Development and Validation of a Copro-Enzyme–Linked Immunosorbent Assay Sandwich for Detection of Echinococcus granulosus–Soluble Membrane Antigens in Dogs

Abstract

Cystic echinococcosis (CE) is a parasitic zoonosis caused by the larval stage of the tapeworm Echinococcus granulosus. Detection of the adult stage in the canine definitive host is essential for estimating infection rates, surveillance and monitoring of CE control programs. This study sought to develop and validate a coproantigen sandwich enzyme-linked immunosorbent assay (copro-ELISA), based on antibodies against E. granulosus-soluble membrane antigens (EGMA), that is capable of distinguishing infected and noninfected dogs. Anti-E. granulosus polyclonal immunoglobulin G antibodies were obtained from rabbit antiserum against EGMA. Optimization of the test was performed with 51 positive and 56 negative stool samples of canine echinococcosis. Specificity, sensitivity, cross-reactivity, intra-and inter-assay precision, and over time detection were evaluated. According to the receiver operating characteristic analysis, the diagnostic sensitivity and specificity were 96.1% (CI: 85.9-99.6) and 98.2% (CI: 89.5-100), respectively. Negative and positive predictive values were 96.5% (CI: 91.7-100) and 98% (CI: 94.1-100), respectively. No cross-reactivity with Taenia hydatigena, Dipylidium caninum, or Toxocara canis was observed. Intra-and inter-assay repeatability showed values of less than 15% of the variation coefficient. The over time detection was from 20 to 27 days postinfection with E. granulosus. The copro-ELISA based on EGMA detection offers a simplified in-house development of diagnostic testing. This assay showed high specificity and sensitivity and had no cross-reactivity with other parasites. Further studies and development of this test in a kit format may be useful for the detection of active infection in dogs living in CE endemic regions.

Control of cystic echinococcosis: Background and prospects

“…In recent years, several coproELISAs have been standardized for use in existing control programmes in endemic areas of the world (Guarnera, Santillan, Botinelli, & Franco, ; Jara et al, ; Morel et al, ; Pierangel et al, ) and they have been included in the surveillance systems of the countries, such as Peru, Argentina, Uruguay, China and Kyrgyzstan (Centro Panamericano Fiebre Aftosa, ; Irabedra et al, ; van Kesteren, Mastin, Torgerson, Mytynova, & Craig, ; Larrieu & Zanini, ).…”

“…In recent years, several coproELISAs have been standardized for use in existing control programmes in endemic areas of the world (Guarnera, Santillan, Botinelli, & Franco, 2000;Jara et al, 2019;Morel et al, 2013;Pierangel et al, 2010)…”

Development of a low-cost copro-LAMP assay for simultaneous copro-detection of Toxocara canis and Toxocara cati

“…Nonetheless, the sensitivity may be improved through the use of new copromicroscopic techniques with higher sensitivity, such as Mini-FLOTAC and FLOTAC, as described by Maurelli et al (2014). Techniques based on the detection of antigens in feces have also been used and have shown a wide range of sensitivity and specificity (Deplazes et al, 1990;Sen et al, 2011;Christy et al, 2012;Shimelis and Tadesse, 2014;Jara et al, 2019). Due to the different sensitivity and specificity presented by both microscopic and antigen detection techniques and with the advent of molecular techniques based on the detection of nucleic acids, certain aspects of sensitivity and specificity were improved (Jacobs et al, 1997;Li et al, 2007;Pinelli et al, 2013;Knapp et al, 2016); however, supplies and equipment are usually expensive and the procedures require human resources with specific training which are not widely available in endemic areas, complicating its implementation.…”

Evaluation of fecal immunoassays for canine Echinococcus infection in China

“…CoproELISA yielded a better sensitivity but included false positives [ 13 ]. CoproELISA was confirmed as an effective, safe, and easy method of detection in Australia [ 14 ], Argentina [ 15 ], Uruguay [ 16 ], or Peru [ 17 ]. This study provides a reference for improving control measures and assessment of the prevalence of echinococcosis in the endemic counties of China, a key step towards elimination.…”