Evaluation of two heterologous recombinant antigens for the serological diagnosis of human polycystic echinococcosis.

Polycystic echinococcosis (PE) is a zoonosis endemic in the Neotropical region of the Americas. It is caused by the larval stage of the cestode Echinococcus vogeli, which develops as harmful cysts that slowly grow in the liver, lungs and other organs of humans and other host species. Human PE diagnosis is usually based on clinical and epidemiological aspects and imaging techniques, often requiring confirmation by immunological assays. The currently available serological tests for detecting antibodies against Echinococcus spp. are mostly based on complex, variable and poorly characterized mixtures of native parasite antigens, which impairs specificity and/or sensitivity. In this scenario, the evaluation of well-characterized alternative antigens is urgently needed for the improvement of PE diagnosis. Here, two subunits (AgB8/1 and AgB8/2) of the major secretory antigen from Echinococcus granulosus (antigen B (AgB)), of diagnostic value for cystic echinococcosis, were validated for PE diagnosis. These antigens, produced as pure recombinant proteins (rAgB8/1 and rAgB8/2) in Escherichia coli, allowed detecting specific immunoglobulin G antibodies in sera from PE patients in an enzyme-linked immunosorbent assay, with sensitivities of 83.72% and 81.40%, respectively, and specificities of 83.12% and 80.09%, respectively. The use of recombinant proteins overcomes difficulties to obtain parasite material and reduced non-specific reactions and costs. Our results demonstrated reproducibility and accuracy high enough to be considered valid according to the acceptance criteria for Food and Drug Administration assay validation. This qualifies rAgB8/1 and rAgB8/2 as potential substitutes for the currently used parasite crude or partially purified antigens.

Digital Image Analysis to Estimate the Minimum Number of Eurytrema Coelomaticum Eggs in the Uterus of Adult Specimens.

This study was carried out to estimate the minimal number of eggs present in adult E. coelomaticum uterus. Samples were collected during post-mortem inspection and were submitted to light microscopy (bright field). The length, width, the total area of the parasite, uterus, and eggs were measured. The ImageJ software was used to calculate the area of the different parameters analyzed in this study. It was possible to observe that the uterus corresponds on average to 51.9 % of the total area of the parasite (ranging from 45 to 64 %). The number of eggs present in the uterus of parasites ranged from 5,946 to 15,813. To estimate the number of eggs three scenarios were considered, where the first taken into account the number of whole eggs observed in the image. In the second way to estimate the number of eggs, all the structures were considered (whole eggs and fractions that could be delimited) and compared with manual counting. Finally, in the last scenario, was considered an occupancy rate of 100 % of the uterine area per eggs, since there are overlapping eggs and these cannot be correctly delimited and accounted for. This study describes an important tool for quantifying eggs in a nondestructive manner and aggregate information until then is not explained by other works.

Morphometric characteristics of the metacestode Echinococcus vogeli Rausch & Bernstein, 1972 in human infections from the northern region of Brazil.

Polycystic echinococcosis, caused by the larval stage (metacestode) of the small-sized tapeworm, Echinococcus vogeli, is an emerging parasitic zoonosis of great public health concern in the humid tropical rainforests of South and Central America. Because morphological and morphometric characteristics of the metacestode are not well known, hydatid cysts from the liver and the mesentery were examined from patients following surgical procedures. Whole mounts of protoscoleces with rostellar hooks were examined under light and confocal laser scanning microscopy. Measurements were made of both large and small hooks, including the total area, total length, total width, blade area, blade length, blade width, handle area, handle length and handle width. The results confirmed the 1:1 arrangement of hooks in the rostellar pad and indicated, for the first time, that the morphometry of large and small rostellar hooks varies depending upon the site of infection. Light and confocal microscopy images displayed clusters of calcareous corpuscles in the protoscoleces. In conclusion, morphological features of large and small rostellar hooks of E. vogeli are adapted to a varied environment within the vertebrate host and such morphological changes in calcareous corpuscles occur at different stages in the maturation of metacestodes.

Co-infections of the cestode Echinococcus vogeli and the nematode Calodium hepaticum in the hystricomorphic rodent Agouti paca from a forest reserve in Acre, Brazil.

