19), fragment amplifications of gdh and tpi genes was observed in 16 (84,2%) and 14 (73,6%), respectively. In total, 30 sequences were obtained. Sequencing analysis showed that for gdh, all isolates were identified as host-specific genotype D, and for tpi, besides host-specific genotype C, were also observed zoonotic genotypes A and B. This study provides, for the first time, current information about genetic characterization of G. duodenalis isolates found in dogs in Minas Gerais state.

Performance of glutamate dehydrogenase and triose phosphate isomerase genes in the analysis of genotypic variability of isolates of Giardia duodenalis from livestocks.

Giardia duodenalis is a small intestinal protozoan parasite of several terrestrial vertebrates. This work aims to assess the genotypic variability of Giardia duodenalis isolates from cattle, sheep and pigs in the Southeast of Brazil, by comparing the standard characterization between glutamate dehydrogenase (gdh) and triose phosphate isomerase (tpi) primers. Fecal samples from the three groups of animals were analyzed using the zinc sulphate centrifugal flotation technique. Out of 59 positive samples, 30 were from cattle, 26 from sheep and 3 from pigs. Cyst pellets were stored and submitted to PCR and nested-PCR reactions with gdh and tpi primers. Fragment amplification of gdh and tpi genes was observed in 25 (42.4%) and 36 (61.0%) samples, respectively. Regarding the sequencing, 24 sequences were obtained with gdh and 20 with tpi. For both genes, there was a prevalence of E specific species assemblage, although some isolates have been identified as A and B, by the tpi sequencing. This has also shown a larger number of heterogeneous sequences, which have been attribute to mixed infections between assemblages B and E. The largest variability of inter-assemblage associated to the frequency of heterogeneity provided by tpi sequencing reinforces the polymorphic nature of this gene and makes it an excellent target for studies on molecular epidemiology.