Characterization of insect-specific Culex flavivirus (Flaviviridae) nucleotide sequences in mosquitoes from urban parks in São Paulo, Brazil.

BACKGROUND: Despite their worldwide occurrence, the distribution and role of insect-specific flaviviruses (ISFs) remain unclear. METHODS: We evaluated the presence of ISFs in mosquitoes collected in São Paulo, Brazil, using reverse transcription and semi-nested polymerase chain reaction (PCR). Some of the positive samples were subjected to nanopore sequencing. RESULTS: Twelve mosquito pools (2.8%) tested positive for flavivirus infection. Nanopore sequencing was successfully performed on six samples. Phylogenetic analysis grouped these sequences into genotype 2 of Culex flavivirus (CxFV). CONCLUSIONS: The identification of CxFV genotype 2 at new locations in São Paulo highlights the importance of understanding the role of ISFs in mosquito vector competence.

Characterization of insect-specific Culex flavivirus (Flaviviridae) nucleotide sequences in mosquitoes from urban parks in São Paulo, Brazil

ABSTRACT Background: Despite their worldwide occurrence, the distribution and role of insect-specific flaviviruses (ISFs) remain unclear. Methods: We evaluated the presence of ISFs in mosquitoes collected in São Paulo, Brazil, using reverse transcription and semi-nested polymerase chain reaction (PCR). Some of the positive samples were subjected to nanopore sequencing. Results: Twelve mosquito pools (2.8%) tested positive for flavivirus infection. Nanopore sequencing was successfully performed on six samples. Phylogenetic analysis grouped these sequences into genotype 2 of Culex flavivirus (CxFV). Conclusions: The identification of CxFV genotype 2 at new locations in São Paulo highlights the importance of understanding the role of ISFs in mosquito vector competence.

Complexity of malaria transmission dynamics in the Brazilian Atlantic Forest.

Plasmodium malariae and Plasmodium vivax are protozoan parasites that can cause malaria in humans. They are genetically indistinguishable from, respectively, Plasmodium brasilianum and Plasmodium simium, i.e. parasites infecting New World non-human primates in South America. In the tropical rainforests of the Brazilian Atlantic coast, it has long been hypothesized that P. brasilianum and P. simium in platyrrhine primates originated from P. malariae and P. vivax in humans. A recent hypothesis proposed the inclusion of Plasmodium falciparum into the transmission dynamics between humans and non-human primates in the Brazilian Atlantic tropical rainforest. Herein, we assess the occurrence of human malaria in simians and sylvatic anophelines using field-collected samples in the Capivari-Monos Environmental Protection Area from 2015 to 2017. We first tested simian blood and anopheline samples. Two simian (Aloutta) blood samples (18%, n = 11) showed Plasmodium cytb DNA sequences, one for P. vivax and another for P. malariae. From a total of 9,416 anopheline females, we found 17 pools positive for Plasmodium species with a 18S qPCR assay. Only three showed P. cytb DNA sequence, one for P. vivax and the others for rodent malaria species (similar to Plasmodium chabaudi and Plasmodium berghei). Based on these results, we tested 25 rodent liver samples for the presence of Plasmodium and obtained P. falciparum cytb DNA sequence in a rodent (Oligoryzomys sp.) liver. The findings of this study indicate complex malaria transmission dynamics composed by parallel spillover-spillback of human malaria parasites, i.e. P. malariae, P. vivax, and P. falciparum, in the Brazilian Atlantic forest.

Aedes aegypti from Amazon Basin Harbor High Diversity of Novel Viral Species.

Viruses are the most diverse and abundant microorganisms on earth, highly adaptive to a wide range of hosts. Viral diversity within invertebrate hosts has gained notoriety in recent years in public health as several such viruses have been of medical importance. Aedes aegypti serves as a vector for several viruses that have caused epidemics within the last year throughout Brazil; including Dengue, Zika and Chikungunya. This study aimed to identify new viral agents within Aedes aegypti mosquito in a city of the Amazonian region, where it is highly endemic. Metagenomic investigation was performed on 60 mosquito pools and viral RNA sequences present in their microbiota were characterized using genomic and phylogenetic tools. In total, we identified five putative novel virus species related to the Sobemovirus genus, Iflavirus genus and Permutatetraviridae family. These findings indicate a diverse taxonomy of viruses present in the mosquito microbiota of the Amazon, the region with the greatest invertebrate diversity in the world.

