Mechanisms of programmed cell death associated to severe dengue in human renal lesions.

Dengue virus (DENV) is a global health problem. Severe dengue can manifest with hemorrhage and signs of organ dysfunction, including the kidneys. The innate immune system is an important barrier against arbovirus infection and, specifically in dengue, the cytokines IL1ß and IL18 and caspase-1 activation make up a set of host immune strategies. Cell death mechanisms include pyroptosis, necroptosis and autophagy, each with peculiar markers: gasdermin, RIPK3/MLKL, LC3, respectively. In DENV infection, necrosis and apoptosis are involved and, when infecting monocytes and macrophages in vitro, DENV is capable of inducing pyroptosis. Our objective was to explore the presence of markers of necroptosis, pyroptosis and autophagy in renal lesions caused by DENV. MATERIAL AND METHODS: twenty specimens of lesions from patients who died due to DENV infection, from the pathology department of Hospital Guilherme Álvaro, Santos, SP, were subjected to histological and immunohistochemical studies. Histological sections were stained with hematoxylin-eosin to evaluate tissue changes or collected for research with antibodies: anti-DENV (Instituto Evandro Chagas-PA), RIPK3 (NBP2-45592), MLKL (ab184718), gasdermin D (#36425 ), LC3 (14600-AP), caspase 1 (#98033), IL1ß (AF201-NA) and IL18 (SC6178). Semi-quantitative analysis was performed on 20 glomeruli and evaluation on tubules and mononuclear cells. This study was approved by the ethics committee of the USP Faculty of Medicine. RESULTS: histological analysis demonstrated glomerular congestion, glomerulitis (medium to severe), acute kidney injury and hyalinization of the glomeruli. Viral antigens were visualized on mononuclear cells. LC3 (autophagy) expression ranged from moderate to intense (++/+++) in glomeruli, tubules and mononuclear cells. The expression of gasdermin (pyroptosis) was mild (+) in most cases in the glomeruli and moderate (++) in the tubules. RIPK3 and MLKL (necroptosis) mild in tubules and mononuclear cells (+). The expression of the cytokines IL1ß and IL18 and caspase 1 was moderate (++). Statistical analysis showed greater expression of LC3 over the others. CONCLUSIONS: Our results contribute to the understanding of the pathogenesis of renal involvement in severe dengue, considering the likely anti-viral mechanism of autophagy. To a lesser extent, pyroptosis is also present, corroborating previous data.

Safety attributes in primary care: understanding the needs of patients, health professionals, and managers.

OBJECTIVES: The aims of this study were (1) to identify attributes for patient safety at a primary healthcare level and (2) to analyze conceptions of patients, professionals, and managers about how these attributes are being addressed. STUDY DESIGN: This was a qualitative study. METHODS: Participants were recruited from three primary care settings in Brazil. A total of 37 subjects (four physicians, three nurses, three dentists, three managers, five community assistants, and 19 patients) participated on interviews about their perceptions of safety attributes at the primary care settings involved in the study. Some of these participants attended a focus group meeting. A thematic categorical analysis was carried out to interpret the interviews. RESULTS: The main attributes for patient safety were valued by the participants. However, barriers such as discontinuity of care, interruptions during consultations, breakdowns in the communication, and ineffective teamwork were reported as frequent sources of patient safety issues. Reports of patients left unattended for excessive time because of the lack of accurate information and disruptions that took up to 35 min show that there is still a long way to go for primary care to be safe and effective in the study settings. CONCLUSIONS: It is necessary that the strategies meet the patient safety needs more effectively and efficiently. Further research is needed to understand the complex nature of the problems that affect patient safety in these settings so that appropriate decisions can be made.

Surveillance of Arboviruses in Primates and Sloths in the Atlantic Forest, Bahia, Brazil.

