Endemic Angiostrongyliasis, Rio de Janeiro, Brazil

Submitted by Sandra Infurna ([email protected]) on 2016-10-06T12:58:28Z No. of bitstreams: 1 fernando_monteiro_etal_IOC_2011.pdf: 214467 bytes, checksum: 7938fea29a355143991cd4f518b2d970 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2016-10-06T13:12:13Z (GMT) No. of bitstreams: 1 fernando_monteiro_etal_IOC_2011.pdf: 214467 bytes, checksum: 7938fea29a355143991cd4f518b2d970 (MD5)Made available in DSpace on 2016-10-06T13:12:13Z (GMT). No. of bitstreams: 1 fernando_monteiro_etal_IOC_2011.pdf: 214467 bytes, checksum: 7938fea29a355143991cd4f518b2d970 (MD5) Previous issue date: 2011Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / Universidade Federal Rural do Rio de Janeiro. Seropédica, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil.Universidade Federal Rural do Rio de Janeiro. Seropédica, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil

Histological assessment of granulomas in natural and experimental <i>Schistosoma mansoni</i> infections using whole slide imaging

<div><p>The pathology of schistosomiasis mansoni, a neglected tropical disease of great clinical and socioeconomic importance, results from the parasite eggs that become trapped in host tissues, particularly in the liver and intestines. Continuous antigenic stimulation from these eggs leads to recruitment of inflammatory cells to the sites of infection with formation of periovular granulomas. These complex structures have variable size and composition and are the most striking histopathological feature of schistosomiasis mansoni. However, evaluation of granulomas by conventional microscopy methods is time-consuming and limited, especially in large-scale studies. Here, we used high resolution Whole Slide Imaging (WSI), which allows fast scanning of entire histological slides, and multiple morphometric evaluations, to assess the granulomatous response elicited in target organs (liver, small and large intestines) of two models of schistosomiasis mansoni. One of the advantages of WSI, also termed virtual microscopy, is that it generates images that simultaneously offer high resolution and a wide field of observation. By using a model of natural (<i>Nectomys squamipes</i>, a wild reservoir captured from endemic areas in Brazil) and experimental (Swiss mouse) infection with <i>Schistosoma mansoni</i>, we provided the first detailed WSI characterization of granulomas and other pathological aspects. WSI and quantitative analyses enabled a fast and reliable assessment of the number, evolutional types, frequency and areas of granulomas and inflammatory infiltrates and revealed that target organs are differentially impacted by inflammatory responses in the natural and experimental infections. Remarkably, high-resolution analysis of individual eosinophils, key cells elicited by this helminthic infection, showed a great difference in eosinophil numbers between the two infections. Moreover, features such as the intestinal egg path and confluent granulomas were uncovered. Thus, WSI may be a suitable tool for detailed and precise histological analysis of granulomas and other pathological aspects for clinical and research studies of schistosomiasis.</p></div

Sequence of steps to acquire and analyze whole slide images.

<p>(A) After loading the slides in the scanner, image acquisition starts with a “prescan” step in which the equipment takes a low resolution grayscale image followed by an automated setup process termed “profile” in which the tissue is detected and focused. (B) The equipment scans all regions of the slide defined by the profile and generates a virtual slide, which is saved on disk. These first steps take just few minutes and many slides can be sequentially scanned. (C, Ci) The operator can then review the virtual slide(s) and work on each one by selecting the area(s) of interest with the use of morphometric software. In the present work, the number and area of granulomas and area of inflammatory infiltrates were quantitated by using <i>Pannoramic Viewer 1</i>.<i>15</i>.<i>2 SP2 RTM</i> software. A representative digital slide shows a section from the small intestine of an <i>S</i>. <i>mansoni</i>-infected mouse in which the above parameters were manually delineated for subsequent automatic quantification.</p

Eosinophil numbers in the natural and experimental infections with <i>S</i>. <i>mansoni</i>.

<p>(A) Representative image of a hepatic granuloma from a naturally infected wild reservoir (<i>N</i>. <i>squamipes</i>) showing accumulation of eosinophils (indicated in high magnification in Ai by arrowheads). (B) Quantitative analyses revealed a lower number of eosinophils per granuloma area (μm²) in all target organs in the natural infection compared to the experimental infections. Different letters indicate significant differences between the means (<i>P</i> < 0.0001 for all comparisons between different letters in the respective groups). (C) Proportion of eosinophils within hepatic granulomas in each model of the infection. (****) <i>P</i> < 0.0001. Eosinophil numbers were quantitated per granuloma area, considering all types of granuloma (B) or per most frequent type of hepatic granuloma (C). Data represent mean ± SEM. Morphometric evaluation was done with the use of <i>Histoquant</i> software. Bar = 400 μm (A), 100 μm (Ai).</p

Granuloma area analyzed in digital slides from target organs of <i>S</i>. <i>mansoni</i>-infected animals.

