Exploring challenges in Giardia cyst visualisation by common microscopy methods

Giardia spp. is an intestinal parasite responsible for worldwide disease outbreaks. Guiding researchers and practitioners to choose among current methods for microscopy detection of the infectious forms may be directly beneficial to public health and the environment. This study provides an overall comparison of brightfield (BF), fluorescence and darkfield (DF) microscopies for detecting Giardia duodenalis and Giardia muris cysts, by illustrating micrographs of such protocols applied to purified samples, as well as discussing advantages and constraints based on secondary information and collected data. BF analysis included Lugol's iodine staining. In fluorescence microscopy, samples were processed by immunofluorescence assay (IFA) with DAPI and by standalone DAPI dye. Cyst suspensions were also analysed by DF microscopy using a recently developed low-cost system. The three techniques enabled detecting Giardia spp. cysts, although they did not provide species identification by morphology. The overview of each method points out some relevant aspects to consider when selecting common optical microscopy techniques, and includes challenges and advantages regarding each of them. HIGHLIGHTS Comparisons of BF, DF, standalone DAPI and IFA-combined are provided.; BF and DF may be alternatives for low-cost detection of Giardia cysts.; Combinations of at least two diagnostic methods are recommended to minimise inherent errors.

Household-scale slow sand filter as an alternative for water treatment with high microbiological risk in isolated communities

O objetivo deste trabalho foi aperfeiçoar o filtro lento em escala domiciliar (FLD) à realidade das comunidades isoladas brasileiras com o uso conjunto de pré-tratamento e pós-tratamento acessíveis. As espécies cactáceas Opuntia cochenillifera e Opuntia ficus indica foram avaliadas em ensaios de tratabilidade como coagulantes naturais em pó. A melhor condição para remoção de turbidez e cor aparente foi com 30 mg.L-1 de O. cochenillifera em pH sem a necessidade de correção (± 6,8). O pré-tratamento adaptado às comunidades isoladas utilizando o coagulante natural apresentou valores compatíveis de turbidez (7,83 ± 2,32 uT) para inserção aos filtros. Os FLDs em fluxo contínuo (taxa de filtração de 1,22 m³/m².dia) e em fluxo intermitente (taxa de filtração máxima de 2,79 m³/m².dia) demonstraram redução da turbidez, da cor aparente, dos coliformes totais e da Escherichia coli e produziram água filtrada com ausência de cistos de Giardia spp. e oocistos de Cryptosporidium spp. O FLD em fluxo contínuo teve a maior eficiência em relação à turbidez (± 81,2%); enquanto que o FLD operado em fluxo intermitente apresentou melhores eficiências para a remoção da cor aparente (48,4%), dos coliformes totais (2,60log ± 0,99) e da E. coli (2,86log ± 0,79). Os principais microrganismos atuantes no schmutzdecke dos FLDs foram ciliados (como, por exemplo, a Vorticella sp.), nematódeos e rotíferos. A esponja-vegetal (Luffa cylindrica) e o quiabo (Hibiscus esculentus) foram ponderados como desinfetantes alternativos para o pós-tratamento aos FLDs. As sementes da L. cylindrica e o fruto da H. esculentus foram avaliados através do preparo do extrato e as folhas da L. cylindrica através do preparo em pó. A melhor condição para inativação de E. coli foi com 1 g.L-1 do extrato de H. esculentus em pH sem a necessidade de correção (± 6,8) durante um tempo de contato de 30 min. Essa condição forneceu 1,12log (± 0,16) para a inativação de E. coli, contudo não afetou a viabilidade dos (oo)cistos de Giardia spp. e Cryptosporidium spp. Em ensaios toxicológicos com Chironomus xanthus, a água filtrada não apresentou toxicidade.The aim of this study was to improve the household-scale slow sand filter (HSSF) to the reality of isolated Brazilian communities with combined use of pre-treatment and post-treatment approachable. Cacti species Opuntia cochenillifera e Opuntia ficus indica were evaluated in Jar tests as powder natural coagulants. The best condition for turbidity and apparent color removal was with 30 mg.L-1 of O. cochenillifera at pH without correction (± 6,8). Pre-treatment adapted to isolated communities using natural coagulant showed compatible turbidity values (7.83 ± 2.32 uT) for insertion into the filters. The HSSFs with continuous (filtration rate of 1,22 m³/m².day) and intermittent operation (maximum filtration rate of 2,79 m³/m².day) demonstrated reductions in turbidity, apparent color, total coliforms and Escherichia coli and produced filtered water without Giardia spp. cysts and Cryptosporidium spp. oocysts. The HSSF in continuous operation had the highest turbidity efficiency (± 81.2%); while the HSSF in intermittent operation had showed better efficiencies for the apparent color (48,4%), total coliforms (2,60log ± 0,99) and E. coli (2,86log ± 0,79). The main microorganisms active in the schmutzdecke were ciliates (for example, Vorticella spp.) nematodes and rotifers. Luffa cylindrica and Hibiscus esculentus were evaluated as alternative disinfectant for post-treatment to HSSF. L. cylindrica seed and H. esculentus fruit were tested as extract and L. cylindrica leave was tested powder. The best condition for E. coli inactivation was with 1 g.L-1 of H. esculentus extract at pH without correction (± 6,8) during 30 min. This condition provided 1,12log (± 0,16) for E. coli inactivation, however did not affect Giardia spp. cysts and Cryptosporidium spp. oocysts viability. In toxicological tests with Chironomus xanthus, the filtered water showed no toxicity

