Parasitiology
Western blot applied to the diagnosis and post-treatment monitoring of human hydatidosis

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Abstract

The serologic diagnosis of hydatidosis (caused by Echinococcus granulosus) can be made by different techniques, although the lack of standardization of the antigens affects the sensitivity, specificity and concordance among the different tests. We have applied the Western-Blot (WB) technique, associated with a purified antigen from sheep hydatid fluid, at 60 samples of serum from 14 patients suffering echinococcosis in different bodily locations, monitored for 3 years. The WB test enabled the detection of antibodies in the pre-surgical samples for proteins of 12–14, 16, 20, 24–26, 34, 39 and 42 kDa in molecular weight in 15–96% of the patients. The combination involving 2 of the 3 proteins of 20, 39 and 42 kDa has made it possible to diagnose 100% of the cases. The antibodies specific to proteins 39 and 42 kDa disappeared in less than one year in the patients cured after surgery, while in patients with persistent or recurrent parasitism the bands present before surgery persisted or other new ones appeared. The WB with purified antigens proved to be highly useful in the diagnosis and post-surgical monitoring of hydatidosis patients. The antigen used is proposed as a standard antigen for the diagnosis and follow-up of pre- and postsurgical hydatidosis.

Introduction

Serologic tests are useful for diagnosing hydatid disease caused by Echinococcus granulosus in humans, since no parasitological diagnosis is possible by non-invasive procedures and the clinical signs of the disease are non-specific. The serologic diagnosis of human hydatidosis is strongly dependent on the antigen used, thus explaining the lack of sensitivity, specificity and concordance among different techniques, standardization of an antigen that enables post-operative follow-up of the disease being necessary. Mercado et al 1988, Orduna et al 1985, Robert-Gangneux and Tourte-Schaefer 1999, Schantz et al 1980, Varela Dı́az et al 1978, Siracusano et al 1991, Leggatt et al 1992.

Almost all the serologic tests available have been used for diagnosing hydatidosis Lightowlers and Gottstein 1995, Babba et al 1994, Zarzosa et al 1999, offering better results, particularly with the use of the B/5-rich fraction or partly purified antigen from hydatid fluid Siracusano et al 1991, Rogan et al 1991, Barbieri et al 1993, Sbihi et al 1996, Sbihi et al 1997.

One key characteristic of human hydatid disease is the frequency of post-surgical relapse. This makes follow-up of the patient necessary for years after surgery, to detect the appearance of new cysts as soon as possible Baldelli et al 1992, Force et al 1992, Guisantes et al 1994, Zarzosa et al 1999. Serology has been one of the methods selected for the post-operative control of hydatidosis. However, the long persistence of anti- E. granulosus antibodies after recovery makes difficult the diagnosis of relapse by serology Todorov and Stojanov 1979, Zarzosa et al 1999. In this sense, many serologic techniques have been evaluated (latex agglutination, passive hemagglutination, immunoelectrophoresis and specific IgE, IgM, IgG enzyme-linked immunosorbent assay) in the post-operative monitoring of hydatid disease patients. In this report, we examine the pattern of antigenic bands, useful for the serologic diagnosis of hydatidosis, revealed by immunoblotting analysis and we report on its the post-operative evolution in patients treated for this disease.

Section snippets

Parasitic material

The hydatid fluid (HF) was taken from the liver and lung cysts from sheep in the province of Zaragoza (Spain). The supernatant was extracted after centrifugation at 4000 g (30 min) and NaN3 (1 g/L) were added together with ethylendiaminotetracetic acid (EDTA, 5 mM) for preventing protease activity. The end product was distributed in aliquots of 20 mL and frozen at −20°C until used.

Antigen preparation

The hydatid fluid was processed according to the procedure described by Rogan et al. (1991), based on the method of

Results

Antibodies against proteins of the following molecular weights were detected: 12–14, 16, 20, 24–26, 34, 39 and 42 kDa. The results of the 13 pre-surgery sera are reflected in Table 1. The bands detected at the greatest frequency were those corresponding to antigens of 39 kDa (92%), 42 kDa (85%) and 20 kDa (69%). All the samples presented antibodies for 2 of these 3 antigens. Analysis of WB evolution in the 13 patients after surgery revealed two different patterns. In 5 patients without cysts

Discussion

Human hydatidosis is characterized by triggering an intense humoral response with a rise in the titers of specific antibodies Matossian et al 1972, Matossian et al 1976, and thus serologic diagnosis is frequent, using ovine hydatid fluid as antigen. The composition of the hydatid fluid presents qualitative and quantitative differences according to its origin, this affecting the quality of the results Janssen et al 1990, Verastegui et al 1992. Therefore, the search for standardized antigens

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