The helminth fauna of Agouti paca (Linnaeus, 1766) has seldom been studied. In this paper, we report an unusual mixed infection of Echinococcus vogeli Rausch & Bernstein, 1972 and Calodium hepaticum (syn. Capillaria hepatica Bancroft, 1863) in free-ranging paca from a forested region in Acre (Brazil). Gross morphological examination revealed that paca liver contained multiple spherical to subspherical white or translucent lesions, which were isolated or frequently contiguous and partially covered by Glisson's capsule. Microscopic examination revealed unilocular cystic structures that contained abundant brood capsules in which numerous protoscolices budded from the inner surface. The protoscolices possessed rostellar hooks (33-41 µm in length), a morphological characteristic of the blade and calcareous corpuscles that is consistent with the metacestode E. vogeli. The diagnosis of C. hepaticum infection was based on the morphology and morphometry of the egg-shaped ellipsoids with bipolar plugs (44.8 ± 1.9 µm (length) × 24.4 ± 2.0 µm (width)) and liver histopathology. This finding expands the known range of C. hepaticum hosts in South America and, to the best of our knowledge, it is the first case of a mixed infection of E. vogeli and C. hepaticum. Furthermore, our data provide evidence that wild animal meat may be a source of C. hepaticum infection.

Do schistosomes grow old? A confocal laser scanning microscopy study.

Infected hosts progressively decrease egg output during the chronic phase of Schistosoma mansoni infection. Ageing may be a factor that results in a progressive decrease in the ability to reproduce. This study was performed to gain insight into the effects of ageing on adult schistosomes, using confocal laser scanning microscopy. Adult worms were recovered from experimentally infected Nectomys squamipes (water rat). Viable eggs were voided in the faeces for 65 weeks and, thereafter, only unviable eggs were eliminated. The rat died after 70 weeks in captivity. Recovered worms (six males and one female) were prepared for confocal microscopy and images were obtained with an LSM 510-ZETA laser confocal microscope. The overall morphology of the adult worms (suckers and tegument) and reproductive organs were the focus of this work. The suckers, ovary, vitellaria and oocytes appeared to be apparently normal. The ootype was formed by flattened cells and unclear nuclei, suggesting that the mechanism for eggshell production by the ootype during organogenesis might have been impaired. Testicular lobes showed empty areas around the germinative cells. Male adult worms showed flaccid musculature of the dorsal surface. Taken together, the present results provided several indications of infertility in older male and female schistosomes.

On the rugose area and caudal papillae distribution in Dirofilaria immitis (Leidy, 1856) Railliet & Henry, 1911, by scanning electron microscopy.

The objective of this study was to obtain a better characterization of Dirofilaria immitis (Leidy, 1856) Railliet & Henry, 1911, in Brazil, ratifying previous accounts and adding new data on the rugose area on the ventral surface of the spiralled posterior portion of males observed by scanning electron microscopy, which presents a different arrangement compared to other species of the genus, and differs also from the genera Litomosoides Chandler, 1931, and Wuchereria Silva Araújo, 1877, adding another taxonomic character to Dirofilariinae and Onchocercinae. Scanning electron microscopy studies also showed some aspects of the distribution of caudal papillae in D. immitis that have not been described before.

Thermal stability studies of hyperimmune horse antivenoms.

Ampoules of horse antivenoms raised against Bothrops spp and Crotalus durissus (final product) produced by Fundação Ezequiel Dias (FUNED) were fractionated on the molecular filtration chromatography (SUPEROSE 12) and the expected MW species of F(ab')2 fragments were observed. It has been known that high temperatures promote aggregation and formation of protein precipitates. Phenol is used in preparations of antivenoms as preservative; however, as thus is a hydrophobic substance, it can also induce an increase in protein denaturation. Sorbitol and glycerol were used as osmolyte (natural substance or organic compound capable of stabilizing proteins) and decreased the formation of protein precipitates in solutions of antibodies; as judged by a spectrophotometric assay (280 nm), by nephelometry or when tested by the ELISA. In 1.0 M concentration, the sorbitol presented the best results when compared with glycerol. Circular Dichroism (CD) was used to study the spectra of antibodies in the presence of PBS, glycerol or sorbitol. Up to 1.0 M concentration of the osmolyte, there is no significant perturbation of the antibodies spectra in the amide region, in control samples not incubated (kept at room temperature with no phenol added) or incubated at 62 degrees C in presence of phenol. Nephelometry and gel SDS-PAGE techniques were used in assays that demonstrated the effects of phenol (denaturing) and the osmolytes (stabilizing) in horse antivenoms in high temperatures.