Household slow sand filters in a continuous flow as an alternative for surface water treatment systems in isolated communities: a comparative assessment for reducing the filter media depth

A carência de serviços de água potável ainda é uma realidade para milhões de pessoas no mundo, sobretudo para aquelas que vivem em comunidades isoladas. Uma das soluções para essas comunidades é capacitar usuários a melhorar a qualidade da água de consumo por meio de tecnologias de tratamento descentralizadas. O Filtro Lento Domiciliar (FLD) é um exemplo de tecnologia descentralizada com diversas vantagens técnicas, construtivas e operacionais, porém, por ser relativamente recente, ainda persistem lacunas relacionadas a sua eficiência. Para a transferência da tecnologia às comunidades isoladas, é fundamental que, além de eficiente, o FLD apresente um tamanho de estrutura adequado ao domicílio dos usuários. Diante disso, este estudo avaliou o impacto da redução da espessura do leito filtrante a fim de torná-lo menor, mais leve e passível de ser alocado em uma bancada de cozinha. Com o intuito de comparar a qualidade e a quantidade das águas filtradas, foram avaliados dois modelos de FLD, um tradicional (FLD-T) com 50 cm de leito e um compacto (FLD-C) com 25 cm de leito, alimentados por águas superficiais do Rio Monjolinho e operados em regime contínuo. Devido às limitações dos FLDs, foram inseridas etapas de pré- e pós-tratamento para avaliar um sistema completo a nível domiciliar. O sistema foi operado por 436 dias consecutivos com 1) pré-tratamento por sedimentação e filtração em mantas, 2) FLDs aplicados a uma taxa de filtração constante de 0,90 m3 m-2 d-1, considerando uma produção individual de 48 L d-1, e 3) pós-tratamento por desinfecção com hipoclorito de sódio 2,5% a 60 mg min L-1. As eficiências dos modelos foram comparadas quanto à remoção de diversos parâmetros de qualidade da água – incluindo turbidez, Escherichia coli, cistos de Giardia spp. e oocistos de Cryptosporidium spp. – bem como às durações das carreiras de filtração, aos perfis de oxigênio dissolvido e às composições dos schmutzdeckes desenvolvidos. Os resultados demonstraram que o pré-tratamento reduziu a turbidez da água do rio em 46 ± 23% diminuindo a sobrecarga e permitindo o funcionamento dos filtros, mas não foi capaz de atenuar variações sazonais e manter a qualidade da água clarificada durante toda a operação. As variações sazonais12 refletiram igualmente nos dois modelos de FLD, pois ambos produziram águas filtradas com qualidade estatisticamente semelhante (p > 0,05) em todos os parâmetros avaliados, indicando que a redução da espessura do leito filtrante não influenciou o desempenho, tampouco a duração da carreira de filtração, o perfil de consumo de oxigênio dissolvido e a composição do schmutzdecke. No entanto, a sazonalidade impactou o desempenho em alguns parâmetros, como turbidez e coliformes totais, de forma que a variação da qualidade da água do rio foi de maior relevância para a qualidade da água filtrada do que a profundidade do leito. Isoladamente, os FLDs removeram 73 ± 20% de turbidez, 1,99 ± 0,66 log de E. coli, 2,82 ± 0,45 log de cistos de Giardia e 1,09 ± 0,23 log de oocistos Cryptosporidium. Considerando as etapas de pré- e pós-tratamento, os sistemas removeram 87 ± 15% de turbidez e 2,91 ± 0,31 log de E. coli, porém não elevaram as remoções de cistos e oocistos. O pós-tratamento foi ineficiente na inativação de bactérias e protozoários, todavia, os sistemas domiciliares propostos