From 2006 through 2014, we conducted seroepidemiological surveys on non-human primates and sloths to investigate the possible circulation of arboviruses in Bahia Atlantic Forest, Brazil. We collected a total of 196 samples from 103 Leontopithecus chrysomelas, 7 Sapajus xanthosternos, 22 Bradypus torquatus and 7 Bradypus variegatus. Serum samples were tested using neutralization test and hemagglutination inhibition test to detect total antibodies against 26 different arboviruses. The overall prevalence of arboviruses was 36.6% (51/139), with the genus Flavivirus having the highest prevalence (33.1%; 46/139), followed by Phlebovirus (5.0%; 7/139), Orthobunyavirus (4.3%; 6/139) and Alphavirus (0.7%; 1/139). Monotypic reactions suggest that the wild animals were exposed naturally to at least twelve arboviruses. Added results from the neutralization test, animals were exposed to thirteen arboviruses. Most of these viruses are maintained in transmission cycles independent of human hosts, although antibodies against dengue virus serotypes 1, 2 and 3 were found in this study. To our knowledge, this is the first study reporting exposure to arboviruses in L. chrysomelas, S. xanthosternos and B. torquatus. Our results also highlight that the Southern Bahia Atlantic Forest has a variety of vertebrate hosts and potential vectors, which may support the emergence or re-emergence of arboviruses, including those pathogenic to humans.

Instruments for evaluation of safety culture in primary health care: integrative review of the literature.

OBJECTIVES: This research aimed to conduct an investigation of the tools used to assess safety culture in the primary health care. STUDY DESIGN: Integrative review of the literature. METHODS: We conducted a literature search using an instrument that included quantitative assessments of safety culture, using the following databases: CINAHL, ScienceDirect, PubMed, BIREME, and SciELO. Retrieved material comprised original articles published from 1998 to 2014, with titles and abstracts available in English, Portuguese, Spanish, and French. RESULTS: The search resulted in seven instruments; however, only three were primary healthcare focused. CONCLUSION: Most of the existing instruments for assessing safety culture have acceptable psychometric properties. The study serves as a source for students, workers, and researchers who want to know more about appropriate instruments for evaluating safety culture in primary care.

Adverse birth outcomes associated with Zika virus exposure during pregnancy in São José do Rio Preto, Brazil.

OBJECTIVES: We aimed to report the first 54 cases of pregnant women infected by Zika virus (ZIKV) and their virologic and clinical outcomes, as well as their newborns' outcomes, in 2016, after the emergence of ZIKV in dengue-endemic areas of São Paulo, Brazil. METHODS: This descriptive study was performed from February to October 2016 on 54 quantitative real-time PCR ZIKV-positive pregnant women identified by the public health authority of São José do Rio Preto, São Paulo, Brazil. The women were followed and had clinical and epidemiologic data collected before and after birth. Adverse outcomes in newborns were analysed and reported. Urine or blood samples from newborns were collected to identify ZIKV infection by reverse transcription PCR (RT-PCR). RESULTS: A total of 216 acute Zika-suspected pregnant women were identified, and 54 had the diagnosis confirmed by RT-PCR. None of the 54 women miscarried. Among the 54 newborns, 15 exhibited adverse outcomes at birth. The highest number of ZIKV infections occurred during the second and third trimesters. No cases of microcephaly were reported, though a broad clinical spectrum of outcomes, including lenticulostriate vasculopathy, subependymal cysts, and auditory and ophthalmologic disorders, were identified. ZIKV RNA was detected in 18 of 51 newborns tested and in eight of 15 newborns with adverse outcomes. CONCLUSIONS: Although other studies have associated many newborn outcomes to ZIKV infection during pregnancy, these same adverse outcomes were rare or nonexistent in this study. The clinical presentation the newborns we studied was mild compared to other reports, suggesting that there is significant heterogeneity in congenital Zika infection.

Age and Environment Influences on Mouse Prion Disease Progression: Behavioral Changes and Morphometry and Stereology of Hippocampal Astrocytes.