A Novel Highly Divergent Strain of Cell Fusing Agent Virus (CFAV) in Mosquitoes from the Brazilian Amazon Region.

Classical insect-specific flaviviruses (cISFs) have been widely detected in different countries in the last decades. Here, we characterize the near full-length genomes of two cISFs detected in mosquitoes collected in the city of Macapá, state of Amapá, Amazon region of Brazil. A total of 105 pools of female mosquitos were analyzed by next-generation sequencing (NGS). Comparative genomics and phylogenetic analysis identified three strains of cell fusing agent virus (CFAV) and two of Culex flavivirus (CxFV). All sequences were obtained from pools of Culex sp., except for one sequence of CFAV detected in a pool of Aedes aegypti. Both CxFV strains are phylogenetically related to a strain isolated in 2012 in the Southeast region of Brazil. The CFAV strains are the first of this species to be identified in Brazil and one of them is highly divergent from other strains of CFAV that have been detected worldwide. In conclusion, CFAV and CxFV, circulate in mosquitoes in Brazil. One strain of CFAV is highly divergent from others previously described, suggesting that a novel strain of CFAV is present in this region.

A survey of flaviviruses in birds in green areas of São Paulo, Brazil.

INTRODUCTION: This study aimed to investigate Flavivirus infection in birds captured in green areas of São Paulo. METHODS: Oropharyngeal swabs, cloacal swabs, and blood samples from 170 birds captured in two green areas in São Paulo, Brazil were subjected to real time-polymerase chain reaction (RT-qPCR) analysis of Flavivirus specific NS5 gene fragment. RESULTS: All samples were negative for the presence of viral ribonucleic acid (RNA). CONCLUSIONS: Despite the negative results, Flavivirus surveillance must be performed regularly due to favorable ecological conditions for virus circulation and transmission among birds in these areas and their close proximity to humans.

Detection of Culex flavivirus and Aedes flavivirus nucleotide sequences in mosquitoes from parks in the city of São Paulo, Brazil.

The dengue viruses are widespread in Brazil and are a major public health concern. Other flaviviruses also cause diseases in humans, although on a smaller scale. The city of São Paulo is in a highly urbanized area with few green spaces apart from its parks, which are used for recreation and where potential vertebrate hosts and mosquito vectors of pathogenic Flavivirus species can be found. Although this scenario can contribute to the transmission of Flavivirus to humans, little is known about the circulation of members of this genus in these areas. In light of this, the present study sought to identify Flavivirus infection in mosquitoes (Diptera: Culicidae) collected in parks in the city of São Paulo. Seven parks in different sectors of the city were selected. Monthly mosquito collections were carried out in each park from March 2011 to February 2012 using aspiration and traps (Shannon and CD C-CO2). Nucleic acids were extracted from the mosquitoes collected and used for reverse-transcriptase and real-time polymerase chain reactions with genus-specific primers targeting a 200-nucleotide region in the Flavivirus NS5 gene. Positive samples were sequenced, and phylogenetic analyses were performed. Culex and Aedes were the most frequent genera of Culicidae collected. Culex flavivirus (CxFV)-related and Aedes flavivirus (AEFV)- related nucleotide sequences were detected in 17 pools of Culex and two pools of Aedes mosquitoes, respectively, among the 818 pools of non-engorged females analyzed. To the best of our knowledge, this is the first report of CxFV and AEFV in the city of São Paulo and Latin America, respectively. Both viruses are insect- specific flaviviruses, a group known to replicate only in mosquito cells and induce a cytopathic effect in some situations. Hence, our data suggests that CxFV and AEFV are present in Culex and Aedes mosquitoes, respectively, in parks in the city of São Paulo. Even though Flavivirus species of medical importance were not detected, surveillance is recommended in the study areas because of the presence of vertebrates and mosquitoes that could act as amplifying hosts and vectors of flaviviruses, providing the required conditions for circulation of these viruses.

