Human serum antibodies against shared antigens of different stages of Trichinella spiralis: relevance of glycan and protein epitopes.

Taking into account that antibodies against the surface antigens of newborn larvae (anti-NBL Abs) present in sera from individuals with chronic trichinellosis recognize antigenic determinants of the excretory-secretory muscle larva products (ML-ESP), and that these products are mainly glycoproteins, the aim of this work was to assess the frequency of anti-NBL Abs in sera from individuals with acute and chronic trichinellosis, to analyse the relevance of glycan and protein epitopes of the ML-ESP in the cross-reactivity phenomenon, and its correlation with the host's serum response towards these products. Anti-NBL surface Abs were determined in sera by indirect immunofluorescence. The degree of recognition by serum and purified anti-NBL Abs was evaluated comparatively before and after chemical deglycosylation of ML-ESP by immunoelectrotransfer blot assay. Results showed that 64% of the sera from individuals with acute trichinellosis and 35% of those belonging to the chronic phase had anti-NBL Abs, and also that the protein epitopes are the major ones responsible for the cross-reactivity phenomenon involving the ML-ESP and the NBL surface during both phases of the infection, while glycan epitopes are immunodominant in the stimulation of the host's immune system. A modulatory phenomenon in the immune response generated towards Trichinella spiralis NBL driven by the ML-ESP is postulated.

Diagnosis of human trichinellosis: pitfalls in the use of a unique immunoserological technique.

Serum samples belonging to three outbreaks in Argentina (47 patients) taken at different times post-ingestion were analysed employing IIF and ELISA simultaneously. Results show that: a) the number of patients diagnosed by a unique technique, especially by ELISA (31 patients), was lower than the one obtained by the simultaneous use of both assays (38 patients); b) four patients out of the seven diagnosed by a unique technique were negative by the other assay over the period of time evaluated. Therefore, it can be concluded that the use of a sole immunoserological technique can not only lead to the delay in the detection but also to the misdiagnosis of this parasitic infection.

Immunoelectrotransfer blot assay in acute and chronic human trichinellosis.

An immunoelectrotransfer blot assay (IETB) using excretory secretory products of muscle larvae of Trichinella spiralis (ML-ESP) and the avidin biotin system was developed in order to characterize reactivity against ML-ESP in sera from patients with acute and chronic trichinellosis. A complete pattern of up to 13 bands was developed by sera from individuals with trichinellosis where doublets, triplets, or single bands were shown to have molecular weights of roughly 66, 55, 45, 36, 29, 24, and 14 kDa. The bands at approximately 55, 36, 29, and 14 kDa proved specific for T. spiralis. The band at approximately 55 kDa was present in all trichinellosis sera, whereas the approximately 14-kDa band was present in only a small percentage of sera. The development of approximately 36- and 29-kDa bands suggests a modulation of the reactivity against ML-ESP over time. IETB proved more sensitive for the population of chronic trichinellosis under study than a conventional diagnostic enzyme-linked immunosorbent assay, allowing negative or borderline serum samples to be determined. Thus, this technique, when applied for human trichinellosis surveillance, should provide a useful tool in endemic areas.

Cytotoxicity-blocking antibodies in human chronic trichinellosis.

Antisurface newborn larva (NBL) antibodies (Abs) were found in sera from individuals chronically infected with Trichinella spiralis. These Abs were incapable of inducing NBL death by activation of normal human leukocytes of peripheral blood as determined by in vitro assays of antibody-dependent cell cytotoxicity (ADCC). Besides, such sera blocked the cytotoxic reaction mediated by Abs produced a few weeks after infection. The blocking activity could not be attributed to any particular isotype by the indirect immunofluorescence technique. Purified antisurface NBL Abs obtained from sera from chronically infected patients recognized antigens of muscle-larva excretory-secretory products (ML-ESP) in an enzyme-linked immunosorbent assay (ELISA) and an immunoelectrotransfer blot assay. Likewise, as did chronic sera, a monoclonal Ab raised against ML-ESP blocked NBL death in ADCC assays. These results suggest that during the course of an infection by T. spiralis, Abs related to ML-ESP provide an immunoevasive mechanism for avoidance by NBL of an important anti-NBL host effector mechanism.

Diagnosis of porcine trichinellosis: parasitological and immunoserological tests in pigs from endemic areas of Argentina.

In order to compare the reliability of serological and parasitological techniques for the diagnosis of porcine trichinellosis from endemic areas in Argentina, 116 pigs were studied: 61 animals from two separate outbreaks and 55 from a small abattoir. Direct diagnostic techniques included trichinoscopy and the artificial digestion method. Indirect diagnostic tests used in this study were the enzyme-linked immunosorbent assay (ELISA), employing the excretory-secretory products of muscle larvae (ML) as antigen, and the indirect immunofluorescence assay using as antigen ML in suspension (IIF-susp), cryostat sections of infected rat muscle or of free ML (IIF-slide). The percentage of parasitologically positive pigs was invariably lower than that of serologically positive animals (IIF-slide), even when digestion studies were carried out individually with a greater amount of muscle sample than required by current regulations. Close correlation was found between IIF using as antigen tissue sections and IIF using free ML sections, while IIF-susp proved unsuitable for diagnosis since this assay presented a high percentage of false negative results (20%). The IIF-slide technique proved positive in all parasitologically positive animals. ELISA rendered a lower percentage of positive reactions than IIF-slide, especially when worm burden was low. Since most parasitologically positive animals rendered at least two positive serological tests (two variations of IIF or IIF plus ELISA), those negative by digestion and positive by two serological methods were strongly suspected of having trichinellosis. Upon studying swine from a abattoir it was found that 9% of the pigs were positive when assayed by two serological techniques, but Trichinella spiralis infection could not be parasitologically confirmed. To sum up, serological methods may be used for screening all pigs and positive findings should be tested by the digestion method by analysing a greater quantity of pork than that required by current regulations, above all in areas with reported clinical trichinellosis in humans, to ensure that the pork is safe for human consumption.

Evaluation of an enzymatic immunohistochemical technique in human trichinellosis.

A comparative study was undertaken between an enzymatic immunohistochemical technique (EIT) developed for the diagnosis of human trichinellosis and the indirect immunofluorescence test (IIF), analysing sera from outbreaks of human trichinellosis in Argentina. The EIT was developed using a biotinylated anti-human gammaglobulin and a preformed macromolecular complex of avidin and biotinylated peroxidase. In both tests, the antigen used consisted of infected rat tissue sections containing muscle larval cysts of Trichinella spiralis. Results showed that the EIT closely correlated with IIF and also allowed diagnosis at an early stage of infection, thus helping to provide effective treatment for the disease. When the test was performed on sera from healthy individuals and those with other parasitic infections, cross-reactions were observed only with sera from patients with toxocariasis (1/8), Chagas' disease (3/17) and four out of 100 healthy individuals. No cross-reactions were observed with sera from patients with toxoplasmosis (0/7) or hydatidosis (0/8). Assay sensitivity was 100% and its specificity 93%. Since it renders no false negative results, EIT is an effective screening tool for detecting infection and should prove to be an important diagnostic technique for trichinellosis in rural areas and for epidemiological surveys.