Because enriched environment (EE) and exercise increase and aging decreases immune response, we hypothesized that environmental enrichment and aging will, respectively, delay and increase prion disease progression. Mice dorsal striatum received bilateral stereotaxic intracerebral injections of normal or ME7 prion infected mouse brain homogenates. After behavior analysis, animals were euthanized and their brains processed for astrocyte GFAP immunolabeling. Our analysis related to the environmental influence are limited to young adult mice, whereas age influence refers to aged mice raised on standard cages. Burrowing activity began to reduce in ME7-SE two weeks before ME7-EE, while no changes were apparent in ME7 aged mice (ME7-A). Object placement recognition was impaired in ME7-SE, NBH-A, and ME7-A but normal in all other groups. Object identity recognition was impaired in ME7-A. Cluster analysis revealed two morphological families of astrocytes in NBH-SE animals, three in NBH-A and ME7-A, and four in NBH-EE, ME7-SE, and ME7-EE. As compared with control groups, astrocytes from DG and CA3 prion-diseased animals show significant numerical and morphological differences and environmental enrichment did not reverse these changes but induced different morphological changes in GFAP+ hippocampal astroglia. We suggest that environmental enrichment and aging delayed hippocampal-dependent behavioral and neuropathological signs of disease progression.

Complete Genome Sequence of the BeAn 58058 Virus Isolated from Oryzomys sp. Rodents in the Amazon Region of Brazil.

Here, we report the complete genome sequence of the BeAn 58058 virus (prototype) strain, isolated from a wild rodent Oryzomys sp. in the Utinga forest, Belém, state of Pará, Brazil in 1963. The genome of this virus showed similarity to the Poxviridae family, suggesting its inclusion in a possible new genus.

Drosha, DGCR8, and Dicer mRNAs are down-regulated in human cells infected with dengue virus 4, and play a role in viral pathogenesis.

Dengue virus (DENV) and its four serotypes (DENV1-4) belong to the Flavivirus genus of the Flaviviridae family. DENV infection is a life-threatening disease, which results in up to 20,000 deaths each year. Viruses have been shown to encode trans-regulatory small RNAs, or microRNAs (miRNAs), which bind to messenger RNA and negatively regulate host or viral gene expression. During DENV infections, miRNAs interact with proteins in the RNAi pathway, and are processed by ribonucleases such as Dicer and Drosha. This study aims to investigate Drosha, DGCR8, and Dicer expression levels in human A-549 cells following DENV4 infection. DENV4 infected A-549 cells were collected daily for 5 days, and RNA was extracted to quantify viral load. Gene expression of Drosha, Dicer, and DGCR8 was determined using quantitative PCR (RT-qPCR). We found that DENV4 infection exhibited the highest viral load 3 days post-infection. Dicer, Drosha, and DGCR8 showed reduced expression following DENV4 infection as compared with negative controls. In addition, we hypothesize that reduced expression of DGCR8 may not only be related to miRNA biogenesis, but also other small RNAs. This study may change our understanding regarding the relationship between host cells and the dengue virus.

Litter size, age-related memory impairments, and microglial changes in rat dentate gyrus: stereological analysis and three dimensional morphometry.

It has been demonstrated that rat litter size affects the immune cell response, but it is not known whether the long-term effects aggravate age-related memory impairments or microglial-associated changes. To that end, we raised sedentary Wistar rats that were first suckled in small or large litters (6 or 12pups/dam, respectively), then separated into groups of 2-3 rats from the 21st post-natal day to study end. At 4months (young adult) or 23months (aged), all individual rats were submitted to spatial memory and object identity recognition tests, and then sacrificed. Brain sections were immunolabeled with anti-IBA-1 antibodies to selectively identify microglia/macrophages. Microglial morphological changes in the molecular layer of the dentate gyrus were estimated based on three-dimensional reconstructions. The cell number and laminar distribution in the dentate gyrus was estimated with the stereological optical fractionator method. We found that, compared to young rat groups, aged rats from large litters showed significant increases in the number of microglia in all layers of the dentate gyrus. Compared to the microglia in all other groups, microglia in aged individuals from large litters showed a significantly higher degree of tree volume expansion, branch base diameter thickening, and cell soma enlargement. These morphological changes were correlated with an increase in the number of microglia in the molecular layer. Young adult individuals from small litters exhibited preserved intact object identity recognition memory and all other groups showed reduced performance in both spatial and object identity recognition tasks. We found that, in large litters, brain development was, on average, associated with permanent changes in the innate immune system in the brain, with a significant impact on the microglial homeostasis of aged rats.

Optimization of dengue virus genome assembling using GSFLX 454 pyrosequencing data: evaluation of assembling strategies.

