María Viviana Bojanich

An evaluation of the dot-ELISA procedure as a diagnostic test in an area with a high prevalence of human Toxocara canis infection

The aim of this work was to evaluate a dot-enzyme-linked immunosorbent assay (dot-ELISA) using excretory-secretory antigens from the larval stages of Toxocara canis for the diagnosis of toxocariasis. A secondary aim was to establish the optimal conditions for its use in an area with a high prevalence of human T. canis infection. The dot-ELISA test was standardised using different concentrations of the antigen fixed on nitrocellulose paper strips and increasing dilutions of the serum and conjugate. Both the dot-ELISA and standard ELISA methods were tested in parallel with the same batch of sera from controls and from individuals living in the problem area. The best results were obtained with 1.33 µg/mL of antigen, dilutions of 1/80 for the samples and controls and a dilution of 1/5,000 for the anti-human IgG-peroxidase conjugate. All steps of the procedure were performed at room temperature. The coincidence between ELISA and dot-ELISA was 85% and the kappa index was 0.72. The dot-ELISA test described here is rapid, easy to perform and does not require expensive equipment. Thus, this test is suitable for the serological diagnosis of human T. canis infection in field surveys and in the primary health care centres of endemic regions.

Infección por Toxocara canis en población adulta sana de un área subtropical de Argentina

Toxocarosis is a sanitary problem extended world-wide. Numerous reports dealing with this subject have been published in Argentina, focusing many of them about its impact on human health. However, as toxocarosis is not a disease of compulsory notification and many cases are asymptomatic, actual prevalence rates are unknown and the disease is not recognised as a public health problem. The aim of this work was to determine the extent of the infection among healthy adult population from a subtropical region of Argentina, where high prevalence in children have been previously reported. For this purpose, we selected 355 sera of blood donors (325 men-30 women) aged 18-68 years, from a private-owned blood bank of Resistencia, a city located in Northeast Argentina. All samples tested negative for the infectious diseases routinely investigated for blood donation. Elisa test employing excretion/secretion antigens of larvae L 2 Toxocara canis was performed and indeterminate cases were confirmed by Western blot. Out of 355 sera, 138 tested positive (127 men-11 women) which means a global prevalence of 38.9%. There was not significant difference in relation to gender (39.1 % for men - 36.7% for women). The results are consistent with the urban and environmental characteristics of the study area and with socio-cultural habits of its inhabitants, and indicate a strong presence of the ascarid with an important degree of impact on the population.

Immunoblotting para diagnóstico de toxocarosis humana en un área subtropical

The diagnosis of toxocarosis is based upon the demonstration of antibodies by ELISA methods,although cross-reaction with other ascarids may occur in populations from tropical areas. For thisreason, some authors proposed western blotting as a confirmatory test. The aim of this work was todevelop an immunoblotting in simpler technical conditions and to compare results with the ELISAtest. With this purpose sera from adults and children with sign and/or symptoms of toxocarosis andliving in the metropolitan area of Resistencia city (Northeast of Argentina) were studied. ELISA testwas performed and 120 positives and 60 negatives sera were selected and analyzed again byimmunoblotting. Positive samples and controls showed a WB pattern with six bands of 67.6 kDa, 55.6kDa, 43.9 kDa, 32.4 kDa, 26.6 kDa and 23.4 kDa, while negative controls from endemic and non-endemic areas of toxocarosis showed no bands. Out of the 180 samples studied, in 172 coincidentresults for both methods were obtained (95.6%), 6 ELISA negative samples were positive for WB(3.3%) and 2 ELISA positive samples resulted negative in the WB (1.1%). The immunoblotting techniquedescribed in this work may constitute an adequate method for the diagnosis of toxocarosis in subtropicalareas, particularly useful in cases with negative or low-titers ELISA test results and with signs orsymptoms of the infection.

Huevos de Toxocara canis como anzuelo para hongos geófilos en una ciudad subtropical

BACKGROUND The use of different isolation techniques allows the recovery of fungi based on their ability to use selective substrates. The sprinkle method is a technique for the recovery of nematophagous fungi in the soil. These fungi are natural predators of nematodes and are widely distributed in nature. AIMS To detect possible fungi with nematophagous ability in the soil of city parks in Corrientes (Argentina). METHODS The soil samples were taken from an area of ground between two trees and to no more than 2cm deep. The isolation was performed according to the sprinkle method with Toxocara canis eggs as bait. RESULTS Eighteen soil samples were collected, and 6 genera and 8 species of fungi were isolated. The sprinkle method, simple and efficient, has the advantage of using a small amount of untreated soil for the isolation of fungi that can grow on the eggs of geohelminths. The genera Bipolaris, Fusarium, Purpureocillium, Curvularia, Phoma and Scytalidium were isolated in this study. CONCLUSIONS No other studies describing the interaction between the genera Curvularia, Phoma and Scytalidium with nematode eggs have been found in the literature, thus more studies are required to determine what is their real action on these eggs.

ASSESSMENT OF THE PRESENCE OF Toxocara EGGS IN SOILS OF AN ARID AREA IN CENTRAL-WESTERN ARGENTINA

With the aim of studying the contamination of soils with eggs of Toxocara spp. in an arid area in the central-western region of Argentina, 76 soil samples were collected from 18 towns belonging to six provinces of central-western Argentina. They were processed by the centrifugal flotation method. No eggs of Toxocara spp. were found. It can be concluded that the negative results are directly related to the characteristics of the environment and climate present in the studied area. The finding of eggs in soils depends on several factors: the presence of canine or feline feces, the hygienic behavior of pet owners, the presence of stray animals without veterinary supervision, the weather and environmental conditions, and laboratory techniques used; and all these circumstances must be considered when comparing the results found in different geographical regions. In order to accurately define the importance of public spaces in the transmission of infection to humans, it is important to consider the role of backyards or green spaces around housing in small towns, where the population is not used to walking pets in public spaces, and in such cases a significant fraction of the population may acquire the infection within households.

In vitro effect of Chrysosporium indicum and Chrysosporium keratinophylum on Toxocara canis eggs

The degree of antagonism exercised by fungi on geohelminth development varies according to the morphological alterations caused by different fungal species. Saprophytic fungi may exert ovicidal or ovistatic effects. The aim of this study was to apply scanning electron microscopy (SEM) to observe the action of two soil saprophytic species of Chrysosporium (C. indicum and C. keratinophylum) on Toxocara canis eggs. The fungal strains to be tested were incubated for 28 days at 28°C in 2% water agar with a suspension of unembryonated T. canis eggs. A suspension of T. canis eggs in 2% water agar was used as control group. The assay was done in triplicate for each fungus and the control group. SEM observations were performed on the 4th, 7th, 14th, 21st, and 28th day after inoculation. The effect of the fungi on eggs was evaluated in accordance with the alterations observed on the surface and the changes in the normal characteristics of the eggs. Hyphae around the eggs, appresoria penetrating the shell and changes in the typical egg membrane were observed in this assay. Type 3 effect (alterations that occur both in the embryo and the shell, and hyphal penetration of the eggs) was the prevalent effect. SEM allowed us to observe clearly the morphological alterations in T. canis eggs due to the effect of C. indicum and C. keratinophylum. Both saprophytic species of Chrysosporium alter the egg structure and alterations increase as exposure increases.