Determination of the relative avidity of the specific IgG antibodies in human toxocariasis.

Human toxocariasis is a zoonosis caused by infection with larvae of the ascarid nematode Toxocara canis and, less frequently, T. cati. Our study developed a method for distinguishing distant from recent human toxocariasis by assessing the avidity of the IgG antibodies. The avidity of specific antibodies increases with time after antigen challenge and assessment of the degree of avidity can be used to discriminate between recent and distant infections. The relative avidity was measured in 150 sera from children with visceral toxocariasis and in 46 sera from children with ocular toxocariasis. The probable time of infection was estimated on the basis of the medical history and clinical syndrome. Our study showed that 94.2% of positive sera collected from patients reporting infection > 6 months ago had high IgG avidity values, confirming distant toxocariasis, whereas 25.9% of positive sera taken < 6 months after infection showed low indices of IgG avidity. Our results suggest that measurement of the specific IgG avidity may assist in discriminating between recent and distant toxocariasis. The method can be used effectively to rule out (because of high avidity) a recently acquired infection. Low avidity is less reliable in discriminating between recent and distant infections.

Detection of Echinococcus multilocularis antigens in faeces by ELISA.

Faecal samples deriving from 391 animals belonging to nine species (polecats, badgers, martens, weasels, rats, dogs, cats, red foxes, raccoon-dogs) were examined by capture ELISA for the presence of the Echinococcus multilocularis coproantigen. The main claim of our studies is the reliable detection of E. multilocularis coproantigens, mainly in the faeces of foxes, dogs and cats. For the first time in coproantigen detection we used a "double-sandwich" ELISA. The main advantage of this method is the higher specificity and better differentiation of positive and negative faecal samples, in comparison with sandwich ELISA. The overall specificity of double-sandwich ELISA was 95.1% with only 16 of 327 E. multilocularis-free animals giving false-positive results. The E. multilocularis coproantigen was detected by double-sandwich ELISA in 37.5% of examined red foxes and in 8.0% of examined raccoon-dogs, compared with a prevalence of just 29.8% in red foxes and 8.0% in raccoon-dogs, as determined by parasitological techniques.

Reduction of muscle larvae burden in rats experimentally infected with Trichinella spiralis.

In Wistar rats infected with 500 to 2,500 Trichinella spiralis larvae the muscle larvae intensity (larvae per gram-l.p.g.) was measured from 20 to 180 day post infection (d.p.i). The l.p.g. increased to day 40-50 p.i. and decreased thereafter. The highest reduction took place between 60 and 120 d.p.i. with intermediate inoculum of T. spiralis larvae. The mechanism of the reduction of T. spiralis larvae in muscles is suggested to depend on pericapsular-intercapsular host cells infiltrations attracted by parasite antigens.

Detection of Trichinella spiralis antigens in urine of men and animals.

The practical inability to diagnose Trichinella spiralis antibodies in man before day 20 post infection (dpi) has stimulated interest in the development of immunodiagnostic test to detect circulating antigens. Our previous experience showed that soon after infection immune complexes as well as uncomplexed parasite antigens in sera of infected rats could be detected. To diagnose the presence of antigen in urine, double sandwich-capture ELISA was applied using a peroxidase-conjugated rabbit immunoglobulin to T. spiralis larval antigens. The plates were coated with metabolic (AES) or somatic (AS) larval antigens. Mice were infected with 500 T. spiralis larvae. The urine samples from experimentally infected mice taken from 1 to 41 dpi. and the urine samples from patients of the Clinical Hospital in Bialystok taken from 3 to 120 dpi were examined. Before testing, the urine samples were heated for 6 min. at 100 degrees C and centrifuged for 6 min. at 5000 g, supernatants were used in ELISA. The presence of T. spiralis antigens in mice urine samples was detected between 6-26 days post infection (dpi) using double sandwich-capture ELISA. All samples taken later were negative as samples taken from uninfected mice. 3 from 9 human urine samples taken 3-10 dpi were positive, the remaining samples taken 3-10 and 10-30 dpi showed values near to "cut-off". In both mice and human urine samples the higher level of antigens was detected in ELISA when somatic larval antigen was used. The T. spiralis antigens were present in urine of infected men and mice in the first phase of infection.

Detection of Taenia saginata antigens in faeces by ELISA.

Stool samples were examined by capture ELISA for the presence of T. saginata antigens. The specimens were from 303 patients infected with T. saginata, 43 samples from individuals suffering from bacterial, fungal, protozoan and nematode infections and 36 ones deriving from uninfected persons. The samples were diluted 1:10 (w:v) in PBS + 0.35% Tween 20, centrifuged twice at 5000 g for 20 min at room temperature and resulting supernatant at 12,000 g for 30 min at 0 degree C. The capturing and detecting polyclonal antibodies were prepared in rabbits immunizing them with T. saginata surface antigens. The stool samples freshly obtained, stored at 4 degrees C not longer than 72 h, or kept at at -20 to -30 degrees C even for 24 months showed reliable results in ELISA. The samples kept for a long time at 4 degrees C, dried and covered with moult were negative in the test. All samples taken from patients infected with other than T. saginata parasites or uninfeced were negative.

Factors conditioning detection of Taenia saginata antigens in faeces.

The conditions which optimize detection of T. saginata antigens in faeces were subject of examination. The most appropriate appeared diluent of PBS + 0.3% Tween 20. The highest effectiveness was achieved using capturing and detecting antibodies to surface antigens in comparison with somatic and metabolic ones. Two step centrifugation and final dilution of samples 1:10 (w:v) were recommended. The stability of freezed faecal supernatants after the first centrifugation was limited to 2 months. The sensitivity of capture ELISA established in our examinations allow to signalize 1 ng/ml antigen in high absorbance values. The antigens recognized by the test had MW bigger than 100,000.