Currently assembling genomes without reference is one of the most important challenges for bioinformaticists all over the world in an attempt to characterize new organisms. The current study has used two dengue virus type 4 (DENV-4) strains recently isolated in Brazil, which have its genomes sequenced using the GSFLX 454 sequencer (Roche, Life Science) by the pyrosequencing method. The GSFLX 454 data were used for testing different genome assembling strategies. We described a pipeline that was able to recover more than 96% of the sequenced genome in a single run and could be helpful for further assembly attempts of other DENV genomes, as well as other RNA virus-like genomes.

A phylogenetic analysis using full-length viral genomes of South American dengue serotype 3 in consecutive Venezuelan outbreaks reveals a novel NS5 mutation.

Dengue virus currently causes 50-100 million infections annually. Comprehensive knowledge about the evolution of Dengue in response to selection pressure is currently unavailable, but would greatly enhance vaccine design efforts. In the current study, we sequenced 187 new dengue virus serotype 3 (DENV-3) genotype III whole genomes isolated from Asia and the Americas. We analyzed them together with previously-sequenced isolates to gain a more detailed understanding of the evolutionary adaptations existing in this prevalent American serotype. In order to analyze the phylogenetic dynamics of DENV-3 during outbreak periods; we incorporated datasets of 48 and 11 sequences spanning two major outbreaks in Venezuela during 2001 and 2007-2008, respectively. Our phylogenetic analysis of newly sequenced viruses shows that subsets of genomes cluster primarily by geographic location, and secondarily by time of virus isolation. DENV-3 genotype III sequences from Asia are significantly divergent from those from the Americas due to their geographical separation and subsequent speciation. We measured amino acid variation for the E protein by calculating the Shannon entropy at each position between Asian and American genomes. We found a cluster of seven amino acid substitutions having high variability within E protein domain III, which has previously been implicated in serotype-specific neutralization escape mutants. No novel mutations were found in the E protein of sequences isolated during either Venezuelan outbreak. Shannon entropy analysis of the NS5 polymerase mature protein revealed that a G374E mutation, in a region that contributes to interferon resistance in other flaviviruses by interfering with JAK-STAT signaling was present in both the Asian and American sequences from the 2007-2008 Venezuelan outbreak, but was absent in the sequences from the 2001 Venezuelan outbreak. In addition to E, several NS5 amino acid changes were unique to the 2007-2008 epidemic in Venezuela and may give additional insight into the adaptive response of DENV-3 at the population level.

[Cocaine: historical background, neurobiology of the addiction and relapse and therapeutic perspectives]. / Cocaína: história, acções neurobiológicas do vício e recaída e perspectivas terapêuticas.

OBJECTIVE: Following more than a century of cocaine hydrochloride extraction from Erythroxylon coca, this drug remains representing a serious social and public health problema around the world. This paper intends to provide a review about the cocaine theme, focusing on historical background and on its different neurotransmission systems, as well as addresses therapeutics aspects about drug addiction. METHOD: Electronic search in databases Medline, Pubmed and Lilacs was accomplished in order to select classics and recent studies relevant to the discussion of issue addressed. DISCUSSION: Previous studies have shown high vulnerability to relapse to cocaine seeking following prolonged withdrawal periods. Such behavioral consequences have been cre-dited to induced changes in brain neurotransmitters provoked by repeated cocaine use. In recent years, the growing abuse of this drug has mobilized researchers worldwide in seeking for new therapies that reduce the behavioral and neurochemical changes resulting from addiction. CONCLUSION: Numerous advances regarding the treatment of cocaine abuse and dependence have emerged in recent years. However, researche aiming at a safe and effective users' pharmacological treatment remain necessary and should be continued.

Characterization of two new rhabdoviruses isolated from midges (Culicoides SPP) in the Brazilian Amazon: proposed members of a new genus, Bracorhabdovirus.

Itacaiunas and Curionopolis viruses were isolated from Culicoides midges in Parauapebas municipality, Pará state, Brazil, in 1984 and 1985, respectively. Itacaiunas virus infected newborn mice and mosquito cells (C6/36), but did not replicate in some mammalian cell lineages; while Curionopolis virus infected only mice. Neither virus showed a serological relationship with any of the 195 known arboviruses circulating in Brazil, nor against 38 other rhabdoviruses isolated worldwide. Both virus particles are bullet-shaped and similar in morphology to that observed for other members of the family Rhabdoviridae. Partial nucleotide sequencing of the N protein showed that those two viruses constitute a separate clade in the family Rhabdoviridae, which we propose to be a new genus, designated Bracorhabdovirus.