melhoraram significativamente a qualidade da água de consumo. Portanto, a implantação de um sistema de tratamento com FLD compacto é executável, porém demanda pesquisas adicionais para produção de águas seguras livres de patógenos.The lack of drinking water services is still a reality for millions of people worldwide, especially for those living in isolated communities. One solution for these communities is to allow users to improve the quality of drinking water through decentralized treatment technologies. The Household Slow Sand Filter (HSSF) is an example of decentralized technology with several technical, constructive, and operational advantages; however, as it is a relatively recent technology, gaps related to its efficiency still persist. For the transfer to isolated communities, it is essential that, besides being efficient, the HSSF has a structure size suitable for the users\' homes. Therefore, this study evaluated the impact of reducing the filter media depth to make it smaller, lighter, and liable to be placed on a kitchen counter. To compare the quality and quantity of filtered water, two models of HSSF were evaluated, a traditional (HSSF-T) with 50 cm of fine sand and a compact (HSSF-C) with 25 cm of fine sand, fed by surface waters of the Monjolinho River and operated in a continuous flow. Due to the limitations of HSSFs, pre- and post-treatment steps were inserted to assess a complete home system. The system was operated for 436 consecutive days with 1) pre-treatment by sedimentation and filtration in blankets, 2) FLDs applied at a constant filtration rate of 0.90 m3 m-2 d-1, considering an individual production of 48 L d-1, and 3) post-treatment by disinfection with 2.5% sodium hypochlorite at 60 mg min L-1. The model\'s efficiencies were compared in terms of water quality parameters - including turbidity, E. coli, Giardia spp. cysts and Cryptosporidium spp. oocysts - as well as the duration of filtration runs, dissolved oxygen profiles, and compositions of the developed schmutzdeckes. The results showed that the pre-treatment reduced river water turbidity by 46 ± 23% – reducing overhead and allowing the filter operation – but it was not able to mitigate seasonal variations and maintain clarified water quality throughout the operation. Seasonal variations were equally reflected in the two HSSF models, as both produced filtered water with statistically similar quality (p > 0.05) in all evaluated parameters, indicating that the reduction in filter media depth did not influence the performance, nor the filter run, the dissolved oxygen consumption profile and the schmutzdecke composition. However, seasonality impacted performance on some parameters, such as turbidity and total coliforms, so that the variance in the river water quality was more relevant to the filtered water quality than the media depth. The FLDs by themselves removed 73 ± 20% of turbidity, 1.99 ± 0.66 log of E. coli, 2.82 ± 0.45 log of Giardia cysts, and 1.09 ± 0.23 log of Cryptosporidium oocysts. Considering the pre- and post-treatment stages, the systems removed 87 ± 15% of turbidity and 2.91 ± 0.31 log of E. coli but did not increase the removal of cysts and oocysts. Post-treatment was inefficient in inactivating bacteria and protozoa; however, the proposed household systems have significantly improved the drinking water quality. Therefore, the implementation of a treatment system with compact HSSF is feasible; nonetheless, further research is required to produce safe water free of pathogens