Prevalence of anemia in non-severe malaria cases in Mâncio Lima, Acre

Malaria, a parasitic disease, is a serious public health problem. In Brazil, the majority of cases are found in the Amazon. The clinical manifestations of malaria depend on several factors and they may be related to the development of anemia. This study evaluated the prevalence of anemia in malaria cases and its associated factors. The study was conducted in Mâncio Lima (Acre, Brazil). Participants were chosen through passive detection of malaria cases in the municipal health services. They were interviewed and blood samples analyzed for Plasmodium detection and for hemoglobin measurement. SPSS 13.0, software was applied for statistical analysis. One hundred and twenty patients with malaria were studied, of which 58.3% male and 41.7% female. There was a 25% prevalence of anemia, mainly among the women, and in those presenting symptoms for more than four days as well as headaches. The prevalence of anemia and its associated factors in these malaria patients may be connected, not only to the infection, but also to previous or overlapping diseases

Detection of assemblages A and B of Giardia duodenalis in water and sewage from São Paulo state, Brazil.

Giardia duodenalis is a protozoan that parasitizes humans and other mammals and causes giardiasis. Although its isolates have been divided into seven assemblages, named A to G, only A and B have been detected in human faeces. Assemblage A isolates are commonly divided into two genotypes, Al and All. Even though information about the presence of this protozoan in water and sewage is available in Brazil, it is important to verify the distribution of different assemblages that might be present, which can only be done by genotyping techniques. A total of 24 raw and treated sewage, surface and spring water samples were collected, concentrated and purified. DNA was extracted, and a nested PCR was used to amplify an 890 bp fragment of the gdh gene of G. duodenalis, which codes for glutamate dehydrogenase. Positive samples were cloned and sequenced. Ten out of 24 (41.6%) samples were confirmed to be positive for G. duodenalis by sequencing. Phylogenetic analysis grouped most sequences with G. duodenalis genotype All from GenBank. Only two raw sewage samples presented sequences assigned to assemblage B. In one of these samples genotype All was also detected. As these assemblages/genotypes are commonly associated to human giardiasis, the contact with these matrices represents risk for public health.

Risk of Giardia infection for drinking water and bathing in a peri-urban area in Sao Paulo, Brazil.

A high incidence of waterborne diseases is observed worldwide and in order to address contamination problems prior to an outbreak, quantitative microbial risk assessment is a useful tool for estimating the risk of infection. The objective of this paper was to assess the probability of Giardia infection from consuming water from shallow wells in a peri-urban area. Giardia has been described as an important waterborne pathogen and reported in several water sources, including ground waters. Sixteen water samples were collected and examined according to the US EPA (1623, 2005). A Monte Carlo method was used to address the potential risk as described by the exponential dose response model. Giardia cysts occurred in 62.5% of the samples (<0.1-36.1 cysts/l). A median risk of 10⁻¹ for the population was estimated and the adult ingestion was the highest risk driver. This study illustrates the vulnerability of shallow well water supply systems in peri-urban areas.

A modified method for detecting Cryptosporidium oocysts using DNA templates extracted from environmental samples / Método modificado para detecção de oocistos de Cryptosporidium utilizando DNA extraído de amostras ambientais

The protozoan parasite Cryptosporidium has emerged as one of the most important water contaminants, causing outbreaks of waterborne diarrhea worldwide. In order to assess the importance for public health of this pathogen’s presence in environmental samples, several methods have been developed to isolate and detect Cryptosporidium oocysts. In the present study, a reliable and reproducible method has been standardized for detecting and identifying Cryptosporidium oocysts in water samples in the State of São Paulo, Brazil as the first step for future genotyping studies. Water samples were concentrated by filtration, and then subjected to ultrasound in Tween 80 0.1%, the obtained sediment was transferred into micro tubes containing 1.0 ml of distilled water and stored at -20ºC. DNA was extracted with the addition of 1% PVP in lysis buffer, the organic extraction was performed in Phase Lock GelHeavy®. There was a 214 bp amplification on the expected fragment in five out of the 11 water samples analyzed. The results of this study demonstrated the application usefulness of the standardized test in epidemiological studies and surveillance programs because the technology allowed to increase significantly the amount of amplified product.