Neurotropism and neuropathological effects of selected rhabdoviruses on intranasally-infected newborn mice.

Viral neurotropism is the ability of viruses to infect neuronal cells. This is well studied for herpesviruses, rabies-related viruses, and a few others, but it is poorly investigated among almost all arboviruses. In this study, we describe both the neurotropism and the neuropathological effects of Amazonian rhabdoviruses on the brains of experimentally infected-newborn mice. Suckling mice were intranasally infected with 10(-4) to 10(-8) LD50 of viruses. Animals were anaesthetized and perfused after they had become sick. Immunohistochemistry using specific anti-virus and anti-active caspase three antibodies was performed. All infected animals developed fatal encephalitis. Survival time ranged from 18 h to 15 days. Viruses presented distinct species-dependent neurotropism for CNS regions. Histopathological analysis revealed variable degrees of necrosis and apoptosis in different brain regions. These results showed that viruses belonging to the Rhabdoviridae family possess distinct tropism for CNS structures and induce different pattern of cell death depending on the CNS region.

Isolation of yellow fever virus from nulliparous Haemagogus (Haemagogus) janthinomys in eastern Amazonia.

In 1998, an epizootic of yellow fever (YF) killed many howler monkeys (Alouatta spp.) in eastern Amazonia near the city of Altamira. An infection level with YF virus of approximately 3.6% was determined from analysis of 456 females of Haemagogus janthinomys Dyar, the main enzootic YF vector in South America. One month later, a second study of 164 females captured in the same place led to infection levels of 0.8% for parous and 2.9% for nulliparous females. These results lead to the conclusion that vertical transmission, one of the key elements in the epidemiology of YF, occurs in South America as it does in Africa.

Epidemic of jungle yellow fever in Brazil, 2000: implications of climatic alterations in disease spread.

Seventy-seven human cases of sylvatic yellow fever were reported in Brazil during the period January-June 2000. The first cases were reported 1 week after New Year's day and originated at Chapada dos Veadeiros, a tourist canyon site in Goiás state, near Brasília, the Brazilian capital. The laboratory procedures used for diagnoses included serology with an IgM capture assay and plaque reduction neutralization test, virus isolation in suckling mice and C6/36 cells, and immunohistochemistry. All cases were diagnosed by at least two different laboratory procedures, with the exception of the first three fatal cases, which were diagnosed on the basis of clinical and epidemiological information. The cases were reported in eight Brazilian states as follows: Goiás with 64.9% (50 cases); Amazonas (1); Bahia (10); Distrito Federal (1); Mato Grosso (4); Minas Gerais (2); Pará (1); São Paulo (2); and Tocantins (6). Patient ages were within the following ranges: 13-74 years old (mean 34.3), 64 (84.4%) were male, especially agricultural workers (n = 30), but tourists (n = 11), carpenters (n = 4), fishermen (n = 4), students (n = 3), truck drivers (n = 3), and other people (n = 22) were also sickened. The case fatality rate was 50.6% (39/77). In Bahia state, a serologic survey that was carried out has suggested a symptomatic/asymptomatic coefficient of 1:4. Field studies developed in Distrito Federal, Goiás, and São Paulo states showed that Haemagogus janthinomys was the mosquito species associated with the transmission. A single strain was also obtained from Aedes scapularis in Bahia. Epizootic occurrence (monkey mortality) was observed in 49 municipalities mainly in Goiás state, where 40 municipalities made reports, 21 of which also diagnosed human cases. Data obtained by the National Institute of Meteorology in Brazil showed an increase in temperature and rain in December 1999 and the first 3 months of 2000 in Goiás and surrounding states, which perhaps has contributed to the intense and widespread transmission of the yellow fever virus. The relatively small number of cases probably reflects the extensive use of yellow fever 17D-vaccine during the last 3 years, in which about 45 million doses were used. During the last months of 1999, 16 and 11 yellow fever cases were reported in Tocantins and Goiás states, respectively. It is noteworthy that the last reported autochthonous cases of sylvatic yellow fever in São Paulo and Bahia, both states outside the endemic/enzootic area, had occurred in 1953 and 1948, respectively.