Exploring challenges in Giardia cyst visualisation by common microscopy methods

Giardia spp. is an intestinal parasite responsible for worldwide disease outbreaks. Guiding researchers and practitioners to choose among current methods for microscopy detection of the infectious forms may be directly beneficial to public health and the environment. This study provides an overall comparison of brightfield (BF), fluorescence and darkfield (DF) microscopies for detecting Giardia duodenalis and Giardia muris cysts, by illustrating micrographs of such protocols applied to purified samples, as well as discussing advantages and constraints based on secondary information and collected data. BF analysis included Lugol's iodine staining. In fluorescence microscopy, samples were processed by immunofluorescence assay (IFA) with DAPI and by standalone DAPI dye. Cyst suspensions were also analysed by DF microscopy using a recently developed low-cost system. The three techniques enabled detecting Giardia spp. cysts, although they did not provide species identification by morphology. The overview of each method points out some relevant aspects to consider when selecting common optical microscopy techniques, and includes challenges and advantages regarding each of them. HIGHLIGHTS Comparisons of BF, DF, standalone DAPI and IFA-combined are provided.; BF and DF may be alternatives for low-cost detection of Giardia cysts.; Combinations of at least two diagnostic methods are recommended to minimise inherent errors.

Brazilian Flora 2020: Leveraging the power of a collaborative scientific network

International audienceThe shortage of reliable primary taxonomic data limits the description of biological taxa and the understanding of biodiversity patterns and processes, complicating biogeographical, ecological, and evolutionary studies. This deficit creates a significant taxonomic impediment to biodiversity research and conservation planning. The taxonomic impediment and the biodiversity crisis are widely recognized, highlighting the urgent need for reliable taxonomic data. Over the past decade, numerous countries worldwide have devoted considerable effort to Target 1 of the Global Strategy for Plant Conservation (GSPC), which called for the preparation of a working list of all known plant species by 2010 and an online world Flora by 2020. Brazil is a megadiverse country, home to more of the world's known plant species than any other country. Despite that, Flora Brasiliensis, concluded in 1906, was the last comprehensive treatment of the Brazilian flora. The lack of accurate estimates of the number of species of algae, fungi, and plants occurring in Brazil contributes to the prevailing taxonomic impediment and delays progress towards the GSPC targets. Over the past 12 years, a legion of taxonomists motivated to meet Target 1 of the GSPC, worked together to gather and integrate knowledge on the algal, plant, and fungal diversity of Brazil. Overall, a team of about 980 taxonomists joined efforts in a highly collaborative project that used cybertaxonomy to prepare an updated Flora of Brazil, showing the power of scientific collaboration to reach ambitious goals. This paper presents an overview of the Brazilian Flora 2020 and provides taxonomic and spatial updates on the algae, fungi, and plants found in one of the world's most biodiverse countries. We further identify collection gaps and summarize future goals that extend beyond 2020. Our results show that Brazil is home to 46,975 native species of algae, fungi, and plants, of which 19,669 are endemic to the country. The data compiled to date suggests that the Atlantic Rainforest might be the most diverse Brazilian domain for all plant groups except gymnosperms, which are most diverse in the Amazon. However, scientific knowledge of Brazilian diversity is still unequally distributed, with the Atlantic Rainforest and the Cerrado being the most intensively sampled and studied biomes in the country. In times of “scientific reductionism”, with botanical and mycological sciences suffering pervasive depreciation in recent decades, the first online Flora of Brazil 2020 significantly enhanced the quality and quantity of taxonomic data available for algae, fungi, and plants from Brazil. This project also made all the information freely available online, providing a firm foundation for future research and for the management, conservation, and sustainable use of the Brazilian funga and flora