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Detecção molecular e monitoramento sazonal de adenovírus em águas fluviais no município de Goiânia, Goiás-Brasil: correlação com parâmetros físico-químicos, bacteriológicos e Metanálise avaliativa de metodologias / Adenovirus molecular detection and seasonal monitoring in bodies of water in the municipality of Goiânia, Goiás-Brazil: correlation with physical-chemical and bacteriological parameters and meta-analysis to evaluate methodologiesSILVA, Hugo Delleon da 27 May 2009 (has links)
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Previous issue date: 2009-05-27 / Although water is of vital importance for living beings, due to antropic action it
becomes a way of dissemination of several microorganisms, which reach aquatic
environments through the faeces of man and other animals and can cause several
illnesses, especially for immunocompromised individuals. During routine
environmental monitoring, coliform bacteria are normally used as a microbiological
parameter of water quality, which does not evidence its contamination by viruses.
Several researchers have proposed the detection of adenovirus (AdVs) by PCR as a
molecular index to monitor other enteric viruses. AdVs are among the most persistent
and ubiquitous enteric viruses present in water and associated with a variety of
clinical manifestations. This study aimed to evaluate the quality of water collected
from lakes and rivers in Goiânia as to the occurrence of AdVs. Water samples were
collected monthly, from December 2007 to November 2008, at five different points in
Goiânia (lakes of Bosque dos Buritis and Vaca Brava park, João Leite and Meia
Ponte rivers downstream and upstream the municipal sewage treatment plant). The
analyses were carried out at the Laboratório de Diagnóstico Genético e Molecular
and Laboratório de Genética Molecular e Citogenética, Universidade Federal de
Goiás. All the samples were filtered in a positively-charged nylon membrane followed
by molecular detection using PCR and semi-nested PCR. Also, we performed
physical-chemical and bacteriological tests to correlate these results with the
occurrence of AdVs. Simultaneously, the Núcleo de Pesquisas em Agentes
Emergentes e Re-emergentes carried out a meta-analysis to evaluate three methods
of concentration of AdVs coupled to molecular detection in samples of untreated
water. Since 29 out of the 54 water samples collected were positive for AdVs
(39.2%), our results suggest the use of the methodology proposed in the present
study for the detection of these pathogens in water. We observed statistically
significant difference between nitrites, phosphates, fixed residues, total residues and
the occurrence of AdVs, whereas no correlation was observed between fecal
coliforms and AdVs. Furthermore, the occurrence of AdVs in the state of Goiás
shows a seasonal trend. Based on the 33 studies selected for the meta-analysis, it
was possible to get to the following interpretations: the most effective method to
detect AdVs in samples from rivers or lakes was ultracentrifugation combined with
nested-PCR; it is advisable to use a combination of microfiltration membrane and
ultrafugation with the subsequent diagnosis using qPCR to detect AdVs in samples of
treated and untreated sewage. This has been the first study carried out for the
detection and monitoring of AdVs in water bodies in the Midwestern Region of Brazil
and the present results may be useful to propose the eco-epidemiological profile of
AdVs or even the routes of some neglected diseases, which points out the need to
define a virus indicator / Embora a água seja de vital importância para os seres vivos, em decorrência da
ação antrópica torna-se meio de disseminação de inúmeros microrganismos, que
chegam aos ambientes aquáticos por meio das fezes do homem e de outros
animais, podendo desencadear diversas doenças, sobretudo em indivíduos
imunocomprometidos. Em rotina de monitoramento ambiental, normalmente são
utilizadas bactérias do grupo dos coliformes fecais como parâmetro microbiológico
de qualidade das águas, o que não evidencia sua contaminação por vírus. Alguns
pesquisadores têm proposto a detecção de adenovírus (AdVs) por PCR como
indexação no monitoramento de outros vírus entéricos. AdVs estão entre os mais