[Intestinal parasitoses in preschool children of the slum communities of the city of Rio de Janeiro, Brazil]. / Enteroparasitoses em pré-escolares de comunidades favelizadas da cidade do Rio de Janeiro, Brasil.

Prevalence of intestinal parasites was investigated in 1381 low-income children under five years of age from March 1990 to October 1991 in the city of Rio de Janeiro, Brazil. Stool tests were run using the Blagg et al. method. Observed prevalence of infection was 54.5%. The most frequent parasites were Giardia lamblia and Ascaris lumbricoides (25.0% of the children). A significant statistical difference (p<0.05) was demonstrated between age and infection according to the species of parasite studied. Infection rates demonstrated the need for control of parasites in these child.

Purification and stability studies of immunoglobulins from Lachesis muta muta antivenom.

Immunoglobulins were isolated from hyperimmune horse plasma against Lachesis muta muta venom by ammonium sulfate precipitation and immunoaffinity technique (Sepharose-venom L. m. muta column). When necessary, limited proteolysis with pepsin was used to generate a bivalent antigen-binding fragment (F(ab')2). Solutions with immunoglobulins or F(ab')2 fragments were fractionated by molecular filtration chromatography (Superose 12) and the expected mol. wt species were observed. The L. m. muta venom shows caseinolytic and haemorrhagic activities. Incubation of the venom with these purified antibodies resulted in a decrease of both activities. High temperatures promote aggregation and the formation of protein precipitates. Sorbitol (1.0 M) was used as an osmolyte (a natural substance or an organic compound capable of stabilizing proteins) and decreased the formation of protein precipitates in solutions of antibodies, as judged by a spectrophotometric assay (280 nm), by nephelometry or when tested by enzyme-linked immunosorbent assay. Circular dichroism was used to study the spectra of antibodies in the presence of phosphate-buffered saline or sorbitol. Up to an osmolyte concentration of 1.0 M, there was no significant perturbation of the F(ab')2 fragments spectra in the amide region. However, whole immunoglobulins in the presence of 1.0 M sorbitol presented a small spectral perturbation, suggesting that the beta-structure was reinforced. The effect of osmolyte on the affinity of antibodies was observed by enzyme-linked immunosorbent assay. There was no significant difference in the results when the antibodies were previously incubated with venom in phosphate-buffered saline or in the presence of 1.0 M sorbitol. In conclusion, an osmolyte (sorbitol) was shown to be capable of stabilizing antibodies at high temperatures, with no significant perturbation in the secondary structure or affinity to L. m. muta venom. These results point to the possibility of using sorbitol, or other osmolytes, as stabilizers of immunoglobulin preparations.

Human pulmonary dirofilariasis: a review.

The authors presented a detailed summary of the geographical distribution, clinical and pathological aspects of human pulmonary dirofilariasis. Although benign, this zoonosis, of which Dirofilaria immitis is the major etiological agent, represents a medical problem since it produces symptoms which may be confused with neoplasia and thus may subject patients to unnecessary thoracic surgery. Of 229 cases cited in the literature, only 17 were reported in Brazil, despite the existence of highly favorable conditions for the transmission of this infection in man. Thus it may well be that this parasitic infection remains underdiagnosed. Finally, the importance of a differential diagnosis between dirofilariasis and pulmonary neoplasia is emphasized in cases where there is a solitary subpleural nodule ("coin lesion") present. In addition, the development and improvement of modern immunological diagnostic techniques are essential to distinguish this benign disease from other pathological conditions and thus avoid unnecessary surgery. These techniques may reveal the true prevalence of this parasitic infection in our environment.