<p>Granuloma area analyzed in digital slides from target organs of <i>S</i>. <i>mansoni</i>-infected animals.</p

Intensity of the granulomatous response in target organs of rodents naturally or experimentally infected with <i>S</i>. <i>mansoni</i>.

<p>(A) Mean percentage of granulomatous response in livers and intestines (mean ± SEM). (B-J) Representative virtual slides of tissues with different levels of granuloma formation. In the liver (B, E, H), the lowest response is shown by the natural infection in <i>N</i>. <i>squamipes</i> (B) compared to the experimental infection in mice (E, H), while in the small intestines (C, F, I), the highest granuloma formation was observed in this wild reservoir (C). Different letters indicate significant differences between the means (<i>P</i> < 0.0001 for all comparisons between different letters in the respective groups). Bar = 1000 μm (B, D, F); 950 μm (H); 750 μm (C); 650 μm (E, G, I, J).</p

Liver and intestine areas taken by inflammatory infiltrates in the natural and experimental infections with <i>S</i>. <i>mansoni</i>.

<p>(A) Representative image of hepatic tissue from a mouse experimentally infected. After acquisition of whole slide images, areas with inflammatory infiltrates (marked in red) outside typical granulomas (marked in green) were measured. In (B), morphometric analyses reveal a very low incidence of infiltrates in the liver of infected wild rodent (natural infection) while mice experimentally infected show a very high proportion of infiltrates. In the small intestine, infiltrates are moderately higher in the natural compared to the acute experimental infection. Different letters indicate significant differences between the means (<i>P</i> < 0.0001 for all comparisons between different letters in the respective groups).</p

Confluent granulomas in livers and small and large intestines of <i>Nectomys squamipes</i> and Swiss mice infected with <i>Schistosoma mansoni</i>.

<p>(A-H) General morphology of confluent granulomas in different organs. Graph I shows the proportions of confluent granulomas in livers and in intestines taken from naturally infected <i>N</i>. <i>squamipes</i> (at day one of capture after confirmation of the infection) and from experimentally infected Swiss mice at days 55 (acute phase) and 120 (chronic phase) of infection. Confluent granulomas were quantified using Pannoramic Viewer software. (***) indicates significant differences between the means (<i>P</i> < 0.001). Bar = 100 μm (A, D), 220 μm (B, C); 150 μm (E, F, G, H).</p

An ecological field study of the water-rat Nectomys squamipes as a wild reservoir indicator of Schistosoma mansoni transmission in an endemic area

Small mammals are found naturally infected by Schistosoma mansoni, becoming a confounding factor for control programs of schistosomiasis in endemic areas. The aims of this study were: to investigate the infection rates by S. mansoni on the water-rat Nectomys squamipes during four years in endemic areas of Sumidouro, state of Rio de Janeiro, using mark-recapture technique; to compare two diagnostic methods for schistosomiasis; and to evaluate the effects of the chemotherapy in the human infected population on the rodent infection rates. The rodent infection rates of S. mansoni increased when rodent population sizes were lower. Coprology and serology results presented the same trends along time and were correlated. Serology could detect recent infection, including the false negatives in the coprology. The chemotherapy in the humans could not interrupt the rodent infection. Rodents can increase the schistosomiaisis transmission where it already exists, they probably maintain the transmission cycle in the nature and can be considered as biological indicators of the transmission sites of this parasite since they are highly susceptible to infection. The water-rats may present different levels of importance in the transmission dynamics of S. mansoni infection cycle for each area, and can be considered important wild-reservoirs of this human disease

Representative types of granulomas and their frequencies in target organs of rodents naturally or experimentally infected with <i>S</i>. <i>mansoni</i>.

<p>Four types of granulomas were identified in virtual slides: Pre-granulomatous exudative (<b>PE</b>); necrotic-exudative (<b>NE</b>); exudative-productive (<b>EP</b>) and productive (<b>P</b>) as described in material and methods. Bar = 250 μm.</p