Hantavirus pulmonary syndrome in Anajatuba, Maranhão, Brazil.

The authors report a confirmed case of hantavirus pulmonary syndrome in the rural area of the municipality of Anajatuba, state of Maranhão. Two other suspected cases from the same region are also described. The confirmed case involved a previously healthy young woman who died with signs and symptoms of acute respiratory insufficiency 5 days after presenting fever, myalgia and a dry cough. The patient was a student who was helping her parents with work in the fields; it was a habit of the family to store rice inside the house. The suspected cases involved two first-degree relatives working as field hands who died of acute respiratory insufficiency 24 and 48 hours, respectively, after presenting fever, myalgia and a dry cough. Both stored rice and corn inside their home. People living in the region reported massive infestations with rats in the woods and fields.

Yellow fever in Pará State, Amazon region of Brazil, 1998-1999: entomologic and epidemiologic findings.

Yellow fever (YF) is frequently associated with high severity and death rates in the Amazon region of Brazil. During the rainy seasons of 1998 and 1999, 23 (eight deaths) and 34 (eight deaths) human cases of YF were reported, respectively, in different geographic areas of Pará State; most cases were on Marajó Island. Patients were 1 to 46 years of age. Epidemiologic and ecological studies were conducted in Afuá and Breves on Marajó Island; captured insects yielded isolates of 4 and 11 YF strains, respectively, from Haemagogus janthinomys pooled mosquitoes. The cases on Marajó Island in 1999 resulted from lack of vaccination near the focus of the disease and intense migration, which brought many nonimmune people to areas where infected vectors were present. We hypothesize that YF virus remains in an area after an outbreak by vertical transmission among Haemagogus mosquitoes.

Serious adverse events associated with yellow fever 17DD vaccine in Brazil: a report of two cases.

BACKGROUND: The yellow fever vaccine is regarded as one of the safest attenuated virus vaccines, with few side-effects or adverse events. We report the occurrence of two fatal cases of haemorrhagic fever associated with yellow fever 17DD substrain vaccine in Brazil. METHODS: We obtained epidemiological, serological, virological, pathological, immunocytochemical, and molecular biological data on the two cases to determine the cause of the illnesses. FINDINGS: The first case, in a 5-year-old white girl, was characterised by sudden onset of fever accompanied by headache, malaise, and vomiting 3 days after receiving yellow fever and measles-mumps-rubella vaccines. Afterwards she decompensated with icterus and haemorrhagic signs and died after a 5-day illness. The second patient-a 22-year-old black woman-developed a sore throat and fever accompanied by headache, myalgia, nausea, and vomiting 4 days after yellow fever vaccination. She then developed icterus, renal failure, and haemorrhagic diathesis, and died after 6 days of illness. Yellow fever virus was recovered in suckling mice and C6/36 cells from blood in both cases, as well as from fragments of liver, spleen, skin, and heart from the first case and from these and other viscera fragments in case 2. RNA of yellow fever virus was identical to that previously described for 17D genomic sequences. IgM ELISA tests for yellow fever virus were negative in case 1 and positive in case 2; similar tests for dengue, hantaviruses, arenaviruses, Leptospira, and hepatitis viruses A-D were negative. Tissue injuries from both patients were typical of wild-type yellow fever. INTERPRETATION: These serious and hitherto unknown complications of yellow fever vaccination are extremely rare, but the safety of yellow fever 17DD vaccine needs to be reviewed. Host factors, probably idiosyncratic reactions, might have had a substantial contributed to the unexpected outcome.

Trocara virus: a newly recognized Alphavirus (Togaviridae) isolated from mosquitoes in the Amazon Basin.

This report describes Trocara virus, a newly recognized member of the genus Alphavirus, that has been isolated from Aedes serratus mosquitoes collected at two widely separated sites in the Amazon Basin. Biological, antigenic and genetic characteristics of the new virus are given. Results of these studies indicate that Trocara virus is the first member of a newly discovered antigenic complex within the family Togaviridae genus Alphavirus. The public health and veterinary importance of Trocara virus is still unknown.