O protozoário parasito Cryptosporidium tem emergido como um dos mais importantes contaminantes da água, causando surtos de diarreia de veiculação hídrica em todo mundo. Para avaliar o significado, para a saúde pública,da presença desse agente patogênico em amostras ambientais, vários métodos têm sido desenvolvidos para isolar e detectar oocistos de Cryptosporidium. No presente estudo foi padronizado um método confiável e reprodutível para detectar e identificar oocistos de Cryptosporidium em amostras de água no Estado de São Paulo, Brasil, comoo primeiro passo para futuros estudos de genotipagem. Amostras de água foram concentradas por filtração,submetidas a ultrasom em solução de Tween 80 a 0.1%; o sedimento obtido foi transferido para microtuboscontendo 1,0 ml de água destilada e conservado a -20ºC. O DNA foi extraído com adição de 1% de PVP no tampão de lise; a extração foi realizada em tubo Phase Lock Gel Heavy®. Houve amplificação do fragmento esperado de 214 bp em cinco das 11 amostras de água analisadas. Os resultados deste estudo demonstraram a utilidade deaplicação do teste padronizado em estudos epidemiológicos e em programas de vigilância, em virtude da técnica ter apresentado sensibilidade para incrementar significativamente a quantidade de produto amplificado.

Caracterização molecular de isolados de Giardia de amostras de água e esgoto provenientes do Estado de São Paulo / Molecular characterization of Giardia isolates in water and sewage samples from São Paulo State

Introdução - Giárdia é um protozoário que parasita o intestino de quase todas as classes de vertebrados, podendo causar giardíase. Dentre os sete agrupamentos da espécie G. duodenalis, nomeados de A a G, apenas A e B foram encontrados no homem. Por se tratar de um patógeno de veiculação hídrica, a pesquisa de cistos desse microorganismo em água e esgoto é de interesse para a saúde pública. Uma vez que os métodos convencionais utilizados para a detecção desse protozoário em amostras ambientais não permitem diferenciar os isolados de Giárdia potencialmente patogênicos para o homem dos demais, o emprego de técnicas que possibilitam a caracterização molecular do parasita em questão se torna necessário, principalmente no Brasil, onde as informações sobre os genótipos de Giárdia do ambiente são escassas. Objetivos - Detectar a presença e identificar os genótipos de Giárdia sp. em amostras de água e esgoto provenientes do estado de São Paulo, discutindo a importância dos achados para a saúde pública, bem como elaborar uma estratégia que possibilite a realização de tal proposta. Método - Amostras de esgoto bruto (5) e tratado (6), de águas superficiais (11), de poço (3) e de nascente (1) foram coletadas e concentradas pela técnica de membrana filtrante modificada ou por centrifugação. O DNA genômico foi extraído pelo método de fenol/clorofórmio/álcool-isoamílico. A amplificação do fragmento de 890pb do gene gdh, que codifica a produção de glutamato desidrogenase, foi realizada por nested PCR, seguida por clonagem e seqüenciamento de nucleotídeos. Resultados - Onze dentre as vinte e seis amostras analisadas (42,3 por cento) foram confirmadas como sendo positivas para a presença de Giardia duodenalis. Os agrupamentos A, genótipo AII, e B foram encontrados. Conclusão - Esses achados indicam que, no estado de São Paulo, isolados de Giardia associados a giardíase humana estão presentes em esgoto tratado e em água superficial e de poço, de modo que o contato com esse tipo...

Proteção de comunidade aquática em efluente tratado por tanques de estabilização de resíduos no rio São Lourenço em São Lourenço da Serra Vale do Ribeira: Estado de São Paulo / Aquatic community protection on treated effluent disposal from waste stabilization ponds on São Lourenço River in São Lourenço da Serra from Vale da Ribeira in the State of São Paulo / Protección de comunidad acuática en disposición de aguas residuales tratada de charcas de estabilización de desecho en del Río São Lourenço en São Lourenço da Serra de Gastos de la Orilla en el estado de San Paulo