persistentes e ubíquos vírus entéricos presentes em águas e associados com uma
variedade de manifestações clínicas. Objetivou-se avaliar a qualidade da água de
rios e lagos da cidade de Goiânia em relação à ocorrência de AdVs. Amostras de
água foram coletadas mensalmente, entre dezembro de 2007 e novembro de 2008,
em cinco diferentes pontos de Goiânia (lagos do Bosque dos Buritis e parque Vaca
Brava e rios João Leite e Meia Ponte a jusante e montante da estação de tratamento
de esgoto municipal). As análises laboratoriais foram realizadas pelo Laboratório de
Diagnóstico Genético e Molecular e Laboratório de Genética Molecular e
Citogenética, Universidade Federal de Goiás. Inicialmente, as amostras foram
filtradas em membrana de nylon carregada positivamente e, em seguida, submetidas
a detecção molecular por PCR e semi-nested PCR. Também foram realizados testes
físico-químicos e bacteriológicos para correlacionar estes resultados com a
ocorrência de AdVs. Concomitantemente, o Núcleo de Pesquisas em Agentes
Emergentes e Re-emergentes conduziu uma metanálise avaliativa de três
metodologias de concentração de AdVs acopladas à detecção molecular em
amostras de águas não tratadas. Das 54 amostras coletas, 29 foram positivas para
AdVs (39,2%), sugerindo o uso da metodologia proposta neste estudo para detectar
estes patógenos em água. Foi observada diferença estatisticamente significativa
entre nitritos, fosfatos, resíduos fixos, resíduos totais e a ocorrência de AdVs, mas
não foi observada correlação entre coliformes fecais e AdVs. Além disso, a
ocorrência de AdVs no estado de Goiás exibiu tendência à sazonalidade. Na
metanálise, a partir dos 33 artigos selecionados foi possível fazer as seguintes
interpretações: para detectar AdVs em amostras de rios ou lagos, o método mais
eficiente foi a ultracentrifugação em combinação com nested-PCR; para a detecção
de AdVs em amostras de esgoto tratado e não tratado é aconselhável utilizar uma
combinação de microfiltração em membrana e ultrafiltração com subsequente
diagnóstico por qPCR. Este foi o primeiro estudo a detectar e monitorar AdVs em
cursos de água na Região Centro-Oeste do Brasil e os presentes resultados podem
ser úteis para propor o perfil eco-epidemiológico dos AdVs ou até mesmo das rotas
de algumas doenças que são negligenciadas, o que demonstra a necessidade de
definir um indicador viral
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Flow Cytometry Sensor System Targeting Escherichia Coli as an Indicator of Faecal Contamination of Water SourcesBenselfelt, Tobias January 2014 (has links)
Poor water quality is a global health concern affecting one billion people around the world. It is important to monitor water sources in order to maintain the quality of our drinking water and to avoid disease outbreaks. Targeting Escherichia coli as a faecal indicator is a widely used procedure, but the current methods are time consuming and not adequate to prevent spreading of faecal influence. This Master thesis demonstrates the development of a near infrared fluorescence flow cytometer sensor system targeting Escherichia coli, using fluorescently labeled chicken IgY antibodies. The near infrared light was chosen to avoid fluorescence from blue-green algae that are present in the water source. The hardware was developed with a 785 nm laser line to detect Alexa Fluor 790 labeled antibodies, using a photomultiplier tube or two different CMOS cameras. The antibodies were labeled using a commercial labeling kit, and evaluated using antibody binding assays and the developed hardware. The IgY antibodies were successfully labeled with Alexa Fluor 790 and the function was maintained after the labeling process. The result demonstrates the principles of the sensor system and how it solved to the problem with fluorescence from blue-green algae. An aperture was used to overcome the suboptimal laser and filter setup, and to increase the sensitivity of the system. However, only a small fraction of the cells could be detected, due to challenges with the focal depth and loss of sensitivity in the photomultiplier tube at near infrared wavelengths. Further development is required to create a working product.
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