An Easy and Efficient Method for Native and Immunoreactive Echinococcus granulosus Antigen 5 Enrichment from Hydatid Cyst Fluid Daniela Pagnozzi 1 *, Grazia Biosa 1 , Maria Filippa Addis 1 , Scilla Mastrandrea 2,3 , Giovanna Masala 2 , Sergio Uzzau 1 1 Porto Conte Ricerche Srl, Tramariglio, Alghero, Sassari, Italy, 2 Centro Nazionale di Riferimento per l’Echinococcosi, IZS ‘‘G. Pegreffi’’, Sassari, Italy, 3 Unita ` Operativa Complessa di Malattie Infettive, Azienda Ospedaliera Universitaria, Sassari, Italy Abstract Background: Currently, the serodiagnosis of cystic echinococcosis relies mostly on crude Echinococcus granulosus hydatid cyst fluid as the antigen. Consequently, available immunodiagnostic tests lack standardization of the target antigen and, in turn, this is reflected on poor sensitivity and specificity of the serological diagnosis. Methodology/Principal Findings: Here, a chromatographic method enabling the generation of highly enriched Antigen 5 (Ag5) is described. The procedure is very easy, efficient and reproducible, since different hydatid cyst fluid (HCF) sources produced very similar chromatograms, notwithstanding the clearly evident and extreme heterogeneity of the starting material. In addition, the performance of the antigen preparation in immunological assays was preliminarily assessed by western immunoblotting and ELISA on a limited panel of cystic echinococcosis patients and healthy controls. Following western immunoblotting and ELISA experiments, a high reactivity of patient sera was seen, with unambiguous and highly specific results. Conclusions/Significance: The methods and results reported open interesting perspectives for the development of sensitive diagnostic tools to enable the timely and unambiguous detection of cystic echinococcosis antibodies in patient sera. Citation: Pagnozzi D, Biosa G, Addis MF, Mastrandrea S, Masala G, et al. (2014) An Easy and Efficient Method for Native and Immunoreactive Echinococcus granulosus Antigen 5 Enrichment from Hydatid Cyst Fluid. PLoS ONE 9(8): e104962. doi:10.1371/journal.pone.0104962 Editor: Kevin K.A. Tetteh, London School of Hygiene and Tropical Medicine, United Kingdom Received March 26, 2014; Accepted July 15, 2014; Published August 13, 2014 Copyright: ß 2014 Pagnozzi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by Regione Autonoma della Sardegna (http://www.regione.sardegna.it/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: pagnozzi@portocontericerche.it Introduction Cystic echinococcosis (CE) is caused by the larval form of Echinococcus granulosus. The life cycle of this cestode involves dogs and other canids as the definitive hosts, while intermediate hosts are usually cattle, sheep, goats and pigs [1–2]. However, due to the contamination of food and water sources with E. granulosus eggs shed with canid feces, man can become an accidental intermediate host and develop CE. The greatest prevalence of CE in human and animal hosts has been recorded in the countries of the temperate zones, including parts of Eurasia, Australia, South America and Africa [3]. In most of these countries the disease has never been eradicated, exposing the population to the risk of reemergence in areas where CE was once believed to be controlled. In Bulgaria, following the reduction in control efforts, the incidence of CE in children peaked from 0.7 to 5.4/100000 between the 1970s and the mid-1990s. Similarly, in Wales the prevalence of infected dogs has more than doubled between 1993 (3.4%) and 2002 (8.1%) [4]. Outside the temperate zones, Italy is also currently considered a medium to high risk country for CE, with areas subjected to a significantly higher prevalence due to extensive sheep farming. In these high-risk areas, CE prevalence can represent a serious public health problem; as an example, the Island of Sardinia records a prevalence of 70–92.8% in sheep, 3–19% in dogs, 9.4% in cattle, 9.4–11.1% in pigs, and 1% in horses [5–6], while in the human population the annual regional average record (Hospital discharge rate) was 9.3 per 100000 inhabitants [7], with the occurrence of over 1000 cases requiring surgery each year in Italy [8]. Early diagnosis in humans is difficult, since the disease is typically asymptomatic for a long period after infection. Diagnosis is usually based on symptomatology, epidemiological data, and the com- bined use of radiologic imaging and immunodiagnostic techniques [9]. Imaging is mostly useful to assess the cyst stage and to differentiate echinococcal cyst from benign cysts, cavitary tuber- culosis, mycoses, abscesses, and benign or malignant neoplasms [10]. On the other hand, serological tests might offer advantages over radiological procedures, including early diagnosis of infection, PLOS ONE | www.plosone.org 1 August 2014 | Volume 9 | Issue 8 | e104962