O tratamento de esgotos de pequenas cidades por lagoas de estabilização é uma maneira simples, eficiente e de baixo custo. Os esgotos são uma fonte de contaminação das águas e solos e, conseqüentemente, contribuem para a transmissão de doenças, além de serem uma ameaça à preservação do meio ambiente. Surge a necessidade de investigar as condições dos efluentes lançados nos cursos de água. O presente trabalho tem como objetivo realizar uma investigação da qualidade das águas residuárias tratadas por lagoas de estabilização de uma estação de tratamento de esgoto localizadas no município de São Lourenço da Serra no Vale da Ribeira no Estado de São Paulo e verificar os riscos sanitários e a comunidade aquática no Rio São Lourenço da Serra. Foram realizadas amostragens para avaliar o conjunto de lagoas anaeróbia e facultativa da estação de tratamento de esgoto do Município de São Lourenço da Serra. Os parâmetros utilizados foram pH, temperatura do ar e da água, condições climáticas, demanda bioquímica de oxigênio e nitrogênio amoniacal, bactérias termotolerantes, pigmentos fotossintéticos e comunidade zooplanctônica. Verificou-se que os sistemas de lagoa anaeróbia e facultativa foram eficientes na produção de efluente e apresentou alguns valores de acordo com a Resolução Conama nº 357, que estabelecem os valores limites para lançamento em corpos d´água. O rio São Lourenço está localizado em uma área de proteção ambiental. Os dados são comparados aos limites estabelecidos na Classe 1 e 2 e demonstram processo de eutrofização, colocando em risco à biodiversidade aquática e a saúde da população.

The treatment of sewer pipes of small cities using waste stabilization ponds is a simple, efficient, and low cost option. Sewer pipes are a fountain of water and ground contamination and consequently they contribute to diseases transmission, besides being a threat to the preservation of the environment. It is thus necessary to investigate the conditions of effluents going to water courses. The present work aims to carryout an investigation on the quality of residual waters treated by stabilization ponds of a sewer pipe treatment facility located in São Lourenço da Serra, Vale do Ribeira, State São Paulo and examine the sanitary risks and the local aquatic community. Samples were taken for evaluating theseveral anaerobic and facultative ponds of sewer pipe of the treatment facility of São Lourenço da Serra. Parameters used were pH, air and water temperature, climatic conditions, biochemical demand of oxygen and ammoniac nitrogen, thermo-tolerant bacteria, photosynthetic pigments and zooplanktonic community. We observed that the anaerobic and optional pond systems were efficient in the production of effluent and presentedvalues in accordance with the Resolution Conama nº 357, which establish limits for waste disposal in water sources. São Lourenço River is located in an area of environmental protection, and data were compared to limits established in Class 1 and 2 and demonstrate a process of euthrophization, putting in risk aquatic biodiversity and the health of the population.

El tratamiento de conductos de alcantarilla de pequeñas ciudades usando charcas de estabilización de desecho es una opción simple, eficiente, y de bajo coste. Los conductos de alcantarilla son una fuente de contaminación del agua y de la tierra y por consiguiente contribuyen a la transmisión de enfermedades, además de ser una amenaza a la preservación del ambiente. Es así necesario investigar las condiciones de aguas residuales que van a cursos acuáticos. El trabajo actual pretende realizar una investigación acerca de la calidad de aguas residuales tratadas por charcas de estabilización de una instalación de tratamiento de conductos de alcantarilla localizada en São Lourenço da Serra, Vale do Ribeira, estado São Paulo e examina los riesgos sanitarios y la comunidad acuática local. Se tomaran muestras para evaluar varias charcas anaerobias y facultativasdel conducto de alcantarilla de la instalación de tratamiento de Sã o Lourenço da Serra. Los parámetros usados fueran pH, aire y temperaturaacuática, condiciones climáticas, demanda bioquímica de oxígeno y nitrógeno amoniaco, bacterias termo-tolerantes, pigmentos fotosintéticos ycomunidad zooplanctónica. Observamos que los sistemas de charca anaerobios y opcionales fueran eficientes en la producción de aguas residuales y presentaron valores de acuerdo con la Resolución Conama nº 357, que establecen límites para la eliminación de deshechos en fuentes acuáticas. El Río São Lourenço está localizado en un área de protección ambiental, y los datos fueran comparados con los límites establecidos para las Clases 1 y 2 y muestran un proceso de eutrofización que pone en riesgo la biodiversidad acuática y la salud de la población.