The diagnosis of human fascioliasis by enzyme-linked immunosorbent assay (ELISA) using recombinant cathepsin L protease

Abstract

Background: Fascioliasis is a worldwide parasitic disease of domestic animals caused by helminths of the genus Fasciola. In many parts of the world, particularly in poor rural areas where animal disease is endemic, the parasite also infects humans. Adult parasites reside in the bile ducts of the host and therefore diagnosis of human fascioliasis is usually achieved by coprological examinations that search for parasite eggs that are carried into the intestine with the bile juices. However, these methods are insensitive due to the fact that eggs are released sporadically and may be missed in low-level infections, and fasciola eggs may be misclassified as other parasites, leading to problems with specificity. Furthermore, acute clinical symptoms as a result of parasites migrating to the bile ducts appear before the parasite matures and begins egg laying. A human immune response to Fasciola antigens occurs early in infection. Therefore, an immunological method such as ELISA may be a more reliable, easy and cheap means to diagnose human fascioliasis than coprological analysis.

Methodology/principal findings: Using a panel of serum from Fasciola hepatica-infected patients and from uninfected controls we have optimized an enzyme-linked immunosorbent assay (ELISA) which employs a recombinant form of the major F. hepatica cathepsin L1 as the antigen for the diagnosis of human fascioliasis. We examined the ability of the ELISA test to discern fascioliasis from various other helminth and non-helminth parasitic diseases.

Conclusions/significance: A sensitive and specific fascioliasis ELISA test has been developed. This test is rapid and easy to use and can discriminate fasciola-infected individuals from patients harbouring other parasites with at least 99.9% sensitivity and 99.9% specificity. This test will be a useful standardized method not only for testing individual samples but also in mass screening programs to assess the extent of human fascioliasis in regions where this zoonosis is suspected.

Figure 1. Analysis of ELISA results using FhCL1 antigen and anti-total-IgG as secondary antibody.

A) Histogram of control and Fasciola-positive serum samples. B) Histogram of normalized total IgG for negative control and Fasciola positive samples. Dash line represents cut-off for negative samples.

Figure 2. Sensitivity and specificity analysis for ELISA using anti-IgG4 as secondary antibody.

A) Histogram showing negative sample where two fall out of the cut-off limit. B) Histogram showing Fasciola-positive and control negative sera. Discrimination between negatives and positives exists; however, a small space exists between them that cannot be noticed from the graph. Dashed line represents the cut-off for negatives sample.

Figure 3. Analysis of ELISA data using FhCL1 antigen and secondary antibodies specific for IgG1, IgG2 and IgG4+IgG1 isotypes.

Histograms of ELISA data obtained for Fasciola-positive and control patient sera using A) anti-IgG1, B) anti-IgG2, and C) IgG4+IgG1 secondary antibodies. Black bars represent sera from control patients while white bars represent sera from positive patients.

Figure 4. Scattergraphs for ELISA data using various secondary antibodies.

Data obtained using secondary antibodies specific for anti-IgG4 (A), anti-IgG2 (B) and anti-IgG1 (C) compared to data obtained using anti-total IgG. Blue circles represent sera from control patients while green circles represent Fasciola-positive patients.

Figure 5. Comparison of ELISA data obtained using secondary antibodies specific for various isotypes.

Absorbance between positive infected sera and negative control sera using anti-total IgG and the different serotypes.

Figure 6. Box plots of ELISA data using sera from non-infected control patients, F. hepatica-infected patients, and patients with various parasitic diseases.

A: ELISA using anti-total IgG as secondary antibody and B: ELISA using anti-IgG4 as secondary antibody. The dashed line represents the cut-off for negative samples. Results are obtained from three independent experiments conducted in duplicate.